Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans

By Muhammad Irfan

Department of Microbiology Quaid-i-Azam University Islamabad, Pakistan 2016

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans

A thesis

Submitted in the Partial Fulfillment of the

Requirements for the Degree of DOCTOR OF PHILOSOPHY

IN

MICROBIOLOGY

By

Muhammad Irfan

Department of Microbiology Quaid-i-Azam University Islamabad, Pakistan 2016

DECLARATION

The material contained in this thesis is my original work and I have not presented any part of this thesis/work elsewhere for any other degree.

Muhammad Irfan

DEDICATED

TO

My Ammi and Abbu

CONTENTS

S. No. Title Page No.

List of Abbreviations i

List of Tables iii

List of Figures iv

Acknowledgements x

Abstract xii

Introduction 1

Aims and objectives 7

Review of Literature 8

2.1 Background 8 2.2 Xylan: An Overview 8

2.3 The Xylanolytic Complex 12

2.4 Xylanolytic Enzymes 13 2.4.1 Endo-1, 4-β-xylanase 13 2.4.2 β-D-Xylosidases 14 2.4.3 Acetylxylan esterase 14 2.4.4 Arabinase 15 2.4.5 α-Glucuronidase 15 2.4.6 Ferulic acid esterase and p-coumaric acid esterase 15 2.5 Synergism between the enzymes of the xylanolytic complex 16 2.6 Xylanase Distribution 16 2.7 Xylanases: multiplicity and multiple domains 17 2.8 Classification of xylanases 18 2.8.1 Glycoside hydrolase families 5, 7, 8, 10, 11 and 43 20 2.8.2 Glycoside hydrolase family 5 21 2.9 Extremophilic Xylanases 22 2.10 Thermophiles 22 2.11 Xylanases Producing microorganisms 24 2.11.1 Fungi 26 2.11.2 Bacteria 27 2.12 Xylanase production 27 2.13 Cloning and expression of xylanases 29 2.13.1 Expression in bacteria 30 2.14 Molecular engineering strategies 31 2.14.1 Directed evolution 31 2.14.2 Site-directed mutagenesis 31 2.14.3 Saturation mutagenesis 32 2.14.4 Truncation 32 2.14.5 Fusion 33 2.15 Applications of xylanases 34 2.15.1 Xylanases in animal feed 35 2.15.2 Manufacture of bread, drinks and food 35 2.15.3 Chemical and Pharmaceutical and applications 36 2.15.4 Textiles 37 2.15.5 Cellulose pulp and paper 38 2.15.6 Bioconversion of lignocellulose in biofuels 40 Materials and methods 41

3.1 Collection of Sampling 41 3.2 Isolation of bacteria 41 3.2.1 Viable cell count or colony forming unit CFU 41 3.2.2 Screening for xylanase producing thermophilic bacteria 41 3.2.3 Screening for cellulase producing thermophilic bacteria 42 3.2.4 Screening for protease and amylase producing 42 thermophilic bacteria 3.3 Identification of xylanase producing thermophilic bacteria 42 3.3.1 Morphological Characteristics 43 3.3.2 Biochemical Characterization 44 3.3.3 Molecular characterization 47 3.4 Optimization Experiments 49 3.4.1 Inoculum preparation 49 3.4.2 Incubation Period effect on Growth and xylanase Production 49 3.4.3 Effect of Temperature on Growth and xylanase Production 49 3.4.4 Effect of pH on Growth and xylanase Production 49 3.4.5 Effect of substrate concentration on Growth and xylanase 50 Production 3.4.6 Effect of Size of Inoculum on Growth and xylanase Production 50 3.4.7 Effect of Age of Inoculum on Growth and xylanase Production 50 3.4.8 Effect of NaCl on Growth and xylanase production 50 3.5 Media, antibiotics, microbial strains and plasmids 50 3.5.1 Preparation of culture media 50 3.5.2 Preparation of antibiotics 50 3.5.3 Preparation of isopropyl-β-D-thiogalactoside 51 3.5.4 Selection of Microbial Strains 51 3.5.5 Selection of plasmids and Vectors 52 3.5.6 Deigning of primers 52 3.5.7 Isolating Genomic DNA of C5 and AK53 strains 52 3.5.8 Gel electrophoresis 54 3.5.9 Quantification of nucleic acids 54 3.5.10 Polymerase Chain Reaction 54 3.5.11 QIAquick Gel Extraction Kit Protocol (QIAGEN) 55 3.5.12 Ligation of DNA molecules (PCR Products) 55 3.6 Cloning and expression experiments 56 3.6.1 Transformation of competent cells with gene of interest 56 3.6.2 Preparation of competent cells 56 3.6.3 Transformation of E. coli competent cells 56 3.6.4 Plasmid isolation of transformed cell GeneJET Plasmid Kit 57 3.6.5 Verification of Cloning 58 3.6.6 Treatment of DNA with restriction enzymes 58 3.6.7 QIAquick Gel Extraction Kit Protocol (QIAGEN) 59 3.6.8 Ligation of double digested pET28a plasmid and insert 59 3.6.9 Transformation of pET28a plasmid + insert in E. coli cells 59 3.6.10 Isolation of plasmid of transformed cell Thermo Scientific 60 GeneJET Plasmid Miniprep Kit 3.6.11 Verification of xylanase Cloning 60 3.6.12 Sequencing of cloned xylanase gene 61 3.7 Bioinformatics analysis 61 3.8 Growth conditions of induced recombinant strains BL21 (DE3) 61 3.9 Purification 62 3.9.1 Ammonium sulphate precipitation 62 3.9.2 Dialysis of recombinant xylanase enzyme 62 3.9.3 DEAE Sepharose chromatography 62 3.9.4 Protein electrophoresis SDS - polyacrylamide gel electrophoresis 63 3.9.5 Staining SDS polyacrylamide gels 63 3.9.6 Zymogram analysis 63 3.9.7 HPLC of recombinant expressed xylanase 64 3.10 Enzyme assay for xylanase 64 3.10.1 Reagents 64 3.11 Characterization of recombinant xylanase 66 3.11.1 Effect of temperature on recombinant xylanase activity and 67 stability 3.11.2 Effect of pH on recombinant xylanase activity and stability 67 3.11.3 Metal ions effect on purified recombinant xylanase activity 67 3.11.4 Effect of Inhibitors on purified recombinant xylanase activity 67 3.11.5 Effect of organic solvents on purified recombinant xylanase 68 activity 3.11.6 Effect of salt concentration on recombinant xylanase activity 68

3.11.7 Determination of Km value 68 3.11.8 Analysis of hydrolysis product 69 3.11.9 Substrate specificity of recombinant xylanase 69 3.11.10 Shelf life Determination of xylanase 69 3.12 Mutational analysis of AK53 and C5 recombinant xylanase 70 3.12.1 Molecular modeling 70 3.12.2 Homology Modeling 70 3.12.3 Fold Recognition 70 3.12.4 Identification of key residues 71 3.12.5 Primer Design for mutation 71 3.13 Plasmid isolation of transformed cell 74 3.14 PCR amplification of G. thermodenitrificans strain C5 xylanase mutants 75 3.14.1 G. thermodenitrificans C5 W137R mutant PCR amplification 75 3.14.2 G. thermodenitrificans C5 D328R mutant PCR amplification 75 3.14.3 G. thermodenitrificans C5 W137R/D328R mutant PCR 76 amplification 3.14.4 G. thermodenitrificans C5 N84R mutant PCR amplification 76 3.14.5 G. thermodenitrificans C5 D339R mutant PCR amplification 77 3.14.6 G. thermodenitrificans C5 R81P and H82E mutant PCR 77 amplification 3.14.7 G. thermodenitrificans C5 W185P, D186E, 78 DM W185P/D186E mutant PCR amplification 3.15 PCR amplification of G. thermodenitrificans strain AK53 xylanase 78 mutants 3.15.1 G. thermodenitrificans strain AK53 W127R mutant 78 PCR amplification 3.15.2 G. thermodenitrificans AK53 D318R mutant PCR amplification 79 3.15.3 Double mutant W127R/D318R mutant PCR amplification 79 3.15.4 G. thermodenitrificans strain AK53 N74R mutant 80 PCR amplification 3.15.5 G. thermodenitrificans strain AK53 D329R mutant 80 PCR amplification 3.15.6 G. thermodenitrificans strain AK53 Double Mutant 81 R71P/H72E PCR amplification 3.15.7 G. thermodenitrificans strain AK53 DM W175P/D176E 81 PCR amplification 3.16 GeneJET Gel Extraction Kit Thermo Scientific 82 3.17 Ligation of mutants W137R, D328R and DM W137R/D328R of C5 82 And W127R, D318R and DM W127R/D318R of strain AK53 xylanase to pGEM®-T Easy Vectors 3.18 Transformation of bacteria with exogenous DNA 83 3.18.1 Preparation of E. coli DH5 competent cells 83 3.18.2 Transformation of E. coli competent cells 83 3.18.3 Plasmid isolation from pGEM®-T Easy Vectors cloned 84 mutant xylanase of strain C5 and AK53 3.18.4 Confirmation of Cloning 85 3.19 DNA treatment with enzymes 85 3.19.1 Digestion of cloned DNA with HindIII enzyme 85 3.19.2 Ethanol Precipitation 86 3.19.3 Digestion with NheI and gel extraction 86 3.19.4 Gel purification through GeneJET Gel Extraction Kit 86 3.19.5 Ligation of double digested pET28a plasmid and mutated insert 86 3.20 Preperation of p15TV-L plasmid for Ligase independent ligation 87 3.21 Ligation with Clontech kit (Ligase independent ligation) 87 3.22 Transformation of bacteria with insert 87 3.22.1 Transformation of E. coli DH5 competent cells 87 3.22.2 Plasmid isolation of transformed cell 88 3.22.3 Confirmation of Cloning 88 3.23 Growth conditions of induced recombinant strains BL21 (DE3) 89 3.24 Purification 89 3.24.1 Ammonium sulphate precipitation 89 3.24.2 Dialysis 89 3.24.3 DEAE Sepharose chromatography 89 3.24.4 Phenyl Sepharose chromatography 90 3.24.5 Purification through His60 Ni Superflow Resin 90 3.24.6 Protein loading and washing 91 3.24.7 Elution of protein 91 3.24.8 Dialysis 92 3.24.9 Molecular weight determination by sodium dodecyl 92 sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) 3.25 Characterization of mutant xylanase 92 3.25.1 Effect of temperature on mutated xylanase 92 3.25.2 Effect of pH on mutated xylanase 92

