Screening Diverse Cellulase enzymes from the white rot ... · personalities involved with me during...
Transcript of Screening Diverse Cellulase enzymes from the white rot ... · personalities involved with me during...
Screening Diverse Cellulase enzymes from the
white rot fungus Phlebia gigantea for high
activity and large scale applications
A thesis submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy
by
Ajay Niranjane
Department of Biotechnology and Environmental Biology
School of Applied Sciences
Royal Melbourne Institute of Technology
March 2006
Thesis Abstract
Cellulosic biomass is the major organic matter produced in the biosphere.
The biodegradation of this cellulosic material is achieved by enzymatic activities of
the cellulose degrading microorganisms. These organisms have evolved a
biochemical system for degrading complex cellulosic substrates. They usually
express a complex extracellular or a membrane bound cellulolytic system comprising
combination of several cellulase enzymes. Cellulases are the group of hydrolytic
enzymes capable of hydrolysing insoluble cellulose to glucose.
Phlebia gigantea is an aggressive white rot basidiomycete that has been
commercially exploited as a biocontrol agent against the invasion of Heterobasidium
annosum. It’s ability to tolerate resinous extracts present on freshly cut wood and
higher specific growth rate helps the fungus to competitively colonise the sapwood
preventing other fungi from becoming established. Biopulping of logs with this
fungus is reported to degrade large quantities of wood extractives resulting in the
reduction of energy consumption during the mechanical refining process. Early
research on the cellulase system of this organism reported the presence of a cellulase
system composed of β-glucosidase, endoglucanase and a cellobiohydrolase enzyme.
Based on these unpublished studies, our initial aim was to obtain a complete sequence
of putative cellobiohydrolase I (CbhI) from this organism. Attempts to identify and
isolate the cellulase gene resulted in an incomplete cDNA sequence of 1154 bp.
In order to fully understand the cellulase system, the expression and
regulation of the cellulase enzymatic activity was examined in a variety of different
growth media and conditions. The study was conducted for 14 days of incubation of
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P. gigantea on substrates glucose, xylose, Avicel, carboxymethyl cellulose (CMC)
and cellobiose. The pH, total protein and biomass production was also monitored
during this period. The result obtained indicated that the capacity of P. gigantea to
degrade cellulose is to a degree dependent upon the nature of the available carbon
source. The P. gigantea multiprotein cellulase complex is acid active and alkali
stable. It is also concluded from the study that the regulation of the cellulase
synthesis is repressed in the presence of simple sugars like glucose and xylose.
The study has also been able to successfully employ the rapid and highly
effective method of purification of cellulase complex by affinity digestion. This two
step purification method purified the cellulase complex in relatively large quantity.
Further characterisation of the kinetic properties of this cellulase complex revealed
that the rate of cellulase catalysis were optimum at pH 5.0 and temperature 50oC. The
purified complex was found to be comprised of multiple proteins and demonstrated
significantly detectable CMCase and CBHase activity on zymogram analysis.
The variety and diversity of the cellulase complex was further studied by
functional proteomics approach. The purified cellulase complex was characterised by
2D gel electrophoresis and further peptide mass finger printing was undertaken by
MALDI-TOF mass-spectrometry analysis. The 2D gel analysis of the purified
cellulase complex showed 15 well separated spots within the range of pI 3.5 to pI7.
The molecular weight of separate protein was calculated to be in the range 20KDa to
100KDa. Three protein spots were selected based on the IEF and SDS zymogram
and further identification analysis was done using MALDI-TOF MS analysis. These
proteins were identified based on the peptide mass data as belonging to the
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6-phospho-α-glucosidase, β-glucosidase and glycosyl hydrolase family 13 α-amylase
or pullulanases. These results indicated diversity in the purified proteins, suggesting
the divergent evolution of specific cellulase proteins.
The result of this study has shown P. gigantea as a potential cellulase
source. It also shows that the cellulase complex secreted in response to the substrate
induction comprises a variety of different enzymes related to hydrolysis of cellulose
biomass. It is evident from this and the earlier studies that P. gigantea cellulase
complex comprises of a specific set of enzymes that not only possess the ability to
degrade crystalline cellulose, but also to be one of the first organisms to colonise
freshly cut wood. Further detailed studies on the cellulase system of this primary
colonist may open up the prospects to utilise this organism as the potential onsite
bioreactor agent, pre-treating the biomass and increasing the economic feasibility of
the industrial bioenergy processes.
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Acknowledgement
The success of any project is never limited to the individual who is undertaking the
work. I take this opportunity to express my gratitude and thank some of the
personalities involved with me during my studies and the completion of this thesis.
First and foremost, I would like to express my sense of gratitude to my supervisor
Assoc. Prof. Trevor Stevenson, for his guidance, encouragement, freedom of work
and the care he provided throughout my research work. His confidence in me and the
invaluable help right from my first day in Australia, has contributed immensely to
develop me personally and professionally.
I am deeply indebted to, Dr. Chitra Ragahavan and Dr Bert Collard for their timely
guidance and help. A special thanks to Priya Madhou, for her support and lots of wise
words.
I sincerely thank Dr. David Stalker, Dr. Greg Nugent, Prof Peter Coloe and Assoc
Prof Ann Lawrie. I am grateful to Dr. John Fecondo for his invaluable comments and
help in the analysis of proteomics data. His cheerful gesture will always be cherished.
I wish to thank my friends and colleagues, Dr Ruchira, Kavitha, Deanne, Manvendra,
Hema, Brooke, Dr. Prashant Sawant and Dr Jason Ross.
Words fail me in expressing my heartfelt thanks to my friends, Anu- Ninad, Sam -
Nayana and Rajshree - Girish for always being there. I thank them for their
understanding, love and support.
I would like to thank Shekhar mama and Jyoti mami, Sanjay, Jaya-Anil Surya,
Vandana and Vijay Palaspagar, Prashant, Ashit, Mummy and Papa for their prayers
and blessings. It wasn’t possible without them.
I thank my soul-mate Preeti, for being extremely patient and tolerant towards my
erratic hours of work and her continued encouragement, being an anchor and
accompanying me through all the highs and lows. She has equally worked hard to
help me develop on all fronts.
Lastly, and most importantly, I wish to thank my parents, Meera and Pundalikrao
Niranjane for everything they did for me. To them I dedicate this thesis.
Ajay Niranjane
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Declaration
I declare that this thesis contains my original work. Information from published or
unpublished sources has been clearly acknowledged within the thesis. None of the
work contained in this thesis has been submitted either in whole or in part to qualify
for any other academic award. The content of the thesis is a result of the work, which
has been carried out during the enrolled period of the program.
AJAY NIRANJANE
March 2006
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List of Publications
1) Niranjane, A.P., Madhou, P. and Stevenson, T.W. (2006) The effect of
carbohydrate carbon sources on the production of cellulase by Phlebia
gigantea. Enzyme and Microbial Technology (In Press).
2) Niranjane, A.P., and Stevenson, T.W. Purification and characterisation of
cellulase produced by Phlebia gigantea (Submitted for publication).
3) Niranjane, A.P., Fecondo, J. and Stevenson, T.W. Rapid mass spectrometric
peptide sequencing and mass matching for characterization of P. gigantea
cellulases isolated by two dimensional PAGE (Manuscript in preparation).
4) Niranjane, A.P., and Stevenson, T.W. (2006) Screening of extracellular
cellulase enzymes of saprophytic fungus P. gigantea by proteomics analysis.
In 5th Discovery Science and Biotechnology Meeting. Melbourne, Australia.
5) Niranjane, A.P., and Trevor Stevenson (2006) Screening of extracellular
cellulase enzymes of saprophytic fungus P. gigantea by proteomics analysis.
In The Australian Health and Medical Research Congress, Third Congress.
Melbourne Convention Centre.
vii
Abbreviations
A280 Absorbance at 280 nm
Ala Alanine
AMP Adenosine monophosphate
amp Ampicillin
Asp Aspartate
ATP Adenosine triphosphate
bp Base pair
BSA Bovine Serum Albumin
CBD Cellulose binding domain
CBHase Cellobiohydrolase
cDNA complementary DNA
CHAPS 3-[(3-cholamidopropyl)dimethylammonio]propane sulfonic acid
cm Centimetre
CMC Carboxy methyl cellulose
CMCase Carboxymethyl cellulases
Da Dalton
DEPC Diethyl pyrocarbonate
DNA Deoxyribonucleic acid
DNSA Dinitro salicylic acid
dNTP Deoxyribonucleotide triphosphate
DTT Dithiothreitol
Exp Experimental
Fig. Figure
GAPDH Glyceraldehyde 3-phosphate dehydrogenase
Glu Glutamate
Hrs Hours
IEF Isoelectric focusing
IPG Isoelectric focusing gel
IU International unit of measurement of enzyme
KCN Potassium cyanide
KDa Kilo Dalton
Km Michellis Menten constant
Abbreviations
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L Litre
MALDI Matrix Assisted Laser Desorption of Ionisation
MgCl Magnesium Chloride
mL Millilitre
mM Millimolar
mm Millimetre
mRNA messenger RNA
MS Mass spectrometry
MW Molecular Weight
NaCl Sodium chloride
nm Nanometer
oA Angstrom unit
oC Degree Celsius
ORF Open reading frame
PAGE Polyacrylamide Gel electrophoresis
PCR Polymerase chain reaction
Pro Proline
RNA Ribonucleic acid
rpm Revolutions per minute
SDS Sodium dodecyl sulphate
Ser Serine
Theo Theoretical
Thr Threonine
TOF Time of flight
UV Ultra violet
V Volt
v/v Volume/Volume (concentration)
w/v Weight/ Volume (concentration)
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Table of contents
Thesis Abstract ........................................................................................................... i
Acknowledgement.................................................................................................... iv
Declaration .................................................................................................................v
List of Publications................................................................................................... vi
Abbreviations .......................................................................................................... vii
Table of contents ...................................................................................................... ix
List of Tables........................................................................................................... xv
List of Figures ........................................................................................................ xvi
Chapter 1 : Literature Review ...................................................................................1
1.1 Introduction ............................................................................................1
1.1.1 Overview on Phlebia gigantea as potential cellulose degrader .............1
1.1.2 Cellulose degrading enzymes and their industrial potential...................3
1.1.2.1 Cellulases ...........................................................................................3
1.1.2.2 Occurrence .........................................................................................4
1.1.2.3 Measurement of cellulose hydrolysis Activity...................................6
1.1.2.4 Induction, regulation and organization of cellulase ...........................8
1.1.2.5 Organization of cellulase gene .........................................................11
1.2 Cellulase Enzyme System ....................................................................13
1.2.1 Fungal Cellulase System ......................................................................14
1.2.2 Bacterial Cellulase Systems .................................................................18
1.3 Structure of cellulase............................................................................20
1.4 Mechanism of Catalysis .......................................................................24
1.4.1 Retaining Mechanism...........................................................................25
1.4.2 Inverting Mechanism............................................................................26
1.5 Glycoside hydrolase classification of cellulases ..................................27
1.6 Genetic and Protein Engineering of Cellulases....................................32
1.7 Industrial applications of Cellulases ....................................................36
1.8 Project Objectives ................................................................................37
Chapter 2 : Materials and Methods.........................................................................39
2.1 Chemical reagents ................................................................................39
2.2 Enzymes and commercial kits..............................................................39
Table of contents
x
2.3 Solutions...............................................................................................39
2.3.1 Buffers ..................................................................................................40
2.3.1.1 Carlson Lysis Buffer –(100 mL) (Carlson et al., 1991) ...................40
2.3.1.2 Citrate Buffer (50 mM) buffer pH 5.5 stock ....................................40
2.3.1.2.1 Stock A Citrate Buffer................................................................40
2.3.1.2.2 Stock B Citrate Buffer................................................................40
2.3.1.2.3 Working 50mM Citrate Buffer...................................................41
2.3.1.3 Dialysis buffer for protein purification ............................................41
2.3.1.4 DNA gel loading buffer....................................................................41
2.3.1.5 Formaldehyde (FA) gel running buffer (1x) ....................................41
2.3.1.6 Phosphate Buffer (10X) ...................................................................41
2.3.1.7 RNA formaldehyde (FA) gel buffer (10 x MOPS) ..........................42
2.3.1.8 RNA gel loading Buffer (5x) ...........................................................42
2.3.1.9 SDS PAGE (Tris Glycine) electrophoresis buffer (10x)..................42
2.3.1.10 SDS sample buffer (5x)................................................................43
2.3.1.11 Sodium Acetate Buffer.................................................................43
2.3.1.12 TBE buffer (1x) 1L ......................................................................43
2.3.2 Electrophoresis solutions......................................................................43
2.3.2.1 Acrylamide (40 %) ...........................................................................43
2.3.2.2 Ammonium acetate (10M) ...............................................................44
2.3.2.3 Ampicillin.........................................................................................44
2.3.2.4 Bovine serum albumin (BSA) ..........................................................44
2.3.2.5 Calcium chloride ..............................................................................44
2.3.2.6 Caesium chloride and EDTA buffer.................................................44
2.3.2.7 Chloroform: Isoamyl Alcohol 24:1 ..................................................45
2.3.2.8 Congo-Red Stain ..............................................................................45
2.3.2.9 Coomassie blue staining solution.....................................................45
2.3.2.10 Destaining Solution for protein SDS gels ....................................45
2.3.3 General reagents and solutions.............................................................45
2.3.3.1 3, 5-Dinitrosalicylic acid (DNSA) ...................................................45
2.3.3.2 Isopropanol (isopropyl alcohol) .......................................................46
2.3.3.3 Miniprep Plasmid Isolation ..............................................................46
2.3.3.3.1 Solution 1 ...................................................................................46
2.3.3.3.2 Solution 2 ...................................................................................46
Table of contents
xi
2.3.3.3.3 Solution 3 ...................................................................................46
2.3.3.4 MOPS (0.1M)...................................................................................46
2.3.3.5 4-methylumbelliferyl-ß-D cellobioside (0.5mM) ............................47
2.3.3.6 NaCl (1.5 M) ....................................................................................47
2.3.3.7 Phenol...............................................................................................47
2.3.3.8 Potassium Acetate (5M) ...................................................................47
2.3.3.9 Potassium Acetate (3M) pH 5.0 .......................................................48
2.3.3.10 RNAase ........................................................................................48
2.3.3.11 Rubidium Chloride (RbCl)...........................................................48
2.3.3.12 Sodium hydroxide ........................................................................48
2.3.3.13 Urea (1M).....................................................................................48
2.4 Media....................................................................................................49
2.4.1 Luria Bertani broth (LB) ......................................................................49
2.4.2 Luria Bertani agar (LB agar) ................................................................49
2.4.3 LB Agar with ampicillin ......................................................................49
2.4.4 LB Agar with Ampicillin /IPTG/X-Gal ...............................................50
2.4.5 Malt extract broth .................................................................................50
2.4.6 Malt extract agar...................................................................................50
2.4.7 SOC broth.............................................................................................50
2.5 Ladders and Markers ............................................................................50
2.5.1 GeneRuler™ 1Kb DNA Ladder, (Fermentas #SM0311/2/3) ..............51
2.5.2 Lambda DNA/EcoRI+HindIII Marker, 3 (Fermentas #SM0191/2/3) .51
2.5.3 PageRuler™ Prestained Protein Ladders (Fermentas #SM0676) .........51
2.6 Methods................................................................................................51
2.6.1 Maintenance of Fungal Strain ..............................................................51
2.6.2 DNA extraction by CTAB method.......................................................52
2.6.3 RNA isolation and quantitation............................................................53
2.6.4 First strand cDNA synthesis.................................................................53
2.6.5 Amplification of cDNA by PCR. .........................................................54
2.6.6 Miniprep Plasmid Isolation ..................................................................54
2.6.7 Cloning by Electrocompetent and heat shock cells..............................55
2.6.7.1 Ligation ............................................................................................55
2.6.7.2 Transformation .................................................................................55
2.6.7.2.1 Electroporation ...........................................................................55
Table of contents
xii
2.6.7.2.2 Heat Shock Transformation .......................................................56
2.6.8 Agarose gel preparation and electrophoresis .......................................56
2.6.9 Visualisation of PCR products .............................................................56
2.6.10 Primer design........................................................................................57
2.6.11 Zymogram analysis ..............................................................................57
2.6.11.1 Activity staining ...........................................................................58
2.6.12 Isoelectric focusing of SDS gel ............................................................58
2.6.13 Protein staining on SDS gel .................................................................59
Chapter 3 : Molecular Characterisation of Cellulase genes..................................60
3.1 Introduction ..........................................................................................60
3.2 Materials and Methods .........................................................................61
3.2.1 Organism ..............................................................................................61
3.2.2 Extraction of fungal RNA ....................................................................61
3.2.3 Quantitation and determination of RNA ..............................................62
3.2.4 Molecular Methods ..............................................................................62
3.2.4.1 Design of PCR primers ....................................................................62
3.2.4.2 Polymerase Chain Reaction .............................................................64
3.2.4.3 Rapid amplification of cDNA ends (RACE) PCR ...........................64
3.2.4.4 Extraction of PCR product from agarose .........................................66
3.2.4.5 Cloning of PCR product ...................................................................66
3.2.4.6 Analysis of cloned PCR product ......................................................67
3.2.4.7 Sequencing of cloned PCR product .................................................68
3.2.4.8 Analysis of Sequences......................................................................69
3.2.5 Transcription analysis of Phlebia gigantea cDNA PCR product for
cellulase expression studies..................................................................69
3.3 Results ..................................................................................................70
3.3.1 Amplification of Phlebia gigantea cDNA using all primers ................70
3.3.2 Cloning of PCR product.......................................................................70
3.3.3 DNA sequencing ..................................................................................73
3.3.4 Sequence homology .............................................................................73
3.3.5 Amplification of Phlebia gigantea cDNA for cellulase expression
studies...................................................................................................75
3.3.6 Amplification of Phlebia gigantea cDNA product using RACE PCR75
Table of contents
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3.4 Discussion ............................................................................................79
Chapter 4 : Effect of carbohydrate carbon sources on the production of cellulase
by Phlebia gigantea.....................................................................................................82
4.1 Introduction ..........................................................................................82
4.2 Materials and methods .........................................................................84
4.2.1 Organism ..............................................................................................84
4.2.2 Inoculum preparation and cultivation ..................................................85
4.2.3 Basal Medium and culture conditions..................................................85
4.2.4 Protein determination ...........................................................................85
4.2.5 Cellulase enzyme assay........................................................................86
4.2.5.1 Carboxymethyl Cellulose Plate diffusion assay...............................86
4.2.5.2 CMC assay .......................................................................................87
4.2.6 Effect of various inducers and repressors on cellulase production ......87
4.2.7 Biomass production..............................................................................88
4.2.8 Study of pH changes on cellulase production ......................................88
4.3 Results ..................................................................................................88
4.4 Discussion ............................................................................................95
Chapter 5 : Purification and characterisation of cellulase produced by Phlebia
gigantea......................................................................................................................101
5.1 Introduction ........................................................................................101
5.2 Materials and Methods .......................................................................104
5.2.1 Fungal Strain and Growth conditions.................................................104
5.2.2 Preparation of inoculum .....................................................................104
5.2.3 Cultivation conditions ........................................................................105
5.2.4 Preparation of Amorphous Cellulose .................................................106
5.2.5 Cellulase purification by affinity adsorption......................................106
5.2.6 Cellulase assays..................................................................................107
5.2.7 Gel electrophoresis and staining ........................................................108
5.2.8 Zymogram analysis of purified protein ..............................................109
5.2.9 pH optimum for purified enzyme.......................................................109
5.2.10 Temperature optimum and stability for purified enzyme...................110
5.2.11 Kinetic properties ...............................................................................110
Table of contents
xiv
5.3 Results ................................................................................................110
5.3.1 Purification of the cellulase complex .................................................110
5.3.2 Gel electrophoresis and staining ........................................................111
5.3.3 Cellulase activity ................................................................................111
5.3.4 Zymogram analysis ............................................................................115
5.3.5 pH optimum........................................................................................116
5.3.6 Temperature optimum........................................................................117
5.3.7 Kinetic properties ...............................................................................118
5.4 Discussion ..........................................................................................120
Chapter 6: Identification of proteins of cellulase complex from two dimensional
gels by molecular mass fingerprinting of peptide fragments ...............................124
6.1 Introduction ........................................................................................124
6.2 Materials and methods .......................................................................127
6.2.1 Protein purification and 2D Analysis by SDS-PAGE.........................127
6.2.2 2D Gel electrophoresis .......................................................................127
6.2.2.1 Sample preparation.........................................................................127
6.2.2.2 First dimension...............................................................................127
6.2.2.3 IPG equilibration for separation by second dimension ..................128
6.2.2.4 Second dimension ..........................................................................128
6.2.2.5 Protein staining methods ................................................................129
6.2.3 Zymogram analysis of IEF gels .........................................................129
6.2.4 Mass Spectrometry.............................................................................130
6.2.5 Protein Identification..........................................................................131
6.3 Results ................................................................................................132
6.3.1 Protein Purification ............................................................................132
6.3.2 2D gel Analysis ..................................................................................133
6.3.3 Zymogram analysis of IEF gels .........................................................135
6.3.4 M S Analysis and protein identification.............................................135
6.4 Discussion ..........................................................................................150
Chapter 7 : Final Discussion and future prospects ..............................................153
Bibliography .............................................................................................................161
Appendix I.................................................................................................................187
Table of contents
xv
Appendix II ...............................................................................................................188
List of Tables
Table1.1 Cellulase families and their clans................................................................. 30
Table 3.1 Primers used for PCR:................................................................................. 63
Table 3.2 Primers used for RACE PCR:..................................................................... 65
Table 5.1 Summary of published cellulase purification schemes from different source
organisms. ......................................................................................................... 103
Table 5.2 Chemically defined cellulase inducing media utilised in P. gigantea
cultivation................................................................................................ 106
Table 5.3 Summary of purification and yield of extracellular cellular from P.gigantea.
................................................................................................................................... 113
Table 5.4 Kinetic constants of P.gigantea cellulase at different concentration of
substrate................................................................................................... 119
Table 6.1 Matching peptide mass of three P. gigantea proteins using Aldente from
SwisprotTREMBL database.............................................................................. 142
Table 6.2 Summary of matching peptides of spot PgCel2 for specific cleavage...... 143
Table 6.3 Summary of matching peptides of spot PgCel5 for specific cleavage...... 144
Table 6.4 Summary of matching peptides of spot PgCel7 for specific cleavage...... 145
Table 6.5 Biological and molecular significance of few selected proteins ............... 146
Table 6.6 Matching peptide mass of PgCel 2 with CBM family from CAZy database
........................................................................................................................... 147
Table 6.7 Matching peptide mass of PgCel 5 with CBM family from CAZy database
........................................................................................................................... 148
Table 6.8 Matching peptide mass of PgCel 7 with CBM family from CAZy database.
........................................................................................................................... 149
xvi
List of Figures
Figure 1. 1 Phlebia gigantea on red pine stump ............................................................1
Figure 1. 2 Fungal Cellulase System............................................................................17
Figure 1.3 Bacterial Cellulase System .........................................................................20
Figure 1.4 Three types of active sites found in glycosyl hydrolases............................24
Figure 1. 5 Retaining and inverting mechanism of enzymatic catalysis ......................26
Figure 1. 6 Ribbon structure of Cellulases. ..................................................................31
Figure 1. 7 Xylanase-cellulase bifunctional fusion protein......................................... 35
Figure 3.1 Amplification of P. gigantea cDNA by using primer D3.......................... 71
Figure 3.2 Cloning of PCR product ............................................................................ 72
Figure 3.3 Transcriptional analysis of PCR product ................................................... 74
Figure 3.4 3’RACE PCR product................................................................................ 76
Figure 3.5 Combined PCR And RACE-PCR Product P223A3S6.............................. 77
Figure 3.6 The amino acid sequence of the product P223A3S6 ................................. 78
Figure 4.1 Mycelial growth curves of P. gigantea...................................................... 90
Figure 4.2 Total protein obtained from P. gigantea in presence of different substrates
..................................................................................................................................... 91
Figure 4.3 Carboxymethyl cellulose plate diffusion assay ......................................... 92
Figure 4.4 Cellulase activity curves of P. gigantea with different substrates............ 93
Figure 4.5 P. gigantea cellulase activity curves of CMC plate assay......................... 94
Figure 4.6 Effect of substrates on pH of P. gigantea media ...................................... 95
Figure 5.1 C: PAGE Analysis of the affinity purified cellulase complex................. 114
Figure 5.2 CMC SDS PAGE CMCase analysis of the cellulase............................... 115
Figure 5.3 MUC-SDS PAGE CBHase analysis of the cellulase............................... 116
Figure 5.4 pH optimum curve of purified P.gigantea cellulase................................ 117
Figure 5 5 Temperature optimum curve of purified P.gigantea cellulase. ............... 118
Figure 5.6 Lineweaver- Burk Plot for kinetics study of P.gigantea cellulase complex
with Avicel as substrate........................................................................... 119
Figure 5.7 Lineweaver- Burk Plot for kinetics study of P.gigantea cellulase complex
with CMC as substrate. ........................................................................... 120
Figure 6.1 2D analysis of affinity adsorption purified cellulase. ...............................134
Figure 6.2 IEF zymogram analysis of P. gigantea cellulase complex. ......................135
List of Figures
xvii
Figure 6.3 MALDI-TOF-MS spectrum of P.gigantea cellulase complex of from 2D
gel spot PgCel2...................................................................................................137
Figure 6.4 MALDI-TOF-MS spectrum of P.gigantea cellulase complex of 2D gel spot
PgCel 5. ..............................................................................................................138
Figure 6.5 MALDI-TOF-MS spectrum of P.gigantea cellulase complex of 2D gel spot
PgCel 7. ..............................................................................................................139
Figure 6.6 MALDI-TOF-MS spectrum of P.gigantea cellulase complex of 2D gel spot
PgCel 5. ..............................................................................................................140
1
Chapter 1 : Literature Review
1.1 Introduction
1.1.1 Overview on Phlebia gigantea as potential cellulose degrader
Phlebia gigantea, (also called as Phanerochaete gigantea, Peniophora
gigantea and Phlebiopsis gigantea) is an aggressive white rot fungus that colonizes
conifer wood. It occurs throughout North America, Central America, Europe, East
Africa, and southern Asia. The fungus plays an important role in the decomposition
of conifer debris and is found on the wood and bark of gymnosperms (notably on
species of Pinus, Picea, Abies, and Tsuga). There have been reports of P. gigantea
causing sap rot of both stored gymnosperm and angiosperm pulpwood and of wood
products (Cram, 1999). P. gigantea produces sexual spores (basidiospores) that
become airborne and spread to woody debris via wind and rain. It also has an asexual
stage that produces spores (oidia) from mycelial fragmentation. These asexual spores
are easily cultured on artificial media in the laboratory, producing large numbers of
brick-like spores by fragmentation of the hyphae (Rishbeth, 1963; Ross, 1973).
Figure 1. 1 Phlebia gigantea on red pine stump
Photo courtesy from USDA- USFS photo file from M. Cram.(Cram, 1999)
Physiological studies indicate that P. gigantea has a strong competitive ability and
high specific growth rate (Adomas et al., 2003), so it will colonize sapwood
Chapter 1 : Literature Review
2
preferably and readily degrade resin and other wood extractives (Behrendt and
Blanchette, 1997).
Fast growth, prolific asexual spore production, potential to fix pulp and
paper and the ability to tolerate resinous extracts present on freshly cut wood provides
the fungus the advantage to quickly colonize sapwood, preventing other fungi from
becoming established (Blanchette et al., 1998). Several studies in England (Rishbeth,
1963) and in the southern United States (Hodeges, 1964; Ross and Hodges, 1981),
showed that the artificial inoculation of pine stumps with P. gigantea significantly
decreases the presence of H. annosum. Dr John Rishbeth at the Cambridge Botany
School found that fresh pine stumps can be colonised by this weakly parasitic fungi
and to subsequently prevent invasion by H. annosum, thereby protecting stump
surfaces without the need for phytotoxic chemicals (Deacon, 1998). P. gigantea has
an interesting mode of action as a biological control agent, the hyphae antagonize the
hyphae of H. annosum (and some other fungi) on contact – a phenomenon termed
hyphal interference. Any hypha of H. annosum that makes contact with a hypha of P.
gigantea shows rapid localized disruption: the protoplasm becomes disorganized and
its membrane integrity is affected (Deacon, 1998; Cram, 1999). P. gigantea is also
effective in colonizing spruce stumps. Presently, P. gigantea is commercially
available in England, Sweden, Norway, Switzerland, and Finland. The English
product, a spore suspension, is sold by Omex International under the name Pg
Suspension. The Finnish product is a dry formula that is sold by Kemia Oy under the
name ‘Rotstop’.
Unlike other biocontrol agents, P. gigantea is not a biocide that kills the
target organism. Rather, it competes for resources that the pathogen would other wise
Chapter 1 : Literature Review
3
use, providing an extension to a naturally occurring process (Pratt et al., 1999).
Biological pulping of logs with this fungi result in the degradation of large quantities
of wood extractives, reducing energy consumption during mechanical refining and
improving paper strength properties (Blanchette et al., 1998). Moreover, it has been
found that this fungus is a high producer of cellulase enzymes (Chee et al., 1998;
Adomas et al., 2003). Research on P.gigantea has provided evidence for the
existence of a cellulase system involving a β-glucosidase, an endoglucanase and a
cellobiohydrolase enzyme (Palaniswamy, 1998).
1.1.2 Cellulose degrading enzymes and their industrial potential
1.1.2.1 Cellulases
The structural complexity and rigidity of cellulosic substrates have given
rise to a remarkable divergence in cellulose degradative enzymes (Mai et al., 2004).
Microorganisms involved in degrading these complex structures have faced many
evolutionary challenges, developing complex enzyme systems to handle these varying
substrates. Organisms usually produce complex extracellular or membrane bound
cellulolytic enzymes comprising a combination of activities. Cellulases are the group
of hydrolytic enzymes that are capable of hydrolysing insoluble cellulose to produce
soluble oligosaccharides. Cellulases are modular enzymes that are composed of
independently folding, structurally and functionally discrete units called domains
(Klyosov, 1995b; Mai et al., 2004).
At least three fundamental enzyme activities are traditionally found within a
cellulase system that demonstrates the degradation of cellulose.
Chapter 1 : Literature Review
4
i) Endoglucanases (Endo-1, 4-β-glucanases/1, 4-β-glucanases/1, 4-β-D-
glucan4-glucanohydrolase, EC 3.2.1.4).
ii) Cellobiohydrolases (Exo-1, 4-β-glucanases/1, 4-β-D-glucan
cellobiohydrolases, EC 3.2.1.91).
iii) Cellobiases (β-glucosidases/β-D-glucoside glucohydrolases, EC
3.2.1.21).
