Professional Documents
Culture Documents
Biotechnology
Biotechnology
Biotechnology
Dr. U. Satyanardyana
M .Sc..P h D .. F.l .C ..F.A ,CB .
Professor of Biochemistry
Siddhartha M edical Colle ge
(NTR University of Health Sciences)
Vijayawada, A.P., India
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expoft and trunslation of this book for all editions and reprints thereof.
Publisher ArunabhaSen
BOOKS AND ALLIED (P) Lro.
8/1 ChintamoniDas Lane, Kolkata 700009
t s BN I t - 8 ? t lt - qo- q
UPPAIA AUTHOR_PUBLISHERINTERLINKS
(A'P')
NagarjunaNagar,MahanaduBoad,Vijayawada520008
D.No.48-16-10,
Biotechnology is an exciting,rapidlydevelopingand revolutionary scientificdiscipline,with its roots
in biologicaland technological sciences. Duringthe pastfew years,the majorityof scientificbreakthroughs
in biologicalscienceshave come from biotechnology, particularlyinvolvinggeneticengineering.Bio-
technologyimpingeson everyone'slife, and is truly regardedas the scientifictechnologyof the twenty-
first century.
THE NECESSITY
FOR A COMPREHENSIVE
EOOK ON BIOTECHNOTOGY
Biotechnology is a subjectlearntby students with differentbackgrounds and post-
at the undergraduate
graduatelevels.lt is truethatthereare somebooksnow availableon biotechnology (by Indianand foreign
authors).Thesebooks,however,deal with one or two specializedareasratherthan cdveringthe broad
spectrumof biotechnology. Consequently, the studentshaveto dependon manybooksfor goodand latest
informationon biotechnology. There is a long-feltneed for a comprehensive book on biotechnology
providingupdatedinformationon the subject.
M A I O N HI GH T IG H T S
OF T H E BOOK ' B I OTE C H N OIOGY '
The presentbook 'BIOTECHNOt-OCy',in colour,with severalnovel features,is comprehensively
written with latestadvancesin the subjectto meet the needsof all categoriesof studentslearning
biotechnology.
'The sectionon 'Basicsto learn Biotechnology'providesthe most essentialinformationneededto
understand biotechnology. Thisis particularly
usefulto students
from diversqbackgrounds who areexposed
to biotechnologyall of a sudden.MolecularBiologyis.dealtwith iri good detail,as it laysthe foundations
for geneticengineeringand modernbiotechnology.
The other sectionsin this book include RecombinantDNA Technology,Medical/Pharmaceutical
Biotechnology,
Microbial/lndustrial
Biotechnology,
Animal Cell/AnimalBiotechnology, Plant/Agricultural
Biotechnologyand Environmental Eachsectionis carefullycraftedto provideextensive,
Biotechnology.
relevantand most recentinformation.
The book is written in a lucid stylewith colour illustrations,
headings,sub-headings,tablesand flou,
charts.Coloursare usefulfor betterunderstanding and easy reproducibilityof the subjectmatter.An
extensiveglossaryis addedto the book to makethe commonlyusedtermsand words in biotechnology
very clear.
(tl
Besidesthe student community, this book will immensely help the personnelworking in biotechnology
laboratoriesand industries.The languageand contentsof this book are made so simple and easy that even
a lay m an wit h a m inim al k nowled g e o f b i o l o g i c a l s c i e n c e sw i l l b e a b l e t o u n d e r s t a n dt h e e s s e n ceo f
biotechnology.
As the subject of biotechnology is multidisciplinaryi books on the subject are written by authors with
different backgrounds-botany, zoology, genetics,pharmacy, agriculture, engineering,etc, This resultsin
an inevitable bias in the books. For instance, an author with a botany background tends to pay more
attention to planVagriculturalbiotechnolgy.The presentbook is free from such bias. Written by an author
with biochemistry background, covering all aspectsof life sciences, it is well balanced in the treatment
of various branches of biotechnology. However, there is a perceptible bias in the book towards recent
information on the applications of different branches of biotechnology to human healthcare,and for the
ultimate benefit of mankind. This is justifiable from the fact that more than TOohof the budget earmarked
for biotechnologl, vst.tt.^ in developing countries goes towards healthcare research.
AN INVITATIONTO READERS
Thisbook is designed
to caterto the needsof all categories
of studentslearningbiotechnology
in various
disciplines-lifesciences,engineeringand technology,medicine,agricultufe,pharmacy,veterinaryetc.
Thus,this book may be regardedas a buffettablewith somethingfor everyone(in everydiscipline).lt is
ultimatelyfor the readerto do appropriateself-service and enjoy the menu.
I invite the readersfor the wonderfuland excitingworld of biotechnology,
and learnthe subjectto
maximumpossiblewith easeand pleasure.
The subjectof biotechnologyis very vast,thereforeit is not an easyjob to preparea concentrated
capsuleof biotechnology. As this book is intendedto caterto the needsof studentslearningbiotechnologv
f r omma n yd i ffe re ndt i s c i p l i n e sth, e r ei s a l ot of scopeto i mprovethe booki n future.Thi spri mari l ydepends
on the feedbackfrom the facultyand studentswith specificrequestsand ideas.
It is truethat I represent a selectedgroupof individualsauthoringbooks,havingsometime at disposal.
besideshard work, determination and dedication.lconsider myselfas an eternallearnerand a regular
studentof biotechnology. However,it is beyondmy capabilityto keeptrackof the evergrowing advances
in biotechnology due to the exponentialgrowthof the subject,arrdthis makesme nervous.I honestlyadmit
that I have to dependon maturereadersfor subsequent editionsof this book.
I welcomeconstructiveand helpfulsuggestions
to improvethe book.
DR. U. SATYANARAYA\,{
(il1
I owe a deep debt of gratitudeto my parents,the late Sri U. VenkataSubbaiah,and Smt.Vajramma,
for cultivatingin me the habit of early rising.The writing of this book would neverhave been possible
without this healthyhabit.I am gratefulto Dr. B. S. NarasingaRao (formerDirector,NationalInstituteof
Nutrition,Hyderbad)for discipliningmy professional life, and to my eldestbrother,Dr. U. Cudaru(former
S angl i ),for di sci pl i ni ngmy personall i fe
, a l c h a n dC o l l egeof E ngi neeri ng,
P r of es s oorf Po w e rs y s te msW
My youngerson, U. Amrutpani,has made a significantcontributionat everystagein the preparation
of this book-writing, verification,proof-reading,
etc. My elderson,Dr. U. Chakrapani M.B.B.S,M.S.,has
helpedme with his creativeideasand suggestions
constructive to orientthe book with adequateemphasis
on human healthcare,besidesinvolving himself in the verificationand proof-reading of the book. I
acknowledgethe help of my friend, Dr. P. Ramanujam(Readerin English,Andhra Loyola College,
Vijayawada), for his help at variousstagesof manuscriptpreparation.
lexpress my gratitudeto Mr. ArunabhaSen (ExecutiveDirector,Books& Allied Pvt. Ltd., Kolkata)
for his whole-heartedcooperationin bringing out this book to my satisfaction.I am gratefulto
Mr. Shyamal Bhattacharya for the excellentpage-makingand graphics-workin the book. I thank
Mr. DineshBhunyafor proof-reading. I alsothank Mr. PrasenjitHalderfor the cover designof the book.
Last but not least, I thank my wife, KrishnaKumari,for her constantcooperation,supportand
encouragement.
Interlinks,Vijayawada,
lam gratefulto UppalaAuthor-Publisher and supportingme to
for sponsoring
write this book.
DR. U. SATYANARAYANA
(llrI
ents in Brief
1l Manipulation in
of CeneExpression
HostCells 137
Introduction to Biotechnology 12 HumanCenomeProiect 148
I of Biotechnology3
TheScope
Organization
of DNA in the cell 19
Structure
of RNA 19
Introduction to Biotcchnologv Typesof RNA 21
Catalvtic
RNAs-ribozvmes 22
1 the Scopeof Biotechnology 3
O ld and n e w b i o te c h n o l o g y 3 3 DNA-Replication,
Recombination
Definition(s) of biotechnology 3 and Repair
A
23
Historyof biotechnology a
Replicationof DNA 23
B iot ec hn o l o g- y a m u l ti d i s c i p l i n a ry
growingtree A Replication in prokaryotes 24
Commercial izationof biotechnology 7 Replication in eukaryotes 27
Publicperceptionof biotechnology 7 Processof replicationin eukaryotes )7
{it
li i l CO NTENTS I N DETAI L
Transcription 39
-franscriptionin prokaryotes 39
Transcription in eukaryotes 4) Genetic Engineering/Recombinant
Post-transcriptionalmodifications +J DNA Technologv
Cellular RNAcontents 45
Reversetranscription +o
6 Introduction
to Genetic 75
Engineering
Translation 46 Briefhistory of recombinant DNA
technology 75
Ceneticcode 46
Molecular toolsof geneticengineering /o
Protein biosynthesis 48
Restriction endonucleases-DNA cuttinB
Requirement of the components 49
enzymes
Activation of aminoacids 50
DNA ligases-DNA joiningenzymes
Proteinsynthesis proper 50
Alkalinephosphatase
lnitiationof translation 51
DNA modifying enzymes
Elongationof translation 53
Host cells- the factoriesof cloning
Termination of translation 53
Prokaryotic hosts
lnhibitorsof proteinsynthesis
Eukaryotic hosts
Chaperones and proteinfolding 55
Vectors- the cloningvehicles
Post-translational modifications
of oroteins 56
Plasmids
Proteintargeting )/
Bacteriophages
Mitochondrial DNA,transcription and
Cosmids
translation 58
Artificialchromosome vectors
5 Regulationof Gene Expression 59 Shuttlevectors
Choiceof a vector
Ceneregulation - general 59
Methodsof genetransfer
Theoperonconcept 60
Transformation
Lactose (lac)operon 60
Conjugation
Tryptophan operon 62
Electroporation
Geneexpression in eukaryotes oz
Liposome-mediated genetransfer
Chromatin andgene
structure
Transduction
expression 63
Enhancers gene Directtransfer of DNA
andtissue-specific
expresston 65 Genecloningstrategies
Combination of DNA elements and proteins Cloning fromgenomic DNAor mRNA?
in geneexpression 65 Geneticengineering of plants
Motifsin proteins andgeneexpression 65 Geneticengineering guidelines
Generegulationin eukaryotes 67 Asilomar recommendations
Methodsto studygeneexpression/regulation 68 NIH guidelines
Ceneanalysisby T-DNAand transposon Thefutureof geneticengineering
tagging 70
Methodsto studyprotein-protein 7 BasicTechniques in
interactions 70 Cenetic Engineering tE
Phage display 70 Agarosegel electrophoresis &:
lmprovingkineticproperties
of enzymes I 35 Ex vivo genetherapy 158
Proteinengineeringby useof gene Vectorsin genetherapy 159
families 136 Viruses 159
Proteinengineering
throughchemical Humanartificial chromosome 162
modifications 136 Bonemarrowcells 162
Proteinengineering
- an ever Selectedexamplesol ex vivo gene
expanding field 136 therapy 162
Therapy for adenosine deaminase
11 Manipulation of GeneExpression in deficiency 162
HostCells 137 Therapy for familialhyper-
of hostcellsfor geneexpression137
Selection cholesterolemia 164
Manipulation of geneexpression in Therapy for Lesch-Nyhan syndrome 164
prokaryotes 138 Therapy for hemophilia 164
Manipulation of geneexpression in Ex vivogenetherapywith non-
eukaryotes 139 autotogous cells 164
Saccharomyces -the yeastin
cerevisiae In vivo genetherapy 164
expressingclonedgenes 140 Cenedeliveryby viruses 165
Otheryeastexpression systems 141 Cenedeliveryby non-viral systems 166
Insectcell expressionsystems 141 Cenetherapystrategies for cancer 167
Mammalian vectors
cell expression 143 Cenetherapyfor AIDS 169
Geneexpression to produceproteins 143 Antigene and antisense therapy 169
Collectionand purificationof recombinant Antisensetherapyfor cancer 170
proteins 144 Antisensetherapyfor AIDS l.l
Purification
of recombinant oroteins 146 Antisenseoligonucleotides as
therapeutic agents 1-'
1.2 Human Genome Project 148 Chimeric oligonucleotides in gene
Thebirthand activityof humangenome correctron 1-'
project 148 Aptamers as therapeutic agents
Mappingof the humangenome 149 Ribozymes as therapeutic agents
l_
Approaches for genomesequencing 149 Thefutureof genetherapy j -_
Human genome sequence-results
summarised 149 14 ONn in DiseaseDiagnosisand
Cenomes of someotherorganismssequenced
153
of humangenome
Benefits/applications
Medical Forensics
Methodsof DNA assav
i-l
sequencing -53
Nucleicacidhvbridization
andhumangenome
Ethics 154 '- tr
DNA orobes
TheDNA chip-microarray of
geneprobes
of infectious
DNA in the diagnosis
llc dical/Pharmaceutical diseases
Biotecluroloey Tuberculosis
(Biotechnologv in Healthcare) Mal ari a
Chaga'sdisease
13 CeneTherapy 157 A cqui redi mmunodefi ci encvsrndrone
for genetherapy
Approaches 1s7 (AIDS)
Corurerurs nr Derntl (vl
of SCPfromhighenergysources375
Production Energy-rich
crops 399
of SCPfrom wastes
Production 378 Thefutureof biomass 399
of SCPfromwooq
Production 379
Production
of SCPfromCO, 379 32 MicrobialMiningand Metal
of SCPfrom sewage
Production 379 Biotechnology 400
Genetically
engineered artificial Bioleaching 400
proteinas animalfeed 380 Commercialprocessof bioleaching 401
Mushrooms 380 Bioleachingof copper 402
Productionof ediblemusnrooms 3Bi Bioleachingof uranium 402
Bioleachingof other metals 403
30 Polysaccharides, Polyhydro- Advantages of bioleaching 403
xyalkanoatesand Lipids 382 Biosorption 403
Polysaccharides 382 Microbial recovery of petroleum 404
Ceneralfeatures of microbial
polysaccharides 383
Xanthan 383
Dextran 385
Animal Cell/Animal Biotechnolos/
Alginate 385
Scleroglucan 385 33 AnimalCellCulture - Fundamentals,
Gellan 386 Facilities and Applications 407
lan
Pollu 386 Facilitiesfor animalcell culture 407
Curdlan 386 Minimalreouirements for cellculture 408
Polyhydroxyalkanoates 386 lnfrastructure 408
PHA-chemistry and properties 386 Equipment 408
Polyhydroxybutyrate(PHB) 3(j/
Culturevesse/s 408
Biosynthesisof otherPHA 389 Useof non-adhesive in
substrates
Biopol- a biodegradable plastic 390 tissueculture 409
Geneticengineering for PHA Contamination, asepticconditions,and
production 390 sterilization 409
Microbiallipids 391 Aseoticconditions 409
Production oils
of single-cell 391 Sterilization 410
Microbialrubber 391 Advantages and limitationsof
Microbialadhesivebiopolymer 392 tissueculture 411
Micobial production of indigo 392 Applications of animalcell cultures 411
Medical/pharmaceutical productsof
3L Biomassand Bioenergy 393 animalcellcultures 412
Biomass 393 Cenetic engineeringof anirnalcells
Sources of biomass
and utilization 394 andtheirapplications 413
Productionof alcoholfrombiomass 395 Risksin a tissueculturelaboratory
Productionof biogasfrom biomass 395 and safety 413
Processof biogasproduction Biohazards 414
(biogas
plant) 397
Hydrogen-a new biofuel 398 34 CultureMedia for Animal Cells 415
Productionof biohydrogen 398 Naturalmedia 415
Ixl CO NTENTS I N DETAI L
culture
Microcarrier 456 Tissue modelling 477
Perfusedmonolaver culture 457 Embryonic stemcell engineering A'7 -7
of media
Constituents 519
Preparation
of media 522
Plant/furicultural Biotechnolo 95, Selectionof a suitablemedium 522
43 PlantTissue
CultureMedia 517 46 Somaclonal
Variations 546
Major typesof media 517 Basisof somaclonalvariations 546
Corurerurs rruDEret lxiiil
of fungi
Characteristics 756
lmportance of fungi 756
Controlof fungi 757
Biotechnologf' and Socief' Viruses / J/
of viruses
Characteristics / J/
6l Biotechnology Risks,
- SocietY,
Biologicalstatusof viruses 757
Ethics,Patenting 739
lmportance of viruses 757
of biotechnologY
Benefits 739
I bio te c h n o l o g y 739
E LS of 64 Bioorganic and Biophysical
Recombinant therapeuticproductsfor Chemistry, Tools758
and Biochemistry
humanhealthcare 740
chemistry
to bioorganic
Introduction 758
andfoodconsumption
Ceneticmodifications 740
Most common orqaniccompoundsfound
Recombinantfoodsand religiousbeliefs 742 in livingsystem 758
Are GM foodssafe? 742 grouPsin
Commonfunctional
Releaseof geneticallyengineeredorganisms 742 biochemistry 758
of humangeneticrDNA research
Applications 743 Commonringstructures in
Humanembryonic stemcell research 744 biochemistry 758
Cloninghumans? 744 lsomerism 759
inventions
biotechnology
Patenting 744 Overviewof biophysicalchemistry 762
BiotechnologyProducts 744 Acidsand bases 762
Biotechnology 744 Buffers 762
Processes
Whatis a patent? 744 Solutions 762
propertyrights
Intellectual 744 Colloidal state /b3
,t.,tllr'it
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BiotechnologY[1]
J he term biotechnology representsa fusion or an DEFTNTTTON(SI OF BTOTECHNOLOGY
I alliance between biology and technology.
T h e t e r m b i o t e c h n o l o g yw a s i n t r o d u c e d i n 1 9 1 7
Fran klyspe ak ing,biot ec hnologyis as old as hu m a n
b y a H u n g a r i a n e n g i n e e r ,K a r l E r e k y . H e u s e d t h e
civilizatio n, and is an int egr al par t of hum an l i f e .
term for large-scale production of pigs by using
Thus, biotechnology is a newly discovered
sugar beets as the source of food. Ereky defined
discipline for age-old practices. There are records
biotechnology as 'all lines of work by which
th at win e an d beer wer e pr epar ed in as ear l y a s
products are produced from raw materials with
6 00 0 8 .C., bre ad and c ur d in 4000 B. C. Toda y ,w e
the aid of living thingt This definition was almost
know that all these are the processesbased on the
ignored for many years. For most people,
n atu ral ca pa bilit iesof m ic r oor ganis m s .
biotechnology represented two aspects of
OL D AND NEW BI O TEG HNO LO G Y engineering-industrial fermentation and study of
the efficiencv at work olace.
Manv authors prefer to use the term old or
traditional biotechnology to the natural processes T h e f a c t t h a t b i o t e c h n o l o g yi s i n t e r d i s c i p l i n a r yr n
that have been in use for many centuriesto produce nature, with a wide range of applications has
beer, wine, curd, cheese and many other foods createdsome confusion with regardto its definition.
This is mainly because scientists from each
The new or modern biotechnology embraces all d i s c i o l i n e h a v e d e s c r i b e dt h e t e r m f r o m t h e i r o w n
the genetic manipulations, cell fusion techniques perspective Around a dozen of the selected
a nd th e im pr ov em ent s m ade in t he o l d d e f i n i t i o n so f b i o t e c h n o l o g ya r e g i v e n i n T a b l e 1 , 1.
biotechnological processes.
T h e E u r o p e a nF e d e r a t i o no f B i o t e c h n o l o g y( E F B )
We have to accept that the present day b r o a d l y c o n s i d e r sb i o t e c h n o l o g ya s " t h e i n t e g r a t i o n
bio techn olo gy is not s om et hing new bu t i t of natural sciences and organisms, cells, parts
representsa series of technologies, some of them thereof, and molecular analoguesfor products ancl
d atin g b ack to t hous andsof y ear s e. g. pr odu c t i o n s e r vi c e s " .
of foods, beverages, modification of plants and
an imals with des ir edt r ac t s .lt is only in r ec ent y e a r s In whichever way the term biotechnology has
that these traditional practices are being subjected been defined, it essentially represents the use of
to scientific scrutiny, understood and improved, at microbial, animal or plant cells or enzymes to
least in some instances. synthesize, breakdown or transform materials.
4 B IOTE C HNO LO CY
T h e r e i s a l m o s t n o d i s c i p l i n e a m o n g t he sci e n ce
subjects that has not contributed either directly or
HISTORY OF BIOTECHNOLOGY indirectly for the growth of biotechnology.About a
dozen specialized branches of science that have
From the h istorical perspective, the
predominantlyprovided the inputsfor biotechnology
biotechnology datesbackto the time (around6000
are shown These may be appropriatelyregardedas
BC)when the yeastwas first usedto producebeer
the roots of biotechnology, and include
and wine, and bacteriawere first usedto prepare
biochemistry,genetics,molecular biology, chemical
yogurt.
engineeringand bioinformatics.A large number of
Some researchersconsider Louis Pasteur, scientists working in these specialities have
w h o i d e n ti fi e dth e ro l e o f mi crooreani sms
i n contributed to the development of biotechnology.
Chaot er1 : T H E S C OP EOF BIOT E C H N OLOC \
Year Development
Tlsrr 1.2 A selected list of historical
foundationsfor the developmentof 1977 Firstgenome
(ofbacteriophage
QX174)
biotechnology sequenced.
Foods
Diagnostics
Therapeutics
Pollution
Vaccines control
Cropyield
Applications of
biotechnology
Environmental
ENVIRON- monitoring
Food quality MENTAL
AGRICUL-
TURAL
MEDICAL
AND
HEALTH
Bioremediation
Animal ffff
/ / //
health
.Protein engineering
. Bioorocessfermentation
technology
. Biosensortechnology
.Monoclonalantibody
technology
GROWINGPLANTWITH Biotechnological
.Cell and tissueculture
STEMS,BRANCHESetc. tools
technology
.Transgenesistechnology
.Antisensetechnology
.DNA chip technology
.lmmunotechnology
. Metabolicengineering
Material
, sclence
Biochemical ooo
engrneenng 1a"; scl€nC€
lmmunology
biology biology
lf
12 B IOTE CHNO LO CY
H o
I
(A) N/"C'
,_ao
*,
H H
Adenine (A) Guanine(G)
H (6-aminopurine) (2-amino6-oxypurine)
I
(B) *tt"-"n
rll
""\ *'tt
I
Fig. 2.1 : General structure of nitrogen bases H
(A) Purine (B) Pyrimidine (The positions are Cytosine (C)
numbered according to the international system). (2-oxy4-aminopyrimidine)
n
tl
ty p e s -p u ri n e s a n d p y ri m i di nes.Thei r general HN)
structuresare depicted in Fig. 2.1. Purinesare l" | , l
n u mb e re di n th e a n ti c l o c kw i sedi recti on w hi l e o4N/
p y ri m i d i n e s a re n u m b e re d i n the cl ockw i se I
direction.And this is an internationally accepted H
systemto represent the structureof bases. Thymine(T) U racil( U)
methylpyrimidine) (2,4-dioxypyrimidine)
(2,4-dioxy-5
Major bases in nucleic acids
Fig. 2.2 : Structures of major purines (A, G) and
T h e s tru c tu reosf m a j o rp u ri nesand pyri mi di nes pyrimidines (C, T, U) found in nucleic acids.
found in nucleicacidsare shown in Fig.2.2. DNA
a n d R N Ac o n ta i nth e s a m ep u ri nesnamel yadeni ne
(A) a n d g u a n i n e (C ). F u rther,the pyri mi di ne The puri ne-guani neand pyri mi di n es- cyt osine,
cytosine (C) is found in both DNA and RNA. The lact am
thymi neand uraci lexhi bi ttautomeri sm.
However,the nucleic acids differ with respectto and lactim forms of cytosineare representedtn
the secondpyrimidinebase.DNA containsthymine Fig. 2.3.
(T) whereas RNA contains uracil (U). As is
At physiologicalpH, the lactam (keto) tauto-
observedin the Fig. 2.2, thymine and uracil differ meric forms are predominantlypresent.
in structureby the presence (in T) or absence(in U)
of a methylgroup. Minor basesfound in nucleicacids: Besides
the
basesdescribedabove,severalminor and unusual
Tautomeric forms of purines bases are often found in DNA and RNA. These
and pyrimidines
The existenceof a moleculein a keto (lactam)
NHa NH,
and enol (lactim) form is known as tautomerism.
c n g so f p u ri nesand pyri mi di nes
T h e h e te ro c y c l i ri tC u,C -
- c c
to\ N'/ N'-
till r t lll
with oxo \-C-l functionalgroupsexhibit tauto-
//
.c-ccc r ,/ \tt
me ri s ma s s i mp l i fi e db e l o w . - x tr r r\
o' Ho
H
OH OH
Lactam form Lactim form
lll I
I
S ugar s of n u c l e i c a c i d s
The five carbonmonosaccharides (pentoses) are
f ound in t h e n u c l e i ca c i d s tru c tu reR. N A contai ns
D-ribose while DNA contains D-deoxyribose.
Riboseand deoxyribosediffer in structureat C -2'
Deoxyribosehas one oxygenlessat C, compared
to ribose Gig. 2.a).
Nomenclature of nucleotides
The additionof a pentosesugarto baseproduces
a nuc leos i d el f. th e s u g a ri s ri b o s e ri
, b o n u cl eosi des
ar e f or m e d .Ad e n o s i n eg, u a n o s i n ec, y ti di ne and
ur idinear e th e ri b o n u c l e o s i d eo sf A , C , C and U
respectively. lf the sugaris a deoxyribose, deoxyribo-
nuc leos id easre p ro d u c e d .
T het er m mo n o n u c l e o ti di e
s u s e dw h e n a si ngl e
phos phat e m o i e tyi s a d d e dto a n u c l e o s i de.Thus
adenosinemonophosphate (AMP)containsadenine
+ ribose+ phosphate.
o
T he pr in c i p abl a s e sth, e i rre s p e c ti vneu cl eosi des
o--P
tl
and nuc le o ti d efo s u n d i n th e s tru c tu reo f nucl erc
acidsaregivenin Table2.1. Notethatthe prefix'd'
I
o-
(e.g.
is us edt o i n d i c a tei f th e s u g a ri s d e o x y ri bose
dA M P ) . OHH
TMP
The binding of nucleotide components
Fig. 2.5 : The structures of adenosine
T he at omsi n th e p u ri n eri n g a re n u m b e redas 1
S'-monophosphate (AMP) and thymidine
to 9 and for pyrimidineas 1 to 6 (Fig.2.1). The
S'-monophosphate (TMP) [*-Addition ol second or
carbons of sugars are representedwith an
third phosphate gives adenosine diphosphate (ADP)
associatedprime (') for differentiation.Thus the and adefiasine triphosphate (ATF) respective,lyl.
oentosecarbonsare 1' to 5'.
14 BIOTECHNOLOCY
(A)
Adenine Deoxyadenosine dAMP
DeoxyadenosineS'-monophosphateordeoxyadenylate
(G)
Guanine Deoxyguanosine dGMP
DeoxyguanosineS'-monophosphateordeoxyguanylate
(C)
Cytosine Deoxycytidine Deoxycytidine or deoxycytidylate
Slmonophosphate dCMP
(T)
Thymine Deoxythymidine Deoxythymidine ordeoxylhymidylate dTMP
Slmonophosphate
OH
o = P-o-
o
G uanine
HH
Major
groove
o = P-o-
l 0.34 nm
o
I
3' end
c
-r l' .^
P
s',
(B)
5' end
3'
Fig. 2.6 : Sttucture of a polydeoxyribonucleotide ^' '/P
J!_
/ tln
Adenine To
Chain Conformations of DNA double helix
H V a r i a t i o n i n t h e c o n f o r m a t i o n o f t h e n u cl e o ti d e s
I of DNA is associatedwith conformational variants
*-". o f D N A . T h e d o u b l e h e l i c a l s t r u c t u r eo f D NA e xi sts
(B)
in at least 6 different forms-A to E and Z. Among
these, B, A and Z forms are important (Table 2.2).
fhe B-form of DNA double helix, described by
Watson and Crick (discussedabove), is the most
predominant Iorm under physiological conditions.
E a c h t u r n o f t h e B - f o r m h a s 1 0 b a s e p a i r s sp a n n i n g
a d i s t a n c eo f 3 . 4 n m . T h e w i d t h o f t h e d o u bl e h e l i x
H
' To
Gu ani ne Chain is 2 nm.
Bent DNA
In general,adeninebasecontainingDNA tracts
are rigid and straight.Bent conformationof DNA
occurswhen A-tractsare replacedby otherbasesor
a collapseof the helix into the minor grooveof A-
t r ac t . B endin gi n D N A s tru c tu reh a s a l s o been
reported due to photochemical damage or
mispairingof bases. Ftg. 2.9 : An outline of Hoogsteen triple helical
r ru g s(e .9 .c i s p l a ti np) ro duce
Cer t ainan ti tu mo d structureof DNA.
bentstructurein DNA. Suchchangedstructurecan
take up proteinsthat damagethe DNA.
The ends of eukaryoticchromosomesnamely
telomeresare rich in guanine,and thereforeform
Triple-stranded DNA
C-tetraplexes.In recent years, telomeres have
Triple-stranded DNA formationmay occur due becomethe targetsfor anticancerchemotherapies.
to additionalhydrogenbonds betweenthe bases. C-tetraplexeshave been implicated in the
Thus, a thymine can selectively form two recombi nati on
of i mmunogl obul i n
genes,and i n
Hoogsteenhydrogenbonds to the adenineof A-T dimerizationof double-strandedgenomic RNA of
pair to form I-A-f. Likewise,a protonatedcytosine
vi rus(H l V ).
the humani mmunodefi ci ency
can alsoform two hydrogenbondswith guanineof
C-C pairs that resultsin C+-C-C. An outline of THE SIZE OF DNA MOLECULE.UNITS
Hoogsteentriple helix is depictedin Fig. 2.9. OF LENGTH
Triple-helical
structureis lessstablethan double D N A mol ecul esare huge i n si ze. On an
helix. This is due to the fact that the three average/a pair of B-DNA with a thicknessof 0.34
negativelychargedbackbonestrandsin triple helix nm has a molecularweight of 660 daltons.
resultsin an increasedelectrostaticrepulsion.
For the measurement of lengths,DNA double-
Four-stranded DNA stranded structure is considered,and expresssed in
the form of base pairs (bp). A kilobase pair (kb) is
Polynucleotideswith very high contents of 103 bp, and a megabase pair (Mb) is 106 bp and a
guanine can form a novel tetramericstructure gi gabase pai r (C b) i s 10e bp. The kb, Mb and C b
called G-guarfefs.Thesestructures are planarand relationsmay be surpmarized as follows :
are connected by Hoogsteenhydrogen bonds 1 kb = 1000 bp
(Fig. 2.10A). Antiparaller four-strandedDNA .l
structures,referred to as G-tetraplexshave also Mb = 1000 kb = 1,000,000bp
been reported(Fig. 2.108). 1 C b = 1000 Mb = 1,000,000,000 bp
Biotechnology
[2]
l8 B IOTE CHNO LO CY
t
Naked DNA l 2nm
doublehelix
'Beads-on-a-string'
form of chromatin
I,.",
30-nm chromalin
fibre composedof
nucreosomes
Chromosomein an
extendedform
(non-condensed
loops)
Condensedform of
cnromosome
J'*0..
Metaphase
chromosome
Fig, 2.12 : Organization of eukaryotic DNA structure in the form of chromatin and chromosomes.
T
11nm
J
lntemucleosome
Messenger
FNA mRNA genetic
Transfers fromgenes
information to
ribosomes proteins.
to synthesize
nuclearRNA
Heterogeneous hnRNA Serves
asorecursorformRNA andotherRNAs.
TransferRNA tRNA Transfers
amino acidto mRNA forprotein
biosynthesis.
Ribosomal
RNA rRNA Provides framework
structural forribosomes.
nuclear
Small RNA snRNA Involved
in mRNA processing.
Smallnucleolar
RNA snoRNA Playsa keyrolein theprocessingof rRNA
molecules.
RNA
Smallcytoplasmic scRNA Involved of proteins
in theselection forexport.
RNA
Transfer-messenger tmRNA Mostlypresent Addsshortpeptide
in bacteria.
tagsto proteinsto facilitate
thedegradation
ol
incorrectly ptoteins.
synthesized
Anticodon
Other R N A s
Fig. 2.14 : Structure of transfer RNA. The variousother RNAsand their functionsare
summarisedin Table2.3.
23
24 B IOTECHNO LO CY
Replication bubbles
The two complementary strands of DNA
separate at the siteof replicationto form a bubble?
Mul ti pl e repl i cati on bubbl es ar e f or m ed I n
eukaryoticDNA molecules,which is essential for a
rapid replicationprocess(Fig.3.3).
RNA primer
For the synthesis of new DNA, a shortfragment
pf RNA (about 5-50 nucleotides,variable with
speci es)i s requi redas a pri mer.This pr im er is
synthesized on the DNA templateby a specificRNA
polymerasecalledprimase.A constantsynthesisand
suppl yof R N A pri mersshoul doccuron t he lagging
Daughter DNA Parent DNA Daughter DNA strandof DNA. This is in contrastto the leading
strandw hi ch has al mosta si ngl eRNA pr im er '
Fig, 3.2 : DNA teplication-semiconservative
DNA synthesis is semidiscontinuous
and bidirectional
which is essential for the survival of the species.
The reolicationof DNA occursin 5' to 3' direc-
Synthesisof a new DNA molecule is a complex
tions,simultaneously, on both the strandsof DNA.
process involving a series of steps.
On one strand,the leading (continuous or forward)
The salient featuresof replication in prokaryotes strand-the DNA synthesisis continuous. On the
are described first. This is followed by some recent other strand, the kqCing (discontinuous or
information on the eukaryotic replication. retrograde) strand-the synthesisof DNA is discon'
tinuous.Shortpiecesof DNA (15-250nucleotides)
REPLICATION IN PROKARYOTES are producedon the laggingstrand.
Replication is semiconselvative
The parent DNA has two strands complemen-
tary to each other. Both the strands undergo
simultaneous replication to produce two daughter
molecules. Each one of the newly synthesized DNA
has one-half of the parental DNA (one strand from
original) and one-half of new DNA (fA. 3.2). This
type of replication is known as semiconservative J
since half of the original DNA is conserved in the
daughter DNA. The first experimental evidence for
the semiconservative DNA reolication was
provided by Meselson and Stahl (195S).
J
Initiation of replication
The initiation of DNA synthesis occurs at a
site called origin of replication. In case of prokaryotes,
there is a single site whereas in eukaryotes, there
Fig. 3,3 : Schematic representation of multiple
are multiple sites of origin. These sites mostly
replicatian bubbles in DNA replication.
consist of a short sequence of A-T base pairs. A
AN
3 : D N A-R EP L IC A T IO N R E C OMB IN A TION
ChA O t Cr , D R E P A IR 25
t he point o f o ri g i n .
Native DNA
Replication fork and DNA synthesis
The seoaration of the two strandsof parentDNA ;-dna A protein
o, ------J
,/---\Sf \'\)-- E,
resultsin the formationof a replicationfork. The
activesynthesis of DNA occursin this region.The E'_ a _a,
E
o
z CTG AACTG
o
ilr il ill il il ilt il lll ll
ilt il lll ll Lt ill ll ll l1
G ACUTG AC
o
(g
o
E
RNAprimer GrowingDNA
activity)to overcomethe problemof supercoils and of eukaryotes.This is depicted in Fig. 3.7, and
thenreseals the strand(ligaseactivity).Typell DNA brieflydescribedhereunder.
topoisomerase (also known as DNA gyrase)cuts
The parentalstrandsof DNA are separatedby
both strandsand resealsthem to overcometne the enzyme hel i case.A
si ngl e-stranded
DNA
pr oblemof s u p e rc o i l s .
binding protein called replicationprotein A (RPA)
REPLICATION IN EUKARYOTES bindsto the exposedsingle-stranded template.This
strandhas been openedup by the replicationfork
Replic at io no f D N A i n e u k a ry o te sc l o sel y (a previouslyformed Okazaki fragmentwith an
resembles that of prokaryotes. Certaindifferences, RNA primer is also shown in Fig. 3.4).
howev erex , ist.Mu l ti p l eo ri g i n so f re p l i c a ti o ni s a
The enzyme primase forms a complex with
characteristic featureof eukaryoticcell. Further,at
DNA polymerase ocwhich initiatesthe synthesis of
least frve distinct DNA polymerasesare known in
Okazaki fragments.The primaseactivity of pol
eukaryotes. Creek lettersare usedto numberthese
u-pri masecompl exi s capabl eof produci ng1O-bp
enz y m es .
RNA primer.The enzymeactivityis then switched
1. DNA polymerased is responsiblefor the from pri mase to D N A pol ymeraseG w hi ch
of R N A p ri me rfo r b o th th e l e a d i n gand elongatesthe primer by the addition of 20-30
s y nt hes is
laggings t r an d so f D N A . deoxyribonucleotides. Thus, by the action of pol
2. DNA po l y m e ra spe i s i n v o l v e di n th e repai r c,-primase complex,shortstretchof DNA attached
of DNA . lt s fu n c ti o n i s c o m o a ra b l ew i th DN A to RNA is formed. And now the comolex
polymeraseI found in prokarybtes. dissociatesfrom the DNA.
3. DNA polymeraseTthis enzymeparticipates The nextstepis the bindingof replicationfactor
in t he r eplic a ti o no f m i to c h o n d ri aDl N A. C (RFC)to the elongatedprimer(shortRNA-DNA).
RFC servesas a clamp loader,and catalysesthe
4. DNA polymerased is responsiblefor the
replicationon the leadingstrandof DNA. lt also assembl yof prol i ferati ngcel l nucl ear anti gen
(PCNA)molecules. The DNA polymerase 6 bindsto
possesses proof-reading activity.
the sl i di ng cl amp and el ongatesthe .Okazaki
5. DNA polymerasee is involved in DNA fragmentto a final lengthof about 150-200 bp.
synthesison the laggingstrandand proof-reading B y thi s el ongati on,the repl i cati on compl ex
f unc t ion. approachesthe RNA primer of the previous
The differences in the DNA replicationbetween Okazakifragment.
bacteria and human cells, attributed to the The R N A pri merremovali s carri edout by a pai r
enz y m es , ' ares u c c e s s fu l l uy s e d i n a n ti b a cteri al of enzymes namely RNase H and flap
therapy to target pathogen(bacterial)replication endonuclease | (FENI).This gap createdby RNA
and s par et he h o s t(h u ma n )c e l l s . removal is filled by continuedelongationof the
new Okazakifragment(carriedout by polymerase
PBOCESs OF NEPL'CAT'ON IN 6, descri bed above).The smal lni ck that remai nsi s
EUKANYOTES fi nal l yseal edby D N A l i gase.
o, Previous
- Okazakifragment
5' Templatetor
lagging
strand
DNA polymerasect-
primasecomplex
RNaseH
DNA ligase
s',
Fig. 3.7 : An outline of DNA replication on the lagging strand in eukaryotes (RPA-Replication protein A;
PCNA-Proliferating cett nuclear antigen; RFC-Reptication factor C; RNase H-Ribonuclease H; FENI-Flap
endonuclease l; Note : Leading sttand not shown)'
AN
Chaot er3 : D N A -R E PL IC AT ION ,R E C OMB IN A TION , D R E P A IR 29
Certain compounds that inhibit human Fig. 3,8 : The cell cycle of a mammalian cell
(M-Mitotic phase; Gr-Gapl phase; G6Dormant phase;
topoisomerases are used as anticanceragentse.g.
S phase-Period of replication; Gp-Gap 2 phase).
adriamycin,etoposide, doxorubicin.The nucleotide
analogst hat i n h i b i tD N A re p l i c a ti o n
a re a l soused
as ant ic an c e r d ru g s € ,8 . 6 -me rc a p t opuri ne,
C ycl i nsand cycl i n-dependent ki nases(C D K 1,
5- fluor our a c i l .
CDK2,CDK4,CDK6)are intimatelyconnectedwith
the progression of cell cycle.For instance, cyclin D
CELL CYCLE AND DNA REPLICATION
levelsrise in late C1 phasewhich activateCDK4
The cell cycle consistsof four distinctphasesin and C D K 6.Thi s resul tsi n the assembl of y nucl ear
higher organisms-mitotic,Cr, S and C, phases proteins in a complex form in late C1 phase.
(Fig.3.A. When the cell is not growing,it existsin
a dor m anto r u n d i v i d i n gp h a s e(C !. C -' p h asei s Gell cycle check points
by activeproteinsynthesis.
characterized A s depi cted i n Fi g.3.B , there occurs a
continuous monitoringof the cell cyclewith respect
of DNA occursonly once in S-phase
Replication
to DNA replication,chromosomesegregation and
and the chromosomesget doubled i.e. diploid
genomegets convertedinto tetraploid.The entire integrity.lf any ddmageto DNA is detectedeither
phaseof the cycle, or if there is a
processof new DNA synthesistakes place in in G1 or C2
formation of defective spindle (i.e. incomplete
about B-10 hours and a large number of DNA
polymerases (500-1,000) are simultaneously chromosomal segregation),
the cell cycle will not
progressuntil appropriatelycorrected.lf it is not
involved in this process. lt is believed that
possibleto repair the damage done, the cells
methylationof DNA servesas a markerto inhibit
undergo apoptosis(programmedcell death).
reolication.
Holliday model
H ol l i daymodel(proposed by H ol l i dayi n 1964)
Recomb inat ionbas ic ally inv olv es t he ex ch a n g e i s the si mpl est among the homol ogous
of genetic information. There are mainly two types recombi nati on model s.l t i s depi ctedi n Fi g. 3.11,
of recombinations. and brieflyexplainedin the next page.
o2 B IOTE C H N O LO CY
Transposition
Transposition primarily involvesthe movement
of specific pieces of DNA in the genome. The
mobilesegments of DNA are called transposonsor
transposableelements.They were first discovered
by B arbaraMcC l i ntock(i n 1950) i n mai ze ,and
thei r si gni fi cancew as i gnored for about t wo
decadesby other workers.
Transposonsare mobileand can movealmostto
any placein the targetchromosome. Thereare two
modesof transposition. One that involvesan RNA
a i.........' ...r b intermediate,and the otherwhich doesnot involve
Recombined
daughter Recombined
daughter RNA intermediate.
DNAstrands DNAstrands
Retrotransposition: Transpositioninvolving
Fig. 3.11 : Hollidaymodelfor homologous
RNA intermediate representsretrotransposition
recombination(Note : Heteroduplexregionsare
(Fig.3.12).By the normalprocessof transcription,
shown in dotted boxes).
a copy of RNA formed from a transposon(also
AN
c hapt er3 : D N A -R E PL IC AT ION ,R EC O MB IN A TION , D R E P A IR 33
Significance of transposition
It is now widely acceptedthat a largefractionof
the human genome has resulted due to the
Transposon copy
(retrotransposon)accumulation of transposons.Shorf intetspersed
elements(SlNEs)are repeatsof DNA sequences
which are presentin about 500,000 copies per
rcpreaentation
Fig. 3,12 : A diagrammatic ot haploid humangenomee.g. Alu seguences,
Long interspersed elements (LrNfs) are also
repeatedDNA sequences and are presehtin about
called as retrotransposon).Then by the enzyme 50,000 copi es i n the human genome e.g. L1
reversetranscriptase,DNA is copiedfrom the RNA. elements.
The newly formed DNA which is a copy of the
Someof the diseasescausedbv mutationsare
transposongets integratedinto the genome.This
due to insertionof transponsinto a genes.
integrationmay occur randomly on the same
chromosomeor, on a differentchromosome. As a
there are now two
resultof the retrotransposition,
copiesof the transposon,at differentpointson the
genome.
DNA transposition: Some transposonsare Being the carrier of genetic information,the
capableof direct transpositionof DNA to DNA. cel l ul ar D N A must be repl i cated(dupl i cated),
This may occur either by replicativetransposition maintained,and passeddown to the daughtercells
or conservative
transposition (Fig.3.13). Both the accurately. In general,the accuracyof replicationis
mechanisms requireenzymesthat are mostlycoded extremely high. However, there do occur
by the geneswithin the transposons. replicationerrors.lt is estimated
that approximately
ln the replicative transposition, a direct one erroris introducedper billion basepairsduring
interactionoccurs betweenthe donor transposon each cycle of replication.The cells do possesthe
capabilityto repair damagesdone to DNA to a
largeextent.
Biotechnology [3]
34 B IOTE CHNO LO CY
REPAIR OF DNA
UCU As alreadystated,damageto DNA causedby
(codonfor Ser)
replicationerrorsor mutationsmay have serious
Tsitent consequences. The cell possesses
an inbuiltsystem
I to repairthe damagedDNA. This may be achieved
rnutation, by four distinctmechanisms (Table3.2).
I
UCA 1. B aseexci si on-repai r
(codonfor Ser)
2. N ucl eoti deexci si on-reoai r
3. Mismatchrepair
4. Double-strand
breakrepair.
9Hs 9Hs
I Singlestrand
cutbVGATCendonuclease
I
9Hg
{' QHe
eronrr,""""
Cuttingat twosites f
to removedefective
oligonucleotide
I DNApolymerase
+
CHa
9Ht t-
Degradationof
defectiveDNA
Resynthesisand
religation | ,,n"."
9H. + ?H.
38
AND TRANSLATION
Chaoter4 : TRANSCRIPTION 39
smallmolecules
tissueor an organismto synthesize
(metabolites)is metabolomics.
Whetherthe centraldogmaof life is represented
in the conventional or more recent form, o-
r eplic at iont r, a n s c ri p ti oann dtra n s l a ti oanrethe key
or core processesthat ultimately control life.
Replicationof DNA hasbeendescribedin Chapter
2, while transcription and translationare discussed
in this chapter.
Core enzyme Sigmafactor
s a p ro c e s si n w h i c h ri b o nucl ei c
T r ans c r i p ti oi n
acid (RNA) is synthesized from DNA. The word
Fig. 4.2 : RNA polymerase of E coli.
gene referslo the functional unit of the DNA that
can be transcribed. Thus,the geneticinformation
storedin DNA is expressed throughRNA. For this
modificationsetc.) commonly known as post-
purpose,one of the two strandsof DNA servesas
transcriptionalmodifications,to producefunctionally
a template(non-codingstrandor sensestrand)and
acti veR N A mol ecul es.
producesworking copiesof RNA molecules.The
other DNA strandwhich does not participatein Thereexistcertaindifferences in the transcription
transcriptionis referredto as coding strand or between prokaryotesand eukaryotes.The RNA
antisensestrand(frequentlyreferredto as coding synthesisin prokaryotesis given in some detail.
strandsincewith the exceptionof T for U, primary Thi si s fol l ow edby a bri efdi scussi on
on eukaryoti c
m RNA c onta i n s c o d o n s w i th th e s a m e base transcri pti on.
sequence).
TRANSCRIPTION IN PROKARYOTES
Transcription is selective A single enzyme-DNA dependent RNA
The entiremoleculeof DNA is not expressed in polymerase or simply RNA polymerase-
RNAsare synthesized
transcription. only for some synthesizesall the RNAs in prokaryotes.RNA
selectedregionsof DNA. For certainother regions polymeraseof E. coli is a complex holoenzyme
of DNA, theremay not be any transcription at all. (mol wt. 465 kDa)with five polypeptidesubunits-
The exact reasonfor the selectivetranscriptionis 2u, 1p and 1B' and one sigma(o) factor(Fig.4.2).
not k nown.Th i sma y b e d u e to s o mei n b u i l ts i gnal s The enzymewithout sigmafactor is referredto as
in t he DNA mo l e c u l e . core enzyme(orpB'1.
The productformed in transcriptionis referredto An overview of RNA synthesisis depicted in
as primary transcript.Most often, the primary RNA Fig. 4.3. Transcriptioninvolves three different
transcriptsare inactive. They undergo certatn stages-i ni ti ati on, el ongati on and termi nati on
alt er at ions( s p l i c i n g , te rmi n a l a d d i ti o n s , base (Fig. 4.4).
Codingstrand
t- RNApotymerase
Template
strand
DNA template
5, M 3'
Newly synthesized
RNA
Termination
A
Rho factor
--mt
o
I
z
a\
-
Fig. 4.4 : Synthesis of RNA trom DNA template (transcription)' -o
Chapt er4 : T R A N S C R IPT ION
AN D T R A NS LA TION 41
-3 5 Pribnow
Sequence oox
Coding 5'- TTGACA TATAAT
strand
Template
strand
Start of
transcription
RNA____-------+5'........AU G C A U G G C A........3'
s',
DNA
c
t
RNApolymerase
ll RNApolymerase
tll
'ly
J I
J
R N A . The p
s n s c ri p ti oann d re l e ases
t e rm i n a tetra 3. RNA polymerase lll participatesin the
factor is also responsiblefor the dissociationof formati onof tR N A sand smal lri bosomalRNAs.
RNA polymerase from DNA.
Besidesthe threeRNA polymerasesfound in the
2. Rho(p) independent termination: The termi- nucleus,there also exists a mitochondrialRNA
nation in this case is brought about by the polymerase in eukaryotes.The latter resembles
formationof hairpinsof newly synthesizedRNA. prokaryotic RNA polymerase in structure and
This occursdue to the presenceoI palindromes.A function.
palindromeis a word that readsalike forwardand
backward e.g. madam, rotor. The presenceof Promoter sites
p a l i n d ro meisn th e b a s es e q u e n ce
of D N A templ ate In eukaryotes, a sequence of DNA bases-which
(same when read in opposite direction) in the is almostidenticalto pribnowbox of prokaryotes-
terminationregionis known.As a resultof this,the is identified(Fig. a.0. This sequence,known as
newly synthesized RNA foldsto form hairpins(due Hognessbox (or TATA box), is located on the left
t o c o mp l e m e n ta ryb a s e p a i ri ng) that cause about 25 nucleotidesaway (upstream)from the
o f tra n s c ri p ti o n .
t e rmi n a ti o n startingsite of mRNA synthesis. There also exists
another site of recognitionbetween 70 and 80
TRANSCRIPTTON IN EUKARYOTES nucleotides upstreamfrom the startof transcription.
This secondsite is referredto as CAATbox. One of
RNA synthesisin eukaryotesis a much more
these two sites (or sometimesboth) helps RNA
complicated process than the transcription
polymerasell to recognizethe requisitesequence
describedabove for prokaryotes. As such, all the
on D N A for transcri pti on.
d e ta i l s o f e u k d ry o ti ctra n s c ri pti on(parti cul arl y
a b o u t te rm i n a ti o n a
) re n o t c l e arl y know n. The
lnitiation of transcription
salientfeaturesof availableinformationare siven
here. The moleculareventsrequiredfor the initiation
of transcriptionin eukaryotesare complex, and
RNA polymerases broadlyinvolvethree stages.
The nuclei of eukaryoticcells possessthree
1. Chromatincontainingthe promotersequence
distinctRNA polymerases (Fig.4.71.
made accessible to the transcriptionmachinery.
1. RNA polymeraseI is responsiblefor the
synthesis of precursors for the largeribosomalRNAs. 2. Bindingof transcription factors(TFs)to DNA
sequences in the promoter region.
2. RNA polymerase ll synthesizes the
o re c u rs o rsfo r m R N Asa n d s ma l lnucl earR N A s. 3. S ti mul ati on by enhancer s.
of transcri pti on
43
--)^to r a T R AN SC R IP T IOANN D T R AN SLA TION
Hogness
CAAT box box
Non-coding5i.- GGCCAATC ATATAA
strancl
Coding
strand
-70 bases i -25 bases
Start of
transcription
hnRNA(preRNA)
End
modifications
-c-#
o-_-=----fl lntrons Cutpieces New chemicalgrouPsadded
cap Poly(A)tail
removeo
nuclearRNl).
Fig. 4'g : An a||ttingof pagt.trensc|iponal mdllieabn$Q| RNA(lnlNA.Heterogeneous
44 B IOTE C H NO LO CY
1 . 5x 1 0 8b p
Messenger RNA
Fig. 4.12 : A diagrammatic representation of RNA content of a cell (Note : RNAs represented in
black are found in all organisms; RNAs in colour and exclusively present in eukaryotes only;
. hnRNA-Heterogeneous nuclear RNA; rFNA-Ribosomal RNA: tBNA-Transfer HNA;
snRNA-Small nuclear RNA; snoBNA-Small nucleolar RNA; scRNA-Small cytoplasmic RNA).
46 B IOTE C H N OLO CY
::f:-."eci Cur ing t he c our s e of ev olut ion. H e n c e of one or two bases will radically change the
r,:"-F:r: :Ce is appropriately regardedas universal. message sequence in mRNA. And the protein
-*r+.= :'e however, a few exceptions. For instance, synthesized from such mRNA will be totally
r - r : :f e c odon f or m et hionine in m it oc h o n d r i a . different. This is encountered in frameshift
-rr: i;Te codon (AUA) codes for isoleucine rn mutations which cause an alteration in the readine
: ,r:: ;s'n. W it h s om e ex c ept ions not e d , t h e frame of mRNA.
: co de is univ er s al.
-:.n : 4. Degenerate : Most of the amino acids have
-" Specificity : A particular codon always codes more than one codon. The codon is degenerateor
'r:':-e redundant,since there are 61 codons available to
sam e am ino ac id, henc e t he genet ic c o d e r s
-:-',. spe c if ic or unam biguous e. g. UCC i s t h e c o d e f o r o n l y 2 0 a m i n o a c i d s . F o r i n s t a n c e ,g l y c i n e
::.:or tbr tryptophan. has four codons. The codons that designate the
same amino acid are called synonyms. Most of the
-1. Non-overlapping : The genetic code is read s y n o n y m s d i f f e r o n l y i n t h e t h i r d ( 3 ' e n d ) b a s e o f
---- a fixed point as a c ont inuous bas e s eq u e n c e .
the codon.
: : n on -over lapping,c om m ales s and wit ho u t a n y
:-^ ciua tion s . For ins t anc e,UUUCUUACAC C C i s The Wobble hypothesisexplainscodon degeneracy
-=ad as UUU/CUUIACA/CCC. Addition or deletion (described later).
Tlslr 4.1 The genetic code along with respective anlno acids
CUU
CUC
CXJA
Leu
ccu
ccA
Pro i
i
i
'i
cAU
cnc
CAA
1,,, C GU
UUU
CGA
Arn
tl
A
vu\f
-l
tr ULI
i
i ^n^
vA\l lo,,.
AUU AGU- I U
ISer
AUc Ite AGCI
I -I
AUA I AGA A
lAro
AUG* Met AGG-]
GUU ITUU GGU U
GUC (,(/(,
Val Gll,
GUA GCA tr \rA A
uuu
*AUG in protein
r.r., as initiating
codon,besidescodingfor methionine
residue synthesis;
UAA,UAGandUGAcalledas nonsense
areresponsible
codons, of protein
fortermination synthesis,
48 B IOl E C H N OLO CY
Anticodon Codon
5' end (/-U
l ll basepairing
) Conventionat
v
il-G base
or A 1 Non-conventional
G - tl or C I pairing
(coloured)
Wobble hypothesisexplainsthe degeneracy of
the geneticcode, i.e. existence of multiplecodons
for a si ngl e ami no aci d. A l thoughthere are 61
codonsfor aminoacids,the numberof tRNAsis far
l ess(around40) w hi ch i s due to w obbl i ng.
Wobble hypothesis
Wobble hypothesis,put forth by Crick, is the The protein synthesis which involves the
phenomenonin which a singleIRNA can recognize translationof nucleotidebasesequenceof mRNA
more than one codon. This is due to the fact that into the languageof amino acid sequencemay be
the third base(3rbase)in the codon often fails to divided into the following stages for the
recognizethe specificcomplementary base in the convenienceof understanding.
anticodon(5'-base). Wobbling is attributedto the
of the comPonents
l. Requirement
differencein the spatialarrangementof the 5'-end
of the anticodon.The possiblepairing of 5'-end l l . A cti vati onof ami no aci ds
]1AOtCT4 : TRANSCRIPTIONAND TRANSLATION 49
Biotechnology
[4]
50 B IOTECHNO LO CY
Amino acid
@-nrrrre-Aminoacid
ri
I
tRNA Aminoacyl
tRNA
Each one of these reactionsconsumestwo hieh synthesis proceeds from N-terminal end to
energyphosphates(equivalentto 2 ATP). C-terminal end. Translationis directional ano
col l i nearw i th mR N A .
6. Proteinfactors : The processof translation
involvesa number of protein factors.These are The prokaryoticmRNAs arepolycistronic,since
neededfor initiation,elongationand terminationof a si ngl emR N Ahasmanycodi ngregi o nst hat code
protein synthesis.The protein factors are more for differentpolypeptides.In contrast,eukaryotic
complex in eukaryotes comparedto prokaryotes. mRNA is monocistronic, sinceit codesfor a single
polypeptide.
II. AC T IVA T IO N OF A M IN O A C ID S
In case of prokaryotes, translationcommences
Amino acidsareactivatedand attachedto tRNAs beforethe transcriptionof the gene is completed.
in a two stepreaction. A groupof enzymes-namely Thus,si mul taneous andtra nslat ion
transcri pti on ar e
aminoacylIRNA synthetases-are requiredfor this possible.This is not so in case of eukaryotic
process.Theseenzymesare highly specificfor the organi sms occursi n t he nucleus
si ncetranscri pti on
a mi n o a c i d a n d th e c o rre s pondi ng
IR N A . whereas translationtakes place in the cytosol.
The amino acid is first attachedto the enzyme Further, the primarytranscript(hnRNA)formedfrom
u ti l i z i n g A T P to fo rm e n z yme-A MP -ami noaci d DNA has to undergo several modificationsto
complex. The amino acid is then transferredto generate functi onalmR N A .
the 3' end of the IRNA to form aminoacvltRNA Proteinsynthesis is comparativelysimplein case
ffig. a.l6). of prokaryotescomparedto eukaryotes.Further,
many steps in eukaryotictranslationwere not
III. PR O T E IN SY N T H ES IS P R OP E R
understoodfor quite sometime.For thesereasons,
The protein or polypeptide synthesisoccurson majorityof the textbooksearlierused to describe
the ribosomes (ratherpolyribosomes). The mRNA is translationin prokaryotes in detail,and give most
read in the 5'-+3' direction and the polypeptide importantand relevantinformationfor eukaryotic
--i.ic'ji.-i 1R\\SCRIPTION AND TRANSLATION 5t
-:i--,:i,-,- ,'.:: t he adv anc esin m olec ularbio l o g y , thentransferredto 43Scomplex.Forthe appropriate
'rr tr-.,:-: :j c r ot ein bios v nt hes isin euk ar v o t e st s associ ati onof 43S prei ni ti ati oncompl ex w i th
Tr'lil"Pr - -'--E-S:OOd nOW. mRNA,energyhas to be suppliedby ATP. I
Frr n : - i rr ith some relevant features of i ni ti ati on comol ex scans the mR N A for the i
iflir* .;-i :i : p rot ein bios y nt hes isTr
. ans lat ionpr o p e r i denti fi cati onof appropri atei ni ti ati on codon. !
5'-AUC is the initiationcodonand its recognitionis i
: r rer rto three stages-initiation, elongation
i'rj i:- ^ a tion ( as it is done f or t r ans c r ipt io n ) . facilitatedby a specificsequenceof nucleotides t
I
g
surroundi ngi t. Thi s marker sequencefor the tr
L
|flTrIATION OF TNANSLATTON identification of AUC is calledas Kozak consensus
sequences. In caseof prokaryotes the recognition
-.^ itjation of t r ans lat ion in euk ar y ote s i s
sequenceof initiationcodon is referredto as Shrne-
-: :\ ;nr,olving at least ten eukaryotic initiation
Dalgarno sequence.
tectors /elFs) Some of the elFs contain multiple
. -i ;-cu nits The pr oc es sof t r ans lat ion init ia t i o n
Formation of 8OS initiation complex
-.- := dirided into four steps (Fig. 4.171
- 4B S i ni ti ati oncomol exbi ndsto 605 ri bosomal
Rib osom aldis s oc iat ion.
subunito t formB 0Si ni ti ati oncompl ex.Thebi ndi ng
- Forma tion of 43S pr einit iat ion c om plex .
i nvol vesthe hydrol ysi sof C TP (boundto el F-2).
: Forma tion of 4BS init iat ion c om plex . This stepis facilitatedby the involvementof elF-5.
j Forma tion of B0S init iat ion c om plex .
A s the B 0S compl ex i s formed,the i ni ti ati on
factorsboundto 4BSinitiationcomplexarereleased,
Ribosomal dissociation
and recycled.Theactivationof elF-2requireselF-2B
805 ribosome dissociatesto form 40S and (al socal l edas guani nenucl eoti de factor)
exchange
-re
:'-S su bu nits. Two init iat ing f ac t or s nam ely e l F - 3 and CTP.The activatedelF-2 (i.e. bound to CTP)
.-: elF-1A bin d t o t he newly f or m ed 40S s ub u n i t , requireselF-2Cto form the ternarycomplex.
.'. th ere by bloc k it s r eas s oc iat ion wit h 6 0 5
=-run it. Fo r th is r eas onrs om e wor k er s nam e e l F - 3 Regulationo? initiation
,: anti-association factor. The elF-4F,a complexformed by the assembly
of three i ni ti ati onfactorscontrol si ni ti ati on,and
Formation of 43S preinitiation complex
process.
thusthe translation elF-4E,a componentof
\ ternary complex containing met-tRNAr and el F-4Fi s pri mari l yresponsi blfor e the recogni ti on
eiF-2 bound to CTP attaches to 40S ribosomar of mR N A cap. A nd thi s stepi s the rate-l i mi ti ng in
s;bu nit to fo r m 43S pr einit iat ion c om plex . T h e transl ati on.
3 resen ceof elF - 3 and elF- lA s t abiliz est his c om p l e x
el F-2w hi ch i s i nvol vedi n the formati onof 43S
,\ote : Me t-tRNA is s pec if ic allyinv olv ed in bin d i n g prei ni ti ati on compl ex al so control s protei n
:o the in itiat ion c ondon AUCs ; henc e t h e
biosynthesis to someextent.
=uperscrip/is used in met-tRNA/).
-rt
a\*\._-*.--- tr .-1.
.;
il !
52 B IOTE C HNO LO CY
s',
cap
Met
@A I
nr o I
)
ADP + Pit'lY
Met
80S initiationcomPlex
Fig. 4.17 : A diagrammatic representation of initiation of protein biosynthesis (translation) in eukaryotic cells
(The eukaryotic initiation factors are represented by symbols n,A, andQ. By prefixing with elE
the fult names of the factors are obtained e.9. E represents elF-S)
fnacner-l : TMNSCRIPTIONAND TRANSLATION 53
80S initiation
complex
AAr
l-
t
AA,
r-..._A A n
Termination
codon
Met
I
AAr
I
AA2
GTP @Translocation t-
AAa
t-
AA"
G DP + Pi i
,-\ AAn
EF-2 ll..UAC/
5'M3',
mR N A Peptide
synthesized
Fig.4 .l a c ontd. ner t c ol um n
HN:
li
R.-9-H i A-Site
ll
C=O i
Peptidyltransferase
I
I
3' m R N A 3'mRNA
Ribosome
Ftg- 4.19 : Formation of peptide bond in translation (P-site-Peptidyt IRNA s'tte;A-site-Aminoacvl IRNA sitd.
Tetra cyc line: lt inhibit st he binding of am i n o a c y l functionally active conformation e.g. danatured
:R\ I to the ribosomal complex. In fact, tetracycline pancreaticribonuclease.However, a vast majority of
-:n also bl oc k euk ar y ot ic pr ot ein s y nt hes i s .T h i s ,
proterns can attain correct conformation, only
^ r',\e ve r, d oes not happen s inc e euk ar y o t i c c e l l through the assistanceof certain proteins referredto
-e mbra ne is not per m eablet o t his dr ug. as chaperones. Chaperones are heat shock proteins
(originally discovered in response to heat shocr,t.
Puromycin : This has a structuralresemblanceto
They facilitate and favour the interactionson the
I RNA. Pur om y c in ent er s t he A s i t e a n d
"^ rin oa cyl polypeptide surfaces to finally give the specific
:ets incorporated into the growing peptide charn
ard causes its release. This antibiotic orevents conformation of a protein Chaperones can
crotein synthesisin 6oth prokaryotesand eukaryotes. reversibly bind to hydrophobic regions of unfolded
proteins and folding intermediates. They can
Chloramphenicol : lt acts as a competitlve stabilize intermediates, prevent formation
of
rhibitor of the enzyme peptidyltransferaseano incorrectintermediates,and also preventundesirable
:rus interfereswith elongation of peptide chain interactionswith other proteins. All these activitres
Erythro m y c in: lt inhibit s t r ans loc a t i o n D y of chaperones help the protein to attain compact
cin din g with 50S s ubunit of bac t er ial r ibos o m e . a n d biologicallyactive conformation.
Polypeptide
Folding
\---------
---t/Proteolytic Intein Chemical(covalent)
splicing modifications
Tertiarystructure
= ren time, only a f r ac t ion of t he genom e i s The genes are generally considered under two
: ' : -a :S€d categones.
--e living cells pos s es sa r em ar k ablepr oper t yt o 1 . Constitutive genes : The products (proteins)
::::r to cha ng es in t he env ir onm ent by r egula t i n g of these genes are required all the time in a cell.
'- sen e e xp r es s ion. For ins t anc e, ins ulin i s Therefore, the constitutive genes (or housekeeping
-
,"^ :^ esize d b y s pec ializ ed c ells of panc r eas a n d genes) are expressedat more or less constant rate in
^ -: ov ce lls of ot her or gans ( s ay k idney , li v e r ) , almostall the cells and, further,they are not subjected
.:-,ruBh th e n uc lei of all t he c ells of t he b o d y to regulatione.g. the enzymes of citric acid cycle
-:a :n th e in s ulin Benes . ' M olec ular r egul a t o r y
2. Inducible genes : The concentration of the
-=cra nisms fa c ilit at et he ex pr es s ionof ins ulin g e n e
p r o t e i n ss y n t h e s i z e db y i n d u c i b l e g e n e si s r e g u l a t e d
- ca ncrea s, w hile pr ev ent ing it s ex pr es s io n i n
b y v a r i o u s m o l e c u l a r s i g n a l s .A n i n d u c e r i n c r e a s e s
.-er cells.
the expression of these genes while a repressor
decreases, e.g. tryptophan pyrrolase of liver is
G ENE REGU LATI O N_G ENERAL
induced by tryptophan.
The regulation of the expression of genes is
acsolutely essential for the growth, development, One cistroh.on€ subunit concept
difierentiation and the very existence of an
T h e c h e m i c a l p r o d u c t o f a g e n e e x p r e s s i o ni s a
lq an ism. Th er e ar e t wo t y pes of gene r egula t i o n -
protein which may be an enzyme. lt was originally
losrtive an d negat iv e.
believed that each gene codes for a specific
1 Positive regulation : The gene regulation is e n z y m e , l e a d i n g t o t h e p o p u l a r c o n c e p t , o n e g e n e -
said to be positive when its expressionis increased o n e e n z y m e . T h i s h o w e v e r , i s n o t n e c e s s a r i l yv a l i d
lr a regulatory element (positive regulator). due to the fact that several enzymes (or proteins)
59
60 B IOTE CHNO LO CY
Ar P U z A
P z A
rTl
|_-1-L
lll
Repressor
tetramer
CAP-cAMP
Glucose
Tryptophan operon regulatlon
J by a repressor
o
Tryptophan acts as a corepressorto shut down
the synthesisof enzymes from tryptophan operon.
T h i s i s b r o u g h t o u t i n a s s o c i a t i o nw i t h a sp e ci fi c
protein, namely tryptophan repressor. Tryptophan
r e p r e s s o r ,a h o m o d i m e r ( c o n t a i n s t w o i d e n ti ca l
s u b u n i t s )b i n d s w i t h t w o m o l e c u l e s o f t r yp to p h a n ,
and then binds to the trp operator to turn off the
transcription.lt is of interestto note that tryptophan
n r e p r e s s o r a l s o r e g u l a t e s t h e t r a n s c r i p t i on o f th e
ffi
lacoperon gene (frpR) responsiblefor its own synthesis.
I Two polycistronic mRNAs are produced from
Polycistronic
mRNA tryptophan operon-one derived from all the frve
structural genes, and the other obtained from the
Fig. 5,2 : Control of lac operon by catabolite gene last three genes.
activator pratein (CAP) and the role of glucose.
Besides acting as a corepressor to regulate
t r y p t o p h a n o p e r o n , t r y p t o p h a n c a n i n h i b i t th e
a c t i v i t y o f t h e e n z y m e a n t h r a n i l a t es y n t h eta seTh . is
regulator for the gene expression. lt is, therefore,
i s r e f e r r e dt o a s f e e d b a c k i n h i b i t i o n , a n d i s b r o u g h t
evident that lac operon is subjectedto both positrve
o u t b y b i n d i n g o f t r y p t o p h a n a t a n a l l o s t e r i csi te o n
(by repressor, described above) and negative
anthrani late synthetase.
r egulat ion.
STRUCTURAL
GE N E S
REGULATORY
ITpTELEMENTS
II __l_r-_T mRNAs
J tl
jiJ
Anthranilate
synthase(CoII) Phosphoribosyl Tryptophan Tryptophan
Phosphoribosyl POLYPEPTIDES
anthranilate synthetaseB synthetasea
anthranilate rsomerase,
transferase Indoleglycerol
phosphate
synthetase
Anthranilatesynthetase Tryptophan
synthetase ENZYME
(Col + CoII) COMPLEXES
(rlz9z\
CATALYSED
REACTIONS
Glutamine L-Serine
Fig. 5.3 : Tryptophan operon in E.coli [regulatory elements are promoter (trpP), operator (trpo),
attenuator (trpa), secondary internal promoter (trpPr) and terminator (trpt); Col, Coll -Component land
component Il; PRPP-5-Phosphoribosyl 1-pyrophosphate;CdRP-Carboxyl-phenylamino 1 deoxyribulose
S-phosphate; lnGP - Indole S-glycerol phosphatel.
Methylation of DNA
Irru 5.1 A selectcdllst of genes{representedlo}r and inactivation of genes
the products)alongwlth rcspective chlonosones
Cytosine in the sequenceCC of DNA gets
Genes Chromosome methylatedto form 5'-methylcytosine. A major
number portion of CC sequences(about 20%) in human
DNA exists in methylated form. In general,
phosphatase
Alkaline 1 methylation leadsto lossof transcriptionalactivity,
Apolipoprotein
B 2 and thus inactivation of genes.This occurs due to
Transfenin bi ndi ng of methyl cytosi nebi ndi ng pr ot eins t o
methylatedDNA. As a result,methylatedDNA is
Alcohol
dehydrogenase ^
not exposedand boundto transcription factors.lt is
HMG CoAreductase interestingto note that methylation of DNA
21-hydroxylase
Steroid correlateswith deacetylationof histones This
Arginase 7 providesa double meansfor repression of genes.
Carbonic
anhydrase I of genes,
The activationand normalexpression
Interferon and gene inactivationby DNA methylationare
I
depicted in Fig. 5.4.
Parathyroid
hormone 11
Glyceraldehyde
3-phosphate
dehydrogenase12
Adenosinedeaminase 13
o,,-Antitrypsin 1n
CytochromePouo 15
Hemoglobina-chain lo
Growlhhormone 17
Prealbumin 18
phosphokinase
Creatine (Mchain) IJ
Adenosinedeaminase 20
dismutase
Superoxide 21
(1.chain)
lmmunoglobulin zz
Glucose6-phosphatedehydrogenase X
Steroid
sulfatase oruRmetnvtation
f
'i-
i-^ancers (or activators)are DNA elementsthat
:aie or enhance gefie expression. The
I
Gene activated
:--:rcers p rov ide binding s it esf or s pec if ic pr o t e i n s
:-= :e gu late tr ans c r ipt ion.They f ac ilit at e bin d i n g
--r t1e transcription complex to promoter
-?- 3ns. Enhancers differ from promoters in two I
I
: ;: 'lct lvays +
Gene activated
.: Enhancersmay be located thousandsof base
:,. 's away from the start of transcription site tz l
Biotechnology [5]
66 B IOTE C HNO LO CY
---_---I
D:n c-,: -:- of t he pr ot ein ac t ually binds t o D N A
fA. t-6D
------I
----=I
-______I
--_-__-I
--€ important features of eukaryotic Sene --_____I
___---I
,r'i[N-=*s on along with the regulatoryaspectsare --------r
:rs:-:ed in the preceeding pages. Besides
-,-,-'iption, eukaryoticcells also employ variety Fig. 5.7 : Diagrammatic representation of
,i -i^ei mechanisms to regulategene expression. gene amplification (the genes are depicted in
- . - E : 1os t i mp o rta n to n e s a re l i s te d b e l o w , and colour shade and colout).
:r =tr describednext.
: Cen e a m p l i fi c a ti o n
now explainedon the basisof generearranSement
L Cene rearrangement or transpositionof genesor somatic recombination
3. P r oce s s i nogf R N A of DNA.
The structure of a typi cali mmunogl obul imoln e-
- 1.A lt e rn a tem R N A s p l i c i n g cule consistsof two light (L) and two heavy (H)
5. Transport of mRNAfrom nucleusto cytoplasm chai ns.E achone of thesechai ns(L or H ) contai ns
an N-terminalvariable(V) and C-terminalconstant
6. Degradation of mRNA. (C ) regi ons.The V regi onsof i mmunogl obul i ns are
responsiblefor the recognitionof antigens.The
Gene amplification phenomenon of gene rearrangementcan be
In this mechanism,the expression of a gene is understood from the mechanismof the synthesis of
rncreased l i ght chai nsof i mmunogl obul i (Fi
ns g.5.8).
severalfold. This is commonlyobserved
during the developmentalstagesof eukaryotic Each light chain can be synthesizedby three
organisms. For instance, in fruit fly (Drosophila), distinct DNA segments,namelythe variable(Vr),
the amplificationof genes coding for egg shell the joining (11)and the constant(CL).The mamma-
proteinsis observedduringthe courseof oogenesls. lian genomecontainsabout 500 V, segments, 6 Jt
T he am pli fi c a ti o no f th e g e n e (D N A) c an be se8ments and 20 C, segments. Duringthe courseof
observedunderelectronmicroscope(Fig.5.V. differentiation of B-lymphocytes, one Vr segment
(out of the 500) is broughtcloserto J1 and C,
The occurrenceof gene amplificationhas also
segments. This occurson the same chromosome.
been reported in humans. Methotrexateis an 100thV L,3tdJ, and 1Oth
Forthe sakeof i l l ustrati on,
ant ic anc e r d ru g w h i c h i n h i b i ts th e e nzyme
C,_ segmentsare rearrangedin Fig. 5.8. The
dihydrofolate reductase. The maliSnant cells
rearrangedDNA (with V1, J,_and C, fragments)is
developdrug resistance to long term administration
then transcribed to producea singlemRNA for the
of methotrexate by amplifyingthe genescodingfor
synthesis of a specificlight chain of the antibody.
dihydrofolatereductase.
B y i nnumerabl ecombi nati onsof V r, l , and C t
Gene rearrangment segments, the body'simmunesystemcan generate
mi l l i ons of anti gen speci fi c i mmunogl obul i n
The body possesses an enormouscapacityto mol ecul es.
synthesizea wide range of antibodies. lt is
estimatedthat the humanbody can produceabout The formationof heavy(H) chainsof immuno-
e apti gen gl obul i nsal sooccursby.rearrangement
10 billion ( 1 0 1 0a) n ti b o d i e isn re s p o n sto of 4 di sti nct
s t im ulat io n sT. h e m o l e c u l a rm e c h a n i s mof thi s genes-variable (Vn), diversity(D), joining (ls) ano
antibody diversity was not understood for long. lt is constant (Cn).
68 B IOTE CHNO LO CY
v (500) c (20)
,, I
-)) 4..#)) O1ginatDNA
r-t-r-l- * RearrangedDNA
Vr oo
Primarytranscript
mRNA
-|-|_
Protein
(lightchainof lg)
Fig. 5.8 : Diagrammaticrepresentationof gene rearrangementfor the synthesis of light chain of immunoglobulin.
Degradation of mRNA
The expression of genesis indirectlyinfluenced C e n e e x p r e s s i o no r g e n e r e g u l a t i o n i s u su a l l y
by the stability of mRNA. Certain hormones s t u d i e d a t t h e t r a n s c r i p t i o n a ll e v e l , i . e . p r o d u cti o n
regulatethe synthesisand degradationof some of mRNA from the gene. The methods to elucidate
mRNAs.For instance,estradiolprolongsthe half- gene expressionare designedto provide information
life of vitellogeninmRNA from a few hours to on one or more of the followine
a b o u t2 0 0 h o u rs .
. Sequence of the gene
a Size of the transcript(mRNA)
a S t a r t i n ga n d f i n i s h i n g p o i n t s o f g e n e s to p r o d u ce
the transcript.
Stem loop a N u m b e r a n d p o s i t i o n o f i n t r o n s o n t he g e n e s.
AU structure a The activity of the promoter.
structure richregion
Some of the importantand general methods
Fig. 5.9 : A diagrammatic representation of a typical
employedto study the gene regulationare briefly
eukaryotic m BNA. (N0S-Non-coding seguences)
descri bed.
- ' " ac r - e' i : R EC U L A T IO OF
N C E N EE XP RE S S ION 69
lforthern blot
\orthern blot specificallydetectsthe size and
Huenc e of th e mR N A . T h e to ta l m R N A i s DNA with exons
:r'i'acted from a cell or tissue suspension,ano mRNA
=carated by agarosegel electrophoresis and then
:etectedby hybridization(ReferChapter7).
Fig. 5,10 : A diagrammatic representation of
Huc leas e S l m a p p i n g mapping of introns by nuclease Sl digestion.
\uclease Sl is an enzymethat can specifically
degradesingle-stranded nucleicacids.NucleaseSl
r.apping is used to determine the number of f t t t 'rt
introns presentin a gene (Fig. 5.10\. LabeledDNA
The mature mRNA is hybridized with its (mRNA)
Testtranscript
c or r es pondi n gg e n e (i .e . g e n o mi c D N A). The
Annealing
portion of the intron on the gene which is not
transcribedis looped out. This looped-outintron (A)
can be specificallydigestedby nucleaseSl, which
degradesthe single-stranded DNA. The number
t t
and presenceof introns can be identified by
analysingthe fragmentedDNAs.
NucleaseSl
Nuclease protection assay
In nucleaseprotectionassay,the test transcript ir 1'
tmRNA)is hybridizedwith excessquantitiesof in
(B) '!, t
vltro synthesizedand radioactivelylabeled DNA
molecules(usuallyobtainedfrom cloned genes). NucleaseDrotected
fragment
T he anneale d h y b ri d s w h i c h a re l a b e l e d, are
subjected to digestion by nuclease Sl which A and B subiectedto
degrades single-strandednucleic acids. The electroporesisand
autoradiography
nuclease-treated and untreated hybridized
moleculesare separated by agargel electrophoresis
and identified(Fig.s.ll).
Nuclease protection assay is a variant of
nuclease5l mappingand providesinformationas
regardsthe presenceof introns, transcriptional
termini and the test transcriptproper.
Primer extension
Fig. 5.11 : A diagrammatic representation of nuclease
Primerextensionmethodis a reliabletechniqueto protection assay (A) Hybridized DNA not treated with
determinethe 5' end of the transcripts.For this nuClease Sl (B) Nuclease Sl trcated hybridized DNA.
purpose,
a synthetic5'-labeled
oligonucleotideprimer
70 B IOTE C HNO LO CY
Promoter TranscriptionTermination
I
stad site site
J 5,DNA
Cene tagging broadly involves the insertion of a
3 'm R N A
I recognizable DNA fragment within a gene so that
primer
Oligonucleotide t h e f u n c t i o n o f a g e n e i s d i s r u p t e d ,a n d t h e g e n e l s
I Reversetranscriptase identified by virtue of the inserted DNA fragment
I
J T-DNA (transferredDNA) is the part of tumor-
3 'm R N A i n d u c i n g p l a s m i d ( T i p l a s m i d )D N A f o u n d i n th e so i l
H bacterium Agrobacterium tumefaciens (For detaiis,
cDNA Refer Chapter 49) Transposons or transposable
e l e m e n t sa r e m o b i l e g e n e t i c e l e m e n t s( D N A p i e ce s)
that can move from one place to another in a DNA
Sequenceanalysisof cDNA
molecule (ReferChapter 3). Transposonsor T-DNA
Fig. 5,12 : A diagrammatic of primer
representation c a n b e u s e d i n g e n e t a g g i n g a n d g e n e a n a l ysi s.
extensiontechniqueto detectthe transcription The transposontagging of a gene is depicted in
starl site. F i g . 5 . 1 3 . Wh e n a t r a n s p o s o n i n a p l a sm i d i s
introduced into a cell, it gets incorporated into
DNA, and the gene gets disrupted' Consequently,
containing complementarybase sequenceto a small transposon insertion produces a mutant (A-). This
porlion of the testtranscriptis used Both are allowed
to hybridize,and the enzyme reversetranscriptasets
used to extend the primer till it reachesthe 5' end of
the mRNA (Fig. 5.12). This resultsin the synthesisof
complementary DNA (cDNA) representing the
distancebetween S'-end of the primer and s'-end of
the mRNA. The cDNA can be separated by
electroohoresisand detected.
Fusedframeof DNA
--e
operation of the genome can be evaluated +
I
^ e stu dy of pr ot eom e. Thus , by s t udy ing th e Transformation
of
E. coli
--: io ns of p rot eins , it is pos s ible t o under s t a n d
- - .,, t1e genome operatesand how a dysfunctional
::-cme activitv c en r es gll in dis eas es t at ess uc h a s
t_
tsxpfe$srcn
:: l C e f
I
PHAGE DISPL AY
Phagedisplaylibrary
Phage display is a novel technique to evaluate
Eenome activity with particular reference to identify
proteins that interact with one another. lt basically
involves insertion of a foreign DNA into phage
genome, and its expressionas fusion product with a
phage coat protein (Fig. 5.1 A) This is followed by
screening of test protein by phage display library
lFig. 5.148).The technique is briefly describedbelow.
A spe cia l ty pe of c loning v ec t or s uc h as a
ba cte riop ha geor f ilam ent ous bac t er iophage ( e . g .
M13 ) are u se d f or phage dis play . A f r agm ent o f
DNA coding for the test protein is inserted into the
vector DNA (adjacentto phage coat protein gene).
After transformation of E. coli, this recombinant
gene (fused frame of DNA) results in the synthesis
of hybrid protein. The new protein is made up of
the test protein fused with the phage coat protein.
The phage particles produced in the transformed
E. coli display the test protein in their coats. Fig. 5.14 : Elucidation of protein-protein interaction by
phage display (A) Production of fusion protein
The test protein interaction can be identified by
displayed on phage (B) Screening of test protein by
u sin g a ph ag e d is play libr ar y For t his pur pos e,t h e
phage display library.
test pro tein is im m obiliz ed wit hin a well of a
72 B IOTECHNO LO CY
*- Transcriotional
activationdomain
Aarnal Bindingpartner
DNAbinding Recombinantgenes
domain protein encodingbait and prey PREY
BAIT introducedinto veast cell
,i t,
I,j
''
73 73
f enet ic e n g i n e e ri n gp ri ma ri l y i n v o l v e s the p S C 1 0 1 D N A . F r o g D N A f r a g m e n t s a n d p l a s m i d
\) manipulation of genetic material (DNA) to DNA fragmentswere mixed, and pairing occurred
ac hiev et he de s i re dg o a l i n a p re -d e te rmi n eway.d between the complementary base pairs. By the
Some ofher terms are also in common use to addition of the enzyme DNA ligase, a recombined
describegeneticengineering. p l a s m i d D N A w a s d e v e l o p e d .T h e s e n e w p l a s m i d s ,
rvhen introduced into E.coli, and grown on a
. Cene manipulation
nutrient medium resulted in the production of an
. Recombinant DNA (rDNA) technology extra protein (i.e. the frog protein). Thus, the genes
. Gene cloning (molecular cloning) of a frog could be successfullytransplanted, and
. Genetic modifications expressedin E.coli. This made the real beginning of
modern rDNA technology and laid foundations for
. New genetics.
the present day molecular biotechnology.
75
76 B IOTECHNO LO G Y
t Multiplication
R E S TR IC TION E N D ON U C LE A S ES_
I Selectionof clones D N A C U TTIN G E N ZY ME S
Restrictionendonucleases are one of the most
importantgroupsof enzymesfor the manipulation
of DNA. Theseare the bacterialenzymesthat can
cut/splitDNA (from any source)at specificsites.
They were first discoveredin E.colirestrictingthe
replicationof bacteriophages, by cuttingthe viral
Ftg. 6.1 : The basic principle of recombinant DNA
DNA (The host E.coli DNA is protected from
technology.
cleavageby additionof methyl groups).Thus,the
enzymesthat restrictthe viral replicationare known
2. Insertion of the selected DNA into a cloning as restrictionenzymesor restrictionendonucleases.
vector (e.g. a plasmid) to create a recomhinant Hundreds of restrictionendonucleaseshave
DNA or chimeric DNA (Chimera is a monster in been isolatedfrom bacteria,and some of them are
Creek mythology that has a lion's head, a goat's commerciallyavailable.The progressand growth
body and a serpent'stail. This may be comparable of bi otechnol ogyi s uni magi nabl ewit hout t he
to Narasimha in Indian mythology). availabilityof restrictionenzymes.
3. Introduction of the recombinant vectors inro
host cells (e.9. bacteria). N omencl ature
4. Multiplication and selection of clones Restrictionendonucleasesare named by a
c ont aining t he r ec om binant m o l e c u l e s . standardprocedure, with particularreference
to the
5. Expressionof the gene to produce the desired bacteriafrom which they are isolated.The first
oroduct. letter(in italics)of the enzymesindicatesthe genus
T O C EN ET ICEN CIN E E R IN C
J - ac t er 6 : I NT R OD U C T IO N 77
Taslr 5.2 Some restrictlon enzynes with sources, recognltlon sequencesand the products fonned
EcoRl . 3'
s' ..ch A-T-T-C,. A-A-T-T-C
(Escherichia
colll . 5',
3' C-T-T-A-Aip. G
G
C-T-T-A-A
Notl 5',..c-cffc-c-c-c-c-c
3' u-u-u-u-u-u .
(Nocardia
otitidis) 3'. ..c-G-c-c-G-Gtc-G5' ii
v-\:...
..t -u
.c-G-c-GG-G,
(l{ole: Sabsorsindicate * Theproducts
thesitesol cleavage. arewithbluntendswhilefot thercst,theWoducts
arewithstbkyendsl.
B IOTE CHNO LO CY
7a
Recognition sequences
Gleavage Patterns
Majority of restriction endonucleases
r]
( par t ic ular lyt y pe ll) c ut DNA a t d e f i n e d s i t e sw i t h i n ol-l I
I
recognition sequence. A selected list of enzymes,
O=P-O-
recognition sequences,and their products formed I I
I
I
is given in Table 6.2.
I
I
The cut DNA fragments by restriction I
ALKALINE PHOSPHATASE
Mix and anneal
Alkalinephosphatase is an enzymeinvolvedin
.4,-_>>
the removalof phosphategroups.This enzyme is
usefulto preventthe unwantedligation of DNA
molecules which is a frequentproblemencountered
in c loningex p e ri me n ts .
When the linearvectorplasmidDNA is treated
rvith alkaline phosphatase, the 5'-terminal
phosphateis removed(Fig 6.fl. This preventsboth
and plasmidDNA dimerformation.
recircularization
It is now possibleto insertthe foreignDNA through Fig. 6.3 : Homopolymer tailing.
the participation
of DNA ligase.
(A) (B) Til
DNA MODIFYING ENZYMES r-l
Someauthorspreferto usethe broadterm DNA
ForeignDNA t]
modifyingenzymesto all the enzymesinvolvedin
recombinant DNA technology.These enzymes
'epresentthe cutting andjoining functionsin DNA
ranipulation. They are broadly categorizedas
rucleases,polymerasesand enzymes modifying
ends of DNA molecules,and briefly described
below,and illustratedin Fig. 6.6. f-T------r-t
I
Nucleases lecoar
Nucleasesare the enzvmes that break the
phosphodiesterbonds (that hold nucleotides
rc
ForeignDNA with
together)of DNA. Endonucleases
act on the internal cohesiveends
phosphodiesterbondswhile exonucleases degrade
DNA from the terminalends(Fig6.64). Restriction
endonucleases,described already, are good
examplesof endonucleases.Someother examples
of endo- and exonucleases
are listed.
Endonucleases
. NucleaseS, specificallyacts on single-stranded
VectorDNA Recombinant
DNA
DNA or RN A m o l e c u l e s .
Fig. 6.4 : Use of linkers (A) and adaptors (B) in the
| (DNasel) cuts eithersingle
. Deoxyribonuclease
formation of recombinant DNA (Note that the linkers
DNA moleculesat random
or double-stranded have blunt ends while adaptors have sticky ends).
sites.
B IOTECHNO LO CY
80
(A) Nucleases
t*"h DNA
HO - P
P - HO Exonucleases
ForeignDNA
fragment r E xonucl easel l l cuts D N A a nd Sener at es
mol ecul esw i th protrudi ng5' -ends'
. N ucl easeB al 31 i s a fastacti ng3'- exonuclease'
Its acti on i s usual l ycoupl edw it h slow act ing
endonucl eases.
A diagrammaticrepresentation of the action of
endo-and exonucleasesis given in Fig' 6'7'
51 .
Nuclease
Sinqle-stranded
DNAoTRNA
DNaseI
DNA
Double-stranded
ffl u
Exonuclease .,
DNA
Double-stranded
NucleaseBal31 .
DNA
Double-stranded
Fig.6.7:Modeofactionofselectedendo_andexonucleases(DNase|_Deoxyribonucleasel)'
o : INTRODUCTIONTO CENETICENCINEERINC
il'h"ir*-=r 8t
Iru 6.3 The most conilonly used enlymes In recombinant DNA technology/genetlc englneerlng
Enzrme Use/reaction
,rlair€ phosphatase Removes phosphate groupsfromS'-ends DNA(orRNA).
of double/single-stranded
h l' 'tuclease Fortheprogressive
shorteningof DNA.
l t,a Ease JoinsDNAmolecules bylorming phosphodiester linkages
between
DNAsegments.
I
lirr oolymerase DNAcomplementary
Synthesizes to a DNAtemplate.
:hes€ | Produces nicksin DNA.
single-stranded
lr:rrdease lll Removes nucleotidesfrom3'-end of DNA.
;r :rSnuclease Removes nucleotidesfromS'-end o1DNA.
br-ucleotidekinase phosphate
Transfers fromATPto 5'-0Hendsof DNAor RNA.
:esrction enzymes Cutdouble-strandedDNAwitha specilic recognition
site.
=euersetranscriptase DNAfromRNA.
Synthesizes
Ei\laseA Cleavesanddigests RNA(andnotDNA).
=ltase H Cleavesanddigests theRNAstrand of RNA-DNA heteroduplex.
-r DNApolymerase Usedin polymerasechainreaction
S ruclease Degrades DNAandRNA.
single-stranded
transferase
-erminal Addsnucleotides
to the3'-ends of DNAor RNA.Useful in homopolymer
tailing.
3ctechnology[6]
a2 B IOTECHNO LO G Y
PROKARYOTIC HOSTS
Tlcrr 6.4 Some examples of host cells used
Escherichia coli in genetic engineering
The bacterium,Escherichiacoli was the first
Group Examples
organismusedin the DNA technologyexperiments
and continuesto be the host of choice by many Prokaryotic
workers.Undoubtedly,E.coli, the simplestCram Bacteria Escherichia
coli
negativebacterium(a commonbacteriumof human Bacillus
subtilis
),a spl ayeda key rol e i n the
a n d a n i ma li n te s ti n e h
Streptomyces
sp
developmentof presentday biotechnology. Under
suitableenvironment,E.colican double in number Eurkaryotic
e v e ry 2 0 mi n u te s T . h u s ,a s the bacteri amul ti pl y, Fungi Saccharomycescerevisiae
th e i r p l a s mi d s(a l o n g w i th forei gn D N A ) al so Aspergillus
nidulans
multiplyto producemillionsof copies,referredto Animals Insect
cells
as colonv or in short clone. The term clone is Oocytes
broadlyusedto a massof cells,organisms or genes
Mammalian cells
th a ta re p ro d u c e db y mu l tipl i cati on
of a si ngl ecel l ,
or 8ene.
Wholeorganisms
o rg a n rs m
Plants Protoplasts
l i m i ta ti o n s o f f. c o l i : There are certai n Intact
cells
i s a host.Thesei ncl ude-
l i m i ta ti o n isn u s i n gE .c o l a Wholeplants
causationof diarrheaby some strains,formation
of endotoxinsthat are toxic, and a low export
ability of proteinsfrom the cell. Another mayor cannotbe synthesized by bacteriacan be produced
drawback is that E.coli (or even other prokaryotic by mammal i an cel l s e.g. ti ssue plasm inogen
organisms) cannot perform post-translational acti vator.Thi s i s mai nl y becauseth e m am m alian
modifications. cells possessthe machinery to modify the protein
to its final form (post-translational modifications).
Bacillus subtilis
It may be notedherethatthe genemanipulation
Bacillussubtilisis a rod shapednon-pathogenic experi ments i n hi gherani mal sand pl a nt sar eusually
bacterium.lt hasbeenusedas a hostin industryfor carried out to alter the genetic make up of the
the productionof enzymes,antibiotics,insecticides organismto createtransgenic animals(Chapter+1;
etc. Some workers consider B.subtilis as an and transgenicplants(Chapter49), ratherthan to
alternative to E.coli. isolategenesfor producingspecificproteins.
EUKARYOTIC HOSTS
Eukaryoticorganismsare preferredto produce
human proteinssince these hosts with complex
structure(withdistinctorganelles) are moresuitable
to synthesize complex proteins. The most Vectorsare the DNA molecules,which can carry
commonly used eukaryoticorganismis the yeasf, a foreign DNA fragment to be cloned.They are self-
Saccharomycescerevisiae.lt is a non-pathogenic replicatingin an appropriatehost cell. The most
o rg a n i s mro u ti n e l yu s e d in brew i ng and baki ng important vectors are plasmids,bacteriophages,
industry.Certainfungi havealsobeenusedin gene cosmidsand phasmids.
c l o n i n ge x p e ri m e n ts .
Characteristics of an ideal vectol
Mammalian cells
A n i dealvectorshoul dbe smal li n size,wit h a
Despitethe practicaldifficultiesto work with si ngl e restri cti onendonucl ease si te , an or igin of
and high cost factor, mammaliancells (such as replicationand 1-2 genetic markers(to identify
mouse cells) are also employed as hosts. The reci pi entcel l scarryi ngvectors).
N atu r allyoccur r ing
advantageis that certaincomplex proteinswhich plasmidsrarelypossess all thesecharacteristics.
a' r aot er6 : I N T R OD U C T IO N
T O C EN ET ICEN C IN E E R IN C 83
P LA S M I DS
Hind lll
P las m ids a re e x tra c h ro mo s o m a l d , o u bl e-
circular,self-replicating DNA molecules.
'ri.anded,
\ lm os t all t he b a c te ri ah a v ep l a s m i d sc o n ta i n i nga
o\ \ ' c opy nu m b e r (1 -4 p e r c e l l ) o r a hi gh
c opy num ber (1 0 -1 0 0 p e r c e l l ). T h e s i z e of
: . e plas m idsv a ri e sfro m 1 to 5 0 0 k b . U s u al l y, PstI
ciasmidscontributeto about 0.5 to 5.0% of the
:otal DNA of bacteria (Note : A few bacteria
contain linear plasmids e.g. Streptomycessp,
Borella burgdorferi).
Types of plasrnids
T her e ar e m a n y w a y s o f g ro u p i n gp l a s mi ds.
They are categorizedas conjugativeif they carry a
;et of transfer genes (fra genes) that facilitates
bacterialconjugation,and non-conjugative,if they Fig. 6.8 : Genetic map of plasmid cloning
do not possess such genes. vector pBR322
( bot h being pr ot eins )and i t s s h a p e i s c o m p a r a b l et o (about 25%) has to be deleted from the vector to
a m iniat ur e hy poder m ic sy r i n g e .T h e D N A , l o c a t e d make space for the foreign DNA (about l Bkb).
in t he head, is a linear m o l e c u l e o f a b o u t 5 0 k b . A t
Replacementvectors : These vectors have a pair
each end of the DNA, there are single-stranded
of restrictionsitesto remove the non-essentialDNA
ex t ens ions of I 2 bas e le n g t h e a c h , w h i c h h a v e
(stuffer DNA) that will be replaced by a foreign
c ohes iv e ( c os ) ends .
DNA. Reolacement vectors can accommodale
On attachmentwith tail to E.coli, phage L injects up to 24kb, and propagate them.
it s DNA int o t he c ell I ns i d e E . c o l i ,t h e p h a g e l i n e a r
Many phage vector derivations (insertion/
DNA cyclizes and gets ligated through cos ends to
replacement) have been produced by researchers
f or m a c ir c ular DNA. T h e p h a g e D N A h a s t w o
f o r u s e i n r e c o m b i n a n t D N A t e c h n o l o g y.
fates-lytic cycle and lysogenic cycle. I
The main advantage of using phage vectors is
Ly t ic c y c le : The c ir c u l a r D N A r e p l i c a t e s a n d
that the foreign DNA can be packed into the phage
it als o dir ec t s t he s y nth e s i s o f m a n y p r o t e i n s
(in vitro packaging), the latter in turn can be
nec es s ar yf or t he head, t a i l e t c , o f t h e p h a g e . T h e
injected into the host cell very effectively (Note :
c ir c ular DNA is t hen c le a v e d ( t o f o r m c o s e n d s )
N o t r a n s f o r m a t i o ni s r e q u i r e d ) .
and pac k ed int o t he he a d o f t h e p h a g e . A b o u t
.l
00 phage par t ic les ar e p r o d u c e d w i t h i n 2 0
Phage Mr" vectors
m inut es af t er t he ent r y o f p h a g e i n t o E . c o l i . T h e
hos t c ell is t hen s ubjec t e d t o l y s i s a n d t h e p h a g e s P h a g e M , , ( b a c t e r i o p h a g e M 1 3 ) i s a si n g l e -
ar e r eleas ed. Eac h pr og e n y p h a g e p a r t i c l e c a n stranded DNA phage of E.coli. Inside the host cell,
inf ec t a bac t er ial c ell. a n d o r o d u c e s e v e r a l M,, synthesizesthe complementary strand to form
hundr eds of phages . l t i s e s t i m a t e d t h a t b y a double-strandedDNA (replicativeform DNA; RF
r epeat ing t he I y t ic c y c l e f o u r t i m e s , a s i n g l e DNA). For use as a vector, RF DNA is isolated and
phage c an c aus e t he d e a t h o f m o r e t h a n o n e a foreign DNA can be inserted on it. This is then
billion bacterial cells. lf a foreign DNA is spliced r e t u r n e d t o t h e h o s t c e l l a s a p la sm i d . Si n g l e -
into phage DNA, without causing harm to phage stranded DNAs are recovered from the phage
genes, the phage will reproduce (replicate the p a r t i c l e s .P h a g e M , ', i s u s e f u l f o r s e q u e n ci n gD N A
foreign DNA) when it infects bacterial cell. This through Sanger'smethod (Refer Chapter 7).
has been ex ploit ed in p h a g e v e c t o r e m p l o y e d
c loning t ec hniques . cosMtDs
Lysogenic cycle : In this case, the phage DNA Cosmids are the vectors possessingthe
I ( ins t ead of independen t l y r e p l i c a t i n g ) b e c o m e s of both plasmidand bacteriophage 1".
I characteristics
integrated into the E.coli chromosome and Cosmidscan be constructedby adding a fragment
r eplic at esalong wit h t he h o s t g e n o m e . N o p h a g e of phagel" DNA includingcos site,to plasmids.A
particles are synthesizedin this pathway foreign DNA (about40 kb) can be insertedinto
cosmidDNA. The recombinant DNA so formedcan
Use of phage )" as a vector be packed as phages and injected into E.coli
Only about 5O"h of phage l, DNA is necessary (Fig. 6.10). Once inside the host cell, cosmids
f or it s m ult iplic at ion and o t h e r f u n c t i o n s . T h u s , a s behave j ust l i ke pl asmi dsand r eplicat e.The
m uc h as 50% ( i. e. up t o 2 5 k b ) o f t h e p h a g e D N A advantagewith cosmidsis thatthey can carrylarger
c an be r eplac edby a dono r D N A f o r u s e i n c l o n i n g fragments of foreignDNA comparedto plasmids.
experiments. However, several restriction sites are
presenton phage )" which is not by itself a suitable Phasid vectors
vector.The l"-basedphage vectorsare modifications
P hasi dsare the combi nati onof plasm id and
of t he nat ur al phage wit h m u c h r e d u c e d n u m b e r o f
phageandcanfuncti onasei therone ( i. easplasm id
r es t r ic t ion s it es . Som e o f t h e m a r e d i s c u s s e o
or phage).P hasi dspossessfunctionalor iginsof
nereunoer. plasmid phage 1., and
replicationof both and
lnsertion vectors : They have just one unique thereforecan be propagated (asplasmidor phage)
cleavage site, which can be cleaved, and a foreign in appropriateE.coli.The vectorsphasidsmay be
DNA lieat ed in. lt is es s e n t i a lt h a t s u f f i c i e n t D N A usedi n many w ays i n cl oni ngexp er im ent s.
. ] \ T R O D U C T IO NT O C EN ET ICEN C IN E E R IN C
o
86 B IO TECHNO LO CY
cos
-+-----.{ 37.5 kb cos The constructionof BACs is based on one
F-pl asmi dw hi ch i s l argerthan the ot her plasm ids
IlPackaoinorn yftro used as cloning vectors.BACs can accept DNA
J insertsof around 3-00 kb. The advantagewith
bacterialartificialchromosome is thatthe instability
problemsof YACscan be avoided.In fact, a major
part of rhe sequencingof human Senome has been
accompl i shed by usi ng a l ibr ar y of BAC
recombinant.
51{UTTLE VECTORS
The plasmid vectors that are specifically
I designedro replicate in two different hosfs (say in
llnfection E.coli and Streptomyces sp)are referredto as shuttle
J vectors.The originsof replicationfor two hostsare
/-rG\.,
( combined in one plasmid.Therefore,any foreign
/ . *Plasmidform
'i ' (orcosmid) DNA fragmentintroducedinto the vector can be
( J
\Y/ expressed shuttlevectorscan
in eitherhost.Further,
\Qplical/ be grown in one host and then shiftedto another
E. coli host (hencethe name shuttle).A good numberof
eukaryoticvectorsare shuttlevectors.
Fig. 6.10 : Cosmids as vectots.
OH OIC E OF A V E C TOR
Amongthe severalfactors,the sizeof the foreign
ART l F I c I Jii'- CF;sli,,Fi.
Jc :.,i{:;
l}1€ V ECTO RS DNA is very importantin the choiceof vectors.The
Hunralr artiflciat cl;rl -ll;rsonne (HAG) efficiency of this process in often crucial for
determiningthe successof cloning. The size of
D e v e l o p e di n 1 9 9 7 (by H . W i l l ard); human DNA insert that can be accepted by different
artificial chromosomeis a syntheticallyproduced vectors is shown in Table 5.5.
vector DNA, possessing the characteristics of
human chromosome.HAC may be consideredas a
self-replicatingmicrochromosomewith a size
ranging from 1/1Oth to 1/5th of a human
chromosome.The advantagewith HAC is that it Introducinga foreign DNA (i.e. the Sene)into
can carry human genesthat are too long. Further, the cells is an importanttask in biotechnology. The
HAC can carry genesto be introducedinto the efficiency of this process is often crucial for
c e l l s i n g e n eth e ra p y . .determi ni ngthe successof cl oning. The m ost
commonly employed gene transfer methods,
Yeast crrlifi*ial ehrn;no;omes (YACs)
namely transform6tion, conjugation, electro-
lntroducedin 1987(by M. Olson),yeastartificial poration and lipofection,and direct transferof
chromosome(YAC)is a syntheticDNA that can DNA are brieflydescribed.
lt @E - : : I \ T R OD U C T IO NT O C EN ET ICEN C IN E E R IN C 87
usediethylaminoethyldextran(DEAE-dextran)for
hr 5"5 Tle different clonlng veetors with DNA transfer.
h cnresponding hosts and the slzes
of foreign insert DNAs coNJucTroh!
Host Foreign insert Conjugation is a natural microbial
DN,4,size recombination process.During conjugation,two
l i ve bacteri a(a donor and a reci pi ent)come
er@g;_ E. coli 5-25kb together,join by cytoplasmicbridgesand transfer
l:rrc i- E. coli 35+5 kb single-stranded DNA (from donor to recipient).
ra-c artifical E. coli 100-300kb Insi de the reci pi entcel l , the new D N A may
integrate with the chromosome(ratherrare)or may
r'rircsome (PAC)
remai nfree (as i s the casew i th pl asmi ds).
b.eral artificial E. coli 100-300
kb
C onj ugati oncan occur among the cel l s from
rnnosome (BAC) different Beneraof bacteria (e.g Salmonella and
r=as1chromosome S. cerevisiae 200-2000
kb Shigellacells).This is in contrastto transformation
w hi ch takespl ace amongthe cel l s of a bacteri al
genus.Thusby conjugation,transferof genesfrom
TRA NS F O RM AT ION two differentand unrelatedbacteriais possible
-'ansformationis the method of introducing The natural phenomenonof conjugation is
r:re:gn DNA into bacterialcells (e.g. E.colD.fhe exploitedfor gene transfer.This is achievedby
- m ak eof plas m i dD N A b y E.c o l ii s c a rri e do u t i n pl asmi d-i nsert
transferri ng D N A from one cel l to
:e-cold CaCl, (0-5"C),and a subsequentheat another.In general,the plasmidslack conjugative
;-.:ck (3745"C for about 90 sec). By this functions and therefore,they are not as such
::r:rnique, the transformation frequency, which capableof transferring DNA to the recipientcells.
-:fers to the fraction of cell population that can be However, some plasmids with conjugative
tansferred,is reasonably good e.g. approximately propertiescan be preparedand used.
c r e c ell f or 100 0 (1 0 -3 )c e l l s .
Transformation efficiency : lt refers to the E LE C TR OP OR f,Ti f
-umber of transformants per microgramof added Electroporation is basedon the principle that
D\A. For E.coli, transformation by plasmid,the hi ghvol tageel ectri cpul sescan i nducecel l pl asma
:.ansformation efficiencyis about 107ro 108 cells membranesto fuse. Thu_s,electroporationis a
per microgram of intact plasmid DNA. The technique involving electric field-mediated
bacterialcellsthat can takeup DNA areconsidered membrane permeabilization.Electric shocks can
as competent.The cornpetencecan be enhanced al so i nduce cel l ul ar uptakeof exogenousD N A
bv alteringgrowth conditions. (believedto be via the pores formed by electric
The mechanismof the transformation processis pul ses) from the suspendi ng sol uti on.
Electroporation is a simple and rapidtechniquefor
not fully understood.lt is believedthat the CaCl,
i ntroduci nggenes i nto the cel l s from vari ous
affectsthe cell wall, breaksat localizedregions,
organi (mi croorgani sms,
sms pl antsand ani mal s).
e r b i n d i n go f D N A to cel l
a nd is als or es p o n s i b lfo
surface.A briefheatshock(i.e.the suddenincrease The basic technique of electroporationfor
in temperature from 5'C to 40"C) stimulatesDNA transferri ng genesi nto mammal i an cel l si s depi cted
uptake. In general, large-sizedDNAs are less i n Fi g, 6.11. The cel l s are pl aced i n a sol uti on
efficientin transforming. containingDNA and subjectedto electricalshocks
to causeholesin the membranes. The foreignDNA
Other chemical methods for fragments enter through the holes into the
transformation cytoplasmand then to nucleus.
Calc ium ph o s p h a te(i n p l a c e o f C a C l r) i s is an effectiveway to transform
Electroporation
oreferredfor the transferof DNA into cultured E .col icel l s contai ni ngpl asmi dsw i th i nsertD N A s
m a y re s u l ti n longerthan 100 kb. The transformation
c ells .S om et im e sc,a l c i u mp h o s p h a te efficiencyis
precipitateand toxicityto the cells.Someworkers around 109 transformants per microgram of DNA
88 B IOTE CHNO LO CY
TR A N S D U C TION
*K Sometimes,the foreign DNA can be packed
i nsi deani malvi ruses.Thesevi rusesca n nat ur ally
Special DNA
cuvetle fragment
a').
,)
a ( o^h^
a DNA enterscell
throughpores
withDNA
Liposome
( @)
DNAenters
nucleus
L IPOS O ME .M ED IAT E D G E N E TR A N S FE R
L i p o s o m e as re c i rc u l a rl i pi d mol ecul es,w hi ch Fusionbetween
plasmamembrane
have an aqueousinterior that can carry nucleic andliposome
acids.Severaltechniqueshave been developedto
encapsulateDNA in liposomes.The liposome-
DNA
mediatedgenetransfer,referredto as lipofection, is nucleus
depictedin Fig. 5.12.
On treatmentof DNA fragmentwith liposomes,
Flg. 6.12 : Liposome-mediated gene transfer
the DNA piecesget encapsulated
insideliposomes.
(Note : For clarity, the native cell DNAis not shown).
Theseliposomescan adherto cell membranes and
Chapt er6 : I N T R O D U C T IO N
T O C E N E T ICE N C IN E E R IN C 89
experiments. This approach is useful if the gene l n 1974, a groupof ten sci enti stled
s by Paul
sequence is short and the complete sequence of Bergwrote a letterthat simultaneously appearedin
am ino ac ids is k nown. the presti geousj ournal s-N ature,Science and
Proceedings of the NationalAcademyof Sciences.
The different strategies for the cloning of
The dangersof DNA technologywere printedout
genom ic DNA and m RNA a r e d e s c r i b e d u n d e r
in that letter(highlightsgiven below) :
gene libr ar ies ( Chapt er 9) .
"Recent advances in techniques for isolation
and rejoining of segmentsof DNA now permit
construction of biologically active recombinant
DNA molecules in vitro. Although such
experimentsare likely to facilitate the solution of
For addit ional inf or m a t i o n o n t h e g e n e t i c
important theoretical and practical biological
problems, they would also result in creation of
engineeringof plants,the reader must refer Chapter
49. Some details on the following aspectsare given
novel types of DNA elements whose biological
in that chapter
propertiescannot be completely predicted. Thereis
a serious concern that some of these DNA
. Cene transfer methods (vector-mediated gene molecules could prove biologically hazardous".
t r ans f er - T DNA, plan t v i r u s e s ; d i r e c t D N A
transfer- physical and chemical methods). The letteralso appealedto molecularbiologists
w orl dw i de for a moratori umon many kinds of
. Marker genes for plant transformation.
recombinant DNA research,particularly those
o Promoters and terminators. i nvol vi ngpal hogeni organi
c sms.
o Transgene stability, expression and gene
s ilenc ing. A S I LOMA R R E C OMME N D A TIO NS
o Chloroplast transformation. ln February1975,a groupof 139 scientists from
17 countriesheld a conferenceat Asilomar,a
conferencecenterin California.USA.Thevassured
the uneasypubl i cthat the mi croorg anismused
s in
DNA experiments were specificallybredand could
not surviveoutsidethe laboratory. Thesescientists
With the success of Boyer-Cohen experiments formulated guidelines and recommendations for
(in 1973), it was realised that recombinant DNA conducting experiments in genetic engineering.
technology could be used to create organismswith
novel genes. This created worldwide commotion N IH GU ID E I.IN E S
(among scientists,public and government officials)
N ati onal l nsti tute of H eal th (NlH) , USA
about the safety, ethics and unforeseen
constituted the Recombinant DNA Advisory
c ons equenc esof genet ic m a n i p u l a t i o n s . S o m e o f
Committee (RAC) which issueda set of stringent
the phrases quoted in media in those days are
guidelinesto conductresearchon DNA. RAC was
8r v en. in fact overseeingthe researchprojectsinvolving
o Manipulation of life. genespl i ci ngand recombi nant
DNA.
. Play ing Cod. S ome of the i mportant original NI H
. M an m ade ev olut ion. recommendations on recombinantDNA researcn
relateto the followingaspects.
o The most threatening scientific research.
. Physical (laboratory)containment levels for
It was feared that some new organisms,created
conductingexpermiments.
inadvertently or deliberately for warfare, would
cause epidemics and environmental catastrophes. . Biological containment-thehost into which
Due to the fears of the dangerous consequences,a foreign DNA is insertedshould not proliferate
cautious approach on recombinant DNA outsidethe laboratoryor transferits DNA into
experiments was suggested. otherorqani sms.
: iapt er 6 : I NT R OD U C T IO N
T O C EN ET ICE NC IN E E R IN C 9l
fi el dori entati on
changes,
the D N A mol ecul es
al i gn
themselves and migratein new directions.
limitation of PFGE: The maior limitationof
pul sed-fi elgel
d el ectrophoresi i ssthat the sampl es
Cover do not mi gratei n strai ghtl i nes.Thi s makesthe
furtheranal ysi sof D N A ratherdi ffi cul t.
Gel
CONTOUR.CLATUIPEDHOMOGENEOUS
+ve
electrode E LE C TR IC A L-FIE LD E LE C TR OP I{ OR E S IS
-ve . . . . . . . .. . . ' - . . I
:lectrocle Migrationof Buffer {cH E Fl
DNA
C H E F i s an i mproved method to appl y
Fig. 7.1 : A diagrammatic representation of agarose alternating electrical fields to separate DNA
gel electtophoresis system. mol ecul es
i n el ectrophoresiIns.thi s approach,
the
reorientationangle of electricalfield is fixed at
120'C (or even at 90') and electrophoresis carried
nove through the gel towards the positive out. By usingCHEF,largeDNA fragments (200-300
e lec t r ode.T he ra te o f mi g ra ti o n o f D N A i s kb) can be routinelyseparated in a matterof hours.
clependent on th e s i z e a n d s h a p e . In g e n e ral ,
..mallerlinearfragmentsmovefasterthan the larger POLYACRYTAMIDE GEL
ones. Hence, gel electrophoresiscan be E LE C TR OP H OR E S IS (P A GE I
conveniently
usedfor the separation
of a mixtureof gel i s composedof chai nsof
P ol yacryl ami de
DNA fragments,basedon their size. acryl ami de monomers cross-l i nked w i th
Agaroseformsgelswith pore sizesrangingfrom methylenebisacrylamide units.The pore sizeof the
100 t o 300 nm i n d i a m e te r.T h e a c tu a lp o re s i ze gel is dependent on the total concentrationof the
dependson the concentration of the agarose.The monomers and the cross l i nks.
size of the pores determinesthe range of DNA Polyacrylamidegel electrophoresis (PACE)is
fragments that can be separated on electrophoresis.used for the separation of single-stranded DNA
For instance,a 0.3'/' agaroseis used for the molecuels that differ in length by just one
separation of DNA fragments between5 and 50 kb, nucleotide. Agarosegels cannot be used for this
rvhile a 5% agarosecan separate100-500 bp purpose.This is becausepolyacrylamide gels have
m olec ules .
The migrationof DNA fragmentsduring the
course of electrophoresis can be monitored by Wellsfor
sampres
usingdyeswith known migrationrates.Thesedyes
Agarosegel
are addedto the DNA samplesbeforeloading.The
bandsof the DNA can be detectedby soakingthe UV transparenl
plasticsupport
gel in ethidium bromide solution.When activated
b y ult r av iolet ra d i a ti o n , D N A b a s e p a i rs i n
wit h e th i d i u m b ro mi d e e mi t o ra n ge
as s oc iat ion | = ,n' o,rrbromi de
fluorescence. And in this way the DNA fragments solution(soaking)
separated in agarose electrophoresiscan be J
identified(Fig.7.2).
P ULS E D" F I E L D GE L EL E C T R O P H OR ES IS
(PFGEI
T he lar ges iz e dD N A mo l e c u l e s(- 1 0 Mb ) c an lTt
UV light
in a g a ro s g
be s epar at ed e e l sb y u s i n gP F G ET. h i si s
madepossibleby periodicallyalteringthe direction Fig. 7.2 : ldentification of DNA bands separated
of DNA migrationby changingthe orientationof by agarose gel electrophoresis.
electricfield with respectto the gel. As the electric
94 B IOTECHNO LO CY
The electrophoretictechnique for protein- P l ant cel l s : P l antcel l s w i th stro ngcell walls
n u c l e i ca c i d i n te ra c ti o n
i n vol vesthe addi ti onof a requireharshtreatmentto breakopen.The cellsare
desiredproteinto double-stranded DNA fragments, frozenand then groundin a morterand pestle.This
separationof this complex and naked DNA by is an effectiveway of breakingthe cellulosewalls.
electrophoresis. The separatedpatternscan be
visualizedto understandthe orotein-nucleicacid Methods to purify DNA
interactions.
There are two differentapproachesto purify
D N A from the cel l ul arextracts.
1. Purificationof DNA by removing cellular
components: Thi s i nvol vesthe de gr adat ionor
I
compl eteremovalof al l the cel l ul a rcom ponent s
I
Al m o s t a l l th e e x p e ri ments deal i ngw i th gene other than D N A . Thi s approachi s' s uit ableif t he
m a n i p u l a ti o nre s q u i rep u reformsof ei therD N A or cel l sdo not contai nl argequanti ti e of s lipidsand
RNA, or sometimeseven both. Hence there is a carbohydrates.
n e e dfo r th e re l i a b l ei s o l a t i onof nucl ei caci dsfrom The cellularextractis centrifuged at a low speed
the cells.The purificationof nucleicacidsbroadly to removethe debris(e.g.piecesof cell wall) that
involvesthree stages. forms a pellet at the bottom of the tube. The
1 . Bre a k i n go r o p e n i n gof the cel l s to expose supernatant is collectedand treatedwith phenolto
n u c l e i ca c i d s . precipitateproteinsat the interfacebetween the
organic and aqueouslayers.The aqueouslayer,
2 . S e p a ra ti o no f n u c lei c aci ds from other
nucl ei caci ds,is collect ed
contai ni ngthe di ssol ved
c e l l u l a rc o mp o n e n ts . (RNase).
and treatedwith the enzymeribonuclease
3 . R e c o v e ry o f n u c l e i caci dsi n a pure form. The R N A i s degradedw hi l e the DNA r em ains
Analyticalproceduresinvolvinga few stepsto i ntact.Thi s D N A can be preci pi ta t ed by adding
severalsteps are in use for the purificationof ethanol and isolated after centrifugation,and
nucleic acids.ln fact, commercialkits are readily suspendedin an appropriatebuffer.
availablethesedaysto enablepurificationof either 2. Direct purification of DNA : In this
DNA or RNA from differentsources. approach,the DNA itself is selectivelyremoved
The basicprinciplesand procedures for nucleic from the cellular extractand isolated.There are
acid purificationare brieflydescribed. two ways for direct purificationof DNA.
- - apt er 7 : B A S ICfEC H N IQ U ESl N C E N E T ICE N C E N E E R IN C 95
High-saltwash
O
/-------\- | Siqnal I
Charger+( e )-l ampliiicationI
--t-----/ | an0 SeleCIlOn i
<)
o Blotting techniques are very widely used
analytical tools for the specific identification of
desired DNA or RNA fragmenfsfrom thousandsof
molecules. Blotting refers to the prccess of
immobilization of sample nucleic acids or solid
support (nitrocellulose or nylon membranes). The
blottednucleicacidsare then usedas targetsin the
hybridization experiments for their specific
detection.An outline of the nucleic acid blotting
techniqueis depictedin Fig, 7.6,
Biotechnologyftl
98 B IOTECHNO LO CY
GenomicDNA
, lResl errao+rrpfe
,Y
\-
\- t'
,' ,t ,l
/ \r
\- \ t /
\\ "'
DNA fragments
A diagrammaticrepresentationof a typical
blottingapparatusis depictedin Fig. 7.7.
SOUTHERN BLOTTING
t
' S o u th e rnb l o tti n gte c h n iquei s the fi rst nucl ei c
acid blotting proceduredeveloped in 1975 by
Southern. lt is depicted in Fig. 7.8, and brietly
described.
{_weight
Nitrocellulose
(or nylon membrane)
or.rn
nrooe
{-Membrane
_______t(_ J
r___________ Gel
Plastic
Filterpaperbutfer
wick
Autoradiograph
Fig.7.7 : Diagrammatic representation
of a typical
blottingapparatus. Fig. 7.8 : Southern blotting lechnique.
C hapt er7 : B A S ICT EC H N IQ U ES
l N C E N E T ICE N C E N E E R IN C 99
The blot + r ans f er r edRNA m o l e c u l e s h y b r i d i z e Direct autoradiography is ideally suited for the
with DNA probes which can be detected by detection of weak to medium strength p-emitting
autoradiography. r a d i o n u c l i d e s e H , 1 4 C , 3 s S ) .I n t h i s t e c h n i q u e , th e
s a m o l e i s p l a c e d i n d i r e c t c o n t a c t w i t h th e fi l m .
Northern blotting is theoretically, a good The radioactiveemissionsresult in the developmenr
t ec hnique f or det er m ining t h e n u m b e r o f g e n e s of black areas.
( t hr ough m RNA) pr es enton a g i v e n D N A B u t t h i s
is not r eally pr ac t ic able s inc e e a c h g e n e m a y g l v e Indirect autoradiography
r is e t o t wo or m or e RNA t r a n s c r i D t s .A n o t h e r
dr awbac k is t he pr es enc eof e x o n s a n d i n t r o n s . For the detection of highly energetic B-particles
''zs},
(e g. 32P, direct autoradiography is not
DCT-BI-OTTING s u i t a b l e . T h i s i s b e c a u s e t h e s e e m i ssi o n s p a ss
through and beyond the film, and a major part of
Dot-blotting is a modification of Southern and the energy gets wasted.
Nor t her n blot t ing t ec hniques d e s c r i b e d a b o v e . I n
this approach, the nucleic acids (DNA or RNA) are I n d i r e c t a u t o r a d i o g r a p h y i s u s e f ul fo r th e
directly spotted onto the filters, and not subjected d e t e c t i o n o f h i g h l y e n e r g e t i c B - p a r t i cl e s. In th i s
t o elec t r ophor es is The
. hy br id i z a t i o n p r o b e d u r e i s technique, the p-particleenergy is first converted to
t he s am e as in or iginal blot t i n g t e c h n i q u e s l i g h t b y a s c i n t i l l a t o r ,w h i c h t h e n e m i t s ph o to n s o n
e x p o s u r e t o p h o t o g r a p h i ce m u l s i o n .
Dot - blot t ing t ec hnique is p a r t i c u l a r l y u s e f u l i n
obt aining quant it at iv e dat a f o r t h e e v a l u a t i o n o f Applications of autoradiography
gene ex pr es s r on.
A s a l r e a d y d e s c r i b e d ,a u t o r a d i o g r a p h yi s cl o se l y
WESTERN associated with blotting techniques for the
BLOTTING
detection of DNA, RNA and proteins.
Western blotting involves the identification of
proteins. lt is to very useful to understand the COLONY AND PLAQUE BLOTTING
nuc leic ac id f unc t ions , par t i c u l a r l y d u r i n g t h e
c our s e of gene m anipulat ions . Colony and plaque blotting is a process of
hybridization for the specific identification and
The technique of Western brlottinginvolves the purification of colonies (e.9. bacterial clones). This
transfer of electrophoresed protein bands from technique is depicted in Fig.7.10, and briefly
poly ac r y lam ide gel t o ny lo n o r n i t r o c e l l u l o s e d e s c r i b e d h e r e u n d e r .
membrane. These proteins can be detected oy
The desired bacteria are grown as colonies on
s pec if ic pr ot ein- ligand int er a c t i o n s .A n t i b o d i e s o r
lec t ins ar e c om m only us ed f o r t h i s p u r p o s e . a n a g a r p l a t e . Wh e n a n i t r o c e l l u l o s ef i l te r p a p e r i s
overlaid on the agar plate, the colonies get
transferred.They are permanentlyfixed to the paper
AUTOAAD'OGBAPHY
b y h e a t . O n t r e a t m e n tw i t h a l k a l i ( N a O H ) , th e ce l l s
Autoradiography is the process of localization lyse and the DNA gets denatured When these
and recording of a radiolabel within a solid DNA prints are exposed to specific probes
specimen, with the production of an image in a ( r a d i o l a b e l ) , h y b r i d i z a t i o n o c c u r s . T h e h yb r i d
photographic emulsion. These emulsions are complex can be localized and detected by
c om pos ed of s ilv er halide c r y s t a l s s u s p e n d e d r n autoradiography.
gelatin
A similar strategy(describedabove for bacteria;
When a B-particle ot a y-tay from a radiolabet can be used for the identification of phages and
pas s es t hr ough t he em uls io n s , s i l v e r i o n s a r e f r a g m e n t so f p h a g e l i b r a r i e s .