3.25.3 Determination of Km value 93 3.25.4 Shelf life Determination of xylanase 93 3.25.5 Half-life of purified mutated xylanase 93 3.26 Effect of C terminus tagged proline on strain C5 xylanase 94 3.26.1 Primer Design 94 3.26.2 Plasmid isolation of transformed cell (Roche plasmid Kit) 94 3.26.3 PCR for amplification of proline with xylanase 95 3.26.4 GeneJET Gel Extraction Kit (Thermo Scientific) 95 3.26.5 Ligation of proline tagged xylanase to pGEM®-T Easy Vectors 96 3.26.6 Bacterial transformation with exogenous insert 96 3.26.7 Preparation of E. coli competent cells using calcium chloride 97 3.26.8 Transformation of E. coli competent cells 97 3.26.9 Plasmid isolation from pGEM®-T Easy Vectors cloned proline 97 tagged xylanase 3.26.10 Confirmation of Cloning 98 3.27 Enzymatic treatment of DNA 98 3.27.1 Digestion of cloned DNA with HindIII enzyme 98 3.27.2 Ethanol Precipitation 99 3.27.3 Digestion with BamHI and gel extraction 99 3.27.4 Purification of digested plasmid and proline tagged xylanse gene 99 product with GeneJET Gel Extraction Kit (Thermo Scientific) 3.27.5 Ligation of double digested pET28a plasmid and proline 100 tagged xylanase insert 3.27.6 Transformation of bacteria with exogenous DNA 100 3.27.7 Preparation of E. coli competent cells using calcium 100 3.27.8 Transformation of E. coli competent cells 100 3.27.9 Plasmid isolation of transformed cells by GeneJET Plasmid Kit 101 3.27.10 Confirmation of cloning 101 3.27.11 Sequencing of Clones 101 3.28 Growth conditions of induced recombinant strains BL21 (DE3) 101 Competent Cell 3.29 Purification 101 3.29.1 Ammonium sulphate precipitation and Dialysis 101 3.29.2 DEAE Sepharose chromatography 102 3.29.3 Phenyl Sepharose chromatography 102 3.29.4 SDS polyacrylamide gel electrophoresis 103 3.29.5 HPLC of proline tagged C5 xylanase 103 3.30 Characterization 103 3.30.1 Effect of Temperature 103 3.30.2 Effect of pH 103

3.30.3 Determination of Km value 104 3.30.4 Analysis of hydrolysis product 104 3.30.5 Polysaccharide-binding properties 104 3.30.6 Shelf life determination of xylanase 104 3.30.7 Enzymatic Treatment of Pulp 104 3.30.8 Kappa number determination 105 Results 106 4.1.1 Collection of Samples 106 4.1.2 Screening of xylanase producing bacterial isolates 106 4.1.3 Identification of xylanase producing bacterial strains 107 4.1.3.1 Morphological Characteristics 107 4.1.3.2 Microscopy 107 4.1.3.3 Biochemical tests 107 4.1.3.4 Molecular identification of bacterial isolates 107 4.1.4 DNA Isolation and Gene Expression 112 4.1.4.1 Isolation of DNA 112 4.1.4.2 Optimization of PCR amplification 112 4.1.4.3 PCR amplification of xylanase gene 112 4.1.4.4 Gel purification of amplified xylanase gene 112 4.1.4.5 Cloning of xylanase gene in cloning vector 112 4.1.4.6 Preparation of pET28a and xylanase gene product for expression 113 4.1.4.7 Double digestion and purification of pET28a vector and xylanase 113 gene product 4.1.4.8 Cloning of xylanase gene in expression vector 114 4.1.5 Bioinformatics analysis 120 4.1.6 Induction and over production of recombinant xylanase enzyme 120 4.1.7 Purification of recombinant enzyme 120 4.1.7.1 Precipitation with ammonium sulphate 120 4.1.7.2 DEAE Sepharose chromatography 120 4.1.7.3 High-performance liquid chromatography (HPLC) 121 4.1.7.4 SDS-PAGE analysis 121 4.1.8 Enzyme assay for xylanase 128 4.1.8 .1 Determination of enzyme activity 128 4.1.8.2 Determination of protein concentration 128 4.1.8.3 Determination of Specific activity 128 4.1.9 Biochemical Characterization and stability studies of purified enzyme 128 4.1.9.1 Effect of temperature on purified xylanase activity 128 4.1.9.2 Study of pH effect on activity of purified xylanase 128 4.1.9.3 Temperature effect on stability of purified xylanase 129 4.1.9.4 Effect of pH on stability of purified xylanase 129 4.1.9.5 Effect of substrate specificity of purified recombinant xylanase 129 4.1.9.6 Effects of metal ion 130 4.1.9.7 Effect of detergents 130 4.1.9.8 Effect of inhibitors 131 4.1.9.9 Effect of organic solvents 131 4.1.9.10 Study of hydrolysis product 131 4.1.9.11 Investigation of shelf life of xylanase 132 4.1.9.12 Kinetics of purified recombinant xylanase 132 4.1.9.13 Effect of salt concentration 132 4.2.1 Bioinformatics analysis 145 4.2.2 Homology Modeling of Strain AK53 Xylanase and Prediction of Mutants 145 4.2.3 Modeling of Strain C5 Xylanase Mutants 146 4.2.4 Isolation of recombinant pET28a Plasmid 151 4.2.5 Overlap extension PCR for mutation 151 4.2.5.1 W137R mutation in strain C5 151 4.2.5.2 Geobacillus thermodenitrificans C5 D328R mutation 151 4.2.5.3 Geobacillus thermodenitrificans Double mutation W137R/D328R 151 4.2.5.4 Geobacillus thermodenitrificans C5 N84R and D339R 152 4.2.5.5 Geobacillus thermodenitrificans C5 R81P, H82E 152 4.2.5.6 Geobacillus thermodenitrificans C5 W185P, D186E, 153 DM W185P/D186E 4.2.6 Generation of strain AK53 mutant models 158 4.2.6.1 D318R Mutation in AK53 158 4.2.6.2 W127R mutation in strain AK53 158 4.2.6.3 Double mutation D318R and W127R in strain AK53 158 4.2.6.4 Geobacillus thermodenitrificans AK53 N74R and D329R 158 4.2.6.5 Geobacillus thermodenitrificans AK53 DM R71P/H72E 159 and W175P/D176E 4.2.6.6 Geobacillus thermodenitrificans AK53 quadrupole 159 mutant R71P/H72E/W175P/D176E 4.2.7 Gel purification of amplified mutated xylanase gene 164 4.2.8 Cloning of mutated xylanase gene of strain AK53 and C5 in cloning vector 164 4.2.9 Preparation of pET28a and mutated xylanase gene product for expression 164 4.2.10 Double digestion and purification of pET28a vector and mutated 165 Xylanase gene product 4.2.11 Cloning of mutated xylanase gene in expression vector pET28a 165 4.2.12 Cloning of mutated xylanase gene in expression vector 166 p15TV-L Vector (derative of pET15b Novagen) 4.2.13 Induction and over production of recombinant mutated xylanase enzyme 166 4.2.14 Purification of recombinant enzyme 172 4.2.14.1 Precipitation with ammonium sulphate 172 4.2.14.2 DEAE Sepharose chromatography 172 4.2.14.3 Phenyl Sepharose chromatography 172 4.2.14.4 Purification through His60 Ni Superflow resin 173 4.2.14.5 SDS-PAGE for mutant proteins 173 4.2.15 Biochemical Characterization and stability studies of purified mutant 182 AK53 and C5 enzyme 4.2.15.1 Effect of temperature on purified C5 mutant xylanase activity 182 4.2.15.2 Effect of temperature on purified AK53 mutant xylanases’ 182 activity 4.2.15.3 Effect of pH on purified C5 mutant xylanases’activity 182 4.2.15.4 Effect of pH on purified AK53 mutant xylanases’ activity 183 4.2.15.5 Stability of purified C5 mutant xylanase at different temperatures 183 4.2.15.6 Stability of purified AK53 mutant xylanase at different 183 temperatures 4.2.15.7 Stability of purified C5 mutant xylanases at different pH 184 4.2.15.8 Stability of purified AK53 mutant xylanases at different pH 184 4.2.15.9 Determination of kinetic parameters of C5 and its mutants 184 4.2.15.10 Determination of kinetic parameters of AK53 mutants 185 4.2.16.1 Half-life of C5 mutant and C5 wild type xylanase 217 4.2.16.2Half-life of AK53 mutant and AK53 wild type xylanase 217 4.2.17.1Hydrogen Bonding Pattern in C5 Mutants 220 4.2.17.2 Hydrogen Bonding Pattern in AK53 Mutants 228 4.3.1 Isolation of recombinant pET28a Plasmid 236 4.3.2 PCR amplification of xylanase gene 236 4.3.3 Gel purification of amplified proline tagged xylanase gene 236 4.3.4 Cloning of proline tagged xylanase gene in cloning vector 236 4.3.5 Preparation of pET28a and xylanase gene product for expression 237 4.3.6 Double digestion and purification of pET28a vector and proline tagged 237 xylanase product 4.3.7 Cloning of xylanase gene in expression vector 237 4.3.8 Induction and over production of recombinant xylanase enzyme 238 4.3.9 Purification of recombinant enzyme 247 4.3.9.1 Precipitation with ammonium sulphate 247 4.3.9.2 DEAE Sepharose chromatography 247 4.3.9.3 Phenyl Sepharose chromatography 247 4.3.9.4 High-performance liquid chromatography (HPLC) 247 4.3.9.5 SDS-PAGE analysis 247 4.3.10 Enzyme assay for xylanase 252 4.3.11 Determination of enzyme activity 252 4.3.12 Determination of protein concentration 252 4.3.13 Determination of Specific activity 252 4.3.14 Biochemical characterization and stability studies of purified enzyme 252 4.3.14.1 Effect of temperature on activity of purified proline 252 tagged C5 xylanase 4.3.14.2 Temperature effect on stability of purified proline tagged 253 C5 xylanase 4.3.14.3 Effect of pH on activity of purified xylanase 253 4.3.14.4 Effect of pH on stability of purified xylanase 253 4.3.14.5 Effect of substrate specificity of purified recombinant xylanase 256 4.3.14.6 Investigation of shelf life of xylanase 256 4.3.14.7 Kinetics of purified recombinant xylanase 256 4.3.15 Polysacchrides Binding capacity GthC5ProXyl and GthC5Xyl 256 4.3.16 Enzymatic Treatment of pulp 257 Discussion 263

