Review of

Literature
 Review of Lllemture

TO meet the objective of this research on L-phenyl acetyl carbinol, it was necessary lo gel

detailed information from the literature to justify thc significmce of the present

investigation. In this regard, relcvant inforn~ationfrom the litcraturc has been collected nnd

critically reviewed under the following sections

P Ephedrine and its importance

> Extraction of ephcdrine from plant

P Synthesis ol'ephcdrinc througli clle~ilicalroute

B Synthesis of ephedrine through Biochcmical route

> Production of L-I'AC is important (sincc I,-I'A(' is an intcr~~~ctIi:ltc

in 111~'

biosynthesis of cphedrinc).

B Analytical procedures of detection of L-PAC'

2.1 EPIiEDRlNE AND ITS IMPORTANCE

The chemical compound cphcdrine is an alkaloid dcrivcd from a shruh it1 Illc

family Ephcdraceue, commonly known as cphcdra. I t is clnscly rclatcd to

methamphetamine and other phenethylamines.

It is a stimulant that acts on the central nervous system, and is widcly used as a

nasal decongestant as a treatment for asthma.

Ephedrine is found in many popular wcight control products, some of which the

FDA believes may be hazardous. Ephedrine HCL, used as a hronchodilator is considered a

drug and not a supplement. Ephedrine is supposed to help stimulating he brain more than

caffeine and has been abused for enhacing the performance.

 The traditional Chinese herb ma huang, or ephedra sinica, contains natural
ephedrine and is often used in health supplements.

Ephedrine is commonly used in clandestine mcthaniphetanrine labs. There are

several methods for convening ephedrine into mcthan~phetemine. It's chemical

composition and other details are provided in Tahlc 2. I .

u,<lVEiiz' : ' I ' " zH\
Table 2.1 Ephedrine Propertics JNT UNIVI-'R'l r ' 'S ~.LY.
.... -- .............. . . . . -- ....-. . . . .
:NAME Ephcdrinc all NO........
- .---+-,-..
4- Nn , .... ....... .............
SYSTEMATIC CHEMICAI,
~ ~ I I I ~ I ~ c ~ I --01
( 1 R , ~ ~ ~ - ? - ~ I ~ I I ~ I -pIicnylpropon-

(rnethyla~~~i~~o)c~hyl]hcn~c~~c~~lctlir~loI

IN AMES
CHEMICAL . -..- . . - ..

a -[I -(mctllylan~ino)ethylJbcnxyl alcohol 1i

Ephedrine is a stimulant that works on the adrenergic receptors of the ccntral

nervous system. It has been shown to increase the level of dopaminc, which is a precursor

to both epinephrine and norepinephrine, which are vital ncurotransmittcrs. Dopaminc is

also an imponant neurotransmitter around thc hypothalamus, an area of the brain that is
 Review ~Ufmtun

important for body arousal. Unlike caffeine, which exerts many of its benefits in muscle

tissue, ephedrine appears to work primarily on the central nervous system.

inhibits the reuptake of norepinephrine, a hormonc sccrcted by tho 111edullaof the

adrenal glands, that acts as a neurotransmitter. The sympatl~ctictltrvous system functio~~~

in response to short-tern1 stress; hence norepincphrine, whosc lcvcls are boosted by

ephedrine, increases thc lieart rate as well as blood pressure. Olbrr actio~ls of

norepinephrine include increased glycogenolysis (the convcrsion of gl!eogal to glucosc)

in the liver, increased lipolysis (the conversion of fats to li~ttyacids) i n adip~sc(lilt)

tissue, and relaxation of bronchial smooth muscle to opcn up the air passilgcs to lhc lu~~gs.

All of these actions represent a mobilisation of tl~cbody's rcsourcts in ordcr to 111ettthr

stressful challenge; such a response is often termed the "flight or light" syndrome.

Basically, ephedrine keeps the body in "combat" niade for of'exlcndcd periods of

time, facilitating weight loss.

2.2 EPHEDRINE FROM PLANT SOURCE

The plants Ephedras are dioccious subshrubs with the same habit as horse~ails.

with slender, angular, and striated branches, and with leaves reduccd lo memhranous

scales. The female flowers are reduced to the ovule and surroundcd by bracts that are red

and fleshy at maturity. The male flowers are grouped in yellowish catkins.

The species that contain substantial quantities of alkaloids arc mostly Asian: 6.
Pakislan.
equisetina E, sinica from China, E. intrrmedicc and O ~rurdiumufrom India

About ten s p s i a are found in Nonh America, for cxamplc 6 nevuden.~i.v.Ephedrm are
 Review ojLNemme

seldom found in Europe (E. nrajor E. procera, E. campylopodrr, E. dimchya, ~ . [ s h b b y

horsetail] in the Atlantic coast and except for E. major, most of these species, like the

North American species. are thought to contain no alkaloids or in negligible concentntion

if present.