There have been a notable differences found in the cellulolytic enzymes isolated from
various sources. The differences were mainly in their polypeptide characteristics such
as capacity to adsorb to cellulose, molecular weight, isoelectric points, carbohydrate
content, catalytic activity, substrate specificity and amino acid composition and
sequence. However, a cellulase system is an efficient hydrolysis of cellulose in a
specific coordinated manner. It is a combination of three representative enzymes with
or without cellulose binding domains (Lynd et al., 2002).
1.1.2.2 Occurrence
Cellulase enzymes are produced by a wide variety of bacteria and fungi,
aerobes and anaerobes, mesophiles and thermophiles but relatively few organisms can
utilize crystalline cellulose as a carbon source (Bhat and Bhat, 1997; Coutinho and
Henrissat, 1999a; Maijala, 2000; Lynd et al., 2002). The distribution of cellulolytic
species across taxonomic groups has revealed that the ability to digest cellulose is
widely distributed among many genera in the domain bacteria and in the fungal
groups within the Eukaryotes. Within the Eubacteria, there is considerable
concentration of cellulolytic capabilities among the aerobic order Actinomycetales
(Phylum Actinobacteria) and the anaerobic order Clostridiales (Phylum Firmicutes).
Fungal cellulose utilization is distributed across the entire kingdom, from the
Chapter 1 : Literature Review
5
primitive, protists like Chytridomycetes, to the advanced Basidiomycetes (Lynd et al.,
2002). Homobasidiomycetes is one of the major class containing over 13,000
cellulolytic species (Hibbett and Donoghue, 2001). Most of the studies with respect
to cellulolytic enzymes and/or wood degrading capability so far have been conducted
on the cellulase systems of Trichoderma, Penicillium, Fusarium, Aspergillus,
Geotrichum, Cladosporium (Deuteromycetes); Phanerochaete, Poria, Schizophyllum,
Coriolus and Serpula (Basidiomycetes); and Bulgaria, Chaetomium, Helotium
(Ascomycetes) (Lynd et al., 2002).
The cellulolytic bacteria however, are grouped into different diverse
physiological groups such as:
i) Fermentative anaerobes, which contains gram-positive Clostridium,
Ruminococcus, Caldicellulosiruptor and gram-negative such as
Butyrivibrio and Acetivibrio
ii) Aerobic gram-positive, Cellulomonas and Thermobifida and
iii) Aerobic gliding bacteria, Cytophaga and Sporocytophaga.
Generally only few of the species within each of the above named genera are
actively cellulolytic. Few favourite organisms studied extensively are: Trichoderma
viride, Trichoderma reesei, Phanerochaete chrysosporium, Sporotrichum
thermophile, Humicola insolens, Neocallimastix frontalis, Thermoascus aurantiacus,
Microbispora bispora, Clostridium thermocellum, Thermonospora fusca, Acetivibrio
cellulolyticus, and Ruminococcus flavefaciens.
Chapter 1 : Literature Review
6
1.1.2.3 Measurement of cellulose hydrolysis Activity
The quantitative measurement of cellulolytic hydrolysis is important from
two perspectives, organization and analysis of fundamental understanding and
designing engineered systems based on the evaluation of this quantitative data (Lynd
et al., 2002). Cellulosic substrates when purified, are present as insoluble
macroscopic particles varying in shape, size and even composition (Lynd et al., 2002).
The intricacy of multiprotein cellulase system and physical diversity of
cellulosic substrates produced by different microorganisms have led to the
development of several assay procedures for the measurement of cellulase activities.
The considerable difference in the nature of substrates used, variation in assay
procedures adopted for measuring different cellulase components, and the synergistic
action of cellulase components have made the comparison of results among
laboratories difficult. In 1984, the International Union of Pure and applied Chemistry
(IUPAC) Commission on Biotechnology published standard assay procedures for
measuring cellulase activities (Sharrock, 1988; Bhat and Bhat, 1997). Some of these
recommendations have been readily accepted by biotechnologists, but many
enzymologists feel that these procedures are quite restrictive and not satisfactory for
understanding the detailed mechanism of action and substrate specificities of
cellulases. Consequently, Wood and Bhat (Wood and Bhat, 1988) reviewed the
cellulase assays used by laboratories working on fungal cellulases and highlighted the
advantages and disadvantages of the various procedures. Although, the assay
procedures described were for Trichoderma cellulases, those methods can be adapted
to other cellulases from other fungal species.
Chapter 1 : Literature Review
7
The main technical use of cellulases lies in the conversion of cellulosic
fractions from various cellulose containing materials into glucose. Therefore, it is
important to measure total cellulolytic activity, the activity of cellulase complex,
which produces glucose from cellulose. However, complexities that arise due to the
formation of enzyme-cellulose complexes are the central features of most quantitation
procedures, and the formation of such complexes is related to the nature of both the
specific enzymes and the substrates.
The enzymes of cellulase complexes often act synergistically, so the activity
measured is greatly influenced by the proportion in which various activities are
present. The soluble and insoluble substrates used are macromolecules, which are
difficult to standardize.
The most widely used substrates for total activity are filter paper,
microcrystalline cellulose, cotton fibers and soluble carboxymethyl cellulose. Each
method usually measures the reducing sugar formed in the enzymatic reaction. The
filter paper hydrolysis method determines the total cellulase activity in units of
micromoles of reducing sugar (measured as glucose) liberated per one minute under
standard assay conditions. The units of measuring this activity are Filter paper units
(FPU), or international Units (IU).
Other important properties that influence cellulase activity and should be
considered while quantifying the cellulase hydrolysis from different organisms are pH
and heat stability, specific molecular inhibitors and activators, adsorption of enzyme
onto substrate, and transglycosylation reactions. Transglycosylation reactions are
Chapter 1 : Literature Review
8
usually the side reactions leading to the formation of unwanted reaction by-products.
The adsorption capacity of cellulases is extremely important factor to consider when
examining potential biotechnological applications of the enzymes to the hydrolysis of
cellulose. The ability of cellulase to hydrolyse cellulose is directly related to its
adsorption capacity. The tighter the adsorption of cellulase to crystalline cellulose,
the higher the rate of reaction and greater the yield of glucose (Klyosov, 1995a, b;
Kawaguchi et al., 1996; Janne Lehtio, 2003). When cellulases are weakly adsorbed,
the degree of hydrolysis of cellulose does not proceed efficiently whereas, when
adsorption is tight, complete hydrolysis can take place. If a cellulase binds weakly to
crystalline cellulose the rate of hydrolysis decreases, irrespective of the amount of
enzyme used and the time given for reaction (Fan et al., 1980; Goto et al., 1992; Janne
Lehtio, 2003). Thus, for the effective and total degradation of cellulose, cellulases
with high rates of catalyses are associated with tight adsorption capacity (Klyosov,
1995b). Therefore in any examination of cellulase enzymes the determination of
adsorption capacity of the cellulase along with the measurement of its hydrolysis
activity is vital.
1.1.2.4 Induction, regulation and organization of cellulase
Cellulase is an inducible enzyme system (Coutinho and Henrissat, 1999a; De
Vries and Visser, 2001; Galbe and Zacchi, 2002; Lynd et al., 2002). All
microorganisms studied to date have produced high levels of cellulase when grown on
cellulose as a sole carbon source. Other substrates such as cellobiose, lactose and
sophorose are also known as inducers of cellulase enzyme activity. Synthetic
compounds like thiocellobiose, palmitate and acetate ester have also found to induce
the cellulase (Bhat and Bhat, 1997). In T. reesei the expression of polysaccharide
Chapter 1 : Literature Review
9
degrading enzymes is found to be induced only in the presence of a complex substrate
such as cellulose and/or cellulose derivatives, but the gene was repressed in the
presence of easily utilizable substrates such as glucose (Ilmen et al., 1997). The
expression of the cellulase transcripts in T. reesei has been shown to be controlled by
the nature of the carbon sources used in the culture medium (El-Dorry et al., 1996).
The mechanism of induction of cellulase is complex because of the insoluble
nature of cellulose which makes it unable to enter the microbial cell and trigger the
gene. The generally accepted theory of induction of cellulase gene expression by
insoluble and polymeric substrate is that, the low levels of cellulase activity is
constitutively produced by the microorganisms, and this low level enzyme activity
initiates hydrolysis of cellulose to soluble sugars (El-Dorry et al., 1996; Bhat and
Bhat, 1997). These sugars are then converted into true inducers, which enter the cell
and either directly or indirectly influence binding protein and promote cellulase gene
expression (Bhat and Bhat, 1997). Induction of the cellulase transcripts by cellulose
thus requires a basal expression of its own genes (Henrique-Silva et al., 1996). In
case of Trichoderma, it has also been suggested that the cellobiohydrolase, bound to
conidia, hydrolyse the cellulose and release cellobiose and cellobiono-δ-1,5- lactone
(CBL), which is ultimately taken up by mycelia and promote increased cellulase gene
expression and enzyme synthesis (Bhat and Bhat, 1997). The study of mRNA levels
of cellulase encoding genes in T. reesei has shown that Solkafloc cellulose and the
disaccharide sophorose induced expression to similar levels (Teeri et al., 1992; Ilmen
et al., 1997). Addition of neither glycerol nor sorbitol promotes expression, but
unlike glucose they do not inhibit the expression either (Ilmen et al., 1997). No
expression of cellulase was found on any medium containing glucose (Lynd et al.,
Chapter 1 : Literature Review
10
2002), and high levels of glucose were also found to suppress the inducing activity of
sophorose. The expression of cellulase as described above is not simply the result of
energy starvation, as the lack of carbon or nitrogen is not sufficient to trigger a
significant amount of gene expression (Ilmen et al., 1997).
It has been noted in T. reesei, that Cbh II (cellobiohydrolase II) and Egl II
(endoglucanase II) are of major importance for the efficient formation of cellulase
inducer from insoluble cellulose and their removal resulted in an inability of T. reesei
to attack crystalline cellulose (Seiboth et al., 1997). It was also been found that
neither Cbh II nor Egl II forms transglycosylation products, the potent inducers of
cellulase formation, suggesting that the action of these two enzymes is important only
to generate the substrates for transglycosylation by other enzymes (Seiboth et al.,
1997). These studies have also indicated the possibility that Cbh I, Egl III and Egl V
act as potential inducer formants. Expression of Cbh I transcripts is influenced by the
physiological state of the fungal mitochondria and this sensitivity is controlled in the
5’- flanking DNA sequence of this gene (J. Abrahao Neto, 1995; El-Dorry et al.,
1996). Cbh I and Egl I transcripts are low in atmospheres of low oxygen tension and
are also repressed by glucose at concentrations known to repress mitochondrial
respiration (J. Abrahao Neto, 1995). The Cbh I promoter is composed of two
regulatory regions adjacent to its TATA box, one region supposedly controls cellulose
induced transcription and the other is required for basal cellulase enzyme expressions
(Henrique-Silva et al., 1996).
Cellulose transcripts are also down-regulated by chemical agents known to
dissipate the proton electrochemical gradient of the inner mitochondrial membrane
Chapter 1 : Literature Review
11
and compounds that block the electron – transport chain such as DNP and KCN (J.
Abrahao Neto, 1995). O-Glycosylation of cellulases, which takes place at the
endoplasmic reticulum has been shown to determine the secretion of cellulases in
Trichoderma (Bhat and Bhat, 1997). A study of four filamentous fungi revealed that
extracellular cellulase was repressed at intracellular ATP concentrations above 10-7
mg/mL and that cyclic AMP (cAMP) played a major role in depression of enzyme
synthesis (Ilmen et al., 1997; Lynd et al., 2002). Also, a motif similar to the sequence
mediating cAMP dependant regulation of rat tyrosyl amino transferase is identified
upstream from the Cbh II gene of T. reesei (Bhat and Bhat, 1997). Very little is
known at the molecular level regarding the mechanism regulating the synthesis of 50
cellulases and further research at the molecular level on cellulase genes from different
microorganisms is necessary for better understanding of the regulation of cellulase
synthesis.
1.1.2.5 Organization of cellulase gene
The genes encoding cellulases are chromosomally located in both bacteria
and fungi. In the fungi, cellulase genes are usually randomly distributed throughout
the genome, with each gene having its own transcriptional regulatory elements (Lynd
et al., 2002). The only exception is in P. chrysosporium where three
cellobiohydrolase like genes are clustered (Covert et al., 1992; Lynd et al., 2002). A
comparison of promoter regions of Cbh I, Cbh II, Egl I and Egl II of T. reesei
revealed the presence of CRE 1 (Catabolite repressor I) binding sites through which
catabolite repression is exerted (Lynd et al., 2002). ACE I (Activator I) and ACE II
(Activator II) activate transcription by binding to the Cbh I promoter region (Aro et
al., 2001; Lynd et al., 2002).
Chapter 1 : Literature Review
12
Both aerobic and anaerobic cellulolytic bacteria as well as fungi, primarily
contain mutlidomain cellulases, with single domain cellulases being the exception as
seen in the Eg III of T. reesei and Eg 28 of P. chrysosporium (Henriksson et al., 2000;
Lynd et al., 2002; Sandgren et al., 2004). The most common modular arrangements
involve catalytic domains attached to a cellulose binding domain (CBD) through a
flexible linker region. The linker regions are 6- 59 amino acid residues, rich in
proline and hydroxyamino acids which are often glycosylated (Gilkes et al., 1991b;
Bhat and Bhat, 1997). The CBD module can be located either at the N or C terminus
of the catalytic cellulase unit (Bhat and Bhat, 1997). The presence of CBD at N or C
terminus is prominently found in non-complex cellulase systems, whereas cellulases
belonging to the complex systems of anaerobic bacteria and fungi are comparatively
more diverse. These complex cellulosomal systems contain a catalytic domain and a
separately folded and functionally independent CBD, linked by a linker
region.(Schwarz, 2001). For example a immunoglobulin- like domain of Cel E
(Cellobiohydrolase E) of C. cellulolyticum (Gaudin et al., 2000; Lynd et al., 2002) and
fibronectin type III domain of Cbh A (Cellobiohydrolase A) of C. thermocellum
(Zverlov et al., 1998; Lynd et al., 2002).
The bacterial CBDs are generally composed of around 100 amino acids and
generally contain within the group greater than 50% sequence homology. The CBD
sequences are mostly composed of a small number of charged amino acids, a high
number of hydroxyamino acids, and all contain conserved tryptophan, asparagine and
glycine residues (Bhat and Bhat, 1997). The CBDs from T. reesei is about 33 amino
acids having a wedge like shape with one flat hydrophilic and a hydrophobic surface
Chapter 1 : Literature Review
13
(Sims et al., 1988; Azevedo Mde et al., 1990; Claeyssens and Henrissat, 1992; Bhat
and Bhat, 1997).
1.2 Cellulase Enzyme System
The ability to decompose and obtain carbon and energy from lignin,
cellulose and hemicellulose is widespread among fungi and bacteria (Klass, 1983).
These microorganisms produces multiple enzymes that are either free or cell
associated, and these enzymes degrade natural cellulosic materials that are
heterogeneously intertwined polysaccharide chains with varying degree of
crystallinity. These multiple enzyme complexes are known as Enzyme Systems (Bhat
and Bhat, 1997; Lynd et al., 2002) and are classified into two major systems are
commonly referred as:
i) Fungal cellulase system (Non-complex systems) - the enzymes in this
system have ability to penetrate cellulosic substrate through hyphal
extensions and do not produce stable high molecular weight complex.
ii) Bacterial cellulase system (Complex systems) - these complexes are
produced and tightly bound on the cell wall of cellulolytic bacteria to
flexibly position the cellulase enzymes on the substrate for cellulose
hydrolysis.
The complex cellulase systems comprises of mostly the anaerobic bacteria
which lack the ability to effectively penetrate the cellulosic materials, and thus have to
develop alternative methods to degrade cellulose and gain access to products of
cellulose hydrolysis with limited ATP for cellulase synthesis and competition from
other microorganisms. The development of such a ‘Complex Cellulase System’, also
Chapter 1 : Literature Review
14
called as ‘Cellulosomes’, can locate the cellulolytic cells at the site of hydrolysis and
are observed in Clostridia and ruminal bacteria. Simultaneously, the cellulosome also
has the capacity to minimize the distance to which cellulose hydrolysis products must
diffuse, thus helping to increase the uptake efficacy of these oligosaccharides by the
host cells (Bayer et al., 1998a; Schwarz, 2001; Eriksson et al., 2002; Lynd et al.,
2002).
The non-complex cellulase systems are present in the filamentous fungi and
actinomycete bacteria, which have the ability to diffuse cellulosic particles by
reaching out the cavities through hyphal extensions (Eriksson et al., 2002; Lynd et al.,
2002).
1.2.1 Fungal Cellulase System
Cellulases from aerobic fungi have been studied extensively and are the
predominant workhorses in industrial processes. Cellulolytic filamentous fungi and
actinomycete bacteria have the ability to penetrate cellulosic substrates through their
hyphal extensions, thus reaching all portions of the cellulosic particles. This ability to
reach out for the substrate with or without the presence of cellulose binding domain
(CBDs) gratifies the efficiency of the cellulose hydrolysis. The enzymes in this
cellulase system do not form stable high- molecular weight complexes and therefore
are termed “Non-complex” systems (Lynd et al., 2002). The extracellular cellulase
components of most fungi are generally found to exist as individual entities
(Coughlan, 1992; Bhat and Bhat, 1997). The cellulose hydrolysis by Non-complex
fungal system is schematically represented in Figure 1.2.
Chapter 1 : Literature Review
15
The cellulase systems of the aerobic filamentous fungi P. chrysosporium
(Broda et al., 1996; Lynd et al., 2002), T. reesei (Bhat and Bhat, 1997; Lynd et al.,
2002), A. niger (De Vries and Visser, 2001), H. insolens (Schulein, 1997; Lynd et al.,
2002), F. oxysporum (Gerlind Sulzenbacher et al., 1996; Bhat and Bhat, 1997), T.
emersonii (Bhat and Bhat, 1997) have been studied in detail. The cellulase system of
these fungi consists of endoglucanases (EC 3.2.1.4), exoglucanases (3.2.1.91) and β-
glucosidases (3.2.1.21). T. reesei produces at least two exoglucanases (Cbh I and Cbh
II), five endoglucanases (Eg I, Eg II, Eg III, Eg IV, and Eg V) and two β- glucosidases
(Bgl I and Bgl II). Generally, the endoglucanases randomly attacks the amorphous
regions of cellulose and release the cello- oligosaccharides (Bhat and Bhat, 1997;
Väljamãe et al., 1999; Valjamae et al., 2001). The exoglucanases hydrolyse the
phosphoric acid swollen cellulose and avicel by sequentially removing the cellobiose
units from both the reducing and non-reducing ends of the cellulose chain (Goto et al.,
1992; Bhat and Bhat, 1997; Väljamãe et al., 1999; Lynd et al., 2002). The
endoglucanases and cellobiohydrolases act synergistically to effect the extensive
hydrolysis of crystalline cellulose, and the β- glucosidase completes the hydrolysis by
converting the resultant cellobiose to glucose (Gusakov et al., 2005; Miettinen-
Oinonen et al., 2005).
Cellobiohydrolase activity is essential for the hydrolysis of microcrystalline
cellulose. Cbh I and Cbh II are the principal components of the T. reesei cellulase
system (Valjamae et al., 1998; Lynd et al., 2002; Gusakov et al., 2005). The need of
these two cellobiohydrolase is accredited to the preferential activity towards the
reducing (Cbh I) and non-reducing (Cbh II) ends of cellulose chains of
microcrystalline cellulose, which has also been supported by the phenomenon of exo-
Chapter 1 : Literature Review
16
exo synergism (Henrissat et al., 1985; Lynd et al., 2002). However, both
cellobiohydrolases are very slow at depolymerising cellulose. Although the exo- endo
synergism is not clear, endoglucanases are thought to be responsible for decreasing
the degree of polymerisation by breaking the internal cellulose chains at amorphous
regions, thus generating cellobiohydrolase susceptible chains of cellulose (Väljamãe
et al., 1999). The production of two β- glucosidases by T. reesei facilitates the
hydrolysis of cellobiose and small oligosaccharides to glucose (Lynd et al., 2002).
The β- glucosidases are present in proximity to the fungal cell wall, which may help
the cell to limit the loss of glucose to the environment and ultimately restrain the
organism from the product inhibition (Lynd et al., 2002).
Another white rot fungus, P. chrysosporium (a model linocellulolytic
organism), produces complex mixtures of cellulases, hemicellulases and ligninases
(Covert et al., 1992; Broda et al., 1995, 1996; Lynd et al., 2002). Cellulose and
hemicellulose degradation in P. chrysosporium occur during primary cell metabolism
whereas lignin degradation, initiated by starvation of carbon, nitrogen or sulphur, is a
process of secondary metabolism (Broda et al., 1996; Lynd et al., 2002). The P.
chrysosporium cellulase system is comprised of one Cbh II and six homologues of
Cbh I. It has also demonstrated the presence of a CBD less, 28- KDa endoglucanases
(Egl 28), which has shown synergistic activity with cellobiohydrolases. Egl 28 is
similar to the Egl II of T. reesei. The Cbh I like genes of P. chrysosporium have been
found to produce differential splicing in the CBD peptide region, ultimately denoting
it a capacity to yield the cellobiohydrolase and endoglucanases activity. P.
chrysosporium also produces cellobiose dehydrogenase, which binds to
microcrystalline cellulose enhancing the hydrolysis activity (Henriksson et al., 1997;
Chapter 1 : Literature Review
17
Lynd et al., 2002). Cellobiose dehydrogenase oxidises cellobiose in the presence of
Oxygen to cellobionolactone, which in turn produces cellobionic acid as a result of a
spontaneous reaction with water (Vallim et al., 1998; Henriksson et al., 2000; Lynd et
al., 2002). Thus cellobiose dehydrogenase is considered a strong supplemental
enzyme activity in lignin and cellulose depolymerisation by helping to generate
hydroxyl radicals (Henriksson et al., 2000)
Figure 1. 2 Fungal Cellulase System
Representation of Hydrolysis of Cellulose By Non-complex Fungal Cellulase System,
figure adopted from (Lynd et al., 2002).
Chapter 1 : Literature Review
18
1.2.2 Bacterial Cellulase Systems
The ability to degrade crystalline cellulose is widespread in both aerobic and
anaerobic bacteria. The bacteria either secrete their cellulases as soluble extracellular
enzymes or assemble them into large complexes called as cellulosomes, which are
attached to the bacterial cell surface (Klass, 1983). Most of the cellulolytic bacteria
mainly produce endoglucanases and some exoglucanases.
Both endo and exoglucanases produced by the bacterium Cellulomonas fimi
show the same general structural features as those of T. reesei. The cellulosome of
the thermophilic anaerobic bacterium, C. thermocellum is the most extensively
studied cellulosome (Klass, 1983; Bhat and Bhat, 1997; Lynd et al., 2002). This
cellulosome is made up of 14–18 complexed polypeptides containing several
endoglucanases accompanied by exoglucanases and xylanases. The cellulosome also
contains polypeptides with no enzymatic activity, which may function in the
organization of the cellulosome or attachment to the cell surface and/or to the
substrate (Klass, 1983). Cellulosomes that have the potential for total degradation of
cellulase are found attached to the cell surface during the exponential phase of
bacterial growth on synthetic medium. However as the cell culture moves into
stationary phase they migrate from the cell surface into the medium (Klass, 1983;
Lynd et al., 2002). The cellulosome components of C. thermocellum are more
efficient at hydrolysing crystalline cellulose as compared to amorphous cellulose.
This high efficiency of the cellulosome has been credited to a) the presence of
different enzymatic activity (cellulolytic or hemicellulolytic) capable of disrupting the
physical obstacles contained in the polysaccharides present in heterogenous plant
materials, b) the correct ratio between catalytic domains that optimise synergism
Chapter 1 : Literature Review
19
between them and c) appropriate spacing between the individual components to
further favour synergism. Cells of C. thermocellum are made up of three layers, the
cytoplasmic membrane, the peptidoglycan membrane and surface protein layer (S-
layer). The cellulosome consists of a large noncatalytic scaffoldin protein Cip A,
which is multimodular and includes nine cohesive domain, four hydrophilic X-
modules, and a family III CBD. The Cip A scaffoldin is anchored to the cell wall via
type II cohesion domains. Cip A contains nine polypeptide domains, each composed
of about 146 residues, separated by Pro- and Thr- rich peptide segments of 17 to 19
residues. These domains recognise a 22- residue catalytic module each of which
exhibit one of the activity as endoglucanases, cellobiohydrolases, β- glucosidases and
xylanases of the cellulosome. Out of these 22, at least 9 of this module exhibit
endoglucanase activity (Cel A, Cel B, Cel D, Cel E, Cel F, Cel G, Cel H, Cel N, and
Cel P), 4 of which exhibits exoglucanases activity (Cbh A, Cel K, Cel O, Cel S), 5 of
which exhibit hemicellulase activity ( Xyn A, Xyn B, Xyn V, Xyn Y, Xyn Z), 1 chitinase
(Man A), and one lichenase activity (Lic B). These catalytic modules posses dockerin
moieties that can interact with Cip A protein to assemble the cellulosome. Cip A and
certain catalytic components of the cellulosome also contains cellulose binding
domains that attach the cellulosome to the surface of crystalline cellulose(Lytle and
Wu, 1998). The major exoglucanases Cel S, which is always present, is a processive
cellulase specifically active on microcrystalline or amorphous cellulose but not on
carboxyl methyl cellulose (CMC) (Lynd et al., 2002). Cellulosome are extensively
glycosylated on the scaffoldin moiety. These glycosyl moieties supposedly protect
the cellulosome against proteases and help the scaffoldin to recognize the cohesion-
dockerin region(Bayer et al., 1998b).
Chapter 1 : Literature Review
20
Figure 1.3 Bacterial Cellulase System
Representation of Hydrolysis of Cellulose By Complex Bacterial Cellulase System,
figure adopted from (Lynd et al., 2002).
1.3 Structure of cellulase
The precise properties of cellulases vary depending on their origin, whereas,
detection of an evolutionary relationship between proteins is done for functional
inferences (Bhat and Bhat, 1997; Bayer et al., 1998b; Yona, 2004). In many cases,
sequences have diverged to such an extent that their similarity is undetectable by
standard sequence comparison, but even such diverged sequences may have similar
Chapter 1 : Literature Review
21
structures and functions (Henrissat et al., 1989; Gilkes et al., 1991a). It has been
inferred that evolutionary pressure acts upon the three dimensional structure of
proteins and their intra-protein interactions and not at the primary sequence (Mornon,
2003). Evidence suggests that 3D structures are more conserved than primary
sequence information and the functional inferences drawn from structure are more
reliable than from primary sequences (Davies and Henrissat, 1995).
The majority of microbial cellulases studied have been shown to be acidic
proteins with significant carbohydrate content (Davies and Henrissat, 1995; Bhat and
Bhat, 1997; Davies, 1998; Henrissat and Davies, 2000) [there are 80 entries in
enzyme class of 3.2.1.4]. Carbohydrates show a wide variety of stereochemical
variation and can be assembled in so many different fashions that there are more than
1012 possible isomers for reducing hexasaccharide (Davies and Henrissat, 1995). As a
result of this diversity in saccharide there is a vast variety of enzymes capable of
hydrolysing the glycosidic bonds contained within them (Davies and Henrissat, 1995).
The general architecture derived for cellulase demonstrates independent globular
modules forming a catalytic domain responsible for hydrolysis reaction, a
proline/serine/threonine rich linker and a cellulose binding module, helping the
adsorption of the enzyme on to the insoluble crystalline cellulose. The cellulose
binding domains do not have any intrinsic catalytic activity (Gilkes et al., 1991a;
Henrissat and Davies, 2000).
Sequence and structure comparison of glycosyl hydrolases and cellulases
have classified them into families sharing similar structures and mechanisms of
chemical reaction. The large number of three dimensional structures of Glycosyl
Chapter 1 : Literature Review
22
Hydrolase (GH) enzymes that have been accumulated so far demonstrated that the
members of GH families share the common motif for catalytic site, similar overall
protein folding patterns and reaction mechanism (Sandgren et al., 2005). Despite
these similarities, several families show a varied divergence in sequence homology.
Most of the effective cellulolytic fungi produce two different cellobiohydrolases
(CBH’s), Cbh I and Cbh II (Teeri, 1997). X- ray scattering studies of Cbh I and Cbh
II structures from T. reesei showed the tadpole-like structure with an isotropic head
and along flexible tail for both of the enzymes (Abuja et al., 1988; Claeyssens and
Henrissat, 1992). Structures of these two domains, determined with the help of
different substrates and inhibitors, exhibit the enclosure of the active site by four and
two long loops of Cbh I and Cbh II respectively, forming a tunnel-like shape (Teeri,
1997). Cbh I have long active site tunnels suitable for the action at chain ends,
whereas, Cbh II which have short active site tunnels, display increasing
endoglucanase activity perhaps due to fewer loops of different length and mobility
(Teeri, 1997).
The catalytic domain of T. reesei Cbh II is a large α/β protein, with the
active site located at the carboxy terminal end of a parallel β- barrel in an enclosed
tunnel (Rouvinen et al., 1990; Bhat and Bhat, 1997). The catalytic residues of Cbh II
are predicted to be the two aspartic acids located in the centre of this active site tunnel
(Rouvinen et al., 1990). The catalytic domain of Cbh I is composed of two large
antiparallel β sheets stacking face to face forming a β sandwich. The active site
tunnel of Cbh I is predicted to possess seven glycosidic binding sites, with two
glutamic acids forming the catalytic residues (Bhat and Bhat, 1997; Divne et al.,
1998). The 3D structure of C. thermocellum Cel D, has a globular and slightly
Chapter 1 : Literature Review
23
elongated shape with two distinct structural domains, a small N- terminal β- barrel
closely packed to the larger α- helical domain. This protein, Cel D of C
thermocellum, which belongs to family 9 of GH folds into a regular (α/α) 6 barrel
formed by six inner and six outer α- helices, and possess at least 5 different substrate
binding subsites. The strictly conserved Glu 95 of this enzyme is centrally located in
the substrate binding cleft attached with the hydrogen bonded to glycosidic oxygen
and presumably works as a proton donor (Dominguez et al., 1995). The catalytic core
structure of endoglucanase V from H. insolens shows barrel topology, with a flattened
sphere shape having six stranded β- barrel domain, belonging to GH family 45
(Rouvinen et al., 1990; Davies et al., 1993). A large deep groove running across the
surface and the location of active site aspartates in this groove, defines the variation in
the type of topology (Davies and Henrissat, 1995). The endoglucanases are predicted
as possessing fully open active sites, denoting a capability to act on internal bonds
along the cellulose chain (Teeri, 1997).