Chapt er7 : B A S ICT E C H N IQU ES
l N C EN ET ICEN GE N E E R IN C l Oi
Double-stranded
DNA
I
Bacterialcolonieson l-
Distributedinto 4 tubes
..t / \\
A +G
(Specificbasesdestroyedand fragmentsformed)
I rrugr"nt.separated
byelectrophoresis
I
+
A +G T +C C
Longer
I
A
g
Specificcolony T
detected
ATACTGCGACT Seouencedstrand
TATGACGCTGA Complementary
strand
Determination of nucleotide sequencein a DNA
m o lecule is the b as ic and f undam ent al r equir em e n t
in b iote ch no log y .DNA s equenc ing is im por t ant t o Fig. 7.11 : Maxam and Gilbert method for DNA
r-inderstandthe functions of genes, and basis of sequenctng.
inh erite d disord er s .Fur t her ,DNA c loning and gen e
m a nip ula tion in v ar iably r equir e k nowledge o f
accurate nucleotide sequence. C i l bert (1977).Thi s method i s bri efl y descri bed
bel ow (Fi g.7.11).
MAXAM AND G I LBERT TECHNI Q UE
A strandof sourceDNA is labeledat one end
The first DNA sequencing technique, using with 32P. The two strands of DNA are then
chemical reagents, was developed by Maxam and The labeledDNA is distributedinto four
seoai'ated.
1|J2 B IOTECHNO LO CY
s am ples ( in s epar at e t ub e s ) . E a c h s a m p l e i s
subjected to treatment with a chemical that
(n) @o-9Hz Base
(A,C, G,T)
specifically destroys one (C, C) or two bases
( A + C, T + C) in t he DNA. T h u s , t h e D N A s t r a n d s
ar e par t ially diges t ed in f ou r s a m p l e s a t s i t e s C ,
A + C, T + C and C. This r e s u l t s i n t h e f o r m a t i o n
of a seriesof labeled fragmentsof varying lengths.
The actual length of the fragment depends on the
(a) @-cHz Base
site at which the base is destroyedfrom the labeled (A,C, G,T)
end. Thus for instance, if there are C residues at
pos it ions4, 7, and 10 away f r o m t h e l a b e l e d e n d ,
then the treatmentof DNA that specificallydestroys
C will giv e lebeled piec es o f l e n g t h 3 , 6 a n d 9 OH H
bases.The labeled DNA fragmentsobtained in the
four tubes are subjected to electrophoresisside by
side and they are detected by autoradiograph.The Fig. 7.12 : Structure of (A) dideoxynucleotide
triphosphate and (B) deoxynucleotide triphosphate
s eouenc e of t he bas es in t h e D N A c a n D e
(Note the difference at s'-cafuon).
constructedfrom the bands on the electroohoresis.
DIDEOXYNUCLEOTIDE METI.IOD
single-stranded DNA to be sequencedis chosenas
Currently, the preferred technique for a template.lt is attachedto a primer(a shortlength
det er m ining nuc leot ide s eq u e n c e i n D N A i s t h e compl ement art oy a sm all
of D N A ol i gonucl eoti de)
one developed by Sanger (1980). This is an sectionof the template.The 3'-hydroxylgroup of
enzymatic procedure commonly referred to as the the pri meri ni ti atesthe new D N A syn t hesis.
dideoxynucleotide method or chain termination
DNA synthesisis carried out in four reaction
method (Note : Fredrick Sanger won Nobel prrze
tubes.Eachtube containsthe primedDNA, Klenow
twice, once for determining the structureof protein,
subunit(the largerfragmentof DNA polymerase of
ins ulin; t he s ec ond t im e f o r s e q u e n c i n g t h e (ddATP,
E. coli), four dideoxyribonucleotides
nuc leot ides in an RNA v ir u s ) .
ddCTB ddCTP or ddTTP). lt is necessaryto
324 the pri mer or one of t he
A dideox y nuc leot ide i s a l a b o r a t o r y - m a d e radi ol abel(w i th
c hem ic al m olec ule t hat lack s a h y d r o x y l g r o u p a t deoxyri bonucleotides.
both the 2' and 3'carbons of the sugar (Fig. 7.12). As the new DNA synthesisis completed,each
This is in contrast to the natural deoxyribo- one of the tubes containsfragmentsof DNA of
nucleotide that possessesat 3' hydroxyl group on varyinglengthboundto primer.Letus considerthe
the sugar. first reactiontube with dideoxyadenosine (ddATP).
In this tube, DNA synthesisterminates whenever
Termination role of dideoxynucleotide the growing chain incorporated ddA
I n t he nor m al pr oc es s o f D N A r e p l i c a t i o n , a n (complementary to dT on the template strand).
inc om ing nuc leos idet r iphos p h a t ei s a t t a c h e db y i t s Therefore, this tube will contain a seriesof different
5'-phosphategroup to the 3'-hydroxyl group of the length DNA fragments, each ending with ddA. In a
last nucleotide of the growing chain (Refer Chapter smilar fashion, for the other 3 reaction tubes,DNA
3) when a dideoxynucleotide is incorporatedto the synthesis stopsasthe respective dideoxynucleotides
gr owing c hain, no f ur t her r e p l i c a t i o no c c u r s . T h i s i s are incorporated.
bec aus e dideox y nuc leo t i d e , lacking a of new DNA fragmentsin the four
The synthesis
3'-hydroxyl group, cannot form a phosphodiester tubes is depictedin Fig. 7.13.
bond and t hus t he DNA s y n t h e s i st e r m i n a t e s .
The DNA pieces are denaturedto yield free
strandswith radiolabel.The samplesfrom eacn
Sequencing method
tube are sepaiated by polyacrylamide gel
The pr oc es s of sequencing DNA by electrophoresis. techniqueresolves
This separation
dideoxynucleotide method is briefly described. A DNA pieces,differentin size even by a single
Chapt er7 : B A S T C
T E C H N Ie U ES
tN C E N E T| CE N C E N E E R TN G 103
Template
G C A TC GA A T5'
5',+
\JN Newlysynthesized
DNA*
Fnmer
Primer+ 9 Primer-CGTAGCTTddA
Primer+ 6 Primer-CGTAGddC
rHd,'---1
i Primer+ 3 Primer-CGddT
Primer+ 7 Primer-CGTAGCddT
Primer+ 8 Primer-CGTAGCTddT
Fis'7'13: svnthesis
of r*tn" sizeofthe
;";.?-,: J:,',#:";;"':::;"::i;::l:i:",::#::1,:"o|ll"'
-
u
-
E A
T
-
u
-
Smallesl
Fig. 7.14 : Sequence ot the newly synthesized DNA fragment (complementary to original strand).
-l
lol
-(_
f 11 I Phage
L_"J
Phage E. coli
lnfe
Single-
strandedDNA
oooo
o o o oo
oo
o oo
oo o
oo o f-\-
Single-
strandedDNA 50-1 00 copies
(double-stranded
DNA)
P1
(A) l-T-lt ,
Plasmid ClonedDNA
DNA
P2
Fig. 7.16 : Primer walking technique for DNA sequencing (A) First round (B) Second round (C) Third
round (P1, Pr, Pr-Primers 1, 2, 3).
AI' T O MA T ED D N A S EQU E N C IN G
DNA sequencingin the recentyearsis carried
out by an automated DNA sequencer.In this Fig. 7.17 : (A) Chromosome walking
technique,flourescenttags are attachedto chain- (B) Chromosome iumping.
terminatingnucleotides(dideoxynucleotides).This
l N C EN ETICE N C E N E E R IN C
Chapt er7 : BA SICT EC H N IQ U ES lo7
DMT-O
T HE P HO S P H O R AMID IT E M ET H O D
Another nucleotide precursor is now added and
T hephos p h o ra mi d im tee th o di s th e te c h n i queof the reactions described in steps 3 and 4 are
choice,currentlyin use,for the synthesis of DNA reDeated.
The synthesis is carriedout on a solidphasesystem
Coupling, oxidation and deprotection are the
g e g ro w i n gD N A s tra n dto a sol i d
i. e.by at t ac h i n th
three steps in the cvclic reaction for the DNA
s uppor t , in a re a c ti o n v e s s e l .T h e p ro c edure
s y n t h e s i sb y p h o s p h o r a m i d i t e m e t h o d . T h e c y c l e s
involvesthe following stages.
are repeated again and again to synthesise the
.l : desired DNA. At the end, the completed
. Nucleosideattachmentto solid support
The initial nucleoside (base + sugar) is attached at o ligonucleotide is removed from the glasssupport
3' end t o an i n e rt s o l i d s u p p o u
rt, s u a l l yto gl ass and the groups protectingthe basesand phosphates
beadswith uniformpores,referredto as controlled are cleaved
pore glass(CPC)beads.
Applications of synthesized
2. Preparationof phosphoramidites : For each oligonucleotides
one of the four bases (A, C, C and T),
phos phor am i d i tecsa n b e s y n th e s i z e dT. h i s i s C h e m i c a l l y s y n t h e s i z e do l i g o n u c l e o t i d e sh a v e a
(DMT)group number of uses in biotechnology.
achievedby attachingdimethoxytrityl
to the 5'-hydroxyl group of deoxyribose and a 1. The linkers and adaptors are the
g roup
diis opr opy la m i ngero l p to th e 3 ' -p h o s p h i te oligonucleotides commonly employed in the
of the nucleoside. A methylresidueprotectsthe 3'- preparation of recombinant DNA (Chapter 6).
phosphite group. The general structure of
phosphoramidite is depictedin Fig. 7.20. 2. Chemically synthesizedDNAs with known
sequencescan be used for the synthesisof proteins/
3. Coupli n g : A n u c l e o ti d ep re c u rs o r(i.e. a polypeptides.
phos phor ami d i te w)i th i ts 3 ' -p h o s p h o rucso upl es
3 . D N A p r o b e s , p a r t i c u l a r l yt h e s i n g l e - s t r a n d e d
e o u ndto
wit h 5' - hv dr o x vol f th e i n i ti a ln u c l e o s i d b
oligonucleotide probes, can be synthesized in the
a CPC bead.
laboratory.This is based on the codon sequenceof
4 Oxidation and deprotection: The unstable mRNA which in turn is dependent on the amino
trivalentphosphiteis oxidizedto pentavalentstable acid sequence
phosphate. This is followedby the removalof DMT
4 . S i n g l e - s t r a n d e do l i g o n u c l e o t i d e sa r e u s e d a s
that protects(deprotection) 5-hydroxylgroup. For
p r i m e r s i n p o l y m e r a s ec h a i n r e a c t i o n ( P C R ) .
conven ience, oxidation and deprotection are
consideredtogether in Fig. 7.21 also, they are 5. For in vitro mutagenesis, single-stranded
actuallvtwo independentreactions. o l i g o n u c l e o t i d e sa r e e m p l o y e d .
B IOTE CHNO LO CY
rto
Base1
oH
I
O cPG bead
to a bead
lnitialbaseattached
OH
H\H
HO-QH2 Base2
H
H H/H
N4eo-il-o-cH2
ct;
oH
I
(cPG-Controlled porc gtass;
Fig. 7.21 : Chemicat synthesisof DNA by phosphoramidite method
DMT-DImethoxytrityl; Me-Methyl; N(iP) t-Diisopropylamine)'
l N C EN ET ICE N C E N E E R IN C
Chapt er7 : B A S ICT E C H N IQU ES ttt
OH OH P 5ott
OH OH OH
Jnnnearino
T4 DNA
ligase
112
C hA P I CT
B : P O L Y M ER AS (D N A A MP LIFIC A TION )
CEH A IN R EA C T ION 113
90
t I Denaturation
J Primerannealing
c
L
g- 80 E
o
1
svntnesis
(g
Jorun
- Originalstrand
ii zn
.(
E -{ ')Long template
,o)
---+...-...F
60 Originalstrand
2345
IJ
t ....-..<--- Longtemplate
Time(minutes)
Biotechnology[8]
114 B IOTECHNO LO CY
Target
DNA UndesiredDNA
rl
,+
ll
J PCR with
first primers
-t ; +l
PCRwith
secono
I
pnmers
The secondprimers
do not bind to undesired
DNA. hence no PCR occurs
modifiedas perthe specificdemandsof the situation. now cut with another restrictionenzyme. This
Thus,thereare manyvariationsin the originalPCR, cleaves only the known sequence.The target
someof them are discussed, hereunder. DNA so formed contains the known sequence
at both the ends"withtargetDNA at the middle.
NESTED PCR The PCR amplification can now be carried
out. lt may be notedthat the primersare generated
Sequencesimilaritiesbetweenthe target DNA in the oppositedirectionto the normal,sincethe
and relatedDNA are very frequentlyseen.As a ori gi nalsequence i s i nvertedduri ngci rcul ari zati on.
result of this, the primersmay bind to both the
DNAs and thereforeeven the undesiredDNA also ANCHORED PCR
gets amplified in PCR. Use of nested primers
In the anchoredP C R , a,smal l sequenceof
increasesthe specificityof PCR, and selectively
nucleotidescan be attached(tagged)to the target
amplifiestargetDNA. NestedPCRis illustratedin
D N A i .e.,the D N A i s anchored. Thi si s parti cul arl y
Fig. 8.3. In the first cycle of PCR,the productsare
usefulwhen the sequenceSurrounding the target
both from target DNA and undesiredDNA. A
DNA is not known.The anchoris frequentlya poly
secondset of internalprimersis now used.They
C tai l to w hi ch a pol y C pri mer i s used. The
will selectively bind to target DNA and
anchoringcan alsobe done by the useof adaptors.
amplificationproceeds.
As the adaptorspossessa known sequence,the
orimercan be chosen.
INVERSE PCR
I DNA (RAPDI
Normally,the objectiveof PCR is to generate
defined fragmentsof DNA from highly specific
primers.In the caseof RAPD(pronounced as rapid),
@ short oligonucleotide primers are arbitrarily
selected to amplify a set of DNA fragments
throughout
randoml ydi stri buted the ge nom e.This
techni que,randomampl i fi edpol ymorphicDNA is
also known as arbitrarily primed PCR(AP-PCR).
The procedureof RAPD is comparableto the
generaltechni queof P C R .Thi s meth odbasically
i nvol ves the use of a si ngl e pri m er at low
stri ngency.A si ngl eshortol i gonucl eo t ide ( usuallya
9-10 base pri mer) bi nds to many sit es in t he
Primer genomeand the DNA fragments are amplifiedfrom
r----+ them. The stri ngencyof pri mer bi nding can be
increasedaftera few PCRcycles.This allows the
{-r Primer amol i fi cati onof best mi smatches. R APD can be
careful l ydesi gnedso that i t fi nal l yyi e ldsgenom e-
Fig. 8.4 : lnverse PCR. specific band patterns that are useful for
comparati veanal ysi s. Thi s i s possible since
genomicDNA from two differentindividualsoften
of f ir s t s t r and of c DNA. The s e i n c l u d e t h e u s e o f producesdifferent amplified patternsby RAPD.
r andom pr im er s , oligo dT pr i m e r a n d a s e q u e n c e Thus, a particularDNA fragmentmay be generated
specific primer (Fig. 8.5). for one i ndi vi dualand not for the other ,and t his
represents D N A pol ymorphi sm w hi ch can be used
ASYMMETRIC PCF as a geneticmarker.
PCRt ec hnique c an als o be u s e d f o r t h e s y n t h e s i s
of s ingle- s t r anded DNA r n o l e c u l e s , p a r t i c u l a r l y
(A) AAAAA mFINA
us ef ul f or DNA s equenc in g ( C h a p t e r 7 ) . l n t h e
{--<-- <-r{--{--
as y m m et r ic PCR, t wo pr im er s i n a r a t i o o f 1 0 0 : 1 cDNA
are used. After 2O-25 cycles of PCR, one primer is
(B) AAAAA mRNA
exhausted.The result is that in the next 5-1 0 PCR
T T TTT cD N A
cycles, only single-strandedDNAs are generated
RA P D is wi d e l y u s e d b y p l a n t m o l e c u l ar s / - - - - G A A T T C T T A A- - - - 3/
biologists for the genetic identification of plant 3/----CTTAAG A A T T- _ _ _ 5 /
species (ReferChapter53).Forthispurpose, different GenomicDNA
cornbinations of nucleotides, mostof them random II
oligonucleotide primershavebeendesignedand are I Restrictionendonucleases
. s e a c h ra n d o m p ri mer
com m er c iallyav a i l a b l e A | (EcoRl and Msel)
+
annealsto a differentregionof DNA, manydifferent AATTC T
regionsof loci on the DNA can be identified.RAPD
is thus usefulfor the construction of geneticmaps
and as a m et ho dfo r g e n o mi cfi n g e rp ri n ti n g .
Limitations of RAPD
The main problemof RAPD is associated with
reproducibility. lt is oftendifficultto obtainsimilar
levelsof primerbindingin differentexperiments. lt Additionof first
is thereforedifficultto correlateresultsobtainedby orimerwithone
selectivenucleotide
differentresearchgroupson RAPD. 5'#A
AATTCT CTTA-
A M P LI F I E D F R A G ME N T L E N GT H
POLYMORPHTSM (AFLPI TTTAAGA GAAT
c +5 ,
AFLP is a very sensivemethod for detecting
p oly m or phis min th e g e n o me .l t i s b a s e do n the Additionof second
primerwiththree
p r inc iple of r e s tri c ti o nfra g m e n t l e n g th p o l y- selectivenucleotides
m or phis m( RF L BR e fe rC h a p te r1 4 ) a n d R A PD . 5.-AAC
AFLPmay be appropriately regarded as a diagnostic r AATTCT CTTA-
fingerprintingtechnique that detects genomic TTAAGA GAAT-
restrictionfragments. -
I AAo sN
I n t he A F LP , PC R a mp l i fi c a ti o nra th e r th an
I selective
Southernblotting(mostlyusedin RFLP)is usedfor I amplification
+
the detectionof restrictionfragments.lt may be AACTTA-
notedthatAFLPis employedto detectthe presence -AATTCTTG
or absenceof restrictionfragments,and not the TTGAATE
-TTAAGAAC
lengths of these fragments. This is the major
I
I
Limitations of RAGE
of PCRare discussed
Other applications
described.
Sincea specificprimeris used,the specificityof at appropriateplaces.
amplificationof RACE may not be very high.
Another disadvantage is that the reverse PCR IN CLINICAL DIAGNOSIS
transcriptase
may not fully reach the S'-endsof
RNA, and this limits the utility of RACE.ln recent The specificityand sensitivityof PCR is highly
years, some modificationshave been done to useful for the diagnosisof various diseasesin
imoroveRACE. humans. These i ncl ude di agnosi so f inher it ed
disorders(geneticdiseases),
viral diseases,bacterial
diseasesetc.
For all such disorders,PCR technique is a real and ampl i fi ed by P C R . More detai l s on thi s
boon, as it providesdirect informationof DNA. technique,referredto as site-directedmutagenesis,
T his is done b y a mp l i fi c a ti o no f D N A o f the are gi ven el sew here(C hapter10). B y usi ng thi s
relevantregion,followed by the direct analysisof method,coding sequencecan be altered(thereby
PCRproducts. changi ngami no aci ds)to synthesi zeprotei n of
genemani pul ati ons
Prenataldiagnosisof inherited diseases: PCR i nterest. Further, are i mportant
in understanding the effects
of promoters, initiators
is em ploy edi n th e p re n a tadl i a g n o s i so f i n h eri ted
etc., i n geneexpressi on.
dis eas esby u s i n g c h o ri o n i c v i l l u s s a m p lesor
c ells f r om a mn i o c e n te s i sT. h u s , d i s e a s esl i ke PCR is importantin the study of mRNAs,the
s ic k le- c ell a n e m i a , p -th a l a s s e m i a and productsof geneexpression. This is carriedout by
pheny lk et on u ricaa n b e d e te c te db y PC Ri n these reversetranscriotion - PCR.
s am ot es .
Diagnosisof retroviral infections : PCR from PCR IN COMPARATIVE STUDIES OF
to o lfo r d i a g n o s iasn dmo n i tori ng GE N OME S
c DNA is a v al u a b l e
of retroviralinfections,
e.g.,HIV infection. The differences in the genomesof two organisms
Diagnosisof bacterialinfections: PCRis used can be measured by P C Rw i th randompri mers.The
for the detection of bacterial infection e.9., products are separatedby electrophoresisfor
tuberculosisby Mycobacteriumtuberculosis. comparative identification.Two genomes from
closely related organismsare expectedto yield
Diagnosisof cancers: Severalvirally-induced
more si mi l ar bands.For more detai l s,refer the
cancers(e.g., cervical cancer causedby human
techni querandom ampl i fi ed pol ymorphi cD N A
papillomavirus)can be detectedby PCR.Further,
(S eep.116).
s om e c anc er sw h i c h o c c u r d u e to c h ro mosomal
t r ans loc at io(nc h ro m o s o me
1 4 a n d 1 B i n fo l l i cul ar PCRis very importantin the studyevalutionary
lymphoma)involving known genesare identified biology, more specifically referred to as
by PCR. phylogenetics. As a techniquewhich can amplify
even minute quantitiesof DNA from any source
PCR in sex determinationof embryos : Sex of
(hai r,mummi fi edti ssues,bone, or any fossi l i zed
human and live stock embryosfertilizedin vitro,
material),PCR has revolutionizedthe studiesrn
can be determinedby PCR,by using primersand
palaentologyand archaelogy. The movie 'Jurassic
DNA probesspecificfor sexchromosomes. Further,
Park',hascreatedpublicawareness of the potential
this technioueis also usefulto detectsex- linked
appl i cati ons
of P C R !
disordersin fertilizedembryos.
12l)
9 : CE N EL IBR AR IES
RE RE RE
SourceDNA
Restrictionendonuclease
(partialdigestion)
2 +3 +4
(a)
1+ 2 3 +4
(b)
1+ 2+ 3 4
(c)
3 +4
(d)
f+z 4
(e)
2+ 3 4
(f)
4
(s)
Fig. 9.1 : Paftial digestion of a DNA by the enzyme restriction endonuclease (RE) at three sites
;
[Note : the coloured fragment represents the desired gene and it is one (shown in f) ;
among several fragments formedl.
I
i
I
E collcells
+
r-/ "O
.- /)
l-/r-./
a)
l-/
Plasmids
1 rg 76
)c
2w uou )7
DNAfragments
lr.'-\ \
'/-\
tn() A
O,cu,,
'40 /z'l
o,
7-
trJ l)
'O
.o 20
a) (
... (-./a
AAAAAA A A A A AA
AAAAAA A A A A AA
-._ TTTTTT
CompletecDNA Incomolete
cDNA
U in the template (mRNA), Ihe corresponding lrnproved method for cDNA synthesis
complementary bases in DNA respectivelyare
To overcomethe limitation describedabove,
T, C, G and A. The newly synthesizedfirst
some improvementshave been made in cDNA
DNA strand has a tendencyto fold back on to
One such i mprovedtechni queis shown
synthesi s.
itselffor a few nucleotidesto form a hairpin loop
in Fig. 9.4.
(Fig. e.3).
As the first strandof cDNA is synthesized, it is
The loop of the first DNA strandservesas the tailed with cytidine residueswith the help of the
templatefor the synthesisof secondDNA strand. enzymeterminaltransferase. The mRNA strandis
By the additionof E. coli DNA polymerase (Klenow hydrol ysed w i th al kal i ,and the ful l l eng t hcDNA is
fragment), the secondDNA strandsynthesis occurs recovered.A syntheticoligo-dC primer is then
startingfrom the end of the hairpin loop. On anneal edto ol i go-dC .Thi s i n turn en ablest he
tre a tm e n tw i th th e e n z v m e R N ase H . mR N A synthesisof the secondstrandof cDNA. By this
m o l e c u l e sa re d e g ra d e dT. h e enzymeS l nucl ease i mproved techni que, a ful l l ength of cDNA
c l e a v e sth e h a i rp i n l o o p s a nd degradessi ngl e- correspondi ng to mR N A (i n turn the gene) is
strandedDNA extensions.The final productsare obtained. But the efficiency of this method rs
c o mp l e m e n ta ry D N A c o p i e s of ori gi nal mR N A , comparativelylower.
some of them are complete while others are
incomplete. Construction of cDNA libraries
limitation of the technique : The main The compl ementaryD N A mol ecu lescan be
disadvantage with the hairpinmethodis the lossof cl onedi n cl oni ngvector(e.g.,pl asmi d),for cr eat ing
a s ma l l s e q u e n c ea t th e 5 ' e n d of cD N A due ro cD N A l i brari es.
ThecD N A i nserti oni nto t he vect or
c l e a v a g eb y S l n u c l e a s e . shouldhavecorrectorientationThis is achievedby
Chapt er9 : CE N EL IBR AR IES 125
3', T T T T5 '
Once a D N A l i brary or a C D N A l i brary i s
I r"rrin"t transferase created,the clones (i.e., the cell lines) must be
+ocre screenedfor identificationof soecificclones.The
J screeningtechniquesare mostly based on the
AA AA sequenceof the clone or the structure/function of
TTTT its product.
I HydrolysemRNA
S C B E E N IN G B Y D N A H Y B R ID IZA TION
(withalkali)
J The target sequence in a DNA can be
TTTT 5' determinedwith a DNA probe (Fig. 9,5). To start
with, the double-strandedDNA of interest is
converted into single strandsby heat or alkali
(denaturation).
Jo"*' TTTT 5'
The two DNA strandsare keptapart
by bi ndi ngto sol i dmatri xS uchas ni trocel l ul ose
nylon membrane.Now, the singlestrandsof DNA
or
"@ 3'
probe(100-1,000bp) labeledwith radioisotope are
c uuu added. H ybri di zati on(i .e., base pai ri ng)occurs
(/ Double-strandedcDNA betweenthe complementary nucleotidesequences
Primer
I
the addition of a syntheticlinker to the double- I Denaturation
membranebinding
strandedcDNA.
J
ln a techniquedevelopedby Okayamaand Berg
(1982),the mRNA is first linked to the plasmid
cloningvectorand then cDNA synthesis is carried DNAs
Single-stranded
out.
RT.PCR as an alternative to
cDNA cloning
Reverse transcription followedby PCR(RT-PCR)
can am plif yt he m R N A to g i v e c D N A (F o rd e tai l s
ReferChapterB).
R
RT-PCRis very rapid hence cDNA molecules
can be obtainedin a shortperiod.Further,
eventhe x x )(
long length mRNAscan be convenientlyused in HybridDNAS
RT-PCR.
Fig. 9.5 : Screening by DNA hybridization
also in RT-PCR.
There are some disadvantages ({ indicates radioisotope label in the DNA probe)
The DNA polymeraseused in RT-PCRis error-
126 B IOTE CHNO LO CY
of the target DNA and the probe. For a stable base 3',
pairing, at least B0% of the basesin the two strands
(target DNA and the probe) should be matching. SourceDNA
The hybridized DNA can be detected by
autoradiography. I uyorioization
primers
Jwith
DNA PROBES
-J
The DNA probes used for screeningpurpose can
be s y nt hes iz edin m any way s
129
Biotechnology [9]
130 B IOTECHNO LO CY
CASETTEE MUTAGENESIS
In casetteemutagenesis, a syntheticdouble-
strandedoligonucleotide(a small DNA fragment
(B) i.e., casettee)containing the requisite/desired
mutant sequence is used. lt replaces the
correspondingsequencein the wild type DNA.
Casetteemutagenesisis possibleif the fragment of
the gene to be mutated lies between two
restriction enzyme cleavagesifes. This intervening
(c) sequencecan be cut and replacedby the synthetic
(w i th mutati on).
ol i gonucl eoti de
The outline of the casettee mutagenesis
techniqueis depicted in Fig. 10.3. The plasmid
DNA is cut with restriction enzymes(suchas EcoRl
and H i nd111l . The syntheti c ol i gonucl eoti de
containingmutant sequenceis also cleavedand
Fig. 10.2 : Variations in oligonucleotide-directed
l i gatedto pl asmi dD N A . The recombi nant
pl asmi ds
mutagenesis (A) Multiple point mutagenesis
(B) lnsertion mutagenesis (C) Deletion mutagenesis. that multiplv in E. coli are all mutants.
Variations in oligonucleotide-directed
mutagenesis
There are some variations in use in the
oligonucleotide-d
irected mutagenesis, as the
situationdemands.
1. Multiple point mutagenesis : Oligo-
nucleotide-directed
mutagenesis can be used to
createDNAswith multiplepoint mutationswith the
requisitenumberof basemismatches (Fig. 10.2A).
2. I ns er t ionm u ta g e n e s i s : In th i s c a s e , th e
mutant oligonucleotidecarriesa sequenceto be
inserted (sandwichedbetween two sites with
complementary sequences). This can bind with the
target DNA on either side (Fig. 10.28).
3. Deletion mutagenesis : The mutant
oligonucleotidebinds to two separatesites on Fig. 10.3 : Casettee mutagenesis.
either side of the target DNA. This enables a
B IOTECHNO LO CY
132
Tube 1
Normalprimers
Primerswith a
l:)
TargetDNA nucleotidemismatch
Originalnucleotide
Strand C
StrandD
LinearDNA
Changed
nucleotide
StrandB
Strand D
a plasmid
Casettee mutagenesis is a simpletechniquewith the targetDNA (gene)is cloned on to
The vector and distributed into two reaction tubes'To
almost 1007oefficiencyto get mutant genes'
two primers (oligonucleotides
drawback is the requirementof restrictionsites eachtube are added
target fragment of DNA svnthesized by using PCR).One primer (A in tube
specificallyflanking the
is complementary to a regionin
a n d th e l i m i ta ti o nto g e t desi redol i gonucl eoti des'1 and C in tube 2)
one strand of the cloned gene except for one
nucleotide mismatch (i.e., the one targetedfor a
PC R .A M PL IF IE D OLIGON U GLE OTID E .
DIRECTED MUTAGENESIS change).The other primer (B in tube 1 and D in
tube 2) is fully complementary to a sequencein the
The polymerasechain reaction(PCR)and its otherstrand,within-oradjacentto the clonedgene'
importancehavebeendescribed(ReferChapterB)' The placementof primersfor hybridization(with
T h e P C Rte c h n i q u ec a n be usedi n ol i gonucl eoti de-the DNA strands)in eachtube is done in opposite
directed mutagenesis.This avoids the use of a direction.Now the PCRtechniqueis carriedout
bacteriophage (M13) system, besides enrichment for amplificationof the DNA molecules'These
of mutated gene. DNAs are actually linear. In Fig. 10.4' they are
for understanding
The PCR-based mutagenesis technique shown as discontinuouscircles
commonlyemployedis depictedin Fig. 10.4' First subsequent steps.
ISN D P R OTE INE N C IN E E R IN C
ED T AC EN ESA
Chapt er10 : S IT E -D IR EC T MU 133
_,.- Serine(359)
394
.-----_-... -
^,--Methionine
(358)
(A)
(B) Valine
(358)
No cleavaoe
Elastase
were found to be in monomeric state with good (K m, speci fi ci ty etc.) through ol i gonucl eoti de-
stabilityand biologicalactivity. directedmutagenesis. This is particularlyrequired
for enzymes w i th i ndustri al and therapeuti c
Tissue plasminogen activator (tPAf appl i cati ons.
Tissueplasminogenactivatoris therapeutically
usedto lysethe blood clotsthat causemyocardial Subtilisin
infarction.Due to its shorter half-life (around 5 Subtilisinis a major industrialenzymesecreted
minutes),tPAhasto be repeatedly administered. By by Cram-positive bacteria(Bacillusspecies).
lt is a
replacing asparagine residue(at position 120) with serine proteasevery widely used as an enzyme
glutamine, the half-life of tPA can be substantially detergent(cleaningagent in laundries).However,
increased. This is due to the fact that glutamineis the l argescal ei ndustri aluseof nati vesubti l i si ni s
lessglycosylated than asparagine and this makesa restricteddue to the oxidativeinactivationof this
differencein the half-lifeof tPA. enzyme.Thi soccursi n a manneranal ogous to that
already discussedfor cx,-antitrypsin. The reason
Hir udin beingthat methionine(at position222) lying close
Hirudin is a proteinsecretedby leech salivary to the active site gefs oxidized, making the enzyme
(i
gland,and is a s tro n gth ro m b i ni n h i b i to r .e .,
acts inactive. Logically,replacementof methionine (at
as an anticoagulant).By replacing asparagine(at position 222) by other amino acids will solve the
47 position) with lysine, the potency of hirudin can problem.This has been done, and in fact, all the
be increasedseveral-fold. 19 otherami noaci dshavebeentri edas substi tutes
for methi oni ne, w i th varyi ngsuccess.
Dihydrof olate reductase i s an enzymethathasbeenextensi vel y
S ubti l i si n
The enzyme dihydrofolatereductase(DHFR) exploitedfor geneticmanipulationsover the past
catalysesthe conversionof 7, B-dihydrofolate to 5, 15 years.The resultis that abouf50% of the native
6, 7, 8-tetrahydrofolate.The lattef coenzyme is amino acidsof this enzyme have been changedby
closelyinvolvedin one carbonmetabolism, which in vitro mutagenesis. And almostevery propertyof
ultimatelyresultsin the synthesisof nucleic acids subti l i si n has been al tered. These i ncl ude i ts
and am ino a c i d s . T h e i n h i b i ti o n o f DH FR stability,substratespecificity,thermaland alkaline
(conventionallyby folate analogues such as i nacti vati on.
methotrexate) will restrictthe growthof tumorcells.
Modifying metal requirementof subtilisin : The
Thereforethe enzymeDHFRhas sometherapeutic enzyme subti l i si n bi nds to cal ci um and gets
applic at ion s . B ut i n many i ndustri es w heresubti l i si n
stabi l i zed.
By employing site-directed mutagenesis, is used,metal-chelatinB agentsare also employed.
replacementof glycine (at position 95) by alanine They bind to calcium and the enzyme becomes
was found to produce DHFR which is completely inactive.This problem can be solved by oligo-
inactive. nucleotide-directed mutagenesis. The nucleotide
sequencein the DNA encoding for the amino
T4 lysozyme aci ds (resi dues 75 to 83) that bi nd to cal ci um i s
Replacement of glycine by any other amino removed. Then, several other amino acids are
acid in the protein structure, in general, decreases modi fi ed i n subti l i si nby tri al and errormethod.In
the stability.On the other hand, proline residues facl, success has been achieved in creating
increase protein stability. In f4 lysozyme, cal ci um-freesubti l i si nw i th good stabi l i ty and
substitutionof glycine(at position77) by alanine, activity.
and alanine(at position82) by prolineare found to
increasethe enzymestability. Asparaginase
This is an enzyme used in the control of
I M P RO V I N G K IN E T IC P R OP ER T IES Ieukemia (uncontrolledgrowth of white blood
OF ENZYMES cells). Intravenousadministrationof asparaginase
It is possibleto improvethe functionalactivities cleaves asparagineto aspartate (the reduced
of enzymes,by improvingtheir kinetic properties avai l abi l i ty
of asparagi ne cel l prol i fi rati on).
restri cts
136 B IOTECHNO LO CY
The bacterium Escherichia coli was the frrsr The yeast Saccharomyces cerevisiae is widely
o rga nism to be us ed in r ec om binant D N A used as a host for the exoression of cloned
technology experiments,and continues to be a fiosf eukaryotic genes.The other yeastsin use include
of choice for commercial production of proteins. Kluveromyces lactis, Schizosaccharomyces pombe
The extensiveuse of E. coli is mainly due to its high and Picha pastoris.
ra te of rep rod uc t ion ( t he c ells double in num b e r ,
The insect cells which are infected bv
every twenty minutes),besidesgood knowledge on
baculovirus are in use as hosts in recent years. The
its biochemistry,physiology and molecular biology.
There are a few disadvantagesalso in using F. coll b a c u l o v i r u ss y s t e mc a n c a r r y a n d e x p r e s sh u n d r e d s
o f g e n e s i n i n s e c t c e l l s . A n o t h e r a d v a n t a g ei s t h a t
as a host. These include a relatively poor export
the safety factor since baculovirusesdo not infect
system for proteins and the production of
h u m a n s , o t h e r v e r t e b r a t e so r p l a n t s .
endotoxins which are often difficult to remove
(from other useful products) Mammalian cells such as mouse cel/s can be
The other host bacterium in use is Bacillus used as hosts to produce complex proteins with
subtilis. This organism is widely employed for optimal biological functions. But the limitations
137
138 B IOTE C HNO LO CY
Stability of proteins
T he half - liv eosf re c o m b i n a nptro te i n sa reh i g hl y '.{- Plasmid
v ar iable, r ang i n g fro m mi n u te s to h o u rs . The
stabilityof proteinscan be increasedby adding
amino acidsat the N-terminalend of the proteins.
T hus ,by at t ac h i n gme th i o n i n es, e ri n ea n d a l a n i ne Chromosomal
DNA
to the N-terminal end, the half-life of
B-galactosidase can be increased from 2 minutesto
20 hours!Frequently, a singleamino acid addition
at N-terminalend stabilizesthe protein The yield
of recombinantDNA proteinscan be enhancedby
inc r eas inghalf -li v e s .
Secretion of proteins
The stabilityof a protein and its secretionare Fig. 11,1 : lntegration of a cloned DNA into
interrelated.An amino acid sequence (signal chromosomalDNA.
peptide)may be attachedto a protein to facilitate
its secretionthroughcell membrane.Recombinant
proteinssecretedinto the growth medium can be metabolicload.Theseincludeincreasedutilization
eas ilypur if ied . of energy for replication and maintenanceof
plasmids,overproductionof proteins(also drains
Integration of cloned DNA into the amino acids, tRNAs),and interference of foreign
host chromosome proteinson the host cell function.
The use of plasmids for transcriptionand
translationof cloned DNA imposesa metabolic
load (discussed later)on the host.In addition,there
is of t en a c han c eo f l o s i n gp l a s mi d sd u ri n g cel l
multiplication.Theseproblemscan be overcome
by integratingthe cloned DNA directly into host
chromosomal DNA. Once the cloned DNA Expression of cloned genesin eukaryoteshas
becomesa part of genome, it can be maintained certain advantages.The most important being
for severalgenerations. the ability of eukaryotic organisms to bring about
post-translational modifications--glycosylation,
Cloned DNA integrationinto the host DNA is
phosphorylation, correctdisulfidebond formation,
possible only when .there is a complementary
proteolytic cleavage etc. Eukaryoticexpression
sequenceof about 50 nucleotidesbetweenthem.
systemsproduce stable and biologically active
The exchangeof DNAs occursby a recombination
proteins.This is in contrastto the prokaryotic
process(Fig. 11.1\.The cloned DNA lies in the
expression of cloned genes.
middle of plasmidDNA. On physicalcontactwith
chromosomalDNA, base pairing occurs between In general,the eukaryoticexpression of cloned
plasmid DNA (x and y) and chromosomalDNA genes is quite comparableto that occurs in the
(x' and y'). And the cloned DNA is transferred to prokaryotes. However, from the technical
host chromosomalDNA by a physicalexchange perspective,it is more difficult to conduct
i.e., recombination. experimentswith eukaryoticcells. Many a times,
vectorswith two distinctoriginsof replicationare
Metabolic load used.They serveas shuttlevectorsand functionin
prokaryoticas well as eukaryotichosts.
The presence of cloned DNA alters the
metabolism and cellular functions of the host The inserlionof a foreign DNA into bacterial
organism.Suchmetabolicchangesare collectively and yeast cells is referred to as transformation
referredto as metabolic load, metabolic drain or (ReferChapter6). The term transfecfionis usedfor
metabolic burden. Thereare severalcausesfor the the introduction of a foreignDNA into animalcells.
14|J BIOTECHNOLOCY
. S. cervisiaeis single-celledthat can be easily Fig, 11.2 : Construction of yeast artificial chromosome
geneticsand physiology (LT-Left teIomere; C-Centromere; RT-Rig ht teIomere).
grown. lts biochemistry,
are quite known.
r lt has a naturallyoccurringplasmidand strong tandemgenearrayshasnot met with success,
since
promotersfor efficient expression. they are unstable.
r 5. cerevisiae can bring about many post- 2. Integratingvectors : They are basicallythe
changesin proteins.
translational integrationof cloned genes with chromosomal
o The secretedrecombinantproteinscan be easily DNA. These are not frequentlyused, since the
proteinproductionis low.
isolated, since very few host proteins are
secreted. 3. Yeast artificial chromosome (YAC) :
. The U.S. Food and Drug Administrationhas lntroduced in 1987, YAC is a fragmentof yeast
certified S. cerevisiaeas a generally recognized DNA that will accept a foreign DNA of about
as a safe (GRA9 organism. 250-500 kb in length.In fact, the yeastDNA is
only about 1% of the total DNA which however,is
As such,S. cerevisiaehasbeen in usefor several very important,since it containsthree essential
decades in baking and brewing industries. genesrequiredfor replication.Theseare the genes
Biotechnologists work quite comfortablywith this for telomere (that protects DNA from nuclease
yeast to produce a large numher of recombinant degradati on and thus mai ntai ns st abilit y) ,
proteins. These include insulin, q-antitrypsin, centromere(forms spindlesduring cell division)
hepatitis B virus surface antigen, platelet and the origin of replication (where DNA
derived growth factor, fibroblast growth factor polymeraseinitiatesreplication). YAC behavesjust
and HIV-l antigens.These products are in use like a chromosomeand replicates.
as diagnosticagents,vaccines,and therapeutic
agents. The construction of the veast artificial
chromosomeis depictedin Fig. 11.2.Two opposite
ends of a yeast chromosomenamely the left
Vectors for S. cerevisiae
telomereand right telomereare chosen.The left
Thereare three typesof vectorsfor S. cerevisiae: telomereis then attachedto a centromere. A large
segmentof the foreign DNA is added and all the
1. Episomalor plasmidvectors. three are ligated.Unlike the plasmidvectors,the
stabilityof YAC increases as the sizeof insertDNA
2. Integrating vectors.
IncreaSes.
3. Yeastartificialchromosomes (YACs).
YACs have not been used for commercial
1. Plasmid vectors : Among the vectors, productionof recombinant proteins.However,they
plasmidswith singleclonedgenesare widely used. have been employed successfullyfor physical
Manipulationwith growth conditionsincreasethe mappi ngof genomi cD N A s,parti cul arly in hum an
vector stabilityand expressionefficiency.Use of genomeproject,(Fordetails,ReferChapter12).
ChA O t CT OF C E N EEX PhE S S ION
11 : M A N IP U L A T IO N IN H OS TC E LLS 141
Post.translational modifications by
S, cerevisiae Table 11.1 Selectedexanples of reconbinant
proteins producedby baculovlrusexpyesslon
The heterologous proteins synthesized by vectoY systen
S. cerevisiaeundergo post-translational changes
while they are beingexportedinto the extracellular Adenosine deaminase
environment.To facilitate protein secretion, a phosphatase
Alkaline
single (leader) peptide is attached to the Amyloidprecursor protein
protein. This peptide is removed by the yeast Anthraxantigen
endoprotease. DNApolymerase a
Erythropoietin
OTHER YEAST EXPRESSION SYSTEMS HIV-lenvelope protein
(a, B)
Interferons
Despitethe very successfuluse of 5. cerevisiae lnterleukin-2
for generatingrecombinantproteins, there are Malariaproteins
certainlimitations.Theseincludea very low or a
Pancreaticlipase
of someproteins
limitedyield,difficultyin secretion
Poliovirusproteins
and hyperglycosylation. Attemptsare being made
Rabiesvirusproteins
to explore the utility of other yeasts for the
production of hepatitis B virus surface antigen Rhodopsin
(HBsAg) and bovine lysozyme. The yeast, Simianrotaviruscapsidanligen
Hansenula polymorpha, is employed for the Tissueplasminogen activator
synthesis of a- and p-globin chains of human
hemoglobin.
cloned gene expresses,and large quantitiesof
recombinantproteinsare produced.Becauseof a
INSECT CELL EXPBESSION SYSTEMS close similarity in the post-translational
Culturedinsectcells are in use for expressing modifications between insects and mammals,
cloned DNAs. Baculovirusesexclusively infect biologicallyactive proteins can be producedby
insect cells. The DNA of these virusesencode this approach.
And in fact, by usingbaculovirusas
for several products and their productivity in an expressionvector system, a good number of
cellsis very high to the extentof morethan 10,000 mammalian and viral oroteins have been
(Table11.1).
t im es c om oare d to ma mma l i a n c e l l s . Be si oes synthesized
carrying a large number of foreign genes, the
baculoviruses can effectivelyexpressand process Baculovirus expression vector system
the productsformed.Anotheradvantage with these The most commonly used baculovirus is
viruses is that they cannot infect humans, other Autographa californica multiple nuclear
vertebratesor plants.Thus, baculovirusesare safe polyhedrosis virus (AcMNPV). lt can grow on the
vectors. insectcell lines(e.g.,derivedfrom fall armyworm)
and produce high levels of polyhedrin or a
Polyhedrin gene of baculovirus recombinantorotein.
The organizationof a baculovirus(AcMNPV)
The polyhedrin gene is responsiblefor the
transfervectoris shown in Fig. 11.3A.lt consistssf
synthesisof a matrix protein-polyhedrin.This
protein is synthesizedin large quantities by an E. coli-basedplasmid vector along with the
D N A of bacul ovi rus.
Thi s i n turn has A cMN P V
baculovirusduring the infectioncycle. Polyhedrin
protects the virus from being inactivated by DNA, a polyhedrinpromoterregion,cloning site
for insertDNA and polyhedrinterminationregion.
environmental agents.The promoterfor polyhedrin
gene is very strong. However,the life-cycleof When the insectculturecells,transfectedwith
baculovirusdoes not depend on the presenceof AcMNPVare mixedwith transfervectorcarryinga
this gene. Polyhedringene can be replacedby a clonedgene,a doublecrossover occurs.The result
cloned gene, and the genetically engineered is that the cloned gene with polyhedrinpromoter
baculovirus can infectthe culturedinsectcells.The and termination sequencesgets integratedinto
142 B IOTE CHNO LO CY
( B)
Transfer
vector
AcMNPV
Recombinant
AcMNPV
Fig. ll,3 : Baculovirus expression vector system (A) Organization of baculovirus transfer vector (B) Replacement
of the polyhedrin gene of baculovirus with a cloned gene from a transfer vector (AcMNPll-Autographa californica
multiple nuclear polyhedrosis virus; Pp-Polyhydrin gene promoter; Cs-Cloning site; Pt-Polyhedrin gene
termination; Note : The coding region of polyhedrin gene not shown in A).
n
tl oI mRNA
spticed
Dihydrofolate
reductase
ITranslation
I
J
As the recombinantproteinsare producedby
the clonedgenes,they startaccumulating. The next
task is to collect and . purify the specific gene
Recombinantprotein product i.e., the requisiteprotein.This is not an
easy job since many a times the recombinant
Fig. 11.5: Coordinatedexpressionof DHFRand a proteinis foreignto the host cell which possesses
cloned gene (DHFB-Dihydrofolatereductase; an enzyme machinery to degrade the outside
p-Pronoter Sequenee:pa-Polyadenylation6equence). protei ns.Thus, human i nsul i n producedin t he
bacterial host cells can be degraded by the
proteases. This problem can be solved by using
Two.vector expression system bacterialstrains(e.9.,E. coli) deficientin proteases.
Two expression vectors,each carryinga cloned But there is a disadvantage sincethe proteases are
genefor each subunit,are co-transfected into host defensiveenzymes and hence the new strains
cells (Fig. 71.6). These genes encode for the lacking these enzymesare susceptiblefor easy
c o rre s p o n d i npgro te i ns u b u n i tsTheythenassembl e destruction.
to form a functional protein. The two-vector
ln an alternative approach, Ihe recombinant
expression systemhas certainlimitations - lossof
proteins are fused with the native host proteins
one vector/overexpression of clonedgenein one of
(Referfusion proteinsp. 138).The fusion proteins
th e v e c to rs .T h i s c a u s e sa n i mbal ancei n tne
are resistant to protease activity. Some times,
fo rm a ti o na n d a s s e m b l o y f s ubuni ts.
foreign proteinsaccumulateas aggregates in the
Two-gene expression vector host organi sm, mi ni mi zi ng the pr ot ease
degradation. The problemwith proteinaggregates
A single vector carryingtwo cloned genes is is that the biologicalactivity may be lost while
used to overcomethe above problem. The twcr extractingthe proteinfrom the aggregates.
IN H OS TC E LLS
OF C E N EEX PR E S S ION
11 : M A N IPU L AT ION
ChA P t CT 145
Clonedgenea
r t
jpa
Jpa RNA
I
+ J
E
Protein
,
subunit
oProtein
subunitp
EO
Assembledorotein
with2 subunits
Fig. 11.6: Two-vector expression system for producing a dimeric protein (p-Promoter sequence;
pa-P olyadenylation sequence)
Cloned Cloned
gene cl geneB
I
+
I
+
IJ - RNA
TO
Protein I I Protein Protein I I Protein
subunit subunit
B subunislt ------T---- l subunipt
----l--- +
flo
EN Assembledprotein
with two subunits
Assembledprotein
with two subunits
Fig. 11.8 : Dicistronic expression vector for producing
Fig, 11.7 : Two3ene expression vector for producing a dimeric protein (p-Promoter sequence;
a dimeric protein (p-Promoter sequence; pa-Polyadenylation sequence; IRES-lnternal
pa-Polyad enylation sequence). ribosomal entrv site).
Biotechnology [10]
146 B IOTECHNO LO CY
t ?_-. II
monoclonal antibodies (MAb) against the marker
peptide, are immobilized on a polypropylene
t o<---------------
I-
L_\
lnterleukin-2
MarkerPePtide
support. These MAb serve as the ligand (an[-
marker peptide antibodies) and selecfively bind to
fusion proteins tagged with marker peptide'
However, the remaining proteins pass through the
Fig. 11.iO : tmmunoaffinity chromatographic immunoaffinity column. The immunopurified
purification of a'fusion Protein fusion proteins can be eluted later from the
column.
<-
Humqn Proiecl
Oenome
144
C hapt er12 : HUM A N GE N OMEP R OJ EC T 149
Genesand related
genesequences
12 00M b
Fig. 12.2 : An overview of the organization of human genome (LlNEs-Long interspersed nuclear elements;
S/NEs-Shorf interspersed nuclear elements; LTR-long terminal repeats).
Up"tr""t t
tt
Start of End of biological
biologicalinformation information
The bioc hem is t r y and gen e t i c s o f m a n y s i n g l e - Many events leading to the mutations can be
gene disordershave been elucidated e g. sickle-cell uncovered with the knowledge of genome.
anemia, cystic fibrosis,retinoblastoma.A majority of of developmental
Better understanding
the common diseases in humans, however, are
biology
poly genic in nat ur e e. g. c a n c e r , h y p e r t e n s i o n ,
By determining the biology of human genome
diabetes At present,w'e have very little knowledge
and its regulatory control, it will be possible to
about the causesof these diseases.The informatron
understand how humans develop from a fertilized
on t he genom e s equenc e w i l l c e r t a i n l y h e l p
eggs to adults.
t o unr av el t he m y s t er ies s u r r o u n d i n g p o l y g e n i c
d iseases. Comparative genomics
C e n o m e s f r o m m a n y o r g a n i s m s h a ve b e e n
lmprovements in gene therapy s e o u e n c e d . a n d t h e n u m b e r w i l l i n c r e a se i n th e
At present,human gene therapy is in its infancy coming years.The information on the genomes of
{ or v ar ious r eas ons .Cenom e s e q u e n c e k n o w l e d g e d i f f e r e n t s p e c i e s w i l l t h r o w l i g h t o n th e m a j o r
will certainly help for more effective treatment of s t a g e si n e v o l u t i o n .
MEDICAL/
PHARMAGEUTICAL
BIOTECHNOLOGY
IBToTEGHNOLOGY
rN HEALTHCABEI
e (e, ?"{
o.,- o
;oo
) G)
155
d va nces i n bioc hem is t r y and nr olec u l a r b o n e m a r r o w c e l l s , b l o o d c e l l s ,s k i n c e l l s , i n t e s t i n a l
I
/ \ bio log y ha v e helped t o under s t andt he gen e t r c c e l l s A t p r e s e n t ,a l l t h e r e s e a r c ho n g e n e t h e r a p y i s
ba sis of in he rit ed dis eas es .lt was a dr eanr of t h e directed to correct the senetic.defects in somatic
researchersto replace the defective genes with c e l l s . I n e s s e n c e ,s o m a t i c c e l l g , e n et h e r a p y i n v o l v e s
good ones, and cure the genetic disorders. the insertion of a fully functional and expressible
gene into a target somatic cell to correct a genetic
Gene therapy is the process of inserting genes
d i s e a s ep e r m a n e n t l y .
into cells to treat diseases.The newly introduced
ge ne s will en c ode pr ot eins and c or r ec t t h e 2. Germ cefl gene therapy ; fhe reproductive
d eficien cie s tha t oc c ur in genet ic dis eas es .Th u s , (sex) cells of an organism constitutegerm cell line
gene therapy primarily involves genetic Cene therapy involving the introduction of DNA
manipulations in animals or humans to correct a into germ cells is passed on to the successrve
d ise ase,an d keep t he or ganis m in good healt h.T h e generations. For safety, ethical and technical
initia l expe riment son gene t her apv ar e c ar r ied o u t reasons, germ cell gene therapy is not being
in a nima ls, a nd t hen in hum ans O bv ious ly , t h e attempted at present
g oa l of the re s ear c her sis t o benef it t he m ank i n d
T h e g e n e t i c a l t e r a t i o n si n s o m a t i c c e l l s a r e n o t
a nd imo rove their healt n.
carried to the next generations.Therefore, somatic
An overview of gene therapy strategies rs cell gene therapy is preferred and extensively
depicted in Fig. I 3.1 . ln gene augmentation studied with an ultimate objective of correcting
therapy, a DNA is inserted into the genome to human diseases.
replace the missing gene product In case of gene
Development of gene therapy in humans for any
inhibition therapy, the antisensegene inhibits the
specific disease involves the following steps. In
e xp ressio nof th e dom inant gene.
fact, this is a general format for introducing any
therapeuticagent for human use.
APPROACHES FO R G ENE THERAPY
1 . ln vitro experiments and research on
There are two approaches to achieve gene
l a b o r a t o r y a n i m a l s ( p r e - c l i n i c a lt r i a l s )
therapy.
.l 2 . P h a s eI t r i a l s w i t h a s m a l l n u n r b e r ( 5 - 10 ) o f
. Somatic cell gene therapy : The non-
human subjects to test safety of the product.
reproductive (non-sex) cells of an organism are
referred tcl as somatic cells. These are the cells of 3 . P h a s e l l t r i a l s w i t h n r o r e h u m a n s u b j e c t st o
an org an ism o ther t han s per m or eggs c ells , e . 9 . , assesswhether the product is helpful.
157
158 B IOTECHNO LO CY
(A)
(B)
4.
Dominant
functionalgene Inhibitoryaction
Breastcancer Multidrug
resistance
I
A I DS rev andenv
Colorectal melanoma,
cancer, renalcancer Histocompatability (HLA-87)
locusantigen-B,
Duchennemusculardystrophy Dystrophin
Shortstature* Growthhormone
Diabetes* Glucose (GLUT-2),
transporter-2, giucokinase
uria*
Phenylketon Phenylalanine
hydroxylase
Citrullinemia* Arginosuccinate
synthetase
* Mostlyconfined
to animalexperiments
efficient only, if the therapeutic gene (remedial material in retroviruses.As the retrovirusenters the
ge ne ) is sta bly inc or por at ed and c ont inuou s l y host cell, it synthesizesDNA from RNA (by reverse
expressed.This can be achieved by use of vectors. transcription) The so formed viral DNA (referredto
as provirus) gets incorporated into the DNA of tne
VECTORS IN GENE THERAPY host cell. The proviruses are normally harmless.
However, there is a tremendous risk, since some of
rhe carrier particles or molecules used to deliver
the retroviruses can convert normal cells inrr-r
genes to somatic cells are referred to as vectors. The
cancerous ones. Therefore, it is absolutely essential
important vectorsemployed in ex vivo gene therapy
to ensure that such a thing does not happen.
are listed below and briefly described next.
o Viruses
Making retroviruses harmless
o Hu man artificial c hr om os om e
Researchers employ certain biochemical
r Bo ne marro w c ells .
methods to convert harmful retrovirusesto harmless
ones, before using them as vectors. For instance,by
VIBUSES artificially removing a gene that encodes for the
The vectors frequently used in gene therapy are viral envelope, the retrovirus can be crippled and
viruses,particularlyretroviruses.RNA is the genetic made harmless. This is because, without tne
160 B IOTECHNO LO CY
&8%
ooo_
in vitro cullure
o 6)
\_-/-
o"
ooo
\:/\J
Geneticallvtransformed
-selected
cells
envelooe, retroviruscannot enter the host cell. The structural protein, a gene pol that codes for reverse
pr oduc t ion of a lar ge num b e r ( b i l l i o n s ) o f v i r a l transcriptase, a gene env coding for envelope
particles can be achieved, starting from a single protein and a 3-LTR sequence.
envelope defective retrovirus (Fig. 13.3). This ls
For use of a retrovirusas a vector, the structural
m ade pos s ible by us ing h e l p e r v i r u s e s w h i c h
genesgag and pol are deleted (besides env gene, as
contain normal gene for envelope formation. Along
describedabove).Thesegenes are actually adjacent
with the helper virus, the vector (with defectrve
to Y region. In addition, a promoter gene is also
envelope gene) can enter the host cell and both of
included (Fig. 13.a8). This vector design allows the
t hem m ult iply . By r epeat ed m u l t i p l i c a t i o n i n h o s t
synthesis of cloned genes. A retroviral vector can
c ells , billions of v ec t or an d h e l p e r v i r u s e s a r e
carry a therapeutic DNA of maximum size of
produced. The vector virusescan be separatedfrom
B kb.
t he helper v ir us es and pur if i e d . l s o l a t i o n o f v e c t o r
v ir us es , t ot ally f r ee f r om h e l p e r v i r u s e s , i s A retroviral vector DNA can be used to
abs olut ely es s ent ial. Cont a m i n a t i o n o f h e l p e r transform the cells. However, the efficiency of
viruses is a big threat to the health of the patients delivery and integration of therapeutic DNA are
undergoing gene therapy. very low. In recent years, techniques have been
developed to deliver the vector RNA to host cells at
Retroviruses in gene therapy a high frequency. For this purposes, packageo
r e t r o v i r a l R N A p a r t i c l e s a r e u s e d . T h is te ch n i q u e
The genetic map of a typical retrovirus is
allows a high efficiency of integration of
depicted in Fig. 13.4A. In general, the retrovirus
pharmaceutical DNA into host genome
particle has RNA as a genome organized into six
regions. lt has a 5'-long terminal repeat (5'-LTR),a Several modified viral vectors have been
non- c oding s equenc e r equir e d f o r p a c k a g i n g R N A developed in recent years for gene therapy. These
designated as psi (Y), a gene gag coding for include oncoretrovirus. adenovirus, adeno-
Hostcell
$
Vectorvirus
(withdefective
envelopegene)
@(r@
;9
@@r
@ Veciorviruses
Host cell
@@@ Helperviruses
Fig. 13.3 : Large scale production of vector viruses by using helper viruses.
Fig. 13.4 : A retrovitus used in gene therapy. (A) General map of a typical retrcvirus (B) Gene map of a
modified retrovirus for use in gene therapy (LTR-Long terminal tepeat; Y-Packaging signal sequence; gag-
Coding sequence for structural protein; pol-Coding sequence for reverse transcriptase; env-Envelope protein
coding sequence; y-Therapeutic gene; p-Pramoter gene).
Biotechnology [11]
162 B IOTECHNO LO CY
VectorDNA
Human
IADA+gene
Retrovirus
containing
ADA'
gene
Transfection
/'tr"-\
-
t-:'/
LCt)
@@
Lymphocytes
with
viralDNAandADA+gene
Childwith SCID
(ADA- gene)
J
Synthesisof
ADA
II
+ expressioninto Cell culturesto
Correction
of patient verifyexpression
SCID of ADA+transgene
Fig. 13.5: Treatment of adenosine deaminase (ADA) deficient patient by somatic ex vivo gene therapy
(SCID-Severe combined immunodeficiency disease).
antibiotic resistance,and then cloned. The Consequently, the ability of the patientto produce
antibioticresistancegene will help to selectthe antibodies is increased.However, there is a
desiredcloneswith ADA gene. limitation.The lymphocyteshave a short life span
(iustlive for a few months),hencethe transfusions
A diagrammaticrepresentation of the treatment
haveto be carriedout freouentlv.
of ADP deficientpatientis depictedin Fig. 13.5.
THERAPY FOR FAMILIAL factor lX were inserted into the liver cells of dogs.
HYPERCI{OLESTEROLEMIA These dogs no longer displayed the symptoms of
hemophilia.
The pat ient s of f am ilial h y p e r c h o l e s t e r o l e m i a
Iack the low density lipoprotein (LDL) receptors
on t heir liv er c ells . As a r es u l t , L D L c h o l e s t e r o l i s EX V'VO GENE THERAPY WITH
not m et abolis ed in liv er . T h e a c c u m u l a t e d L D L - NON.AUTOLOGOUS CELLS
c holes t er olbuilds up in t he c i r c u l a t i o n , l e a d i n g t o The ex vivo gene therapiesdescribed above are
arterial blockage and heart diseases.Attempts are basedon the iransplantationof geneticallymodified
being m ade by gene t her ap i s t st o h e l p t h e v i c t i m s cells for the production of desired proteins.
of f am ilial hy per c holes t er o l e m i a I. n f a c t , t h e r e i s However, there are several limitations in using the
s om e s uc c es sals o. I n a wo m a n , 1 5 % o f t h e l i v e r patient's own cells (autologous cells) for gene
was removed. The hepatocytes were transduced t h e r a p y . T h e s e i n c l u d e l a c k o f e n o u gh ce l l s fr o m
with retrovirusescarrying genes for LDL receptors. target tissues,defective uptake of genes and therr
These genetically modified hepatocytes were inadequate expression. To overcome these
infused into the patient's liver. The hepatocytes problems, attempts are on to develop methods to
es t ablis hed t hem s elv es i n the liver and use non-autologous cells (i.e., cells from other
pr oduc ed f unc t ional LDL- r e c e p t o r s .A s i g n i f i c a n t i n d i v i d u a l s o r a n i m a l s ) . T h e o u t li n e o f th e
im pr ov em ent in t he pat ient 'sc o n d i t i o n , a s a s s e s s e d procedure is briefly described below.
by es t im at ing t he lipid par a m e t e r si n b l o o d , w a s
observed Further, there were no antibodres T i s s u e - s p e c i f i cc e l l s c a p a b l e o f g r o w i n g i n
pr oduc ed agains t t he LD L - r e c e p t o r m o l e c u l e s , culture are selected.These include fibroblastsfrom
c lear ly s howing t hat t he gen e t i c a l l y m o d i f i e d l i v e r skin, hepatocytesfrom liver, myoblastsfrom muscle
cells were acceoted. and astrocytesfrom brain. These cells are cultured
a n d g e n e t i c a l l ym o d i f i e d w i t h t h e t h e r ap e u ti cg e n e .
THERAPY FOR LESCH.NYHAN They are then encapsulatedin artificial membrane
SYNDROME composed of a synthetic polymer (e.g., polyether
s u l f o n e , a l g i n a s e - p o l y L - l y s i n e - a lg i n a te ) . Th e
Les c h- Ny han s y ndr om e i s a n i n b o r n e r r o r I n p o l y m e r i c m e m b r a n e s a r e n o n - i mm u n o g e n i c,
purine metabolism due to a defect in a gene that
therefore the patient can accept non-autologous
encodes for the enzyme hypoxanthine-g,uanine
e n c a p s u l a t e dc e l l s . F u r t h e r , b e i n g s e m i p e r m e a b l e
phosphoribosyl transferase (HGPRT). In the
in nature, these membranes allow the nutrients to
absenceof HCPRT, purine metabolism is disturbeo
enter in, and the encoded protein (by the
and ur ic ac id lev el builds u p , r e s u l t i n g i n s e v e r e
therapeutic gene) to pass out.
gout and k idney dam age T h e v i c t i m s o f L e s c h -
Ny han s y ndr om e ex hibit s y m p t o m s o f m e n t a l
Experimentsconducted in animals have shown
retardation,besidesan urge to bite lips and fingers,
some encouraging resultsfor using non-autologous
c aus ing s elf - m ut lat
i ion.
c e l l s i n g e n e t h e r a p y . T h e e n c a p s u l a te dce l l s w e r e
By using retroviral vector system, HCPRT found to proliferate and produce the required
producing genes were successfully inserted into protein. However, the successhas been very limited
c ult ur ed hum an bone m ar r o w c e l l s . T h e m a i o r in human trials.
pr oblem in hum ans is t he i n v o l v e m e n t o f b r a i n .
Ex per im ent sc onduc t ed in an i m a i s a r e e n c o u r a g i n g .
However, it is doubtful whether good successcan
be achieved by gene therapy for Lesch-Nyhan
s v ndr om e in hum ans , in t he n e a r f u t u r e .
fhe direct delivery of the therapeutic gene
(DNA) info the target cells of a particulartissue
THERAPY FOR HEMOPHILIA
of a patient constitutesin vivo gene' therapy
Hemophilia is a genetic disease due lack of a (Fig. 13.6). Many tissues are the potential
gene that encodes for clotting factor IX. lt is for thi s approach.Thesei ncludeliver ,
candi dates
c har ac t er iz ed by ex c es s iv e b l e e d i n g . B y u s i n g a muscl e,ski n,spl een,l ung, brai n and blood cells.
retroviral vector system, genes for the synthesisof Cene deliverycan be carriedout by viral or non-
:apt er 1l : G E NET H ER AP Y 165
Lipoplexes
-Therapeutic
fhe Iipid-DNA complexesare referred to as
or morecommonlyliposomes.
lipoplexes Theyhave Poly L-
t Cell
gene
DNA.molecular coniugates
conJugate
The useof DNA-molecular conjugatesavoidsthe
lysosomalbreakdownof DNA. Anotheradvantage
of using conjugatesis that large-sizedtherapeutic
DNAs (> 10 kb) can be deliveredto the target
tissues. The most commonly used synthetic
conjugateis poly-L-lysine,boundto a specifictarget
The therapeutic
cell receptor. DNA is then madeto
combinewith the conjugateto form a complex(Fig.
13.4. fhis DNA molecular conjugatebinds to
specific cell receptoron the target cells. lt is
engulfedby the cell membraneto form an endosome
which protectsthe DNA from beingdegraded. The
DNA released fromthe endosome entersthe nucleus
wherethe therapeutic gene is expressed.
Nucleotide
Falsenucleotide II
J
Cancer
cells die
Fig. 13.8 : The action of ganciclovir mediated by thymidlne kinase to inhibit the growth of cancer cells.
(surgery, chemotherapy, radiation therapy). Cene ganciclovir (GCV) bears a close structural
therapy is the latestand a new approach for cancer r e s e m b l a n c et o c e r t a i n n u c l e o s i d e s( t hym i d i n e ) .By
treatment. Some of the developments are briefly mistake, TK phosphorylates ganciclovir to form
described hereunder. triphosphate-CCV a false and unsuitablenucleotide
{or DNA synthesis. Triphosphate-GCV inhibits
Tumor necrosis factor gene therapy DNA polymerase (Fig. 13.0. fhe result is that the
e l o n g a t i o n o f t h e D N A m o l e c u l e a b r up tl y sto p s a t
Tumor necrosis factor (TNF) is a protern
a p o i n t c o n t a i n i n g t h e f a l s e n ucl e o ti d e ( o f
produced by human macrophages. TNF provides
ganciclovir). Further,the triphospate-CCVcan enter
defense againstcancer cells. This is brought out by
a n d k i l l t h e n e i g h b o u r i n g c a n ce r ce i l s, a
enhancing the cancer-fighting ability oI tumor-
phenomenon referred to as bystander effect. fhe
infiltrating lymphocytes (TILs), a special type of
u l t i m a t e r e s u l t i s t h a t t h e c a n c e r ce l l s ca n n o r
im m une c ells .
m u l t i p l y , a n d t h e r e f o r e d i e . T h u s, th e d r u g
The tumor-infiltrating lymphocytes were g a n c i c l o v i r c a n b e u s e d t o k i l l t h e c a n ce r ce l l s.
transformed with a TNF gene (along with a
Canciclovir is frequently referredro as a prodrug
neomycin resistantgene) and used for the treatment
and this type of approach is called prodrug
of malignant melanoma (a cancer of melanin
activation gene therapy. Ganciclovir has been used
pr oduc ing c ells , us ually oc c u r s i n s k i n ) . T N F a s
for treatment of brain tumors (e.9., glioblastoma, a
s uc h is highly t ox ic , and f o r t u n a t e l y n o t o x i c s i d e
c a n c e r o f g l i a l c e l l s i n b r a i n ) , a l t h o u g h w i th a
effects were detected in the melanoma patients
limited success.
injec t ed wit h genet ic ally a l t e r e d T l L s w i t h T N F
gene. Some improvement in the cancer patients ln the suicide gene therapy, the vector used is
was observed. herpes simplex virus (HSV) with a gene for
t h y m i d i n e k i n a s e ( T K ) i n s e r t e d i n i ts g e n o m e .
Suicide gene therapy N o r m a l b r a i n c e l l s d o n o t d i v i d e w h i l e th e b r a r n
t u m o r c e l l s g o o n d i v i d i n g u n c h e c k e d . Th u s, th e r e
The gene encoding the enzyme thymidine
i s a c o n t i n u o u s D N A r e p l i c a t i o n i n t u m o r ce l l s. By
kinase is often referred to as suicide gene, and is
using CCV-HSVTK suicide gene therapy, some
used for the treatment of certain cancers.
reduction in proliferatinBtumor cells was reported.
Thym id ine kinase (f K) phosphorylates Several new strategies are being developed to
nuc leos idest o f or m nuc leot i d e sw h i c h a r e u s e d f o r i n c r e a s et h e d e l i v e r y o f H S V T K g e n e t o a l l th e ce l l s
t he s y nt hes isof DNA dur ing c e l l d i v i s i o n . T h e d r u g t h r o u e h o u t a t u m o r .
Chapt er13 : C E N ET H ER AP Y 169
Two-gene cancer therapy Researchershave used HIV lacking rev and env
genesfor therapeuticDUrposes. T-Lymphocytesfrom
For treatment of certain cancers/ two Sene
HIV-infected patients are removed, and mutant
systemsare put together and used. For instance,IK
viruses are inserted into them. The modified
suicide gene (i.e., CCV-HSVTK) is clubed with
T-lymphocytesare cultivated and injected into the
interleukin-2 gene (i.e. a gene promoting
patients.Due to lack of essentialgenes,the viruses
immu no the rap y ) .I nt er leuk in- 2pr oduc ed m obili z e s
(HlV) cannot multiply, but they can stimulatethe
immu ne re sp ons e. lt is believ ed t hat c er t a i n
production of CDu (cluster determinant antigen B)
proteins are releasedfrom the tumor cells on their
c e l l s o f T - l y m p h o c y t e s .C D u c e l l s a r e t h e k i l l e r
death. These proteins, in associationwith immune
lymphocytes. It is proved in the laboratory studies
ce lls, re ach the t um or and init iat e im m unolog i c a l
that these lymphocytes destroy the HIV-infected
reactions directed against the cancer cells.
c e lIs .
Two-gene therapies have been carried out in
expe rimen tal a nim als wit h c olon c anc er and li v e r Genes of HIV proteins
ca ncer, a nd th e r es ult sar e enc our aging.
Some genes synthesizing HIV proteins are
attached to DNA of mouse viruses. These
Gene replacement therapy
g e n e t i c a l l y - m o d i f i e dv i r u s e s a r e i n j e c t e d t o A I D S
A gene named t'3 codes for a protein with a p a t i e n t sw i t h c l i n i c a l m a n i f e s t a t i o n so f t h e d i s e a s e .
molecular weight of 53 kilodaltons (hence ps3).ps3 I t i s b e l i e v e d t h a t t h e H I V g e n e s s t i m u l a t e n o r m a l
is considered to be a tumor-suppressor gene, since b o d y c e l l s t o p r o d u c e H I V p r o t e i n s . T h e l a t t e r i n
the pro tein it en c odes binds wit h DNA and inhi b i t s t u r n s t i m u l a t et h e o r o d u c t i o n o f a n t i - H l V a n t i b o d i e s
re plicatio n. Th e t um or c ells of s ev er al t is s u e s w h i c h p r e v e n tt h e H I V r e p l i c a t i o n i n A I D S p a t i e n t s .
(breast, brain, lung, skin, bladder, colon, bone)
were found to have altered genes of p53 (mutated Gene to inactivate gpl 2O
psr, synthesizing different proteins from the
o rigin al. Th ese alt er ed pr ot einsc annot inhibit D N A gpl 20 is a glycoprotein (molecular weight 120
replication. lt is believed that the damaged ps3 kilodaltons) present in the envelope of HlV. lt is
gene may be a causative factor in tumor absolutely essentialfor binding of virus to the host
develooment. c e l l a n d t o b r i n g r e p l i c a t i o n . R e s e a r c h e r sh a v e
synthesized a gene (called F105) to produce an
Some workers have tried to replacethe damaged antibody that can inactivate gp12o. In the anti-
ps3 gene by a normal gene by employing adenovirus AIDS therapy, HlV-infected cells are engineered to
vector systems (See p. 165). There are some produce anti-lllV antibodies when injected into
encouragingresultsin the patientswith liver cancer. t h e o r g a n i s m .
The antisense therapy for cancer is discussed Studies conducted in experimental animals
e lse whe re (See p. 170) . showed a drastic reduction in the synthesis of
gp120 due to anti-A|DS therapy. The production of
GENE THERAPY FOR AIDS
HIV particles was also very reduced. There are
AIDS is a global dis eas e wit h an alar m i n g some attemptsto prevent AIDS by antisensetherapy
increasein the incidence every year. lt is invariably (Seep. 171).
fatal, since there is no cure. Attempts are
being made to relieve the effects of AIDS by gene
therapy. Some of the approaches are discussed
hereunder.
ANT I S E NS E T H ER AP Y F O R AID S
{ttempts are also being made to preventHIV
I Targetcell
rrection through antisensetherapy. The basrc ,geneticallyengineered)
crincipleis brieflydescribed(Fig. 13.10)., |
\r_
The targetcellsfor HIV infectionare genetically
engineeredto contain a gene that can expressa
Jlnfection
complementarycopy of HIV genome.This gene -=--=--------Vi ral RNA
producesantisense RNA.Whenthe cellscontaining
antisenseRNA are infectedby HIV it bindsto viral
RNA forminga double-stranded RNA-RNAhybrid Double-stranded
m olec ulesT. his d o u b l e -s tra n d emo
d l e c u l ec a n n ot RNA-RNAhybrid
be used by the enzyme reversetranscriptase.
AntisenseRNA
Consequently, a DNA copy of the HIV genome
cannotbe madeand incorporated into the genome. Geneticallymodified
targetcell infectedwith HIV
A NT I S E NS E OL IGON U C L EOT ID F S A S
THERAPEUTIC AGENTS
Fig. 13.10 : Antisense therapy to prevent AIDS
It is found that oligodeoxynucleotides (with (HI V-Human immunodeficiency virus)
a b out 15- 20 uni ts ) c a n h y b ri d i z ew i th s p e c i f i c
mRNAand blocktranslation with an ultimateeffect
of reducingthe specificprotein.Oligonucleotides Possible inhibition of smooth muscle cell
can hybridize with different types of RNA proliferation: Proliferation of smoothmusclecells
transcripts-mRNAs, intron-exons,double-stranded (along with secretion of extracellular matrix)
RNAs. The major limitation of using naturally has been implicated in hypertension,failure of
occurringoligonucleotides is thatthey aredegraded coronary bypass grafts, hypertension and
b y int r ac ellular
nu c l e a s e s . atherosclerosis.There exists a possibility of
Certainmodificationsin the nucleotides(bases controlling smooth muscle cell proliferationby
or sugars, phosphate)without affecting their usingantisense therapy.
hybridizationabilityhavebeen prepared. However,
these modified oligonucleotidesare resistantto C H IME R IC OLIGON U C LE OTID E S IN
degradationby nucleases.The most r,r,idelyused GE N E C OR R E C TION
antisenseoligonucleotidehas a sulfur group In Many geneticdiseasesare due to single base
place of the free oxygen in phosphodiester bond. pair mutations.A strategyhas been devisedto
The modified bond is referred to as correct such defects by using a chimeric
phosphorothioate linkage. These phosphorothioate oligonucleotides composed of RNA-DNA
antisense oligonucleotides are resistant to ol i gonucl eoti dew i th 6B uni ts. Thi s chi meri c
degradationby nucleases,besidesbeing water ol i gonucl eoti de has hai rpi ncaps and methyl ated
so luble. S om e o th e r mo d i fi c a ti o n so f o l i g o- ribose sugars(at 2nd carbon).The hairpin caps
nucleotidesare with phosphoramidite Iinkageand protect the molecule from the degradationby
2-O-methylribose(Fig. 13.11). The modified exonucleaseswhile methylated ribose prevents
antisenseoligonucleotidesare in fact used as digestionby RNase.
therapeuticagents in the clinical trials for the The correctionof a singlebasepair mutationby
treatmentof certaincancers,bowel disease,AIDS, usi ng a chi meri c ol i gonucl eoti de i s depi ctedi n
m alar iaand v ir al i n fe c ti o n s . Fi g. 13.12.The R N A -D N A mol ecul es(chi meri c
Thefreeoligonucleotides do not readilyenterthe oligonucleotides)
bind more readily w'ith duplex
cells.Therefore, they are encapsulated in liposomes DNA and bringaboutthe correctionin the mutated
for efficientdeliveryto inhibitthe mRNAtranslation. basepair.
172 B IOTECHNO LO CY
Base Base T
I I A
Sugar Sugar
I I TargetDNA with single
o o base pair mutation
I I .....",.TT
O:P-S TT_,_._.._.._..-G-
I I TT T-f
o o
I I Chimericolioonucleotide
Sugar' Sugar
i I Fig. 13.12: Chimericoligonucleotide in gene
Base Base
correction(The dotted lines representribonueleotides
Phosphodiester Phosphorothioate
linkage linkage while the continuouslines correspondto
deaxyribanucJeotidea; the calouredbase:pair is the
Base conecf one tb r€plaaptli?, nutated Wset pair) '
I
Sugar
I THE FUTURE O F G E N E T H E R A PY
N
I
O:P-O Theoretically, gene therapy is the permanent
solution for genetic diseases.But it is not as simple
o a s i t a p p e a r ss i n c e g e n e t h e r a p y h a s s e ve r a l i n b u i l t
I c o m p l e x i c i t i e s . C e n e t h e r a p y b r o a d l y i n vo l ve s
Sugar
I i s o l a t i o n o f a s p e c i f i c g e n e , m a k i n g i ts co p i e s,
Base inserting them into target tissue cells to make ihe
Phosphoramidite desired protein. The story does not end here. lt is
linkage
absolutely essential to ensure that the gene is
h a r m l e s s t c t h e p a t i e n t a n d i t i s a p p r o p r i a te l y
Fig. 13,11 : Certain modifications in antisense expressed(too much or too little will be no good).
oligonucleotides. Another concern in gene therapy is the body's
immune systemwhich reactsto the foreign proteins
produced by the new genes.
APTAMERS AS THERAPEUTIC AGENTS
A special type of oligonucleotides that can The public, in general, have an exaggerated
specifically bind to target proteins and not to expectations on gene therapy. The researchers, at
nucleic acids are referredto as aptamers.Aptamers least for the present, are unahle to satisfy them. As
are useful as therapeutic agents. For instance, an per the records, by 1999 about 10C0 Americans
apt am er t hat c an bind t o t hr o m b i n i n h i b i t s b l o o o h a d u n d e r g o n ec l i n i c a l t r a i l s i n v o l v i n g va r i o u s g e n e
c lot t ing. Suc h oligonuc leot i d e s c a n b e u s e d i n therapies. Unfortunately, the gene therapists are
s ur gic al pr oc edur es . u n a b l e t o c a t e g o r i c a l l yc l a i m t h a t g e n e th e r a p y h a s
permanently cured any one of these patients!Some
RIBOZYMES AS THERAPEUTIC AGENTS p e o p l e i n t h e m e d i a ( l e a d i n g n e w s pa p e r s a n d
magazines) have openly questioned whether it rs
RNA molecules that can serve as enzymes worth to continue researchon gene therapy!!
(biocatalysts)are referred to as ribozymes. The
nat ur allyoc c ur r ing r iboz y m e sa r e t o b e m o d i f i e d s o It may be true that as of now, gene therapy due
t hat t hey c an s pec if ic ally h y b r i d i z e w i t h m R N A to several limitations, has not progressedthe way it
s equenc es and bloc k protein biosynthesis should, despite intensive research. But a hreak-
( t r ans lat ion) . Ther e is a lim i t a t i o n h o w e v e r , i n through may come anytime, and of course, this rs
dir ec t ly ins er t ing r iboz y m es i n t o t a r g e t c e l l s , a s o n l y p o s s i b l e w i t h p e r s i s t e n tr e s e a r c h .An d a d a y
the,riare easily degraded This can be overcome by may come (it might take some years) when almost
binding r iboz y m e wit h an o l i g o d e o x y n u c l e o t i d e . every disease will have a gene therapy, as one of
Ther e is a pos s ibilit yof t r eat in gc e r t a i n c a n c e r sa n d the treatment modalities. And gene therapy will
v ir al dis eas esby genet ic allye n g i n e e r e dr i b o z y m e s . r e v o l u t i o n i z et h e p r a c t i c e o f m e d i c i n e !
ia gn osis of dis eas es due t o pat ho g e n s a set of genes of the organism. Likewise an
(ba cte ria, v ir us es , f ungi et c . ) or due t o inherited genetic defect can be diagnosed by
inherent genetic defects is necessaryfor appropriate identifying the alterations in the gene. In the
treatment Traditional diagnostic methods for m o d e r n l a b o r a t o r y d i a g n o s t i c s ,D N A a n a l y s i s i s a
o ara site inf ec t ions inc lude m ic r osc o p r c very useful and sensitivetool
examination, in vitro culture, and detection of The basic principles underlying the DNA
a ntib od ies in s er um . And f or t he genet ic dis e a s e s , diagnosticsystems,and their use in the diagnosisof
the orocedures such as estimation of metabolites
certalnpathogenicand geneticdiseasesare described.
(b loo d/u rine ) and enz y m e as s ay s ar e em plo y e d
Besides these, the various approaches for DNA
The se lab ora t or y t ec hniques ar e indir ec t and n o t f i n g e r p r i n t i n g( o r D N A p r o f i l i n g )a r e a l s o d i s c u s s e d .
a lways spe cific .Sc ient is t sar e in c ont inuous s e a r c h
fo r spe cific, s ens it iv e and s im ple diagn o s t i c
technioues for identification of diseases.
173
174 BIOTECHNOLOCY
{- DNA probe
Substrate
Light-emitting
product
Adv ant ages : The biot in- l a b e l e d D N A i s q u r t e Target DNA fragments with complementary
stable at room temperaturefor about one year. The sequences bind to DNA probes. The remaining
det ec t ion dev is es us ing c he m i l u m i n e s c e n c e a r e DNA fragmentsare washed away. The target DNA
preferred,since they are as sensitiveas radioisotope p i e c e s c a n b e i d e n t i f i e d b y t h e i r f l u o r e sce n ce
det ec t ion, and m or e s ens i t i v e t h a n t h e u s e o f e m i s s i o n b y p a s s i n g a l a s e r b e a m . A co m p u te r i s
chromogenic detection systems. used to record the pattern of fluoresenceemission
and DNA identification.
PCR in the use of DNA probes
T h e t e c h n i q u e o f e m p l o y i n g D N A ch i p s i s ve r y
DNA pr obes c an be s uc c e s s f u l l yu s e d f o r t h e
r a p i d , b e s i d e s b e i n g s e n s i t i v ea n d s p e ci fi c fo r th e
ident if ic at ion of t ar get D N A s f r o m v a r i o u s several DNA fr a g m e n ts
identification of
s am ples- blood, ur ine, f e c e s , t i s s u e s , t h r o a t
s i m u l t a n e o u s l y . S c i e n t i s t s a r e t r y i n g to d e ve l o p
was hingswit hout m uc h pur ifi c a t i o n .T h e d e t e c t i o n
C e n e c h i p s f o r t h e e n t i r e g e n o m e o f a n o r g a n i sm .
of target sequence becomes quite difficult if the
quant it y of DNA is v er y lor v . I n s u c h a c a s e , t h e
Applications of DNA chip
poly m er as ec hain r eac t ion ( P C R ) i s f i r s t e m p l o y e d
to amplify the minute quantities of target DNA T h e p r e s e n c em u t a t i o n s i n a D N A s e q u e n ceca n
( Chapt er E) , and ident if ied b y a D N A p r o b e . b e c o n v e n i e n t l y i d e n t i f i e d . I n f a c t , G e n ech i p p r o b e
array has been successfullyused for the detection
DNA probes and signal amplification o f m u t a t i o n s i n t h e p 5 3 a n d B R C A I ge n e s. Bo th
Signal am plif ic at ion is an a l t e r a t i v et o P C R f o r t h e s e g e n e s a r e i n v o l v e d i n c a n c e r ( d e ta i l s g i ve n
t he ident if ic at ion of m inut e q u a n t i t i e s o f D N A b y later).
us ing DNA pr obes . I n c as e o f P C R , t a r g e t D N A i s
am plif ied, while in s ignal a m p l i f i c a t i o n i t i s t h e
target DNA bound to DNA probe that is amplified
There are two general methods to achieve signal
am olif ic at ion
1 . Separate the DNA target-DNA probe
T h e u s e o f D N A a n a l y s i s ( b y e m p l o yi n g D N A
c om plex f r om t he r es t of t he D N A m o l e c u l e s , a n d probes) is a novel and revolutionary approach for
t hen am plif y it . specifically identifying the d i s ea se - ca u si n g
2. Amplify the DNA probe (bound to target pathogenic organisms This is in contrast to the
DNA) by us ing a s ec on d p r o b e . T h e R N A t r a d i t i o n a l m e t h o d s o f d i s e a s e d i a g n o si s b y
complementary to the DNA probe can serve as the detection of enzymes, antibodies etc., besides the
s ec ond pr obe. The RNA- DN A - D N A c o m p l e x c a n m i c r o s c o p i ce x a m i n a t i o no f p a t h o g e n s .Al th o u g h a t
be separated and amplified. The enzyme O-beta p r e s e n t n o t i n w i d e s p r e a d u s e , D N A a n a l ysi s m a y
r eplic as e whic h c at aly s es R N A r e p l i c a t i o n i s soon take over the traditional diagnostictests in the
c om m only us ed. years to come. Diagnosis of selected diseases by
genetically engineered techniques or DNA probes
THE DNA CHIP.MICROARRAY OF or direct DNA analysis is briefly described.
G ENE PRO BES
The DNA chip or Genechip contains thousands T U B E R C U L O S I S
of DNA probes (4000,000 or even more) arranged
T u b e r c u l o s i s i s c a u s e d b y t h e b a cte r i u m
on a s m all glas s s lide of t h e s i z e o f a p o s t a g e
Mycobacterium tuberculosis. The commonly used
s t am p. By t his r ec ent and a d v a n c e d a p p r o a c h ,
diagnostic tests for this disease are very slow and
thousandsof target DNA molecules can be scanned
sometimesmay take severalweeks. This is because
s im ult aneous ly .
M. tuberculoslsmultiplies very slowly (takes about
Technique for use of DNA chip 2 4 h r s . t o d o u b l e ; E . c o l l t a k e s j u s t 2 0 m i n u te s to
double).
The unk nown DNA m o l e c u l e s a r e c u t i n t o
fragmentsby restrictionendonucleases.Fluorescent A novel diagnostic test for tuberculosis was
markersare attachedto these DNA fragments.They d e v e l o p e d b y g e n e t i c e n g i n e e r i n g ,a n d is i l l u str a te d
are allowed to react to the probes of the DNA chip. in Fig. 14.3. A gene from firefly, encoding the
14 : DNA IN DI SEASEDI ACNO SI S AND M E D I C A L F O R E N S I C S
ChA OICT 177
CHAGAS' DISEASE
-4otechnology [12]
178 B IOTE CHNO LO CY
HIV existsas a segment of DNA integratedinto the by certain bacteria.At leastthree distinct speciesof
T-lyrnphocytesof tlre host. The T-lymphocytesof a bacteria have been identified and DNA pr,obes
suspectedAIDS patient are isolated and disrupted developed for their detection. Early diagnosis of
to releaseDNA. The so obtained DNA is amplifieo p e r i o d o n t a l d i s e a s e w i l l h e l p t h e tr e a tm e n t
by PCR, and to this DNA probes are added. lf the modalities to prevent the tooth decay.
HI V DNA is pr es ent , it h y b r i d i z e s w i t h t h e
complementary sequence of the labeled DNA MNA PF3@BES FGR OTHER DISEASES
probe which can be detected by its radioactivity.
In principle, almost all the pathogenic
The advantage of DNA probe is that it can detect
organisms can be detected by DNA probes. Several
the virus when there are no detectable antibodres
DNA probes (more than 100) have been developecl
in t he c ir c ulat ion.
and many more are in the experimental stages.The
HIV diagnosis in the raewborep ultimate aim of the researchersis to have a stock of
probes for the detection of various pathogenic
Detection of antibodies is of no use in tne organisms-bacteria, viruses, parasites.
newborn to ascertain whether AIDS has been
transmitted from the mother. This is because the The other important DNA probes in recent years
antibodies might have come from the mother include for the detection of bacterial infections
but not from the virus. This problem can be solved caused by E. coli (gastroenteritis) Salmonella typhi
by using DNA probes to detect HIV DNA in the (food poisoning), Campylobacter hyoitestinalis
newborn. (gastritis).
recessive disease that appears between 3 and 5 clinical manifestationsof this Ciseaseare observed
years of age. The affected chilcirenare unsteadyon after middle age, and by then the person might
their feet as they lose the strength and control of have already passed on the defective gene to his/
t heir m us c les . By t he age o f t e n , t h e v i c t i m s o f her offsprings.
DMD are confined to wheel chair and often die
before reaching 20 years age. FRAGILE X SYNDROME
studies however, may be needed to confirm this. gerre sodf . The deleterious effects of free radicals
The occurrence of triple repeat diseasesindicates can be reduced by administering certain
that the structure of DNA may be rather unstable compounds such as vitamins C and E
and d yn amic. This is in c c nt r as tt o what m olec uiar Another group of workers have reportedthat the
biolog ists ha ve be en t hink ing all along. defective superoxide dismutase cannot control a
transporter protein responsible for the removal of
ALZHEIMER'S DISEASE t h e a m i n o a c i d g l u t a m a t ef r o m t h e n e r v e c e l l s . A s
Alzheimer's disease is characterized by loss of a result, large qr-rantities of glutamateaccumulate in
memo ry a nd im pair ed int ellec t ual f unc t ion t h e n e r v o u st i s s u e l e a d i n g t o d e g e n e r a t i v ec h a n g e s .
(de men tia). Th e vic t im s of t his dis eas e c annot
CANCERS
properly attend to their basic needs, besides being
unable to speak and walk. It is nov., agreed that there is some degree of
genetic predisposition for the occurrence of
The patients of Alzheimer's diseasewere founo cancers, although the influence of environmental
to have a specific protein, namely amyloid in the factors cannot be underestirnated.In fact, cancer
plaqu es (o r. clump s ) of dead ner v e f iber s in
susceptible genes have been identified in some
their brains. A group of researchershave identified f a n r i l i e s e . 9 . , g e n e s f o r m e l a n o m a s u s c e p t i b i l i t yi n
a specific gene on chromosome 21 that is humans are located on chromosomes 1 and 9
believed ro be responsible for familial Alzheimer's
disease. p53 gene
A DNA probe has been developed to locate the The gene p53 encodes for a protein with a
genetic marker for Alzheimer's disease.l'he present m o l e c u l a r w e i g h t 5 3 k i l o d a l t o n s ( h e n c e t h e n a m e ) .
belief is that many environmental factors, and a It is believed that the protein produced by this gene
virus may also b e res pons iblef or t he dev elopm ent h e l p s D N A repair and suppresses cancer
of this d ise ase. lt m ay be pos s ible t hat in t he development. Certain damages that occur in DNA
individuals with genetic predisposition,the outside m a y l e a d t o u n l i m i t e d r e p l i c a t i o na n d u n c o n t r o l l e d
factors nray be stimulatory for the onset of the multiplication of cells. In such a situation, the
d isease. protein encoded by ps3 gene binds to DNA and
blocks replication. Further, it facilitates the faulty
A]I'IYOTROPHIC LATERAL SCLEROSIS DNA to get repaired. The result is that the
cancerous cells are not allowed to establishano
Amyotrophic lateral sclerosis (ALS) is multiply. Thus,
f3 is a cancer-suppressorgene and
characterized by degenerativechanges in the motor acts as a guardian of cellular DNA.
neurons of brain and spinal cord. A gene to explain
Any nrutation in the gene fi is likely to alter its
the inherited pattern of ALS was discovered.
tumor suppressor function that lead to cancer
The gene, known as sodl, encoding for the development And in fact, the altered forms of
enzyme superoxide dismutase is located on ps3 recovered from the various tunror cells (breast,
chromosome 21. T.his gene was found to be b o n e , b r a i n , c o l o n . b l a d d e r ,s k i n , I u n g ) c o n f i r m t h e
defective in families suffering from amyotrophic protective function of ps3 gene against cancers.
laterai sclerosis.In fact, certain point mutations in It is believed that the environrnentalfactors mav
the sodl resulting in single amino acid charrgesin
cause mutations in ps3 gene which may ultinrately
superoxide dismutase have been identified. lead to cancer. Some of the mutations of p53
Sup-^roxide ciismutase is a key enzyme in g e n e m a y b e i n h e r i t e c i ,w h i c h p r o b a b l y e x p l a i n s
elim in atin g th e hig hly t ox ic f r ee r adic als t hat t h e o c c u r r e n c e o f c e r t a i n c a n c e r s i t t s o m e l 'a m i l i e s .
damage the cells (free radicals have been
implicated in aging and severaldiseasee.B. cancer. Genes of breast cancer
cataract,'Parkinson'sdisease,Alzheimer's disease). Two genes, namely BRCN and BRCNI,
On the basis of the function of superoxide implicated ir-r certain hereditary forms of breast
dismutase, it is presumed that ALS occLtrs as a cancer in rvomerr, have beerr identified. lt is
'fre e estimated that abolrt Bj'k of irrherited breast
result o f ra di c al ac c um ulat ion due t o a
defective enzyme (as a consequence of mutatecl c a n c e 1 5a r e d u e t o m u t a t i o n s i n e i t h e r o n e o f t h e s e
182 B IOTECHNO LO CY
Diagnos t ic t es t s f or t he a n a l y s i s o f t h e g e n e s D I A B E T E S
BRCAI and BRCAII were developed. Unfortunately,
t heir ut ilit y is v er y lim it ed, s i n c e t h e r e c o u l d b e D i a b e t e s m e l i i t u s i s a c l i n i c al co n d i ti o n
hundr eds of v ar iat ions in t h e b a s e s e q u e n c e o f characterized by increased blood glucose level
t hes e genes . : (hyperglycemia) due to insufficient or inefficient
( i n c o m p e t e n t ) i n s u l i n . I n o t h e r w o r d s , i n d i vi d u a l s
Genes ef colon cancer with diabetes cannot utilize glucose properly in
their body.
The oc c ur r enc e of c olon c a n c e r a p p e a r s t o b e
genet ic ally link ed s ir r c e it r u n s i n s o m e f a m i l i e s . A rare form of type ll diabetes (i.e., non-insulrn
Some researchershave identified a gerre lir.rked d e p e n d e r r td i a b e t e s n r e l l i t u s , N I D D M ) i s m a tu r i ty
with hereditary nonpolyposis colon cancer or onset diabetes of the young (MODY) MODY
HNPCC (some times called lynch syndrome). fhis o c c u r r i n g i n a d o l e s c e n t sa n d t e e n a g e r s,i s fo u n d to
gene enc oded a pr ot ein t hat a c t s a s a g u a r d i a n a n d have a genetic basis A gene, synthesizing the
br ings about DNA r epair w h e n e v e r t h e r e i s a enzyme glucokinase, located on chromosome 7, is
danrage to it. However, as and when there is a found to be defective in MODY patients
mutation to this protective gene, an altered protetn
C l u c o k i n a s e i s a k e y e n z y m e i n g l u co se
r s pr oduc ed whic h c annot u n d o t h e d a m a g e d o n e
m e t a b o l i s m . B e s i d e s i t s i n v o l v e m e n t i n th e
t o DNA. This leads t o HNP C C . l t i s e s t i m a t e dt h a t
n r e t a b o l i s m , g l u c o k i n a s e i n t h e p a n cr e a ti c ce l l s
t he oc c ur r enc e of t his alt er e d g e n e i s o n e i n e v e r y
serves as a detector for glucose concentration in
200 people in gener al popu l a t i o n .
t h e b l o o d T h i s d e t e c t i o n s t i m u l a t e sB - ce l l s o f th e
p a n c r e a s t o s e c r e t e i n s u l i n . A g e n e m o d i fi ca ti o n
Micresatellite marker genes
t h a t r e s u l t si n a d e f e c t i v eo r a n a l t e r e d g l u co ki n a se
Microsatellites refer to the short repetitive h a m p e r s p a n c r e a t i c i n s u l i n s e c r e t i o n. L a te r r n ,o r k
s equenc es of DNA t hat c a n b e e m p l o y e d a s h a s s h o w n t h a t g l u c o k i n a s eg e n e i s d e fe cti ve i n th e
nr ar k er sf or t he ic lent if ic at i o no f c e r t a i n g e n e s . F o r common form of type ll diabetes.
c olon c anc er , nr ic r os at eil i t e m a r k e r g e n e s h a v e
been ident if iedon c lr r om oso m e2 i n h u m a n s .T h e r e DNA probes for type ll diabetes
is a lot of v ar iabilit y i n t h e s e q u e n c e o f
T h e g l u c o k i n a s eg e n e s f r o m n o r m al a n ci typ e l l
nr ic r os at ellit es( as is t he c as e w i t h R F L P sd e s c r i b e o
d i a b e t e s p a t i e n t s w e r e c l o n e d a n d s ca n n e d w i th
on p 185) .
DNA probes. lt was found that a single base
Ear ly det ec t ion of t he r is k f o r c o l o n c a n c e r b y mutation of the gene led to a defective glucokinase
DNA analy s isis a boon f or t h e w o u l d b e v i c t i m s o { production that is largely responsible for MODY,
t his dis eas e The s us pec t e d i n d i v i d u a l s c a n b e a n d a l s o a m a j o r i t y o f i n d i v i d u a l s w i th typ e l l
per iodic ally m onit or ed f or t h e s i g n s a n d t r e a t e d diabetes.Later,some workers reported a possibility
appr opr iat ely Unlik e m an y o t h e r c a n c e r s , t h e o f a t l e a s ta d o z e n m u t a t i o n s( t h o u g h I essi m p o r ta n t
c hanc es of c ur e f or c oion c a n c e r a r e r e a s o n a b l y t h a n t h e o n e d i s c u s s e d )i n B l u c o k i na seg e n e fo r
good. type ll diabetes.
C hapt er14 : DN A l N D ISE ASD
E IAC N OS IS
AN D ME D i C A LFOR E N S IC S 183
Genes responsible for type I diabetes will ultimately help in the specific diagnosis of
Type I d iab etes or ins ulin- dependentdiabet e s these diseases before their actr-raloccurrence. In
mellitu s (IDDM) m ainly oc c ur s in c hildhood , a d d i t i o n t o h u m a n d i s e a s e sd e s c r i b e da b o v e , s o m e
.l more are given below.
particularly between 2-1 5 years of age. IDDM rs
chara cte rize dby a lm os t t ot al def ic ienc y of ins ulin . Deafness
Researchershave identified at least 18 Cifferent
chromosome regions linked with type I diabetes. T h e d e a f n e s s ,i n h e r i t e d i n s o m e f a m i l i e s , h a s
These DNA seouences are located on genetic basis. A team of workers have iclentifieda
.l.l gene on chromcsome 5, encoding a protein tl-rat
chromosomes 6, and 18.
facilitatesthe assemblyof actin (protein) rrolecules
OBESITY i n t h e c o c h l e a o f i n n e r e a r . T h e a s s o c i a t i o no f a c t i n
Obesity is an abnormal increase in the body is very essentialfor the detection of sound waves
weight due to fat deposition. Men and women are by the ear. A mutation of the gene on chromosome
con sid ere d ob es e if t heir weight due t o f a t , 5 results in a defective rrrotein svnthesisand non-
respectively exceeds more than 2Oo/oand 25"h of a s s e m b l yo f a c t i n m o l e c r - r l ew
s hich causedeafness.
the body weight. Obesity increasesthe risk of high Some other genes, besidesthe one described here,
blood pressure,diabetes, atherosclerosisand other have also been found to be associated with
I ife-th rea ten
ing co ndit ions . deafness.
Hemochromatosis
Hem oc hr om at os isis an ir o n - o v e r l o a dd i s e a s ei n
whic h ir on is dir ec t ly depos i t e d i n t h e t i s s u e s( l i r r e r ,
s pleen, hear t , panc r eas and s k i n ) A n a b n o i " m a l E n v i r o n m e n t a lp o l l u t i o n ( p a r t i c u l a r l yw a te r a n cl
gene on c hr onr os om e 6 i s l i n k e d w i t h h e m o - foods) by path<-rgenic nricroorganismsand viruses is
cl.rromatosis.The amino acid tyrosine, in the a c o m m o n o c c u r r e n c e . r n t h e t r a d i t i o n a l a p p r o a ch ,
normal protein encoded by this gene is replaced by t h e p a t h o g e n sa r e i d e n t i f i e d b y c u l t i v a ti n g th e m tn
cysteine. This abnormal protein is responsiblefor t h e l a b o r a t o r y I n r e c e n t y e a r s , t e c h n i q u e s h a ve
excessiveiron absorption irom the irrtestinewhich been developed for their detection by DNA
ar : c um ulat esin t he v ar ious ti s s u e sl e a d i n g t o t h e r r a n al y s i s .
dam age and r nalf r r nc t ion.
is genet ic ally
de te rmi n e da,n d th u s th e fi n g e rp ri nt R1 R2 R3
. c o n tra s t,th e D N A
is uniquef or an i n d i v i d u a l In
fingerprint is an analysisof the nitrogenous base DNA 1
sequencein the DNA of an individual.
DNA MARKERS IN DISEASE DIAGNOSIS A DNA molecule can be cut into different
A ND F I NG E RP R IN T IN G fragments by a group of enzymes called restriction
endonucleases(For details, Refer Chapter 6). These
The DNA markers are highly useful for genetic fragments are called polymorphisms (literally
mapping of genomes.There are four types of DNA means many forms).
seouenceswhich can be usedas markers.
A n o u t l i n e o f R F L Pi s d e p i c t e d i n F i g . 1 4 '5 . T h e
fra g m e n tl e n g th p o l y m o rp h i sms
1. Res t r ic t i o n DNA molecule t has three restriction sites (Bi, R,
(RFLFs,
pronouncedas rif-lips).
Rr), and when cleaved by restrictionendonucleases
s r v a ri a b l e n u m b e r ta n d em forms 4 fragments. Let us now consider DNA 2
2. M inis at e l l i te o
(VNIRs,
repeats pronounced as vinters). with an inherited mutation (or a genetic change)
that has altered some base pairs. As a result, the
3. Microsatellitesor simple tandem repeats site (Rr) f or the recognition by restriction
(5fRs).
e n d o n u c l e a s ei s l o s t . T h i s D N A m o l e c u l e 2 w h e n
4. Single nucleotide polymorphisms(SNPs, cut by restriction endonuclease forms only 3
p r onounc ed
as s n i p s ). fragrnents(instead of 4 in DNA 1).
186 B IOTE CHNO LO CY
Rr Re Ra
t-
- DNAwith
(A) - restriction(R) sites
DNAprobe One band
Two bands
Nylonmembrane
Fig. 14.6 : Two common methods used for scoring restriction fragment length polymorphism (RFLP)
(A) BFLP by Southern hybridization (B) RFLP by polymerase chain reaction (PCH)'
As is ev ident f r om t he ab o v e d e s c r i o t i o n . a m e m b r a n e . A D N A p r o b e t h a t s p a n st h e su sp e cte d
stretch of DNA exists in fragments of various r e s t r i c t i o n s i t e i s n o w a d d e d , a n d t h e h vb r i d i ze o
lengths (polymorphisrns), derived by the action of bands are detected by autoradiograph. lf the
restriction enzymes, hence the name restriction r e s t r i c t i o n s i t e i s a b s e n t , t h e n o n l y a si n g l e
f r agm ent lengt h poly m or phis m s . restrictionfragment is detected.lf the site is present,
then two fragments are detected (Fig. 1a.6A).
RFLPs in the diagnosis of diseases
2. Polymerase chain reaction : RFLPscan also
lf t he RFLPlies wit hin or ev e n c l o s e t o t h e l o c u s b e s c o r e d b y P C R . F o r t h i s p u r p o s e , P C R p r i m e r s
of a gene t hat c aus es a par t i c u l a r d i s e a s e , i t i s t h a t c a n a n n e a l o n e i t h e r s i d e o f t h e su sp e cte d
pos s iblet o t r ac e t he def ec t iv eg e n e b y t h e a n a l v s i s r e s t r i c t i o ns i t e a r e u s e d .A f t e r a m p l i f i c a t i o n b y PC R ,
of RFLP in DNA. The oer s o n 's c e l l u l a r D N A i s t h e D N A m o l e c u l e s a r e t r e a t e d w i t h r e str i cti o n
isolated and treated with restriction enzymes The e n z y m e a n d t h e n a n a l y s e d b y a g a r o se Be l
DNA fragn.rentsso obtained are separated by e l e c t r o p h o r e s i sl.f t h e r e s t r i c t i o ns i t e i s a bse n t o n l y
electrophoresis.The RFLP patterns of the disease o n e b a n d i s s e e n w h i l e t w o b a n d s a r e f ou n d i f tn e
s us pec t edindiv idualsc an be c o m p a r e d w i t h t h a t o f site is found (Fig. 1a.68\.
nor m al people ( pr ef er ablywit h t h e r e l a t i v e si n t h e
Applications of RFLPs: The approach by RFLP
s am e f am ily ) By t his appr o a c h , i t i s p o s s i b l e
is very powerful and has helped many genes to be
t o det er m ine whet her t he i n d i v i d u a l h a s t h e
m a p p e d o n t h e c h r o m o s o m e s e . g . si ckl e -
m ar k er RFLP and t he dis eas e g e n e . Wi t h 9 5 %
cell anemia (chromosome 11), cystic fibrosis
certainity, RFLPs can detect single gene-based (chromosome
7), Huntington'sdesease(chromosome
diseases.
4 ) , r e t i n o b l a s t o m a( c h r o m o s o m e 1 3 ) , Al zh e i m e r 's
Methods of RFLPscoring : Two methods are in d i s e a s e( c h r o m o s o m e2 1) .
common use for the detection of RFLPs(Fig. 1 .5)
VAR'ABLE NUMBEN TANDEM BEPEATS
1. Sout her n hy br idiz at ion : T h e D N A r s
(VNTRS)
diges t ed wit h appr opr iat e r es t r i c t i o n e n z y m e , a n d
separated by agarose gel electrophoresis.The so V N T R s , a l s o k n o v v na s m i n i s a t e l l i t e s li , ke R FL Ps,
obtained DNA fragmentsare transferredto a nylon a r e D N A f r a g m e n t s o f d i f f e r e n t l e n g t h Th e m a r n
ChA P I CT
14 : DN A IN D IS EA SE
D IA C N O S IS
A ND ME D IC A LFOR E N S IC S 147
DNA 1 (A)
-cAGCTGTCGAT-
GGCGAGAGAGAGATC -CAGoTCTCGAT
(5 repeatingunitsof GA)
(B)
I
?"/'
\--
v --S N P
- OAGCTGTCGAT - TagetDNA
--- DNA2
-..-.' lllllllllll
GGCGAGAGAGAGAGAGAGAGAGATCT...-.. - GTCGACAGCTA- oligonucleotide
(10 repeatingunitsof GA) +L Matchedbase
Fig. 14.9 : Two alleles of DNA molecules representing Completearid stable
5 and 10 dimer repeating units. hybridizationof base pairs
I
rSNP
Y
MICBOSATELLITES -CAGCTGTCGAT - TagetDNA
(stMPLE TANDEM REPEATS)
-GTCGAGAGCTA - oligonucleotide
M ic r os at ellit es ar e s hor t r epe a t u n i t s ( 1 0 - 3 0 fL-
Mismatchedbase
co pies ) us ually c om pos ed of d i n u c l e o t i d e o r
Hybridizationnot formed
tetr anuc leot ideunit s . Thes e s im ple t a n d e m r e p e a t s due to mismatchbase pair
) e m or e popular t han m inis a t e l l i t e s( V N T R s )
(S TRs ar
as DNA markers for two reasons. Fig. 14.10 : (A) An illustration of single nucleotide
polymorphism (SNP) (B) Oligonucleotide hybridization
1 M ic r os at ellit es ar e ev en l y distributed to detect SNP.
thr oughout t he genom e
The p ha rmaceut ic al pr oduc t s of r ec om bina n t The hormone insulin is produced by the p-cells
DNA techn olo gv ar e br oac lly div ided int o t h e o f i s l e t so f L a n g e r h ; i n so f p a n c r e a s .H u m a n i n s u l i n
follo wing th ree c at egor ies and br ief ly dis c us s e d c o n tains 51 amino acids, arranged in two
with impo rtan t ex am ples . polypeptide chains. The chain A has 21 amino
189
190 B IOTE CHNO LO CY
Growth
defects growth
Growlhhormcne, factorVlll
Coagulatlon Hemophilia
A
hormone'releasing
factor, factorlX
Coagulation Christmas
disease
somatomedin-C DNaseI fibrosis
Cystic
Heartattacks Prourokinase
Erythropoietin Anemia,
kidney
disease
HemophiliaA Factor
Vlll
Glucocerebrosidase Gaucher's
disease
HemophiliaB FactorlX
Growth
hormone Growth in children
defects
Hepatilis
B F.lonatitic R rrennino
Granulocyte
colony Cancer
Hypoalbuminemia Serumalbumin
factor
stimulating
lmmune disorders Interleukins,
B-cellgrolvth
factors Hepatitis
B surface Hepatitis
B
(vaccine)
antigen
Kidneydisorders Erythropoietin
l nsul i n Diabetes
mellitus
LowGehrig's disease Brain-derived
neurotropic
(amytrophiclateralsclerosis)factor Interferon
a Leukemia,kaposisarcoma
genitalwarts,hepatitis
B
Multiplesclerosis Interferons
(u, 0, y)
Nervedamage Nervegrowthfactor interferon
B Multiple
sclerosis
Osteomalacia Calcitonin y
Interferon granulomatous
Chronic
drsease
Pa i n Endorphins and
enkephalins Interleukin-2 Renalcellcarcinoma
Rheumatic disease Adrenocorticotropic Interi euki n-10 Thrombocytopenia
n0rm0ne Somatotropin Growthdefects
Ulcers Urogastrone Tissueplasminogen Aculemyocardial
infarction,
Viralinfections (u, 0, y)
Interferons activator pulmonary
embolism
Ch a pt er15 : P HA R MA C E U T IC AL
P R OD U C fSO F DN A TE C H N OLOC Y 191
) nu*" I/ B'"ilfi
damage (retinopathy),nerve diseases(neuropathy)and
circulatory diseases(atherosclerosis,stroke). In fact,
diabetes is the third leading cause of death (afterheart
diseaseand cancer) in many developed countries.
Plasmid plasmid
In the early years, insulin isolated and purified
from the pancreasesof pigs and cows was used fcr
| ,r"n"rorminto I
t he trea tmen t o f d iabet ic s . Ther e is a s light E.coti
difference (by one to three amino acids) in the
J J
st ructure o f a nima l ins ulin c om par ed t o hum an
insu lin. Th is re su lted in aller gy in s om e of t he
diabe tics whe n a nim al ins ulin was adm inis t er eo.
A nothe r pro ble m wi t h anim al ins ulin is t hat lar ge
numb er of a nima ls hav e t o be s ac r if ic ed f or
extracting insulin from their pancreases. For
instance, about 7O pigs (giving about 5 kS
E. coli E. coli
pancre atictissue )ha v e t o be k illed t o get ins ulin f or
treating a single diabetic patient iust for one year! Culturecellsin
a nutrientmedium
containinglactose.
Production of recombinant insulin lsolateand purify
A and B chains
Attemps to produce insulin by recombinant
DNA technology started in late 1970s. fhe basic
technique consisted of inserting human insulin -/--.--l.t-'-'t-'
B chain
gene and the promoter gene of lac operon on to
the plasmids of E. coli. By this method human
insulin wa s pro du c ed. lt was in J uly 1980, Joiningof chains
seventeen human volunteers were, for the first and
reconstitution
t ime , a dmin iste red r ec om binant ins ulin f or
treatment of diabetes at Cuy's Hospital, London.
And in fact, insulin was the f irst ever
pharmaceu tica l p roduc t of r ec om binant DNA
t ech no log y ad minist er edt o hum ans . Rec om binant Humaninsulin
insulin worked well, and this gave hope to scientists
that DNA technology cbuld be successfully Fig. 15,1 : The production of recombinant insulin in
employed to produce substancesof medical and E, coli (l-lnducer gene, P-Promoter gene,
commercial importance. An approval, by the
concerned authorities, for using recombinant
insulin for the treatment of diabetes mellitus was
given in 1982. And in 1986, Eli Lilly company
received approval to market human insulin under F i g . 1 5 . 1. T h e g e n e s f o r i n s u l i n A c h a i n a n d B
the trade name Humulin. chain are separatelyinsertedto the plasmids of two
different E. coli cultures. The /ac operon system
Technique for recombinant insulin production :
(consisting of inducer gene, promoter gene,
The orginal technique (described briefly above) of
operator gene and structural gene Z lor
insulin synthesis in E. coli has undergone several
change s, fo r impro v ing t he y ield. e. g. addit ion of B-galactosidase)is used for expressionof both the
genes. The presence of lactose in the culture
signal pe ptid e, synt hes is of A and B c hains
m e d i u m i n d u c e s t h e s y n t h e s i so f i n s u l i n A a n d B
seDaratelvetc.
c h a i n s i n s e p a r a t ec u l t u r e s . T h e s o f o r m e d i n s u l i n
The procedure employed for the synthesis of chains can be isolated,purified and joined together
t wo insulin cha ins A and B is illus t r at ed in t o g i v e a f u l l - p l e d g e d h u m a n i n s u l i n .
192 B IOTE C H N O LO CY
cDNAfor
hGH
Sequencefor Sequencefor Sequencefor 167
signalpeptide 24 amino acids aminoacidsof hGH
(26 aminoacids) of hGH I
I ECoRI
Jcreavage
:
Deletionof sequencefor 50
amiho acidd(26 + 24) I
+
Sequencefor 167aminoacids
- of hGH
ffi ---l
Ligation
cDNAencodingfor \
24 amino acids I
of hGH +
cDNAfor hGH alono
with expression-
veclor genome
I tnsertion
intoa
vector
J
E. coli
acids) is cut by restriction endonuclease ECoRl. Gontroversy over the use of hGH
Now a gene (cDNA) for 24 amino acid sequenceof
Recombinant hCH can be administered to
hCH (that has been deleted) is freshly synthesized
children of very short stature. lt has to be given
an d liga ted to th e r em aining hCH c DNA. The s o
daily for many years with an annual cost of about
constitued cDNA, attached to a vector, is inserted
$ 20,000. Some workers have reported substantial
into a bacterium such as E. coli for culture ano
increase in the height of growth retarded children.
pro du ctio n o f hGF l. I n t his m anner ,t he biologic all y
One group of workers observed that the normal
f unction al hGH c an be pr oduc ed bv DNA
growth pattern in children was not restoredon hCH
technology.
administration,although there was an initial spurt.
Recombinant hGH was approved for human Another big question raised by the opponents of
use in 1985. lt is marketedas Frotropin by Cenetech hCH therapy is whether it is necessaryto consider
company and Humatrope by Eri.Lilly company. short stature as a disorder at all for treatment!
Biotechnology [i3]
r94 B IOTE CHNO LO CY
Degraded Soluble
products
Synthetic
plasmid
Fig. 15.3 : Role of tissue plasminogen activator in
dissolving blood clots.
Chemically,thrombusconsistsof a networkof
fibrin, formed from the fibrinogen.In the normal
circumstances,plasmin degrades fibrin and
d is s olv esblood c l o ts . T h i s p l a s mi n i s a c tu a l l y
producedby activationof plasminogenby tissue
plasminogenactivator (Fig. 15.3). The natural
biological systemsis however,not that efficientto
remove the blood clots through this machinery.
Tissueplasminogenactivatoris very useful as a
therapeutic agentin dissolving blood clots(thrombi)
by activating plasminogen.By removing the
arterial,thrombi, the possibledamagecausedby
them on heartand brain could be reduced.
Antibody.plasminogen
activator coniugates
An ant ibody agains t f ibr in ( a n t i f i b r i n a n t i b o d y )
c an be c onjugat ed wit h t i s s u e p l a s m i n o g e n
ac t iv at or t his c onjugat e is a p p r o p r i a t e l y r e g a r d e d
as im m unot l. r er apeut ic t hr o m b o l y t i c a g e n t . l t
quic k ly and s pec if ic ally bind s i o f i b r i n c l o t s a n d
loc ally inc r eas est he c c nv er s i o n o f p l a s m i n o g e nt o
plasmin to dissolve fibrin (Fig. lS.S) In fact,
ant if ibr in m onoc lonal ant i b o d i e s h a v e b e e n
synthesized, conjugated with tPA and tried for
s olubilis ing blood c lot s
Advantages of tPA as
thrombolytic agent
Fig. 15.5 : Action of antifibrin antibody-tPA
lis s ue plas m inogenac t iv a t o ra c t s o n b l o o d c l o t s conjugate in dissalving blood clot
( s olubiliz es by degr adat ion)w i t h o u t r e d u c i n g t n e (tPA-tissue plasminogen activator).
blood c lot t ing c apabilit y el s e w h e r e l - h i s i s i n
contrast to the action of urokinase or streptokinase
whic h ar e m or e gener alis edin t h e i r a c t i o n . F u r t h e r , Mechanism of action of interferons
t PA c an be adm inis t er ed i n t r a v e n o u s l y w h i l e
I n t e r f e r o n sa r e p r o d u c e d b y m a m m a l i a n ce tt:
ur ok inas e and s t r ept ok in a s e h a v e t o oe when infected by viruses. As the virus releasesi:'
adm inis t er eddir ec t ly t o t he b l o c k e d b l o o d v e s s e r .
n u c l e i c a c i d i n t o c e l l u l a r c y t o p l a s m , i t sti m u l a te .
Another merit of using tPA is tlrat its action is mucn
the host DNA to produce interferons. The:e
faster than other thrombolytic agents with much
interferons, secreted by the cells, bind to the
reduced side effects.
a d j a c e n t c e l l s . H e r e , t h e y s t i m u l a t e t h e ce l l u l a '
D N A t o p r o d u c e a s e r i e so f a n t i v i r a l e n z ym e s.Th e
I NTERFERO NS
s o f o r m e d p r o t e i n s i n h i b i t v i r a l r e p l i ca ti o n a n .
Interferon is an antiviral substance, and is the protect the cells (Fig. 15.6). lt is believed that the
first line of defense against viral attacks. -fhe term p r o t e c t i v e ( e n z y m e s ) p r o t e i n s b i n d t o m R N A c'
interferon has originated from the interference of v i r u s e sa n d b l o c k t h e i r p r o t e i n s y n t h e s i s.Th e a cti o ^
t his m olec ule on v ir us r eplic a t i o n . l t w a s o r i g i n a n y of interferonsappears to be species specific. Thu.
dis c ov er ed in 1957 by Ali c k l s a a c s a n d J e a n h u m a n i n t e r f e r o n so p e r a t ei n h u m a n s .O th e r a n i m "
Lindem ann and was c ons id e r e d t o b e a s i n g l e (dog, mouse) interferonsare ineffective in man.
s ubs t anc e.I t is now k nown t h a t i n t e r f e r o na c t u a l l y
consistsof a gi'oup of more than twenty substances lsolation of interferons in
with molecular weights between 20,000-30,000 the early years
dalt ons . All t he inier f er onsar e p r o t e i n s i n n a t u r e
B l o o d w a s t h e o n l y s o u r c e o f i n t e r f e r o n se a r l i e -
and m any of t hem ar e gly c o p r o t e i n s . T h e y a r e
T h e p r o c e d u r ew a s v e r y t e d i o u s a n d t h e q u a n ti t\ ,:-
broadly categorized into three groups based on
i n t e r f e r o n si s o l a t e d w a s v e r y l i t t l e . T h u s, a s m u c-
t heir s t r uc t ur eand f unc t r on
a s 5 0 , 0 0 0 l i t r e so f h u m a n b l o o d w a s r e q ui r e dto e=-
Interferon-cr(l FN-cx) .l
just 00 mg of interferons!Therefore, it was re-
I nt er ier on- p( lFN- p) d i f f i c u l t t o c o n d u c t r e s e a r c ho r u s e i n t e r fe r o n s .-
Interferon-y(lFN-y) therapeuticpurposes.
Chapt er15 : P H A R MA C E U T IC AL
PR O D U C T S
OF D N A TE C H N OI-OC Y 197
64:
Adjacentcell
Blockviral reolication
years1986,1993and 1990.A S w i ssbi otechnol ogy
firm was the first to market infurteron-a with a
trade name lntron. Interferonsare used for tne
treatmentof a largenumberof viral diseasesand
cancers.The cancersi ncl ude l eukemi a.kaoosi s
Fig. 15.6 : The mechanism of action of interferons. sarcoma,bladdercancer,head and neck cancer,
renal cel l carci noma,ski n cancer and mul ti pl e
myeloma.The other diseases employinginterferon
Production of recombinant inter{erons therapyare AIDS,multiplesclerosis, genitalwans,
hepatitisC, herpeszosteretc.
The complementary DNA (cDNA) was synthe-
sized from the mRNA of a soecific interferon.This is
insertedto a vector (say plasmid)which is introduced
into E. coli or-other cells. The interferon can be RE 1 RE2
is ola tedfro m the c ult ur e. m edium .This is t he bas r c \P \P
m e ch an ismo f p roduc ing r ec om binantint er f er ons. r--- -------A------A-- IFN-cqgene
Interferonsare also employed in the treatmentof (released on bacterial lysis). Adnrrnistration of the
c om m on c olds and inf luenz a . F o r t h i s p u r p o s e , enzyme DNase I to the lungs of CF patients
interferonscan be used as nasal sprays. decreases the viscosity of the mucus, and the
breathing is made easier. lt must be remembered
The basic mechanism of action of interferons
that DNase I cannot cure cystic fibrosis. lt can onlr
against viruses has already been described.
relieve the severe symptoms of the disease in mosi
lnterferonsare found to causethe death of cancerous
oatients.
c ells .This is br oughtout by s t im u l a t i n gt h e a c t i o n o f
natural killer (NK) cells, a specialised form of
lymphocytesthat can destroy cancerouscells. ALGINATE LYASE
199
200 BIOTECHNOLOCY
HepatitisB virus
HEPATITIS B zA\
tl f- \
He pa titis B is a wides pr ead dis eas e in m a n ' l t \\ ) l+-- HBsAssene
primarily affects liver causing chronic hepatitis,
cirrhosis and liver cancer. Hepatitis B virus is a 42
nm particle, called Dane particle. lt consists of a
core containing a viral genome (DNA) surrounded
by a ph osph olipid env elope c ar r y ing s ur f a c e
de@&
antigens (Fig, 16,1A). Infection with hepatitis B
virus produced Dane particles and 22 nm sized
oarticles.The latter contain surface antigenswhich
are more immunogenic. lt is however, very difficult
to gro w h ep atit isB v ir us in m am m alian c ell c u l t u r e Grow in tryptophan-free
and produce surface antigens. mediumand selectcells
The gene encodipg for hepatitiS B surface
antigen HBsAg) has been identified. Recombinant
hepatitis B vaccine as a subunit vaccine, is
produced by cloning HbsAg Sene in yeast cells'
@36/
Saccharomyces cerevisiae, a harmless baking and
Culturecells
brewing yeast, is used for this purpose (Fig- 16.18)' I
The gene for: HBsAg is inserted (pMA 56) which is
+
Lyseyeast cells
linked to the alcohol dehydrogenase promoter'
These plasmids are then transferred and cultured'
The cells grown in tryptophan, free medium are +
I
selected and cloned. The yeast cells are cultured'
PurifyHBsAg
1-heHBsAg gene is expressedto produce 2nm sized (as 22 nm particles)
particles similar to those found in patients infected
with hepatitis B. (These particles are
Fig. 16.1 : (A) Hepatitis B virus-Dane particle (42 nm
immu no rea ctiv e wit h ant i- HBs Ag ant ibod i e s ) '
Th e su bu nit HBs Ag as 22 nm par t ic les c a n b e
isola ted a nd us ed t o im m uniz e indiv iduals ag a i n s t
heoatitis B.