Clrenrical Cun~posiliua.Flavonoids and proanthocyanidins havs hern idcntiiicd in

the drug, but nitrogen conlaining substances-proloalk,7loids-an: 111s fbcus of at~cntioa.

These are phenethylamine-typc derivatives and their concaitration. wliicli varics as ;I

function of the species, can exceed 2%. The chicf cons tit urn^ is alnlost alwitys (- )

ephedrine, which represents from 40 to 90% of the total alkaloids. ( )-llplvdrinc 1;. (1 K ,

2s)-1-pheny I-2-methylaminopropan-1 -011 occurs alongside (+)-psa~dophedrinc(which

has the IS,2s configuration) and the corresponding nor and N.N-dimcthyl daivativcs. All

of the Asian ephedras contain alkaloids, but their concentration varics dcpcnding on llic

species: E. sinica ( I .3%), E.cquiselina (2.2%),E. monnsprmrr (2.8%),E in~crmcdicr( I . l -

1.6%). Ephedrine is tile major compound in most species, cxccpt in 1;. interntcdilr where

pseudoephedrine is dominant.

Pharmacological activio Ephedrine is an indirect sympathomimetic. S~ructurally

very close to adrenaline, it triggers the release of endognous catecholamines from thc

post-ganglionic sympathetic fibers. It slimulales cardiac automaticity and has a positive

ionotropic activity; it accelerates respiralion and increases its intensity; it is a

bronchodilator and a stimulant of the brainstem respiration center; it decreases the

contractility of the bladder. It is not metabolised much, can bc used orally, and its duration

of action is longer illan that of adrenaline. It is well rcsorbed and highly lipophilic; it
 Review oJ Llleraturc

crosses the bhod-brain barrier and, by releasing mediators centrally, has a stimulating
psychic effect: stimulation of the attention and ability to concentrate, decrease in the

sensation of fatigue and the need for sleep. High doses can cause headaclies. anxiety,

tremors, insomnia, and psychotic manifestations; redness of the face; nausea: tachycardia

and pecordial pain; sweating; urinary retentitrn. and morc.

Epliedroxane and (+)-pseudoephedrinearc espcrinlcntal anti-itifla~ii~nutoq

agents

Uses of eplredrus :In France its uses arc vcry limitcd. In (icrnial~p.I:' Si~rrt~tr
c;~nhr usrtl

by the oral route, but only'for a short time. I t is believed Illat in Asia, tlw tlrug Ili~sI~ce11

used for about 5 millenia. Mahuang consists of the stenls of B sirtic-(I. (:' i t ~ ~ ~ ~ r r t rand
r~rl~~i,

E. equisetina, and is official in the Peoplc's Repuhlic of Cllina whcrc it is ussd ils iin

antiasthmatic, diuretic, and sudorific. 'l'llc ('hinese Pharniacopocia i~lso dcscrillcs

mahuanggen (ephedra root), a drug reputed 10 br an antisudorificand uscd as such.

2.3 SYNTHESIS OF EPHEDRINE THROUGlI CHEMICAL KOCJI'K

Of the many routes of synthesis of (+) and (-) xphcdrinc thc one givcn by Niryiri

et. a1.(1929) Scheme 1 is of commercial interest. Condensation of 13en7aldchydc with

nitroethane in the presence of potassium carbonate yields a diastcm) mixture of

nitroalcohols. Reduction of this nitroalcohols yields a mixture. (5).. Norephedrine can be

separated from mixture. Methylation of separated from mixture yiclds (k) ephedrine that

can be resolved into optical antipodes without difficulty.

 Review of UIennrn

Scheme 1:

c6H&H0 + CZHSNOZ i(rCO3 + CrjlhCH (011) CH (CH1) NOz H~il't +

~enuldehydb Nitro ethane Nitro alrol~olr

C6H5CH(OH) CH (CH3) NHz Srparate+ (+) Nurephcdrinc

(1) + (11) (11)

C6HsCH (OH) CH (CH3) NHCHj

-
(i.) Epbcdrine

Figure (2.1) Ephcdrinc Synthesis by N a g ~ei / ~ /

Another synthesis was given by Manske CI.at.. (1929) ( Scheme 2). Whcrc thcy

reported its synthesis by the catalytic reduction of' I-l'l~cnyl I'ropanonc-I. 2-dioric

(Benzoyl acetyl) in the presencc of Methylamine in Methanol solution.