Conclusions 285 Future prospects 286

References 287

Appendices 325

List of published papers 372

List of Abbreviations

°C Degree celsius A Adenosine A˚ Angstrom

AgNO3 Silver nitrate AlCl3 Aluminium chloride Amp Ampicillin APS Ammonium persulfate ATP Adenosine-5’-triphosphate BLAST Basic Local Alignment Search Tool BLASTN BLAST search using a nucleotide query BLASTX BLAST search using a translated nucleotide query BLASTp BLAST search using a protein query bp Base pairs C Cytosine CAZymes Carbohydrate-Active enZYmes CBM carbohydrate binding module

CoCl2 Cobalt(II) chloride CTAB Cetyl trimethylammonium bromide CuSO4 copper sulphate D Aspartic acid DEPC Diethylpyrocarbonate DNA Deoxyribonucleic acid DMSO Dimethyl sulfoxide DMF Dimethylformamide DNS 3-Amino-5-nitrosalicylic acid dNTP Deoxynucleoside triphosphate DOPE Discrete Optimized Protein Energy DTT Dithiothreitol E Glutamic acid e.g. Exempli gratia, for example EDTA Ethylenediaminetetraacetic acid et al. et alii/alia, and others Fe Iron Fig. Figure G Guanine GRAS Generally recognized as safe GthAK53Xyl Recombinantly expressed xylanase of G. thermodenitrificans AK53 GthC5ProXyl Proline tagged C5 xylanase GthC5Xyl Recombinantly expressed xylanase of G. thermodenitrificans C5 H Histidine HPLC High-performance liquid chromatography i.e. id est, that is IPTG Isopropyl β-D-1-thiogalactopyranoside

i

Kan Kanamycin KCl potassium chloride KMnO4 potassium permanganate MDS Molecular dynamic simulation MgCl2 Magnesium chloride MgSO4 Magnesium sulfate MnCl2 Manganese(II) chloride MOPS 3-(N-morpholino)propanesulfonic acid N Asparagine NaCl Sodium Chloride NBS N-Bromosuccinimide NCBI National Center for Biotechnology Information NEM N-Ethylmaleimide P Proline PCR Polymerase Chain Reaction pH Power of Hydrogen PDB Protein Data Bank PMSF phenylmethylsulfonyl fluoride R Arginine rRNA Ribosomal ribonucleic acid SDM Site directed mutagenesis SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis T Thymine TAE Tris acetate EDTA TAPPI Technical Association of the Pulp and Paper Industry TBE Tris borate EDTA TEMED Tetramethylethylenediamine TLC Thin-layer chromatography TSC Trisodium citrate VMD Visual molecular dynamics W Tryptophan X-gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside ZnSO4 Zinc sulfate

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List of Tables

No. Title Page No. 3.1.1 Antibiotic used in this study 51 3.1.2 E. coli strains used for cloning in the present work. 51 3.1.3 List of plasmid used in this study 52 3.1.4 Primer used in this study 52 3.2.1 Primer used for C5 xylanase and their mutation 73 3.2.2 Primer used for AK53 xylanase and their mutation 74 3.3.1 Proline primer 94 4.1.1 Physical characteristics of samples isolated from Hot springs of 106 Pakistan 4.1.2 Physiological and biochemical characteristic of Geobacillus 108 thermodenitrificans AK53 and C5 4.1.3 Summary of GthAK53Xyl purification steps 127 4.1.4 Summary of GthC5Xyl purification steps 127 4.1.5 Substrate specificity of Geobacillus thermodenitrificans C5 and 136 Geobacillus thermodenitrificans AK53 4.1.6 Shelf life determination of C5 and AK53 recombinant expressed 143 xylanase 4.2.1 Ramachandran values for sequence AK53 149 4.2.2 Ramachandran values for sequence C5 150 4.2.3 Half-life of AK53 and their mutant 218 4.2.4 Half-life of C5 and their mutants 219 4.3.1 Overall purification scheme for recombinant xylanase 251 GthC5ProXyl 4.3.2 Shelf-Life of proline tagged C5 xylanase 258 4.3.3 Biobleaching of C5 and proline tagged C5 xylanase 260 4.3.4 Biobleaching of C5 W137R/D328R xylanase 261 4.3.5 Biobleaching of AK53 and AK53 W127R xylanase 261 4.3.6 Kappa number determination of xylanase 262

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List of Figures

S.No Page Title No. 2.1 Structure of xylan complex and the sites of attack by xylanolytic 10 complex enzymes 2.2 Molecular engineering methods and procedures to improve the 33 catalytic performance of industrial enzymes 4.1.1 Screening of thermozymes from hot spring’s isolates 109 4.1.2 Phylogenetic tree demonstrating the location of G. 110 thermodenitrificans AK53 4.1.3 Phylogenetic tree of Geobacillus thermodenitrificans C5 111 4.1.4 Gel electrophoresis of isolated genomic DNA 115 4.1.5 Optimization of PCR conditions (55°C to 62°C) 115 4.1.6 PCR amplification of AK53 and C5 xylanase 116 4.1.7 Gel purification of PCR amplified AK53 and C5 xylanase gene 116 product 4.1.8 Blue/white screening of pGEM®-T transformed cell with strain 117 AK53 and C5 xylanase gene 4.1.9 Digestion of strain C5 and AK53 xylanase cloned pGEM®-T vector 117 with EcoR1 restriction enzyme 4.1.10 Isolation of xylanase cloned pGEM®-T plasmid and pET28a 118 plasmid 4.1.11 Digestion of pGEM®-T cloned plasmid and pET28a plasmid with 118 HindIII restriction enzyme 4.1.12 Double digestion of xylanase cloned pGEM®-T plasmid and 119 pET28a plasmid with NheI and HindIII 4.1.13 Gel extraction of double digested pET28a vector, strain AK53 and 119 C5 xylanase gene product 4.1.14 Phylogenetic relationship of G. thermodenitrificans AK53 xylanase 122 (GthAK53Xyl) with other xylanase available at NCBI database 4.1.15 Phylogenetic relationship of xylanase of G. thermodenitrificans C5 123 with other xylanases available at NCBI database 4.1.16 Expressed recombinant xylanase of Geobacillus thermodenitrificans 124 C5 and AK53 4.1.17 DEAE-Sepharose chromatography of the endoxylanase produced by 124 G. thermodenitrificans AK53 4.1.18 DEAE-Sepharose Ion-exchange chromatography of the 125 endoxylanase produced by Geobacillus thermodenitrificans C5 4.1.19 HPLC profile of the purified recombinant G. thermodenitrificans 125 AK53 xylanase 4.1.20 HPLC profile of the purified xylanase from G. thermodenitrificans 126 C5 4.1.21 SDS-PAGE of crude and purified xylanase from G. 126 thermodenitrificans AK53 (GthAK53Xyl) 4.1.22 SDS PAGE showing purified recombinant G. thermodenitrificans 127 C5 Xylanase 4.1.23 Effect of temperature on the activity of purified recombinant AK53 133 and C5 xylanase

