Isolation and characterization of chitinolytic

bacteria and its chitinase(s)

A dissertation work submitted in partial fulfillment for the degree of

Master of Science (M.Sc.) in Plant Biology and Biotechnology

By
Prashanta Kumar Behera
Regd. No.: 16LPMS15

Supervisor
Dr. Jogi Madhuprakash
Assistant Professor, Department of Plant Sciences

School of Life Sciences, University of Hyderabad,

Gachibowli, Hyderabad – 500046
Telangana

April, 2018
 University of Hyderabad
(A Central University established in 1974 by an act of parliament)
HYDERABAD- 500046, INDIA
__________________________________________________
CERTIFICATE
This is to certify that Mr. Prashanta Kumar Behera, bearing regd. no.
16LPMS15, has carried out the project work embodied in the present thesis under
the guidance of Dr. Jogi Madhuprakash for one academic year 2017–2018 of
Master of Science of this university. We recommend his thesis entitled “Isolation
and characterization of chitinolytic bacteria and its chitinase(s)” for
submission for the degree of Master of Science in Plant Biology and
Biotechnology.

Prof. Ch. Venkata Ramana Dr. Jogi Madhuprakash

(Head, Dept. of Plant Sciences) (Project Supervisor)
 University of Hyderabad
(A Central University established in 1974 by an act of parliament)
HYDERABAD - 500046, INDIA
__________________________________________________
DECLARATION
This is to declare that the work embodied in this thesis entitled “Isolation and
characterization of chitinolytic bacteria and its chitinase (s)” has been carried
out by me under the supervision of Dr. Jogi Madhuprakash, Assistant
Professor, Department of Plant Sciences, School of Life Sciences. The work
presented in this thesis is a bonafide research work and has not been submitted
for any degree or diploma in any other University or Institute.

Dr. Jogi Madhuprakash Prashanta Kumar Behera

(Project Supervisor) Regd. No. 16LPMS15

Date :
Place :
Dedicated To My Beloved Parents & My
Guide
For Their Belief, Support & Inspiration.
 ACKNOWLEDGEMENT

I would like to take this opportunity to extend my humble gratitude to my

supervisor, Dr. Jogi Madhuprakash for his unrelenting encouragement and
constant support, without whom this endeavour would not have been possible.
Working under him gave me true sense of freedom, broadened my scientific
outlook and inculcated a spirit of positive attitude in me. I am deeply indebted of
his unfailing support in every scientific aspects like writing, communication skill,
presentation skill which enabled me to successfully complete my master degree
project work and his extended support throughout the tenure of this project work.
I would like to thank Prof. Ch. Venkata Ramana, HOD, Department of
Plant Sciences, for his support and guidance.
I would like to take this opportunity to extend my humble gratitude towards
Prof. Appa Rao Podile, Dr. Kodetham Gopinath and Dr. Rahul Kumar for
allowing me to use their lab facilities.
My special thanks to Dr. Swaroopa Rani for her support and
encouragement as well as for her guidance during the course of the project.
I would like convey my heartfelt gratitude to Mr. Saumashish Mukherjee
for his support, guidance and encouragement till the completion of the project
work. He had extensively helped me in experiments and preparatory phases of
the project work. Working with him was an enjoyable and unforgettable
experience with friendliness.
I would like to convey my gratitude to Ms. Bhavana Karlapudi for her
unrelenting encouragement and constant support. I am deeply indebted of her
unfailing support in every aspects throughout the tenure of the project. Working
with her was an enjoyable and unforgettable experience.
I would like convey my sincere gratitude to my labmates Mr. Rajat, Mr.
Akash, Mr. Nandkiran, and Ms. Mounika for their constant support and
cooperation throughout the tenure of the project work.
I express my humble regards and gratitude to my parents, for their love,
affection, belief, cooperation, encouragement and inspiration during the long and
tiring hours of study which had always boosted my morale and always set me on
the right.
 I find profound ecstasy to express my gratitude towards to all my friends
of the university and outside, for their motivation and love that has helped me to
keep going and complete my project on time.
Last but not the least, I express my deepest sense of gratitude and devotion
to god for being by my side and showering me with blessings that has helped me
a lot to complete my project work properly, successfully and on time.

Date:
Place: (Prashanta Kumar Behera)
 ABSTRACT

Chitin is a linear polymer of β-1, 4 linked N-acetylglucosamine. It is the most abundant

aminopolysacharide in nature and the key building component of the exoskeleton of
crustaceans, fungi, algae and insects. Chitinases (E.C. 3.2.2.14) are glycosyl hydrolase that
depolymerize chitin to generate chitooligosaccharides, together which has enormous
biotechnological applications. Chitin and its related materials are also used in wastewater
treatment, drug delivery, wound healing, and dietary fiber. Longer-chain chitooligosaccharides
have potential applications in agriculture sectors and they do act as antimicrobial compounds.
Whereas, chitinases have been implicated in plant resistance against fungal pathogens because
of their inducible nature and antifungal activities in vitro. Soil harbours numerous chitinolytic
bacteria. The present work focusses on screening and isolation of potential chitinolytic bacteria
from environmental samples and among the different bacteria screened, the isolate JM1 was
found to be a potential target. Growth curve experiments with different polymeric chitin
substrates revealed that the JM1 utilized colloidal chitin and β-chitin with almost equal
preference, followed by α-chitin. Phylogenetic analysis revealed that Paenibacillus pabuli was
the nearest neighbour of the isolate JM1. Carbohydrate Active enZymes (CAZy) database
revealed the presence of four chitinases in the genome of P. pabuli. A candidate chitinase gene
i.e. ChiC was cloned from the genome of JM1. Furthermore, protein expression standardization
was done to obtain the maximum yield of soluble protein. The recombinant protein was purified
to its homogeneity using Ni-NTA affinity chromatography and was found to be chitin active.