Schcmc 2

C6HSCOCOCHJ+ CH3NHr CH30H + CrllsCOC (zNCIIJ)Cllt 111Il't ,

Benzylacetyl Methylamint Intermediate

C~HSCBOHCH(CHJ)NHC& Hnalvcd By M r m 0
~1 Manddic Acld
+
(i)-Ephedrine

-
(-) Ephedrine + (+)- Ephedrine

Figure (2.2):Ephedrine Synthesis by Manskc et al

 Review of UICMRCM

The raeemic mixture is resolved inlo optically active ephedrine by means of msmdelic

acid.

Among the various strategies for synthesis of ephedrine alkaloid, 111cfollowing are

worthy of mention. Spath and Gohring (Scheme 3)synthcsized k>tli riic~micfornls of

ephedrine from propionic aldchydc. bromine, and phenylmagncsiun~bromide:

Scheme 3:

Figure (2.3): Ephedrine Synthesis by Spath & (;ohring

They obtained DL-pseudoephcdrine, which was isonlcrizcd to cphcdrinc. lilwryard

started with propiophenone; the u-methylan~inopropiophcnoncli)rnicd was rcduccd by

sodium amalgam in presence of dilutc hydrochloric acid and by hydrogen in prescncc of

palladium on wood charcoal. The main product was pseudocphedrinc. In 1929 1:ourneau

condensed benzene with the acid bromide of a-bromopropionic acid. 'Ihe resultant a -

bromopropiophenone was converted into the secondary amine, which yieldcd cphcdrinc on

reduction with hydrogen (over platinum black catalyst).

Propionic acid was used as the starting material for synthesis ol'cphcdrine, as this

acid is a by-product in the manufacture of acetoacetic ester and can be casily obtained from

acrylonitrile, which is produced in our country on a large scale.

The rcaction scheme(4) given below shows that propionic acid is chlorinated by

phosphorus trichloride; phosphorous acid is separated from the resultant solution of

propionyl chloride in bcnzene, and the bcnzene solution reacts funhcr with alun~inum

chloride to give propiophenone in 83-85% yield, colculatcd on tlle propionic i~cid~iikcn(a).

Scheme 4:

Figure 2.4: Ephedrine Synthesis using Propionic acid as starti~rfirnrleri:~l.

From the aqueous alkaline solution after amini~tion I I g of' a rnixturc of

methylamine hydrochloride and hydrobromide was isolated.

The a -bromopmpiophenone (111) formed by reaction (b) is n~cthylarninatcdin the

same vessel.; this reaction gives a -methylaminoethyl phcnyl ketone (cphctlronc) in 70-74%

yield on the propiophenonc taken. Ephedrone is reduced by molcct~lar hydrogcn in

presence of m e y nickel catalyst (c). The yield of Ill.-cphedrinc in this rcaction,

calculated on the ephedrone taken, is 64%; 19% of DL-pscudophcdrinc. to hc convcrtcd

into DL-ephedrine, is also obtained.

 Review of LIIcroIure

2.4 SYNTHESIS OF EPHEDRINE THROUGH BIOCHEMlCAL ROUTE

Biosynthesis of Ephedrine and related alkaloids:

These alkaloids are formed by a union of a C6'CI unit a~ldC2unit. For many years

it has been known lhat phenylalanine is the precursor for cYb-CI C4'r("
. .. being
. . . nloicry.

first to benzaldehyde or benzoic acid. Recently Grue-Sorenscn i111dSpnscr u tng

..-
"6-arid "(7
'~-labelledprecursors in feeding experiments with E.g~ri~rllitn?cr
lisvc shown that bcnzoic

C k C O group of pyruvic acid to Ihr~iicplicdrinc and related

ilcid combines with the intact--.

alkaloids with I -phenylpropan-l 2-dionc and (5)-(-)-2 l -pl~cnyprop;rn-1

il~lli~lcb- -oar
A,.

(cathinone) serving as intermediates. The route is illustrated in I:ig.( 7.1) I -llhcnylpropan-

1 ,Zdione and cathinone are known constituents of crt~hirzrlrtlis hilt 111~'prcvious non

isolation of cathinone from ephedra is attributed to its high c l f i ~ i ~ nof~incorporation

y illfa

non-alkaloids.