Cellulases show different folds, α8/ β8- barrel in GH- A and β- sandwich in
GH- C clans and in general, have a long substrate binding groove running along one
side of the protein that binds several consecutive sugar residues (at least two glucose
units on each side of the catalytic centre) (Davies and Henrissat, 1995; Davies, 1998).
The binding groove is lined by hydrogen bonding residues, and a few aromatic
residue side chains are often found as hydrophobic platforms that form discrete sugar
binding subsites. Endoglucanases generally form an open binding cleft, where as
CBHs have extended loops that surround the cellulose polymer chain forming a
substrate binding tunnel (Davies et al., 1993).
Chapter 1 : Literature Review
24
Figure 1.4 Three types of active sites found in glycosyl hydrolases.
a) The pocket (glucoamylase from A. awamori. b) The cleft (endoglucanase E2 from
T .fusca). c) The tunnel (cellobiohydrolase II from T. reesei). The proposed catalytic
residues are shaded in red. Picture from(Davies and Henrissat, 1995).
1.4 Mechanism of Catalysis
Cellulases mostly employ two different types of catalytic mechanisms for
the hydrolysis of glycosidic bonds, the retention or the inversion mechanism (Bhat
and Bhat, 1997; Teeri, 1997; Coutinho and Henrissat, 1999a). These mechanisms
involve either the retention or inversion of the configuration of the anomeric carbon,
which in both cases are catalysed by the two carboxyl groups present in the active site
of the enzyme (Mai et al., 2004).
Chapter 1 : Literature Review
25
1.4.1 Retaining Mechanism
The retaining mechanism cleaves the anomeric substrate at the C1 position
via a double displacement mechanism. The two steps of this mechanism,
glycosylation and deglycosylation, help to retain the β- configuration at the anomeric
carbon after hydrolysis. The mechanism involves first glycosylation, where one of
the two carboxylic groups, acid catalyse the substrate, while the second carboxyl
group provides a nucleophilic attack on the anomeric carbon to form a transient
covalent enzyme- substrate intermediate. Next in the deglycosylation step the first
carboxyl group activates an incoming nucleophile, a water molecule in hydrolysis and
a new glycosyl hydrolase group in case of transglycosylation, by extracting a proton
from it. This activated nucleophile then hydrolyses the substrate-enzyme complex
(Sandgren et al., 2005). During the deglycosylation the substrate undergoes a ring
distortion within the active site of the enzyme in order to attain a ‘twisted boat’
conformation (Mai et al., 2004).
Chapter 1 : Literature Review
26
Figure 1. 5 Retaining and inverting mechanism of enzymatic catalysis
(a) The retaining mechanism in which the glycosidic oxygen is protonated by the acid
catalyst (AH), and nucleophilic assistance to a glycon departure is provided by the
base B. The resulting glycosyl enzyme is hydrolysed by a water molecule and this
second nucleophilic substitution at the anomeric carbon generates a product with the
same stereochemistry as the substrate. (b) The inverting mechanism in which
protonation of the glycosidic oxygen and a glycon departure is accompanied by a
concomitant attack of a water molecule that is activated by the base residue (B). This
single nucleophilic substitution yields a product with opposite stereochemistry to the
substrate(Davies and Henrissat, 1995).
1.4.2 Inverting Mechanism
The inverting enzymes works via a single step concentration mechanism,
where the hydrolysis of a β- glycosidic bond of the substrate at anomeric carbon C1
creates a product with a α- configuration. This cleavage is formed via a single
Chapter 1 : Literature Review
27
nucleophilic displacement. An enzyme performing the activity by this method
involves two catalytic carboxylates. This carboxylates helps to remove a glycosidic
oxygen by its protonation which is assisted by a nucleophilic attack of a water
molecule activated by the base residue (Beguin and Aubert, 1994; Davies and
Henrissat, 1995; Sandgren et al., 2005).
1.5 Glycoside hydrolase classification of cellulases
The cellulase enzyme systems discussed have been based on substrate
specificities, categorising cellulases as either endoglucanases or exoglucanases
(cellobiohydrolases) based on their ability to cleave β- 1, 4 glycosidic bond internally
or at the one end of a polymeric substrate. As such the cellulases are grouped along
with hemicellulases and other polysaccharide degrading enzymes such as O-
glycoside hydrolases (EC 3.2.1.x), and with the other auxiliary enzymes including
hemicellulose hydrolases (EC 2.4.1.x) (Lynd et al., 2002). But with the exponentially
increasing genomic data, an alternative classification based on amino acid sequence
similarities, of their catalytic domains has been suggested (Henrissat, 1997; Lynd et
al., 2002). Whilst this system of classification was very effective, it was unable to
easily accommodate enzymes that displayed both modes of catalysis, further dividing
classification towards more conclusive structural and mechanistic properties (Mai et
al., 2004). As a result, a classification of glycoside hydrolases into families based on
amino acid sequence similarity, exploiting the direct relationship of amino acids
sequences with enzyme folding was proposed (Coutinho and Henrissat, 1999a; Lynd
et al., 2002; Mai et al., 2004; Sandgren et al., 2005). The large number of three
dimensional structures of GH enzymes studied verified similarities in protein folding
characteristics by members of GH families (Mai et al., 2004; Sandgren et al., 2005).
Chapter 1 : Literature Review
28
Bernard Henrissat and Pedro M. Coutinho, maintain and updates a site of
Carbohydrate-Active Enzymes server at URL: http://afmb.cnrs-mrs.fr/CAZY/
(Coutinho and Henrissat, 1999a; Lynd et al., 2002; Mai et al., 2004; Sandgren et al.,
2005), presently there are more than 16,000 protein sequences of GHs grouped into
96 active families and 14 known clans.
As has been discussed cellulases are mostly the modular structures,
composed of a catalytic module attached to one or several associated non- catalytic
domains. Whilst catalytic modules can be classified into several families based on
their structural and amino acids sequence similarities, the classification of non-
catalytic modules, independent and complimentary to their catalytic modules, is yet to
be achieved completely. The well known cellulose binding domains at present
constitute 43 families, based on structure and function. There are also several other
cellulosomal non- catalytic cohesions and dockerins containing a growing number of
modules with unknown function that are yet to be classified (Coutinho and Henrissat,
1999a).
Glycoside hydrolases are enzymes which hydrolyse the glycosidic bond
between two or more carbohydrates or carbohydrate and non carbohydrate moiety
(Coutinho and Henrissat, 1999a). These glycoside hydrolases are classified into
evolutionary and structurally related families by Bernard Henrissat and co-workers
(Coutinho and Henrissat, 1999a; Sandgren et al., 2005). Cellulases are grouped into
12 different GH families further classifying them into 4 of the 14 clans based on
structural similarities (Table 1.1) (http://afmb.cnrs-mrs.fr/CAZY/) (Sandgren et al.,
2005). Cellulases are found in several different GH families 5, 6, 7, 8, 9, 10, 12, 44,
Chapter 1 : Literature Review
29
45, 48, 61, and 74 suggesting convergent evolution of different folds resulting in the
same substrate specificity. Family 9, which contains cellulases of bacteria (aerobic
and anaerobic), fungi, plants and animals are thus proved to be evolutionarily deeply
rooted. Whereas, family 8 contains only bacterial enzymes and family 7 consist of
only fungal cellulases. Cellulases from different families can form different folds
with either an inverting or retaining mechanism that can be observed in
microorganisms. For example, the cellulosome complex of C. thermocellum contains
endoglucanases and exoglucanases from families 5, 8, 9 and 48 respectively (Shoham
et al., 1999).
Hydrophobic cluster analysis (HCA) allows the classification of glycoside
hydrolases and transglycosidases to be made (Henrissat et al., 1989; Henrissat et al.,
1995). Three-dimensional structural analysis, as well as HCA of the regions around
the catalytic residues of glycosidases, have shown that several of the sequence-based
families can be grouped into superfamilies or 'clans' (Henrissat et al., 1995) sharing
not only the same global fold, but also the same molecular mechanism and the same
catalytic machinery. Some of the representative ribbon structures of cellulases and
their respective clans are shown in Figure 1.6. The largest of these clans, clan GH-A,
grouped together families 1, 2, 5, 10, 17, 26, 30, 35, 39, 42, 51, 53, 59, 72, 79, and 86
[http://afmb.cnrs-mrs.fr/CAZY/] (Henrissat et al., 1995; Coutinho and Henrissat,
1999b; Lynd and Zhang, 2002), where the catalytic acid–base and nucleophilic
Chapter 1 : Literature Review
30
Table1.1 Cellulase fam
ilies and their clans
Cellulase fam
ilies and their respective clans as classified at http://afm
b.cnrs-m
rs.fr/CAZY/ (as on date 1 August 2005).
Cellulase
Family
Clan
Former name
Mechanism
Fold
Nucleophile
Proton
donor/Acid
catalyst
Total entries
5
A
Cellulase A
Retaining
(β/α) 8
Glu
Glu
634
6
- Cellulase B
Inverting
- Asp
Asp
103
7
B
Cellulase C
Retaining
Β- Jelly Roll
Glu
Glu
131
8
M
Cellulase D
Inverting
(α/α) 6
Asp
Glu
81
9
- Cellulase E
Inverting
(α/α) 6
Asp
Glu
242
10
A
Cellulase F
Retaining
(β/α)8
Glu
Glu
241
11
C
Cellulase G
Retaining
Β- Jelly Roll
Glu
Glu
201
12
C
Cellulase H
Retaining
Β- Jelly Roll
Glu
Glu
78
26
A
Cellulase I
Retaining
(β/α)8
Glu
Glu
63
44
- Cellulase J
Inverting
- -
- 15
45
- Cellulase K
Inverting
- Asp
Asp
73
48
M
Cellulase L
Inverting
(α/α)6
- Glu
21
61
- -
- -
- -
53
74
- -
Inverting
7-fold β-
Propeller
Asp
Asp
22
Chapter 1 : Literature Review
31
Figure 1. 6 Ribbon structure of Cellulases.
A) 1cz1 GH5 Exo1, 3 glucanase of Candida albicans at 1.85A resolution. B)
1eg1GH7 from T reesei cellulose degradation endoglucanase 1. C) 1h12 GH 8
Structure of cold- adapted family 8 xylanases. D) 1us2 GH10 Xylanase10c from
Cellovibrio japonicus. E) 1xyn GH11 Xylanase. F) 1r7o GH26 crystal structure of
apo- mannase from Pseudomonas cellulosa. G) 1l2a GH48 crystal structure of
cellobiohydrolase CelS from Clostridium thermocellum. H) 1ks5 GH12
endoglucanase of A. niger. I) 1dys, cellulase GH6 endoglucanase Cel6b from
H.insolens. J) 1ia6 GH9 crystal structure of cellulase Cel9m of C. thermocellum. K)
1hd5 GH45 H. insolens endoglucanase at 1.7A resolution. Glycosyl hydrolase family
6, 9, and 45 are yet to be classified in clans (Davies and Henrissat, 1995).
Chapter 1 : Literature Review
32
residues were located respectively at the C-termini of strands β4 and β7 of a common
(β/α)8 barrel structure (Woodcock et al., 1992; Henrissat et al., 1995; Howard R.L.,
2003).
1.6 Genetic and Protein Engineering of Cellulases
The use of multienzyme complexes at both laboratory and industrial scale
and the capacity of extracellular cellulolytic systems to degrade native cellulose to
glucose have generated the need for the exceptional cellulase with overall enzymatic
perfection. Recombinant DNA technology and protein engineering are dynamic tools
with the potential to make significant improvements in naturally occurring enzyme
systems (Schulein, 2000). Some sort of improvements that may be possible to
achieve with cellulases with respect to increase in stability, half life, high level of
expression in host organism, it’s specific activity and variations in substrate
specificity (Bhat and Bhat, 1997; Schulein, 2000).
The enzymes with multiple activities can be created by artificial gene
fusions. An expression vector pAMH110, containing the promoter region of the
strongly expressed Cbh I gene has been used to over express a cDNA coding for Eg I
of T. reesei (Bhat and Bhat, 1997). Using the above approach, novel strains with
either mixture of CBHs, endoglucanases and xylanases (exo- endo glucanases fusion
(Warren et al., 1987; Howard R.L., 2003), xylanases–endoglucanases fusion (Tomme
et al., 1994) or mixtures completely free of CBHs and strains over- expressing the
endoglucanases has been produced (Bhat and Bhat, 1997; Schulein, 2000).
Chapter 1 : Literature Review
33
Protein engineering of cellulases has involved the mutagenesis of potential
active site residues and their subsequent kinetic analysis based on the identification of
invariant residues picked up from the sequence based family classification (Schulein,
2000). Protein engineering has focussed on the catalytically important amino acids,
specific amino domains within cellulases and the role disulphide bridges have in the
stability of the enzyme. In order to understand the active site architecture and modify
catalytic function by directed mutagenesis it is important to identify the catalytically
important amino acids (Ozaki et al., 1994; Damude et al., 1995; Schulein, 2000).
Multiple amino acid exchanges by site directed mutagenesis of Ser 287 and Ala 296
of family 5, two catalytically active Asp’s of family 6, identified and revealed the
catalytic machinery of family 5 and 6 cellulases (Park et al., 1993; Schulein, 2000).
The putative catalytic residues of family 9 cellulases from C. stercorarium
Cel Z, Asp 84 and Glu 447, are located within the N- terminal of the modular protein.
Replacement of either one or both of these catalytic residues completely removed the
ability of Cel Z to attack the insoluble Avicel, allowing the identification of the
catalytic activity residues of this specific cellulase family (Schulein, 2000). The study
of this active site amino acid and its importance in the catalytic reaction has provided
an insight into the steps required to move towards the “perfect cellulase (Shakin-
Eshleman et al., 1996; Riedel and Bronnenmeier, 1999; Schulein, 2000; Jeoh et al.,
2002; An et al., 2005; Mamoru Nishimoto et al., 2005; Sandgren et al., 2005; Violot
et al., 2005).
Whole domains of cellulases can either be exchanged and/or engineered to
develop hybrid enzymes (Bhat and Bhat, 1997). The CBD from the C. fimi
Chapter 1 : Literature Review
34
endoglucanases is at N-terminus and at the C-terminus of the exoglucanases (Beguin
and Aubert, 1994; Kormos et al., 2000). This particular exoglucanase displays
extremely tight binding to cellulosic substrates, even in the presence of high salts,
suggesting a hydrophobic substrate–enzyme interaction (Gilkes et al., 1991a). The
conservation of this hydrophobicity within exoglucanases is due to the presence of
four tryptophan residues within the CBD. This CBD has been used to prepare
chimeric fusion proteins (Warren et al., 1987; Schulein, 2000; Levy et al., 2003)
resulting in enzyme complexes that bind more tightly to cellulosic substrates and at
the same time deliver high activity of hydrolysis (Gilkes et al., 1988). Recently a
xylanase-cellulase bifunctional fusion protein has been designed (Park et al., 2002;
An et al., 2005). The Xylanase enzyme Xyn X from C. thermocellum and Cellulase
Cel 5Z::Ω from P. chrysanthemi were fused together to construct xylanase- cellulase
fusion enzyme. The CBD and Ser/Thr- rich linker region from C- terminus of Cel5Z
is completely removed, as the activity of Cel5Z is seen to be enhanced by removing
the C- terminal region (Park et al., 2002; An et al., 2005). Then this Cel 5Z without C
terminal end is fused at the C- terminus of xylanse Xyn X, resulting in the functionally
expressible and active fusion protein (Figure 1.7) (An et al., 2005).
Disulfide bonds are commonly found in extracellular proteins, and it is
widely accepted that they contribute to the stability of the native conformation of
proteins (Bhat and Bhat, 1997). To enhance the thermal stability of cellulases, a
method for introducing disulfide bonds, replacing a pair of nearby residues with
cysteine residues, can be employed (Pons et al., 1995; Bhat and Bhat, 1997).
Chapter 1 : Literature Review
35
Figure 1. 7 Xylanase-cellulase bifunctional fusion protein
Schematic representation of xylanase Xyn X gene from C. thermocellum and Cel5Z::Ω
from P .chrysanthemi cellulase fusion enzyme. P1, P2, P3, P4 indicates primers used
for the amplification, SP, signal peptide, TSD, thermo stabilizing domain; CD,
catalytic domain; CBD, cellulose binding domain; SLD, S-layer like domain; LR,
linker region. Figure adapted from J. M. An et al., 2005 (An et al., 2005)
The expanding database of three dimensional structures of cellulase enzymes can be
exploited to introduce cysteine residues such that the newly formed disulphide bonds
will not interfere with the active site of enzyme and at the same time minimize the
disruption of the protein structure. The contribution of the disulfide bonds and
individual cysteine residues to cellulase protein stability has been confirmed by the
protein engineering investigation of Cysteine- 61 and Cysteine- 90 from the B.
licheniformis 1,3- 1,4- β- glucanase (Pons et al., 1995; Bhat and Bhat, 1997).
Chapter 1 : Literature Review
36
1.7 Industrial applications of Cellulases
Cellulases have a wide range of applications for the following industries:
chemicals, textiles, food and pharmaceutical.
i) In the food industry, cellulases are used in the extraction of juices and
oils, gelatinisation of seaweed, digestion of ball milled lignocellulose
for food additives, pre-treatment of soybeans, isolation of starch from
corn and sweet potato (Bhat and Bhat, 1997; Nazareth and Sampy,
2003; Mosier et al., 2005).
ii) In the fuel and chemical industry, the current application is production
of ethanol and other commodity products from cellulosic biomass
(Himmel et al., 1999; Lynd et al., 2002).
iii) In the paper industry, cellulases are used in pulp refinement and in
paper waste recycling.
iv) In the textile industry, cellulases are used as agents for fibre and fabric
surface modification. The few important uses are destaining of denims
for a prefaded look, restoring the colour and brightness of cotton
fabrics by removing and renewing the fabric surfaces by removing
microfibrils and fuzz fibers (Beguin and Aubert, 1994).
v) In the brewery and wine industry, cellulase is used to enhance the
aroma of wines (Beguin and Aubert, 1994).
vi) In pharmaceutical industry cellulases have been used in the separation
of enantiomers from racemic drug mixtures.
vii) Also, cellulases and glucanases mixture have been used for the
production of plant and fungal protoplasts for producing hybrid strains
(Bhat and Bhat, 1997).
Chapter 1 : Literature Review
37
1.8 Project Objectives
Whilst many organisms have the capacity to colonize on cellulose only very
few are actually capable of completely hydrolysing cellulose to glucose (Lynd et al.,
2002). Glucose evolved as part of this breakdown can then be further utilized for
production of alcohol or for other industrial process.
One of the primary objectives in cellulase research has been the
development of enzymes with sufficient activity to be used in industrial applications.
In such industrial settings one of the major challenges has been to obtain enzymes
with sufficient activity and in large enough quantities to make them commercially
viable. As a result of this need significant effort has been focussed on the search for
new sources of cellulase enzymes or to engineer new more efficient and robust
enzymes. P. gigantea has the ability to colonize on sap wood and degrade resins and
other extractives of the wood (Behrendt and Blanchette, 1997) demonstrating an
ability to degrade the wood and the associated cellulose components. Whilst it has
been exploited industrially as biocontrol agent (Ross and Hodges, 1981) and was
found to be very efficient at utilising complex cellulosic resources, no studies on
cellulase system of this organism has been reported in the publicly available
databases. However, Collet first suggested P. gigantea as cellulase producer (Collett,
1983) and K. Palaniswamy further studied the organism and reported the presence of
potential cellulase system although this work remains unpublished in referred
literature. He also reported the partial sequence of gene cellobiohydrolase (Cbh I) in
this organism which showed low homology sequence match with T. reesei and P.
chrysosporium CbhI gene (Palaniswamy, 1998).
Chapter 1 : Literature Review
38
The basis for the work presented in this thesis involved building on these
preliminary studies and to perform a detailed examination of P. gigantea as a source
of cellulase enzymes. More specifically the aim was to examine and characterise at
the biochemical and molecular level the putative cellulase from this organism. Based
on the unpublished studies of K. Palaniswamy our initial aim was thus to obtain the
complete sequence of this putative CbhI gene from P. gigantea and using the DNA
and inferred amino acid sequences examine the molecular architecture of this putative
“cellulase”.
In order to more fully understand the cellulase system the expression and
regulation of the cellulase activity was examined in a variety of different growth
media and conditions. After examining and optimising cellulase expression the
P.gigantea was assayed and subsequently purified (Chapter 5). The purified cellulase
was further analysed by obtaining internal peptide sequence information by Matrix-
assisted laser desorption/ionisation (MALDI) mass spectrometry.
The information gained from these studies has provided a more detailed
understanding of the molecular basis of cellulose digestion by the little studied
cellulolytic fungi P.gigantea.
39
Chapter 2 : Materials and Methods
2.1 Chemical reagents
All common chemicals were purchased from Sigma Chemical Co., BDH
Chemical Ltd., and Oxoid Laboratories. Protein purification and analysis related
chemicals were bought from Bio-Rad Laboratories unless specified in methods. All
primers were synthesised by GeneWorks Pty Ltd., Australia
2.2 Enzymes and commercial kits
Clonetech RACE PCR kit
Invitrogen Taq DNA polymerase
Invitrogen 10 mM dNTP Mix (Invitrogen #18427-088)
Invitrogen 10 mM individual dNTPs (dATP, dTTP, dGTP, dCTP)
Invitrogen PCR buffer 10x (670 mM)
Promega pGEM® - T Easy Vector systems (Promega #A1360)
Promega M-MLV reverse transcriptase (Promega #M1701)
Promega RT- PCR kit
Promega RNasin® Plus RNAase Inhibitor (Promega #N2611)
Qiagen QIAquick PCR Purification Kit (Qiagen #28104)
Qiagen RNeasy plant mini kit (Qiagen #74904)
2.3 Solutions
Solutions used in the present study are listed below, alphabetically within
each section. Final concentrations of the stock solutions are given for most solutions.
Amounts and/or volumes used in preparing solutions are given in some cases. All
stock solutions were prepared in Milli-Q® grade distilled water. All the solutions used
Chapter 2 : Materials and Methods
40
for RNA and mRNA preparations were either prepared in Diethyl pyrocarbonate
(DEPC) treated sterile Milli-Q® water or the solutions were DEPC treated and
sterilised prior to use. The stock solutions and or the Milli-Q® water were DEPC
treated by mixing 0.2 (v/v) DEPC solutions. The mixture was left at room
temperature overnight to denature the RNAase and sterilised to inactivate the
remaining DEPC.
2.3.1 Buffers
2.3.1.1 Carlson Lysis Buffer –(100 mL) (Carlson et al., 1991)
Tris (100 mM), pH 9.5 70mL
EDTA (20 mM) 0.76g
NaCl (1.4 M) 8.18g
CTAB (Hexadecyltrimethylammonium bromide, Sigma H-5882) (2 %) - 2g
PEG 8000 or 6000 (polyethylene glycol) (1 %) - 1g
Stir until dissolved (sometimes overnight). Bring to volume. Add 2ul β-
mercaptoethanol per 1 mL of buffer just before use.
2.3.1.2 Citrate Buffer (50 mM) buffer pH 5.5 stock
2.3.1.2.1 Stock A Citrate Buffer
Citric Acid monohydrate (0.1 M citrate) 21.01g/L
2.3.1.2.2 Stock B Citrate Buffer
Sodium Citrate dihydrate (0.1 M sodium citrate) 29.41 g/L
Chapter 2 : Materials and Methods
41
2.3.1.2.3 Working 50mM Citrate Buffer
Stock A 28mL
Stock B 72mL
Make upto 1L with Milli-Q® water, pH to 5.5
Store at 2 to 8° C
2.3.1.3 Dialysis buffer for protein purification
CaCl2 (10mM) 400mg
Tris (50mM) 6.06g
DTT (5mM) 770mg
Make upto 1L with Milli-Q® water, and filter sterlize.
Store at 2 to 8° C
2.3.1.4 DNA gel loading buffer
0.25%(w/v) bromophenol blue, 0.25% (w/v) xylene cyanol FF and 30%
(v/v) glycerol were dissolved in sterile Milli-Q® water and stored at 4
oC.
2.3.1.5 Formaldehyde (FA) gel running buffer (1x)
FA gel buffer (10x) 100mL
Formaldehyde (37% of 12.3M) 20mL
RNAase free water 880mL
2.3.1.6 Phosphate Buffer (10X)
KH2PO4 27.2g
Chapter 2 : Materials and Methods
42
Na2HPO4 · 7H20 53.6g
Make volume upto 4 L with Milli-Q® water
2.3.1.7 RNA formaldehyde (FA) gel buffer (10 x MOPS)
MOPS (3N-Morpholino-propane-sulphonic acid) (200 mM) 41.86 g
Sodium acetate (50mM) 4.1 g
EDTA (10mM) 3.72 g
Dissolve in 1liter of Milli-Q® water and adjusted to pH 7.0 with NaOH
2.3.1.8 RNA gel loading Buffer (5x)
Bromophenol blue (aqueous) 16 µL
EDTA pH 8.0 (500mM) 80 µL
Formaldehyde (37% of 12.3M) 720mL
Glycerol (100%) 2mL
Formamide 3.08mL
FA gel buffer (10x) 4mL
Make up the volume upto 10mL RNAase free water
2.3.1.9 SDS PAGE (Tris Glycine) electrophoresis buffer (10x)
Tris 30.2g
Glycine 144.0g
SDS 10g
Make up the volume to 1L with Milli-Q® water.
pH should be 8.3 without adjustment.
Chapter 2 : Materials and Methods
43
2.3.1.10 SDS sample buffer (5x)
Tris Cl pH 6.8 (1 M) 0.6mL
Glycerol (50 % (v/v) 5 mL
SDS (10% (w/v) 2mL
β-mercaptoethanol (14.4mM) 0.5mL
Bromophenol blue (1%) 1mL
Sterile Milli-Q® water 0.9mL
Stable for weeks at 4 °C and for months at -20°C
2.3.1.11 Sodium Acetate Buffer
Sodium acetate (0.1M) 6.81g
Milli-Q® water to 400mL
Glacial acetic acid to required pH
Milli-Q® water (after pH adjust) to 500mL
2.3.1.12 TBE buffer (1x) 1L
Tris hydroxymethyl aminomethane 10.8 g
Boric acid 0.98 g
EDTA disodium salt 0.55 g
2.3.2 Electrophoresis solutions
2.3.2.1 Acrylamide (40 %)
29.2% (w/v) acrylamide and 0.8% (w/v) bis-acrylamide were dissolved in
Chapter 2 : Materials and Methods
44
Milli-Q® water filtered through a 0.45µm Whatman filter paper and stored in a dark
glass container at 4oC.
2.3.2.2 Ammonium acetate (10M)
Ammonium acetate (10M) was dissolved in Milli-Q® water and sterilised.
Ammonium persulphate was made fresh weekly by dissolving in sterile Milli-Q®
water and stored at 4°C.
2.3.2.3 Ampicillin
Ampicillin sodium salt was dissolved in 1 M sodium bicarbonate and is
stored at -20°C in aliquots of 100 µg/mL
2.3.2.4 Bovine serum albumin (BSA)
1 mg/ml of BSA dissolved in sterile Milli-Q® water and stored at -20°C.
2.3.2.5 Calcium chloride
1M Calcium chloride was dissolved in Milli-Q® water, filtered through a
0.22 µm filter and stored in 1ml aliquots at -20°C.
2.3.2.6 Caesium chloride and EDTA buffer
5.7 M Caesium Chloride and 0.1 M EDTA were dissolved in Milli-Q® water
adjusted to pH 7.4 with NaOH, DEPC treated and sterilised.
Chapter 2 : Materials and Methods
45
2.3.2.7 Chloroform: Isoamyl Alcohol 24:1
96 ml Chloroform is mixed with 4 mL isoamyl alcohol.
2.3.2.8 Congo-Red Stain
Make 1% Congo-red in Milli-Q® water (1.0g in 100mL).
2.3.2.9 Coomassie blue staining solution
Coomassie brilliant blue R-250 (0.1%) 1.3g
Methanol (50%) 500 mL
Acetic acid (10%) 100 mL
Make upto 1litre with Milli-Q® water
2.3.2.10 Destaining Solution for protein SDS gels
Methanol (20%) 500mL
Acetic acid (10%) 500mL
2.3.3 General reagents and solutions
2.3.3.1 3, 5-Dinitrosalicylic acid (DNSA)
Dissolve 1g of 3, 5, DNSA and moisten it with few drops of water. Add 20
mL of 1N NaOH followed by 30g of Rochelle’s salt (Sodium-Potassium tartarate).
Make the volume upto 100 mL with Milli-Q® water. Filter with Whatman paper no.
1®. Store in amber coloured bottle for further use. Keep it air tight as CO2 from
environment may tend to lower the alkalinity levels.
Chapter 2 : Materials and Methods
46
2.3.3.2 Isopropanol (isopropyl alcohol)
Analytical grade Isopropanol is used.
2.3.3.3 Miniprep Plasmid Isolation
2.3.3.3.1 Solution 1
Sterile Glucose (40%) 2.27 mL (50 mM)
EDTA (0.5 M), pH 8 2.0 mL (10 mM)
Tris-HCl (1M), pH 8 2.5 mL (25 mM)
Sterile Milli-Q® water 93.23 mL
Use all sterile stock solutions. Store at 4°C
2.3.3.3.2 Solution 2
SDS (10%) 1mL
NaOH (10M) 0.2 mL
Milli-Q® water 8.8 mL
2.3.3.3.3 Solution 3
Potassium acetate (5 M) 60 mL
Glacial acetic acid 11.5 mL
Milli-Q® water 28.5 mL
Make up the volume to 100mL and filter sterilize.
2.3.3.4 MOPS (0.1M)
MOPS 1.05g
Dissolve in 45 mL Milli-Q® water, adjust pH to 7.5 with5M NaOH, and
bring volume to 50 mL with Milli-Q® water, filter sterilised and stored at -
20°C.
Chapter 2 : Materials and Methods
47
2.3.3.5 4-methylumbelliferyl-ß-D cellobioside (0.5mM)
4-methylumbelliferyl-ß-D cellobioside (MUC) 125mg
Milli-Q® water 100mL
Filter sterilised and stored at 4°C
2.3.3.6 NaCl (1.5 M)
Dissolve 87.66gm of NaCl in 1000mL of Milli-Q® water
2.3.3.7 Phenol
Phenol was prepared by melting 500g of crystals at 68oC for 1 h and mixed
with 0.1% (w/v) 8-hydroxyquinoline. To the melted phenol an equal volume of 59mM
Tris base was added and stirred for 10 min. The aqueous phase was removed and
replaced with an equal volume of 50mM Tris/HCL, pH 8.0. The mixture was stirred
and the procedure was repeated until the pH of the phenol reached 8.0. The saturated
phenol was stored in a dark glass container at 4oC with an overlay of TE (pH 8.0)
buffer phenol was stored in a dark glass container at 4oC with an overlay of TE (pH
8.0) buffer.