KEY WORDS: Chitinases, glycosyl hydrolases, chitooligosaccharides

 TABLE OF CONTENTS

S.No. Topic Page No.

1. Introduction 1-6
2. Aim & Objectives 7
3. Methodology 8-12
4. Result & Discussion 13-22
5. Conclusion 23
6. References 24-26
7. Appendix 27-31

LIST OF TABLES
S.No. Table No. Description Page No.
Summary of expression standardization
1. 1. 21
for ChiC
 LIST OF FIGURES

S.No. Figure No. Description Page No.

Schematic representation of
1. 1. 5
enzymatic degradation of chitin
Applications of chitinases and
2. 2. 7
chitooligosaccharides
Uninoculated and degraded CC
3. 3. 13
plate by JM1
PCR amplification of 16S rRNA
4. 4. 14
gene of JM1
5. 5. Phylogenetic tree of JM1 14
Growth of JM1 of different
6. 6. 15
polymeric chitin substrates
Total cell protein estimation for
7. 7. JM1 growing on different chitin 16
substrate.
Chitinase activity of JM1 on
8. 8. 16
different chitin substrates
CAZy database showing the
9. 9. presence of different chitinases in 17
the genome of Paenibacillus pabuli
10. 10. PCR amplification of ChiC 17
11. 11. Vector map of pET-28a (+) 17
ChiC clone confirmation by double
12. 12. 18
digestion
LB-Kan plate showing transformed
13. 13. 18
colonies of BL21DE3 plysS.
14. 14. Hourly expression of ChiC 19
Expression of ChiC in BL21DE3
15. 15 (a,b) 20
plysS
Expression of ChiC in Rosettagami
16. 16. 20
II DE3
17. 17. Purification profile of ChiC 21

18. 18. Gel profile of concentrated ChiC 22

19. 19. Dot blot assay of ChiC 22

 LIST OF ABBREVIATIONS

S.No. Abbreviation Full Form

1. APS Ammonium Persulfate

2. BLAST Basic Local Alignment Search Tool

3. bp base pair

4. BSA Bovine Serum Albumin

5. CAZy Carbohydrate Active enZymes

6. CBP Chitin Binding Protein

7. CBM Carbohydrate Binding Module

8. CHOS Chitooligosaccharides

9. C-terminal Carboxy terminal

10. DA/FA Degree of Acetylation/ Fraction of Acetylation

11. dNTPs deoxy-nucleotide triphosphates

12. DP Degree of polymerization

13. D-UNIT Deacetylate ‘D’ unit (GlcN)

14. FnIII Fibronectin type-III

15. g Gram

16. GH Glycosyl Hydrolase

17. GlcN Glucosamine

18. GlcNAc N-acetyl glucosamine

19. h Hour

20. IPTG Isopropyl β-D -1-thiogalactopyranoside

21. kb Kilobase
22. kDa Kilo Dalton

23. L Litre

24. LB Luria Bertani

25. M Molar

26. mg Milligram

27. mL Millilitre

28. mM Millimolar

29. Mw Molecular weight

30. NCBI National Center for Biotechnology Information

31. Ni-NTA Nickel-nitrilotriacetic acid

32. N-terminal Amino terminal

33. OD Optical Density

34. PA Pattern of acetylation

35. PAGE Polyacrylamide Gel Electrophoresis

36. PCR Polymerase Chain Reaction

37. PMSF Phenylmethylsulfonyl fluoride

38. rpm Revolutions per minute

39. SDS Sodium dodecyl sulphate

40. Tris Tris-hydroxymethylaminomethane

41. UV Ultra violet

42. µg Microgram

43. µL Microlitre

44. µM Micromolar
o
45. C Degree Celsius
 1. INTRODUCTION
Recent advances in the area of biotechnology is leading to the gradual replacement of synthetic
polymers with biodegradable polymers obtained from natural resources. Natural biopolymers
have several advantages like availability from natural resources, biodegradability and
biocompatibility. Use of biodegradable resources lead to ecological safety and derivatives of
natural resources may have specific applications. Chitin is the second most abundant
polysaccharide in nature after cellulose. Chitin and its derivatives are class of natural
macromolecules which have the potential to be bioactive by the action of enzymes and
chemicals. Chitin, chitosan and chitooligosaccharides have linearity in their biological
applicability.

Chitin and Chitosan

Chitin is the most abundant natural amino-polysaccharide and the key building material of the
exoskeleton of arthropods and the cell wall of fungi. It is a linear polysaccharide consisting of
(1-4) linked units of 2-acetamido 2-deoxy-β-D –glucopyranose (N-
acetylglucosamine/GlcNAc/A-unit). The consecutive sugar units in chitin are rotated 180° and
are relative to each other. In nature, depending on the arrangement of individual chitin chains,
three different crystalline polymorphic forms of chitin exists with different orientation of the
microfibrils, which are α -, β-and γ- chitin. In α- chitin, the chains are arranged alternatively in
an antiparallel arrangement. It is the most stable form of chitin in nature, which is prevalent in
crustaceans. In β-chitin, the chains are arranged in a loose packing parallel-chain arrangement
and are rare in nature. This type is found in pen squids. γ -chitin is a variant, mixture of two
parallel chains with a single anti-parallel chain, found in cocoons of insects (Rinaudo and
Domard, 1989). Arrangement of individual chitin in different forms clearly explains that, the
chains are densely packed in α-chitin making it a rigid structure with inability to swell in water
while in β-chitin giving a looser packing and soft chitinous structure. Even though it has few
applications, but it is used as a starting material for chitosan and chitooligosaccharides.
Chitosan, a water-soluble heteropolymer of β (1, 4) - linked GlcNAc (A-units) and D-
glucosamine (GlcN or D-unit), is a natural nontoxic biopolymer which is produced by the
partial deacetylation of chitin by the action of chitin deacetylases. Chitosan has three types of
reactive functional groups, an amino group and both primary and secondary hydroxyl groups
at C-2, C-3 and C-6 positions respectively. The chemical composition of chitosans depends on