n 4
(IS,25).(+).p*udocphcdnnc (IL~SH*>- 1tR.m-b- (4UH--

FIGURE 2.5 : Biosynthcsis of ephedrine and rclaicd alkaloids

lt has been shown by ''c nuclear magnetic resonance spcclroscopy that the labelled
c2fragment of . $ . l ' ~ ~ >pyruvir acid is ~ransfcrrcdinlact into thc C-111ethyl group and the
adjacent carbon atom of the ephedra alkaloids, norephedrine, ephedrine,

nor~seudoephedrine,and pseudoephcdrine, in growing plants of Ephcdru gcr~rdiona.nis

finding serves to identify pyruvate as the elusive precursor of the alipl~aticC2 terminus of

the skeleton of the alkaloids. In earlier experi~ncntswith I4c-labcllcd suhsmtes. labcl from

c3-I4c>
pyruvic acid was incorporated mainly. but not exclusively. into tllc C-methyl

group of ephedrine, and label fro111 <'-I4c> PYnl\'iitt was incorporiitcd similarly into tl~c

carbon atom adjacent to the C-n~cthylgr1)up.A C6'r1 unit rtlatcd I(, I>cnzaldellydcor

benzoic acid has long bccn known to generate tl~chcnzylic lii\glncnt of 111sc a r h w skcloton

of the ephedra alkaloids. It is likcly that the carbon skclcton ol'cphcdrinr is pcnerntcd liom

pyruvate and either bcnzaldehydc or bcnzoic ;~cid.hy a reaction ~III~II(I~()IIS to the ti~r~niition

of acetoin or diacetyl from pyruvate and acetaldchydc or acetic acid, rcspcclivcly.

of 111c hcrh Mrr

Ephedrine is obtained commercially not only fro111 cxirncti~)~~

Huong but also by a synthetic process in which I-I-hydroxy-l-phc1~yI-2-propi~none

(L-PAC) obtained by action of berlzaldchydc on L'cnncnting sugar solut~ons,i s allowcd to

react with methylamine, and the resulting Schill' h:ac is rcduccd to I-cphcdrinc. 'l'his

process has the advantage that no resolution is ncccssary. Mechanism of I,-I'AC synthesis

by microbial reactions is indicated in Fig 2.6.

 Review of U~CMIUIZ

-r
Oxidation Glycolysis / EMP Pathway

Cytosol P y r u ~ v ~ r

oxidalivc
Dectrrhoxy.
Acetyl CoA biorl 1)ccarhoxyliac

el Citrate
NAD
Acctaldehydc

NAD1i, 1
Iicnzrldel~yde condcnsiition
(Co-suhslralc)
added
0 1 10

H
1 1

Mitochondria Ethanol

(:I llNllz, 14dl'l chemiccrl

synthesis
Kreb's cycle1
TCA cycle/
Citric acid cycle

I ,-Iphcdrine

FIGURE 2.6 :Mechanism uf I.-PAC: formation

Early Studio on production of L-PAC : Smith and Hendlin (195)) had shorn 1h.t the
conversion of bewldehyde to phenylacetyl carbinol by yea& is accompanied by a

concomiant reduction of portion of added aldehyde to bcnzyl alcohol. It was noted that

their common requirement was for cocnzynlc I (DPN). Oxidized fom~of DPN serves as

I]+ acceptor in dismutation of pyruvic acid and reduced DPN acts iis 114 donor in the

reduction of benzaldehyde by alcohol dehydrogcnase.

Smith. and Hendlin., (1954) obscrvcd that cenain structuri~lannlogs of ~iicotinic

acid, such as 3-acctyl pyridine and nicotinamidc. ilrc cffcctivc. in supprrssing 11icundesird

reduction of benzaldehyde to benzyl alcohol by lllc pyridinc-linked--_.-

dcliytlrotsnasc of I-.-

Ose. . and Hironaka , (1957) produced phcnyl acctyl carbinol by Noiihcrg"~mcthod

and increased the yield by addition of acctaldchydc \vlullich acls as hydrogc.cn ixceptor and , I,
inhibits hydrogenation of bcmldehydc. lli~ncr.1 al(1057)
--." huvc invcstigtcd wlrther yeast '2 v

carboxylase catalyses the biosynthesis of plrunpl acctyl carbinnl. They Ibund that pl~cnyl

acelyl carbinol results fmm benzaldehydc and pyruvic acid by rhc catelysis of yeasl

carboxylase without thc carboligase as assunled by Ncubcrg. Jaromcr Chylik. (1960)

repofled that constant yields of phenyl ace~ylcarbinol arc dcpndent o11 ihc gcncralivc,,hi u ;
, .,,,,\
degree o f ~ and~ their - . . ,. ..el
s resistance to acid media. Iliina Bccvarova , .al --
.. (1963) found that a

the ability of yeast to melabolise ben7aldchyde Scll rapidly with time. I'henylace~yl

carbinol was formed most from fresh yeast. 'lbey found addition of acetic acid

obtained by them -
to fermentation medium raised the yield of phenylacetyl carbinol. Maximum yield

- -
39.2% when 6ml of ben~aldchydeWUC addcd to 3 I.el' ~ O ~ ~ S S C S
medium. O W et a1 (1963) studied the tolerance level of benzaldehyde by six yeast n
species. Highest decarboxylase activity in presence of benzaldehydc was observed with

Hanscnula m?omala. Saccltur~rnyccscarlbergcnsis and socclt~~romyccs

ccrcvisiac. These

46-51.5% phenyl acetyl carbinol on the added bcrrzaldclryde.