2|J2 B IOTE C H N OLO CY
TUBERCULOSIS
(A)
Cell surface
Bindingof gp41
Attachmentof virus to host cell for
to hostcellby 9p120 virus entry
(B)
F-CD+ receptor
Anti-9p120antibody . Anti-qp41
-/ anttbodY
V-t
t/
-\*
()-so+t
)-
_/
Anti-9p120
antibody Anti-9p41antibody
preventsbindingof bindsto gp41 and blocks
9p120to CD4 receptor fusionof HIV to host cell
Fig. 16.4 : Subunit recombinant vaccines against AIDS. (A) The functions of 9p120 and gp41 (B) The
MemorvB-lvmPhocvte
YYY (protecisagairistfu[ureinfection)
YYY Antibodies
Antigen
HUMORALIMMUNITY
CELLULARIMMUNITY
Fragments
of antigen
Killpathogenic
ceils
T-lymphocyte
boundio antigen Activatedcytotoxic
T-lymPhocYtes
"B[1"' Nucteus ---l
m
rf
lnoculated T
cell Z
--
(MHC-Maior histocompatabilitycomplex molecule) -o
Fig. 16.s : DNA vaccne and mechanism of its action in devetoping immunity
Chaot er' 16 : R E C OMB IN A NVA
T C C IN E S 2lJ7
a c t a s a n i r n m u n i z i n g a g e n t . T h i s t y p e o f a p p r o a ch
is almost outdated now.
edible subunit vaccines
lt is now possible to genetically engineer the
Antigen Host plant organisms (bacteria or viruses) and use them as
Iive vaccines, and such vaccines are referred to as
glycoprotein
Habies Tomato attenuated recombinant vaccines. The genetic
virus(VPl)
Footandmouth Arabidopsis m a n i p u l a t i o n sf o r t h e p r o d u c t i o n o f t h e s e v a cci n e s
are broadly of two types
Herpes
virusB surface
antigen Tobacco
1 . Deletion or modification of viruience genes
toxinB subunit
Cholera Potato
of pathogenic organisms.
Human glycoprotein
cytomegalovii'us B Tobacco
2 . C e n e t i c m a n i p u l a t i o n o f n o n - p a t h og e n i c
organisms to carry and express antigen
d e l e r m i n a n t sf r o m p a t h o g e n i c o r g a n i s m s .
expressing antigens derived from animal viruses
(ra bies v ir us , her pes v ir us ) . A s e l e c t e d l i s t o f l-he advantage with attenuated vaccines is that
re c om binant v ac c ines agains t a n i m a l v i r u s e s the native conformation of the immunogenic
prcduced in plants is given in l-able 16.2. determinants is preserved, hence the immune
responseis substantiallyhigh. This is in contrast to
Edible vaccine production and use purified antigens which otten elicit poor
in-rmunological response.
The production of vaccine potatoes is iilustrated
in Fig. 16.6. The bacterium, Agrobacterium Some of the imoortant attenuated vaccines
tumeiaciens is commonly used to deliver the DNA developed by genetic manipulatior.rsare briefly
(g enet ic m at er ial)f or bac t er ial or v i r a l a n t i g e n s .A described.
p las m id c ar r y ing t he ant igen gene a n d a n a n t i b i o t i c
CHOLERA
resistancegene are incorporated into the bacterial
cells (A. tumefaciens). The cut pieces of potato Cholera is an intestinaldiseasecharacterizedby
le av es ar e ex pos ed t o an ant ibiot ic w h i c h c a n k i l l d i a r r h e a ,d e h y d r a t i o n ,a b d o m i n a l p a i n a n d f e ve r . l t
th e c ells t hat lac k t he new genes . T h e s u r v i v i n g is caused by the bacterium, Vibrio cholerae. This
cel ls ( i. e. , gene alt er ed ones ) c an m u l t i p l y a n c if o r m p a t h o g e n i c o r g a n i s m i s t r a n s m i t t e d b y d r i n ki n g
a c allus ( c lum p of c ells ) . This c allu s i s a l l o w e d t o water contaminated with fecal matter. Cholera
sprout shoots and roots, which are grown in soil to epidemics are frequently seen in developing
form plants. In about three weeks, the plants bear countries where the water purification and sewage
potatoes with antigen vaccines. disposal systemsare not well developed.
The first clinical trials in humans, using a plant- On entering the small intestine, V. cholerae
de riv ed v ac c ine wer e c onduc t ed i n 1 9 9 7 . f h i s colonizes and starts producing large amounts of a
involved the ingestion of transgenic potatoes with toxic protein, a hexameric enterotoxin. This
a toxin of E. coli causing diarrhea. Some success e n t e r o t o x i n s t i m u l a t e s t h e c e l l s l i n i n g i n t e sti n a l
was reported. For some more details on edible w a l l s t o r e i e a s e s o d i u m , b i c a r b o n a t e a n d oth e r
vaccines, the reader nrust refer Chapter 51. i o n s . Wa t e r a c c o m p a n i e s t h e s e i o n s l e a d i ng to
severe diarrhea, dehydration, and even death.
The currently used cholera vaccine is composed
of ohenol-killed V. cholerae. The imrnuno-
protection, !astingfor 3-6 months is just moderate.
Attempis aie being made to develop better
In the early years of vaccine research,attenuated vacctnes.
strains of some pathogenic organisms were The DNA technologistshave identified the gene
prepared by prolonged cuitivation - weeks, encoding enterotoxin (toxic protein). Enterotoxin,
months or even years.Although the reasonsare not an hexamer, consists of one A subunit and five
known, the infectious organism would lose its i d e n t i c a l B s u b u n i t s . T h e A s u b u n i t h a s tw o
ability to cause diseasebut retains its capability to functional domains-theA, peptide which possesses
Chaot er16 : RE C OMB IN A NVTAC C IN ES 249
Plasmid
Plantcell
Genetransfer
Potato
Potato
the toxic activity and A, peptide that joins A 2. The DNA sequence of A, peptide is
. y g e n e ti ce n g i n e e ri n g,
s ubunitt o B s u b u n i tsB it incorporatedinto a plasmid,cloned and digested
was possibleto deletethe DNA sequenceencoding with restrictionenzymes(ClaI and XbaD. In this
A. peptideand createa new strain of V. cholerae. manner,the A.' peptidecodingsequenceis deleted
This strain is non-pathogenic,since it cannot (the DNA encodingfor 183 of the 194 aminoacids
produceenterotoxin.The genetically engineeredV of the A, peptideis actuallyremoved).By usingTo
cholerae is a good candidate to serve as an D N A l i gase,the pl asmi d i s reci rcul ari zed.
Thi s
attenuated vaccine. plasmid containsa small portion of A., peptide
coding sequence.
Gieating a new strain ol V, cholerae
3. The pl asmi ci ,contai ni ngthe del eted A ,
The develooment of a new strain of Vibrio
peptidesequenceis transferred by conjugationinto
choleraethat can effectivelyserveas an attenuated
the V. cholerae strain carrying a tetracycline
recombinantvaccine is depicted in Fig. 16.7, anc)
resistance gene.
brieflydescribedbelow.
1. A tetracyclineresistance gene was inserted 4. Recombinationcan occur between the
into the A, peptide sequence of V. cholerae plasmid (containinga srnall portion of peptide
chromoSome.This destroysthe DNA sequence A 1 codi ng sequence)and the.chromosomeof
encodingfor Ar peptide,besidesmakingthe strain V. cholerae (carryingtetracyclineresistancegene).
resistant to tetracycline. Unfortunately, the The resultof this double crossoveris the formation
tetracycline gene
resistant is easily lost and the of V, cholerae containing a chromosomal DNA
enterotoxinactivityis restored.Because of this,the lacking A, peptide DNA sequence. As the
new strainof V. choleraeas suchcannot be usedas bacterium,V. choieraemultiplies,the plasnridsare
a v ac c t ne. lost in the next few generations.
Biotechnology
[14]
21'J B IOTE C H N OLO CY
SALMONELI.A SPECIES
The differentstrainsof Salmonellagenus are
responsiblefor causingtyphoid, entericfever,food
poisoning and infant deafh. Immunoprotection f**b-chromosomat DNA
againstSalmonellapathogensis really required. t-.--J
Someworkershave been successful in deleting
,'-l\
aro genesand pur genesin Salmonella.Aro genes ( )+- Plasmid
encode for the enzymes responsiblefor the \___--l
and hygromycin. This new strain of L. major against pathogenic organisms Another advantage
invaria bly req ui r est hy m idine in t he m edium f or i t s of vaccinia virus is the possibility of vaccinating
g rowth a nd mu lt iplic at ion.The at t enuat eds t r ain o f individuals against different diseases
L. major can survive only a few days when simultaneously.fhis can be done by a recombinant
administeredto mice. This short period is enough to vaccinia viruses which carries genes encoding
ind uce immun it y in m ic e agains t t he les ions o f d ifferent antigens.
leishma nia .Ho wev er , m or e ex per im ent son anim a l s
Antigen genes for certain diseases have been
ha ve to be ca r r ied out bef or e t he leis hm a n i a
successfullv incorporated into vaccinia virus
a tten ua tedvacc ine qoes f or hum an t r ials .
genome and expressedT . h u s , v e c t o r v a c c i n e sh a v e
b e e n d e v e l o p e d a g a i n s th e p a t i t i s ,i n f l u e n z a , h e r p e s
s i m p l e x v i r u s , r a b i e s , a n g u l a r s t o m a t i t i sv i r u s a n d
malaria. However, none of these vaccineshas been
Iicensed for human use due to fear of safety lt is
Some of vectorscan be geneticallymodified a r g u e dt h a t r e c o m b i n a n tv a c c i n i a v i r u s m i g h t c r e a t e
and em ploy eda s v a c c i n e sa g a i n spt a th o g e n s . l i f e t h r e a t e n i n gc o m p l i c a t i o n s i n h u m a n s .
Advantages
1. Authenticatedantigensthat closelyresemble
naturalantigenscan be produced.
=-r772--T-l
Vacciniavirus genornewith 3 insertedgenes Other viral recombinant vaccines
213
214 BIOTECHNOLOCY
-
precursors Tetrahydrofolate
t The establishment of hybridomas and
productionof MAbs involvesthe following steps
(Fig. 17.2).
o e .rovo
^ ' ---
I 1. l mmuni zati on
|
^.Y,, - 2. C el l fusi on
-J"t4es;"
-l Nucreotides- - - ->DNA 3. Selectionof hybridomas
the products
4. Screening
5. Cloningand propagation
6. Characterization and storage.
Hypoxanthine 1. lmmunization : The very first step in
Thymidine hybridomatechnologyid to immunizean animal
(usuallya mouse),with appropriateantigen.The
Fig. 17,1 : Pathways for the synthesis of nucleotides
antigen, along with an adjuvant like Freund's
complete or incomplete adiuvant is injected
subcutaneously (adjuvants are non-specific
potentiatorsof . specific immune responses). The
folate. The formation of tetrahvdrofolate(and injectionsat multiple sites are repeatedseveral
therefore nucleotides) can be blocked by the ti mes. Thi s enabl es i ncreasedsti mulat ion of
inhibitor aminopterin. B-lymphocyteswhich are responding to the
anti gen.Threedayspri orto ki l l i ngof the anim al,a
The salvage pathway involves the direct final doseof antigenis intravenously administered.
conversionof purines and pyrimidinesinto the The immune-stimulatedcells for synthesisof
c o rre s p o n d i nngu c l e o ti d e s.
H ypoxanthi ne guani ne antibodieshavegrown maximallyby this approach.
phosphoribosyltransferase(HCPRT) is a key The concentrationof the desired antibodiesis
enzyme in the salvagepathway of purines. lt assayedin the serum of the animal at frequent
convertshypoxanthine and guaninerespectively to intervalsduring the courseof immunization.
i n o s i n e mo n o p h o s p h a t e and guanosi ne
When the serum concentration of the
monophosphate. Thymidinekinase(TK), involved
antibodiesis optimal,the animal is sacrificed. The
in the salvagepathway of pyrimidinesconverts
spleen is asepticallyremoved and disruptedby
th y m i d i n eto th y m i d i n emo n ophosphate (TMP ).A ny
mechanicalor enzymaticmethodsto releasethe
mutationin eitherone of the enzymes(HCPRTor
cells. The lymphocytesof the spleenare separated
TK) blocksthe salvagepathway.
from the rest of the cells by density gradient
When cells deficient(mutatedcells) in HCPRT centrifugation.
a re g ro w n i n a me d i u mc o n tai ni nghypoxanthi ne 2. Cell fusion : The thoroughly washed
a m i n o p te ri na n d fh y m i d i n e(H A T medi um),they lymphocytesare mixed with HGPRT defective
cannot survive due to inhibition ol de novo myelomacells.The mixtureof cells is exposedto
synthesisof purine nucleotides(Note : Salvage polyethylene glycol (PEC)for a shortperiod(a few
pathwayis not operativedue to lack of HCPRT). minutes),since it is toxic. PEC is removed by
T h u s ,c e l l sl a c k i n gH C P R I g row ni n H A T medi um washingund th" cells are kept in a freshmedium.
die. These cells are composed of a mixture of
The hybridoma cells possessthe ability of hybridomas(fusedcells),free myelomacells and
myeloma cells to grow in vitro with a functional free lymphocytes.
HCPRT gene obtained from lymphocytes(with 3. Selectionof hybridomas: When the cellsare
which myeloma cells are fused).Thus, only the culturedin HAT medium (the principledescribed
hybridomacells can proliferatein HAT medium, above),only the hybridomacells grow, while the
and this procedureis successfully used for their rest will slowly disappear.This happensin 7-.10
selection. days of culture.
17: M O N OC L O N AL A N T IB O D IES (HY B R ID OMA TE C H N OLOC Y )
lmmunizedanimal
Spinnerculture
Selectionof
hybridsin HATmedium
/I
l-
l<- | AssayantibodY
I
+
Freeze '71\".
'-+
l{------1
lRecloning
Characterizeclones
Selectvariants
Tumoursof cells
producingantibody
216 B IOTE CHNO LO CY
+
I
Screenfor
antigenbinding
+
I
M o n o c l o n a l a n t i b o d i e sw i t h s p e c i f i c i t ya n d h i g h
lncorporateH and L purity have a wide range of applications,which
sequencein a plasmid
can be broadly categorized as follows.
I
+ 1 Diagnosticapplications
E coiltransformed
with H-L DNA construct 2 Therapeutic uses
+
I 3. Protein purification
4 . M i s c e l l a n e o u sa p p l i c a t i o n s .
Productionof
Fv fragmentin E. coli A summary of the important applications of
MAbs is given in Table 17.1, and briefly discussed
Fig. 17.6 : Production of monoclonal antibodies
below.
in E. coli. ,
1. DIAGNOSTIC APPLICATIONS
ADVANTAGES OF MONOCLONAL M o n o c l o n a l a n t i b o d i e s h a v e r e v o l u t i o n i z e dt n e
ANTIBODIES l a b o r a t o r y d i a g n o s i s o f v a r i o u s d i s e a s e s .F o r t h i s
purpose, MAbs may be employed as diagnostic
Monoclonal antibodies truely represent a
reagents for biochemical analysis or as tools for
homogeneous sfafe of a single molecular species
diagnostic imaging of diseases.
Each MAb is s pec if ic t o a giv en ant ige n i c
d ete rmina nt.This is in c ont r as tt o t he c onv ent io n a r
a ntiseru mtha t c ont ains poly c lonal ant ibodies .T h e lAl MAbs IN BTOCHEMTCALANALYSTS
wide range of applications of MAbs are described Diagnostic tests based on the use o{ MAbs as
Iater. r e a g e n t sa r e r o u t i n e l y u s e d i n r a d i o i m m u n o a s s a y s
220 B IOTE C H N OLO CY
i applications
Oiagnostic
(A) Biochemical for the diagnosis
analysis of pregnancy,cancers, disorders,
hormonal diseases.
infectious
imaging(immunoscintigraphy)
(B) Diagnositic of
tor the detection myocardial deep
infarction, veinthrombosis,
cancers,
atherosclerosis, bacterialinfections.
applications
2. Therapeutic
(A) Directuseas therapeuticagents disease-causing
to destroy in thetreatment
organisrns, in the
of cancers,
oforgan
immunosuppression inthetreatment
transplantation, ofAIDS, andautoimmune diseases.
(B) As targetingagentsin thercpyas immunotoxins(forireatment
of cancers), fordissolving
in drugdelivery,
bloodclots, (of
in tadioimmunotherapy tumors).
myocard ial in farction. lm aging of r adiolabeled (lung cancer/ breast cancer/ ovartran cancer/
MA b, is u su ally d one af t er 24- 48 hour s of m a l a n o m a , c o l o r e c t a lc a n c e r )b y e m p l o y i n g N l A b s
int raven ou sa dmin is t r at ion.This is c ar r ied out eit her About B0 per cent specificity has been achieved for
by p lan ne r ga mma c am er a or s ingle phot on detecting cancers by this approach
emission co mpu ted t om ogr aphy ( SPECT) . lt is
A n i o d i n e ( 1 3 1 1 )l a b e l e d m o n o c l o n a l a n t i b o o y
possible Io detect the location and the deg,ree of
s p e c i f i ct o b r e a s tc a n c e r c e l l s w h e n a d m i n i s t e r e dr <t
damage to the heart by using radiolabeled
the patients detects (by imaging) the spread of
antimyosin MAb. fhus, this technique is useful for
cancer (metastasis)to other regions of the body.
the diagnosis of heart attacks.
This is not possibleby scanningtechniques
Deep vein thrombosis (DVT) : DVT refersto the
The imaging technique by using MAb can also
f ormatio n o f b loo d c lot s ( t hr om bus ) wit hin t ne
be used to monitor therapeutic responses of a
blood vein s, prima rily in t he lower ex t r em it ies .For
the detection of DVI radioisotope labeled MAb cancer.There are certain limitations in using MAb
i n c a n c e r d i a g n o s i s a n d p r o g n o s i s .T h e s e i n c l u d e
directed against fibrin or platelets can be used.
t h e d i f f i c u l t y i n t h e s e l e c t i o no f a s p e c i f i c M A b a n d
T he ima gin g is u s ually done af t er 4 hour s of
the access of MAb to the target site of the tumor
injectio n. Fibrin sp ec if ic M Abs ar e s uc c es s f ully
which may be lessvascularized.
used fo r th e de tection of c lot s in t high, pelv is , c alf
anci knee regiorrs. MAbs in immunohistopathology of cancers :
Ath ero sclero sis : Thic k ening and los s of The pathological changes of the canceroustissue
elasticity of arterial walls is referred to as can be detected bv immunohistochemical
at hero sclero sis. Ath er os c ler ot ic plaques c aus e t e c h n i q u e s .T h i s c a n b e d o n e b y u s i n g M A b a g a i n s t
dise ases o f co ron ar y and per ipher al ar t er ies . a specific antigen.
A t hero sclero sis ha s been im olic at ed in t ne MAbs in hematopoietic malignancies :
development of heart diseases.MAb tagged with a Hematopoietic stem cells in bone marrow are
radiolabel directed against activated platelets can the precursors for different blood cells, B- and
be used to localize the atherosclerotic lesions by T-lymphocytes which are produced in a stepwise
imaging, technique. t r a n s f o r m a t i o n .D u r i n g m a l i g n a n c y ,t r a n s f o r m a t i o n
of lymphocytes stops at a particular stage of
Gancers maturation. This can be detected by using stage-
specific MAbs.
Mon oclon al an tibodies agains t m any t y pes of
huma n can ce rsare n ov v av ailable.A s elec t edlis t of
Bacterial infections
tumor markers (along rvith the associatedcancers)
t hat can be u se d for M Ab im aging is giv en in In recent years, attempts are made to detect the
Table 17.2. Tumors can be located in patientsusing sites of infections by using MAbs. This is made
radioisotopelabeled MAbs specificto the protein(s), possible by directing MAb against bacterial
particu larly o f me m br ane or igin. I t has been antigens. Further, monoclonal antibodies against
possible to detect certain cancers at early stages inflammaton, leucocvtes which accumulate ar
222 B IOTE C H N OLO G Y
infectionsite are also usefulto specificallydetect bone marrow cells, due to non-availabilityof a
localizedinfections. sui tabl edonor).
\/
gpl20
-----* Cell-mediated
lysis
Fc
Fc
T-cellsinfected
with HIV
T h e f o l l o w i n g a r e s o m e e x a m p l e s o f e n z y me s
that have been used in ADEPT.
I
Normal cell death
carboxyl prodrugs to active drugs.
o Lactamase for hydrolysing B-lactam ring
containing antibiotics.
J
Discarded
blockage in cornary or cerebral artery by a blood
(A) {-Drug
Tumor-specific Conjugation
MAb Monoclonal
antibody
Cell surface
protein
(B)
--'-}- Enzyme
Prodrug Drug
Monoclonal
antibody
Cell surface
protein
M o n o c l o n a l a n t i b o d i e sc a n b e p r o d u c e d f o r a n y
protein. And the so produced MAb can be
clot (thro mbu s ) .Fibr in is t he m ajor c ons t it ue n t o f c o n v e n i e n t l y u s e d f o r t h e p u r i f i c a t i o no f t h e p r o t e i n
blo od clot whic h get s dis s olv ed by plas m i n . a g a i n s tw h i c h i t w a s r a i s e d .M A b s c o l u m n s c a n b e
Plasmin in tu r n is f or m ed by t he ac t iv at io n o f prepared by coupling them to cyanogen bromide
pla smin og en by plas m inogen ac t iv at or T h e activated Sepharose(chromatographicmatrix). The
blockage of arteries occurs due to inadequate immobilized MAbs in this manner are very useful
d issolu tion o f blood c lot s . Tis s ue plas m in o g e n for the purification of proteins by immunoaffinity
activator (tPA)can be used as a therapeutic agent to method.
remove the blood clots.
Biotechnology [15]
226 B IOTE C H N OLO CY
4. MISCELLANEOUS APPLICATIONS L e r n e r a n d h i s a s s o c i a t e sc a r r i e d o u t p i o n e e ri n g
CATALYTTC MAbs (ABZYMES, work in the development of abzymes. They could
c r e a t e a l a r g e n u m b e r o f i m m u n o g l o b u l i n - ge n e
Catalysis is the domain of enzymes. The most l i b r a r i e sf o r t h e p r o d u c t i o n o f a n t i b o d i e st h a t c o u l d
important common chdracter between enzymes be screened for their catalvtic function. Abzymes
and antibodies is that both are proteins. Further.rne represent a major biotechnological advancement
bin din g of an ant ibody t o it s ant igen is c o m p a r a b l e t h a t w i l l h a v e a w i d e r a n g e o f a p p l i c a t i o n s( c u t ti n g
to the binding of an enzyme to its substrate.In both of peptides and DNAs, dissolution of blood clots,
in sta nc es ,t he binding is s pec if ic wit h h i g h a f f i n i t y k i l l i n g o f v i r u s e se t c . )
and involves weak and non-covalent interactions
(electrostatic,hydrogen and van der Waals forces).
Advantages of abzymes
The striking difference is that the enzyme alters the
substrate(to a product) while the antigen bound to T h e n u m b e r o f n a t u r a l l yo c c u r r i n g e n z y m e s a n d
an tibo dy r em ains unalt er ed. t h e i r c a t a l y t i c f u n c t i o n s a r e l i m i t e d . A n t i b o d i e s ,o n
and may be
Certain similarities between enzyme-substrate t h e o t h e r h a n d , a r e u n l i m i t e d ,
developed to possess recognized site structures,
interaction and antibody-antigen interaction have
tempted researchersto explore the possibility of appropriate for the catalytic functions. This makes
using ant ibodies in c at aly s is . T h e a n t i b o d y a b z y m e s v e r s a t i l e w i t h w i d e r a n g i n g c a t a l yti c
technology
enzymes, appropriately regarded as abzymei, are applications. Thus, the area of al:zyme
i s v e r y promising, although the studies are at
the catalytic antibodies.
preliminary stages.
There is a difference in the antibody recognition
of an antigen and enzyme recognitionof a substrate.
Limitations of abzymes
While the antibodies recognize in ground sfate, the
enzymes recognize in a transition state (associated Despite the progress made in the production
with a conformational change of protein). In fact, it and utility of abzymes, it is doubtful whether they
is in t he t r ans it ionc ondit ion t he c at al y s i so c c u r s . l f w i l l e v e r m a t c h t h e n a t u r a l e n z y m e s i n . t he i r
a molec ule r es em bling t he t r ans it i o n s t a t e a n d catalytic function. However, the abzymes will be
conformation (betweensubstrateand product)could certainly useful for a variety of reactionswhere the
be used as a hapten the antibodies so produced natural enzymes do not have the desired
sh ou ld br ing about c at aly s is .This is w h a t p r e c i s e l y s p e ci f i c i t i e s .
is done to create abzymes.
ca the ter. For c er t ain anim als wit h s m a l r e r As the embryos are split, each hemispherical I
reproductive tract, surgical procedures may be cell mass reforms spheres.These split embryos can {
needed to expose the oviduct and recover the be transferred into the oviducts of synchronized
embryos. These embryos are examined recipients The pregnancy rate with split embryos is
microscopically and icjentified. a b o u t 5 - 1 0 % l e s st h a n t h e t h e o r e t i c a lc a l c u l a t i o n s .
Thus, about 9 progencies (insteadof only 5 in the
The embryos are then transferred into a
original embryo transfer)can be developed through
synchronized recipient (i.e. the foster mother) by
MOET
using a pro ce dur e,whic h is c om par ablet o ar t i f i c i a l
insemination. Alternately, the embrvos can oe By MOET, it is possible to produce at least four
frozen and storedfor use at an appropriatetime and i d e n t i c a l o f f s p r i n g si n a n i m a l s a t a t i m e .
place later. About 50-60% of pregnancy could be
achieved in cattle with transferredembryos.Thus, a Embryo biopsy
superovulatedfemale may result in 5-6 pregnancies.
Embryo biopsy involves the removal of a ferv
The enrbryos may also be subjected to cells frorn the embryo (mostly from the trophoblast
ma nip ula tion s .as des c r ibed below. cells of the trophectoderm)for analysisto determine
t h e s e x . I n r e c e r r ty e a r s ,t h i s t e c h n i q u e i s b e c o m i n g
Embryo splitting popular for the detection of genetic diseases.By
It is po ssiblet o inc r eas et he num ber of pr og e n i e s using embryo biopsy, it is possible to stop tl're
(a lmost to do uble) by s plit t ing t he em br y o s . I n transferof embryos with genetic abnormalitiesand
a dd ition , e mbr y o s plit t ing r es ult sin ident ic al t w r n s undesirable traits.
for various purposes(particularlygenetic researcn).
Embryo sexing
Embryo splitting techniques have been refined,
and are routinely used these days. The embryos are Before the transfer or implantation of the
su sp en de d in a hy per t onic ( high os m ot ic ) s u c r o s e embryo, the progeny sex can be determinecl.
230 B IOTE C H N OL O CY
E m b ry o s e x i n g i s b a s e d o n th e pri nci pl e of
The occurrenceof a large number of antral
detecting the presence or absence of Y folliclesthroughoutreproductive life in the ovaries
of cattle and sheepare known. lt is theoretically
c hr o mo s o m ei n th e e mb ry o b i o psy cel l s. The
absence of Y chromosome indicates that the possibleto remove them by biopsy and induce
embryo is a female. oocyte maturationin vitro. This may ultimately
resultin the productionof hundredsand thousands
In recentyears,the presenceDNA sequences
of eggsfrom a singlefemale.However,the potential
specificto Y chromosomeare being detectedfor
of in vitro maturationof oocvtes is limited for
embryosexing.Anotherdevelopmentis the use of
variousreasons-degeneration of follicles,unknown
polymerasechain reactionto amplify the DNA
metabol i c
and hormonalcondi ti ons.
sequence(of Y chromosome)even from a single
c ell a rrdd e te rm i n a ti oth
n e sei. Fertilization of eggs
EMBRVO C LO NI NG
A clone represents a population of cells or ry
org an isms d er iv ed f r om a s ingle anc es t o r c e l l Ovarian
Clon ing ba si c allym eanst he pr oduc t ion of ide n t i c a l ligament
co pie s of an indiv idual.
It wo uld be ac iv ant ageoust o inc r eas e . t h e
number of embryos from a pai'ticularembryo which
possessesthe desired characters.Two approaches
Cervix
are in use fo r em br y o c lonr ng.
1 . Nuclear transfer. Vagina
Azoospermia : Total lack or very low o ln vitro fertilization and embrvo transfer(lVF and
concentration
of motile sperm. ET).
TNTRAUTERTNE (rUrl
TNSEMTNATTON Although it is not always possible to have a
choice in the selection of subjects, the follovring
The infertile women (due to endometriosis, criteria are preferred.
idi opat hic inf er t ilit y ) wit hout bloc k a g e o r d a m a g e
to fallopian tubes can be effectively treated by o Woman below 35 years.
intr aut er ine ins em inat ion. The w o m e n w i t h . Presenceof at least one functional ovary.
adequate ovulation and below the age of 40 years
are c ons ider ed f or I Ul. . H u s b a n d w i t h n o r m a l m o t i l e s p e r m c o u n t ( i .e .
normal seminogram).
The wom en ar e us ually s u p e r o v u l a t e d b y
ad m inis t er ing gonadot r ophins . T h i s r e s u l t s i n . The couple must be negative for HIV and
mult iple egg dev elopm eni. The IU I i s t i m e d t o hepatitis.
co inc ide wit h ov ulat ion.
METHODOLOGY OF
The s em en is was hed and t he h i g h l y m o t i l e 'VF
sperms are separated. By using a thin and soft The in vitro fertilization boardlv involves the
catheter,the sperms are placed either in the cervix following steps.
or in uterine cavity. The women subjects are 1. lnduction of suoerovulation.
advised to remain lying down for about 15-30
minut es f ollowing lUl. 2. Monitoring of ovarian response.
3. Oocyte retrieval.
I ns em inat ions hould be c ar ef ull y t i m e d f o r g o o d
success. lf it is done, a little before the expected 4. Fertilization in vitro.
time of ovulation, the chances for fertilization are
5. Embryo transfer.
muc h higher lUl is us ually s uc c e s s f u li n t h e f i r s t
3-4 attempts. In any case, this approach is not
Induction of superovulation
recommended for more than a maximum of 6
o vu lat ion c y c les . It is well known that the success rate IVF is
much higher when more embryos (3-5) are
The successrates of lUl vary considerably and
transferred. This is possible only with controlled
are in t he r ange of 15- 30% . (COH). The other
ovarian hyperstimulation
advantages of COH include improvement in the
IN V'TRO FERTILIZATION AND quality of oocyte, control of ovulation timing,
EMBRYO TRANSFER (lVF and ETf besidesovercoming the ovulatory dysfunction. The
following drug regimes are in use to induce
ln vitro fertilization broadly deals with
superovulation.
the removal of eggs from a women, fertilizing
them in the lahoratory, and then transferring the . Clomiphene citrate (CC).
fertilized eggs (zygotes) into the uterus a few days
. CC + human menopausal gonadotrophin (hMC).
Iater.
. CC + follicle stimulatinghormone (FSH).
Indications for IVF
. H u m a n m e n o p a u s a lg o n a d o t r o p h i n .
Int-ertilitydue to the following causes may be
. Follicle stimulatinghormone.
considered for lVF.
. Conadotrophin releasing hormone agonists
. Failed ov ulat ion induc t ion
(CnRHa) + hMC (or FSH).
. Tubal diseases
It is now common to use CnRH agonists to
. C er v ic al hos t ilit y induce ovulation.
Chapt er1B : A SS IST ER
DE PR O D U C T IVE
T E C H N OLOC Y 233
These compounds act through a process called of embryo may be transferred. (Note : Excess
do wnre gu lation of t he phy s iologic hy pot ha l a m i c - oocytes and embryos are cryopreservedfor further
p ituitary-ovarian feedback mechanism to effectively u s e . T h i s w i l l r e d u c e t h e c o s t , b e s i d e st h e r i s k o f
suppressspontaneousovulation. ovarian hyperstimulation).
. Increasedrisk of abortion
Fertifization in vitro
. Multiple pregnancy
The semen specimensare collected (just prior to
o Ectopic pregnancy
oocyte retrival) via masturbation, processed, and
in cu ba ted in pr ot ein- s upplem ent ed m edi a f o r . Low birth weigh baby
3-4 ho urs pr ior t o f er t iliz at ion. The inc ub a t r o n r Prematuredelivery.
results in sp er m c apac it at ion.
The retrieved oocytes are also cultured in THE WORLD'S PTCTUBE OF
protein-supplementedmedia for about 6-8 hours. TEST TUBE BAB'ES
For th e pur pos e of lVn 50, 000- 1 , 0 0 , 0 0 0 By employing in vitro fertilization and embryo
ca pa cita ted s per m s ar e plac ed in c ult ur e w i t h a transfer, the world's first test tube baby (Lourse
single oocyte. The signs of fertilization may be Brown) was born in UK on 2}th luly 1978. fhe
.l w o r l d 's s e c o n d t e s t t u b e b a b y ( K a n u p r i y a a l r a s
demonstrated 6-20 hours later bv the presenceof
two p ron uclei wit hin t he dev eloping em br y o . Durga) was born in Kolkata on 3td Cctober 1978.
A t e a m l e d b y S u b h a s hM u k h e r j e e c a r r i e d I V F a n d
There is no need to change the regime for a
E T i n I n d i a . S c i e n t i s t sr e s p o n s i b l ef o r t h e b i r t h o f
single failure of lVF. Many a times, successoccurs
test tube babies were severely criticized then
in th e sub se quentc y c les
ln fact, IVF turned out to be one of the major
The two most importanl criteria for the success
achievements of medical sciences in the last
of IVF are sperm density and motility.
century. lt has become a novel way of treating
i n f e r t i l i t y .T o d a y ,t h e r e a r e m o r e t h a n a m i l l i o n t e s t
Embryo transfer
tube babies born all over the world.
Embryo at a stage between pronuclei and
I n 2 0 0 3 , t h e w o r l d c e l e b r a t e dt h e s i l v e r l u b i l e e
blastocyststage are transferred.Conventionally, 4-
of IVF with much fanfare.
B cell stageembryos are transferredbetween 48-60
ho urs fo llowing ins em inat ion. The t r a n s f e r
GAMETE INTRAFALLOPIAN TRANSFER
procedure is carried out by use of a catheter Not
(GrFTl
more than three embryos are transferred(per cycle)
to min imize m ult iple pr egnanc ies .Howev er , i n t h e Camete intrafallooian transfer involves the
women above the age of 40 years, higher number transfer of both sperm and unfertilized oocyte into
234 B IOTE C H N OLO CY
I CRYOPRESERVATION
Intracytoplasmic
sperm injection Preservation in a frozen state is regarded as
cryopreservation.Cryopreservationis verlzu5sfLrl1n
assistedreproductive technology.
Zona oellucida . Semen can be cryopreserved.This may be frorn
Fig. 18.3 : Micromanipulation for fertilization of an the donors, cancer patients (before the
egg (micrcsurgically assisted fertitization). commencement of treatment).
spermatozoon can be directly injected into the . Embryos can also be preservedfor transfer at a
cytoplasm of the oocyte through the micropuncture later stage.
oI zona pellucida. A micropipette is used to hold
Human enrbryos have been successfully
the oocyte while the spermatozoon is deposited
preserved in the presence of cryoprotectants ('l,
inside the ooplasm of the oocyte.
2-propanediol/dimethyl sulfoxide/glycerol) and
Beside s using nor m al s per m s , r ound- hea d e d s t o r e d a t - 1 9 6 'C u n d e r l i q u i d n i t r o g e n . A t
sperrns, sperms collected directly form the appropriate time, the embryos are thawed,
ep idid ymis an d pr ev ious ly c r y opr es er v ed s pe r m s crvoprotectants removed and then transferreo.
ca n be u se d in lCSl. Many test tube babies in fact have been born as a
Arrong the micromanipulation techniques ICSI result of application of freezing technology.
is the most successful one with a fertilization rate
of about 65%. Attempts are on to improve this ASSISTED HATCHING (AHI
further. In fact, lCSl has revolutionized assistant lmproper implantation of the embryo in the
reproductive technology by utilizing the sperms of uterus is one of the limiting factors in the success
husbands who were once considered to oe of ART in humans. Assisted hatching is a novel
un su itab lefo r fer t iliz at ion pr oc es s . approach for the proper implantation of the embryo
in the endrometrium.
Sub zo na l in s er t ion ( SUZI l
The embryos in the uterus possess an outer
In sub zo na l ins er t ion, t he z ona pelluc ida i s coating namely zona pellucida (the shell). These
cun ctu red a nd s per m s ( 1- 30 in num ber ) a r e embryos must be hatched to remove the shell,
^iected into an area between the zona and the a step necessary for implantation. In certain
=rl. lt is expected that one of the sperms will women, particularly above 40 years age, natural
'-': .:ze th e e 88 hatching does not occur, and requires outside
-lre ma ior lim it at ion of SUZI is poly s per n r y assistance.
^ ce it is no t o os s ible t o c ont r ol t he num ber o f A s s i s t e dh a t c h i n g i s c a r r i e d o u t b y u s i n g a L a s e r
-
;:€11s that enter the egg. to make a small hole in the shell of the embryo.
These embryos when transferred into the uterus,
Ro un d spe rmid nuc leus iniec t ion hatch and get implanteo.
(ROSNT)
During the course of AH for 3-4 days, the
Trere are a few men who cannot manufacture women are kept on steroids (to suppressmother's
::,.-'-r's and therefore they have a zero sperm immunity) and antibiotics (to counter infections).
----ri. For the se m en, it is pos s iblet o t ak e out t h e Better results are reported with this approach.
' ( im m at ur e c ells ) dir ec t ly f r om t h e
--a spe rmati ds
236 B IOTE C H NO LO C\
237
i-, o r ma ny c ent ur ies , m an has been ex ploi t i n g
: microo rga nis m s f or t he pr oduc t ion of f o o d s
(bread, cheese, yoghr,rrts,pickles) and beverages
(beer, wine). Howevet; the organized use of I he heart of fermentatiorr (or bioprocessing)
micro org an ismf or indus t r ial pur pos esis about o n e technology is the fermenter (or bioreactor) A
an d a ha lf ce nt ur y old. bioreactor is basicallv a device in which the
organisms (cells) are cultivated and motivated to
The word fermentation originates from a Latin form the desired product(s). lt is a containment
verb fervere which literally means to boil. During system designed to give right environment for
the p rod uction of alc ohol ( t he f ir s t t r u e l y optimal growth and metabolic activity of the
ind ustrialisedp r oc es s ) ,t he gas bubbles ( of C O r ) orSanrsm.
ap pe ar at th e s ur f ac e of t he boiling liq u i d .
A fermenter usually refers to the containment
Fermentation in a strict sense rs a biological
system for the cultivation of prokaryotic cells
process that occurs in the absence of oxygen
(bacteria, fungi), while a bioreactor grows the
(anaerobic). This definition however, is no more
eukaryotic cells (mammal ian, insect).
valid, since the term industrial fermentation is now
used for large-scalecultivation of microorganisms, Traditionalfermentersare open vats made up of
even though most of them are aerobic (use wood or slate. In recent years, stainless steel
oxyg en ). b i o r e a c t o r sa r e i n u s e . A h i g h q u a l i t y s t a i n l e s ss t e e l
that does not corrode or leak toxic metals into the
Bioprocess technology is a more recent usage
growth medium is used.The size of a bioreactoris
to replace fermentation technology. Bioprocessing
highly variable, ranging from 20 litres to 250
broadly invo'lvesa multitude of enzyme-catalysed
million litres or even more.
re action s ca rried out by liv ing c ells ( or c ell- f r e e
systems) for industrial purposes. Some workers T Y P E S O F B I O R E A G T O R S
prefer to use bioprocess technology for industrial
use ol higher plant and animal cells while Based on the designs of the bioreactors,
fermentation technology is confined to microbial they can be grouped into the following types
( F i g s . 19.1-19.4)
use. This demarcation is however, not very rigid.
1. Continuous stirredtank bioreactors
Bioprocessfermentation technology is very
ivid ely e xp loited f or indus t r ial applic at ions . T h e 2. Bubble column bioreactors
broad range of fermentation products are listed rn 3. Airlift bioreactors
Table 19.1. 4. Fluidized bed bioreactors
24(, B IOTE C H NO LO CY
Group Products
Foods Dairyproducts(cheese,
yogurt)
Vitamins(Br,Brz)
Amino (glutamic
acids acid,lysine)
Glucoseandhighfructosesyrup
Mushroom products
Baker'syeast
Foodadditives(antioxidants, flavours)
colours,
Beverages(beer,wine,whisky)
Chemicals
(bulk)
Organic Ethanol,butanol,acetone, acids(citricacid,gluconic
organic
acid,lacticacid)
(fine)
Organic Enzymes, polymers (xanthan,
dextran)
Inorganic Bioaccumulationandleaching(Cu,U)
Pharmaceuticals( healthcare) Antibiotics
Vaccines
Steroids
Diagnostic
enzymes
Monoclonalantibodies
Enzyme inhibitors
Agriculture protein
Single-celi
pesticides
Microbial
Composting processes
Plantcellandtissueculture
ln all types of bioreactors,the ultimate aim is to In stirred tank bioreactors or in short stirred tank
ensure that all parts of the system are sublected to reactors (STRs), the air is added to the culture
the s am e c ondit ions . m e d i u m u n d e r p r e s s u r e t h r o u g h a d e v i c e ca l l e d
sparger.The spargermay be a ring with many hotes
Gontinuous stirred tank bioreactors o r a i u b e w i t h a s i n g l e o r i f i c e . T h e s p a r g e ra l o n g
A continuous stirred tank bioreactor consistsof with impellers (agitators) enables better gas
a cyiindrical vessel with motor driven central shaft distribr,rtion system throughout the vessel. The
that supports one or more agitators (impeller) fhe bubbles generated by sparger are broken down to
shaft is fitted at the bottom of the bioreactor s m a l l e r o n e s b y i m p e l l e r sa n d d i s p e r s e dt h r o u g h o u t
(Fig. 19.1A). The number of impellers is variable t h e r n e d i u m . T h i s e n a b l e st h e c r e a t i o n o f a u n i fo r m
and depends on the size of the bioreactor i.e., a n d h o m o g e n e o u s e n v i r o n m e n t t h r o u g h o ut th e
height to diameter ratio, referred to as aspect ratio. bioreactor.
The aspect ratio of a stirred tank bioreactor is Advantages of STRs : There are many
usually. between 3-5. However, for animal cell advantagesof STRs,overother types. These incluoe
culture applications,the aspect ratio is less than 2. the efficient gas transfer to growing cells, good
Th e diam et er of t he im peller is usu a l l y {r d o f t h e mixing of the contents and flexible operating
vessel diametei'. The distance between two c o n d i t i o n s , b e s i d e s t h e c o m m e r c i a l a v a i l a b i l i ty o f
impeller s is appr ox im at ely 1. 2 im p e l l e r d i a m e t e r . the bioreactors.
ChA P I CT
19 : B I O P R OC ES S/F E R ME N T A TTIO N N OLOC Y
E CH 241
Jll
t
Airlift bioreacfols are commonly employed for
aerobic bioprocessing technology. They ensure a
controlled liquid flow in a recycle system by
pumping. Due to high efficiency, airlift bioreactors
are sometimespreferrede.g., methanol production,
waste water treatment, single-cell protein
(D) production. In general, the performance of the
, airlift bioreactors is dependent on the pumping
Fig. 19.1 : Types of bioreactors (A) Continuous (injection.o) f air and the liquid circulation.
stirred tank biareactar (B) Bubble column bioreactor
(C) lntemalloop airlift bioreactor (D) Externalloop Two.stage airlift bioreactors
r ,, r ,.' :,airt!ftbioreactgT , i ,, ,
,,; Two-stage airlift bioreactors are used for the
temperature dependent formation of products.
G r o w i n g c e l l s f r o m o n e b i o r e a c t o r ( m a i n t a i n e da t
Bubble column bioreactors
temperature 30'C) are pumped into another
th e a i r o r g a s bioreactor (at temperature 42oC). There is a
In t he bubblec o l u m nb i o re a c to r,
is introducedat the base of the column through necessityfor the two-stageairlift bioreactor,since it
perforatedpipes or plates,or metal microporous is very difficult to raise the temperature quickly
spargers(Fig. 19.18).The flow rate of the airlgas from 30oC to 42"C in the same vessel. Each one
influencesthe performancefactors-O, transfer, of the bioreactors is fitted with valves and they
mixing. The bubble column bioreactorsmay be are connected by a transfer tube and pump
fitted with perforated plates to improve (Fig. 19.2A). The cells are grown in the first
performance.The vesselused for bubble column bioreactor and the bioprocessproper takes place in
bioreactorsis usually cylindricalwith an aspect the second reactor.
ratio of 4-6 (i.e.,heightto diameterratio).
Tower bioreactors .'
Airlift bioreactors
A pressure-cycle fermenter with large
In the airlift bioreactors,the medium of the dimensions constitutes a tower bioreacror
vesselis divided into two interconnected
zonesbv (Fig. 19.28). A high hydrostatic pressuregenerated
Biotechnology
[16]
242 B IOTE C H NO LO C\
Transfer
tube
Fig. 19.2 : Types of bioreactors (A) Two-stage airlift bioreactor (B) Tower bioreactot.
out
Settlingzone
Fluidizedbiocatalyst
Nutrientbroth
Packedbed with
biocatalyst
Outlet
(B)
Photobioreactors
These are the bioreactors specialisedfor
fermentation that can be carried out either by
exposing to sunlight or artificial illumination. Since
ar t if ic ialillumi n a ti o ni s e x p e n s i v eo, n l y th e out
door photobioreactorsare preferred. Certain
importantcompouncis are producedby employing
photobioreactors e.g.,B-carotene, asthaxanthin.
The different types of photobioi'eactors are
depictedin Fig. 19.4.Theyare madeup of glassor
more commonlytransparentplastic.The array of
t ubes or f la t p a n e l s c o n s ti tu tel i g h t re c e i vi ng
systerns(solar receivers).The culture can be
circulatedthroughtlre solar receiversby methods
s uc has us ingc e n tri fu g aplu m p so r a i rl i ftp u m ps.l t
is es s ent ialth a t th e c e l l s a re i n c o n ti n uous
c ir c ulat ionw i th o u t fo rm i n g s e d i m e n ts .F u rther
adequat e pe n e tra ti o no f s u n l i g h t s h o u l d be (D)
maintained.The tubes should also be cooled ro
preventrise in temperature. Fig. 19.4 : Types of phatobiarcactors (A) Continuous
run tubular loop (B) Multiple parallel tube (C) Helical
Photobioreactors are usually operated in a
wound tubular loop (D) Flat panel conliguration.
continuousmode at a temperature in the rangeof
244 B IOTE C HNO LO CY
Air exhaust
Removabletoo
Sightglass
Foam beaker
Connectionsfor acid, alkaliand
antifoamchemicals
lnoculumconnection
Sparger
Jacket -
conneclton
Motor
Reducinggear box
Harvestnozzle
25-4O'C. Microalgae and cyanobacteria are motor at the bottom.The reactionvesselhas side
nor r nally us ed. The or ganis m s g r o w d u r i n g d a y portsfor pH, temperature and dissolvedO, sensors.
light while t he pr oduc t sar e pr od u c e d d u r i n g n i g h t . Above the liquid level of the reaction vessel,
connecti onsfor aci d, al kal i , anti foamchem icals
A COrtNxiESlTICIESALBIOREACTOR - and i nocul umare l ocated.
G O M M O f { FEATI I RES
The bioreactoris usuallydesignedto work at
The different types and designs of bioreactors higher temperature(150-1B0"C), higher pressure
are described. The most common features of a 377-412 kP a).The reacti onvesseli s al sod esigned
typical bioreactor are diagrammaticallyrepresented to w i thstandvaccum,or el sei t may col l apsewhile
in Fig. 19.5, and briefly described hereunder. cooling.The matei'ialsusedfor the construction of
bioreactormust be non-toxicand must withstand
Conv ent ional bior eac t or sar e c y l i n d r i c a l v e s s e l s with high pressure steam.
the repeatedsterilization
r,vith domed top and bottom. The reaction vessel,
surrounded by a jacket, is provided with a sparger The bioreactorvessel is usually made up of
at the bottom through which air (or other gases steel.lt shouldbe free from crevicesand
stainless
s uc h as CO , and NH, f or pH m a i n t e n a n c e )c a n b e accumulate.
stagnantareasso that no solids/liquids
introduced. The agitator shaft is connected to a Easyto cleanchannelsand weldedjoints(instead of
Chapt er19 : BIOP R OC ES S/F E R ME N TA TION
TE C H N OLOC Y 245
arepreferred.
couplings) Transparent materialshould I N O C U L A T I O N A N D S A M P L I N G
be usedwhereverpossible,
sinceit is advantageous
The bioreactor with the growth nredium under
to inspectmediumanclculturefrequently.
aseptic conditions is ready for inoculation with the
production organism.The size of the inoculum is
generally'l-10% of the total volume of the medium.
A high yielding production strain of the organism
taken from a stock culture (lyophilized and stored
in a deep freezer or irr liquid nitrogen)is used.
D u r i n g t h e c o u r s e o f f e r m e n t a t i o n ,s a r n p l e sa r e
The o pe r at ion of a bior eac t or bas ic ally in v o l v e s
regularly drawn from the bioreactor.-fhis is
the following steps.
required to check the contamination (if any) and
1 . Ste riliz at ic ln measurementof the oroduct formed.
2. Ino cu lat ion and s am pling
AERATION
3. Aera t ion
Aeration of the fermentationmedium is required
4. Control systerns to supply O, to the production organisms and
renrove CO, frotn the bioreactor. -lhe aeration
5. Clea ning.
system is designed for good exchange of gases.
Oxygen (stored in tanks in a compi'essedform) is
STERIL IZAT I O N
introducecjat the bottom of the bioreactor through
Aseptic conditions are the basic requirernents a sparger.The small bubbles of the air passthrough
for successfulfermentation That is the bioreactor t h e m e d i u m a n d r i s e t o t h e s u r f a c e T h e b i o r e a c t o r
and its accessories,the grol,r,thmediurn and the arr usually has about 20"k of its volume as vacant
su pp lied d ur ing f er m ent at ion m us t be s t er il e . space on the upper part which is referred to as
head space.The bioreactor has about BO'h working
In situ sterilization v<tlume. The gases released during fermentatron
accumulate in the headspace which pass out
Th e b iore ac t or f illed wit h t he r equir ed m e d i u m
through an air outlet.
is injected with pressurizedsteam into the jacket or
coil surrounding the reaction vessel fhe whole
Air-lift system of aeration
system is heated to about 120"C and held at this
temDerature for about 20 minutes. ln situ ln this type of aeration, sparging of air is done at
ste riliza tionhas c er t ain I im it at ions .lt is not e n e r g y - the bottom of the fermenfer. This allows an uoward
e fficie nt (i.e . , ener gy is was t ed)s inc e t he bio r e a c t o r flow of air bubbles. The more is the aeration
has to be heated fr,rr a long period to rise the capacity of the fermenter,the more is the dissoived
te mpe ratu re of t he whole s y s t em t o 1 2 0 'C . O, in the nredium. Further,the aeration capacity of
Prolonged heating may destroy vitamirrs, besides the air-lift system is directly proportional to the arr-
pre cip itatin gt he m edium c om ponent s . flow rate and the internal pressure Oxygen demand
refers to the rate at n,hich the growing culture
Gontinuous heat sterilization requires Or. For all the aerobic organisms, the
aeration capacity should be more tlran ihe oxyg,en
In th is tec hnique, em pt y bior eac t or i s f i r s t
demand or else the growth of the organismswili be
ste rilize d by injec iing pr es s ur is ed s t eam . T h e
inhibited due to oxygen depletion (starvation).
medium is rapidly heated to 140"C for a short
period, by injecting the pressurised steam
Stirred system of aeration
Alternately, the medium can be sterilized by
p assin g th rough a heat ex c hanger heat e d b y The aeration capacity of the mediurn can be
pressurisedsteam. Subjecting ihe rnediunr to high enhanced by stirring. This can be dc-,neby using
temperature for a short period does not precipitate impellers driven by a motor. The aeration capacity
-'ec/iurrt components. Further there is no enerqy of the stirred fermenter is proportional to the stirring
.: -i-. ;n cont inuous heat s t er iliz at ionm et h o d . speed, rate o{ air {low and the i'nternal pressure
246 B IOTE C H N OLO CY
Stirred fermenters are better suited than air-iift nutrients and O2, besides preventing the
fermentersto produce better aeration capacities, accumulationof toxic metabolic byproducts(if
any). C ood mi xi ng (by agi tati on)al so creat es
CONTRO L SYSTEM S favourable environment for optimal and
It is essential to maintain optimal growth
homogeneousgrowth environment, and good
product formation. However,excessiveagitation
environment in the reaction vessel for maximum
product formation. Maximal efficiency of the may damage microbial cells and increasethe
fermentation can be achieved by continuously
ternperatureof the medium,besidesincreasedfoam
formation.
monitoring the variahles such as the pH,
ternperature, dissolved oxygen, adequate mixing,
nutrient concentration and foam formation.
Nutrient concentration
lmp r ov ed s ens or sar e now av ailable f o r c o n t i n o u s The nutrient concentrationin a bioreactoris
a nd aut om at ed m onit or ing of t hes e v a r i a b l e s ( i . e . , limitedso that its wastageis prevented. In addition,
on line m eas ur em entof oH) . l i mi ti ng concentrati onsof nutri ents may be
M os t of t he m ic r oor ganis m s e r n p l o y e d i n advantageous for optimal productformation,since
fermentation grow optimaliy beiween pH 5.5 and high nutrient concentrations are often associated
8.5. In the bioreactor,as the microorganismsgrow, with inhibitory effect on microbial growth.lt is now
they reiease metabolites into the medium w,hich
possi bl e
to do on-l i ne moni tori of the nut r ient
ng
cl.range pH. Therefore, the pH of the mediurn concentrati on,and sui tabl y modi fy as per t he
sh ou ld be c ont ir r uous lym onit or ed a n d m a i n t a i r . r e d reouirements.
at th e opt inr al lev el. This c ar r be d o n e b y t h e
addition of acici or alkali base (as r.reeded)ano a Foam formation
tlrorough nrixirrg of the fernrentation contents. The media used in industrialfermentationis
Som et ir r es ,an ac id or alk aline m ediu m c o m p o n e n t
general i yri ch i n protei ns.W hen agi tateddu r ing
ca n be us ed t o c or r ec t pH, besi d e s p r o v i d i n g
aeration. it invariably results in froth or foam
n utr ient st o t he gr owing nr ic r oor gan i s m s .
formati on that bui l ds i n head space of t he
bioreactor.Antifoamchenlicalsare usedto lower
Temperature surfacetensi onof the medi um, besi descau sing
Temperaturecontrol is absolutely essentialfor a foam bubblesto collapse.Mineral oils basedon
good fermentation process. Lower temperature si l i coneor vegetabl e oi l s are commonl yused as
causes reduced product formation while higher antifoamagents.
ternperature adversely affects the growth of Mechanicalfoam controldevices,referredto as
micr oor Ranis m s . ' i- he bior eac t or s a r e n o r m a l l y mechanicalfoam breakers, can also be used.Such
e qu ipped wit h heat ing and c ooling s y s t e m s t h a t devices.fittedat the too of the bioreactorbreakthe
can be usecias per the requirement,io maintain the foam bubbles and the throw back into the
reaction vessel at optima! temperature. fermentati onmedi um.
Dissolved oxygen C LE A N IN G
O x y gen is s par ingly s oluble in w a t e r ( 0 . 0 0 8 4
As the fermentationis compiete,the bioreactor
g/1 at 25'C\. Continuous suppiy of oxygen in the
is harvested i.e. the contents are removed for
form of s t er iliz edair is done t o t he c u l t u r e m e d i u m .
processing. The bioreactoris then prepar€dfor the
Th is is c ar r ied out by int r oduc ing a i r i n t o t h e
next round of fermentation after cleaning
bioreactor in the form of bubbles. Continuous
(technicallycalled turn round).The time taken for
rnonitoring of dissolved oxygen concentration is
turn round, referredto as down time, should be as
done in the bioreactor for optimal product
short as possi bl e(si nce i t i s non-produ ct ive) .
folmation.
Due to large size of the bioreactors,it is not
possi bl eto cl ean manual l y.The cl eani ngof t he
Adequate m:xing
bioreactorsis carried out by using high-pressure
Cont inuous and adequat e m i x i n g o f t h e water jets from the nozzlesfitted into the reaction
micr obial c ult ur e ens ur es opt im a l s u p p l y o f vessel.
ChA O t Cr TE C H N OLOC Y
19 : BIOP R OC ES S/F E R ME N T A TION 247
r P r e c i s e m o n i t o r i n g o f S S F ( e . g . , O z a n d C O,
levels, moisture content) is not possible
. T h e o r g a n i s m s g r o w s l o w l y a n d c o n s e q u e n tl y
there is a limitation in product formation.
I n t h e l a b o i 'a t o r y ,p u r e d e f i n e d c h e m i c a l s m a y
b e u s e df o r c u l t u r i n g m i c r o o r g a n i s m sH . o w e v e r , fo r
i n d u s t r i a l f e r m e n t a t i o n s , u n d e f i n e d a n d c o m pl e x
substrates are frequently used for economic
reasons.Cheaper substratesare advantageoussince
t h e y m i n i m i z e t h e p r o d u c t i o n c o s t o f t h e f e r m e nte d
products. Wastesfrom agriculture,and byproducts
of other industriesare generally preferred,although
they are highly variable in composition. Raw
materialsused in fermentation largely depend on
Fig. 19,6 : Bioreactors for solid-substrate their cost at a particular time, since there are
fermentatian. s e a s o n a lv a r i a t i o n s .
T h e c h o i c e o f t h e m e d i u m i s v e r y c r i t i c a l fo r
s u c c e s s f u l p r o t l u c t f o r m a t i o n . F o r i n d u str i a l
. Manv dom es t ic , indus t r ialand agr ic u l t u r a lw a s t e s [ e r n r e n t a t i o n , t h e m i c r o o r g a n i s m s , i n g e n er a l ,
can be f r uit f ully us ed in SSF utilize a luxury metabolism. Therefore, good
production yields are expected with an aburrdant
Limitations of SSF
s u p p l y o f c a r b o n a n d n i t r o g e n s o u r c e s , b e si d e s
. The m ic r oor ganis m s t hat t oler a t e o n l y low requisite growth factors. The media used in
mois t ur e c ont ent c an be us ed. fermentation processes rnay be synthetic or crude.
TE C H N OLOGY
Chapt er1 9 : B IO P R OC ES S/F E R ME NTA TION 249
Glucose formed from glycogen and trehalose A brief account on the sterilization of tne
during yeast extraction is a good carbon source. bioreactor has already been described (See p...). A
Yeast extracts are produced from baker's yeast bioreactor can be sterilized by destroying the
th rough aut oly s is ( at 50- 55' C) o r t h r o u g h organisms by heat/chemicals/radiation or sometimes
p ias m oly s is ( high c onc ent r at ion o f N a C l ) . Y e a s t by physical procedures such as filtration.
extractsare very good sourcesfor many industrially Sterilizationof media and air are discussedbelow.
important microorganisms.
Soy meal : After extractingthe soy bean oil from STERILIZATION OF CULTURE MEDIA
the so1,bean seeds,the left out residue is soy rneal. The constituents of culture media, water and
It i s r ic h in pr ot eins ( about 50% ) a s w e l l a s containers contribute to the contamination by
carbohydrates (abor-rt307o) contents. Soy meal is vegetativecells and spores.The mecjiamust be free
otien used in antibiotic production. from contamination before use in fermentation.
Peptones : I he protein hydrolysates are Sterilization of the media is most comnronly
collectively referred to as peptones, and they are achieved by applying heat, and to a lesser extent
good sources for many microorganisms. The by other means (physical methods, chenrical
sourcesof peptonesinclude meat, soy meal, peanut treatment, radiation).
seeds, cotton seeds and sunflower seeds. The
proteins namely casein, gelatin and keratin can Heat sterilization
also be hydrolysed to yield peptones. In generat,
Heat is the mosf widely used sterilization
peptones derived from animal sources have more
technique. The quality and quantity of
nitrogen content while those frorn plant sources
contamination (i.e., the type and load of
have more carbohydrate content. Peptones are
microorganisms),compositiorr of the media and its
relatively more expensive, hence not widely used
pH and size of the suspended particles are the
in i ndus t r ies .
imoortant factors that influence the suciess of heat
sterilization. In general, vegetative cells are
SOURCES OF GROWTH FACTORS
destroyed at lower temperature in a short time
Sorne of the microorganismsare not capable of (around 60'C in 5-1 0 minutes). However,
synthesisingone or more growth factors such as destruction o{ spores requires higher temperature
vitamins. These growth factors are very expensive and relatively longer time (around BO"C for 15-20
in pure form, hence crude sources are preferred. minutes). Spores of Bacillus stearothermophilus are
Yeast extract is a rich source of almost all growth the most heat resistant. In fact, this organism is
factors. exploited for testing the sterility of fermentation
equipment.
Cenerally, the substratesderived from plant or
animal sources in a crude form are reasonablyrich
in mineral conteht. Sometimes, however mineral Physical methods
(phosphate, sulfate) supplementation may be
The physical methods such as filtration,
reouired.
centrifugation, and adsorption (to ion-exchangers
or activated carbon) are in use. Among these,
filtration is most widely used. Certain constituents
(vitamins,blood components, antibiotics)of culture
media are heat labile and therefore, are destroyed
by heat sterilization. Such components of the
For successful fermentation, it is absolutely medium are completely dissolved (absolutely
esserrtialto ensure. essentialor eise they will be removed along with
microorganisrns) and then subjected to filter
. Sterility of the media containing the nutrients.
sterilization. There are a couole of limitations of
o St er ilit y of inc om ing and out goin g a i r . filtration technique.
. Sterility of the bioreactor.
1 . A p p l i c a t i o n o f h i g h p r e s s u r e i n f i l t r a t io n r s
.. Preventionof contamination during fermentation. u n s u i t a b l et b r i n d u s t r i e s .
TE C H N OLOC Y
Chapt er19 : BIOP R OC ES S/F E R ME N T A TION 251
---)A i rout
Diffusion
varia tion in t he quant it y of s us pendedp a r t i c l e sa n d recent years, glassfiber filter cartridges(that do not
microb es in t he at m os pher ic out do o r a i r . T h e h a v e t h e l i m i t a t i o n s o f g l a s s w o o l f i l t e r ) a r e b e in g
microo r ganis m s m ay r ange f r om 1 O- 2 , O O O / n 3 useo.
wh ile t he s us pendedpar t ic les m ay be 2 O - 1 0 0 , 0 0 1
Membrane cartridge filters : These are
m3 . Am ong t he m ic r oor ganis m spr es e n t i n t h e a r r ,
removable pleated membrane filters made up of
the fungal spores(50%) and Cram-negativebacteria
cellulose ester, nylon or. polysulfone. Membrane
(4 0% ) dom inat e.
c a r t r i d g e f i l t e r s a r e s m a l l e r i n s i z e , s i m p l e r fo r
Air or ot her gas esc an be s t er iliz ed b y f i l t r a t i o n , operation and replacement.
h ea t, UV r adiat ion and gas s c r ubb i n g . A m o n g
T h e m o s t i m o o r t a n t l i m i t a t i o n o f a i r s t e r i l i z a ti o n
th ese, heat and f ilt r at ion ar e m os t c om m o n l y u s e d . 'is
that there is no filter that can remove
bacteriophages. Bacteriophages are capable of
Air sterilization by heat
crippling the i n d u s t r i a l f e r m e n t a t i o n . e .8 .,
In the early years, air was passed over bacteriophages interfere in the production of
e lectric ally heat ed elem ent sand s t er il i z e d .B u t t h i s glutamic acid by Corynebacteriumglutamicum.
is qu it e ex pens e, henc e not in us e t he s e d a y s .
4 A erosoldi l uti on
Tasrr 19.3 A selected list of important
Flotation
fiiicyoorganismsand their products
o. C entri fugati on.
Microorganism Product
F o r f u l l d e t a i l s o n t h e i s o l a t i o n p r o c e d u r e s ,t h e
Algae reader must refer books on microbiology. The
Chlorellasorokiniana protein
Single-cell actual technique for the isolationof nricroorganisms
maxima
Spirulina protein
Single-cell depends on the source and the physiological
properties of microorganisms.The general scheme
Bacteria
adopted for isolating microorganismsfrom soil or
Acetobacteraceti Aceticacid
water source is given below.
Acetobacterwoodii Aceticacid
Bacillussubtilis Bacitracin . The sample (soil or water) is diluted with sterile
w a t e r t o w h i c h a n e m u l s i f y i n g a g e n t ( T w e e n )i s
B. brevis Gramicidin
added.
B. thuringiensis Endotoxin
Clostridiumaceticun Aceticacid . Sample is throughly mixed and allowed to stand
at room temperature
Methylophil
us nethylotroph
us Glutamicacid
Pseudomonas denitrificans VitaminB.,, . S u p e r n a t a n ti s d i l u t e d , 1 6 - 1 1 o 1 6 - 1 0 .
Actinomycetes o V a r i o u sc u l t u r e m e d i a a r e i n o c u l a t e dw i t h d i l u t e d
aureof
Streptomyces aciens Tetracycline s a m p l e sa n d i n c u b a t e d .
S. griseus Streptomycin . Colonies fronr the plates are isolated and
S. tradiae Neomycin i d e n t i fi e d .
Nocardianediterranei Rifamycin o The required pure strains are maintained and
M!,!g'?l??l?i?
P!w7? Gentamycin preserved.
Fungi
Enrichment methods
Aspergillus
niger Citricacid
for isolation of microorganisms
A oryzae Amylase, cellulase,
'l-he culture conditions can be appropriately
protein
single-cell
Candidalipolytica Lrpase modifiedto isolatecertaintypesof microorganisms.
protein The typesof organisms that can be isolatedby use
C. utilis Single-cell
of enrichmentmethodsis given in Table19.4.For
Penicillium
chrysogenum Penicillin
instance,thermophilescan be isolatedby using
Saccharomyces cerevisiae Ethancl,wine, hi ghtemperature w hi l e aci dophi l es
can be i sol ated
protein
single-cell i n aci di c pH . E nri chmentmethodsare certai nl y
S. lipolytica Citricacid, useful for quick isolation of specific types of
protein
single-cell orS anrsms.
Rhizopusnigricans Steroids
Gibberellafujikuroi Gibberellin Strains of microorganisms
Trichoderna vuide Cellulase from unusual environments
Biotechnologistsoften prefer to isolate
bacteria, 104-106 actinomycete spores and
microorganisms from very extreme and unusual
102--104fungal spores. The common techniques environments. This is done with a hope that such
e mplo ye d f or t he is olat ion of m ic r oor gan i s m sa r e strainsmay be capableof
producingnew products
given below. of i ndustri al
i mportance. The unusualenvi ronments
such as cold habitats,high altitudes, deserts,deep
1 . Dire c t s ponge of t he s oil
seaand petroleum fields areconstantlybeing tried
2 . Soil dilut ion for this purpose.The enrichmentmethodsdescribed
3 . Crad ient plat e m et hod ( pour plat e an d s t r e a k above(Iable 19.4)will be very usefulfor isolating
p late techn ique) Lrnusual strai ns.
254 B IOTE C H N OLOCY
Thermophiles (42-100"C)
Hightemperature
Psychrotroohs Lowtemperature
(5-15'C)
Acidophiles LowpH (2-4)
Halophiles HighNaClconcentration
The microorganisms possess tremendous
Anaerobes N, atmosphere
capacityto producea wide rangeof productsthat
Actinoplanes grains
Pollen havecommercialvalue.The primaryand secondary
metabolisms and bioconversions of microorganisrns
Myxobacteria Woodbark
with specialreferenceto their importancefor the
formationof biotechnologicallyimportantproducts
Screening of metabolites for isolation are di scussedhereunder(Fi g. 19.9). Mi crobial
of microorganisms
growth in relation to primary and secondary
metabolismsis depictedin Fig. 19.10.
'fhe
microorganisms can be tested directly for
the product formation, and isolated. In fact, the PRIMARY METABOLITES
water or soil samples can be directly used or
Primary metabolism, also referred to as
su itab ly dilut ed f or m et abolit e s c r e e n i n g . A g a r
trophophase,is characterizedby balancedgrowth
plates can t-'eused for screeningmetabolitesforrned
l t occursw hen al l the nutri e nt s
of mi croorgani sms.
from the m ic r oor ganis m s . For ins t a n c e , i f t h e
needed by the organismsare provided in the
required product is an antibiotic, then the test
medium. Primarymetabolismis essentialfor the
system consists of the strains of organisms which
verlr existenceand reproductionof cells. In the
inh ibit t he z ones , on t he agar plat es .Th e i n h i b i t o r y
trophophase, the cells possess optimal
activitv indicates the possible presence of some
concentrations of almost all the macromolecules
an tibio t ic being pr oduc ed by t he m icr o o r g a n i s m s .
(proteins,DNA, RNA etc.).
Ano the r ex am ple is t he is olat ion of m ic r o o r g a n i s m s
p rod ucing am y las es .W hen gr own on a g a r p l a t e s It i s duri ng the peri od of trophophasea, n
co nta ining s t ar c h, and t hen s t ained w i t h i o d i n e , exponential growth of microorganismsoccurs.
amylase- or oduc ingor ganis m sc an be id e n t i f i e da n d Severalmetabol.ic products,collectivelyreferredto
isolated. as primary metabolites, are produced in
trophophase (i.e.,duringthe periodof growth).The
Screening for new metabolites, primarymetabolitesare divided into two groups.
and isolation of microorganisms 1. Primaryessentialmetabolites: Thesearethe
Ind us t r ial m ic r obiologis t sc ont inue t h e i r s e a r c h
compounds produced in adequate quanties to
for newer metabolitesproduced by microorganisms.
sustain cell grawth e.g. vitamins, amino acids,
Research work is particularly directed for
nucleosidesThe nativemicroorganisms usuallydo
ide ntify ing
not overproduceessentialprimary metabolites,
c hem ot her apeut ic ally importarrt
products for the treatment of tumors, bacterial since it is a wasteful exercise. However, for
d ise ases( newer ant ibiot ic s agains t r es i s t a n ts t r a i n s )
i ndustri al overproducti on, the regul at or y
mechani sms are sui tabl vmani oul ated.
and viral diseases,besidesseveral other substances
(e .g. h or m ones , enz y m e inhibit or s ) . I n a d d i t i o n , 2. Primary metabolic end products : Theseare
iso lati on of m ic r oor ganis m s f or im pr o v e m e n t o f the normal and traciitional end products of
fooC indusiry, and for efficient degradation of the fermentation processof primary metabolism. fhe
e nvironm ent al pollut ant s and haz ar do u sc h e m i c a l s end productsmay or may not haveany significant
a lso ass um ess ienif ic anc e function to perform in the nricroorganisms,
Chapt er19 : B IO P R OC ESF
S/ER M EN T ATION
TE C H N OLOC Y 255
S U B S TR A TE
Prima r y m et abolit es Se c o n d a r v m e t a b o l i t e s B i o c o n v er s i o n s
although they have many other industrial In the above example, an unbranched
applicationse.g. ethanol,acetone,lactic acid. pathway is shown. This type of manipulation for
overproduction of metabolites can be done for
Carbondioxide is a metabolicend oroduct
branched metabolic pathways also.
of Saccharomyces cerevisiae.This CO, is essential
for leaveningof dough in baking industry. 2. Mutant microorganisms with antimetabolite
limitations in growth : Due to insufficienV resistance which exhibit a defective metabolic
limited supply of any nutrient(substrate or even regulation can also overproduce primary
Or), the growth rate of microorganismsslows metabolites.
t,
down. However,the metabolismdoes not stop. lt l
a
continuesas long asthe cell lives,but the formation SECONDARY METABOLITES I
of oroductsdiffers. As the exponential growth of the
microorganisms ceases (i.e. as the trophophase
Overproduction of primary metabolites ends), they enter idiophase. ldiophase is
Excessiveproductionof primary metabolitesis characierized. lsy secondary metabolism wherein
very importantfor their largescaleusefor a variety
of purposes.
Overproduction of severalmetabolites
hasbeensuccessfully accomplished by elimirrating
the feedbackinhibitionas brieflydescribedbelow.
1. B y u s i n ga u x o tro p h i cmu ta n tsw i th a bl ock
in one of the steps in the biosyntheticpathway
concernedwith the formationof prinrarymetabolite
(this should be an intermediateand not the final
end product).In this manner,the end product(E)
formationis blocked,henceno feedbackinhibition. Hi:
But overproductionof the requiredmetabolite(C) E o
occursas illustratedbelorv. o
o
Feedbackregulation :
Biotechnology
[17]
25ft B IOTE C H N OL O C\
Bandom screening
2. lsolation of antimetabolite resistant strains :
The mutated strains are rarrciomlyselected and A n t i m e t a b o l i t e s w h i c h h a v e s t r u c t u r a l s i m i l a ri ti e s
checked for their ability to produce the desired with metabolites can block the norrrral metabolic
industrial oroduct. This can be done with model o a t h w a v s a n d k i l l t h e c e l l s . T h e m u t a n t s t ra i n s
fermentation units. The strains with maximum yield resistant to antirnetabolites can be selected for
can be selected. Random screening is costly and industrial purposes. In the lable 19.5, a selected
te dio us pr oc edur e. But m any a t im e s , t h i s i s t h e l i s t a n t i m e t a b o l i t e s u s e d f o r s c r e e n i n g th e
only u,ay to find the right strain of mutants metabolites is given.
develooed.
3. Isolation of auxotrophic mutants : An
Selective isolation of mutants auxotrophic mutant is characterizedby a defect in
one of the biosynthetic pathways. As a result, it
There are many rnethods for selective isolatron
requiresa specific compound for its normal growth.
of imoroved strains.
For instance, tyr rnutants of Corynebacterium
1. lsolation of antibiotic resistant strains : The glutamicus require tyrosine for their growth'while
mutated strains are grown on a selective rnedium t h e y c a n a c c u m u l a t e p h e n y l a l a n i n e .T h e i s o l a tr o n
con t aining an ant ibiot ic . The wild s tr a i n sa r e k i l l e d of such mutants can be done by grbwing them on
while t he m ut ant s t r ainswit h ant ibi o t i c r e s i s t a n c e a c o m p l e t e a g a r m e d i u m t h a t c a n s p e c i f ica l l y
can gr ow. Suc h s t r ainsm ay be us ef u l i n i n d u s t r i e s support the biochemically defective mutant.
c hapt er19 : BIo P R OC ES S/F E R ME N T A TION
TE C H N OLOC Y 259
I
o
E
E c)
c
C)
c
()o
Time--+
Time--------J
Fig. 19.12 : Pattern of microbial cell growth in batch
(A) culture or batch feimentation.
C ON T IN U O U S C U L T U R E OR
C ON T IN U O U S F ER M EN T ATION
Continuousfermentationis an open system.lI
involves the removal of culture medium
continuously and replacement of this with a fresh
sterile medium in a bioreactor.Both addition ano
removal are done at the same rate so that the
w o rk i n g v o l u m e re m a i n s c o n stant.Further,to
m a i n ta i na s te a d ys ta tec o n d iti on i n conti nuous Nutrient
process,it is advisablethat the cell lossas a result medium
of outflow is balancedby growthof the organisms.
The two commontypesof continuousfermentation
and bioreactorsare describedbelow (Fig. 19.13).
Turbidity
Homogeneously mixed bioreactors recorder
ln thistype,theculturesolutionis homogeneously
nrixed,and the bioreactorsare of two types
'fhe
Chemostatbioreactors: concentrationof
any one of the substrates (carbohydrate,nitrogen
source,salts,C)2)is adjustedto corrtrolthe cell
growth and maintairra steadystate.
Turbidostatbioreactors: In this case,turbidity
me a s u re m e nits u s e d to mo ni tor the bi omass Nutrient
concentration.The rate of addition of nutrient medium
solutioncan be appropriately adjustedto maintarn Turbidity
a constantcell growth. recoroer
Industrial applications of
continuous fermentation
Continuousfermentationprocesseshave been
used for the production of antibiotics, organic
solvents, single-cell protein, beer and ethanol,
besideswaste-water treatment.
2. The yield of the product is more consistent After 2nd generation, the cell number will be
sincethe physiological stateof the cells is uniform. N o x 2 2 .
3. The 'down time' between two successrve After 3rd generation, Nn x 23, and so on. Thus,
fermentationsfor cleaning and preparing the the number of cells after a given time (Nt) will be
bioreactor for reuse is avoided in continuous as follows :
fermentation. N t =N o x 2 n
4. Continuousfermentationcan be run in a where n is the number of generations.
cost-effective manner.
The term douhling firne (td) ot mean generation
fime (MCT) refers to the time taken for doubling
Disadvantages of the cell number or biomass. The specific growth
continuous fermentation rate constant expressedby [, is the direct measure
Despite many advantages of continuous of rate of growth of the organism. If N is the
fermentation above),it is not veryvvidely number of ceils at a given time, then the increase
(described
used in industries.Some of the drawbacksare in the number of cells (growth rate) with time rs
Iisted. given by the formula.
.l dN =
(1)
. Continuous fermentation may run lrN
dt
continuouslyfor a period of 500 to 1,000 hours.
Maintenance of sterile conditions for such a long lf X is the biomassconcentrationat a given time,
period is difficult. then the increase in the biomass (growth rate) with
time is given by
2. T he re c o mb i n a n t c e l l s w i th pl asmi d
constructs cannot function continuously and dx =
pX (2)
thereforethe productyield decreases. dr
lrr
3. It is not easyto maintainthe samequalityof In general, the specific growth rate (p) is a lrtl
t he c ult u rem e d i u mfo r a l l th e a d d i ti o n s.N utri ent function of the concentration of limiting substrate l ll
1, ll
variationswill alter the growth and physiologyof (S),the maximum specific grorvth rate (p-u*) and a t i r.
the cells,and consequently the productyield. substratespecific constant (Kr). Their relationship
was expressed by Monond by the following
In addition to the disadvantageslisted above, equation
industrialbiotechnologistsare rather reluctantto
S
switch over to continuousfermentationfrom the !t = F.u* (3)
K r+S
batch fermentation.However; it is expectedthat
continuousfermeltationwill alsobecome,popular Both S and K, are expressedas concentrationse.g.,
in due course. in moles or grams per liter.
Aftercompletionof lag phase,the cell enterslog In batch culture, the substrateis initially present
phase which is characterizedby exponential at a higher'concentration i.e. (5) > Kr, hence the
growth (See Fig. 19.12). lf the initial number of equation (3) is approximately 1.
cells is No, then s 1 -=
K +S
Atter 1st generation,the cell number will be
Nn x 21. Thus, p = F_u*.
26,4 B IOTE C H N O LO CY
CLASSIFICATION OF
FEBMENTATION PROCESSES
There are different ways of classifyingthe
s5 fermentation processes.
The major classification as
S, batch,fed-batch,semi -conti nuousand conti r r uous
S3 fermentationprocessesare already describedin
6 some detai l (S eep. 259). A nothercl assi fi c at ion,
-cl basedon the productformationin relationto energy
5
c metabolismis brieflydiscussed below (Fig. 19.15).
I
Type I fermentation
When the product is {ormed directly from the
primary metabolismusedfor energyproduction,it
Time _____+ is referredto as type I and may be representedas.
Type ll fermentation
!n type ll category,the product is also formed
from the substrate used for primary energy
] u."* l- metabolism.However.the product is produced in
0)
g
the secondarypathway, as illustratedbelow.
-c
= A + B -+ C -+ D'..Primarymetabolism
Substrate
(5 I
+ E + F+ C -+ P roduct
Ks
Substrateconcentration(S) At the beginning, the gi'owth of the
microorganisrns is accompaniedby high substrate
(B)
utilizationwith littleor no productformation.Now
the growth is slowed down but the substrate
Fig. 19.14 : Growth curves for unicellular organism in consumpti oni s hi gh, and thi s i s coupl ed wit h
batch culture (A) tllith increasing ccncentrations of a productformation.As is evidentfrom Fig. 19.158,
substrate (S7<S?<E<S4<S) (B) The effect of a given in type ll fermentation,the trophophaseand
substrate concentration on growth rate (Ks = substrate idiophaseare separate.Productionof some amino
concentratjon to prodyce half-maximal growth rate) aci ds, ci tri c aci d and i taconi c aci d are g ood
examplesof type ll fermentation.
o
I t
jit
industrialfermeniationsunder anv one of these
types(1,ll, lil) due to complexnatureof the process
e.g. mycel i um produci ng mi croorgani smsi n
i:
rel ati onto anti bi oti coroducti on.
-c
o
()
o TH E FE R ME N TA TION P R OC E S S
The fermentationprocessbasically consistsof
inoculum preservation, inoculum build-up,
T]me-----*
prefermenter culture and finally production
(B)
fermentation A brief accountof the four stagesof
fermentationis given below.
Inoculum preservation
(culture maintenancef
The preservationof high-yieldingstrains of
microorganisms for fermentationis very important
for productformationin substantial amounts.The
ultimatepurposeof preservation is to maintainthe
o strains,as long as possible,without cell division.
g
Thereare differentmethodsof preservation.
E
o
o
Storage at low (2-6'C) temperature : In this
o
o method,the microorganisms can be stored in a
refrigeratorin liquid culture or as stab culture.
Althoughthis is the easiestmethodof preservation,
llm€ -_--) therei s a hi gh ri sk of contami nati on.
(c)
Storageby freezing: The nricrobialculturescan
be frozen and preservedfor severalyears.In the
Fig. 19.15: Typesof fermentations in rclationto freezers,the preservationcan be done at - 1BoCor,
product formation(A) Type I fermentation(B) TVpell
at -B0oC. For preservationat -1 96'C, liquid
fermentation(C) Typelll fermentation(- represents
nitrogenmust be used.It is very importantthat the
grawthrate; -------correspondto substrate
freezing(and laterthawingwhen required)is done
consumption;- indicatesproduct fomation).
slorvly (usually with a change of 1'Clmin) to
266 B IOTE C H N OLO G Y
Pre ssure : Appr opr iat e m aint enanc e o f (reference electrode, glass electrode) are being
hydro sta tic p r es s ur e, par t ic ular ly in lar ge s i z e d used. In fact, electrodes are also available for
bio rea cto rs is v er y im por t ant . This is beca u s e m c a s u r i n g s e v e r a lo t h e r i n o r g a n i c i o n s .
pre ssureinflu enc est he s olubilit y of O r and COr i n
O, and CO, measurement : Oxygen electrodes
th e cultu re medium . An ov er pr es s ur ein t he r a n g e
and CO, electrodes can be used to measure O,
C).2-0 .5b ar is gener ally us ed
and COz concentrations respectively. The
Aeration : A bioreactor gets aerated by the electrodes are amperometric in nature They are
su pp lv o f O, and t her ef or e, adjus t m ent m us t b e h o w e v e r , s u s c e p t i b l ef o r d a m a g e o n s t e r i l i z a t i o n
ma de to furn is h r equir ed am ount of O " t o t h e
In a commonly used technique, O, and CO,
micro org an is m s .Us ually ,t he aer at ion r at e is i n t h e
respectively can he measured by the magnetic
ra ng e of 0.2 5- 1 . 25 v v m ( v olum e of air / v olum e o f
liq uid /minu te) .
Stirring : The type and the speed of impellers Tns$ 19.6 Inportant parametersthat can be
de termin es the s t ir r ing r at e in a f er m ent e r . I n neasured durlng bloprocessing
ge ne ral,th e im peller s peed dec r eas esas t he s i z e o f
the fermenter increases.Thus, for a small bioreactor Physical parameters
(size 1-2 0 litr es ) ,t he im peller s peed is in t he r a n g e Temperature
of 25 0-3 50 rpm , while f or a lar ge bior eac t or ( s i z e Pressure
around 450 litres, the i.'npeller speed is 60=120 Flowrates
r0 m. Viscosity
OF
l urD r0[y
MEASUREME NT AND CO NTRO L
BIOPROCESS PARAMETERS Powerconsumption
Th ere are a lar ge num ber of phy s ic al, c hem i c a l Chemical parameters
and biological parameters that can be measured pH
during fermentation/bioprocessing(Table | 9.6) for Substrateconientration
data analysisand appropriatecontrol. Some specral
Productconcentration
sensors have been developed to carry out
(dissolved)
O, concenlration
mea su reme nts in t he bior eac t or s . The b a s i c
Waste gasesconcentration
(e.9.CO2)
req uire men to f all t he s ens or sis t hat t hey m u s t b e
sterilizab le. The m eas ur em ent sof t he par am e t e r s lonicstrength
(liste d in Tab le) c an be done eit her dir ec t ly in t h e
Riological parameters
bioreactor or in the laboratory.
Activities
of specific
enzymes
pH measurement : There are pH electrodesthat
Proteinconcentration
can withstan d high t em per at ur e ( s t er iliz a t i o n )
Energetics(ATPconcentration)
p ressu rean d m ec hanic al s t r es s esand
, y et m ea s u r e
DNAiRNA content
tb e o H a c c ur at elv . Com binat ion elec t r o d e s
268 B IOTE C HNO LO CY
o For the purpose of storage and transport. o Freeze dryers (to dry soup ingradients, beverage
. To proiect from contamination extracts).
270
P R OC ES SIN C
Chapt er20 : D OW N ST R EA M 271
s u b j e c t i n gt o s o l i d - l i q u i d s e p a r a t i o n .D e t a i l s o f c e l l
disruption are described later). Some authors use
the term harvesting of microbial cells for the
seoaration of cells from the culture medium.
FLOTAT'ON
CEL LDISRUP TI O N
(physical,chemical Wh e n a g a s i s i n t r o d u c e d i n t o t h e l i q u i d b r o t h ,
enzymaticmethods) i t f o r m s b u b b l e s .T h e c e l l s a n d o t h e r s o l i d p a r t i c l e s
get adsorbed on gas bubbles. These bubbles rise to
the foam layer which can be collected and
BROTHWITHSOLIDS
AND LI Q UI D removed. The presence of certain substances,
referred to as collector substances,facilitates stable
foam formation e.g., long chain fatty acicis,amines
SO LI D_LI Q UI D FLOCCULATION
SEPARATION
(flotation,f locculation, ln flocculation, the cells (or cell debi'is) form
filtration,centrifugation) large aggregatesto settle down for easy removal.
The process of flocculation depends on the nature
o f c e l l s a n d t h e i o n i c c o n s t i t u e n t so f t h e m e d i u m .
Addition of flocculating agents (inorganic salt,
CONCENTRATION organic polyelectrolyte, mineral hydrocolloid) is
(evaporation,liquid- often necessaryto achieve appropriateflocculation.
liquidextraction,
membranefiltration, FILTRAT'ON
precipitation,
adsorption)
F i l t r a t i o ni s t h e m o s t c o m m o n l y u s e d t e c h n i q u e
for separatingthe biomass and culture filtrate. The
efficiency of filtration depends on many factors-
the size of the organism, presence of other
PURI FI CATI OBY N organisms, viscosity of the medium, and
CHROMATOGRAPHY
temperature. Several filters such as depth filters,
(gel{iltration,
absolute filters, rotary drum vacuum filters and
ion-exchange,affinity,
hydrophobicinteraction) membrane filters are in use.
Depth filters : They are composed of a
filamentous matrix such as glass wool, asbestosor
filter paper. The particles are trapped within the
FORMULATION matrix and the fluid passesout. Filamentousfungi
(drying,
freeze-drying, can be removed by using depth filters.
crystallization)
Absolute filters : These filters are with soecific
pore sizes that are smaller than the particles to be
removed. Bacteria from culture nredium can be
removed by absolute filters.
FI NALPRO DUCT
Rotary drum vacuum filters : These filters are
frequently used for separation of broth containing
10-40% solids (by volume) and particles in the size
Fig. 20.1 : A summary of the major steps in
of 0.5-1 Opm. Rotary drum vacuum filters have
downstream processing.
been successfullyused for filtration of yeast cells
272 B IOTE C H N O LO C\
CENTBIFUCAT'ON
The techniqueof centrifugaticn is basedon the
principle of density differences between the
particlesto be separatedand the medium.Thus,
centrifugationis mostly used for separatingsolid
particles from liquid phase (fluid/particle
Fig. 20.2 : Diagrammatic representation of a rotary
separati on).U nl i ke the centri fugati onthat is
drum vacuum filter.
convenientlycarried out in the laboratoryscare,
therearecertainlimitationsfor largescaleindustrial
centrifugation. However, in recent years/
and fi l a m e n to u sfu n g i . T h e e q u i p menti s srmpl e
continuousflow industrialcentrifugeshave been
with low power consumptionand is easy to
deveiopeci.There is a continuousfeedingof the
operate.The filtration unit consistsof a rotating
sl urryand col l ecti onof cl ari fi edfl ui d, w ' hi let he
drum partiallyimmersedin a tank of broth (Fig.
solidsdepositedcan be removedintermittently. The
20.2).As the drum rotates,it picks up the biomass
different types of centrifugesare depicted in
which gets depositedas a cake on the drum
Fig. 20.4, and brieflydescribedhereunder.
surface.This filter cake can be easilyremoved.
Tubular bowl centrifuge(Fig.20.44) : This is a
Membrane filters : ln this type of filtration,
si mpl eand a smal lcentri fuge,
commonl yused r n
membraneswith specificpore sizescan be used.
pilot plants. Tubular bowl centrifuge can be
However,cloggingof filters is a major limitation.
operatedat a high centrifugalspeed,and can be
Therearetwo typesof membranefiltrations-sfatic
run in both batch or continuousmode.The solids
filtration and cross-flow filtration (Fig, 20.3). ln
are removedmanually.
cross-flowfiltration,the culturebroth is pumpedin
a crosswisefashion acrossthe membrane.This Disc centrifuge (Fig. 20.48) : lt consists of
reducesthe cloggingprocessand hencebetterthan severaldiscs that separatethe bowl into settling
the staticfiltration. zones.The feed/slurry
is fed througha centraltube.
Culturesolution
Biomass
concentrate
Membrane
Membrane
Filtrate
(A)
Fig. 20.3 : Filter systems for separation of biomass and culture fiitrate (A) Static-tlow filtration
(B) Crass-flovt filtration.
Chapter20 : DOWNSTREAMPROCESSINC 273
Microfiltration pm
0.1-10 Cellsor cellfractions,
viruses.
Ultrafiltration pm
0.001-0.1 Compoundswithmolecular greater
weights
than1000(e.9.enzymes).
3. Reverse
osmosis pm
0.0001-0.001 Compounds weights
withmolecular less
(hyperfiltration) than1000(e.9.lactose).
Clarified
fluidout Fluid
tn
Cells
Cells
ano
Fluidin solids Clarified Gellsand
(A) (c) fluid out solids
(B) (D)
Fig. 20.4 : Centrifuges commonly used in downstream processing (A) Tubular bowl centrifuge (B) Disc
centrifuge (C) Multichamber centrifuge (D) Scroll centrifuge (decante).
Biotechnology [18]
274 B IOTE C H NO LO CY
*
Ultrasonication Alkalies
I
Osmoticshock Organicsolvents L Glucanase,mannanase
and protease
Heat shock Detergents
(thermolysis)
High-pressure +
*
homogenisation
lmpingement*
Grindingwith glass beacls*
Fig. 20.5 : Major melhods for cell disruption to release the intracellular prcducts
(* indicate fieahanicat methods while all the remaining are non-mechanical).
CELL DTSBUPT'ON i o s t r i k e o r h i t ) . T h e c e l l s a r e d i s r u p t e d b y th e
forces created at the point of contact.
Physical methods of cell disruption
Microfluidizer is a device developed based on the
The m ic r oor ganis m sor c ells c a n b e d i s r u p t e d b y p r i n c i p l e o f i m p i n g e m e n t l t h a s b e e n s u c ce ssfu l l y
c er t ain ohv s ic al m et hodst o r elea s et h e i n t r a c e l l u r a r used for breaking F. coli cells. The advantagewith
oroducts. i m p i n g e m e n t t e c h n i q u e i s t h a t i t c a n b e e f fe cti ve l y
used for disrupting cells even at a low
Ult r as onic at ion : Ult r as onic d i s i n t e g r a t i o n r s
concentratron.
widely employed in the laboratory. However, clue
to high cost, it is not suitable for large-scaleuse rn Grinding with glass beads : The cells mixed
indus t r ies . with glass beads are subjectedto a very high speed
i n a r e a c t i o n v e s s e l . T h e c e l l s b r e a k a s t he y a r e
O s m ot ic s hoc k : This m et h o d i n v o l v e s t h e
forced against the wall of the vessel b1' the beads.
s us pens ionof c ells ( f r ee lr om gr o w t h m e d i u m ) r n
'fhe Severalfactors influence the cell breakage-sizeand
20% buffered sucrose. cells are then transferreo
quantity of the glass beads, concentration and age
to water at about 4'C. Osmotic shock is used for
of cells, temperature and agitator speed. Under
the r eleas e of hy dr oly t ic enz y m e s a n d b i n d i n g
o p t i m a l c o n d i t i o n s , o n e c a n e x p e c t a ma xi m a l
proteins from Cram-negative bacteria.
breakage of about BO% of the cells
Heat shock (thermolysis) : Breakageof cells by
A diagrammatic representation of a cell
subjecting them to heat is relatively easy and
disrupter employing glass beeds is shown In
c heap. But t his t ec hnique c an be u s e d o n l y f o r a
Fig. 20.6. It contains a cylindrical body rn,ith an
v er y f ew heat - s t ableint r ac ellula rp r o d u c t s .
inlet, outlet and a central motor-drivenshaft.To this
High pressure homogenization : This technique shaft are fitted radial agitators.The cylinder is fitted
inv olv es f or c ing of c ell s us pens io na t h i g h p r e s s u r e w i t h g l a s s b e a c j s . T h e c e l l s u s p e n s i o n i s a d d e d
through a very narrow orifice to come out to t h r o u g h t h e i n l e t a n d t h e d i s r u p t e d c e l l s c om e o u t
a t m os pher ic pr es s ur e.This s udde n r e l e a s eo f h i g h t h r o u g h t h e o u t l e t . T h e b o d y o f t h e c e l l d i s ru p te r r s
pressure creates a liquid shear that can break the kept cool while the operation is on.
c el Is .
Mechanical and non-mechanical
lmpingement : In this procedure, a stream of
methods
s us pendedc ells at high v eloc it y a n d p r e s s u r ea r e
forced to hit either a stationarysurfaceor a second Among the physical methods of cell disruption
s t r eam of s us pendedc ells ( im pin g e l i t e r a l l y m e a n s d e s c r i b e d a b o v e , u l t r a s o n i c a t i o n , h i g h - p r e ssu r e
Cha pte r 20 : D O W NSI REAM PRO CTSSI NC 275
Cellsuspension
Disruptedcells
I
I I
+
{- Coolant
O0
oo
o
o oo Glass beads
Oo
0
{- Shaft
0
0
o
o
ni Bodyof disrupter
Radialdisc
Baffleto
preventoutflow
ho mog en isa tion, im pigem ent and gr inding w i t h membrane proteins and lyse the cells. Non-ionic
glass beads ate mechanical while osmotic shock cietergents(although less reactive than.ionic ones)
and heat shock are non-mechanicnT.rh" chemical are also used to some extent e.g., Tritdn X-l00 or
and enzymatic methods (described belc.rw)are non- lween. The problem with the use of detergentsis
mechanical in nature that they affect purification steps, particularly the
s a l t p r e c i p i t a t i o n .T h i s l i m i t a t i o n c a n b e o v e r c o m e
Ghemical methods of cell disruption by using ultrafiltration or ion-exchange chromato-
graphy for purification.
Tre atme nt wit h a lk alies , or ganic s olv e n t s
and detergents can lyse the cells to release the
contents. Enzymatic methods of cell disruption
Alkalies : Alkali treatment has been used for the Cell disruption by enzymatic methods has
extraction of some bacterial oroteins. However. the c e r t a i n a d v a n t a g e si . e . , l y s i s o f c e l l s o c c u r s u n d e r
alkali sta bilityo f t he des ir ed pr oduc t is v er y c r u c i a l m i l d c o n d i t i o n s i n a s e l e c t i v em a n n e r .T h i s i s q u i t e
fo r th e su cces sof t his m et hod e. 9. , r ec om bi n a n t advantageous for product recovery
growth hormone can be efficiently released from
Lysozyme is the most frequently used enzyme
E. coli by treatment with sodium hydroxide at
and is commercially available (produced from hen
p H 1 1.
egg white). lt hydrolysesB-1, 4-glycosidic bonds of
Orga nic solv ent s : Sev er al wat er m is c i b l e the rnucopeptide in bacterial cell walls. The Cram-
orq .rnic so lve nt s c an be us ed t o dis r upt t he c e l l s positive bacteria (with high content of cell wall
: q , meth an ol, et hanol, is opr opanol, br , r t a n o l . mucopeptides)are more susceptibiefor the action
Th ese comp ou nds ar e inf lam m able, henc e r eq u i r e of lysozyme. For Cram-negativebacteria, lysozyme
.:re cia lise de qu ipm ent f or f ir e s af et y .The or ga n i c i n a s s o c i a t i o nw i t h E D T A c a n b r e a k t h e c e l l s A s
: .e'rt tolu en e is f r equent ly us ed. lt is belie v e d the cell wall gets digestedby lysozyme,the osmotic
--.- :oiu en e d is s olv esm em br ane phos pholipidsa n d effects break the periplasmic membrane to release
- -:-:i€Snreffibrane pores for releaseof intracellular t h e i n t r a c e l l u l a rc o n t e n t s .
Certain other enzymes are also used, although
Detergents : Detergentsthat are ionic in nature, less frequently, for cell disruption. For the lysis of
:atio nic-ce tvl tr im et hy l am nr onium br om ide o r yeast cell walls, glucanase and mannanase in
rrro nic-.od ium laur y l s ulf at e c an denatu r e combination with oroteasesare useo
276 B IOTE C HNO LO CY
like carboxymethyland sulfonate, and anion- concentrating them with removal of most of the rl
Drying r n a i n l y b e c a u s e f r e e z e - d r y i n g u s u a l l y do e s n o t
Dr y ing is an es s ent ial c om p o n e n t o f p r o d u c t cause loss of biological activity of the desired
f or m ulat ion. lt bas ic ally inv ol v e s t h e t r a n s f e r o f oroduct.
heat to a wet product for removal of moisture.Most L y o p h i l i z a t i o n i s b a s e d o n t h e p r i nci p l e o i
of the biological products of fermentation are s u b l i m a t i o n o f a l i o u i d f r o m a f r o z e n s t ate . In th e
sensitiveto heat, and thereforerequire gentle drying a c t u a l t e c h n i q u e , t h e l i q u i d c o n t a i n i n g t h e p r o d u ct
methods. is frozen and then dried in a freeze-dryer under
v a c u u m . T h e v a c u u m c a n n o w b e r e l ea se d a n d
Based on the method of heat transfer, drying
t h e p r o d u c t c o n t a i n i n g v i a l s c a n b e se a l e d
devices may be categorized as contact-,
e . g . , p e n i c i l l i n c a n b e f r e e z e d r i e d d i r e ctl y i n
convection-, radiation dryers. These three iypes of
a m p ur e s .
dr y er s ar e c om m er c ially av aila b l e .
i
f
n zymes ar e t he bioc at aly s t s s y nt hes i z e d b y A s per recentesti mates,
and i n sci enti fi cresearch.
L- living c ells . They ar e c om plex p r o t e r n a great majority of industrially produced enzymes
mole cu les t hat br ing about c hem ic al r e a c t i o n s are useful in processes related to foods (45"/"),
co ncern ed wit h lif e. The gener al f eat u r e s o t detergents(35'/"), textiles(10%) and leather (3"k)
en zymes ar e giv en in Chapt er 66. For detai l s on the appl i cati onsof i ndi vi dual
enzymes,Tables21.1-21.3 @iven later)must be
It is fortunat et hat enz y m es c ont inue t o f u n c t i o n
referred
(bring out catalysis)when they are separatedfrom
the cells i.e. in vitro. Basically,enzymes are non-
to xic an d b iodegr adable.They c an be pr od u c e d i n
la rge amo unt s by m ic r oor ganis m s f or in d u s t r i a l
ap plicatio ns .
2',1
282 B IOTE C H N OLO CY
Enzyme Source(s) Application(s) Aninral organs and tissuesare very good sources
for enzymes such as lipases, esterases and
p-Amylase Barley,
soybean Baking,preparation proteases.
The enzyme lysozyme is mostly obtained
of maltcse
syrup from hen eggs. Some plants are excellent sources
Bromelain Pinoqnnlo Baking for certain enzymes-papain (papaya), bromelain
Esterase Wheat Esterhydi'olvsis ( p i n e a p p l e ) .
Ficin Fig Meattenderiser l-imitations : There are several drawbacks
Papain Papaya Meattenderiser, associatedwith the manufacture of enzymes from
tanning,
baking a n i m a l a n d p l a n t s o u r c e s .T h e q u a n t i t i e sa r e l i mi te d
Peroxidase Horseradish Diagnostic a n d t h e r e i s a w i d e v a r i a t i o n i n t h e i r d i s t r i b u ti o n .
Urease Jackbean Diagnostic The most important limitations are the difiicuities
i n i s o l a t i n g , p u r i f y i n g t h e e n z y m e s , a n d t h e co st
factor. As regards extraction of industrial enzymes
Commercial enzymes can be produced from a
from bovine sources, there is a heavy risk of
wide range of biological sources.At present,a great
ccntaminatic,rnwith bovine spongiform encephalo-
majority (80%) of them are from microbial sources.
p a t h y ( B S Ei s p r i o n d i s e a s ec a u s e d b y i n g e s t i o no f
T he dif f er ent or ganis m s and t h e i r r e l a t i v e abnornral proteins)'.For these reasohs, nticrobial
contribution tbr the oroduction of commercial production of enzymes is preferred.
enzvmes are siven below.
Enzymes from mammalian cell cultures
Fungi - 60%
T h e r e e x i s t s a p o s s i b i l i t v o f p r o d u ci n g
Bacteria - 24"/o
c o m m e r c i a l e n z y m e s d i r e c t l y b y m a m m a l i a n ce l l
Yeast - 4% cultures. But the main constraintwill the cost facror
Streptomyces - 2o/o w h i c h w i l l b e e x t r e m e l y h i g h . H o w e v e r , c e r ta i n
Higher anim als - 6% therapeutic enzvmes such as tissue plasminogen
activator are produced by cell cultures.
Higher plants - 4"/o
A r eai br eak t hr ough f or lar ge sc a l e i n d u s t r i a l Enzymes from microbial sources
production of enzymes from rnicroorganisrns Microorganisms are the most significant and
occurred after 'l 950s. c o n v e n i e n t s o u r c e s o f c o m m e r c i a l e n z ym e s.
They,can be made to produce abundant quantities
Enzymes from animal and
o f e n z y m e s u n d e r s u i t a b l e g r o w t h c o n d i t i o n s.
plant sources
M i c r o o r g a n i s m s c a n b e c u l t i v a t e d b y u si n g
lr r t he ear ly day s , anim al and p l a n t s o u r c e s inexpensive media and production can take place
largely contributed to enzymes. Even now, for i n a s h o r t p e r i o d . I n a d d i t i o n , i t i s e a sy to
certain enzymes they are the major sources. m a n i p u l a t e m i c r o o r g a n i s m si n g e n e t i c e n g i n ee r i n g
Preserved
inoculunr
FinalpuriJication Cool
(chromatographyetc.) srorage
Fiq.21.1 : An outline of the flow chart for the production of enzymes by m'rcroorganisms.
REGULATI O N O F M I CRO BI AL
ENZYME PRODUCTION Tlrrr 21.4 $electedexanplesof lnduclb-le
_GEN ERAL CO NSI DERATI O NS enrynesalongwith the inducers
The native metabolism of microorganism is so Enzymes are the functional products of genes.
devised that there occurs no production of Therefore, theoretically, enzymes are good
un ne c es s ar y enz y m es . I n ot her w o r d s , t h e candidates for improved production through
microorgnaismsdo not synthesizeenzymes that are genetic engineering
no t r equir ed by t hem , s inc e t his i s a w a s t e f u l
D u r i n g t h e p a s t 1 5 y e a r s , t h e a d v a n c e s i n th e
e xe rc is e. The inhibit ion of unwan t e d e n z y m e
recombinant DNA technology have certainly
production is done by nutrient repression. The
h e l p e d f o r i n c r e a s i n gt h e m i c r o b i a l p r o d u c t i o n o f
nutrients may be carbon, nitrogen, phosphate or
commercial enzymes. lt is now possible to transfer
su lfat e s upplier s in t he gr owt h m edi u m . F o r l a r g e
the desired enzyme genes from one organism to
scale production of enzymes, nutrient repression
the other Once an enzyme with a potential use
must be overcome.
i n i n d u s t r y i s i d e n t i f i e d , t h e r e l e v a n t g e n e ca n
Glucose repression is a classical example of be cloned and inserted into a suitable production
n utr ie nt (more app rop ri ately catabol ite) rep ression. host.
247
Chapter21 : E N Z YMET E C H N OL OC Y
Selectionof microorganism
with an enzymeof
industrialaPPlication
tn its
properties make lipolase a stronB candidate for
use fabric w a s h i n g .
\-)'\ /'/
-=-.=..--=.-_--./-----
I
tdentit*ationof
cDNAclones production.
with Probes)
(bYhYbridization
I for modification of
Protein engineering
I industrial enzymes
J
Transformationof
industrialhost organism
(e.g' A' oryzae1
I
I
J
IndustrialProduction
of desiredenzyme
Cloning strategies
Microencapsulation
(B)
u m i c r o e n c a p s ul a t i o n .
tr 1. Building of special membrane reactors.
u n 2. Formationof emulsions.
3. Stabilization of e m ul s i o n s to form
n n m i c r o c a p s ul e s .
Govalent binding
irt=:s"ology [19]
290 B IOTE C H N O LO CY
1. Cyanogenbromide activation : The inert involves the risk of denaturation of the enzyme by
supportmaterials(cellulose,sepharose, sephadex) the polyfunctional reagent.
containingglyccllgroups are activatedby CNBr,
whic h th e n b i n d to e n z v m e sa n d i m mobi l i zethem CHOICE OF IMMOBILIZATION
( F ig .2 t.6 A ). TECHNIOUE
HCI
* fnzir pq.
=N:N ------l
\-r
Oligogluta-
raldehyde
Fig. 21.6 : lmmobilization of enzymes by covalent binding (A) Cyanogen bromide activation, (B) Diazotation,
(C) Peptide bond formation, (D) Activation by bifunctional agent.
292 B IOTE CHNO LO CY
Stabilization by rebuilding
the stabilityof the enzymesis due
Theoretically,
F to hydrophobicinteractionsin the core of the
l1 enzyme.lt is therefore,proposedthat enzymescan
frr be stabi l i zed by enhanci ng hydr ophobic
interactions.For this purpose,the enzyme is first
Fig, 21,7 : Immobilization of enzyme molecules by unfold and then rebuilt in one of the following
cross linking. ways (Fig. 21.8).
o The enzymecan be chemicallytreated(e.9.urea
and a disulfide)and then refolded.
Stabilization by salts
. The refoldingcan be done in the presence
of low
Stabilityof metalloenzymes can be achievedby mol ecul arw ei ghtl i gands.
adding saltssuch as Ca, Fe, Mn, Cu and Zn e.g.
r For certain enzymes, refolding at higher
proteasescan be stabilizedby addingcalcium.
temperatures (around50"C)stabilizesthem.
*gP
Refoldingin
the presence
Unfold of a ligand
Native enzyme
Escherichia
coli Forthesynthesis acidfromfumaric
of L-aspartic acidandNH,
Escherichia
coli Forthe production
of L-tryptophan
fromindoleandserine
Pseudomonas
sp Production fromglycineandmethanol
of L-serine
Sacch ae
aronycescerevisi Hydrolysis
of sucrose
sp
Saccharomyces Largescaieproduction
of alcohol
Zymononas
mobilis Synthesis andgluconic
of sorbitol acidlromglucose
andfructose
Anthrobacter
sinplex of prednisolone
Synthesis fromhydrocortisone
Pseudononas
chlororaphis Production
of acrylamide
fromacrylonitrile
Hunicolasp Fortheconversion
of rifamycin
B to rifamycin
S
andyeasts(several
Bacteria sp) In biosensors
Membrane reactors
Severalmembraneswith a variety of chenrical
compositions can be used. The commonly used
membrane materials include poivsulfong,
polyamide and cellulose acetate The biocatalysts
(enzvmes or cclls) are normally retained on the
membranes of the reactor. The substrate is
introduced into reactor while the product passes
o u t . C o o d r n i x i n g i n t h e r e a c t o rc a n b e a c h i e v e d b y
using stirrer (Fig. 21.10A\. In a continuous
membrane reactor, the biocatalysts are held over
membrane lavers on to w,hich sr:bstratemolecules
are passed (Fig. 21.108\.
APPLICATIONS OF IMMOBILIZED
ENZYMES AND CELLS
Magnetic
stirrer lmmobilized enzymesand cells are very widely
used for industrial, analytical and therapeutic
Fig. 21.10 : Membrane reactors (A) Batch membrane purpose, besides their involvement in food
reactor, (B) Continuous membrane reactor, production and exploring the knowledge of
(C) Recycle membrane reactor (Coloured lines biochemistry, microbiology and other allied
indicate membranes). specialities. A brief account of the industrial
applications of immobilized cells is given in
Table 21.5.
there occurs inadequate product formation in flow
type reactors. Further, plug flow reactors are also MANUFACTURE OF COMMENCTAL
useful for obtaining kinetic data on the reaction PRODUCTS
systems.
A selected list of important immobilized
enzymes and their industrialapplications is given
Continuous reactors in Table 21 .6. Some details on the manufacture of
L-amino acids and high fructose syrup are given
In coniinuous enzyme reactors,the substrateis
hereunder.
a dd ed cont inuous ly while t he pr oduc t is r e m o v e d
simulta neous ly .I m m obiliz ed enz y m es c a n a l s o b e
Production of L.amino aeids
used for continuous operation. Continuous reactors
have certain advantagesover batch reactors.These L - A m i n o a c i d s ( a n d n o t D - a m i n o a c i d s )a r e v e r y
includ e c ont r ol ov er t he pr oduc t f o r m a t i o n , important for use in food and feed supplementsand
convenient operation of the system and easy medical purposes. The chernical methods
automation of the entire process.There are mainly employed for their production result in a racemic
two tvoes of continuous reaclors-continuous stirred mixture of D- and L-amino acids. Thev can be
296 B IOTE C H N OLO CY
acvlated to form D, L-acyl amino acids. The High fructose syrup can be produced from
immobilized enzyme aminoacylase (frequently glucose by employing an immohilized enzyme
imm obiliz ed on DEAE s ephadex ) c a n s e l e c t i v e l y glucose isomerase. The starch containing raw
hydrolyse D, L-acyl amino acids to produce materials(wheat, potato, corn) are subjectedto
L-a m ino ac ids . hydrolysisto produceglucose.Glucoseisomerase
then isomerises glucoseto fructose(Fig.21.11\.fhe
Aminoacylase
D, L-Acyl > L-Amino acids + product formed i s H FS contai ni ng about
a m ino ac ids D, L- Ac v l a m i n o a c i d s 50ozb fructose.(Note : Someauthorsuse the term
high fructose corn syrup i.e. HFCSin place of HFS)
The free L-amino acids can separatedfrom rne
unhydrolysed D-acyl amino acids. The latter can Gl ucose i somerase: Thi s i s an i ntracellur ar
be recemized to D, L-acyl amino acids and enzymeproducedby a numberof microorganisms.
recycled through the enzyme reactor containing The species of Arthrobacter, Bacillus and
imm obiliz ed am inoac y las e. Huge q u a n t i t i e s o f Streptomycesare the preferredsources.Being an
L -met hionine, L- pheny lalanine L- t r y p t o p h a n a n d i ntracel l ul arenzyme, the i sol ati on of gl ucose
L -va linear e pr oduc ed wor ldwide by t h i s a p p r o a c h . isomerasewithout loss of biological activity
requi resspeci aland costl y techni ques.Man y a
Production of high fructose syrup ti mes, w hol e cel l s or partl y broken cel l s ar e
i mmobi i i zedand used.
Fructose is the sweetest among the
monosaccharides,and has twice the sweetening
strengthof sucrose.Clucose is about 757o as sweet ENZYMES AND CELLS.
as sucrose. Therefore,glucose (the most abundant 'MMOB'LTZED APPL'CAT'ONS
ANALYT'CAL
monosaccharide)cannot be a good substitute for In biochemical analysis
sucrose for sweetening. Thus, there is a great
demand for fructosewhich is very sweet, but has the
l mmobi l i zedenzymes(or cel l s)can be usedf or
same calorific value as that of glucose or sucrose
the developmentof preciseand specificanalytical
techniques for the estimation
of severalbiochemical
High fructose syrup (HFS) contains compounds.The pri nci pl e of anal yti cala ssay
approximately equivalent amounts of glucose and pri mari l yi nvol vesthe acti on of the i mmob ilized
fructose. HFS is almost sirnilar to sucrose from enzymeon the substrate. A decrease in the substrate
nutritional point of view. HFS is a good substitute concentration or an increasein the productlevelor
for sugar in the preparationof soft drinks, processed an alterationin the cofactorconcentration can be
foods and bakine. used for the assay.A selectedlist of examplesof
Chapt er21 : EN Z YMET E C H N OL OC Y 297
Starch
(wheat,potatoes,corn)
| ,r-Rmvlase
I and glucoamylase
+
Glucose
Thermistorlmmobilized
u**"*
I isomerase enzyme
J
High fructosesyrup (B)
(fructose/glucose" 50/50)
Electrode
Fig. 21.11 : Production of high fructose syrup from
statch (glucose isomeraso is the immobifized
enzyffie).
{-lmmobilized
immobilizedenzymesused in the assayof some enzyme
substancesis given in Table 2I .7. fwo types of
detectorsystemsare commonlyemployed.
Glass
Thermistorsare heat measuringdeviceswhich (c) electrode
can record the heat generatedin an enzyme
catalysedreaction.Electrodedevicesare used for
measuringpotential differencesin the reaction
system.ln the Fig. 21.12, an enzyme thermistor
and an enzyme electrode, along with a specific
ureaseelectrodeare depicted.
Glucoseoxidase Glucose
Urease Urea
A biosensoris an analyticaldevice containing
Cholesterol
oxidase Cholesterol
an immobilized biological material (enzyme,
Lactate
dehydrogenase Lactate anti body,nucl ei c aci d, hormone, organel l eor
Alcohol
oxidase Alcohol whole cell) which can specificallyinteract with
Hexokinase ATP an analyte and produce physical, chemical
Galactoseoxidase Galactose or electrical signals that can be measured.
Penicillinase Penicillin An analyte is a compound (e.9. glucose,urea,
Ascorbic
acidoxidase Ascorbicacid drug, pesticide) whose concentration has to
be measurecl.Biosensorsbasically involve the
L-Aminoacidoxidase L-Aminoacids
quantitative analysis of various substancesby
Cephalosporinase Cephalosporin
convertingtheir biologicalactionsinto measurable
Monoamine oxidase Monoamine s.
si gnal
29', B IOTE C HNO LO CY
-----)l Processor
I
l)
z)
-4
>.i
(
Analyte Opticallyo!'electronicallY
aclrvesunace
Bound
analyte
Biological
component
(e.9.enzyme)
Tt
-tl _ A
A
T- A
TT A A Current
A A
tI
I
Product
ll
Electrode
Substrate
Enzyme
(immclbilized
on
membrane)
I
I
T TT A
A
trI A ,,, \ H+
------------f
Potential
AA
T
Substrate
I
Product Potentiometric
electrode
Enzyme
the reference electrode can be measured. lt is thermometric biosensors.They are more commonly
proportionai to the concentration of the substrate. referred to as thermal biosensors or calorimetric
The major limitation of potentiometric biosensorsis biosensors. A diagrammatic representation of a
the sensitivitv of enzvmes to ionic concentrations thermal biosensor is depicted in Fig. 21 ,16. lt
such as H+ and NHl. consists of a heat insulated box fitted with heat
e x c h a n g e r( a l u m i n i u m c y l i n d e r ) .T h e r e a c t i o n ta ke s
Ion-selective field effect transistors (ISFET) are
place in a small enzyme packed bed reactor.As the
the low cost devices that can be used for
substrate enters the bed, it gets converted to a
miniaturization of ootentiometric biosensors. A
product and heat is generated The difference in the
good example is an ISFETbiosensorused to monitor
temperature between the substrateand product is
intramyocardial pH during open-heart surgery.
measured by thermistors. Even a small change in
Gonductimetric biosensots
OPTICAL B'OSE'VSOBS C O 2 +A c e t a t e +H 2 O
Trru 21.8 A selected list of organisms along with the analytes and the types of biosensors
Optical fibre sensing devices are in use for WT'OLE CELT- B'OSEN'SOFS
mea s ur ingpH, pCO , and pO , in c r iti c a l c a r e , a n d Whole cell biosensors are particularlyusefulfor
su rgi c al m onit or ing. multi-stepor cofactor requiringreactions.These
biosensors may employ live or dead microbial
P'EZOELECTR'C BIASEfi'SOBS cells.A selectedlist of someorganismsalongwith
the analytesand the types of biosensorsused is
Piezoelectric biosensors are based on the
given in Table21.8.
principle of acoustics (sound vibrations), hence
they are also called as acoustic biosensors.
Adtramtages 0f m;crobial eell
Piezoelectric crystals form the basis of these
blosensErs
biosensors.The crystalsr,vithpositive and negative
charges vibrate with characteristic frequencies. The mi crobi alcel l s are cheaperw i th l onger
Adsorption of certain molecules on the crystal half-lives. Further, they are less sensitive to
surface alters the resonancefreouencieswhich can variationsin pH and temperaturecompared to
be measured by electronic devices. Enzymeswith isolatedenzymes.
9BPl"'?-1E N Z Y M ET EC H N O L OC Y 303
/\.:,',,,
.r'"', , \
-)
'.--t,/t'..
ni'
(B)
ii'",
,-, o^ on
Z,o,-"
Ag-Ab
(c) a@ @ 00r@
+A A A
+/\
,M' ,t__\
A +
.&-,
Ag-Enz + Ag
(D)
Fig. 21.18 : Diagrammatic representation of selected immunobiosensors (A) Direct binding of antigen to
immobilized antibody, (B) Antigen-antibody sandwiches (immobilized antigen binds to antibody and then to a
second antigen), (C) Antibody binds to immobilized antigen which gets partially released by a competitive free
antigen, (D) lmmobilized antibody binds to free antigen and enzyme labeled antigen (in competilion).
l=iin itatip ns of r nic r obia! c ell hios ens or s based on amperometricor potentiomeiricbio-
sensors.There are severalpossibleconfigurations
The who le c ells , in gener al, r equir e lon g e r
for i mmunobi osensors and some of them are
p erio dsfo r cata ly s is .I n addit ion, t he s pec if ic it ya n d
depi cted i n Fi g. 21.18, and bri efl y descri bed
sensitivity of whole cell biosensorsmay be lower
hereunder.
compared to that of enzymes
1. A n i mmobi l i zedanti bodyto w hi ch anti gen
il,tMUNaSxosE^tsoBs can di rectl ybi nd (Fi g.21.18A ).
lmmunobiosensors
or immunochemical
bio- 2. A n i mmobi l i zed anti gen that bi nds to
sen so rs wo rk on t he pr inc iple of im m unologi c a l anti bodyw hi ch i n turn can bi nd to a free secono
'cecif
icitv, co upled wit h m eas ur em ent ( m os t l y ) anti gen(Fi g.21.18A .
304 B IOTE C H NO LO CY
tt
it
3 . A n a n ti b o d yb o u n d to i mmobi l i zedanti gen
wh i c h c a n b e p a rti a l l yre l e a s e b
d y competi ngw i th
free antigen(Fig.21.18Q. biosensorapplications
storagedisease(Pompe'sdisease).
lmmobilization of artificial cells
;;
A n arti fi ci alcel l pri mari l yconsi stsof a spheri cal ii:
lmmobilized semipermeable membrane with , comparable lr:
glucoseoxidase
di mensi ons of a l i vi ngcel l .The bi ol ogi calmateri al s
suchas enzymesenclosedwithin the artificialcells
can be i mmobi l i zed. The so i mmobi l i zedcompact
in pH
Insulin arti fi ci alcel l scan functi onas arti fi ci alorgans.The
sensitive
polymer important artifical organs constructed include
artificial kidney, artificial liver, blood detoxifiers
and immunosorbents. Theirfunctioningis however,
very l i mi ted.
systemcan be i mmobi l i zedi n the
Mul ti enzyme
form of artificial cells for the conversion of
Fig. 21.20: A diagrammaticrepresentationof
a substrateto a product, through a series of
immobilized glucose oxidase for insulin delivenJ.
reactions.
Biotechnology [20]
f \ ; , | ic r oor ganis m s pos s es s t h e c a p a b i l i t y t o h "i,s *5 $jr!'' S,i 1; ["i i.i{ !'J$ ir$ (ritl! & F E# Hd
i V 6 enz y m at ic allym odif y a wid e r a n g eo f o r g l n i c t;li':+{:"i'[ #:l Ii,
cornpourrds. Biotransfornrations(biconversions or
M a n y t y p e s o f c h e m i c a l r e a c t i o n s occu r i n
microbial transformations) broadly refer to the -fhese
biotransformations. include oxidatian.
processes in u,hich nticroorganisms convert
reduction, hydrolysis, condensation, isomerization,
organic compounds into structurally related
formation of new C-C bonds, synthesis of chiral
products. In other words, biotransformationdeals
compounds and reversal of hydrolytic reactions.
wit h m ic r obial ( enz y m at ic ) c : o n v e r s i o n o f a
A m o n g t h e s e , o x i d a t i o n , i s o m e r i z a ti o n a n d
s ubs t r at eint o a pr oduc t wit h a li m i t e d n u m b e r ( o n e
h y d r o l y s i s r e a c t i o n sa r e n r o r e c o m m o n l y oi r se n ve d
or a f ew) enz y m at ic r eac t ions .T h i s i s i n c o n t r a s tt o
in biotransforrnatior-rs Many a times bro-
fermentation which involves a large number
t r a n s f o r m a t i o n s i n v o l v e m o r e t h a n o n e tvo e o f
reactions (often complex in nature).
r e a c t i o r . r . A s e l e c t e d l i s t o f i m p o r t an t b i o -
Alt hough t her e ar e hu n d r e d s o f bio- t r a n s f o r m a t i o n r e a c t i o n s a l o n g w i t h t h e m i cr o -
t r ans f or m at ions k nown, only a s e l e c t e d f e w o f o r g a n i s m si n v o l v e d i s g i v e n i n T a b l e 2 2 . 1
t hem ar e us ef ul f or t he s y nt hes i so f c o m m e r c i a l l y
T h e c o n v e r s i o n t i m e r e o u i r e d fo r b i o -
im por t ant pr oduc t s . The significance of
transfornratior-r rs related to the tvoe of reaction, the
bioc onv er s ion r eac t ions b e c o m e s obvious
s u b s t r a t e c o n c e n t r a t i o n a n d t h e m i c r o o r e a n i sn - r
when t he pr oduc t ion of a pa r t i c u l a r c o m p o u n d
r u s e d .I n g e n e r a l , o x i d . . r t i o n ,h y d r o l y s i s a n d d e h y-
is eit her dif f ic ult or c os tl y b y c h e m i c a l
d r a t i o n r e a c t i o n sa r e c o m p l e t e d i n a f e w h o u r s
m et hods . Fur t her , biot r an s f o r m a t i o n s a r e
gener ally pr ef er r ed t o c hem ic al r e a c t i o n s b e c a u s e
SOUROES OF BIOCATA!-Y$T$
of s ubs t r at e s pec if ic it y , s t e r e o s p e c i f i c i t y a n d
A f {P T E G F I N I G U E S F O R
m ix ed r eac t ion c ondr t ior r s ( p H , t e n r p e r a t u r e ,
B I O T R A h {S F O R M A T 9 C I F {
pr es s ur e) . The env ir onm ent a l l t o l l u t i o n c i u e
t o biot r ans f or m at ion is alnr os t i n s i q n i f i c a n t o r A r v i d e v a r i e t y o f b i o l o g i c a l c a t a l y s ts ca n b e
negligible. ln addit ion, it i s e a s y k ) a p p l y t u s e df o r b i o t r a n s f o r m a t i o nr e a c t i o n s T h e se i n cl u d e
r ec onr binant DNA t ec hnology t o m a k e d e : s i r e d growing cells, resting cells, killed cells,
im pr ov em ent s in biot r ans f o r m a t i o n s . A n o t h e r immobilized cells, cell-free e\tr.rcts, enzymes .rnd
pr ac t ic al adv ant ageof biot r ansf o r m a t i o n si s t h a t r t immobilized enzymes. The most important sources
is eas y t o s c ale- up t he pr oc es s e sd u e t o l i m i t e d of biocatalysts and the procedures employed for
num ber of r eac t ions . b i o t r a n s f o r r n a t i o na r e b r i e f i v d e s c r i b e d .
306
: B IO T R AN SF OR M AIION S
flPryP:l9igrf:r_- Pentachroroanirine
st!| l:1,Y,u ?,:i:11:!