1
the number of residues (the degree of polymerization, DP), the relative proportion of acetylated
and deacetylated residues (the degree of acetylation, DA) and their distribution along the chain
(the pattern of acetylation, PA). The solution properties of chitosans depend on the degree of
acetylation, the distribution of acetyl groups along the main chain, and the chain length
(Rinaudo and Domard, 1989; Aiba, 1991; Kubota and Eguchi, 1997).
Chitosan is chemically more active than chitin due to the presence of primary and
secondary hydroxyl groups on each unit, and the amino group on each deacetylated unit. These
reactive groups are readily subjected to chemical modification to alter mechanical and physical
properties of chitosan.

Chitinolytic Bacteria
Chitinolytic bacteria are capable of decomposing chitin in aerobic and anaerobic condition.
They are abundant in environment with high amount of chitin. Soil bacteria capable of
degrading chitin include Bacillus, Cytophaga, Pseudomonas and so on (Schelgel et. al, 1993;
Brzezinska et.al, 2014), while in aquatic environment, chitin is degraded by bacteria of the
genera Aeromonas, Enterobacter, Serratia, Erwinia, Chromobacterium, Flavobacterium and
so on (Brzezinska et.al, 2014). Cellulomonas (ATCC 21 399) is capable of rapid degradation
of cellulose and chitin both aerobically and anaerobically. Chitinolytic bacteria are involved as
a natural agent in the suppression of plant pathogen and insect pests. Studies have shown that
the combination of two chitinolytic bacteria Paenibacillus sp. 300 and Streptomyces sp. 385 is
more effective against F. oxysporum causing cucumber wilt than individual strains or other
combinations (Brzezinska et.al, 2014). The combination of S. marcescens, Streptomyces
viridodiasticus, and Micromonospora carbonacea strains effectively inhibited the growth
of Sclerotinia minor responsible for vegetable rot (El Tarabily et.al, 2000).

Chitinases
Chitin is insoluble in water and resistant to moderate acid, alkaline and many organic solvents
and therefore, microbial degradation is the most important degradation pathway (Hoell et. al.,
2010). The main degradation pathway of chitin in nature is by the action of chitinases, that
causes hydrolysis of 1, 4 β- glycosidic bonds between the GlcNAc. The products of this enzyme
activity are monomers of GlcNAc (Beier & Bertilsson, 2013).
Chitinases are encoded in a wide range of organisms including bacteria, fungi, insects,
viruses, animals and humans for different purposes. According to amino acid sequence
homology chitinases are grouped into different families of glycosyl hydrolase (GH). Bacterial

2
chitinases come under the family-18 and 19 of GHs. Many chitinolytic bacteria produce only
family 18 chitinases, while Streptomyces species are known to produce family 19 chitinases.
Based on site of action or functional pattern, chitinases are classified into
endochitinases and exochitinases (Henrissat & Davies 2000). Endochitinases randomly cleave
chitin resulting in dimer di-acetyl chitobiose and soluble low molecular mass oligomers of
GlcNAc such as chitotriose and chitotetraose. Exochitinases include chitobiosidases and β –N-
acetyl hexosaminidase, where the former produces GlcNAc dimers (chitobiose) by cleaving
the non-reducing end of chitin while the latter hydrolyzes chitobiose, chitotriose and
chitotetraose, resulting in the release of N-acetyl glucosamine (Bhattacharya et. al., 2007;
Tronsmo & Harman, 1993).

The structure of chitinases generally consist of a catalytic domain (GH), chitin binding
domain (CBD) and fibronectin III (FnIII) domain for successful enzymatic action. Generally,
GH18 chitinases have all the domains. Its catalytic domains have a (β/α)8 TIM barrel fold with
crucial catalytic residues being located on β-strand number 4, having deep substrate-binding
cleft on top of it. A hydrophobic stacking interaction between the aromatic residues in this cleft
and oligosaccharides, leads to holding of this moiety at this position during the catalytic
reactions. The chitin-binding domain facilitates correct positioning of the catalytic domain for
processive action and local de-crystallization of the substrate. Even though the fibronectin
domain does not involve directly in chitin binding, but it affects the overall enzyme structure
and spatial localization of the catalytic domain and the chitin-binding domain, during
enzymatic action (Yan et.al, 2015). Enzymatic action of chitinases leads to formation of
derivatives of chitin, which are chitooligosaccharides (CHOS) having wide application in
industry, agriculture and biomedicine.

Catalytic Mechanism of Chitinases

During hydrolysis of the glycosidic linkage, a nucleophilic substitution occurs at the anomeric
carbon leads to either retention or inversion of the anomeric configuration (Sinnott, 1990).
Generally this hydrolysis reaction takes place by acid catalysis which requires a carboxylic
acid at the active site of the enzyme. One of the carboxylic acid acts as a proton donor and the
other as a base.
Both the retaining and the inverting mechanisms are also called as single displacement
and double displacement reaction, respectively. The inverting mechanism is a “one-step”
reaction, where the protonation of the glycosidic oxygen occurs simultaneously with a

3
nucleophilic attack on the anomeric carbon by an activated water molecule. This water
molecule is located between a carboxylic group and the anomeric carbon and it is activated by
the carboxylic group that acts as a base. Since the water molecule approaches the anomeric
carbon from the side of the catalytic base, this mechanism leads to inversion of the anomeric
configuration. The retaining mechanism is a two-step reaction, where the first step involves the
protonation of the glycosidic oxygen (by the catalytic acid) and a congruent nucleophilic attack
on the anomeric carbon atom by the nucleophile (the second carboxylic acid). This attack leads
to breakage of the glycosidic linkage and the formation of a covalent linkage between the
anomeric carbon and the catalytic nucleophile (Vocadlo et al., 2001). Subsequently, this
intermediate is hydrolyzed by a water molecule that approaches the anomeric carbon from a
position close to that of the original glycosidic oxygen, leading to retention of the anomeric
carbon configuration.