(1966) studied the influence of various 1Ltors on the co~ivcrsiotiof

7
benzaldehyde into phenyl acetyl carbinol. Whey and Decr worr stin~ulatedphenyl acetyl

carbinol synthesis. High phenyl acetyl carbinol conccn~ratio~~s

i~lliibitedfcrmcntatioti to ;t

to 1)c 5.XX gll. in

marked degree. Maximum yield of phcnyl acetyl carbitlo1 is li~u~ld il
Q.D hM
medium containing 20% molasses by Vocts ct a1

-
obtain 5.24 g/I, of phenyl acetyl carhinol. 'l'hey sliuwcd that itroculatio~isizc 01' 10% iilld
2 -, ,,j P,i.',
Nctrvnl a al'(1982)
PH 5.0 are important in the forn~ationof phenyl acctyl ci~rhi~lol.

studied the production of phcnyl acctyl carbinol in 38 yeast species. 'fllc higlicst phcnyl

acetyl carbinol production 6.3 mdml was reachcd in the srtair~.s~~~'c*ltr~ronr)~i~~~

~.ctrll~er.~~~~w

is Budavar.
.I, +
4
Cc.
2.
2.5 PRODUCTION OF L-PAC (+!.* I r, '/

-. .-have investigated tllc conversion of aromatic

Long, James and Ward (1988)

aldehyde to L-acetyl aromatic carbinals and aromatic alcohols by Atcchuromyces

cerevisiae. Typical fermentation raw materials are molasses which provide a source of

hexoses for glycolysis, and benjraldehydc. Fermentation was carried out in 250 MI.

Erlenmeryer Flasks on an orbital shaker. After 6 hours fermenlation 10.1 to 10.2 mg/MI,

L-PACwas produced from benzaldchyde.

 Long and Ward (1989) reported Biotransformation of bcmldehydc by semi-
continuous method: In the biotransformation experiments an increase in benzaldehyde

concentration resulted in decreased yeast cell viability, a cessation in PAC production and

reduced sucrose metabolism. By recovery of yeast cells, which l~adlost viability and

reinoculation into fresh biotransfomation media. it was confirnied th;at when cell viability

is diminished, the cells are no longer capable of producing significant amounts of I)/\(' and

benzyl alcohol. The initial biotranslbrmation rate was Lbrllrd to he optim;ll ar thc

benzaldehyde concentration of 6gll.. PAC' was substanti;tlly rcsist;arlt to hetizi~ltlchycts

denaturation at concentration as high as 7~11,.

Biotransformation using free yeast ctlls: ___.,._.__

Rogers, Shin anil W i ~ ! s(1996) carried
. - ...-----

out a fed- batch process for L-PAC production by suhdividin~it into thrce hasic phiacs.

1. Growth of the yeast under panial fermentation conditions to produce sufficient

biomass for the biotransformation process, and also lo facilitate the ilccun~ulntion

of pyruvate for subsequent L-PAC production.

2. Optimal induction of PDC for biotransfomadon.

3. Programmed feeding of benzaldehyde to maintain concentrations of' 1 -2d1, and the

',:. .
subsequent production of L-PAC.
,t i
1,

. .Krishna
Ellaiah and - (1987)
- studied on thc relationship ktwccn added
I-.

benddehyde by % bioconversion with different mutant strains of S.ccrcvisiue. Optimum

concentration of benzaldehyde that can be added to the medium of 4dI.. Among the

various mutagens used like benzaldehyde, 1.-PAC, UV, nitrous acid, sodium cyanide. IJV

did not result in much improvement over the parent strain. 'l'rcatmcnt with sodium cyanide
 RNJov of LlYnIure

~ieldedthree better strains enhancing the conversion by 3.0-8.2%. Nitrous acid mutant

gave more conversion than the parent strain.