::_'
Hydrolysis ------+f.tr.ly.lir.
Anhydrotetracycline Streptomyces
aureofaciens
Menthyl ---+ Menthol
laureate Mycobacteriun
phlei
Condensation ------+Streptomycin-phosphate
Streptomycin griseus
Streptonyces
tl
I Stigmasterol
i Chemical
i reactions
Progesterone
Diosgenin
l_ nigricans
I Rhizopus
+
11 o-Hydroxyprogesterone
i Chemical
i reactions
Y
Corynebacterium simplex
Reichstein's (cortisol)
Hydrocortisone Prednisolone
S
substance
(11-deoxycortisol) i Chemical
i reactions
Corynebacterium simplex
Coftisone Prednisone
BIOTRANSFORMATION
OF ANTIBIOTIC S G
Penicillin
Production of new antibiotics or modifications
in the existing ones for more effective treatment Phenyr,aceric
of the diseases is always on the priority of
""'o7
W:-
t he p ha rmaceu t ic al indus t r y . Fur t her , ant ibiot i c s
with wide r an t im ic r obial s pec t r um , r educe d
toxicity, low allergic reactions and decreased 6-Aminopenicillan
ic
\
Benzylpenicilloic
acid acid
resistance are highly advantageous. Biotrans-
formation reactions significantly contribute for I cr'eri.at
improving the pharmaceutical products. I reacvlation
J
Semisynthetic
Direct biotransformation penicillin
Acyla tion a nd deac y lat ion, phos phor y lat io n , Fig. 22.3 : Biotransformationof penicillin G.
ade nylatio n a nd hy dr oly s is ar e s om e of t h e
310 B IOTE CHNO LO CY
3tt
312 B IOTE C H N OLO CY
Glucose
i Glycolysis
+ Acetoacetic
acid
J
Aceticacid CrotonylCoA I
l+
'| CH3-CO-CH3
ButyrylCoA Acetone
I
+l +
Butyricacid
'j lsoProPanol
cH3-cH2-cH2-cH2-oH
Butanol
Fig. 23,3 : Biosynthesis of acetone and butanol (Note : Acetic acid and butyric acid
can also be produced by this pathway)
.-.arrich raw materials (potato, molasses)and the The sequence of reactions leading to the
-'-anism Clostridium acetobutylicum. After World formation of acetone and butanol, along with acetic
,',a' ll, acetone and butanol became readily acid and butyric acid is depicted in Fig. 23.3.
316 B IOTE C H N OLO CY
Glucose
+
1,G-bisphosphate
Fructose
3-phosphate
Glyceraldehyde phosphate
Dihydroxyacetone
NAD+-! I
Dihydroxy- I,-NADH + H+
NADH+ H* +4 acetone (
+ . phosphate l\runO*
1,3-Bisphosphoglycerate qenyorogenase
I
+
Y Glycerol
3-phosphate
Pyruvate
I
+
cH2-oh
ncetailenyde
I
NADH+ H*-11 Sodium
t-
CH-OH
i bisulfite
NAD+</l blocks I
cH2-oh
+
Ethanol GlYcerol
318
Chapt er24 : M IC R O B IALPR O D U C T IO N
OF OR C A N ICA C ID S 319
Glucose
MEDIUM
Pyruvate CYTOPLASM
pyruvare
Ror---.]
I Jcarboxylase
MITOCHONDRION
Citricacid
MEDIUM
Citricacid
Fig. 24.1 : An outline of metabolic pathway for the biosynthesis of citric acid
(TCA cycle-Tricarboxylic acid cycle).
Dissolved O, Solidmedium
Liquidmedium
The yield of citric acid production substantially
increaseswhen the dissolvedO, tension is higher.
Fig. 24.3 : The industrid processes for the
This can be achievedby strong aerationor by production of citric acid.
spargingwith pure Or. lt has been observedthat
s uddenint er ru p ti o ni ns O , s u p p l y(a so c c u rsduri ng
powerbreakdovvns) causedrasticreductionin citric wheat bran or pulp from sweetpotato starchare
acid productionwithout harmingthe growthof the used,as cul turemedi a.The pH of the medi umi s
orSanrsm. adlusted to 4-5, and then sterilized.Now the
inoculumin the form of sporesol A. nigeris spread
Nitrogen source as l ayers(3-6cm thi ckness) and i ncubated at 28" C .
The growthof the organisms can be accelerated by
Ammonium salts, nitrates and urea are the
the additionof cr-amylase. Solid-state fermentation
nitrogensourcesused in the media for citric acid
takes about B0 to 100 hours for maxirrtal
pr oduc t ion.Al l th e th re ec o mp o u n d sa re e qual l y
productionof citric acid.At the end of the process,
good sources,as long as they do not adversely
citric acid can be extractedinto hot water and
effectthe pH of the medium.lf molasses are used
i sol ated.
for nutrientsupply,additionof extranitrogensource
is not required. However, some workers have
Liquid surface fermentation
shown that exogenousaddition of ammonium ions
stimulates citric acid production. Surfacefermentationusing liquid as nutrient
medium is the oldest method for citric acid
PRODUCTION PROCESSES producti on.l t i s sti l l i n use due to a si mpl e
F O R CI T RI C A C ID technology, low energy costs and h igher
reproducibility.Further,the interferenceof trace
There are two processes by which citric acid
metal sand di ssol ved O, tensi onare mi ni mal .The
can be industriallyproduced-the surfaceprocess
labour costs are however, higher since the
and submergedprocess(Fig. 24.3).
manpowerrequirements are more for cleaningthe
The surfaceprocess: This is characterized by systems. About 20% of the citric acid in the world
growing rhe microorganismsas a layeror a film on is producedby surfaceprocesses.
a surface in contact with the nutrient medium,
The nutrient supply for surfacefermentation
which may be solid or liquid in nature.Thus,the
normally comes from beet molasses. The
surfaceprocesshas supported-growth systems.
fermentati on i s usual l ycarri edout i n al umi ni um
T he s ubme rg e dp ro c e s s : In th i s c a s e , the trays fi l l ed w i th steri l e nutri ent medi um. The
organismsare immersedin or dispersedthroughout inoculumin the form of sporesis sprayedover the
the nutrient medium. There are two types of medi um.A steri l eai r i s passedfor suppl yi ngO, as
submerged fermenters (bioreactors) stirred well as cooling. The temperatureis maintained
bioreactors and airlift bioreactors. around 30"C during fermentation.As the spores
germinate (that occurs within 24 hours of
SURFACE PBOCESSES i nocul ati on),a l ayerof mycel i umi s formedover
the medi um.The pH of the nutri entmedi umfal l sto
Solid surface fermentation
less than 2, as the mycelium grows in size and
Surface processesusing solid substratesare forms a thick layer on the surfaceof the nutrient
particularlycarriedout in lessdevelopedareasof solution.The fermentationis stopped after 7-15
rcme Asiancountries.The solid substrates such as days.
)cr=:,hnology[21]
322 B IOTE C HNO LO CY
-o
5
Air tn F
-
L
Nutrient
medium
(raw material)
Surfacefermentation
Air in
C)
c
(g
-c
c)
I
c
o
6
()
g)
or submerged processes N
Fig. 24.5 : Flow chart for industrial praduction of citric acid by surface GI
324 BIOTECHNOLOCY
(A)
POQ , Glucbse',, ,
rdehydrsgenase Heo
(bacteria)
D-Glucose D-Gluconolactone
S Gluconicacid
Lactonase
(fungi)
(B)
1, 3-Bisphosphoglycerate
Lactic acid occurs in two isomeric forms i.e.
L(+) and D(-) isomers, and as a racernic mixture Fig. 24.7 : An outline of the pathway (glycolysis) for
(DL-la ctic acid). The is olat ion of lac t ic ac id f r o m the biosvnthesis of lactic acid.
milk wa s d on e in 1798. lt was t he f ir s t or ganic ac i d
produced by microorganisrnsin 1880. Today, lactic
acid is competitively produced both by Lactobacillus sa are used for lactic acid
micro bio log ica l and c hem ic al m et hods procjuction. However, there are variations in the
substratesutilised as indicated below.
Applications of lactic acid
L. delbrueckli l
The re are d iff er ent gr ades of lac t ic ac id m ain l y . I Clucose
L- letcnamannt )
based on the percentageof lactic acid. The grades
and their applicatiorrsare giverr in Table 24.2. L. bulearicus l
I Whey (lactose)
L. netveilt )
Microorganisms for production of
L. lactis - Maltose
lactic acid
L. amylophilus - Starch
Lactic acid producing bacteria are broadly
L. pentosus - Sulfitewaste liquor
categorized into two types.
Biosynthesis of lactic acid : The synthesis of
Heterofermentative bacteria-produce other
lactic acid occurs through glucose oxidation by
byproducts, besides lactic acid, and therefore are
glycolysisto produce pyruvate which on reduction
not useful for industrial production of lactic acid.
gives laciic acid. The reducing equivalents
These bacteria are employed in food or feed
( N A D H ++ H +) p r o c i u c e d d u r i n g t h e o x i d a t i o n o f
oreservation.
glyceraldelyde 3-phosphate are utilised by the
Homofermentative bacteria-specialised lor enzyme lactate dehydrogenase to form lactate
exclusive oroduction of lactic acid and therefore Gig. za,V. Most of the lactic acid producing
a re su itab le fo r indus t r ial pur pos e. m i c r o o r g a n i s n r sn o r m a l l y p r o c i u c eo n l y o n e i s o m e r
of lactic acid L(+) or D(-). However, some bacieria
which usually occLrras infection can form racenric
Tastl 24.2 Commerclalgrades of lactic acid nrixture.
along with their applications
Production process for lactic acid
Grade (% lactic acid) Application(s)
T h e f e r m e n t a t i o n m e d i u m c o n t a i n s 1 2 - - 1 5 'h o f
grade
Technical Estermanulacture, glucose, nitrogen and phosphate containing salts
(20-50%) industry
textiie anci nticronutrients.The orocess is carried out at
Foodgrade (sour
Foodadditive oH 5.5-6.5 and temoerature 45--50"C for about 75
(>80%) flouranddough) h o u r s . C e n e r a l l y , t h e s t r a i n s o p e i - a t i n ga t h i g h e r
temperature (45-60'C) aie preferred, since it
grade
Pharmaceutical lntestinal
treatment
r e d u c e st h e n e e d f c r m e d i u m s t e r i l i z a t i o r rA . s tne
(>90%) (metalionlactates) lactic acid is oroduced, it has to be removed since
326 B IOTE C H N O LO CY
Production of vinegar
Vinegaris an aqueoussolutioncontainingabouf
4% by volume aceticacid and small quantitiesof
The productionof acetic acid, in the form o{ alcohol,salts,sugarsand esters.lt is widely usedas
vinegar(usedas a refreshing
drink),from alcoholic a flavouringag,entfot processedliquid foods such
liquidshas been known for centuries. as saucesand ketchuos-
Ervviniasp
2, s-Diketo- Acetobactersp
D-Glucose Glucuronic
acid
gluconic
acid Gluconobactersp
I c"tutyti"
reduction
J
Glucuronolactone
2-KetoL-
gluconicacid
:
L-Gulonolactone
2-Keto-Lgulonicacid I
ACI D
L -ASCOBBI C I
+
I
l.^
Enolformof 2-ketoL-gulonic
acid i L-uuronoraclone
dehydrogenase
I
l, e " id ,
Jtreatment
J
L.ASCORBICACID ACID
L-ASCORBIC
Reichstei n-Grussner sy nthesis l I
Fig. 24.10 : Pathways for the commercial production of ascorbic acid. (A) Two-step fermentation process
(B) Reichstein.Grussner synthesis (C) Production via L-gulonolactone.
Glucose Itaconicacid
MEDIUM
CYTOPLASM Calcium
Glucose Itaconicacid
^^l
v v 2\ | Itaconic
) Acotinicacid acidoxidase
decarboxylase
YY I
Pyruvate Pyruvate Cls-Aconiticacrd
Itatarta-ric
acid
L- as c or bicac id. Nor m ally , about 1 0 0 g o f a s c o r b i c process(Fig. 24.10A) has been reduced to one. The
a c id is pr oduc ed f r om 200 g o f g l u c o s e i n genetically engineered Erwinia cells were able to
Reicl'rstein-Crussner synthesis. convert D-glucose directly to 2-keto-L-gluconic
Two-step fermentation process : In th is, a c i d . T h i s i s c e r t a i n l y a d v a n t a g e o u s s i nce th e
D- gluc os e is c onv er t ed t o 2, 5 - d i k e t o g l u c o n i c metabolic caoabilities of two different micro-
acid by Erwinia, Acetobacter or Gluconobacter sp. o r g a n i s m sc o u l d b e c o m b i n e d i n t o o n e o rg a n i sm .
ln the second step, Corynebacterium sp converts 2, However, the yield of ascorbic acid by the hybrid
5 - dik et ogluc onic ac id t o 2- k et o - L - g i u c o n i c acid, strain rvas very low. Scientistsare now trying to
(Fig. 24.10A). lt is also possible to invclve Bacillus a l t e r c e r t a i n a m i n o a c i d s i n 2 - 5 d i k e t o g l u c on i ca ci d
nnegateriunl for converting L-sorbose to 2-keto-L- reductaseand increasethe catalvtic activitv of this
g luc onic ac id. The lat t er , by c h e m i c a l r e a c t i o n s , e n z y m e .
be converted to ascorbic acid.
^" nfiltrt$:an
/ilfrf'" F _ * -,F
\ Production via L-gulonolactone
Ascorbic acid can also be synthesized via-
gulonolactone which can be directly converted to Itaconic acid is used in plastic industry, paper
L-ascorbic acid by the enzyme L-gulonolactone industry and in the manufacture of adhesives.
dehydrogenase(Fig. 24.1 OCt.
I t a c o n i c a c i d c a n b e c o m m e r c i a l l y p r o d uce d b y
Direct production of ascolbic acid by Aspergillus itoconicus and A. terreus. The
fermentation biosynthesis of itaconic acid occurs by way of
Krebs cycle. The metabolite cis-aconitic acid
Several workers are trying to produce ascorbic
(formed from citric acid) undergoesdecarboxylation
acid directly from glucose. Microalgae o{ Chlorella
catalysedby the enzyme cls-aconiticdecarboxylase
have shown some promising results, although the
(Fig. 24.11). ltaconic acid is oxidised to itatartaric
yield is very low.
acid by itacorric acid oxidase. Tl-risenzyme has to
Genetic engineering for ascorbic acid b e i n h i b i t e d f o r a m a x i m u m y i e l d o f i t a c o ni c a ci d .
production This can be achieved by adding calcium.
329
330 B IOTE C H N OL O G Y
APPLICATIONS OF ANTIBIOTICS
The commercialoroductionof antibioticsis a
Ant ibiot ic s ar e par t ic ular ly im p o r t a n t a s
hi ghl y profi tabl ei ndustryw orl dover.A nnualsales
antimicrobial agents for chemotherapy. A large
w i l l run i nto severalbi l l i onsof dollar s
of anti bi oti cs
n umb er of bac t er ial dis eas es hav e b e e n b r o u g h t
with an annualgrowth potentialof about 10%.
u nd er c ont r ol bv us e of ant ibiot ic s .T h e s e i n c l u o e
pneumonia, cholera, tuberculosisand leprosy. The Antibiotics may be produced by microbial
antifungal antibiotic griseofulvin has controlled the fermentation, or chemical synthesis, or a
de bil it at ingf ungal s k in dis eas ess uc h a s r i n g w o r m . combination of both. For certain antibiotics,the
Chaot er25 : MIC R OB IALP R OD U C T IO N
O F A N TIB IOTIC S 331
/-\
Penicillin
G
(benzylpenicillin)
P enic illi n sa re a g ro u p o f p -l a c ta mc o ntai ni ng \_/
bac t er ic idaal n ti b i o ti c sBe
. i n gth e fi rs t a m o ngthe
ant ibiot ic s to b e d i s c o v e re d ,p e n i c i l l i ns are
historicallyimportant.The structuresof important
ll-"-cH,-co Penicillin
V
Action of penicillins
HO cH-co- Amoxicillin
Natural penicillins(penicillinsV and C) are I
NHe
effective against several Gram-positive bacteria.
T hey inhib i t th e b a c te ri a l c e l l w a l l (i .e.
peptoglycan) synthesis and causecell death.Some Oxacillin
persons(approximately 0.5-2% of population)are
aller gict o p e n i c i l l i n .
Natural penicillins are ineffective against
microorganisms that produce pJactamase, since
t his enz y m e c a n h y d ro l y s e p e n i c i l l i ns e.B . Cloxacillin
Staphylococcus aureus. Several semi-synthetic
penic illinst h a t a re re s i s ta nto t B -l a c ta m a se have cHs
been developedand successfullyused againsta
large number of Cram-negative bacteria.
Clox ac illina, m p i c i l l i nfl, o x a c i l l i na n d a z l o ci l l i nare
s om eex am p l e o s f s e mi -s y n th e tipce n i c i l l i n s. These
co- Floxaciflin
are quite comparablein actionto cephalosporins.
F r omt he h u g eq u a n ti ti eosf p e n i c i l l i n ps ro duced cHs
by ferrnentation,about 40"/" are used for human
healthcare, 15o/o for animalhealthcareand45o/o for
Ticarcillin
t he pr epar a ti oonf s e mi -s y n th e tipce n l c i l l i ns.
Nutrients
iI
il:11.,'""
phase can be extendedto 150-180
o Recovery of penicillin
(U
E
c
q) As the fermentationis complete, the broth
c contai ni ngabout 1% peni ci l l i n i s processedfor
O extraction.The myceliumis removedby filtration.
Penicillinis recoveredby solvent(n-butylacetate or
methylketone) extraction at low temperature
(< 10' C ) and aci di c pH (< 3.0).B y thi s w ay, the
chemi caland enzymati c(bacteri alpeni ci l l i nase)
degradati ons of peni ci l l i ncan be mi ni mi zed.
The peni ci l l i ncontai ni ngsol venti s treatedw i th
Fig. 25.4 : Penicillin production in relation to activated carbon to rernove imourities and
substrates utilization and biomass formation.
pi gments.P eni ci l l i ncan be recoveredby addi ng
potassiumor sodium acetate.The potassiumor
sodi umsal tsof peni ci l l i ncan be furtherprocessed
Penicillin production is an aerobic process ancl (i n dry sol ventssuch as n-butanolor i sopropano l)
therefore,a continuous supply of O, to the growing
to remove i mpuri ti es.The yi el d of peni ci l l i n i s
culture is very essential.The required aeration rate
around907o.
is 0 .5-1.0 v v m . The pH is m aint ained ar o u n d 6 . 5 ,
and the optimal temperature is in the range of As the water is totally removed,penicillinsalts
2 5-2 7"C. Penc illin pr oduc t ion is us uall y c a r r i e d can be crystallizedand dried under required
out by submerged processes. pressure.This can be then processedto finally
producethe pharmaceutical dosageforms.
The medium used for fermentation consists of
corn steep liquor (4-5% dry weight) and carbon
sou rce (us ually lac t os e) . An addit ion o f y e a s t
extract, soy meal or whey is done for a good supply
o f nitro ge n.Som et im es ,am m onium s ulf at ei s a d d e d
for the supply of nitrogen. Phenylacetic acid (or
phenoxyaceticacid) which servesas a precursorfor
pe nicillin bios y nt hes isis c ont inuous ly f ed . F u r t h e r ,
continuous feeding of sugar is advantageousfor a
g oo d yie ld of penic illin. The penic illin pr o d u c t i o n
profiles are depicted in Figs. 25.4 and Fig. 25.5.
Penicillins C and H are the fermented products Several mutants of C. acremonium have been
obtained from the fungus Penicillium chrysogenum. developed for improved production of
cephalosporin. Mutants with defective sulfur
PRgDUCTTON OF 6.AM!NO metabolism or those with resistance to sulfur
PENIC I LLANI C ACI D analogs have high yielding capacity. Certain
Th e penic illins C and H ar e nr os t ly u s e d a s t h e regulatory genes of cephalosporin biosynthesis
starting materials for the production of several ( e . g . , i s o p e n i c i l l i n N s y n t h e t a s e )h a v e b e e n c l o n e d
synthetic penicillins containing the hasic nucleus a n d g e n e t i c m a n i p u l a t i o n sc a r r i e d o u t f o r i n c r e a s e d
namely 6-amino penicillanic acid (6-APA). About production of cephalosporins.
10 yearsago, only chemical methods were available
Biosynthesis of cephalosPorin
for hydrolysisof penicillins to produce 6-APA. Now
a days, enzymatic methods are preferred. The early stages of the biosynthetic pathway
for cephalosporin are the same as that for
lmm obiliz ed penic illin am idas esen z y m e s h a v e
penicillin synthesis(SeeFig. 25.2). As the tripeptide
been developed for specific hyclrolysisof penicillin (aminoadipylcysteinylvaline) is formed, it
C an d penic illin V. Penic illin s alt of ei t h e r C o r V p r o d u c e i s o p e n i c i l l i n N.
undergoes c y c l i z a t i o n t o
can be us ed f or hy dr oly s isby im m obili z e d e n z y m e
B y t h e a c t i o n o f e p i m e r a s e ,p e n i c i l l i n N i s f o r m e ci
system. The pH during hydrolysis is kept around
f r o m i s o p e n i c i l l i n N . T h e n , p e n i c i l l i n N g e ts
7-8, and the product 6-APA can be recovered by
converted to cephalosporin C by a three stage
b ring ing down t he pH t o 4. At pH 4 , 6 - a m i n o
reaction catalysed by three distinct enzymes
penicillanic acid gets precipitatedalmost completely
n a m e l y e x p a n d a s e , h y d r o x y l a s e a n d a c e tyl
in the oresenceof a water immiscible solvent.
transferase (Fig. 25.71.
In general, the enzymatic hydrolysis is more Regulationof biosynthesis: A low concentration
e fficie nt f or penic illin V t han f or p e n i c i l l i n C . of lysine promotes cephal<lsporin synthesis. The
Howe v er , penic illin C is a m or e v e r s a t i l e inhibitory effect of lysine at a higher concentration
compound, as it is required for ring expansions. can be overcome by adding L-aininoadipic acid.
The carbon sources that get rapidly degraded (e.g.
glucose, glycerol) reduce c e p h a l o s p or i n
production. Methionine promotes cephalosporin
T he p h a rm a c e u ti c aul s e s o f p e ni ci l l i ns are synthesis in C. acremonium, but has no influence
associated with allergic reactions in some on Streptomycetes.
indiv id u a l sT. o o v e rc o m eth e s ea l l e rg i cprobl ems,
PRODUCTION PROCESS
cephalosporins were developed. They have
OF CEPHALOSPORIN
improved stability againstB-lactamases, and are
more active against Cram-negative bacteria. The fermentation process concerned with the
Cephalosporins production of cephalosporin is similar to that of
are broadspectrumantibioticswith
low toxicity. p e n i c i l l i n . T h e c u l t u r e m e d i a c o n s i s t so f c o r n s t ee p
liquor and soy flour-based media in a continuous
The structures of different cephalosporins
feeding system. The other ingradients of the
are shown in Fig. 25.6. Basically cephalosporins
m e d i u m i n c l u d e s u c r o s e /g l u c o s e a n d a m m o n i um
have a p-lactam ring fused with a dihydrothiazine
s a l t s . M e t h i o n i n e i s a d d e d a s a s o u r c e o f s u l f u r.
ring
The fermentation is carried out at temperature
Organisms for cephalosporin 25-28"C and pH 6-7. fhe growth of micro-
production o r g a n i s m s s u b s t a n t i a l l y i n c r e a s e s w i t h g o o d Ot
a l t h o u g h d u r i n g p r o d u c t i o n p h a s e , O,
CephalosporinC was first discoveredin the s u p p l y ,
c o n s u m o tion declines.
cultures of fungus Cephalosporiurnacremonium
(later renamedas Acremoniumchreysogenum)and Cephalosporin C from the culture broth can be
this organismcontinuousto be used even today. recovered by ion-exchange resins, and by using
The other organismsemployedfor cephalosporin column chromatography.Cephalosporin C can be
production are Emericellopsissp, Paecilomycessp precipitated as zinc, sodium or potassiumsalt, and
and Streptomycessp. isolated.
r 25 : M IC R O B IALPR O D U C T IO NO F A N TIB IOTIC S
p-Lactam
nng Dihydrothiazine
flng
R1 R2
Cephalosporin
co- -cHg
Cephalexin NHz
:
/ \ .co-
Cefadroxil Ho-(\ -cHs
,r/{lrH,
\___-,
-cl
Cefaclor
N-:--N
Cefazolin
,l=- ,1,.--."o- -tyt\.,-
N_N
L-Aminoadipicacid
I
_. l, L-Valine
L-Cysteine
Amirroglycosides are oligosaccharide (carbohy-
Y drate) antibiofics. They contain an aminocyclo-
J h e x a n o l m o i e t y w h i c h i s b o u n d t o o t h e r a m i no
Tripeptide
(aminoadipylcysteinylvaline) sugars by glycosidic linkages. More than 100
a m i n o g l y c o s i d e s a r e k n o w n e . g . ' s t r e p t o m y c in ,
lCyclase neomycin, kanamycin, gentamicin, hygromycin,
+ stsomtctn.
lsopenicillin
N
I A m i n o g l y c o s i d e sa r e v e r y p o t e n t a n t i b i o t i c sa n d
lEpimera$e act atainst Crarn-positive and Cram-negatrve
+ b a c t e r i a . b e s i d e s m v c o b a c t e r i a . A t t h e m o l e c u l ar
Penicillin
N l e v e l , a m i n o g l y c o s i d e sb i n d t o 3 0 S r i b o s o m e a n d
t_
I EXpanoase
block protein biosynthesis. Prolonged use of
J a m i n o g l y c o s i d e sc a u s e s d a m a g e t o k i d n e y s , a n d
Deacetoxycephalosporin
C hearing impairment.
I For the treatment of severe and chronic
I Hydroxylase
+ i n f e c t i o n s , a m i n o g l y c o s i d e sa r e t h e a n t i b i o t i c s o f
c h o i c e . S t r e p t o m y c i nw a s t h e f i r s t a m i n o g l y c o s i de
Deacetylcephalosporin
C
t h a t w a s s u c c e s s f u l l yu s e d t o t r e a i t u b e r c u l o s i s( i . e .
IAcetyltransferase against Mycobacterium tuberculosis). Usually,
I
J aminoglycosidesare regardedas reserveantibiotics,
CEPHALO SPO RIC
N s i n c e r e s i s t a n c em a y d e v e l o p e a s i l y .
PBODUCTION PROCESS
OF STREPTOMYCIN
Biotechnology[22]
338 B IOTE C HNO LO CY
Ra g(cHe)z
H2
OH o OH o
Tetracycline R1 R2 Ft^
''J R, Examples of producing
organrsms
I
I'
ir b y weak c at ionic ex c hange r e s i n s i n a n i o n - treatment of human and veterinary deseases,
Ir
I I exchange column. Treatment with activated of fish,meatand poultry
besidesin the preservation
I (in some countries)
carbon is often necessary to remove impurities.
Streptomycin can be precipitated in the form of
sulfate salt. Organisms for tetracycline production
Recgvery of chlortetracycline
Fig. 25.11. There are at least 72 intermediates At the end of the fermentation, the cuiturebroth
formed during the course of chlortetracycline is filteredto removethe mycelium.The filtrateis
biosynthesis,some of them have not been fully treatedwith n-butanolor methvlisobutvlketone in
characterized. aci di c or al kal i ne condi ti on for extracti ngthe
antibiotic.lt is then absorbedto activatedcharcoar
Polyketide antibiotic synthesis : The term rs
to remove other i mpuri ti es.C hi ortetraycl i ne
polyketiderefersto a group of antibiotics that are
elutedancicrystallized.
synthesized by successivecondensation of small
carboxylic acids such as acetate, butyrate,
PRODUCTION OF TETRACYCLINE
propionate and malonate. The synthesis of
-DIFFERENT PROCESSES
polyketideantibioticsis comparableto that of long
chainfattyacids.That is the carbonchaingrowsby The productionof tetracyclinecan be achieved
cvclic condensation process. The synthesisof by one or more of the following ways.
340 B IOTE C H N O LO CY
Recovery
and
purification
a By chemical treatment of chlortetracycline. butyratewhile the sugar units are derived from
a By carrying out fermentation in a chloride-free gl ucose.Macrol i de bi osynthesi si s a com plex
cult ur e m edium . processand a good exampleof polyketide synthesis
By em ploy ing m ut ant s in whic h c h l o r i n a t i o n
which is analogousto fatty acid biosynthesis. The
reaction does not occur.
enzymelactonesynthase is a multienzyme complex
w hi ch i s comparabl ei n i ts structureand funct ion
By bloc k ing c hlor inat ion r eac t ion b y t h e a d d i t i o n
to fatty acid synthasecomplex. An outline of
o f inhibit or s e. g. t hiour ea, 2{ hiou r a c i l .
the bi osynthesi sof erythromyci ni s gi ven I n
Fig. 25.13.
Regulation of biosynthesis: End product
i nhi bi ti on of erythromyci n synthesi si s well
Macrolides are a group of antibiotics with large documented. E rythronol i de
B i nhi bi tsthe enzym e
Iactone rings (i.e. macrocylic lactone rings). They lactonesynthase.The final product erythromycrn
co ns is t of 12- , 14- , or 16- m em ber e d l a c t o n e r i r r g s hasal sobeenshow nto i nhi bi tcertai nenzymesof
with 1- 3 s ugar s link ed by gly c os i d i c b o n d s . l - h e the pathway (e.g. transmethylase). Addition of
sugars may be 6-deoxyhexoses or aminosugars. propanol to the cul ture medi um i nduces t he
Erythromycin and oleandomycin are 14-membered synthesiso{ acetyl CoA carboxylase,and almost
(lac t one r ing c ont aining) m ac r o l i d e s w h i l e doubl esthe producti onof erythromyci n.
le uc om y c in and t y los in ar e ex am pl e s t , o r 1 6 - m e m -
be r ed m ic r olides . PRODUCTION PROCESS
OF ERYTHROMYCIN
E r y t hr om y c in and it s der iv at iv e c l a r i t h r o m y c i n
are the most commonly prescribedmicrolides.They producti onof erythromyci ins ca r r ied
Industri al
are effective against Gram-positive bacteria, and out by aerobic submerged fermentation. fhe
are f r equent ly us ed t o k ill pen i c i l l i n - r e s i s t a n t of soy mealor cor n
cul turemedi ummai nl yconsi sts
org anis m s . Clar it hr om y c in is c ur r e n t l y u s e d t o steepliquor,glucose(or starch),yeastextractand
combat stomach ulcers caused by H. pylori. fhe ammoni umsul fate.Fermentati on i s carri edout at
macr olides inhibit t he pr ot ein b i o s y n t h e s i s b y 30-34'C for about3-7 dat1s.Conventional methods
bin ding t o 50S r ibos om e. are used for the recoverv and purification of
erythromycin.
Polyene macrolides is the term applied for very
large ring macrolides that many contain lactone
ring s in t he r ange of 26- 28. e . g . n y s t a t i n ,
amp hot er ic in. Thes e poly ene m a c r o l i d e s a r e Tmrr 25.3 lmportant macrolide antibiotics
antifungal.
with the organlsmsproducingthem
C h l o ? a m p h e n i c o lb i n d s t o 5 0 S r i b o s m a l s u b u n i t
1 molecule 6 molecules and blocks (peptidyltransferasereaction) protein
dTDP-Glucose b i o s y n t h e si s .
Lactonesynthase
J
dTDp_4_ Polyketideintermediate Production of chloramphenicol
fet-o-O--deoxy- (enzYmebound)
D glucose- Chloramphenicol can be produced by
dTDp_ + Streptomyces venezuelae and 5. omiyanesis.
I L-Mycarose J
I However, chemical synthesisis mostly preferredfor
| \ ErvthronolideB
the commercial production of chloramphenicol.
L+orop-o---
Desosamine
\ 3-L-Mycarosyl- GRISEOFULVIN
\ erythronolideB
--------t-l C r i s e o f u l v i ni s a n a n t i b i o t i c t h a t a c t ss p e c i f i c a l l y
on fungi with chitinous cell walls. lt is used in the
J treatment of various fungal skin infections. Further,
Erythromycin
D
griseofulvin is also employed in the treatment of
Methylation
-&:in;' ,/\ plant diseases caused by Biotrytis and Alternaria
/ \Hvdroxvration solani.
t\
Erythromycin
B Erythromycin
C Although the exact mechanism of action of
tr"n"- griseofulvin is not known, it is believed that chitin
I
(SAM) biosvnthesisis adverselv affected.
Imethylation
:RYTHROMYCINA
CHLOFAM PHENI CO L
Production of griseofulvin
o f g ri s e o ful viins carri ed
C o m m e rc i apl ro d u c ti o n
out by employing Penicillium patulum. The
chemicalsynthesisis lessfrequentlyused due to
high cost.
Mycelialfilaments
The fermentationis carriedout by an aerobic
submergedprocesswith a glucoserich medium. l_
I Enzymallc
Nit r o g e ni s s u p p l i e db y s o d i u mn i tra te.Theopti mal
Itreatment
conditions for fermentation are-temperature +
23-26"C,pH 6.8-7.3,aeration0.8-1 vvm, and the \J l) v/-\
^
per i o di s 7 -10 d a v s . Xoo,Po"r-t
vn
,"r\J r-.,-
v^v v
a-!],
Protoplasts
I
DNA
{-lnsert
There are several antibiotics (more than 200 or glycol
Polyethylene
so ) w hic h hav e nuc leos ide lik e s t r u c t u r e s e . g . I
pu rom y c in, blas t ic idin S. Nuc leosi d e a n t i b i o t i c s
have diverse structuresand biological activities.
Puromycin Streptomycesalboniger
N e p l a mo s i A
n AmpulIarielIa reguIaris Desiredcell
in a selective
B l a s ti c i d i S
n S. griseochromogenes medium
Polyoxins S. cacaoi
Fig. 25.15 : A diagrammaticrepresentationol
transformation rn Streptomyces sp.
Transformation of stleptomyces
Streptomycesstrains exist as aggregatesof
A greatmajorityof antibioticsare producedby
mycel i alfi l amentsand not as i ndi vi dualcel l s.This
Streptomycessp, a Cram-positivebacteria. Of
is in contrastto E. co!i. lt is thereforeessential
course,thereare someother bacteria(both Cram-
that the cell walls of Streptomycesare broken to
positiveand Gram-negative)and fungithat can also
releaseprotoplastsfor transformation(Fig. 25.15).
produceantibiotics.
B y addi ng the desi redD N A (i n pl asmi ds)and
Cenetic manipulationsin Streptomyceshave polyethyleneglycol, the cells can be transformed.
been extensivelycarried out to enhancethe yield These protoplastsare grown on a solid medium
of antihiotics and reduce cost of production, to regenerate the cell walls. The transformed cells
besides developing newer and more effective rvith desiredpropertiescan be isolatedfor further
ant ib i o ti c s . use.
Chapter2s : tvttcRogtAl pRoDUCTtoN oF ANT|BIOT|CS 343
applications are of L-category. However, for the certain amino acids have been isolated e.g.
synthesis of glycine (optically inactive) and some gl utami caci d, al ani ne,val i ne.
ot her am ino ac ids whic h c an b e u s e d i n L - o r
In order to achievean overproductionof any
D-form (D, L-alanine, D, L-methionine) for certatn
amino acid by a microorganism, methodshave to
purposes,chemical methods are employed.
be devisedfor the eliminationof the metabolic
3. Microbiological production : For the large- processes.
regulatory/control In fact, severalamino
scale production of amino acids, microbiological acid-producinI microorganisms have been
rnethods are employed. There are three different developed by mutagenesis and screening
approaches. proqrammes.
(a) Direct fermentation methods : Amrncr The following are the major ways of strain
acids can be produced by microorganisms developmeirt. In f act, several methods are
by ut ilis ing s ev er al c ar b o n s o u r c e s e . g . combinedto successfullv developa new strainfor
glucose,fructose,alkanes,ethanol,glycerol, produci ngami noaci ds.
pr opionat e. Cer t ain indu s t r i a l b y p r o d u c t s Auxotrophic mutation : These mutants are
like molassesand starch hydrolysate can characterizedby a lack of the formation of
als o be us ed. M et hano l , b e i n g a c h e a p regulatoryend product(i.e. repressor
or regulatory
carbon source, is tried for amino acid effector). The intermediatesof the metabolic
pr oduc t ion, but wit h I im i t e d s u c c e s s . pathwaysaccumulateand get excreted.
(b) Conversion of metabolic intermediates Genetic recombination : Mutants can oe
into amino acids : In this approach, the developed by genetic recombinationfor over-
microorganisms are used to carry out productionof amino acids. Protoplastfusion in
selected reactions for amino acid certainbacteriais usedfor developmentof hybrids
pr oduc t ion e. g. c onv er s i o n o f g l y c i n e t o e.g. Corynebacteriumglutamicum and Bacillus
s enne. flavum.
(c) Direct use of microbial enzvmes or RecombinantDNA technology: The classical
immobilized cells : Sometimes resting techniquesof geneticengineering can be usedfor
c ells , im m obiliz ed c e l l s , c r u d e c e l l strain development. Strains with increasing
extracts or enzyme-membrane reactors activities of rate-limiting enzymes have been
can be used for the productiolr of amino developed.In one of the techniques,E. coli and
. acids. Some examples are given below. cloning vector pBR322were usedto increasethe
genes for the production of amino acids e s
Amino acid dehvdrogenases from certain
gl utami caci d, l ysi ne,phenyl al ani ne,
val i ne.
bacteria (e.9. Bacillus megaterium) can be used for
the amination of cr-keto acids to produce L-amino
Functional genomics : a new approach
a c ids e. g. alanine ( f r om py r uv a t e ) , l e u c i n e ( f r o m
rx - k et ois oc apr oicac id) and phe n y l a l a n i n e ( f r o m Analysisof genomesfrom the wild and mutant
p heny lpy r uv at e) . lm m obiliz ed c e l l s o r e n z y m e - strai nsof mi croorgani sms w i l l hel p i n cr eat ing
membrane reactors can be used. improvedstrains.Once the entiresequenceof the
chromosomes in the organisms (e.9.C. glutamicum,
Enz y m esor im m obilis ed c ells a r e a l s o e m p l o y e d E. coli) is established,effortscan be made to carry
for t he pr oduc t ion of s ev er alot he r a m i n o a c i d s e . g . out geneti c mani pul ati ons for e f f icient
tr y pt ophan, t y r os ine, ly s ine, v alin e . overproductionof desired amino acids. Chip
technologycan be used to detect new mutations
STRAIN DEVELOPMENT FOR and consequently the fermentationprocesses.
AMINO ACID PRODUCTION
Glucose
I
I
I
I
I
Phosphoenol
pyruvate Pyruvate
/.\^-
Oxaloacetate
SuccinylCoA 0,-Ketoglutarate
L.GLUTAMIC
ACID
bacterium, Corynebacteriumglutamicum, that was strains to produce and excrete more and more of
first used for largescale manufactureof glutamic glutamic acid. These include the strainsthat can
acid continuesto be successfully usedeven today. tolerate high concentrations of biotin, and
The other importantorganisms(althoughusedto a lysozyme-sensitivemutants with high yield.
lesser extent due to low yield) employed for
glutamic acid production belong to genera Biosynthesis of L.glutamic acid
M i crobacteri um, Brevi bacteri um and A rthrobacter. The pathway for the synthesisof glutamic acid
All these organismshave certain morphological with glucose as the carbon source is depicted in
and physiological characters comparable to Fig. 26.1 . Clucose is broken down to phosphoenol
C. glutamicum. Biochemically, glutamic acid- pyruvate and then to pyruvate. Pyruvate is
producing bacteria have a high activity of convefted to acetyl CoA. Phosphoenolpyruvate (by
glutamate dehydrogenaseand a low activity of the enzyme phosphoenol pyruvate carboxylase)can
a-ketoglutarate dehydrogenase.They also requrre be independently converted to oxaloacetate. Both
t he v it a mi nb i o ti n . these carboxylation reactionsare quite critical, and
require biotin as the cofactor.
lmproved production strains
The next series of reactionsthat follow are the
Several improvements have been made, familiar citric acid (Krebs)cycle reactions wherein
particularlyin C. glutamicum,for improvingthe the k"y metabolite namely o-ketoglutarate is
348 B IOTE C H NO LO CY
_ N A D P +\
\
\
Isocitrate Glutamate
Nutrients
J
lDissolv r nql .f--
- l- - - - \ . lBuf f er l f c.{r-l ai-,b. )
L_t"t__J-[''"1'""', -tjalJ I seoaratorl
\l \:i ,
'l exchanqa-'
Sterileair
I
L-GLUTAMICACID
(as monosodiumglutamate)
Process of production and recovery divertedfor the synthesisof 3 amino acids.The enzyme
Some important information on the production dihydrodipicolinate synthase converts aspartate
of glutamic acid by Brevibacterium divaricatum rs semialdehyde (and pyruvate) to piperideine 2,
eiven below. 6-dicarboxylate.There are two distinct enzymes
succinylasevariant (catalyses4-step reaction) and
Carbon source G l u cose(12% ) dehydrogenase variant(catalysesa singlestepreaction)
Nitrogen source A m moni um that can conveft piperideine2, 6-dicarboxylateto D,
acetate(0.5%) L-diaminopimelate which later forms L-lysine.
pH 7 .8
Temperature 3 B" C Regulation of L.lysine biosynthesis
Period for fermentation 3 0 -35 hours
The following are the regulatoryprocessesin the
Yield of glut am ic ac id 1 00 g/l medi um.
production of lysine (Fig. 26.Q.
A schematic representation of glutamic acid
Aspartate kinase : This enzyme is controlled by
production plant is shown in Fig. 26.3. As the
feedback inhibition of the end products. Three
fermentation is complete, the cells are separated,
isoenzymes of aspartate kinase have been
the c ult ur e br ot h is pas s ed t h r o u g h a n i o n
i d e n t i f i e d - o n e r e p r e s s e d b y L - m e t h i o n i n e , th e
exch anger .The glut am ic ac id bound t o t h e r e s i n si s
second one repressed by L-threonine and
elu ted in NaO H, while t he am m onia r e l e a s e dc a n
L - i s o l e u c i n e ,a n d t h e t h i r d o n e b e i n g i n h i b i t e d a n d
b e r eus ed. W it h NaO H, glut am ic a c i d f o r m s
repressedby L-lysine.The amino acid sequenceand
mo no s odium glut am at e ( M SC) wh i c h c a n b e
purified by passingthrough anion exchanger.MSC structureof asoartatekinasehave been elucidated.And
by genetic manipulations, it has been possible to
can be subjectedto evaporation and crystallization.
create mutants (of aspartate kinase) that are
i n s e n s i t i v et o f e e d b a c k r e g u l a t i o n b y L - l y s i n e .
Glucose
Phosphoenolpyruvate
+
Pyruvate
Oxaloacetate
:1
Citricacid
i Krebs
Malate cvcle
t-
0,-Ketoglutarate
Asparticacid
"'
I n"p"n","
Krnase
I
Aspartyl
phosphate
+ Homoserine
+ dehydrogenase
Aspartate -----+ -----+ ----J L-Methionine
semialdehyde L-Threonine
I L-lsoleucine
Pyruvate | Dihydrodipicolinate
- ) synthase
+
J
2, 6-
Piperideine
pimelate Ala-Glu-DaP
t
L-LYSINE
(peptidein cell wall)
T
L-Threonine is manufactured industrially by
employing either E coli or C. glutamicum. With
c
the mutant strains of E. coli, the product yield rs
E better
E
0)
c
Biosynthesis of L-threonine
O
The metabolic pathrvay for the synthesis of
L-threonine is depicted in Fig. 26.6 Some of the
reactions of this pathway are common for ihe
Fermentationtime ______+ b i o s y n t h e s i so f L - l y s i n e a n d m e t h i o n i n e , b e s i d e s
isoleucine (Refer Fig. 26.4 also). Starting lvith
Fig. 26.5 : Production of L-lysine n relation to
a s p a r t i ca c i d , i n a s e q u e n c eo f f i v e s t e p s ,t h r e o n i n e
substrate (glucose) and biomass concentration.
is produced
Biotechnology
[23]
354 B IOTE C H N OLO CY
The productionof tryptophanby C. glutamicum lmmobilizedE. coli cells with good activityof
was increasedby introducing a second gene are also usedfor aspartateproduction.
aspartase
\ /itamins are organic compounds that pefbrm erythrocytes and neurological manifestations,has
V specific biological functions for normal b e e n k n o w n {o r s e v e r a l d e c a d e s . l t w a s i n 1 9 2 6
nraintenanceand optimal Browth of an organism some workers reoorted the liver extracts could
These vitamins cannot be synthesized by the cure pernicious anemia. The active principle was
higher organrisrnt including man, and therefore later identified as vitamin 8,,, a water solubie
they have to be suppliedin small amountsin the diet. B - c o n r o l e xv i t a m i n .
355
356 B IOTE CHNO LO CY
2 AcetylCoA
+
I carotenoids
Acetoacetyl
a:i Carotenoid Organism Production
^ .
CoA yield (g/l)
lr-Acetyl
+ p-Carotene Blakeslea
irispora 3.0
B-HydroxyB-methyl (mixed of
cultures
glutarylCoA
+ and- sexualforms)
+
I Lycopene Blakeshlea trispora 0.4
(mixedculture)
Mevalonicacid5-PP
I Streptomyceschrestomyceticus 0.5
Dimethylallyl-PPH
J s-PP
lsopentenyl
Zeaxanthin Flavobacterium
sp 0.4
+
I cultures of both sexual forms, (+) and (-) strainsof
B. trispora.The vield of B-carotene is significantly
phytoene (Cas) h i g h e r w i t h m i x e d c u l t u r e s , c o m p a r e d to + o r -
J
I strains (Frg. 27.4). fhis is due to the fact that
Phytofluene
+
I
Neurosporene
c
E
c
Lycopene Zea carotene 0)
+
I +
I c
o
o
c)
p-Carolene 6-Carotene c
0)
t
I
+
I 6
Zeaxanthin o-Carotene o
I
362
OF FOOD SA N D B E V E R A C E S
Chapt er28 : M IC R O B IALP R OD U C T IO N 363
Dairy products
(worldwide)
Cheese Milk Streptococcus
sp
Penicilliunroquefortii,
P. camembertii
Yogurl(worldwide) Mitk Streptococcus thermophiIus
Lactobacillus
bulgaricus
Kefir(Russia) Mitk Lactobacillus
sp, Candidasp
Vegetarianproducts
Cocoabeans(worldwide) Cocoafruit Candidakrusei,
Geotrichun sp
Coffeebeans(worldwide) Coffeecherries Edwiniadissolvens,
Saccharonycessp
(lndonesia)
Tempeh Soybeans Rhizopusoryzae,Lactobacillus
delbrueckiia
Soysauce(worldwide) Soybeans Aspergillus
orzyae,
A. soyae
(Europe)
Sauekraut Cabbage Leuconostocnesenteroides,
L.plantarum
Breads(worldwide) Whealflour Saccharomycescerevisiae
Rolls,cakes(worldwide) Wheatflour Saccharomycescerevisiae
ldli(lndia) Riceandblackgram Leuconotocmesenteroides
Non-vegetarian products
(worldwide)
Drysausages Beef,pork Pedicoccuscerevisiae
(worldwide)
Fishsauces Small
fish Halophilic
sp,Eacil/us
sp
Country-cured
ha?ns(worldwide) Pork,hams Aspergillus
sp,Penicillium
sp
Coagulum
formation
r-Casein
Caseinin degraded
solublestate
. Cood texture.
SAUEKRAUT
The yeast fermented bread has the above
Sauekrautis prepared in most western countries
c h a r a c t e r i s t i c s .T h i s i s i n c o n t r a s t t o t h e b r e a d
It is a fermented and preserved form of cabhage.
p r o d u c e d w i t h b a k i n g p o w d e r w h i c l - ra l s o p r o d u c e s
The sh red ded c abbage is m ix ed wit h s a l t
(approximatelyat 2.5'/o concentration)and packed COr. Bui this does not have the same flavour and
texture as that produced by yeast. Thus the yeasr,
anaerobically. High salt concentration promotes
which is appropriately referred to as baker,s yeast
leakageof sugarsfrom the cabbage while reducing
is a package of enzymes to give a desired product.
the water activity. As the growth of the lactic acid
b acteriao ccur s , t he pH is lower ed. At t his low p H , In recent years, some workers have reported the
the putrifying bacteria cannot grow In this way, development of genetically engineered strains of
sauekraut can be preserved for long. Sauekraut is Saccharomyces cerevisiae with improved
n utritio us a s w ell as delic ious . ferrnentation properties Such organisms, when
used in baking industry, are believed to furtner
The traditional pkkles (mango, Iemon, etc.) in
enhance the quality of bread with regardto flavour,
lndia are also good examples of fermentation and
texture etc.
preservation, based on the principles of
biotechnology. Sour-dough breads : In some parts of the world,
sour breads are prepared by using the yeast,
BREAD Candida milleri and bacterium, Lactobacillus
Brea d making f r om t he dough by em pl o y i n g sanfrancisco.
micro org an is m sis one of t he oldes t ex am pl e s o f
fermentationp!'ocessesknown to mankind. There is BAKER'S YEAST
evidence that bread was prepared in Egypt in 3000 The living cells of aerobically grown
BC. Saccharomyces cerevisiae are collectively referrecl
Primarily, bread is a fermented product of to as baker's yeast. Baker's yeast is commercially
cereal flours srrch as wheat and rye. The cerear available eiiher as a dried powder i.e. dry yeast
flour mixed with water, salt, sugar, fat and other with about 95"k dry u,eight or in the form of cakes
ingradientstas desired for enrichment of bread) is (about 25-30% dry weight). These commercially
subjected to fermentation by yeast, Saccharomyces available yeast preparationscan be used in breao
cerevisiae(top fermenting strain).The main reaction making.
366 B IOTE C H N O LO C\
Nutrientmedium
n
I Ll*ll
T
Frozen
culture
Freshyeast
Fig. 28.3 : Flow chart for the production of baker's yeast (1 is the inoculation reactor,
2-5 are production reactors).
Production of baker's yeast : The medium for This block can be cut into small pieces,wraped
baker's yeast production contains molasses, and stored (at -4'C) until used. The samplesof
am m o n i u m s a l ts (o r a mmo ni a), vi tami ns, yeastare usuallytestedfor bakingproperties
before
phosphatesand antifoam agents.Sugar cane or they are put in use.
sugarbeet molassescan be used.A commercially
availablemolasseswith a sugarconcentrationof OTHER VEGETARIAN PRODUCTS
45-50% is usually preferred. Baker's yeast
production is carried out by an aerobic fed-batch Besides the bread which is consumed
worldwide, there are several other vegetarian
process.
fermented foods. These include rolls and cakes
The flow chart for the productionof baker's (worldwide)soy sauce(worldwide)idli (lndia) and
yeastis depictedin Fig.28.3.The actualproduction tempeh(lndonesia).
processand the strainof Saccharomyces cerevisiae
useddependson the company.The desiredstrainis Goffee, tea and cocoa
maintainedin a frozen state. The inoculum is
preparedin stagesso that largevolumesare finally Coffee,tea and cocoa are very popular non-
obtained.Fromthe inoculation (fermenter),
reactor alcoholic fermented beverages,and extensively
the culture is transferredto productionreactors. consumedthroughoutthe world.
The orocessis carried out in air-lift or bubble
When the tea leavesare crushed,the chemical
column type reactors at pH 4-5, temperature
componentsof tea are releasedby enzymatic
28-30"Cfor about 12-1B hours.The yeastcellsare
activity. For coffee and cocoa, a natural
washed, centrifugedand then dewateredon a
fermentationprocess(by bacteria,yeastand fungi)
rotatingdrum. The fresh yeast obtained can be
on the pulp surroundingbeans results in the
eitherdirectlyusedor dried and stored.
developmentof flavour and aroma. The exact
The yeastcells may be mixed with plasticizer natureof the fermentationprocessesinvolved in
(e.g.vegetableoil) and preparedin a block form. tea, coffee and cocoa are not fully known.
Chapt er28 : MIC R OB IALP R OD U C T IO N
OF FOOD SA N D B E V E R A C E S 367
The dried products namely tea leaves, and thaumatin production was very low. Attempts were
coffee and cocoa beans are commercially available also made to express thaumatin genes in yeasts
for beverage preparation. such as S. cerevisiae and Kluyveromyces lactis.
Some successhas been reported in this direction.
SWEETENERS
MONELL'N-A CANDIDA|E FON
Sweetenersoccupy a prominent place in food
SUGAR SUBST'TUTE
con su mptio n, by all t he people. The m o s t
predominantly used natural sweetener is sucrose Monellin is a protein found in the fruit of an
(obtained from sugar cane or beet). Sweetenersare African plant Discorephyllum cumminsii.lt is about
invariably required for the preparation of soft 100,000 times sweeter than sucrose (on molar
d rinks, ice c r eam s , jam s , jellies , s auc es , b r e a d , b a s i s ) .M o n e l l i n i s d i m e r c o m p o s e d o f a n A - c h a i n
confectionery, pickles etc. Hence there is a (45 amino acids) and a B-chain (50 amino acids).
continuous search for low calorie sweeteners. This proiein is optimally sweet when both tne
chains are held together and not when they are
Saccharin, a chemically synthesizedsweetener separated.
(300 times sweeter than sucrose) was used for
several years, and its use is now being restricted I t h a s b e e n p o s s i b l et o c h e m i c a l l y s y n t h e s i z ea
due to certain adverse health effects. High fructose g e n e t h a t e n c o d e s b o t h A a n d B c h a i n s a s a s i n g l e
corn syrup (HFC\, produced from sucrose rs polypeptide. This gene was in fact introduced and
sweeter (1.5 times) than sucrose. Aspartame expressed in tomatoes for the production of
(a sp artylp heny lalanine)is a pr oduc t of inn o v a t i v e monellin. However, the success has been very
biotechnology. lt is about 200 times sweeter than limited.
sucrose and is truely a low calorie sweetener.
Aspartame is approved for human consumption FLAVOUR ENHANCERS
a nd is widely us ed in s of t dr ink and o t h e r Taste and flavour are very important for the
i nd ustries. acceptability of foods. Monosodium glutamate is
the most commonly used flavour enhancer. lts
THAUMAT'N-A SWEET PROTE'N production and other aspects are described
Thaumatin is a orotein extractedfrom the berries elsewhere (Chapter 26). The degraded products of
ol the Thaumatococcus daniellii, a native plant in proteins and RNAs also impart flavour. Thaumatin
Africa. Thaumatin is aboul 3000 times sweeter and monellin (described above) are also flavour
than sucrose. Thaumatin certainly appears to be a e n h a n c i n g a g e n t s , b e s i d e ss w e e t e n i n g .
good sugar substituteand a low-calorie sweetener. The other flavouring agents are citric acid
It is in fact being used as a sweetener and flavour (produced by Aspergillus niger), acetone and
enhancer of various foods. At least five differenr butanol (produced by Clostridium acetobutylicum).
forms of thaum at ins wit h a m olec ular wei g h t s r n The lactones formed by some microbial
the range of 20,OOO-22,000have been isolatedand fermentations also enhance flavour e.g.
characterized. y-butyrolactone produced in yeast fermentation of
wine and beer. Mention must be made abour
Genetic engineering alcohol also which can increase flavour and taste
to produce thaumatin when added at appropriate concentrations e.g.
Natural production of thaumatin is very limited certain medicinal svruos.
and therefore can never meet the demands of
consumption. Biotechnological production of this
:\.,eeteneris the method of choice for its laree scale
-ianufacture.
Tha uma t in- enc odingm RNA s pec ies hav e b e e n Alcoholic beveragesare producedthroughout
:clated, identified and converted to cDNA and the world. The type of the beverageand the
'-allv to do uble- s t r andedDNA. Thes e DNA s w e r e usedfor its productionmostlydependson
substrate
: and transferredto E. coli. But the yield of the crops grown in the region. For instance,rn
'rned
368 B IOTE C H N O LO CY
Biotechnology
[24J
37(J B IOTE C HNO LO CY
Industry Enzymes
Dairy (animal/microbial),
Chymosins lipase,lactase,
lysozyme
Baking protease,
u-Amylase, phospholipases,
xylanase
Starchandsugar Amylases (o- andB-), glucoamylase,
pollulanase,
invertase,
glucoseisomerase,
xylanase
processing
Fruitandvegetable pectinlyase,hemicellulases,
Pectinesterase, polygalacturanase
Meatprocessing papain
Proleases,
Animalandlishboneprocessing Alkaline
ohosohatase
Eggwhiteprocessing Glucose
oxidase,
catalase
Brewingproduction
of beerand (a- andB-), proteases,
Amylases papain,
cellulases,
alcoholic
beverage aminoglucosidases,
xylanase
p r o t e i n s o f s o y b e a n ; p r o t e i n ( g l u t e n )o f d o u g h i n
preparing bread.
Removal of glucose and oxygen from sp, Campylobacter sp and Listetia sp. The food
foodstuffs toxins are derived from bacterial (endototoxins)ano
fungal (mycotoxins)sources.
The presenceof glucose and oxygen in the
foods is often associatedwith certain amount of Several novel and innovative techniques have
damage.Their removalenhancesthe storabilityof been developed in recent years for testing the
foods.Glucosefrom the foodstuffscan be removeo safety of foods. For instance, the presence of
by the combined action of glucoseoxidaseand Salmonella in foods can be detected by an
catalase,as illustratedbelow. immunoassyon the same day (This is in contrastto
Clu co se o xid a se the traditional methods that take 5-7 days).
^t
UfUCOSe * U2 -
C l u c oni cacfd + H rO, ln fact, kits are available for the detection of
Catalase concentrations of several undesirable compounds
2H2O2 ----------------+2HrO + O, in the foods e.g. endotoxins, mycotoxins,
The egg white used in bakingindustryis made antibiotics, hormones.
freefrom glucoseby the enzymatictreatment,given
above. This pair of enzymes(glucoseoxidaser FOOD BIOTECHNOLOGY.PUBLIC
catalase)is also useful for the removal of O, ACCEPTANCE
presentin the head spaceof bottledand canned
ln general, the public have a negative attitude
foods and drinks.
towards the biotechnology based foods, particularly
involving genetic manipulations. For this reason,
DIAGNOSTICS IN FOOD
t h e a p p l i c a t i o n o f g e n e t i c e n g i n e e r i n gt e c h n i q u e s
BIOTECHNOLOGY
for food biotechnology is rather slow. Several
It is essential
to ensurethat the foods oroduced factors influence the public acceptance. These
by biotechnological processesarefreefrom disease- include the type of biotechnology adopted, tne
causingorganisms and toxins.Then only the foods regulatory procedures, economics of the people
are suitablefor human consumotion.The most and consumer acceotance.For more details on this
common pathogenspresentin foods are Salmonella aspect, refer Chapter 61 .
r ,::r :t .i r ,. - , :.- i t;,i ,.r ..i - l ,- * r i r ;1.:+tgi ,;tl n;58" r " !$
ing ,le- c ellpr ot ein ( SCP)r c t . er st o t he t l i c r o l l i r l .
'
.-,ce lls or t ot al pr ot eit r c x t r ac t c ' d f r o r l r p L r r c ' ' j-lr
.
373
374 B IOTE C H N OLO CY
final cost of SCP.The most commonlv used raw 2. Short chain alkanes with carbon length in
materials may be grouped in the following the range of C',o-C''r, isolated from gas oil bv use
categories. of molecular sieves.
.l
methane,
. High-energysourcese.g. alkanes, Airlift bioreactor system with continuous
m et hanole, th a n o l g
, a so i l . o p e r a t i o n w a s o n c e u s e d ( i n F r a n c e a n d B r i t a i n )t o
wheyisewage, produce SCP from gas oil employing the or5lanism
2. Wasteproductse.g.molasses,
animal manures/straw,begasse. Saccharomycopsis lipolytica. But this is now
d i s c o n t i n u e df o r o o l i t i c a r r e a s o n s .
3. Agricultural and foresty sources e.g.
c ellulos el,i g n i n . Degradation of alkanes : Alkanes harreto be first
brolien down to appropriate metabolites for their
4. Carbondioxide,the simplestcarbonsource.
utilization to form SCP.The most important step in
Some details on the oroductionof SCP from t h i s d i r e c t i o n i s t h e i n t r o d u c t i o n o f o x y g e n i n t o
most importantraw materialsare brieflydescribed. a l k a n e s w h i c h c a n b e b r o u g h t o u t b y t w o
pathways-terminal oxidation and subterminal
PRODUCTION OF SCP FROM HIGH oxidation (Fig. 29.1).
E NE RG Y S O U R C ES
ln terminal oxidation, the terminal carbon gets
Thereare a largenumberof energy-rich carbon oxidized to the corresponding monocarboxylic
comooundsor their derivatives which serveas raw acid. The latter then undergoesB-oridation to form
m at er ialsfo r S C P o ro d u c ti o n . T h e s e i ncl ude a c e t i c a c i d . I n s o m e m i c r o o r g a n i s m st,h e o x i d a t i o n
alkanes, methane, methanol, ethanol and gas oil. may occur at both the terminal carbon atoms (by a
Bacteriaand yeastsare mostly employedfor SCP process referred to as crr-oxidation)to form a
production from high energy sources. Some dicarboxylic acid. This can be further broken down
scientistsquestion the wisdom of using (rather to acetate and succinate bv B-oxidation. Terrninal
m is us ing ) h i g h -e n e rg y c o mp o u n d s for the oxidation is the predominant pathway occurring in
production of food, since they regard it as a majority of yeasts and bacteria
wastefulexercise.
Suhterminal oxidation involves the oxidation of
interminal carbon atoms (any carbon other
Production of SCP from alkanes
than terminal i.e. C2, C3, C4, and so on).
Alkanes can be degraded by many yeasts, The corresponding ketone produced undergoes
certainbacteriaand fungi. The major limitationof a-oxidation, decarboxylation, and finally
alkanesis that they are not easilysoluble,hence p-oxidation to form acetate and propictnate.
they cannot enter the cells rapidly.lt is believed The individual enzymes responsiblefor terminal
thatthe cellsproduceemulsifying substances which oxidation or subterminal oxidation have not been
convert insoluble alkanes into small droplets fully identified.
0.01-0.5pm) that can enter the cells by passrve
dittusion.lt is observedthat when cells are grown Limitations of SCP production from
on a m ediu mo f a l k a n e se n ri c h e dw i th l i p i ds,the alkanes
dirfusionof alkanesinto the cells is enhanced.
The production of SCP from alkanes is a very
Certainyeastshave been successfully used for complex
biotechnological process and has been
producingSCPfrom alkanese.g.Saccharomycopsisextensivelystudied. The malor dral,r,back
of alkanes
Iipolytica, Candida tropicalis, Candida oleophila. as substratesis the formation of carcinogens,along
Petroleum products for SCP production : w i t h S C Pw h i c h a r e h i g h l y h a r m f u l . F o r t h i s r e a s o n ,
Severaloil companieshavedevelopedfermentation many countries have discontinued alkane-based
systems,employingpetroleumproductsfor large production of SCP.
scalemanufactureof SCPby yeasts.Two typesof
petroleumproductsaremainlyusedfor this purpose. Production of SGP from methane
1. Gas oil or dieseloil containing 1O-25"hol lzlethaneis the chief constituentof natural gas in
wit h c a rb o nl e n g thC ,' r-C ro(i .e .l o ngchai n many regions.Although methane can be isolated in
alk anes
aIkanes). p u r e g a s f o r m , i t c a n n o t b e l i q u i f i e d . T h e 'h a n d l i n g
376 B IOTE C H N OLO CY
cH3-(cH2)n-cH2-cH3
cH3-(cH2)2-cH2-cooH fol
acid
Monocarboxylic llll
-(cH2)n-+!ll-
cH3 cH3
i\
i \ r+Oxldation Ketone
iF-oxidation
i\ \ I u-Oxroation
Y
n""I"t" HooC-(CH2)n-CH2COOH
tol
1 ilI
Dicarboxylic
acid cH3-(cH2)n---lel--coo
H
I
Decarboxylation
i P-OxiOatlon +I
CH3-(CH2)n-COOH + COt
Fig. 29.1 : Oxidation of alkanes by yeasts (e.9. Saccharomycopsislipolytica/ to produce single-cell protein.
and transportation of methane (an explosive gas) Hansenula sp, Torulopsis sp) and fungi
are very difficult and expensive. (Tric hoderma Iignorum, GIiocIadi um deIi nquescens)
Certain bacteria that can utilize methane for are capable of producing SCP from methanol.
SC P pr oduc t ion hav e been i d e n t i f i e d e . g . Bacteriaare mostlypreferredbecausethey require
Methylococcus capsulatus, Methylomonas simple fermentationconditions,grow rapidly and
methanica, Methylovibrio soehngenii. So far, yeasts possesshigh contentof protein.
that can utilize methane have not been identifieo. Oxidationof methanol: Methanolgetsoxidized
The bacterial enzyme methane oxygenase to formaldehyde,then to formic acid and finallyto
o xi diz es m et hane t o m et hanol, w h i c h c a n b e carbondioxide,as depictedin Fig. 29.2.
converted to formaldehyde and then to formic acid.
The productsobtainedfrom methanolhave to
Although methane was extensively researched
form C, compounds(such as pyruvate)for final
for its use as a source of SCB it is not widely used
oroductionof SCP.Carbon dioxide formed from
du e t o t ec hnic al dif f ic ult ies . by photosynthetic
methanol can be utilized
organi sms for the formati on of ri bulose
Production of SCP from methanol
diphosphate. Alternately, formaldehyde may
Methanol is a good substratefor producing SCP
condense w i th ri bul ose 5-phosphateto f or m
Methanol as a carbon source for SCP has several
3-keto 6-phosphohexulosewhich then gives
advantagesover alkanes and methane. Methanol is fructose 6-phosphateand finally pyruvate.This
ea s ily s oluble in aqueous p h a s e a t a l l pathway is referredto as ribulose monophosphate
concentrations,and no residue of it remains in the (or
Quayle) cycle.
h ar v es t ed biom as s . Tec hnic allv , m e t h a n o l c a n b e
e as ily handled. The s our c es f or m e t h a n o l a r e Formaldehydecan condensewith glycine to
n atur al gas , c oal, oil and m et hane.M a n y s p e c i e so f form serinewhich in a seriesof reactionsforms
bacteria (Methylobacter, Arthrobacter, Bacillus, phosphoenolpyruvate.This is referredto as serine
Pseudomonas, Vibrio) yeasts (Candida biodinii, pathway.
Chapt er2 9 : S IN C L E-C ELPLR OT E INA, ND MU S H R OOMS 377
lCl pruteen
fhe single-cell protein produced by lCl from Downflow
methanol and ammonia using M. methylotrophus tube
was referredto as ICI pruteen. This SCP was
Heat
ex c lus iv e luy s e dfo r a n i ma lfe e d i n g l.C l i nvesteda excnanger
huge am o u n t (a ro u n df4 0 mi l l i o n ) i n 1 979 and
ins t alleda c o n ti n u o u sc u l tu re s y s te mfor S C P
production.Thiswas the world'slargestcontinuous
airliftfermenter.Unfortunately, the plant could not Nutrient
be operatedfor long due to economicreasons. For solution
instance,in 1984the cost of soy meal was around
Fig. 29.3 : A diagrammatic representation of ICI
$125- 200p e r to n w h i l e l C l p ru te e nw a s sol d at
pressure cycle fermenter to produce SCP from
$600 per to n ! T h i s i s m a i n l yb e c a u s eo f the hi gh
methanol.
cost of methanolwhich represents approximately
378 B IOTE C H N OLO CY
Symba process
Glutamine
synthase Symba processis a novel technology developed
i n S w e d e n t o p r o d u c e S C P b y u t i l i s i n g s t ar ch y
wastes by employing two yeasts, Endomycopsis
Glutamate A Glutamlne fibuligira and Candida utilis. The Symba process is
Glutamineketo- carried out in three phases.
glutaratetransaminase
PhaseI : The waste material containing starch is
sterilized by passing through a heat exchanger.
However, the large scale production of artificial Nutritive value of edible mushrooms
protein by rumen bacteria is yet to be clearly Some people regard €dible mushrooms as
e si ablis hedand c om m er c ialis ed. vegetable meat. Mushrooms contain 80-90%
water, depending on the growth conditions
( t e m p e r a t u r eh
, u m i d i t y ) . E d i b l e m u s h r o o m sa r e r i ch
sources of protein (35-45"/' of dry weight).
However, all these proteins are not easily digestible
b y h u m a n s . M u s h r o o m s a l s o c o n t a i n f a t s a n d fr e e
Mushrooms are fungi belonging to the classes
fatty acids (7-1O%), carbohydrates (5-1 5%) and
basidiomycetes (Agaricus sp, Auricularia sp,
m i n e r a l si n g o o d c o n c e n t r a t i o n .C e r t a i n u n d e si r a b l e
Tremella sp) and ascomycetes (Morchella sp, Tuber
substances may also be present in edible
sp ) . M ajor it y of edible m us hr oom s a r e t h e s p e c i e s
mushroomse.g. cadmium, chromium.
of basidomyces.lt is estimatedthat there are around
4 ,000 s pec ies of bas idiom y c es O f t h e s e , a r o u n d M a n y d e l i c i o u s r e c i p e s o f e d i b l e m u s h ro o m s
200 are edible, and a dozen of them are cultivated can be prepared. This actually depends on the
on lar ge s c ale. Som e of t he m os t i m p o r t a n t e d i b l e dietary habits of the people. Some of the common
mus hr oom s , t hbir c om m on na m e s a n d t h e recipes are mushroom soup, mushroom paneer,
substratesused are given in Table 29.3. mushroom puiao, and mushroom omelette.
PR
Chapt er2 9 : S IN GL E -C E L L O T E INAN
, D MU S H R OOMS 381
mostly carried out by batch culture fermentation. producti on, are gi veni n the
and thei r appl i cati ons
By manipulatingthe nutrient supply, differential Table 30.1. Some of the imoortantfeaturesof
synthesisof polysaccharides can be achieved.By i ndi vi dualmi crobi al pol ysacchari des are bri efl y
lim it ing n i tro g e ns u p p l y i n th e me d i u m , mostl y descri bedhereunder.
neutralpolysaccharides are produced.When metal
ionsar e lim i te d ,a c i d i cp o l y s a c c h a ri daere
s mai nl y XANTIIAN
synthesized. Molecularoxygen supply of around
907" saturationis ideal for good growth and Xanthan or more frequently referred to as
polysaccharide synthesis. xanthangumwasthe firstpolysaccharide available
commerci al l y.
l t i s a w el l studi edand mostw i del y
Biosynthesis of polysaccharides: Micro-
used hexopolysaccharide.
organisms are capableof producinga largenumber
of polysaccharides.The pathways for their Chemistry: Xanthanhas a molecularweight in
biosynthesis are comparableto the processess that the range of 2-15 x 104 dal tons.The basi c
occur for the formationbacterialcell wall. lt is repeati nguni t of xanthan i s a pentasacchari de
estimatedthat there are well over 100 enzymatic contai ni nggl ucose (C l c), mannose(Man) and
reactions,directly or indirectly involved in the glucuronic acid (ClcA) with acetate (Ac) and
synthesisof polysaccharides.
Startingwith glucose, pyruvate(Pyr)as depictedbelow.
appropriate sugars (by transforming glucose to
others) are incorporated in the formation of
polysaccharides.
Recovery of polysaccharides : As the
productionincreases,
polysaccharide there occurs Pyr
a markedincreasein viscosityof the culturebroth.
46
The polysaccharidescan be precipitatedby salts,
acids or organic solvents, and recovered by p-Man-( 1----+4)-p-G lcA-( 1-----;2)-o-Man-6-O-Ac
employingappropriatetechniques.
B a s i c a l l y ,x a n t h a n i s a b r a n c h e d p o l y m e r w i t h
Microbial polysaccharides versus plant (1
0 -++; linked glucan (glucose polymer) backbone
polysaccharides b o u n d t o a t r i s a c c h a r i d e( M a n , C l c A , M a n ) s i d e
c h a i n o n a l t e r n a t eg l u c o s e r e s i d u e s .T h e m a n n o s e
Thereis a lot of comoetitionbetweenmicrobial
and plant polysaccharides for industrial has either acetate or pyruvate groups. The number
applications. Productionof plant polysaccharides is of acetate or pyruvate molecules in xanthan rs
r elat iv elyc h e a p ,a l th o u g hi t i s u n c o n trol l edand variable and is deoendent on the bacterial strain
used. The culture conditions and the recovery
occurs for a short period in a year. In contrast,
processesalso influence the quantities of pyruvate
productionof mic,robialpolysaccharides is well
and acetateresidues.lt is believed that the viscosity
controlled and can be continued throughout the
gum is influenced by the contents of
year. However, fermentation processes for of xanthan
(from plant pyruvate and acetate.
manufacture of cheap sources)
polysaccharides
is not advisable. Applications : Xanthan gum is used as a food
additive for the preparation of saft foods (ice
G E NE RAL F EA T U R ES OF cream, cheese). lt is also used in oil industry for
MICROBIAL POLYSACCHARIDES enhancing oil recovery. Further, xanthan is useful
Of the several microbial polysaccharides,for the preparation of tooth pastes and water based
As already o a i n t s .
around2O areof industrialimportance.
stated,the commercialvalueof a polysaccharideis
' Biosynthesis: For the biosynthesisof xanthan,
mostlydependenton its rheologicalpropertiesi.e.
t h e m o n o m e r sa r e b o u n d t o a c a r r i e r l i p i d m o l e c u l e
its ability to modify the flow characteristics
of
and then transferredto a growing polymer chain.
s olut ions .
The activated monosaccharide nucleotides (e.g.
A selected list of commercially important u r i d i n e d i p h o s p h a t eg l u c o s e , U D P - g l u c o s e )s u p p l y
polysaccharides, usedfor their energy for the formation of glycosidic bonds
the microorganisms
384 B IOTE C H N OLO CY
the E. coli laczy genes (encoding the enzyme It is also possibleto use a cell free systemfor the
p-galactosidaseand lactose permease respectively) production of dextrans. The extracellular enzyme
were cloned under the transcriptional control of dextrasucrasecan transform sucrose into dextran rn
X campestris bacteriophage promoter. This a cell-free nutrient solution. This reaction rs
construct was first introduced into E. coli, and then o p t i m u m a t p H 5 0 - 5 . 5 a n d t e m p e r a t u r e2 5 - 3 0 'C .
transferred to X. campestris. The genetically
engineered strains of X. campestris expressedthe ALGINATE
genes and produced high quantitiesof the enzymes Alginate is a linear polymer composed of
B-galactosidaseand lactose permease. These new mannuronic acid and glucuronic acid (both of
stra ins u tiliz e lac t os e or whey v er y ef f ic ien t l y f o r them being uronic acids) in a proportion ranging
the ind ustrialpr oduc t ion of x ant han. This is a g o o d from 4 : I to 20 : 1. Some of the mannuronic acid
example of successfully converting a waste product residues are acetylated. Alginate is commercially
(whey) into a commercially important and valuable produced by Gram-negativebacteria, Pseudomonas
product (a biopolymer namely xanthan gum). aeruginosa and Azobacter vinelandi i.
The type of organism used and the culture
DEXTBAN conditions determine the relative orooortion of
m a n n u r o n i c a c i d a n d g l u c u r o n i c a c i d r e s i d u e sa n d
Chemically, dextrans are glucans (polymers of
the degree of acetylation in alginate.Alginateswith
glu co se ) co nt aining 1 - + 6 gly c os idic lin k a g e s .
high contents of mannuronic acid are elastic in
Some dextrans also have cx1 -+2, a.l -+3 and
a1 +4 linkages .The m olec ular weight s of de x t r a n s
nature while those with high concentration of
glucuronic acid are strong and brittle.
tl
a re in th e ra nge of 15, 000 500, 000. ll
A l g a l ( s e a w e e d )a l g i n a t e s a r e a l s o p o l y m e r s o f rl
Applications : Dextrans are used as blood mannuronic acid and glucuronic acid, and
plasma expanders, for the prevention of comparable in structure with bacterial alginates.
thrombosis and in wound dressing. In addition, However, algal alginates lack acetylation
d extran s are us ef ul in t he labor at or y ana l y t i c a l
For commercial purposes,seaweed alginatesare
techn iqu es for pur if ic at ion of biom olec ules .
m o r e c o m m o n l y u s e d t h a n b a c t e r i a la l g i n a t e s .T h i s
Production : Dextrans can be produced by a i s m a i n l y b e c a u s e b a c t e r i a l a l g i n a t e sa r e r e l a t i v e l y
wid e ra ng e of G r am - pos it iv e and Cr am - ne g a t i v e u n s t a b l e a n d g e t e a s i l y d e g r a d e d . A l g i n a t e s a r e
bacteria e.g. Leuconostoc mesenteroides and useful as thickening agents in food industry, and
Streptococcus mutans. ln contrast to other for immobilization of cells and enzymes.
e xo po lysacc har ides( whic h ar e s y nt hes iz edw i t h i n
SCLEROGLUCAN
the cells), dextrans are produced by extracellular
en zyme in t he " m edium . The enz y m e t s Scleroglucan is a glucose polymer (glucomer). lt
dextransucrase(a transglucosidase)which acts on i s a n e u t r a l p o l y s a c c h a r i d e w i t h p 1 - +3 g l u c a n
sucroseand brings about polymerisationof glucose backbone and single glucose (Glc) residuebranches
re sid ue s,a nd s im ult aneous lyliber at esf r ee f r u c t o s e ( B 1 - +6 l i n k a g e ) .T h e b r a n c h i n g o c c u r s a t a r e g u l a r
in to th e med ium . s e q u e n c ea t e v e r y t h i r d g l u c o s e u n i t i n t h e p o l y m e r
backbone chain.
The commercial production is carried out by -----+3)-B-Glc ( 1-----+3)-B-Glc (1----+3)-B-G lc (1-----+
using lactic acid bacterium, L- mesenteroidesby a I
batch fermentation process. Besides sucrose, the _r_
cu lture me dium c ont ains or ganic nit r ogen s o u r c e 1I
and inorganic phosphate. The crude dextran =
p rod uced is pr ec ipit at ed by alc ohol and t h e n I
sub jected to ac id hy dr oly s is . ln r ec ent y ea r s , t h e 0-Glc
alcohol precipitated polymeric dextran is subjected S cl erogl ucani s a fungalheoxpol ysacchari de.lt
to enzymatic hydrolysis by using exo- or endo-u is commercially produced by Sclerotium
dextranasesto get dextrans of desired molecular glucanicum,S. rolfsii and S. delphinii.Scleroglucan
',veight.The resultant dextrans can be fractionated is useful for stabilizing latex paints, printing inks
a nd d ried . and dri l l i ngmuds.
Bictechnology [25]
386 B IOTE CHNO LO CY
G E L L AN
' Cellan is a linear heteropolysaccharide. The
re p e a ti n gu n i t o f g e l l a n i s composedof tw o
o-1"-r.rr,-8-]
g l u c o s e ,o n e g l u c u ro n i ca c i d and one rhamnose (A)Generalstructureof a
monomerin PHA
molecules.Cellan is produced by Pseudomonas
elodea f c H" ol
A deacetylatedgellan which forms firm and ll" t t l
brittlegels underthe trade name Gelrite has been {_o-cH-cHz-4n
developedby a reputedcompany in USA (Kalco (B) Polyhydroxybutyrate
l n c ).
CHa
C e l l a n i s u s e d i n fo o d i n d ustry.E venat a l ow t-
c o n c e n tra ti o ni t, i s a th i c k n er. CHr
t-
CH2
POL L U L AN t-
CHr
Po l l u l a ni s a n c ,-g l u c o speol ymer(o-gl ucan)
w i th t-
a 1-+4, and a few s., 1-+6 glycosidic bonds. f CHc ol
Po l l u l a n i s p ro d u c e d b y usi ng the fungus, l t- il 1
Aureobasidiumpollulans.lt is estimatedthat about t-o-cH-cHz-cln
7O"h oI glucose (the substrate)is convertedto (C) PHA with 3-hydroxyoctanoate
p o l l u l a n d u ri n g fe rm e n ta ti on, al thoughthe ti me
taken is ratherlong (5-7 days).
Po l l u l a ni s m a i n l y u s e d i n food coati ngand I CH" Ol I
?'.
CH, Ol
packaging. l r" illl r- tr l
t-o-cH-cHz-cJ t-o-cH-cHz-c+
C U R D L AN (D) 3-Hydroxybutyrate-co-3-hydroxyvalerate
C u rd l a ni s a B-g l u c o speo lymer(F-gl ucan).The
g l u c o s e re s i d u e sa re h e l d t ogetherby p 1-+ 3 Fig. 30.1 : Structures of polyhydroxyalkanoates(PHA).
g l y c o s i d ib
c o n d sT
. h ee x o p o lysaccharicurdl
de ani s
commerciallyproducedby employingAlcaligenes
faecalis. Curdlan-like polysaccharidesare also of yearsand are a major sourceof environmental
produced by other microorganismssuch as pol l uti on,often resul ti ngi n ecol ogi caim
l balance.
Agrobacterium rhizogenes and Rhizobium trifolii. A heavydemandfor biodegradable plasticmaterials
exists. There are some attemptsto chemically
Curdlanformsstronggelswhen heatedto above
synthesize biodegradable polyesters e.g. polylactic
55"C. Therefore,it is used as a gelling agentfor
aci d and pol ygl ycol i caci d. The pro duct ionof
cookedfoods.In addition,curdlanis alsoemployed
polyhydroxyalkanoates by fermentation is preferred
fo r i m m o b i l i z a ti oonf e n z y m es.
for use as biodegradable plastics.
Homopolymer PHA-polyhydroxybutyrate
cn3-do-scon
The most common PHA is polyhydroxybutyrate Acetyl CoA
which is frequently referred to as PHB. lt is a CHq-CO-SCoA----l
-
polyester with 3-hydroxybutyrate as the repeating ) 3-Ketothiolase
CoA{/l
unit (Fig. 30,1B). PHB is the homopolymer PHA.
J
PHB, a s such, is r at her har d and inf lex ible.
o o
Be ing a hig h m olec ular weight c om pound , t h e
a ccumu latio n of PHB in huge quant it iesals o d o e s CH3-C-CH2-C-SCoA
not significantly effect the osmotic pressurewithin AcetoacetylCoA
the cell. The reservecarbon comoound PHB can be NnDeH--11
oxidized to carbon dioxide and water, releasing NAD'*, Acetoacetyl
CoAreductase
large amount of energy. Bacteria require energy, +
although they are not growing, to maintain pH OH
gradient and concentration gradient of several
CH3-CH-CH2-C-SCoA
compounds. This energy, referred to as maintenance
energy essentialfor the survival of the cells, is met CoA
3-Hydroxybulyryl
by the reservematerial PHB.
I eory1e-nyoroxybutyric
CoA<lsynthase
acid) tll
!ll
Heteropolymers of PHA J
Majority of polyhydroxyalkanoates, with the I cu. o- ]
exception of PHB, contain two or more different lr"ill
monomers and are referred to as heteropolymers. t-o-cH-cHzaJ
These heteropolymers are usually composed of a Poly3-hydroxybutyric
acid
random sequence of monomers, and not different
monomers, in different chains. Besides 3-hydroxy Fig. 30.2 : Biosynthesis of polyhydroxybutyric
acids, several other hydroxy acids are found in the acid (PHB).
structure of PHA. e.g. 4-hydroxybutyrate (4HB). In
fact, it has been found that the bacteria are capable
of in co rpo rat ing m or e t han 100 dif fe r e n t eutrophia produces a copolymer of 3-hydroxy-
hydro xylate d m onom er s int o PHA. This dep e n d s butyrate and 3-hydroxyvalerate(abbreviatedPHB/
on the organism and the nature of the carbon V). PHBas suchis hardand brittlebut the presence
sou rce sup pli ed dur ing t he ac c um ulat ion of t h e 3-hydroxyvaleratemonomersmakesPHBIr'flexible
DOtVmer. The properties
and stronger. of PHBIr'are similarto
those of polypropylene, and therefore it is
The properties of PHA mostly depend on the
commerci al l more
y useful .
na ture o f th e m onom er s it c ont ains . I n gen e r a l ,
PHA with longer s ide c hains ( e. g. 3- hy d r o x y -
POLYHYDROXYBUTYRATE (PI{BI
octanoate containing PHA) and heteropolymeric
PHA are more flexible and soft. Biosynthesis of PHB
Among the severalPHA, the pathwayfor the
3.Hydroxybutyrate'cG (PHB)hasbeen
biosynthesisof polyhydroxybutyrate
3.hydroxyvalerate
thoroughlyinvestigated.Startingwith acetyl CoA,
As already stated, by selecting a specific P H B i s synthesi zedi n three reacti on steps
org an ism a nd by m anipulat ing t he c om pos it i o n o f (Fig. 30.21.Acetyl CoA is convertedacetoacetyl
the me diu m, the c hem ic al c ons t r uc t ionof t he P H A CoA bv the enzvme3-ketothiolase which is then
ca n b e alte red. Thus , when t he m edium c on t a i n s reducedto 3-hydroxybutyryl CoA by acetoacetyl
qlu co sea nd p r opionic ac id, t he or ganis m Rals t o n r a CoA r.eductase. The reducing equivalentsare
388 B IOTE C H NO LO CY
Glucose
i
+
Pyruvate
l/'
I
l>COe
+
Acetyl CoA
OU O A
CoA
NADPH
_--- a.rrr"*
// .dynthase :
p+ Oxaloacetate Cikate
/\
/\
I
/T
I
I
Biosynthesis of PHBAI
Some strainsof Ralstoniaeutropha(formerly
c
known as Alcaligeneseutrophus)are capable of
o synthesizing poly (hydroxybutyrate-co-hydroxy-
c
0) valerate)(PHB/V).For the formation of PHB/r/,
glucose and propionic acid are required as
substrates Propionic acid as propionyl CoA rs
responsible for the synthesis of 3-hydroxyvalerate
The three enzymesinvolvedin the synthesis of 3-
hydroxybutyrate(3-HB) also participate in the
formation of 3-hydroxyvalerate(3-HV). The
pol ymer P H B /V contai ns 3-H B and 3-H V
monomersi n a randomseouence(no i ndi vi dual
pol ymersof 3-H B or 3-H V exi st).The rel atrve
concentrati ons of gl ucose(precursor for 3-H B )and
propi oni caci d (precursorfor 3-H V ) i n the cul ture
medi um determi nethe chemi calcomoosi ti onof
pftR /\/
Fig. 30.4 : Production of polyhydroxybutyrate (PHB).
The biosynthetic pathway for the formation of
PHBI/ is depicted in Fig. 30.5.
Production of PHB
Polyhydroxybutyrate is mostlymanufactured by
batch culture.PHB oroductionoccurswhen there
> an excesssupplyof carbonsource,and limitation
: i s om eot her e s s e n ti anl u tri e n ts u c h a s n i tro gen,
r hos phor usor s u l fu r s o u rc e . T h e p ro d u c ti on/
:ccumulation of PHB is depicted in Fig. 30.4.
Trere are two distinct phases-a growth phaseand
Acetyl CoA
= polymer accumulation phase. As the growth
: ' as e c eas esd, u e to n u tri e net x h a u s ti o ns,y n t hesi s
: poly m er( P H B)c o mme n c e sl t. i s a l s op o s s i bl e
to
I
+ 3-KetovalerylCoA
g e o x y g e ns u p p l yto AcetoacetylCoA
: ' : ' duc e P HB b y re s tri c ti nth
. = ' obic bac t eri a .
I
+
A pplic at ions o f PH B 3-Hydroxybutyryl
CoA
r HB c an b e i mp l a n te di n th e h u m a n body
'. :^out rejection.This is becausePHB does not
' - , juc e any i mmu n e re s p o n s ea n d th u s i t i s CoA
3-Hydroxyvaleryl
Poly (3-hydroxy-
biocompatible. PHB has several medical butyricacid)
: : : r c at ionse.B. a s d u ra b l e b o n e i mp l a n ts,for
, - . lr d dr es s i n g s .A tte m p ts to u s e PH B as Poly (3-hYdroxy-
butyricacid)
: = : - . : dables u tu re sa n d o th e r i m p l a n tsh a s not synthase
- =: ,r ith successdue to very slow degradationof
Poly (3-hydroxybutyrate-co
'3-hydroxyvalerate)
B I O S Y NT HES IS O F O T H ER PH A
^e bios yn th e s i so f o th e r PH A i s ' q ui te Fig. 30.5 : Biosynthesis of poly (3-hydroxybutyrate-co-
s-hydroxyvalerate) (Note : The same 3 enzynes are
- : : . able t o th a t d e s c ri b e dfo r P H B Some
required for the synthesis of poly
-:, iant featureswith regardto the synthesisof
3-hydroxybutyric acid; For details refer Fig. 30.2)
- - c or t ant P H A a re d e s c ri b e d .
390 B IOTE CHNO LO CY
The biopolymer,adhesiveproteinproducedby
the blue musselMytilus edulis is very strong and lndole
water proof. lt is usefultbr the musselto attacn
lndole2-3-
I
itself to different surfaces.As the adhesive is
dihydrodiol Ixyene
secreted,it gets randomlycross-linked,hence it oxidase
cannot be sequenced.Fortunately, the immediate I
precursorfor the formationof adhesiveproteinhas
+
Indoxyl
been isolatedand characterized. lt is a 130-kDa
p ro te i n ri c h i n c e rta i n a mi n o aci ds l i ke seri ne
I
lo z
threonine,tyrosine,lysineand proline.Most of the J
proline and tyrosine residuesare hydroxylated. Indigo
F u rth e r,b i o c h e mi c a la n a l y s i sreveal edthat thi s
precursorprotein contains repeatingunits of a Fig. 30.6 : Biosynthesis of indigo from tryptophan by
rlprrnoniirlp
393
394 B IOTE C HNO LO G Y
Naturalvegetation Combustion
(grass,timber,foliage) (electricity,
steam)
SOURCES UTILIZATION
C o v e rn me n ts i n ma n yd e v e l o p i ng countri es
al so Cellulose Starch Proteins Lipids
o f b i o g a spl ants.In Indi a,
en c o u ra g ei n s ta l l a ti o n
Covernment provides 25% subsidy, besides Hydrolytic phase
encouraging banksto offerloansfor construction of
. h e y a re b e c o m i ngpopul ari n rural
bio g a sp l a n ts T
areas.Pakistanalso has a good numberof gobar
gas plants.
Acidifying phase
=--+Gas outletpipe
Pulley
Usedslurry
(manure)
PROCESS OF BIOGAS PRODUCTION w el l s i nto the top of the l andfi l l .(Formore detai l s
(BIOGAS PLANTI ReferChaoter5B).
Biogas poduction from biomass is an anaerobic
process.The anaerobic digestion is usually carried Factors affecting biogas (methanef
o ut by us ing air t ight c y lindr ic al t ank s w h i c h a r e
production
referred to as anaerobic digesters. A digester may
The factorsaffectingmethaneproduction,with
be made up of concrete bricks and cement or steel,
special referenceto biogas plant, are briefly
u su ally bu ilt under gr ound.The diges t erha s a n i n l e t
descri bed.
attached to a mixing tank for feeding cow dung
(Fig. 31 .3). The methanogenic bacteria from 1. Temperature and pH : The idealtemperature
another digester .are also added with cow dung. i s 30-40' C , w hi l e the pH i s 6-8, for good yi el o.
The digester is attached to a movable gas holding
or storage tank with a gas outlet. The used slurry 2. Slurrycomposition: The ratiobetweensolid
(spent cow dung) comes out from the digester and w ater composi ti oni n the sl urry shoul d be
through an outlet. This can be used as a manure. around 1 : 1. A carbon nitrogenratio of 30 : 1 in
The anaerobic digester described above, is a low the sl urryresul tsi n opti mal methaneproducti on.
technology gobar gas plant, commonly used for C ood mi xi ng and sol ubi l i zati onof the organi c
do mestic pur pos es in r ur al ar eas in I n d i a . T h e constituents is reouired.
processof digestion usually takes about 2-3 weeks
lvhen cow dung is used as the substrate. 3. Anaerobicconditions: The digestershould
be completelyairtight, so as to create suitable
Landfill sites for methane production anaerobi ccondi ti ons.
Landfill sites are low cost digesters huilt 4. Presenceof inhibitors: Ammonium sulfate
underground for the digestion of solid wasfes (of and anti bi oti cs i nhi bi t methane producti on.
ind ustries and m unic ipalit ies ) . As t he a n a e r o b i c A gri cul turalw astes, pi g and chi cken manure
d ige stio no f s olid or ganic m at er ial oc c ur s , m e t h a n e (generating ammonia)and wastesfrom paper(rich
gas is generated.lt can be recovered by boring gas i n sul fate)i nhi bi tbi ogasproducti on.
398 BIOTECHNOLOGY
gas, a byproduct of nitrogen metabolismis lost in rubber produced from petroleum is preferredfor
the soil. lt is estimatedthat from a soybeancrop in use in automobiliesand planes,due to low-cost
one hectarefield, about 30 billion m3 hydrogenis and high elasticity.BesidesHevearubber,there are
generatedand lost annually.As such,thereare no some other plants for the productionof naturar
methodsavailableto trap such huge quantitiesof rubber e.g. Parthenium agrentatum (guayule)
hydrogenproducedin agriculturalfields. Taraxacumkoksaghyz(Russiandandelion) grown
in Mexico and some partsof USA.
Wood.rich plants
Some plants grow very fast and they serve as
good suppliers of wood. e.g. Eucalyptus,Butea,
Besidesits utility for the generationbiofuels
Melia, Casurina.Theseplantsare importantsources (alcohol,
methane),biomassis also used for the
of firewood.lt is estimatedthat approximately 50% production of butanol, acetone, single-cell
protein
of the total wood harvestedannuallyis utilizedfor and many
other producfs (Chapter29).
the purposeof firewood.Wood is also usefulfor
the supplyof pulp for papermanufacture. As such, the contributionof biomassto the
world's requirements of energy is very low. lt rs
Petroleum plants around5% i n the U .S .A .,and may be a l i ttl ehi gh er
Thereare certainplantswhich can accumulate in the developingcountries.However, being a
high molecular weight hydrocarbons. They are renewable source of energy, biomass will have
referred to as petro-crops or gasoline plantations. immensevalue in future. This is particularlytrue as
The productsof thesehydrocarbon-rich the world's non-renewable fuels (gasand oil) get
plantscan
depleted.There is a growing realisation
on the fuel
serve as good substitutes of conventional
petroleum and petroleumproducts. value of biomass. In the coming years, biomass
productionand utilizationstrategies will be fully
The rubber plant (Hevea rubber), grown in exploited.In addition,furtherimprovements in the
South"East Asia is the principal sourceof rubber. biotechnological processes for bettermanagement
Rubberis collectedin the form of latex from the and uti l i zati on of i ndustri al ,agri cul turalan o
stemsof trees.This plant meetsaboutone third of domesticwasteswill also solve the problem of
the total world's demandof rubber.However,tne world energycrisis.
o il m ic r oor ganis m sar e v er y c lose l y i n v o l v e d a s
Q
J cat aly t ic agenl. sin m any geolog i c a l p r o c e s s e s .
Th es e inc lude m iner al f or m a t i o n , m i n e r a l
d eg r adat ion, s edim ent at ion and g e o c h e m i c a l ln microbial leaching (bioleaching),metals can
cycl ing. be extracted from iarge quantities of low grade
ores Although recovery of metals (e.g. copper)
In r ec ent y ear s , a new dis c ipl i n e o f m i n e r a l
from the drainage water of mines has been known
science namely biohydrometallurgy or microbial
f o r c e n t u r i e s ,t h e i n v o l v e m e n t o f m i c r o b e s i n th i s
mining (mining with microbes) is rapidly growing.
process was recognized about 40 years ago.
Broadly s peak ing, biohy dr om et allu r g y d e a l s w i t h
the application of biotechnology in mining T h e b a c t e r i aw h i c h a r e n a t u r a l l y a s s o c i a t e dw i th
industry. In fact, microorganisms can be t h e r o c k s c a n l e a d t o b i o l e a c h i n g b y o n e o f th e
successfully used for the extraction of metals (e.9., following ways.
co pper ,z inc , c obalt , lead, ur anium ) f r o m l o w g r a d e
1 . Direct action ol bacteria on the ore to extract
ore s . M ining wit h m ic r obes is bot h e c o n o m i c a l a n d
metal.
e nv ir onm ent al f r iendly .
2. Bacteria produce certain substancessuch as
The term metal is used to any substancethat is
s u l f u r i c a c i d a n d f e r r i c i r o n w h i c h e x t r a c tt h e me ta l
hard, possessing silvery lusture, and is a good
(indirect action).
conductor of heat and electricity. Some of the
metals, however, are relatively soft, malleable and ln practice, both the methods may work
d uct ile e. g. s ulf ur . An or e is a nat u r a l l y o c c u r r i n g together for efficient recovery of metals.
sol id m iner al aggr egat ef r om which o n e o r m o r e
minerals can be recovered by processing. Organisms for bioleaching
400
Chaot er32 : MIC R OB IAL
MIN IN C A N D M E TA LTE C H N OLOC Y 4(|1
ffilttr4nEnrffiurrr,:i'
402 B IOTE C HNO LO CY
S e l e c t e de x a m p l e s o f m i c r o b i a l b i o l e a c h i n g a r e
briefly described below.
(B)
B'OLEACHING OF COPPEB
C o p p e r o r e s ( c h a l c o p y r i t e , c o v e l l i te a n d
chalcocite) are mostly composed of other metals,
b e s i d e s c o p p e r . F o r i n s t a n c e ,c h a l c o p y r i t e m a i n l y
contains 26"h copper, 26"h iron, 33olosulfur and
2.5"/" zinc.
(c) B i o l e a c h i n g o f c o p p e r o r e ( c h a l c o p yr i te ) i s
widely used in many countries. This is carried out
by the microorganism Thiobacillus ferrooxidans
w h i c h o x i d i s e s i n s o l u b l e c h a l c o p y r i t e( C u F e Sr )a n d
converts it into soluble copper sulfate (CuSOo).
S u l f u r i c a c i d , a b y p r o d u c t f o r m e d i n t h i s r e a cti o n ,
Metal m a i n t a i n sa c i d i c e n v i r o n m e n t ( l o w p H ) r e q u i r e d fo r
recovery growth of the microorganisms.
Heap leaching : ln this case, the ore is arranged In situ bioleaching technique is commonly used
in large heaps (Fig. 32.28) and subjected to f o r e x t r a c t i n gu r a n i u m . I n t h e t e c h n i q u e e m p l o ye d ,
treatmentsas in slope leaching. t h e i n s o l u b l et e t r a v a l e n tu r a n i u m i s o x i d i z e d ( i n th e
presence of hot HrSOo/Fe3* solution) to soluble
In sifu leaching : The ore, in its original natural
h e x a v a l e n tu r a n i u m s u l f a t e .
place is subjected to leaching (Fig. 32.2C). WaIer
c ont aining t he m ic r oor ganis m sis p u m p e d t h r o u g h UO, + Fer(SOa)3--------) UO2SO4 + 2FeSO.
Ch APICT32 : M I CRO BI AL M I NI NC AND M ET A L T E C H N O L O C Y 403
The process of removal of sulfur containing 1. Removalof toxic metalsfrom the industrial
pyrite (FeS2) from high sulfur coal by effluents.
microorganisms is referred to as biodesulfurization. 2. R ecoveryof val uabl ebut toxi c metal s.
Hig h su lfur c oal, when us ed in t her m al po w e r
Boththe aboveorocesses are concernedwith a
stations, emits sulfur dioxide (S02) that causes
poi soni ng/pol l uti on.
reducti oni n envi ronmental
en viro nme nta lpoll ut ion.
A wide rangeof microorganisms(bacteria,algae,
By using the microorganisms Thiobacillus
yeasts,moulds) are employed in biosorption.In
ierrooxidans and T. thiooxidans, the pyrite which
fact, some workers have developed biosorhent-
contains most of the sulfur (80-90%) can be
based granules for waste water/industrial effluent
remo ve d. Thu s , by em ploy ing bioleac hing, h i g h
treatment, and metal recovery.
.u lfur co al can be f r uit f ully ut iliz ed in a n
:-.''onment friendly manner. ln addition, this ln general, the microbial cell membranesare
;-., - :.': i; o ui t e ec onom ic al als o. negativelychargeddue to the presenceof carboxyl
40'4 B IOTE C HNO LO CY
(COO ), hydroxyl (OH-) phosphoryl (POi-) and instance,Chlorella vulgaris and C. regularis can
s ulf hy dr y l ( HS- ) gr oups . This ena b l e st h e p o s i t i v e l y certai nmetal sl i ke P b,H g, C u, M o and
accumul ate
charged metal ions (from solutions)to be adsorbed U. The green algae Hydrodictyon reticulatum
on t o t he m ic r obial s ur f ac es . adsorbsand accumul ates hi gh quanti ti es of Pb, Fe
and Mn.
The different groups of microorganismsused in
biosorption processesare briefly described below. Someworkersare in fact trying to use marine
algae (e.9., Luminaria, Ulva, Codium sp) as
Bacteria bioaccumulatorsto reducethe metal pollution in
fl vers.
Several bacteria and actinomyceles adsorb and
accumulate metals such as mercurv cadmium, lead,
Higher plants in control of
z inc , nic k el, c obalt and ur aniu m . F o r e x a m p r e ,
metal pollution
Rhodospirullumsp can accumulate Cd, Pb and Hg.
Bacillus circulans can adsorb metals such as Cu, Besidesihe microorganisms describedabove,
Cd, Co, and 7n. By use of electron microscopy, thereare somehi gheraquati cpl ants(i .e.,aquat ic
deposition of metals on the bacterial cell walls was macrophytes) that can accumulatepotentialtoxic
recorded. lt appears that the cell wall composition wastes including many metals. Water hyacinth
plays a key role in the metal adsorption. (Eichornia crassipes),duck weeds (Spirodel sp),
water lettuce (Pistia stratiotes)and ceriain ferns
Fungi (Saiviniasp) are importantin the control of metal
pollution.For more details,the readermust refer
There is a large scale productior.r of fungal C haoter54.
biom as s in m any t . er r nent at io ni n d u s t r i e s . T h i s
bior,nasscan be utilized for metal biosorption from
industrial effluents. lmmobilized fungal bionrass rs
more effective in biosorpticln due to increased
density, mechanical strength and resistance to
c hem ic al env ir onr ner r t . Fur t h e r , i m n t o b i l i z e d
B y t h e c o n v e n t i o n a l t e c h n i q u e u s e d i n th e o i l
biomass can be reused after suitable processing.
f i e l d s , a p p r o x i m a t e l y o n e - t h i r d o f t h e o i l ca n b e
The fungus Rhizopus arrhizus can adsorb several recovered. However, the oil recovery can be
m et allic c at ions e. g. ur anium , t h o r i u m . P e n c i l l i u m e n h a n c e d b y u s i n g s o l v e n t s , a n c j s u r fa cta n ts.
lapidorum, P. spimulosum are useful for the Certain polymers produced by microorganisms
biosorption of metals such as Hg, Zn, Pb, Cu. (e.g., xanthan gum), when added to oil wells are
Several fungi were tried with some degree of capable of increasing oil recovery Xanthan gum
s uc c es s t o s elec t iv ely ads or b u r a n i u m e . g . can pass through small pore spaces and promote
Aspergillus niger, A. oryzae, Mucor haemalis, the releaseof more trapped oil.
Penici II i u m chrysogenum.
In recent years, oil technologists are trying to
Edible mushrooms were also found to adsorb directly use microorganisms in situ Ior increasing
the oil recovery. In this process, there is no
certain metals. For instance,fruit bodies o{ Agaricus
bisporus can take up mercury while Pleurotus sajor-formalised use of a bioreactor. The naturar
caju can adsorb lead and cadmium. geological site itself is the bioreactor. This allows
the water and microorganismsto flow over the ore
Many yeasts, commonly used in fermentation which are collected after seepageand outflow. The
industries, are capable of adsorbing and microorganisms,through surfactantproduction, gas
accumulating metals. For instance, Saccharomyces formation or by other microbial activities reduce
cerevisae and Sporobolomyces salmonicolour can the viscosityof the oil so as to enhance its recovery.
respectivelyadsorb mercury and zinc. However, the success in this direction is not very
commendable. -
Algae
Continued further researchmay one day help to
Severalspecies of algae (fresh.water or marine) use microbes for commercial releaseof oil from oil
can serve as bioaccumulators of metals. For w e l l s o r t a r s a n d s .
(t)
33 A ni mal C el l C ul ture -
Fundamental s,Faci l i ti esand
A ppl i cati ons 4O7
34 C ul ture Medi a for
A ni mal C el l s 4' 15
35 C ul tured C el l s- B i ol ogy and
Characterization 423
36 P ri mary C ul ture and
C el l Li nes 437
37 A ni mal C el l C ul ture-C eneral
C onsi derati onsand S cal e-up 450
3B C el l V i abi l i ty and C ytotoxi ci ty 459
ANIMAL CELL/ 39 C el l Transformati onand C el l
ANIMAL C l oni ng 463
40 Organ and H i stotypi c C ul tures,and
BIOTEGHNOLOGY Ti ssueE ngi neeri ng 468
41 Transgeni cA ni mal s 480
405
I nim al c e l l c u l tu re b a s i c a l l yi n v o l v e sthe i n Histotypicculture: The culturingof the cellsfor
/ | vitro (in the laboratory)maintenanceand their reaggregation to form a tissue-like structure
pr opagat ioonf a n i m a lc e l l s i n a s u i ta b l en utri ent represents histotypicculture.
media.Thus,culturing is a processof growing cells
artificially.Cell culturehasbecomean indispensible Organotypi ccul ture : Thi s cul turetechni que
t ec hnologyin v a ri o u sb ra n c h e o i nvol vesthe recombi nati on
of di fferentcel l tvoesto
s f l i fe s c i e n ces.
form a more defi nedti ssueor an organ
His t or ic al b a c k g ro u n d Primaryculture : The culture producedby the
I t was in 1 9 0 7 ,R o s sH a rri s o nfi rs td e v e l opeda freshl y i sol atedcel l s or ti ssuestaken from an
. e p ro b a b l ychose
f r og t is s uec u l tu rete c h n i q u e H organi smi s the pri mary cul ture.Thesecel l are
f rog for two reasons-beinga cold-bloodedanimal, heterogenousand slow growing, and represent
no inc ubat i o ni s re q u i re da n d ti s s u ere g e n e rati on rs the ti ssue of thei r ori gi n w i th regardto thei r
f as tin f r og.In 1 9 4 0 ' sc h i c k e m b ry oti s s u eb e came properties.
a f av our it e fo r c u l tu re te c h n i q u e s .In te restrn
C el l l i ne: The subcul turi ngof the pri mary
c ult ur inghu ma n ti s s u e ss ta rte di n 1 9 5 0 ' safter i t
culturegivesriseto cell lines.The term continuous
was demonstrated(by HeLa; Cey) that human
cell linesimplies the indefinitegrowth of the cells
t um orc ellsc o u l dg i v e ri s eto c o n ti n u o u cs e l l l i nes.
i n the subsequent subcul turi ng.
On the otherhand,
A m ongt he v a ri o u sa n i ma lc e l l c u l tu re smo
, usecel l
finite cell lines representthe death of cells after
c ult ur esar e th e m o s t c o mmo n l y u s e d i n the
severalsubcul tures.
laboratory.
T er m inolog y i n c e l l c u l tu re
The term tissue culture is commonly used to
inc ludebot h o rg a nc u l tu rea n d c e l l c u l tu re.
Organ culture : The cultureof nativetissue(i.e
undisaggregated tissue)that retainsmost of the in While designing the laboratory for animal cell
t,ivo histological features is regarded as organ
culture technology, utmost care should be taken
c uit ur e. with regard to the maintenance of aspetic
Celf cufture : This refers to the culture of conditions. The facilities required with regard to
- .::i'rsed(or disag,g,regated) cells obtainedfrom the infrastructure and equipment are /isted in the next
. : ^al t is s ueo, r fr6 m a c e l l l i n e . pa8e.
4|J7
408 B IOTE C H N O LO CY
. P r epar at ionf ac ilit ies The fol l ow i ngare the commontypesof cult ur e
vessets.
. A nim al hous e
. Mul ti r,velpll ates
. M ic r ohiology labor at or y
o P etri di shes
. St or age f ac ilit ies ( f or glas s w a r e , c h e m i c a l s ,
l iquids , s m all equipm ent ) . . Fl asks
o S ti rrerbottl es.
EOUTPMENT
The actualchoi ceof sel ecti nga cul turevessel
Laminar-flow, sterilizer, incubator, refrigerator
dependson severalfactors.
and f reezer (-.20'C), balance, COz cylinder,
centrifuge, inverted microscope, water purifier, 1. The w ay cel l s grow i n cul ture-mon olayer
hemocytonreter, liquid nitrogen freezer, slow- or suspensron.
cooling device (for freezing cells), pipette washer,
2. The quanti tyof the cel l srequi red.
de ep was hing s ink .
3. The frequencyof sampl i ngfor the desir ed
Bes ides t he bas ic and m inim a l r e q u i r e m e n t s
W OT K
listed above, there are many more facilities that
may be benef ic ial or us ef ul f or t i s s u e c u l t u r e s . 4 . T h e p u r p o s e f o r w h i c h t h e c e l l s a r e 8 r o \ /n .
Th es e inc lude air - c ondit ionedr oom s , c o n t a i n m e n t
5. The cost factor.
room for biohazard work, phase-contrast
microscope, fluorescence microscope, confocal ln general,for monolayer cultures, the cell yield
microscope, osmometer, high capacity centrifuge is almost proportional to the surface area of the
an d t im e laps e v ideo equipm ent . c u l t u r e v e s s e l .T h e f l a s k s a r e u s u a l l y e m p l o ye d fo r
this purpose.
CULTURE YESSEI.S
Any type of culture vessel can be used to grow
I n t he t is s ue c ult ur e t ec hnology,t h e c e l l s a t t a c h suspension cr-rltures.lt is necessaryto slowly and
to the surface of a vessel which serves as the c o n t i n u o u s l y a g i t a t e t h e s u s p e n d e d c e l l s in th e
substrate, and grow. Hence there is a lot of vessel.
importance attached to the nature of the materials
used and t he qualit y of t he c ult ur e v e s s e l s . Treatment of culture vessel surfaces
The term anchorage dependent cells is used For improving the attachment of cells to the
wh en t he c ells r equir e an at t ac h m e n t f o r t h e i r surfaces, arrd for efficient growth, some devices
g rowt h. O n t he ot her hand , s o m e c e l l s have been developed. lt is a common observation
undergo transformation, and become anchorage that the growth of the culture cells is better on the
independent. surfaces for second seeding. This is attributed to
matrix coating of the surfaces due to the
Materials used for culture vessels accumulation of certain compounds like collagen
and fibronectin releasedb,7the cells of the previous
Glass : Although glasswas the original substrate
c u l t ur e .
used f or c ult ur ing, it s us e is alm o s t d i s c o n t i n u e d
no w. This is m ainly bec aus e of t h e a v a i l a b i l i t y o f T h e r e a r e n o w c o m m e r c i a l l y a v a i l a b l e m atr i ce s
more suitakrleand alternate substrates. (e.g. matrigel, pronectin, cell-tak).
Thereare severalroutesof contaminationin the single species. Despite utmost care taken, no
tissueculturelaboratory(Table33.1).Theseinclude laboratory can claim to be totally free from
the various materials (glassware, pipettes), contami nati on.l t i s necessaryto conti nuousl y
equipment (incubators,refrigerators,laminar-flow monitorfor contaminationand eliminatethe same
hoods), reagents(media, solutions),contaminated at the earliest.
cell lines anclpoor technigues.
ASEPTIC CONDITIONS
The routes of contamination are mosfly
associatedwith the laboratorvenvironment,and Maintenanceof proper aseptic conditions is
operatingtechniques. necessarvto eliminate various cantaminants (due
to different microorganisms and viruses). The
Types of microbial contamination fol l ow i ngmeasures
are suggestedfor mi ni mi zi ng
contami nati on,and mai ntenance of asepti c
Severalspeciesof bacteria,yeasts,fungi, molds
condi ti ons.
and mycoplaimas,besidesvirusesare responsible
for contamination.Major problems of conta- o Strict adherenceto standardsteriletechniques
minationare linkedto the repeatedrecurrence of a and code of oractices.
410 B IOTE C H NO LO CY
b i o l o g i c a l c o m p o u n d s b y t h e m i c r o o r g a ni sm s i s
,3 Summaryof the applications described in Chapters 23-30.
of animal cell cultures
A s e l e c t e dl i s t o f a n i m a l c e l l c u l t u r e p r o du cts o f
Category Applications cornnrercial importance is given in Table 33.4.
Intracellularactivity Studies
relatedto cellcycle
Production of vaccines
anddifferentiation,
transcription,
translation,
energy metabolism, M o n k e y k i d n e y o r c h i c k e m b r y o c e l l s, o r
1,,,^
ur uv il lvtquuilJil
-^r^L^l;^- t. r e c e n t l y h u m a n d i p l o i d c e l l s a r e i n u s e fo r th e
o r o d u c t i o n o f v a c c i n e s . T h e v a c c i n e m a n ufa ctu r e
lntracellular
flux Studies involving
hormonal i n a n i m a l c e l l c u l t u r e s i s r a t h e r c o m p l e x wi th r i sk
receptors,metabolites,
signal of contarnination, and safetv aspect. For these
transciuction,
membrane reasons, production of vaccines by recombinant
trafficking D N A t e c h n o l o g y e m p l o y i n g b a c t e r i a o r v e a sts r s
.l
Cell to cell interactionStudies
dealingwithcell preferred (Refer Chapter 6).
adhesion
andmotility,
matrix
Interaction,
morphogenesis, Production of high value therapeutics
paracrine metabolic
control, M a n y h u m a n p r o t e i n s w i t h h i g h t h e ra p e u ti c
cooperation potential are often in short sLrpply e.g tissue
p l a s m i n o g e na c t i v a t o r ,c l o t t i n g f a c t o r s ( V l l l a n d l X) ,
E n V i ro n me n ta l Studies relatedto drugactions,
infections, e r y t h r o p o i e t i n . T h e r e i s a m a j o r l i m i t a ti o n to
interaction cytotoxicity,
produce human proteins that undergo post-
mutagenesis, carcinogenesis
t r a n s l a t i o n a lm o d i f i c a t i o n s ( g l y c o s y l a t i o n ,c a r b o xy-
Genetics Studies dealing withgenetic l a t i o n e t c . ) i n b a c t e r i aa n d y e a s t s .T h i s i s d u e to th e
analysis, transfection, fact that these organisms do not possess the
transformation, immortalization,m a c h i n e r y t o p e r f o r m p o s t - t r a n s l a t i o n a cl h a n g e s.
q A N A E 'A N A A
However, pharmaceutical proteins that do not
Cell products Widerangeof applications of r e q u i r e p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n s ca n b e
thecellular productsformed p r o d u c e d b y b a c t e r i a o r y e a s t s e . g . i n su l i n ,
(Referlable 33.4)e g. vaccines, l b u m i n , g r o l v t h h o r m o n e .
a
hormones,
interferons
etc. Animal cell cultures (particularly mammalran
c e l l c u l t u r e s )a r e u s e f u l f o r t h e p r o d u c t i o n of m a n y
. St udies dealing wit h genet i c s e . g genetic p h a r m a c e u t i c a l l y / m e d i c a l l y i m p o r t a n t p r o te r n s
(Table 33.4) These include the following.
analy s is ,im m o( aliz at ion, s ene s c e n c e
r Laboratory production of o Plasminogen
medical/pharma-
ceutical compounds for wide range of .lnterferons
applic at ionse. 8. ' r ac c ines ,int er f e r o n s ,h o r m o n e s . . Blood clotting factors
. Hormones
There are however, several limitations on the
us e of anim al c ell c ult ur es . This i s m o s t l y d u e t o . Monoclonal antibodies
the differencesthat exist between Ihe in vivo and in . Ervthroooietin
vit r o s y s t em s , and t he v alidit y o f t h e s t u d i e s
conducted in the laboratory. Purification of pharmaceutical products
A s t h e d e s i r e d p r o d u c t i s p r o d u c e d i n th e ce l l
MEDICAL / PHABMACEUTICAL c u l t u r e m e d i u m , i t s p u r i f i c a t i o n , i s o l a t i on a n d
PRO DUCTS O F ANI M AL CE L L storage (collectively re{erred to as downstream
C ULTURES p r o c e s s i n g )a s s u m e ss i g n i f i c a n c e .T h e f i n a l pr o d u ct
The m os t im por t ant applic at io n o f a n i m a l c e l l for therapeutic applications is expected to satisfy
the following criteria.
cult ur es is t he pr oduc t ion of a w i d e r a n g e o f
com m er c ial c om pounds f or m edi c a l a n d p h a r m a - . T h e o r o d u c t s h o u l d h a v e a s t a b l e s t r u c t u r ew i th
ceut ic al us e. The c om m er c ial pr o d u c t i o n o f s e v e r a l ootimal activitv
Ch ap ter 33 : ANI M AL CELL CULTURE- FA
FU N D A M E N T A L S , C I L I T I EASN D A P P L I C A T I O N S 413
-f h e se lect ionof an appr opr iat e gr owt h m e d i u n r 1950 The nrinimal criteria needed for choosing a
E fo r th e in v it r o c ult iv at ion of c ells i s a r r r r e r l i r : n r f o r a n i m a l c e l l r . r - r l t u r casr e l i s t c d b e l o w .
imp orta nt an d es s ent ials t ep The nr anr m alia nc e l l s . T h e n r c c l i t r r l s h o r - r l cpl r o v i d e a l l t h e n u t r i e n t s t o
of a n o rga n in t he body r ec eiv e nut r ient s f r o n r
the cells
Irloo d circu lat ion For c ult ur ing,t hes e c ells in v i t r r , t ,
o M a i n t a i n t h e p h y s i o l o g , i c ap
l H arouncl 7 0 with
it is expected that they should be provided with
acJequatebuffering
the components similar to those present in blood
In ge ne ral, t he c hoic e of t he m edium m o s t l y o T h e m e d i u m m u s t b e s t e r i l e ,a n d i s o t o n i c t o t h e
de pe nd so n the t y pe of t he c ells t o be c r , r lt ur e da, n d ceils.
th e p urp ose of t he c ult ur e ( gr owt h, dif f er ent i a t i o n , The basis for the cell culture media was the
p rod uction o f des ir ed pr oduc t s ) The c ult ur e m e d i a balanced salt solution which was originally used to
may be na tur al or ar t if ic ial. create a physiological pH and osmolarity required
to maintain cells in vitro For 1lromolinSJgrr>wth
iIIATURAL M EDI A and proliferation of cells, vartous constituents
. (glucose, amino acids, vitamins, growth factors,
:{ In the ea r ly y ear s , t he nat ur al m edia obta i n e d
s
:t from vario us biologic al s our c eswer e us ed antibiotics etc.) were added, and several media
{ developed Addition of serumto the various media
Bod y fluid s : Plas m a, s er um , ly m ph, am n i o t i t
is a common practice However, some workers in
fluid , a scitic and pleur al f luids , aqueous hu m o u r
r e c e n t y e a r s h a v e s t a r t e d u s i n g s e r u m - f r e em e d t a
from e ye s an d ins ec t hem oly m ph wer e in c om m o n
use. Th ese fluids wer e t es t ed f or s t er ilit y a n c l The physicochemical properties of media
toxicity be for e t heir ut ilit y required for tissue cultures are briefly desc.ribed.
t n b a l a n c e ds a l t
T h i s i s f o l l o w e d b y a b r i e f a c c o L r no
Tissueextracts : Among the tissueextracts,chick
solutions,commonly used culture media and the
embryo extract was the most commonly employed.
s e r u m - f r e em e d i a
The e xtra cts of liv er , s pleen, bone m ar r ow a n d
le ucocyte sw er e als o us ed as c ult ur e m edia PHYS!COCHEMICAL PROPERTIES
So me wo rk er s s t ill pr ef er nat ur al m edia f or o r g a n OF GULTURE MEDIA
cu lture T h e c u l t u r e m e d i a i s e x p e c t e dt o p o s s e s sc e r t a i n
physicochemical properties (pH, O2, COz,
ARTIFICIAL M EDI A b u f f e r i n g ,o s m o l a r i t y ,v i s c o s i t y ,t e m p e r a t u r ee t c ) t o
The artifi c ial m edia ( c ont aining par t ly de f i n e d support good growth and proliferation of the
co mpo ne nts)hav e been in us e { or c ell c ult ur e s i n c e cultured cells
415
416 B IOTE CHNO LO CY
pH f a c t m a d e p o s s i b l eb y a c o n t i n u o u s s u p p l y o f O, to
t h e t i s s u e sb y h e m o g l o b r n .
Most of the cells can grow at a pH in the range
of 7. 0- 7. 4, alt hough t her e a r e s l i g h t v a r i a t i o n s T h e c u l t u r e d c e l l s m o s t i v r e l v o n t h e d i sso l ve o
depending on t he t y pe of c ells ( i . e . c e l l l i n e s ) T h e O , i n t h e m e d i u m w h i c h m a y b e t o xi c a t h i g h
indicator phenol red is most commonly used for concentrationdue to the generationof fi'eeradicals
v is ible det ec t ion of pH of t he m e d i a . l t s c o l o u r a t i o n T h e r e f o r e , i t i s a b s o l u t e l y n e c e s s a r y to su p p l y
at the different pH is shown below. a d e q u a 'r e q u a n t i t i e s o f O , s o t h a t t he ce l l u l a r
AI pH 7.4 ReC requirementsare met, avoiding toxic effects.Some
workers add free-radical scavengers (glutathione,
At pH70 - Orange
m e r c a p t o e t h a n o l t)o n u l l i f y t h e t o x i c i t y . A d d i ti o n o f
At pH 6. 5 Ye l l o w s e l e n i u m t o t h e m e d i u m i s a l s o a d v o ca te d to
At pHZB - Pu r p l e r e d u c e O . t o x i c i t v T h i s i s b e c a u s e s e l en i u m i s a
c o f a c t o r f o r t h e s y n t h e s i so f g l u t a t h i o n e.
CO, bicarbonate and buffering
I n g e n e r a l , t h e g l y c o l y s i s o c c u r r i n g i n cu l tu r e d
Car bon diox ide in t he m ed i u m i s i n a d i s s o l v e d c e l l s r s m o r e a n a e r o b i c w h e n c o m p a r e d to i n vi vo
state, the concentration of which depends on the cells. Since the depth of the culture nredium
atmospheric CO, ter.rsionand temperature.CO, in i n f l u e n c e st h e r a t e o f O . d i f f u s i o n , i t i s a d vi sa b l e
t he nr edium ex is t s as c ar boni c a c i d ( H 'C O . ) , a n d to keep the depih of the'medium in the range 2--5
bic ar bonat e( HCO 3) and H+ io n s a s s h o w n b e l o w . mm.
Biotechnology [27]
418 B IOTE CHNO LO CY
. POI-, SOI- and HCO5 ions are involved in the The oresent recommendation is that for the
main ten anc e of int r ac ellular c har ge, b e s i d e s routine culture of cells, antibiotics should not be
serving as precursorsfor the production of certain added. However, they may be used for the
important compounds e.g. POj- is required for development of primary cultures.
ATP synthesis.
SERUM
Glucose
Serum is a naturalbiological fluid, and is rich in
Ma iority of c ult ur e m edia c ont ain gluc os ew h i c h
various components to support cell proliferation.
serves as an important source of energy. Clucose
The major constituentsfound in different types of
is degraded in glycolysis to form pyruvate/lactate.
sera are listed in Table 34.3. The most commonly
Th ese co moounds on t heir f ur t her m et abolis me n t e r
used sera are calf serum (CS), fetal bovine serum
citric acid cycle and get oxidized to COr. However, (FBS),horse serum and human serum. While using
exoe rimen t al ev idenc e indic at es t ha t the
human serum, it must be screenedfor viral diseases
contribution of glucose for the operation of citric ( h e p a t i t i sB , H l v ) .
a cid cycle is v er y low ln v it r o ( in c ult ur e c e l l s )
compared to in vivo situation. Glutamine rather Approximately 5-20% (v/v) of serum is mostly
than glucose supplies carbon for the operation of used for supplementingseveral media. Some of the
citric acid cycle. And for this reason, the cultured imoortant features of the serum constituents are
ce lls req uir e v er y high c ont ent of glut am in e . briefly described.
Hormones
Tasle34.3 Major constituentsof serum
Hydrocortisone promotescell attachment,while
Proteins insulinfacilitatesglucoseuptakeby cells. Crowth
hormone,in association with somatomedins (lGFs),
Albumin
Globulins promotescell proliferation.
Fetuin
Fibronectin lnhibitors
Translenin S erum may al so contai n cel l ul ar gr owt h
Proteaseinhibitors inhibitingfactors.Majorityof them areartefacts e.g.
(cr''
-antitrypsin) bacterialtoxins,antibodies. The natural serum also
grow th i nhi bi to rnam ely
contai nsa physi ol ogi cal
Amino acids
transforming growth factor p (TCF-P). Most of
Almostall the20
thesegrowthinhibitoryfactorsmay be removedby
tipids heat inactivation(at 56'C for 30 minutes).