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4.1.24 Effect of pH on the activity of purified recombinant AK53 and C5 133 xylanase 4.1.25 Effect of temperature on the stability of purified GthAK53Xyl. 134 4.1.26 Effect of temperature on the stability of purified GthC5Xyl 134 4.1.27 Effect of pH on stability of purified GthAK53Xyl 135 4.1.28 Effect of pH on stability of purified GthC5Xyl 135 4.1.29 Substrate specificity of Geobacillus thermodenitrificans C5 and 137 GeobacillusthermodenitrificansAK53 4.1.30 Effect of various metals ion on GthAK53Xyl activity 137 4.1.31 Effect of various metals ion on GthC5Xyl activity 138 4.1.32 Effects of various Detergents on GthAK53Xyl activity 139 4.1.33 Effects of various Detergents on GthC5Xyl activity 139 4.1.34 Effects of various Inhibitors on recombinant purified AK53 140 Xylanase activity 4.1.35 Effects of various Inhibitors on recombinant purified C5 Xylanase 140 activity 4.1.36 Effects of organic solvent on GthAK53Xyl activity 141 4.1.37 Effects of organic solvent on GthC5Xyl activity 141 4.1.38 TLC analysis for hydrolysis products released from oat spelt xylan 142 by GthAK53Xyl 4.1.39 TLC analysis for hydrolysis products released from oat spelt xylan 142 by xylanase from Geobacillus thermodenitrificans C5 4.1.40 Lineweaver-burk plot of AK53Xylanase (Km and Vmax values were 143 determined according to Linewear-burk plot 4.1.41 Lineweaver-burk plot of C5Xylanase (Km and Vmax values were 144 determined according to Linewear-burk plot 4.1.42 Effect of NaCl on purified xylanase GthAK53Xyl and GthC5Xyl 144 activity 4.2.1 The proposed Three dimensional structure of Geobacillus 147 thermodenitrificans C5 xylanase. 4.2.2 The proposed Three dimensional structure of Geobacillus 148 thermodenitrificans AK53 xylanase. 4.2.3 Gel electrophoresis of D328R, W137R and W137R/D328R mutant 154 fragment 4.2.4 Gel electrophoresis of D328R, W137R and W137R/D328R 155 complete xylanase gene amplification 4.2.5 Gel electrophoresis of N84R and D339R mutant xylanase gene 155 product 4.2.6 Gel electrophoresis of R81P, H82E, W185P, D186E, W185P/D186E 156 mutant xylanase fragment product 4.2.7 Gel electrophoresis of strain C5 triple mutant 157 4.2.8 Gel electrophoresis of mutant R81P, H82E, W185P, D186E, 157 W185P/D186E xylanase gene product 4.2.9 Gel electrophoresis of AK53 D318R, W127R and W127R/D318R 160 mutant fragment 4.2.10 Gel electrophoresis of AK53 D318R, W127R and W127R/D318R 160 mutant complete xylanase gene 4.2.11 Gel electrophoresis of AK53 N74R and D318R mutant fragment 161 4.2.12 Gel electrophoresis of mutant AK53 N74R and D339R full xylanase 161

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gene amplification 4.2.13 Gel electrophoresis of mutant AK53 R71P/H72E, W175P/D176E 162 and Quadrupole mutant R71P/H72E/ W175P/D176E 4.2.14 Gel electrophoresis of mutant DM R71P/H72E, W175P/D176E and 163 R71P/H72E/W175P/D176E xylanase gene product 4.2.15 Blue white screening of strain AK53 and C5 mutant xylanase 168 4.2.16 Gel electrophoresis of pGEMT cloned mutated xylanase plasmid 168 4.2.17 Digestion of pGEM®-T Vector cloned mutant xylanase with EcoRI 169 4.2.18 Gel electrophoresis of digested pGEMT cloned mutated plasmid and 170 pET28a plasmid 4.2.19 Gel electrophoresis of double digestion of pGEM®-T cloned mutant 171 plasmid and pET28a plasmid with Nhe1 and Hind111 4.2.20 Gel electrophoresis of p15TV-L plasmid digested and un digested 171 plasmid 4.2.21 DEAE-Sepharose Ion-exchange chromatography of Geobacillus 174 thermodenitrificans C5 W137R 4.2.22 DEAE-Sepharose Ion-exchange chromatography of Geobacillus 174 thermodenitrificans C5 D328R 4.2.23 DEAE-Sepharose Ion-exchange chromatography of Geobacillus 175 thermodenitrificans C5 DM W137R/D328R 4.2.24 Phenyl-Sepharose Ion-exchange chromatography of Geobacillus 175 thermodenitrificans C5 W137R 4.2.25 Phenyl-Sepharose Ion-exchange chromatography of Geobacillus 176 thermodenitrificans C5 D328R 4.2.26 Phenyl-Sepharose Ion-exchange chromatography of Geobacillus 176 thermodenitrificans C5 DM W137R/D328R 4.2.27 DEAE-Sepharose Ion-exchange chromatography of Geobacillus 177 thermodenitrificans AK53 W127R 4.2.28 DEAE-Sepharose Ion-exchange chromatography of Geobacillus 177 thermodenitrificans AK53 D318R 4.2.29 DEAE-Sepharose Ion-exchange chromatography of Geobacillus 178 thermodenitrificans AK53 DM W127R/D318R 4.2.30 Phenyl-Sepharose Ion-exchange chromatography of Geobacillus 178 thermodenitrificans AK53 W127R 4.2.31 Phenyl-Sepharose Ion-exchange chromatography of Geobacillus 179 thermodenitrificans AK53 D318R 4.2.32 Phenyl-Sepharose Ion-exchange chromatography of Geobacillus 179 thermodenitrificans AK53 DM W127R/D318R 4.2.33 SDS-PAGE showing purified recombinant C5 mutated xylanase 180 enzyme obtained from ion-exchange column chromatography by DEAE Sepharose and Phenyl sepharose 4.2.34 SDS-PAGE showing purified recombinant C5 mutated xylanase 180 enzyme obtained from Ni column 4.2.35 SDS-PAGE showing purified recombinant AK53 mutated xylanase 181 enzyme obtained from ion-exchange column chromatography by DEAE Sepharose and phenyl Sepharose. 4.2.36 SDS-PAGE showing purified recombinant AK53mutated xylanase 181 enzyme obtained from Ni column 4.2.37 Effect of temperature on the activity of purified recombinant mutant 186

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C5 xylanases 4.2.38 Effect of temperature on the activity of purified recombinant mutant 186 AK53 xylanase 4.2.39 Effect of pH on the activity of purified recombinant mutant C5 187 xylanase 4.2.40 Effect of pH on the activity of purified recombinant mutant AK53 187 xylanase 4.2.41 Temperature stability of mutant W137R of C5 xylanase 188 4.2.42 Temperature stability of mutant D328R of C5 xylanase 188 4.2.43 Temperature stability of mutant W137R/D328R of C5 xylanase 189 4.2.44 Temperature stability of mutant N84R of C5 xylanase 189 4.2.45 Temperature stability of mutant D339R of C5 xylanase 190 4.2.46 Temperature stability of mutant R81P of C5 xylanase 190 4.2.47 Temperature stability of mutant H82E of C5 xylanase 191 4.2.48 Temperature stability of mutant W185P of C5 xylanase 191 4.2.49 Temperature stability of mutant D186E of C5 xylanase 192 4.2.50 Temperature stability of mutant W185P/D186E of C5 xylanase 192 4.2.51 Temperature stability of mutant H82E/W185P/D186E of C5 193 xylanase 4.2.52 Temperature stability of mutant W127R of AK53 xylanase 193 4.2.53 Temperature stability of mutant D318R of AK53 xylanase 194 4.2.54 Temperature stability of mutant W127R/D318R of AK53 xylanase 194 4.2.55 Temperature stability of mutant N74R of AK53 xylanase 195 4.2.56 Temperature stability of D329R mutant of AK53 xylanase 195 4.2.57 Temperature stability of R71P/H72E mutant of AK53 xylanase 196 4.2.58 Temperature stability of W175P/D176E mutant of AK53 xylanase 196 4.2.59 Temperature stability of R71P/H72E/W175P/D176E mutant of 197 AK53 xylanase 4.2.60 pH stability of mutant W137R of C5 xylanase 197 4.2.61 pH stability of mutant D328R of C5 xylanase 198 4.2.62 pH stability of mutant W137R/D328R of C5 xylanase 198 4.2.63 pH stability of mutant N84R of C5 xylanase 199 4.2.64 pH stability of mutant D339R of C5 xylanase 199 4.2.65 pH stability of mutant R81P of C5 xylanase 200 4.2.66 pH stability of mutant H82E of C5 xylanase 200 4.2.67 pH stability of mutant W185P of C5 xylanase 201 4.2.68 pH stability of mutant D186E of C5 xylanase 201 4.2.69 pH stability of mutant W185P/D186E of C5 xylanase 202 4.2.70 pH stability of mutant H82E/W185P/D186E of C5 xylanase 202 4.2.71 pH stability of mutant W127R of AK53 xylanase 203 4.2.72 pH stability of mutant D318R of AK53 xylanase 203 4.2.73 pH stability of mutant W127R/D318R of AK53 xylanase 204 4.2.74 pH stability of mutant N74R of AK53 xylanase 204 4.2.75 pH stability of mutant D329R of AK53 xylanase 205 4.2.76 pH stability of mutant R71P/H72E of AK53 xylanase 205 4.2.77 pH stability of mutant W175P/D176E of AK53 xylanase 206 4.2.78 pH stability of mutant R71P/H72E/W175P/D176E of AK53 206 xylanase 4.2.79 Kinetic parameters of mutant W137R of C5 xylanase 207