2.3.3.8 Potassium Acetate (5M)
Potassium Acetate 29.4g
Glacial Acetic Acid 11.5mL
Make upto 100mL with Milli-Q® water.
Sterile filter and store at room temperature.
Chapter 2 : Materials and Methods
48
2.3.3.9 Potassium Acetate (3M) pH 5.0
Potassium Acetate 147.2g
Glacial Acetic Acid 57.5mL
Make volume upto 500 mL Milli-Q® water.
2.3.3.10 RNAase
10 mg/mL RNAase was dissolved in Milli-Q® water. The solution was
heated at 80oC for 15 min to inactivate the contaminating DNAses and stored at -
20oC.
2.3.3.11 Rubidium Chloride (RbCl)
RbCl 5g
Sterile Milli-Q® water 10mL
Store -20°C.
2.3.3.12 Sodium hydroxide
10 M Sodium hydroxide pellets were dissolved in sterile Milli-Q® water.
2.3.3.13 Urea (1M)
1 M urea was dissolved in Milli-Q® water, filtered through a 0.45 µm
cellulose acetate filter into a sterile container.
Chapter 2 : Materials and Methods
49
2.4 Media
All media were made up in Milli-Q® water and either autoclaved or filter-
sterilised prior to use. For agar used for bacterial growth 15 mg/mL bacto-agar was
added to the appropriate media. Antibiotics were added to media as appropriate to the
final concentrations.
2.4.1 Luria Bertani broth (LB)
Bacto-tryptone 10 g
Yeast extract 5 g
NaCl (pH 7.4) 5g
Dissolve in 1L of Milli-Q® water and adjust the pH to 7.0 with NaOH and
autoclave sterilise.
2.4.2 Luria Bertani agar (LB agar)
Bacteriological tryptone (Oxoid) 10 g
Yeast extract (Oxoid) 5 g
Sodium chloride 5g
Bacteriological agar (Oxoid) 1.5g
Dissolved in 1L of Milli-Q® water and adjusted to pH7.0 with NaOH and
sterilised.
2.4.3 LB Agar with ampicillin
The LB agar was prepared as described in Chapter 2 (Section 2.3.1.3) and
cooled to 50oC. Ampicillin stock (100 mg/mL) was added to a final concentration of
µg/mL was added to the molten media before it was poured into sterile plastic Petri
dishes.
Chapter 2 : Materials and Methods
50
2.4.4 LB Agar with Ampicillin /IPTG/X-Gal
The LB agar plates with ampicillin were prepared as described in 2.3.1.3.
Prior to use, the plates were spread with 100µL of 100 mM of IPTG and 20µL of 50
mg/mL of X-Gal over the surface of the agar and allowed to absorb the solutions for
30 min at 37oC prior to use.
2.4.5 Malt extract broth
Malt extract 2g
Was dissolved in 1L Milli-Q® water and sterilised.
2.4.6 Malt extract agar
Malt extract 2g
Bacteriological agar 1.5g
Milli-Q® water 1L
Sterilised, cooled and dispensed into sterile plastic petri dishes.
2.4.7 SOC broth
2.0% (w/v) bacteriological tryptone, 0.5% (w/v) yeast extract , 1% (v/v) 1 M
sodium chloride and 0.25% (v/v) 1M potassium chloride were dissolved in Milli-Q®
water and adjusted to pH 7.0 with NaOH. The mixture was sterilised, cooled to 50oC
and mixed with 1% (v/v) 1M filter sterilised magnesium chloride/ magnesium
sulphate concentrated solution and 1% (v/v) 2 M filter sterilised glucose prior to use.
2.5 Ladders and Markers
DNA, RNA and protein molecular weight markers were from Fermentas
Life Sciences., USA.
Chapter 2 : Materials and Methods
51
2.5.1 GeneRuler™ 1Kb DNA Ladder, (Fermentas #SM0311/2/3)
Range14 fragments (in bp): 10000, 8000, 6000, 5000, 4000, 3500, 3000,
2500, 2000, 1500, 1000, 750, 500, 250.
2.5.2 Lambda DNA/EcoRI+HindIII Marker, 3 (Fermentas #SM0191/2/3)
Range13 fragments (in bp): 21226*, 5148, 4973, 4268, 3530*, 2027, 1904,
1584, 1375, 947, 831, 564, 125 (it makes 0.3%).
2.5.3 PageRuler™ Prestained Protein Ladders (Fermentas #SM0676)
The PageRuler™ Prestained Protein Ladder is a mixture of 10 recombinant
highly purified, coloured proteins. The molecular weights range of protein ladder is
10 to 170KDa. A blue chromophore is coupled to these proteins, and the reference
band protein of ~70KDa is coloured with an orange dye.
2.6 Methods
All general molecular biology protocols were followed from the Molecular
Cloning: A Laboratory Manual by Joseph Sambrook and David W Russell (Sambrook
and Russell, 2001) and protein related protocols were followed from Protein Methods
by Daniel Bollag et al., (Bollag et al., 1996) unless specified in methods.
2.6.1 Maintenance of Fungal Strain
The Phlebia gigantea, (DFP 14356) used in this study was obtained from
CSIRO, Forest Production Division, Melbourne, Australia. The culture was
maintained on 5% (w/v) malt extract agar (MEA) plates grown at 22°C and stored at
4°C. The culture was routinely maintained by periodic transfers.
Chapter 2 : Materials and Methods
52
2.6.2 DNA extraction by CTAB method
DNA was isolated using a CTAB (Hexadecyltrimethylammonium bromide)
protocol derived from Saghai-Maroof et al., (1984).
250 mg of fungal mycelial tissue was placed in the mortar and ground to a
fine powder in liquid N2. The powdered fungal tissue was scraped into a 1.5µL
microfuge tube and 750µL Carlson lysis buffer was added. The sample was then
incubated at 74° C for 30 min in heating block. Samples in the heating block were
inverted every 10 minutes throughout the incubation time. Samples after the specified
incubation time were centrifuged at 10,000 rpm for 10 min at room temperature to
sediment cell debris. The supernatant was collected into a new tube and 1 volume of
chloroform: isoamyl alcohol (24:1), [750 µL/tube] was added. The samples were then
gently mixed by inverting the tube several times and centrifuged for 10 mins at
10,000g. The upper aqueous phase was transferred to clean tube and an equal volume
of isopropanol was added. The tube was inverted several times to mix and encourages
the DNA to clump and to improve recovery. Centrifuge 10 mins at 10,000 g to
sediment DNA. The DNA pellet was washed with ice cold 70% ethanol (ETOH), 250
µL by spinning at 4 C for 10 mins. To wash of the polysaccharides present in most of
the fungal samples add ice-cold 70% ethanol, vortex and spin for a minute in the
centrifuge to re-pellet the DNA. This process was repeated several times to recover
clean DNA. Pour off the ethanol and turn the tube upside down to dry. After a few
minutes, blot any remaining liquid from the sides of the microfuge tube and add 100
µL TE to the nucleic acid pellet. Mix until the pellet goes into solution. 0.5µL of
RNAase was added and the sample was incubated overnight in the refrigerator.
Chapter 2 : Materials and Methods
53
2.6.3 RNA isolation and quantitation
Total RNA was extracted from 100mg of fungal mycelia (from 3 flasks
consisting of 100mL of media each) using the RNeasy plant mini kit (Qiagen #
74904). The three RNA samples served as biological replicates and were used to
check reliability of the experiments.
The quantity and quality of total RNA was checked by spectrophotometry
and gel electrophoresis. Total RNA was quantified by measuring absorbance at 260
nm and 280 nm using a spectrophotometer. An OD of 1 at 260 nm equals 40µg RNA
per mL and the A260/A280 ratio of RNA samples between 1.8 and 2.1 indicates high
purity of RNA. The quality of 1µL total RNA (~0.5µg) was checked on a 1% agarose
gel pre-stained with 1µL ethidium bromide (10 mg/mL), alongside a 1Kb
(GeneRuler) molecular marker. The gels were run in 1x TBE buffer at 80V.
2.6.4 First strand cDNA synthesis
Approximately 2µg of total RNA was incubated in a sterile RNAase-free
microcentrifuge tube, with 0.5µg of the primer or primer-adaptor per microgram of
the mRNA sample in a total volume of ≤15µL in water. The sample was heated to
70°C for 5 minutes to melt secondary structure within the template. It was then
immediately placed on ice to prevent secondary structure from reforming. The
sample was vortexed briefly to collect the solution at the bottom of the tube. To this
solution the following components were added in the order shown.
M-MLV 5X Reaction Buffer 5µL
dNTP, 10mM 6µL
Recombinant RNasin® Ribonuclease Inhibitor 25 units 0.6µL
Chapter 2 : Materials and Methods
54
M-MLV RT 200 units 1.0µL
RNAase-Free Water 0.9µL
This total 25µL volume of solution was mixed gently by flicking the tube and
incubated for 60 minutes at 42°C.
2.6.5 Amplification of cDNA by PCR.
1-3ng/µL of cDNA was amplified in a reaction volume of 25µL as required.
Reactions contained 2µL of 10mM of each oligonucleotide primer, 2.5µL of 10x PCR
buffer (Invitrogen), 1.5µL of 50mM MgCl2, 1.2µL of 10mM of deoxyribonucleoside
triphosphate (dNTP), 0.2µL of 1U of TaqPolymeraseTM
(Invitrogen). Unless specified
otherwise, cycling conditions were as follows:
Initial denaturation for 4 minutes at 94o
C followed by 35 cycles of: 30
seconds at 94o
C, 1min at 55 o
C [primer-specific annealing temperature], 1min at 72o
C,
followed by a final extension step of 5 minutes at 72o
C.
PCR products were separated on 1.0% agarose gels as described in Chapter2
(Section 2.6.8) and visualised by ethidium bromide staining as described in Chapter2
(Section 2.6.9).
2.6.6 Miniprep Plasmid Isolation
The colony with positive clone inoculated in 1.5mL of LBamp broth was
centrifuged for 5min at 5000rpm to spin down the cells. These cells were then
resuspended in 200µL of Solution 1 and 400µL of Solution 2, and were mixed by
vortexing. After incubating this reaction for 5min at room temperature 300µL of
solution 3 was added. The reaction was incubated at -20°C for 5min and then
Chapter 2 : Materials and Methods
55
centrifuged at 10,000 rpm for 10min. The supernatant (approximately 700µL) was
mixed with equal volume of isopropanol and was further incubated for 30min at room
temperature. The reaction was re-centrifuged at 10,000rpm for 10min and the
supernatant was discarded to collect plasmid as pellet. After the tube was air dried in
laminar air flow hood the plasmid was suspended in 100µL of TE buffer.
2.6.7 Cloning by Electrocompetent and heat shock cells
The purified PCR product was cloned into pGEM®
- T Easy Vector
(Promega) according to the instructions.
2.6.7.1 Ligation
The ligation was conducted in 5µL volume containing 2x Rapid ligation
buffer (60mM Tris-HCl (pH 7.8), 20mM MgCl2, 20mM DTT, 2mM ATP, 10% PEG-
8000), 50ng of pGEM® - T Easy Vector, 3 Unit of T4 DNA ligase (Promega) and
15ng of purified PCR product. The mixture was incubated overnight at 4°C.
2.6.7.2 Transformation
2.6.7.2.1 Electroporation
The 4µL of ligation mixture was added to 40µL of electrocompetent E.coli
DH5α SURE cells thawed on ice and is gently mixed. The mixture was then added to
electroporation cuvette and the high intensity electric field was applied enabling
transient permeability of the cell membranes and uptake of molecules from the
surrounding medium. The Gene-Pulsar® (BioRad) electroporation machine set at
200Ω, 25µF and 2.8 volts was used for the electroporation purpose. The cells were
Chapter 2 : Materials and Methods
56
then immediately transferred to 1mL of SOC broth and incubated for 2 hours at 37°C.
After the incubation the cells were plated on LBamp/X-Gal/ IPTG plates and were
incubated overnight at 37°C in upside down position. The plates were then used for
blue/white screening. White colonies generally contain the insert.
2.6.7.2.2 Heat Shock Transformation
The ligation mixture was gently flicked and 5µL was mixed with 100µL of
E.coli JM109 heat shock competent cells thawed on ice. The mixture was flicked
gently and was incubated on ice for 20minutes and then incubated for 90 seconds in
the water bath at 42°C without agitation and transferred on ice for another 2 minutes.
The mixture was then added to 950µL of SOC broth and incubated for 1.5 hours at
37°C with gentle shaking. Using a sterile spreader, 100µL aliquots were spread onto
LBamp/X-Gal/IPTG plates. The plates incubated overnight at 37°C were then used
for blue/white screening. White colonies generally contain the insert.
2.6.8 Agarose gel preparation and electrophoresis
Agarose gels were prepared in 1x TBE containing 250ng/µL ethidium
bromide and the appropriate percentage of agarose according to the size of fragments
being separated: 2.5 % agarose gels were used for electrophoresis of fragments below
1Kb; 1.0% agarose gels were used for analysis of larger fragments. Electrophoresis
was performed at 50 - 90 V for 15 - 45 minutes depending on the separation required.
2.6.9 Visualisation of PCR products
The amplified PCR products were run on a 1% agarose gel prestained with
1% ethidium bromide (10 mg/mL) and visualised using Gel Doc (BioRad) system.
Chapter 2 : Materials and Methods
57
2.6.10 Primer design
Primers were designed manually using the following guidelines:
1) As far as possible, sequences chosen were 18 - 25bp in length.
2) Sequences were chosen to avoid areas of simple sequence showing non-
representative use of the bases and obvious repetitive sequence i.e., runs of single
nucleotide (e.g. TTTT) or double nucleotide (CGCGC) motifs.
3) Sequences were chosen to exclude palindromes which will form
inhibitory secondary structure, especially at the 3' ends (e.g. GACGTC).
4) As far as possible, sequences were chosen with a GC content of at least
50%.
5) Sequences were chosen to avoid complementarity between pairs of
primers, especially at the 3’ end, which could result in primers annealing to each other
and forming primer dimers.
2.6.11 Zymogram analysis
The activity of the purified enzyme was determined by zymogram analysis
on polyacrylamide gel electrophoresis (PAGE) using 10% separating gel and 4%
stacking polyacrylamide gels without β- mercaptoethanol, according to standard
protocol (Laemmli, 1970). The separating gels were incorporated with either 1%
CMC for CMCase assay or 1% MUC for CBHase analysis. The gels were then run in
Mini Protean® (Bio-Rad)
gel running box at 70V for 45 minutes in 1X Tris-glycine
buffer. The gels were run at 70V for 90 minutes.
Chapter 2 : Materials and Methods
58
2.6.11.1 Activity staining
Activity staining was performed by washing SDS by sodium phosphate
buffer pH 7.2 containing 40% isopropanol for 60 minutes at room temperature. The
gel was washed again with Sodium phosphate buffer without isopropanol for another
60 minutes. After removal of SDS, renaturation of enzyme was done by treating the
gel overnight at 4°C with 5mM β- mercaptoethanol and 1mM EDTA in sodium
phosphate buffer pH 7.2.
After overnight incubation the gels were then incubated on glass plate at
42°C for 2-3hours. The CMC gels were stained thereafter with 1% Congo red stain
for 30 minutes and destained in 1M NaCl for 15 minutes. The MUC gels were
visualised and photographed using Gel Doc (BioRad) system after incubation at 42°C
for 2-3hours. The Molecular weight was estimated by comparison to Fermentas
prestained protein ladder (SM0676).
2.6.12 Isoelectric focusing of SDS gel
Proteins were separated by electrophoresis according to charge. Isoelectric
focusing was carried out with 232µg of protein. Proteins were separated using gel
strips forming an immobilized non-linear pH gradient from 3 to 10 (ReadyStrip® IPG
strips, pH 3-10 NL, 11 cm, (Catalogue number: 1632016, Bio-Rad Laboratories Pty
Ltd.). After the rehydration of strips with sample in rehydration buffer for 12 hours at
room temperature, isolectricfocusing was performed in the Protean® IEF Cell (Bio-
Rad Laboratories Pty. Ltd.) at 20oC and 50µA current per strip. Proteins were
separated using the multistep focusing conditions under stepwise voltage changes.
The programme used includes first step of 250V for 20 minutes at linear ramp, second
Chapter 2 : Materials and Methods
59
step of 8000V for 2.5 Hrs for another linear ramp and final step of 8000V for 20,000
Volt hours with rapid ramp. Ramping or change in voltage helps to stabilize the
resistance of sample in run.
2.6.13 Protein staining on SDS gel
Gels were stained with colloidal Coomassie Blue (Bio-Safe™ Coomassie,
Bio-Rad Laboratories Pty. Ltd.). Bio-Safe™ Coomassie stains broadest spectrum of
proteins and is ready to use, single reagent protein stain. The gels were stained
overnight in Bio-Safe™ Coomassie on shaker and then subsequently destained thrice
with MilliQ® water.
The stained gels were scanned with GS-800™ densitometer (Bio-Rad
Laboratories Pty. Ltd.).
60
Chapter 3 : Molecular Characterisation of Cellulase genes
3.1 Introduction
Current studies form various cellulose degrading microorganisms are very
extensive as several cellulase genes from various bacteria, fungi and other
microorganisms have been cloned and sequenced (Azevedo Mde et al., 1990; Ahsan
et al., 1996; Teeri, 1997; Spiridonov and Wilson, 1998; Bhat, 2000; Gusakov et al.,
2005). Most fungi typically produce two or more cellobiohydrolases and several
endoglucanases that are secreted to the culture medium (Teeri et al., 1992; Bhat,
2000; Maheshwari et al., 2000; Lynd et al., 2002). The most efficient producers of
extracellular cellulases are the different fungal strains of Trichoderma reesei and
Phanerochaete chrysosporium,(Teeri et al., 1992; Bhat and Bhat, 1997; Lynd et al.,
2002). T. reesei contains four distinct cellulase genes which encodes
cellobiohydrolase I (CbhI), cellobiohydrolase II (CbhII), endoglucanase I (EgI) and
endoglucanase III (EgIII) (Covert et al., 1992). There is a close evolutionary
relationship between cellobiohydrolase I (CbhI) and endoglucanase I enzymes of T.
reesei and these two types of enzymes are known to have significant amino acid
sequence homology between them (Sims et al., 1988; Covert et al., 1992).
Here, I report the identification, isolation and analysis of the
cellobiohydrolase gene of the white rot fungus Phlebia gigantea using the sequences
of previously obtained partial cellobiohydrolase I gene from P. gigantea in our
laboratory (Palaniswamy, 1998) and the sequences of closely related basidiomycetes
P. chrysosporium fungi (Tempelaars et al., 1994; Vallim et al., 1998). PCR
amplification of cDNA prepared from the RNA of the mycelium of the fungus grown
in cellulose rich medium was performed and the PCR amplified cellulase gene was
Chapter 3 : Molecular Characterisation of Cellulase genes
61
cloned into pGEM® - T Easy vector and transformed into E. coli JM 107. The
recombinant plasmid was purified, analysed for the presence of the clone and the gene
was then sequenced. The gene sequence was further analysed by comparing the
nucleotide sequence with other known cellulase genes using Gene bank.
Based on the earlier data of partial CbhI gene sequence from P. gigantea by
Palaniswamy (Palaniswamy, 1998), a simple and efficient rapid amplification of
cDNA ends (RACE) PCR cloning strategy to obtain full length CbhI product was
employed. The RACE PCR strategy was first described in 1988 by Michael Frohman
and Gail Martin (Frohman et al., 1988). The original protocol used a gene specific
primer and polyadenylation of the extended cDNA at the 3’ end with terminal
deoxytidyl transferase (TdT) (Frohman et al., 1988). RACE PCR provides the most
efficient and simple method for performing both 5'- and 3'-rapid amplification of
cDNA ends. RACE PCR can be used to construct a full-length cDNA with the
knowledge of very few nucleotides of target gene's sequence.
3.2 Materials and Methods
3.2.1 Organism
The white rot fungus P. gigantea strain DFP 14356 was used in this study. The
stock culture was maintained on 2% (w/v) malt extract agar medium.
3.2.2 Extraction of fungal RNA
The white rot fungus P. gigantea strain DFP 14356 was cultivated in a
cellulase inducing medium to study the expression of cellobiohydrolase gene. The
fungus was cultivated for this study as described in chapter 2. The mycelium was
Chapter 3 : Molecular Characterisation of Cellulase genes
62
harvested after 6 days of cultivation period and the RNA was isolated using Qiagen
RNeasy plant mini kit for total RNA isolation as described in chapter 2. The first
strand cDNA sample was synthesised as described in chapter 2. The fungus was also
grown in a medium containing glucose and CMC and was harvested after 6 days, and
the first strand cDNA was prepared as described in chapter 2. The cDNA samples
were used as a DNA template to PCR amplify the cellobiohydrolase gene and the
PCR amplification of cellobiohydrolase gene was performed as described in chapter
2.
3.2.3 Quantitation and determination of RNA
The total RNA concentration was determined by measuring the absorbance
at 260nm (A260) in a spectrophotometer (Chapter 2). First strand cDNA was
synthesised from the total RNA according to the manufacturer’s protocol with the
Promega first strand cDNA synthesis kit (Promega).
3.2.4 Molecular Methods
3.2.4.1 Design of PCR primers
Similar primers were designed and used as described by K. Palaniswamy to
isolate the P. gigantea cellobiohydrolase gene sequence primer Cbhl.1u and Cbhl.1d
(D1) (Table 3.1). We also designed the gene specific primers from P. gigantea
PcbhI.1U and Pcbhl.1d (D3) (Table 3.1) Apart from this we also used primers
CbhI.3u, CbhI.3d, cdh.Wu and cdh.Wd, which has been previously used for isolation
of cellobiohydrolase gene (Vallim et al., 1998). The primers gpdhF and gpdhR (Table
3.1) were designed from GAPDH gene of P. chrysosporium and are used as the
housekeeping gene for transcriptional analysis.
Chapter 3 : Molecular Characterisation of Cellulase genes
63
Table 3.1 Primers used for PCR:
Primer Sequence
5’ to 3’
Annealing
Temperature
(oC)
Refrence
D1: Cbhl.1u
(forward)
ACAATGTTCCGCACTGCTACTT 53 (Palaniswamy,
1998)
D1: Cbhl.1d
(Reverse)
AGGGTGCCCGCGGAGGTGCC 64 (Palaniswamy,
1998)
D2: CbhI.3u
(forward)
AAGATCGTGCTCGACGCGAACTG 54 (Vallim et al.,
1998)
D2: CbhI.3d
(reverse)
CTCGAAGCCCTTCACCGTTGTCAC 54 (Vallim et al.,
1998)
D3: Pcbhl.1U
(forward)
AGAGGCTGAGGAAGAAGTAGC 61 From P.gigantea
CbhI seq
(Palaniswamy,
1998)
D3: Pcbhl.1d
(reverse)
AGGGTGCCCGCGGAGGTGCCAG 59 From P.gigantea
CbhI seq
(Palaniswamy,
1998)
D4: cdh.Wu CAGTGCGGTGGCATTGGCTG 58 (Vallim et al.,
1998)
D4: cdh.Wd CTGGTCGACGATGAAGGTCG 56 (Vallim et al.,
1998)
gpdh F
(forward)
CGTATCGTCCTCCGTAATGC 54
gpdh R
(reverse)
ACTCGTTGTCGTACCAGGAG 54
From P
.chrysosporium
(NCBI:M81754)
Chapter 3 : Molecular Characterisation of Cellulase genes
64
3.2.4.2 Polymerase Chain Reaction
PCR was carried out in 25µL reaction volume which contained 50mM
MgCl2, 10mM dNTP, 20µL 10 x PCR buffer, 10mM primers, and 1 U of Taq DNA
polymerase.
The PCR reaction was subjected to thermo cycling parameters, the annealing
temperature was set at 550 C with a constant annealing time of 1 min. The PCR
reactions were performed under the optimal concentrations of magnesium chloride,
primers and deoxynucleotide triphosphate. In all of the PCR amplifications, the
concentration of the Taq DNA polymerase was constant at 1 U and the method was
followed as described in chapter 2. Following the completion of the PCR reactions,
the PCR mixture was carefully removed from the tube and extracted with chloroform:
isoamyl alcohol to concentrate the PCR product. An aliquot of the PCR product was
resolved on agarose gel electrophoresis, stained with ethidium bromide and visualised
under UV light as described in chapter 2. The PCR mix in the negative control
contained all the necessary PCR ingredients except the DNA template (cDNA).
3.2.4.3 Rapid amplification of cDNA ends (RACE) PCR
The RACE PCR was carried out using BD SMART ™ RACE cDNA
amplification kit (catalogue no. K1811-1) according to the manufacturer’s protocol,
performing both 5’- and 3’- rapid amplification of cDNA ends. The RACE ready
cDNA was obtained following reverse transcription reaction using BD PowerScript™
reverse transcriptase. The gene specific primers (GSP) for RACE PCR were designed
as per the manufacturer’s protocol. The gene specific primers described in Table 3.2
Chapter 3 : Molecular Characterisation of Cellulase genes
65
Table 3.2 Primers used for RACE PCR:
Primer Sequence
5’ to 3’
Annealing
Temperature
(oC)
Extension
end
pGp22GSP2N
1 (forward)
GGCTCAAACTTCCAGGCGC 62 5’ - end
pG22FGSP2A GAGGTTTGGGGCTCCGCACGTTGTC 64 3’-end
pGp22GSPN1 AGGTTGCGGAAGCTCCGCCATAGTG 63 3’- end
VvGSP2a AACAAGAGTACGGGCCAGGGTTGGA 61 3’-end
P22N GSP2 GCGCCGCAGTGGGCATTGGTAGTC 64 5’-end
Chapter 3 : Molecular Characterisation of Cellulase genes
66
has been designed from the 750bp P22 DNA fragment obtained from the earlier PCR
reaction. The RACE PCR was carried out in 25µL reaction volume which contained.
2.5µL of 10 X Advantage 2 PCR buffer, 1µL of 10 mM DNTP, 0.5µL of 50 X
Advantage 2 polymerase mix, 1µL of 10µM GSP 1, 2.5µL universal primer mix
(UPM) and 2.5µL of 5’- or 3’- RACE ready cDNA.
The PCR thermocycle parameters were 40 cycles of 94oC for 5 seconds,
68oC for 10 seconds and 72
oC for 3 minutes. A negative control consisting of all
reagents except the DNA was always included in thermal cycling. The products were
analysed on 1% agarose gel pre-stained with ethidium bromide.
3.2.4.4 Extraction of PCR product from agarose
The amplified PCR products were electrophoresed on 1% agarose gel. After
staining with ethidium bromide, the gel was visualised over UV light and the PCR
product were quickly excised with a clean scalpel. DNA was extracted from the gel
slices by using the QIAquick gel extraction kit (Qiagen) according to the
manufacturers’ protocol. 50µL of the eluted DNA was further concentrated by
mixing with 2µL of 5M NaCl, 100µL ice cold ethanol, vortexed and centrifuged at
10,000g for 5 minutes. Supernatant was discarded and the pellet was dissolved in
10µL of sterile Milli-Q® water.
3.2.4.5 Cloning of PCR product
The amplified PCR product was cloned into pGEM® - T Easy vector using
the Promega cloning protocol (Promega Corp.,). The vector has a 3’ terminal
Chapter 3 : Molecular Characterisation of Cellulase genes
67
thymidine attached to both ends, which greatly improves the efficiency of ligation of a
PCR product (Promega Corp.). The vector also carries an antibiotic resistance gene
for ampicillin for screening the recombinants. The PCR product was ligated within
the vector between the EcoR1 and Spe1 restriction sites (Appendix I). The vector
contains T7 and SP6 polymerase promoters flanked by a multiple cloning site (MCS)
within the α-peptide gene, producing white colonies rather than blue colonies which
aides in the screening the recombinants by blue and white colony selection.
Screening is also improved by culturing the recombinants on ampicillin plates.
The transformation by electroporation is done by mixing 4µL of ligation
product with 44µL of thawed competent cells of E. coli JM 107 in electrophoresis
cuvette. The Gene pulsar (Bio-Rad) electroporation machine set at 200Ω, 25µF and
2.8V was used for the transformation purpose. The cell mix was then transferred
immediately to 1mL of SOC broth and incubated for 2 hours at 37oC. 10µL and 50µL
transformation reaction were spread onto LB plates containing 100µg/mL ampicillin
with 16µL X-Gal (50 mg/mL) and 40µL of IPTG (100 mM). The plates were
incubated overnight at 37oC. White colonies were picked for analysis.
3.2.4.6 Analysis of cloned PCR product
A sterile toothpick was used to pick the white colonies from the plates. The
toothpick was immersed in PCR master mix containing T7 and SP6 primers, touched
on to LB amp/X-Gal/IPTG replica plate and finally immersed in 3mL of LBamp broth
for further cultivation. Plasmid DNA from the clones was extracted using mini prep
plasmid isolation techniques as described in chapter 2. Positive clones containing the
Chapter 3 : Molecular Characterisation of Cellulase genes
68
inserts were identified on the replica plate and were electrophoresed on 1% agarose
gel.
3.2.4.7 Sequencing of cloned PCR product
The positively identified PCR products were used for sequencing. The
sequencing reaction consisted of 6µL big dye terminator, 1µL of 50 ng/µL of T7
primer and 2 µL of plasmid DNA containing the insert. Thermal cycling consisted of
25 cycles of 96oC for 10 seconds (denaturation), 50
oC for 5 seconds(annealing), 60
oC
for 4 minutes (extension) and 4oC (hold). After thermal cycling the 15µL volume of
sequencing reaction was made upto 20µL with sterile Milli-Q® water. The post
reaction purification was done by adding the entire 20µL of sequencing reaction to
1.5mL Eppendorf tube containing 2µL of 3M sodium acetate (pH 4.6) and 50µL of
95% ethanol. The tube was left on ice for 10 minutes, centrifuged at 23,000g for 20
minutes and the supernatant was discarded. The pellet was rinsed by adding 250µL of
70% ethanol and centrifuged at 23,000g for 5 minutes at room temperature. The
supernatant was discarded and the sample was air dried overnight in laminar air flow.
The recombinant plasmid prepared for DNA sequencing was sequenced in
both directions using an automatic ABI model DNA sequencer at Monash Sequencing
services, Melbourne, and at the sequencing facility provided by School of
Biomolecular and Bio-Medical Sciences, Griffith University, Nathan, Queensland.