Biotransformation using - immobilized yeast cells: Immobili~~d

yeast cells can

reduce the toxic effects of benzaldehyde by virtue of diffusional limitations and toxic

substrate gradients that are established in the immobilizing matrix. Waafa M.Mahrmoud ct

al., (1989) reported yields of 90%L-PA(' using 0.6% bcni?;ildehydc in ~rlcdiun~

with

immobilized cells of S.cerevisiae, in contrast to about 10% with f r c ~cclls whicli tend to

farm pellets in the presence of benzaldehpde. Slrin Rogers ( t ' ) l ) ( r ) reported I.-llA(' ler.rls

of 10-15 glL with the frec cells of C:Utilis while for the immohilizcd cell systcn~the

maximum concentration was only 5 dL. For both frec i~ndimmohilizcd yeast cclls tlic
biotransformation is a relatively low efficiency process in which ~hcreis a significiait

diversion of benzaldehyde to benzyl alcohol and a loss of' upto 30-40 % due to fornlalion

of byproducts. This diversion was attributed to the activity ol' AD11 in YciisI. Studies hy

Nikolava and Ward, using mutant strains of yeast tliiit lackcd AI)II-I, 11, 111 dc~~~o~istriitcd

that these strains were still able to produce ethanol and bcnzyl alcoliol and suggcstcd that

other oxidomductases were catalyzing these reductive hiotransl'ormations.

The influence of an organic solvent such as ethanol in enhancing thc hcn~~ldel~ydc

solubility and increasing the L-PAC production rate was cvaluatcd. 'fhc sclcction of'

ethanol a water miscible solvent was based on the Pdct that POC is rclativcly

resistant to denaturation of &an01 up to a concentration of 3M. III)C: has highly

hydrophobic substrate binding sites and the presence of' cthanol could assist enzyme-
 01L&erafure
Rmmm

substrate interaction. An increase in 30-40% in the rate could be achieved by h e addition

of 2-3 M ethanol.

Biotransformation with purified PDC- frcc cnqme: Onc of the problems of

using yeast whole cells for biotransformation process is that colisidcrnble amounts of

benzaldehyde are converted to the unwanted product benzyl alcohol. The use of purilicd

I'DC offers the possibility of overcoming this problem. Bur pyruvate has to bc supplied as

a substrate. It is likely that the pyruvate added will bu removed via dccarboxylation ti)

acetaldehyde and to acctoin. Bringer-Mycr and Sahn~(1988) purified I'1)C' obti~incdl'rotn

-S.car1bergensis showed low substrate aflinity and conscqucnr I.-llh(' concc~l~ra~icrns

wcrr

relatively low. Shin HS,Rogers PL (1996) purified I'DC from cells of ( 'undicicr rrrilis and

evaluated operating conditions and substrate concentratio~ls.'Ihe Kni value of 111)(' fbr

pyruvatc was determined to be 2.2mM at 25' C and pll 6.0. with sat~~rationar

concentration in excess of 10 mM pyruvate. The cffccts of various fiidcton on I.-I'AC'

formation are as follows: a temperature of 4' C was selected for enzyme hiotranslbr~llatic~~~

the optimum pH was 7.0, the inhibition constant Kp for acctaldehydc was of thc order of

20mM(0.9g/L). PDC immobilized in spherical polyacrylamide bcads wcrc found to havc a

longer half life compared to that of free enzyme They carricd out comp;rrison of various

immobilization techniques and concluded that entrapment in polyacrylamide gel provided

a higher immobilization capacity for PDC when comparcd with adso~rionon various

cationic resins. From their results, they concluded that relatively high concentration of 1,-
PAC can be achieved either with whole cells of C. U1ili.r in a controlled Fed-Batch process

or pmially or immobilird PDC. These concentrations resultcd from optimal

environmental conditions & the use of a high I.-PAC producing strain of ('.(l/ili.~.
~dvantagesof using whole cells wen thal pyruvale required for the b i o ~ f o r m a t i o n sof

benddehyde to L-PAC are produced by yeast itself. Ethanol, which enhances

benzaldehyde solubility and facilitates biotransfomlions is slm produced by the

of glucose.

Shin and Rogers, (1995) reported the production of L-PAC frcltil hcnz~ldchydc

using partially purified PDC fiom Cundidn utilis. 11has heen reportcd thilt relatively higli

yields of L-PACcan be obtained under optin~ulconditions using partially purilied nrxymc

from Candida urilis.

Biatransformation using purified PIK- immohilizcd on diffcrcnt mclriccs:

Cell and enzyme immobili7ation in suitable gels and matriccs could ~nininiizcsubstratc

inhibition effects by means of diffusional lirnira~ionsand tlic suhstrarc grcldiaits. which

exist in immobilizing material. The developmen1 of un immobilized system providcs rhc

technology for long-term continuous operation. provided tlrat enzymc stability can hc

maintained. It had been reported by Shin and Wang (1995) that cnrnpmcnt in calcium

polyacrylamide gel provided a higher activity than adsorption on cithcr Amberli~c112-200

or CM-Sephadex, and that the addition of 0.2-0.3% glutaraldchydc to polyacrylamide gcl

enhanced the PDC binding capacity and increased PDC activily by 40%.