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4.2.80 Kinetic parameters of mutant D328R of C5 xylanase 207 4.2.81 Kinetic parameters of mutant W137R/D328R of C5 xylanase 208 4.2.82 Kinetic parameters of mutant N84R of C5 xylanase 208 4.2.83 Kinetic parameters of mutant D339R of C5 xylanase 209 4.2.84 Kinetic parameters of mutant R81P of C5 xylanase 209 4.2.85 Kinetic parameters of mutant H82E of C5 xylanase 210 4.2.86 Kinetic parameters of mutant W185P of C5 xylanase 210 4.2.87 Kinetic parameters of mutant D186E of C5 xylanase 211 4.2.88 Kinetic parameters of mutant W185P/D186E of C5 xylanase 211 4.2.89 Kinetic parameters of mutant H82E/W185P/D186E of C5 xylanase 212 4.2.90 Kinetic parameters of mutant W127R of AK53 xylanase 212 4.2.91 Kinetic parameters of mutant D318R of AK53 xylanase 213 4.2.92 Kinetic parameters of mutant W127R/D318R of AK53 xylanase 213 4.2.93 Kinetic parameters of mutant N74R of AK53 xylanase 214 4.2.94 Kinetic parameters of mutant D329R of AK53 xylanase 214 4.2.95 Kinetic parameters of mutant R71P/H72E of AK53 xylanase 215 4.2.96 Kinetic parameters of mutant W175P/D176E of AK53 xylanase 215 4.2.97 Kinetic parameters of mutant R71P/H72E/W175P/D176E of AK53 216 xylanase 4.2.98 Structural changes in C5 W137R 221 4.2.99 Structural changes in C5 D328R 222 4.2.100 Structural changes in C5 R81P 223 4.2.101 Structural changes in C5 H82E 224 4.2.102 Structural changes in C5 N84R 225 4.2.103 Structural changes in C5 W185P 226 4.2.104 C5 D186 wild type hydrogen bond pattren 227 4.2.105 Structural changes in C5 D186E mutant 227 4.2.106 Structural changes in AK53 W127R 229 4.2.107 Structural changes in AK53 D318R 230 4.2.108 Structural changes in AK53 R71P 231 4.2.109 Structural changes in AK53 H72E 232 4.2.110 Structural changes in AK53 N74R 233 4.2.111 Structural changes in AK53 W175P 234 4.2.112 Structural changes in AK53 D176E 235 4.3.1 Isolation of pET28a recombinant plasmid 239 4.3.2 PCR amplification of C5 with amplified with XGeoT-F and Xyl 239 ProC5 4.3.3 Gel extraction of C5 xylanase amplified with XGeoT-F and Xyl 240 ProC5 primer 4.3.4 PCR amplification of wild type (GthC5Xyl ) and proline tagged 240 xylanase (GthC5ProXyl) 4.3.5 Gel extraction of proline tagged C5 xylanase gene product 241 4.3.6 Transformation of pGEM®-T ligated proline tagged xylanase gene 241 product in JM 101 4.3.7 isolation of proline tagged xylanase cloned pGEM®-T vector 242 4.3.8 Digestion of proline tagged xylanase cloned pGEM®-T vector with 242 EcoRI restriction enzyme 4.3.9 Isolation of pET28a vector 243 4.3.10 Digestion of proline tagged xylanase cloned pGEM®-T vector and 243

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pET28a vector with NheI restriction enzyme 4.3.11 Precipitation of proline tagged xylanase cloned pGEM®-T vector 244 and pET28a vector digested with NheI restriction enzyme 4.3.12 Digestion of proline tagged xylanase cloned pGEM®-T vector with 244 BamHI restriction enzyme 4.3.13 Gel extraction of double digested pET28a vector and double 245 digested proline tagged xylanase gene product 4.3.14 Transformation of pET28a ligated proline tagged xylanase gene 245 4.3.15 Digestion of pET28a ligated proline tagged xylanase gene with 246 BamHI and NheI restriction enzyme 4.3.16 DEAE Sepharose chromatography of C5 proline tagged xylanase 249 4.3.17 Phenyl Sepharose chromatography of C5 proline tagged xylanase 249 4.3.18 HPLC profile of the C5 proline tagged purified xylanase 250 4.3.19 SDS-PAGE showing purified recombinant GthC5ProXyl 250 4.3.20 Effect of temperature on activity of proline tagged C5 recombinant 254 xylanase activity 4.3.21 Effect of pH on activity of proline tagged C5 recombinant xylanase 254 activity 4.3.22 Effect of temperature stability on activity of proline tagged C5 255 recombinant xylanase activity

4.3.23 Effect of pH stability on activity of proline tagged C5 recombinant 255 xylanase activity 4.3.24 Lineweaver-burk plot of GthC5ProXyl 258 4.3.25 TLC analysis for hydrolysis products released from oat spelt xylan 259 by proline tagged C5 xylanase from Geobacillus thermodenitrificans 4.3.26 Binding affinity of GthC5ProXyl xylanase towards various 259 substrates

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Acknowledgements

Praise to ALMIGHTY ALLAH, whose blessings enabled me to achieve my goals. Tremendous praise for the Holy Prophet Hazrat Muhammad (Peace Be upon Him), who is forever a torch of guidance for the knowledge seekers and humanity as a whole.

I have great reverence and admiration for my research supervisor, Dr. Aamer Ali Shah, Department of Microbiology, Quaid-i-Azam University, Islamabad, Pakistan, for his scholastic guidance, continuous encouragement, sincere criticism and moral support throughout the study. His guidance helped me in all the time of research and writing of this thesis, with his patience and immense knowledge.

I do not find enough words to express my heartfelt gratitude for Prof. Dr. Ali Osman Belduz, Faculty of Science Department of Molecular Biology Karadeniz Technical University, Turkey. He supervised me during my studies in Karadeniz Technical University,Trabzon, Turkey during TUBITAK scholarship. This experience would not have been as valuable without the guidance, support and inspiration provided by him. I am impressed by his scientific thinking and politeness.

I am deeply grateful to Dr. Claudio Gonzalez, associate professor at University of Florida for his patience, guidance at support during my research on IRSIP grant at USA. I consider myself very fortunate for being able to work with considerate and encouraging professor like him. I would have not been able to finish my study at University of Florida without his help and support.

I wish to express my most sincere appreciation to my chairperson, Professor Dr. Fariha Hasan, Department of Microbiology, Quaid-i-Azam University Islamabad Pakistan, for her valuable guidance, patience, support, criticism, encouragement and constructive suggestions throughout the course of this study.

I am also thankful to Dr. Graciela Lorca, Fernando Pagliai, Lei Pan, and Flavia Loto at Cancer and Genetics Research Complex University of Florida, and Halil Ibrahim Guler, Merve Tuncel Sapmaz,, Aysegul OZER, Fulya Ay Sal, Kadriye Inan, and Dr.

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SABRİYE ÇANAKÇI for their care and immense help during my entire stay at Karadeniz Technical University, Trabzon turkey.

I would also like to thank TUBITAK Scientific and Research Council of Turkey for Turkish scholarship and Higher Education Commission, Pakistan, for providing me grant under the Project “International Research Support Initiative Program (IRSIP)” and Indigenous Scholarships.

I am extremely grateful to the entire faculty at the Department of Microbiology, Quaid-i-Azam University, Islamabad. I feel thankful to the non-teaching staff, Shabbir Sahib, Shafaqat sahib, Sharjeel, Tanveer and Shahid Department of Microbiology, QAU, Islamabad, for their kind assistance.

I extend my great depth of loving thanks to all my friends and lab mates (seniors and juniors) especially Asad Zia, Ikram Ullah, Abdul Haleem, Sahib Zada, Arshad, Abdul Haq, Ghufran, Matiullah, Wasim, Waqas, Hameed Wazir, Wasim Hukam, Manzoor, Umair, Akhtar Nadhman, Saad, Shahid, Barkat, Ayesha, Shama, Muzna and Qurrat Ul Ain Rana for their help and care throughout my study.

I would like to thank my class fellows Muhammad Rafiq, Imran, Abdul Rehman and Zia Ullah for their help and support.

I would also like to thank Muhammad Rafiq, Mehmood Qadir and Wasim Hukam who assisted in sample collection.

A non-payable debt to my loving Ammi, Abbu, brothers and sisters for bearing all the ups and downs of my research, motivating me for higher studies, sharing my burden and making sure that I sailed through smoothly. Completion of this work would not have been possible without the unconditional support and encouragement of my loving family members

Finally, I express my gratitude and apology to all those who provided me the opportunity to achieve my endeavors but I missed to mention them personally.

Muhammad Irfan

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Abstract

Geobacillus thermodenitrificans AK53 and C5 xyl gene encoding xylanase were isolated, cloned and expressed in Escherichia coli. High level of xylanase activity produced by G. thermodenitrificans AK53 (952 IU/mL) and G. thermodenitrificans C5 (994.50 IU/mL) originated gene was detected when it was expressed in E. coli BL21 host. After purifying recombinant xylanase from G. thermodenitrificans AK53 (GthAK53Xyl) and G. thermodenitrificans C5 (GthC5Xyl) to homogeneity by ammonium sulfate precipitation and ion exchange chromatography, molecular mass was determined as approximately 45 kDa and 44 kDa, respectively, by SDS-PAGE. The specific activity of purified GthAK53Xyl and GthC5Xyl was up to 1113 and 1243.125 IU/mg with 7.38 and 9.89-fold purity, respectively. The kinetic studies for

GthAK53Xyl showed Km value to be 4.34 mg/mL and Vmax value to be 2028.9 μmoles –1 –1 mg min . Similarly, kinetic studies for GthC5Xyl indicated Km value to be 3.90 –1 –1 mg/mL and Vmax value to be 1839.86 μmoles mg min . The optimal temperature and pH for GthAK53Xyl and GthC5Xyl enzyme activity were found out to be (70°C, pH 5.0) and (60°C, pH 6.0), respectively. GthAK53Xyl showed the highest sequence similarity with the xylanases of G. thermodenitrificans JK1 (JN209933) and G. thermodenitrificans T-2 (EU599644). While GthC5Xyl showed the highest sequence similarity with the xylanases of Geobacillus sp. TC-W7 (GQ857066) followed by G. thermodenitrificans strain JK1 (JN209933).