Chitooligosaccharides
Chitooligosaccharides (CHOS) are the degraded products of chitosan or chitin prepared by
enzymatic or chemical hydrolysis. CHOS have a degree of polymerization higher than 20%
and an average molecular weight less than 3900 Da (Lodhi et al., 2014). Still, chitosan and
CHOS can be very diverse in several features like sequence, degree and pattern of N-
acetylation, fraction of N-acetylated residues, degree of polymerization, molecular weight and
polydiversity. Chitooligosaccharides can be generated from chitin by the action of different
classes of chitinases or from chitosan (partially deacetylated chitin) by the action of
chitosanases.

4
 Figure 1: Schematic representation of enzymatic degradation of chitin

Applications
Chitin, chitosans and CHOS are showing unique biological activities. As starting material,
chitin itself has been centre of many therapeutic application and suitable for tissue engineering
and stem cell technology. Oral administration of chitin (α and β forms) are beneficial for food
allergies (Bae et al., 2013). Application of chitin is limited because of being chemically inert
and insolubility in water and acids as compared to chitosans. Chitinases are useful for the
production of single cell protein (SCP). The end product of chitin hydrolysis by Penicillium
ochrochloron chitinase was mainly GlcNAc, its further utilization as a substrate for SCP
production (Patil et. al., 2014). Chitinases have also found application in chitin waste
management from sea-food industry. The conventional method of seafood processing includes
chitin disposal by ocean dumping, incineration and land filling that causes environmental
accumulation leading to pollution. Bioconversion of such wastes into value-added products by
chitinases causes bioremediation and at the same time develops industrial importance of such
wastes (Patil et.al, 2014).

5
 Figure 2: Applications of chitinases and chitooligosaccharides

CHOS and derivatives of CHOS have been the subject of increasing attention due to high
solubility, non-toxic and their positive physiological effect in pharmaceutical and medicinal
applications. CHOS has anti-tumour effect by enhancing natural killer activity in intestinal
intraepithelial lymphocytes and reduce tumour growth in mice (Qin et al., 2002). It is used as
a vector for gene therapy in place of chitosan to avoid the formation of chitosan complex
aggregates. CHOS are reported to accelerate wound healing by enhancing the function of
inflammatory and repairing cells. CHOS are efficient to manage diabetes in alexan induced
mice (Katiyar et al., 2011). Even recent advances fosters many health benefits of CHOS like
lowering blood cholesterol, lowering blood pressure, increasing calcium uptake, controlling
arthritis and protective against infection etc.

6
 2. AIMS & OBJECTIVES
Chitinases and the chitin derivatives have wide-scale application. In agriculture, chitinases are
potential alternatives to chemical pesticides and fungicides. Chitinase mediated bioremediation
of crustacean wastes from sea-food industry is another application. Also chitinases have
impacted food industry for the production of single cell proteins from yeast and along with this,
chitooligosaccharides have shown promising impact in the field of biomedicine. Thus,
employment of chitinases and chitin derivatives may be a boon towards an eco-friendly and
sustainable approaches in the near future.

The aim of the present work is the isolation and characterization of a potential chitinolytic
bacteria and its chitinases. On the basis of this, the following objectives have been framed:

 Isolation and screening of potential chitinolytic bacteria.

 Growth pattern of the selected isolate on different chitin substrates.
 Cloning, expression and purification of a potential chitinase from the selected isolate.

7
 3. METHODOLOGY
2.1 Screening and isolation of chitinolytic bacteria
Screening and isolation of potential chitinolytic bacteria was done by suspending collected
environmental sample in sterile double distilled water, followed by serially diluting the
suspension into different folds of dilution. The dilutions were spread onto M9 minimal media-
agar plates supplemented with 0.3 % colloidal chitin (CC) and incubated at 28 °C till
development of colonies and zone of degradation, if potential chitinolytic bacteria are present
in the sample. Pure culture was obtained through streaking on fresh CC plates.

2.2 Bacterial identification and phylogenetic analysis

Bacterial identification was done by performing 16S rRNA gene amplification by colony PCR
using the universal primers 27F and 1492R (Eurofins Genomics India Pvt. Ltd., Bengaluru,
India), followed sequencing supported by AgriGenome Labs Pvt. Ltd., Kochi, India.
The sequencing result was analyzed through NCBI nucleotide BLAST (blastn) tool and
phylogenetic tree was constructed using MEGA 6.0 by neighbour joining algorithm.