Different Microorganisms used for Biotransfarmation

Netrval and Vojtisek, in 1982 studied the production of I'AC.' in 38 yeast species.

The highest PAC production 6.3mglml was oblaincd in Ihc strain Soccharomyce.~

Hana Becvarova and Ohanc, in 1963 studied the tolerance level of

~~rlbergen~is''B~&ar'.

benzaldehyde by six yeast species Highest dccarhoxylar activity in presence of

~enzaldeh~dewas 0bsmed with Saccharomyces cerevisiac, Saccharomyces
carlbergensi~,.KIyveromyces lacris, These cultures formed 46-5 1.5 % PAC on the added

~enzaldehyde. The following microorganisn~s have been used for study of thcir

bioconversion potential ( Table 2.2)

TABLE :2.2 : Microorganismz used to study thcir bioconvrrsion polential

Yeast Specics Collcctinn Itcfcrmce No
Saccharomyces carlshergerisis "Budvar " ( '( '1.
(Saccharornycr.~rtr0nruni)
Sacchurornyces kl~~yvcri 21-5-1
Sacchuromyces chc~~alicri ?/-//-I
Saccharomyces bayanu.~ 1'1-13-1
Saccharornyces oviformis 21-?I-/
Saccharomyces italicus .?1-.f3-1
Succharomyces diastaric~rs ?/-Lf-/
Cundidi guilliermondii ' 29-4-23
Cundidu ~ropicalb 19-7-24
Candida humicola 29-11-1
Candida curvata 29-18-1
Candida arborea(Candida ritilis) 29-27-1
Candida macedonicnsis 29-29-1
Candida utilis 29-38-l.2,3,4,
5.16.17.1(1,22
Cundidu mycodermu(c.vulidu) 2'1-39-4
Candida tamarindi(l,'.krusei) 29-40-1
Candida majoricen.fi.v 29-51-1
Candida vinariu 29-52-1
Candidapelliculosa (Hanscnulaanomalu) 29-6-1
~ ~ ~ d i d ~ ~ u l c h e r r i r n a ( M c r . ~ cpulchcrrim(rl
nik~wia 29-2-2
Candjda reukaufii (Mershnikowia reukoufiil 24-1-22
Hansenula anomala var.anomula 18-1-17
Pichia m~mbrunaefucien~ SY- 1-3
Pichia polyrnorpha 39-7-1
Pichia etchelsii 39-22-1
Pichia guilliermondii 39-23-1
39-30-1
Pichia ohmeri
Saccharomyes luhigii 34-1-1
~chizosaccharomycesja~nicus 14-5-1
Studies On addition of various additives: Mon widely know and used growth regUIatm
in ~lantsis 2,4-dichlorophenoxy acetic acid. One of the most striking Mponser to 2.4-

dichlorophen~xyacetic acid is the abnormal growh produced by some organs a d lissucs

following treatment with this material. Litvinenko (1964) studied 2.4-dichlorophenoxy

acetic acid effects on streptomycin production. Growth and production increased in the

presence of 0.00001% of 2, 4-dichlorophenoxy acetyl plycinc, and 2.4- dichlorophcnoxy

ace~ylisoleucine. All three increased the growth rate nrld rcsultcd in 38% higher yields

72 hours of fermentation. Illlaiah et a]., (1987) studied ~ h rcl'ficl of usillg 2.4-

dichlorophenoxy acetic acid on this fcrmcntation. 'I'bc otllrr plant grc~wllirvguliitors like (1-

naphthoxy acetic acid was also tricd out in a similar way. I'o clicli~rctlic rticti~llic

impurities, which might interfere with fermentation. I<I)'I'A has bccrl tricd out. Mizrclri and

Miller found EDTA stimulated alkaloid synthesis. So with 1111' S;IIIIC COIICCPI 1311'1A has

been tried out. Wafaa M.Mahmood, ct.al., (1989) studicd tllc ratc and cxtcnt of microbial

transformation of higher concentntions of bcnzaldcllydc substrtltc lo I.-phcnyl acctyl

carbinol (L-PAC) by immobilized cells of ,~(~cchoron!,sc~s

crrevi.riae and rcportcd that

L-PAC production was markedly stimulated by addition ot' diffcrcnt concentrations of p-

cyclodextrin (BCD) to the fermentation medium.

2.6 ANALYTICAL PROCEDURES FOR DETECTION AND ESTIMATION OF

LPAC

Po1arimetly: The quantitative estimation of L-PA(.' was carrid Out by Pnl.rImCtQ'. '1hc

optical mtation caused by various samples obtained by hioconvcrsion W ~ studied

S and the

results were compared. L-pAC is a yellow coloured liquid and by ils name it is

levorotatory in nature. Phenyl a ~ t ycarbinol

l rotates the plane of polarized light fo thc lefl

and hence it is levorotatory.