Cholesterol
Phospholipids S E LE C TION OF ME D IU M A N D S E RUM
Fattyacids As already stated,there are around a dozen
Carbohydrates media for the cell cultures.The selectionof a
h
rh Glucose parti cul armedi umi s basedon the cel l l i ne and t he
lh purpose of cul turi ng.Fori nstance, for chi ckem br yo
Hexosamine
fi brobl astsand H eLa cel l s, E ME M i s used. The
lP
I
Otherorganiccompounds medium DMEM can be usedfor the cultivationof
Lactic
acid neurons. A sel ected l i stof cel l sand cel l l i n esalong
Pyruvicacid w i th the medi a and sera used i s given I n
h
rb Polyamines Table34.4. In fact, informationon the selectionof
4, Urea appropri atemedi um for a parti cul arcell line is
avai l abl efrom l i terature.
Vitamins
A
Vitamin The selectionof serumis alsobasedon the type
Folicacid of cells being cultured.The following criteriaare
w hi l e choosi ngser um .
takeni nto consi derati on
Growth factors
r Batchto batch variations.
Epidermalgrowthfactor
growthfactor
Platelet-derived . Quality control.
growthfactor
Fibroblast to promotegrowthand preservation
. Efficiency of
Hormones cel l s.
Hydrocortisone r Sterility.
Thyroxine r Heat inactivation.
Triiodothyronine
In recent years, there is a tendency to
lnsulin
discontinuethe use of serum,and switch over to
Inorganics more clearlydefinedmedia (describedlater).
Calcium
Sodium S U P P LE ME N TA TION OF TH E ME DI UM
Potassium WITH TISSUE EXTRACTS
Chlorides B esi desserum,the cul turemedi a can also be
tr0n supplementedwith certain tissue extracts and
Phosphates The examplesare-chick
microbialcultureextracts.
Zinc embryo extract, proteolytic digestsof beef heart,
Selenium bactopeptone, lactalbuminhydrolysate, tryptose.
ChA P I CT
34 : C U L T U R E
M ED IAF ORAN IM ALC E LLS 421
The chick embryo extract was found to contain Contamination : lt is rather difficult to get serum
bot h high m o l e c u l a rw e i g h t a n d l o w m o l ecul ar totally free from all pathogens,particularly viruses.
weight compounds that support growth and
proliferatior.t of cells. - Presenceof growth inhibitors : In general, the
concentration of growth promoters in the serum is
m u c h h i g h e r t h a n t h e i n h i b i t o r s . B u t s o n 'r e t i m e s ,
the growth inhibitors such as TCF-B may dominate
and inhibit cell oroliferation.
'serum
Addition of to the culture media has been
, Availability and cost : There is a dependenceon
an age-old practice. However, in recent years, t h e c a t t l e f o r t h e s u p p l y o f s e r u m . H e n c e t h e
certain serum-free media have been develooed. lt availability may be restrictedon several occasions
is worthwhile to know the disadvantagesassociated f o r p o l i t i c a l a n d e c o n o m i c r e a s o n s F . u r t h e r ,c o s t a l s o
with the the use of serum, and the advantagesand i s a n o t h e r f a c t o r f o r d i s c o u r a g i n gt h e u s e o f s e r u m .
disadvantagesof serum-free media.
Downstream processing: The presenceof serum
Disadvantages of serum in media in the culture medium interferes with the isolation
and purification of cell culture products. For this
Variable composition : There is no uniformity rn
reason,severaladditional steps may be required for
the co mpo sition of t he s er um . lt is highly v ar i a b l e
(source, batch, season/ collection method, t he isolationof the desired oroduct.
p rocessin g).Suc h dif f er enc es in t he c om po s i t r o n
ADVANTAGES AND DISADVANTAGES
sig nifican tly i nf luenc e t he c ells in c ult ur e.
OF SERUM.FREE MEDIA
Quality control : To maintain a uniform quality
Advantages
of the serum, special tests have to be performed
with each batch of serum, before its use. T h e l i m i t a t i o n sa s s o c i a t e dw i t h t h e u s e o f s e r u m
i n t h e m e d i a ( d e s c r i b e da b o v e ) a r e e l i m i n a t e d i n
the serum-free media. In addition, there are two
more distinct advantages.
lines along with the media and seyum rscd
Selection of media with defined composition :
The main advantage of serum-free medium is to
Cells or cell line Medium Serum control growth of the cells as desired, with a well
defined medium This is in contrast to the use of
Chickembryo E M EM CS
serum wherein the growth frequently proceeds in
fibroblasts
an uncontrolled fashion.
Chinese hamster EMEM,
Ham'sF12 CS
ovary(CHO) Regulation of differentiation : lt is possible to
HeLacells E M EM CS use a factor or a set of factors to achieve
Human leukemia RPMI1640 FB differentiation of cells with the desired and
s o e c i a il s e d f u n c t i o n s .
Mouse leukemia Fischer's
medium, FB,HoS
RPMI1640
Disadvantages
Neurons D M EM FB
Mammary epithelium R PMI1 6 4 0D, ME M FB Slow cell proliferation : Most of the serum-free
Hematopoietic
cells RPMI1640, FB media are not as efficient as serum added media rn
medium
Fischer's the growth promotion of cells.
muscle
Skeletal D M EM,F 12 F B ,H OS Need for multiple media : A large number of
Glialcells ME MF, 1 2 ,D ME M FB serum-freemedia need to be developed for different
3T3cells ME M , M EM
D CS cell lines. This may create some practical
d i f f i c u l t i e s i n a l a b o r a t o r ys i m u l t a n e o u s l yh a n d l i n g
(EMEM--Eagle's minimalessentialmedium;RPMI 1640-Medium
cell lines. Another limitation of serum-free
from to BosewellPark Memoriallnstitute;DMEM-Dulbecco'ss e v e r a l
of Eagle'snediun; CS-Calfserum;FB-Fetalbovine m e d i u m i s t h a t a g i v e n m e d i u m m a y n o t b e a b l e t o
modification
serum:HoS-Horse serum) support the different stages of development even
422 BIOTECHNOLOCY
423
424 B IOTE C HNO LO CY
As is evident from the above, different and limited supply in the normal culture conditions(i e.
alm os toppos in gc o n d i ti o n sa re re q u i re dfo r cel l atmospheric oxygen and a submerged culture)
oroliferation. and for cell differentiation. Therefore creating an anaerobic situation. Lactate, secreted
is requiredtwo distinctsetsof
if cell differentiation i n t o t h e m e d i u m , a c c u m u l a t e s .S o m e a m o u n t o f
conditionsare necessary pyruvate produced in glycolysis gets oxidized
.l through Krebs cycle. A small fraction of glucose
. To optimizecell proliferation.
(4-9%) enters pentose phosphate pathway Io
2. T o oot im i z ec e l l d i ffe re n ti a ti o n .
supply ribose S-phosphate and reducing
e q u i v a l e n t s( N A D P H ) f o r b i o s y n t h e t i cp a t h w a y se . g .
Maintenance of differentiation
s y n t h e s i so f n u c l e o t i d e s .
lt is now recognizedthat the cells retain their
Clutamine is an important source of energy for
native and original functions for long when their
the cultured cells. By the action of the enzyme
three dimensional structures are retained. This is
g l u t a m i n a s e ,g l u t a m i n e u n d e r g o e sd e a m i n a t i o n t o
possible with organ cultures. However, organ
p r o d u c e g l u t a m a t ea n d a m m o n i u m i o n s . C l u t a m a t e ,
culturescannot be propagated.In recent years,
on transamination (or oxidative deamination)
someworkersaretryingto createthreedimensional
forms cr-ketoglutarate which enters the Krebs
by pe rfu s i n m
st r uc t ur es g o n o l a y ecr u l tu re sF. u rther,
cycle. Pyruvate predominantly participates in
in v it r oc ult ur in go f c e l l so n o r i n s p e c i ama
l tri ces
t r a n s a m i n a t i o nr e a c t i o n t o o r o d u c e a l a n i n e , w h i c h
( e. g. c ellulos e , c o l l a g e n g e l , m a tri x of
is easily excreted into the medium In the rapidly
g ly c opr ot einsa) l s o re s u l ts i n c e l l s w i th th ree
g r o w i n g c u l t u r e d c e l l s , t r a n s a m i n a t i o nr e a c t i o n i s a
d im ens ional s t ru c tu
re s .
dominant route of glutamine metabolism.
(o)
C o
(c)
(c) C o
+)
( o) 0 o
Stemcell C C
regeneration
Differentiation
(B) cell
Differentiated
Stem cells
t9
----1e,/-A
-tr7
@
(o) @
o) @
( o) @
@
@
Stemcell
regeneration @ Differentiation
Fig. 35.2 : Differentiation of cetls (A) ln vivo differentiation of stem cells (B) Blocked differentiation in cultured cells'
GLUCOSE
+
I
Glucose6-phosphate G
lL
+y
Fructose
f-OhosRhate B
J
Fructose1, 6-bisphosphate
Glyceraldehyde
{- Dihydroxyacetone
3--phosphaie pnosfinate S
J
:nol pyruvate
+
ruvate.
J \
rylCoA
iitrate
iREBS \ \
)YCLE
)YCLE tT \
+ lsocitrate \
I t \ A LA N TN E
\ J \/
Fumarate o-Ketoglutarate'!-:
\ / - *---7- Glutamate
\, ^
sudcinate
/ coA +_ ?' * ";
succinlii NH; 6LUTAM1NE
Fig' 35.3 : An outline of the glucose and glutamine metabolism in cultured mammalian cells,
1020
1018 Continuous
C U IIU T C c e l ll i n e
o
(d 10 1 6
a
o
o 10 1 4
o
Senescenceand
E
J 1012 cell death
c
Subculture
q) interval
O
10 1 0
Explantation
2nd subculture
108 J
106
?
I
o n the cultu r e env ir onm ent . For ins t anc e , t h e as cell markers for identification. Some examples
e pith elia l ce lls gr owing at t he c ent er ( of t he c u l t u r e ) are given below.
a re reg ula r p oly gonal wit h c lear ly def ined e d g e s ,
. Albumin for hepatocytes.
while tho se g r owing at t he per ipher y ar e ir r e g u l a r
. Melanin for melanocytes
an d disten de d ( s wollen) . The c om pos it ion o f t h e
cu lture me diu m , and t he alt er at ionsin t he s ub s t r a t e . Hemoglobin for erythroid cells
also influ en ce t he c ellular m or phology . I n a t i s s u e o Myosin (or tropomyosin)for muscle cells.
cu lture la bo r at or v . t he t er m s f ibr oblas t ic a n d Enzymes as tissue markers : The identificatron
e pith elia l are c om m only us ed t o des c r ibe t h e
of enzymes in culture cells can be made with
a pp ea ran ceo f t he c ells r at her t han t heir or ig i n .
referenceto the following characters.
Fibroblastic cells : For these cells, the length is r
C o n s t i t u t i v ee n z y m e s .
usua lly mo re t han t wic e of t heir widt h Fibr ob l a s t i c
. Inducible enzymes.
cells a re b ipo lar or m ult ipolar in nat ur e.
. lsoenzymes.
Epith elia l cells : Thes e c ells ar e poly gon a l i n
The comnronly used enzyme markers for cell
na ture with regulardim ens ionsand us ually gr o w i n
l i n e i d e n t i f i c a t i o n a r e g i v e n i n T a b l e 3 5 . 1.
mon ora ye rs.
Tyrosine aminotransferase is specific for
The terms fibroblastoid (fibroblast-like) and
h e p a t o c v t e s ,w h i l e t y r o s i n a s e i s f o r m e l a n o c y t e s .
epitheloid (epithelial-like)are in use for the cells
C r e a t i n e k i n a s e ( M M ) i n s e r u m s e r v e sa s a m a r k e r
that do not possessspecific charactersto identify as
for muscle cells, while creatine kinase(BB) is used
fibro bla stic o r eoit helial c ells
for the detection of neurons and neuroendocrrne
cells.
SPECIES OF O RI G I N O F CELLS
Filament proteins as tissue markers : The
The ide ntific at ionof t he s pec iesof c ell line s c a n
i n t e r m e d i a t ef i l a m e n t p r o t e i n sa r e v e r y w i d e l y u s e d
be done by.
as tissue or lineage markers.For example-
. Chro mosom al analy s is .
a Ele ctro ph or es isof is oenz y m es .
o A combination of both these methods.
Tlelr 35.1 Some common enryne narkers for
cell lin6 identification
ln re ce nt vear s , c hr om os om al ident if ic at i o n i s
be ing do ne b y em ploy ing m olec ular pr obes . I nzy i nc u-.ell ivpr:
Tyrosine
aminotransferase Hepatocytes
I DE NT I F I CA T ION O F Tysosinase Melanocytes
T I S S UE O F O R IG IN
Glutamylsynthase Brain(astroglia)
The identificationof cell lines with regardto kinase
Creatine Musclecells
tissueof origin is carriedout with referenceto the (isoenzymeMM)
following two characteristics.
kinase
Creatine Neurons,
1. T he line a g eto w h i c h th e c e l l sb e l o n g . (isoenzymeBB) neuroendocrine
cells
s f th e c e l l si .e .s te mc e l l s ,p re cursor
2. T hes t at u o Non-specific
eslerase Macrophages
c elIs . DOPA-decarboxylase Neurons
phosphatase
Alkaline typell
Enterocytes,
T is s ue m ark e rs fo r c e l l l i n e pneumocytes
ident if ic at io n Angiotensin-converti
ng Endothelium
S om eof t h e i mp o rta ntit s s u eo r l i n e a g emarkers enzyme
f or c ell line id e n ti fi c a ti oanre b ri e fl yd e s c ri b ed Sucrase Enterocytes
Differentiatedproductsas cell markers: The Neuron-specific
esterase Neurons
c ult ur edc ells,o n c o mp l e tee x p re s s i o n
a ,re c a p abl e DOPA-Dihydroxy
phenylalanine;
MM-Twopolypeptidesubunitsol
of producingdifferentiation markers,which serve muscle;BB-Twopolypeptide of brain
subunits
430 B IOTE C HNO LO CY
o Crowth rate
a Mode of growth
used for tte detertlon of cell types a Longevity
No of coloniesformed
Platingefficiency = x 100
N o. of cel l s seeded
2468 10
Days of subculture
Biotechnology
[28]
434 B IOTE C I.I NO LO CY
T U N E L i s a n a b b r e v i a t i o n f o r T dT- m e d i a te d
Role of caspases in apoptosis
d U T P n i c k e n d - l a b e l i n g a s s a y .T U N E L i s ve r y fa st
A group of enzymes namely activated proteases and effective for the determination of DNA
play a c r uc ial r ole in t he pr o g r a m m e d c e l l d e a t h . fragmentsformed by endogenous nucleaseactivity.
These proteases are actually cysteinyl aspartate The apoptotic nuclei can be identified by a
specific proteinasesor in short, commonly referred f l u o r e s c e n t technique using fl u o r e sce i n
to as caspases.There are about ten different types i s o t hi o c y a n a t e( F I T C )a n d 4 , 6 - d i a m in o p he n yli n d o l e .
of caspasesacting on different substratesultimately
leading t o c ell deat h For i n s t a n c e , c a p s a s e I DNA laddering test
.l
cleaves interleukin B.
During the courie of apoptosis,the genomic
D N A i s c l e a v e d t o m o n o - a n d o l i g o nu cl e o so m a l
Inhibition of caspase activities
DNA fragments.These fragmentscan be separated
Sinc e t he c as pas es ar e c l o s e l y i n v o l v e d i n by agarose electrophoresis, and detected. The
apoptosis, it is possible to prevent cell death by nucleosomal fragments of apoptotic cells give a
inhibit ing t heir ac t iv it ies Ce r t a i n s p e c i f i c p e p t i d e s characteristicladder pattern on electrophoresis.
that can inhibit caspases,and thus apoptosis have
been identified. Limitations of the test : DNA laddering test is
not verv soecific since several cells that have
undergoneapoptosismay not show DNA laddering.
MEASUREMENT OF APOPTOS'S
Further,some cells not subjected to apoptosis may
A simple and easy way of detecting dead or also show DNA ladders, For these reasons, DNA
dy ing c ells is t he dir ec t m ic r o s c o p i c o b s e r v a t i o n . laddering test is coupled with some other test for
fhe dying cells are rounded with dense bodies measurementof apoptosis.
I
primary culture reters to the starting culture substanti al l y l ow er (when compared to
/ \of cells, tissues or organsl taken directly from sr,rbcul tures)
an o rga nism .Thus , t he pr inr ar y c ult ur e is t lr e i n i t i a l . T h e t i s s u e ss h o u l d b e p r o c e s s e dw i t h m i n i m L r m
cu lture b efo r e t he f ir s t s ubc ult ur e The t er m c e l l
damage to cells for use in primary culture
linc is used for the propagation of cultures alrer
F u r t h e r ,t h e d e a d c e l l s s h o u l d b e r e m o v e d .
th e first su bc ult ur e Som e bas ic and f unda m e n t a l
aspe (itso f p r im ar y c ult ur e and c ell lines ar e b r i e f l v . S e l e c t i o no f a n a p p r o p r i a t em e d i u m ( p r e f e r a b l ya
de scribe d. n u t r i e n t r i c h o n e ) i s a d v i s a b l e .F o r t h e a d d i t i o n o f
serum, fetal bovine source is preferred rather
than calf or horse serum.
r lt is necessaryto remove the enzymes used for
d i s a g g r e g a t i o no f c e l l s b y c e n t r i f u g a t i o n .
As alre ady s t at ed, pr im ar y c ult ur e b r o a d l y
involves the culturing techniques carried following TECHNIQUES FOR PRIMARY CULTUBE
the isolation of the cells, but before the first Among the various techniques devised for the
subculture. Prinrary cultures are usually prepared p r i m a r y c u l t u r e o f i s o l a t e dt i s s u e s ,t h r e e t e c h n i q u e s
from la rge ti s s ue m as s es .Thus , t hes e c ult ur e s m a y
are most comrnonly used.
con tain a v ar iet y of dif f er ent iat ed c ells e . g .
fibro bla sts, l y m phoc y t es , m ac r ophages , epi t h e l i a l l. Mechanical disaggregation.
cells. 2. Enzymalic disaggregation.
With th e ex per ienc esof t he per s onnel wo r k i n g 3. Primary explant technique.
in tissu ecu lt ur e labor at or ies t, he f ollowing cr i t e r i a / An outline of these techniques is depicted in
cha racterist ic s ar e c ons ider ed f or eff i c i e n t F i g . 3 6 . 1, a n d t h e p r o c e d u r e sa r e b r i e f l y d e s c r i b e d .
de ve lop men t of pr im ar y c ult ur es .
. Emb ryo nic t is s ues r at her t han adult t is s u e s a r e MECHAN'CAL D'SAGGBEGATION
p refe rredfor pr im ar y c ult ur es .This is due t o t h e
For the disaggregationof soft fissues(e 9,.spleen,
fa ct tha t t he em br y onic c ells c an b e
brain, embryonic liver, soft tLrmors),mechanical
disag gre gat edeas ily and y ield m or e v iable c e l l s ,
technique is usually employed. This technique
besides rapidly proliferating in vitro.
basically involves careful chopping or slicing of
o The qu an t it y of c ells us ed in t he pr im ar y c u l t u r e tissue into pieces and collection of spill out cells.
sh ou ld b e higher s inc e t heir s ur v iv al r a t e i s The cells can be collected bv two wavs.
437
438 B IOTECHNO LO CY
Primaryexplanttechnique
r Pressing the tissue pieces through a series of effi ci ent w i th hi gher yi el d of cel l s by use of
s iev eswit h a gr adual r educ t i o n i n t h e m e s h s i z e enzymes.This is due to the presence of lessfibrous
connecti ve ti ssue and extracel l ular m at r lx.
. Forcing the tissue fragments through a syringe
Enzymaticdisaggregation can be carried out by
and needle.
usi ngtrypsi n,col l agenase or someotherenzym es.
Alt hough m ec hanic al di s a g g r e g a t i o n i n v o l v e s
the risk of cell damage, the procedure is less Disaggregation by tryPsin
ex pens iv e, quic k and s im p l e . T h i s t e c h n i q u e r s
The term trypsinization is commonly used for
par t ic ular lyus ef ulwhen t he a v a i l a b i l i t yo f t h e t i s s u e
disaggregationof tissuesby the enzyme,trypsin.
is in plenty, and the efficiency of the yield is not
Many worker;spreferto use crude trypsin rather
very crucial. lt must however, be noted that the
than pure trypsinfor the followingreasons.
v iabilit y of c ells obt aine d f r o m m e c h a n i c a l
t ec hniques is m uc h lower t h a n t h e e n z y m a t i c . The crude trypsin is more effectivedue to the
t ec hnioue. presenceof other proteases
. Cellscan toleratecrude trypsinbetter.
E NZYM AT'C D'SAG G R EG AT' O N
. The residualactivity of crude trypsin can be
Enzymatic disaggregationis mostly used when easi l y neutral i zedby the serumof t he cult ur e
high recovery of cells is required from a tissue. media (when serum-freemedia are used, a
Disaggregation of embryonic tissues is more trypsi ni nhi bi torcan be usedfor neut r alizat ion) .
ChA P I CT
36 : P R IM AR Y AN D C E L LL IN E S
CULTURE 439
Disaggregation of cells can also be carriedout ineffective for certain tissues (e.g. fibrous
b y us ingpur et r y p s i nw h i c h i s l e s sto x i c a n d m o re connective tissue). Hence other enzymes are also
soecificin its action. i n u s e f o r d i s s o c i a t i o no f c e l l s .
The desiredtissueis choppedto 2-3 mm pieces
and then subjectedto disaggregation by trypsin. Disaggregation by collagenase
There are two techniquesof trypsinization-warm Collagen is the most abundant structuralprotetn
trypsinizationand cold trypsinization(Fig.36.2). i n h i g h e r a n i m a l s . l t i s m a i n l y p r e s e n ti n t h e e x t r a -
Warm trypsinization(Fig. 36.2A) : This method cellular matrix of connective tissue and muscle.
is widely used for disaggregation of cells. The The enzyme collagenase (usually a crude one
choppedtissueis washedwith dissectionbasalsalt contaminated with non-specific proteases)can be
solution (DBSS),and then transferredto a flasr effectively used for the disaggregationof several
containingwarm trypsin(37oC). The contentsare tissues(normal or maligndnt) that may be sensitive
stirred,and at an intervalof every thirty minutes, to trypsin. Highly purified grades of collagenase
th e s uper nat an
co g e d i s s o c i a tecde l l sc an
t n ta i n i n th have been tried, but they are less effective when
be collected.After removalof trypsin,the cellsare compared to crude collagenase.
dispersedin a suitablemedium and preserved(by The important stages in collagenase dis-
keepingt he v ial o n i c e ). aggregation, depicted in Fig. 36.3, are briefly
The processof additionof freshtrypsin(to the described hereunder.
tissue pieces), incubation and collection of
The desired tissue suspended in basal salt
c ells(a t 3 0 mi n u te si n te rv a l si s) c a rried
d is s oc iat ed
solution, containing antibiotics is chopped into
out for about 4 hours.The disaggregated cells are
pieces. These pieces are washed by settling, and
pooled, counted, appropriatelydiluted and then
t h e n s u s p e n d e di n a c o m p l e t e m e d i u m c o n t a i n i n g
incubated.
collagenase. After incubating for 1-5 days, the
Cofdtrypsinizatlon(Fig.36.28): Thistechnique tissuepieces are dispersedby pipetting.The clusters
is more appropriatelyreferredto as trypsinization of cells are separated by settling. The epithelial
with cold preexposure.The risk of damageto the cells and fibroblastic cells can be separated.
cells by prolongedexposureto trypsinat 37oC(in
war m t r y ps iniz a ti o nc)a n b e mi n i mi z e d i n th i s Collagenase disaggregation has been
techn ioue. successfully used for human brain, Iung and
several other epithelial tissues, besides various
After choppingand washing,the tissuepieces human tumors,
and other animal tissues. Addition
are kept in a vial (on ice) and soakedwith cold o f a n o t h e r e n z y m e h y a l u r o n i d a s e ( a c t s
on
trypsin for about 6-24 hours. The trypsin is carbohydrate residues on
cell surfaces) promotes
removedand discarded.However,the tissuepieces d i s a g g r e g a t i o n .C o l l a g e n a s e i n c o m b i n a t i o n w i t h
l p s i n .T h e s eti s s u ep i e c e si n a hyaluronidase is found
c ont ainr es idua try to be very effective for
mediumare incubatedat 37oCfor 20-30 minutes. dissociatingrat or rabbit liver. This can be
done by
The cellsget dispersedby repeatedpipettings. The perfusing the whole
organ in situ.
dissociatedcells can be counted, appropriately
dilut edand t hen u s e d . Some workers use collagenase in conjunction
with trypsin, a formulation developed in chick
The cold trypsinization method usually results
serum, for disaggregationof certain tissues.
in a higher yield of viable cells with an improved
survivalof cellsalter24 hoursof incubation.This
Use of other enzymes
methoddoes not involvestirringor centrifugation,
in disaggregation
and can be convenientlyadoptedin a laboratory.
The major limitationof cold trypsinization is that it Trypsin and collagenase are the most widely
is not suitablefor disaggregation of cellsfrom large used enzymes for disaggregation.Certain bacterial
quant it ies
of t is s u e s . proteases (e.g. pronase, dispase) have been used
with limited success. Besides hyaluronidase
Limitations of trypsin disaggregation (described above), neuraminidase is also used in
Disaggregation by trypsin may damage some conjunction with collagenase for effective
c ells ( e. g. epit h e l i a lc e l l s ) o r i t ma y b e a l most degradation of cell surface carbohydrates.
5
D
o
After 30 minutes
piecessettle Tissuepieces viai kept
in a vial
I
Add iresh
trypsinand
continueincubation
RemovetrYPSin
and incubate
at37"C for 20-30 min
Cellsin a medlum
Dilutein a
suitablemedium
o
-o
m
I
Z
o
-o
basa! salt solution)',
^^1, '+ianl o
(A) warm trypsinization (B) cotd trypsinization (DBs'-Dissectron
Fig. 36.2: preparation of primary curture by trypsin disaggregation
Chapt er36 : P R IM AR Y AN D C E L LLIN E S
CULTURE 441
Choppedinto --------------- f
piecesand
washed by settling
Fibroblastic
I
Pind++6d t^
cells
tissues
disperse
Separationby
Epithelial settling
cells
Fig. 36,3 : lmporlant stages in collagenase disaggregationof tissue for primary culture (BSS-Basal salt solution)
It is a common practice to remove the non- 1. The consent of the patient and/or relatives
viab le cells wh ile t he pr im ar y c ult ur e is pr ep a r e d for using tissuesfor researchpurposes.
442 B IOTECHNO LO CY
3 . C o n s e n t f o r g e n e t i c m o d i f i c a t i o n o f th e ce l l
Ii n e s .
6 . P a t e n t r i g h t s f o r a n y c o n r m e r c i al u se o f ce l l
Ii n e s .
I n t h e g e n e r a l p r a c t i c e o f c u l t u r e te ch n i q u e s
u s i n g h u m a n t i s s u e s ,t h e d o n o r a n d / o r re l a ti ve sa r e
asked to sign a disclaimer statement (in a prescribed
p r o f o r m a ) b e f o r e t h e t i s s u e i s t a k en . By th i s
a p p r o a c h , t h e l e g a l c o m p l i c a t i o n s a r e mi n i n r i ze d
Safety measures
Tissuepieceson
growthsurface
+
I
T h e d e v e l o p m e n t a n d v a r i o u s o t h er a sp e cts o f
Incubateand change o r i n r a r v c u l t u r e a r e d e s c r i b e da b o v e T he te r m ce l l
mediumat line refers to the propagation of culture after the
weeklyintervals first subculture, In other words, once the primary
c u l t u r e i s s u b c u l t u r e d , i t b e c o m e s a ce l l l i n e . A
J
I given cell line contains several cell lineages of
either similar or distinct phenotypes.
t _l-r
rxpranrs
i I I t i s p o s s i b l et o s e l e c ta p a r t i c u l a rc e l l l i n e a g eb y
J
I c l o n i n g o r p h y s i c a l c e l l s e p a r a t i o no r so m e o th e r
selection method Such a cell line derived by
selection or cloning is referred to as cell strain.
C e l l s t r a i n s d o n o t h a v e i n f i n i t e l i f e , a s th e y d i e
after some divisions.
T h e c e l l s i n c u l t u r e d i v i d e o n l y a l i m i te d n u m b e r
of times, before their growth rate declines arrd they
Freshculture eventually die. The cell lines with limited culture
vessel Iife spans are referred to as finite cell lines. fhe
c e l l s n o r n r a l l yd i v i d e 2 0 t o 1 0 0 t i m e s ( i .e . i s 2 0 - 1 0 0
Fig. 36.4 : Primary explant technique for
p o p u l a t i o n d o u b l i n g s )b e f o r e e x t i n c t i o n Th e a ctu a l
Drimarv culture.
n u m b e r o f d o u b l i n g s d e p e n d s o n t h e sp e ci e s,ce l l
Chapt er36 : P R IMA R Y
CULTURE
AN D C EL LL IN E S 443
lin ea ge diffe renc es , c ult ur e c ondit ions et c . T h e It must be noted that the passagenumber and
h uma n ce lls ge ner ally div ide 50- 100 t im es , wh i l e generation number are not the same, and they are
mu rine ceils di v ide 30- 50 t im es bef or e dv ine totallv different.
3. Ce ll typ e : Em br y onicc ells , t r ans f or m edc e l l s There are two types of subcultures-monolayer
an d co ntin uo us c ell lines gr ow r apidly and r eq u i r e subcul ture subcul ture.
and suspensi on
more freq ue nt subc ult ur ingand c hange of m edi u m
Th is is in co nt r as t t o nor m al c ells , whic h g r o w MON OLA Y E R C U LTU R E S
slowly. When the bottom of the culture vessel is
4. Morphological changes:Frequentexaminatton covered with a continuous layer of cells, usually
o f ce ll mo rph ology is v er y im por t ant in c ul t u r e one cell in thickness,they are referred to as
techniques. Anv deterioration in cell morphology monol ayer cul tures.The attachment of cel l samong
may lead to an irreversibleciamage to cells. Change themselves and to the substrate(i.e. culturevesser,l
of the medium has to be done to completely avoid is mediated through surface glycoproteins
(cell
the risk o f cell dam age. adhesi onmol ecul es) and cal ci um i ons (C az* )'For
subcul turi ng of monol ayercul tures, i t i s usual ly
necessary to removethe mediumand dissociate the
cel l s i n the monol ayerby degradi ngthe cel l
adhesi onmol ecul es, besi desremovi ngC az+ .
Confluent
monolayercells
Cellsrounding
Resuspended up
cells
r Maintenanceis easy.
o B ulk pr oduc ti o no f c e l l s c a n b e c o n v e n i entl y
ac hiev ed. The cells that retain their proliferative capacity
throughout life are regarded as sfem cells. When
Criteria for suspension subculture the stem cells divide, they can generate
The criteriaadopted for suspension subculture differentiatedcells and/or some more stem cells.
are the same as that already described for Thesestemceilsare capableof regenerating tissue
m onolay ers ub c u l tu re(Se
s ep . 4 4 6 ).T h e fo l l o wi ng after injury. The lack of tissue-specific
asDectshaveto be considered. differentiationmarkersis a characteristic featureof
stemcel l s.
o Culturedensity.
. pH changerepresenting mediumexhaustion. E MB R Y ON IG S TE M (E S I C E LLS
T h e b a s i c c r i t e r i a t o m a i n t a i n s t e m c e l l i n vi tr o
is to ensure that they possess the same
Stirrerflask
c h a r a c t e r i s t i c sa n d d i f f e r e n t i a t i n g a b i l i ti e s w h e n
they are present in the tissue in vivo. fhe
maintenance of epidermal and non-epidermal
e p i t h e l i a l c e l l s i n t h e i n v i t r o c u l t u r e s i s b r i e fl y
loaa
alo described.
. a .. Cellsin
-aoa
aa
suspensron
o ro a Epidermal stem cells in culture
t ._---{t Rotating
. Y. pendulum The epidermal stem (or keratinocyte stem) cells
a//\a
c a n b e s u c c e s s f u l l y m a i n t a i n e d b y c o - cu l tu r i n g
w i t h 3 T 3 f e e d e r l a y e r . B y t h i s t e c h n i qu e , i t i s
p o s s i b l et o a c h i e v e l o n g t e r m m a i n t e n a n ceo f ce l l s,
b e s i d e s r e t a i n i n g t h e i r c a p a c i t y fo r b o th
Fig. 36.7 : Suspension culture in a stirrer flask. p r o l i f e r a t i v e a n d d i f f e r e n t i a t i n g c h a r a c te r i sti cs.l t
has been demonstratedthat the so maintained stem
||i c e l l s w h e n p l a c e d i n t o n u d e m i c e c ou l d fo r m
s Advantages of ES cells
stratified and differentiatedepithelium.
;
In general, the cultures fron'r enrbryonic tissues
Serum-free meciia rvith added growth factors
t survive, and proliferate better than those from the
w e r e f o u n d t o b e m o r e e f f i c i e n t i n n r a i n ta i n i n gth e
adult . This is due t o t he f ac t t h a t E S c e l l s a r e l e s s
eoidermal stem cells in culture.
s pec ializ edwit h higher pr olif e r a t i v ep o t e n t i a l .
epithelialcells(of cytokeratin
family)arealsouseful Applications of tissue
for their identification. specific stem cells
Biotechnology [29]
n t he l. r bor at or iess, m all s c al e c u l t u r e s o f c e l l s r n maintenance of cells in culture. A good
I
l f las k s ( us ually 1- 5 lit r e v olu m e ) a r e d o n e f o r u n d e r s t a n d i n go f t h e s e p a r a m e t e r s ,l i s t e d b e l o w r s
? es t ablis hingt he c ell lines . Suc h c e l l c u l t u r e s a r e a l s o n e c e s s a r yf o r s c a l e - u p
* us ef ul f or s t udy ing t he m o r p h o l o g y , g r o w t h , . Cell quantitation.
m et abolis m et c . Lar ge- s c alec u l t u r e s a r e r e q u i r e d
e Equipmentand medium.
f or s em i- indus t r ial( 100- , 1, 000| c a p a c i t y ) a n d l a r g e -
scale industrial (5,000-20,000 | capacity) use of . pH and buffer systems.
cells for production of wide range of biologically . Oxygen
important compounds (e g. enzynres, antibodies, . Crowth kinetics.
hormones, interferons, plasminogen activator,
o Typesof culture processes.
i nt er leuk i ns ) .
. Other practical considerations.
The terms fermenter and bioreactor are in
common use while dealing with the industrial use
CELL QUANTITATION
of cells. A fermenter usually refers to the
c ont ainm ent s y s t em f or t h e c u l t i v a t i o n o f T h e t o t a l n u m b e r o f c e l l s i n a c u l t u r e ca n b e
pr ok ar y ot icc ells ( bac t er ia,f ung i ) ,w h i l e a b i o r e a c t o r m e a s u r e d b y c o u n t i n g i n a h a e m o c y t o me te r l t i s
gr ows t he euk ar y ot ic c ells ( m a m m a l r a n ,i n s e c t ) . h o w e v e r , n o t p o s s i b l e t o i d e n t i f y t h e v ia b l e a n d
Scale-up refers to the process of developing the non-viable cells by this method
culture systems in stages from a laboratory to the C e l l v i a b i l i t y : T h e v i a b i l i t y o f c e l l s ca n b e
industry. Scale-up although tedious, labour detected by use of dyes e g. tryphan blue. The
int ens iv e and ex pens iv e, is r e q u i r e d f o r t h e p r i n c i p l e i s b a s e d o n t h e f a c t t h a t t h e d ye i s
pr oduc t r on of c om m er c ially im p o r t a n t p r o d u c t s . p e r m e a b l e t o d e a d c e l l s w h i l e t h e v i a b l e ce l l s d o
For a bet t er under s t andingo f s c a l e - L r p c, e r t a i n not take up dye
bas ic and f undam ent al c onc e p t s o f c e l l c u l t u r e lndirect measurements for cell viability : The
s hould be c lear v i a b i l i t y o f c e l l s c a n b e m e a s u r e d b y th e i r
metabolic activity Some of the most commonly
used parametersare listed
. Clucose utilization
. Oxygen consumption.
450
Chapt er37 : A N IN 4 A LC EL LC U L T U R E -C E NE R A C
L ON S ID E R A TION
ASN D S C A LE -U P 451
In recentyears,many laboratories hal'e started The commonly used buffer of Ihe in vitro culture
measuringthe activity of lactate dehydrogenasecarbon dioxide-bicarbonatesystem(2-5% CO, with
( LDH) t o det e c tc e l l v i a b i l i ty .D e a d c e l l s re l ease 1 0 - 2 5 m M N a H C O 3 ) i s c o m p a r a b l e t o t h e b l o o d
LDH and therefore,this enzyme can be used to buffer. The presenceof phosphatesin the medium
quant it at iv elm
y e a s u reth e l o s so f c e l l v i a b i l i ty improves the buffering capacity. Some laboratories
u s e H E P E Si n s t e a do f b i c a r b o n a t ef o r m o r e e f f i c i e n t
E O UI P M E NT A N D ME D IU M buffering.
I
'6
that the cell proliferationis much higherthan the
batch culture.Thus, in the fed batch culturethere
is an increasein the volume of culture.
o
Semi-continuous batch culture
o
A portionof the culturemediumis intermittently
replacedwith an equal volume of fresh medium.
The growth patternof the cells is fluctuating,with
a rapid increase in" cell density after each
replacement of the medium.
perfusion culture
ol differentcultureprccesses. Continuous
Fig. 37.1: Comparison
'':
Thereis a continuousadditionof the mediumto
the cultureand a withdrawalof an equalvolumeof
Gro w th o f c e l l s u s u a l l ymeansan i ncreasetn used bell-freemedium.The continuousperfusion
cell numbers.However,increasein cell massmay processmay close or open for circulationof the
o c c u r w i th o u tre o l i c a ti o n . medi um.
The following terms are in common use to Continuous-flow cultute
growth of culturedcells.
represent
In the continuous-flowculture, a homeostatic
Specific growth rate : The rate of cell growth condi ti onw i th no change i n the cel l num ber s,
p e r u n i t a mo u n to f b i o m a s s . nutrientsand metabolitesis attained.This is made
i Doubling time : The time required for a possible by a balancebetweenthe additionof the
populationof cellsto double in numberor mass. medium and withdrawal of medium along with
a cells.This is mostlysuitablefor suspensioncultures.
Degreeof multiplication(numberof doublings):
T h e n u mb e r o f ti me s a g i ven i nocul um has OTHER PRACTICAL CONSIDERATIONS
reolicated. Besidesthe parameters describedabove,there
I
{ are severalother practicalconsiderations for in
TYPES OF CULTUBE PROCESSES vitro cultureand scale-up.Someimportantonesare
The differentculture processes and the growth given below.
patternsof cells (represented
by cell density)are Culture surfacearea i The availablesurfaceis
depictedin Fig 37.1. They are brieflydescribeo. important for the cells.to grow. In general, the
culture processes are plannedin such a way that
Batch culture the surfacearea is not a limiting factor.
l n th i ste c h n i q u ew, h e n th e cel l sare i nocul ated Inoculationdensityof cells : As such,there is
i n to a fi x e dv o l u meo f th e me di um,they uti l i zethe no set rule for the densityof inoculation.However,
nutrientsand grow,and simultaneously accumulate inoculationwith high densityis preferredfor better
metabolites. As the nutrientsget exhausted,toxic growth.
waste products accumulate and the cell
Growth phase of cells : Cells in the late
multiplicationceases. Further, the cell densitydrops
exponential (log) phase are most suitable for
due to deathof the cells.
inoculation.The cellsat the stationaryphaseshould
Batch culture is a standardtechnique.Several be avoidedsince they have either prolongedlag
modifications have been made to increase phaseor no growth at all.
Chaot er37 : A N IMA LC E L LC U L T U R E-C EN E R A C AS
L ON S ID E R A TION N D S C A LE -U P 453
t t t
t
a a at
Scale-up involves the development of culture ta
' at a
'
systems in stages from (small scale) laboratory to ta
a a ' a
'
(large scale) industry. The methodology adopted to
t .----l t
increa se the s c ale of a c ult ur e depends on t h e .
pendulum
pro lifera tion of c ells and is br oadly div ided i n t o
two categories.
5%co2
u*
"3 *-Filter
Cell
Magne.tized suspensron
rotating
pendulum bath
31'c Medium
Cell
Cell
suspensron
in medium
Cells
The monolayer culture are anchbrage-
Bafile dependent. Therefore, for the scale-up of
Air bubble monolayer cultures, it is necessaryto increase the
surface area of the substrate in proportion to the
number of cells and volume of the medium.
Advantages
NASA bioreactor
NASA (National Aeronautics SPace
Administration,USA) constructed a bioreactorto
grow the cellsat zero gravityby slowly rotatingthe
chamber (Fig. 37.5). The cells remain stationary
and form three-dimensional aggregatesand this
enhancesthe product formation. In the NASA
Rotatingculture
bioreactor,thereis almostno shearforce;hencethe chamber
cells are not damaged.
As the culture chamberstopsits rotations,the Suspendedcell
sedimentand the medium can be
cell aggregates aggregates
replaced.
O T HE R S Y S T E M S F OR SU SP EN SION
CULT URE
Rotatingchambers: The mixingand aerationof
the culture medium can be achievedby 2 or 3
TI
Fresn- Spent
rotatingchambers.The chambersare so designed medium medium
t hatt he c ell s usp e n s i oann d m i x i n ga re h i g h i n o ne input and productoutpul
chamber rvhile the product and spent medium
,remainin the other chamber.Thesechambersare
separatedby semipermeable membrane.
456 B IOTE CHNO LO CY
MULTISURFACE CULTURE
The most commonl y used mu lt isur f ace
Roller propagatorof monolayer is Nunclon cell factory
bottle (in short Nunc cell factory). lt is composed of
Cells rectangularpetri dish-likeunits with huge surface
area (1,000-25,000 cm2). The units are inter-
cap
connectedat two adjacentcornersby verticaltubes
(Fig, 37.V. The medium can flow between the
Medium comoartments from one end.
The cell factory is almost like a conventional
petridishor a flaskwith multiplayerunits.
The main limitationof cell factory is that it is
Rollers very difficult to monitor the growth of cells. The
majoradvantage however,is its simpleoperationto
Fig. 37.6 : Roller bottle culture. producel argenumber' ofcel l s.
MULTIARRAY DISKS,
. T h e c e l l p ro d u c t fo rma ti o n (pharmaceuti cal l ySPIRALS AND TUBES
importantcompoundse.g interferon,antibodies) The surface area for growth of monolayer
i s mu c h h i g h e r. cul turescan be i ncreasedby usi ngdi sks,spir alsor
o The samesetup and apparatus can be repeatedly tubes. They are however, not in common use as
usedwith differentmedia and cells. thei r commerci i
al moortance
i s l i mi ted.
- oo o o
5
Water iackel Water
jacket
Glassbeads Glass
OO beads
--+
Mediumand t
I
inoculationinlet
Mediumand
inoculationinlet
(A) Fixed-bed reactor (B) Fluidized-bed reactor
Medium
co2---+
Samplestakenout for
Numberof cells
Monitoring by Productsformed
probes Leftovernutrients
Volume Contaminationcheck
pH {-
vv2
^n
Water jacket
O2
Glucose
Bioreactor
Ternperature
Turbidity
MONITORING OF MONOLAYER
C U LTU R E S
It is ratherdifficult to monitor monolavercell
culturesfor scale-up.This is due to the fact that in
most of the techniquesemployedfor monolayer
Mcnitoringof the progressof cell growth and cultures,the cells cannot be observeddirectly to
the culturesystemsare very importantin scale-up. monitorthe progressof the culture.
In recent years, nuclear magnetic resonance
MON IT O R IN G OF
(NMR)techniqueis usedto assaythe contentsof
SUSPENSION CULTURES
NMR spectragenerated
culture.The characteristic
The progressof suspensioncultures can be by specificmetabolitesenablesthe identification
monitored in situ by measuringglucose, 02, CO? and quantitationof metabolites,
besidesdetecting
pH or metabolitesproduced (lactate,ammonia)or the progressof cell growth.
I s the celis are removed from the living (in vivo) with special reference to systemic responses,
Ae nviron men t and s ubjec t ed t o ex per im en t a l metabolism and pharmacokinetics.
manipulations in the culture systems(in vitro), Ihetr
viab ility assu mess ignif ic anc e.Viabilit y of t he c e l l s Tissue and systemic responses
represents the capability of their existencel
The cultured cells reoresent the cells in
survival and development.
i s o l a t i o n ,a n d n o t a n i n t e g r a l p a r t o f a t i s s u eo r a n
Man y e xp eri m ent sar e c ar r ied out wit h c ells i n organ. As already stated, the nature of the in vitro
the cu lture ra ther t han us ing t he anim al m ode l s . effect is measured by cell survival or by an
Th is is p artic ular ly s o wit h r egar d t o th e altered metabolic effect. On the other hand,
determination of safety and cytotoxicity of several the in vivo reactions are complicated that may
comp ou nd s (ph ar m ac eut ic alsc,os m ot ic s ,ant ic an c e r lead to tissue responses (fibrosis, inflammatory
drugs, food additives). ln vitro testing Ior reaction) or systemic responses (pyrexia,
cytotoxicity and safety evaluation is in fact vascular dilation). Therefore, the in vitro
cost-effective, bresides reducing the use of cytotoxic responses have to be considered
animals. Studies on cytotoxicity broadly involve carefully.
the metabolic alterations of the cells, including
the death of cells as a result of toxic effects In recent years/ some attempts are made to
of the compounds. For instance, in case of anti- create in vitro organotypic cultures by assembly of
cancer drugs, one may look for death of different cell types. Even with this approach, it is
not possible to observe all the tissue, and systemic
ce lls, wh ile f or c os m et ic s t he m et abo l i c
responses.
alterations and allergic responses may be more
rmoortant.
Metabolism
LIMITATION OF V'TBO
The whole body metabolism is complex and
CYTOTOXICITY "V
STUDIES
well integrated. Some of the compounds that are
To xicity of a giv en c om pound is a c om pl e x toxic in vitro are detoxified by the liver. On the
process as it occurs in vivo. This may result in other hand, certain non-toxic or less toxtc
d irect da mag e to c ells , alt er at ionsin phy s iologi c a l compounds may be converted to more toxic ones
a nd b ioche mica l f unc t ions , inf lam m at or y c hang e s in the liver. For these reasons, it is necessarythat
and other systemic effects, not only at the site of the in vitro cells are exposed to the same type of
a pp lica tion b ut als o at t he ot her s it es .Som e of t h e compound that is formed in liver after
important in vitro limitations are briefly described detoxification
460 B IOTE CHNO LO CY
METABOLIC ASSAYS
The metabolic assays are based on the
measurements of metabolicresponsesof the cells.
o Thesetestare carriedout afterexposureof the cells
I 0 .1 to cytotoxicdrugs(eitherimmediatelyor after2-3
o populationdoublings).
The most commonly used metabolic
measurements are DNA, RNA or proteinsynthesis
$ n n{ (by estimatingtheir concentration),besidesthe
assay of certain dehydrogenase enzymes.Some
U) detailson the estimationof DNA and proteinare
al readygi ven (S eep. 493).
limitationsof metabolicassays: The estimation
of the total contentof DNA proteinmay or may not
Concentrationof compound ----J
be i ndi cati veof i ncreasei n cel l numbe r .This is
Fig, 38.1 : Survival curve in clonogenic assay. becausetheseassayscannotdiscriminatebetween
the proliferativeand metabolic activity of cells.
Some workers, therefoie prefer to confirm the
. C e l l d e n s i tyd u ri n ge x p o s u re. metabolicmeasurements by colonogenicsurvival
. C e l l d e n s i tyd u ri n gc l o n i n g . a5say.
Transformation is associated with genetic The finite life spanof culturedcells is regulated
immortalization, aberrant growth by about 1O senescence genes.Thesedominantly
instabitity,
463
464 B IOTE CHNO LO CY
Biotechhology
[30]
466 B IOTE CHNO LO CY
STIMULATION OF PLATING
___t
t---------------
w.r.r.7fifi.m.t
EFFICIENCY
COLONIESISOLATED
JJ Addition of metabolites: Supplementingthe
medi um w i th i ntermedi ary metabo lit es ( e. 9.
pyruvate, o-ketoglutarate) and nucleosides
stimulatesplatingefficiency.
The clones can be isolateddirectly from the Use of purified trypsin : Some workers prefer
multiwelldishes(by trypsinization),
by cloningring to use purified trypsin insteadof crude trypsin
techniquefrom petridishes,and by irradiatingthe for trypsinizationso that the plating efficiencyis
plasticbottle. better.
ChA P t CT
39 : CE LLT R AN SF OR M AT ION
A N D C ELLC LON IN C 467
Gonditioned medium
A medium that has already been used for the
growth of other cells contains certain metabolites,
growth factors and other products that stimulate CellsIn suspensron Cellsin monolayer
culture culture
growth. Such a medium, referredto as conditioned
me diu m, whe n a dded t o c ell c loning m edium II I
improves plating efficiency. + +
Counting Trypsinization
Feeder layers (---- andcounting
I
A layer growth-arrestedliving cells, referred to ---r
as feeder layer, promotes plating efficiency. This rs
because the feeder cells provide nutrients, growth
factorsand matrix constituentsthat support survival,
growth and proliferation of cells.
S USPENSION CLO NI NG
Stockof cells
Certa in ce lls, par t ic ular ly t he t r ans f or m ed I
fibroblasts and hematopoietic stem cells. can oe I
mo re co nven ien tlyc loned in s us pens ionr at hert han +
monotayers. Seiialdilutions
468
r 40 : OR G A NAN D H IST OT Y PIC
C U L TU R E S
A,N D TIS S U EE N C IN E E R IN C
Organculture
. Cell layer grown on the matrix with interactive epithelium, - stratified epidermis, intestinal
cell layer on the underside of the filter epithelium and renal (kidney) epithelium.
ffig. ao.2D).
HISTOTYPIC CULTURES
Filterwell-insertswith differentmaterials(ceramic,
Crowth and propagationof cell lines in fhree-
collagen, nitrocellulose)are now commercially
dimensional matrix to high cell density represent
availablefor use in culture laboratories.
histotypiccultures.The advantage with this culture
Filter-well inserts have been successfully used systemis that dispersedmonolayerculturescan be
to develop functionally integrated thyroid usedto regenerate tissue-likestructures.
Petridish or
multiplatewell
Cell layer
Collagen
Filter or Matrigel
Petridish or -)
multiplatewell
Cell layer
Collagen
Filter or Matrigel
lnteractive
cell layer
(C) Interactive cell layer Medium (D) Interactivecell layer on the
on the underside of filter undersideof filter with matrixcoating
Fig. 40.2 : Diagrammatic representation of organ culture in filter-well insefts.
Chapt er40 : O R C A N AN D H IST OT Y PIC A,N D TIS S U EE N C IN E E R IN G
C ULTU R E S 471
.)o o o
d,?..o-q
n
v
(J^" (J
()n
oo -ov^(
iz"::$
NEst
Capillary
Fig. 40.3 : A diagrammatic representation of a tumor in comparison with a multicellular tumor spheroid.
1. Tensionalforces.
CELL ORIEI{TATION
forces.
2. Compressional
The orientationof cells with regardto specific
shapeand spatialarrangement is influencedby the 3. Shearforces.
followingenvironmental factors.
or contactguidance.
1. Substrate CELL SUPPORT MATERIALS
2 . C h e m i c a gl ra d i e n ts . The supportmaterialsof cells largelydetermine
the nature of adherentcells or cell types, and
3 . Me c h a n i c acl u e s .
consequently tissueengineering.Thereare a large
numberof supportmaterialswhich may be broadly
Substrate guidance
categorizedas follows.
The topographical features of the substrate
. Traditionalabiotic materials.
determinethe contact guidance.These features
may be in the form of ridges,alignedfibersetc. lt . Bioprosthesis
materials.
is possible to use differential attachment to
substratesas a means of producing different r Syntheticmaterial.
a l i g n m e n t o f c e l l s . l n re cent years, syntheti c . N aturalpol ymers.
polymer substratecollagenfibrils and fibronectin
are used as bioresorbabletemplatesfor tissue . S emi -natural
materi al s.
e n Sr n e e nn 8 .
Traditional abiotic materials
Ghemical gradients
The traditionalabioticsupportmaterialsinclude
Developmentof chemicalgradientsis required plastics,ceramics and metals. These materials
for cellular orientation and for the stimulation of cannot be resorbed or become biologically
cellular functions. Certain growth factors integratedinto the tissues. it is preferable
Therefore,
and extracellularmacromoleculesare capable to avoid the traditional materials in tissue
of creating chemical gradients e.g. vascular enSrneenn8.
Chapt er40 : O R C A N A N D H IS T O T Y PIC A,N D TIS S U EE N C IN E E R IN C
CU LTU R E S 475
Synthetic materials
DESIGN AND ENGINEERING
A wide range of synthetic bioresorbable
OF TISSUES
polymersare availableas supportmaterials.The
most commonly used polymers in tissue The following surgical criteria are taken into
engineeringare poly (glycolic'acid)(PCA), poly c o n s i d e r a t i o nw h i l e d e a l i n g w i t h t i s s u ee n g i n e e r i n g .
( lac t ic ac id ) (P L A),a n d c o p o l y me rPL GA (pol y
. Rapid restorationof the desired function.
( lac t ic - c o-g l y c o l iacc i d ). T h e c o mp o s i ti onand
dimensions of thesepolymerscan be so adjustedto . E a s eo f f i x i n g t h e t i s s u e .
make them stable ln vlvo, besidessupportingin
r Minimal patient discomfort.
vivo cell growth.
Thereare certainadvantages in usingsynthetic For designing tissue engineering, the source of
donor cells is very critical (See cell sources
BOrymers.
p . a 7 3 ) . U s e o f p a t i e n t so w n c e l l s ( a u t o l o g o u sc e l l s )
. Productionis easyand relativelycheap.
is favouredto avoid immunological complications.
o Compositionof polymersis reproducibleeven in A l l o g e n e i c c e l l s a r e a l s o u s e d , p a r t i c u l a r l y w h e n
largescaleproduction. the TE construct is designedfor temporary repair. It
There are however, some disadvantagesalso. is observed that when the cells are cultured and/or
preserved(i.e. cryopreservation),the antigenicity of
o Com pati b i l i ty
w i th c e l l si s n o t a s g o o da s natural
allogeneic cells is reduced.
poly m er s .
. On degradation,they may form some products Another important criteria in TE is the support
whic h c a u s eu n d e s i ra b lcee l l u l a re ffe c ts. material, its degradation products, cell adhesion
characteristicsand mechanical cues.
Natural polymers The design and tissue engineering with respect
The mostwidely usednaturalpolymermaterials to skin, urothelium and peripheral nerve are briefly
are collagen-chondroitin sulfateaggregates. These described hereunder.
m at er ialsa re c o m m e rc i a l l a
y v a i l a b l ew i th varyi ng
compositionunder the trade name Integra. The Tissue engineered skin
other naturalpolymersfor cell supportare usually
lt was first demonstrated in 1975 Ihat human
obtainedby their aggregation in cultureas it occurs
keratinocytes could be grown in the laboratory in
in vivo e.g. collagengels,fibrin glue,Matrigeland
a form suitable for grafting. Many improvements
somepolysaccharides. Amongthe polysaccharides,
have been made since then. lt is now possible to
chitosanand hyaluronanare usedas hydratedgels.
grow epithelial cells to produce a continuous sheet
T he natu rapl o l y m e rsma i n l ya c t o n th e p ri nci pl e which progresses to form cornified layers. The
of intermolecuiar interaction within the polymersto major difficulty with TE skin is the dermal layer
476 BIOTECHNOLOCY
Additions
Conduit
material
Regenerating
nerve
Fig, 40.4 : A diagrammatic representation of the basic design for peripheral nerve implant.
Retinoicacid
Earlyembryo
(blastocyte)
Retinoicacid,insulin,
thyroidhormone
CulturedES cells
Retinoicacid ---l
m
o
T
Z
--
a\
various cells. o
Chapter40 : ORCAN AND HISTOTYPIC
CULTURES,
AND TISSUEENCINEERINC 479
480
Chapt er4 1 : T R A N S C E N IC
A N IMA L S 481
Biotechnology
[31]
482 B IOTE CHNO LO CY
@@@@
procedureusi ng a markerB ene or P CR analysis
(describedbelow). The transfectedcells can be
cul tured, i ntroduced (by mi croi nj ect ion)int o
Transgenic blastocystand then implantedinto foster mother
(i.e.,pseudopregnant femalemouse).By this way,
transgenicfoundermice are produced.Transgenic
Fig. 41.1 : DNA microinjection method to produce l i nes can be establ i shedby sui tab le br eeding
transoenic mice. strategies of the foundermice.
ES cells
Et thymidine to produce thymidine monophosphate
(dTMP) which is finally converted to thymidine
I
triphosphate (dTTP).
-Transoene
ia The gene that encodes the enzyme thymidine
, kinase can be used as a marker to determine
JTransfection
@ @ @_ES whether the transgene has been inserted. This is
cel with
l'a;;s';;; illustrated in Fig. 41.3. The mammalian cells are
@O@-O
capable of synthesizirrgdTTP by salvage pathway
I Selectionandculture and endogenous synthetic pathway. The cells
I to enrichtransfected
EScells lackingTK gene cannot produce dTTP lf such cells
J
are cultured in a HAT mediurn containing
(o(o@
v/-a\v/-^\\
hypoxanthine(H), aminopterine(A) and fhymidine
\Y-\Y-l (T), they cannot grow and therefore die. This is
ES cells with transgene
b e c a u s et h y m i d i n e c a n n o t b e u t i l i z e d i n t h e s a l v a g e
into
I Microinlection pathway due to lack the enzyme thymidine kinase.
blastocYst Further, aminopterine blocks the endogenous
J
pathway (by inhibiting the enzyme dihydrofolate
reductase,required for one carbon metabolism).
If a transgene is joined to a TK gene, inserted
into a mammalian cell (TK-)and then the cells can
g r o w i n H A T m e d i u m . T h i s i s p o s s i b l eo n l y i f t h e
T K g e n e i s i n c o r p o r a t e di n t o t h e m a m m a l i a n c e l l s .
Recipientblastocyst
A n d l o g i c a l l y ,t h e c e l l s t h a t s u r v i v e i n H A T m e d i u m
carry the transgene. In this fashion, thymidine
kinase can be effectively used as a marker gene.
There are other enzymes that serve as markers
for identifying transgeneinsertion. These include
dihydrofolate reductase (resistantto methotrexate)
and neomycin phosphotransferase (resistant to
antibiotic C41B) and PCR analysis for selecting
Fostermother transgene containing cells. The last one is a more
+
I direct and recent method, and is successfullvuseo
f o r d e t e c t i n g t r a n s g e n ec o n t a i n i n g c e l l s .
ffi,\ffinffi-\
q.44V t}
Promoter sequence to
I n t h e e a r l y e x p e r i m e n t s o n t r a n s g e n e s i s ,t h e
Transgenic mouse metallethionein (MMT) gene promoter was
founder
used. MMT gene encodes for a metal binding
protein that is involved in metal homeostasis.The
Fig. 41.2 : Embryonic stem cell (ES) nethod to
foreign gene (i.e., a rat growth hormone gene) can
produce transgenic mice.
be linked to a promoter sequence of MMT. By
484 B IOTECHNO LO CY
(A) Normal mammalian cell (B) Mammalian cell lacking TK gene (C) Mammalian cell (TK-)
with transgene and TK gene
Fig.41.3: Thymidine kinase (TK) marker gene for selecting transgene containing cells (X-represents the genes
for synthesizing dTTP by endogenous synthetic pathway).
doing so, the promoterswitcheson the growth knockout an existing function can be blocked hy
hormonegene when the MMT is activatedby a destroying a specific gene. The target gene
me ta l i n th e e n v i ro n m e n(e
t .g.,cadmi um).Thus, disruption can be carried out by incorporatinga
the metal inducer(Cd)can stimulatethe promoter DNA sequence,usually a selectablemarker gene
(MMT promoter)to facilitatethe transgene (growth (smg)into the coding region.This is depictedin
hormonegene)to express.Therefore,additionof Fig.41.4. The chromosome carryingthe targetgene
cadmiumtriggersthe growth hormoneproduction. (with 4 exons)with flankingsequences is subjected
to homologous recombination with a Vector
carryinga selectable markergene.The homologous
recombi nati onresul ts i n gene kno ckout i. e. ,
disruptionof the targetgene.
By inserting a transgene (foreign gene) into a ln the gene knockout, the loss-of-function
c hr om os om e, a new f unc t io n i s i n t r o d u c e d w h i l e occurs in transgenicanimals.This is in contrastto
producing transgenic animals (described above). gain of functionthat takesplace by introducinga
On the other hand, in a processreferredto as gene foreigngene.
Chromosome
t
Homologous
Target gene t
Homologous
reglon regron
J + I
Recombination
J
with geneknockedout
Chromosome
Fig. 41.4 : Gene knockout by homologous recombination
41 : TRANSCENI CANI M ALS
Ch AOICT 485
T H E A L Z H E I M E R 'S MOUSE
Alzheimer's disease affects about 1% of the
population between 60-65 years old, and about
Mou se , a ltho ugh not c los e t o hum ans in i t s 30"/" of the populatiCrnover B0 years old This
biology, has been and continues to be the most disease is characterized by progressive loss of
exp loite d an im al m odel in t r ans gene s i s memory and personality changes (decline in
experiments. The Common feature between man t h i n k i n g , j u d g e m e n t e t c . ) . T h e p o s t m o r t e ma n a l y s i s
an d mo use is tha t bot h ar e m am m als . Tr ans ge n t c o f b r a i n s o f A l z h e i m e r 's p a t i e n t s r e v e a l e d p l a q u e s
mice are extensively used as animal models for of dead nerve cells entangled in a protein called
understandine human diseases and for the amyloid. This protein w'as purified and its amino
486 B IOTE CHNO LO CY
TH E ON C OMOU S E
The animal model for cancer is the oncomouse
(onco refersto cancer).Firstdevelopedfor breast
cancer,the oncomouseis usefulfor r-inderstanding
of cancer and evol vi ne modal i ti esf or cancer
therapy.
fhe oncogenec-myc in associationw'ith mouse
mammarytumor (MMT) virus was found to be
responsible for breastcancerin animals.Transgenic
mice were producedby introducingchimericDNA
consistingof c-myc gene and sectionsMMT virus
i nto ferti l i zed mouse egg cel l s. B reastcancer
develooedin adult femalemice and the trait was
passedon to the offspring.
Oncomouse (the mouse that is genetically
alteredand is susceptiblefor cancer)was patented
by U.S. Patentoffice in 1988. In fact, it was the
lmmaturehuman
immunecells very first animal to be patented.
mice have been developed. lt is not an Knockout mouse with memory loss
exaggerationthat'the knockout mice have become
The memoryprocesses
in brain are believedto
as common as a test tube in the laboratory for a
be carried out by a specialized area called
biotechnologist. lt must be noted that not all the
hi ppocampus.B y a gene knockout techni que,
knockout mice are immediately useful for human
researchers have developed mice that lack
health and welfare. A selectedfew of the knockout
hippocampus.These knockout mice lacked the
mice are described.
ability to remember.
SGID mouse
Knockout mouse with
Se ve reco m bined im m unodef ic ienc y( SCID ) i s a retinitis pigmentosa
condition with a total lack of immune system.SCID
mice were developed by eliminating a single gene By inactivatingthe mouserhodopsingene,rods
and the resultant mice lost the ability to produce cells of the retinal deterioratein the transgenic
B-lymphocytes and T-lymphocytes. The human mice. This retinal degenerationis comparableto
mouse was later developed from the SCID nrouse human retinitis pigrnentosa. The rhodopsin
(Se e p . 48 5) . knockout mouse is useful for understanding the
retinal degeneration, besidesevolvingtherapeutic
Knockout mouse for allergy strategies.
The receptorsiteson certainbody cells for lgE
TR A N S GE N IC MIC E FOR
antibodies are believed to be responsible for
H U MA N D IS E A S E S
triggeringallergy reactions.Knockoutmice were
developedfor allergyby removingthegeneencading Transgenic mice are important for the
for receptor protein. The result is that antibodies devel opmentof therapeuti cdrugs and possi bl e
cannotbind to cellsdue to lackof receotors and the gerretherapies,besidesthe understanding of the
mice are unaffectedby allergic reactions.lt is humandi sease. Many transgenimic cew i th human
expectedthat somebreakthrough may occur in the diseaseequivalentshave been developedand a
near future to benefitthe millions of sufferersof selectedfew of them are given in Table41.1.
spreadthroughoutthe world.
allergicreactions,
Transgenlcanimals as
of therapeutlcally inportant proteins
Transgenesisin chicken can be used to develop use. In fact, any protein synthesizedin the human
low fat and cholesterol,and high protein containing b o d y c a n b e m a d e i n t h e t r a n s g e n i c a n i m a l s,
eggs. Transgenic chickens that are resistant to viral provided that the genes are correctly programmed.
and bacterial diseases have also been developed. T h e a d v a n t a g e w i t h t r a n s g e n i c a n i ma l s i s to
Some attempts have also been made to develop p r o d u c e s c a r c e h u m a n p r o t e i n s i n h u g e q u a n ti ti e s.
pharmaceutical proteins in the eggs of transgenic Thus, the animals serving as factories for
c h ic k ens . production of biologically important products are
referred ro as animal bioreactors ol, sometimes
TRANSG ENI C FI SH pharm animals. Frankly speaking, transgenic
Several transgenic fish (catfish, salmon, trout a n i m a l s a s b i o r e a c t o r s c a n b e c o nr m e r ci a l l v
etc.) have been developed with increase in their exoloited for the benefit of mankind.
growth and size. This was carried out by
Once developed, animal bioreactors are cost-
introciucing growth hormone transgene (by
effective for the production of large quantities of
microinjection or electroporation). The fertilized
h u m a n p r o t e i n s . R o u t i n e b r e e d i n g a n d h e a l th fu l
eggs with inserted transgene are incubated rn
l i v i n g c o n d i t i o n s a r e e n o u g h t o m a i n t a i n tr a n sg e n i c
temperature-regulatedholding tanks. (Note : The
animals.
fish egg development is external in contrast to the
mammalian embryogenesis).The efficiency of fish The importance of transgenic animals has
(ransgenesisis as high as 70"/o. already been discussed under the respective
i n d i v i d u a l a n i m a l s . A s e l e c t e d l i s t o f th e
It was found that the transgenicsalmon fish (with
therapeutically important proteins produced by
growth hormone transgene)were 10 times heavier
animal bioreactors is given Table 4l .2.
than the normal ones, at the end of one year
rA
As reportedby Wilmut and Campbell,theyfused
277 ovum cel l s,achi eved13 pregnanci es,
and of
theseonly one pregnancyresultedin live birth of
\Y/ the offspring-Dolly.
Ovum with nucleus
I
l-* aO
a)
Some of the companies involved in transgenic
experimentshave startedcloning pet animals like
cats and dogs. Little Nicky was the first pet cat that
was cloned at a cost of $50,00 by an American
\-/ company (in Dec. 2004). More cloned cats and
Enucleatedovum dogs will be made available to interested parties
(who can afford) in due course.
PIGS IN XENOTRANSPLANTATION?
Some workers are actively conducting research
to utilize organs of pigs in xenotransplantation'lt is
now identified that the major reasonfor rejection of
pig organs by primates is due to the presence of a
s p e c i a l g r o u p o f d i s a c c h a r i d e s( C a l - c r 1 ,3 - C a l ) i n
pigs, and not in Primates.
The enzyme responsible for the synthesis of
s p e c i f i c d i s a c c h a r i d e si n p i g s h a s b e e n i d e n ti fi e d ' l t
is u 1,3-galactosyltransferase, present in pigs and
not in primates. Scientists are optimistic that
knackout pigs lacking the gene encoding the
enzyme ct, 1,3-galactosyltransferase can be
developed in the next few years. Another approach
is to introduce genes in primates that can degrade
Transgenic calves produced bY or modify Cal-c 1,3-Cal disaccharidegroups (of
pigs).This will reduce immunogenecity.
AN IM AL S
Chaot er41 : T R AN SC EN IC 493
il
42 P l ant Ti ssueC ul ture-C eneral 497
43 P l ant Ti ssueC ul ture Medi a 517
44 P rotopl astC ul ture and S omati c
H ybri di zati on 523
45 P roducti onof H apl oi d P l ants 538
46 S omacl onalV ari ati ons 546
o$ 47 Clonal Propagation
(Mi croproP agati on) 552
48 C ermpl asnt C onservati onand
C ryopreservati on 565
49 C eneti c E ngi neeri ngof P l ants-
Methodol ogY 572
PTANT/ 50 A ppl i cati ons of P l ant
ic
Transformation,rTransgen
AGRICULTURAI- P l ants 596
51 Transgeni cP l antsas
BIOTECHNOLOGY Bioreactors 622
52 C rorvth P romoti ng B acteri a i n
P l ants 639
53 Mol ecul ar Marker-A i dedP l ant
B reedi ng 648
\K
/ /'
w
at
-.'
l,/
1
/-
/i/
/
/
| ' / /\ l // i
\
t-i
l. )
:J--r (.-:)) l\.,)
I
+
I
..-'-
r\
\
495
iilihliiuiifri,-l, ,
merist€m,nucellus)
Cell culture
Protoplast culture
p lant tissue culture broadly refersto the in vitro pathogen-free plants, besides the synthesis of many
I cultivation of plants, seeds and various parts biologically important compounds, including
of th e p lan ts (o r gans ,em br y os ,t is s ues ,s ingle c e l l s , pharmaceuticals Because of the wide range of
pro top lasts) The c ult iv at ion pr oc es s is inv ar ia b l y applications, plant tissue culture attracts the
ca rried o ut in a nut r ient c ult ur e m edium un o e r attention of molecular biologists, plant breeders
asep tic co nd itions and industrialists.
497
[32]
-::echnology
498 B IOTECHNO LO G Y
C ON VE I{ T IO N AL PL A N T B R E E D IN G
A N D PL A N T T IS SU E C U LTU R E
Si n c e th e ti m e i m m e m ori al ,man has beerr
Fig. 42.2 : A diagrammatic representatian of important
of pl antsto
c l o s e l yi n v o l v e di n th e i m p rovement Darts in a flower.
meet his basic needs.The conventional methoos
fhapt er 42 : P LA N TT ISS U E
C U L IU R E-C EN ER A L 499
i a suitableculiuremediumrevertto meristematic
=:ate
Callusculture
Redifferentiationr The abiiityof tl.recalluscells
into a plant organor a whole plant
:o differentiate Organculture
s regardedas redifferentiation. (embryo,seed,
Anthel ovary,
Totipotency: The abilityof an individualcell to meristem,nucellus)
:er elop int o a who l ep l a n ti s re fe rre d
to a s c e l l u l ar Ceilculture
:rtifrotency.The inherentcharacteristic featuresof
r ant c ells na n re l y d e d i ffe re n ti a ti o n a n d Protoplastculture
':ditferentiation are responsible for the
Fig. 42.3 : Major types of plant tissue cultures.
rlenomenon of totipotency.
The othertermsusedin planttissuecultureare
Organ culture
e r . plained places.
at appro p ri a te
Cultureof isolatedplant organsis referredto as
BF I E F HI S T O RY O F P L AN T T IS SU E organ culture. The organ used may be embryo,
CULT URE seed, root, endosperm, anther, ovary, ovule,
meri stem(shootti p) or nucel l us.
About 250 years ago (1756), Henri-Louis
The organ culture may be organizedor un-
D uham el du M o n c e a u ' d e mo n s tra te dc a l l u s
organi zed.
formationon the decorticated regionsof elm plants.
vlany botanistsregardthis work as the fcrwardfor Organized organ culture : When a well
.l organized structureof a plant (seed,embryo) is
th e dis c ov erof y pla n tti s s u ec u l tu reIn
. 8 5 3 ,T re c ul
p ublis hedpic t ur e so f c a l l u sfo rm a ti o ni n p l a n ts . used in culture, it is referredto as organized
culture.ln this type of culture,the characteristic
Cerman botanist Gottlieb Haberlandt (1902), individualorgan structureis maintainedand the
regardedas the father of plant tissue culture, first progenyformedis similarin structureas that of the
developedthe concepto{ in vitro cell culture.He ori gi nalorgan.
was the first to culture isolated and {ully
Unorganizedorgan culture : This involvesthe
plan t c e l l s i n a n u tri e n tme d i u m .
d i f f er ent iat ed
isolationof cells or tissuesof a part of the organ,
Dur ing 1934-1 9 4 0 , th re e s c i e n ti s tsn a mel y and their culture in vitro. Unorganizedculture
Cautheret, White and Nobecourtlargelycontributed resul tsi n the formati onof cal l us.The cal l uscan be
to the developments made in plant tissueculture. dispersed into aggregates of cellsand/orsinglecells
to gi ve a suspensi on cul ture.
Cood progress and rapiddevelopments occurred
a f t er 1940 in p l a n t ti s u e c u l tu re te c h n i q u es. Cell culture
Stewardand Reinert(1959)firstdiscovered sornatic
embryo productionin vitro.Maheswariand Cuha Thecul tureof i sol atedi ndi vi dualcel l s,obtai neo
(1964)from India 'werethe first to developanther from an expl antti ssueor cal l usi s regarded as cel l
culture and poller culture for the productionof cul ture. These cul tures are carri edout i n di spensi on
h aploidplant s . medium and are ret'erred to as cell suspension
cultures.
TYPES OF CULTURE
Protoplast culture
Thereare differenttypesof plant tissueculture (i .e.,cel l sdevoi dof cel l w al l s)
P l antprotopl asts
te c hniques m
, ain l y b a s e d o n th e e x p l a n t u s ed are also used in the laboratory
for culture.
ffig. a23).
BASIC TECHNIQUE OF PLANT
Gallus culture TIS S U E C U LTU R E
This involvesthe cultureof differentiated trssue The generalprocedureadoptedfor isolationand
from explantwhich dedifferentiates in vitroto form cultureof planttissuesis depictedin Fig.42.4 and
cailus . brieflydescribedin the next page.
502 B IOTECHNO LO CY
Th e no rmal inc ubat ion t im e f or t he s us pe n s i o n The batch cul tures can be mai nta!ned
cultures is in the range of 21-28 days. smal lamountsof the
y transferri ng
conti nuousl by
susoensi onmedi unr (w i th i nocul unr)to fresn
TYPES OF SUSPENSION CULTURES medi umat regul ari nterval s(2-3 days).
Th ere are m ainly t wo t y pes of s us pe n s i o n Batchculturesare qharacterized
by a constant
cu lture s- b atc h c ult ur es and c ont inuous c ultu r e s . cl.range in the pattern of cell growth and
metabol i sm. For thi s reason,thesecul turesare not
Batch cultures i deal l y sui ted for the studi esrel atedto cel l ul ar
A bat c h c u l tu re i s a c e l l s u s p e n s i o n c u l ture behaviou r.
grown in a fixed volume of nutrient culture
m edium .I n b a tc h c u l tu re ,c e l l d i v i s i o na n d cel l Gontinuous cultures
growth coupled with increasein bicmassoccur In conti nuouscul tures, there i s a regul ar
unt il one of t h e fa c to rsi n th e c u l tu i ' ee n v i rc nment addition of fresh nutrient medium and draining
( nut r ientO
, , s u p p l y )b e c o m e sl i m i ti n g .T h e cel l s
out the used mediuntso that the culturevolume is
exhibit the following five phasesof growth when normallyconstant.Theseculturesare carriedout in
the cell number in suspension culturesis plotted speciallydesignedculturevessels(bioreactors).
againstthe time of incubation(Fig.42.6).
C onti nuouscul tures are carri ed out under
1. Lag phase characterizedby preparationof defined and controlled conditions-cell density,
c ellst o div ide . nutri ents,Or, pH etc. The cel l s i n thesecul tures
2. Log phase(exponential phase)wherethe rate are mostlyat an exponentialphase(log phase)of
i s h i g h e s t.
of c ell m ult ip l i c a ti o n growth.
3. Linearphaserepresented by slownessin cell Continuousculturesareof two types-open and
div is ionand i n c re a s ei n c e l l s i z ee x p a n s i o n. cl csed.
4. Deceleration phase characterized by Open continuouscultures: In thesecultures,the
in c e l l d i v i s i o na n d c e l l e x p a n s i o n '
dec r eas e inflowof freshnrediumis balancedwith the ouiflow
5. Stationaryphase representeC by a constant of the vol umeof spentmedi umal ongw i th the cel l s.
num berof c e l l sa n d th e i r s i z e . The additionof fresh mediumand cultureharvest
504 B IOTECHNO LO CY
are so adjusted that the cultures are maintained Several methods are in use to bring out
indefinitely at a constant growth rate. At a steady s y n c h r o n i z a t i o no f s u s p e n s i o nc u l t u r e s. Th e y m a y
state, the rate of cells removed from the cultures b e b r o a d l y d i v i d e d i n t o p h y s i c a l a n d ch e m i ca l
equals to the rate of formation of new cells. methods.
B e r g m a n n ( 1 9 6 0 ) d e v e l o p e d a t e ch n i q u e fo r
c l o n i n g o f s i n g l e c e l l s . N o w a d a y s , Be r g n ta n n 's
plating technique is the mosf wiclely used method
f o r c u l t u r e o f i s o l a t e d s i n g l e c e l l s . T h i s m e th o d i s
depicted in Fig. 42.8 and briefly describedhereunder.
Outer chamber
Singlecell
in a drop ol
medium
Plant tissue cultures are associated with a
Fig. 42.7 : Techniques for the culture of single cells wide range of applications--the most important
(A) Filter paper raft-nurse tissue technique being the production of pharmaceutical, medicinal
(B) Microchanber technique (side u,iew), and other industrially important compounds. ln
(C) Microdrop method (Refer Fig. 42.8 for addition, tissue cultures are useful for several other
Bergmann's plating technique). purposes listed below.
'!
. To study the respiration and metabolism of
Dlants.
s lide c an be dir ec t ly us ed. A d r o p o f t h e m e d i u m
c ont aining a s ingle c ell i s p l a c e d i n t h e 2. For the evaluation of organ functions in
m ic r oc ham ber .A dr op of m i n e r a l o i l i s p l a c e d o n olants.
either side of the culture drop which is covered 3. To study the various plant diseasesanci work
with a coverslip (Fig. 42.78). On incubation, slngle out methods for their elimination.
cell colonies are forme<-r.
4 . S i n g l e c e l l c l o n e s a r e u s e f u l fo r g e n e ti c,
m o r p h o l o g i c a l a n d p a t h o l o g i c a l s t u d ie s.
Microdrop method
5 . E m b r y o n i c c e l l s u s p e n s i o n sc a n b e u se d fo r
For t he c ult ur e of s ing l e c e l l s b y m i c r o d r o p large scale clonal prapagation.
m et hod, a s pec ially des igne d d i s h ( c u p r a k d i s h ) i s
6 . S o m a t i c e m b r y o s f r o m c e l l s u sp e n si o n sca n
useci.lt has a snrall outer chamber (to be filled u,ith
s t er ile dis t illed wat er ) and a l a r g e i n n e r c h a m b e r be stored for long term in germplasm banks.
with a number of rnicrowells (Fig. a2.7A. The cell 7 . l n t h e p r o d u c t i o n o f v a r i a n t c l o n e s w i th n e w
density of the medium is adjusted in such a way characteristics, a phenomenon referred to as
that it contains one cell per droplet. somaclonal variations.
Chapt er42 : PL A N TT IS SU E
C U L T U R E-GE N E R A L 507
PRODUCTION OF SECONDARY
Tleu 42.1 A selected llst of secondary METABOLITES
metabolltesobtained from plant cell cultures
along with their application(s) The process of in vitro culture of cells for the
l a r g e s c a l e p r o d u c t i o n o f s e c o n d a r y m e t a b o l i t e si s
Product Uses complex, and involvesthe following aspects
Shikonine Lilhospemun Dye, .l
Selection of cell lines for high yield of
erythrorhizon pharmaceutical
secondary metabolites.
Codeine, Papaversomniferun Analgistic
2 Largescale cultivation of plant cells
m0rpnrne
Quinine Cinchonaofticinalis Antimalarial 3. Medium composition and effect of nutrients.
Atropine Atropabelladcnna Muscle 4. Elicitor-induced production of secondary
relaxanl metabolites.
Digoxin n;-;t^t:^:^^^.^
urguauJ tat ra@ Cardiovascular
disorders 5. Effect of environmental factors.
Reserpine Rauwolfia seryentina Hypotensive 6 . B i o t r a n s f o r m a t i o nu s i n g p l a n t c e l l c u l t u r e s .
Diosgenin Dioscorea deltoidea Antitertility 7 . S e c o n d a r y m e t a b o l i t e r e l e a s ea n d a n a l y s i s .
Vanillin Vanilla sp Vanilla
Jasmrne Jasniumsp Perf
ume SELECTION OF CELL L'NES FOB H'GH
Vinblastine, Catharanthus Anlicancer YIELD OF SECONDARY METABOLITES
ajmalicine, r0seus
vincristine The very purpose of tissue culture is to produce
high amountsof seccndarymetabolites.However, in
Taxol Taxusbrevifolia Anlicancer
g e n e r a l , m a j o r i t y o f c a l l u s a n d s u s p e n s i o nc u l t u r e s
Baccharine Baccharis negapotanica Anticancer
produce less quantities of secondary metabolites.
Cesaline Caesalpinia gillisesii Anticancer
This is mainly due to the lack of fully differentiated
Fagaronine Fagara zanthoxyloides Anticaner
c e l l s i n t h e c u l t u r e s .S o m e s p e c i a l t e c h n i q u e sh a v e
Maytansine Maytenus bucchananii Anticancer been devisedto select cell lines that can oroduce
Harring tonine Cephalotaxus harringtoniaAntrcancer higher amounts of desired metabolites. These
ThalicarpineThalictrum dasycarpun Anticancer methods are ultimately useful for the separation of
Ellipticine, Ochrosia moorei Anticancer producer cells from the non-producer cells. fhe
3-deoxycolchine techniques commonly employed for cell line
Pyrith rins Ttdolttc araala Insecticide s e l e c t i o n a r e c e l l c l o n i n g , v i s u a l o r c h e m i c a l
Chrysanthemun a n a l y s i sa n d s e l e c t i o nf o r r e s i s t a n c e .
cincerariefoliun
Rotenoids Derriselliptica lnsecticide Gell cloning
Tephrosia sp
This is a simple procedure and involves the
Nicotine Nicotiana tabacum Insecticide growth of single cells (taken from a suspension
Nicotiana rustica c u l t u r e s )i n a s u i t a b l em e d i u m . E a c hc e l l p o p u l a t i o n
Saff ron Crocus sativus Foodcolour is then screened for the secondary metabolite
andllavouring f o r m a t i o n . A n d o n l y t h o s e c e l l s w i t h h i g h - y i e l d i n g
agenr a b i l i t y a r e s e l e c t e da n d m a i n t a i n e d 'b y s u b c l o n i n g .
Stevioside Steviarabaudiana Sweetener
Thaumatin Thaumatococcus danielli Sweetener Single cell cloning : There are certain practical
difficulties in the isolation and culture of single
v dPJ dt uil l Capsicum frutesus chiili
cells.
Rosamarinic Coleus blunei Spice,
acrd antioxidant Cell aggregatecloning : Compared to single cell
Anthraquinones Morindacitrifolia Laxative,
dye cloning, cell aggregate cloning is much easier,
Berberine a^^t;^;^^^^i^a
wuPuJ Jdyut rtua Antibacterial hence preferred by many workers.
Sarcoplasmine Daturastramoniun Treatment
of A schematic representation of cell aggregate
(hyoscine) nausea yielding
cloning for the selection of cells high
510 B IOTECHNO LO CY
Som e wor k er s us e s i m p l e . s e n s i t i v e a n d
inex pens iv e c hem ic al an a l y t i c a l m e t h o d s f o r Growthof cell
quantitative estimation of desired metabolites. aggregares
Analy s is is c ar : r iedout in s o m e c o l o n i e s d e r i v e d
f r om s ingle c ell c ult ur es . Ra d i o i m m u n o a s s a yi s t h e
m os t c om m only us ed a n a l y t i c a l m e t h o d .
Microspectrophotometry and fluorescent antibody Subculture Subculturefor
t ec hnioues ar e als o in us e. for gro\4/th metaboliteanalysis
lines(highyielding)
Selection for resistance
Cell lines selected for resistance of 5-methyl- Fig. 42.9 : A schematic representation of cell
tryptophan (analogueof tryptophan)produce strains
which can overproduce tryptophan. These
C U L T U R-C
" : P LAN TT ISS U E
Chapt er42 E ENERAL 511
Catheranthus
roseus Entrapment
in alginate,
agarose, Sucrose Ajmalicine
carrageenin
Petuniahybrida Entrapment
in hollow
fibres Sucrose Phenolics
Morindacitrifolia Entrapment
in alginate Sucrose Anthraquinone
Solanum aviculare polyphenylene
Attachment beads Sucrose glycosides
Steroid
Glycinemax Entrapment fibre
in hollow Sucrose Phenolics
Biotechnology
[33]
514 B IOTECHNO LO CY
Th e co mpos it ion of t he c ult ur e m edi a r s N6 medium : Chu formulated this medium ancr
p rimarily de pe ndent on t wo par am et er s . it is used for cereal anther culture, besidesother
.l tissuecultures.
The p ar t ic ular s pec iesof t he plant .
2 . The tvp e of m at er ial us ed f or c ult ur e r . e N i t s c h 's m e d i u m : T h i s m e d i u m w a s d e v e l o p e d
cells, tissu es,or gans , pr ot oplas t s . by Nitsch and Nitsch and frequently used for anther
cu ltures.
Thu s, th e c om oos it ion of a m edium i s
fo rmula ted co ns ider ingt he s pec if ic r equir em en t so f Among the media referredabove, MS medium is
a given cultu r e s y s t em . The m edia us ed m ay b e most frequently used in plant tissue culture work
solid (solid medium) or liquid (liquid medium) n d u e t o i t s s u c c e s sw i t h s e v e r a l p l a n t s p e c i e s a n d
na ture . The selec t ion of s olid or liouid m ediu m i s culture systems.
dependent on the better responseof a culture
Synthetic and natural media : When a medium
is composed of chemically defined components,it
MAJOR TYP ES O F M EDI A
is referred to as a synthetic medium. On the other
Th e co mpo s it ion of t he m os t c om m only u s e d hand, if a medium contains chemically undefined
tissue culutre media is given in Table 43.1, and compounds (e.9.,vegetableextract,fruit juice, plant
briefly discribed below. extract), it is regarded as a natural medium
517
518 B IOTECHNO LO CY
Sirnthetic media have almost replaced the natural as mass values (mg/i or ppm or mg l-1;. However,
m c dia f or t is s ue c ult ur e. as per the recommendations of the International
Expression of concentrations in media : The Association of Plant Physiology,the concentrations
c onc ent r at ions of inor g a n i c a n d organrc of macronutrientsshould be expressedas mmol/l
c ons t it uent sin c ult ur e m edi a a r e u s u a l l v e x o r e s s e d and micronutrients as umol/l-
: r aDt er43 : P L A N TT IS SU E M ED IA
CULTURE 519
s p e c i e s .T h e y u s u a l l y i n h i b i t a d v e n t i t i o u sr o o t a n d
H N -C H "
shoot formation
Abscisic acid (ABA) : The callus growth of
cultures may be stimulated or inhibited by ABA.
This largely depends on the nature of the plant
species. Abscisic acid is an important growth
I regulationfor induction of embryogenesis.
H H
An auxin A cytokinin Solidifying agents
(lndole ^
(No-Methylaminopurine)
aceticacid)
F o r t h e p r e p a r a t i o no f s e m i s o l i d o r s o l i d t i s s u e
culture media, solidifying or gelling agent-s are
OH
r e q u i r e d . I n f a c t , s o l i d i f y i n g a g e n t se x t e n d s u p p o r t
t o t i s s u e sg r o w i n g i n t h e s t a t i c c o n d i t i o n s
Agar : Agar, a polysaccharide obtained frorn
seaweeds, is most commonly used as a gelling
agent for the follou'ing reasons(next page).
A uxi ns
Amon g th e c y t ok inins , k inet in and be n z y l - IAA Indole 3-aceticacid
a mino pu rine ar e f r equent ly us ed in c ult ur e m e d i a .
IBA Indole 3-butyric
acid
Ratio of auxins and cytokinins : The relative NAA 1-Naphthyl aceticacid
concentrationsof the growth factors namely auxtns 2, 4-D 2, 4-Dichlorophenoxyaceticacid
a nd cyto kin ins ar e c r uc ial f or t he m or phogen e s i so f 2, 4, 5-I 2, 4. S-Trichlorophenoxy
acetic
culture systems. When the ratio of auxins to acid
cytokinins is high, enibryogenesis, callus initiation 4--CPA 4-Chlorophenoxy aceticacid
and root initiation occur. On the other hand, for aceticacid
NOA 2-Naphthyloxy
axillary and shoot proliferation, the ratio of auxins
MCPA 2-Methyl4-chlorophenoxy acelic
to cvtokinins is low. For all practical purposes, it is
acid
considered that the formation and maintenance of
Dicamba 2-Methoxy 3, 6-dichlorobenzoic
ca llus cultu res r equir e bot h aux in and c y t o k i n i n ,
acr0
while au xin is neededf or r oot c ult ur e and c y t o k i n i n
Picloram 4-Amino 2. 5, 6-trichloropicolinic
for sh oo t cultur e.
acrd
The actual concentrations of the Srowth
Cytokinins
re gu lato rsin c ult ur e m edia ar e v ar iable depe n d i n g
BAP
on the typ e o f t is s ue ex plant and t he plant s p e c i e s .
aminopurine
6-Benzyl
BA Benzyl
adenine
Cib be rel!i ns : About 20 dif f er ent gibber e l l i n s 2 iP (rPA) adenine
N6-(2-isopentyl)
have been identified as growth regulators.Of these,
DPU Diphenyl
urea
gib be rellin43 ( CA3) is t he m os t c om m only us e d f o r
Kinetin 6-Furfuryl
aminopurine
tisue culture. CA, promotes growth of cultured
growth and induces dwarf
Zealin 4-Hydroxy
3-methyltrans
cells, enhances callus
plantlets to elongate. 2-butenyl
aminopurine
Thidiazuron 1-Phenyl yl)
3-(1,2, 3-thiadiazol-5
Cib be relli ns ar e c apable of pr om ot in g o r
urea
inh ibitin g tissue c ult ur es , depending on t he p l a n t
522 B IOTE CHNO LO CY
1. lt does not r eac t wit h m e d i a c o n s t i t u e n t s . Most of the growth regulatorsare not soluble rn
water.They have to be dissolvedin NaOH or alcohol.
2 lt is not digested by plant enzymes and is
stable at culture temperature. Dry powders in media preparation
Agar at a c onc ent r at ion o f 0 . 5 t o 1 % i n t h e The conventional procedure for media
m edium c an f or m a gel. p r e p a r a t i o ni s t e d i o u s a n d t i m e c o n s u mi n g N o w a
days, plant tissue culture media are commercially
Celatin : lt is used at a high concentration(.10%)
prepared, and are available rn the market as dry
wit h a lim it ed s uc c es s . This i s m a i n l y b e c a u s e
p o w d e r s .T h e r e q u i s i t em e d i u m c a n b e p r e p a r e db y
gelat in m elt s at low t em p e r a t u r e ( 2 5 'C ) , a n d
d i s s o l v i n g t h e p o w d e r i n a g l a s s d i sti l l e d o r
c ons equent lyt he gelling pr op e r t v i s l o s t .
d e m i n e r a l i z e dw a t e r .S u g a r o
, r g a n i cs u p p l e m e n tsa n d
O t her gelling agent s : Bio g e l ( p o l y a c r y l a m i d e agar (melted) are added, pH adjusted and
pellet s ) , phy t agel, gelr it e a n d p u r i f i e d a g a r o s e t h e m e d i u r nd i l u t e d t o a f i n a l v o l u m e ( u s u a l l y1 l i tr e ) .
ar e ot her s olidif y ing age n t s , a l t h o u g h l e s s
f r equent ly us ed. lt is in f ac t a d v a n t a g e o u st o u s e Sterilization of media
s y nt het ic gelling c om pounds, s i n c e t h e y c a n f o r m
T h e c u l t u r e m e d i u m i s u s u a l l y s t e r i l i ze d i n a n
gels at a r elat iv ely low c onc e n t r a t i o n ( 1 . 0 t o 2 5
autoclave at 121"C and 15 psi for 2O minutes.
c l- 1) H o r m o n e s a n d o t h e r h e a t s e n s i t i ve o r g a n i c
c o m p o u n d s a r e f i l t e r - s t e r i l i z e d ,a n d a d d e d to th e
pH of medium
autoclaved medium.
The optimal pH for most tissue cultures is in the
range of 5.0-6.0. The pH generally falls by 0 3-0.5 SELECTION OF A SUITABLE M E D IU M
unit s af t er aut oc lav ing.Bef or e s t e r i l i z a t i o n ,p H c a n l n o r d e r t o s e l e c t a s u i t a b l e m e di u m fo r a
be adjus t ed t o t he r equir ed o p t i m a l l e v e l w h i l e p a r t i c u l a r p l a n t c u l t u r e s y s t e m , i t i s c usto m a r y to
pr epar ingt he m edium . lt is us u a l l y n o t n e c e s s a r yt o
s t a r t w i t h a k n o w n m e d i u m ( e . g . M S me d i u m , 8 5
use buffers for the pH maintenance of culture m e d i u m ) a n d t h e n d e v e l o p a n e w m e d i um w i th th e
m edia. desired characteristics.Among the constituentsof a
At a pH higher t han 7. 0 an d l o w e r t h a n 4 5 , t h e m e d i u m , g r o w t h r e g u l a t o r s( a u x i n s ,c y t o ki n i n s) a r e
plant c ells s t op gr owing in c u l t u r e s . l f t h e p H h i g h l y v a r i a b l e d e p e n d i n g o n t h e c u l t u r e syste m .l n
f alls dur ing t he plant t is s ue c u l t u r e , t h e n f r e s h practice, 3-5 different concentrations of growth
m edium s hould be pr epar ed.I n g e n e r a l , p H a b o v e r e g u l a t o r s i n d i f f e r e n t c o m b i n a t i o n s a r e u se d a n d
6. 0 giv es t he m edium har d a p p e a r a n c e , w h i l e the best among them are selected.
pH below 5. 0 does not a l l o w g e l l i n g o f t n e For the selection of appropriate concentrations
m edium . o f m i n e r a l s a n d o r g a n i c c o n s t i t u e nts i n th e
m e d i u m , s i m i l a r a p p r o a c h r e f e r r e d a b o ve , ca n b e
PREPARATI O N O F M EDIA employed.
523
524 B IOTECHNO LO CY
Fig. 44.1 : A diagrammatic representation of mechanical method for the isolation of protoplasts.
Leaf sterilization
Differentiation
of
callustissue
r Peeledleaf segment
\
@@
Plasmolysedcells
J
Cellsin
enzyme mixture
/
Ce ll wa ll
reseneration\a";5 (e @,
Cellwall digestion
^ 6 [-l and releaseof protoplasts
\=7*3p=^o--M
(,xo-{_
-.____'-_/
Platingof
*
Protoplasts
protoplasts lsolated Celldebris
protoplasts
Washingol
protoplasts
Fig. 44,2 : Major steps involved in protoplast isolation, culture and regeneration of plants.
1 . Two step or sequential method : The iissue lsolation of protopfasts from leaves
is first treated with pectinase (macerozyme) to
Leaves are most commonly used, for protoplast
sep ara tecell s by degr ading m iddle lam ella. T h e s e
i s o l a t i o n ,s i n c e i t i s p o s s i b l et o i s o l a t eu n i f o r m c e l l s
free cells are then exoosed to cellulase to release
in large numbers. The procedure broadly involves
protoplasts.Pectinasebreaks up the cell aggregat€s
the following steps (Fig. 44.2).
in to in dividu al c ells while c ellulas e r em ove s t h e
cell w,all proper. 1 . S t e r i l i z a t i o no f l e a v e s .
Purification of protoplasts P r o t o p l a s t sa r e c u l t u r e d e i t h e r i n s e mi so l i d a g a r
o r l i q u i d m e d i u m . S o m e t i m e s ,p r o t o p l astsa r e fi r st
The enz y m e diges t ed p l a n t c e l l s , b e s i d e s
allowed to develop cell wall in liquid medium, and
pr ot oplas t s c ont ain undige s t e d c e l l s , b r o k e n
then transferredto agar medium.
pi' ot oplas t sand undiges t edt is s u e s T h e c e l l c l u m p s
and undiges t edt is s uesc an be r e m o v e d b y f i l t r a t i o n .
Agar culture
This is f ollowed by c ent r if uga t i o na n d w a s h i n g s o f
the protoplasts.After centrifugation,the protoplasts Agarose is the mosf frequently used agar to
are recovered above Percoll. s o l i d i f y t h e c u l t u r e m e d i a . T h e c o n c e n t r a ti o no f th e
a g a r s h o u l d b e s u c h t h a t i t f o r m s a s o ft a g a r g e l
Viability of protoplasts w h e n m i x e d w i t h t h e p r o t o p l a s t s u s p e n si o n .Th e
p l a t i n g o f p r o t o p l a s t si s c a r r i e d o u t b y B e r g m a n n 's
I t is es s ent r al t o ens ur e t h a t t h e i s o l a t e d c e l l p l a t i n g t e c h n i q u e ( R e f e r4 2 . 8 ) l n a ga r cu l tu r e s,
protoplastsare healthy and viable so that they are t h e p r o t o p l a s t sr e m a i n i n a f i x e d p o s i ti o n , d i vi d e
c apable of under going s us t ai n e dc e l l d i v i s i o n s a n d a n d f o r m c e l l c l o n e s . T h e a d v a n t a g e w i th a g a r
regeneration.There are several methods to assess c u l t u r e i s t h a t c l u m p i n g o f p r o t o p l a s t sis a vo i d e d .
t he pr ot oplas tv iabilit y .
Liquid culture
I . Fluorescein diacetate (FDA) staining
m et hod- The dy e ac c um u l a t e s i n s i d e v i a b l e L i q u i d c u l t u r e i s t h e p r e f e r r e d me th o d fo r
protoplastswhich can be detected by Iluorescence o r o t o p l a s t c u l t i v a t i o n f o r t h e f o l l o w i n g r e a so n s.
mrcroscoov.
1 lt is easy to dilute and transfer.
2. Phenosafraninestain is selectively taken up
2 D e n s i t y o f t h e c e l l s c a n b e m a n i p u l a te d a s
by dead pr ot oplas t s( t ur n r ed) w h i l e t h e v i a b l e c e l l s
desired
r em ain uns t ained.
3 . F o r s o m e p l a n t s p e c i e s , t h e c e l l s ca n n o t
3. Ex c lus ion of Ev ans b l u e d v e b v intacr
d i v i d e i n a g a r m e d i u m , t h e r e f o r el i q u i d m e d i u m i s
meml]ranes.
the only choice.
4. Measurement of cell wall formation-
Calc of iuor whit e ( CFW ) s t ain b i n d s t o t h e n e w l y 4 . O s m o t i c p r e s s u r eo f l i q u i d m e d iu n .rca n b e
f or m ed c ell walls whic h em it f l u o r e s c e n c e . altered as desired.
2 . The con c ent r at ion of c alc ium s hould o e Feeder layer technique
2-4 -times h igh er t han us ed f or c ell c ult ur es .Th i s i s
For culture of protoplastsat low density feeder
needed for membrane stabiliiy.
l a y e r t e c h n i q u e i s p r e f e r r e d .T h i s m e t h o d i s a l s o
3 High a ux in/ k inet in r at io is s uit ablet o ind u c e i m p o r t a n t f o r s e l e c t i o no f s p e c i f i c m u t a n t o r h y b r i d
ce ll divisio ns while high k inet in/ aux in r at i o i s c e l l s o n p l a t e s .T h e t e c h n i q u e c o n s i s t so f e x p o s i n g
req uire d fo r re gener at ion. p r o t o p l a s tc e l l s u s p e n s i o n st o X - r a y s ( t o i n h i b i t c e l i
division with good nretabolic activity) and then
4 Clucose is the preferred carbon soulce by
plating them on agar plates.
pro top lasts al t hough a c om binat ion of s u g a r s
(glu co sean d suc r os e)c an be us ed.
Co-culture of protoplasts
-5 The vita m ins us ed f or pr ot opias tc ult ur e s a r e
th e same a s u s ed in s t andar dt is s ue c ult ur e m e d i a . Protoplasts of two different plant species (one
slow growing and another fast growing) can be co-
Osmoticum and osmotic pressure cultured. This type of culture is advantageoussince
the growing speciesprovide tlre growth factors and
Osmoticum broadly refers to Lhe reagents/ o t h e r c h e m i c a l s w h i c h h e l p s i n t h e g e n e r a t i o no f
chemicals that are added to increase the osmotic cell wall and cell division. The co-culture method
pressure of a liquid. i s g e n e r a l l y u s e d i f t h e t w o t y p e s o f p r o t o p l a s t sa r e
The isola tion ar r d c ult ur e of pr ot oplas t sr eq u i r e m o r p h o l o g i ra l l y d i s t i n c t
osrrroticp rote c t ion unt il t hey dev elop a s t r ong c e l l
wall. In fa ct, i f t he f r es hly is olat ed pr ot oplas t sa r e Microdrop culture
d irectly a dd ed t o t he nor m al c ult ur e m edium , t h e y
S p e c i a l l y d e s i g n e dd i s h e s n a m e l y c u p r a k d i s h e s
will bu rst Th us , addit ion of an os m ot ic um r s
with outer and inner chambers are used for
e ssen tialfor bot h is olat ion and c ult ur e m edi a o f
microdrop culture. The inner chamber carries
protoplast to prevent their rupture The osmotica
s e v e r a l w e l l s w h e r e i n t h e i n d i v i d u a l p r o t o p l a s t si n
a re o f two tvp es- non- ionic and ionic .
droolets of nutrient medium can be added. The
Non -ion ic os m ot ic a : The non- ionic s ubs t a n c e s o u t e r c h a m b e r i s f i l l e d w i t h w a t e r t o m a i n t a i n
mo st co mmon ly us ed ar e s oluble c ar bohy d r a t e s h u m i d i t y . T h i s m e t h o d a l l o w s t h e c u l t u r e o f f e w e r
such a s ma nnit ol, s or bit ol, gluc os e, f r uc t o s e , protoplastsfor droplet of the medium.
g ala cto se an d s uc r os e M annit ol, being
me tab olically iner t , is m os t f r equent ly us ed.
| (.])
\ \_-/ /
Cytoplast
t9
Miniprotoplas
So matic h ub ridiz at ion inv olv es t he f ollowing The protoplasts can be pushed together
asDects: mechanically to fuse. Protoplasts of Lilium and
'I Trillium in enzyme solutions can be fused by gentle
. Fusion of protoplasts
t r a p p i n g i n a d e p r e s s i o ns l i d e . M e c h a n i c a l f u s i o n
2. Sele ctio n o f hy br id c ells may damage protoplastsby causing injuries.
3. lde ntificatio nof hv br id olant s .
Induced fusion
F USION OF PR O TO PLASTS
Freshly isolated protoplasts can be fused by
As the isolated protoplasts are devoid of cell
induction. There are several fusion-inducing agents
walls, their in vitro fusion becomes relatively easy
which are collectively referred to as fusogense.g.
T he re a re no b ar r ier s of inc om pat ibilit y ( at
NaNOr, high pH/Ca2*, polyethylene glycol,
Interspecific,intergenericor even at interkingdom
p o l y v i n y l a l c o h o l , l y s o z y m e ,c o n c a v a l i nA , d e x t r a n ,
levels) for the protoplast fusion.
dextran sulfate,fatty acids and esters,electrofusion.
Pro top last fusion t hat inv olv es m ix ing of Some of the fusogens and their use in induced
protoplasts of two different genomes can be' fusion are described. A diagrammatic
ac hie ve d by spo ntaneous ,m ec hanic al, or induc ed representationof protoplast fusion is depicted in
fusion methods. Fig. 44.4.
Biotechnology [34]
530 B IOTECHNO LO CY
T h e m e t h o d c o n s i s t so f i n c u b a t i n g p r oto p l a stsi n a
s o l u t i o n o f 0 . 4 M m a n n i t o l c o n t a i n i n g 0 .0 5 M
.l
CaCl, at pH 0 . 5 ( g l y c i n e - N a O H b u ffe r ) a n d
t e m D e r a t u r e 3 7 'C f o r 3 0 - 4 0 mi n u te s Th e
protoplasts form aggregates, and fusion usually
o c c u r s w i t h i n 1 0 m i n u t e s . B y t h i s m e th o d , 2 0 - 5 0 %
of the protoplastsare involved in fusion
o l t r e s u l t s i n a r e p r o d u c i b l e h i g h - fr e q u e n cy o f
heterokaryon formation.
I . P E C - i n d u c e df u s i o n i s n o n - s p e c i f i ca n d th e r e fo r e
c a n b e u s e d f o r a w i d e r a n g e o f p l a n ts
E l e c t r o f u s i o n: I n t h i s m e t h o d , e l e ctr i ca l fi e l d i s
used for protoplastfusion. When the protoplastsare
Hybrid o l a c e d i n a c u l t u r e v e s s e l f i t t e d w i th m i cr o -
electrodes and an electrical shock is applied,
p r o t o p l a s t s a r e i n d u c e d t o f u s e . El e ctr o fu si o n
Fig. 44,4 : A diagrammatic representation of t e c h n i q u e i s s i m p l e , q u i c k a n d e f f i c i e n t a n d h e n ce
protoplast fusion. preferred by many workers. Further, the cells
formed due to electrofusiondo not show cytotoxrc
responsesas is the case with the use of fusogens
Treatment with sodium nitrate : The isolated (including PEC). The major limitation of this
protoplasts are exposed to a mixture of 5.5% method is the reguirement of specialized and
NaNO , in 10% s uc r os e s o l u t i o n I n c u b a t i o n i s costly equipment.
c ar r ied out f or 5 nr inut es a t 3 5 'C , f o l l o w e d b y
centrifugation (200 xg for 5 min). The protoplast
Mechanism of fusion
pellet is kept in a water bath at 30'C for about 3C
m inut es , dur ing whic h pe r i o d p r o t o p l a s t f u s i o n T l r e f u s i o n o f p r o t o p l a s t si n v o l v e s th r e e p h a se s
occurs. NaNO, treatment resultsin a low frequency a g g . l u t i n a t i o n , p l a s m a m e m b r a n e fu si o n a n d
of heterokaryon formation,' particularly when formation of heterokaryons.
mesopfryll protoplastsare fused.
1 . Agglutination (adhesion) : When two
High pH and high Ca2* ion treatment : This protoplasts are in close contact with each other,
method was first used for the fusion of tobacco a d h e s i o n o c c u r s . A g g l u t i n a t i o n c a n b e i n d u ce d b y
protoplasts,and is now in use for other plants also. f u s o g e n se . g . P E C , h i g h p H a n d h i g h C a 2 +.
A N D SOMA TICH Y B R ID IZA TION
Chapt er44 : P R OT OP L ASCTU L T U R E 531
2 . M e c h a n i c a l i s o l a t i o n : T h e v i s u a l l y i d e n ti fi e d
heterokaryons under the microscope can be
No divisions
Smallcolonies Largecolonies and no growth i s o l a t e d b y m e c h a n i c a l m e a n s . T h i s i n vo l ve s th e
use of a special pipette namely Drummond pipette.
+
I The so isolated heterokaryons can be cloned to
Nofurther FurthergroMh finally produce somatic hybrid plants. The major
growth limitation of this method is that each type of hybrid
+
I
Callus
I
+
SpeciesA
(NR nia-)
SpeciesB
(NR cnx-)
Somatic
hybridplants
II II
Fig. 44.6 : A diagrammatic reprcsentation of drug
T J
Protoplasts Protoplasts
sensitivity method for the isolation of hybrid cells.
SpeciesA SpeciesB i d e n t i f i c a t i o no f h y b r i d p l a n t s a r e b r i e f l y d e s c r i b e d .
(Green) (Albinoor non-green)
+
I I
+ Morphology of hybrid plants
Protoplasts Protoplasts
Morphological features of hybrid plants which
usually are intermediate between two parents can
be identified. For this purpose, the vegetative and
floral charactersare considered.These include leaf
shape, leaf area, root morphology, flower shape, its
structure, size and colour, and seed capsule
Culturedon a selectionmedium morphology.
II II ,I A l t h o u g h t h e g e n e t i c b a s i so f t h e m o r p h o l o g i c a l
I
+ + + charactershas not been clearly known, intermediate
No further Greencallus Whitecallus morphological features suBgestthat the traits are
growth u n d e r t h e c o n t r o l o f m u l t i p l e g e n e s .l t i s p r e f e r r a b l e
II
t o s u p p o r t h y b r i d m o r p h o l o g i c a l c h a r a c t e r sw i t h
+ evidence of genetic data.
So m a tic
h yb r id p la n ts
lsoenzyme analysis of hybrid plants
Fig. 44.8 : A diagrammatic representation of visual
selection of hybrids coupled with growth on T h e m u l t i p l e f o r m s o f a n e n z y m e c a t a l y s i n gt h e
selection medium. same reaction are referred to as isoenzymes.
Electrophoreticpatterns of isoenzymes have been
widely used to verify hybridity. Somatic hybrids
c ell r equir esa s p e c i a l c u l tu re me d i u m fo r i ts posses specific isoenzymes (of certain enzymes) of
growth. This can be overcome by employing one or the other parent or both the parents
m ic r odr opc ultu reo f s i n g l ec e l l su s i n gfe e d e rl a yers s i m ul t a n e o u s l y .
( Ref erp. 506) .
T h e r e a r e m a n y e n z y m e s p o s s e s s i n gu n i q u e
Gytometric methods isoenzymes that can be used for the identificationof
somatic hybrids e g amylase, esterase,aspartate
Some workers use flow cvtometrv and aminotransferase, phosphodiesterase, isoperoxidase,
fluorescent-activated cell sortingtechniquesfor the and hydrogenases(of alcohol, lactate, malate). lf
analy s isof pla n tp ro to p l a s ts
w h i l e th e i rv i a b i l i tyrs t h e e n z y m e i s d i m e r i c ( h a v i n g t w o s u b u n i t s ) ,
m aint ained.Th e s a me te c h n i q u e sc a n a l s o be s o m a t i c h y b r i d s u s u a l l y c o n t a i n a n i s o e n z y m ew i t h
appliedfor sortingand selectionof heterokaryons.a n i n t e r m e d i a t em o b i l i t y p r o p e r t i e s .
T he hy br idc el l sd e ri v e dfro m s u c hs e l e c ti o nhave
s
proved useful for the developmentof certain The isoenzymes are often variable within the
s om at ichy br idp l a n ts . same plant. Therefore, it is necessaryto use the
same enzyme from each plant (parentsand somatic
|DE NT |F T CAT | ON OF H YB R T D (C E L L SI hybrids), from a specific tissue with the same age.
P LA NT S
Chromosomal constitution
The development of hybridcellsfollowedby the
generationof hybridplantsrequiresa clearproofof The number of chromosomes present in the
genetic countribution from both the parental hybrid cells can be directly counted. This provides
protoplasts.
The hybriditymustbe established only information on the ploidy state of the cells. The
from euploidand not fromaneuploidhybrids.Some somatic hybrids are expected to possess
of the commonly used approaches for the chromosomesthat are equal to the total number of
534 B IOTECHNO LO CY
ltetE 44.3 A selected list of interspecific hybrlds produced through protoplast fusion
along with the chromosomenumbers in the hybrids
Plant species with their chromosome number Chromosome nuntber(s) in the hybrid(s)
c hr om c s om es or iginally pr e s e n t i n t h e p a r e n t a l s p e c i e so f p l a n t s r e s u l t i n f o r m a t i o n o f a sym m e tr i c
protoplasts.Sometimes, the hybrids are found to hybrids
contain more chromosonresthan the total of both the 5 A s y m m e t r i c h y b r i d s m a y b e d u e to u n e q u a l
par ent s .The pr es enc eof c h r o m o s o m a i m a r k e r s i s r e p l i c a t i o n o f D N A i n t h e f u s i n g p r o t o p l a sts.
gr eat lyus ef ulf or t he genet ica n a l y s i so f h y b r i d c e l l s
t i . P r o t o p l a s t i s o l a t i o n a n d c u l t u re m a y a l so
l e a d t o s o m a c l o n a l v a r i a t i o n s , a n d t h u s va r i a ti o n s
M olec ular t ec hniques
in chromosome number
M any r ec ent dev elopm en t si n n r o l e c r - r l abri o l o g y
A selected list of interspecifichybrids produced
hav e im pr ov ed t he unde r s t a n d i n g o f g e n e t i c
t h r o u g h p r o t o p l a s tf u s i o n a l o n g w i t h t he n u m b e r o f
c ons t it ut ionof s om at ic plant h y b r i d s .S o m e o f t h e m
c h r o m o s o m e si n t h e h y b r i d s i s g i v e n i n Ta b l e 4 4 .3 .
are listed below.
l. Dit Ter enc es in t he r e s t r i c t i o n p a t t e r n s o f Symmetric and asymmetric hybrids
c hlor oplas t and m it oc hondr i a l D N A s . lf Ihe chromosome number in the hybrid is the
2. M olec ular m ar k er s s u c h a s R F L R A F L P , sum of the chromosomes of the two parental
RAPD and m ic r os at ellit es . protoplasts, the hybrid is said ro be symmetric.
S y m m e t r i c h y b r i d s b e t w e e n i n c o m p a ti b l e sp e ci e s
3. PCR t ec hnology .
a r e u s u a l l y 's t e r i l e .T h i s m a y b e d u e t o p r o d u cti o n
o f 3 n h y b r i d s b y f u s i n g 2 n o f o n e s p e ci e sw i th n o f
CHRO M O SO M E NUM BER IN
another species.
SO M ATI C HYRBI DS
Asymmetric hybrids have abnormal or wide
The c hr om os om e num ber i n t h e s o m a t i c h y b r i d s variations in the chromosome number than the
is generally more than the total number of both of exact total of trvo species.These hybrids are usually
the parental protoplasts. However, wide variations formated with full somatic complement of one
ar e r epor t ed whic h m ay be d u e t o t h e f o l l o w i n g p a r e n t a l s p e c i e s w h i l e a l l o r n e a r l y a l l o f th e
reasons chromosomes of other parental species are lost
1 . Fusion gf more than two protoplasts. d u r i n g m i t o t i c d i v i s i o n s . A s y m m e t r i c h yb r i d s m a y
be regarded as cybrids but for the introgressed
2. lr r egular it iesin m it ot i c c e l l d i v i s i o n s .
8enes.
3. I n f us ogen or elec t r o- i n d u c e df u s i o n s , a b o u t
As given in Table 44.3, protoplast fusion
one third of the fusions occur between more than
between N. tabacum (2n = 48) and N. nesophila
two protoplasts.
( 2 n = 2 4 ) r e s u l t s i n a s y m m e t r i c h yb r i d s, w h i l e
4. Differences in the status of protoplasts asymmetric hybrids are formed when B. napus and
( ac t iv ely div iding or quie s c e n t ) f r o m t h e t w o B. iunea are fused.
44 : P.R O T OP L AS
ChA P I CT CU A N D S O MA TICH Y B R ID IZA TION
T LTURE 535
Methodology of cybridization
X-rays irradiated protoplasts for more efficient
A diagrammatic representaiionof the formation formation of cybrids.
of hybrids and cybrids is given in Fig. 44.9.
4 . l t i s p o s s i b l et o s u p p r e s sn u c l e a r d i v i s i o n r n
As the formation of heterokaryonoccurs during some protoplasts and fuse them with normal
hybrid iza tion , the nuc lei c an be s t im ulat ed t o protoplasts.
segregateso that one protoplast contributes to tfre
cvto ola sm whil e t he ot her c ont r ibut es nuc le u s
Genetic recombination in
a lon e (o r bo th nuc leus and c y t oplas m ) .I n t his w a y
asexual or sterile plants
cybrid iza tion c an be ac hiev ed. Som e of th e
approachesof cybridization are given hereunder. There are many plants that cannot reproduce
s e x u a l l y .S o m a t i c h y b r i d i z a t i o n i s a n o v e l a p p r o a c h
1 . The protoplastsof cytoolasm donor species
through which two parental genomesof a sexual or
are irradiated with X-rays or Trays. This treatment
sterile plants can be brought together. Thus, by
re nd ers the pro t oplas t sinac t iv e and non- div idi n g ,
fusing parental protoplasts, fertiie dipioids and
b ut th ey are ef f ic ient donor s of c y t oplas m r c
polyploids can be produced.
constituentswhen fused with recipient protoplasts.
Brief history
IN VTVA AND VITRO APPROACHES
The ex is t enc e of haploid s w a s d i s c o v e r e d ( a s 'fV
T h e i m p o r t a n c e o f h a p l o i d s i n t h e f i el d o f p l a n t
szply as 1921) by Bergner in Datura stramonium. b r e e d i n g a n d g e n e t i c sw a s r e a l i s e dl o n g a g o Th e i r
Plant breeders have been conducting extensrve practical application, however, has been restricted
r es ear c ht o dev elop haploids . T h e I n d i a n s c i e n t i s t s d u e t o v e r y a l o w f r e q u e n c y ( <0 . 0 0 1 % ) o f th e i r
Guha and Maheswari (1964) reported the direct f o r m a t i o n i n n a t u r e . T h e p r o c e s s o f ap o m i xi s o r
development of haploid embryos and plantlets parthenogenesis (development of embryo from an
from microspores of Datura innoxia by the cultures
unfertilized egg) is responsible for the spontaneous
of ex c is ed ant her s . Subs eq u e n t l y , B o u r g i n a n d natural production of haploids. Many attempts
Hit s c h ( . 1967) obt ained t he f ir s t f u l l - p l e d g e dh a p l o i d
were made, both by in vivo and in vitro methods to
plants from Nicotiana tabacum. Thereafter,much
d e v e l o p h a p l o i d s .T h e s u c c e s sw a s m u ch h i g h e r b y
progress has been made in the anther cultures of
in vitro techniques.
wheat , r ic e, m aiz e, pepper a n d a w i d e r a n g e o f
ec onom ic ally im por t ant s pec i e s . ln vivo techniques for haploid
production
G r ouping of haploids
There are several methods to induce haploid
Haploids m ay be div id e d i n t o t w o b r o a d production in vivo. Some of them are listed below.
cateSofles. .l
. Androgenesis : Development of an egg cell
.l
. Monoploids (monohaploids) : These are the containing male nucleus to a haploid is referredto
haploids t hat pos s es s ha l f t h e n u m b e r o f as androgenesis. For a successful in vivo
c hr om os om es f r om a diploid s p e c i e s e . g . m a i z e , a n d r o g e n e s i st,h e e g g n u c l e u s h a s t o b e i n a cti va te d
bar ley or eliminated before fertilization.
538
ChA P t Cr
45 : P R O D U C T IO N
O F H AP L O IDPL A N TS 539
2. Gynogenesis : An unfertilized egg can lre at the correct stage,each anther is gently separated
m an ipu late d (by delay ed pollinat ion) t o dev el o p (from the filament) and the intact anthers are
in to a h ap loid p lant . inoculated on a nutrient medium. Injured anthers
should not be used in cultures as they result in
3. Distant hybridization : Hybrids can be
callusingof anther wall tissue.
prod uced b y e lim inat ion of one of t he par en t a l
ge no mes a s a r es ult of dis t ant ( int er s pec if ic o r T h e a n t h e r c u l t u r e sa r e m a i n t a i n e d i n a l t e r n a t i n g
inte rge ne riccro s s es )hy br idiz at ion. p e r i o d s o f l i g h t ( 1 2 - 1 8 h r ) a n d d a r l <n e s s( 6 - 1 2 n r s t
at 28"C. As the anthers proliferate, they produce
4lrra dia tion ef f ec t s : Ult r a v iolet r ay s o r
callus which later forms an embryo and then a
X-ravs mav be us ed t o induc e c hr om os om a l
haploid plant (Fig. 45.1).
brea ka ge a nd t heir s ubs equent elim inat ion t o
prod uce h ap loid s .
POLLEN (MTCROSPORE) CULTURE
5 Ch emical t r eat m ent : Cer t ain c hem ic a l s
Haploid plantscan be produced from immature
( e g ., ch lora mph enic ol, c olc hic ine, nit r ous ox id e ,
pollen or microspores(male gametophytic cells).
m ale ic hydra z ide) c an induc e c hr om os om a l
The pollen can be extracted by pressing and
elimin atio n in s om at ic c ells whic h m ay r es ult r n
s q u e e z i n gt h e a n t h e r s w i t h a g l a s s r o d a g a i n s t t h e
hao loid s.
s i d e s o f a b e a k e r .T h e p o l l e n s u s p e n s i o ni s f i l t e r e d
to remove anther tissue debris. Viable and large
ln vitro techniques for haploid
pollen (smaller pollen do not regenerate) are
production
concentrated by filtration, washed and collected.
In th e p lan t b iot ec hnologypr ogr am m es ,haplo i d These pollen are cultured on a solid or liquid
production is achieved by two methods medium The callus/embryoformed is transferredto
1 . And rog en es is : Haploid pr oduc t ion oc c u r s a suitable medium to finally produce a haploid
thro ug h an the r or pollen c ult ur e, and t hey a r e plant (Fig. 45.1), and then a diploid plant (on
referred to as androgenic haploids. colchicine treatment).
There are two approaches in androgenesis- Many workers prefer pollen culture, even
a nth er cu lture a nd pollen ( m ic r os por e) c ult ur e . though the degree of successis low, as it offers the
You ng pla nts, gro wn under opt im al c ondit ions o f following advantages
ligh t, temp era tur e and hum idit y , ar e s uit able f o r
. Undesirable effectsof anther wall and associateo
androg en esi s.
t i s s u e sc a n b e a v o i d e o .
. A n d r o g e n e s i s ,s t a r t i n gf r o m a s i n g l e c e l l , c a n b e
ANTHER CULT URE
better regulated.
The selected flower buds of young plants are
. l s o l a t e dm i c r o s p o r e s( p o l l e n ) a r e i d e a l f o r v a r i o u s
surface-sterilizedand anthers removed along with
genetic manipulations (transformation,
the ir filame nts. The ant her s ar e ex c is ed unde r
mutaSenesrs)
aseoticconditions,and crushed in 17oacetocarmine
to test the stage of pollen development. lf they are . The yield oi haploid plants is relatively higher.
540 B IOTE CHNO LO CY
D E V E LOP ME N T OF A N D R OGE N IC
H A P LOID S
The processof in vitro androgenesis for the
o{ hapl oi dpl antsi s d epict edin
ul ti mateproducti on
-----__}
J-- Fig. 45.2.
n
ww
n
Anthers
ss
Anthers
The cul turedmi crosporesmai nl y f ollow f our
distinctpathwaysduringhe initial stagesof in vitro
androgenesi s.
J J Pathway | : The uninucleate microspore
undergoesequal division to form two daughter
cellso( equal size e.g. Daturainnoxia.
Platingof Pathwayll : In certainplants,the microspore
anthers Extractionof pollen
di vi desunequal l yto gi vebi ggervegeta t ive cell and
J J a smallergenerativecell. lt is the vegelativecell
,d_\ that undergoesfurtherdivisionsIo form callusor
r^€&\
qry embryo.The generati ve cel l , on the o t her hand,
degeneratesafter one or two divisions-e.g.,
Proliferating Nicotianatabacum,Capsicumannuum.
anthers
Pollencollected Pathway lll : In this case, the microspore
J (afterwashing)
undergoesunequal division. rhe embryos are
_1')-a
q ', <
(ry
I formed from the generative cell while the
vegetativecell does not divide at all or undergoes
Callus \i limitednumberof divisionse.g. Hyoscyamus niger.
P athw ayl V : The mi crospore di vi desunequally
I Pollenculture
(solid/liquid
medium) as i n pathw aysl and l l . H ow ever,i n thi scase,bot h
t\
u
1 ,.1l
Embryo \i
and contribute to the development of haploid
plant e.g. Datura metel, Atropa belladonna.
development \__-/ At the initial stages,the microsporemay follow
Pollen
embryo
any one of the four pathwaysdescribedabove.As
the cel l s di vi de, the pol l en grai n becom es
and burstopen.Thi smul ti celluar
mul ti cel l ul ar m ass
may form a calluswhich laterdifferentiates into a
Haploid plantlet plant (through callus phase). Alternately,the
massmay producethe pl antt hr ough
mul ti cel l ul ar
I cot.hi"in"
direct ernbryogenesis(Fig. 45.1).
J treatment
FA C TOB S A FFE C TIN G A N D R OGENESI S
A good knowledgeof the variousfactorsthat
i nfl uenceandrogenesiwsi l l hel p to i mpr ovet he
productionof androgenichaploids.Someof these
factorsare brieflydescribed.
