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Based on these unpublished studies, our initial aim was to obtain a complete sequence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

ii

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

iii

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.

iv

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

v

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

vi

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

viii

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)

ix

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

xiii

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 6.5 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


PgCel 7. ..............................................................................................................139


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


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


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.


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


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.


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.


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


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


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.,


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


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).


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


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


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.


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 cellooligosaccharides (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-


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;


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).


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


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).


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


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


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


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).


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).


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).


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


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).


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 noncatalytic

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 noncatalytic 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,


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


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


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).


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).


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


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).


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).


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).


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).


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


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


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


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.


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


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.


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.


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


SDS (10%) 1mL

NaOH (10M) 0.2 mL

Milli-Q® water 8.8 mL


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.


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.


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.


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.


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.


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.


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.


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


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


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


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.


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.


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


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


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


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.


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)


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


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


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


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


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.


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.


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


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


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


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.


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


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


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.


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


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.


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).


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


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).


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.


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


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


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.


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


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


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


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


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.


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


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


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


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).


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.,


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


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


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).


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


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


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.


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


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


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


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).


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


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


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.


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


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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


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.


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.


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.


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.


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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.


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)


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


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


123

with complex substrates like wheat straw and cotton are needed to verify this

hypothesis.

124


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


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


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.


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


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


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.)


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


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


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.


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.


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


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.


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


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


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.


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.


138

Figure 6.4 M

ALDI-TOF-M

S spectrum of P.gigantea cellulase complex of 2D gel spot PgCel 5.

MALDI-TOF-M



ALDI was perform


an A

pplied B

iosystem


S m


ode in the



139

Figure 6.5 M

ALDI-TOF-M


MALDI-TOF-M



ALDI was perform


an A

pplied B

iosystem


S m


ode in the



140

Figure 6.6 M

ALDI-TOF-M


MALDI-TOF-M



ALDI was perform


an A

pplied B

iosystem


S m


ode in the



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).


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.


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)


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)


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)


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)


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)


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


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


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


148

Table 6.7 M


ily from CAZy database

Exp M

m

Theo M

m

∆ M

m


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


149

Table 6.8 M


ily from CAZy database.

Exp M

m

Theo M

m

∆ M

m


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


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


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


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


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).


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


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


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).


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


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


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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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

screening diverse cellulase enzymes from the white rot ... · personalities involved with me during...

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