 Review of literature

In 1962 Flana Becvmva and Hano have determined keml either polamgraphically
and polarimetrically. Fermentation medium was twice shaken with an equal volume of

ether, combined etheric extracts evaporated in vacuum and the residue dissolved in 10 ml

ethanol. The sample is evaluated polarographically at the same time its optical activity was

measured on the circular polarimetcr of Schmit-Hocnsch (uo20of D (-)- pl~snylaceryl

carbinol = - 180'). In this arrangement PAC yielded a wavc at half-wave potc~ltialof -1.73

V and benzaldehyde at - 1.4 1 V against a calomcl elcctrodc. In 1987 lillaiall c't al.. ~lscd

polarimeter with 2 decimeter tube for the estimation of1.-PAC.

lodometry: Paulsmith and David Hendlin (1954) dctcrmincd PAC' hy tliis ~nctlicld

developed in Merck & Co.lnc., New Jersy. An aliquot of' filtered broth is rrcatd with

excess 0.5 N Iodine. Excess 10 N NaOH is added and thc precipitated iodofbrm wasl~cd

with 1.0 N HCI. The iodoform, dried over calciun~chloride in tarred cenrrili~gcruhcs. is

weighed and the amount of kctol calculated. No componcnl of the brotll olllcr tlla~lp l ~ c ~ ~ y l

acetyl carbinol gives an iodoform reaction.

OH
I
C6Hs-C-c=0
I
H
I
CH,
+ NaOH + 312 - C6H5cH0 + CHI,+
1
HCOONa + JHI

lodoform (yellow ppt)

L-Phenyl Acetyl carbinol

Figure (2.7) Iodoform reaction of LPAC

In fermentation industry specific gravity calcula~ionis one of the idcntilication tests

canied out for identifying L-PAC. The standard value fur I.-PAC is 0.93 a Room
Temperature.
 Review o / l i ~ e m f ~ r e

Thin Layer Chromatogra~h~:

The quantitative estimation of midual substrate and the

product fomned was carried out by Groger and Erge in 1965. The Rf value of L-PAC was

to be 0.23.Solvent system is chloroform, and identification reagent is iodine.

Silica gel sluny was made with chloroform + methyl alcohol (2:l ratio)

HPLC: Tripathi and Aganval ct al., cstimated L-PAC using Dcckman 110 nroctcl I{I'I.<'
with 160-absorbance detector (* wavelength 25 nm). The column was PAC' 18 300 A (3.0 "
x 30 cm, 15pm, spherical) and the mobilc phase was acetonitrilc and watcr (30;70:vlv) at

a flow rate of 1.5 ml/min. The rctenlion times of' bcnxoic acid, h c ~ ~ ~ i l l I.-Phi'
~ ~ l ~ (aod
~I.

benzaldehyde were 2.3. 3.1, 3.6 and 4.9 min respectively. I,ong. Janlcs. and Wart1 uscd kt

Bondpak TMjC18 reverse phasc sleel column, Measurement was madc hc.li)rc atlili~ion01'

the enzyme and at the end of thc reaction.

Gas Chromatography: Long, James. Ward (1988) dctern~incd aromittic alcohols

produced in the fermentation and solution of purified corbinols by gas chromalography.

The GC analysis was carried out on a Perkin-Elmcr model F17 gas chron~atogrirphyusing

a glass column 6 ft long X 0.25 packed with 30 % siliconc clusromcr 11301 on chromosorh

WHP 60-80. Oven temperature was maintained constant at 195 O C:, (increasing at a rate of

3' Cjmin). Standards and femnentation broth (IOrnL) were extracted with ether cxtracl

made up to 20 mL, One-micro liter volumes were injected. l'cilk areas wcrc measured

using a Carlo Reba SP 4270 integrator. Hyoun Shin and Peter Rogers hcrc 111' series 427

gas chromatograph was used, the concentrations of hn7~ldehydcand I.-l'M:wcrc

determined by comparison with standard samplcs of L-PAC: providcd by I(:I Australia I'vt.

Ltd.
 Review of Literature

colorimetry: Groger and Erge (1965) L-Acetyl aromatic corbinols produced by

fermentation and also recovered purified carbinols were estimated by colorinietric method.

Acetyl benzoyl was used as a standard using the appropriate correction factor. This n~cthod

is being used till today for estimating L-PAC. Several workers like Wafan. Mahamood.

~ b d u lHalim, El. Sayed and Robert (1989) used tl~csame colorinictric mcthod Ibr Ihc

estimation of L-PAC.