(A)
FI RST MITOSIS
(B)
e n r \ r , i ^ h \ / i; /h a r r l n i ,l \
The selection of anthers at an ideal stage of ln general, the production of haploids is better
microspore development is very critical for haploid in light. There are however, certain plants which
production. In general, microspores ranging from can grow well in both light and dark.
tetrad to binucleate stages are more responsive. lsolated pollen (not the anther) appears to be
Anthers at a very young stage (with microspore s e n s i t i v e t o l i g h t . T h u s , l o w i n t e n si ty o f l i g h t
mother cells or tetrads) and late stage (with promotes development of embryos in pollen
binuc leat e m ic r os por es )ar e u s u a l l y n o t s u i t a b l ef o r cultures e.g. tobacco.
androgenesis.However, for maximum productron
of andr ogenic haploids , t h e s u i t a b l e s t a g e o f Effect of culture medium
m ic r os por e dev elopm ent is de p e n d e n t o n t h e p l a n t
T h e s u c c e s so f a n t h e r c u l t u r e a n d a n d r o g e n e si s
species, and has to be carefully selected
i s a l s o d e p e n d e n t o n t h e c o m p o s i ti o n o f th e
medium. There is, however, no single medium
Phy s iologic al s t at us of a d o n a r p i a n t
s u i t a b l ef o r a n t h e r c u l t u r e so f a l l p l a n t sp e ci e s Th e
The plant s gr own u n d e r b e s t n a t u r a l c o m m o n l y u s e d m e d i a f o r a n t h e r c u l t ur e s a r e M 5 ,
env ir onm ent al c ondit ions ( l i g h t , t e m p e r a t u r e , Wh i t e 's , N i t s c h a n d N i t s c h , N 6 a n d 8 5 . Th e se
nut r it ion, CO , et c . ) wit h goo d a n t h e r sa n d h e a l t h y m e d i a i n f a c t a r e t h e s a m e a s u s e d i n p l a n t ce l l a n d
m ic r os por es ar e m os t s uit a b l e a s d o n o r p l a n t s . tissue cultures. ln recent years, some workers have
Flowers obtained from young plants, at the d e v e l o p e d s p e c i a l l y d e s i g n e d m e d i a fo r a n th e r
beginning of the flowering season are highly cultures of cereals.
responsive.The use of pesticidesshould be avoided S u c r o s e , n i t r a t e , a m m o n i u m s a l t s , a m i n o a ci d s
at leas t 3- 4 week s pr ec eedin g s a m p l i n g . a n d m i n e r a l s a r e e s s e n t i a l f o r a n d r o g e n e si s. In
some species, growth regulators- auxin and/or
Pretreatment of anthers cytokinin are required for optimal growth.
The bas ic pr inc iple of nat i v e a n d r o g e n c s i si s t o l n c e r t a i n p l a n t s p e c i e s ,a d d i t i o n o f g l u ta th i o n e
stop the conversion of pollen cell into a gamete, and ascorbic acid promotes androgenesis.When
and f or c e it s dev elopm ent in t o a p l a n t . T h i s i s r n t h e a n t h e r c u l t u r e m e d i u m i s s u p p l e m e n te d w i th
f ac t an abnor m al pat hway i n d u c e d t o a c h i e v e a c t i v a t e d c h a r c o a l , e n h a n c e d a n d r o g e n e si s i s
in vitro androgenesis. Appropriate treatment of observed. lt is believed that the activated charcoal
ant her s is r equir ed f or good s u c c e s s o f h a p l o i d removes the inhibitors from the medium and
production. Treatment methods are variable and f a c i l i t a t e sh a o l o i d f o r m a t i o n .
lar gely depend on t he donor p l a n t s p e c i e s .
.l
Chemical treatment : Certain chemicars
ar e k nown t o induc e par t hen o g e n e s ies . g . 2 - c h l o r o -
et hy lphos phonic ac id ( et hr e l ) . Wh e n p l a n t s a r e
Haploid plants can be developed from ovary or
t r eat ed wit h et hr eal, m ult in u c l e a t e d p o l l e n s a r e
ovule cultures. lt is possible to trigger female
pr oduc ed. Thes e pollens whe n c u l t u r e d m a y f o r m
gametophytes (megaspores) of angiosperms to
embryos.
develop into a sporophyte.The plants so produced
2. Tem per at ur e inf luenc e : I n g e n e r a l , w h e n are r€ferred ro as gynogenic haploids.
the buds are treated with cold temperatures(3-6"C) Gynogenic haploids were first developed by San
f or about 3 day s , induc t ion o c c u r s t o y i e l d p o l l e n Noem (1976) from the ovary cultures of Hordeum
embryos in some plants e g. Datura, Nicotiana. v u l g a r e .T h i s t e c h n i q u e w a s l a t e r a p p l i e d fo r r a i si n g
Further, induction of androgenesis is better if haploid plants of rice, wheat, maize, sunflower,
anthers are stored at low temperature, prior to sugar beet and tobacco.
c ult ur e e. g. m aiz e, r y e.
In vitro culture of unpollinatedovaries(or ovules)
There are also reports that pretreatment of i s u s u a l l y e m p l o y e d w h e n t h e a n t h e r cu l tu r e s g i ve
ant her s .of c er t ain plant s at h i g h e r t e m p e r a t u r e s unsatisfactoryresultsfor the production of haploid
( 35' C) s t im ulat esandr ogenesi se . g . s o m e s p e c i e so f plants. The procedure for gynogenic hdploid
Brassica and Capsicum. oroduction is brieflv described.
O F H AP L O IDPLA N TS
Chaot er45 : P R OD U C T IO N 543
Limitations of gynogenesis
In practice,productionof haploid plantsby ovary/
ovule cultures is not used as frequently as anther/ Fig. 45.3 : A diagrammatic representation of
po llen cultu res in c r op im pr ov em ent pr ogr am m e s . important pafts in a flower.
Th e majo r limit at ionsof gy nogenes isar e lis t ed .
1 . The diss ec t ion of unf er t iliz ed ov ar ies a n d
ovule s is ra the r dif f ic ult . The above markers have been used for the
2 . The p resenc eof only one ov ar y per f low e r i s development of haploids of maize.
another disadvantage.In contrast, there are a large It may be noted that for the detection of
n umb er o f mic r os por esin one ant her . androgenic haploids, the dominant gene marker
However, the future of gynogenesismay be more should be presentin the female plant.
p romising with im pr ov ed and r ef ined m et hods .
Two approaches based on morphology and As described in the preceeding pages, haploid
genetics are commonly used to detect or identify plants are obtained either by androgenesis or
ha plo ids. gynogenesis.These plants may grow up to a flowering
stage, but viable gametes cannot be formed due to
MORPHOLOGICAL APPROACH lack of one set of homologous chromosomes.
The vegetativeand floral parts,and the cell sizes Consequently,there is no seed formation.
o f h ap loid p lant s ar e r elat iv ely r educ ed w h e n Haploids can be diploidized (by duplication of
comp are d to diploid plant s . By t his way hap l o i d s chromosomes)to produce homozygousplants.There
can be de tec t ed in a populat ion of diplo i d s . are mainly two approaches for diploidization-
Morphological approach, however, is not as c o l c h i c i n e t r e a t m e n ta n d e n d o m i t o s i s .
effective as genetic approach.
COLGHICINE TREATMENT
GENETIC APPROACH
Colchicine is very widely usedfor diploidization
Cenetic markersare widely used for the specific
identificationof haoloids.Severalmarkersare in use' of homologous chromosomes.lt acts as an inhibitor
of spindle formation during mitosis and induces
. 'a.,' ma rke r f or br own c olour ed aleur one.
chromosome duplication. There are many ways of
. 'A' marker for purple colour. colchicine treatment to achieve diploidization for
. 'Lg' marker for ligulelesscharacter. production of homozygous plants.
544 BIOTECHNOLOCY
TABrr45.1 A selected llst of inproved varietles of crops developed by using anther culture
ln g en era l, the new c r ops ar e high- y ieldin g 9 The doubling of haploids (diploidization)
varietieswith diseaseresistance.Some of them are may not always lead to the formation of
resistance to cold, salt etc. Many plant breeders homozygousplant.
t he se d ays u se ant her c ult ur es in t heir r egul a r 10 ln vitro haploid production is not
bree din g p rog ram m es . economi cal lvi
y abl edue to very l ow success
rate.
Biotechnology [35]
-f- he genetic variations found in the in vitro regeneratedplants from gametic cells (e.g., anther
I cultured cells are collectivelyreferredto as c u i t u r e s ) . F o r t h e p l a n t s o b t a i n e d f r o m p r o to p l a st
. he pl ants deri ved from
s o ma c l o n a vl a ri a ti o n sT cultures, protoclonal variations is used.
such cells are referredto somaclones.
Some authors use the Ierms calliclones and BASIS OF SOMACLONAL V A RIATION S
protoclones to represent cultures obtained from S o m a c l o n a l v a r i a t i o n s o c c u r a s a r e su l t o f
c allus and pr ot oplas t sr es p e c t i v e l y . genetic heterogeneity (change in chromosome
The growth of plant cells in vitro is an asexual n u m b e r a n d / o r s t r u c t u r e ) i n p l a n t t i ssu e cu l tu r e s.
pr oc es s inv olv ing only m i t o t i c d i v i s i o n o f c e l l s . This may be due to
Thus , c ult ur ing of c ells is t h e m e t h o d t o c l o n e a . E x p r e s s i o no f c h r o m o s o m a l m o s a i c i smo r g e n e ti c
particular genotype lt is therefore expected that
d isorders
plant s ar is ing f r om a giv en t i s s u ec u l t u r e s h o u l d b e
t he ex ac t c opies of t he p a r e n t a l p l a n t . T h e . S p o n t a n e o u sm u t a t i o n sd u e t o c u l t u r e co n d i ti o n s.
oc c ur r enc e of phenot y pi c v a r i a n t s a m o n g t h e
T h e g e n e t i c c h a n g e sa s s o c i a t e dw i th so m a cl o n a l
r egener at edplant s ( f r om t i s s u e c u l t u r e s ) h a s b e e n
variations include polyploidy, a n e u p l o i d y,
known for several years. These variations were
c h r o m o s o m a lb r e a k a g e ,d e l e t i o n ,t r a n sl o ca ti o n a , nd
ear lier dis m is s edas t is s ue c u l t u r e a r t e f a c t s .
g e n e a m p l i f i c a t i o n s , b e s i d e s s e v e r a l m u ta ti o n s In
The t er m s om ac lonalv a r i a t i o n sw a s f i r s t u s e d b y fact, the oresence of several chromosomai
Lar k in and Sc owc r af t ( 198 1 ) f o r v a r i a t i o n s a r i s i n g a b e r r a t i o n s - r e c i p r o c a l t r a n s l o c a t i o n , d e l e ti o n s,
due to culture of cells. i.e., variability generated by i n v e r s i o n s , c h r o m o s o m a l b r e a k a g e, m u l ti ce n tr r c,
a tissue culture. This term is now universallv acentric fragments have been found among the
accepted. s o m a c l o n e so f b a r l e y , g a r l i c a n d o a t.
546
Chapter46 : SOMACLONALVARIATIONS 547
Callus
+
I
WITHOUT IN V'TRC SELECTION
Tobacco Phytophthoraparasitica
Proliferation
+
and
J
Toxicitydetermination
Apple Phytophthoracactorun maintenanceof callus for lethalconcentration
Banana Fusariunoxysporum
Lettuce Lettuce
mosaicvirus
Alfalfa Fusariumsolani
+I
A diagrammatic representation of in vitro f Selection
protocol for the isolation of disease resistance + cycles
plants with in vitro selection approach is given in
J
lsolationof tolerantcalli
Fig. 46.2.
J
Regeneration
The differentiated callus, obtained from an
ex plant is ex pos ed in t he m e d i u m t o i n h i b i t o r s l i k e
t ox ins , ant ibiot ic s , am ino a c i d a n a l o g s . S e l e c t i o n
J
Plantlet
cycles are carried out to isolate the tolerant callus
cultures and these calli are regeneratedinto plants.
J
vlvoscreeningagainst
/n
The plants so obtained are in vitro screenedagainst toxin/pathogen(R6 or SC1 plants)
t he t ox in ( or pat hogen or a n y o t h e r i n h i b i t o r ) . T h e I
plants resistantto the toxin are selectedand grown Self I Vegetative
pollinationI propagation
further by vegetativepropagationor self pollination. +
The subsequent generations are analysed for Progenyclonesfrom each plant
disease resistant plants against the specific +
pathogenic organism. Testfor diseaseresistance
A selected list of disease resistant crop plants +
I
obtained from somaclonal variations by in vitro Generationof disease
resistantplants(R1 or SC2 plants)
s elec t ion along wit h pa t h o g e n i c o r g a n i s m s a n d
selection agents is given in Table 46.2. +
I
Besides the disease resistant olants (described Agronomictrials
above), plants with herbicide resistance and
Fig. 46.2 : A diagrammatic representation of isolation
antibiotic resistancehave also been developed with
of disease resistant,plants with in vitro selection.
in vitro selection approach.
Chapter46 : SOMACLONALVARIATIONS 549
o f r a c h i s a n d p e t i o l e a r e m u c h h i g h e r ( - 5 0 %)
Tewe 46.2 A selccted list of disease resistant compared to those regenerated from callus of
crop plants obtained from somaclonalvariations l e a v e s ( - 1 2 %) .
with in yifro selection along with pathogenic
organismsand selectionagents
Duration of eell culture
Crop Pathogenic Selection I n g e n e r a l ,f o r m a n y p l a n t c u l t u r e s ,s o m a c l o n a l
organism(s) agent variations are higher with increased duration of
Hice Xanthomonas oryzae Bacterial
cells cultures. For example, it was reported that
Wheat Helninthosporium genetic variability increased in tobacco protoplasts
sativum Crudetoxin
from 1.5 to 6"/o by doubling the duration of
Pseudononas syringae Syringomycin
cu ltures.
Barley Fusarium sp Fusaricacid
Heln intho
sporiun sativun Crudetoxin Growth hormone effects
Maize Helminthosporium naydis HmT toxin
T h e p l a n t g r o w t h r e g u l a t o r si n t h e m e d i u m w i l l
SugarcaneHelminthosporium sacchari Toxin influence the karyotypic alterations in qultured
Potato Fusariunoxyspotum Culturefiltrate cells, and therefore development of somaclones.
Ercoiniacarotovora Pathogen Crowth hormones such as 2, 4-dichlorophenoxy
Tomato Pseudononas solanacearumCrudefiltrate acetic acid (2, 4-D) and naphthalene acetic acid
(NAA) are frequently used to achieve chromosomal
Tobacco mosaic virus Virus
variability.
Alfalfa Foxysporum sp Crudeliltrate
Tobacco Tobacco mosaic virus Virus Besidesthe factors discussedabove, selection of
Pseudononas somaclones with in yifro selection are dependent
syringae Methionine
on the following parameters.
sulfoximine
a S e l e c t i o n p r o p a g u l e ( c e l l s , p r o t o p l a s t s ,c a l l i ) .
AdvantaEes of with in vitra selection a Selection agent (toxin, herbicide, amino acio
a p p r aoc h analogue).
The major advantageof with in vitro selection a Technique used for selection.
methodis the specificselectionof the desiredtrait a Stability of resistantsubstance.
ratherthan a generalvariationfound at the plant
l e v el.T hispr oc edu rei s l e s sti m e c o n s u mi n g
when o ln vivo testing procedure.
comparedto without in vitro selectionapproach. a Ability for regenerationof plants.
FA CT O RS A F F E C T IN G PR O D U C T IO N O F
SO M A CLO NA L V AR IAN T S
Some of the importantfactorsthat influence
Jevelopment of somaclonalvariantsby without ln
: itro selectionand with in vitroselectionare briefly
:escribed. Somaclonal variations (and also gametoclonal
variations, described later) are highly useful in
Ge n ot y pe and e x p l a n t s o u rc e plant breeding programmes.The genetic variations
with desirable (or improved characters),besidesthe
The natureof genotypeof the plants influences
existing favourable characters can be introduced
the frequency of regeneration and frequency of
into the plants In general, Ihe methodology
production of somaclones.
adopted for induction of somaclonal variations are
Erplantscan be takenfrom any part of plant- simpler and easier compared to recombinant DNA
..,es, roots,internodes,ovariesetc. The sourceof technology. Hence, they are preferred by some
n 'r ant is v er y c r it i c a lfo r s o ma c l o n avl a ri a ti o n s . workers. The important applications of somaclonal
:,- instance,potatoplantsregenerated from callus variations are briefly described.
550 B IO TECHNO LO CY
Resistance to diseases
Tlslr 46.3 A selected list of somaclonal variants
Somaclonal variations have largely contributed
obtained from different cop species with their
morphological characters towards the development of disease resistancein
many crops e.g. rice, wheat, maize, sugarcane/
Crop Character(s) tobacco, apple, tomato.
Bice period,
Flowering panicle
size;tillernumber; Selected crops somaclonal variants, with
plantheight; shapeandcolour; increaseddisease resistancedeveloped, without ln
leaflength,
fl:gy:l_.t
{ lg.tiLg
tgg9: TYl?lll
:lglil't)/ vitro selection and with in vitro selection are
respectivelygiven in Tables 46.1 and 46.2.
Wheat graincolour;
tillernumber;
Plantheight;
seedstorageprotein.
Resistance to abiotic s t r e sse s
Maize Reduced pollenfertility
andmalesterility;
I t h a s b e e n p o s s i b l e t o d e v el o p b i o ch e m i ca t
twinsfalksfroma singlenooe.
mutants with abiotic stressresistance.
Sugarcane Highsugaryield;increased
stalklength,
. F r e e z i n gt o l e r a n c e e . g . w h e a t .
diameter,
weightanddensity.
Barley Increased heading . S a l t t o l e r a n c e e . 9 . , r i c e , m a i z e , to b a cco .
grainyield;leafshape;
date;ashcontent. o A l u m i n i u m t o l e r a n c e e . 9 . , c a r r o t, so r g h u m ,
Oats Plantheight; headingdate;morphology
and tomato.
fertility.
Resistance to herbicides
Soybean Vaiiable
height; seedprotein
maturity; and
oilconteni. C e r t a i n s o m a c l o n a l v a r i a n t s w i th h e r b i ci o e
resistancehave been developed. Selectedexamples
Potato yield;growth
Higher habit,maturity
and
are Srven
m0rpn0r0gy.
. Tokraccoresistantto glyphosate,sulfonylureaand
Tomato Dwar{habit;earlyflowering;
orangefrLiil
c0t0ur. oicloram.
Carrot Higher
carotene
content. . Carrot resistantto glyphosate.
Brassica Multiple
branching
stem;alteredleaf;slow quality
lmproved seed
growth;
failure
to floweror delayin
largepollengrains.
flowering; A new variety oI Lathyrussativa seeds (Lathyrus
Bio L 2.12) with a low content of neurotoxin has
Tobacco yield;plantheight;
Increased leafnumber,
been developed through somaclonal variations
widthandyield;typeof inflorescence
shape,
( N o t e : L a t h y r i s m i s a c r i p p l i n g d i se a seca u se d b y
andvield.
c o n s u m p t i o n o f L a t h y r u s s e e d s i .e . ke sa r i d a l , i n
many parts of central India).
2 Meiotic crossing over is the recombination 2. Variations may be induced while doubling
process observed in gametoclonal variations. the haploid chromosomes.
+
I MU LTIP LIC A TION B Y A X ILLA R Y B U D S
StagelV Transferof plantletsto A N D A P IC A L S H OOTS
sterilizedsoil for hardening
undergreenhouse
environmenl Quiescent or actively dividing meristems are
presentat the axillaryand apicalshoots(shoottips).
The axillarybuds locatedin the axilsof leavesare
Fig. 47.1 : Major stages involved in micropropagation.
capable of developing into shoots. ln the in vivo
state,how everonl y a l i mi ted numberof axi l l ary
meristems can form shoots. By meansof induced
Stage ll :lt is in this stage,the major activity of
in vitro multiplicationin micropropagation, it is
micropropagation occurs in a defined culture
possibleto developplantsfrom meristemand shoot
me diu m. Sta gell m ainly inv olv es m ult iplic ati o n o f
tip culturesand from bud culture!s.
shoots or rapid embryo formation from the explant.
Stage lll : This stage involves the transfer of Meri stem and shoot ti p cul tures
shoots to a medium for rapid development into
Apical meristemis a dome of tissuelocatedat
shoots. Sometimes,the shoots are directly planted
the extremetip of a shoot. The apical meristem
in soil to develop roots. ln vitro rooting of shoots
alongwith the young leaf primordiaconstitutes
the
is p refe rredw hile s im ult aneous lyhandling a l a r g e
shoot apex. For the developmentof disease-free
n umb er of spec ies .
plants,meristemtips shouldbe cultured.
Stage lV : This stage involves the establishment
Meristemor shoottip is isolatedfrom a stemby
o f p lan tletsin s oil. This is done by t r ans f er r i n gt h e
a V-shapedcut. The size (frequently0 2 to 0.5
plantlets of stage lll from the laboratory to the
mm) of the ti p i s cri ti calfor cul ture.In generalthe
,
environment of greenhouse. For some plant
larger the explant (shoottip), the better are the
species, stage lll is skipped, and unrooted stage ll
chancesfor culture survival. For good resultsof
shoots are planted in pots or in suitable compost
micropropagation, explantsshould be taken from
mixture .
the acti vel ygrow i ngshootti ps,andthe i dealti mi ng
The different stages described above for is at the end of the plantsdormancyperiod.
, micropropagation are particularly useful for
The mosi widely used media for meristem
comparison between two or more plant systems,
cul tureare MS medi umand W hi te' smedi um. A
besides better understanding. lt may however, be
diagrammatic representationof shoot tip (or
noted that not all plant species need to be
meri stem) i s gi ven i n
cul turei n mi cropropagati on
propagated in vitro through all the five stages
Fig 47.2, and brieflydescribedhereunder.
referred above.
In stagel, the cultureof meristemis established.
Micropropagation mostly involves in vitro clonal
Addition of growth regulatorsnamely cytokinins
propagation by two approaches.
(ki neti n,B A )and auxi ns(N A A or IB A )w i l l support
1 . Multiplication by axillary buds/apicalshoots. the growth and development.
554 B IOTECHNO LO G Y
Pottedplantlet Plant
/
I
/
Rootedplantlet Primaryculture
Rootingof Shootdevelopment
shoots (withaxillaryshoot
proliferation)
Fig. 47.2 : A diagrammatic representation of shoot tip (or meristem) culture in micropropagation
(Note : l, ll and lll represent stages in micropropagation)
(A)
(B)
Node isolated
and cultured
Fig, 47.4 : A diagrammatic representation of micropropagation of plants by axillary bud (or shoot tip)
method (A) Rosette plant (B) Elongate plant.
556 B IOTECHNO LO CY
1 . Genotype of the plant : Selectionof the right For efficient in vitro rooting during micro-
genotype of the plant species (by screening) is propagation, low concentration of salfs (reduction
necessary for improved micropropagation In to half to one quarter from the original) is
general, plants with vigorous germination and advantageous lnduction of roots is also promoted
branching capacity are more suitahle for micro- by the presence of suitable auxin (NAA or IBA).
propagation.
ORGANOGENESIS
2. Physiologicalstatus of the explants : Explants
(plant materials) from more recently produced parts Organogenesisis the process of morphogenesis
of plants are more effective than those from older involving the formation of plant organs i.e. shoots,
r egions . Cood k nowledge of d o n o r p l a n t s ' n a t u r a l roots, flowers, buds from explant or cultured plant
propagation process with special reference to tissues. lt is of two types- direct organogenesis
gr owt h s t age and s eas onalin f l u e n c e w i l l b e u s e f u l and indirect organogenesis.
in s elec t ingex plant s .
Direct organogenesis
3. Cult ur e m edia : The s t a n d a r d o l a n t t i s s u e
c ult ur e m edia ar e s uit able f o r m i c r o p r o p a g a t i o n Tissues from leaves, stems, roots and
during stage I and stage il. However, for stage lll, inflorescences can be directly cultured to produce
c er t ain m odif ic at ions ar e r e q u i r e d . A d d i t i o n o f plant organs. In direct organogenesis,the tissue
growth regulators (auxins and cytokinins) and undergoesmorphogenesiswithout going through a
alt er at ions in m iner al c om p o s i t i o n a r e r e q u i r e d . c a l l u s o r s u s p e n s i o nc e l l c u l t u r e s t a g e. Th e te r m
This is lar gely dependent on t h e t y p e o f c u l t u r e direct adventitious organ formation is also used for
(meristem, bud etc). direct organogenesis.
Chapter47 : CLONALPROPACATION
(MICROPROPACATTON) 557
Automated micropropagation
APPL ICATION S OF
MICROPROPA G ATI O N It has now become oossible to automate
microprcpagation at various stages. In fact, bio-
Micro pro pa gat ion has bec om e a s uita b l e
reactors have been set up for large scale multi-
alternative to conventional methods of vegetative
plication of shoots and bulbs. Some workers
propagation of plants. There are several advantages
e m p l o y r o b o t s ( i n p l a c e o f l a b o u r e r s )f o r m i c r o -
of micropropagation
propagation, and this further reduces production
cost of plants.
High rate of plant propagation
1 . M o s t o f t h e v i r u s e s a r e n o t s e n s iti veto h e a t
Apic al m er is t em s wit h l o w
treatment.
c onc ent r at ion of v ir us es
In general,the apical meristemsof the pathogen 2. Many plant species do not survive after
inf ec t ed and dis eas e har bou r i n g p l a n t s a r e e i t h e r thermotherapy.
free or carry a low concentration of viruses,for the With the above disadvantages,heat treatment
f ollowing r eas ons . h a s n o t b e c o m e p o p u l a r f o r v i r u s e l i m i n a ti o n .
. Abs enc e of v as c ular t is s u e i n t h e m e r i s t e m s
t hr ough whic h v ir us es r ead i l y m o v e i n t h e p l a n t Meristem-tip culture
body. A general description of the methodology
. Rapidly div iding m er is t em a t i c c e l l s w i t h h i g h adopted for meristem and shoot tip cultures has
metabolic activity do not allow virusesto multiply. been described (see Fig 47.2).
(MICROPROPACATION)
Chapter47 : CLONALPROPACATTON 561
Terla 47.1 A selected list of the plants wlth vlrus elimitation by neristem cultures
Biotechnology [36]
562 B IOTECHNO LO CY
Endosperm
with failed Normal
development enoosperm
'Hybrid Normal
embryo emoryo
Ovulewithhybridembryo Ovule with normalembrvo
j
I
J
t
Hybridembryo
Normalendosoerm
Fig. 47.7 : Embryo-endosperm transplant technique used in embryo rescue (or immature embryo culture).
F or ade q u a ten u tri ti o n a sl u p p o rto f i m mature 1 . Heterotrophic phase : This is an early phase
em br y osemb
, ry o -e n d o s p e trarm n s p l a nits used. and the embryo is mostly dependent on the
endosperm and maternal tissuesfor nutrient supply.
Embryo-endosperm transplant: The endosperm
t r ans plantt e c h n i q u eu s e d fo r c u l tu ri n gi mmature 2. Autotrophic phase : This phase is
embryosis givenin Fig.47.7,and brieflydescribed characterized by the metabolic capability of the
below. embryo to synthesize substances required for its
growth which slowly makes it independent.
T he hy b ri d e mb r:y ofro m th e o v u l e i n w hi ch
endospermdevelopmenthasfailed is takenout by The critical stage is the intervening phase
excision.Another normallvdevelopedovule with between the heterotrophic and autotrophic phases.
endos per me n c l o s i n ga n e m b ry o i s th o s e n. Thi s The nutrient supply is highly variable at this phase
ovule is dissected and the normalembryois pressed which mostly depends on the plant species.
w i th a n exi t
out . T his le a v e sa n o rm a le n d o s p e rm
hole. Now, the hybridembryocan be insertedinto In general,the composition of the medium for
t he nor m a l e n d o s p e rmth ro u g h e x i t h o l e . Thi s culturing immature embryos is more complex than
rm n s p l a nwt h ich can
r es ult sin em b ry o -e n d o s p e tra that required by mature embryos which can grow
be c ult ur edi n a s u i ta b l em e d i u m. o n a s i m p l e i n o r g a n i c m e d i u m F u r t h e r ,t h e t r a n s f e r
of embryos from one medium to another is
By using embryo-endosperm transplant,many frequently needed in order to achieve full
interspecificand intergeneric plants have been develooment of embrvos.
r ais ede. g. ,h y b ri dp l a n tso f l e g u m e s .
Composition of the medium : Some salient
NUT RI T I O N A L R EOU IR E M EN T S OF features of medium and culture conditions are
E M B RY O C U L T U R E S listed below
Tlow 4:7.2A selectedlist of dlstant plant speciessossed and the reslstancctralts developed
throughembryorescuetechnique
Distant plant species crossed Resistancetrait(s)
Oryza sativax O.minuta Bacterial andblast
blight
Solanum tuberosum x S. etuberosum leafrollvirus
Potato
Solanun melanogena x S. khasianum Brinjal
shoot andlruitborer
Brassicanapus x B. oleracea vduudvs
^^hL^^^ ^^hiillu
oPl
565
t
I
566 B IO TECHNO LO CY
Certain seeds are heterogeneousand therefore, Among these, the most commonlY used
are not suitable for true genotype maintenance. cryopreservation is by employing liquid nitrogen.
At the temperatureof liquid nitrogen (-1 96'C), the
c e l l s s t a y i n a c o m p l e t e l y i n a c t i v e s ta te a n d th u s
In vitro methods for germplasm
can be conserved for long periods. In fact,
conservation
cryopreservationhas been successfullyapplied for
In vitro methods employing shoots, meristems germplasm conservation of a wide range of plant
and embryos are ideally suited for the conservation s p e c i e s e . g . r i c e , w h e a t , p e a n u t, ca ssa va ,
of germplasm of vegetatively propagated plants. sugarcane,straberry,coconut. Severalplants can be
The plants with recalcitrant seeds and genetically regenerated from cells, meristems and embryos
engineered materialscan also be preservedby this stored in cryoPreservation
in vitro approach.
Mechanism of cryoPreservation
There are several advantagesassociatedwith in
The technique of freeze preservationis based on
v itro germplasm conservation
the transfer of water present in the cells from a
. Large quantities of materialscan be preservedin liquid to a solid state. Due to the presenceof salts
s m all s pac e a n d o r g a n i c m o l e c u l e s i n t h e c e l l s , th e ce l l w a te r
requires much more lower temperature to freeze
. The ger m plas m pr es er v e dc a n b e m a i n t a i n e d t n
(even up to -68'C) compared to the freezing point
an environment, free from pathogens
of pure water (around 0"C). When stored at low
r lt can be protected against the nature's hazards temperature, the metabolic processes and
b i o l o g i c a l d e t e r i o r a t i o n si n t h e c e l l s/ti ssu e sa l m o st
r From the germplasm stock, large number of
come to a standstill.
plants can be obtained whenever needed.
Precautions/limitations for successful
. Obstaclesfor their transportthrough national and
cryopleservation
int er nat ional bor der s a r e m i n i m a l ( s i n c e t h e
ger m plas m is m aint a i n e d u n d e r a s p e c t i c Cood technical and theoretical knowledge of
c ondit ions ) . I i v i n g p l a n t c e l l s a n d a s w e l l a s c r yo p r e se r va ti o n
AND CRYOPRESERVATION
CONSERVATION
Chaoter48 : CERMPLASM 567
t2 ------l
tr
Shoottips
Pregrownon medium
with DMSO
in culturemedium
Ampoulethawing
Ampoulestored
in liquidnitrogen
Shoottips in ampoule
frozenin liquidnitrogen
Fig. 48.1 : An outline of the protocol for cryopreservation of shoot tip (DMSO-Dimethyl sulfoxide).
technique are essential. Other precautions (the cryopreservationof plant cell culture followed by
limita tion stha t s hould be ov er c om e)f or s uc c es s f u l the regeneration of plants broadly involves the
cryopreservationare listed below following stages
.l
. Forma tion ice c r y s t als ins ide t he c ells s hould b e . Development of sterile tissue cultures
prevented as they cause injury to the organelles 2. Addition of cryoprotectantsand pretreatment
a nd th e cell.
3. Freezing
. Hig h in tracel lular c onc ent r at ion of s olut es m a y
4. Storage
a lso da mag e c ells .
5. Thawing
o Sometimes,certain solutesfrom the cell may leak
o ut d urin g fre ez ing- 6. Reculture
An outline of the protocol for cryopreservation The selection of plant species and the tissues
of shoot tip is depicted in Fig. 48.1 . The with particular referenceto the morphological and
568 B IOTECHNO LO CY
phy s iologic alc har ac t er sla r g e l y i n f l u e n c et h e a b i l i t y freezing technique is used for the cryopreservation
of the explant to survive in cryopreservalion.Any of shooi tips and somatic embrYos.
tissue from a plant can be used for cryopreservation 3. Stepwise freezing method I This is a
, br y os ,en d o s p e r n t so, v u l e s ,s e e d s , combination of slow and rapid freezing procedures
e. g. m er is t em sem
c ult ur ed plant c ells , pr oto p l a s t s ,c a l l u s e s . A m o n g (rvith the advantagesof both), and is carried out tn
these, meristematic cells and suspension cell a s t e p w i s em a n n e r .T h e p l a n t m a t e r i al i s fi r st co o l e d
cultures,in the late lag phaseor log phaseare most t o a n i n t e r m e d i a t e t e m p e r a t u r e a nd m a i n ta i n e d
suitable. t h e r e f o r a b o u t 3 0 m i n u t e s a n d t h e n r a p i d l y co o l e d
b y p l u n g i n g i t i n t o l i q u i d n i t r og e n . Ste p w tse
; \ odlt ion of c r y opr ot ect a n t s and freezing rnethod has been successfully used for
pretrelatment
c r y o p r e s e r v a t i o n o f s u s p e n s i o n c u l tu r e s, sh o o t
Crvoprotectants are the compounds that can aoices and buds
prevent the damage caused to cells by freezing or 4. Dry freezing method : Some workers have
thawing. The freezing point and supercooling point reported that the non-germinated dry seeds can
of water are reduced bv the presence ot survive freezing at very low temperature in contrast
cryoprotectantsAs a result,the ice crystalformation t o w a t e r - i m b i b i n g s e e d s w h i c h a r e su sce p ti b l eto
is retarded during the process of cryopreservation c r y o g e n i c i n j u r i e s . I n a s i m i l a r f a s h i o n , d e h yd r a te d
Ther e ar e s ev er al c r y opr o t e c t a n t sw h i c h i n c l u d e cells are found to have a better survival rate after
dim et hy l s ulf ox ide ( DM S O ) , g l y c e r o l . e t h y l e n e , cryopreservatton.
pr opy lene,s uc r os e,m ann o s e ,g l u c o s e , p r o l i n e a n d
acetanride. Among these, DMSO, sucrose and Storage
glycerol are most widely used. Cenerally, a mixture
M a i n t e n a n c eo f t h e f r o z e n c u l t u r e sa t th e sp e ci fi c
of cryoprotectantsinstead of a single one, is used
temperatureis as important as freezing. ln general,
for more effectivecryopreservationwithout damage
the frozen cells/tissues are kept for storage at
to cells/tissues.
temperatures in the range of -70 to -196'C.
However, with temperatures above -1 30'C, ice
Fl a+ ; illr g
c r y s t a l g r o w t h m a y o c c u r i n s i d e t he ce l l s w h i ch
The serrsitivityof the cells to low temperature is r e d u c e sv i a b i l i t y o f c e l l s S t o r a g ei s i d e a l l y d o n e i n
v ar iable and lar gely depe n d s o n t h e p l a n t s p e c i e s . l i q u i d n i t r o g e nr e f r i g e r a t o r - a t 1 5 0 'C i n th e va p o u r
Four different types of freezing methods are used. p h a s e ,o r a t - 1 9 6 'C i n t h e l i q u i d p ha se .
1 . Slow-freezing method : The tissue or the The ultimate obiective of storage is to stop all
r equis it e plant m at er ial is s l o w l y f r o z e n a t a s l o w t h e c e l l u l a r m e t a b o l i c a c t i v i t i e sa n d m a i n ta i n th e i r
cooling rates of 0.5-5oC/min from 0'C to -100"C, r,iability For long term storge, temperature at
and t hen t r ans f er r ed t o l i q u i d n i t r o g e n . T h e -196"C in liquid nitrogen is ideal. A regular and
advantage of slow-freezing method is that some c o n s t a n t s u p p l y o f l i q u i d n i t r o g e n to th e l i q u i d
amount of water flows from the cells to the outside. nitrogen refrigeratoris essential.lt is necessaryto
This pr om ot es ex t r ac ellu l a r i c e f r o m a t i o n r a t h e r c h e c k t h e v i a b i l i t y o f t h e g e r m p l a s mp e r i o d i ca l l y i n
t han int r ac ellularf r eez in g . A s a r e s u l t o f t h i s , t h e some samples.
plant c ells ar e par t ially d e h y d r a t e d a n d s u r v i v e
Proper documentation of the germplasm storage
better. The slow-freezing procedure is successfully
has to be done. The documented information must
used for the cryopreservation of suspension
b e c o m p r e h e n s i v ew i t h t h e f o l l o w i ng p a r ti cu l a r s.
cultures.
. T a x o n o m i c c l a s s i f i c a t i o no { t h e ma te r i a l
2. Rapid freezing method : This technique is
o History of culture
quit e s im ple and inv olv e s p l u n g i n g o f t h e v i a l
c ont aining plant m at er i a l i n t o l i q u i d n i t r o g e n . . Morphogenic potential
During rapid freezing, a decrease in temperature o C e n e t i c m a n i p u l a t i o n sd o n e
-300" to -1000"C/min occurs. The freezing process
. Somaclonalvariations
is c ar r ied out s o quic k ly th a t s m a l l i c e c r y s t a l sa r e
f or m ed wit hin t he c ells . F u r t h e l t h e g r o w t h o f . Culture medium
int r ac ellular ic e c r y s t als i s a l s o m i n i m a l . R a p i d o Growth kinetics
ChAPICT
48 : CERMPLASM
CONSERVATION
AND CRYOPRESERVATION 569
Thawing
Tlrrr 48.1 A selected list of plants in varlous
Thawingis usuallycarriedout by plungingthe
forms that are successfullycryopreserved
frozen samples in ampoules into a warm water
(temperature 37-45"C)bathwith vigorousswirling. PIant material Plant species
By this approach,rapidthawing(atthe rateof 500-
750" C m in- l) o c c u rs ,a n d th i s p ro te c tsth e c el l s Cellsuspensions 4^,-^
vr yzd ^^t:.,^
JdIIva
570 B IOTECHNO LO CY
Other
gases (2%)
z
E
c)
o
6 seo
o
o-
0
Fig. 48.2 : A graphic representation of tissue culture storage under normal atmospheric pressure,
low-Pressure,and low-oxYgen'
The partial pressureof oxygen below 50 mm Hg 4. Recalcitrant seeds can be maintained for
re du ce s p lan t t is s ue gr owt h ( or ganiz ed o r ron8.
u no rga nizedtiss ue) .This is due t o t he f ac t t hat wi t h
5. Conservation of somaclonal and game-
r e du ce d availa bilit y of O r , t he pr oduc t ion of CO,
toclonal variations in cultures.
is low. As a consequence, the photosynthetic
activity is reduced, thereby inhibiting the plant 6. Plant materials from endangered species can
tissue growth and dimension. be conserved.
Limitations of LOS : The long-term conservation 7. Conservation of pollen for enhancing
of plant materialsby low-oxygen storageis likely to longevity.
in hib it the pla nt gr owt h af t er c er t ain dim ens ion s .
B Rare germplasmsdeveloped through somatic
h y b r i d i z a t i o n a n d o t h e r g e n e t i c m a n i p u l a t i o n sc a n
be stored
572
Chapt er49 : C EN ET ICEN C IN EE R INO
CF PLA N TS -ME TH OD OLOC Y 573
o Insecticidal activity
The gene transfer techniques in plant genetic
. lmp roved nu t r it ional qualit y
transformation are broadly grouped into two
o Altered flower pigmentation categories
574 B IOTECHNO LO CY
Wound
Crown
galltumor
f i.o<- Aorobacterium
"o" iumefaciens
Roots
Fig. 49.1 : Formation of a crown gall tumor in a plant infected n4th Agrobacterium tumefaciens.
NH a l s o t r a n s f e r r e dt o t h e p l a n t c e l l s . l t i s n o w c l e a r l y
il establishedthat the right border is more critical for
H2N-C-NH-(CHz)s -cH-cooH T - D N A t r a n s f e ra n d t u m o r i g e n e s i s .
I
NH 2. Virulence region : The genes responsiblefor
I the transferof T-DNA into the host olant are located
cH-cooH outside T-DNA and the rigion is referredto as viror
I virulence region. Vir region codes for proteins
CHs
OctoPine involved in T-DNA transfer.At least nine vir-gene
operons have been identified These include vir A,
NH
vir C, vir Br, vir C1, vir Dt,Dzand D4, and vir E,
I
H2N-C-NH-(CHz)s -cH-cooH and Et.
I 3. Opine catabolism region : This region codes
NH
for proteins involved in the uptake and metabolisms
I of opines.
CH-COOH
I
(CN)z Besides the above three, there is ori region rhal
i s r e s p o n s i b l ef o r t h e o r i g i n o f D N A r e p l i c a t i o n
I
COOH which permits the Ti plasmid to be stably
Nopaline maintained in A- tumefaciens.
t he r angeof 12 to 2 4 k b , w h i c h d e p e n d so n the
bac t er ials t r ai n fro m w h i c h T i p l a s mi d sc o me.
Nopalines t r ai n so f T i p l a s mi dh a v e o n e T -D N A
with lengthof 20 kb while octopinestrainshave
two T-DNA regionsreferredto as T, and T* that
ar e r es pec t iv e l1ya k b a n d 7 k b i n l e n g th . (24 bp repeat)
A diagrammatic of a Ti plasmidis
representation
depicted tn Fig, 493. fhe Ti plasmid has three
importantregions. Virulence
regron
1. T-DNA region: This regionhasthe genesfor (y/rgenes)
the biosynthesisof auxin (aux), cytokinin (cyt) and
opine (ocs),and is flankedby leftand rightborders.
Thesethree genes-aux,cyto and ocs are referredto
as oncogenes,as they are the determinantsof the (ori gene)
t um or phenot y p e .
Fig. 49.3 : A dagrammatic representation of a
T-DNA borders- A set of 24 kb sequences Ti plasmid.
presenton eitherside (rightand left)of T-DNA are
576 B IOTE CHNO LO CY
Woundedplantcell
Phenoliccompounos
and sugars
Nuclear
,1
vir D1/D2
#
I OO
+r 09
virDllD2 virE2
Virulenceproteins
( Dr , Dz , E2,B e t c . )
Translation
+
I
Agrcbacteriumsp Attachment
Productionof
auxin,cytokinin
and opine
+
I
Autoetirnulation
,otceHdivision
Plant cell
Fig. 49,4 : A diagrammatic representation of T-DNA transfer and its integration into host plant cell genome
(pTi-Ti plasmid; RB-Right border; LB-Left border; ss-Single-stranded).
Biotechnology
[37]
r-7
574 B IOTECHNO LO CY
Homologous
DNAsequence
Fig. 49.5 : Cointegrate vector system (vi-Ti plasmid virulence region; pBR322-Bacterial plasmid 322; LB-Left
border; RB-Right border; MCS-Multiple cloning site; PTM-Plant transformation marker; RES-Bacterial resistance
marker; col E,-Origin of a replication from col E, plasmid; orif -Origin of transfer site for
conjugative plasmid mobilization).
Fig. 49.6 : Binary vector system (vir-Ti plasmid virulence region; LB-Left border; BB-Right border; MCS-Multiple
cloning s!!e; PTM:Planltransformatianmarker; RE9-Bacterialrcsistancemarker; oril.Origin of transfet site lar
of! ftom
The cloning process is carried out in E. coli, the gene is retained).lt may be noted that both of them
bacterium wl.rerethe cloning is most efficient. The a r e n o t p h y s i c a l i y l i n k e d ( o r i n t e g r a t e d ) .A b i n a r y
intermediatevector is mated with Agrobacteriumso vector with T-DNA can replicate in E. coll and
that the foreign gene is mobilised into the latter. Agrobacterium.
The transformed Agrobacterium cells with receptor
A diagrammatic representation of a typical
Ti plasmid and intermediate vector are selectively
binary vector system is depicted in Fig. 49.6 fhe
iso late d wh en gr own on a m inim al m ed i u m
binary vector has the following components
con tain ing spec t inom y c in. The s elec t ion pr o c e s s
becomes easy since E. coli does not grow on a 1 Left and right borders that delimit the
minimal medium in which Agrobacterium grows. T-DNA region.
Within the Agrobacterium cells, intermediate
plasmid gets integratedinto the receptorTi plasmid 2. A plant transformationmarker (PTM) e.g. npt
// that confers kanamycin resistance in plant
to p rod uce coint egr at e plas m id. This pla s m i d
t r a n s f o r m e dc e l l s .
containing plant transformationmarker (e.g. npt ll)
gene and cloned target gene between T-DNA 3 . A m u l t i p l e c l o n i n g s i t e ( M C S )f o r i n t r o d u c i n g
borders is transferredto plant cells. The transformed target/oreign genes.
pla nt ce lls can be s eleCt edon a m edium c ont ai n i n g
kanamycin when the plant and Agrobacterium cells 4. A bacterial resistancemarker e.g. tetracycline
resistancegene for selecting binary vector colonres
are incubated together.
in E. coli and Agrobacterium.
Advantages of cointegrate vector 5. oriT sequence for conjugal mobilization of
r Target genes can be easily cloned the binary vector from E. coli to Agrobacterium.
. The pla smid is r elat iv ely s m all wit h a num be r o f 6 . A b r o a d h o s t - r a n g eo r i g i n o f r e p l i c a t i o ns u c h
restriction sites. as RK, that allows the replication of binary vector
o In terme dia teplas m id is c onv enient lyc loned i n E . in Agrobacterium.
coli and transferred to Asrobacterium. " Production and use of binary vector : The target
(foreign)gene of interestis insertedinto the multiple
Binary vector
cloning site of the binary vector. ln this way, the
The binary vector system consists of an target gene is placed between the right and left
Agrobacterium strain along with a disarmed Ti border repeats and cloned in E. coli. By a mating
plasmid called vir helper plasmid (the entire process,the binary vector is mobilised from E. coli
T-DNA region including borders deleted while vir to Agrobacterium.Now, the virulence gene proteins
s80 B IOTECHNO LO G Y
PLANT
USING
TRANSFORMATION
AGROBACTEB'UM
TECHNIOUE @@
Agrobacterium-mediatedtechnique is the most Agrobacterium
widely used Ior the transformation of plants and tumafaciens
gener at ion of t r ans gen i c p l a n t s . T h e i m p o r t a n t
requirements for gene transfer in higher plants / ,rt et
\)
/1
\
/\{--'\\
i \-//-\ '
through Agrobacterium mediation are listed. L,K
. The ex plant sof t he pla n t m u s t p r o d u c e p h e n o l i c
compounds (e.g. autosyringone)for activation of
Normalplant
c Q9e
"Xo"
v ir ulenc e genes .
Advantages ol Agrobactefium
mediated transformation
o This is a natural method of gene transfer.
c Agrobacterium can conveniently infect any Transformedplant
explant (cellsltissues/organs).
Fig. 49.7 : Transformationtechnique using
Even large fragments of DNA can be efficiently Agrobacterium -mediatedgene transfer.
'
transferred.
Chapt er49 : CE N ET ICEN C T N EE R T OF
N G PL A N TS -TV IE TH OD OLOC Y 581
582 B IOTECHNO LO CY
T h e g e m i n i v i r u s e sc a n i n f e c t a w i d e r a n g e o f
crop plants (monocotyledons and dicotyledons)
which attract plant biotechnologists to employ
these virusesfor gene transfer.Curly top virus (CTV)
and maize streak virus (MSV) and bean golden
mosaic virus (BCMV) are among the important
gemtnrvrruses.
C o m p l e m e n t a r y D N A ( c D N A ) co p i e s o f R N A
GEMINIVIRUSES AS VECTORS virusesare preparedin vitro. The cDNA so Senerated
The gem iniv ir us esar e s o n a m e d b e c a u s e t h e y can be used as a vector for gene transferin plants
hav e gem inat e ( Cem ini l i t e r a l l y m e a n s h e a v e n l y This approach is t.ediousand cumbersome.However,
t wins ) m or phologic al par t i c l e s i . e . t w i n a n d p a i r e d some successhas been reported.A gene sequence
capsid structures.These viruses are characterized e n c o d i n g c h l o r a m p h e n i c o l r e s i sta n ce ( e n zym e -
by pos s es s ingone or t w o s i n g l e - s t r a n d e dc i r c u l a r chloramphenicolacetyltransferase) has been inserted
DNAs ( s s DNA) . O n r epl i c a t i o n s ,s s D N A f o r m s a n into brome mosaic virus genome. This Sene
intermediate double-stranded DNA. expression,however,has been confinedto protoplasts.
CF PLA N TS -ME TH OD OLOC Y
Chapt er49 : C E N E T ICEN C IN EE R INO 583
tI _ . .Testinofor
ptants**
I transgeniJ
+
T0 seeds
The term director vectorless transferof DNA is
+
I
used when the foreign DNA is directly introduced
Transformedplants
into the plant genome. Direct DNA transfer screenedin the
methodsrely on the deliveryof naked DNA into next generation
t he olant c e l l s . T h i s i s i n c o n tra s t to the
+
I
Agrobacteriumor vector-mediatedDNA transfer T1 seeds
which may be regardedas indirectmethods.
Majorityof the directDNA transfermethodsare Fig. 49.9 : An overview of the protocol for the
simpleand effective.And in fact,severaltransgenic
plantshave been developedby this approach.
Rupturedisc
Maclocarrier
Vector DNA-coated
particles(microcarriers)
Stoppingplate
Plant material
Holdingpipette
Liposomes
I o@@ o@o @ @c.r
9.99ie-
(?(U (9 ptalmids
@@@ @@o @@ 6
Plasmids
----+
Nucleus
. DNA is stableand can be storedfor sometimein . The embryonicplant cellsare hard and compact
liposomesprior to transfer. and are resistantto SCFpenetration.
. A pplic ablet o a w i d e ra n g eo f p l a n t c e l l s In recentyears/some improvements have been
. Thereis good reproducibilityin the technique. made in SCF-mediated transformation.This has
helpedin the transformation of rice, wheat,maize
Limitations of liposome fusion and barl eyby usi ngthi s techni que.
T he m ajor pro b l e m w i th l i p o s o m e -m e d iated
transformation is the difficultyassociated with the C H E MIC A L GE N E TR A N S FE R ME TH OD S
regeneration of plantsfrom transformed protoplasts.
PO LYETHYLEN E G LYCO L.MED'AT ED
TNANSFORMATION
S'L'CON CABB'DE F'BBE.MED'ATED
TBATVSFOBMAT'ON Polyethyleneglycol (PEG),in the presenceof
divalent cations (using Ca2+) , destabilizesthe
T he s ilic on c a rb i d e fi b re s (S C D a re a b out
plasma membrane of protoplasts and renders it
0. 3- 0. 6pm in dia m e te a r n d 1 0 -10 0 p m i n l e n gth.
permeable to naked DNA. In this way, the DNA
Thesefibresarecapableof penetrating the cell wall
entersnucleusof the protoplasts and getsintegrated
and plas m am em b ra n ea, n d th u sc a n d e l i v e rD NA
w i th the genome.
i nt o t he c ells .
T he DNA c o a te d s i l i c o n c a rb i d e fi b re s a re The procedure i nvol ves the i sol ati on of
vortexedwith plant material(suspension culture, protopl astsand thei r suspensi on,addi ti on of
) . ingth e mi x i n g ,D N A a d h e ri n gto th e
c allus es Dur plasmid DNA, followedby a slow additionof 4O'/.
fibresentersthe cellsand getsstablyintegrated with P.EC-4000 (w/v)dissolvedin mannitoland calcium
the hostgenome.The siliconcarbidefibreswith the ni trate sol uti on.A s thi s mi xture i s i ncubated,
trade name Whiskersare availablein the marker. protoplasts get transformed.
Hygromycin phosphotransferase
(hpt genel
T h e a n t i b i o t i c h y g r o m y c i n i s m o r e to xi c th a n
In general, genetic transformation of plants is
neomycin and therefore can kill non-transfornted
a low-frequency event. Some methods for
p l a n t c e l l s m u c h f a s t e r . H y g r o m y c in p h o sp h o -
selecting the transformd plant materials (cellsl
transferase(hpf) gene thus provides resistanceto
tissues) have been devised by using a set of
transformed cells.
genes referred to as marker genes. These marker
genes are introduced into the plant material along
Aminoglycoside adenyltransferase
with the target gene The marker genes are of two
types. laadA gene|
Aminoglycoside3'-adenyltransferase (aadA)gene
I Selectable marker senes
confers resistanceto transformedplant cells against
Il. Reporter genes t h e a n t i b i o t i c s s t r e p t o m y c i na n d s p e c t i o n o m yci n .
Chaot er49 : C EN ET ICE N C IN E ER INOF
C P L AN TS -ME TH OD OLOC Y 589
Tmrs 49.3 A selected list of selectable marker genes used for gene transfer In plants,
' their source and substrates used for their selection
Herbicideresistance
Phosphinothricin
acelyltransferaseuauPqr nyces hygroscopicusi Glufosinate,
Strepto L-phosphinothricin,
S viridochronogenes Bialophos
Enolpyruvyl
shikimatephosphate epsps/aroA Agrobacteriunspl
synthase Petuniahybrida Glyphosate
Acetolactase
synthase qle
Arabidopsi
s sp/maize/tobacco
Sulfonylureas
Glyphosate
oxidoreductase g0x Achromobacter LBAA Glyphosate
Bromoxynil
nitrilase bxn pneumoniae
Klebsiella Bromoxynil
Others
B-Glucuronidase gus/uidA E. coli glucuronide
Cytokinin
Xylose
isomerase xylA Thermoanaerobcterium Xylose
urogenes
thermosulf
Mannose isomerase pmi/manA
6-phosphate E. coli lvlannose
Betaine dehydrogenasebadh
aldehyde Spinach Betaine
aldehyde
chromatography
B-Glucuronidase gus/uidA E. coli Fluorometric
orhistochemical
or colorimetric
Greenlluorescentprotein stp victoria(jellytish)
Aequorea Fluorescence
(bacterial)
Luciterase luxNIuxB Vibrioharveyi Bioluminescence
(firefly)
Luciferase luc Photonus pyralis Bioluminescence
acetyltransferase v a t
Chloramphenicol E. coli Autoradiography
Op-ine synthase (ocs, nos genesl i n many i nstances, GFP has repl acedC U S si nce
assays of GFP are easier and non-destructive.Thus,
The common opinespresentin T-DNA of Ti or
screening of even the primarytransplants can be
Ri plasmidsof Agrobacteriumare octopine and
done by C FP w hi ch i s not possi bl ew i th other
nopaline,respectivelyproducedby the synthase
genesocs and nos. The transformedstatusof the reporterBenes.
plant cells can be easilydetectedby the presence C ene for C FP has been i sol atedfrom j el l y fi sh
of these opines. Opines can be separated by Aequoreavictoriawhich is a luminescent organism.
electrophoresisand identified. Alternately, the The original gfp gene has been significantly
enzymeactivitiesresponsible for the productionof modifiedto makeit more usefulas a reportergene.
opinescan also be assayed. C FP emi ts fl uorescence w hi ch can be detected
undera fl uorescent mi croscooe.
B.Glucuronidase lgusluidA gene)
genes|
p-Clucuronidase producinggene (gus/uidA)ts Bact'erial luciferase lluxNIuxB
the most commonly usedreporter genein assessing The bacterialluciferasegenes(/uxA and luxB\
plant transformation for the following reasons. have originatedfrom Vibrio harveyi.They can be
. p-Clucuronidase assaysare very sensitive. detectedin someplanttransformation vectors.The
detection assayof the enzyme is based on the
o Quantitative estimation of the enzyme principleof bioluminescence. Bacterialluciferase
can be done by fluorometric method (using catalyses the oxidation of long-chain fatty
substrate4-methylu mbelIiferryl B-D-glucuronide al dehydes of l i ghtw hi ch
thatresul tsi n the eri i i ssi on
which is hydrolysedto 4-methylumbelliferone). can be measured.
. Qualitativedataon the enzymecan be obtained
l e a n s (e n z y mel o c a l i z a ti on Fireffy luciferase Uuc genel
by his t oc he m i c am
can be detectedby chromogenicsubstance such The enzymefirefly luciferase,encodedby the
as substrate X-gluc). geneluc, catalyses the oxidationof D-luciferin(ATP
. No needto extractand identifvDNA. dependent)which resultsin the emissionof light
that can be detectedby sensitiveluminometers.
Green fluorescent {gfp genel
protein
The firefly luciferasegene, however, is not
Creen fluorescentprotein(CFP),coded by gfp widely usedas a markergenesincethe assayof the
qene,is beingwidely usedin recentyears.ln fact, enzymeis rathercumbersome.
592 B IOTECHNO LO C\
Biotechnology [38]
594 B IOTECHNO LO CY
' Pbst-transcriptionalgene silencing is due to the and exciting field in modern biotechnology as it
production of double-strandedRNA either in the offers the following advantages.
RNAis
T h e doubl e-stranded
n u c l e u so r c v to p l a s m. 1 . Chloroplasts are maternally inherited, hence
formedwhen antisense RNA is produceddue to the
there is no danger of gene transferthrough pollen
activityof RNA dependentRNA polymerase. to related weeds. This is because pollen does not
contain transgenes.
Strategies to avoid gene silencing
2 . M u l t i g e n e t r a n s f e r c a n b e co n ve n i e n tl y
T h e c o n tro lo f g e n es i l e nci ngi s not an easyj ob,
o u t i n c h l o r o p l a s t sw h i c h i s r a th e r d i ffi cu l t
s i n c ei t o c c u rsi n a n u n p redi ctablfashie on.S ome c a r r i e d
with nuclear genome.
generalrecommendations, are, however,made to
m i n i m i z eth e i mp a c to f g e nesi l enci ngi n transgeni c 3. Chloroplasts genome is fu n cti o n a l l y
plants comparable to prokaryotic Senome. A single
of of group
o Reductionin the numberof transgenes inserted promoter can control the expression
genes (transgenes). lt is therefore possible to
(i .e .re d u c e dc o p y n u m b er).
i n t r o d u c e d e s i r a b l e m u l t i p l e g e n e s w h i ch ca n
o Avoiding the use of promotersand transgenes
be expressed under the control of a single
with high degreeof homology. promorer.
. M i n i m i z i n g /a v o i d i nthg e use of mul ti pl ecopi es
4. High level of transgeneexpressionis possible
of the samepromoteror terminator.
. h e r e a r e a b o u t . l 0 0 ch l o r o p l a sts
w i t h c h l o r o p l a s t sT
Gene silencing is not observed in chloroplast p e r c e l l , e a c h c o n t a i n i n g a b o u t 1 0 0 co p i e s o f
(plastid) transformation,hence it is preferred in g e n o m e . T h u s , t h e r e i s p o s s i b i l i t yo f 1 0 ,0 0 0 co p i e s
recentyears. o f t r a n s g e n e sp e r c e l l ! T h i s i s a t r e m e nd o u sn u m b e r
of transgenescarried by transformed chloroplasts.
There is a tremendous potential for a very high
Ievel of gene expression and large scale productron
of active oroteins.
The chloroplasts (plastids) and mitochondriaare
5. Chloroplast transformation is nof associated
believedto have evolvedfrom prokaryotes during
with gene silencing which is a major problem with
the courseof evolution.Boththeseorganelles have
nuclear Benome transformation.
th e i r o w n g e n o me ,a l th oughi t i s much si mpl er
when comparedto nucleargenome.Further,many 6. Antibiotic resistancegenes need not be used
of the proteinsthat function in chloroplastsand as selectable markers. Even if used, thev can oe
mi to c h o n d ri a re e n c o d edby nucl eargenesand easily excised.
then transported to the organelle.
7. Toxicity associated with foreign protetn
p r o d u c t i o n i n c h l o r o p l a s t s i s m u ch l e ss w h e n
Ghloroplast genome
compared to nuclear-controlledforeign proteins.
M o s t o f th e h i g h e r pl ants have about- 100
p e r l e a fc e l l . Eachchl oropl ast
c h l o ro p l a s ts contai ns Design of vectors for chloroplast
a p p ro x i m a te l y1 0 0 c o p i e s of chl oropl astD N A transformation
genome.The chloroplastgenome(theplasfome)is
a circular double-stranded DNA molecule (or A diagrammaticrepresentation of two vector
chromosome)located in the stroma.Majority of constructsfor chloroplast is depicted
transformation
c h l o ro p l a sgte n o me a s re i n the si zeof 120-160 kbp in Fig. 49.13.
a n d c o n ta i n a b o u t 1 2 0 - 140 genes.A bout 100
1. A construct for expressionof a single
chloroplastgenesare known to code for proteins. gene : The vectorfor chloroplasttransformation is
The proteinsynthesis in chloroplasts resemblesthat
based on the selectablemarker gene aadA that
of prokaryotes. providesresistance The
to antibioticspectinomycin.
singleforeign(desirable) geneis fusedto regulatory
CHLOROPLAST ENGINEERING sequences (promoterand terminator)which in turn
Ceneticengineering DNA ( Cp
of chloroplastthat leadsto i s fl ankedon ei thersi de by chl oroplast
is an important DNA) (Fig.49.13A).
chloroplast(plastid)transformation
49 : CE N E T ICE N C IN E ER INOF
C P L AN TS -ME TH OD OLOC Y
(A)
Foreign
gene 1
(B)
Foreigngenes
2. A construct for expressionof multiple DNA sequenceson the vector and those of on tne
genes: In this case,the selectablemarker is the g e n o m e . T h i s i s a s i t e - s p e c i f i ci n t e g r a t i o na n d t h u s
betaine-aldehydedehydrogenase (badh)gene. avoids the frequent problems associated with
randon insertion of foreign genes into nuclear
I t is f lank edb y a p ro m o te ra n d th e m u l ti p l e
transgenesare flanked by a terminator.At both S e n o m e .
e nds c hlor oplasD t N A s e q u e n c eas re p re s e n t.In The regenerated plants derived from the
betweenthe transgenes, theseare ribosome-binding m o d i f i e d p l a s t o m e ( c h l o r o p l a s t g e n o m e ) a r e
sites (one between two transgenes)to ensure regarded as transplastomic plants.
efficienttranslation(Fig.a9J 38).
The future of chloroplast
Introduction of foreign genes into transformation
chloroplast genome
T h e t e c h n o l o g y o f c h l o r o p l a s t t r a n s f o r m a t i o nr s
Most of the methodsused for introducingthe in the developing stages. In fact, it has not become
foreigngenesinto nucleargenomeare not useful as routine as transformationof nuclear genomes of
The mostsuccessful o l an t s .
for chloroplasttransformation.
methodfor insertingforeigngenesinto chloroplasts
Chloroplast engineering,however, holds a great
is particle gun bombardment.
promise in plant biotechnology being an efficient,
After the bombardment, homologous c l e a n a n d e n v i r o n m e n t a l - f r i e n d l ya p p r o a c h f o r t h e
re c om binat iono c c u rs b e tw e e n th e c h l o ro p l ast p r o d u c t i o n o f t r a n s g e n i cp l a n t s .
genet ic m anipula t i o n sc a r r i e d o u t i n p l a n t s l ti / i, ABIOTIC
J he S T F r IISLS STRESSES
I f or t he pr oduc t ion o f t r a n s g e n i c p l a n t s h a v e
been des c r ibed ( Chapt er4 9 ) . T h e u l t i m a t e g o a l o f Insects-+ Herbicides
t r ans genic s( inv olv ing int r o d u c t i o n ,i n t e g r a t i o n ,a n d Viruses-+
expression of foreign genes) is to improve the Fungi-)
crops, with the desired traits. Some of the
-+
Bacteria
im por t ant ones ar e lis t e d .
-)
Wounds
r ft,esistanceto biotic stresses r.e resistance to
Ozone
dis eas es c aus ed by in s e c t s , v i r u s e s , f u n g i a n d
Intenselight
bac t er ia
. Res is t anc e t o abi o t i c stresses-herbicides,
t em per at ur e ( heat , c h i l l i n g , f r e e z i n g ) , d r o u g h t , Floods
s alinit y , oz one, int ens e l i g h t . Heavymetals
r lm pr ov em ent of c r op y i e l d , a n d q u a l i t y e . g .
storage, longer shelf life of fruits and flowers. Fig. 50.1 : Biotic and abiotic stresses that affect
plant growth, development and yield.
. Tr ans genicplant s wit h i m p r o v e d n u t r i t i o n
. Trans8enic plants as bioreactors for the
A l most al l the stresses,ei ther dir ect ly or
m anuf ac t ur e of c om m e r c i a l p r o d u c t s e . B .
pr ot eins , v ac c ines , and b i o d e g r a d a b l ep l a s t i c s .
indirectly,leadto the productionof reactiveoxygen
species(ROS)that createoxidativestressto plants
Thi s damagesthe cel l ul arconstit ut entofs plant s
Env ir onm ent aE s r t l' e s s e s t o p l a n t s
w i th a reductionin plantvield.
w hi ch i s associ ated
The different tvoes of external stresses that
The major objective of plant biotechnology is to
inf luenc e t he plant gr ow t h a n d d e v e l o p m e n t a r e
develop plants that are resistant to biotic and
depicted in Fig. 50.1, These stressesare grouped
abiotic stresses.
based on their characlers-biotic and abiotic
sfresses.The biotic stressesare caused by insects,
pat hogens ( v ir us es ,f ung i , b a c t e r i a ) , a n d w o u n d s RESISTANCETO BIOTICSTRESSES
The abiot ic s t r es s esar e d u e t o h e r b i c i d e s , w a t e r
def ic ienc y ( c aus ed by d r o u g h t , t e m p e r a t u r e , C e n e t i c e n g i n e e r i n g o f p l a n t s h a s l e d to th e
s alinit y ) ,oz one and int e n s e l i g h t development of crops with increased resistanceto
596
Chapter50 : APPLICATIONS
OF PLANTTRANSFORMATION/|RANSCENIC
PLANTS 597
Tlrre 50.1 A selectedlist of common insect pests along with the major crops damagedby them
Bt toxin genes
Severalstrainsof B. thuringiensrsproducing a
wide range ol crystal (cry) proteins have been
identified.Further,the structureof cry genesand
their correspondingtoxin (6-endotoxin)products
have been characterized.The cry genes are
c l a s s i fi e d(l a te s ti n 1 9 9 8) i nto a l argenumberof
distinct families (about 40) designatedas cry
1 ......c ry4 0 , b a s e coJ n t hei r si ze and sequence
s i mi l a ri ti e sAn. d w i th i n each fami l y, there may
be sub-families.Thus, the total number of Parasporalcrystal
genesproducingBt toxins (Cry proteins)is more
.l Io'nuti
than 00. +
Thereare differences in the structureof different o
250-kDasubunitprotoxin
Cry proteins,besidescertainsequencesimilarities.
The molecularweights of Cry proteinsmay be
e i th e rl a rg e(-1 3 0 K D a )o r smal l(-70K D a).D espi te
the differencesin the Cry proteins,they sharea
common activecore of three domains.
Bt based genetic transformation stems and fruits. This is not possible by any
of plants chemical pesticide.
It has been possibleto geneticallymodify (GM) . Toxic proteins are produced within the plants;
plants by inserting Bf genes and provide pest hence they are environmental-friendly.
resistanceto these transformedplants. For an
" Bt toxins are rapidly degraded in the
effective pest resistance,the bacterial gene in environment.
tra ns genic plan ts m u s t p o s s e s s h i g h l e v el
expression. Thisobviouslymeansthatthe transgene The problem of insect resistance
transcriptionshouldbe underthe effectivecontrol to Bt crops
of promoterand terminatorsequences.
The major limitation of Bt-gene possessing
The earlyattempts to expresscry 1A and cry 34 transgenicplantsis the developmentof Bt-resistant
oroteins under the control of CaMV 355 or insects.
The Bttoxin is a orotein.and the membrane
AgrobacteriumT-DNA promotersresultedin a very receptor (of the gut) through which the toxin
low expressionin tobacco,tomatoand potatoplants. mediates itsactionis alsoa protein.lt is possible
that
the appropriate mutationsin the insectgenecoding
Modificationol Bt uy 1A gene : The wild type
for receptorproteinmay reducethe toxin binding
transgene Bf cry 1A(b)wasfoundto express at a very
and renderit ineffective. This may happenwithin a
l o w lev els in t r a n s g e n i cp l a n ts .T h e n u c l e o ti de
few generations by repeated growingof BI crops.
sequence of this genewas modified(G + C content
altered,severalpolyadenylation signalsremoved, Several approachesare made to avoid the
ATTTA sequence deleted,etc). With appropriate developmentof resistance in insects.
sequence changes, an enormousincrease (about100
. lntroductionof two differentBt toxin genesfor
iold) in the Bt toxin oroductformationwasobserveo.
the same target insect.
The transgenicBt crops that were found to . Development of transgenicplantswith two types
provide effectiveprotectionagainstinsect damage of insect resistancegenes e.g. Bt gene and
were given approvalfor commercialplantingby protei nasei nhi bi torgene.
U S A in t he m i d -1 9 9 0 s (T a b l e 5 0 .A . So me
o RotatingBt crops with non-Bt crops may also
biotechnological companieswith their own trade
namesintroducedseveraltransgenic cropsinto the prevent the build-up of resistancein insect
Among these, maize and cotton Bt crops popul ati on.
fields. only
are currentlyin use in USA.The other genetically
modifiedolantsmet with failurefor variousreasons. The environmental impact oI Bt crops
The most serious impact of Bt crops on
Advantages of transgenic plants environment is the build-up of resistancein the
with Bt genes pest population.
r Bt genescould be expressedin all partsof the l n 1999, anotheri ssuew as broughtto l i ght
plants,includingthe rootsand internalregionsof aboutBf crops.lt was reportedthat the pollenfrom
600 BIOTECHNOLOC\
transgenlcplantswfth lnsectresistance
Proteaseinhibitors
CpTi apple,rice,
Potato, Trypsin LePidoPtera
Coleoptera,
wheat,tomato
sun{lower,
CII Tobacco,potato protease
Serine LepidoPtera
Coleoptera,
PI-IV Potato,tobacco protease
Serine Lebidoptera
o Gl Tobacco, oilseedrape protease
Cysteine Homoptera
Coleoptera,
CMe Tobacco Trypsin !:-li-d'p':ll
a-Amylaseinhibitors
a-Al-Pv Pea,tobacco o-Amylase Coleoptera
WMAI-1 Tobacco o,-Amylase Lepidoptera
Lectins
GNA rice,sugarcane
Potato, Lectin Lepidoptera
Homoptera,
sweetpotato,tobacco
WGA Maize Agglutin _1. ptgi1 9_gl
p,d. f
9_.pt'
Others
BCH Potato Chitinase Lepidoptera
Homoptera,
TDC Tobacco decarboxylase Homoptera
Tryptophan
Bt maize might be toxic to the larvaeof Monarch Some of the importantones are
microorganisms.
butterfly.This generatedconsiderableoppositionto Iisted.
Bf crops by the public, since Monarchbutterflyis '1. Cholesterol oxidase of Strepotmycesculture
one of the mostcolorfulnativesin USA.It was later filtratewas found to be toxic to boll weevil larvae.
proved that the fearsabout the impact of 8t crops Cholesterol oxidasegenehas been introducedinto
on the monarchbutterflywere withoutthe required tobaccoto developa transgenicplant.
scientificevidence.
2. Isopentenyl transferase gene from Agro-
The lessonlearnt from the monarch butterfly bacterium tumefacienshas been introduced into
episodeis that the risks of CM crops should be tobacco and tomato. This gene codes for an
thoroughlyassessed before they are reported. importantenzymein the synthesis of cytokinin.The
transgenic plants with this transgene were found
Usageof 8f : The usageBt is commonlyusedfor to reduce the leaf consumption by tobacco
a transgeniccrop with a cry Senee.g. Bt cotton.In
hornworm and decreasethe survival of peach
the same way, Cry proteinsare also referredto as potatoaphid.
Bf proteins. lt may also be stated here that the
authorsusefour differentnamesfor the samegroup R E S IS TA N C E GE N E S FR OM
of proteins-6-endotoxin,insecticidalcrystalprotein
HIGHER PLANTS
(lCP),Cry and norv Bt.
Certaingenesfrom higherplantswerealsofound
to result irr the synthesisof productspossessing
Resistance genes from
insecticidalactivity.Someauthorsregardthem as
other microotganisms
non-Bt insecticidalproteins.A selectedlist of plant
The Bt toxin genes from B.thuringiensishave insecticidal(non-Bt)genes used for developing
beendescribedin the preceedingpages.Thereare transgenicplants with insect resistanceis given
certain other insect resistantgenes from other Table50,3.Someof them are brieflydescribed.
PLANTS
OF PLANTTRANSFORMATIONIRANSCENIC
chapter 50 : APPLICATIONS 60r
3', AntisensemRNA
Fig. 50.3 : Hybridization of antisenEe nRNA with virus RNA to block replication.
Targetvirus RNA
Ribozyme(antisenseRNA)
$
,iI
i
I (*
Cleavedviral RNA
Defensins m o l e c u l a r w e i g h t a n d a n t i m i c r o b i a l i n n a tu r e . Th e
p h y t o a l e x i n su s u a l l y p r e s e n t i n s p e c i a l i ze dce l l s o r
Def ens ins ar e ant im ic r obi a l p e p t i d e s ( 2 6 - 5 0
o r g a n e l l e s a r e m o b i l i z e d w h e n i n f e c t i o n o ccu r s.
am ino ac id r es idues )f ound in a l l t h e p l a n t c e l l s .
F u r t h e r ,d u r i n g i n f e c t i o n t h e r e o c c u r s i n d u cti o n o f
They at t ac k t he m ic r obial p l a s m a m e m b r a n e ,
g e n e s f o r i n c r e a s e d p r o d u c t i o n o f p h yto a l e xi n s.
however this is not adequate to provide resistance
Stilbene synthaseis a key enzyme for the synthesis
to pathogens
o f a c o m m o n p h y t o a l e x i n .T h e g e n e c o d i ng sti l b e n e
In recent years, an artificial defensin gene has synthase has been isolated from peanut and
been developed and introduced into potatoes. introduced into tobacco, rice and Brassica napus.
These potatoes developed resistance to the The transgenicplantscarrying stilbenesynthasegene
bacterium Eswinia carotovora. were resistantagainst some fungi.
A selected list of transgenic plants developed,
Thionins along with the genes transferredand the controlled
Thionin proteins also offer protection against pathogens is given in Table 50.6.
bac t er ia. Thionin c oding g e n e s h a v e b e e n
introduced into tobacco and the transgenicplant so NEMATODE RESISTANCE
developeci showed resistance to Pseudomonas N e m a t o d e sa r e s i m o l e w o r m s f o u n d i n th e so i l .
syr!nqae. Thev possessa complete digestivetract. The annual
crop loss of the world due to nematode
PHYTOALEXINS ( r o u n d w o r m ) i n f e s t a t i o ni s v e r y h i g h .
is proposedthat this gene encodesa proteinthat from crop plants. For this reason,the crops are also
detects the pests (nematodes)and triggers a affected by herbicides, hence the need to develop
r eac t io n si n th e p l a n t.l t i s b e l i e v e dth a t
d ef ens iv e herhicide-resistant plants.Thus, these plants provide
som ec hem ic alc o m p o u n d sth a t d e s tro yth e g u t o f an opportunity to effectively kill the weeds (by
the nematodeare oroduced. herbicides)without damaging the crop plants.
Weeds (wild herbs) are unwanted and useless 4. Mutation of the target protein: The target
plants that grow along with the crop plants. Weeds protein which is being affected by the herbicide
compete with crops for light and nutrients,besides can be suitably modified. The changed protein
harbouring various pathogens. lt is estimated that s h o u l d b e c a p a b l e o f d i s c h a r g i n gt h e f u n c t i o n s o f
t he wo rld's cro p yi eld is r educ ed by 10- 15% due t o the native protein but is resistantto inhibition by
the presence of weeds. To tackle the problem of the herbicide. Once the resistant target protein
weeds, modern agriculture has developed a wide gene is identified, it can be introduced into the
range of weedkillers which are collectively referred p l a n t g e n o m e s , a n d t h u s h e r b i c i d e - r e s i s t a npt l a n t s
to as herbicides. In general, majority of the can be developed.
herbicides are broad-spectrum as they can kill a For success in the development of herbicide
wide range of weeds. A good or an ideal herbicide resistant plants, good knowledge of the target
is expected to possess the following characteristics. protein and the action of herbicides is required.
r Capable of killing weeds without affecting crop Some of the developments made in the herbicide
o lan ts. resistanceof plant are briefly described.
. No t to xic to a ni m als and m ic r oor ganis m s .
GLYPHOSATE RESISTANCE
. Rapidly translocatedwithin the target plant.
Clyphosate, is a glycine derivative. lt acts as a
. Ra pid ly de gra de d in t he s oil.
broad-spectrumherbicide and is effectiveagainst76
, No ne of th e co m m er c ially av ailable her bic ide s of the world's worst 78 weeds. Clyphosate is less
f ulfils all th e a bo v e c r it er ia.The m ajor lim it at ion of t o x i c t o a n i m a l s a n d i s r a p i d l y d e g r a d e d b y
the herbicidesis that they cannot descriminateweeds microorganisms.In addition, it has a short half-life.
608 B IOTECHNO LO CY
Phosphinothricin
Alanine
I
Alanine
Bialaphos
I
Glutaminesvnthase
+ NHa-_#
L-Glutamate L-Glutamine
glyphosate. The reasonfor this was not immediately thricin is moreeffectiveagainstbroad-leafed weeds
identified.lt was later known that the shikimate but least effective against perennials.(Note :
pathway occurs in the chloroplastswhile the Phosphinothricin and glufosinate are two namesfor
glyphosate resistantEPSPS was producedonly in the the same herbicide.However,to avoid confusion
cytoplasm.This enzymewas not transported to the between glyphosateand glufosinate,phosphino-
chloroplasts,hence the problem to provide thricin is more commonlyused. BastaAventisand
resistance. Thiseoisodemadescientists to realizethe Libertyare the trade namesfor phosphinothricin).
importance of chloroplasts in geneticengineering.
Phosphinothricin-a natural herbicide
ln later years,the mutant EPSPSgene was
tagged with a chloroplast-specific transit peptide P hosphi nothri cii snan unusualherbi ci de, bei ng
sequence.By this approach,the glyphosate-resistant a derivative of a natural product namely bialaphos.
EPSPS enzymewas directedto freely enterchloroplast Certainspeciesof Streptomyces produce bialaphos
and confer resistanceagainst the herbicide w hi ch i s a combi nati on of phosphi nothri ciboundn
to tw o al ani neresi dues, formi nga tri pepti de.B y
3. Det ox if ic a ti o no f g l y p h o s a te:T h e s o i l the aciionof a peptidase, bialaphosis convertedto
microorganismspossessthe enzyme glyphosate activephosphinothricin (Fig 50.V.
oxidasethat convertsglyphosateto glyoxylateand
a m inom et hy lph o s p o naic i d . T h e g e n e e n c o d i ng Mechanism of action of
glyphosateoxidasehas been isolatedfrom a soil phosphinothricin
organism Ochrobactrum anthropi. With suitable
modifications, this gene was introducedinto crop P hosphi nothri ci n as a competi ti ve
acts i nhi bi tor
plantswere of the enzyme glutamine synthase (Fig5O.l.fhis ts
plantse.g.oilseedrape.The transgenic
possible since phosphinothricin has some structural
found to exhibitvery good glyphosate resistance in
the f ield. similarity with the substrateglutamate.As a
consequence of the inhibitionof glutaminesynthase,
Use of a combined strategy: More efficient ammoni aaccumul ates and ki l l s the ol ant cel l s.
resistanceof plants against glyphosatecan be Further,disturbancein glutaminesynthesisalso
providedby employinga combinedstrategy. Thus, inhibitsphotosynthesis. Thus,the herbicidalactivity
resistant(i.e. mutant)EPSPS gene in combination of phosphinothricin is due to the combinedeffectsof
with glyphosateoxidasegene are used. By this ammoniatoxicityand inhibitionof photosynthesis.
approach,there occursglyphosateresistance (due
to mutantEPSPS gene)as well as its detoxification Strategy for phosphinothricin
(due to glyphosateoxidasegene). resistance
The natural detoxifying mechanism of
PHOSPHINOTHRICIN RESISTANCE phosphinothricinobservedin Streptomyces sp has
(or
Phosphinothricin glufosinate)is also a broad prompted scientists to develop resistant
spectrum herbicide like glyphosate.Phosphino- pl ants agai nst thi s herbi ci de. The enzyme
Biotechnology
[39]
610 B IOTECHNO LO CY
Glyphosate of EPSPS
lnhibition Soybean,
tomato
Glyphosate by glyphosate
Detoxification oxidase.
1 l ^ i -^
tvrqr4u, ouyu94rl
^a.,haan
(EPSPS-5-Enoylpyruvytshikinate
3-phosphate 2,4-D-2,4-Dichlorophenoxy
synthase; aceticacid)
aceticacid;2,4,5-T-2,4,S'Trichlorophenoxy
S P L AN TT R A N S FOR MA TION | IR A N S GEPNLA
Chapt er50 : A P P L IC A T IO NOF ICN TS 611
availability (shortage due to drought), and salinity therefore water deficit. lt is therefore, logical to
influe ncethe p lan t gr owt h, dev elopm entand y ield . think of genetic engineering strategies for the
The abiotic stressesdue to temperature, drought increased production of osmoprotectants.
and sa linity are c ollec t iv ely r egar ded as wat e r
S o m e p r o g r e s sh a s b e e n n r a d e i n t h i s d i r e c t i o n .
deficit stresses(See Fig 50.7).
The biosynthetic pathways for the production of
manv osmoprotectantshave been establishedand
Causes of water deficit
genes coding key enzymes isolated. In fact, some
Water deficit may occur due to the following progress has been made in the development of
ca uses. transgenic plants with high production of
. Reduced soil water ootential. osmoorotecta nts.
Effects of water deficit Some of the key enzymes for the production of
glycine betaine have been identified e.g. choline
. Resultsin osmotic stress.
m o n o o x y g e n a s e ,c h o l i n e d e h y d r o g e n a s e ,b e t a r n e
. ln hib its oh oto sv nt hes is aldehyde dehydrogenase.The genes coding these
. Increasesthe concentrationof toxic ions (reactive enzymes were transferred to develop transgenrc
oxyg en spe cie s )wit h, in t he c ells . Dlants
. Loss of water from the cell causing plasmolysis By using choline oxidase gene from Arthrobacter
a nd fin aliy cell deat h sp, transgenic rice that produces higher glycine
betaine (which offerstolerance againstwater deficit
Tolerance to osmotic stress stress)has been developed.
Chlorophyll
---\
\
\ Polygalacturonase
\ lz
\ / -/
\
,/ / ........"+.......,.,-"'
\
I \
\
E
- -t,ht -v. l-e n e
tl-.
. , / . . "j ." '*'
//r'
I
Lycopene
o \,,',\t"
c
t1
i ,,.1i\.
| /.r
'.j
\- .
\- r
I '.j \- - -
-_---
10
Ceneticengineeringwork has been extensively (PC) and pectin methyl esferase. The
carried out in tomatoes, and some of the phytohormoneethylene production is intimately
developmentare described. linked to fruit ripeningas it triggersthe ripening
processof fruit. Addition of exogenousethylene
BIOCHEMICAL CHANGES DUR'NG promotesfruit ripening,while inhibitionof ethylene
TOMATO BIPEN'NG biosynthesisdrasticallyreducesripening.
Tomato plant
I *g#
DNA strand DNA
Complementary
Fig. 50.9 : Genetic manipulation of the enzyme polygalacturonase (PG) by antisense RNA approach
(Production of Flavr Savr tomato plant)
Phenylalamine
+
i
4-Coumaryl-CoA
lrz 3X Malonyln
CoA
I Cnalconcsynthase
Niringen
in-chalcone
Naringenin
I
I Ravone&hydroxylase
+
Dihydrokaempferol
Dihydroquercetin Dihydromyricetin
I
I D.Ffi I ot*
| 3cr J3Gr
Pelagonidin-3-glucoside Cyanidin3-glucoside o"toni il'ilr Iucoside
(brickred/orange) (red) "
"ll
Fig. 50.11 : Biosynthesis of anthocyanins.
in tomato have been described. The same GE N E TIC E N GIN E E R IN G FOR FLO WER
approachesin fact can be successfullyused for PIGMENTATION
other fruits, vegetablesetc., to achievelonger shelf-
Thereare continuousattemptsin flower industry
Iife
to make the ornamentalflowersmore attractive(by
improving or creating new colours), besides
G E N E T IC EN G IN EE R IN G FOR prolonging post-harvestlifetime. The cut flower
PREVENTING DISCOLORATION
industryis mostly (about 70'/") dominatedby four
Discoloration is a major plants-roses,tulips,chrysanthemums
of fruitsand vegetables and carnations.
postharvestproblem encounteredin food industry.
The most common type of flower pigmentsare
Certain food additives are added to prevent
anthocyanins,a Broup of flavonoids.They are
discoloration.However,theseadditivesmay cause
synthesizedby a seriesof reactions,startingfrom
h e a l thc o mp l i c a ti o nisn h u mans.
the amino acid phenylalanine(Fig. 50.11).The
Biochemically, discoloration of fruits and colour of the flower is dependenton the chemical
vegetables is mainlydue to the oxidationof phenols natureof the anthocyaninproduced.
(mono-and diphenols)to quinones,catalysedby a . Pelagonidin3-glucoside - brick red/orange.
group of enzymes namely polyphenol oxidases.
Theseenzymesare localizedin the membranes of . C yani di n3-gl ucosi de - red.
mitochondriaand chloroplasts. . D el phi ni di n3-gl ucosi de-bl ue to pur ple.
Ceneticmanipulations usingantisenseapproach
Manipulation of anthocyanin pathway
of polyphenoloxidasehas
to inhibit the synthesis
enzymes
been carried.Somesuccesshas been reportedin
preventing the discoloration of potatoes by this for differentreactions,
The enzymesresponsible
strategy. in the anthocyaninpathwayhave been identified.
OF PLANTTRANSFORMATIONIRANSCENIC
Chapter50 : APPLICATIONS PLANTS 617
B y genet ic m a n i p u l a ti o n sa n d m u ta ti o n s,i t i s t r a n s g e n i cp l a n t s c o n t a i n i n g r i b o n u c l e a s ei n h i b i t o r
possible
to developflowerswith the desiredcolours.
Most of the flowers (roses, carnations
c hr v s ant he m u m sl a) c k b l u e c o l o u r d u e to the
absenseof the key enzyme flavonoids 3', 5'-
hy dr ox y las(F
e 3 ' 5 ' H ) th a tp ro d u c eds e l p h i ni di ne
3
gluc os ide. O n e c o m p a n yb , y th e n a m e F l o ri gene,
has geneticallymanipulatedand introducedthe Cenetic manipulations for improving the
geneencodingthe enzymeF 3' 5' H (fromPetunia nutritional quality of plant products are of great
hybrida)into the following plants. importance in plant biotechnology. Some success
has been achieved in this direction through
The world's first genetically modified (GM)
conventional cross-breeding of plants. However,
flower was introducedin '1996.lt was a mauve
this approach is very slow and difficult, and many
( bluis h)c olo u re d c a rn a ti o nw i th a tra d e name
a times will not give the traits with the desired
Moondus{'. Subsequently,many other flowers
improvements in the nutritional quality. Selected
have been oroducedand marketed.
examples of genetic engineering with improved
Gan GM-flowers be eaten? nutritional contents are described.
In tobaccoplants,male sterilitywas introduced The four essential amino acids namely lysine,
by using a mitochondrialmutatedgene encoding m e t h i o n i n e ,t h r e o n i n e a n d i s o l e u c i n ea r e p r o d u c e d
t he enz y m e ri b o n u c l e a s eT. h e g e n e e n c odi ng from a non-essential amino acid aspartic acid
ribonucleasenamely barnasegene from Bacillus (Fig. 50.12).The formation of lysine is regulatedby
amyloliquefaciens was transferred to tobacco feedback inhibition of the enzymes aspartokinase
' plant s . T he ri b o n u c l e a sies to x i c to ta p e ta lcel l s, (AK) and dihydrodipicolinate synthase (DHDPS).
and thus preventsthe developmentof pollen, Theoretically, it is possible to overproduce lysine
ult im at ely le a d i n g to ma l e s te ri l i ty . By thi s by abolishing the feedback regulation. This is what
approach,transgenic plantsof tobacco,cauliflower, has been accomplished.
cotton,tomato,corn,lettuceetc.,with malesterility The lysine feedback-insensitivegenes encoding
have been developed. the enzymes AK and DHPDS have been
It is possibleto restoremale sterility in the respectivelv isolated from E.coli ano
aboveplantsby crossingthem with a secondsetof Cornynebacterium with appropriate genetic
618 B IOTECHNO LO CY
Aspartic
l
B-
ami no aci ds i n the desi red propor t ion.Som e
successhasbeenreportedin the productionof one
syntheti c protei n contai ni ng 13% m et hionine
,semialdehYder Dihvdrodioico-
tinatesynfhase residues.
,/ \
{)
Homoserine 2, 3-Dihydropicolinate GE N E TIC E N GIN E E R IN G FOR
/\
/\
J\
II IMPROVING PALATABILITY OF FOODS
More than the nutritivevalue,tasteof the food
Methionine Threonine
I I
Lysine
is importantfor attractinghumans.lt is customary
+ to make food palataHe by adding salt, sugar,
lsoleucine fl avorsand many other i ngradi ents. lt would be
ni ce i f a food has an i ntri nsi ca llyappet izing
Fig. 50.12 : An overview of the biosynthetic pathway character.
ol some essentia! amino acids derived from aspartic
acid (Note : Lysine regulates its own synthesis by A orotein monellin isolatecifrom an African
feedback inhibition) plant (Dioscorephyllum cumminsil is about
100,000 sweeter than sucroseon molar basis.
Monel l i n gene has been i ntroduce dint o t om at o
manipulations, these genes were introduced into and lettuceplants.Somesuccesshasbeenreported
s oy bean and c anola plant s . T h e t r a n s g e n i c p l a n t s i n the producti onof monel l i n i n t hese plant s,
so developed produced high quantities of lysine. i mprovi ngthe pal atabi l i ty.
lsoprenoid units Golden Rice has met almost all the objections
i raised by the opponents of GM foods. However,
i Sequential
addition many people are still against the large scale
v
p r o d u c t i o n o f C o l d e n R i c e , a s t h i s w i l l o p e n d o or
Geranylgeranyldiphosphate
to the entrv of manv other CM foods. Another
IPhytoenesynthase argument put forth against the consumption of
I
Colden Rice is that it can supply only aboul 2O"h
J of daily requirement of vitamin A. But the
Phytoene
proponents justify that since rice is a part of a
I
I Carotenedesaturase m i x e d d i e t c o n s u m e d ( a l o n g w i t h m a n y o t h er
Y
I foods), the contribution of provitamin A through
Lycopene Colden Rice is quite substantial.
I
I
Recently (in 2004), a group of British scientists
I Lycopenep-cyclase
J have developed an improved version of Colden
p-Carotene R i c e . T h e n e w s t r a i n ,C o l d e n R i c e 2 c o n t a i n s m o re
(provitamin
A) t h a n 2 0 t i m e s t h e a m o u n t o f p r o v i t a m i n A t h a n i ts
p r e d e c e s s o rl.t i s c l a i m e d t h a t a d a i l y c o n s u m p t i on
Fig. 50.13 : An outline of pathway for the
of 70 g rice can meet the recommended dietary
biosynthesis of provitamin A (B-carotene).
aliowance for vitamin A.
Tlsle 50.10 Some examplesof transgeniccrop plants (( plantsl at the developmental stages
Others
Tobacco
andsoybean Pouo
Cytochrome Synthesis of epoxyfattyacidsfor
manufacture oJadhesivesandpaints
Rice Nicotianamine
aminotransferase Tolerance to lowironavailability
Tobacco Nitroreductase Reduces landcontaminationbv
trinitrotoluene
(potato and cotton) were, for the first time, made o Modified sensory attributes e.g. increased
availableto farmersin USA. By the Vear1998-99, sweetness as in thaumatin.
five major transgenic crops (cotton, maize,
soybean,canola and potato)were in widespread Concerns about transgenic plants
use. They accountedfor about 75"/oof the total The fearsabout the harmfulenvironmental ano
area plantedby crops in USA. hazardoushealth affectsof transgenicplantsstill
A selected list of transgenic crop plants exists,despitethe fact that there have been no
(approvedin USA)for the commercialuse is given reportsso far in this regard.
in Table50.9. Someexamplesof transgeniccrop The transferof almostall the transgenicplants
plants,which are at the developmental stagesare from the laboratoryto the crop fields is invariably
given in Table 50.10. These plants are carefully associatedwith legal and regulatory hurdles,
designedto give rise to products,which will besi desthe soci aland economi cconcerns.
im pr ov ehum a nh e a l tha n d i n c re a s eo f c ro p yi el d.
The major concern expressedby public (also
Goals of biotechnological acknowledged by biotechnologists) is the
improvements in crops development of resistancegenes in insects,
generationof super weeds etc. Severalremedial
There are about 30-40 crops that have been measures are advocated to overcome these
genet ic allym o d i fi e d ,a n d m a n y m o re a re b ei ng oroblems.
added. However, very few of them have got the
The farmersi n devel opi ngcountri esare much
c lear anc ef or c o m m e rc i a ul s e . A s e l e c te dl i st i s
worried about the seed terminator technology
alreadygiven in Table50.9.
which forcesthemto buy seedsfor everynew crop.
The ultimategoalsof geneticallymodified(CM) Thesefarmersare traditionallyhabituatedto use
crop plantsare listedbelow. the seedsfrom the previouscrop which is now not
o Resistance to diseases (insect,microorganisms). possibledue to seedtermintortechnology.
o lmprovednitrogenfixing ability.
o Highery iel d i n gc a p a c i ty .
e Resistance
to droughtand soil salinity.
o Betternutritionalproperties.
o lmprovedstoragequalities. A nother i mportantappl i cati onof geneti cal l y
transformedplantsis their utility as bioreactors
to
a Production of pharmaceutically important producea wide rangeof metabolicand industrial
c om pound s . products.Theseaspectsare describedin the next
Absenceof allergens. chaDter.
f enet ic ally m odif ied pla n t s c a n b e m a n i p u l a t e d STARCH
\J to act as bioreactorsto produce a wide range
Starchis a polymerof glucose,and is composed
of biologic ally im por t an t c o m p o u n d s . T h e s e
of amyl oseand amyl opecti n. S tarchis used as a
inc lude c ar bohy dr at es ,lipid s a n d p r o t e i n s , b e s i d e s
food, feed and for industrialpurposes.Cenetic
t he s ec ondar y pr oduc t s . The c o m m e r c i a l p r o d u c t s
engi neeri ng
techni ques can be usedto m anipulat e
of plant s ar e us ef ul f or indu s t r i e so r f o r i m p r o v i n g
the quanti tyand qual i tyof starch.
t he hum an or anim al health .
The terms molecular farming/pharming or Increasing the production of starch
metabolic engineering of plants are also used to
B i osynthesiofs starch i n pl ants is a highly
c ollec t iv ely r epr es ent t he m o i e c u l a r b i o l o g i c a l
regulated process involvingthe key enzymeADP-
techniquesfor the synthesisof commercial products
glucose pyrophosphorylase. This enzyme rs
in plant s . M et abolic engi n e e r i n g o f t r a n s g e n i c
al l osteri cal l control
y l ed(feedbacki nhibit ion)by
plant s is gaining im por t ance i n r e c e n t y e a r t i r u n
attractive alternative to animals and micro-
metabolites such as phosphate.
or ganis m s f or t he m anufa c t u r e o f b i o l o g i c a l l y ADP-glucosepyrophosphorylase in E.coli was
important products. mutatedto alterits allostericproperties. Thismutant
gene was transferredand expressedin potato
plants.The transgenicpotato plants produced high
quantities of starch.
622
Chaot er51 : T RA N S C E N IC
P L AN T SA S T | OR EA C TOR S 62a.
Cytosol
Sucrose
UDP-Glucose
1
Glucose1-phosphate
TREHALOSE
1
Glucose6-phosphate MANNITOL
1 myalnositol
Fructose6-phosphate
t +
I
I
I P IN ITOL
Triose-phosphate
,
GLYCOLYSIS
Fiq.51.1 : An outline of the metabolic pathway for the biosynthesis of carbohydrates and
theit imoorlant derivatives in a plant cell.
antisense approach, the enzyme C BSS was gene coding for this enzyme was transferredto
inhibitedin potatoplants Consequently, amylose- potatoplants.However,the transgenic potatoplants
free starchi.e. starchcontainingonly amylopectin did not show any enhancement in the synthesis
of
was produced. cycl odextri ns
due to vari ousreasons.
Commerciallyimportant
nolecular fannlng (metabollc eEglncerlngl
Acetate
Plastid
I PalmitoylACP (C16)
(c1s)
i--+StearoVlACP Endoplasmic
OleoylACP (C16: 1) reticulum
Triacylglycerol
\ioesterase
"/
lipids Free fatty acid i
Diacylglycerol
1
Phosphatidicacid
Fattyacid modified
(elongation,desaturation,
hydroxylationetc.) Glycerol3-phosphate
Cytoplasm
many industries - for the production of soaps, Some attemptsare being made to genetically
detergents,cosmeticsetc. manipulatecrops to yield oils with high contents
eruci c aci d. The approachesi ncl ude to
It is possibleto terminatethe hydrolysisof acyl- of
overexpress genesencodingthe enzymeselongases
ACP by specificthioesterases and produce high
proportionof selectedfatty acids. One acyl-ACP and transfer of genesto produceenzymesthat can
preferentially incorporate erucic acid into
thioesterase that specifical ly hydrolyses lauroyl-ACP
tri acyl gl ycerol s.
has been isolated from California bay tree
(lJmbellulariacalifornica).The gene encodingthis
enzvmehasbeenclonedand transferred to oilseed PRdDUCTION OF FATTY AcIDs wITH
MOD IFIE D D E GR E E OF S A TU R A TION
rape.The transgenicplantswere found to produce
oils with high proportionof lauric acid (12-carbon A fatty acid is said to be saturatedif it has no
fatty acid). double bonds.Saturatedand unsaturated (with on
or more double bonds)fatty acidsoccur in nature.
P RO DUCT I O N O F L ON G E R C H A IN It is possibleto geneticallymanipulatethe degree
FATTY ACIDS of saturationin fatty acids.
F or t he a p p l i c a ti o n a s i n d u s tri a l o i l s,
triacylglycerolswith longer chain fatty acids Production of unsaturated fatty acids
(> Cre) are preferred.Thus, erucic acid (22C)
Ol ei c aci d (C ,u:1)ri ch oi l s are usefulas food,
containingoils are usefulin industries.However, feed and in some industries. Therehas been some
e r h u ma nc o n s u mp ti on. successin the transferof antisense
e r uc icac id is un s u i ta b l fo gene encoding
By conventionalplantbreedingtwo distinctcrops the enzyme desaturase in oilseedrapeand soybean
of oilseedrape have been developed- high-erucic plants. These transgenicplants were found to
acid rape (HEAR)and low-erucic acid rape (LEAR). produceoils with very highproportionof oleicacid.
Biotechnology
[40]
626 B IOTE CHNO LO CY
(A)
, CoA reducrase
pnaal
+I
: 3-Hydroxy
I butyrylCoA
I PHuryntnu""
(phaQ
.f
J
Polyhydroxy- Flavonoids
butyrate
(B)
Plastid
Cytoplasm
AcetylCoA---- AcetylCoA
./
| ,-*"ro,n,o,u." ./
(PhaA) / \
|
AcetoacetylCoA MalonylCoA
AcetoacetylCoA
.
I RcetoacetylCoA
I reductase
@haB)
J
3-Hydroxy
butyrylCoA +
j PHnryntn"." lsoprenoids rlavoloios
j (Phac)
Polyhydroxybutyrate
Fig. 51.3 : Development of transgenic Arabidopsis for the biosynthesis of polyhydroxybutyrate (PHB)
(A) Cytoplasmic synthesis of PHB (B) Chloroplast (plastid) synthesis of PHB (Note : phaA, phaB and phaC
represent genes for the corresponding enzymes)
Many proteinshave been producedin leaves w i th di sul fi debondsand sui tabl ymodi fi edami no
( par t ic ular ly
u s i n g to b a c c o p l a n t) e .g . l y s o somal aci dsi s i mportantfor [hi s purpose.
enz y m es ,m a mma l i a na n ti b o d i e s h , u ma n s erum
The changesthat occurafter(or sometimes even
album in.
during)the protein is synthesized are collectively
In recentyears,seedshavebecomean attractive referredto as post-translational modificationse.g.
high pr ot einp ro d u c ti o n(e .g .h i ru d i n )v e h i c l esdue phosphoryl ati on,hydroxyl ati on, carboxyl ati on,
to good storagecapacity and stability.lt is no glycosylation, proteolyticprocessing etc. Variations
surprisethat someof the seedproteinsretaintheir in post-translational changesare often limitingthe
biologic alac t i v i tyfo r s e v e rayl e a rs O . n e l i mi t ati on successfulprroductionand use of recombinanr
<lf seedsis that it is sometimesdifficultto recover proteinseither in plantsor even in other systems
the protein. (transgenianic mal sor cel l cul tures).
'fhe major limitations plants with regardto
Expression strategies of
post-translational modificationsof proteins are
There are two major approachesfor the
Iisted.
transgene expression in plantsto produceproteins-
stableintegration approachand useof plantviruses . Clycosylation (incorporationof carbohydrate
as transientvectors. moiety)is differentin plants.
1. Stableintegrationapproach: This strategyis . Plant glycosylationresultsin the productionof
most suitablefor the bulk productionof proteins complex glycanscontainingfucose or xylose,
par t ic ular lyin l e a v e s . l n th i s a p p ro a c h , the w hi ch do not occur i n humans.
transgeneis regulatedby a strong constitutive
promotersuchas 35Spromoter.Anotheralternative r Plantscannotperformspecializedcarboxylation
is to usetissue-specific promoter.By restricting the necessary for certainclottingfactors(ll, Vll, lX,
transgeneexpressionto a particulartissue (e.g. and X) and enzymes(proteinC).
seed),the yield of the protein can be substantially Due to abovelimitations,many a timesproteins
increased.In addition,the protein is stablefor a of transgenicplantsare inefficientor lessefficient
long time from weeksto years. i n thei r functi on, besi desexhi bi ti ng i mmuno-
A diagrammaticrepresentation of stablegene genecity.
expressionapproach for protein production is In recent years,cloning of genes responsible
depictedin Fig 51.4. for post-translationalmodificationsof proteins(e.g.
2. Transientexpressionby using plant viruses: glycation,carboxylation)have met with successto
Bioproduction strategiesinvolvingvirusesare useful producemore bi ol ogi cal l yacti veprotei ns.
for the protein productionat the desiredperiod
(discreteperiod).By genetic manipulations,it is Recovery strategies
possibleto insertthe desiredgene(for protein)into (purif ication strategiesf
the coat proteingeneof the virus genome.The so
A rational approach for a cost-effective
develooedinfectiousRNA virusesare introduced purificationstrategyis desirablefor the recoveryof
into the plantsfor the productionof proteins.
the proteins(downstream processing) producedin
Transient
expression systemby viruseshas been transgenic plants.The degreeof purityis dependent
used in tobaccoplantsby employing
successfully on the purposefor which the proteinis produced.
tobaccomosaicvirus (TMV).Tobaccoplantsat an For instance,industrialenzymesin general,do not
appropriateage were inoculatedwith genetically require high degree of purity. Sometimes,
modified TMV. Recombinantorotein could be purificationmay not be necessary at all. A good
extractedwithin 2-3 weeksof harvesting. exampleis the productionof the enzymephytasein
seeds.Phytaseacts on phytateand increases the
Post-translational processing avai l abi l i tyof phosphateto ani mal s. For thi s
The ultimate purposeof the use of plants as purpose,milledtransgenic seedscontainingplrytase
bioreactorsis to produce proteins with native can be directly added to the feed. This totally
conformation, good biological activity and avoids the purification/downstream processing
The specificfoldingof the protein
biocompatibility. costs.
630 B IOTECHNO LO G Y
lissue specific
promoter Terminator
Transgenicplant plant
Transgenic
For.a great majority of proteins,particularly can bi nd to l i gand,and thus the prot eincan be
pharmaceuticalproteins, purificationstrategyis i sol ated.Fol l ow i ng puri fi cati onof t he desir ed
absolutelyrequired.lt is thereforenecessaryto protein, the affinity tag can be removed.Some
develop efficient methods for downstream workers have been successfulin purifying the
processing.Some of the recoverystrategiesare enzyme glucocerebrosidase by this approach.
brieflydescribed. Purification through compartmentalization: lt
Affinity tag-based purification : The use of is possibleto directthe accumufationof proteinsin
affinity tags for the purificationof recombinant a specificsub-cellularorganelle.This is achieved
proteinshas been describedelsewhere(Chapter by usingsignalpeptidesor fusion peptides.Once
worksin plantsystems
1l), and a similarstrategy as the protein is localized,the technique of sub-
well. The techniqueinvolvesthe fusionof desired cellularfractionationcan be used for preliminary
proteinwith anotherproteinor peptide(tag),which isolationof proteinfollowed by its purification.
: TRANSGENIC
PLANTSAS BIOREACTORS
ttlr
Oleosinpromoter Oleosin Desiredprotein Terminator
Structure of a transgene
I
I Genetransfer
+
TransgenicPlant
I
Plantcrushed
I
+
Centrifuged
I Desired
YI
oleosin Protein
n
-F-E R n nfnTnffitriacylglycerol
{ (I (] {I
{\ i | \ l ] i I ll(|| i
\ \\--- lr -rl ,/
( Phospholipid
'monolayer
btdy-
I transterred
+
-;il
I Treatedwith
Protease
J
)
An oil bodY
Chapter51 : TRANSCENIC
PLANTSAS BIOREACTORS 633
B-Glucuronidase ln diagnostic
kits T h e t w o e n z y m e s n a m e l y c e l l u l a s ea n d x y l a n a s e
Ligninperoxidase In papermanufacture are used in several industries- bioethanol, textile,
paper and pulp. In all these processes,they are
Phytase lmproved phosphate
basically involved in the degradation of plant
(byphytate
utilization
m a t e r i a l s ( p r e d o m i n a n t l yc e l l u l o s e ) .
breakdown)
By a novel approach, it is possible to produce
Trypsin Pharmaceutical
cellulaseand xylanasein the plants for which the
Xylanase Biomassprocessing,paper target itself
is the digestion of plant cell. The risk of
andtextileindustries autodigestionof the plant cells (by these enzymes)
is overcome by genetic manipulation to produce
thermostable enzymes. Thus, cellulase and
A selectedlistof industrialenzymesproducedin xylanase, produced
by transgenic plants, are
plantsand their importantapplications
transgenic is inactive at the temperatures
at which plants
given in Table 5I .2. Some of the important normally grow.
The activity of these enzymes is
enzymesare described. restored on heating the plant extracts. And these
extracts are very successfully used in many
Avidin and B.glucuronidase i n du s t r i e s .
The first proteins/enzymes that were produced While the recovery of enzymes from transgenic
in t r ans ge n i cp l a n ts (ma i z e ) a re a v i d i n and plants is often required, there are some instances
p- gluc ur on i d a sTeh. e y a re u s e di n d i a g n o s ti cki ts. where the crude extracts
can be directly used. The
industrial applications of cellulase, xylanase and
Trypsin phytase(describedabove) are some good examples.
Trypsinis an imptrrtantproteolyticenzyme and
its production by conventional recombinant PRODUCTION OF LYSOSOMAL
approachesis rather difficult. Trypsin is now ENZYMES IN PLANTS
produced in plants. lt has a wide range of Lysosomes are the cellular organelles
the productionof insulin,vaccines,
applications-for responsiblefor the degradationof macromolecules.
wound care etc. The degradative enzymes present in lysosomes
include proteases,nucleases,lipases,glycosidases,
Phytase phosphatases, phospolipases and sulfatases
Deficiency of a specific lysosomal enzyme results
Phytaseis a hydrolyticenzymethatcatalyses
the
in the accumulation of undegraded substrate
hydrolysisof phytate(inositolhexaphosphate) to
causing clinical manifestations.Some examples of
inositoland inorganicphosphate.
lysosomaldisordersand the correspondingenzyme
Phytateis presentin high quantitiesin many deficiencies are listed below.
plant seedsused as a feed to pigs and poultry
r Tay-Sachsdisease- c-hexosaminidase.
( m onogas t ri ca n i ma l s ).T h e s e a n i m a l s d o not
possessthe enzyme phytase;hence they cannot . Caucher's disease- glucocerebrosidase.
derive the nutrient phosphatefrom phytate.The . H u r l e r s y n d r o m e- i d u r o n i d a s e .
634 B IOTECHNO LO CY
Heavy
Cross3
IN
Single-chainFv (scFv)
scFv(T84.66) Viralantigens Cereals cancer
Carcinoembryonic
scFv(38C13) Viralantigens Tobacco Lymphoma
world, and eaten raw. Another advantage is that The future of edible vaccines : Despite several
child ren (to whom v ac c ines ar e m os t needed) l i k e limitations, biotechnologistssee a great hope for
e atin g b an an a. the new wave of transgenic veggie vaccines. But it
may take severalyears before the banana or tomato
The procedures adopted for the production of
replaces the needle as a vehicle for vaccrne
vaccines by plants, and the use of plants as edible
delivery!
su bu nit vaccin es ar e giv en in Chapt er 16.
The possibility of allergic reactions as they enter Chloroplasts are capable of folding the foreign
circulatio n. proteins, besides bringing out most of the post-
638 B IOTECHNO LO CY
639
640 B IOTECHNO LO CY
Soil
Nitrosomonassp
Nitrobactersp
Nitrogenase
+H 2
Nitrogenaseis a complex enzyme containing
two oxygen sensitivecomponents.ComponentI
has two o-protein subunits and two B-protein n
subunits,24 moleculesof iron, two rnoleculesof
molybdenum and an iron molybdenum cofactor NITROGENASE
COMPLEX
(FeMoCo).Component ll possesses t\,vo o-protein
subunits(differentfrom that of componentl) ano a
Fig. 52.2 : Nitrogen fixation in symbiotic bacteria.
lar genum berof i ro n m o l e c u l e s .
ComponentI of nitrogenase catalyses the actual
conversionof N, to ammoniawhile componentll
Hydrogenase
donateselectronsto component I (Fig. 52.2).
During the course of nitrogen fixation by
Leghemoglobin nitrogenase, an undesirablereactionalso occurs.
A proteincomparable of hemoglobinin animals That is reduction of H+ to H2 (hydrogengas).For
has been identified in the nodules of leguminous the production of hydrogen,ATP is utilized,rather
planfs.Leghemoglobin (tHb) containsiron and is red wasted. Consequentlythe efficiencyof nitrogen
in colour.lt is an oxygenbindingprotein.The heme fixation is drastically lowered. lt is possible
partof leghemoglobin is synthesizedby the bacterium theoreticallyto reduce the energy wastage by
while the protein(globin)portionis producedby the recyclingH, to form H+.
hostplant.Leghemoglobin is absolutelynecessaryfor ln f act, some strains of Bradyrhizobium
nitrogenfixation.The noduiesthat lack LHb are not japonicum in soybean plants were found to use
capableof fixingnitrogen. hydrogenas the energysource.Thesestrainswere
It is LHb that facilitatesthe appropriatetransfer found to possessan enzyme namely hydrogenase.
of oxygen(by formingoxylHb) to the bacteriafor Recyclingof the hydrogengas that is formed as
respiration to produceATP.And energyin the form a byproduct in nitrogen fixation is shown in
of ATP is absolutelyrequiredfor nitrogenfixation. Fig. 52.3.
Another imoortantfunction of LHb is that it It is advantageousfor nitrogenfixation if the
preventsthe damagingeffectsof direct exposureof symbiotic bacteria possess the enzyme
O, on nitrogenase. hydrogenase.However, the naturally occurring
strainsof Rhizobium and Bradhyrhizobiumdo not
ln Fig.52.2,the fixationof nitrogenby symbiotic
normallypossess the gene encodinghydrogenase.
bacteriais depicted.As the oxylHb supplies02,
bacterialrespirationoccurs.The ATP generatedis
usedfor fixing nitrogento produceammonia.The GE N E TIC MA N IP U LA TION S FOR
complexreactionis summarizedbelow. NITROGEN FIXATION
N, + B H+ + Be - + 1 6 A T P The nitrogen-fixingbacteria that are closely
associatedwith the world'sfood supplyare among
2NH-
JZ
+ H " t + 1 6 AD P + 1 6 P i the favouredorganismsfor geneticmanipulations.
a':technology [41]
642 B IOTECHNO LO CY
It is absolutely necessaryto identify, isolate and r The nitrogenaes(nifl genes are located as a single
characterize the nftrogen-fixing genes, nifgenes to cluster, occupying approximately 24kb of the
undertake any kind of genetic manipulations. bacterial genome.
Genetic complementation is the common . There are seven distinct operons that encode 20
technique used for the isolation nif genes. The different oroteins.
l ra p t er 52 : CRO W T H -P R OMOT INBA l N P LA N TS
C C T E R IA 643
K. pneumonia
(Nif*cells)
K. pneumonia
(Nif- cells)
Conjugated,grown on
mediumlackingnitrogen
K. pneumonia (Nit-)
transformants
I
+
,iffi"
isolated
Fig. 52,4 : An outline of the method for the isolation of nit genes from K. pneumonia
by genetic complementation.
o :, the nif Benes transcribe and translate in a instance, insertion of more nifA genes into
. el I-coordinatedfashion. Rhizobium meliloti resulted in an increased
. -'e nif genes are under the regulatorycontrol of b i o m a s s p r o d u c t i o n i n a l f a l f a p l a n t s .T h i s i s d u e t o
-.., qenes namely nifA and nifL. the fact that nitrogen fixation in these plants is
-r m u c h i n c r e a s e d .B u t t h e m a j o r l i m i t a t i o n o f t h i s
: :1ough the nifgenes have been characterized
approach is that the geneticallyengineeredbacteria
have a diminished growth rate. As a result, these
: -" iru tion of this or ganis mf or biologic al nit r ogen newly developed bacteria loss their efficiency as
" ,- ,,r is almost insignificant. However, the nif plant-growth promoting agents.
-
-: : j. of K. pneumonia have been used as
' --: za tion p rob es t o ident if y nif genes f r om t he Despite continuous efforts by several groups of
- \ . : clon e ba nks of diaz ot r ophic or ganis m s workers, no significant success has been reported
in the transfer of nif genes into plants. The major
-,- --;larly Rh izo bium s p) . lt is now k nown t hat
limitationsare listed.
, -- .: all the nitrogen-fixing bacteria possess
. - a: type o f n ifge ne c lus t er s . . Transferof 24 kb nif gene cluster has not been
effective since the normal cellular concentration
Hanipulation of nif genes of oxygen would inactivate nitrogenaseenzyme.
Sonre workers have been successful In A n y r e d u c t i o n i n O , r e s u l t si n c e l l d e a t h .
-'-rciucing extra copies of nitrogenaseregulatory . Transferof one or two genes of nlf gene cluster is
For usetess.
-=-es namely nifA and nifL into diazotrophs.
644 B IOTECHNO LO CY
. Thereare no plantpromotersthat can respondto genes (about 20 nodA- nodX have been
nifA regulatoryprotein. Consequently,it is not identified in diazotrophs. The nod genes are
possibleto turn on the nlf genesin transgenic broadly divided into three groups.
plants. . Common genes
. The plant cells cannot process nif gene r Host-specificgenes
which are multigenicin nature.
transcripts,
o Regulatorygenes.
For the variousreasonsgiven above,it has not
been so far possible to introduce functional The functions of each one of the nod genes in
nitrogenfixationcapabilityinto plants. nodulation have not been clearly identified. Further,
many more new nod genes are being discovered
every year.
GENETIC ENGINEER'NG OF
HYDBOGENASE GENE
Manipulation of nod genes?
The role of the enzyme hydrogenasein
The process of nodulation is complex thlough
promoting nitrogen fixation has already been
the participation of a large number of nod genes,
described(Seep. 641).Hydrogenase is synthesized
besides various other factors-concentration of
by hup (fiydrogen uptake)genes, which are not
nutrients,soil temperature,light, CO, concentration
p re s e nitn th e n a tu ra l l yo ccurri ngR hi zobi alstrai ns.
etc.
Considerable variationshave been identifiedin
Despite attempts by severalworkers, no success
hydrogenases from differentorganisms.There are
has been reported to enhance the ability oi
different types of hydrogenases, which usually
Rhizobium sp for nodulation through genetic
contain subunits. Different eenes code these
manioulations.
subun its.
m ic r obial c el l . T h i s i s re o u i re d s i n c e i ron i s
s i n c e 1 9 8 9 . T h i s t r a n s g e n i co r g a n i s m p r o d u c e st h e
essential, and cannotdirectlyenterthe bacteriadue
antibiotic agrocin 84 that is toxic to crown gall
to a very low solubility. disease-causingAgrobacterium tumefaciens. By this
r
biocontrol approach, the crop yield of almond trees
The growth-promoting bacteria, through
and peach trees can be improved by preventing
can take up largequantitiesof iron
siderophores
crown gall disease.
from the soil, and make it unavailablefor the
growth and existenceof fungal pathogens.
This is
ENZYMES
possiblesincethe siderophoresproducedby fungi
have a very low affinity when compared to Certain plant growth-promoting bacteria are
sideroohoresof bacteria. capable of synthesizing some enzymes that can
T her eis no h a rm to th e p l a n tss i n c eth e y can degrade fungal cell walls and lyse them. These
grow at a much lower concentration of iron in the enzymes include chitinase, p-l,3-glucanase,
s oil. protease and lipase.
It is now clearly established that siderophores The genes responsiblefor encoding the enzyme
can preventthe proliferationof phytopathogens. lt n a m e l y c h i t i n a s e a n d B - g l u c a n a s e h a v e b e e n
is thereforelogicalto think of siderophoregenesfor isolated and characterized. Chitinase gene has
more effectivebiocontrolof plant pathogens. been isolated from Serralia mercescens ano
introduced into Rhizobium meliloti and
Pseudobactinis a siderophoresynthesizedby Trichoderma harzianum. The genetically
plant growth-promotingbacteria Pseudomonas engineered organisms displayed an increased
putida. By genetic complementationand other antifungal activity. By this biocontrol approach, the
techniquesof molecular biology, at least five phytopathogen Rhizoctonia solani has been
separategene clustersinvolved in pseudobactin effectively control led.
oroductionhave been identified.
Ceneticmanipulationfor improvedsynthesis of
siderophore is not an easyjob, sinceit is a complex
pr oc es sinv olvi n ga l a rg en u m b e ro f g e n e s .S ome
success, however,hasbeenreportedin cloningthe It i s esti mated thatmorethan 100 mi l l i ontonsof
genesfor iron siderophorereceptorsfrom certain fixed nitrogen are needed for global food
plant growth-promoting bacteria,and introducing production.The useof chemical/synthetic fertilizers
them into other bacteria. is the common practiceto increasecrop yields.
Besidesthe cost factor, the use of fertilizersis
A NT I B I O T I C S associ ated w i th envi ronmental
ool l uti on.
Certainantibioticsproducedby plant growth- Scientists are on a constantlook for alternate,
promotingbacteria,can preventthe growth and cheap and envi ronmental -fri endlsources y of
proliferationof plant pathogens.Some of the nitrogenand other nutrientsfor plants.The term
antibiotics synthesizedby pseudomonadsare blofertilizersis used to refer to the nutrient inputs
agrocin 84, agrocin434, herbicolin,phenazine, of biological origin to support plant growth. This
oom y c in,py r ro l i n i tri n . can be achieved by the addition of microbial
inoculantsas a sourceof biofertilizers.
Genetic manipulations for antibiotics
B i oferti l i zersbroadl y i ncl udesthe fol l ow i ng
It is possibleto enhancethe growth-promoting tategories.
activityof the bacteriaby insertinggenesencoding o Symbioticnitrogenfixers
the synthesis of antibiotics.
o Asymbioticnitrogenfixers
ln f act, a geneticallyengineeredbiocontrol
. Phosphate solubilisingbacteria
bacteriumof Agrobacteriumradiobacferhas been
dev eloped,an d i s b e i n g ma rk e d c o m m e r ci al l y o Organi cferti l i zers.
646 B IOTECHNO LO CY
1. M or phologic al m ar k e r s
2. Bioc hem ic al m ar k er s
648
Chaot er53 : M OL E C U L AR
MA R K ER -AID ED
P LA N TB R E E D IN C 649
MARKERS BASED ON
DNA HYBRIDIZATION
The DNA piece can be cloned, and allowed to
hybridize with the genornic DNA which can be
detected Marker-based DNA hybridization is
widely used. The rnajor limitation of this approach
is that it requires large quantities of DNA and the
use of radioactivity (labeled probes).
Microsatellites MOLE C U LA R B R E E D IN G
Microsatellites are the tandemly repeated With rapid progressin molecularbiology and
multicopies of mono-, di-, tri- and tetra nucleotide geneticengineering, there is now a possibilityof
motifs.In someinstances, the flankingsequenceof
improvingthe crop plantswith respectto yield and
the repeatsequences may be unique.Primerscan quality.The term molecularbreedingis frequently
be designedfor such flankingsequences to detect used to represent the breeding methods that are
the sequencetagged microsatellites (STMS).This
coupled with genetic engineering techniques.
can be done by PCR.
lmprovedagricultureto meetthe food demands
Sequence characterized amplified of the world is a high priority area. For several
regions (SCARsl years,the conventionalplantbreedingprogrammes
(althoughtime consuming) havecertainlyhelpedto
SCARsare the modifiedforms of STSmarkers. improve grain yield and cereai production.The
They are developedby PCRprimerthat are made
develoomentof dwarf and semi-dwarfvarietiesof
for the endsof RAPDfragment.The STS-converted
riceand wheathavebeenresoonsible for the 'Green
RAPDmarkersare sometimes referredto as SCARs. Revolution',which has helped to feed millions of
SCARsare usefulfor the rapid developmentof STS poverty-strickenpeoplearoundthe world.
marKers.
Many developmenton the agriculturefront are
expected in the coming years as a result of
mol ecul arbreedi ng.
G57
3 ttechnology [42]
658 B IOTECHNO LO CY
pollution
1. Airlatmosphere moni tori ng of pol l uti on w hi ch i s a gl obal
2. W at eroollu ti o n phenomenon.W orl d H eal th Organi zati onand
U ni ted N ati on E nvi ronmentalP rosrammeare
3. Land/ s oilpo l l u ti o n
activelyinvolved.
4. Nois eoollu ti o n
5. T her m alpo l l u ti o n BIOTECHNOLOGICAL METHODS FOR
6. Radioac t ive
o o l l u ti o n . ME A S U R E ME N T OF P OLLU TION
T he det ailsof a i r p o l l u ti o na n d w a te rp o l l u ti o n In recent years, envi ronmental pol l uti on
are describedelsewhere(Chapters 55 and 56). detecti on and moni tori ng i s bei ng done by
approaches involvingbiosystems. Forthis,purpose,
NATURE OF POLLUTANTS several groups of pl ants, ani mal s and
The pollutantsthat occur in the environment microorganismsare utilized. The environmental
protection agencis (EPAs)consider biomonitoring
, i o l o g i c aal n d p h y s i c a il n th e i r
ma y be c hem ic al b
nature. of pollution as a useful device to monitor
envi ronmentalpol l uti on from the poi nt of
Chemicalpollutants: Caseouspollutants(sulfur diagnostic,preventiveand remedialmeasures.
dioxide,nitrogendioxide),toxic metals,pesticides,
herbicides, hydrocarbons, toxins,acidicsubstances, CBITER'A FOR B'OMONITOB'ilG
carctnoSens. OF POLLUTION
Biological pollutants : Pathogenicorganisms,
The parametersor the criteria chosen for
p roduc t sof biolo g i c aol ri g i n .
of pol l uti onare very cruci al .They
bi omoni tori ng
Physical pollutants : Heat (thermal),sound, should be reliable,reproducibleand cost-effective.
odours,radiationand radioactivesubstances. Three types of criteria are mostly adopted for
bi omoni tori ng of pol l uti on-vi sual rati ng,
genotoxicityratingand metabolicrating.