Chapter 3 : Molecular Characterisation of Cellulase genes
69
3.2.4.8 Analysis of Sequences
The DNA sequence was analysed for homology with other known cellulase
sequences using Genebank and BLAST search databases in the NCBI website
(www.ncbi.nlm.nih.gov) using the Blastn, Blastx and tBlastx programs. The
sequences were also analysed using the Bio-manager program provided by Australian
National Genomic Information Service (ANGIS) at www.angis.org.au. The sequence
was translated into amino acids and the amino acid sequence was also analysed for
homology using Genebank and European Molecular Biology Laboratories (EMBL)
databases in the ExPASy (Expert Protein Analysis System) proteomics server of the
Swiss Institute of Bioinformatics (SIB) at http://au.expasy.org/sprot/.
3.2.5 Transcription analysis of Phlebia gigantea cDNA PCR product for
cellulase expression studies
To study the expression of cellobiohydrolase gene, the fungus P. gigantea
was grown in microcrystalline cellulose (Avicel), carboxymethyl cellulose (CMC) or
glucose as a sole carbon source. The mycelium of the fungus, which was grown in
both the carbon sources, was harvested at different stages of the cultivation cycle.
The first strand cDNA was prepared from the total RNA (Chapter 2) and it is served
as DNA template in the PCR amplification using the cellobiohydrolase primers type I
(CbhI.1). An aliquot of the PCR product from both microcrystalline cellulose and
glucose was resolved on agarose gel electrophoresis, prestained with ethidium
bromide and visualised under UV light as described in chapter 2.
Chapter 3 : Molecular Characterisation of Cellulase genes
70
3.3 Results
3.3.1 Amplification of Phlebia gigantea cDNA using all primers
The first strand cDNA was synthesised from the total RNA of P. gigantea
mycelium, grown in microcrystalline cellulose (Avicel). The first strand cDNA was
PCR amplified using D3 primers (PcbhI.1U/Pcbhl.1d). Other Primers such as D1
(Cbhl.1u/Cbhl.1d), D2 (Cbhl.3u/Cbhl.3d) and D4 (cdh.Wu/cdh.Wd) were also used to
amplify the cDNA but there was no specific PCR product obtained, even after several
attempts to optimise PCR reaction conditions for these primers. A 750bp PCR
product was amplified from P. gigantea cDNA using D3 primers (Fig. 3.1, lane 2 and
3). No product was observed with all the negative controls indicating the absence of
contamination from other sources. In all further experiments only the D3 primers
were used to amplify P. gigantea cDNA
3.3.2 Cloning of PCR product
The recombinant vector was transformed into competent cells of E. coli JM
107 as described in chapter 2. The transformants were plated and screened as
described in Chapter 3 (Section, 3.2.4.5). Positive clones of E. coli JM 107 were
randomly selected to determine the presence of the P. gigantea cDNA PCR product
insert. The clones were confirmed again by subculturing on ampicillin/ X-Gal/ IPTG
plates twice. Fifty clones appeared as white colonies after subculturing twice and
these selected as positive recombinant clones.
One of the recombinant clones was selected and plasmid DNA was prepared
from the positive clone. The plasmid DNA was restriction digested with EcoRI and
analysed on an agarose DNA gel. The plasmid DNA was subjected to PCR using T7
Chapter 3 : Molecular Characterisation of Cellulase genes
71
Figure 3.1 Amplification of P. gigantea cDNA by using primer D3.
Lane 1-1 kb DNA ladder, Lane 2 and 3 – 750 bp PCR product, Lane4-PCR using D2
primer and Lane 5- Negative control
and SP6 polymerase promoters as primers to confirm the ligated product. In figure
3.2, lane 3 shows the 3.0Kb vector and lane 1 and 4 shows a much smaller fragment
of 750bp PCR products. This supports the successful ligation of the cDNA PCR
product into the pGEM®- T Easy vector and transformation into E. coli JM 107. The
recombinant plasmid (10ng) containing the cDNA PCR products were also subjected
to PCR amplification using PcbhI.1 U/d primers under the optimised PCR parameters.
The PCR amplified recombinant plasmid was analysed on DNA grade agarose gel.
Presence of 750bp DNA fragment in both the restriction digested recombinant
Chapter 3 : Molecular Characterisation of Cellulase genes
72
plasmid and the PCR amplified recombinant plasmid (Fig.3.2, lane 4) also confirms
the ligation and transformation of the PCR amplified cDNA of the white rot fungus P.
gigantea.
Figure 3.2 Cloning of PCR product
Lane 1-1Kb Ladder, Lane 2- Restriction enzyme digested plasmid product, Lane 3-
3Kb vector; Lane 4 – PCR amplified recombinant plasmid
Chapter 3 : Molecular Characterisation of Cellulase genes
73
3.3.3 DNA sequencing
The recombinant plasmid carrying the PCR product was prepared for
sequencing using an ABI PRISM Dye terminator cycle sequencing ready reaction kit
and sequenced on ABI DNA sequencer at Monash DNA sequencing facility. The
sequencing results of the main strand are presented in Appendix II. A 750bp final
product P22 was obtained after the sequencing.
3.3.4 Sequence homology
The DNA sequence of the PCR product was analysed for homology with
other cellulase genes. The sequence homology was analysed using Genebank and
NCBI blast search. The nucleotides sequence was translated into amino acids and the
amino acid sequence was also analysed for homology. When the 750bp nucleotide
sequence was translated, it gave a 263-amino acids protein that do not possess good
open reading frame with ‘Flip 6 frame’ programme using Biomanager at ANGIS.
However, a low homology match was found with a Volvaria volvacia
cellobiohydrolase I (CbhI) gene using the tBlastx programme provided by NCBI site.
Chapter 3 : Molecular Characterisation of Cellulase genes
74
Figure 3.3 Transcriptional analysis of PCR product
Lane 1-1Kb Ladder, Lane 2, 4 and 6- Housekeeping gene GAPDH in Avicel, CMC
and glucose grown cDNA respectively, Lane 3- Avicel, Lane 5- CMC, Lane 7 –
Glucose, Lane 8 – 100bp DNA ladder
Chapter 3 : Molecular Characterisation of Cellulase genes
75
3.3.5 Amplification of Phlebia gigantea cDNA for cellulase expression studies
To study the expression of cellobiohydrolase gene, the fungus Phlebia
gigantea was grown in microcrystalline cellulose (Avicel) or glucose as a sole carbon
source. The mycelium of the fungus, which was grown in both the carbon sources,
was harvested at different stages of the cultivation cycle. The first strand cDNA was
prepared from the total RNA and it is served as DNA template in the PCR
amplification using the cellobiohydrolase primers D3 (PcbhI.1U/ PcbhI.1d). The
agarose gel electrophoresis of the PCR amplified product using primer D3
demonstrates that there is no PCR product in the size range of 750bp in the glucose
grown culture (Figure. 3.3, lane 7) whereas, the desired PCR product of approximate
size 750bp was found to be amplified in the avicel and CMC grown culture (Figure.
3.3, lanes 3 and 5). The results clearly indicate that the cellobiohydrolase gene was
induced in the presence of microcrystalline cellulose and suppressed when the fungus
P. gigantea was grown in the presence of glucose.
The synthesis of cellulases by other organisms is generally induced by
cellulose and repressed by other readily metabolisable sugars such as glucose, which
is also true in P. gigantea as it was observed that there is a PCR product of 750 bp in
the microcrystalline cellulose grown cultures whereas there is no PCR product in the
glucose grown cultures.
3.3.6 Amplification of Phlebia gigantea cDNA product using RACE PCR
The RACE ready first strand cDNA was synthesised from the total RNA of
P.gigantea mycelium, grown on avicel. The amplification of 5’-end was performed
using 5’RACE ready cDNA and the 5’ gene specific primers pGp22GSP2N1 and
Chapter 3 : Molecular Characterisation of Cellulase genes
76
P22GSP2. The 3’- end was amplified using 3’RACE ready cDNA and the gene
specific primers for pG22FGSP2, pGp22GSPN1 and VvGSP2a, designed specifically
for the amplification of 3’end. A 900bp PCR product was obtained with 3’-RACE
PCR (Figure 3.4, lane 2 and 4) using the primer pG22FGSP2. No P22 amplifying
5’RACE PCR product was obtained using the 5’ gene specific primers.
Figure 3.4 3’RACE PCR product
Lane 1-1 Kb Ladder, Lane 2, 900 bp RACE Product, Lane 3- DNA ladder
LambdaEco Hind III marker, Lane 4 900 bp RACE product.
Chapter 3 : Molecular Characterisation of Cellulase genes
77
5’-GAGGACATGATGTCCGCTCACAGTGCCCTGTCGATGCTGAACGTTTGTT
GGGTACTATcGGCTGGCTGAGGGCTATCACGACTGCGTTCTTCTATCCGGA
AGCCCGGTGTGACTTTGTGGGTGCTTCTTCCTTTCCCTGACCCCGAATACC
TTCACCGCACGGGACGTACGAGTACGGTGCCGTCCCGGGTAAGAGGGCTA
TCCTTGTTATCCTGGAAGAGATCAAGGGAGGCAGGGTGTGCACAGATGAG
TATACCCCGCTCAAGAGGGTCGATGAAGCGGACGGTCGGGTATGATGACC
TCGAAACTCCACTTGCTTGGATGTGGGTATGtAGTGGATGCGGCCTGACCC
GCGCGATCGGGACAATAGCTGGCGCACAAACACTGCTCGGACTTTCTGTA
TGGTTCCCTGCAGTACCAGAGGTCAGCCGCCGCGAAGGTACCAGAAATTC
CTCATTGACATACCATTGGATGGGACGTGATTTGAGTGGCGAAGTGCTTC
CACATCCCCtCTGGCCTATGGCGCGAGCGATCTGTATGAACGGCTCAAACT
TCCAGGCGCCGCAGTGGCATTGGTtATCGCCTATAGCGAGGACCGGTCTGC
GCCTGTCAATTTTGGAGGTGTGGCAGTGtTTATTCTTCCCTCGTAGTGCCGA
CAACGTGCGGGAGCCCCAAACCTCCCTAGAACATAGCACTAGGGTACTAT
TAAGCATGCGTGTCCACTTGCGAGGCGGCAAGGCAGGATCCTCCTTTTCCT
CCTGCAACTCGGAACCTAGTCCGCCCATGTTCGTGACACGCTCCCACTAG
ATGAATGAGTACTCACCAATCCACAGGGCCCCTACGCTGACCGCGAACAA
GGCACTATGGCGGAGCTTCCGCAACCTTAAGTGGATTAACCCCAAAGTTG
CAGCCATGTTCAGTTCACGCCTCCCACTAGAGTGCTGGAATCTCACCAACC
ACAAGGGCCTCTCTACCGTGACGGGGACAAGAACATTGGCGGACCTCCGA
CCTAATGGATTACCCAAGTTGCACCTGAAAAGTGTAGCCTTGCAAGCGTT
GCCCCGGTTCTTAAAGTATCTTTTTCGGCTGAAGAGTGCATACCTGCCGAA
GAGCTGTACCCGCCGCTCCCTACCGCTACTTCTTCCACAGCCTCT-3’
Length 1154 nucleotides
Figure 3.5 Combined PCR And RACE-PCR Product P223A3S6
Chapter 3 : Molecular Characterisation of Cellulase genes
78
EDMMSAHSALSMLNVCWVLSAG*GLSRLRSSIRKPGVTLWVLLPFPDPEYLH
RTGRTSTVPSRVRGLSLLSWKRSREAGCAQMSIPRSRGSMKRTVGYDDLETP
LAWMWVCSGCGLTRAIGTIAGAQTLLGLSVWFPAVPEVSRREGTRNSSLTYH
WMGRDLSGEVLPHPLWPMARAICMNGSNFQAPQWHWLSPIARTGLRLSILE
VWQCLFFPRSADNVREPQTSLEHSTRVLLSMRVHLRGGKAGSSFSSCNSEPSP
PMFVTRSH*MNEYSPIHRAPTLTANKALWRSFRNLKWINPKVAAMFSSRLPL
ECWNLTNHKGLSTVTGTRTLADLRPNGLPKLHLKSVALQALPRFLKYLFRLK
SAYLPKSCTRRSLPLLLPQP
Length: 384 amino acids
Figure 3.6 The amino acid sequence of the product P223A3S6
* Denotes stop codon.
Chapter 3 : Molecular Characterisation of Cellulase genes
79
3.4 Discussion
In recent years, the genes encoding a number of fungal and bacterial
cellulolytic enzymes have been isolated, cloned and expressed in E. coli. Cellulase
genes were also isolated by polymerase chain reaction (PCR) using known cellulase
primers(Broda et al., 1995; Vallim et al., 1998; Wymelenberg et al., 2002). Strategies
based on the efficient induction of cellulolytic enzymes have also been used to isolate
genes encoding for both exo- and endoglucanases from filamentous fungi (Teeri et al.,
1992; Tempelaars et al., 1994; Lynd et al., 2002).
The homologous relationship between cellulase families aids in their
identification and isolation of cellulase gene of unknown origin by PCR using primers
made from known gene sequences representing cellulase gene types (Henrissat et al.,
1995; Ilmen et al., 1997; Maheshwari et al., 2000; Lynd et al., 2002).
In order to employ a PCR based strategy to clone a cellulase gene(s) from P.
gigantea it was necessary to design and use internal primers specific for cellulase
genes. Given that there have been no reports of P.gigantea cellulase genes published
nor are there any genes from this organism available from publicly accessible
databases we designed degenerate primers from the predicted sequence of a putative
cellulase gene previously isolated from our laboratory (Palaniswamy, 1998). We also
used the set of preused primers to isolate the cellulase gene from P. chrysosporium
(Vallim et al., 1998). Using these primers and cDNA generated from P. gigantea
total RNA several gene fragments were obtained and subsequently cloned and
sequenced. Amplification of cDNA at 55oC with D3 primers resulted in a PCR
product of 750bp similar to that obtained by K.Palaniswamy (Palaniswamy, 1998).
Chapter 3 : Molecular Characterisation of Cellulase genes
80
New sets of gene specific primers were designed based on the sequence and these
primers were then used to amplify cDNA ends using the RACE ready cDNA and
RACE-PCR to amplify the 5’ and 3’ regions of the putative cellulase gene. The final
1154bp predicted cellulase gene, P223A3S6 was obtained (Figure 3.5) which was
translated into a 384 amino acids product with multiple stop codons (Figure 3.6).
In order to examine the regulation of the P223A3S6 gene in response to
cellulose P. gigantea mycelium was cultured in liquid medium supplemented with
either glucose or Avicel as a sole carbon source. The RT-PCR was performed using
primers specific to P223A3S6 and also primers specific to the house keeping gene
GAPDH. Electrophoresis and visualisation of the RT-PCR products demonstrated
that the level of cellulase P223A3S6 gene transcript increased in the mycelium grown
in cellulose compared to the mycelium grown in media with glucose as the sole
carbon source. This pattern of expression/ induction in media containing cellulose as
a sole carbon source is consistent with P223A3S6 expression being regulated by
cellulose and that the gene may be involved in cellulose utilization by the P.gigantea.
The sequence P223A3S6 comprises 1154bp and contained a predicted 384
amino acid protein. Blast search analysis with this predicted protein revealed little in
terms of the homology with other proteins in the publicly accessible databases.
The PCR amplification of P. gigantea cDNA resulted in an 1154bp DNA
fragment and the cloning and sequence analysis revealed the absence of open reading
frame (ORF) in the genomic data obtained herewith. Thus, the sequence obtained
herewith may represent an incomplete sequence of the P. gigantea cellulase degrading
Chapter 3 : Molecular Characterisation of Cellulase genes
81
gene. However, the transcription analysis reveals that this putative cellulase gene of
P. gigantea was induced in the presence of microcrystalline cellulose as a sole carbon
source whereas it was suppressed in the presence of glucose, which is in agreement
with other cellulolytic fungus such as P. chrysosporium (Sims et al., 1988).
The complete sequence of this putative cellulase is important to completely
classify the gene within a cellulase family. The complete gene sequence would also
be helpful in studying the gene in order to exploit the fungus P. gigantea for the
industrial production of the cellulase enzyme. However with the accumulation of vast
amounts of DNA sequences in databases, merely having complete genomic sequence
is not enough to denote and identify the biological functions and the presence of ORF
alone does not confirm the existence of functional gene (Pandey and Mann, 2000).
Thus a functional proteomics approach was further taken to accomplish the aims of
this project. In this approach the proteins are affinity purified exploiting the specific
cellulose binding and degrading function and were further identified by mass
fingerprinting approach (Andersen and Mann, 2000).
82
Chapter 4 : Effect of carbohydrate carbon sources on the production of cellulase
by Phlebia gigantea
4.1 Introduction
Phlebia gigantea is an aggressive white rot fungus that colonizes conifer
wood (Behrendt et al., 2000) with demonstrated competitive ability to colonize freshly
cut wood and produce extracellular cellulase (Collett, 1983; Blanchette et al., 1998;
Palaniswamy, 1998). P. gigantea is also able to tolerate resinous extracts present on
freshly cut wood allowing the fungus to quickly colonize sapwood and preventing
other fungi becoming established (Hodeges, 1964). Moreover, this filamentous wood
decay fungus is a common inhabitant of forest litter and fallen trees (Ross and
Hodges, 1981). The diverse capacities of this fungus and its interactions in nature
with different substrates, suggests that this fungal enzyme system has some
commercial potential for the production of efficient cellulases.
Cellulose degradation and its subsequent utilisations are important for global
carbon sources (Schwarz, 2001; Criquet, 2002). The value of cellulose as a renewable
source of energy has made cellulose hydrolysis the subject of intense research and
industrial interest (Bhat, 2000). Over the years, a number of organisms, in particular
fungi, possessing cellulose-degrading enzymes have been isolated and studied
extensively (Bhat and Bhat, 1997; Nowak et al., 2005), however, the major constraints
in the economic feasibility of using cellulase industrially involves four major
interrelated factors (i) the substrate (ii) the microorganisms used to perform the
digestion (iii) the cellulose degrading enzymes, and (iv) the end products (Coughlan,
1992). The interaction between these factors has a marked effect on the production of
commercially potential cellulases (Bhat and Bhat, 1997). Previous studies based on
Chapter 4 : Effect of carbohydrate carbon sources on the production of cellulase by Phlebia
gigantea
83
other fungi (Bhat and Bhat, 1997; Kirk and Cullen, 1998; Maheshwari et al., 2000;
Lynd and Zhang, 2002; Howard R.L., 2003; Mai et al., 2004) have suggested that
overall, at least three types of enzymes are traditionally components of a cellulase
system that performs complete degradation of cellulose to glucose (Kirk and Cullen,
1998). These enzymes are the endoglucanases (EC 3.2.1.4); cellobiohydrolases (exo-
glucanases, EC 3.2.1.91) and cellobiases (β- glucosidases, EC 3.2.1.21)
Fungal cellulase is an inducible enzyme system which is induced in the
presence of cellulosic substrate (Coutinho and Henrissat, 1999a; De Vries and Visser,
2001; Galbe and Zacchi, 2002). The most commonly known cellulase inducers are
cellobiose, lactose and sophorose (Bhat and Bhat, 1997; Lynd et al., 2002). For
example, in T. reesei the expression of cellulase gene Cbh I, Cbh II, Egl I and Egl II is
found to be induced in the presence of cellulose and is co-ordinated through
transcription factor (Covert et al., 1992; J. Abrahao Neto, 1995; El-Dorry et al., 1996;
Ilmen et al., 1997; Wymelenberg et al., 2002). This implies that the nutrients and
their mutual interaction as well the other chemical, biological and physical conditions
play a major role in the disruption of cellulose in the cellulosic organisms.
In P. gigantea cellulase is produced during the exponential and stationary
phases of cultivation (Palaniswamy, 1998) and therefore the optimisation of cellulase
production will necessitate an optimisation of fungal growth. Fungal growth is
dependent on several physical and chemical parameters such as biomass production,
pH and substrate induction and specificity. It is found that there is a system for
regulation of gene expression by extra cellular pH which promotes producing extra
cellular enzymes under the conditions of pH where they can function (Denison, 2000).
Chapter 4 : Effect of carbohydrate carbon sources on the production of cellulase by Phlebia
gigantea
84
Such pH regulatory systems as are very common in fungi and has been well described
in A. nidulans, S. cerevisiae, P. chrysogenum, Y. lipolytica and C. albicans (Denison,
2000).
The parameter optimisation work completed to date has identified the
optimum temperature for both P. gigantea growth and cellulase production to be 250C
(Palaniswamy, 1998) while the optimum pH for the production of cellulase ranges
from pH 4.5 to pH 3.0 (Palaniswamy, 1998). The cellulase production by P. gigantea
has been shown to be strongly induced by the microcrystalline cellulose and
sophorose while glucose, xylose and lactose have been found to repress the cellulase
production (Palaniswamy, 1998). The effects of growth parameters and inducers
outlined above indicate the regulation of the cellulase gene is influenced by these
parameters. Based on these studies, investigation of the effects of different substrate
in promoting the production of the cellulase enzyme by the fungus P. gigantea is
studied in this chapter. A conventional approach of classical enzyme study is
undertaken herewith to study the effects of different substrates on production and
purification of cellulase.
4.2 Materials and methods
4.2.1 Organism
P. gigantea, DFP 14356 (CSIRO, Forest Production Division, Melbourne,
Australia) was used to study the effects of different substrates on growth of fungus
and production of cellulase. It was cultured at 22°C and was routinely maintained on
Malt Extract Agar (MEA) medium by periodic transfers as described in Chapter 2.
Chapter 4 : Effect of carbohydrate carbon sources on the production of cellulase by Phlebia
gigantea
85
4.2.2 Inoculum preparation and cultivation
To prepare inoculum for liquid culture, a 5mm mycelial disc was cut from
agar slab and inoculated in sterile 2% (w/v) of 100mL of Malt Extract Broth (MEB).
Incubation was carried out at 25°C for 6 days at 110rpm (Palaniswamy, 1998). After
6 days of incubation, 2mL of inoculum was removed and reinoculated in sterile 2%
(w/v), 100mL MEB broth. The mycelia obtained herewith were used as an inoculum
for further shake flask cultivation experiments. Flask cultivation was performed using
250mL flasks with 100mL culture volumes. The shake flasks were maintained at
25°C at 110rpm for 12 days. Based on preliminary studies in our laboratory to
determine optimal growth and cellulase production the medium was adjusted to pH
5.5 prior to inoculation (Palaniswamy, 1998).
4.2.3 Basal Medium and culture conditions
Basal medium used was composed of 18mM (NH4) H2PO4, 5mM KH2PO4,
0.3% urea (70% (w/v)), 2mM MgSO4.7H2O, 2mM CaCl2, 0.05% peptone, 17mM
FeSO4.7H2O, 15mM ZnSO4.7H2O, 5.5mM MnSO4.H2O, 5mM CoCl2.6H2O, 100µg
thiamine-HCl (Palaniswamy, 1998).
4.2.4 Protein determination
The extracellular soluble protein was measured by the dye binding method
of Bradford (Bradford, 1976) using the Bio-Rad® dye reagent concentrate (500-
0006,Bio-Rad) in microtiter plates and read using the Dynatech MR 7000 Elisa reader
(Hutronics Ltd.,) A standard curve was generated using solutions containing 1µg/µL
bovine serum albumin (BSA). Absorbances were measured at 595nm after 5 minutes
Chapter 4 : Effect of carbohydrate carbon sources on the production of cellulase by Phlebia
gigantea
86
of incubation at room temperature and individual samples were performed in
triplicate.
4.2.5 Cellulase enzyme assay
Two basic approaches to assay cellulase activity were undertaken in this
study. First based on measuring the individual activity of component enzyme and
second based on measuring the activity of the total cellulase complex.
Carboxymethyl cellulose plate diffusion assay was used to measure the endoglucanase
activity. Measuring the reducing sugars released from substrates like CMC and
Avicel is used to measure the total cellulase complex activity. All the assays are
performed at least three times.
4.2.5.1 Carboxymethyl Cellulose Plate diffusion assay
The cellulase activity was determined using agar diffusion technique for
assay. Culture supernatant obtained after centrifugation at 17,700g for 10 minutes
were used to determine the cellulase activity. A series of 90mm Petri dishes
containing 1.5% (w/v) agar and 2% (w/v) of carboxymethyl cellulose as the substrate
were prepared. Wells (0.6mm diameter) were made in the agar plates and these wells
were inoculated with 20µL of culture supernatant. Commercial T. reesei RUT C30
cellulase was used as positive control and sterile water as the negative control. The
plates were incubated at 420C for 16 hours and the cellulase activity was indicated as
clear orange halos after staining with 1% Congo red solution for 10 minutes and
washing several times with 1M NaCl (Shoseyov and Doi, 1990). Presence of zones of
clearance around the inoculated wells indicated cellulase activity and the magnitude
of the activity were calculated by measuring the diameter of the zones. Comparison
Chapter 4 : Effect of carbohydrate carbon sources on the production of cellulase by Phlebia
gigantea
87
of the zones of clearance with those of the commercial T. reesei RUT C30 was then
made.
4.2.5.2 CMC assay
To further confirm the cellulase activity, a second assay was conducted.
Cellulase activity was measured as per the 3,5- dinitrosalicylic acid (DNS) method
(Mandels et al., 1976) by determining the amount of reducing sugars released during
30min in a reaction mixture. The cellulase activity was assayed by mixing 0.5mL of
culture supernatant with 0. 5mL of 1% carboxymethyl cellulose solution in 50mM
citrate buffer (pH 4.8) and incubated for 10 minutes at 600C in an oven. One unit (IU)
of enzyme activity was defined as the amount of enzyme releasing 1µmole reducing
sugar in 1 min reaction; D- glucose was used as the standard.
4.2.6 Effect of various inducers and repressors on cellulase production
Growth and productivity in the presence of a variety of sole carbon sources
was studied using the basal medium supplemented with either 2% (w/v) of glucose,
microcrystalline cellulose, carboxymethyl cellulose (CMC), or cellobiose. The effects
of the different substrates on the cell growth and the cellulase production of P.
gigantea were examined based on the determination of cell biomass, total extra
cellular soluble protein, media pH profile and cellulase activity assays. These
parameters were determined every alternate day starting from the day of inoculation
for a period of 12 days. The inoculation period was based on the preliminary
screening of P. gigantea cellulase activity on CMC plate assay. All shake flask
cultivations were performed in triplicate and data are presented as means of standard
errors.
Chapter 4 : Effect of carbohydrate carbon sources on the production of cellulase by Phlebia
gigantea
88
4.2.7 Biomass production
Growth was determined by measuring dry weight of mycelium by filtering
through pre-weighed 40µm Whatman® filter paper circles with the aid of suction. The
mycelial cells and non-degraded cellulose were washed with 200mL of sterilized
water and then was oven dried at 700C for 48 hours. The supernatant was used to
assay the cellulase activities as described in Chapter 4 (Section 4.2.5.1).
4.2.8 Study of pH changes on cellulase production
The change in pH of the cultivation medium of P.gigantea was studied in
basal medium with 2% (w/v) of glucose, microcrystalline cellulose, carboxymethyl
cellulose (CMC), or cellobiose as the sole carbon source. The initial pH of medium
was adjusted to sterilised media (pH 5.5) was then inoculated with the mycelium and
incubated as described in Chapter 4 (Section 4.2.2). During cultivation the broth was
sampled every alternate day until the end till 14 days. The mycelium and unused
substrate is removed as described in Chapter 4 (Section 4.2.7). Once the solid
particles had settled to the bottom of the tube the pH of the medium supernatant was
measured using a pH meter. The supernatant was also used to assay the cellulase
activities as described in Chapter 4 (Section 4.2.5).
4.3 Results
In the presence of glucose, both the cell biomass (Figure 4.1) and total
protein concentration (Figure 4.2) were found to increase linearly. However, no
cellulase activity was detected with either the CMC (Figure 4.3) or the cellulase
assays (Fig 4.4 and 4.5). Growth profile for the xylose was similar to that of glucose
and a significant increase in cell biomass and total protein was recorded. Cellulase
Chapter 4 : Effect of carbohydrate carbon sources on the production of cellulase by Phlebia
gigantea
89
activity in the present of xylose substrate was below the limits of determination. The
increase in cell biomass (Figure 4.1) and total protein concentration (Figure 4.2)
through the 12 day period in the presence of glucose was found to be significantly
higher as compared with the growth and the total protein production in the presence of
Avicel, CMC, cellobiose and xylose.
There was a significant increase in cell biomass (Figure 4.1) and the
concentration of total protein (Figure 4.2) when P. gigantea was cultured on the
Avicel substrate. Both the CMC plate assay and the cellulase assay indicated
enhanced effect on cellulase activity of growing P. gigantea on the Avicel substrate.
The CMC plate assay (Figure 4.3) showed an increase in activity beginning at day 4
to peak at day 10 and then decreased slightly thereafter (Figure 4.5). The cellulase
DNS assay also indicated that activity began on day 4; however activity using this
assay showed a consistent rise in activity during the 14 days period (Figure 4.4).
Significant production of the cellulase enzyme by P. gigantea in the
presence of CMC was recorded as shown by both the CMC plate assay (Figure 4.5)
and cellulase assay (Figure 4.4). The maximum activity of 103.5IU/mL in the
presence of CMC was obtained on the day 12 after an initial lag phase during the first
4 days. In the presence of the substrate cellobiose, cellulase activity showed similar
pattern to that of CMC and Avicel. The cellulase activity started emerging on day 4
and then gradually reached its peak on day 10. Concomitant with this, a significant
increase in total protein concentration and cell biomass were registered in both CMC
and cellobiose (Figure 4.4).
The effect of pH of the culture medium on cellulase activity in presence of
different substrates was also studied. When basal medium was supplied with CMC
the pH increased consistently throughout the 12 day incubation period whereas the pH
Chapter 4 : Effect of carbohydrate carbon sources on the production of cellulase by Phlebia
gigantea
90
for medial containing cellobiose decreased till the day 8 then showed a slight increase
from the day 10 until the end of the cultivation period. In media supplemented with
Avicel, the pH increased until day 10 and decreased thereafter. The pH of glucose
containing medium increased consistently (Figure 4.6) for 12 days while there was no
significant change in pH in the xylose consisting medium.