Metal cations Mg2+ and Mn2+ were found to be required for the enzyme activity of GthAK53Xyl, however, Co2+, Hg2+, Fe2+ and Cu2+ ions caused inhibitory effect on it. GthAK53Xyl showed resistant to SDS, Tween 20, Tween 40, Triton X-100, β-

Mercaptoethanol, DTT, and TSC. The activity of GthC5Xyl was stimulated by CoCl2,

MnSO4, CuSO4, MnCl2 but inhibited by FeSO4, Hg, CaSO4. Similarly GthC5Xyl showed resistance to SDS, Tween 20, Triton X-100, β- Mercaptoethanol, PMSF, DTT, NEM, DEPC, and EDTA. A greater affinity for birchwood and beechwood xylan was exhibited by GthAK53Xyl and GthC5Xyl, respectively. GthAK53Xyl and GthC5Xyl were less active on arabinoxylan. The activity portrayed by GthC5Xyl was found to be acellulolytic with stability at high temperature (50°C-70°C) and low pH (4.0 to 8.0). GthAK53Xyl had no cellulolytic activity and degraded xylan in an endo- fashion with stability at temperature (50°C-80°C) and pH (4.0 to 7.0). Xylobiose and

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xylopentose were the end products of oat spelt xylan hydrolysis by GthAK53Xyl and GthC5Xyl. High SDS resistance and broader stability of GthC5Xyl proves it to be worthy of impending application in numerous industrial processes especially textile, detergents and animal feed industry. GthAK53Xyl results are suggestive of a xylanase exhibiting desirable kinetics, stability parameters and metal resistance required for the efficient production of xylobiose at industrial scale.

Protein engineering is frequently applied to enhance the strength of enzymes for its stability and activity at extreme temperature. In this study, three amino acid were recognized to mark the temperature stability of G. thermodenitrificans AK53 and C5 xylanase through multiple sequence analysis (MSA) and molecular dynamic simulations (MDS). Site-directed mutagenesis was employed to construct 11 mutants of G. thermodenitrificans C5 by exchanging these residues with arginine, proline and glutamic acid (W137R, D328R, W137R/D328R, N84R, D339R, R81P, H82E, W185P, D186E, DM W185P/D186E, H82E/W185P/D186E). 07 mutant models of G. thermodenitrificans AK53 were created (W127R, D318R, W127R/D318R, N74R, D329R R71P/H72E, W175P/D176E, and R71P/H72E/W175P/D176E). Mutant and wild-type enzymes were expressed in E. coli BL21 host. Mutant enzymes displayed enhanced thermal properties as compared to the wild-type. The activity and stability assays showed that the mutations at positions W137R/D328R, H82E and H82E/W185P/D186E increased the thermal performance more than the mutations at positions N84R, D339R, W185P and D186E in C5 xylanase. The mutants with combined substitutions (W137R/D328R and H82E/W185P/D186E) showed the most pronounced shifts in temperature optima. Half-life was increased 13 times at 60°C, 15 times at 65°C, 9 times at 70°C and 5 times at 75°C by H82E/W185P/D186E mutant. The activity and stability assays of AK53 mutant models showed that the mutations at positions W127R, D318R, double mutant W127R/D318R, R71P/H72E, W175P/D176E and quadruple mutant R71P/H72E/W175P/D176E increased the thermal performance more than that of the mutations at positions D329R and N74. The mutants with combined substitutions in AK53 xylanase (W127R/D318R, W175P/D176E, R71P/H72E and R71P/H72E/W175P/D176E) showed the most pronounced shifts in temperature optima. Half-life was increased 11 times at 60°C and 65°C, 7 times at 70°C and 3 times at 75°C by R71P/H72E and R71P/H72E/W175P/D176E. Similarly the mutation combinations of W127R and

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D318R, H72E, W175P and D176E of AK53 in adjacent positions more than doubled the effect of single mutations. These mutations probably rigidified the local structure. The rigidified main chain and filling of groove by the mutations on the xylanase enzyme may stabilize the mutants and thus improve their thermostability.

Efficient utilization of hemicellulose entails high catalytic capacity containing xylanases. Proline rich sequence was fused together with a C-terminal of xylanase gene from G. thermodenitrificans C5 and designated as GthC5ProXyl. Both GthC5Xyl and GthC5ProXyl were expressed in Escherichia coli BL21 host in order to determine effect of this modification. The C-terminal oligopeptide had noteworthy effects and instantaneously extended the optimal temperature and pH ranges and high specific activity as compared to GthC5Xyl. GthC5ProXyl revealed improved specific activity, a higher temperature (70°C versus 60°C) and pH (8 versus 6) optimum, with broad ranges of temperature and pH (60–80°C and 6.0–9.0 versus 40–60°C and 5.0– 8.0, respectively) stability. More than 80% activity was retained by the modified enzyme for 200 min as compared to wild type with only 45% residual activity, Our study demonstrated that proper introduction of proline residues on C-terminal xylanase of Geobacillus thermodenitrificans C5 is very important for xylanase thermostability. Moreover, this study reveals an engineering strategy to improve the catalytic performance of enzymes.

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Chapter 1 Introduction

Introduction In metabolism, enzymes are considered as catalytic linchpin. With the rapid advent of bio industrialism in world, apart from biological researchers, research on enzymes has also become a source of interest for process engineers, process designers, chemical engineers and researchers of other scientific fields. The most commercially exploitable properties owned by enzymes are their high degree of selectivity, milder reaction conditions and easy biodegradability with being environmentally safe. Enzymes are currently being utilized in detergent, food, fine chemicals, diagnostics and pharmaceutical industries. As reported by Business Communications Company Inc. in 2004, the total estimated industrial enzymes global market was about $2 billion and in 2010 it was about $3.3 billion with the predicted growth rate annually lying in between 4 and 5%. By 2016 it is expected that this market will reach up to $4.4 billion and $6.2 Billion by 2020 (Enzymes for Industrial Applications Business Communications Company, Inc; RC-147U; 2004 http://www.bccresearch.com). Europe and North America are the largest users of industrial enzymes although the economic strengths of countries from Asia Pacific region are undergoing a rapid rise in enzyme demand including Japan, China and India. The global market of industrial enzymes includes competitors like Novozymes, followed by DSM, and DuPon. Quality of product, intellectual property rights, innovations and performance are the main factors of competition among companies.

Due to their exceptional characteristics enzymes have acquired attention in all biochemical and industrial processes. As enzymes can perform multitude of biochemical reactions under ambient conditions and are environmental friendly, they are regarded as the best replacement of hazardous environmental polluting chemical agents. Enzymes also provide effects analogous to that of chemical additives. In addition to this, enzymes are natural products and are much safer additives than chemical additives. Enzyme are being utilized to produce over 500 different products with 150 applications in different industries including feed enzymes, food enzymes and technical enzymes (mainly use in starch, detergent, pulp/paper, personal care products and textile industry). Enzyme utilization is expanding day by day with advancement in molecular biology bioinformatics, protein biochemistry and bioanalytical techniques (Penstone, 1996). Recent studies proposed substitution of

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 1 Chapter 1 Introduction

chlorine bleaching by enzymatic treatment for the residual lignin removal from pulp (Viikari et al., 1994). United States alone use more than 2x106 tons of chlorine and chloride derivatives for the purpose of pulp bleaching yearly. This process generates chlorinated lignin derivatives such as dioxins, which is the major environmental hazard posed by pulp and paper industry (McDonough, 1992). Craft pulp when treated with xylanases, removes xylan and residual lignin from pulp without causing unwanted removal of other pulp contents. In this process enzyme acts as an ecofriendly replacement of chlorine (Beg et al., 2001).

Out of all the known enzymes only few are available commercially at the moment. With respect to industrial utilization of enzymes, the key markets include feed processing and food industry (Bull, 2001). However the demand is increasing in paper pulp, leather and textile industry. List of enzymes chiefly focused for large scale production includes proteases and carbohydrates ruling the world wide enzyme sale markets because of their extensive utilization in industry. The majorities 65% of the industrial enzyme is used in the hydrolysis of polymer, 25% are used in food processing industry and 10% are used in supplementation of animal feed (Cherry and Fidantsef, 2003). It has been observed that microbial enzymes are much more convenient to use than the enzymes from animal or plant origin because microbial enzymes are much more stable, have higher viability, greater yield, can be easily genetically manipulated and have higher catalytic power. Microbial enzymes suffer no seasonal fluctuations and microorganisms can be rapidly grown on comparatively cheap media (Wiseman, 1995).