Visual rating
ln the visual rating, the growth rate and
Environmental pollutionposesa big threatto the productivity are considered.When m icroorganisms
h e alt hyex is t encoef h u m a n k i n dT. h e C o v e rn me nts are used in the test assay,the growth can be
worldover pay seriousattentionto continuously measuredby turbidometricanalysis.In case of
monit orand m ini m i z ep o l l u ti o n .T h e p u b l i c a n d higherplants,growth rate of differentparts,visual
non-governmental organizations(NCOs) are also damageto leaves,seed viability and germination
activelyinvolvedin this venture.Broadly,thereare frequencyare taken into account.
four levels of pollution monitoring agences or
A s regardsani mal s(fi shesare commonl yused),
environmental protection agencies(EPAs\.
the conceptof LDro is usedi.e. the doseat which
Primary level : This is at the districtor block 50% of the test organismis affected.
level.The non-governmental organizations and the
Sometimes,the presenceor absence of a
rural development agencies are involved in
particularspeciesof an organismservesas an
p ollut ionm onit or i n g . pol l uti on.
i ndi catorfor the envi ronmental
Secondary level : Existing at the state level, the
monitoringis done by the respective statepollution
Genotoxicity rating
control boards.
Genotoxicity tests measure the extent of
Tertiarylevel : This is at the national/country damage causedto an organism by environmental
level. Each country has its own environmental pollution at the cellular and sub-cellular levels.
protectionagencyto monitorpollution. The genotoxic lesions may be detectedon the
Quaternary level : International/inter-cel l ul arorganel l es(membranes commonl yused),
Covernmentalbodies are closely associated with genomes, immune systems, biomolecules, etc.
660 BIOTECHNOLOGY
Cytotoxic tests such as measurementof The mostcommonl yusedpl antsan d anim alsin
chromosomaldamage(includingbreakage),sister the bioassays
are brieflydescribed.
c h ro ma ti d e x c h a n g e (SC E ) and mi cronucl ei
counting are also useful for pollution detection. Plant test systems in bioassays
The cell viability can be measuredby detecting
Certain algae, bacteria, lichens, mosses and
in vitro lysosomalviability.In recentyeas, DNA
vascular macrophytesare commonly used in
probesare in use for the identificationof disease-
bioassavs.
causingorganismsin water.
Algal bioassays: Amongthe plantsystems, algal
Metabolic rating bioassays are the most commonlyused.Algaeare
fhe biochemical changeswith environmental consi deredto be rel i abl ei ndi cator sof pollut ion
pollution can be measured (qualitativelyand due to their high sensitivityand easy availability,
quantitatively) in selectedorganisms. The cr it er ia
In fact,certain besi dessi mpl e cul turi ngtechni ques.
metabolicparameterscan be usedas biomarkersto adopted for algal bioassaysare the growth rate,
assessfhe pollution sfress.The biomarkersused in biomass accumulation and ohotosvnthetic
metabolic rating include chlorophyll, proteins, efficiency.
n u c l e i c a c i d s (D N A a n d R N A ) and chansesi n The algae used in the test assays include
enzymeactivities. Chlorella, Microcystis, Spirulina, Navicula,
The biotechnoiogicalmethods adopted for Scenedesmus, Anabaena,Ulva, Codium,Fucusand
pollutionmeasurement are brieflydescribedin the Laminaria.In water, organic pollution can be
followingorder. detectedby usingthe blue greenalgae,Microcystis,
.l w hi l e metal pol l uti on can be m easur edby
. Generalbioassays
Navicula.
2 . C e l l b i o l o g i c aal s s a y s
Bacterialbioassays : Theseare commonlyused
3 . Mo l e c u l a rb i o l o g i c aassays
l for the detectionof fecalpollutionin potablewater,
4 . Bi o s e n s o rs . the most widely employedtest being coliform test.
Ames test that detects mutagenicpollutants is
E'OISSATS'N ENV'NONME}JTAL carried out by the bacteriumSalmonella.
MON'TON'NG Bacterialbioluminescenceis a recenttechnique
ln the earlyyears,the conventionalphysicaland usedfor the measurementof gaseous pollutantsand
chemical methodswere used for the detectionof othercompoundse.g.sulfurdioxide,formaldehyde,
environmentalpollution. Bioassaysare preferred ethylacetate.Photobacteriumphosphoreumis the
these days, since the biological responsesthat organismof choice for bacterialbioluminescence.
reflect the damagesto the living organismsare
Lichensin bioassays : Lichensare widely used
very crucial for the actual assessmentof
for the detection of atmosphericgas pollution,
p o l l u ti o n .
particularlyin cities.Lichensare very sensitivefor
The organismsemployed in the bioassaysfor the measurement of sulfurdioxide.
pollution detection are expectedto satisfy the
Mosses in bioassays: Environmentalmetal
followingcriteria.
pollution can be detectedby using certainforest
o lt shouldreadilytake up the pollutant(absorption and aquaticmossese.g. Stereophyllum,Sphagnum,
or adsorption). Brvnus.
o The organismshouldbe sensitive to the pollutant. Vascular macrophytes in bioassays : Water
o lt should possessmeasurablefeaturesto detect hyacinth (Eichormia crassipes)and duck weed
p o l l u ti o n . (Lemnaminor) are in use to detect aquatic metal
pollution.In fact, certainbiochemicalparameters
. The organismshouldhavewide occurrence,and
of macrophytesare usedto serveas biomarkersof
availableround the year.
pollutione.9., peroxidase activityincreases
due to
. The bioassay shouldbe simple,reproducible
and metal pollution; inhibition of nitrate reductase
cost-effective. activity by mercury. The other commonly used
Chaot er54 : EN VIR O N M EN T AL -C E N E R A L
POL I-U T I ON 661
is the useof human lymphocytecultureto monitor Recently, the yeast cells (Saccharomyces
the personsexposedto toxic pollutants. cerevisae)are also used for the detection of
chemicalcarcinogens.
Gytogenetic bioassays
MOLECULAR B'OLOGY
The geneticdamagesof the cells,as reflectedby
'N
ENV'BONMENTAL MON'TOB|NG
changesin the chromosomes, can be effectively
used in biomonitoring of pollution. For this The use of molecular probes and immunoassays
purpose,animals(e.9.insectDrosophila) and plants i n moni tori ng pol l uti o nis gaining
of envi ronmental
(e.8. Arabidiopsrs)with shorter life cycles are importancein recentyears.Molecularbiological
preferred.
Other plantssuchas pea,maizeand soy bioassays are particularlyusefulfor the detectionof
bean are also used in cytogeneticbioassays. bacteria,virusesand other pathogenicorganisms
that causediseases.
Chromosomal damage : The pollutants may
cause several types of chromosomaldamages-
DNA probes
fragmentation, bridgeformation,disruptionin cell
division. The chromosomal alterations can be DNA probes and polymerase chain reaction
effectively usedfor pollution detection lt has been (PCR) can be effectively used for water quality
clearlyestablished thatthe severitvof chromosomal monitoring, particularly potable water. However,
damagedependson the chemical nature of the thesetechniquesare expensiveand not practicable
polIutant. at all olaces.For more detailson DNA probesand
PCR,ReferChapters9 and 8 respectively.
Micronucleus test : Severe damage to
chromosomesby pollutantsmay result in large lmmunoassays
scalefragmentationof chromosomes, followed by
micronucleiformation.The degreeof micronuclei lmmunologicaltechniquesare useful for the
developmentis directly relatedto the severityofdetectionof pollutants(pesticides,
herbicides)and
the damage.Micronucleustest (MNT) is used for identification of pathogens that exhibit
screeningof mutagenic compounds. immunological properties.
lmmunoassays are in use
for the measurementof several pesticidese.g.
Sister-chromatid exchange : The damages aldrin, triazines DDI glyphosate. Metabolic
caused by pollutantsresultsin misexchangeof productsof certainbacteriacan also be detectedby
chromosomalsegments(chromatids)during cell immunoassays. For instance/assaysystemshave
division.The sisterchromatedexchange(SCE)can been developed for the detection of toxins of
be detectedby usinga fluorescentdye technique. cholera and Salmonella.
ln recent years, use of monoclonal antibodies
Ames test in bioassays
(MAbs) in the detection and biomonitoringof
Ames test can be used for the detection of environmentalpollution is gaining importance.In
chemicalmutagensand their carcinogenecity.This fact, assaytechniquesare availablefor detectionof
is very widely used bioassayfor screeningof pesticideand herbicidecontaminationin water.
virious pollutants,drugs,cosmotics,food additives
and metals. Bioluminescent bioassays using
Ames test employsthe use of a specialmutant Lux reporte; genes
strainof bacteriumnamelySalmonellatyphimurium Certaingenes,referredto as Lux reportergenes,
(His-).This organismcannot synthesizehistidine, on the plasmids produce assayable signals.
hencethe sameshouldbe suppliedin the medium Wheneverthesegenesare expressedin luminescent
for its groMh. Addition of chemicalcarcinogens bacteria like Photobacterium and Vibrio. Some
causesmutations(reversemutation)restoringthe bacterialstrainshavebeendevelopedthroughgene
ability of this bacteriumto synthesizehistidine cloning (employingLux reportergenes)for the
(His+).By detectingthe strainof Salmonella(His+) detectionof pollutantsand their degradation.For
in the colonyof agarplates,the chemicalmutagens instance,geneticallyaltered Pseudomonas can be
can be identified.The Ames assaycan detect about used for detectingnaphthalene,xylene, toluene
90o/oof the chemical carcinogens. and salicvlate.
54 : EN VIR O N M EN T AL
ChA O t CT -C E N E R A L
POL L U TION 663
B'OSENSORs'IV ENVIRONMENTAL
MONITON'NG Tlru 54.1 A selected llst of pollutants
measured by enploylng biosensors
A biosensor is an analytical device containing
an immobilized biological material (enzyme, Pollutant measured Biasensor-
o rga ne lle, c ell) whic h c an s pec if ic ally i n t e r a c t biological componenl
rvith an analyte (a compound whose concentration BOD Trichosparoncutaneun
is to be det er m ined) and pr oduc e p h y s i c a l ,
Soz Thiobacillus
sp
che mica l o r elec t r ic als ignalst hat c an be m e a s u r e d .
Biosen so rsar e highly s pec if ic and ac c ur at e i n t h e r r
cH+ Methanomonas flagellae
f u nctron . coz Pseudononas sp
Nitrate Azobactervinelandi
The details on biosensors-principles of
NH,andNO, Mixednitrifying
bacteria
working, types, various applications are described
Nervegas Acetylcholine
esterase
elsewhere (Refer Chapter 21) Some of the
imoo rtan t bios ens or s us ed in env ir on m e n t a l Ethanol NADHanddehydrogenase
po llutio n m onit or ing ar e br ief ly des c r ibed . Phenol Phenoloxidase
Parathion to parathion
Antibody
BOD biosensor Herbicide-induced
eleclrontransport
Biolo gic al ox y gen dem and ( BO D5) is a w i d e l y
chaininhibition Synechococcus
used test for the detection of organic pollution. This
test reo uir es f iv e dav s of inc ubat ion. A B O D
biosensor using the yeasl Trichosporon cutaneum Biosensorsfor the detection of polychlorinated
rvith oxyg en pr obe t ak es jus t 15 m inu t e s f o r b i p h e n y i s( P C B s )a n d c h l o r i n a t e dh y d r o c a r b o n sa n d
d ete ctin g o r ganic pollut ion. certain other organic compounds have been
developed.
Gas biosensors
Phenol oxidase enzyme (obtained from potatoes
Microbial biosensorsfor the detection of gases and mushrooms)containing biosensor is used for
su ch as su lf ur diox ide ( SO r ) , m et hane and c a r b o n the detection of ohenol.
dioxide have been developed. Thiobacillus-based A graphite electrode with Cynobacterium and
bio se nsor c an det ec t t he pollut ant SO r , w h i l e Synechococcushas been developed to measurethe
meth an e (CHr ) c an be det ec t ed by im m o b i l i z e o degree of electron transport inhibition during
Methalomonas. For carbon dioxide monitoring, a photosynthesis due to certain pollutants e.g.
particular strain of Pseudomonasis used. herbicides.
A selected list of environmental pollutants
lmmunoassay biosensors measured by employing biosensors is given in
lmmunoelectrodes as biosensorsare useful for Table 54.1.
the detection of low concentrations of pollutants.
Pesticide specific antibodies can detect the
presence of low concentrations of triazines,
ma lathion and c ar bam at es , by em p l o y i n g
rmmu no ass ay s .
The different approachesfor the managementof
Other biosensors environmental oollution are described in the
Biosensors employing acetylcholine esterase respectivechapters for air pollution, refer Chapter
55, and for water pollution Chapter 56.
lobtained from bovine RBC) can be used for the
d ete ctio n of or ganophos phor us c om pou n d s I n The general concepts of the biotechnological
rvater. In fact, portable pesticide monitors are approaches for the abatement of pollution n,ith
comme rcia lly av ailable in s om e de v e l o p e d special reference to the following aspects are
cou ntries. described in this chapter.
664 BIOTECHNOLOCY
EUTROPHICATION AND
PHOSPHORUS POLLUTION
Highenergy \
comPounos Polyphosphates
---J-*;"",
carbon
PolYPhosPhates
|
r"-Enerov<--4
JJ
"| *r-l
"otpornOt
.Highenergy P; Metabolism P;
carDoncompounos \
P;
Anaerobic condition Aerobic condition
The aq ua t ic plant s and m ic r oor ganis m s m a y The immobilized cells are useful for the waste
-3Ke up the metal pollutants through absorption water treatment, and for the recovery of metals
668 B IOTECHNO LO CY
669
670 B IOTECHNO LO CY
Biotechnology
[43]
B IOTE CHNO LO CY
674
Water outlet
(D) SpraY tower
To high.voltage
caDle Shaking
arrangement
--) Clean
alr
-) Cleanair
Fabricfilters
U !U ! U
(E) ElectrostaticPreciPitator
Electrostatic precipitators
TmLr 55.6 A selected list of adsorbents and
Electrostaticprecipitators(ESPs)are very efficient their major applications
and versatile. They work on the basis of
electrostatic attraction. An ESP consists of a thick Adsorbent Major application(s)
cylinder fitted with an inlet at bottom and an outlet
or zeolates
Silicate of SOr,NO, and
Control
a t the top (Fig .55. 1D. An elec t r ode( m or e t han o n e
(asmolecular
sieves) mercury
electrodeare commonly used)fitted at the centre of
the cylinder is connectedto a high voltage cable. As
Activated
alumina air,gasesandliquids
Drying
the dust laden air passesthrough ESP,larger sized Activated
carbon Removing purification
odours,
particles settie down due to gravity. The smaller of gases
charged particles are attached to the oppositively Silicagel Dryingandpurifying
gases
chargedelectrodeswhich graduallyfall down to the Bauxite Drying of gasesandliquids,
bottom. The dust free air comes our. treatment of petroleum
Electrosiatic precipitators are said to be dry fractions
precipitators if the dust particles can be removed Fuller's
earth Refining
of oilsandfats
by scrapping. In case of wet electrostatic
precipitators, the particles are removed by using
water or any otlier fluid. Wet precipitatorsare more lf the adsorption is purely a physical
phenomenon (held by van der Waal's forces), it is
efficient, however, dry precipitatorsare preferably
referred to as physical adsorption. This process is
used for oractical reasons.
rapid and easily reversible. ln chemical adsorption
(chemisorption), the pollutant gas molecules form
Fabric filters
c h e m i c a l b o n d s w i t h t h e a d s o r b e n t .C h e m i s o r o t i o n
Fabric filters (Fig. 55.1D are the most efficient is very slow, usually requires the supply of energy
a nd can sep ara t epar t ic leswit h s iz es les s t han 0 . 5 and is irreversible. Pressure and temDerature
prm in diameter. As the air or gas is passed over significantlyinfluence chemical adsorption.
fabric like mats of wool, celluloss etc., the dust is
trapped while the gas passes out. Collection of In the Table 55.6, a selected list of tne
dust on the fabric filter resultsin the formation of a c o m m o r f l y u s e d a d s o r b e n t sa l o n g w i t h t h e i r m a j o r
dust layer (commonly referred to as filter cake). applications is given. The equipment used for
This in turn servesas a more efficient dust collector adsorption, referred to as adsorbers, may be fixed,
and helps to capture even fine dust particles. The moving or fluidized beds.
fabric filters have to be frequently cleaned Several
Multiple fixed bed adsorber : In this, adsorbent
models of fabric filters have been developed for
is arranged in the form of beds or layersas depicted
comme rcia l ap plic at ions .
in Fig. 55.2A. As the adsorption occurs, the
adsorbent gets saturated with the adsorbate. For
CONTROL DEVICES FOR GASEOUS reuse, the gas collected on the adsorbent must be
POLLUTANTS removed. lf this is not possible,the adsorbent must
The prin cip le gas pollut ant sar e SO r , N02, N2 O , be reolaced.
CO, hydrocarbons,and organic and inorganic acid
gases. The control devices used for gaseous Absorption
pollutants are based on the principles of adsorption,
absarption, condensation and combustion. fhe The pollutant gas (absorbate),when brought in
irrportant aspectsof selecteddevicesfor controlling contact with a solvent or liquid (absorbent),gets
gaseouspollutants are briefly described. adsorbed.The processof absorption may occur due
to physical or chemical phenomena.
Adsorption Absorption process is used to control the
When a stream of gas is passed through a pollutants SOr, NOz,HrS and hydrocarbons.
porous solid material (absorbent),it can attract and Ammonia is used as an absorbentfor controlling
hold the gas (adsorbate)molecules. soz'
676 B IOTE CHNO LO CY
Cleanair
-----J Steam
(duringcleaning)
Adsorbed
beds
--)Clean
all
Heated-----J
system
(forcleaning)
(A) Multiple fixed bed adsorber
(B) Perforatedtray tower
:{- Liquid
inlet
bp,"y
Packing
material
Air with
pollutants
J IUE
Liouidoutlet Coolingliquid Condensate
outlet outlet
(C) Packed tower
(D) Surface condenser
Cleanair
{- Cold water
inlet
{- Air with
----* Clean
all
Waterand vapour
condensate Preheater
(E) Contact condenser (F) Thermal incinerator
55 : A I R P O L L U T IO NA N D C ON T R OL
ChA O t Cr 677
678 B IOTECHNO LO CY
Water is required for development and progress A minimum level of dissolved oxygen at a
.' people, and therefore a nation. In fact concentration around a mg/cll is considered to be
^ ;iorically, the places of availability of water were necessary to support healthy aquatic life. If good
'-=
"rajor determinantsfor the settlementof people. aerobic conditions are not maintained, anaerobic
^ 5 is because besides regular use, water ts microorganisms take over and cause harmful
::::^iial to raise the crops It is an accepted fact effects And this is what happens in polluted 'waters,
-i. r co mmun it y wit h good wat er s upply has g o o d particularly from the industrial and municipar
:- '..f , progressand prosperity. wastes.
679
>tr
Determinationof dissolved oxygen is very The salient features of these pollutants and the
importantin water pollution.This is mostlyceirried sources of their contribution are briefly described.
out by measuring biochemical oxygen demand
for the oxidation ORGANIC POLLUTANTS
(BOD), utilizing r.'ricroorganisms
of organicmatter(Seep. 683). c o m p o u n d s a s s u c h a r e a b so l u te l y
Qrganic
esse'ntialfor the existence of life. However, their
entry into water causes pollution resulting in bad
o d o u r a n d u n p l e a s a n tt a s t e . I n a d d i t i o n , so m e o f
the organic compounds may be toxic or even
c a r c i n o g e n i ct o h u m a n s .
Sev er al k inds of nat ur a l , a n d m a n m a d e
ac t iv it ies ( dom es t ic , indus t r i a l , a g r i c u l t u r a l e t c . ) Sources of organic pollutants
c ont r ibut e t o wat er pollut ion . Wa t e r p o l l u t i o n i s
characterized by certain observable disturbances in There are several natural sources of organic
the normal properties and functions of fresh water. p o l l u t a n t s . T h e s e i n c l u d e t h e d e c a y o f l e a ve s,
These include offensiveodours, decreasein aquatic plants, and dead animals, besides the release of
life (e.g. fishes),bad taste and unchecked growth of o r g a n i c m a t e r i a l s f r o m a q u a t i c pl a n ts a n d
aouat ic weeds . mrcroorganisms.
Bacteria
Escherichia
coli Gastroenteritis Dianhea
(enteropathogenicl
Salmonellatyphi Typhoid Highfever,diarrhea
paratyphi
Salmonella Paratyphoid Fever
Salmonellasp Salmonellosis Foodpoisoning
Vibriocholerae Cholera Diarrhea,
dehydralion
Shrge//a
sp Shigellosis Bacillary
dysentery
pneunophila
Legionella Legionellosis Acuterespiratory
illness
Viruses
Adenovirus Respiratory
diseases Breathing
difficulty
Enteroviruses Gastroenteritis Diarrhea
Reovirus Gastroenteritis Dianhea
Rotavirus Gastroenteritis Diarrhea
HepatitisA Infectious
hepatitis Jaundice
Protozoa
Entanoebahistolytica Amoebic
dysentery Prolonged
diarrhea
Giardialanblia Giardiasis Milddianhea,
nausea
Balantidiuncoli Balantidiasis Dysentery
Helminths
Ascarislumbricoides Ascariasis infestation
Roundworm
Schistosoma
sp Schistosomiasis Hematuria,
dianhea
Fasciolahepatica Fascioliasis Jaundice
T::!!i:?s_i:?11
, Taeniasis Intestinal
infection
Algae
aeruginosa
Microcystis Gastroenteritis Dianhea
Aphanizomenon flosaquae Gastroenteritis Diarrhea
calciola
Shizothrix Gastroenteritis Diarrhea
Total nitrogen .U 45 on
Organic N 10 15 40
InorganicN (asfreeNH' 10 30 50
Total phosphorus + 8 16
Organic 1 6
Inorganic I
Chlorides 25 100
(asCaCO3)
Alkalinity 50 100 200
Grease 50 50 100150
Volatile (pg/ l)
compounds
organic <100 100-400 >400
Biochemicaloxygen demand(BOD) 100 400
Chemicaloxygendemand(COD) 250 1000
Total coliform(no/dl) 106-107 1o7-108 108-10e
* The unitsfor volatileorganb compounds
and totalcolitormare givenalongwith then.
The organic matter present in the sewage is 3. For measuring BOD of toxic waste water,
regarded as biologically active, if it can be oxidized pretreatment is necessary.
by the bacteria. On the other hand, the organrc 4. Requires long period of incubation i.e.5
matter resistant to bacterial degradation rs 0ays.
considered as biologically inactive. For the purpose Despite these drawbacks, BOD is very widely
of sewagetreatment,the biologically active organic used worldover for oractical and economic reasons.
matter is imoortant.
The commonly used laboratory methods for the Chemical oxygen demand (COD)
measurementof organic matter in sewage are given
Chenrical oxygen demand refers to the oxygen
below. equivalents of organic matter that can be oxidized
o Bioche mica l ox y gen dem and ( BO D) by using strong chemical oxidizing agents. [.lsually,
. Che mica l o x y gen dem and ( CO D) potassiumdichromate in the presenceof a catalyst,
i n a c i d i c m e d i u m i s e m p l o y e d f o r t h i s p u r p o s e .T h e
o Total organic carbon (TOC)
overall reaction is given below'.
. Theoretical oxygen demand (ThOD).
Organic matter + CrrO2r-+ H+ ------+
Biochemical oxygen demand (BODI C r 3 ++C O r +H r O
Bioche mic al ox y gen dem and is t he m os t wi d e l y When compared with BOD, COD oxidizes more
used parameter Io measure the organic pollution n organic compounds, hence COD valuesare higher
sewage as well as surface water. BOD basically than BOD values. Chemical oxygen demand can
involves the measurement of dissolved oxygen be determined in lust three hours, in contrast to
(DO) utilized by the microorganisms for the BOD requiring five days. Some workers determrne
biochemical oxidation of organic matter. fhe COD and calculate BOD. This is possible since
demand for oxygen and the process of oxidation there exists a reasonablygood correlation between
depends on the type and quantity of organic matter, COD and BOD.
temperature and type of the organism used.
Total organic carbon (TOC)
In ge ne ral, bioc hem ic al ox y gen dem an d i s
measured for an incubation period of five days Measurementof total organic carbon is required
(hence appropriately referred to as BODt) aI a when the concentration of organic matter is very
temperature of 20"C. lf the organic content of the low. TOC can be determined by oxidizing organic
sewage is high, it needs to be diluted for the carbon, in the presenceof a catalystto COr, which
measurementof BOD. Further,for waste water with can be measured
less p op ula tion of m ic r oor ganis m s ,s eeding w i t h
bacterial culture is necessary. Theoretical oxygen demand (ThODl
BOD indicates the amount of organic matter The organic matter of sewage is mainly
present in the sewage. Thus, the more is organic composed of carbohydrates, proteins, fats and
content, the higher is the BOD (Refer lable 56.2for products of their decomposition. lf the chemical
BOf) values of different types of sewages).lf the formulae of the organic matter (i.e. individual
a va ilab le oxy gen ( dis s olv ed 02) is les s t han t h e compounds) are known, the theoretical oxygen
BOD, the organic matter decomPoses demand can be calculated.
644 B IOTE C HNO LO CY
Innerfermentation
tube
Fig. 56.1 : Multiplelube fermentation technique for the detection of coliform bacteria.
686
Chapter57 : SEWAGEAI/ASTE
WATERTREATMENT 687
Fine screen
As alreadystated,preliminarytreatmentinvolves
the removalof floating materials(leaves,papers,
rags)and settleableinorganicsolids (sand,grit),
besidesoily substances(fats,oils, greases).The
three major types of equipment-screeners, grit
Sewage
c ham ber s ,and s k i mmi n g ta n k s , e m p l o y e d i n outlet
(B)
pr elim inar sy c re e n i n g
a re b ri e fl yd e s c ri b e d .
Fig. 57.1 : Diagrammatic representation of screeners
Screeners (A) Fixed bar screen (coarse or medium)
A s c r eene irs a d e v i c ew i th o p e n i n g s(u s u al l y (B) Shredder.
uniform in size)to removethe floating materials
and suspendedparticles.The processof screening
can be carried out by passingsewagethrough Grit chambers
different types of screeners(with different pore The heavy inorganic materiai.ls (specificgravity
sizes). The screeners are classified as coarse, 2.4-2.7)like sand,ash and otherscan be removed
medium or fine, depending on the size of the by usinggrit chambers.Thistechniqueis basedon
openings.The coarsescreenhas largeropenings the processof sedimentation due to gravitational
( 75- 150 m m ) . T h e o p e n i n g sfo r m e d i u ma n d fi ne forces.Crit chambersmay be kepteitherbeforeor
screensrespectively are 20-50 mm and lessthan afterthe screens(described above).A diagrammatic
20 m m . representation of a typicalgrit chamberis depicted
in Fig. 57.2A.
Different types of screens-fixedbar screen
(coarseor medium) disc type fine screen,drum
Skimming tanks
type fine screen are in use. A diagrammatic
Several grcasy and oily materials (fats, oils,
reoresentationo( a fixed bar screen is shown rn
waxes,soapsetc.)from the domesticor industrial
Fig. 57.1A.
outletsfind their entry into the sewage.They can
A shredderor comminutor is a special screen be removedby using a skimmingtank which is
that can cut and retain the floatingand suspended fitted with baffle walls that divide the tank
materials(Fig.57.1B). (Fig. 57.28). The skimmingtank is divided into
688 B IOTE CHNO LO CY
Principle of sedimentation
Types of settling
Air pushed
up-waros There ar four major types of settlinS-discrete
s e t t l i n g , f l o c c u l a n t s e t t l i n g , h i n d e r e d o r zo n e
s e t t l i n g a n d c o m p r e s s i o n . T h i s c a t e g or i za ti o n i s
(B) mainly based on the tendency of the particles to
interact and form solids.
Fig. 57.2 : (A) Grit chamber (B) Skimming tank
Discrete settling : The particles which do not
(cross section).
change their size, shape and weight are referredto
as discrete particles or granular particles. The use
three comoartmentsthat are interconnected.As the of grit in sewage may be consideredas an example
compressedair is pushed from the floor of the tank, of discrete settling.
t he r ais ing air bubbles c oagu l a t e a n d s o l i d i f y t h e Flocculant settling : The flocculant particlescan
oily and greasy materials present in the sewage. change their size, shape and weight, and thus lose
This material is pushed to the side compartment their identity. These particles actually coalesce
referred to as stilling compartment from where it d u r i n g s e t t l i n g . S e t t l i n g o f b i o f l o c s , a n d ch e m i ca l
c an be r em ov ed m anuallv or m e c h a n i c a l l v . flocs in secondary sedimentation tanks are good
examplesof flocculant settling.
Chemical.aided' sedimentation
Biotechnology
[44]
690 B IOTECHNO LO CY
-l
-----------* ----____-l>
Erf
ruenl
lr."jltr
Excessand
waste sludge
Factors affecting performance : There are done by step aeration, tapered aeration, high rate
several factors that influence the efficiency of aeralion by complete mixing and extended
activated sludge process,the most important being aeration.
the type of the reactor, aeration, food
microorganism (F/M) ratio, nutrients, sludge AERATED LAGOONS
recirculation rate, besides pH and temperature.
Aerated lagoons, also called as aerated ponds,
Advantages : The activated sludge process is a are the faculative stabilization pcnds wherein
very compact, low-cost and an efficient biological surface aeratorsare installed to overcome the bad
treatment system for sewage treatment. lt is worked adours (due to overload of organic materials).The
o ut th at un de r ideal c ondit ions , up t o 95% o f microbiological treatment of aerated ponds is
BOD', 98% of bacteria (particularly coliform) and comparable to the activated sludge process. The
95% of suspendedsolids can removed by activated major difference is the large surfacearea in aerated
sludge process.The excessand waste sludge has a p o n d s a n d t h i s i s m o r e s u s c e p t i b l ef o r t e m p e r a t u r e
higher fertilizer value compared to other treatment effects.
processes.
It is possibleto carry out continuous nitrification
Disadvantages : There is production of large in aerated lagoons. This however, depends on the
r olumes of sludge which sometimes becomes design and operating conditions of the pond
ctifficultto ha nd le. Power c ons um pt ion is r elat iv e l y (particularly the temperature).
h igh for o pe ratio n.Super v is ionby s k illed per s on n e l
s necessary. SEQUENCING BATCH REACTOR
S e q u e n c i n gb a t c h r e a c t o r( S B R )i s a m o d i f i c a t i o n
Conventional activated sludge process
of activated sludge treatment system.The processes
ln the normal treatment of sewage,the activated namely aeration and sedimentationare carried out
sludge is preceededby primary sedimentationtank. in both the systems.The major difference is that
The conventional activated sludge system consists while in the conventional activated sludge system,
of a separationtank, settling or sedimentationtank a e r a t i o na n d s e d i m e n t a t i o no c c u r s i m u l t a n e o u s l yi n
and sludge removal line (Fig. 57.4\. fhe sewage separatetanks, these two processesare carried out
after the primary treatment is introduced at the sequentially in the same tank in SBR. Thus, the
h ea d of the tan k . lt is des ir able t o s upply O, sequencing batch reactor may be regarded as fill-
u nifo rmly thro ug hout t he t ank . and-draw activated sludge process.
Air onlolt
A m o n g t h e s e ,t r i c l <l i n gf i l t e r i s m o s t w i d e l y u se d .
TRICKLING FILTERS
Influentin--__)
(sewage)
i:ri ifu
li :.:;:.:.<-
Biologicalfilm
, r,
', .l:l ,r,,ii;;
,,r:ri ( m i c r o b i asll i m e )
+ ||
OO O
a--\ O
^
o Ao n
^
"UVV QO O
o 00 0 oo ooo
Porusbed
The waste waters obtained in milk processing, reduce the organic matter in downstream
paper mills and pharmaceutical industries are p r o c e s s i n g ,b e s i d e s n i t r i f i c a t i o n a p p l i c a t i o n s . D u e
treated by trickling filters. to high hydraulic loading, there is a continuous
s l o u g h i n go f t h e b i o l o g i c a l f i l r n . I n s u c h a c a s e ,t h e
Types of trickling filters unsettled filter effluent can be recycled, and this
increasesthe efficiency of the treatment process.
The trickling filters are classifiedas low rafe (the
The retention time on the biofilm being less,
converrtional one), high rate and super rate. This
organic materials that are not readily degradable
ca teg orizati on is m ainly bas ed on t he hy d r a u l i c
remain unaffected.
and organic loading rates of the sewage. The low
rate filters are suitable for the treatment of domestic
BOTATING BIOLOGICAL
sewage,while high rate filters and super rate filters
GoNTACTORS (RBCI
are useful for industrial sewage.
Rotating biological contactor (RBC) is a recent
Factors affecting performance of device for the biological treatment of sewage. lt
trickling filt er s operates on the principle of aerobic attached-
The type of the media and its depth, organic and growth system operated on the moving media.
hydraulic loading, filter staging, recirculation rate RBC are suitable for the treatment of domestic ano
and flow distribution are the important factors that indusirial sewage in small and medium towns.
influence the perforn.ranceof the trickling filters. A rotating biological contactor is composed of a
Advantages : Trickling filters are simple, occupy series of closely spaced and light weight circul'ar
less space and the operating costs are low. They discs (Fig. 57.V. They are made up of inert
operate efficiently in hot climate and thus are materials such s polystyrene or polyviriyl chloride
suitable for most developing countries (like India) (PVC) or polyethylene.These discs are mounted on
a horizontal shaft in a tank through which waste
Disadvantages : Removal of BOD is moderate
water flows. The shaft is rotated slowly (less than
around TOoh), and disposal of excess sludge is
10 revolutions per minute) by a low speed motor
ne ce ssary.Pr im ar y s edim ent at ionis r equir ed, s i n c e
The discs of the shaft, i'eferred to as biodiscs, are
trickling filter s c ar r not handle r aw s ewage. partially (40-60%) submerged in sewage. As the
biodiscs are rotated, the biornassattached tc them
ROUGHING FI LTERS
is alternately submerged in sewage. This enables
Rou gh ing f ilt er s ar e a s pec ial t y pe of t r ic k l i n g the discs to pick up a thin layer of sewage, and
filtersthat are designedto operate at high hydraulic then to oxidize the absorbed substrates The
Ioading rates. These filters are mostly used to unoxidized substratesfall back into the sewage.
694 B IOTECHNO LO CY
Common Rotation
shaft (B)
Biodisc
Shaft
Sewage
Fig. 57.7 : A diagrammatic reprcsentation of (A) rotating biological contactor (HBC) and (B) A single biodisc.
And this processin repeatedagain and again by the The sewage along with air (or pure oxygen)
rotating biodiscs. is introduced from the bottom of the reactor
(Fig. 57.8). The main advantage with packed bed
Rotating biological contractor is basically a film
reactors is that they have high surface area of
flow bioreacfor. The microbial biofilm is built
biofilms for unit of reactor.
upon the partly submerged support medium
c ont aining biodis c s
The RBC is very efficient for the removal of
organic matter in the sewage (about 90% of BOD).
As and when there is an excessgrowth of biomass,
it has to be removed. This process can be carried
in a s im ilar f as hion, as it is d o n e f o r t h e t r i c k l i n g
A n a e r o b i c p r o c e s s e s b a s i c a l l y i nvo l ve th e
filter (described already).
d e c o m p o s i t i o n o f o r g a n i c a n d i n o r g n i c m a tte r i n
RBC are commonly used for the treatment of the absence of oxygen. Anaerobic processing
municipal waste water. In addition, they are widely systemsare important for the treatment of sludges,
used for the biological processing of industrial
wastes, coming from several industries such as
vegetables, pulp, meat and textiles.
Factors affecting performance of RBC : The
treatment process in RBC is influenced by rotation
speed of shaft, waste water retention time, Overflow
temperature, disc submergence, organic loading,
media density, and number of stages.
Adv ant ages : RBC is c o m p a c t a n d r e q u i r e s
moderate energy input. lt has high BOD removal
efficiency.
Disadvantages: Disposal of sludge formed is a
major problem with RBC. The treatment process is
frequently associated with odour formation. RBC
oper at ion r equir es s k illed pe r s o n n e l .
PACKED.BED REACTORS
Sewage+ Air
Packed-bed reactors or fluidized bed reactors
are used for the removal of BOD and nitrification.
Fig. 57.8 : A diagrammatic tepresentation of a
A reactor is oacked with a medium to which tne packed-bed reactor
microorganisms get attached and form biofilms.
ChAOICT
57 : SEWACEAVASTE
WATERTREATMENT 695
Fermentation products
(propionate,butyrate,
lactate,succinate)
Methanogenicsubstrates
(methanol,acetate,H2, CO2)
Methanogenesis
METHANOL+ CO2
Fig. 57.10 : A diagrcmmatic view of the degradation of organic materials in anaerobic digestion.
696 B IOTECHNO LO CY
. et hane gas i s h i g h l y i n s o l u b l e a n d
ac idogenes is M ( s e w a g e ) f l o w s u p w a r d s t h r o u g h t h e co l u m n
its departure from the digester represents the c o n t a i n i n g a n a e r o b i cb a c t e r i a .D u e t o th e p r e se n ce
stabilization of sewage or sludge. of solid media, the bacteria are retained in the
column. This makes the treatment process more
Microorganisms to degrade organic efficient.
matter of sludge (or sewagef
EXPANDED-BED PROCESS
A consortir-rmof anearobic microorganismswork
together for degradation of sludge (or sewage) The sew'age can be treated by pumping lt
organic matier. They may be categorized into two through a bed of inert materials (sand or coal
rypes. expanded aggregates)on which the bacteria have
grown and formed a film. The effluent that comes
1 . Acid-forming bacteria : These are also
out can be recycled to maintain the flow rate.
known as acidogens or non-methanogenic
bacteria. They bring out the hydrolysis of
m ac r om olec ules ( e. B c ar b o h y d r a t e ) t o s i m p l e
substrates(e.g. monosaccharides),and the latter to
acids e.g. Clostridium sp, Corynebacterium sp,
Lactobacillus sp, Actinomyces sp, Staphylococcus Pond treatment processesfor the treatment of
sp, Peptococcus anaerobus, Escherichia coli. sewage (containing biodegradable wastes) are
2. Methanogenic bacteria : These bacteria,also c a r r i e d o u t b y s p e c i a l l y d e s i g n e d a n d co n str u cte d
referred to as methanogens ot methane formers are ponds. These ponds, referred to as stabilization
r es pons ible f or t he c onv e r s i o n o f a c e t i c a c i d ponds, are large, shallow earthen basins. The
and hydrogen to methane and carbon dioxide. treatment process is a natural one involving the
The most important methanogens belong to combineci use of bacteria and algae. The
the genera Methanobacterium, Methanobacillus, stabilization ponds are classified as aerohic,
Methanococcus and Methanosarcin a. anaerobic and facultative ponds.
T h e a e r o b i c s t a b i l i z a t i o np o n d s c o n ta i n b a cte r i a
a n d a l g a e i n s u s p e n s i o n T h e y a r e p a r ti cu l a r l y
useful for the treaiment of soluble wastes.
T h e a l g a e a n d b a c t e r i a e x h i b i t a s ym b i o ti c a n d
There are mainly two treatment processesunder
c y c l i c r e l a t i o n s h i pi n t h e a e r o b i c p o n d s. Th e a l g a e
the anaerobic attached-growth treatment system-
can carry out photosynthesisand releaseoxygen to
anaerobic filter processand expanded bed process.
m a i n t a i n a e r o b i c c o n d i t i o n s i n t h e p o n d . Th e
bacteria degrade the organic matter to produce
AhI AERO BI C FI LTEB PR O C E S S
CO, and other nutrierrts to be utilized by algae
Anaer obic f ilt er c ons is t so f a c o l u m n f i l l e d w i t h (Fig. 57.11). Some higher organisms like protozoa
solid media for the treatment of organic matter in and rotifers present in the pond are responsiblefor
sewage. In this process system, waste water t h e p o l i s h i n g o f t h e e t T l u e n t .
WATERTREATMENT
Chapter57 : SEWAGEMASTE 697
In facultative ponds, the treatment of sewage is Advantages: For the facultativeponds, the initial
carried out by a combination of both aerobic and and operating costs are low. There is no need for
anaerobic processes. Three types of microorganisms s k i l l e d personnel.
aerobic, anaerobic and facultative (both aerobic
Disadvantages : Unpleasant odours and
and anaerobic) are employed in facultative ponds.
mosquite breeding are frequently seen in facultative
The\erm oxidation pond or stabilization pond ponds. The requirement of land area for
is frequently used for facultative ponds. construction of these oonds is more.
B IOTECHNO LO CY
698
Sunlight
H 2S , C H 4
Sewage
o'nf"
----*"""'
a't^
oeaJcerts
/ \
coz O2
Settleable
solids
\
\ ------...--- /-orsanic
matter
Bacteriak---t'
'->
t-t
Newcells Deadcells
BOTTOMSLUDGE
Cranular activated carbon contactors (GAC) are As the name indicates, ion-exchange involves
the most widely used for the advanced treatment of the displacement of one ion by another. The
sewage. The CAC consists of a tank (usually exchange occurs between the ions of insoluble
cylind rica l)whic h is loaded wit h gr anular a c t i v a t e d exchange material (ion-exchangematerials)and the
carbon (Fig.57.13). The waste water/sewage ions of different speciesin solution (i.e. waste water
(influent) is passed from top through the activated for advanced treatment).
carbon bed and the effluent comes out from the
The ion-exchange process is carried out by
bottom. The solids attached to the carbon bed
employing two types of ion-exchange materials-
hamper further adsorption process.These particles
cation exchangers and anion exchangers
can be removed by backwashing.
(Fi9.57J4. The synthetic resins with strong
The activated carbon requires periodical acidic (H+) and basic (OH-) functional groups serve
regeneration. This can be done by heating in a as ion exchangers.The cation exchangers(with H+
furnance at about 800'C in the absence of Or. At or Na*) can replace the positively charged
this temperature,the adsorbed organic compounds ions (Ca2+, Mg2*; in water by hydrogen ions.
are converted to gases and released. The carbon This is what is done for removinq the hardnessof
granulates are reactivated for reuse. water.
700 B IOTE C H N OLO CY
Ca2+ so+F
M92+ ct-
c"?-
Fig. 57.14 : Ion-exchange process for the removal of dissolved solids-
Ammonianitrogen
N
I
T
R
I
Lysisand F
autooxidation I
Organicnitrogen C
(bacterialcells)
T
S
S o
I N
[,1
I
A D
T E
I N
o
N T
Organlcnitrogen R
(net growth) I
F
I
C
T
I
N
presence of organic carbon is essential. The The flcu, diagramfor the removal of biological
presence of even minute quantities of O2 nitrogen is depicted in Fig. 57.16. The process
suppresses denitrification. The heterotrophic primarily consistsof the removal of organic carbon
bacteria can reduce nitrate in the following stages, (aerobic), followed by nitrification (aerobic) and
to fina lly nitr ogen gas . denitrification (anaerabic).
Effluent{-
.-'. .-.'->
Wastesludge
Phosphorus
Phosphorusstripped enrichedsludge
returnsludge
(";;),
phosphorLrs
i /
\Jrflpper/
Supernatant
return
J
Wastesludge
I
I
Fig. 57.17 : Biological phosphorus removal by Phostrip (phosphate sttipper) process.
I
I
Chapter57 : SEWAGEA//ASTE
WATERTREATMENT 7o3
l*-- Preliminary
-) { Primary.-,J {- Secondary
' lreatment treatment treatment
Seconciary
Fig. 57,18 : A diagrammatic representation of a flow chart of conventional sewage treatment plant
aerobic pond. The treated water coming out of the The treatnrentprocess is carried out in two phases
a naer obic pond is r ic h in nut r ien t s ,h e n c e i s u s e f u l ffig. s7.21).
for irrigation purposes.At the end of the two-stage
Phase | : This phase involves biological
treatment, abour 95"h of the BOD material rs
acidification and production of sulfides from
removed from the waste water.
sulfates present in the waste water. The sulfide
recovered in the form of sodium hydrogen sulfide ls
WASTE WATER TREATMENT
reused in tanneries.
FO R DI STI LLERY
P h a s e l l : T h i s i s c a r r i e d o u t i n a n a na e r o b tc
The waste water from distilleriesmostly consists
sludge blanket reactor. ln this phase, the organic
of dissolved O, and organic wastes (predominantly
fermented starches and organic nitrogen). The
treatment is carried out by a two-stage bacterial
oxidation in anaerobic reactors. The complex
organic wastes are first degraded to organrc
m olec ules ( ac ids , aldehy des , a l c o h o l s , k e t o n e s
et c . ) . Thes e m olec ules ar e f ur t h e r o x i d i z e d t o
s m aller or ganic ( CHo) and inor g a n i c ( C O 2 , N H 3 ,
H25, H2O) compounds (Fig. 57.20)-
I
57 : SEWACEA//ASTE
ChAPICT WATERTREATMENT 7o5
TREATMENT OF ANTIBIOTICS
IN WASTE WATER
Biotechnology [45]
706 B IOTECHNO LO CY
O XI DATI O N DI TCH
Fig. 57.22 : A diagrammatic representation of
O x idat ion dit c h m et hod , f i r s t d e v e l o p e d I n oxidation ditch-
Netherlands,is a suitable method for the treatment
of s ewage in s m all c om m un i t i e s . T h i s i s b a s i c a l l y
an aeration type of activated sludge process (See Septic tanks work on the principle of anaerobic
p. 590) wit h a m ec hanic a l s y s t e m o f a e r a t i o n . digestion.This occurs as the solids of the sewage
Howev el t her e is no pr im a r y s e d i m e n t a t i o n o f settle at the bottom of the tank. Under anaerobic
s ewage,c ons equent lyt he pr o b l e m o f h a n d l i n g a r r d c o n d i t i o n s , t h e b i o d e g r a d a b l e o r g a n i c m a tte r i s
t r eat m ent of pr im ar y s ludge i s e l i m i n a t e d . converted to gases(CH4, CO2, HrS etc.) and liquid
c o m p o u n d s . T h i s r e s u l t s i n a d r a s t i c re d u cti o n tn
O x idat ion dit c h c ons is t s o f a e r a t i o n u n i t s ,
t h e v o l u m e o f s l u d g e .A t h i c k c r u s t o f s cu m fo r m e d
nam ely dit c h c hannels ( 2 or m o r e ) c o n s t r u c t e ds i d e
on the surface of septic tank maintains anaerobic
by side (Fig. 57.22). The sizes of the ditch channels
c o n d i t i o n s . T h e e f f l u e n t c o m i n g o u t of th e se p ti c
ar e v ar iable- lengt h150- 1 000 m , w i d t h I - 5 m a n d
t a n k c o n t a i n s s o m e o r g a n i c s o l i d s a n d p a th o g e n s
dept h 1- 5 m . They ar e c ons t r u c t e d w i t h b r i c k o r
Therefore, disposal of the effluent from septic tank
stone masonary. A special type of rotors (cage
has to be-dealt with very carefully.The septic tanks
rotors) are fitted into each ditch channel
a r e d e s l u d g e d a n d c l e a n e d a t r e g u l a r i n te r va l s,
f or c ont inuous ly agit at ing a n d c i r c u l a t i n g t h e
u s u a l l y o n c e i n 2 - 5 y e a r s ( d e p e n d i n g o n th e ta n k
s ludge,bes idess upply ingO r . T h e s l u d g e i s a l l o w e d
size and its use).
to settle down in a separate sedimentation tank'
The activated sludge is returned to ditch channels.
Gonstruction and operation of
lnstead of using a separatesedimentation tank, septic tanks
the sewage may be allowed to settle down in
tl-re ditch channels by stopping the rotors S e p t i c t a n k s a r e u s u a l l y c o n s t ru cte d w i th
( us ually dur ing night ) The s u p e r n a t a n t f r o m t h e b r i c k s , o r s t o n e m a s o n a r y . T h i c k - w a l l p o l yth e n e
dit c h c hannelsc an be t ak en o u t . T h e e x c e s ss l u d g e a n d f i b r e g l a s s t a n k s a r e a l s o i n u se i n r e ce n t
c ollec t ed ( in t he s edim en t a t i o n t a n k o r a t t h e y e a r s . Wh a t e v e r m a y b e t h e c o n s t r u c ti o n m a te r i a l
bot t om of dit c h c hannels )ca n b e s t a b i l i z e d . u s e d , t h e s e p t i c t a n k m u s t b e w a t e r - ti g h t a n d
m u s t f u n c t i o n e f f i c i e n t l y . T h e s i z e o f th e ta n k i s
v a r i a b l e , d e p e n d i n g o n t h e n u m b e r o f u se r s. Fo r
SEPTI C TANKS
a f a m i l y o f f i v e m e m b e r s , a t a n k w i t h a l e n g th o f
Sept ic t ank s ar e r ec om m e n d e d f o r i n d i v i d u a l 1 . 5 m a n d a b r e a d t h o f 0 . 7 5 m i s r e co m m e n d e d .
hous es and f or s m all c om m u n i t i e s a n d i n s t i t u t i o n s F o r s u c h a t a n k , t h e c l e a n i n g i n t e r va l i s 2 - 3
wit h a c ont r ibut ing populati o n a r o u n d 3 0 0 . years.
ChAPtCT
57 : SEWACEA//ASTE
WATERTREATMENT 7(,7
Sludge
IMHOFF TANKS
r Sludge- pr oc es s ing
unit s . Sometimes, removal of grit (degritting) is
necessary for the effective treatment of sludge.
The details on the above processeshave already D e g r i t t i n g c a n b e c a r r i e d o u t b y a p p l yi n g
been described. The important sludges and their centrifugal force to the flowing sludge. Cyclone
characteristics are given in Table 58.1. The degrittersare usually employed for this purpose to
characters of sludge are variable and depend on separategrit particles from the organic sludge.
t he or igin of t he s ludge, a n d t h e a g i n g a f t e r i t i s
produced Sludge blending
Sludge produced in primary (settleablesolids),
Chem ic al c om pos it ion of sludge
secondary (settleablesolids and biological solids)
The c hem ic al c om pos it i o n o f s l u d g e i s v a r i a b l e . and tertiary (biological solids and chemical solids)
The major constituentsof sludge are proteins and treatmentsis mixed (blended)to produce a uniform
ot her nit r ogen c ont aining c o m p o u n d s , g r e a s e a n d mixture.
708
58 : S L U D C EAN D SOL IDWA ST E S-TR E A TME NATN D D IS P OS A L
C hA P I CT 709
SLUDGETHICKENING
(coNcENTRATTONI
characteristics
The solid content of the sludge is variable and
SIudge Characteristics may range from 0.5 to 12oh. It is necessary to
c o n c e n t r a t et h e s l u d g e ( p a r t i c u l a r l yw i t h l o w s o l i d
Screenings Containsorganic
and
content) by removing some amount of liquid.
inorganicmaterials
thatcan
Physical methods are employed for sludge
be easilyremoved.
thickening.
Grit Richin heavierinorganic
solidsanda goodquantityof Gravity thickening
organicmatter(fats,grease).
Cravity thickening is the most commonly used
Primarysludge Obtained fromtheprimary m e t h o d f o r t h e t h i c k e n i n g o f p r i m a r y s l u d g e .l t c a n
settling
tanks,greyin colour be carried out in the conventional sedimentation
andoffensive in odour. t a n k ( c i r c u l a r t a n k i s p r e f e r r e d ) .A s t h e s l u d g e g e t s
Activatedsludge concentrated by gravity, the supernatant can be
Brownishto darkin colour
(i.e. primary settling
appearance, r e t u r n e dt o t h e t r e a t m e n tp l a n t
withflocculant
hasearthy t a n k )
usually odourand
canbe digestedeasily.
Trickling-filter
sludge Brownish
in colourwith
Tnslr 58.2 Sludgetreatment and disposal
odour(fresh
inoffensive
methods
easily
sludge), digestible.
Scum materials
Floatable skimmed Type of process Treatment methods
fromthesurfaces of primary Preliminary
operationsGrinding,
degritting,
blending,
andsecondary settlingtanks. storage
Scumcontains oils,fats, Thickening Gravity
thickening
foodwastes,plastic
Flotation
thlckening
materials,
cottonetc.
Centrif
ugalthickening
Aerobically
digested Brownishto blackin colour Rotarydrumthickening
sludge withflocculant
appearance. Stabilization Limri
ilairitiiiiiil
Nooflensive
odour. Heattreatment
Anaerobically
digested Darkbrownto blackin Anaerobicdigestion
sludge colour,
containslarge Aerobicdigestion
quantities
of gas.0n drying Composting
thegases
thesludge, Conditioning Chemicalmethod
escape. Heattreatmentmethod
Septage Sludgeof septictanks,black Disinfection Pasteurization
in colour.
Usuallywell Dewatering Vacuum filter
digesteddueto longstorage. Centrif
uge
Offensiveodourdueto Sludge dryingbeds
hydrogensulfide. drying Flashdryer
Slowdryer
Rotarydryer
Sludge storage
Thermal reduction Multiple-hearth inciner
ator
Storageof sludge becomes necessarywhen the Fluidized bedincinerator
p ro ce ssingun its ar e not in oper at ions ( night s hif t s ,
Ultimatedisposal Landapplication
rveekends). Short{erm storage of sludge rs
Distribution andmarketing
a ccomp lish ed in s et t ling t ank s while f or long -
Landf
ill
term storage, specially designed tanks have to be
Lag00nrng
u s eo .
71(, B IOTECHNO LO CY
Sludge requires stabilizationto achieve the S i ngl e-stagehi gh rate di gesti on: This is
followingobjectives. characterized by high rate of sludgeloading,and
i ts di gesti onunder anaerobi ccon dit ions.The
. To reducethe load of disease-causing organisms
sludgeis continuouslymixed by gas, heatedand
(pathogens).
recirculated for optimal digestion.
. To inhibit the potentialfor putrefaction.
Two-stagedigestion : There are two digestion
o To eliminateoffensiveodours.
tanks used in this processof high-ratedigestion.
Sludge stabilizationcan be carried out by The first tank is used for digestionproper (with
appropriatebiologicaland chemical means.The mi xi ng and heati ngfaci l i ti es)w hi l e t he second
techniquesusedfor sludgestabilization are- lime digesteris employedfor storageand concentration.
stabilization, heat treatmenl anaerohic digestion,
Thermophilicanaerobicdigestion: Sometimes,
aerohic digestion and composting.They are briefly
sludgedigestioncan be carried out at relatively
described. higher temperature (45-60"C) by thermophilic
bacteria.In fact, the digestionprocessis fasterwith
Lime stabilization
increased bacterial destruction and improveo
When lime, in the form of hydrated lime dewateringat higher temperature.But the major
l C a (O H )rlo r q u i c k l i m e (C aO) i s added to the limitation is the requirementof high energy for
58 : SL U D C EA N D S O L IDWA STE S -TR E A TME NATN D D IS P OS A L
M ECHANI SM O F CO M PO S T I N G
ChaPter
in'vesse':
..J[::,J:Jil!,'T#'J".'IT:H'J' tt/ t''
|owi"*".":l':f::"i:9,::l$T":li::
Plug-f
aretwo tvpesot l:T-';;"J u"a
tiii. sa.se
ComPosting
ml x
cvlindrical'o*"t "no,r,'if,''"' .i'.j'"thip between
r"f
is.sn).In.both:f :itfi.;i"s-mass is maintained
theparticles n, thesewase rs
the same
'"'l]:.:T5;;.Err.
throughoutrnc of first
oc i;:;"n" principle
---* Outfeed
fed, the comPosting
in and first out'
Dvnamic, '1;-]:;
inlv1*'.:"1H,T[t'T:f lnteed
Soillayer
Gas outlet
Monitor Sealinglayer
J I Leachateto
treatment
First layer
Secondarylayer
Leachatecollection
ln some countries, there are strict regulationsto . Land application (surface or subsurface).
us e landf ills f or s ewagedis pos a l .T h e s e i n c l u d e t h e
c Co-treatment with waste water (biological or
air- and water tight sitesto protect the environment
chemical treatment processes)'
Bioremediation refers to the process of using Members of the genus Pseudomonas (a soil
microorganisms to remove the environmental microorganism) are the most predominant
pollutants i.e. the toxic wastesfound in soil, water, microorganismsthat degrade xenobiotics. Different
air etc. fhe microbes serve as scavengers n strains of Pseudomonas, rhal are capable of
bioremediation. The removal of organic wastes by d e t o x i f y i n g m o r e t h a n 1 0 0 o r g a n i c c o m p o u n d s,
microbes (or environmental clean-up is the essence have been identified. The examples of organic
of bior em ediat ion.The ot her na m e s u s e d ( b y s o m e c o m p o u n d s a r e s e v e r a l h y d r o c a r b o n s, p h e n o l s,
authors) for bioremediation are biotreatment, o r g a n o p h o s p h a t e s i p o l y c h l o r i n a t e d b i p h e n yl s
bioreclamation and biorestoration. ( P C B s )a n d p o l y c y l i c a r o m a t i c s a n d n a p hth a l e n e .
A b o u t 4 0 - 5 0 m i c r o b i a l s t r a i n s o f m i cr o -
I t is r at her dif f ic ult t o s h o w a n y d i s t i n c t i o n
o r g a n i s m s ,c a p a b l e o f d e g r a d i n g x e n o b i oti cs h a ve
bet ween biodegr adat ionand bio r e m e d i a t i o n ,h e n c e
been isolated. Besides Pseudomonas,other good
they are consideredtogether in this chapter.Further,
examples are Mycobacterium, Alcaligenes, ano
in biotechnology, most of the reactions of
N o c a r d i a .A s e l e c t e dl i s t o f m i c r o o r g a n i sm sa n d th e
biodegr adat ion/ bior em ediat ioinn v o l v e x e n o b i o t i c s .
xenobiotics degraded is given in Table 59.1 .
Thus , t his c hapt er m ay be apor o p r i a t e l yc o n s i d e r e c l
as m ic r obial degr adat ion of x e n o b i o t i c s . F o r t h e Consortia of microorganisms for biodegradation :
bioremediation of metal polluiants, the reader must A p a r t i c u l a r s t r a i n o f m i c r o o r g a n i s mm a y d e g r a d e
Ref er Chapt er 32) . o n e o r m o r e c o m p o u n d s . S o m e t i m e s , fo r th e
718
Chaot er59 : B IO D EC R A D A T IOANN D B IO R E ME D IA TION 719
Corynebacterium
sp hydrocarbons, . M o l e c u l a r o r i e n t a t i o n o f a r o m a t i c c o m p o u n d s
Halogenated
phenoxyacetate. influences biodegradation i.e. ortho > para >
meta.
Bacillus
sp Longchainalkanes,
phenylurea. o The presence of halogens (in aromatic
compounds) inhibits biodegradation.
Jdttutua >V Polychlorinated
biphenyls
lspergillussp Phenols Besidesthe factors listed above, there are two
recent developmentsto enhance the biodegradation
,Qnthomonas
sp hydrocarbons
Polycyclic
by microbrganisms.
S:'eptomyces
sp Halogenated
hydrocarbons,
phenoxyacetate. B i o s t i m u l a t i o n : T h i s i s a p r o c e s sb y w h i c h t h e
-,sanunsp microbial activity can be enhanced by increased
Propanil
supply of nutrients or by addition of certain
sp
-^,tninghamella Polycyclic
aromatics, stimulating agenfs (electron acceptors, surfactants).
polychlorinated
biphenyls.
Bioaugmentation : lt is possible to increase
biodegradation through manipulation of genes.
'.:'ad atio n o f q s ingle c om pound, t he s y ner g e t i c M o r e d e t a i l s o n t h i s g e n e t i c m a n i p u l a t i o n i . e .
, - :: of a fe w m ic r oor ganis m s( i. e. a c ons or ti u m g e n e t i c a l l ye n g i n e e r e dm i c r o o r g a n i s m s( G E M s ) ,a r e
- rcktail of microbes) may be more efficient. For d e s c r i b e d l a t e r B i o a u g m e n t a t i o n c a n a l s o b e
- r-; -ce, the ins ec t ic ide par at hion is m o r e a c h i e v e d b y e m p l o y i n g a c o n s o r t i u m o f m i c r o -
,- - :n tly de gr aded by t he c om bined ac t ion o f o r g a n r s m s .
: .:
- --crnonas aeruginosa and Psudomonas stulzeri.
Enzynre systems for biodegradation
fo-metabolism in biodegradation : In general,
'. --:abolism (breakdown) of xenobiotics is not Several enzyme systems (with independent
, ,:eo with any advantage to the enzymes that work together)are in existence in the
- -." -:an ism. That is t he pollut ant c hem ic a l microorganisms for the degradation of xenobiotics.
72(J B IOTE CHNO LO G Y
ln situ bioremediation
RECALCITRANT XENOBIOTICS
ln situ bioremediation involves a direct
There are certain compounds that do not easily
approach for the m icrobial degradation of
undergo biodegradation and therefore persist in
xenobioticsat the sitesof pollution (soil, ground
the environment for a long period (sometimes irr
water).Additionof adequatequantitiesof nutrients
years).They are labeled as recalcitrant.There may
at the sitespromotesmicrobialgrowth.When these
be several reasons for the resistance of xenobiotics
microorganismsare exposed to xenobiotics
t o m ic r obial degr adat ion.
(pollutants),they develop metabolic ability to
. They m ay c hem ic ally and b i o l o g i c a l l y i n e r t degradethem. The growth of the microorganisms
( highly s t able) . and their ability to bring out biodegradation are
dependenton the suppl y of essenti alnut r ient s
. Lack of enzyme system in the microorganismsfor
(nitrogerr, phosphorusetc.)./n situ bioremediation
biodegradation.
has been successfully applied for clean-upof oil
. They c annot ent er t he m icr o o r g a n i s m s b e i n g spillages,beachesetc. There are two types of rn
large molecules or lack of transport systems si fu bi oremedi ati on-i ntri nsi
andc engi neer ed.
. The c om pounds m ay be highl y t o x i c o r r e s u l t r n
Intrinsic bioremediation : The inherent
the formation highly toxic products that kill metabolicabilityof the microorganisms to degrade
m ic r oor ganr s m s . certai npol l utantsi s the i ntri nsi cbi oremediat ion.
In
fact, the microorganisms can be tested in the
There are a large number of racalcitrant
laboratory for their natural capability of
xenobiotic compounds e.g. chloroform, freons,
biodegradation and appropriatelyutilized.
insecticides (DDI, lindane), herbicides (dalapon)
and synthetic polymers (plastics e.g. polystyrene, Engineered in sifu bioremediation : The inherent
polyethylene, polyvinyl chlorine). ability of the microorganisms for bioremediation is
Chaot er59 : BIOD EC R A D A T IOAN
N D B IO R E ME D IA TION 721
gener allys l o w a n d l i m i te d . H o w e v e r,b y usi ng not always possible This is evident from the
suitable physico-chernical means (good nutrient different types of metabolic effects as shown below.
and O, supply, addition of electron acceptors,
Detoxification : This process involves the
optimal temperature), the bioremediationprocess
m i c r o b i a l c o n v e r s i o no f t o x i c c o m o o u n d t o a n o n -
can be engineered for moreefficientdegradation of
toxic one. Biodegradation involving detoxification
oollut ant s .
is highly advantageous to the environment and
Advantagesof in situ bioremediation population.
1. Cost-effective, with minimal exposurercr Activation : Certain xenobiotics which are not
pub l i c o r s i te p e rs o n n e l . toxic or less toxic may be converled to toxic or
n ma i nm i ni mal l y more toxic products. This is dangerous.
2. S ite so f b i o re me d i a ti ore
disru p te d . Degradation : The complex compounds are
'
Disadvantages
of in sifu bioremediation degraded to simpler products which are generally
harmless.
1. V eryti m e c o n s u mi n gp ro c e s s .
Conjugation : The process of conjugation may
2. Sites are directly exposed to involve the conversion of xenobiotics to more
environmentalfactors (temperature,O, complex compounds. This is however, not very
s up p l ve tc .). common.
3. M ic ro b i a l d e g ra d i n g a b i l i ty vari es
seasonally. TYPES OF REACTIONS IN
BIOBEMEDIATION
Ex situ bioremediation
Microbial degradation of organic compounds
The wasteor toxic materialscan be collected primarily involves aerobic, anaerobic and
with
from the oollutedsitesand the bioremediation sequential degradation.
the requisite microorganisms (frequently a
consortiumof organisms)can be carried out at Aerobic bioremediation
designed places. This process is certainly an
improvementover in situ bioremediation,and has Aerobic biodegradation involves the utilization
been successfullyusedat some places. of O, for the oxidation of organic pompounds.
These compounds may serve as substratesfor the
Advantagesof ex situ bioremediation supply of carbon and energy to the microorganisms.
1. Better controlled and more efficient Two types of enzymes namely monooxygenases
orocess. and dioxygenases are involved in aerobic
biodegradation. Monooxygenasescan act on both
2. Processcan be improvedby enrichment
aliphatic and aromatic compounds while
with desiredmicroorganisms.
d i o x y g e n a s e so x i d i z e a l i p h a t i c c o m p o u n d s .
3. T im e re o u i re di n s h o rt.
of ex sifu bioremediation
Disadvantages Anaerobic bioremediation
3crechnology[46]
722 B IOTECHNO LO CY
P e s t i c i d e sa n d h e r b i c i d e s a r e r e g u la r l y u se cito
Biodegr adat ion of aliph a t i c
c o n t a i n v a r i o u s p l a n t d i s e a s e s a n d im p r o ve th e
hy dr oc ar bons
crop yield. In fact, they are a part of the modern
The upt ak e of aliphat ic h y d r o c a r b o n s i s a s l o w a g r i c u l t u r e , a n d h a v e s i g n i f i c a n t l y c o n tr i b u te d tc,
pr oc es s due t o t heir low s o l u b i l i t y i n a q u e o u s green revolution. fhe common herbicides anc;
m edium . Bot h aer obic and a n a e r o b i c p r o c e s s e sa r e p e s t i c i d e s a r e p r o p a n i l ( a n i l i d e ) , p r o p h a n '
oper at iv e f or t he degr a d a t i o n o f a l i p h a t i c ( c a r b a m a t e )a, t r a z i n e ( t r i a z i n e ) ,p i c l o r a m ( p yr i d i n e
hy dr oc ar bons . For inst a n c e , u n s a t u r a t e d d i c h l o r o d i p h e n y l t r i c h l o r o e t h a n e ( D D T) m o n o -
hydrocarbons are degraded in both anaerobic and c h l o r o a c e t a t e ( M C A ) , monochloropropionaie
aer obic env ir onm ent s , while s a t u r a t e d o n e s a r e (MCPA) and glyphosate lorganophosphate).
degraded by aerobic process.
M o s t o f t h e p e s t i c i d e sa n d h e r b i c i d e s a r e to \ -
Som e aliphat ic hy dr o c a r b o n s w h i c h a r e a n d a r e r e c a l c i t r a n t ( r e s i s t a n tt o b i o de g r a d a ti o n
reclacitrant to aerobic process are effectively Some of them are surfactants(activeon the sui'fact
c iegr adedin anaer obicenv ir o n m e n te g . c h l o r i n a t e d and retained on the surface of leaves.
aliphat ic c om pounds ( c ar bo n t e t r a c h l o r i d e ,m e t h y l
c hlor ide, v iny l c hlor ide) . Biodegradation of halogenated
aromatic cornpounds
Biodegradation of aromatic
M o s t c o r n n r o n l y u s e d h e r b i c i d e s a n d p e sti ci c;.'.
hydrocarbons (predominar--
are arornatic halogenated
' M ic r obial
degr adat ionof a r o m a t i c h y d r o c a r b o n s c h l o r r n a t e d )c o m p o u n d s . T h e b i o d e g r a d a ti vep a - - -
oc c ur s t hr ough aer obic and a n a e r o b i c p r o c e s s e s . ways of halogenated compounds df€ corllpdrd: :
The m os t im por t ant r nic r oor g a n i s mt h a t p a r t i c i p a t e s y r i t h t h a t d e s c r i b e c f o r t h e d e g r a d a ti o n o f l c- -
in these processesis Pseudomonas. h a l o g e n a t e da r o m a t i c c o m p o u n d s ( F i g s .5 9 .1, 5 9 .)
59.3 and 59.4). The rate of degradation
The biodegr adat ion of a r o m a t i c c o m p o u n d s
h a i o g e n a t e dc o m p o u n d s i s i n v e r s e l y r e l a te d to :- .
bas ic ally inv olv es t he f oll o w i n g s e q u e n c e o f
n u m b e r o f h a l o g e n a t o m s t h a t a r e o r i g i n a l l y p r e :r - '
reactions.
o n t h e t a r g e t m o l e c u l e i . e . c o m p o u n d s w i th h r ,:'-
1 . Rem ov al of t he s ide ch a i n s . n u m b e r o f h a i o g e n s a r e l e s s r e a d i l y d eg r a cl e d
AN D B IO R EME D IA TION
-r apt er 59 : B I O D E C R AD AT ION 723
Toluene L-Mandelate
I J
Benzoylalcohol Benzoylformate
1-------)
Benzaldehyde Anthracene
- +
I
Benzoate Salicylate{- Nephthalene
+ *- Phenanthrene
./l -.rt
\
BenzenePhenol Anthranilate
ll
++
Shikimate p-Hydroxybenzaldehyde
+----<------p-Toluate
ll
x "f
rJ
S-Dehydro- P-Hydroxybenzoate{- Benzoate
shikimate
\
\
\ k-
{-
PROTOCATLCHU,T,Ti:. Vanillate
\.+ I I
4-Oxoadipate
enol lactone
J
Oxovalerate
J
Oxovalerate
I II
.'-t \
I \ J
P rl rLrr,:-,te A cetal dehyde 2 Pyruvate
Fig. 59.3 : Conversion of catechol and Fig. 59.4 : Conversion of catechol and
protocatechuate to acetyl CoA and succinate prctocatechuate to pyruvate and acetalciehyde
by ortho-cleavage PathwaY. by meta-cleavage Pathway'
724 B IOTE CH\ C_ -
A \." B
\.,/
+]conlrgation
CAM_OCT XYL
plasmid plasmid
XYL plasmid
CAM-OCT
plasmid NAH plasmid
Fig. 59.5 : Creation of the superbug by transfer of plasmids (A, B, C, D, E, F and G are the different
slrains ol bacteria egn!?iningth.e.plasmidsshow1, Slrain G is the gupq{bug.,)
c oex is t in t he s am e bac t e r i u m T h i s n e w l y ,
produced bacterium contains genes for the Trru 59.3 A selectedllst of genetical$l
degr adat ionof x y lene and n a p h t h a l e n e .
englneelednicloorganisrns(GEltlslwith the
potentlalxenobloticsthat can be degraded
The next and final step is the conjugation of
bac t er ium c ont aining CAM - OC T p l a s m i d w i t h t h e G eneticalIy engin eered Xenobiotic
ot her bac t er iumc ont aining X Y L a n d N A H p l a s m i d s . microorganism (CEMs)
The newly created strain is the superbug that
diminuta
Pseudononas Parathion
carries CAM-OCT plasmid (to degrade camphor
and octane), XYL (xylene-degrading) plasmid and P. oleovorans Alkane
NAH (naphthalene-degrading) plasmid.
P. cepacia 2, 4, 5-Trichlorophenol
Because of the risks involved in the use of result is that there occurs biostimulationby effective
GEMs, so lar no GEM has been allowed to enter b i o r e m e d i a t i o no f p o l l u t e d s o i l o r w a s t e l a n d .
the environmental fields. Thus, the use of CEMs
has been confined to the laboratories, and fully Bioaugmentation ln soil biorennediation
controlled processes of biodegradation (usually Addition of specific microorganisms to the
employing bioreactors). Further, several pre- polluted soil constitutes bioaug,'nentation. The
ca utio na ry meas ur es ar e t ak en while c r ea t i n g pollutants are very complex molecules and the
GEMs, so that the risks associatedwith their use are native soil microorganisms alone may not be
min imal. capable of degrading them effectively. The
Some researchersare of the ooinion that CEMs examples of such pollutants include
will create biotechnological wonders for the p o l y c h l o r o b i p h e n y l s ( P C B s ) ,t r i n i t r o t o l u e n e ( T N T ) ,
environmental management of xenobiotics, in the polyaromatic hydrocarbons (PAHs) and certain
pesticides.
next few decades.This may be possible only if the
a ssociate d risk s of eac h CEM is t hor oug h l y Based on the researchfindings at the laboratory
evaluated, and fully assured of its biosafety. level (with regard to biodegradation), it is now
possible to add a combination of microorganisms
referred to as consortium ar cocktail of
m i c r o o r g a n i s m s ,t o a c h i e v e b i o a u g m e n i a t i o n .
Activated
carbonfilter
Nutrients
Clean
Soil sudace water
Clean
water Watertable Injection
weil
Flecovery---;, Injection
well well
I
Bioactivezone
Pollutedground
waler
Directionof waterflow
(A) (B )
Fig. 59.7 : Bioremediation of ground water (A) Pump- and lreat technique (B) Biofencing technique.
730
Cha ote r 60 : G LO BAL ENVI RO NM ENI AL PR O B L E N 4 S 731
3. Exposureto UV-B radiation is also associated sulfuric acid and nitric acid, and to a lesser extent
with several other health complications-damageto b y h y d r o c h l o r i c a c i d a n d o r g a n i c a c i d s.
eyes, decreased immune response, increased
incidence of several infections. DEVELOPMENT OF ACID RAIN
2. The qualit y and qua n t i t y o f thousands of kilometers by the winds. Thus, the
foods are
adversely affected. export of the acid forming pollutants may occur
from one region to another region, and from one
3. Retardation in the growth of forests. country to another country. For this reason, acid
rain is a global problem.
Effects on aquatic ecosystems
The following reactionssummarisethe formation
1. The aquat ic lif e is v er y v u l n e i - a b l et o U V - B of acid rain from the oxides of sulfur (SO") and
r adiat ions , par t ic ular ly up t o a d e p t h o f 2 0 m r n oxides of nitrogen (NO*)
clear waters and a depth of 5m in unclear waters.
SO* + O, + HrO ---------) H2SO3 + HrSOo
2. Harmful effectshave been observed in fishes,
{Su lfurous (Su Ifu ric
c r abs , s hr im p and z ooplank t o n s .
acid) a ci d )
3. Ther e oc c ur s a reduction in the
photosynthetic efficiency of phytoplankton NO* + Or+ HrO -------+ HNO2 + HNO,
(Nitrous ( N i tr i c
M EASURES TO CO HTRO L acid) a ci d )
O ZO NE DEPLETI O N
In the development of acid rain, there occurs
The only effective way of controlling ozone diffusion of SOr, and diffusion of HNO, gas into
depletion is the complete elimination of the factors the cloud particles/droplets.
responsible for it. As already described oxides of
nitrogen, chlorofluorocarbons and halons largely EFFECTS OF ACID RAIN
contribute to ozone destruction. These are the
A c i d r a i n d i s t u r b s t h e e n v i r o n m e n t . T h e a ctu a r
env ir onm ent al pollut ant s and r e d u c t i o n i n t h e i r
effects of acid rain depends on the degree of the
pr oduc t ion/ ut iliz at ionwill lar g e l y h e l p t o c o n t r o l
acidity and the nature of the environment-aquatic,
oz one deplet ion.
terrestrial,human population and materials. Some
of the important aspects are described.
ON
BIOTEGI{NOLOGY
AruDSOGIETY
,\ dvan ce s in biot ec hnology , and lhe i r Ii is a fact that modern technology in various
/-a .rpp lica tion s ar e nr os t f r equent ly as s oc . r a t e d f o r n r s i s w o v e n t i g h t l y i n t o t h e f a b r i c o f o u r l i v e s
' ,iith con troversies Bas ed on t heir per c ept ion t o O u r d a y - t o - d a y l i f e i s i n s e p a r a b l ef r o m t e c h n o l o g y .
: r io techn olo gy,th e people m ay be gr ouped int o l m a g i n e l i f e a b o u t l - 2 c e n t u r i e s a g o w h e r e t h e r e
' r rp e h rn :d r-rl e o nr ies w a s n o e l e c t r i c i t y ,n o r u n n i n g w a t e r , s e w a g e i n t h e
streets,unpredictablefood supply and an expectecl
1 Strong opponents who oppose the new
years Undoubtedly,
- ..clrno log y,a s it will giv e r is e t o pr oblem s , is s u e s l i f e s p a n o f l e s s t h a n 4 0
technology has largely contributed to the present
- :rcl co ncern s h um ans hav e nev er f ac ed bef or e day world we Iive in. Many pepople consider
- .e v
con sid er biot ec hnology as an unnat ur a l
biotechnology as a technology that will improve
- -,in ipu lativetechnology
the quality of life in every country, besicles
) Strong proponents who consider that the m a i n t a i n i n g I i v i n g s t a n d a r d sa t a r e a s o n a b l yh i g h e r
- :techn olo gy w ill pr ov ide unt old benef it s t o tevet.
. iie ty. The y a rgue t hat f or c ent ur ies t he s oc ie t y
- -. safe ly u se d t he pr oduc t s and pr oc es s es o f The probable positive and negative effects oi
,:e cnno log y. b i o t e c h n o l o g y ,w i t h s p e c i a l r e f e r e n c et o d e v e l o p i n g
c o u n t r i e s a r e g i v e n i n T a b l e 6 1. 1
) A neutral group ol people who [ave a
- . .:nced ap pro ac h t o biot ec hnology This gr or - r p
ELSI OF tsIOTEGHNOLOGY
e \ es th at res ear c h on biot ec hnology ( wit h
---. rtory system s ) ,and ex t ending it s f r uit s t o t h e Wh y s o m u c h u p r o a r a n d n e g a t i v i t y t o
. ...'\ sh ou ld be pur s ued wit h a c aut io u s b i o t e c h n o l o g y ?T h i s i s m a i n l y b e c a u s e t h e m a j o r
' -.rach The r is k s and benef it s of t h e p a r t o f t h e r n o d e r n b i o t e c h n o l o g y d e a l s w i t h
: r)l)nrcntsof biot ec hnology m ay not be m uc h g e n e t i c m a n i p u l a t i o n s . T h e s e u n n a t u r a l g e n e t i c
.'--':,n tfrom tha t of any ot her br anc h of s c ience . m a n i p u l a t i o n s ,a s m a n y p e o p l e f e a r , m a y l e a d t o
u n L n C r WnC O n S e q U e n C e s .
E ENEFITS OF BI O TECHNO LO G Y
ELSI is the short form to represent the ethical,
- -.' irr,rits legal and social implications of biotechnology. ELSI
o f biot ec hnology ar e benef ic ial t o t h e
- i,i he alth c ar e,agr ic uit ur e, f ood pr oduc iio n , broadly covers the relationship between bio-
-. -f,ctureof indus t r ialenz y m es and appr opr ia t e t e c h n o l o g y a n d s o c i e t y w i t h p a r t i c u l a r r e f e r e n c et o
- -me nta l managem ent . ethical and legal aspects.
739
744 B IOTECHNO LO CY
r e s e a r c hd e a l i n g w i t h D N A m a n i p u l a ti o nw e r e ve r y
Tnsre61.2 The probablc positive and negative s t r i n g e n ti n t h e e a r l i e r Y e a r s
effects of biotechnology (particularly in the
So far, risk assessmentstudies have failed to
developingcountries)
demonstrateany hazardous propertiesacquired bv
Positiveeffects host cells/organismsdue to transferof DNA. Thus,
the fears of genetic manipulations may be
. lmprovedhealthand life span(throughmoreelfective
unfounded to a large extent. Consequently, there
vaccines
pharmaceuticals, etc.).
has been some r e l a x a t i o n i n t he r e g u l a to r y
. increasedcropproductionandmoreiood. g u i d e l i n e sf o r r e c o m b i n a n tD N A r e s e ar ch( Fo r m o r e
. Decreased
dependence and d e t a i l s , R e f e r C h a P t e r 6 ) .
on importof food,fertilizers
chemicals
I t i s n o w w i d e l y a c c e p t e dt h a t b i ote ch n o l o g y i s
. Enhanced production
biomass for energy. c e r t a i n l y b e n e f i c i a l t o h u r n a n s . B u t i t sh o u l d n o t
. Useo{ plants as bioreactors.
andanimals cause problems of safety to people and
environmerrta , n d c r e a t eu n a c c e p t a b leso ci a l , m o r a l
. lmproved facilities.
toodstorage
and ethical issues. The public fe a r s o f
. Increased
production
ancjhealthof ltvestock. b i o t e c h n o l o g y ,b e s i d e s s o m e o f t h e r i sks,so ci a l a n cl
ethical issuescan be better understoodwith special
Negativeeffects
reference to the follorving
. More.dependence cn developedcounlriesand private
lor and
technology resources 1 T h e r a p e u t i c p r o d u c t s f o r u s e i n h e a l th ca r e
companies
. Increase
in unemployment is
as the labourrequirement 2 . C e n e t i c m o d i f i c a t i o n s o f f o od s a n d fo o d
less i n g r a d i e n t sa n d t h e i r c o n s u m p t i o n .
. Reduction
in natural ecosyslems. 3 R e l e a s eo f g e n e t i c a l l y e n g i n e e r e do r g a n i sm s
andnatural
biodiversity
. Morelegalandfinancial
complications. into the environment.
Germ 'line gene therapy : This is not being body. B y thi s approach,i t mi ght be one day
c ar r ied out at pr es entdue t o t e c h n i c a l , e t h i c a l a n d possi bl eto repai rti ssuedamagesan d cur e m anv
s oc ial r eas ons .M anipulat io n o f g e r m c e l l s w i l l l e a d di seasese.g. P arki nson'dissease, m ellit us
di ab et es
to serioi.rsproblems and complications.
At one international meeting on biotechnology
the following was the final message given to
biotechnology companies on the applications of
genet ic engineer ingt o hum a n s .
'Provide the information and listen to the Biotechnologyinvolves the production of a large
publiC. n u m b e r o f c o m m e r c i a l p r o d u c t s of e co n o m i c
importance. Broadly, biotechnology inventions can
HUMAN EMBRYONIC STEM CELL be categorized into two forms - products and
RESEARCH orocesseS.
The hum an em br y onic s t e m c e l l ( E S C ) l i n e s
BIOTECHNOLOGY PRODUCTS
wer e es t ablis hec iin Nov em b e r 1 9 9 8 . T h e s e c e l l s
are capable c-rfgiving rise to any human cell type. 1. living entities : The various forms of life-
ESC lines open t he pos s ibi l i t i e so f t r e a t i n g d i s e a s e s existing and life-supporting systems derived from
with cell therapy. f)isordersthat involve the loss of l i v i n g o r g a n i s m s a r e r e g a r d e d a s l i vi n g e n ti ti e s.
nor m al c ells s uc h as diabe t e s m e l l i t u s , P a r k i n s o n 's T h e s e i n c l u d e m i c r o o r g a n i s m ,a n i m a l s a n d p l a n ts,
dis eas e, Alz heim er ' s dis e a s e c o u l d p e r h a p s b e c e l l l i n e s , o r g a n e l l e s ,p l a s m i d s a n d g e n e s.
corrected with celltherapy. This may however, take
m or e t im e. 2. Naturally occurring products : The primary
a n d s e c o n d a r y m e t a b o l i t e s p r o d u c e d b y l i vi n g
There are many ethical and legal issuesinvolved s y s t e m se . g . a l c o h o l , a n t i b i o t i c s .
in ESC research, besides several obiections raised li
of hairy-cell leukemia, a rare form of cancer. His the researchers.Watson and Crick who discovereo
spleen was removed, and Mo cell lines developed. DNA structure never had a thought of patenting!
These cell lines were patented by the researchers
The situation has now changed. A good
and their associatedorganizations.Moore, however
proportion of researchis either being conducted or
was not involved in the patenting.
f i n a n c i a l l y s u p p o r t e d b y p r i v a t e co m p a n i e s/
Moore filed a suit in the court on the ground universities.lt is quite natural for these companies
that he should also be a party to the profits derived to expect returns for their investments.There is a
by us ing his t is s ue.The c our t a l l o w e d h i s c l a i m t o lot of change in the attitude of researchersalso.
share the profits. For many scientists, financial gains have become
more important than academic recognition.
PLANT BREEDERS' RI G H T S Consequently, some people carry out research
secretly and opt for patenting of their discoveries
Agriculture for the first time was included (in
ratherthan publishing.
1994) in the trade-related intellectual property
rights (TRIPS). TRIPS is a major concern for We have to accept the fact that the progressof
dev eloping c ount r ies . r e s e a r c h i s u s u a l l y m u c h f a s t e r i n p r i va te
companies compared to many government
The plant varieties in many countries (not in
o r g a n i z a t i o n s . T h i s m a y b e d u e t o th e h i g h e r
India) are protected through plant breeders' rights
f i n a n c i a l r e t u r n s a n d e f f i c i e n t a d m i n i s t ra ti o n .
(PBR) or plant variety rights (PVR). PVR provides
legal protection to the original breecier,orowner of Probably, the biotechnological research might
t he plant v ar iet y . lt is belie v e d t h a t P B R w i l l not have progressed to the same extent as it is
encourage innovative research and plant breeding today without the dreams of getting the patents by
programmes, in view of the expected financial scientistsand researchfunding organizations.
tl
ill
if
returns.
747
-fl"he cell is t he s t r uc t ur al and f unc t ional un i t o f 1 . Prokaryotes (Creek : pro-before; karyon -
I tife. tt may be also regardedas the basic unit of n u c l e u s )l a c k a w e l l d e f i n e d n u c l e u s a n d p o s s e s s
biological activity. relatively simple structure These include the
various bacteria.
Th e co ncept of c ell or iginat ed f r om t h e
con tribu tion s of Sc hleiden and Sc hwann ( 18 3 8 ) . 2. Eukaryotes(Creek : eu-true; karyon-nucleus)
However, it was only after 1940, the complexities possess a well defined nucleus and are more
of cell structure were exoosed c o m p l e x i n t h e i r s t r u c t u r ea n d f u n c t i o n . T h e h i g h e r
organisms (animals and plants) are composed of
PROKARYOTI C AND eukaryotic cells
EUKARYOTIC CELLS
A comparison of the characteristics between
The cells o f t he liv ing k ingdom m ay be div i d e d prokaryotes and eukaryotes is listed in Table
into two categories 62.1.
S iz e Small 1 10pm)
(generally Large
(generally pm)
10-100
Cy t oplas m Organelles
andcytoskeleton
absent Contains
organelles
andcytoskeleton
(anetwork
oftubules
andfilaments)
749
750 BIOTECHNOLOCY
Plasmamembrane
Mitochondrion
Lysosome
Vacuole
Rough- Ribosomes
endoplasmic Nucleus
reticulum
Nucleolus
Golgi
apparatus Smoothendoplasmic
reticulum
Peroxisome
Cytosol
Cytoskeleton
1.0 x 3 pm. About 2, 000 m it oc hondr ia, oc c u p y i n g disrupted to form small vesicles known as
about l/uth of the total cell volume, are present in microsomes. lt may be noted that microsomes as
a typical ce ll. such do not occur in the cell.
The mitochondria are composed of a double The smooth endoplasmic reticulum does not
membrane system. The outer membrane is smooth c o n t a i n r i b o s o m e s .l t i s i n v o l v e d i n t h e s y n t h e s i so f
a nd co mple t ely env elops t he or ganelle. The i n n e r l i p i d s ( t r i a c y l g l y c e r o l s p
, h o s p h o l i p i d s , s t e r o l s )a n d
membrane is folded to form cristae (lafin-crests) metabolism of drugs, besides supplying Ca2+ for
which occupy a larger surface area. The internal t h e c e l l u l a r f u n c t i o n s .
chamber of mitochondria is referred to as matrix.
Golgi apparatus
The components of electron transportchain and
oxidative phosphorylation (flavoprotein, Eukaryotic cells contain a unique cluster of
cytochro mes b, c r , c , a and a, and c o u p l i n g membrane vesicles known as dictyosomes which,
factors) a re bur ied in t he inner m it oc ho n d r i a l i n t u r n , c o n s t i t u t e C o l g i a p p a r a t u s ( o r C o l g i
me mbra ne . The m at r ix c ont ains s ev er al en z y m e s c o m p l e x ) . T h e n e w l y s y n t h e s i z e d p r o t e i n s a r e
concerned with the energy metabolism of handed over to the Golgi apparatuswhich cataryse
ca rbo hydra t es ,lipids and am ino ac ids ( e. g., c i t r i c the addition of carbohydrates, lipids or sulfate
acid cycle, B - ox idat ion) .The m at r ix enz y m e s a l s o m o i e t i e s t o t h e p r o t e i n s . T h e s e c h e m i c a l
pa rticip ate in t he s y nt hes is of hem e and u r e a . modifications are necessary for the transport of
Mitocho nd ria ar e t he pr inc ipal pr oduc er sof A T P i n proteins across the plasma memorane.
the aerobic cells. ATP, the energy currency,
generated in mitochondria is exported to all parts Cer:tainproteins and enzymes are enclosed in
of th e cell to pr ov ide ener gy f or t he c ellular w o r k . membrane vesiclesof Colgi apparatusand secreted
from the cell after the appropriate signals. The
Th e mito c hondr ial m at r ix c ont ains a ci r c u r a r digestiveenzymes of pancreasare produced in this
do ub le-stran ded DNA ( m t DNA) , RNA a n o f a s h i o n .
ribosomes. Thus, the mitochondria are equipped
with an independent pr ot ein s y nt he s i z i n g Colgi apparatus are also involved in the
machinery. lt is estimated that about 10% of the membrane synthesis,particularly for the formation
mitochondrial proteins are produced in the o f i n t r a c e llular organelles (e.g. peroxisomes,
mitocho nd ria. lysosomes).
A large portion of the ER is studded with The pH of the lysosomal matrix is more acidic
ribosomes to give a granular appearance which is (pH < 5) than the cytosol (pH-7) and this facilitates
'eferred to as rough endoplasmic reticulum. the degradation of different compounds. The
Ribosomesare the factoriesof protein biosynthesis. lysosomal enzymes are responsiblefor maintaining
)uring the processof cell fractionation, rough ER is t h e c e l l u l a r c o m p o u n d s i n a d y n a m i c s t a t e ,b y t h e i r
752 B IOTE CHNO LO CY
Cell wall
Cell membrane
Nucleus
Smoothendoolasmicreticulum
Roughendoplasmicreticulum
Mitochondrion Nucleolus
Chloroplast
Centralvacuole
Cellsap
Tonoplast Golgiapparatus
Microfilaments
Cytoplasm
Ribosomes
i s i nextensi bland
e determi nes
the fi nal shaoeano
Tlorr 62.2 A comparison between plant and si zeof the pl antcel l .
animal cells
B esi descel l ul oseand l i gni n, hemi cel l ul oses,
Character Plant cell Animal cell pecti ns,and extensi ns(ol i gosacchari des)are al so
Size Generally
larger Smallerthan oresenti n the cel l w al l
plantcells
C hl oropl asts
Cellwall Sunounded by rigid Absent
cellwallof cellulose The chl oropl asts are found onl y i n the pl ant
cel l s, and are the si tes of photosynthesiThe s.
Vacuole Matureplantcells Absent
generalterm plastidsis often used to collectively
possess a rargevacuore
representchl oropl asts(greenpl asti dscontai ni ng
Plastids Chloroplasts found Absent chlorophylls) , chromoplasfs(yellow to reddish
in greenplants col our pl asti ds contai ni ng carotenoi ds)and
Celldivision Divisionof cytoplasm Division occurs leucoplasts(colourless plastids).
duringcelldivision by constriction
C hl oropl asts
have a doubl e membranesystem.
occursby theformation
Internally, chloroplasts containa systemof flattened
of a cellplate
membranousdiscs called thvlakoids. Piles of
thyl akoi dsare l ocatedi n the centralregi oncal l ed
Ge ll wall stroma.C hl orophyl lthe, green
sunl i ghtcapturi ng
pi gmenti s presenti n the thyl akoi ds.
Th e pla nt c ell wall is us ually r igid, non- liv i n g
an d p erme ab lec om ponent , s ur r oundingt he pla s m a Vacuoles
membrane. Cell walls are of two types - primary
P l antcel l shave membrane-bound, l i qui d fi l l eo
and secondary.
vesi cl escal l ed vacuol es,w hi ch may occupy as
fhe primary cell wall is the one that is formed much as 90"k ol the cells total volume. The
d urin g the co ur s e of c ell div is ion. lt is m a i n l y vacuoles may contain wide range of dissolved
comp osed o f c ellulos e. and is f lex ible in natu r e . mol ecul es suchas sal ts,sugars, pi gments and toxrc
The secondary cell wall is rigid and more complex wastes.The pigmentsof vacuolescontributeto the
in na ture . Ch em ic ally , it is m ade up of m o r e coloursof flowersand leaves.The physicalsupport
cellu lose, an d h igh c ont ent of lignin. Lignin is t h e of pl ant ti ssuescomes from the hi gh i nternal
major component of wood. The secondarycell wall pressureof water maintainedwithin the vacuoles.
3 ctechnology [48]
( Cr eek , m ikr o s - s m a l l ; b i o s - l i f e ) c e l l w a l l c o m p a r a b l e t o t h a t f o u n d i n p l a n ts. Th e
[ / ic r obiology
I Y I is the science of small or microscopic a v e r a g e s i z e o f a b a c t e r i u m i s a r o u n d 2 p m Th e
organisms. The most important microorganisms b a c t e r i a m a y b e s p h e r i c a l , r o d - l i k e , s p i r a l l y co i l e d
relevant to biotechnology include bacteria, fungi, o r f i l a m e n t l i k e . C e r t a i n b a c t e r i a m a y o ccu r i n
and v ir us es . M ic r oor ganis m s a r e v e r y w i d e l y more than one form. A typical structure of a rod-
distributed, and are found almost everywhere rn shaped bacterium is depicted in Fig. 63.1 .
nature. ln general, the conditions for their growth
and m ult iplic at ion( f ood,t em p e r a t u r e ,m o i s t u r ee t c . ) Gram-positive and
ar e s im ilar t o t hat of hum ans . H e n c e , t h e y a r e m o s t Gram'negative bacteria
abundantly present at places wl-rerepeople live. B a s e d o n t h e r e s p o n s e t o C r a m 's sta i n , th e
A very brief description of the microorganisms, bacteria are grouped as Cram +ve or Cram -ve. By
relevent to biotechnology is given in this chapter. C r a m 's s t a i n , s e v e r a l d i s t i n g u i s h i n g fe a tu r e s o f
bacteria can be identified. For instance,Gram +ve
bacteria possess single-layered cell wall while
Cram -ve bacteria have a double-layered one.
754
Chapt er63 : M IC R OOR C A N IS M S 755
Cellwall
Plasmamenrbrane
Flagellum
Ribosonres
Food Cytosol
Plasmid
reserve
Pillus
causedby microorganisms
Mycology,a branchof microbiology, dealslvith
Diseasesof humans Pathogenic microorganism the studyof fungi. Fungiare a groupof eukaryotic,
Tuberculosis acteriun tubercuIosis
Mycob heterotrophicorganisms.They are dependenton
organi ccompoundsfor thei r nutri tion.I n gener al,
Lepr0sy M. leparae
fungi can w i thstand extreme envir onm ent al
Typhoid Salmonella
typhi conditionsbetterthan mostof the microorganisms.
Cholera Vibriocholerae
C H A R A C TE R IS TIC S OF FU N GI
Diptheria Corynebacteriu ae
n diphtheri
Syphillis pallidum
Treponema Fungicells are usuallylargerthan the bacteria.
Thesi zesmay range.l-5 pm i n w i dth and 5- 35 pm
Tetanus Clostridiuntetani i n l ength. Fungal cel l s may be elongat edor
Bacterial
dysentery Shigelladysenteriae spheri cal .
Gonorrhoea gononhoeae
Neisseria sincethey cannot
The fungi are heterotrophic,
s'ynthesizetheir own food from the inorganic
Diseasesof animals
compounds.
Anthrax Bacillusanthracis
Pneumonia Dipiococcus
oneunoniae IMP OR TA N C E OF FU N GI
Blacklegol cattle Clostridium
chauvei As is the case with bacteria,fungi are both
fri endsand enemi esof humans.
Diseasesof plants
galls
Crown Agrobacterium
tumefaciens Beneficial aspects of fungi
Pearblight Pseudomonas
amylovorus The yeasts are useful for the following
Ringrot of potato Corynebacte
riun sependonicun . Alcohol fermentation e.g. Saccharomyces
Citruscanker Xanthomonas citri cerevlslae
a e.g. Candidasp.
Citric acid fermentation
s y m b i o ti c n i tro g e n -fi x ingbacteri a. In addi ti on, a Baker'syeaste.g. S. cerevisiae.
nitrifying bacteria (convert ammonia to nitrates) fhe application of molds are listed
and ammonifyingbacteria (convertamino acidsto
ammonia)also contributeto agriculturalpractices. a Productionof enzymese.g. Aspergillussp.
o Citric acid fermentatione.g. Aspergillusniger.
GONTBOL OF PATHOGENIC BACTERIA
a Penicillinproductione.g. Penicilliumnotatum.
As alreadystatedabove,bacteriacauseseveral o e.g. Rhizopussp.
Steroidtransformation
diseases.l'here are differentaooroaches
to control
pathogenicbacteria a Cluconic acid productione.g. Aspergillusniger.
. Use of antibiotics
Harmful aspects of fungi
. Treatment
with antiseptics
and disinfectants. . Spoilage of foods e.g. moldy bread, rot of fruits
. Sterilization and autoclaving and vegetables.
. Radiation treatment o Deterioration of textiles made up of cotton.
. Biologic al c ont r ol. . Dama8e to paper.
In addition to the above, vaccines have been . Diseasescaused by fungi e.g. ring worm of the
developed against certain pathogens to control scalp in children caused by Microsporum
dis eas esin hum ans and a n i m a l s . audouinii.
Chapt er63 : MIC R OOR C A N IS M S 757
CONTROL O F FUNG I
TffiLr63.2 A selectedlist of conrmon
Th e fu ng al gr owt h c an be c ont r olled by us i n g
viral diseases
p he no l a nd its der iv at iv ese. g. c r es ol, et hy lphe n o l ,
pro pylph en ol, b ut y lphenol. Chlor ine and c hlo r r n e Diseasesof humans Virus
co mpo un ds are als o us ef ul in t his r egar d.
Common cold Rhinoviruses
VegetativecelJsof yeastsand other fungi can be pox
Sntall Pox virus
destroyedby moist heat at 50-60'C for about 5-10
Rabies Rabiesvirus
min ute s. Sp or es , howev er r equir e hig h e r
temperature 20-80"C.
Infectious
hepatitis Hepatitisvirus
Measles Measlesvirus
Poliomyelitls Poliovirus
Infiuenza lnfluenzavirus
A virus m ay be r egar ded as a s n r a l l
Encaphalitis Encephalitis
virus
rricrocrga nisrnw it h a nuc leopr ot ein ent it y whi c h MY'p1 Mumpsvirus
lives an d multi plies in t he liv ing c ells of ot h e r
Diseasesof plants
o rga nisms.Th us , v ir us es as ar e unable t o liv e in a
cell-free environment, ar-rd become active and Tobacco
mosaic Tobacco
mosaicvirus
nru ltiply a s the y ent er iiv ing c ells . Potatoleafroll Potatoleaf roll virus
leafcurl
Tomato Tonatoleafcurl virus
CHARACTERIS TI CS O F VI RUSES
t--,
I ii l
NU
Aldehyde R-C-H Aldehyde glucose
Glyceraldehyde,
o Lr
I lI
,C -I
Keto R1-C-R2 Ketone Fructose.
sedoheptulose
o
I I
Carboxyl -C-OH R-C -.ali--l Carboxylic
acid Aceticacid.oalmitic
acid
o
-c-o-R1
i
Ester R2-C--O---R1 Ester Cholesterol
U o
i lt -
J ,R , li . fl l
Amido -C-N1 .R , R"-C-N( Amide N-Acetylglucosamine
',2
Pyran structure is found in hexoses. Pyridine and chemical properties. Isomerism is partly
ru cle us is a par t of t he v it am ins - niac in a n d responsihlefor the existenceof a large number of
c','rido xin e. Pyr im idines ( c y t os ine, t hy m ine) a n d organic molecules.
:.rrin es (ad en ine, guanine) ar e t he c ons t it uent so f
Consider the molecularformula-CrHuO.Thereare
-:cleo tide s an d nuc leic ac ids . I ndole r ing is f ou n d
two important isomers of this- ethyl alcohol
- the amin o a c id t r y pt ophan. Pur ine and indo l e
(C 2H ' OH )and di ethylether(C H TOC H T)
as show n
:.re examples of fused heterocyclic rings.
below :
ISOMERISM
H OH H H
Tre co mpo unds pos s es s ingident ic al m olec u l a r ltt I
':'r.,ulae but different structuresare referred to as H-C_G_H H_C-.O -c-
isomers. The phenomenon of existence of isomers
' ltl I
. called isomerism (Greek : isos-equal; meros-
HH F. H
::Tiri lsomers differ from each other in physical Ethylalcohol Diethylether
760 B IOTECHNO LO CY
o
(A)
o
Benzene Phenol Benzoquinone Naphthalene a-Naphthol
U
Naphthoquinone Anthracene Phenanthrene CycloPentane
(B)
[_\,
,\ //-\
\o / t*/
'*/
I I
H H
Furan Pyrrole Thiophene lmidazole
lttl
a-\
tI
NA
\o / \- t2 \,)
Fig. 64.1 : Common ting structures found in biomolecules (A) Homocyclic rings (B) Heterocyclic rings.
It must, however, be remembered that D- and Sorenson (1909) introduced the term pH to
L- do not representthe direction of the rotation of express H+ ion concentration . pH is defined as the
plane of polar iz ed light . negative logarithm of H+ ion concentration.
p H = - l o g [ H +]
Existence of chiral biomolecules
is d issolvedis s olut e and t he m edium t hat dis s o l v e s Colloidal state is characterizedby the particle size
the solute is referredto as solvent. The oarticle srze o f 1 t o 1 0 0 n m . Wh e n t h e p a r t i c l e s i z e i s <1 n m ,
.l
o f a solu te in s olut ion is < nm . i t i s i n t r u e s o l u t i o n . F o r t h e p a r t i c l e s i z e s>. 10 0 n m ,
t h e m a t t e r e x i s t s a s a v i s i b l e p r e c i p i t a t e .T h u s , t h e
The rela'tiveconcentrations of substancesin a
colloidal state is an intermediate between true
solution can be measured by several ways
solution and precipitate.
Per cent concentration : This representsparts
p er -l00 . Th e m os t f r equent ly us ed is weight p e r Phases of colloids : Colloidal state is hetero-
g e n eouswith two phases.
volu me (w/v) e. g. 9% s aline ( 9 gl1O 0 m l s olut i o n ) .
1. Dispersed phase (internal phase) which
Parts per million (ppm) : This refers to the
c o n s t i t u t e st h e c o l l o i d a l p a r t i c l e s .
n umb er o f p ar t s of a s ubs t anc ein one m illion p a r t s
of the so lutio n Thus 1O ppm c hlor ine m eans 1 0 p g 2. Dispersion medium (external phase) which
of chlorine in 1 g of water. refers to the medium in which the colloidal
p a r t i c l e sa r e s u s p e n d e d .
Molarity (M) : lt is defined as the number of
mole s of so lut e per lit er s olut ion. NaCl ha s a
Classification of colloids
mole cu lar wei ght of 58. 5. To get one m olar ( 1 M )
o r o ne mo le so lut ion of NaCl, one gr am m olec u l a r B asedon the affi ni tyof di spersi on medi umw i th
we igh t (58 .5 g ) of it s hould be dis s olv ed in t h e dispersed phase,colloidsareclassified as lyophobic
solvent (HrO) to make to a final total volume of .l and lyophilic colloids.
lite r. For sma ller c onc ent r at ions , m illim ole a n d
1. l-yophobic(Creek : solvent-hating) : These
micromo le are us ed
colloids do not have any attraction towaros
Molality : lt representsthe number of moles of di spersi onmedi um. W hen w ater i s used as
so lute p er 1,0 00 g of s olv ent O ne m olal s olu t i o n di spersi on
medi um,the col l oi dsare referredto as
ca n be p rep ar ed by dis s olv ing 1 m ole of s olu t e i n hydrophobic.
1 ,00 0 g o f sol v ent .
2. tyophilic (Greek : solvent-loving): These
Norma lity : M olar it y is bas ed on m olec u t a r col l oi ds have di sti nctaffi ni tytow ardsdi spersron
we igh t wh iie nor m alit y is bas ed on equiv a l e n t mediunr. The term hydrophilic is used for the
we igh t. One gr am equiv alent weight of an elem e n t col l oi dsw hen w ater i s the di spersi on
medi um.
or compound representsits capacity to combine or
rep lace 1 mole of hy dr ogen. B i ol ogi cal i mportance of col l oi ds
1 Biologicalfluids as colloids: Theseincluoe
COLLOIDAL STATE bl ood,mi l k and cerebrospi nal
fl ui d.
Th oma s Craham ( 1861) , r egar dedas t he ' f a t h e r 2. Biologicalcompoundsas colloidalparticles:
of collo ida l che m is t r v ' ,div ided s ubs t anc esint o t w o The compl exmol ecul es of l i fe,the hi gh mol ecul ar
classes-crystalloids and colloids. w ei ght protei ns, compl ex l i pi ds and
pol ysacchari des
exi sti n col l oi dalstate.
Crystallo idsar e t he s ubs t anc eswhic h in. s olu t i o n
can freely pass (diffuse)through parchment"mem- 3. Fat digestionand absorption: The formation
bra ne e .g. su ga r ,ur ea, NaCl. Colloids ( G r eek : g l u e - of emul si ons,faci l i tatedby the emul si fyi ng
agents
like), on other hand, are the substancesthat are bile salts,promotesfat digestionand absorptionrn
reta ine db y p ar c hm entm em br ane e. g. gum , gel a t i n , the intestinaltract.
a lbu min. Th e a bov e c las s if ic at ionof Cr aham is n o 4. Formationof urine : The filtrationof urine is
lon ge r ten ab le, s inc e any s ubs t anc e c an b e basedon the pri nci pl eof di al ysi s.
co nverte d in to a c olloid by s uit able m eans . F o r
in sta nce, sod ium c hlor ide in benz ene f or m s a D IFFU S ION
co lloid .
The mol ecul es i n l i qui ds or gases are i n
Colloidal state : As such, there are no group of continuousmotion. Diffusion may be regardedas
substancesas colloids, rather, substancescan exist the movement of solute molecules from a higher
in the form of colloidal state or colloidal system. concentration to a lower concentration Diffusion
7fi4 B IOTECHNO LO CY
i s m o re ra p i d i n g a s e sth a n i n l i qui ds.The smal l er 2. Redblood cellsand fragility: When the RBC
particlesdiffusefasterthan the largerones. The are kept in hypotonic solution(say0.4% NaCl),the
greaterthe temperature, the higher is the rate of cells bulge due to entrv of water which often
d i ffu s i o n . causes rupture of plasma membrane of RBC
(hemolysis).
Applications of diffusion
3. Transfusi on: l sotoni c sol uti o nsof NaCl
1. Ex c h a n g e o f O , a n d C O, i n l ungsand i n (0.9%)or glucose(5'k) or a suitablecombinationof
ti s s u e so c c u rsth ro u g hd i ffu s i on. these tw,o are commonly used in transfusionin
hospitalsfor the treatmentof dehydration,burns
2. Certainnutrientsare absorbedby diffusionin
etc.
the gastrointestinal tract e.g. pentoses,minerals,
w a te r s o l u b l ev i ta m i n s . 4. Action of purgatives: The mechanismof
acti on of purgati vesi s mai nl y due t o osm ot ic
3. Passage of the wasteproductsnamelyammo-
phenomenon. epson(Mg SO oTHr O )
For i nstance,
n i a , i n th e re rra tu l b u l e so c cursdue to di ffusi on.
or Gl auber' (N
s arS O*10H 2O)sal tsw i th dr aww'at er
from the body, besidespreventingthe intestinal
osMosts water absorption.
Osmosis (Greek : push) refersto the movement
5. Edemadue to hypoalbuminemia : Disorders
of solvent (most frequently water) through a
semipermeable membrane.
such as kw ashi orkorand gl omerul onephr itar is e
associ ated w i th l ow ered pl asma album r n
The flow of solvent occurs from a solution of concentrationand edema. Edema is caused by
low c onc ent r at ion t o a s o l u t i o n o f h i g h reducedoncoti cpressure of pl asma,l ea dingt o t he
concentration, when both are separated by a of excessfl ui d i n ti ssuespaces.
accumul ati on
s emioer m eable m em br ane.
vrscosrrY
Osmotic pressure
Li qui dor fl ui d hasa tendencyto fl ow which is
Osmoticpressure
maybe definedasthe excess referredto as fluidity.The term viscositymay be
pressure that must be applied to a solution to defined as the internal resistanceoffered by a
prevent the passage of solvent into the solution, liquid or a gas to flow. fhe propertyof viscosityis
when both are separated by a semipermeable due to frictionalforcesbetweenthe laverswhile
m em br ane. their movementoccurs.
The s olut ions t hat ex er t t h e s a m e o s m o t i c V i scosi tyof col l oi dal sol uti ons,par t icular ly
oressure are said to be isoosmofic. The term l yophi l i c col l oi ds,i s general l yhi gher t han t r ue
isotonic is used when a cell is in direct contact sol uti ons.
wit h an is oos m ot ic s olut ion ( 0 . 9 % N a C l ) w h i c h
does not change the cell volume and, thus, the cell Unitsof viscosity: The unit of viscosityis poise,
t one is m aint ained. A s olu t i o n w i t h r e l a t i v e l y who firstsystematically
afterthe scientistPoiseuille,
greater osmotic pressure is referred to as studied the flow of liquids. A poise represents
hypertonic. On the other hand, a solution with dynes/cm2.
relatively lower pressure is hypotonic.
Applications of viscosity
Units of osmotic pressure : Osmotic pressureof
biologic al f luids is f r equ e n t l y e x p r e s s e d a s 1. Viscosityof blood : Blood is about 4 times
milliosmoles. The osmotic pressure of plasma (of rnoreviscousthan water.The viscosityof blood is
hum an blood) is 280- 300 m i l l i o s m o l e s / l . mainly attributedto suspendedblood cells and
col l oi dalpl asmaprotei ns.
Applications of osmosis
2. Viscositychangein muscle: Excitation of the
1. Fluid balanc e and bloo d v o l u m e : T h e f l u i d muscleis associated with increasein the viscosity
balance of the different compartments of the body of the muscl efi bres.Thi sdel aysthe ch angein t he
is m aint ained due lo os m os i s . tensionof the contractingmuscle.
Chapt er64 : BIOOR C A N IC
A N D BIOP H Y SIC ACLH E MIS TR A
YN. D B IOC H E MIS TRTOOLS
Y 765
ll
I tic
"iu.n
---|> Paper chromatography
{---]r
SingledimensionalTwodimensional
I nscenoing
IL
Descending
Thin layerchromatography
Gas-liquidchromatography
2. The precursor-product relationshipin several related compounds from a mixture. These include
metabolic pathways has been investigatedby proteins,peptides,amino acids, lipids, carbohy-
radioisotopes. e.g. Krebscycle, p-oxidationof fatty drates,vitaminsand drugs.
acids,urea cycle, fatty acid synthesis.
Principles and classification
3. Radioisotopes are convenientlyused in the
studyof metabolicpools (e.g.aminoacid pool)and Chromatography (Creek : chroma- colour; gra-
metabolicturnovers(e.g.proteinturnover). phein- to write),usuallyconsistsof a mobile phase
a stationaryphase.The mobile phaserefersto
4 . C e rta i n e n d o c ri n e a nd i mmunol ogi cal and
(to dissoved
studiesalsodependon the useof radioisotopes e.g. the mixtureof substances be separated),
in a phaseis a porous
!iquidor a gas.The stationary
radioimmunoassay.
solidmatrixthroughwhich the samplecontainedin
5. Radioisotopes are employed in elucidating the mobile phase percolates.The interaction
d ru g m e ta b o l i s m. betweenthe mobileand stationary phasesresultsin
the separation of the compoundsfrom the mixture.
These interactions include the physico-
chemical principlessuch as adsorption,partition,
ion-exchange,molecular sieving and affinity.
The foundations for the present(and the future,
The interactionbetweenstationaryphase and
of course!)knowledgeof biochemistry are basedon
mobilephaseis oftenemployedin the classification
the laboratorytools employed for biochemical
chromatographye.g. partition, adsorption,ion-
experimentation. The basic principlesof some of of chromato-
exchange.Further,the classification
the commonlyemployedtools are describedhere. graphy is also basedeither on the natureof the
stationaryphase(paper,thin layer,column),or on
CHROMATOGRAPHY the nature of both mobile and stationaryphases
Chromatography is one of the most usefuland (gas-liquidchromatography). n summary of the
popular tools of biochemistry.lt is an analytical differentmethods(classes) of chromatographyis
technique dealing with the separationof closely given in Fig. 54.3.
64 : B I OOR C A N IC
C hA P I CT A N D BIOP H Y SIC AL
C H E MIS TR A
Y N D B IOC H E MIS TRTOOLS
Y 767
1. Partition chromatography: The molecules 2. l soel ectri cfocussi ng: Thi s techni que i s
of a mixtureget partitionedbetweenthe stationary primarily based on the immobilization of the
p has eand m obilep h a s ed e p e n d i n o g n th e i rre l a ti v e moleculesat isoelectricpH during electrophoresis.
aninityto each one of the phases. StablepH gradientsare set up (usuallyin a gel)
coveringthe pH rangeto include the isoelectric
2. Adsorption column chromatography . pointsof the components
in a mixture.lsoelectric
Th e ads or bent ssu c h a s s i l i c a g e l , a l u mi n a , focussing
can be conveniently used for the
charcoal powder and calcium hydroxyapatite puri fi cati on of protei ns.
a r e pac k edint o a c o l u m n i n a g l a s stu b e . T h i s
servesas the stationaryphase.The samplemixture 3. lmmunoelectrophoresis: This technique
i n a s olv ent is l o a d e d o n th i s c o l u mn . T h e i nvol ves combi nati on of the pri nci pl es of
individualcomponentsget differentiallyadsorbed el ectrophoresi sand i mmunol ogi cal reacti ons.
on to the adsorbent. The elutionis carriedout by a lmmunoelectrophoresis is usefulfor the analysisof
b uf f er s y s t em ( m o b i l e p h a s e ). T h e i n d i v i d u al compl exmi xturesof anti gensand anti bodi es.
compoundscome out of the column at different
rates which may be separatelycollected and PHOTOMETRY-COLORIMETER
i d e nt if ied.F or in s ta n c e ,a mi n o a c i d s c a n b e AND SPECTROPHOTOMETER
i d e nt if iedbv ninh v d ri nc o l o ri me tri cme th o d .A n
Photometrybroadlydealswith the studyof tne
automatedcolumn chromatographyapparatus -
phenomenonof l i ght absorpti onby mol ecul esi n
fraction collector- is frequentlyused nowadays.
solution.The specificityof a compoundto absorb
3. High performanceliquid chromatography light at a particularwavelength(monochromatic
(HP t C) : I n g e n e ra l , th e c h ro ma to g ra p hi cligh! is exploitedin the laboratoryfor quantitative
te c hniquesar e s l o w a n d ti me c o n s u mi n g .T h e measurements. From the biochemist's perspective,
separationcan be greatly improvedby applying photometryforms an importantlaboratorytool for
h i g h pr es s ur ein th e ra n g e o f 5 ,0 0 0 -1 0 ,0 0 0p si accurateestimationof a wide varietyof compounds
(poundsper s qua rei n c h ),h e n c eth i s te c h n i q u ei s i n bi ol ogi calsampl es.C ol ori meterand spectro-
also referred(lessfrequently)to as high pressure photometerare the laboratoryinstruments usedfor
liquid chromatography. this purpose.T[ey work on the principlesdiscussed
below.
Moreinformation on chromatography with special
reference to gel-filtration chromatography, ion- W hen a l i ght at a parti cul arw avel engthi s
exchange chromatography, affinity chromatography passed through a solution (incident light), some
and hydrophobic interaction chromatography is amount of it is absorbedand, therefore,the light
givenunderdownstream processing (Chapter20). that comes out (transmittedligh0 is diminished.
The nature of light absorptionin a solution is
ELE CT RO P HO B ES IS governedby Beer-Lambertlaw.
that the amount of transmitted
rhe movement of charged particles (ions) in an ,,_,?"::': 11_states
light decreases.exponentially with an increase in
electric field resulting in their migration towaJs
the concentration of absorbing material (i'e' the
the oppositely charged electrode is known as
amount ligtrt absorbed depends on the
electrophoresis. Molecules with a net positiv; :f
conceltrlti3n of the absorbing molecules)' And
charge (cations) move towards the negative cathoie ,
--, according to Lambert's law, the transmitted light
\\'nile tnose wrtn net neEailve cnarSe (anronS/
exponentially with increase in the
migrate towards positive anode. Electrophoresisis a ilSases
\ltoety useo anatyncat tecnnrque ror Ine separalron
. :- amount of light absorbed is dependent on the
ot Dro tog rca tmo tec ut es s uc n as pr as m a pr oler ns' , , .:
t h i c k n e s so t t h e m e d i u m ) .
ttp op rote rnsa no rm m uno8t oouilns .
The ratio of transmittedlight (l) to that of
Different types of electrophoresis incidentlight (Io)is referredto as transmittance
(T).
I
1. Zone electrophoresis: An inert supporting T=
materialsuch as paperor gel are used. Io
768 B IOTECHNO LO CY
Principle
I zooo * to min
J R a d i o i m m u n o a s s a yc o m b i n e s t h e p r i n c i p l e s o f
radioactivity of isotopes and immunological
reactionsof antigen and antibody; hence the name.
Siotechnology [49]
77(, B IOTECHNO LO CY
E N Z Y M E.L IN KE D
IMMUNOSORBANT ASSAY
Enzyme-linked immunosorbant assay(ELISA)is
a non-isotopicimmunoassay. An enzymeis usedas
a label in ELISAin place of radioactiveisotope
employedin RlA. ELISA is as sensitiveas or even
more sensitivethan RlA. In addition,there is no
riskof radiationhazards(asis the casewith RIA)in
E L ISA .
Principle
E L ISA i s b a s e d o n t he i mmunochemi car F.d,fi]
principlesof antigen-antibody reaction.The stages
of ELISA,depictedin Fig. 64.6, are summarized. Fig. 64.6 : Diagrammatic representation of enzyme-
linked immunosorbant assay (ELISA).
1. The antibody against the protein to be
determinedis fixed on an inert solid such as
polystyrene. 5. The enzyme activity is determined by its
action on a substrateto form a product (usually
the protei n
2 . T h e b i o l o g i c asl a mp l econtai ni ng
coloured). This is related to the concentration of
to be estimatedis appliedon the antibodycoated
the protein being estimated.
surface.
3. The proteinantibodycomplexis then reacted Applications
with a secondproteinspecificantibodyto which
E L I S A i s w i d e l y u s e d f o r t h e d e t e r m i n a ti o n o f
an enzyme is covalentlylinked. Theseenzymes
small quantities of proteins (hormones, antigens,
must be easily assayableand produce preferably
antibodies) and other biological substances.The
coloured products. Peroxidase,amylase and
most commonly used pregnancy test for the
a l k a l i n ep h o s p h a ta saere c o mmonl yused.
d e t e c t i o n o f h u m a n c h o r i o n i c g o n a d o tr o p i n ( h C C
4 . A fte rw a s h i n gth e u n b oundanti bodyl i nked i n u r i n e i s b a s e d o n E L I S A .B y t h i s t e st, p r e g n a n c\
enzyme,the enzymeboundto the secondantibody can be detected within few days after conception
complex is assayed. E L I S Ai s a l s o u s e f u l f o r t h e d i a g n o s i sof AID S.
Iiving ma tler is t om pos c d of m ainly s ix CHEMICAL MOLECULES OF LIFE
Jhe
I elenrents- carbon, hydrogen, oxygen,
Life is composed of lifeless chemical molecules.
nitrogen, phosphorus and sulfur. These elements
A single cell of the bacterium, Escherichia coli
together constitute about 90% of the dry weight of
c o n t a i n s a b o u t 6 , 0 0 0 d i f f e r e n to r g a n i c c o m p o u n d s
ihe h uma n bo dy . Sev er al ot her f unc t ionally .1
It is believedthat man mav contain about 00.000
, mpo rtan t ele rne nts ar e als o f ound in t he c ells .
different types of molecules although only a few of
T hese includ e Ca, K, Na, Cl, M g, Fe, Cu, Co, 1, 7n,
them have been characterized.
F. N1o and Se.
Carb on is th e m os t pr edom inant and v er s at ile The organic compounds such as amino acids,
element of life. lt possessesa unique property to nucleotides and monosaccharides serve as the
iorm in finite nu m ber of c om pounds . This is monomeric units or building blocks of comptex
attributed to the ability of carbon to form stabte biomolecules-proteins, nucleic acids (DNA ano
cova len t b on ds a nd C- C c hains of unlim it eo RNA) and polysaccharides, respectively. The
ength. lt is estimated that about 90% of i m p o r t a n t b i o m o l e c u l e s ( m a c r o m o l e c u l e s )w i t h
:ompounds found in living system invariably their respectivebuilding blocks and major functions
: cnta in ca rbo n. are given in Table 65.1. As regardslipids, it may be
Pr ot ein Amino
acids Fundamentalbasisofstructure
and
function
ofcell(static
anddynamic
lunctions).
acid (DNA)
Deoxyribonucleic Deoxyribon
ucleotides Repository
of hereditary
information
acid (RNA)
Ribonucleic Ribonucleotides Essentially forprotein
required biosynthesis.
(glycogen)
Polysaccharide (Elucose)
Monosaccharides Storage
formofenergy
to meetshortterm
0eman0s.
L i pids glycerol
Fattyacids, Storage
formofenergylo meetlongterm
demands:structural
components ofmembranes.
771
772 B IOTE CHNO LO CY
Functions of carbohYdrates
participatein a wide range of
Carbohydrates
functi ons
Constituent Percent (%o) Weight (kg) 1. Theyarethe mostabundantdietarysourceof
Waler o t.o 40 energy(4 Cal/g)for all organisms.
Protein 17.0 11 2. Carbohydratesare precursorsfor many
Lipid 1 3 .8 9 organiccompounds(fats,amino acids).
Carbohydrate ' 1 .5 1 (as glycoproteinsand glyco-
3. Carbohydrates
Minerals o. l 4 lipids)participatein the structureof cell membrane
suchascel l grow th,adhesion
andcel l ul arfuncti ons
and ferti l i zati on.
noted that they are not biopolymersin a strict
sense,but majorityof them containfatty acids. 4. Carbohydratesalsoserveas the storageform
of energy(glycogen)
to meetthe immediateenergy
Structural heirarchy of an organism demandsof the body.
Oligosaccharides
OJigosaccharides (Greek : oligo-few) contain
Carbohydrates are the most abundantorganic
2-10 monosacchari demol ecul es which ar e
moleculesin nature.They are primarilycomposed
liberatedon hydrolysis.Basedon the number of
of the elemenrscarbon,hydrogenand oxygen.fhe
monosaccharide unitspresent,the oligosaccharides
n a me c a rb o h y d ra tel i te ra l l y means' hvdratesof
are further subdivided to disaccharides, tri-
carbon.'
saccharidesetc.
Carbohydratesmay be defined as polyhydroxy-
aldehydes or ketonesor compounds which produce Polysaccharides
them on hydrolysk. The term 'sugar' is applied to (Creek : poly-many)are poly-
Polysaccharides
carbohydrates unitswith high molecular
solublein water and sweetto taste. mersof monosaccharide
C h a pt er65 : B I O M O L E C U L ES 773
Functions of lipids a\
Lipidsperformseveralimportantfunctions
cH2-o- c-R1
fuel reserveof the
1. Theyare the concentrated
I
body (triacylglycerols). R'-c-o-?H
2. Lioids are tlre constituentsof membrane ll
structureand regulatethe membranepermeability CH2-O-C-R3
(p h o s p h o l i p i dasn d c h o l e sterol ).
Triacylglycerol
3 . T h e ys e rv ea s a s o u rceof fat sol ubl evi tami ns
(A, D, E and K). Fig, 65.2 : General structure of triacylglycerol.
4 . L i p i d s a re i mp o rtantas cel l ul ar metabol i c
regulators(steroidhormonesand prostaglandins)
Essential fatty acids
FATTY ACIDS The fattyacidsthatcannotbe synthesized by the
body and,therefore,shouldbe suppliedin the diet
Fatty acids are carboxylic acids with
are knownas essential fattyacids(EFA). Cfremically,
hydrocarbon sidechain.Theyarethe simplestform
they are polyunsaturatedfatty acids, namely
of lipids.
Iinoleic acid (18 : 2; 9, 12) and linolenic acid
Even and odd carbon fatty acids (18 : 3; 9,12, 15).A rachi doni caci d ( 20: 4; 5, B,
11, 14) becomesessenti ali f, i ts precur sorlinoleic
Most of the fatty acids that occur in natural acid is not orovided in the diet in sufficient
l i p i d sa re o f e v e nc a rb o n s(usual l y14C -20C )Thi s amounts.
is due to the fact that biosvnthesis of fattv acids
m a i n l y o c c u rsw i th th e s equenti aladdi ti onof 2 TRIACYLGLYCEROLS
c a rb o nu n i ts .P a l mi ti ca c i d (16C )and steari caci d
(18 C )a reth e m o s tc o mmon.A mongthe odd chai n Triacylglycerols (formerlytriglycerides) are the
fatty acids, propionic acid (3C) and valeric acid esters.ofglycerol with fatty acids.The fats and oils
(5 C )a re w e l l k n o w n . that are widely distributedin both plants and
ani mal sare chemi cal l ytri acyl gl yc er ols.