Figure 4.1 Mycelial growth curves of P. gigantea
P. gigantea grown on CMC (×); microcrystalline cellulose (); glucose (♦); xylose
(); cellobiose (). Dry weight of mycelium was measured after filtering through
40µm Whatman filter paper and oven dried at 700C for 48 hours
Chapter 4 : Effect of carbohydrate carbon sources on the production of cellulase by Phlebia
gigantea
91
Figure 4.2 Total protein obtained from P. gigantea in presence of different substrates
Total protein obtained in the presence of CMC (×); microcrystalline cellulose ();
glucose (♦); xylose (); cellobiose () of P. gigantea The extracellular soluble
protein was measured by the dye binding method of Bradford using the Bio-Rad dye
reagent concentrate in microtiter plates. Absorbance was measured at 595nm
Chapter 4 : Effect of carbohydrate carbon sources on the production of cellulase by Phlebia
gigantea
92
Figure 4.3 Carboxymethyl cellulose plate diffusion assay
The cellulase activity was determined by incubating the CMC agar plates at 420C for
16 hours with culture supernatant. The cellulase activity was indicated as clear
orange halos after staining with 1% Congo red solution. The magnitude of the
activity was calculated by measuring the diameter of the zones. Sample S,
Commercial T. reesei RUT C30 cellulase was used as positive control; sample B,
sterile water as the negative control; sample A, 20µL culture supernatant of Avicel
medium; sample C1, 20µL supernatant of CMC medium; sample C2, 20µL culture
supernatant of cellobiose medium; sample G, 20µL culture supernatant of glucose
medium; sample X, 20µL culture supernatant of xylose medium.
Chapter 4 : Effect of carbohydrate carbon sources on the production of cellulase by Phlebia
gigantea
93
Figure 4.4 Cellulase activity curves of P. gigantea with different substrates
Cellulase activity curves of P. gigantea grown on CMC (×); microcrystalline
cellulose (); glucose (♦); xylose (); cellobiose () (the results represent the
average of 3 assays). The activity was measured as per DNS method by determining
the amount of reducing sugar. One unit (IU) of enzyme activity was defined as the
amount of enzyme releasing 1µmole reducing sugar in one minute reaction. No
cellulase activity is detected with glucose and xylose
Chapter 4 : Effect of carbohydrate carbon sources on the production of cellulase by Phlebia
gigantea
94
Figure 4.5 P. gigantea cellulase activity curves of CMC plate assay
Cellulase activity curves of P. gigantea grown on CMC (×); microcrystalline
cellulose (); glucose (♦); xylose (); cellobiose () (the results represent the
average of 3 assays). The magnitude of the activity was calculated by measuring the
diameter of the zones on the CMC agar plates indicated as clear orange halos after
staining with 1% Congo red solution. No cellulase activity was detected with glucose
and xylose
Chapter 4 : Effect of carbohydrate carbon sources on the production of cellulase by Phlebia
gigantea
95
Figure 4.6 Effect of substrates on pH of P. gigantea media
Effect of sole carbon source on the of pH of medium supernatant of P. gigantea over
the incubation period. Fungi were grown on 2% (w/v) CMC (×); microcrystalline
cellulose (); glucose (♦); xylose (); cellobiose ()
4.4 Discussion
In the inducible enzyme system of cellulases, induction, catabolite
repression, product inhibition and restricted carbon source supply have been reported
to be involved in the regulation mechanism (Bhat and Bhat, 1997). In most
organisms that digest cellulose, cellulose is found out to be the best carbon source for
Chapter 4 : Effect of carbohydrate carbon sources on the production of cellulase by Phlebia
gigantea
96
the production of high levels of cellulase whereas glucose repressed the production of
these enzymes (Bhat and Bhat, 1997; Lynd et al., 2002). Although, it has been
suggested that the cellulase formation is regulated at the transcriptional level (Kubicek
et al., 1993; El-Dorry et al., 1996; Ilmen et al., 1997; Wymelenberg et al., 2002), there
is considerable variation in the level of individual transcript, as well which specific
cellulase gene is transcribed, depending on the carbon source used for growth (Bhat
and Bhat, 1997; Spiridonov and Wilson, 1998; Lynd et al., 2002).
This investigation, studied the effects of different substrates in promoting the
activity of the cellulose-degrading enzymes by the fungus P. gigantea. Cellulase
enzyme activity was determined by (i) the ability of this organism to grow in the
presence of the different substrates as indicated by corresponding increase in cell
biomass (ii) production of extracellular protein (iii) changes in the pH of the different
media over the time course of fungal growth and (iv) measurement by using two
different invitro assays which quantitated cellulose degradation by cellulases.
Relative to the other substrates, there was a higher increase in cell biomass
when the P. gigantea grown in glucose as the sole carbon source. Glucose, a simple
sugar is readily metabolised, and as such the P. gigantea do not have to expend a
large amount of energy to use this glucose as carbon source. Thus this may account
for the higher biomass increase registered in our study. In contrast, substrates Avicel,
cellobiose, and CMC are chemically more complex requiring significant enzymatic
degradation prior to being in a form which the organism can utilise as a carbon or
energy source (Ng and Zeikus, 1982).
Chapter 4 : Effect of carbohydrate carbon sources on the production of cellulase by Phlebia
gigantea
97
A number of studies have shown that cellulase biosynthesis is higher when
organisms are exposed to complex substrates as opposed to when they are grown in
the simple substrates(Bisaria and Mishra, 1989; El-Dorry et al., 1996; Henriksson et
al., 2000; Wymelenberg et al., 2002). In most organisms, cellulase production is
repressed in the presence of readily metabolisable carbon sources such as glucose but
is induced by low molecular weight compounds such as cellobiose (Lynd et al., 2002).
In addition, a study conducted by El- Gogary (1989) (El-Gogary et al., 1989) has
shown that the gene coding for the cellulase enzyme in T. reesei, Cbh1, showed an
increase in expression in the presence of Avicel whereas the addition of glucose
repressed the synthesis of transcript encoding this enzyme (El-Gogary et al., 1989).
In the presence of glucose and xylose, no cellulase activity was recorded in our
experiments whereas substrates such as CMC and Avicel proved to be the strongest
inducers of the cellulase enzyme activity. The cellulase activity in cellobiose
containing medium was slightly less compared with the activity in the presence of
CMC and Avicel, and this may be due to the ability the organism to repress enzyme
synthesis or activity in the presence of high concentration of cellobiose (Spiridonov
and Wilson, 1998).
Studies have shown that the fungal degradation of cellulose is a synergistic
sequence of reaction (Beguin and Aubert, 1994; Bayer et al., 1998b; Bhat, 2000; Lynd
et al., 2002). In the first instance, an endo-1, 4-β- glucanases cleaves cellulose
randomly within the polymer chain, releasing chains with the non-reducing ends.
These chains are then hydrolysed by the enzymes exo-β-glucanases or
cellobiohydrolases to give cellobiose. Cellobiose is thus further broken down to two
glucose units by 1, 4- β- glucosidases (Henriksson et al., 1995; Henriksson et al.,
Chapter 4 : Effect of carbohydrate carbon sources on the production of cellulase by Phlebia
gigantea
98
2000; Mai et al., 2004). A number of studies have reported that during this enzymatic
sequence of reactions the production of glucose from cellulose in turn results in a
decrease in cellulase activity (Sherif M.A.S. Keshk and Sameshima, 2005). This drop
in cellulase activity may be as a result of the formation of cellobiose, an intermediate
metabolic product of cellulase hydrolysis reactions (Lynd et al., 2002; Lynd and
Zhang, 2002). P. gigantea grown on Avicel and on CMC substrates showed
decreased cellulase activity towards the day 10 of culture while the cellulase activity
for the cellobiose-containing medium began to decline on day 8. These observations
may be as a result of the accumulation of glucose, an end-product of cellobiose
cleavage by cellobiose phosphorylase, and this increased glucose inhibits further
production of the cellulase enzyme. A decrease in cellulase activity was recorded
earlier for the cellobiose substrate as this substrate is an intermediate in the conversion
of cellulose to glucose. Glucose accumulation hence occurred earlier in that medium
as only the single cleavage reaction was required to produce glucose resulting in an
earlier decrease in cellulase activity than in Avicel or CMC.
The optimum pH for fungal cellulases and crude protein production varies
from species to species though in most cases, the optimum pH ranges from pH 3.0 to
6.0 (Garg and Neelakantan, 1981). Preliminary studies in this laboratory on the effect
of pH on the culture medium have shown a drop in pH of the medium as cellulase
enzyme was produced by the P. gigantea. In this study, the media was adjusted to pH
5.5 on day 1 and subsequently the pH was recorded during the 14 days period for
growth on the different substrates. There was an increase in pH during culture in the
presence of both CMC and glucose as substrates. However, pH of Avicel and
cellobiose containing medium dropped after reaching the cellulase activity peak
Chapter 4 : Effect of carbohydrate carbon sources on the production of cellulase by Phlebia
gigantea
99
(Figure 4.6). Xylose containing medium showed no significant pH change over the
course of the experiment. The drop in pH may arise as a result of formation of
cellobiose, oxidising to cellobionolactone by cellobiose dehydrogenase (CDH) (E.C.
1.1.3.25), and cellobionolactone is subsequently hydrolysed to carboxylic acids
(Henriksson et al., 2000). Bao et al. (1994) reported a strong influence of the pH
value and the buffer system used on cellobiose dehydrogenase (CDH) production by
P. chrysosporium, with lower pH values being harmful for enzyme production (Bao et
al., 1994). The drop in the pH and cellulase activity of P. gigantea in presence of the
substrates cellobiose and Avicel is consistent with these reports.
Surprisingly, the pH of the medium with CMC showed a consistent increase
even though significant cellulase activity was recorded (Figure 4.6). This study was
conducted for 14 days and it could be hypothesised that during the 14 day incubation,
P. gigantea may not produce enough cellulase to cause a decrease in pH of medium.
As expected, the glucose substrate did not induce any detectable cellulase activity and
the pH in the presence of glucose increased steadily till the end of the cultivation
(Coutts and Smith, 1976).
On the basis of data obtained in this study, it can be concluded that the
capacity of P. gigantea to efficiently degrade cellulose is dependent upon the nature
and concentration of the carbon source added. Microbial cellulases are generally
active in acidic to neutral pH range, and consistent with that the enzyme preparation
obtained in the present studies was found to be acid active and alkali stable, indicating
a potential for industrial process application. In conjunction with earlier studies by K.
Palaniswamy, it is believed that P. gigantea utilises cellulose for growth and
Chapter 4 : Effect of carbohydrate carbon sources on the production of cellulase by Phlebia
gigantea
100
regulation of cellulase synthesis, whereas simple sugars like glucose and xylose
represses the synthesis of cellulase (Palaniswamy, 1998). The drop in pH of medium
in presence of Avicel was also recorded by Palaniswamy and is thought to be indicate
playing a role in inactivation/or synthesis of the cellulase enzyme. However, the
consistent increase in pH of medium and demonstrated high cellulase activity in CMC
as compared to other substrates is consistent with the finding in T. reesei that
intermediate transglycosylation products of CMC probably induced the cellulases
(Zhu et al., 1982).
This knowledge of cellulase synthesis can be used to increase cellulase
production and will be helpful in the genetic analysis of the white rot fungi
P.gigantea. This study on synthesis and regulation of cellulase in P.gigantea has
provided an insight for understanding the basic biological process of gene expression
and thus used as a platform for further study in this thesis.
101
Chapter 5 : Purification and characterisation of cellulase produced by Phlebia
gigantea
5.1 Introduction
Cellulases are the group of hydrolytic enzymes capable of hydrolysing
insoluble cellulose to glucose. Cellulase systems require at least three types of
enzyme for the complete degradation of cellulose to glucose: endoglucanases (EC
3.2.1.4); ); cellobiohydrolases (exo-glucanases, EC 3.2.1.91) and cellobiases (β-
glucosidases, EC 3.2.1.21), and these enzymes have been found to show different
types of synergistic interactions when hydrolysing crystalline cellulose (Henrissat et
al., 1989). Moreover, the catalytic domains of all cellulases are known to be
reasonably large and can represent more than 70% of cellulase protein. Cellulases are
modular enzymes composed of independently folding, structurally and functionally
discrete units called as domains or modules (Klyosov, 1995b; Mai et al., 2004).
Analysis of catalytic domains based on their amino acid sequence shows considerable
diversity between cellulases (Bhat and Bhat, 1997)
Microorganisms that are required to utilize these complex structures as
carbon sources had many evolutionary challenges to develop a compatible system to
use these substrates (Morag (Morgenstern) et al., 1992; Bhat, 2000; Zhang and Lynd,
2003). Cellulose degrading organisms often produce complex extracellular or
membrane bound cellulolytic systems comprising a combination of several enzymes
(Bhat, 2000).
A cellulase system is not just the conglomeration of the three representative
enzymes with or without cellulose binding domains; rather it is a complex that
Chapter 5 : Purification and characterisation of cellulase produced by Phlebia gigantea
102
catalyses the efficient hydrolysis of cellulose in a well coordinated manner (Lynd et
al., 2002). There have been a large number of studies reporting the purification and
characterisation of cellulase components, their mechanism of substrate specificities
and their mode of action (Klass, 1983; Persson et al., 1991; Klyosov, 1995a, b; Bhat,
2000; Maheshwari et al., 2000; Lynd et al., 2002; Mai et al., 2004; Palonen et al.,
2004). Some of the major purification schemes from different source organisms is
summarised in Table 5.1.
The extracellular supernatant produced by P. gigantea when cultured on
different substrates can catalyse the breakdown of cellulose to its simpler derivatives
(Chapter 4). This extracellular fluid is therefore believed to be responsible for the
breakdown of cellulosic materials to smaller oligosaccharides and glucose that can be
used by the fungus as a carbon source. To confirm this, a cellulase active fraction of
P. gigantea extracellular fluid was purified and characterised. The results of the
purification procedure and enzyme characterisation are summarised in this chapter.
The purification procedure used is a modified version of that proposed by
Morag et al., (1992) for the purification of cellulase from culture filtrate by affinity
adsorption (Morag (Morgenstern) et al., 1992). This purification protocol is based on
the substrate based affinity adsorption for the near total recovery of the cellulase
(Morag (Morgenstern) et al., 1992).
Chapter 5 : Purification and characterisation of cellulase produced by Phlebia gigantea
103
Table 5.1 Summary of published cellulase purification schemes from different source
organisms.
Organism Purification steps Yield
%
Year Reference
Trichoderma
viride
Ammonium ppt
Sephadex G50
DEAE Toyopearl
PBE 94
TSK-G300 SW
14% 1992 (Goto et al.,
1992)
Acremonium
persicinum
Ultrafiltration
Ammonium ppt
Mono Q
Superose 12
13% 1997 (Pitson et al.,
1997)
Thermotoga
neapolitana
FFQ Sepharose
Biogel P-60
Mono Q
Preparative PAGE
17.5% 1998 (Bok et al., 1998)
Schizophyllum
commune
Ammonium ppt
DEAE Sephadex A50 (pH 5)
DEAE Sephadex A50 (pH 6.5)
Sephadex G-100
16% 1998 (Fang et al.,
1998)
Phlebia gigantea Ammonium ppt
Sephadex S-300
DEAE Sephadex A50
HPLC gel
8% 1998 (Palaniswamy,
1998)
Phanerochaete
chrysosporium
Ammonium ppt
Sephacryl S-200
Mono Q (FPLC)
32% 2000 (Li et al., 2000)
Thermotoga
neapolitana
AEC-Q- Sepharose
GF-Biogel P60
HIC-Phenyl Sepharose
AC-galactose-agarose
AEC-Mono-Q
Preparative PAGE (native)
32% 2000 (Yernool et al.,
2000)
Bacillus strain
CH43
Acetone ppt.
HIC
SEC
IEC
12% 2000 (Mawadza et al.,
2000)
Haliotis discus
hannai (Abalone)
Toyopearl CM 650
Hydroxyapatite column
Sephacryl S-200
7% 2003 (Suzuki et al.,
2003)
Ampullaria
crossean
(Mollusca)
Ammonium ppt.
DEAE sepharose (fast flow)
Phenyl Sepharose CL-4B
Biogel P-100
DEAE sepharose (fast flow)
1.48% 2005 Yan-Hong Li et
al.,
AEC – anion exchange chromatography; AC- affinity chromatography; HIC-
hydrophobic interaction chromatography; GF- gel filtration; SEC- size exclusion
chromatography; IEC- ion exchange chromatography
Chapter 5 : Purification and characterisation of cellulase produced by Phlebia gigantea
104
Unlike the other multi-step purification procedures using combination of
ultrafiltration, ion exchange and hydrophobic interaction chromatography (Table 5.1),
this procedure used in the present study is based on two step purification. This
procedure has shown a capacity to obtain higher yields of high specific activity
cellulase complexes (Morag (Morgenstern) et al., 1992; Zhang and Lynd, 2003) from
several source organisms.
This chapter outlines the purification of the cellulase complex from P.
gigantea. Furthermore the identity of the purified cellulase was confirmed through
characterisation of the molecular mass, isoelectric point, cellulase activity assays,
kinetic properties, temperature and pH optimum of the enzyme. The purified
cellulase was subsequently used in a series of kinetic studies to determine the utility
of this enzyme to degrade various cellulose substrates.
5.2 Materials and Methods
5.2.1 Fungal Strain and Growth conditions
The P. gigantea, (DFP 14356) used in this study was obtained from CSIRO,
Forest Production Division, Melbourne, Australia. The culture was maintained on 5%
(w/v) malt extract agar (MEA) plates grown at 22°C and stored at 4°C. The culture
was routinely maintained by periodic transfers.
5.2.2 Preparation of inoculum
For the production of mycelial inoculum, 100mL conical flasks containing
50mL of 2% (w/v) malt extract broth (pH 5.5) were autoclaved and inoculated with 5
Chapter 5 : Purification and characterisation of cellulase produced by Phlebia gigantea
105
mm mycelial disc from P. gigantea malt extract agar plate. Incubation was at 25°C
for 6 days at 110 rpm. Aliquots of 1mL of mycelium were then transferred to sterile
centrifugation tubes and centrifuged at 10000×g for 10 minutes. The supernatant was
discarded and mycelium was repeatedly washed with sterile Milli-Q® water. The
washed mycelial pellet was used as an inoculum for all experiments. The washed
mycelia was further snap frozen in liquid nitrogen and stored at -80°C until further
processing.
5.2.3 Cultivation conditions
For cellulase production, 10mL of washed mycelia was used for the
inoculation of 1L of culture medium contained in 2L shake flask. The composition of
the liquid media is detailed in Table 5.2. The pH of the medium was adjusted to 5.5
with 8 M of NaOH. Batch cultivations for protein purifications were carried out in 2L
flasks, with a working volume of 1.5L. The cellulase production was induced by
addition of 2% (w/v) Avicel, used as substrate inducer. The shake flasks were
incubated at 25°C at 110 rpm throughout the growth period of 14 days. All shake
flasks cultivations were performed in triplicates.
Chapter 5 : Purification and characterisation of cellulase produced by Phlebia gigantea
106
Table 5.2 Chemically defined cellulase inducing media utilised in P. gigantea
cultivation.
Basic Nutrients
NH4H2PO4
KH2PO4
70% Urea
MgSO4.7H2O
10% CaCl2
Peptone
2 g.L-1
0.6 g.L-1
0.3%
0.5 g.L-1
55mg.L-1
0.5g.L-1
Substrate Inducer Avicel 2% (w/v)
Trace elements FeSO4.7H2O
ZnSO4.7H2O
MnSO4.7H2O
CoCl2.6.H2O
4.6 g.L-1 (1mg.L
-1 of Fe
++)
4.4 mg.L-1 (0.8mg.l
-1 of Zn
++)
0.93 gm.L-1 (0.5 mg.L
-1 of Mn
++)
1 mg.L-1 (0.5 mg.L
-1 of Co
++)
Vitamins Thiamine HCl 100 µg.L-1
5.2.4 Preparation of Amorphous Cellulose
Amorphous cellulose was prepared by dissolving 10gm of Avicel (Merck) in
500mL concentrated phosphoric acid at 25oC for 2 hours and then adding 5 volumes
of distilled water. The precipitated amorphous cellulose was washed by centrifuging
at 14,000g for 10 min and the pellets were resuspended in distilled water. After
washing the amorphous cellulose with distilled water for 5 times, the pH was adjusted
to 6.5 with 1M NaOH. The final concentration of amorphous cellulose thus prepared
was estimated to be 12 mg/mL (Lamed, 1985).
5.2.5 Cellulase purification by affinity adsorption
Cellulase purification was carried out by affinity adsorption followed by
digestion as described by Morag et al. (1992) (Morag (Morgenstern) et al., 1992) and
Chapter 5 : Purification and characterisation of cellulase produced by Phlebia gigantea
107
Zhang et al.(2003) (Zhang and Lynd, 2003). Further to this procedure a final step of
concentration of purified cellulase complex by ultrafiltration was performed.
On day 10 of cultivation, when cellulase activity reached its peak, the media
was harvested and cells were separated from the medium by centrifugation at
17,700g, for 20 mins at 4oC (Zhang and Lynd, 2003). The centrifugation was
repeated twice and the pH of the supernatant was adjusted to pH 7.0 using 1M NaOH.
2% (v/v) of 12 mg/mL of amorphous cellulose was added to the supernatant at 4oC,
stirring constantly. After overnight treatment with amorphous cellulose the
suspension was centrifuged at 17,700g for 20 mins at 4oC. The pellet was suspended
in the dialysis buffer containing 10mM CaCl2, 50mM Tris and 5mM DTT (pH 7)
(Zhang and Lynd, 2003). The suspension was dialysed in 10KDa (MWCO) dialysis
sac at 55oC against distilled water. Distilled water was changed every two hours to
prevent cellulase inhibition by hydrolysis. When the suspension had become
transparent (about 8 hours) it was centrifuged at 5000g for 20 mins at room
temperature. The supernatant was collected and stored at 4oC until further use and
was considered as purified cellulase multi-protein complex.
Protein concentration was determined by Bradford method (Bradford, 1976)
using Bio-Rad dye reagent concentrate (Bio-Rad 500-0006) with bovine serum
albumin as standard protein. Homogeneity of the enzyme was confirmed by sodium
dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970).
5.2.6 Cellulase assays
The cellulase activity was analysed with the 3,5- dinitrosalicylic acid (DNS)
method (Mandels et al., 1976) by determining the amount of reducing sugars released
Chapter 5 : Purification and characterisation of cellulase produced by Phlebia gigantea
108
during 30 min in a reaction mixture containing 2% (w/v) Avicel and 50mM citrate-
phosphate buffer pH 4.8 at 60oC. One unit (IU) of enzyme activity was defined as the
amount of enzyme releasing 1µmole reducing sugar in 1 min reaction; D-glucose was
used as the standard. CMCase activity was tested on agar plates containing 1% CMC.
Twenty µL of enzyme solution was spotted and incubated for 16 hours at 50oC; then
the plates were stained with 1% Congo red solution for 10 minutes and destained
several times by washing with 1M NaCl. Orange halos on red background indicated
CMCase activity (Shoseyov and Doi, 1990).
Cellobiohydrolase (CBHase) activity was measured on agar plates
containing 0.5mM 4-methylumbeliferyl, β–cellobioside. Twenty µL of enzyme
solution was spotted and incubated for 2 hours at 42oC. CBHase activity was
photographed as the fluorescent spots upon UV illumination (Shoseyov and Doi,
1990).
5.2.7 Gel electrophoresis and staining
The molecular mass of the purified enzyme was determined by sodium
dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using 12%
polyacrylamide gels according to standard protocol (Laemmli, 1970). Molecular
weight was estimated by comparison to Fermentas prestained protein ladder
(SM0676). Cellulase (10µL) was boiled for 10 minutes with 0.5µL of β-
mercaptoethanol and 2µL of 10% (w/v) SDS to denature the enzyme. Isoelectric
point and isozyme pattern were determined by isoelectric focusing gel analysis using
BIO-RAD 3-10nl strips and 4-12% Bis-Tris ready gels as described in Chapter 2
Chapter 5 : Purification and characterisation of cellulase produced by Phlebia gigantea
109
(Section 2.6.12). All gels were stained using Coomassie blue as described in Chapter
2 (Section 2.6.13).
5.2.8 Zymogram analysis of purified protein
The purified protein complex was identified to be a multiprotein cellulase
enzyme by Native-PAGE zymogram (Schwarz et al., 1987; Sharrock, 1988). Two
cellulase zymogram techniques were utilized, the first used CMC (Sigma) as the
substrate, while the second used 4-Methylumbelliferyl-β-Cellulose (MUC) (Jung et
al., 1998) fluorescent tagged cellulose (Sigma) as the substrate. A cellulase fraction
of 10µL was used without denaturation to retain its activity. Native PAGE and
activity staining with Congo red stain was carried out as described in Chapter 2
(Section 2.6.11). The fluorescence of MUC activity is measured under UV
illumination.
5.2.9 pH optimum for purified enzyme
To estimate the pH optimum of the enzyme, activity was measured using 1%
(w/v) CMC as the substrate at 50oC in 100mM sodium acetate buffer (pH 2.0 to pH
8.0). The enzyme stability at different pH levels was determined by measuring the
activity after incubating the enzymes for 60 minutes at 50oC at pH 2.0 to 8.0. After
the preincubation, the enzyme solution was subjected to the normal cellulase assay as
described in Chapter 4 (Section 4.2.5.2).
Chapter 5 : Purification and characterisation of cellulase produced by Phlebia gigantea
110
5.2.10 Temperature optimum and stability for purified enzyme
The temperature optimum was determined with the standard CMC assay as
described in Chapter 4 (Section 4.2.5.2), over a temperature range of 4oC to 70
oC. To
determine the temperature stability of P. gigantea cellulase complex, the activity of a
purified sample (6.54 U.mL-1) was determined after preincubation for 60 minutes at
temperatures of 4oC, 10
oC, 20
oC, 40
oC, 50
oC , 60
oC and 70
oC in 100mM sodium
acetate buffer (pH 5.0).
5.2.11 Kinetic properties
Kinetic constants such as Km, Vmax were determined under steady state
conditions using various concentrations of CMC and Avicel as substrates. The Km
and Vmax for the effect of concentration of CMC (0.5mg/mL, 1mg/ mL, 2mg/mL,
4mg/mL, 6mg/mL and 8mg/mL) and Avicel (0.1mg/mL, 0.5mg/mL, 1mg/mL,
2mg/mL, 4mg/mL, 6mg/mL) by purified P.gigantea cellulase complex were
determined at 50oC with 100mM sodium acetate buffer at pH 5.0. Values for Km and
Vmax were obtained from Lineweaver-Burk plot.
5.3 Results
5.3.1 Purification of the cellulase complex
The culture supernatant obtained after the cultivation of P.gigantea for
cellulase production was separated from the cell mass and was treated as the crude
cellulase extract. This crude cellulase extract was used in further purification
processes. The extra cellular cellulase complex was isolated from the crude protein
mixture by taking advantage of the extremely high affinity of the complex for the
cellulose. Repeated dialysis at activity temperature against the large volumes of water
Chapter 5 : Purification and characterisation of cellulase produced by Phlebia gigantea
111
helped to trap the cellulase complex (Table 5.3). As observed in Table 5.3, after the
first step of affinity binding, there was 85% recovery of total enzyme activity, which
is in accordance with earlier reports (Morag (Morgenstern) et al., 1992; Zhang and
Lynd, 2003). The specific activity of purified cellulase preparation was 17.5 IU/mg
of protein which increased from 6.3 IU/mg. The yield of cellulase on activity basis
was 62% after concentration by ultrafiltration and 78% after activity dialysis.
5.3.2 Gel electrophoresis and staining
The purity of cellulase complex was confirmed by SDS-PAGE analysis.
The cellulase complex resolved by SDS-PAGE and stained by Coomassie blue
showed three major protein bands. The molecular masses were estimated as 100KDa
(designated Pg 100), 55KDa (designated Pg 55) and 38KDa (designated Pg 38) and at
least five other minor proteins were recorded (Figure 5.1 B, Lane 1). Concentration
by ultrafiltration revealed that the protein further migrated as three major bands in
native PAGE (Figure 5.1 A, Lane 3) which further indicates that the P gigantea
cellulase was a multi-protein complex composed of different subunits. The 2D
analysis showed the IEF of the protein bands in the range of pI 4 to pI 7 (Figure 6.1,
Chapter 6).
5.3.3 Cellulase activity
The observation of Congo red stained CMC decolourising halos in solid
medium is a quick and fast practical way to verify presence of cellulase. The
enzymatic activity on the CMC plate was found to increase from day 2 onwards
reaching its peak on day 10 with the diameter of the zone of clearance of 1.5cm. The
increasing size of the zone of clearance demonstrates the cellulase activity being
Chapter 5 : Purification and characterisation of cellulase produced by Phlebia gigantea
112
further increased through the purification steps to reach 2.2cm. The Avicelase
specific activity measured by DNSA method was measured as 6.3 IU/mg after day 10
which continuously improved throughout the purification steps to reach the maximum
of 17.5 IU/mg.
Chapter 5 : Purification and characterisation of cellulase produced by Phlebia gigantea
113
Table 5.3 Summary of purification and yield of extracellular cellulase from P.gigantea.
Purification Steps
Total protein(m
g)
Total Activity(U)
Specific Activity(U/mg)
Yield (%)
Purification factor
Crude culture filtrate
126
795
6.3
100
1
Amorphus cellulose treatment
89
680
7.6
85
1.2
After Dialysis
48
620
12.9
78
2.04
Concentration by ultra filtration
28
490
17.5
62
2.8
Chapter 5 : Purification and characterisation of cellulase produced by Phlebia gigantea
114
Figure 5.1 PAGE Analysis of the affinity purified cellulase complex.
(Α) SDS PAGE: Lane 1, molecular mass marker; Lane 2, Standard commercial T. reesei RUT C30 cellulase used as control; Lane 3,. Purified
cellulase after concentration by ultrafiltration (B) Native PAGE; Lane 1, the affinity purified cellulase of P. gigantea
Chapter 5 : Purification and characterisation of cellulase produced by Phlebia gigantea
115
5.3.4 Zymogram analysis
A test for CMCase activity was performed on the proteins separated by SDS-
PAGE (Figure 5.2, Lane2). The major CMCase activity was associated with 38KDa
and 12KDa proteins (Pg12).
A test for CBHase activity was also performed on the proteins separated by
SDS (Figure 5.3, Lane 2). The major CBHase activity was associated with 68KDa
(Pg 68), 66KDa (Pg 66) and 45KDa (Pg 45). The other major protein of 190KDa (Pg
190) has shown activity band of less intensity as compared to Coomassie blue band.
The native and zymogram SDS results showed a 100KDa (Pg 100) protein as a major
protein which does not have any detectable hydrolytic activity but possess the highest
affinity for the cellulose (Figure 5.2, Lane 2; Figure 5.1.B, Lane 1).
Figure 5.2 CMC SDS PAGE CMCase analysis of the cellulase.