2.3 Growth pattern on different chitin substrates

2.3.1 Qualitative analysis
M9 minimal media supplemented with 0.5 % of chitin substrate i.e. α-chitin, β-chitin and
colloidal chitin was prepared and also, M9 supplemented with 0.5 % glucose was kept as a
positive control and M9 alone as a negative control. Overnight culture of the isolate JM1 was
centrifuged and the pellet was resuspended in M9 medium and the absorbance was measured
at 600 nm. Based on obtained OD, required inoculum amount was calculated to get O.D as 0.1.
900 µL of inoculum was used in each setup in biological triplicates and growth of the isolate
in each was analyzed by measuring culture absorbance at 600 nm. 1mL culture from each
sample at each time interval were collected and centrifuged at 10000 rpm for 12 minutes. The
pellet and supernatant were collected and stored at -20 °C for further analysis.
2.3.2 Estimation of total cell protein
Stored pellets were treated with 0.2 N sodium hydroxide solution and boiled at 120 °C for 10
minutes. Boiled samples were centrifuged at 12000 rpm for 15 minutes at 4 °C. Total protein
estimation was performed using the supernatant obtained by Bradford’s method, taking

8
absorbance at 595 nm. Obtained absorbance was plotted against BSA standard curve to
estimate total cell protein concentration for each sample.
2.3.3. Chitinase Assay
Reaction mixture consisting of 50 mM sodium acetate buffer, pH 5.6, 0.5 % colloidal chitin
substrate and 50 µL of culture supernatant was incubated at 37 °C for 1 hour. Incubated samples
were centrifuged at 12000 rpm for 10 minutes and the supernatant was taken for chitinase
assay. 300 µL of Schale’s reagent was added to 100 µL of supernatant, boiled at 120 °C for 15
minutes. 200 µL of each sample was loaded in 96-well plate in triplicates and absorbance was
measured at 420 nm.

2.4 Identification, selection and cloning of a candidate chitinase gene from JM1
The Carbohydrate Active enZymes (CAZy) database was searched for presence of candidate
chitinase genes in the nearest homologous of isolate JM1. Four candidate chitinase genes were
found, out of which ChiC was selected as potential target.
PCR amplification of ChiC gene was performed using specific primers (Eurofins Genomics
India Pvt. Ltd., Bengaluru, India) and analyzed on 1 % agarose gel. The single band of DNA
obtained were excised out from the gel and was extracted using Mackery-NagelTM (Germany)
NucleoSpin Gel and PCR Clean-up kit. The eluted DNA concentration was checked at 260 nm
using Nanodrop (Thermofisher Scientific, India Ltd.).
The eluted DNA (insert) was double digested using NcoI and XhoI (Thermofisher Scientific,
India Ltd.) and the reaction was incubated for 8 hours at 37 °C. Simultaneously, the expression
vector pET-28a (+) was also double digested using the same restriction enzymes and incubated
for 1 hour at 37 °C. Ligation of the insert and the vector (3:1) was performed using ligase
(Thermofisher Scientific, India Ltd.) and reaction was incubated at 22 °C for 30 minutes. The
ligated product was then transformed into the cloning host DH5α.
Clone confirmation was performed by plasmid isolation using GSureTM Plasmid Mini Prep Kit
(GCC Biotech, Kolkata, India), followed by double digestion of the isolated plasmid with NcoI
and XhoI, incubated at 37 °C for 2 hours and analysis on 1% agarose gel. Final confirmation
was done by sequencing of the clone with the help of Eurofins Genomics India Pvt. Ltd.,
Bengaluru, India.

9
2.5 Preparation of BL21DE3 plysS and Rosetta gami II DE3 competent cells
BL21DE3 plysS and Rosetta gami II DE3 are the two commonly used E.coli expression hosts.
The strains that were used in the present work were devoid of any antibiotic resistance gene.
1 % of inoculum from overnight culture was inoculated into 50 mL LB broth and was incubated
at 37 °C under shaking till the OD of the culture reaches 0.5 at 600 nm. The culture was kept
in ice for 30 minutes and centrifuged at 7000 rpm for 10 minutes at 4 °C. The pellet was
resuspended in filter sterilized cold 100 mM CaCl2 and centrifuged at 7000 rpm for 10 minutes
at 4 °C. The pellet obtained was resuspended in a mixture of 100 mM CaCl2 and glycerol (21:4).
150 µL of resuspended sample was aliquoted into sterile 1.5 mL micro-centrifuge tubes
(MCTs) and frozen with liquid nitrogen for 2 minutes followed by immediate storage at -80
°C.

2.6 Transformation
The competent cells for BL21DE3 plysS and Rosetta gami II DE3 were thawed on ice.
Recombinant plasmid was added into the competent cells and incubated on ice for 30 minutes.
Heat shock was given at 42 ºC for 75 seconds and immediately transferred into ice for 5
minutes. 850 µL of LB broth was added into it and the culture were incubated at 37 °C for 1
hour under shaking. After 1hr incubation, the cultures were centrifuged at 11000 rpm for 2
minutes and the pellet was resuspended in 150 µL of LB broth. The suspensions were spread
onto LB agar plates supplemented with kanamycin (50 µg/µL) and incubated overnight at
37 °C.

2.7 Expression of ChiC

Expression of ChiC was standardized under different conditions in BL21DE3 plysS and
Rosetta gami II DE3 simultaneously.
Primary culture of both the transformed cells were prepared by inoculating a single
transformed colony into 5 mL of LB-Kan broth and incubated at 37 °C under shaking for
overnight. 1 % inoculum from the primary culture was inoculated into 50 mL of LB-Kan broth
and was subjected to different conditions for protein over-expression. These conditions include
induction type i.e. IPTG induction or auto-induction, induction temperature and time of
incubation after induction. The culture was harvested by centrifugation at 10000 rpm for 10
minutes at 4 °C. The pellet obtained was dried and resuspended in lysis buffer for sonication.
Sonication was carried out at 30 % amplitude and the sonic on/off timing was set to 20s/40s
respectively. The cell lysate was centrifuged at 10000 rpm for 40 minutes at 4 °C followed by

10
separation of the sonicated pellet and supernatant, which were then subjected to SDS-PAGE
using 12 % polyacrylamide gel. 250 kDa protein ladder (Genetix Laboratories India Pvt. Ltd.)
was used. After electrophoresis, the gels were stained with Coomassie Brilliant Blue G-250
(Sigma Aldrich, USA) for visualization of the protein bands.
Based on the over-expression and yield of ChiC into the soluble fraction, the best
condition was chosen for over-expression of ChiC in bulk. In case of bulk expression, the
sonicated supernatant was subjected to purification by Ni-NTA affinity chromatography.