The most frequent quoted reasons behind reduced enzyme utilization in industry are their high cost, poor stability of enzyme at high pH and temperature, limited enantiomer selectivity for artificial substrates, solubility in organic-water mixtures or organic solvents and less shelf life (Bowers et al., 2009). Reaction rate, pH, temperature optima, specificity, substrate affinity and stability are some of the factors that highly affect the choice of enzyme for large-scale industrial production. Personnel’s safety while enzyme production process and consumer’s safety while utilizing products based on enzymes are also factors requiring consideration (Bull, 2001). pH and thermostability are the two crucial factors which ensure the adaptability of enzymes for industrial applications. Due to these reasons, researchers

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 2 Chapter 1 Introduction

are still engaged in discovering new and novel biocatalysts which can survive in extreme conditions, also known as extremozymes. Enzymes derived from alkaliphilic sources propose successful functioning at higher pH in industry (Ramchuran et al., 2005). The β-glucuronidase isolated from Thermotoga maritima was found to be quite stable towards heat denaturation, at 85°C with a half-life of 3 hours (Salleh et al., 2006). Xylanase isolated from Aspergillus kawachii had an optimum pH of 2.0 and was stable at pH 1.0 (Fushinobu et al., 1998).

Xylanases are the xylosidic hydrolase enzymes commonly found in microorganisms like bacteria, fungi, actinomycetes, intestine of termites, marine algae, protozoons, insects, crustaceans, seeds and snails, which hydrolyze the glycoside bond of xylans forming hemi acetyls and glycans (Corral and Ortega, 2006; Sharma and Kumar, 2013; Motta et al., 2013; Butt et al., 2008; Qiu et al., 2016). Xylanases hold promising applications with immense potential in various biotechnological platforms. Some of their applications includes pulp pre-bleaching, plant fibers degumming, feed stocks and cereal food products enhancement, treatment of lignocellulosic biomass to convert it as a substrate for biofuel and beer manufacturing (Lee et al., 2005; Sanghi et al., 2009). The noteworthy application of this enzyme lays in the enzyme-aided paper bleaching. It is estimated that the paper and paperboard demand will grow from 300 million tones to over 490 million tones by the year 2020. With this increase in demand there will also come rise in pulp wood cost (Agnihotri et al., 2010; Vena et al., 2012). When paper pulp is pre bleached with acellulolytic xylanases the re- precipitated lignin and xylan-carbohydrate complexes get hydrolyzed leading to easy chemical extraction of lignin present in crude pulp. This results into substantial savings of chemicals which are traditionally used for bleaching (Viikari et al., 1994). The kraft pulping process takes place at elevated temperature and pH thus it requires alkaline thermophilic xylanases for cooked pulp pretreatment. Quest for alkaline thermostable acellulolytic xylanases producing novel microorganisms has already begun.

Thermostable xylanases possess evident advantages in industries that operate at high temperature where the process of cooling is wearisome and costly or where high temperature poses advantages of improved substrate solubility/bioavailability, decreasing contamination risk and reducing viscosity (Collins et al., 2005).

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 3 Chapter 1 Introduction

Thermophilic and mesophilic xylanases have somewhat similar structures. Thermophilicity can be achieved in mesophilic xylanases by simply putting in trivial array of modification without any substantial tampering with the backbone conformation (Collins et al., 2005; Hakulinen et al., 2003; Umemoto et al., 2009; Wang et al., 2012; Zhang et al., 2010, Waning et al., 2014). These alterations improve intra- and intermolecular interactions resulting in a more stable and rigid enzyme structure. It has been observed that arginine present at the surface of protein are crucial to protein stability at high temperature and replacing other amino acid residues with arginine helps in increasing stability of the enzyme as arginine forms hydrogen bonds and salt bridges with nearby groups (Chan et al., 2011; Lam et al., 2011; Matsutani et al., 2011; Mrabet et al., 1992; Strub et al., 2004; Turunen et al., 2002; Vogt et al., 1997., Wenqin et al., 2014).

Xylanases not only finds successful applications in pulp and paper industry but it also has potential applications in other industries as well. Food industry also requires solutions via biotechnology to the problems it is currently facing in animal feed and agrowaste. Xylanases hold impending applications in clarification of juices, animal feed digestibility and beer consistency improvement and lignocellulosic materials bioconversion to utilizable fermentative products (Vena et al., 2013). Sacccharification of xylan present in agrofoods and agrowastes also augments to the necessity of taking advantage from xylanases. In all the above mentioned scenarios xylan hydrolysis is the primary factor.

Xylanase isolated from different organism from different natural sources lack the capabilities to work optimally at industrial level, however with the help of direct evolution and site directed mutagenesis much progress has been made in enzyme activity and characteristic improvement which makes the future possibilities look endless. When looking to improve and alter a specific protein function, researchers have been extra-ordinarily more successful at modifying or altering the existing catalytic activities or characteristics properties of that protein, as opposed to engineering an entirely novel function into the protein (Shao and Arnold, 1996). This can become a problem sometime, as there is complete information available for a few of well-studied protein system, however a little of information is available for the vast majority of potentially industrial attractive enzymes. Working on a new yet to be

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sequenced protein and enzyme is deter by the lack of full information of that enzyme. Rational design is a powerful technique that predicts which nucleotide codon and its respective amino acid is necessary for protein structure function relationship.

To make the enzyme application in industry successful, its properties should be understood explicitly. Purification of xylanases from different microorganisms has been reported since last three decades (Esteban et al., 1982). However, in order to purify xylanase from Bacillus spp. Specific contemplation and assimilation of various techniques is required. Characterization of these purified enzymes generates data which helps in classification of the proteins as well as understanding their nature. An alkaliphilic Bacillus strain B. stearothermophilus T-6 is reported which secretes xylanase that can bleach pulp with pH 9 and temperature 65°C as optimum parameters (Khasin et al., 1993). There are certain conditions, which the xylanase must fulfill for this conceivable market: 1. They must be acellulolytic so that they do not cause cellulose fibers hydrolysis. 2. They must have lower molecular mass so that they can easily diffuse in pulp fibers. 3. They must remain active and stable at elevated temperature and pH. 4. Enzyme yield should be high at very low cost.

With the dawn of recombinant DNA technology, there came uplift in the production of enzymes that can be utilized well in industry (Headon, 1994). Moreover, the specificity, stability, economy in short biotechnological and industrial application of enzymes on the whole has improved drastically with the advances in gene and protein engineering. Protein expression technology plays a major role in harnessing novel proteins and developing high-production expression systems among which bacterial and yeast expression systems are the most commonly used (Lee, 1996).

Direct evolution is also a new protein modification, a power tool for making enzyme having the desired capabilities especially for industrial processes. Various enzymes with improved structure and function relationship were created by this method (Roodveldt et al., 2005). But it is based on Darwinian principle of selection and mutation. The technique starts with a set of genes or a specific gene and finally

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creating a mutant gene library by random mutagenesis or genetic recombination process. The mutant gene library is then cloned and expressed in expression organisms. Mutant strain having improved function ability is recognized through careful designed screening or selection process (Johannes and Zhao, 2006).

Another important approach for improving thermal stability of mesophilic enzymes is protein stability. The commonly used strategies include disulfide bridges, salt bridges and hydrogen bonds introduction and N-terminus modifications (Li et al., 2005). Many examples of successfully engineered proteins with increased thermal stability have been published (Wintrode et al., 2001).

Rational design follows the site directed mutagenesis (SDM) techniques which alters the specific codon of amino acid on the basis of data produced by computer assisted software modeling, determining which codon of amino acid will cause a structure changes of protein allowing the overall function changing of that protein (Blackburn, 2000), mostly it is dependent on amino acid location in protein structure, single amino acid substitution some time may have little effect on enzyme activity it may increase or hinder enzyme specific function. Researcher focuses amino acid residue near to the active site of enzyme if they need altered catalytic activity of enzyme, as this area amino acid is responsible for the catalytic activity of enzyme for their respective substrate.

Present study is aiming to engineer xylanases’ genes using advanced recombinant DNA technology which will result in thermostable xylanases in paper and pulp industry thus resulting in energy efficient and cost effective ways while insisting on environment friendly (green) standards. Acellulolytic xylanase is mandatory for paper pulp bio-bleaching. Thus choice of a bacteria producing large amount of Acellulolytic xylanase would be valuable. Acellulolytic xylanase purification is convenient thus leading to economically reasonable process. The choice of a thermophilic bacterium like Geobacillus thermodenitrificans is perfect to achieve the said objectives.

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Aim of study The aim of present study was production of thermostable xylanase from a thermophilic bacterium that would be applied in paper industry for bio-bleaching of pulp. In order to achieve this goal, following objectives were set:

Objectives  Isolation of xylanase producing bacteria from hot springs;  Cloning and over expression of xylanase producing genes;  Purification and characterization of the recombinant xylanase;  Site directed mutagenesis of recombinant xylanase with enhanced thermal properties;  Application of thermostable xylanase for bio-bleaching of paper pulp.

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Literature review

2.1 Background

Nature has evolved enzymes to accelerate the speed of chemical reactions in all life forms. Various enzymes are serving as industrial biocatalysts and their use in industrial synthetic processes is on the verge of substantial progression in industries like leather textile food, paper, detergents, pharmaceuticals and feed. Among the different enzymes like proteases and amylases, members of class carbohydrate-active enzymes (CAZymes) are being used extensively in industrial processes. 1-5% genome of living organism comprises of genes encoding CAZymes. CAZymes are involved in the synthesis and breakdown of polysaccharides, oligosaccharides and glycoconjugates which play important part in cellular recognition events as well as structural and energy reserve components. Important members of the CAZymes family include glycosyltransferases, glycoside hydrolase and polysaccharide lyases (PLs).