Lane 1, Protein ladder; Lane 2, Purified P. gigantea cellulase complex; and Lane 3,
Commercial T. reesei RUT C30. The CMCase activity is observed as clear band.
Chapter 5 : Purification and characterisation of cellulase produced by Phlebia gigantea
116
Figure 5.3 MUC-SDS PAGE CBHase analysis of the cellulase
Lane 1, Protein ladder; Lane 2, Purified P. gigantea cellulase complex; and Lane 3,
Commercial T. reesei RUT C30. The CBHase activity can be seen as bright band
Zymogram analysis.
5.3.5 pH optimum
The activity pH profile of the purified cellulase was studied by incubating
the enzyme fraction with 1% CMC over the pH range of pH 2- pH 8. The optimum
pH of the purified cellulase was recorded as pH 5.0 as depicted in Figure 5.4.
Chapter 5 : Purification and characterisation of cellulase produced by Phlebia gigantea
117
pH optimum Curve
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
2 3 4 5 6 7
pH
Activity (U/mg)
activity in IU/mg
Figure 5.4 pH optimum curve of purified P. gigantea cellulase.
The effect of pH on hydrolysis of CMC by P. gigantea cellulase complex. The
enzyme stability at different pH levels was determined by measuring the activity after
incubating the enzymes for 60 minutes at 50oC at pH 2.0 to 8.0.
5.3.6 Temperature optimum
The effect of temperature on the cellulase activity was studied by pre-
incubating the enzyme at range of temperatures from 4oC-70
oC for 120 minutes. The
cellulase activity of thus activated cellulase was measured by standard assay as
described in Chapter 4 (Section 4.2.5.2). The optimum temperature for the maximum
detectable cellulase activity was determined to be 50oC as shown in Figure 5.5.
Chapter 5 : Purification and characterisation of cellulase produced by Phlebia gigantea
118
Temperature Optimum Curve
0
0.1
0.2
0.3
0.4
0.5
0.6
4 10 20 40 50 60 70
Temperature (oC)
Activity (U/m
L)
Activity in
IU/mL
Figure 5 5 Temperature optimum curve of purified P. gigantea cellulase.
The effect of temperature on hydrolysis of CMC by P. gigantea cellulase complex,
the activity was determined after preincubation of cellulase complex and 1% (w/v)
CMC for 60 minutes at different temperatures in 100mM sodium acetate buffer (pH
5.0).
5.3.7 Kinetic properties
The measurement of initial rate of reducing sugars production from Avicel
and CMC at different substrate concentration yielded linear Lineweaver-Burk plots
Figure 5.6 and Figure 5.7 respectively. The Km, Vmax values obtained from these
plots with different substrates are presented in Table 5.4.
Chapter 5 : Purification and characterisation of cellulase produced by Phlebia gigantea
119
Table 5.4 Kinetic constants of P.gigantea cellulase at different concentration of
substrate.
Substrate Km (mg/mL) Vmax (IU/mg) Avicel 0.29 0.547
CMC 1.9 0.391
Figure 5.6 Lineweaver- Burk Plot for kinetics study of P. gigantea cellulase complex
with Avicel as substrate.
Chapter 5 : Purification and characterisation of cellulase produced by Phlebia gigantea
120
Figure 5.7 Lineweaver- Burk Plot for kinetics study of P. gigantea cellulase complex
with CMC as substrate.
5.4 Discussion
The use of an affinity digestion procedure to perform a rapid and effective
way of cellulase purification has previously been reported for C. thermocellum
(Morag (Morgenstern) et al., 1992; Zhang and Lynd, 2003). The adoption of this
procedure for application to cellulase enzyme in P. gigantea enabled purification with
a yield of 62%, achieving yields greater than reported for other fungal and bacterial
cellulases (Table 5.1) and was similar to the activity recovery as reported by Morag et
al.(Morag (Morgenstern) et al., 1992).
The two step purification procedure of affinity adsorption was adequate to
purify cellulase complex. The 38KDa molecular weight of the purified Pg 38 is
similar to molecular mass of endoglucanase V of Aspergillus aculeatus (36KDa)
Chapter 5 : Purification and characterisation of cellulase produced by Phlebia gigantea
121
(Muller et al., 1998) and endoglucanase IV of Trichoderma viride (38KDa) (Kim et
al., 1998). Other purified cellulases from this white rot fungus P.gigantea showed
consistency with 13KDa of endoglucanase of Trichoderma koningii (Churilova et al.,
1980), 45KDa in Piromyces Spp (Teunissen MJ et al., 1992).
The purified enzymes are acidic in nature with isoelectric point (pI) in the
range of pI 3.4 to 7.0. The isoelectric points are also comparable with pI of T. reesei
β- glucosidase (pI 4.5) (Chen et al., 1994) and endoglucanase III and IV of N. crassa
(pI 5.5 and 6.5) (Yazdi et al., 1990).
The enzyme complex shows some similarities to the commercial T. reesei
RUT C-30 cellulase complexes (Figure 5.1 a, Lane 2) used in this work as control
cellulase. The enzyme complex contains true cellulase activity and is composed of
multiple sub-units. However, the Pg 12 with a molecular mass of 14KDa although
smaller in size showed the strongest CMCase activity after the SDS exposure. This
smaller size, the relative ease of purification, the stability at room temperature, the
recovery of the CMCase and CBHase activities after SDS exposure makes the P.
gigantea cellulase complex a favourable cellulase system for study.
The proteins Pg 190, Pg 68 and Pg 60 have shown a significantly detectable
CBHase activity. The presence of multiple CMCase and CBHase activities in SDS-
PAGE system can be interpreted as a result of partial degradation of cellulase
complex into smaller, but still active fragments. All the cellulase containing factors
detected as different bands on the SDS-PAGE proved to be necessary for the cellulase
activity. Thus it is clear that in P. gigantea more than one type of CMCase and
Chapter 5 : Purification and characterisation of cellulase produced by Phlebia gigantea
122
CBHase is associated with the cellulase complex and that these cellulases are in a
different stoichiometric relationship. The detection of CBHase activity from its
subunits is similar to an active cellulase from C. thermocellum on native gel and the
observation that there was no detectable activity from protein bands after SDS PAGE
fractionation is similar to the observations reported by Shoseyov and Doi (Shoseyov
and Doi, 1990).
The purified enzyme complex has shown to have the maximum activity at
pH 5.0 and 50oC. The optimal conditions for different cellulases purified have been
shown to vary in the range of pH 4.0-pH 5.0 (Goto et al., 1992; Bok et al., 1998;
Palaniswamy, 1998; Li et al., 2000) and temperatures from 50oC -60
oC (Goto et al.,
1992; Pitson et al., 1997; Fang et al., 1998; Yernool et al., 2000). The Km and Vmax
values obtained with P. gigantea using CMC and Avicel as substrate were in the
range of 0.15-5.6mg/mL obtained in various studies (Goto et al., 1992; Bok et al.,
1998; Fang et al., 1998; Schulein, 1998).
In conclusion it is indicated that all the different proteins in the cellulases
mixture play an important role in hydrolysing cellulose substrate. Most of the
cellulase obtained was cell free with the high cellulase production in the filtrate. P.
gigantea is able to hydrolyse microcrystalline cellulose, but cellulase activity in cell
free filtrate was lower than when cultured in the soluble substrates CMC and
cellobiose. Thus, the results obtained highlights the fact that P. gigantea is a potential
cellulose degrader with wide substrate specificity with desirable features for various
industrial applications. Notwithstanding, further studies on this cellulase complex
Chapter 5 : Purification and characterisation of cellulase produced by Phlebia gigantea
123
with complex substrates like wheat straw and cotton are needed to verify this
hypothesis.
124
Chapter 6: Identification of proteins of cellulase complex from two dimensional
gels by molecular mass fingerprinting of peptide fragments
6.1 Introduction
Proteomics is the large scale study of proteins involved in the identification
of purified proteins necessary in many areas of bio-chemical research (Henzel et al.,
1993). The word proteomics has been associated with displaying a large number of
protein from a given cell line or organism on two dimensional polyacrylamide gels
electrophoresis (2D PAGE) (Andersen and Mann, 2000; Pandey and Mann, 2000;
Sechi, 2002). For most protein based approaches of gene function, mass-spectrometry
(MS) is the method of choice for rapid identification of the proteins (Perkins et al.,
1999; Andersen and Mann, 2000). MS is very sensitive and offers the possibility of
high throughput analysis. One of the crucial steps in proteomics is obtaining and
handling the protein sample of interest. One of the major challenges in proteomics is
the complexity of the initial samples to be analysed, with the dynamic range of
abundance of proteins in biological samples potentially as high as 106. The best 2D
gel electrophoresis can resolve only about 1000 proteins hence if a crude protein
mixture is used only the most abundant protein can be visualised. To get a better
resolution on 2D gels or for further analysis, the use of affinity based protein
purification strategies can be employed, reducing the complexities and differences in
abundance of proteins in complex samples.
The separation of protein using two-dimensional sodium dodecyl sulfate
polyacrylamide gel electrophoresis (2D-SDS-PAGE) was first described in 1975
(O'Farrell, 1975; Pandey and Mann, 2000; Sechi, 2002). In 2D-PAGE, the proteins
are separated with respect to two independent properties, isoelectric point (pI) and
Chapter 6: Identification of proteins of cellulase complex from two dimensional
125
molecular weight. Due to differences in pI and size the proteins appear in different
areas of the gel, where they are monitored as spots. Isomers of the same proteins
often appear as separate spots on the gel, since the pI is influenced by post-
translational modifications, and truncations alter protein size. After separation of the
proteins, the content of the spots can be examined taking different approaches.
Generally, the proteins are digested by an enzyme to yield shorter peptides, after
which peptide masses from each spot are detected by mass spectrometry. Each
protein gives rise to a characteristic peptide pattern, and the protein can be identified
by database searches against the detected peptide masses.
One of the most significant breakthroughs in proteomics has been mass-
spectrometric identification of gel separated proteins. This approach relies on
digestion of gel separated proteins into peptides by a specific protease such as trypsin.
The reason for analysing peptides rather than proteins is that the gel separated proteins
are difficult to elute and to analyse by MS, and the molecular weight of proteins is not
usually sufficient for database identification. Peptides are easily eluted from gels and
even a small set of peptides from a protein can provide sufficient information of
identification. In the ‘peptide-mass mapping’ approach suggested by Henzel et al.,
(1993), the mass spectrum of the eluted peptide mixture is acquired which results in
‘peptide-mass finger printing‘ of desired protein (Henzel et al., 1993; Pandey and
Mann, 2000). The masses of set peptides can be measured by matrix assisted laser
desorption ionisation (MALDI), where a co-precipitate of light absorbing matrix
(usually α-cyano-4-hydroxycinnamic acid or dihydroxy benzoic acid) and the peptide
solution is irradiated by a short pulse of UV light in a vacuum. Some of the released
peptides are ionised by attachment of protons and are accelerated in a strong electric
Chapter 6: Identification of proteins of cellulase complex from two dimensional
126
field. After being turned around by an energy correcting ion mirror, they are then
detected on a channeltron detector. The result of this measurement is a time of flight
(TOF MS) distribution of the peptide in the supernatant of the trypsin digested
protein. After determining the peak centroids and calibrating the spectrum a set of
highly accurate peptide masses is obtained. With state of the art MALDI mass
spectrometers, peak resolution of about 10000 and a mass accuracy of a few parts per
million can be obtained. The MALDI peptide mass mapping or mass finger printing
method has advantage of being scalable. Many samples can be deposited on a single
probe holder and measured in a single run.
The aim of this part of study was to characterise the cellulase degrading
proteins from the P. gigantea. This study is based on two facts; firstly, there are no
reports of P. gigantea cellulase genes, available from publicly accessible database.
And secondly, the comparative genomic approach to identify the cellulase gene
(Chapter 3) was not able to reveal much from the sequence homology with other
proteins in the databases. Based on these facts, the functional proteomics approach to
identify individual genes using MS is undertaken. The general approach is to
compare the experimental data of MS analysis for purified protein with the calculated
peptide mass values obtained, by applying appropriate cleavage rule to the entries in
the sequence database. The proteins are identified based on the corresponding
statistically significant mass values counted or scored by the peptide or protein
matches in the database.
Chapter 6: Identification of proteins of cellulase complex from two dimensional
127
6.2 Materials and methods
6.2.1 Protein purification and 2D Analysis by SDS-PAGE
Cellulase purification was carried out by affinity digestion as described in
Chapter 5 (Section 5.2.5). The purified cellulase complex was analysed by 2D gel
electrophoresis as described in (Section 6.2.2). The commercial T. reesei RUT C 30
cellulase mix was also analysed by 2D gel electrophoresis as a positive control for
comparative studies.
6.2.2 2D Gel electrophoresis
The protein fraction to be loaded on a 2D PAGE gel must be in low ionic
strength denaturing buffer that maintains the native charges.
6.2.2.1 Sample preparation
A sample volume of 100µL equivalent to 232µg of protein was mixed with
85µL of ReadyPrep™ rehydration buffer containing 10mL of 8M urea, 2% CHAPS,
50mM DTT, 0.2% (w/v) Bio-Lite®, 3/10 ampholytes and trace amount of
bromophenol blue. The sample is placed in the rehydration tray and is loaded with
the pH 3-10, 11 cm ready strip IPG Strips.
6.2.2.2 First dimension
Proteins were separated by electrophoresis according to charge. Isoelectric
focusing was carried out with 232µg of protein. Proteins were separated using gel
strips forming an immobilized non-linear pH gradient from 3 to 10 (ReadyStrip® IPG
strips, pH 3-10 NL, 11 cm, (Catalogue number: 1632016, Bio-Rad Laboratories Pty
Chapter 6: Identification of proteins of cellulase complex from two dimensional
128
Ltd.). After the rehydration of strips with sample in rehydration buffer for 12 hours at
room temperature, isolectricfocusing was performed in the Protean® IEF Cell (Bio-
Rad Laboratories Pty. Ltd.) at 20oC and 50µA current per strip. Proteins were
separated using the multistep focusing conditions under stepwise voltage changes.
The programme used includes first step of 250V for 20 minutes at linear ramp, second
step of 8000V for 2.5 Hrs for another linear ramp and final step of 8000V for 20,000
Volt hours with rapid ramp. Ramping or change in voltage helps to stabilize the
resistance of sample in run.
6.2.2.3 IPG equilibration for separation by second dimension
It is important to equilibrate proteins separated on IPG strips to solubilize the
focused proteins and to allow binding to SDS in second dimension gel. The gel strips
were equilibrated twice for 10 minutes each in 4mL of equilibration buffer I with 2%
(w/v) DTT and in equilibration buffer II containing 2.5% (w/v) iodoacetamide. Both
the equilibration buffer I and buffer II were made in 10mL of equilibration buffer base
containing 6M urea, 2% (w/v) SDS, 50% (w/v) glycerol, 1.5M Tris-Cl (pH 8.8) as per
the Bio-Rad 2D starter kit instruction manual (catalogue number: 163-2105, Bio-Rad
Laboratories Pty. Ltd.).
6.2.2.4 Second dimension
The proteins resolved in IPG strips in the first dimension were applied to
second-dimension gel and separated by molecular weight perpendicular to the first
dimension. The IPG strips were placed on top of Criterion® Bis-Tris 4-12%
polyacrylamide gels (catalogue number: 3450127, Bio-Rad Laboratories Pty. Ltd.)
Chapter 6: Identification of proteins of cellulase complex from two dimensional
129
and the gels were overlayed with agarose as per the manufacturers instructions. The
gels were then run in Criterion® gel running box at 200V for 45 minutes in 1X MES
buffer (catalogue number: 161-0789, Bio-Rad Laboratories Pty. Ltd)
6.2.2.5 Protein staining methods
Gels were stained with colloidal Coomassie Blue (Bio-Safe™ Coomassie,
Bio-Rad Laboratories Pty. Ltd.). Bio-Safe™ Coomassie stains broadest spectrum of
proteins and is ready to use, single reagent protein stain. The gels were stained
overnight in Bio-Safe™ Coomassie on shaker and then subsequently destained thrice
with MilliQ® water.
The stained gels were scanned with GS-800™ densitometer (Bio-Rad
Laboratories Pty. Ltd.).
6.2.3 Zymogram analysis of IEF gels
The zymogram analysis of the separated purified cellulase complex on IEF
gels was performed as described by Biely et al., (Biely et al., 1985). The purified
cellulase complex was separated by isoelectric focusing using pH 3-10, 11 cm,
ReadyStrip® IPG (catalogue number: 1632016, Bio-Rad) as described in Chapter 2
(Section 2.6.12). The overlay agar replica gel containing 1% (w/v) carboxymethyl
cellulose (CMC), 3% (w/v) agarose and 50mM sodium acetate buffer pH 5.5 was set
on the glass plates. The set overlay CMC agar gel was kept warm to about 40oC in
incubator and the IEF gel with separated proteins was laid immediately after
electrophoresis. The gel was incubated further for another 60 minutes at 40oC. The
IEF gel was removed and the CMC overlay agar gel was incubated into pre-warmed
50mM sodium acetate buffer (pH 5.5) for another 60 minutes. The gel was then
Chapter 6: Identification of proteins of cellulase complex from two dimensional
130
transferred for staining into the 1% congo red staining solution for 30 minutes
followed by repeated wash of 1M sodium chloride for destaining. The zones
corresponding to cellulase activity appeared as pale red or completely destained areas
on the transparent red background.
6.2.4 Mass Spectrometry
The spots of interest determined from zymogram analysis were excised from
the two-dimensional gels and underwent reduction (25mM dithiothreitol/25 mM
ammonium bicarbonate) at 56oC for 30 minutes and alkylation (55mM
Iodoacetamide/ 25mM ammonium bicarbonate) at room temperature for 30 minutes.
The samples were then subject to a 16 hour tryptic digest at 37oC. Samples were
concentrated and desalted using a Millipore C18 ZipTip and 1µL aliquot was spotted
onto a sample plate with 1µL of matrix (α-cyano-4-hydroxycinnamic acid, 4 mg/mL
in 70% v/v can, 1% v/v TFA) and allowed to air dry. Matrix assisted laser desorption
ionisation (MALDI) mass spectrometry was performed with an Applied Biosystems
4700 Proteomics Analyser with TOF/TOF optics in MS mode. An Nd:YAG laser
(355 nm) was used to irradiate the sample. The spectra were acquired in reflectrom
mode in the mass range 750 to 3500 Th.
The instrument was then switched to MS/MS (TOF/TOF) mode where the
eight strongest peptides from the MS scan were isolated and fragmented (by collision
induces dissociation with filtered lab air), then re-accelerated to measure their mass
and intensities. A near point external calibration was applied and will give a typical
mass accuracy of ~50 ppm or less.
Chapter 6: Identification of proteins of cellulase complex from two dimensional
131
The data was exported in a format suitable for submission to the database
search program Mascot (Matrix Science Ltd, London UK). The data was screened
with High scores in the database search indicating a likely match. The MS analysis
result was facilitated by access to the Australian Proteome Analysis Facility (APAF)
under the Australian government’s major national Research facilities programme.
6.2.5 Protein Identification
Database searching was carried out using the Aldente programme available
via internet at http://www.expasy.org/tools/aldente/. The parameters for Aldente
searches were as followed: 1 missed cleavage; 2 and 0.4 Da mass accuracy allowed
for parent and the fragment ions, respectively, carbamidomethyl as fixed
modification, oxidized methionine, acetylation of the peptide or the N-terminus as
variable modifications. Masses larger than 800 and less than 3500 Da obtained from
MALDI mass spectrometry were input into the Aldente program. A molecular mass
range based on estimates from the second gel dimension (Figure 2) was used to limit
the size of the proteins searched in the database by the computer program. This value
was obtained by increasing the observed molecular mass by 10-20% to allow for
potential posttranslational modifications. The program default threshold value of
slope maximum +/- 200 ppm, internal errors +- 25 ppm, and shift max +- 0.2 Da was
used. The process filter was set for maximum 4 hits and pValue of 1.
Identification is based on experimental MS peaks to be matched with
theoretical peptide masses. Proteins were identified with mass measurement accuracy
of +/- 10 ppm and pValue below 1.
Chapter 6: Identification of proteins of cellulase complex from two dimensional
132
All database searches were performed using a generic database at
UniProtKB/ Swiss-Prot or UniProtKB/TrEMBL (http://au.expasy.org/databases/),
with no species specified. Positive identification was checked with the percentage of
sequence covered and the masses match the significant peaks in the MS spectra.
The identified protein was then classified according to the Glycosyl
Hydrolase family and the theoretical peptide mass for the identified protein was
obtained using the PeptideMass (http:/au.expasy.org/tools/peptide-mass.html). The
theoretical peptide mass is then matched with the related P. gigantea protein peptide
mass using FindPept (http://au.expasy.org/tools/findpept.html) and FindMod
(http://au.expasy.org/tools/findmod/) programme at publicly accessible SwissProt
website. The similar proteins from the identified Glycosyl Hydrolase family were
selected from CAZy database (http://afmb.cnrs-mrs.fr/CAZY/). The theoretical
peptide mass of this selected protein from Glycosyl Hydrolase family was matched
with the respective P. gigantea purified protein peptide mass.
The parameters used for FindPept and PeptideMass tools were kept similar
to those as in Aldente program
6.3 Results
6.3.1 Protein Purification
The cellulase complex was purified as described in Chapter 5 by affinity
adsorption method. The purified cellulase complex was analysed by SDS gel
electrophoresis (Figure 5.1, Chapter 5) and zymogram analysis on SDS gel using
Chapter 6: Identification of proteins of cellulase complex from two dimensional
133
CMC (Figure 5.2, Chapter 5) and MUC analysis (Figure 5.3, Chapter 5) as described
in Chapter 5 (Section 5.27 and 5.2.8).
6.3.2 2D gel Analysis
The 2D gel analysis of the purified cellulase complex has shown 15 well
separated spots in the second dimension SDS gel (Figure 6.1). The pI of separated
spots, range from pI 3.5 to pI 7. The molecular weight of the separated proteins was
calculated from 190KDa to 14KDa using protein ladder (Seablue® Pre-stained
standard, Invitrogen Life Technologies). The gel was scanned in densitometer and the
spots were excised for further identification by MS analysis. Three spots, PgCel 2
(spot 2), PgCel 5 (spot 5) and PgCel 7 (spot 7) were selected based on the IEF
zymogram (Figure 6.2) and the SDS zymogram analysis (Figure 5.2 and Figure 5.3,
Chapter 5). The estimated experimental molecular weight and the pI of the selected
spots were calculated as described in Table 6.1. The commercial cellulase complexes
of T. reesei RUT C30 showed a similar pattern of separated spots as the purified
cellulase complex of P gigantea.
Chapter 6: Identification of proteins of cellulase complex from two dimensional
134
Figure 6.1 2D analysis of affinity adsorption purified cellulase.
M denotes molecular mass marker. The Isoelectric Focussing (IEF) range from pI 3 to pI 10 runs from right to left
Chapter 6: Identification of proteins of cellulase complex from two dimensional
135
Figure 6.2 IEF zymogram analysis of P. gigantea cellulase complex.
The activity of P. gigantea cellulase complex and of commercial T. reesei cellulase as
a positive control on the CMC gels can be seen as the bright yellow spots on the red
background.
6.3.3 Zymogram analysis of IEF gels
The cellulolytic enzyme complex of P.gigantea purified by affinity
adsorption was resolved by isoelectric focusing and the activity was demonstrated by
means of carboxy methyl cellulose containing agar replicas (Figure 6.2). The
cellulase activity was detected by clear halos or spots on the agar replica gels at pI
value of 3.9 to 5.0.
6.3.4 M S Analysis and protein identification
The three excised spots from the 2D gels were analysed by mass
spectrometry after reduction and alkylation. Figures 6.3, 6.4, and 6.5 shows the
spectra of the tryptic digest of spots PgCel 2, PgCel 5 and PgCel 7 respectively after
reduction and alkylation. Figure 6.6 shows spectra of the tryptic digest of spots PgCel
Chapter 6: Identification of proteins of cellulase complex from two dimensional
136
5 before reduction and alkylation. A summary of the output of the Aldente program
obtained from analysis of spots PgCel 2, PgCel 5 and PgCel 7 is shown in Tables 6.2,
6.3 and 6.4. These tables list all observed masses that were used by the program and
the peptide sequences that matched each mass value.
Masses that are not listed in table did not match with any of the predicted
masses but did not prevent the program from identifying the protein. Although most
peptides in Table 6.2, 6.3 and 6.4 contain masses more accurate than ±4 Da, we chose
this value to reflect the largest error observed. Larger mass tolerances increase the
possibility of obtaining proteins that are unrelated to the sample. Decreasing the mass
tolerance results in higher confidence in the matches but also increases the chance of
missing a match. By using this search strategy to analyse the data from the protein
spots by Aldente, few proteins were usually identified from the >91,000 proteins in
the database. The most perfect match from this result with respect to activity,
molecular mass and pI values are selected to validate further. The spots PgCel 2,
PgCel 5 and PgCel 7 resulted in proteins from more than one species of protein being
identified, since the masses used result from sequences which are invariant between
species. The Aldente programme thus identified Spot PgCel 7 peptides was matching
with 52-kDa β-glucosidase A (EC 3.2.1.21), spot PgCel 5 with α-glucosidase and spot
2 with α-amylase protein (Table 6.1). All the three proteins identified based on
significant peptide mass matches belong to the glycosyl hydrolase family as described
in CAZy web site. The proteins PgCel 2, PgCel 5 and PgCel 7 were found to be from
GH family 13, 1 and 4 respectively.
Chapter 6: Identification of proteins of cellulase complex from two dimensional
137
Figure 6.3 M
ALDI-TOF-M
S spectrum of P.gigantea cellulase complex of from 2D gel spot PgCel2.
MALDI-TOF-M
S spectrum of the peptides identified from a tryptic digest of protein spot PgCel2 taken from 2D gel of P. gigantea cellulase
complex (Figure 6.1) after reduction and alkylation reaction. M
ALDI was perform
ed by the Australian Proteome Analysis Facility (APAF) with
an A
pplied B
iosystem
s 4700 Proteomics Analyser with TOF/TOF optics in M
S m
ode. The spectra were acquired in reflectrom m
ode in the
mass range 750 to 3500 Th.
Chapter 6: Identification of proteins of cellulase complex from two dimensional
138
Figure 6.4 M
ALDI-TOF-M
S spectrum of P.gigantea cellulase complex of 2D gel spot PgCel 5.
MALDI-TOF-M
S spectrum of the peptides identified from a tryptic digest of protein spot PgCel5 taken from 2D gel of P. gigantea cellulase
complex (Figure 6.1) after reduction and alkylation reaction. M
ALDI was perform
ed by the Australian Proteome Analysis Facility (APAF) with
an A
pplied B
iosystem
s 4700 Proteomics Analyser with TOF/TOF optics in M
S m
ode. The spectra were acquired in reflectrom m
ode in the
mass range 750 to 3500 Th.
Chapter 6: Identification of proteins of cellulase complex from two dimensional
139
Figure 6.5 M
ALDI-TOF-M
S spectrum of P.gigantea cellulase complex of 2D gel spot PgCel 7.
MALDI-TOF-M
S spectrum of the peptides identified from a tryptic digest of protein spot PgCel7 taken from 2D gel of P. gigantea cellulase
complex (Figure 6.1) after reduction and alkylation reaction. M
ALDI was perform
ed by the Australian Proteome Analysis Facility (APAF) with
an A
pplied B
iosystem
s 4700 Proteomics Analyser with TOF/TOF optics in M
S m
ode. The spectra were acquired in reflectrom m
ode in the
mass range 750 to 3500 Th.
Chapter 6: Identification of proteins of cellulase complex from two dimensional
140
Figure 6.6 M
ALDI-TOF-M
S spectrum of P.gigantea cellulase complex of 2D gel spot PgCel 5.
MALDI-TOF-M
S spectrum of the peptides identified from a tryptic digest of protein spot PgCel5 taken from 2D gel of P. gigantea cellulase
complex (Figure 6.1) after reduction and alkylation reaction. M
ALDI was perform
ed by the Australian Proteome Analysis Facility (APAF) with
an A
pplied B
iosystem
s 4700 Proteomics Analyser with TOF/TOF optics in M
S m
ode. The spectra were acquired in reflectrom m
ode in the
mass range 750 to 3500 Th.
Chapter 6: Identification of proteins of cellulase complex from two dimensional
141
These respective GH families were then further screened to validate the
identification by using programme PeptideMass, FindPept and FindMod based on the
molecular mass match and pI similarities. The results are shown in Table 6.2, 6.3 and
6.4 respectively. This table lists all observed masses that were used by the program
and the peptide sequences those match with each mass value after specific cleavage.
The peptide mass match analyses of PgCel 5 and PgCel 7 were identified as α-
glucosidase and β-glucosidase from glycosyl hydrolase family 4 and family 1
respectively. The protein PgCel 2 was identified as Glycoside hydrolase from family
13. The identified proteins were cross checked for their presence in carbohydrate
binding module (CBM) families in CAZy web site using FindMod programme
keeping the same constraints of search as those used earlier with Aldente, and
FindPept programme (Table 6.6, 6.7 and 6.8). The basis of selecting data from the
database was limited to the experimental molecular mass and pI of the selected
proteins. Further the enzymic assay and zymogram displayed the cellulase activity
confirming the identifications to be true.
All the proteins identified were proteins associated with hydrolase activity
and in carbohydrate metabolism. For example, α-glucosidase (pullulanase) is
associated with the endohydrolysis of 1-4, α-glucosidic linkages in oligosaccharides
and in hydrolysis of (1-6) - α -D-glucosidic linkages in pullulan, and the alpha- and
beta-limit dextrins of amylopectin and glycogen (Table 6.5) (Boeckmann B et al.,
2003; Bairoch et al., 2005).
Chapter 6: Identification of proteins of cellulase complex from two dimensional
142
Table 6.1 M
atching peptide mass of three P. gigantea proteins using Aldente from SwisprotTREMBL database
Sample
Accession
No.
Exp
MW
KDa
Exp
pI
Description
Cov.
%
Theo.
MW
KDa
Theo
pI
Taxon
pValue
PgCel5(6)
Q9AGA6
52
5.5
6-phospho-alpha-glucosidase (EC 3.2.1.122)
20
49
5.7
Other
Bacteria
1.9 e-4
PgCel7
Q08638
52
5.9-
6.0
Beta-glucosidase A (E C 3.2.1.21 ( Gentiobiase)
(Cellobiase)
15
52
5.6
Other
Bacteria
0.013
PgCel2(4)
Q414G3
98
3.5
Glycoside
Hydrolase, family13 N-terminal: alpha
amylase catalytic region
10
80.234
4.9
Other
Bacteria
0.037
Exp M
W K
Da- Experim
ental molecular weight in K
Da; Exp pI-Experim
ental pI; C
ov.%
- Sequence C
overage percentage; Theo. MW K
Da-
Theoretical molecular weight in KDa; Theo pI- Theoretical pI.