2.8 Purification by Ni-NTA affinity chromatography

Packing of the column was done by adding 4 mL of Ni-NTA resin slowly onto the column base
and the flow rate of the column was checked and adjusted. The column was equilibrated with
60 mL lysis buffer (10 mM imidazole) for removal of ethanol from the resin and bringing the
resin into the buffer. The sonicated supernatant was added slowly (1-2 mL at a time) and the
flow through (FT) was collected. This was followed by passing wash buffer (20 mM imidazole)
through the column to remove non-specific proteins from the resin, and the wash through (WT)
was collected. This process was continued until all of the sonicated supernatant was passed
through the column.
Elution of protein were done by using elution buffers of increasing imidazole
concentration i.e elution buffers I, II and III containing 50 mM, 150 mM and 250 mM of
imidazole respectively. Fractions for each elution were collected for analysis by SDS-PAGE.

2.9 Concentration of ChiC

The purified protein fractions were pooled and concentrated using centricon (Sartorius, UK) of
suitable Molecular Weight Cut-Off (MWCO) i.e. 10 kDa MWCO . Eluted samples were added
in order (E1, then E2, then E3) into the centricon and centrifuged at 3200 rpm for 40 minutes
at 4 °C. The eluted flow-throughs (FTs) were collected and labelled according to the samples
added. Centrifugation was continued till the volume of sample comes to 1–1.5 mL. Buffer
exchange of the sample was done with Tris Cl (50 mM, pH 8.0) and centrifuged under same
conditions as before till the sample volume comes to 500-700 µL. Final concentrated protein
was collected into 2 mL eppendorf tube and stored at 4 °C. Purity of the concentrated protein
was checked by SDS-PAGE.

11
2.10 Activity analysis of concentrated protein by dot-blot assay
12 % native polyacrylamide gel was prepared with glycol chitin as substrate. Positive control
(known chitinase present in lab), sonicated pellet and ChiC were spotted carefully on the gel
and was incubated at 37 °C for 10 hours. After incubation, the gel was stained with calcofluor
white M2R and kept under shaking for 20 minutes. The gel was destained with double distilled
water and visualised under UV transilluminator.

12
 4. RESULTS & DISCUSSION
3.1 Isolation and screening of potential chitinolytic bacterial isolates
Bacterial isolates were screened as potential targets based on the zone of clearance produced
on 0.3 % colloidal chitin plate. The zone of clearance clearly demarcates the ability of the
isolate(s) to hydrolysis the polymeric chitin. Out of the different positive isolates obtained,
isolate JM1 was found to be more efficiently degrading chitin as it zone of clearance could be
visualized within a short span of incubation i.e. 3-4 days at 28 °C.

Figure 3: From the left, uninoculated colloidal chitin agar plate, followed by plate showing
chitin degradation by JM1.

3.2 Identification of potential isolate by 16S rRNA gene sequencing and phylogenetic
analysis

PCR amplification of 16S rRNA gene of the isolate JM1 was observed on 0.8 % agarose gel
as a single band corresponding to 1.5 kb (Figure 2). On analysis of the sequencing results using
nucleotide BLAST (blastn) tool of NCBI showed that the isolate JM1 was homologous to
Paenibacillus sp. showing nearly 99.5 % identity with Paenibacillus pabuli. Using the blastn
data, phylogenetic tree for JM1 was constructed using MEGA 6.0 by neighbour joining
algorithm, which showed that nearest neighbours of the isolate JM1 were Paenibacillus pabuli,
Paenibacillus tundrae, Paenibacillus xylanexedens and Paenibacillus amylolyticus (Figure 3).

13
 Figure 4: PCR amplification of 16S rRNA gene of JM1

Figure 5: Phylogenetic tree of JM1

3.3 Growth pattern on different chitin substrates

Not much visible change in the JM1 inoculated α-chitin flasks could be seen throughout the
period of 10 days, which indicates that JM1 might not be capable of degrading α-chitin
effectively. This may be because α-chitin has the highest crystallinity among the other
polymeric chitin substrates.
Isolate JM1 however shows prominent degradation of β–chitin and colloidal chitin. β-chitin is
a less crystalline polymorph than α-chitin and visible degradation was seen at the onset of 4th
day itself and by the end of 10th day, the entire substrate was degraded.

14
Colloidal chitin is the least crystalline form, produced by the acid hydrolysis of α-chitin, was
also found to be highly preferred by the isolate as the substrate degradation was prominently
visible from the 3rd day of incubation and no substrate was found in the culture flasks by the
end of 10th day.

Figure 6: Growth of JM1 on different polymeric chitin substrates. R1, R2 and R3

represents growth on α-chitin, β-chitin and colloidal chitin, respectively.

On estimating the total cell protein using the pellets obtained from each setup, including the
positive and negative controls, it was observed that protein content in the cells growing on β-
chitin and colloidal chitin was much higher and approximately equal as compared to α-chitin.
This indicates that cell growth has been more in β-chitin and colloidal chitin than α-chitin and
hence, can be predicted that the former two are more preferred substrates as compared to the
latter.

15
 Figure 7: Total cell protein estimation for JM1 growing on different chitin substrates

Chitinase activity assay for JM1 using Schales’ reagent revealed that activity of the isolate was
higher and approximately equal in colloidal chitin and β-chitin, than α-chitin in which the
activity was observed to be much lower.