They ar e
Saturated and unsaturated i nsol ubl ei n w aterand non-pol ari n char act erand
fatty acids commonlyknown as neutralfats.
Saturatedfatty acids do not contain double Fatsas storedfuel : Triacylglycerols
arethe most
bonds,while unsaturated fattyacidscontainone or abundantgroupof l i pi dsthat pri ma r ilyf unct ionas
moredoublebonds.Bothsaturated and unsaturated fuel reserves of animals.The fat reserveof normat
fatty acids almost equally occur in the natural humans (men 20oh, women 25% by weight) rs
l i p i d s .F a ttya c i d sw i th o n e doubl ebondare know n sufficientto meetthe body caloricrequirements for
as monounsaturated and those with 2 or more 2-3 months.
d o u b l e b o n d s a re c ol l ecti vel y know n as
Structuresof acylglycerols: Monoacylglycerols,
polyunsaturated fatty acids (PUFA).
diacylglycero_lsand triacylglycerols,respectively'
Shorthand representation of fatty acids consistingof one, two and three moleculesof fatti'
acids esterifiedto a molecule of glycerol, are
Insteadof writingthe full structures, biochemists known.Amongthese,triacylglycerols are the most
employ shorthand notations (by numbers) to i mportantbi ochemi cal l y.
representfatty acids.The generalrule is that the
Triacylglycerolsof plants have higher content
total number of carbon atoms is written first,
of unsaturatedfatty acids comparedto that of
followed by the number of double bonds and
ani mal s.
fi n a l l yth e (fi rs tc a rb o n )p osi ti onof doubl ebonds,
startingfrom the carboxylend.Thus,saturated fatty
a c i d , p a l m i ti ca c i d i s w ri t tenas 16:0, ol ei c aci d P H OS P H OLIP ID S
a s 1 8 : 1 ; 9 , a ra c h i d o n i aci c d as 2O : 4:5, B , 11, These are compl ex or compound lipids
14. contai ni ngphosphori caci d, i n addit iont o f at t r
l r apt er 65 : B I O MOL E C U L ES 777
ca
20 zo
1B 25
17
27
o NHs
lBa
-ooc-cH2-cH2-cH-coo-
12. Glutamic
acid Glu E yCarboxyl
NH;
.----\
1 7. Ph en yla lanine
Phe (, >cH2-cH--coo- or phenyl
Benzene
\-// - |
I
NHi
'18.Tyrosine
,t ----\
lyr HO <' \,/.1
)-Cr-1.-cH-coo- Phenol
:l+
NHg
cH2--cFrcod
-l+
19.TryptophanTrp W Indole
NHi
Fis. 55.5) :
Peptide bond
- Primary str uc t ur e: The linear s equenc e o f
The amino acids are held together in a protein
,- ro acids form ing t he bac k bone of pr ot ein s
by covalent peptide bonds or linkages.These bonds
:,- 1rpe ptid es).
are rather strong and serve as the cementing
I Secon da ry s t r uc t ur e: The s pac i a l material between the individual amino acids.
.'-angement of protein by twisting of the
: ,- \pe ptid e cha in. F o r m a t i o n o f a p e p t i d e b o n d : Wh e n t h e
amino group of an amino acid combines with
,r Tertiary structure : The three dimensional
the carboxyl group of another amino acid, a
---rctlrre of a fu nc t ional pr ot ein. peptide bond is formed (Fig. 65.6). Note that a
-l Quaternary structure : Some of the proteins dipeptide will have two amino acids and one
, - = co ,np osed o f t wo or m or e poly pept ide c hain s peptide (not two) bond. Peptides containing
.l
to a s su bunit s .The s pac ial ar r angem ento f more than 0 amino acids (decapeptide) are
- 'e rre,lbu nits
' -:se:u is k nown as quat er nar y s t r uc t ur e referred to as polypeptides.
742 B IOTE C HNO LO CY
TE R TIA R Y S TR U C TU R E OF P R OTE IN
fhe three-dimensionalarrangement of protein
structureis referredto as tertiarystructure.lt is a
compact structurewith hydrophobicside chains
hel d i nteri orw hi l e the hydrophi l i cgroupsare on
the surfaceof the protein molecule.This type of
arrangement ensuresstabi l i tyof the mol ecul e.
Bonds of tertiary structure: Besides the
hydrogenbonds, disulfide bonds (-S-S), ionic
interactions(electrostatic
bonds)and hydrophobic
interactions
also contributeto the tertiarystructure
of oroteins.
Domains:The term domainis usedto reoresent
/" the basic units of protein structure(tertiary)and
N functi ons.A pol ypepti dew i th 200 ami no aci ds
t\: normallyconsistsof two or more domains.
i QU A TE R N A R Y S TR U C TU R E OF P B OTE IN
I
"- r---\r^r
A great majorityof the proteinsare composed
N
of singlepolypeptidechains.Someof the proteins,
?,t'i-
6 Hi /L however, consist of two or more polypeptides
\rH which may be identicalor unrelated.Suchproteins
' No/
\o are termed is oligomers and possessquaternary
j-----=-r- c C structure.The individual polypeptidechains are
known as monomersl protomers or subunits. A
) dimer consitsoI two polypeptideswhile a tetramer
has four.
Bondsin quaternarystructure: The monomeric
subunitsare held togetherby non-convalent
bonds
Fig. 65.7 : Diagrammatic representation of secondary namelyhydrogenbonds,hydrophobicinteractions
structure of protein - a right handed a-helix and i oni c bonds.
H
I
(l-lndicate -C-R groups of amino acids;
dotted coloured shadel are hydrogen bonds; Note
that only a few hydrogen bonds shown for clarity).
Proteinsare classifiedin severalways. Three
major types of classifyingproteinsbasedon their
5. o-Helix is a stable conformation formed functi on,chemi calnatureand sol ubi l i typroperti es
.pontaneously with the lowest energy. and nutritionalimoortanceare discussedhere.
Nucleoproteins,
Scleroproleins
Glycoproteins
Albumins Collagens Mucoproteins Coagulated Proteoses
Globulins Elastins Lipoproteins proteins Peptones
Glutelins Keratins Phosphoproteins Proteans Polypeptides
Prolamines Chromoproteins
Histones Metailoproteins l\4etaproteins Pantidaq
Globins
Protamines
Tetrapyrroles
Tnru 65.5 Classificatlon of terpenes wlth
selected examples The mosi abundant coloured comoound in the
world is chlorophyll, the photosynthetic pigment.
Class Basic structure Example There are different types of chlorophylls (c, d, e, a)
tsoprene Structure with slight variation in colours-green, greenish
units b l u e , g r e e n i s hy e l l o w .
Hemiterpenes 1 csHe rs0prene Structurally, chlorophylls are composed of
Monoterpenes 2 u1on16 Limonene tetrapyrroles (pyrrole rings) with their nitrogen
Sesquiterpenes nu
v15"24 Abscisic
acid iinked to magnesium.
Diterpenes A
czoHsz Gibberellin Tetrapyrrolesare also found in heme in certain
Triterpenes a u
v30"48 Stigmasterol p r o t e i n s .T h e s e i n c l u d e h e m o g l o b i n , c y t o c h r o m e s ,
?traterpenes n nu
v40"64 Carotenes catalase and peroxidase
Pclyterpenes n (c5Hs)n Rubber
Tetraterpenes (carotenoids)
unit. A majority of the isoprenoidsare formed by The colour of carotenoids is variable, generally
' oinin g o f isop ren e unit s head t o t ail as depic t ed yellow, orange or red. A large number of
Derow carotenoids (about-600) have been identified in
ia plant kingdom e.g. B-carotene, xanthophylls,
tt
c_c_c-c-c-c-c-c
lycopene.
Biotechnology [50]
f nz y m es ar e bioc at aly s t s - t h e c a t a l y s t so f l i f e . c l a s s i f i c a t i o na n d n o m e n c l a t u r eo f e n z y m e s. Si n ce
LA catalvst is defined as a subsfance that 1964, the IUB system of enzyme classification has
increasesthe velocity or rate of a chemical reaction been in force. Enzymes are divided into six major
wit hout it s elf under goingany ch a n g e i n t h e o v e r a l l c/asses (in that order). Each class on its own
orocess. represents the general type of reaction brought
Enzymes may be defined as biocatalysts about by the enzymes of that class.
synthesized by living cells. They are protein in 1 . O x i d o r e d u c t a s e s : E n z y m e s i n vo l ve d i n
nature, colloidal and thermolabile in character, oxidation-reduction reactions.
and specific in their action.
2 . T r a n s f e r a s e s : E n z v m e s t h a t c a ta l vse th e
In recent years, certain non-protein enzymes
transfer of functional groups.
( c hem ic ally RNA) hav e als o be e n i d e n t i f i e d .
3. Hydrolases: Enzymes that bring about
NO M ENCLATURE h y d r o l y s i so f v a r i o u s c o m p o u n d s .
AND CLASSI FI CATI O N 4. lyases : Enzymesspecialisedin the addition
ln the early days, the enzymeswere given names or removal of water, ammonia, CO, etc.
by t heir dis c ov er er s in an ar b i t r a r y m a n n e r . F o r 5 . t s o m e r a s e s : E n z y m e s i n v o l v e d in a l l th e
ex am ple, t he nam es pep s i n , t r y p s i n a n d isomerization reactions.
chymotrypsin convey no information about the
function of the enzyme or the nature of the 6. ligases : Enzymes catalysing the synthetic
substrateon which they act. reactions (Creek : ligate-to bind) where two
m o l e c u l e s a r e j o i n e d t o g e t h e r a n d A T P is u se d
Enzymes are sometimes considered under two
broad categories: (a) lntracellular enzymes-They lThe word OTHLIL (first letter in eacl.r class)
ar e f unc t ional wit hin c ells w h e r e t h e y a r e may be memorised to remeinber the six classesof
synthesized. (b) Extracellular enzymes-These enzymes in the correct orded
enzymes are active outside the cell; all the digestive
E a c h c l a s s i n t u r n i s s u b d i v i d e d i n t o m a n y su b -
enz y m es belong t o t his gr oup
c l a s g e s w h i c h a r e f u r t h e r d i v i d e d . A fo u r d i g i t
The I nt er nat ional Union of B i o c h e m i s t r y ( l U B ) Enzyme Commission (E. C.) number is assigned to
.l
appoint ed an Enz y m e Com m i s s i o n i n 9 6 1 . T h i s each enzyme representingthe class (firstdigit), sub-
committee made a thorough study of the existing c l a s s ( s e c o n d d i g i t ) , s u b - s u b c l a s s ( t h i r d d i g i t) a n d
enz y m es and dev is ed s om e ba s i c p r i n c i p l e s f o r t h e the individual enzyme (fourth digit).
786
Chaoter66 : ENZYMOLOCY 747
ln rhe Table 66. 1, selecteclexamples for the six activity. The important factors th?t influence the
cla sseso f en zym es ar e giv en. velocity of the enzyme reaction are discussed
hereunder
CHEMICAL NATURE O F ENZYM ES
l. Goncentration of enzyme
All the e nzy m es ar e inv ar iably pr ot eins . I n
As the concentrationof the enzyme is increased,
recent years, however, a few RNA molecules have
the velocity of the reaction proportionately
been shown to function as enzvmes. Each enzvme
increases (Fig.66.1). In fact, this property of
has its own tertiary structure and specific
e n z y m e i s m a d e u s e i n d e t e r m i n i n gt h e a c t i v i t i e so f
conformation which is very essentialfor its catalytic
serum enzymes for diagnosis of diseases.
activity.The functional unit of the enzyme is known
as holoenzyme which is often made up of
2. Concentration of substrate
apoenzyme (the protein part) and a coenzyme
(non-protein organic part). Increasein the substrateconcentrationgradually
increases the velocity of enzyme reaction within
Holoenzyme ----+ Apoenzyme + Coenzyme the limited range of substratelevels. A rectangular
(activeenzyme) (prot6inpad) (non-proteinpaft) hyperbola is obtained when velocity is plotted
against the substrate concentration (Fig. 66.2).
FACTORS AFFECTING Three distinct ohases of the reaction are observeo
ENZYME ACTIVITY in the graph.
The contact between the enzyme and substrate Enzyme kinetics and K- value : The enzyme (E)
is the most essential pre-requisite for enzyme and substrate(S) combine with each other to form
I Oxidoreductases
Alcohol (alcohol
dehydrogenase : NAD+ E. C. 1.1.1.1.),
oxidoreductase Oxidation ----+ Reduction
cytochrome L- andD-amino
oxidase, acidoxidases A H r+ B -----+ A + B H ,
2. Transferases
(ATP: D-hexose
Hexokinase E. C. 2.7.1.1.),
O-phosphotransferase, Grouptransfer
phosphorylase
transmethylases,
transaminases, A -X + B ---+ A + B -X
Hydrolases
Lipase(triacylglycerol
acylhydrolase
E. C. 3.1.1.3),
cholirre
esterase, Hydrolysis
acidandalkalinephosphatases,
pepsin,urease A -B + H rO----+ A H + B OH
4. Lyases
(ketose
Aldolase 1-phosphate lyase,E. C. 4.1.2.7),
aldehyde Addition_+ Elimination
fumarase,
histidase A -B + X -Y -----+ A X -B Y
5. lsomerases
Triosephosphate (D-glyceraldehyde
isomerase keloisomerase,
3-phosphate Interconversion
of isomers
E.C.5.3.1.1),
retinol phosphohexose
isomerase, isomerase A -+ A '
a. Ligas es
Glutamine (L-glutamate
synthetase ligase,
ammonia E. C. 6.3.1.2), (usually
Condensation onATP)
dependent
acetylCoAcarboxylase,
succinate
thiokinase A + B ----4,-----+ A -B
ATP ADP+ Pi
* Fu oneenzynein eachclass,systenatic
namealongwithE.C.numberis givenin the brackets.
7a8 B IOTECHNO LO CY
I Vr""
fI
o
o II
c)
E 1 I
N
J\/
2
f max -ft
c
ul >
o
c)
Substrate -+
concentration
Fig. 66.1 : Effect of enzyme concentration on Fig. 66.2 : Effect of substrate concentrationon enzyme
enzyme velocity. velocity (A-linear; B-cu rue; C-almost unchanged).
a c i d p h o s p h a t a s e( 4 - 5 ) a n d a l k a l i n e p h o s p h a t a s e
(i0-11) for optimum pFr.
I
)
5. Effect of product concentration
T h e a c c u m u l a t i o no f r e a c t i o n p r o d u c t sg e n e r a l l y
decreasesthe enzyme velocity. For certain enzymes/
the products combine with the active site of
c)
enzyme and form a loose complex and, thus,
o)
E inhibit the enzyme activity. In the living system,
N
this type of inhibition is generally preventedby a
uJ
quick removal of products formed.
6. Effect of activators
Some of the enzymes require certain inorganic
20 30 40 50 60 metallic cations like Mg2+, Mn2+, Zn2+, Ca2+,
Temperature('C) C o z *, C u 2 *, N a +, K + e t c . f o r t h e i r o p t i m u m a c t i v i t y .
R a r e l y ,a n i o n s a r e a l s o n e e d e d f o r e n z y m e a c t i v i t y
Fig, 66.4 : Eftect of temperature on enzyme velocity. e.g. chloride ion (Cl-) for amylase.
ACTIVE SITE
iro m the inte rc ept on x - ax is whic h is - ( ' llKm ) . Enzymesare big in size compared to substrates
Furth eri the do uble r ec ipr oc al plot is us ef ul i n which are relatively smaller. Evidently, a small
u nd erstan din g the ef f ec t of v ar ious inhibit io n s portion of the huge enzyme molecule is directly
'd iscusse d la ter ) . i n v o l v e d i n t h e s u b s t r a t eb i n d i n g a n d c a t a l y s i s .
The active site (or active ce:ntre) of an
3. Effect of temperature
enzyme is defined as the small region at which
Velocity of an enzyme reaction increaseswith the substrate(s) binds and participates in the
n cre asein te mper at ur eup t o a m ax im um and t h e n catalysis.
cie clin es.A b ell - s haped c ur v e is us ually obs er v e d
Salient features of active site
Fig. 66.a).
.l
. The existence of active site is due to the
The optimum temperature for most of the
tertiary structure of protein resulting in three-
enzymes is between 40"C-45"C. However, a tew
dimensional native conformation.
:n zymes (e .g. v enom phos phok inas es , m us c l e
.l
:denylate kinase) are active even at 00"C.
' I he
2. ac t iv e s it e is m ad e u p o f a m i n o a c i d s regarded as a substrate analogue. The inhibitor
(known as catalytic residues)which are far from comoetes with substrateand binds at the active site
eac h ot her in t he linear s eq u e n c e o f a m i n o a c i d s of the enzyme but does not undergo any catalysis.
(primary structure of protein). For instance, the A s l o n g a s t h e c o m p e t i t i v e i n h i b i t o r h o l d s th e
enz y m e ly s oz y m e has 129 am i n o a c i d s . T h e a c t r v e active site, the enzyme is not available for the
s it e is f or m ed by t he c ont r i b u t i o n o f a m i n o a c i d substrateto bind.
r es iduesnum ber ed 35, 52, 6 2 , 6 3 a n d 1 0 1.
The relative concentration of the substrateand
3 The ac t iv e s it e is not r i g l d i n s t r u c t u r e a n d i n h i b i t o r a n d t h e i r r e s p e c t i v e a f f i n i ty w i th th e
shape. lt is rather flexible to promote the specific enzyme determines the degree of competitive
s ubs t r at ebinding. i n h i b i t i o n . T h e i n h i b i t i o n c o u l d b e o v e r co m e b y a
4. Cenerally, the active site possesses a high substrate concentration. In competitive
substrate hinding site and a catalytic site. The inhibition, the K,n value increases r,r,hereasV,,,u,
latter is for the catalysis of the specific reaction remains unchanged.
DihydrofolatereductaseDihydrofolic
acid Aminoplerin, Employed in thetreatment
of leukemia
and
amethopterin, olhefcancers.
methotrexate
Th e inh ibito r gener ally binds wit h t he e n - to be inhibited e.g., allopurinol, an inhibitor of
zyme as we ll a s t he ES c om plex . xanthine oxidase, gets conve!"tedto alloxanthine, a
more effective inhibitor of the enzvme.
For non-competitive inhibition, the K- value is
unchanged while V^", is lowered.
ENZYME SPEGIFICITY
Heavy metal ions (Ag+, Pb2+, Hg2+ etc.) can
E n z y m e sa r e h i g h l y s p e c i f i c i n t h e i r a c t i o n w h e n
no n-comp etitively inhibit t he enz y m es by bindi n g
compared ivith the chem ical catalysts. The
rvith cysteinyl sulfhydryl groups.
occurrence of thousands of enzymes in the
biological system might bre due io the specific
2. lrreversible inhibition nature of enzymes. Thiee types of enzyme
Th e in hib itor s bind c ov alent lv wit h t he enz v m e s specificity are wel l-recognised
a nd in activa te them , whic h is ir r ev er s ible.The s e 1. Stereospecificity, 2. Reaction specificity,
inh ibito rs are usually t ox ic s ubs t anc eswhic h m a y
b e pre se nt n atu r ally or m an- m ade. 3. Substratespecificity,
Fig. 66.6 : Diagrammatic representation of stereo- (c) Broad specificity : Some enzymes act on
specificity (a',b',c')-three point attachment of c l o s e l y r e l a t e d s u b s t r a t e s wh i ch is
substrate to the enzvme (a, b, c). commonly known as broad substrate
specificity,e.g. hexokinaseacts on glucose,
f r u c t o s e ,m a n n o s e a n d g l u c o s a m i n ea n d n o t
Hexokinase acts on D-hexoses; on Salactose.
Cluc ok inas e on D- gluc o s e ;
Am y las e ac t s on u- gly c o s i d i c l i n k a g e s ; COENZYMES
pyrophosphate
Thiamine (TPP) Thiamine Aldehyde
or keto Transkelolase
Flavin (FMN)
mononucleotide Riboflavin Hydrogen
andelectron L-Amino
acidoxidase
Flavin
adenine (FAD)
dinucleotide Riboflavin D-Amino
acidoxidase
Nicotinamide
adenine
dinucleotide(NAD) Niacin Lactate
dehydrogenase
Nicotinamide
adenine
dinucleotide
phosphate
(NADPJ Glucose
6-phosphate
dehydrogenase
Lipoic
acid Lipoic
acid Pyruvate
dehydrogenase
complex
phosphate
Pyridoxal (PLP) Pyridoxine Amino
cr keto Alanine
transaminase
A (CoA)
Coenzyme Pantothenic
acid Acyl Thiokinase
(FH.)
Tetrahydrofolate Folicacid Onecarbon Formyl
transferase
(formyl,
methenyl
etc)
Biotin
coenzyme Biotin Pyruvate
carboxylase
Methylcobalamin
; Deoxyadenosyl
cobalamin Cobalamin Methyl/rsomerisation Methylmalonyl
CoAmutase
d ioxide etc. Coe nz y m es play a dec is iv e r ole i n hydrogen transfer. The specificity of the enzyme is
enzyme function. mostly dependent on the apoenzyme and not on
the coenzyme
Coenzymes from B-complex vitamins : Most of
the coenzymes are the derivativesof water soluble
MECHANISM OF ENZYME ACTION
B-co mple x vitam ins . I n f ac t , t he bioc hem ic a l
functions of B-comolex vitamins are exerted Catalysis is the prime function of enzymes.
through their respective coenzymes. The chapter T h e n a t u r eo f c a t a l y s i st a k i n g p l a c e i n t h e b i o l o g i c a l
on vitamins gives the details of structure and system is similar to that of non-biological
fun ctio n of th e c oenz v m es . ln Table. 66. 3, a catalysis. For any chemical reaction to occur, the
summary of the vitamin related coenzymes with reactants have to be in an activated state or a
the ir fun ctio ns is giv en. transition state.
Non-vitamin coenzymes : Not all coenzymes Enzymes lower activation energy : The energy
are vitamin derivatives. There are some other required by the reactantsto undergo the reaction is
organic substances,which have no relation with known as activation energy. The reactants when
vitamins but function as coenzymes. They may be heated attain the activation energy. The catalyst (or
considered as non-vitamin coenzymes e.g. ATP, the enzyme in the biological system) reduces the
CDP, UDP etc. activation energy and this causes the reaction to
Nucleotide coenzymes : Some of the coenzymes proceed at a lower temperature. Enzymes do not
possess nitrogenous base, sugar and phosphate. a l t e r t h e e q u i l i b r i u m c o n s t a n t s ,t h e y o n l y e n h a n c e
Such coenzymes are, therefore, regarded as the velocity of the reaction.
n ucleo tide s e .g. NAD+ , NADP+ , FM N, FAD ,
The role of a catalvst or an enzvme is
co en zyme A, UD PC et c .
comparable with a tunnel made in a mountain to
Coenzymes do not decide enzyme specificity : reduce the barrier as illustrated in Fig. 66.7. fhe
A particular coenzyme may participate in catalytic enzyme lowers energy barrier of reactants,thereby
reactions along with different enzymes. For making the reaction go faster.The enzymes reduce
instance, NAD+ acts as a coenzyme for lactate the activation energy of the reactantsin such a way
dehydrogenaseand alcohol dehydrogenase.ln both that all the biological systems occur at body
the enzymatic reactions, NAD+ is involved in temperature (below 40"C).
794 B IOTE CHNO LO CY
E+ Sr - - - . ES- - - - - +E +P
A few theories have been put forth to explarn
mechanism of enzyme-substratecomplex formation
(Fig. 66.8).
Amylase Acutepancreatitis
Serum pyruvate
glutamate (SGPT)
transaminase Liverdiseases (hepatitis)
Serum glutamate
oxaloacetate (SGOT)
transaminase Heartattacks (myocardialinfarction)
phosphatase
Alkaline Rickets,
obstructive jaundice
Acidphosphatase Cancer of prostategland
Lactate (LDH)
dehydrogenase Heartattacks, liverdiseases
phosphokinase
Creatine (CPK) Myocardial infarction(earlymarker)
Aldolase Muscular
dystrophy
5'-Nucleotidase Hepatitis
y-Glutamyl (GGT)
transpeptidase Alcoholism
IN T R OD U C T IO N T O ME TA B OLIS M
Me ta b o l i s m i s b ro a d l y di vi ded i nto tw o to generatethe substances (precursors) requiredfor
categories(Fig.67.1). the synthesisof complex molecules.Catabolism
1. Catabolism : The degradativeprocesses occurs in three stages(Fig. 67.2).
concerned with the breakdown of comptex 1. Conversionof complexmoleculesinto their
m o l e c u l e sto s i m p l e ro n e s , w i th a concomi tant buildingblocks: Polysaccharides are brokendown
releaseof energy. to monosaccharides, lipids to free fatty acids and
2. Anabolism : The biosynthetic reactions gl ycerol and , protei nsto ami no aci ds.
from
i n v o l v i n gth e fo rma ti o no f c o m pl exmol ecul es
2. Formation of simple intermediates: The
s i mp l ep re c u rs o rs . bui l di ngbl ocksproducedi n stage(1)aredegr aded
A clear demarcation between catabolism to simpleintermediates suchas pyruvateand acetyl
and anabolismis ratherdifficult,since there are CoA. These intermediates are not readily
common
severalintermediates to both the processes. identifiable as carbohydrates,lipids or proteins.A
small quantity of energy(as ATP) is capturedin
C a ta b o l i s m stage2.
The very purposeof catabolismis to trap the 3. Finaloxidationof acetylCoA : Acetyl CoA
energyof the biomoleculesin the form of ATPand i s compl etel yoxi di zedto C Or, l i beratingNADH
796
Chaot er67 : ME T AB O L IS M S 797
Stage1 +
I J I
M o n o sa cch a r id e s -
Stage 2
AcetylCoA
NADH
FADH2
Stage 3
2. Utility of metabolic probes : Two types of 4. Clycolysis is very essentialfor brain which
metabolicprobesare commonlyusedto trace out is dependent on glucosefor energy.The glucosein
biochemical pathways. These are metabolic brain hasto undergo glycolysisbeforeit is oxidized
inhibitors and mutations. to C O, and H rO.
I I
A IH \' H e x o k i n a s e o r
+
Glucose1- tr,,t92+Jgtucokinase
phosphate nopvI
Glucose 6-phosphate
Pyruvate (enol)
1I Phosphohexose I
isomerase
I J,spoganeou$
I
Pyruvate (keto)
Fructose 6-phosphate
I NADH + Ht
ATP
\l Lactale
FhoeP-
ho{rtratbkinase
I dehydrogenase
ADP<J NAD-
Fructose 1, 6- L-Lactate
bisphosphate
Fig. 67.4 : The reactions in the pathway of glycolysis
(The three steps calalysed by hexokinase,
Aldolase phasphofructoki nase and pyruvate
kinaoe are iriaversible).
DihvdroxvacetonePhosphotriose Glyceraldehyde
ptrdsptraie , isomerase $phosphate l i p o i c a c i d l i n k e d t o e - a m i n o g r o u p o f l y s i n e ) .T h e
- overall reaction of PDH is
PDH
Pyruvate + NAD+ + CoA
' Acetyl CoA
Pi + CO, + NADH + H+
+
NAD
CITRIC ACID CYCLE
The citric acid cycle (Krebscycle or tricarboxylic
NA DH+ H + acid-TCA cycle) is the most important metabolic
pathway for the energy supply to the body. About
1, $Bisphosphoglycerate
65-700/"of the ATP is synthesized in Krebs cycle.
Citric acid cycle essentially involves the oxidation
of acetyl CoA to CO, and HrO.
The citric acid cycle is the final common
oxidative pathway for carbohydrates, fats and
amino acids. This cycle not only supplies energy
8-Phosphoglycerate but also proVides many intermediatesrequired for
^I the synthesis of amino acids, glucose, heme etc.
I Phosphogtycerate Krebs cycle is the most important central pathway
I mutase connecting almost all the individual metabolic
+
2-Phosphoglycerate pathways (either directly or indirectly).
"rr-3-.oo-
pyruvate
Pyruvate
Cehydrogenase
CH.-C-S Con
AcetylCoA
o I coasH
t i-'" cHp-coo-
c-coo- -# Ho_f_coo_
]H2-COO- svnthase
) CH2_COO-
Oxatoacetate Citrate
J/r. \ JrO
/ w^l^r" nconiraseY
,1 dehydrogenase \
HO_CH_COO_ OH2_COO_
rl
CH2-COO- c-coo-
L-Malate CH-COO-
Cls-Aconitate
f
,..,^...'
, \;, Hzo
' '2" --/Frt"r"r" Aconitase{
I
l+
+ OH2-COO-
H_C_COO- I
li cH-coo-
ooc-c_H I
HO_CH_COO_
Fumarate
,socitrate
a
\/
{.\ Succinate tsocitrate l. ,
cetrydrogenase dehydrogenase
(\l A , ,. ,-,,, , ,,
.L
CH2-COO-
CH2-COO-
I
cH2-coo- cH-coo-
Succinate O = C_COO-
Succinate
A- tiriotrinase - Oxalosuccinate
coAs;\ )\ cH2-coo
r - ^o** ,
..."r?
CHz
,..o\[roo",r"
.vru ^r2-vvv
_ . . '. . l d " '''. /
O =
' 0-l(or^-'
k' "tt
J;;,:.T^-!K5L!"-- -
O = C-COO-
o-Ketoglutarate
"o'
COz CoA SH
Biotechnology [51]
8(J2 B IOTE C H NO LO CY
RibuloseS-pnosptrate
Riboses-phosphate
ketoisomelase
Ribose 5-
phosphate
Fructose 6-phosphate
TPP
Tran$kelolase Glyceraldehyde
Glyceraldehyde 3-phosphate
3-phosphate
t
I Fleversalof
It *. olvcolvsis
Fructose 6-phosphate
Erythrose 4-phosphate Fructose 6-phosphate
B. lmportance of NADPH u n s a t u r a t e d l i p i d s , p r o t e i n s a n d D N A . T h i s i s,
however, prevented to a large extent through
1 . N A D P H i s re q u i re d fo r the reducti ve
antioxidant reactions involving NADPH. Gluta-
biosynthesis of fatty acids and sferoids, hence
thione mediated reduction of HrO, is given
H MP s h u n ti s m o rea c ti v ei n th e ti ssues concerned
hereunder
w i th l i p o g e n e s i e
s ,.g . a d i p o s eti ssue,l i ver etc.
2 . N AD PH i s u s e d i n th e s ynthesiof
s certai n
amino acids involving the enzyme glutamate
dehydrogenase.
3 . T h e rei s a c o n ti n u o u ps ro d ucti on
of H rOri n
t h e l i v i n g c e l l s w h i c h c a n c h emi cal l ydamage
C hapt er62 : M E T A BOL IS M S 803
Fattyacid
Y
I
+
AcetylCoA
6x
Aconitase
l$ocitratelyase
compounds(Fig.67.9).The Calvincyclestartswith
lPhosphoglyc"r"r\t to
a reactionof CO, and ribulose1, S-bisphosphate
form two molecules 3-phosphoglycerate. This
1, 3-Bisphosphoglycerate
3-phosphoglycerate can be convertedto fructose
\
ATP./\
I l;NAoeH
6-phosphate,
comoounos.
glucose6-phosphate and othercarbon
I
Y
Glyceraldehyde
Ribulose 3-phosphate
5-phosphate .,....-.-'
'-"Fructose(
6-phosphate Li pi dsare i ndi spensablfor
e cel l st r uct ur eand
functi on.D ue to thei r hydrophobi and
c non- polar
Fig. 67.8 : An outline of the Calvin cycle ot nature, lipids differ from rest of the body
photosynthesis. compoundsand are uni quei n thei r act ion
The important pathwayskycles of lipid
metabolismare brieflydescribed.
c h i o ro p l a s tsT. h i s re s u l t si n the producti onof
excitationenerSywhich is transferredfrom one
FATTY ACID OXIDATION
l o l e c u l eto a nother,unti l i t i s trapped
c h l o rc p h y lm
by a reactior.r center.The light-activated transferof The fatiy acidsin the body are mostlyoxidized
an electronto an acceptor(photosystems) occursat by p-oxidation.B-Oxidationmay be definedas the
the reactioncenter. oxidationof fatty acidson the B-carbonatom. Thrs
resultsin the sequentialremovalof a two carbon
Pirotosynthesis primarilyrequiresthe interactions
fragment,acetyl CoA (Fig. 67.10).
of two ciistinctphotosystems (l and ll). Photosystem I
generatesa strong reductantthat resultsin the Fatty acid oxidation-
formationof NADPH. Photosystem ll producesa stages and tissues
strong<.rxidant thatformsO, from HrO. Further, the
generationof ATP occurs as electronsflow from The p-oxidationof fatty acids involvesthree
photosystemll to photosystem| (Fig. 67.6. fhus, stages
light is responsible for the flow of electronsfrom l. Activationof fatty acids occurringin the
HrO to NADPH with a concomitantgenerationof cytosol;
ATP. ll. Transportof fatty acids into mitochondria;
Il l . p-Oxi dati onproper i n the m it ochondr ial
The Calvin cycle matri x.
The dark phaseof photosynthesisis referredto as Fattyacidsareoxidizedby mostof the tissuesin
C a l v i nc y c l e . In th i s c y c l e,the A TP and N A D P H the body. However,brain, erythrocytesand adrenal
producedin the light reaction(described above)are medulla cannot utilize fatty acids for energy
utilizedto convertCO, to hexoses andotherorganic requirement,
""'-"'-'NADPH
Strongreductant
Lioht ----------+t -- ---,1
(<706nm) HnolosyslemII
|
Weak oxidant
>ATP
Weak reductant
-------J
,.o|J311', fl
Fr.'"*Y"*,'
Strongoxidant-7=+@
Hzo
o
R-CH2-CH2-CH2-C-O
Fattyacid
ATP CoASH
Mg-
A M P + PP i a - Thiokinase
o
tl
R-CH2-CH2-CH1-C-S CoA
Acyl CoA
- I cYrosol
ll ca;,tin"tr;l-.poItrrtut
i MlrocHoNDRloN
1t. o
Bcrll
Ftg. 67.10 : p-Oxidation of fatty acids : Palmitoyl CoA (16 carbon) undergoes seven cycles to yietd B acetyt CoA
.'-lctivation; ll-TransporT; lll-B Oxidation proper-(l) Oxidation, (2) Hydratiotl, (3) Oxidation and (4) Cleavagel
806 B IOTE CHNO LO CY
CH3-C-SCoA
@yslsH
(
9l i
)
q97-s- c-cH3
Irans A2-EnoylACP
Acetyl-S-ACP j Transferof acetyr
tocysierne
J
o
s- c-cH3
Jt1 rrfl
Acetyl S-enzyme
FC-CH2-CH2-CH3
o
ll
-OOC-CH2-C-S
Acyl-ACP (butyryl-ACP)
CoA
MalonylCoA
t_
I r ranslerof caroon
MalonylCoA-ACF I chainfrornACPto Gys
iransaoylase +
CoASH a)
t1
s-c-cH2-cH2-cH3
SH
co,- J3),.]g-Ketoacyt-AoP
| synthase
sto
rl
FC-CH2-C-CH3
I
p-Ketoacyl-ACP HzO\ Palmitoyl
Jrnroesrerase
6GfsH
( + C H 3-C H 2-(C H 2)13-C OO-
)
\ACP}-SH
\--l PALTvITATE
Fig. 67.1 I contd. next column
Fig, 67.11 : Biosynthesis of long chain fatty acid-palmitate. (Cys-Cysteine: ACP-Acyl carrier protein; The
pathway repeats 7 times to produce palmitate; the first two carbans at the methyl end are directly from acetyl
CoA, the rest of the carbons come from malonyl CoA).
808 B IOTECHNO LO CY
Mevalonate(6C)
lsoprenoidunits
(5C;buildingblocks)
T h e a m i n o a c i d s u n d e r g o c e r t ai n co m m o n
i 6 units
reactions like transamination followed by
.|i condense
Squalene(30C) deamination for the liberation of ammonia. fhe
a m i n o g r o u p o f t h e a m i n o a c i d s i s u ti l i ze d fo r th e
i
formation of urea which is an excretory end
\7
product of protein metabolism.The carbon skeleton
Lanosterol(30C)
of the amino acids is first converted to keto acids
(by transamination)which meet one or more of the
*' following fates.
(27C)
Cholesterol
1 . U i i l i z e d t o g e n e r a t ee n e r g y .
2. Llsed for the synthesisof glucose.
Fig. 67.12 : Outline of cholesterol biosynthesis.
3. Diverted for the formation of fat or ketone
bod ies.
\ ,1
Protein Synthesisof ,
{- Aminoacids -----)
synthesis N-compounds
Proteins are the most abundant organic
compounds and constitutea major part of the body cr-Ketoglutarate
dr y v ^r eight( 10- 12 k g in a d u l t s ) . T h e y p e r f o r m a
wicie variet'y of static (structural) and dynamic Transamination
(enzymes.hormones,clotting factors,receptorsetc.)
iunctions. About half of the body protein (predo-
m inant ly c ollagen) is pr es e n t i n t h e s u p p o r t i v e
tissue (skeleton and connective) while the other
half is int r ac ellular .
lhe proteins on clegradation(proteolysis)release
indiv idual am ino ac ids . Am i n o a c i d s a r e n o t j u s t
the structural components of proteins. Each of the
20 nat ur ally oc c ur r ing am i n o a c i d s u n d e r g c e s i t s
Energy Glucose Fat Non-essential
own metabolism and performs specific functions. aminoacids
Some of the amino acids also serve as orecursors
for the synthesis of many biologically important
Fig. 67.13 : An overuiew of amino acid metabolism.
c om pounds ( e. g. m elanin, s e r o t o n i n ,c r e a t i n e e t c . ) ,
Chapter67 : METABOLISMS 809
CO2+ NHI
ZA I r=\
V I
rrn n 'l C arbamovl
N"9'/
ot'oiPhatl sYnthasel+
2A D P+ P i + ' + '
OO
ltl
H2N-
-l
c -o-P-o
d
CarbamoylPhosphate
N Hz
I
I
HN
NHT ,/ I
CHz
I
CHz
QHz
CHz
I,
H-C-NHi
I
"f-**A
I
coo-
Citrulline
ATP
o j^a\r1-
Argino-
tl succlnale
H2N- C -N I synthase
Urea NHi NH; CH..
Arginase IL ll-l- A MP+ P P i
c -NH2
I
I
HN HT'J coo
I I Aspartate
CHz CHu
I t-
cHz Argino' CHc
I succlna9e l-
CHe v11.t
lt.-
l-
H-C-NHi H-c-NHa
I
coo- I
coo I coo-
H-C Argine
Arginine
il succinate
c-H
I
coo-
Fumarate
group ts
(NAG-N-acetlrlgtutamate; (in the formati.onof urea, one amino
i,n. ur.ru: Beactionsof urea cycle carbon^is obtained from co';
derivec from freeur*on,u* ion white the otier is from aspartate;
* mitochondrialenzymes, the rest of the enzymes are cytosomal)
Chant er67 : M E T A BOL IS M S 811
Proteins
Purines
Serine G (Cq, Cs, N7 atoms) Proteins T
R
L Glutathione
Oxalate Y Glucose P NAD-,NADP-
T (coenzymes
of niacin)
C Conjugation
o
Glucose (bileacids,detoxif
ication) Fat
I P Serotonin
H
NHs N Heme
Indoleacetic A t,Ar
E acid N Melatonin 5-Hydroxy-
Creatine indoleacelicacid
Formate
+
One-carbonpool
Fig. 67.16 : Overview of glycine metabolisrn. Fig. 67,18 : Overview of iryptophan metabclism.
The p red omin ant m et abolis m of pheny lalani n e propionic acid. Tryptophan is both glucogenic and
occurs through tyrosine.Tyrosineis incorporatedinto ketogenic in nature. lt is a precusorfor the synthesis
pro tein sa nd is in v olv ed in t he s y nt hes isof a v ar ie t y of important compounds, namely NAD+ and
of biologically important compounds-epineph rine, NADP+ (coenzymes of niacin), serotonin and
norepinephrine, dopamine (catecholamines), thyroid melatonin tFig. 67.1 B\.
hormones-and the pigment melanin (Fig. 67.1/
Durin g the cou rseof degr adat ion,pheny laianinean d SULFUB AMINO ACIDS
tyrosine are converted to metabolites which can
T h e s u l f u r - c o n t a i n i n ga m i n o a c i d s a r e m e t h i o -
serve as precursorsfor the synthesisof giLrcoseand
nine, cysteine and cystine. Among these, only
fat. Hence, these amino acids are both glucogenic
methionine is essential. lt serves as a precursor for
and ketogenic.
t h e s y n t h e s i so f c y s t e i n e a n d c y s t i n e w h i c h a r e ,
therefore, non-essential. An overview of the
TRYPTOPHAN
m e t a b o l i s mo f t h e s u l f u r a m i n o a c i d s i s d e o i c t e d i n
Tryptophan (Trp, W) was the first to be identified Fig.67./9.
as an essential amino acid. lt contains an indole
-ing a nd che mic ally it is c r - am ino p- indole GLUTAMATE AND GLUTAMINE
M
E Proterns
T Transmethylation L] y-Aminobutyric
acid
H NHs L (GABA)
I U
Proteins Cystathionine Glucose Glutathione
o T
N Histidine
I Polyamines M N-Acetylglutamate
N Proline A
T
E I
"y-Carboxyglutamate
Arginine E (clottingfactors)
Proteins C Glutathione
S Taurine
T r:
Purines(Ne,Ns)
E CoenzymeA
i
'.-J
I
Proteins (N3)
Pyrimidines
Glucose N Active sulfate
i: NHs
M
I Aminosugars
<-F!s rnl]
Proteins
I +
Urea i'l
r Detoxification
Fig. 67.19 : Overview of the metabolism Fig. 67.20 : Overview of glutamate and glutamine
of sulfur amino acids. metabolism.
Ot
Tlslr 67.1 Classlflcatlon of anlno acids t-
EnergV-----.5 CO2 + H2O
based on the fate of carbon skeleton supprv \/ ./ \I
/v a.i i hi a\ 'Yl /
I
I
IL +
Fattyacids
--+Hzo
I feh"29K:"n
//)1
/ A D P +P i
l l l . Gl y c i n e
1 3 . G l y c i n u ri a in renalreabsorption
Defect
14. Primary
hyperoxaluria Glycine
transaminase
Tryptophan
15. Hartnup's
disease intestinal
Defective absorption
V. Branchedchain amino acids (valine,leucineand isoleucine)
16. Maplesyrupurinedisease Branchedchaina-ketoaciddehydrogenase
17. Intermittent chainketonuria Variant
branched of theaboveenzvme (lesssevere)
18. Hypervalinemia Valine
transaminase
19, lsovaleric
acidemia lsovaleryl
CoAdehydrogenase
vl. Histidine
20. Histidinemia Histidase
vll. Proline
typeI
21. Hyperprolinemia Proline
oxidase
at 6
C haot er68 : I MMU N OL OC Y 417
a,:r+:--,oiogy[52]
818 B IOTE C HNO LO C\
Interchain
disulfidebonds
NH;
Fab
S*
Hingeregion Papain
S-
Fc
coo- coo-
ijg. 68.2 : Diagrammatic representation of human lgG molecule (V-VariaHe region, C-Constant region;
CHO-Carbohydrate; Each heavy chain is composed of four units-Vr, Cr,, Crr, Cr, while light
chain consists of two units-V,, C,)
-'
-=" and is the most effective against invading SY'VTHEs'S OF'MMANOGLOBULINS
-: , c roorganisms. T h e r e a r e m i l l i o n s o f d i f f e r e n ta n t i g e n s l t w a s a
big puzzle for a long time how an individual can
- mun og lob ulin D ( lgDl produce so many antibodies to protect against
antigens. lt is now recognized that a gene
-- s comp osed of a s ingle Y- s hapedunit an d
:::-i in a lo w c onc ent r at ionin t he c ir c ulat io n . r e a r r a n g e m e n ti n v o l v i n g a c o m b i n a t i o n o f s e v e r a i
-- :cr-rle sare pr es enton t he s ur f ac eof B c ells . g e n e s i s r e s p o n s i b l ef o r g e n e r a t i n g a n e x t r e m e l y
'. :ttion , h owev er , is not k nown f or c er t ain . l a r g e n u m b e r o f a n t i b o d i e s .( F o r m o r e d e t a i l s R e f e r
Chapter 5).
: ---r€r-sb eliev e t hat lgD m ay f unc t ion a s
i--ttt re ce pto r
MAJOR HISTOCOMPATIBI LITY
COMPLEX
r* -u no glo bu lin E ( lgEl
The major histocompatibilitycomplex (MHQ
-i .- . rgle Y - s hapedm onom er . I t is nor m al l y represents a special group of proteins, present on
-' - ^ ''-rte c onc ent r at ionin blood. lgE lev e l s
the cell surfacesof T-lymphocyfes. MHC is involved
,-- r r individuals with allergies as it is i n t h e r e c o g n i t i o no f a n t i g e n so n T - c e l l s .l t m a y b e
:^ :he body ' s aller gic r es pons es Th
. e noted here that the B-cell receptors(antibodies)can
. :: : :' r t l\ bind wit h m as t c ells whic n r e c o g n i z ea n t i g e n sa n d o n t h e i r o w n , w h i l e T - c e l l s
: -' ^ : . : 1t l c aus e aller gy . can do so through the mediation of MHC.
82tJ B IOTE CHNO LO C\
I n hum ans , t he M HC pr ot ei n s a r e e n c o d e d b y a
cluster of genes located on chromosome 6 (it is on
c hr om os om e 17 f or m ic e) . T h e m a i o r h i s t o -
c om pat ibilit y c om plex in hum a n s i s r e f e r r e dt o a s
human leukocyte antigen (HLA). fhree classes of C l a s sI M H C
M HC m olec ules ( c hem ic ally g l y c o p r o t e i n s ) a r e
Antigenfragment
k nown in hum an. Clas s I m ol e c u l e s a r e f o u n d o n
T-cellreceptor
alm os t all t he nuc leat ed c ells o f t h e b o d y . C l a s s l l
m olec ules ar e as s oc iat ed on l v w i t h l e u k o c v t e s Ts cell
inv olv ed in c ell- m ediat ed im m u n e r e s p o n s e .C l a s s
lll m olec ules ar e t he s ec r et ed p r o t e i n s p o s s e s s i n g
im m une f unc t ions e g. c om p l e m e n t c o m p o n e n t s
(C2, C4), tumor necrosisfactor.
Therapeutic uses of cytokines viral or any foreign antigen. Some of the epitopes
of foreign atrtigens are similar (homoiogous) to
lt is now possibleto produce cytokines in vitro.
epitopes present on certain host proteins. This
Some of the cytokines have potential applications
results in cross reaction of antigens and antibodies
in t he p ractice o f me dic ine. For ins t anc e, lL- 2 is
w h i c h m a y l e a d t o a u t o i m m L r n ed i s e a s e s .
used in can ce r im m unot her apy , and in t he
t reat m e nt o f immu nodef ic ienc y dis eas es . lL- 2
ORGAN TRANSPLANTATION
induces th e p rolife r at ion and dif f er ent iat ion of
T -and B-cells, b esides inc r eas ing t he c y t ot ox ic T h e p h e n o m e n o n o f t r a n s f e r o f c e l l s , t i s s u e so r
caoacitv of na tura l killer c ells . organs from one site to another (in the sarne
organism, autograft or from another organism
A group of cytokines namely interferons can
allograft) is regarded as organ transplantation.In
comba t viral infe ctio nby inhibit ingt heir r eplic at ion.
case of humans, majority of organ
t r a n s p i a n t a t i o n s a r e a l l o g ra f t s ( b e t w e e n t w o
i n d i v i d u a l s ) .T h e t e r m x e n o g r a f t i s u s e d i f t i s s u e s /
organs are transferred from one species to
another e.B. from pig to man (For more details on
xenotransplantation Refer Chapter 41).
The prime function of imune system is to protect Organ transplantation is associated with
t he ho st a ga instth e inv ading pat hogens .The body i m m u n o l o g i c a l c o m p l i c a t i c n s ,a n d t i s s u e r e j e c t i o n .
tries its best to overcome various strategies of This is because the host body responds to the
infec tiou s a ge nts (ba c t er ia, v ir us es ) ,and pr ov ides t r a n s p l a n t e dt i s s u e i n a s i m i l a r w a y a s i f i t w e r e a n
immunity. i n v a d i n g f o r e i g n o r g a n i s m .M a i o r h i s t o c o m p a t i b i l i t y
complex (MHC) is primarily involved in allograft
S o me of th e impo rt ant im m unologic al as pec t sr n rejection. This is due to the fact that MHC proteins
human h ea lth an d d is eas ear e br ief lv des c r ibed are unique to each individual, and the immune
system responds promptly to foreign MHCs.
A UT OIMMUNE DISEASES
Organ transplantation between closely related
In general, the immune system is self-tolerant family members is preferred,since their MHCs are
r e not responsive to cells or proteins of self. also likely to be closely related. And major
Sometimes, for various reasons, the immune system immunological complicationscan be averted.
fails to discriminate between self and non-self. As
a consequence,the cells or tissuesof the body are CANCERS
attacked. This phenomenon is referred to as
autoimmunity and the diseases are regarded as Crowth of tumors is often associatedwith the
autoimmu ne disea se s .The ant ibodies or oduc ed t o formation novel antigens. These tumor antigens
(also referred to as oncofetal antigens e.g.
self molecules are regarded as autoantibodies:
S ome examp les of aut oim m une dis eas es ar e a-fetoprotein) are recognized as non-self by the
I isted. immune systems However, tumors have developed
several mechanisnrsto evade immurre responses.
. I nsulin -de pe nd en t d iabet es ( panc r eat ic p- c ell
autoreactive T-cells and arrtibodies). AIDS
. Rheuma toid arth ritis ( ant ibodiesagains t pr ot eins Acquired immunodeficiency syndrome (AIDS),
pres en t in jo ints). caused by human immunodeficiency virus, is
r Mya sth en ia gra vis ( ac et y lc holine r ec ept or c h a r a c t e r i z e d b y i m m u n o s u p p r e s s i o n ,s e c o n d a r y
auto an tibo dies). neoplasma and neurological manifestationsA . lDS
p r i m a r i l y a f f e c t st h e c e l l - m e d i a t e d i m n r u n e s y s t e m
o A utoirn mun e he moly t ic anem ia ( er y t hr oc y t e
which protectsthe body from intracellularparasites
autoantibodies).
s u c h a s v i r u s e s ,a n d b a c t e r i a .M o s t o f t h e i m m u n o -
Mechanism of autoimmunity : lt is widely d e f i c i e n c y s y m p t o m so f A I D S a r e a s s o c i a t e dw i t h a
accepted that autoimnrunity generally occurs as a reduction in CDo (cluster determinant antigen 4)
consequence of body's responseagainst bacterial, c e l l s .
enetics is the study of heredity. lt is the first time reported the fundamental laws of
appropriately regarded as the science that inheritance. He conducted several experinrentson
ex plains t he s im ilar it iesand dif f e r e n c e sa m o n g t h e the breeding patterns of pea plants. Mendel put
related orqanisms. forth the theory of transmissible facfors which
states that inheritance is controlled by certain
The blood theory of inheritance factors passedfrom parentsto offsprings.His results
in humans were published in 1866 in an obscure journal
Proceedingsof the Society of Natural Sciences.
For many centuries, it was customary to explain
inheritance in humans through blood theory. For about 35 years, the observations made by
People used to believe that the children received Mendel went unnoticed, and were almost forgotten.
blood from their parents, and it was the union of Two European botanists (Correns and Hugo de
blood that led to the blending of characteristics. V r i e s ) i n 1 9 0 0 , i n d e p e n d e n t l y a n d s i m u l ta n e o u sl y
That is how the terms 'blood relations', 'blood will rediscoveredthe theoriesof Mendel. The year 1900
tell', and 'blood is thicker than water' came into is important as it marks the beginning the modern
existence.They are still used, despite the fact that era of genetics.
blood is no more involved in inheritance.With tne
advances in genetics, the more appropriate terms The origin of the word gene : In the early years
should be as follows of twentieth centurv. it was believed that the
Mendel's inheritancefactorsare very closely related
. Gene relations in place of blood relations.
to chromosomes (literally coloured bodies) of tne
. Genes will tell instead of blood will tell. cells. lt was in 1920s, the term g'ene (derived from
a Creek word gennan meaning to produce) was
BRI EF HI STO RY AND DEV E L O P M E N T introduced by Willard Johannsen. Thus, gene
O F G ENETI CS replaced the earlier terms inheritance factor or
Cenet ic s is r elat iv elyy oung, n o t e v e n 1 5 0 y e a r s . inheritance unit.
The blood theory of inheritancewas questioned in Chemical basis of heredity : There was a
1850s, based on the fact that the senren contained
controversy for quite sometime on the chemical
no blood. Thus, blood was not being transferredto
basis of inheritance. There were two groups-the
the offspring. Then the big question was what was protein supportersand DNA supporters.lt was in
the hereditary substance. 1944, Averyand his associatespresentedconvincing
Mendel's experiments : lt was in 1866, an evidence that the chemical basis of heredity lies in
Austrian monk named Gregor Johann Mendel, for DNA, and not in protein. Thus, DNA was finally
422
Chapter69 : CENETICS 823
identifiedas the genetic material. lts structurewas femaleshaveXX.The sexof the child is determined
elucidatedin 1952 by Watsonand Crick. by the father.
Autosomalrecessive
Sickle-cell
anemia 1 : 100(inAfricans) Severe lifethreatening
anemia;
confers
resislance to malaria
Cysticfibrosis 1 : 2500(inCaucasians) Defective severelung
iontransport;
infectionsandearlydeath(beforethey
reach30 years)
Phenylketonuria 1 : 2000 Mental
retardation
dueto braindamage
deficiency
cr,-Antitrypsin 1 : 5000 Damage to lungsandliver
Tay-Sachs
disease 1 : 3000 Nervous
disorder; and
blindness
(inAshkenazi
Jews) pararysrs
immunodeficiency
Severecombined Rare(only100cases Highly immune
defective system;
early
(SCID)
disease reported
worldwide) death
Sex-linked
Colour
blindness 1 : 50 males Unable
to distinguish
colours
(li/B)
Hemophilia 1 : 10,000
males Defective
bloodclotting
Duchenne dystrophy
muscular 1 : 7000males Muscle
wastage
Mitochondrial
opticneuropathy
Leberhereditary Not known Damage mayleadto
to opticnerves,
blindness
827
a28 B IOTE CHNO LO CY
Medical informatics : This involves tne fransfer protocol) that can identify the protocol for
m anagem ent of biom edic al d a t a w i t h s p e c i a l communication over www.
referece to biomolecules. in vitro assavs ano
c linic al t r ials . BIOLOGICAL DATABASES
The collection of the biological data on a
CO M PO NENTS O F BI O I NF O R M A T I C S computer which can be manipulatedto appear in
Bioinformatics comprises three components varying arrangements and subsets is regarded as a
database.The biological information can be stored
1. Creation of databases : This involves tne
in different databases.Each database has its own
organizing,'storageand managementthe biological
w e b s i t e w i t h u n i q u e n a v i g a t i o nt o o l s .
data sets. The databases are accessible ro
researchersto know the existing information and The biological databases are, in general,
submit new entries. e.g. protein sequence data p u b l i c l y a c c e s s i b l eS
. e l e c t e de x a m p l e so f bi o l o g i ca l
bank f or m olec ular s t r uc t ur e.D a t a b a s e sw i l l b e o f databasesare briefly described (Table 70.1).
no us e unt il analy s ed.
2 Development of algorithms and statistics : Nucleotide sequence databases
This inv olv es t he dev elopm e n t o f t o o l s a n d
r es our c est o det er m ine t he r ela t i o n s h i pa m o n g t h e T h e n u c l e o t i d e s e q u e n c ed a t a s u b m i t te d b y th e
members of large data sets e.g. comparison of s c i e n t i s t sa n d g e n o m e s e q u e n c i n gg r o u p s i s a t th e
pr ot ein s equenc e dat a wit h t h e a l r e a d y e x i s t i n g d a t a b a s e s n a m e l y C e n B a n k , E M B L (Eu r o p e a n
protern sequences. M o l e c u l a r B i o l o g y L a b o r a t o r y ) a n d D DBJ ( D N A
Data Bank of Japan).There is a good coordination
3. Analysis of data and interpretation : The
between these three databases as they are
appr opr iat e us e of c om ponen t s 1 a n d 2 ( g i v e n
s y n c h r o n i z e do n d a i l y b a s i s .
above) to analyse the data and interpret the results
in a biologic ally m eaningf ul m a n n e r . T h i s i n c l u d e s Besides the primary nucleotide databases
DNA, RNA and pr ot ein s e q u e n c e s , p r o t e i n (referred above), there are some other databases
structure,gene expressionprofiles and biochemical also to provide information on Benes,genomes and
pathways. ongoing research projects.
Other nucleotidesequencedatabases
UniGene Thenucleotide
sequences
of GenBank
in theformof ciusters,
(www.
ncbi.
nih.goviUniGene/) genesareavailable.
representing
GenomeBiology Theinformation
aboutthecompleted
genomes
is available.
(www.
ncbi.nlm.
nih.gov/Genomes/)
EBIGenomes Provides
dataandstatistics
for thecompleted
genomes,
besides
(wwwebi.
uk./genomesi) theinlormation
on theongoing projects.
Protein sequencedatabase
SWISS-PROT Provides thedescrlption of a protein,
of thestructure its domains
(www.
expasy.ch/sport) structure,post-translational
modifications,
variants
etc.lt hashigh
levelof integration
withotherdatabasesandminimal levelof
reoun0ancy
PIR Protein
information
resource
(PlR)is a database
by the provided
(pir.georgetown.edu) *'l'::1':"::::'1 us'r
"::':'l:::T:":l-aRD
:n
Protein sequencemotif databases
PROSITE Provides
information
on proteinfamilies
anddomains.lt alsohas
(www.expasy.ch/prosite/) patterns
andprofilesfor sequences
andbiological
functions.
Pfam A database
of protein
families
defined
intheformof domalns.lt has
(www.cgr.ki.se/Pf
am/) multiple
alignmentof a setof sequences.
Macromolecular databases
P DB Thisis theprimary
database (3-D)slructures
for 3-dimensional
(www.rcsb.org/pCb) ol biological
macromolecules
(deterrnined
by X-rayandNMR
studies).
SCOP Provides
informalion
on thestructural of proteins
classification
(www.
mrc.
lmb.cam.
ac.uUscop/) The
er:l'::' "l ln'o"''l '_l"u'olll'
lscoP] ::':n'"]:o
Other databases
KEGG TheKyotoEncyclopedia of GenesandGenomes (KEGG)is a
genome.ad.jp/kegg/)
www. database withlatestcomputerised
informationon biomolecules
ano
cellbiology.
KEGGprovides detailson information
pathways,
interacting
molecules andtheconnecting linkswithgenes.
830 B IOTE CHNO LO CY
The advent of bioinformatics has revolutionized . To trace the evolutionary trees of genes.
t he adv anc em ent s in biolog i c a l s c i e n c e . A n d
biot ec hnology is lar gel y benefited by . F o r t h e p r e d i c t i o n o f 3 - d i m e n s i o n a l str u ctu r e o f
bioinf or m at ic s The bes t ex am p l e i s t h e s e q u e n c i n g prorerns.
of hum an genom e in a r ec or d t i m e w h i c h w o u l d
o M o l e c u l a r m o d e l l i n g o f b i o m o l e c u l e s.
not hav e been oos s ible wit ho u t b i o i n f o r m a t i c s .A
s elec t ed lis t of applic at ions o f b i o i n f o r m a t i c s r s o D e s i g n i n g o f d r u g s f o r m e d i c a l t r e a t me n t
given below.
. H a n d l i n g o f v a s t b i o l o g i c a l d a t a w h i c h o th e r w i se
. Sequenc em apping of biom o l e c u l e s( D N A , R N A ,
is not possible.
oroteins).
o ldent if ic at ion of nuc leoti d e seeuences of . D e v e l o p m e n t o f m o d e l s f o r t h e f un cti o n i n g
f unc t ional genes . v a r i o u s c e l l s , t i s s u e sa n d o r g a n s .
. Finding of s it es t hat c an b e c u t b y r e s t r i c t i o n The above list of applications however, may be
enz y m es . treated as incomplete, since at present there is no
. Des igning of pr im er s eque n c e f o r p o l y m e r a s e f i e l d i n b i o l o g i c a l s c i e n c e st h a t d o e s n o t i n vo l ve
c hain r eac t ion. bioinformatics
:,1
t.t:tltttll
:l :i , I I I , I I l
,i,.1'':,:'l'
'l ',,,, r
Clossary 83
,t"l' .' , ,
,.,, ,,.
Abbreviations
tt,
-lr , I ' , I i I I I l
,1,,,1
831
Agarose gel electrophoresis Electrophoresis carried
out on agarosegel to separate DNA fragments.
{ bz y m es Th e c at aly t ic ant ibodies or ant ibo d y Agrobacterium tumefaciens A rod shaoeo
zymes. bacterium that causes crown gall disease by
-^
{biotic stress The stress caused to plants clue to inserting its DNA into plant cells.
-:'bicides, water deficiency ozone, intense light
AfDS Acquired immunodeficiency syndrome, a
disease caused by a retrovirus resulting In
{bscisic acid An important plant growth regulator diminished immune response, and increased
' - th e ind uction of em br y ogenes is . s u s c e p t i b i l i t yt o v a r i o u s i n f e c t i o n s a n d c a n c e r s .
{ cid ra in Wh en t he pH of t he r ain wat er is less Aleurone The outmost layer of the endosperm in a
' - :n 5 .5, it is con s ider ed as ac id r ain.
seed.
\ctive site Th e sit e on t he enz y m e at whic h t h e
Algalization The process of cultivation of brue-
.
-:rstra teb ind s. green algae (cynobacteria)in the fields as a source
{ dap tor A synt het ic , double- s t r anded oligo - of biofertilizer.
' -.:leo tide used t o at t ac h s t ic k y ends t o
a blun t
Allele Alternative forms (one or mor:e)of a gene
--::e d mo lecule .
occupying a given locus on the chromosome.
{erated lagoons (aerated ponds) Facultatrve
.- ponds with surface aerators to Aflogeneic cells The cells taken from a person
-otlization
.eTco meba d od our s ( due t o ov er load of or ganr c o t h e r t h a n t h e p a t i e n t f o r u s e i n c u l t u r e , t i s s u e
^^a terials). e n g i n e e r i n ge t c .
833
834 B i OTE C HNO LO CY
Androgenesis Development of plants from male Autosomes Chromosomesthat are not involved tn
sex determination
Bametophytes.
Autotroph An organism that is capable of
A neuploidy An abnor m a l c o n d i t i o n o f
s y r r t h e s i z i n g i t s o w n f o o d e . g . p l a n t s th r o u g h
c hr om os om es , dif f er ing f r om t h e u s u a l d i p l o i d
phctosynthesis.
c ons t it ut ion.This m ay be due t o a l o s s o r g a i n c f
c hr om os om es . A u x i n s A g r o u p o f p l a n t g r o w t h r e g u l a t or sw h i ch
a r e i n v o l v e d i n c e l l e l o n g a t i o n , r o o t i n i t i a ti o n e tc.
A nnealing The pair ing of c om p l e m e n t a r y s i n g l e
e.g indole aceiic acid.
s t r andsof DNA t o f or m a doubl e h e l i x .
Auxotroph A mutant microorganismthat can grow
A nt ibiot ic A biologic al m olec ul e t h a t i s p r o d u c e d
w h e n a n u t r i e n t i s s u p p l i e d ( t h i s n u t r i e nt i s n o t
by one or ganis m whic h c an k ill/i n h i b i t t h e g r o w t h
needed by the wild type).
of anot her or ganis m .
Ant ibody A pr ot ein ( im m unoglo b u l i n ) s y n t h e s i z e d
by B-lymphocytesthat recognizes ar.rtigen.
B
Ant ic odon A s et of t hr ee nuc l e o t i d e s i n I R N A
molecule that are complementary to a set of three Bacillus thuringiensis A rod shaped bacterium
nuc leot ides ( c odon) in m RNA. w h o s e t o x i c c r y s t a l s a c t a s a n i n s e c t i c i d e a g a i n st
certain species of arthropods
Ant igen A s ubs t anc et hat elic it s i m m u n e r e s p o n s e
a nd induc es t he pr oduc t ion of a n t i b o d i e s . Bacterial artificial chromosome (BAC) : A vector
system based on the F-factor plasmid of E. coli.
Antioxidants A group of substances that can
BAC is used for cloning large (100-300 kb) DNA
pr ev ent ox idat ion e. g. v it am in E
segments.
Antisense therapy The in vlvo treatment of a Bacteriophage A virus that infects a bacterium
genet ic dis eas e by bloc k ing t r a n s l a t i o n ( p r o t e i n
Also called phage.
s y nt hes is )wit h a DNA or RNA s e q u e n c e t h a t i s
B a k e r 'sy e a s t T h e l i v i n g c e l l s o f a e r o b i c a ll y g r o w n
c om plem ent ar y t o s pec if ic m RN A .
yeast, Saccharomyces cerevisiae, used in bread
Apoptosis Programmed cell death. making.
Aptamers A special type of oligonucleotides that Base pair (bp) Ihe hydrogen bonded structure
can specifically bind to target proteins and not to formed between two complementary nucleotides
n uc leic ac ids . ( i . e . p a r t n e r s h i po f A w i t h T o r o f C w i t h C ) i n D N A
Aspartame A low-calorie artificial sweetener used structure.
i n s of t - dr ink indus t r y . Chem ic a i l y , i t i s a s p a r t y l - Base ratio The ratio of A to I or C to C in a
p heny lalanine m et hy l es t er d o u b l e - s t r a n d e dD N A
Assisted reproductive technology (ART) The Batch culture (batch fermentation) A cell or
n r anipulat ions of r epr oduc t ion i n a n i m a l s a n d m i c r o b i a l c u l t u r e t h a t i s g r o w n f o r a l i m i t e d ti m e . l t
h u m ans . follows a sigmoid pattern of growth
Attenuation A regulatory process used by some Bergmann's plating technique The most widely
b ac t er ia t o c ont r ol t he ex pr es s io no f c e r t a i n a m r n o used method for culture of isolated single plant
a c id ooer ons . cells.
CLOSSARY 835
Binary vector system A two plasmid vector system Bionretry Application of statisticalmethodsto
for transferring T-DNA into plant cells. l-he studybiologicalproblems.
vir ule nce g en es ar e on one plas m id while t he
BiopesticidesThe toxic compoundsproducedby
engineered T-DNA region is on the other plasmid.
l i vi ng organi smsthat can speci fi cai l yki l l a
Bioaccumulation (biomagnification) Increasingthe particularpestspecies.
concentration of chemical agents by way of BioplasticsThe biodegradable
plastics,chemically
increasingthe quantities in the organismsof a food composedof polyhydroxyalkanoates (PHAs).
cha in.
Biopol A biodegradableplastic composed of
BiodegradationBiological transformationof organic pol yhydroxybutyriaci
c d and val eri caci d.
comp ou nd s by living or ganis m s , par t ic ular ly t he
Bioprocesstechnology A more recent usage to
mrcro org an rSms
replacefermentation technologythat involveslarge-
Biofertilizers The nutrient inputs of biological scale cultivationof microorganisms for industrial
origin that support piant growth. purposes.
Bioaugmentation The addition of microorganisms Bioprosthesismaterials The natural materials
to waste sites so that the hazardous wastes are modi fi edto becomebi ol ogi cal liynerte.g.col l agen-
rendered harmless. based connective tissue forming porcine heart
valves.
Biofiltration The process of removing complex
rvastes (from domestic or industrial sources) by Bioreactor A growth chamber or a vessel
usr n Smrcro orSa nts m s . (i'ermenter)
for cellsor microorganisms.The cellsor
cell extractscarry out biological reactionsirr a
Biogas A mixture of gases mainly composed of bioreactor.
rrethane, COr, nitrogen and hydrogen sulfide. lt is
used for cooking purposes,generationof electricity
BioremediationThe biologicalprocessinvolving
Iiving organismsto remove unwantedsubstances
etc.
(contaminants or pollutants)from soil or water.
Biohazards The accidents or risks associatedwith
Biosensor An analyticaldevice containing an
biolo gical ma teria ls .
bi ol ogi calmateri al(enzyme,
i mmobi l i zed anti body,
Bioinformatics A science of using information organel l eor w hol e cel l ) w hi ch reactsw i th an
technology to understandbiological phenomena. lt analyteto producesignalsthat can be measured.
may be regarded as a marriage between biology Biosorption The processof microbialcell surface
and information technology. adsorptionof metals.
Bioleaching The use of bacteria to recover valuable BiosphereAll the zoneson the earthin which liie
metals {rom ores. is present.
Biolistics The process of introducing DNA into Biostimulation Addition of soecific nutrientsto
plan t an d a nim al c ells , and or ganelles by enhance. the growth of naturally occurring
bombardment of DNA-coated pellets under microorganismsthat converttoxic compoundsto
pressure a t h igh s peed. This is als o c alled as non-toxi ccompounds.
m i c rop roj e ctil e bombard ntent. B i otechnol ogy The appl i cati onsof bi ol ogi cal
Biochemical oxygen demand (BOD) The oxygen principles,organismsand products to practical
requil'ed to meet the metabolic needs of aerobic purposes.
orga nismsin wate r c ont aining or ganic c om pounds' BioticstressThe stresscausedto plantsby insects,
pathogens(viruses,fungi, bacteria),woundsetc.
Biological databases The collection of biological
data on a cofitputer in different arrangementsand BiotransformationThe use of biologicalsystems
sub)sets. of bi omol ecul es.
for the conversi on
Bionrass The organic rnassthat can be used as a BlastodermA stagein embryogenesis in which a
source of energy. Biomass also refers to the cell layerof nucleior cellsaroundthe embryosurround
ma ss pro du ce d b y a populat ion of liv ing or ganis m s. the i nternalmassof yol k.
836 BIOTECHNOLOCY
Broth Any fluid mediumsupportingthe growthof C el l l i nes A ni mal or pl ant cel l s that can be
microorganisms. cultivatedunder laboratoryconditions.
Bt plants The plant carryingthe toxin producing Cell-mediatedimmune response The activationof
gene from Bacillus thuringiensis,and capable of the T-lymphocytesof the immune system in
protectingthemselvesfrom insectattack. responseto a foreignantigen.
Chromatin The complex of DNA and protein rn a Composting The processof biological degrao'ation
cel l. of solid organic wastes to stable end products.
Chromatography An analytical technique dealing Concatemer A DNA molecule composed of a
with the separation of closely related compouncts n u m b e r o f i n d i v i d u a l p i e c e s ( o r s e q u e n c e s )j o i n e d
from a mixture. together via cohesive enos.
Ch romo so m e A phy s ic ally dis t inc t unit o f t h e Congenital The term used to describe genetic
Senome/ carrytng many genes. abnormalities present at birth.
Chromosome jumping A technique used for the Conjugation The transfer of DNA from one
identification of two segments of DNA in a bacterium to another through cell-to-cell contact.
chromosome separatedby thousandsof base pairs.
Constitutive genes Also called as house-keeping
Chromosome walking lt is a technique used to genes. They are expressed as a function of
identify the overlapping sequences of DNA in a interaction between RNA polymerase and the
chromosome in order to identify a particular locus promoter without any additional regulation.
of interest.
Contaminant A substancenot present in nature but
Cistron The smallestfunctional unit (sequence)of released due to human activities e.g. methyl-
DNA) that encodes a polypeptide chain. rsocyanate.
Clean gene technology The processof developing Contig map A chromosomemap in which various
transgenicplants without the presenceof selectable DNA segmentshave been joined together to form
marker genes or by use of more acceptable genes. a continuous DNA molecule.
Degeneracy This term is used in relation to genetic DNA sequencers The machines that are used for
code representingthe fact that there may be more the determination of specific sequences of
than one codor-rfor most amino acids. n u c l e o t i d e si n a g i v e n D N A m o l e c u l e .
Genetically modified (GM) foods The entry of Golden rice The geneticallyengineeredrice with
transgenicplantsarld animalsinto the food chain provitaminA (p-carotene)
enrichment.
representsGM foods.
GRAS This is a shortform for generallyrcgarded
Geneticallymodified organisms(GMOs) A term as safe,and is in usein somecountriesto represent
usedto represent an organisms that are genetically the safety(no historyof causingillnessto humans)
engineer ed l.t u s u a l l y d e s c ri b e sth e tra n sgeni c of foods,drugsand other materials.CRAS is also
plant sand tra n s g e n iac n i m a l s . usedto representthe hostorganismsemployedin
Genetic code The total set of 64 codonsthat code geneticengineeringexperiments.
for 20 amino acids,and threeterminationcodons. Gratuitousinducer A substancethat can induce
Genetic disease A diseasecaused by defective transcription of gene(s),
but is not a substrate
for the
gene(s) from one generatron
that can be transmitted inducedenzyme(s).
to the next. Greenhouseeffect The ohenomenonof retention
Genetic engineering Broadly involvesall the in of earth'sheat by the atmosphere.
vitro geneticmanipulations. Greenhousetechnology The growingof plantsin
Genetic portfolios The identificationof all the greenhouses
beforethe plantculturesaretransfered
geneticportfolios. to fields.
genesin individualsrepresents
Genetic immunization The immune responseof Creen revolution The improvement in crop
the body stimulatedby DNA vaccines. varietiesand their manaqementfor increasein
world'sfood supply.
Genetics A branchof biology involvingthe study
of genes. CynogenesisOvary or ovule culturethat resultsin
the productionof haploids(gynogenichaploids).
Genome The total contentof DNA represented by
t he genesc o n ta i n e di n a c e l l .
Genomic library A collection of clones H
the entiregenomeof an organism.
representing
Hairy root diseasesA plantdiseasecausedby the
Genomics The studyof the structureand function infection ol Agrobacterium
rihizogens.
of genomes.
Haploid Containing one set of chromosomes
Genotype The geneticconstitutionof an organism. (opposite
to diploid).
(oppositeof phenotype).
Hel-acells A pure cell line of humancancercells
Germ cell A reproductive cell which on maturation
usedfor the cultivationof viruses.
can be fertilizedto producethe organism.
Helix-loop-helix motif A domaincommonlyfound
Germ line Reproductive cellsthat producegametes
i n D N A -bi ndi ngprotei ns.
which in turn give riseto spermsand eggs.
Helix-turn-helixmotif The structuralmotif for the
GermplasmThe hereditarymaterialtransmitted to
attachment of a proteinto DNA molecule.
the offspringthroughgerm cells.
Gibberellins A group of plant hormonesthat Herbicides The weedkillers that destrov the
induc ec ell e l o n g a ti o na n d c e l l d i v i s i o n . unwantedand uselessplants.
Global warming The retentionof eafth's heat by Heritabletraits The characteristics that are unoer
atmosphere(greenhouseeffect) resultsin global the control of genes, and transmitted from one
war m r nS . generation to another.
Motif The part of a protein that mediates the Nodule A tumor like growth on the roots of
binding of regulatoryproteins (transcriptionfactors) legumes(beans,peanuts)that containssymbiotic
to DNA. nitrogenfixing bacteria.
Multicellular tumor spheroids (MCTS) ln vitro Northern blotting The transferof RNA from an
cellu lar th ree- dim ens ionalpr olif er at ingm o d e l s f o r electrophoresisgel to a membraneto perform
the stu dy of t um or c ells . N orthernhybri di zati on.
Multiple alleles The alternative forms of a gene NorthernhybridizationThe techniqueusedfor the
tha t h as mor e t han t wo alleles . detecti on of speciifc R N A mol ecul e through
Multiple myeloma A cancer of B-lymphocytes. Northernblotting.
Multiple ovulation See superovulation. Nucleoid A term used to reoresentthe DNA-
Mu sh roo ms The f ungi belonging t o t h e c l a s s contai ni ngregi onof a prokaryoti cel
c l.
b asido myc et es . Som e of t hem ar e edib l e e . g .
Agaricus bisporus (button mushroom).
Mutagenesis The changes in the nucleotides of
o
DNA o f an or ganis m by phy s ic al or c h e m i c a l Okazaki fragment A short segment of DNA
treatments. synthesi zed
duri ngrepl i cati on
on the l aggi ngstrand
Mutagens The agents that increase the rate of of doubl ehel i x.
mu tatio n by induc ing c hanges in DNA. Old biotechnologySeetraditionalbiotechnology.
Mutation The changes in the base sequencesof
Oleosin partition technology The purificationof
DNA that are heritable
forei gn protei nsthrough thei r producti oni n oi l
Mu tein s The s ec ond gener at ion r ec om b i n a n t bodi es.
therapeutic proteins are collectively referred to as
Oleosins The oil body proteins that are
mu tein s.
hydrophobicin nature, and are associatedwith
Myasthenia gravis An autoimmune diseasecausing ol antseeds.
neuromusculardisorders.The diseaseoccurs due to
OligonucleotideA smallpieceof syntheticsingle-
the production of antibodies againstacetyl choline
strandedD N A mol ecul e.
receptors.
Myelo ma A t um or c ell line der iv ed f r o m a Oligonucleotide-directed mutagenesis A technique
lymp ho cyte whic h us ually pr oduc es a s ing l e t y p e to al terone or morespeci fi cnucl eoti desi n a gene
(DNA sequence)so that a protein with specific
o f immu no globulin.
ami no aci d changei s produced.
Mycelium A mass of interwoven thread-like
filaments of a fungus or bacteria. Oncogene A genethat promotescell proliferation
to resultin uncontrolledgrowth.
Oncomouse The animal model of mouse for
N cancer.lt was grantedU.S.patentin 1988,the first
animal to be patented.
Natural selection The preservationof favourable
a llele s whi le r ejec t ing t he injur ious one s i n a Oocyte lt is a stagein the developmentof female
natural way. gameteor ovum (egg).The wordsoocyteand ovum
are used interchangeably.
Nick Th e oos it ion in a double- s t r ande d D N A
where one of the polynucleotides is broken due to Operon A clusterof bacterialgenes under the
lack of phosphodiesterbond. control of a sigle regulatorysequence.
Nitrogen fixation The process of conversion of Opine An amino acid derivativeformed in some
atmo sp he r ic nit r ogen t o am m onia. Bio l o g i c a l plantsby the condensation
of an amino acid with
nitrogen fixation occurs in prokaryotes and is a sugaror a keto acid.
catalysed by the enzyme nitrogenase. Organ culture The in vitro cultureof an organso
Nodulation The formation of nodules on the roots as to achievethe developmentand/orpreservation
of plants by symbiotic bacteria. of the ori gi nalorgan.
846 B IOTE C HNO LO CY
Primer A s hor t s equenc e of oligonuc leo t i d e st h a t RACE See rapid amplification of cDNA ends.
hybridizes with template strand and provides R a d i o i m m u n o a s s a y( R l A ) A n a n a l y t i c a lt e c h n i q u e s
in itiatio n f or t he nuc leic ac id s y nt hes is . b a s e d o n t h e p r i n c i p l e s o f r a d i o a c t i v i t yo f i s o t o p e s
Primer extension Synthesisof a copy o{ a nucleic and immunological reactions of antigen and
acid from a prirner. antibody. RIA is a sensitive technique for the
estimationof severalbiological compounds.
Primer walk ing A t ec hnique f or s equen c i n g l o n g
(>1 kb) p iec es of DNA. Random amplified polymorphic DNA (RAPD) A
P C R - b a s e dm e t h o d o f D N A p r o f i l i n g . l t b a s i c a l l y
Primordium The earliest detectable stage of an
i n v o l v e st h e a m p l i f i c a t i o n o f D N A s e q u e n c e su s i n g
org an in plant s .
r a n d o m p r i m e r s , a n d u s e o f g e n e t i c f i n g e r p r i n t st o
Prion A proteinaceous infectious agent which i d e n t i f y i n d i v i d u a l o r g a n i s m s( m o s t l y p l a n t s ) .
be ha ve sl ik e an inher it ablet r ait ( alt hough n o D N A /
RAPD See random amplified polymorphic DNA.
RNA is present).
Rapid amplification of cDNA ends (RACE) A
Prob e A labeled m olec ule us ed in hy b r i d i z a t i o n
technique that employs reversetranscription and
te ch niq ue.
PCR for the rapid amplification of cDNA ends.
Prokaryote An organism whose cells lack a
Recalcitrant xenobiotics The xenobiotics that ocr
memb ran e bound or dis t inc t nuc leus .
not easily undergo biodegradation, and therefore
Protein engineering Ceneration of proteins with p e r s i s ti n t h e e n v i r o n m e n t f o r a l o n g p e r i o d .
subtly modified structures conferring improved
pro pe rties e. g. higher c at aly t ic f u n c t i o n , Recombinant vaccines The new generation of
vaccines produced by employing recombinant
thermostability.
DNA technology.
Protein targeting The process of transport of
proteins.from one compartment to other within a Recombination Exchange of genetic information
cell. Also called as protein sorting. between two different DNA molecules
Proteome The total population of protetns Regeneration Development and formation of new
p rod uced by a c ell. orSans.
Recombinant DNA (rDNA) technology The Satellite DNA ReoetitiveDNA that forms a satellite
techniquesinvolvedin the construction, and useof band in a density gradient.
re c o m b i n a nDt N A m o l e c u l es.
Scale-up The expansion of laboratory experiments
Recombinantprotein A proteinthat is produced to full-sized industrial processes.
by the expression of a cloned gene of a
S C P S e e s i n g l e - c e l lp r o t e i n .
re c o m b i n a ncte l l .
Secondary metabolite A metabolite that is not
Reportergene A gene whose phenotypecan be
required for the growth and maintenanceof cellular
assayedwhich in turn can be usedto determinethe
functions.
functionof regulatoryDNA sequence.
Senescence The biological aging of ce l l s,
R e p re s s i o nIn h i b i ti o no f transcri pti on
by bl ocki ng
characterizedby loss of functions and degradatron
the binding of RNA polymeraseto transcription
of biological molecules.
i n i ti a ti o ns i te .
Septic tanks Anaerobic digestersof solids of the
Restriction endonuclease An enzyme that
sewage settled at the bottom of tanks.
s p e c i fi c a l l y c u ts D N A mol ecul e at speci fi c
n u c l e o ti d es e o u e n c e s . S e w a g e T h e l i q u i d w a s t e a r i s i n g ma i n l y fr o m
domestic and industrial sources.
Restrictionfragmeni length potymorphism(RFLP)
A restrictionfragmentwith variablelengthsdue to Sex chromosomes The non-autosomal X and Y
the presenceof polymorphicrestrictionsitesat one c h r o m o s o m e s i n h u m a n s t h a t d e t e r m i n e th e se t o f
or both ends. i n d i v i d u a l s . M a l e s a r e X Y , f e m a l e s X X.
Salvagepathway The directconversionof purines Single-locus probe Probe used in DNA finger-
and pyrimidines into the corresponding printing that identifies a single sequence (locus) in
n u c l e o ti d e s . the qenome.
CLOSSARY 849
Single nucleotide polymorphisms (SNPs) The Sub-protoplasts The fragments derived from
po sitio ns on t he genom e wher e s om e i n d i v i d u a l s protoplaststhat do not contain all the contents of
ha ve one nuc leot ide ( e. g. C) while ot h e r s h a v e a the plant cells.
d iffere nt nuc leot ide ( e. g. C) .
Subunit vaccine An immunogenic protein
Site-directed mutagenesis The techniques used to produced from a cloned gene or purified from a
produce a specified mutation at a predetermined d i s e a s e - c a u s i n go r g a n i s m .
po sitio n in a DNA m olec ule.
Suicide gene A gene that producesa protein which
Slud ge The s em i- s olid m as s pr oduc ed d u r i n g t h e i s c a p a b l e o f d e s t r o y i n gt h e c e l l b y d i r e c t o r i n d i r e ct
course of sewage/wastewater treatment processes. m e a n s .
SNPs S ee s ingle nuc leot ide poly m or p h i s m s .
S u p e r b u g T h e f i r s t g e n e t i c a l l ye n g i n e e r e do r g a n i sm
Solid substrate (state) fermentation Fermentatron (bacterialstrain of Pseudomonadthat was oatented.
processwherein the growth of the microorgantsms It carries different hydrocarbon-degradinggenes on
is carri ed out on s olid s ubs t r at es . p l a s mi d s .
Somatic cell Any body cell as opposed to germ Synchronization A term used to representthe cells
ce ll. Som at ic c ell is non- r epr oduc t iv e,a n d d i v i d e s when they are at the same stageof cell division.
by mito s is .
T
Somatic cell gene therapy The delivery of gene(s) I
to somatic cells to correct genetic defects.
Somatic embryogenesis Formation of embryos Tandem repeat A repeat consisting of an array of
from a s ex ual c ells . DNA sequencethat is repeatedcontinuously in tne
same direction.
Southern hybridization A technique used for the
detection of specific DNA sequences (restriction T-cells See T-lymphocytes.
fragments). T-DNA The part of the Ti plasmid that is transferred
Sparger A device that introduces air into a t o t h e p l a n t D N A .
bioreactor in the form of a fine stream. Telomere The natural end DNA sequence in a
Splicing A term used to describe the removal of cnromosome.
intro ns and joining of ex ons in RNA. Th u s , i n t r o n s Template The polynucleotide strand (of nucleic
are spliced out wh'ile exons are spliced together acid) that determines the sequence of nucleotides
Stem cell A progenitor cell that is capable of i n a n e w l y s y n t h e s i z e dn u c l e i c a c i d . T e m p l a t e i s
dividin g c ont inuous ly t hr oughout t he l i f e o f a n used by polymerases(DNA or RNA) for new strano
org anrs m . synthesis.
Stirred tank fermenter A fermentation vessel in Termination codons See stop codons.
which t he c ells or m ic r oor ganis m sar e m i x e d b y
Thaumatin A protein extractedfrom berries whicn
mecha nic ally dr iv en im peller s .
is about 3000 times sweeter than sucrose.
Stop codons The three triplets (UAA, UAC, UCA)
Ti plasmid The large-sizedtumor inducing plasmid
wh ich ter m inat e pr ot ein bios y nt hes is( tr a n s l a u o n l .
found in Agrobacterium tumefaciens. lt directs
Stuffer DNA The non-essentialDNA of a vector crown gall formation in certain plant species.
that can be replaced by a foreign DNA.
Tissue culture A process where individual celrs,
Subculture The transfer of cells from one culture or tissues of plants or animals are grown
vessel to another culture vessel. artificially.
Biotechnology [54]
850 B IOTE CHNO LO CY
of a polypeptide,the
TranslationThe biosynthesis Vitrification An undesirable
conditionin in vitro
sequencebeing determinedby codonsof mRNA. tissues,characterizedby brittlenessand glassy
appearance.
TransposonA geneticelementthat is capableof
moving from one positionto another in a DNA VNTRs Seevariablenumbertandemrepeats.
m o l e c u l el.t i s a l s oc a l l e da s transposableleement.
Trickling filters Oxidation units used for the W
biological treatment of sewage in small
c o mmu n i ti e s . Westernblotting The transferof proteinfrom a gel
Triplex A type of DNA structurewith three to a membrane.
p o l y n u c l e o ti d e s . Wild type The term usedin geneticsto describea
Trisomy The occurrenceof three copies of a commonly observedphenotype(in the normall
h o rn o l o g o ucsh l o m o s o mei n a nucl eus. nativestate)of an organism.
Trypsinization Disaggregation
of tissuesby the Wobble hypothesisThe processin which a single
e n z y m etry p s i n . tRNA can decodemorethanone codon(of mRNA).
CLO S S A R Y 851
X-ra y crys t allogr aphy A t ec hnique r r s e c l t o Zoo blotting A biotting techniquc used to
s lr Lr c t ur co f l . r n , e
d ete rmirret he t hr ee- c linr ens ional rleternrine the gerres oi relatecl organisms by
moie cules h v l t r i ciiz . t t i o n
Ye ast arti f ic ial c hr om os om e ( YAC) A c l o n i n g Zygote intrafallopian transfer (ZIFT) The transfer
vector constructeclfrom the conroonentsof a veast o f t h e f e r t i l i z e d e g g s ( z y g o t e s )w i t h i n 2 4 h o u r s i n t o
chro mosom e the fallopian tube by using laparoscopy
carboxylic acid
ACC 1- Am inoc y c lopr opane- .1 CFCs Chlorofluorocarbons
Ac M NPV Aut ogr aphac alif or ni c a m u l t i p l e n u c l e a r
regulator'
CFTR Cyslicfibrosistransmembrane
poly hedr os isv ir us . homogeneou elect
C H E F C ontour-cl amped s r ic-
ACP Ac y l c ar r ier Pr ot ein field electrophoresis.
ADEPT Antibody-directedenzyme prodrug therapy. c e steri l i tY
C MS C vtoP l asmimal
ADA Adenos ine deam inas e. C MV C ucumbermosai cvi rus
AFLP Am plif ied f r agr lent leng t h p o l y m o r p h i s m C OD C hemi caloxY gendemand
AH As s is t edhat c hing. C OH C ontrol l edovari anhypersti mul a t ion
AI DS Ac quir ed im m unodef ic i e n c y s y n d r o m e . CPPP Cyclopentenoperhydrophenanthrene
AK Aspartokinase CpTi Cowpeatrypsininhibitor
A[S Acetolactate sYnthase CSTR Continuousstirredtank reactor
AP-PCR Arbitrarily Primed PCR. CT Cytoplasmictransfer
ART Assisted reproductive technology' ammoni
C TA B C etyl tri methY l um
ATPS Aqueous two-Phase systems. C S C al f serum
BAC Bac t er ialar t if ic ial c hr om o s o m e . D E A E D i ethyl ami noethY l
BCG Bac illus Calm et t e- Cuer i n . DHFR Dihydrofolatereductase
BNR Biologic al nit r ogen r em o v a l ' synthase
DHPDS Dihydropicolinate
BO D Bioc hem ic al ox y gen de m a n d . DMD Duchenne'smuscular dYstroPhY
bp Base pair. modification
DMEM Dulbecco's medium'
of Eagle's
BPTI Bov ine panc r eat ict r y psi n i n h i b i t o r . DMSO Dimethylsulfoxide
Bt Baccillus thuringiensis D N A D eoxyri bonucl eiaci
c d
CAM s Cell adhes ion m olec ul e s DSBs Double-stranded breaks
CaM V Caulif lower m os aic v ir u s DVT Deerrvein thrombosis
c DNA Com plem ent ar YDNA. ' E A A s E ssenti al
ami no aci ds
CDK Cy c lin- dependentk inase . virus
EBV Epstein-Bar
CDRs Com plem ent det er m in i n g r e g i o n s ' E.C. E nzymecomml ssron
CF Cystic fibrosis EGF Epidermalgrowthfactor
852
A B B RE V IAT ION S 853
Hl-A Histocompatability locus antigen MODY Maturity onset diabetes of the young
mRNA Messenger
RNA rRNA RibosomalRNA
MSG Monosodiumglutamate RT-PCR Reversetranscriptionpolymerasechain
mtDNA MitochondrialDNA reaction.
MTF Multiple-tubefermentation SARs Scaffoldattachmentregions
NASA NationalAeronautics SpaceAdministration SCF Siliconcarbidefibres
NHGRI National Human Genome Research SCF Sisterchromatidexchange
. Institute fluid
SCF Suoercritical
NI I D M N o n -i n s u l i nd e p e n d e ndt i abetesmel l i rus SCID Severecombinedimmunodeficiency
OHSS Ovarianhyperstimulation syndrome SCS Soecializedchromatinstructure
p Promotersquence SCP Single-cellprotein
pa Polyadenylation
sequence slgA SecretoryimmunoglobinA
PAGE Polyacrylamidegel electrophoresis SINEs Shortinterspersed nuclearelements
PBR Plantbreeders'rights S IP C S urfacei mmobi l i zedpl ant cel l
PCBs Polychlorinated
biphenyls S N P s S i ngl enucl eoti depol ymorphi sms
PCNA Proliferating
cell nuclearantigen SPECT Single photon emission computed
PCR Polymerase
chain reaction tomography
PCV Packedcell volume bi ndi ng
S S B S i ngl e-stranded
PDGF. Platelet-derived,growth
factor ssDNA Single-stranded DNA
P D T Po p u l a ti o n
d o u b l i n gti me SSF Solid substrate(solidstate)fermentation
PEC Polyethylene glycol STRs Shorttandem repeats;stirredtank reactors
gel electrophoresis
PFGE Pulsed-field SUZI Subzonalinsertion
PC Prostaglandins;
Polygalacturonase T-DNA TransferredDNA
PGD Preimplantationgeneticdiagnosis TE Tissueengineering
PHA Polyhydroxyalkanoates TEUSA ThermometricELISA
PHB Polyhydroxybutyrate ThOD Theoreticaloxygendemand
PPi Pyrophosphate TCF-p Transforminggrowth factor p
P Q Q Py rro l o q u i n o l i nqeu i n o n e TGS Transcriptionalgenesilencing
PR Pathogen-related TlLs Tumorinfiltratinglymphocytes
PTGS Posttranscriptional
gene silencing TK Thymi di neki nase
PUFA Polyunsaturated
fatty acids Tm Meltingtemperature
RAC RecombinantDNA AdvisoryCommittee TMV Tobaccomosaicvirus
RACE Rapidamplificationof cDNA ends TNF Tumornecrosisfactor
RAIT Radioimmunotherapy TOC Totalorganiccarbon
RAPD RadomamplifiedpolymorphicDNA tPA Tissueplasminogenactivator
rDNA RecombinantDNA intellectualpropertyrights
TRIPS Trade-related
RFC ReplicationfactorC IRNA TransferRNA
RFLP Restriction
fragmentlengthpolymorphism TTC Triphenyltetrazoliumchloride
RI A R a d i o i m m u n o a s s a y TUNET ldt-mediated dUTP nick end-labeling
RlPs Ribose-inactivating proteins assay
RN A R i b o n u c l e i ac c i d VEGF Vascularendothelialgrowth factor
ROSNI Roundspermidnucleusinjection VNTRs Variablenumbertandemrepeats
RPA ReplicationproteinA XP Xerodermapigmentosum
RPMI RoswellParkMemorialInstitute YAC Yeastartificialchromosome
Index
855
856 BIOTECHNOLOCY
Ba cu lovirus,
14 1 B io in fo r m ati cs-appl i cati ons,830 B i osphere,658
Baker'syeast,365 Bio le a ch ing,400 Biostat, 454
Ba lan ce dsa lts olut ion,417 Bio fe a ch ing o( copper, 402 B i osti mul ati on, 719, 727
g en e,183
Ba ldn ess Bio le a ch ing of urani um, 402 B i otechnol ogi cal rnethods for
pol l uti on management, 663
Baseexcisionrepair,35 Bio listics,584
Bases,762 B i otechnol ogy ar-rddevel opi ng
Bio lo g ica l cal ci fi cati on, 664
countri es,746
BastaAventis,609 Bio lo g ica l databases,828
B i otechnol ogy and soci ety, 739
Batchculture/fermentation,
259 Bio lo g ica l fi l m, 692
B i otechnol ogy tree, 6
BCC vaccine,2O3 Biological nitrogen fixation, 639
B i otechnol ogy-deti ni ti ons, 3
Beer,369 Bio fo g ica f ni trogen removal , 700
B iotechnoiogy-history, 4
Beer-Lambert
law, 767 Bio lo g ica l phosphorus
I i otechnol ogy-publ i c
Beerwort, 369 r e m o val , 731 percepti on, 7
Be ntDNA, 17 Bio lo g ica l treatment of B i oti c el i ci tors, 5i 4
sewage, 689
Be nth ics,66 6 B i oti c stresses,596
Bio lo g ica l w arfare, 743
platingtechnique,506
Bergmann's B i otransformati onof anti bi oti cs,
Bio m a g n ificati on,666 309
Bia lph os,60 9
Bio m a r ke r sof pol l uti on, 660 B i otransformati onof steroi ds,308
Binaryvector,579
Bio m a ss,3 93 B i otransforrnati ons,306
Bio accu mula t ion,
666, 720
Bio m e th ylati on, 666 B i otri ckl e fi l ters, 678
Bioassays
in environmental
monitoring,660 t' | o m o te cu tes, ././| Biovenling, 727
Bioaugmentation,
7 19, 727 Bio m o n ito ri ng of ai r pol l uti on, 672 B i rnboi m and D ol y method, 95
Bioblaster,
584 Bio m o n ito ri ng of pol l uti on, 659 B i speci fi c monocl onal anti bodi es,
Biochemicaloxygendemand,683 Bio p e sticide,59B 218
B l ood gas moni tori ng, 304
Biocon ve rsio ns , 256 Bio p h o to ly si s,398
B l ood gl ucose bi osensor,299
plastics,386, 390,
Biodegradable Bio p la stics,626
626 B l ue-green al gae, 646
Bio p o l, 3 9 0
polymers,386
Biodegradable B l unt ends, 78
Bio p r o ce sstechnol ogy, 239
Bio de gra da tion,
718 B -Lymphocytes,21 7
Bio p r o sth esi smateri al s,475
Biodegradationof contaminated B OD bi osensor,663
Bioreactor-operalion, 245
soils,72 7 Bovine growth hormone, 741
Bio r e a cto r s,239
Biodegradation
oi herbicides,722 Bovine somatotropin story, 24l
Bio r e cla m ati on, 718
Biodegradation
of B oyer and C ohen experi ments, 75
hydrocarbons,722 Bio r e m e ciiati on,7.18
Brassica napas, 631
Biodegradation Bio r e m e d iati on of contami nated
of organic B read, 365
matter,695 so|s, / l/
B reast cancer, 181
Bio r e m e d iati on of ground
of pesticides,
Biodegradation 722
waIer, 728 B rew i ng, 369
Bioethanol,
399
Bio r e m e d iati on of w aste B romoxyni l ni tri l ase, 59C
Biofencingtechnique,728 ta n o s, / z/ B t resi stanti nsects,599
Biofe rtilize rs,645 Bio r e so r b abl epol ymers, 475 B t toxi n, 597
Biofilte rs,6 TS Bio sca ve n gersof metal s, 666 B t toxi n genes, 598
Biofuels,395, 398 Bio scr u b b ers,678 B ubbl e col umn bi oreactors,241
Biogasplant,397 Bio se n so r s ,29T B ud cul tures, 554
Biogas,395 Buffer action, 762
Bio se n so r s -appl i cati ons,
304
Bioh yd rog en ,39S Buffers, 762
Bio se n so r si n evi ronmental
Biohydrometall
ugy, 4OO m o n ito ri ng, 663 B utanol , 315
b to tn Io rma U CS , ull .l
Bio so r p tio n, 404 Bystander effect, 68
854 BIOTECHNOLOCY
Ca llu s, 4 9 8 Cellularsenescence,
435 Citric acid cycle, 799
Ca lvin cycle , 8 0 4 C e n t r ad
l o g m a ,1 1, 2 3 Clone, 82
Ca n ce r , 8 2 1 Centrifugal
elutriator,
466 Cfoning efficiency, 466
.l
antisense therapy, 70 Cephalosporins,
334 C l oni ng of fetal cel l s, 491
d ia g n o sis, 1 8 1
Cerezyme,666 C foni ng of humans, 744
gene therapy strategies, 16/
m o n o clo n a l a n tib o d ie s, 2 2 1
Chaga'sdisease-diagnosis,
177 C l oni ng of pet ani mal s, 47 1
.l
Ca n ce r a n d ce ll cycle , 2 9
Chainterminationmethod, 07 C l onogeni c assays,461
Carbohydrates, 772
Chaperones,
55 Clostr id i u m acetobutyl i cu m, 3 16
Ca ta lytic M Ab s, 2 2 6 Chimericoligonucleotides,
171 C ol l oi dal state, 763
Ca ta lytic RNA, 2 2 Chiral, 761 C ol l oi ds, /63
Ceneticrecombination,
259 C o l d e n R i c e2 , 6 1 9 -l84
Hemochromatosis,
Cenetics,822 751
Colgi apparatus, H e m o p h i l i a1, 6 4 , 1 9 4
Cenom e,38, 59 hormone,228
Conadotrophic HepatitisB, 201
149
Cenomesequencing, Cram-negativebacteria,754 HepatitisB vaccine,2O2
Cenom iclibr ar ies120
, Cram-positivebacteria,754 HepatitisB
Cenomesof organisms,I53 Cranularactivatedcarbon surfaceantigen,201
Leachate,715 L y o p h i l i cc o l l o i d s ,7 6 3
lsomers,758
786 Leafprimordia,498 Lyophobiccolloids,763
lsoprenoids,
95 Leber'shereditaryoptic Lysine,350
lsopycniccentrifugation,
neuropathy/ 5B Lysine-export,351
lsotopes,765
Lectins,60.1 Lysosomalenzymes,633
Itaconic acid, 328
641
Leghemoglobin, 751
Lysosomes,
cr-L-lduronidase,634
L e i s h m a n i a s i2s1, 0 L y s o z y m e2,7 5 , 6 0 5
Lesch-NyhansYndrome, 164 B-Lymphocytes, 817
Leucinezipper motif,66 817
T-Lymphocytes,
L e u k e m i a1, 3 5
J 753
Leucoplasts,
761, 773
Levorotatory,
Messenger
RNA, 21, 44, 96 Microbialenzymeproduction- Mitochondria,650
Metabolic assaysfor cel regulation,286 MitochondrialDNA, 58
viab ility,4 62 Microbialleaching,400 Molality,763
Metabolicdrain/burden,
139 M ic r obia ll i p i d s ,3 9 1 Molarity,763
of
Metabolicengineering Microbialmetabolicproducts,254 Mofasses,
249, 314
622
carbohydrates, M ic r obia lm i n i n g ,4 0 0 Molds,756
Metabolicengineering
of Microbialproductionof amino Molecularbreeding,652
lipids, 624 acids, 344
Molecular farming,622
Metabolicengineering
of Microbialproductionof
plant
Molecularmarker-aided
plants,622 antibiotics,329
breeding,648
Metabolicengineering
of Microbialproductionof foodsand
Molecularmarkerassisted
proteins,628 beverages,362
selection,652
lvletabolicload, 139 Microbialproductionof
Molecularmarkers,648
Metabolicpathways,796 indigo ,3 9 2
Molecularpharming,622
Metabolicpathways- Microbialproductionof organic
ac ids ,3 18 M o n e l l i n ,3 6 7 , 6 1 8
integration,
813
Microbialproductionof Monocistronic
mRNA,50
Metabolicpathways-
v it am i n s , 3 5 5 214
Monoclonalantibodies,
re gu latio n,
815
Microbialrecoveryof Monoclonalantibodies-
Metabolismof amino acids,808
petroleum,403 a p p l i c a t i o n s2,1 9
798
Metabolismof carbohydrates,
Microbialrubber,391 Monogenicdisorders,
823
806
Metabolismof cholesterol,
slime,692
Microbiological Monolayercultures,455
Metabolismof lipids,804 pollutants,680
Microbiological Monopartiteviruses,682
Metabolisms,
796 Microcarrierculture,456 Monoploids,538
Metabolite.796 technique,505
Microchamber Monosaccharid
es, 773
Metabolomics,
38 Microdrop method, 506 Monosodiumglutamate,367
Metacleavagepathway,722 289
Microencapsulation, Moondust,617
Metal biotechnology,400 Microfluidizer, 274 MTT-basedcytotoxic assays,462
Metallothioneins,
667 89, 48"1,586
Microinjection, arides,775
Mucopolysacch
Metal pollution-managment,
566 Microinjectionof DNA, 586 Multicellulartumor spheroids,
471
Metal scavenging,66T 234
Micromanipulation, Multichambercentrifuge,273
Methane,396 test,662
Micronucleus Multipartiteviruses,682
production,375
Methane-SCP Microorganisms as oil Multiplefixed bed adsorber675
Methanogenesis,
496, 695 f ac t o r i e s , 3 9 l M u l t i p l em y e l o m a 2
, 13
production.377
Methanol-SCP Microorganisms-growth Multipleovulation,228
kinetics,263
Methanogenic
bacteria,696 Multipleovulationwith
Microorgan 252
isms-isolation, embryo transfer,229
Methylationof DNA, 64
Micioprojectile bombardment,584 Multiple-tubefermentation
constant,7BB
Michaelis-Menten
584
Microprojectiles, technique,684
Microalgalphotosynthesis,
664
Micropropagation,
552 Multisurface
culture,456
Microarrays,
1O8,176
559
Micropropagation-applications, Murine leukemiaviruses,161
Microbialadhesive
Microprotoplasts,
528 Mushrooms,380
biopolymer,392
markergenes,182
Microsatellite Mutations,34, 258
Microbialbiofilm,694
1BB,652
Microsatellites, Muteins,192, 198
Microbialculturesystems,
259
Microsporeculture,539 Myocardialinfarction,195
Microbialenhancedoil
recovery,382
528
Miniprotoplasts, Mycophage,'l 77
Mismatch repair,37 Mycoprotein,379
Microbial enzyme production-
geneticengineering,286 Missensemutations,35 Mycolhizas, 646
Biotechnology [55]
866 BIOTECHNOLOCY
Ovariancancer,236
N o Ovarianhyperstimulation
syndrome,236
Ovary culture,542
Na r b o m ycin , 3 .10 Obesitygene,183
Ovule culture,542
NASA bioreactor, 455 Octopines,575
Oxidationditch, 706
Na tu r a l cu ltu r e m e d ia , 4 1 5 Okazakipieces,25
p-Oxidationof fatty acids,804
Ne m a to d e r e sista n ce ,6 0 6 3
Old biotechnology,
Ne o m ycin p h o sp h o tr a n sfe r a se5, 8 8
Ozone,732
63.1
Oleosinpartitiontechnology,
Ne ste d PCR, 1 1 5 Ozone depletion,733
O l e o s i n - h i r u d ifnu s i o n
nif genes, 642 protein,63.l Ozone hole, 733
NIH g u id e lin e s, 9 0 Oleosins,624, 631
Nitr ifica tio n , 6 9 2 , 7 OO Oligomers,783
Nitr ifyin g b a cte r ia , 7 5 6 rected
Ol igonucleotide-di
Nitr o g e n cycle , 6 3 9 130
mutagenesis,
Nitr o g e n fixin g b a cte r ia , 6 3 9 TT2
Oligosaccharides,
P
Nitrogen-fixing genes, 642 Oligospermia,23l
.l
Nitr o g e n a se ,6 4 l chip, i-08
Oligonucleotide ps3 gene, 8.1
nod genes, 644 Oncomouse,486 Packed bed bioreaclors, 242
No d u la tio n g e n e s, 6 4 4 Oocyte recovery,230 P acked cel l vol ume, 505
No n - a u to lo g o u s ce lls, 16 4 Oocyteretrieval,233 P acked tow ers, 677
No n - co m p e titive in h ib itio n , 7 9 0 Operon concept,60 Packed-bed reactors, 694
No n - fe ca l b a cte r ia , 6 8 5 P aki l o,378
Opines,574, 591
No n - h o m o lo g o u s r e co m b in a tio n ,3 2 Palatability of foods-genetic
591
Opine synthase,
No n - m e th a n o g e n ic b a cte r ia , 6 9 6 engi neeri ng, 6'l 8
30-l
Optical biosensors,
No n se n se m u ta tio n s, 3 5 P arasi tes,755
761
Optical isomerism,
No p a lin e , 5 7 5 P arki nson'sdi sease, 183
Optical isomersof amino
No r m a lity, 7 6 3 P arti cl e bombardment, 58 4
acids,778
No r th e r n b lo Vb lo ttin g , 5 9 , 9 9
791
Opticalspecificity, P arti cl e B un, 584
Nuclear transfer, 492
Organicacids-microbial P arti cul ateai r pol l utants, 670
Nuclease protection assay, 69 production,3lB P atenting bi otechnol ogy
Nu cle a se Sl m a p p in g , 6 9 Organcultures,468 i nventi ons, 744
Nu cle a se s,7 9 222, 821 Pathogenesis-relatedproteins, 605
Organtransplantation,
Nu cle ic a cid b lo ttin g
Organicair pollutants,
620 P athogeni corgani sms i n
te ch n iq u e , 9 7
Organic fertilizers,647 sewage, 684
Nu cle ic a cid h yb r id iza tio n , - 1 7 3
Organicslurries,708 pB R 322 pl asmi d, 83
Nu cle ic a cid p u r ifica tio n , 9 4
Organicsolvents-microbial P C R -ampl i fi edol i gonucl e oti de-
Nu cle ic a cid th e r a p y, 1 7 0 di rected mutagenesi s,132
- l.l production,3.11
Nu cle ic a cid s- co m p o n e n ts,
Organicwater pollutants,680 P C R -appl i cati ons,l 19
Nu cle ic a cid s- fu n ctio n s,11
553, 556
Organogenesis, P ecti n methyl transferase,6.13
Nu cle o lu s, 7 5 0
irect,556
Organogenesis-d Pekilo, 378
Nu cle o p la sm , 7 5 0
ndirect,557
Organogenesis-i P eni ci l l i ns,33l
Nu cle o sid e a n tib io tics, 3 4 2
Organotypiccullures,472 Peptide bond, 78.l
Nu cle o so m e , 1 9
Orthocleavagepalhway,722 P epti de bond formati on, 53
Nu cle o tid e e xcisio n r e p a ir , 3 6
611
Osmoprotectants, P epti de vacci nes, 2O2
Nu cle o tid e s, i.l
Nu cle u s, 7 5 0 Osmosis,764 Peptidyltransferase,53
anemia,355
Pernicious Plantcell culture,502 mRNA,50, 60
Polycistronic
752 biotransformation, 516 P o l y c l o n aal n t i b o d i e s2, 1 3
Peroxisomes,
measurement of growth,505
Perstraction,277 Polyenemacrolides, 340
measurement of viability,505
Petvaporation,277 secondarymetabolites, 507 P o l y e t h y l e ngel y c o l - m e d i a t e d '
suspension cultures,502 transformation,587
Pestresistance,599
s y nc h r o n i z a t i o n , 5 0 4 6.13
Polygalacturonase,
Petroleumplants,399
Plantcell culturesand 823
Polygenicdisorders,
Petroselenicacid, 626 5.16
biotransformation, P o l y h a p l o i d s5,3 8
pH, 762
Plantc ell - m q s cs u l t i v a t i o n5, 1 1 Polyhedrin g e n e ,1 4 1
Phagedisplay,70 752
Plantcefl-structure, kanoate
Polvhydroxyal
PhageM,., 84 Plantgrowthregulators,513,520 627
co-polymers,
Phagemiddisplay,72 Planttissueculturemedia,517 386, 626
Polyhydroxyalkanoates,
PhageI, 83 Planttissueculture- tyate, 387, 626
Polyhydroxybu
Pharmanimals,490 appli c a t i o n s , 5 0 6 Polyketide
antibiotics,339
Phasidvectors,84 Planttissueculture-basic chain reaction,112
Polymerase
Phe nylala ni ne,
353, 809 t ec hn i q u e , 4 9 9
Polymerases,
81
497
Planttissueculture-benefits,
Pho sp ha te
so lubiliz ing Polyphenoloxidase,616
bacteria,646 499
Planttissueculture-history,
Polyribosome,
49
Phosphatestripper,702 Pfantvariety tights,746
382, 774
Polysaccharides,
Phosphinothricin,
609 Plant ibo d i e s , 6 3 4
Pofyunsaturatedlatty acids, 776
acetyl-
Phosphinothricin 628
Plantsas bioreactors,
Pomatoes,533, 536
transferase,
589 Plantsas proteinfactories,628
Pond treatmentprocesses,
696
609
resistance,
Phosphinothricin 596
Planttransformation,
Populationdoublingsof cells,435
Phospholipids,
776 572
Plant t!'ansgenesis,
Positioneffects,490
method,109
Phosphoramidite Plasmidcopy number,95
linkage,l 71
Phoslhorothioate
pollution,665
Phosphorus PlasmidDNA purification,95
Post-harvest
spoilage,615
Phostrip, 7O2 Plas m id s8,3
gene
Post-transcriptional
242, 664
Photobioreactors, 753
Plastids, silencing,593
Photometry,767 Plastome,
594 ptional
Post-transcri
394, 664, BO3
Photosynthesis, Platetowers, 677 modifications,43
Plaquelift technique,127 Post-translational 56
modifications,
bacteria,398
Photosynthetic
Platingefficiency,466 Potentiometricbiosensors,299
Photosystems,
803
p-Pleatedsheet,783 Powderedactivatedcarbon, 699
Physicalgene(plant)'transfer,
583
262
Plugflow bioreactors, Pre-embryonicdetermined
Phytase,633
Plug-flowin-vessel
composting cells,558
514, 606
Phytoalexins,
reactors,113 genetic
Preimplantation
661, 667
Phytochelatins, 236
diagnosis,
Pluropotency,490
520
Phytohormones, Point mutations,34 treatmentof
Preliminary
Phytopathogens,
644 sewage,687
Pollenculture,539
666
Phytoplanktons, fermenter,24.1
Pressure-cycle
Pollulan 3
, 86
T28
Phytoremediation, Pollutants,659, 669 Pribnorvbox, 41
Piezoelectric 302
biosensors, Pollutioninducedpeptides,661 Primaryair pollutants,
670
Pig in organfarms,489 Pollut io nm o n i t o r i n g6, 5 9 Primarycell culture,437
785
Pigments, Polly,49'l Primaryexplanttechnique,441
Pituitarydwarfism,192 Primarylymphoidorgans,B-l7
Poly-Atail, 44
254
Primarymetabolism,
Plant breeders'ti1hrs,746 167
Poly-L-lysine,
gel electro- 254
Primarymetabolites,
498,
Plantbreeding-conventional, Polyacrylamide
) / z^ phoresis,93 of
Primarystructure protein,781
a68 BIOTECHNOLOCY
Primarytranscript,39, 43 718
Pseudomonas, tPA, 195
Recombinant
Primarytreatmentof sewage,688 Pseudomonassyringae,612, 742 vaccines,199
Recombinant
Primase,24 8',,
Pseudomonas-vitamin vacciniaviruses,211
Recombinant
production,357 194
Recombinate,
Prematuremenopause,236
Primaryexplanttechnique,441 Pulsed-fieldgel electrophoresis,
93
Recombinationof DNA, 31
Primerextension,69 Pump and treat technique,728 31
Recombination,
Primerextensiontechnique,104 P u r i n e sl,l
Recombivax,202
11
Pyrimidines, (plant cells),499
Primers,24, 114 Redifferentiation
107
Pyrosequencing,
Primerwalking,104 Regulatablepromoters,138
Prions,56 synthesis,327
Reichstein-Crussner
Procrit,198 Rennet,287, 364
Prograrnmedceli death, 435 Replicaplating,126
Prokaryotes,749 a bubble,24
Replication
Prokaryotichosts,82, 137 Replicationfork, 25
Promotersof transcription,592 Replicationin eukaryotes,27
Quantitativetrait loci, 652
310
Prostaglandins,
Quaternarystructureof Replicationin prokaryotes,24
Prostatemouse, 487 protein, 783 33
transposition,
Replicative
Proteaseinhibitors,601 Quaylecycle,376 Reportergenes,590
ProteinDNA interactions,65 553
Quiescentmeristems, 76, 136
endonucleases,
Restriction
133
Proteinengineering, Quinoid pigment,785 Restrictionenzymes,76
Proteinengineering-disulfide Quorn, 378
bonds , 133 Restrictionfragmentlength
polymorphisms, 185, 649
ProteinenBineering-
asparagine,
l33 Reteplase,196
57
Proteinlocalization, 182
Retirroblastoma,
32
Retrotransposition,
Proteinproductionin plants,628 ll
Proteinpurification-use
of Retroviralvector method,481
MAbs, 243 Retrovirusvector system,165
R A C E ,1 1 8 , 1 2 3 , 7 2 1
Proteinsorting,57 46,757
Retroviruses,
Radioactiveisotopes,765
Proteintargeting,57 Reversemiceller systems,277
pollutants,
Radioactive 681
Proteinase
inhibitors,601 46, 115
Reversetranscriptase,
769
Radioimmunoassay,
ProIeins,777 Reversetranscription,46
225
Radioimmunotherapy,
783
Proteins-classification, Reverse PCR,115,
transcription
Randomamplifiedpolymorphic
781
Proteins-structure, 125
DNA, I16
P_roteome,
38 of cDNA
Rapidamplification Rheologicalproperties,383
Proteomics/
38 ends,70, 1'lB Rheology,382
Protocatechuate,
722 Real-timequantitative
PCR,116 Rho dependenttermination,41
Protoclonal
variations,
546 Recalcitrant 720
xenobiotics, termination/42,
Rho independent
Protoplastcoculture,527 Recemicmixture,761 123
Protopfastculture, 523, 526 DNA Advisory
Recombinant Rhodopsinknockout mouse,487
Protoplast
fusion,529 Committee,90, I58 Ri plasmids,577
isolation,523
Protoplast DNA, 75
Recombinant Riboflavin,357
Protoplastregeneration/527 Recombinant
toods,742 P, 22
Ribonuclease
Protropin,193 hCH, 192
Recombinant RibosomalRNA, 22, 45
ProvitaminA, 618 insulin,191
Recombinant nactivating
Ribosome-i
A enrichedrice, 618
Provitarnin Recombinant 197
interferons, proteins,605
Pseudobactin,
645 proteins,144
Recombinant Ribosomes,49
INDEX 869
Ribostamycin,
310 SCPfrom methane,375 Shikimicacid pathway,608
Ribozymes,
22, 53, 172, 604 SCPfrom methanol,377 Shine-Dalgarno sequence,
51
Rice actin promoters,592 SCPfrom sewage,379 Shoottip culture,553
Ricin,2 23 SCPfrom wastes,378 Short interspersed nuclearelements
Risksof biotechnology,740 SCPfrom wood, 379 ( S l N E s )3,3
RNA plant virusesas vectors,582 Screeners,
687 Shotgunapproach,120
RNA polymerases,
42 Scrollcentrifuge,
273 Shreddar,'687
RNA primer,24 air pollutants,670
Secondary Shuftlevectors,86
RNA structure,19 Sickle-cellanemia,48, 179
Secondarymetabolism,254
type s.21 Siderophores,
644
Secondarymetabolites,254
RNA vaccines,207 Signalpeptide,57, 146
Secondarymetabolites-el icitor
Roller bottle culture,456 in d u c t i o n5, 1 4 Silentmutation,35
Rotarydrum vaccum filler, 271 Secondarystructureof Siliconcarbidefibres,587
Rotatingbiologicalcontactors,
693 protein, 782 Siliconcarbidemediated
Secondarymetabolites(of plant transformation,
587
Roughingfilters,693
cultures),507 Simplecopy segments
of
Roundspermidnucleus
Secondarytreatmentof genomes,65l
injection,235
sewage,689 Simplesequencelength
Round up, 608
SecretorylgA, 634 polymorphisms,
651
Rubberplant,399
Sedimentation
coefficient,768 Simpletandemrepeats,1BB
Rubisco,626
Sedimentation
tanks, 688 Singlecell clones(of plants),505
Single-cell
oils, 391
Seedterminatortechnology,621
Singlenode culture,554
Selectablemarkergenes,588
Singlenucleotide
Selectivebreedingmethods,480
s Semi-aridplant biotechnology,
653
p o l y m o r p h i s m s1,5 1 , 1 7 9 , 1 8 8
S i n g l e - c eol li l s , 3 9 1
Semi-aridregion,653
protein,373
Single-cell
Saccharin,
367 Semi-continuous
culture/
protein-use
Single-cell of
Saccharification,
368 fermentation,
261
microorganisms,374
Saccharomycescerevisiae,137, Senescence,
31, 435 Sisterchromatidexchange,652
140, 365, 368 Senescence
and cancer,3 l Site-directedmutagenesis,
129
Safeagriculturalpractices,602 Sept a g e , 7 1 6 Skimmingtanks,687
Salmonella, 210 Septagedisposal,716 Slopeleaching,402
Salvagepathway,2'14 Septictanks,707 Sludge,708
SatelliteRNAs,603 Sequencecharacterizedamplified Sludgeconcentration
Sauekraut,365 regions,652 (thickening),
709
Scaffoldaftachmentregions,593 Sequencetaggedsites,651 Sludgeconditioning,7'l4
Scale-up,453 Sequencingbatch reactor,691 Sludgedisinfection,
714
Scale-upin monolayer,
455 Serum-free
culturemedia,421 S l u d g ed i s p o s a l7, 1 5
Scale-upin suspension,
453 Severecombined Sludgestabilization,
710
Scale-upof fermentation,268 im m u n o d e f i c i e n c y6, 2
'l , 2 1 7
Slurry-phase
bioreactors,
728
Scale-upmonitoringof cell Sewage,682,686 Small sewagetreatment
growth, 458 Sewagesludge,708 systems,706
Scleroglucan,
385 Sewagetreatment,686 Solidphasesoil reactor,728
Schistosomiasis,
493 Sex chromosomes,823 Solid substrate(state)
SCID mouse,217, 487 Sex-linkedinheritance,
824 fermentation, 247
SCPfrom alkanes,375 Shelf-lifeof fruits-extend.O, S o l u b l eR N A , 2 1
Ur,
SCPfrom COz, 379 :. _
ot) >o t u u o n s , . / b 2
870 BIOTECHNOLOCY
geneexpression,
Tissue-specific 65 method, 482
TK suic idegene,169 for human di seases,487
mi croi nj ecti on method, 481
u
To mat or ipening,613 retroviral vector method, 481
Topatoes,533 Transgeni cmosqui toes,493 Ubiquitinpromoter,592
Totalorganiccarbon,683 Transgeni cpi gs, 488 768
Uftracentrifugation,
Totipotency,490, 497 Transgeni cpl anl s, 572, 585, 622 Uniformresourcelocator,828
Totipotent,572 Transgeni cpl ants as bi oreactors, Urea biosensor,
300
Toxoids,200 622 Urea cycle,809
Tradesecrets,745 Transgenicplants-therapeutic Urokinase,196
Traditionalbiotechnology,3 proteins,638
'lransgenic
Traditionalvaccines,199 sheep,488
Transamination,
809 Transgenic
snails,493
Transcriptional
genesilencing,593 Transgenic
tsetseflies,493
Transcriptionfactors,43 Transgenic
veggievaccines,637 v
Transcription
inhibitors,45 Translation,
46
Transcription,
39 Transposable
elements,32 Vaccines,l98
in eukaryotes,
42 Transposition,
32 Vaccinesin plants,636
in prokaryotes,
39 Transposition
of genes,67 V a c c i n i av i r u s , 2 1 1
Transcriptome,
38 I'ansposons,
32 Vacuoles,753
Tra ns c r ipt om ic s , 3S Transposontagging, TO Variablenumber tandem repeats,
Transduction,88 fray towers, 677 186
TransferredDNA, 70 Trehalose,624 Vectorrecombinant
vaccines,211
Transfer
RNA, 21, 45 l r l ac y l gl y c er O l S/ / /t) Vectorvaccine,211
Transformation,
87 Tricklingfilters,692 Vector-mediated(plant)gene
Transformation
assays,462 Triosephosphate isomerase, 133 rransret5/J
Transformation
efficiency,87 Triple repeatdiseases,-180 VectorlessDNA transfer,583
Transformation
of cells, 463 Triple-stranded
DNA, 17 Vectors, 82
Transformedcells, 430 Trophophase,
254 Vegetablevaccines,636
Transformed
cells-characteristics, Trypsinization,438 Veggievaccines,636
463 Tryptophan, Vermicomposting,
714
353, 811
Transgene,480, 572 Viable celfs-separalion,
44'l
Tryptophanoperon, 62
Transgenesis,
480 Vinegar,325
Tryptophanrepressor,62
Transgenesis
in largeanimals,487 v r r a to r s e a s e s , , / 5 /
Tuberculosis-diagnosis,
176
in plants,572
Transgenesis Virapap,178
Tubercu
losis-recombi
nant vaccine,
Transgene
stability,593 203 Virus coat proteins/602
Transgenic
animals,480 Tubularbowl centrifuge,
272 Virus resistanceof plants,602
Transgenic
bollworms,493 Tumornecrosisfactor,168 gene transfer,58'l
Virus-mediated
Transgenic
Btplants,743 Tumor-infiltrating
lymphocytes,168 Viruses-characteristics,
757
Transgeniccattle, 488 Tumor-suppressor gene, .l69 Viruses-importance,
757
Transgenic
ihickens,489 Tumorigenicity,
465 Virusesin gene therapy,159
crop plants,620
Transgenic TUNELassay,436 Viscosity,764
Transgenicfish, 49O Turbidbstatbioreactors,262 Vital force theory, 758
Transgenicgoats,486 Two-geneexpressionvector,144 V i t a m i nB r 2 ,3 5 5
Transgerric
medflies,493 Two-vectorexpressionsystem,144 VitaminC, 327
Transgenicmice, 2"17,481 Tyrosine,€09 Vitamins-microbial
production.355
applications,
485
embryonicstemcell TyrosylI-RNA synthetase,
136 Volatileorganiccompounds.618
6?2 BIOTECHNOLOCY