Chapter 6: Identification of proteins of cellulase complex from two dimensional
143
Table 6.2 Summary of matching peptides of spot PgCel2 for specific cleavage
Protein
identified
by
Protein
Identified/Acc.
No./Mass/pI
GH
famil
y
MALDI
Mass (Da)
Exp
MALDI
Mass (Da)
Theo
∆ mass
(Da)
Peptide sequence consistent with mass
Aldente
Alpha amylase
Q414G3 /
80KDa / 4.9
13
963.414
1007.435
1349.817
1349.817
1714.784
2211.0806
3095.6196
3252.7578
3409.593
963.489
1007.517
1349.644
1349.682
1715.007
2211.0788
3095.5341
3252.6404
3409.653
0.075
0.081
-0.172
-0.134
0.223
-0.08
0.12
0.09
0.060
(R)/GQQVQEFK/(S)
(R)/YGYRVHGR/(F)
(R)/ISGSSDLYAHSGR/(K)
(R)/VHGRFHPESGAR/(C)
(R)/ELLAFTQRVVNLRR/(T)
TLGEFASRISGSSDLYAHSGR
HPHVLQLIM
DSLRYWVTEMHHVDGFR
HYFDTTGTGNSLLMRHPHVLQLIM
DSLR
(R)/YWVTEMHVDGFRFTLAATLARQFHEVDR/(L)
GH Family
Pullulanase/
Q93UZ9/
128KDa/ 4.4
13
890.406
2045.039
2470.305
2722.283
2722.283
890.498
2045.066
2470.319
2722.398
2722.446
0.092
0.027
0.013
0.115
0.162
(K)/IA
LDPYAK/(S)
(R)/GVWEVQLTKENTGLDSLR/(G)
(K)/EPKEPKEPKEPKEPKDPKDPK/(D)
(R)/LGNLMTLTSQGIA
FLHAGQEYGRTK/(Q)
(K)/KLID
EIH
SRGMGVVLDVVYNHTAR/(V)
GH Family
Glucodextranase/
O00019/
74KDa/ 4.48
13
1007.435
2045.039
2408.057
3409.593
3409.593
1007.552
2045.132
2408.264
3409.679
3409.943
0.116
0.093
0.207
0.086
0.349
(R)/KLEEIY
GR/(D)
(K)/LKHMIR
KLEEIY
GRDK/(I)
(K)/ATSVHIA
AEGANQGRNLKDIN
TK/(V)
(K)/VPKDGEASASSFSTPTSILSNADMGNNISSLLAK/(K)
(R)/QRTDPVLHIELRRWADILVVAPLTANTLAK/(I)
Chapter 6: Identification of proteins of cellulase complex from two dimensional
144
Table 6.3 Summary of matching peptides of spot PgCel5 for specific cleavage.
Protein
identified
using
Protein
identified/ Acc.
No. Mass/pI
GH
family
MALDI
Mass (Da)
Exp
MALDI
Mass (Da)
Theo
∆ mass
(Da)
Peptide sequence consistent with mass
Aldente
Alpha
glucosidase/Q9AG
A6 /
49KDa / 5.69
4
1967.951
2225.1113
2284.1338
2300.134
2593.2322
2807.279
3084.396
3084.396
1967.9296
2225.0784
2284.1764
2300.1713
2593.2626
2807.512
3084.499
3084.4020
0.02
0.03
-0.04
-0.04
-0.03
0.233
0.102
-0.01
YYLFPDYVVAHSNPER
YYLFPDYVVAHSNPERTR
GLMSQQVAVEKLVVDAWEQR
GLMSQQVAVEKLVVDAWEQR
YSPNAWMLNYSNPAAIV
AEATRR
(K)/KFSVVIA
GGGSTFTPGIV
LMLLANQDR/(F)
(R)/EQDEKIPLRHGVLGQETCGPGGIA
YGMR/(S)
EYVAKYGYVPPSNDPHTEASWNDTFAK
GH Family
Alpha glucosidase/
O86960/
52KDa/ 6.02
4
1962.940
3033.343
1962.920
3033.662
-0.019
0.318
(K)/AVGFCHGHYGVMEIIEK/(L)
(R)/FVIN
IPNKGIIQGID
DDVVVEVPAVVDR/(D)
GH Family
Beta-glucosidase
Q9X108/
47 KDa/ 5.76
4
869.481
1210.659
1314.804
1857.962
2084.035
3033.343
869.484
1210.701
1314.690
1857.978
2084.164
3033.667
0.002
0.042
-0.114
0.015
0.192
0.323
(R)/VHTLSQGK/(G)
(K)/KISTHELRAR/(E)
(K)/DLLEEILEANR/(E)
(R)/EVMKIEKELFEKYR/(T)
(K)/KISTHELRAREVMKIEK/(E)
(K)/KISTHELRAREVMKIEKELFEKYR/(T)
Chapter 6: Identification of proteins of cellulase complex from two dimensional
145
Table 6.4 Summary of matching peptides of spot PgCel7 for specific cleavage.
Protein
identified
using
Protein
identified/ Acc.
No. Mass/pI
GH
family
MALDI
Mass (Da)
Exp
MALDI
Mass (Da)
Theo
∆ mass
(Da)
Peptide sequence consistent with mass
Aldente
Beta- glucosidase
A /Q08638 /
52KDa / 5.6
1
1383.6776
1540.7887
2083.0029
2564.1863
1383.7740
1540.8281
2083.0141
2564.2830
-0.10
-0.04
-0.01
-0.10
EDIEIIEKLGVK
IDYLKAHIG
QAWK
DGKIG
IVFNNGYFEPASEK
IDFVGLNYYSGHLVKFDPDAPK
Aldente
Oligo-1,6-
glucosidase/
13
973.5192
1179.5878
1591.775
2408.0566
2564.1863
2766.3547
973.5224
1179.5956
1591.717
2408.0972
2564.1806
2766.3505
-0.00
-0.01
-0.058
-0.04
0.01
0.00
ASLSRWQK
DFYIW
RPGK
(R)/DNARTPMQWDETK/(H)
FDSIEQYQDIETLNMYKEK
AIM
DEFGTMADWETLLAEIH
TR
GLQDIG
WNSLYWNNHDQPRIV
SR
GH Family
Beta-
glucosidaseA/
O33843/ 51KDa/
5.67
1
1142.560
1323.637
2448.161
1142.679
1323.706
2448.264
0.119
0.068
0.103
(R)/AHAKSVKVFR/(E)
(R)/VLFENFGDRVK/(H)
(K)/VHDQNRID
YLRAHIEQVWR/(A)
GH Family
Beta-
glucosidaseA/
O52505/ 51KDa/
5.67
1
1142.560
1323.637
2448.161
1142.679
1323.706
2448.264
0.119
0.068
0.103
(R)/AHAKSVKVFR/(E)
(R)/VLFENFGDRVK/(H)
(K)/VHDQNRID
YLRAHIEQVWR/(A)
GH Family
Beta-
glucosidaseA/
O93784/ 51KDa/
5.67
1
2347.983
2408.076
3339.780
2348.370
2408.076
3339.641
0.386
0.018
-0.138
(R)/YPKKSAKSLKPLFDSLIR
KE
(K)/YFNDYVRAMAAAVAEDGCNVR/(G)
(K)/IY
VTENGTSLKGENDMPLEQVLEDDFRVK/(Y)
Chapter 6: Identification of proteins of cellulase complex from two dimensional
146
Table 6.5 Biological and m
olecular significance of few selected proteins
Family
Known Activity(CAZy)
Accession
No.
Molecular function
Biological Process
-glucosidase
Q9AEN6
hydrolase activity, hydrolysing O-glycosyl compounds
carbohydrate m
etabolism
-glucosidase
O93784
Hydrolase Activity
carbohydrate m
etabolism
1
-glucosidase
Q08638
Hydrolysis of term
inal, non reducing β-D
-glucose.
Cellulose degradation
-glucosidase
Q9AGA6
Hydrolyzes a wide variety of 6-phospho--D
-glucoside including m
altose-6’P
isomaltose, malilitol, trehalose &
6 phosphorylated derivatives of the five
linkages isomeric -D
-glucosyl-D-fructoses
-glucosidase
O86960
hydrolase activity
carbohydrate m
etabolism
4
β-glucosidase
Q9X108
hydrolyse cellobiose – 6-p to glucose 6-p &
glucose
Pullulanase
Q93UZ9
alpha-amylase activity, carbohydrate binding, hydrolase activity
carbohydrate m
etabolism
Glucodextranase
QC8438
Protein binding
Protein phosphatase inhibitor activity
G I/S transition of mitotic cell
cycle
monovalent
inorganic
cation homeostasis.
13
-glucosidase
P38939
Endo hydrolysis of1-4-- glucosidic linkages in oligosaccharides &
polysaccharides, hydrolysis of (1-6)
--D glucosidase linkages of pullulan and
in amylopectin and glycogen
-amylase
Q414G3
amylase activity, glycogen debranching activity, hydrolase activity,
hydrolysing O-glycosyl compound.
carbohydrate
metabolism
,
glycogen m
etabolism
Chapter 6: Identification of proteins of cellulase complex from two dimensional
147
Table 6.6 M
atching peptide mass of PgCel 2 with CBM fam
ily from CAZy database
Exp M
m
Theo M
m
∆ M
m
Peptide sequence consistent with mass
C B M
MW
KDa
pI
Description
Acc. No
963.414
1581.936
2768.389
3095.620
963.5145
1581.8423
2768.2831
3095.662
0.101
-0.093
-0.105
0.046
DALDFAALK
VGLPWHVVESYPAK
DGSLNIPESGNGYPDILDEARYNMK
FISISKAQGYGVPLEEKVISSPFDASVVK
4
94
5.08
cellulase
Q46002
2045.039
2045.005
-0.032
(R)/AVQVMFWAHEWAKEQGK/(E)
2
107
4.39
β- 1- 4 exocellulase
Q9XCD4
1085.472
1085.592
0.120
MNFKKYVR/(K)
3
85
4.96
Cellulase
Q9EYQ4
1349.817
1349.707
-0.109
(K)/ALLHNQSGWPAR/(V)
3
107
4.68
endoglucanase/
Avicellase
P23659
800.360
1405.128
800.455
1404.748
0.095
-0.379
(K)/ITLYYK/(G)
GVADHPVVISAPDK
3
75
4.68
1, 4- β -
cellobiohydrolase
Q9L3J2
1277.636
1277.6161
-0.019
QLYTFADTYR
3
90
4.4
endoglucanese/cellul
ose
P26221
815.360
1085.472
2225.102
2663.362
815.4410
1085.6023
2225.0583
2663.3504
0.081
0.13
-0.043
-0.011
FVGLDHK
IMLDIH
SIK
NYGKKDYPLLFDENYMSK
YKNVVTNVTFWGLKDDYSWLSK
3
113
5.55
Β glucanase
O24828
Chapter 6: Identification of proteins of cellulase complex from two dimensional
148
Table 6.7 M
atching peptide mass of PgCel 5 with CBM fam
ily from CAZy database
Exp M
m
Theo M
m
∆ M
m
Peptide sequence consistent with mass
CBM
MW
KDa
pI
Description
Acc. No
895.507
2084.0
895.4632
2083.9535
-0.043
-0.08
YSGNKAGAK
CTSSGCSDVKGSVVID
ANWAR
1
53
5.93
exoglucanase
P46238
869.481
1857.962
869.447
1857.7279
-0.033
-0.233
DPSAPGVAR
NTGLCDHGDGCDFNSFR
1
54
4.73
cellulase
Q09431
1314.804
1543.774
1314.7164
1543.6668
-0.087
-0.106
IELASHATAVFR
13
57
4.85
α- galactosidase
P28351
1835.975
1835.8891
-0.085
MQMPADQYKLQQQAR
2
52
5.39
β – 1, 4-
endoxylanase
P74912
2225.111
2224.9676
-0.142
DCAAAASNGEWAIA
NNGANNYK
1
52
5.58
Avicelase
Q9C1S9
Chapter 6: Identification of proteins of cellulase complex from two dimensional
149
Table 6.8 M
atching peptide mass of PgCel 7 with CBM fam
ily from CAZy database.
Exp M
m
Theo M
m
∆ M
m
Peptide sequence consistent with mass
CBM
MW
KDa
pI
Description
Acc. No
836.272
1591.775
2448.161
836.4117
1591.5747
2448.1174
0.14
-0.199
-0.043
GGMKQMGK
CEGDSCGGTYSNER
FIN
GEANIEGWTGSTNDPNAGAGR
1
54
4.84
exoglucanase
P15828
1475.751
1837.936
2083.003
2664.373
1475.8216
1837.8385
2083.0355
2664.3277
0.071
-0.096
0.032
-0.044
SNGLYNLVVDLLR
TGNVSPPSQFTLNGATCS
DYIA
HAFRKAHEVDPDAK
GQYNWSQADNIINFAKANNQIV
R
2
52
5.39
β-1,4 endoxylanase
P74912
2408.057
2922.368
3097.629
2408.0823
2922.3951
3097.3818
0.025
0.027
-0.246
LSMYIQ
EDGYETNPRGFTDR
SGAVFQPGTCASGGPWSGSSLKASGQWVR
GMSTHGIQ
WFDHCLTDSSLDALAYDWK
2
46
5.00
Endoglucnase/cellul
ase E5
Q01786
886.973
2039.945
2517.300
886.5356
2040.0185
2517.3095
-0.436
0.073
0.009
LKDEIIR
GQGIPTFKPYNLSDAEFR
YAPLIQ
GGVTPPPGKPNAPTDVDGR
12
52
6.19
Chitinase
Q54442
1277.697
1890.965
1277.5731
1890.9959
-0.123
0.031
QQWGDWCNIK
AASVANIPTFTWLDSVAK
1
48
5.04
Exocellobiohydrola
se
Q02321
973.519
3061.607
973.5312
3061.4393
0.012
-0.167
TSLPEVATR
MIA
GQQDLTWKDAID
MYQRVFDDTGK
10
53
4.97
Endo-β-1,4-
mannanase
Q840B9
Chapter 6: Identification of proteins of cellulase complex from two dimensional
150
6.4 Discussion
Mass spectrometry is a technique that helps to understand protein structural
information such as peptide masses or amino acid sequences (Perkins et al., 1999;
Graves et al., 2002; Smith, 2002). This information of peptide mass can be used to
identify the protein by searching protein database (Perkins et al., 1999; Pandey and
Mann, 2000). One of the method of protein identification is peptide mass
fingerprinting (Sechi, 2002). In this method the masses of the peptides obtained from
the proteolytic digestion of the unknown protein are compared to the predicted masses
of peptides from the theoretical digestion of proteins in the database. Protein
identification is validated based on the number of matches in real and the theoretical
mass spectrum (Andersen and Mann, 2000; Sechi, 2002). The identification is also
dependent on the quality of the purified cellulase complex and the affinity purification
process with a specific function in mind to obtain purified multi-protein complex is
successfully employed in this study and is screened accordingly with the specific
assays and zymogram analysis (Perkins et al., 1999; Andersen and Mann, 2000;
Marmagne et al., 2004). The proteins identified from the MALDI-TOF analysis
exhibited negative or near zero P values. The cysteine alkylation procedure was used
to increase the number of peptides observed in the MALDI-TOF spectra and
constraining the database search for improving the process of protein identification by
peptide mapping.
Cellulases are the subsets of glycosidases, which are involved in the
hydrolysis of glycosidic bonds and carbohydrate metabolism (Davies and Henrissat,
1995; Coutinho and Henrissat, 1999a). All the three proteins identified in this study
based on peptide mass match data were found to be included in the glycosyl hydrolase
Chapter 6: Identification of proteins of cellulase complex from two dimensional
151
families, and has also showed the matches in the cellulose binding module family at
CAZy web site (Table 6.6, 6.7 and 6.8) (Coutinho and Henrissat, 1999a; Coutinho and
Henrissat, 1999b). Although, only one of the purified protein (PgCel 7) was located a
match in cellulase containing family 1, the other two proteins PgCel 2 and PgCel 5
identified in family 4 and family 13 enzymes. These families are well related to the
carbohydrate metabolism and hydrolysing activity as shown in Table 6.5. The
possible explanation for finding such diversity in the purified proteins lies in the
diversity found in glycosyl hydrolases. It has been found that several glycoside
hydrolase families (families 4, 5, 10, 13, 37) contain enzymes with different substrate
specificities suggesting divergent evolution of a basic fold at the active site to
accommodate different substrates (Lynd and Zhang, 2002). At the same time the
cellulases were found to be accommodated in different families (families 5, 6, 8, 9,
10, 11. 12) (Coutinho and Henrissat, 1999a) suggesting the convergent evolution of
different folds in resulting in the same substrate specificity (Lynd and Zhang, 2002)
It is also evident from the study on the model white rot fungus,
Phanerochaete chrysosporium that this wood decay fungus excretes multiple and
diverse extracellular enzymes belonging to glycoside hydrolase group in presence of
cellulose as the sole carbon source to completely degrade the major polymers of wood
such as cellulose, hemicellulose and lignin (Lynd et al., 2002; Howard R.L., 2003;
Martinez et al., 2004; Wymelenberg et al., 2005). The three proteins identified after
selective purification based on affinity adsorption has displayed this diversity by
identifying the matches with different range of glycoside hydrolases. However, the
further studies on the remaining other proteins isolated on 2D gels will be able to
Chapter 6: Identification of proteins of cellulase complex from two dimensional
152
reveal more detailed mechanism of the cellulose degradation by this potential
saprophytic fungus, P. gigantea.
As found in the P. chrysosporium (Wymelenberg et al., 2005), it is predicted
that P. gigantea must be expressing the families of structurally related genes which
can be identified in different glycoside hydrolase families. Although the
identification of the 3 enzymes studied does not confirm the substrate based
differential regulation among closely related genes in P. gigantea, this study suggests
the use of mass spectrometry analysis technique as a tool to study the cellulose
degradation mechanism of P. gigantea.
Since our strategy for this study was based on obtaining the sequence
information from low quantity of purified protein complex, the identified peptide
sequence will enable us to design oligonucleotide probes for gene cloning.
153
Chapter 7 : Final Discussion and future prospects
Biomass is the energy trapped and converted by photosynthesis and it
comprises upto half of all the organic matter produced in the biosphere (Henriksson et
al., 1995; Lynd et al., 2002; Lynd and Zhang, 2002). The major and universal
component of biomass is lignocellulose (Beguin and Aubert, 1994; Bayer et al.,
1998b; Bhat, 2000). Utilization of the vast resource of available biomass will help
deal with our dependence on the fossil fuels as well as food and animal feed shortages
(Bhat and Bhat, 1997; Lynd et al., 2002; Howard R.L., 2003). Organisms with the
ability to utilise this chemically complex energy source are widespread in nature in
the form of a wide variety fungi and bacteria species. However, there are four
interrelated constraints that currently restrict the progress in industrial application of
the degradation of lignocellulose by enzymes, and these are i) the nature of the
substrates, ii) the microorganisms themselves, iii) the characteristics of natural
enzymes and iv) the chemical nature of intermediates and end products of
lignocellulose digestion and the effects these intermediates and end products can have
on degradative enzyme systems (Klyosov, 1995a, b; Lee, 1997; Lynd et al., 2002).
Natural cellulose is a crystalline polymer generally associated with
hemicellulose and lignin in a matrix that as a substrate is highly resistant to enzymatic
attack (Beguin and Aubert, 1994; Bayer et al., 1998b; Mai et al., 2004). To use the
cellulosic material in biomass as a renewable source of energy it is necessary to
decrease the crystallinity and degree of polymerisation. However the efficient
degradation of lignocellulose is affected by degradation intermediates that can attach
to lignin to further increase the structural complexity of the substrate (Klyosov,
1995a; Howard R.L., 2003). It is evident that for such a complex structure to be
Chapter 7 : Final Discussion and future prospects
154
efficiently and completely degraded there is a requirement for a group of enzymes
acting synergistically rather than a single enzyme acting in isolation (Hibbett and
Donoghue, 2001; Leonowicz et al., 2001; Xun Sun, 2004; D'Annibale et al., 2005).
A large number of microorganisms are capable of biodegradation of
lignocellulose material, using variety of different hydrolases to breakdown the
polysaccharides present in lignin to smaller units (Bhat and Bhat, 1997; Himmel et al.,
1999; Henrissat and Davies, 2000; Lynd et al., 2002; Howard R.L., 2003; Xun Sun,
2004). In taxonomic terms the degradation of wood in nature is predominantly
performed by the basidiomycetes group of fungi (Beguin and Aubert, 1994; Hibbett
and Donoghue, 2001; Watkinson, 2001). Basidiomycetes are often considered as
secondary colonists during microbial attack on wood, their colonisation often tends to
follow organisms that remove wood extractives and other toxic compounds (Behrendt
and Blanchette, 1997; Behrendt et al., 2000).
P. gigantea is an aggressive white rot basidiomycete reported to play a
major role in the decomposition of coniferous debris (Behrendt and Blanchette, 1997;
Cram, 1999; Behrendt et al., 2000). This fungus has been successfully employed as
an industrial product, having been used as a biocontrol agent to protect stump surfaces
from hazardous effect of Heterobasidium annosum (Deacon, 1998). P.gigantea
preferably colonises sapwood, degrade toxic wood extractives and resin (Rishbeth,
1963; Ross and Hodges, 1981) and has also been reported possessing strong
competitive ability and higher specific growth rate relative to other colonising fungi,
demonstrating potential as a candidate for use in industrial bioprocesses (Ross and
Hodges, 1981; Adomas et al., 2003).
Chapter 7 : Final Discussion and future prospects
155
The ability of white rot fungal enzymes to degrade cellulose has been
extensively studied by a number of researchers (Danneel et al., 1993; J. Rogalski et
al., 1993; Beguin and Aubert, 1994; Broda et al., 1996; Behrendt and Blanchette,
1997; Chee et al., 1998), with work focused on white rot fungal enzymes as catalyst
for the degradation of cellulose and lignin, predominantly on the white rot
basidiomycetes, Phanerochaete chrysosporium (Azevedo Mde et al., 1990; Covert et
al., 1992; Tempelaars et al., 1994; Broda et al., 1996; Rensburg et al., 1996; Li et al.,
2000; Wymelenberg et al., 2002). The success of these studies encouraged further
research into white rot fungal cellulose degradation systems, and at the same time the
problems of the enzyme production capacity and the slow rates of enzyme catalysed
degradation has encouraged researchers to investigate the use of new white rot fungal
organism and enzymes as potential sources of industrial lignocellulose degradative
enzymes.
The reports of presence of cellulase system in white rot basidiomycete P.
gigantea by K. Palaniswamy (Palaniswamy, 1998) and P. Collett (Collett, 1983)
encouraged further study of the nature of cellulolytic capacity of this organism. This
Ph. D. project objective was subsequently narrowed to an investigation of the ability
of P. gigantea as a source of cellulase enzyme.
To identify the cellulase enzymes and to study the activity of the cellulase
complexes of P. gigantea, a series of experiments were conducted in this study.
During the initial period of this research work (Chapter 3), attempts were made using
data published in previous Ph. D. thesis to identify and isolate cellulase gene from P.
gigantea. The PCR amplification of P. gigantea cDNA resulted in the incomplete
Chapter 7 : Final Discussion and future prospects
156
sequence of 1154bp gene product (P223A3S6). The analysis of transcription of this
product found gene expression to be induced when the fungus was grown in the
presence of microcrystalline cellulose (Avicel) and carboxymethyl cellulose (CMC).
Transcription was suppressed in the presence of simple sugars such as glucose and
xylose, which is similar to results reported regarding transcription of cellulase enzyme
in a previous study on P. chrysosporium (Sims et al., 1988). Whilst some sequence
information was obtained which indicated the fragment may be a fragment of
cellulase gene, it is not enough to denote the biological function with any degree of
confidence. Whilst sufficient DNA sequence was obtained to permit a tentative
identification of the biological function of the cloned cDNA, the assignment of
definitive biological function to a DNA sequence clearly relies on further functional
evidence, to this end, research was directed towards the study of production,
purification and characterisation of this fungal cellulase complex.
In order to more thoroughly understand the nature of the P. gigantea
inducible cellulase complex, the production of cellulase in the presence of different
substrate was examined (Chapter 4). This investigation revealed that P. gigantea
cellulase complex is quite inducible in the presence of cellulosic substrates such as
Avicel and CMC, whereas it is clearly repressed in the presence of simple sugars such
as glucose and xylose. As discussed in Chapter 4, the hydrolysis of cellulose by the
synergistic sequence of reaction produces a variety of intermediate substrates, and
consisting with findings examining the expression of cellulase gene in other cellulose
degrading organisms (Zhu et al., 1982; Spiridonov and Wilson, 1998; Wymelenberg
et al., 2002), after a certain incubation time, these intermediates in the degradative
process seem to somehow mediate a decrease in cellulase activity. The changes in the
Chapter 7 : Final Discussion and future prospects
157
substrate structure over the extended incubation time as a result of the enzymatic
hydrolysis of the larger cellulose polymers, gradually turns the inducing carbon
structures to forms that somehow repress the expression of the cellulase complex.
This is evident from the rise and fall of cellulase activity over the period of
incubation, as recorded in chapter 4. Clearly the results of the present study indicate
that P. gigantea has the capacity to efficiently degrade cellulose, and that this capacity
depends on the nature and concentration of the carbon source added.
Work detailed in this thesis indicates that P. gigantea was able to utilise
cellulose for growth and regulation of cellulase synthesis whereas simple sugars like
glucose and xylose represses the synthesis of the cellulase complex. It is also evident
from this study that the regulation of P.gigantea cellulase system is most probably
due to the intermediate transglycosylation products which may be involved in
induction, inactivation and/or synthesis of cellulase enzyme, as has been found in the
other fungal cellulase systems (Zhu et al., 1982; Bisaria and Mishra, 1989; Covert et
al., 1992; Beguin and Aubert, 1994; Spiridonov and Wilson, 1998).
This study successfully employed the rapid and highly effective method of
affinity digestion to purify the P. gigantea cellulase complex, the first reported use of
the affinity digestion technique to purify a cellulase complex from any fungi. The
results indicated that purification by affinity digestion provided higher values of
cellulase activity than any other purification methods previously reported (Goto et al.,
1992; Bok et al., 1998; Fang et al., 1998; Li et al., 2000; Yernool et al., 2000).
Chapter 7 : Final Discussion and future prospects
158
The P. gigantea purified cellulase complex was positively screened by both
enzymatic assays and zymogram and has been shown to be similar to the cellulases
previously purified from T. reesei (Sims et al., 1988), N. crassa (Yazdi et al., 1990),
T. viride (Kim et al., 1998), A. aculeatus (Muller et al., 1998), and C. thermocellum
(Morag (Morgenstern) et al., 1992; Zhang and Lynd, 2003). The purification and
biochemical characterization study revealed that P. gigantea cellulase complex
possess more than one type of CMCase and CBHase in different stoichiometric
relationship. The optimum pH of P. gigantea cellulase was found to be pH 5.0 and
the optimum reaction temperature to be at 500C. It is also evident from this study that
P.gigantea is potential cellulase degrader with wide substrate specificity, with the
inherent capacity to produce variety of different enzymatic activities simultaneously.
The ability to purify cellulase complex in relatively large quantities provided
the platform for the further detail protein characterisation study reported in Chapter 6.
This functional proteomics approach has the capacity to reveal the important data
related to the sequence and protein functionality of the purified protein components.
The 2D gel electrophoresis successfully separated 15 proteins, but due to time and
economic constraints only three protein fractions based on the zymogram and
enzymic assay reports, were further characterised. These three proteins identified
after the selective purification displayed the diversity of P. gigantea cellulase
complex by identifying matches with a range of glycoside hydrolases. This study
therefore revealed that P. gigantea, in the presence of cellulose as a sole carbon
source, secreted a cellulase complex that comprised multiple and diverse extracellular
enzymes belonging to the glycoside hydrolase family. As found in the P.
chrysosporium (Wymelenberg et al., 2005), this filamentous fungus efficiently
Chapter 7 : Final Discussion and future prospects
159
degrades the plant polymers with the help of variety of the protein components with
the capacity to degrade cellulose. The induction and expression of such a diverse
array of protein components may be due to proactive responses of the polymer
structures, the various oligomeric and monomeric sugars, acids and phenolic
compounds created through the enzymatic actions and subsequent metabolic
responses in the fungus.
In summary, the cellulase complex of P. gigantea is not an artefact but a
structural entity and the existence of this complex is dependent upon the presence of
specific substrate inducers and the secretion and activity of this enzymatic functions
can be eliminated by repressors, possibly intermediates or the final products of
degradative process. This complex is comparable to the known cellulases from other
fungal species, possessing true cellulase activity and is made up of variety of different
protein entities.
P. gigantea competes for the nutrient resources with other fungal plant
pathogens (Cram, 1999). As compared to the other basidiomycetes that infect the
wood as secondary colonist, P. gigantea has the specialised quality to colonise wood
prior to many other species, perhaps due to the capacity to deal with toxic resin and
wood extractives.
The utilisation of biomass as renewable energy resource is dependent on the
important and expensive process of pre-treatment (Klyosov, 1995a, b; Galbe and
Zacchi, 2002). The presence in this organism of an effective multiprotein cellulase
system, as reported in this study, indicates that it may be worth further characterising
Chapter 7 : Final Discussion and future prospects
160
the biochemical and molecular nature of the cellulose degrading enzyme complex and
exploring the use of this organism as an on-site source of cellulase which may be used
to eliminate the expensive industrial pre-treatment procedures.
The next stage of this project should focus on attaining an improved
understanding of P. gigantea biokinetics and the regulation of cellulase expression.
The future studies should also concentrate on the isolation and identification of the
cellulase gene by using molecular as well as proteomics approaches. The industrial
applications of the cellulases are many and varied and the availability of cellulase
system in fungus P. gigantea surely holds the capability to reveal novel cellulases to
handle multitasks demanded by the industry. Future work is also needed to explore
the molecular and structural architecture of the P. gigantea cellulases, which in turn
may help to provide an insight to engineer these enzymes enabling them to be
exploited in agricultural and industrial process to its extent.
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161
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Appendix I
The promoter and multiple Cloning sequence of the pGem®-T Easy Vector
pGEM®-T Easy Vector circle map and sequence reference points
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Appendix II
Electropherogram of PCR products of Phlebia gigantea
• P. gigantea 750bp P22 product as 3A3T7 and 3A3S6
• Combined full length product P223A3S6