Figure 8: Chitinase activity of JM1 on different chitin substrates

3.4 Identification, selection and cloning of a candidate chitinase gene from JM1
The nearest homologue of JM1 was found to be Paenibacillus pabuli with 99.5% identity.
Carbohydrate Active enZymes (CAZy) database revealed in the presence of four candidate
chitinase genes in the genome of Paenibacillus pabuli i.e. ChiA, ChiB, ChiC and ChiD. Out of
these, all the chitinases are multi-domain enzymes except ChiC. Moreover, ChiC was found to
be less identical than other well characterized chitinases from other bacterial species. Hence,
ChiC was selected as a potential target chitinase gene to be cloned. Primers for PCR
amplification of ChiC were designed using the online bioinformatics tools SMART database

16
(smart.embl-heidelberg.de) and JustBio (www.justbio.com). The gene size for ChiC was 1.63
kb.

Figure 9: CAZy database showing the presence of

different chitinases in the genome of Paenibacillus pabuli

Successful PCR amplification of ChiC was indicated as the appearance of single band just
above the marker of 1500 bp. The amplicons were extracted from gel, double digested using
Nco I and Xho I, ligated into the expression vector pET-28a (+) and transformed into the
cloning host DH5α.

Figure 10: PCR amplification of Figure 11: Vector map of pET-28a (+)
ChiC

17
Double digestion of the recombinant plasmid for clone confirmation revealed the appearance
of two bands, one corresponding to size of ChiC (~1.6 kb) and the other to the size of the vector
backbone (~5.3 kb). This indicates successful cloning of ChiC, however final confirmation was
done by sequencing.

Figure 12: ChiC clone confirmation by double digestion; DD

indicates double digested recombinant plasmid

3.5 Transformation of ChiC plasmid into expression hosts

The ChiC plasmid was successfully transformed into both the expression hosts i.e. BL21DE
plysS and Rosettagami II DE3 cells. Only the transformed cells having the pET-28a(+)
recombinant plasmid conferring resistance to kanamycin grew on the LB-Kan plates.

Figure 13: LB-Kan plate showing transformed

Figurecolonies
11: LB-Kan plate showing
of BL21DE3 plysS. transformed

18
3.6 Expression standardization of ChiC
3.6.1 Hourly expression
The secondary culture was allowed to reach the O.D. of 0.4–0.6 at 600 nm at 37 °C. 1 mL of
culture was collected as control before induction and the remaining culture was induced with
0.7 mM IPTG, followed by incubation at 37 °C for 4 hours. 1 mL sample was collected at every
hour till 4th hour and these along with control were centrifuged at 11000 rpm for 2 minutes and
the pellets obtained were air-dried. The remaining culture was harvested, sonicated and run on
SDS-PAGE along with the control and hourly pellets. Protein over-expression was observed
as thick band corresponding to 54.7 kDa (size of ChiC protein) but nothing could be seen in
the soluble fraction.

Figure 14: Hourly expression of ChiC

3.6.2. Expression for ChiC in BL21DE3 plysS

Expression standardization for ChiC in BL21DE3 plysS was done using different induction
types, induction temperature and time of incubation. A visibly good yield of protein in the
soluble fraction was obtained in the condition of 0.7 mM IPTG induction at 18 °C for 12 hours.

19
Figure 15a: Expression of ChiC in BL21DE3 plysS;
Lane 1 & 2 represents auto-induction condition (26 °C,
24 hrs);
Lane 3 & 4 represents auto-induction + 0.1 mM IPTG
condition (26 °C, 24 hrs).
Figure 15b: Expression of ChiC in
BL21DE3 plysS by induction with 0.7
mM IPTG at 18 °C, 12 hrs

3.6.3 Expression for ChiC in Rosettagami II DE3

Expression standardization for ChiC in Rosettagami II DE3 was done using different induction
types, induction temperature and time of incubation. All the three conditions tested produced
moderate yield of protein in the soluble fraction.

Figure 16: Expression of ChiC in Rosettagami II DE3.

Lane − 1 & 2 – Autoinduction at 26 °C, 24 h
Lane − 3 & 4 – Autoinduction+0.1 mM IPTG at 26 °C, 24 h
Lane − 5 to 7 – 0.7 mM IPTG induction at 37 °C/18 °C

20
 Table 1: Summary of expression standardization for ChiC
The highlighted condition in Table 1 was found to produce the maximum yield of ChiC
protein in the soluble fraction, hence was used for over-expression of ChiC in bulk.

3.7. Purification of ChiC by Ni-NTA affinity chromatography

Figure 17: Purification profile of ChiC;

E1- elution I fractions (50 mM), E2 – elution II fractions (150 mM), E3 – elution III fractions (250
mM)

The purification gel profile of ChiC indicates successful purification of the protein due to
appearance thick single band corresponding to 54.7 kDa, devoid of non-specific bands. High
yield of pure protein was observed in the elution II fractions.

21
3.8 Concentration of ChiC
ChiC was concentrated using Amicon ultraspin centricon and later buffer exchanged with 50
mM Tris Cl (pH 8.0). A thick band of pure concentrated protein was observed on 12 % SDS-
polyacrylamide gel as shown in Figure 16.

Positive
control

Expression
pellet

Purified
ChiC
Figure 18: Gel profile Figure 19: Dot blot assay of ChiC
of concentrated ChiC

3.9. Dot blot assay for activity study of ChiC

Glycol chitin is a soluble substrate for chitinases used for assessing the in-gel activity of the
enzyme. Calcofluor white M2R is a dye that stains carbohydrate polymers. Figure 17 represents
the dot blot assay for ChiC where dark blue spots could be visualized under UV for the positive
control and ChiC. This deep blue coloration indicates the hydrolyzed region of glycol chitin.
Hence, it is imminent that ChiC is active after purification and concentration.

22
 5. CONCLUSION
Isolate JM1 seems to be a promising potential chitinolytic bacterial isolate. Identification by
16S rRNA sequencing and phylogenetic analysis shows it is 99.5 % identical with
Paenibacillus pabuli. The isolate was observed to be have almost equal preference for colloidal
chitin and β- chitin than α-chitin. Based on CAZy database, the genome of Paenibacillus pabuli
consist of four chitinase encoding genes, of which, ChiC was selected as a target due to its less
identity with already characterized chitinases from other bacterial species. ChiC was
successfully cloned into pET-28a(+) vector and transformed into expression hosts BL21DE3
plysS and Rosettagami II DE3. Standard condition of expression for ChiC was observed with
IPTG induction at 18 °C for 12 hours, further which the protein expressed in bulk, purified and
buffer exchanged. Dot blot assay represents activity of concentrated ChiC. Moreover, in order
to understand ChiC better, biochemical characterization of the enzyme such as pH and
temperature optimization, Michaelis Menten kinetics under optimal conditions, activity on
various polymeric substrates of chitin as well as on chitooligosaccharides are yet to be done.

23
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Bon, E. P., ... & Seldin, L. (2011). Cellulolytic potential of a novel strain of
Paenibacillus sp. isolated from the armored catfish Parotocinclus maculicauda
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26
 7. APPENDIX
Autoinduction Medium
Component-I

Na2HPO4.2H2O : 1.25 M

KH2PO4 : 1.25 M

NH4Cl : 2.5 M

Na2SO4 : 0.25 M

Component-II

Glycerol : 25 %

Glucose : 2.5 %

Lactose : 10 %

Colloidal Chitin Agar

Na2HPO4 : 47.7 mM

KH2PO4 : 22 mM

NaCl : 8.55 mM

NH4Cl : 18.7 mM

Agar powder :2%

Colloidal chitin : 0.3 %

Adjust pH to 8.0 using NaOH.

Coomassie Blue G – 250 Staining Solution

(NH4)2SO4 : 0.08 % (w/v)
Coomassie Blue (G-250) : 0.08 % (w/v)
O-phosphoric acid : 25 %
Methanol : 20 % (v/v)

27
Elution Buffer - I
NaH2PO4.2H2O : 50 mM
NaCl : 300 mM
Imidazole : 50 mM

Elution Buffer - II
NaH2PO4.2H2O : 50 mM
NaCl : 300 mM
Imidazole : 150 mM

Elution Buffer – III

NaH2PO4.2H2O : 50 mM
NaCl : 300 mM
Imidazole : 250 mM

Lysis Buffer
NaH2PO4.2H2O : 50 mM
NaCl : 300 mM
Imidazole : 10 mM
Adjust pH to 8.0 using NaOH.

Lysis Buffer for Plasmid Isolation

NaOH : 200 mM

SDS :1%

M9 Minimal Medium (for growth analysis)

Component - I

Na2HPO4.2H2O : 47.7 mM

KH2PO4 : 22 mM

NaCl : 8.55 mM

NH4Cl : 18.7 mM

28
Component - II

MgSO4. 7H2O : 100 µL/100 mL

CaCl2 : 30 µL/100 mL

Biotin : 1 mg/mL

Thiamine-Cl : 1 mg/mL

Trace elements : 100x

Neutralisation Buffer for Plasmid Isolation

Potassium acetate (pH- 5.5) : 3M

Adjust pH to 5.5 with glacial acetic acid

Resuspension Buffer for Plasmid Isolation

Tris-Cl (pH -8.0) : 50 mM

EDTA : 10 mM

RNase : 100 µg/mL

Schale’s Reagent
K4Fe(CN)6.3H2O : 0.5 g/L
Na2 CO3 : 0.5 M

SDS-Polyacrylamide Gel
12% Resolving Gel (10 mL)

Double distilled water : 3.3 mL

Acrylamide mixture (30%) : 4.0 mL

Tris-Cl (1.5 mM, pH 8.8) : 2.5 mL

SDS (10 %) : 0.1 mL

APS (10 %) : 0.1 mL

TEMED : 0.004 mL

29
Stacking Gel (4 mL)

Double distilled water : 2.7 mL

Acrylamide mixture (30%) : 0.67 mL

Tris-Cl (1 mM, pH 6.8) : 0.5 mL

SDS (10 %) : 0.04 mL

APS (10 %) : 0.04 mL

TEMED : 0.004 mL

SDS-PAGE Loading Buffer (1X)

Tris Cl (pH – 6.8) : 50 mM
Dithiothreitol : 100 mM
SDS (electrophoresis grade) : 2 % (w/v)
Bromophenol blue : 0.1 %
Glycerol : 10 % (v/v)

Sodium Acetate Buffer (50 mM, pH 5.0)

Sodium acetate : 4.1 g
Adjust pH to 5.5 with glacial acetic acid

Make up final volume to 100 mL with double distilled water.

Trace Elements (100X)

EDTA : 13.1 mM

FeCl3.6H2O : 3.1 mM

Zn Cl2 : 0.62 mM

CuCl2.2H2O : 76 µM

CoCl2.2H2O : 42 µM

H3BO4 : 162 µM

MnCl2.4H2O : 8.1 µM

30
Tris Glycine Buffer (5X)
Tris : 0.125 M
Glycine : 1.25 M
SDS : 10 %

Wash Buffer
NaH2PO4.2H2O : 50 mM
NaCl : 300 mM
Imidazole : 20 mM
Adjust pH to 8.0 using NaOH.

31