Xylanases are actually glycosidase (O-glycoside hydrolases) (EC 3.2.1.x) which act upon xylan. In xylan, xylanases act upon 1, 4-β-D-xylosidic linkages and catalyze their endohydrolysis. Xylanase are a broad group of enzymes which are responsible for the production of xylose from xylan. Xylose is a primary source of carbon for cellular metabolism. A plentitude of organisms are involved in the production of xylan. These include bacteria, fungi, algae, arthropods and gastropods. Xylanases were first identified in 1955 (Whistler and Masek, 1955). A large number of microorganisms produce enzymes which are capable of degrading xylan. This was first identified by Hoppe-Seyler in 1889 (Hoppe-Seyler, 1889). He discovered these enzymes by identifying the production of gas in a wood xylan suspension and river mud microbes. Submerged or solid-state fermentation is often used to induce microorganisms for the production of xylanases by providing them with xylan derivatives (Kalim et al., 2015).

2.2 Xylan: An Overview

In 1891 Schulze (Schulze, 1891) first coined the term hemicellulose for that fraction of plant material which was soluble in dilute alkali and was subsequently isolated on

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 8 Chapter 2 Review of Literature that basis. D-xylose, D-mannose, D-galactose and L-arabinose are the principle monomers constituting hemicellulose whereas the main heteropolymers are xylan, mannan, galactan and arabinan. D-xylose and traces of L-arabinose compose the monomeric unit of xylan. D-galactosyl units are present in galactan, D-mannosyl units are present in mannan whereas arbinan comprises of L-arbinose (Sharma and Kumar, 2013).

In plants, xylan is the major structural polysaccharide whereas in nature it is the second most abundant polysaccharide (Zheng et al., 2014). It constitutes about one by third of the total renewable organic carbon existing in world (Collins et al., 2005; Scheller and Ulvskov, 2010; Bai et al., 2014). It is the major constituent of hemicellulose and along with cellulose (1,4-β-glucan) and lignin (a complex polyphenolic compound) it forms the plant cell wall’s major polymeric constituents (Collins et al., 2005). Xylan, cellulose and lignin interact with each other through covalent and non-covalent linkages in plant’s cell wall structure. As xylan is found at the interface of cellulose and lignin, it is supposed to be providing fiber cohesion and cell wall integrity (Beg et al., 2001).

Hardwoods from angiosperms contain about 15-30% of xylan as their cell wall content, softwoods from gymnosperm contain about 7-10% of xylan whereas annual plants contain about >30% (Singh et al., 2003). Primarily xylan is present in the secondary cell wall of plants but in some monocots it is also located in primary cell wall (Wong et al., 1988). It is highly branched complex heteropolysaccharide which differs in structures with different plant species. The homopolymeric backbone chain of 1,4-linked-β-D-xylopyranosyl units in xylan can be substituted to different levels by glucuronopyranosyl, 4-O-methyl-D-glucuronopyranosyl, α-L-arabinofuranosyl, acetyl, feruloyl and/or p-coumaroyl side-chain groups (Wong et al., 1988; Kulkarni et al., 1999; Collins et al., 2004).

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Fig 2.1 (a); Structure of xylan complex and the sites of attack by xylanolytic complex enzymes. (b) Hydrolysis of xylo-oligosaccharide by β-xylosidase. Adapted from Collins et al., 2005

β (1-4) linked xylopyranoses linked in chains forms the backbone of xylan. Sidechains of arabinosyl, methylglucronosyl, glucuronosyl acetyl, feryolyl and p- coumaroyl residues can substitute the backbone as sidechains. Xylanases hydrolyze this backbone by acting on β-xylosidases linkages and release xylooligomers and xylobiose. Side chain catalysis is carried out by α-L arabinoses, acetylxylan esterases, p-coumaric acid esterases and ferulic acid esterases (Pastor et al., 2007).

Wood xylan occurs in various forms. In hardwood it exists as O-acetyl-4-O- methylglucuronoxylan, in soft wood as arabino-4-O-methylglucuronoxylan and in grasses and annual plants as arabinoxylans. In esparto grass, tobacco and marine algae linear unsubstituted form of xylan has also been found. Marine algae contains xylan in the form of xylopyranosyl residues linked by both 1,3-β and 1,4-β-linkages. Xylan also differs with respect to the extent of polymerization. For example hardwood xylan consists of about 150–200 β-xylopyranose subunits whereas softwood contains only about 70-130 (Kulkarni et al., 1999).

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Xylan is a complex heterogenic polymer and its complete hydrolysis requires a large number of enzymes working cooperatively (Harris and Ramalingam, 2010). Random cleavage of xylan backbone is brought about by endo-1,4-β-D-xylanases (EC 3.2.1.8) (kavya and Padmavadhi, 2009), xylose monomers at the non-reducing end of xylo- oligosaccharides and xylobiose are cleaved by β-D-xylosidases (EC 3.2.1.37) and side group removal is done by α-L-arabinofuranosidases (EC 3.2.1.55), α-D- glucuronidases (EC 3.2.1.139), acetylxylan esterases (EC 3.1.1.72), ferulic acid esterases (EC 3.1.1.73) and p-coumaric acid esterases (EC 3.1.1.1). Among microorganisms bacteria, actinomycetes and fungi are the major producers of complete xylanolytic enzyme systems which have all of the above mentioned properties. Some important microorganisms with respect to xylanolytic enzyme production are Ruminococci, Fibrobacteres, Clostridia, Bacilli, Aspergillis, Trichodermi, Streptomycetes, Phanerochaetes, Chytridiomycetes. These microorganisms have a widespread and diverse habitat including environments where plant material tends to accumulate as well as rumen of the ruminants (Subramaniyan and prema, 2002).

Xylanolytic enzymes are collectively called as xylanases. Secretion of xylanase often accompanies high or low amount of cellulase. For pulp treatment, cellulase free xylanase is preferably used because cellulase has adverse effects on paper pulp quality (Saitou and nei, 1987; Nakamura, 1993; Gessesse and Mamo, 1998; Shallom and Shoham, 2003; Ninawe et al., 2008; Sharma and Kumar, 2013). The best practical approach for this purpose is to screen out those microorganisms, which under optimized fermentation conditions secrete cellulase free xylanase. For its use in bleaching industry, xylanase produced should be stable at high temperature and pH (Sharma and Kumar, 2013). However the use of xylanase for enzymatic hydrolysis in industry is costly which limits its application in industry. This can be improved by searching for more potent microbial strains or producing mutant strains that secrete high amount of enzyme (Dekker and Richards, 1976).

Xylanase immobilization can be done by covalently attaching the enzyme to a carrier (Maalej-Achouri et al., 2009). In this way enzyme can easily be recovered and reused hence, reducing the cost of large-scale enzyme production effectively. With the ability to reuse enzyme as well as longer half-life of immobilized enzyme, a process for

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 11 Chapter 2 Review of Literature continuous production of enzyme can also be developed (Maalej-Achouri et al., 2009; Kar et al., 2008). Moreover as endo-1,4-β-xylanase is secreted extracellularly, for continuous production of this enzyme, immobilization of whole microorganism can also be considered.

2.3 The Xylanolytic Complex

Xylan hydrolysis is catalyzed by xylanases. Microorganisms secrete these enzymes and breakdown polysaccharides of plant cell wall. Xylanases sometimes also degrade stored xylan present in germinating seeds for example barley grain malting. In addition to microorganisms, xylanases are also secreted by plethora of other organisms such as marine algae, protozoans, insects, crustaceans, snails etc. They are also found in land plant especially in their seeds (Sunna and Antranikian, 1997). Amongst microorganisms, high level of xylanase is secreted by filamentous fungi. As compared to bacteria and yeast, the level of xylanase production in filamentous fungi is much higher. Moreover, bacterial xylanases are limited to only intracellular or periplasmic fraction. Xylanase encoding gene from a variety of microbial genera has been isolated and its expression in different E. coli host was reported, but the enzymes expressed in bacteria do not undergo post-translational modifications like glycosylation. Extracellular xylanase expression in E. coli which expresses the genes from thermophilic and akaliphilic Bacillus and Aeromonas species have been reported (Kulkarni et al., 1999a; Horikoshi, 1996). Among yeast, Bacillus endoxylanase coding gene (xyn A) was expressed in Saccharomyces cerevisiae (Nuyens et al., 2001). S. cerevisiae has also been found to express endoxylanase genes of fungal origin such as that from Trichoderma reesei and Aspergillus kawachii (Dalboge, 1997). Xylan is a complex heterogenic polymer therefore; its degradation requires activity of an enzyme complex. Endoxylanases and the β-xylosidases are those constituents of this complex which have been extensively studied. P-coumaric acid esterase, acetylxylanesterase, ferulic acid esterase and α-glucuronidase were discovered in late 1980s.Their late discovery is may be due to unknown suitable substrates. Only a few of these are purified and had their physical and chemical properties analyzed.

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2.4 Xylanolytic Enzymes

2.4.1 Endo-1, 4-β-xylanase

The glycosidic backbone of xylan is cleaved by Endo-1, 4-β-xylanase (1, 4-β-D-xylan xylanohydrolase (EC 3.2.1.8), thus bringing about reduction in its polymerization extent. This cleavage is not brought about randomly but the selection of bonds for hydrolysis depends upon molecular nature of substrate i.e. length of chain, number of branches and the occurrence of substituents (Reilly, 1981; Puls and Poutanen, 1989; Li et al., 2000). The main product of hydrolysis at the initial stages is β-D- xylopyranosyl oligomers but during the later stages smaller molecules like mono-, di- and trisaccharides of β-D-xylopyranosyl may also be produced. The equation for endoxylanase hydrolysis of xylan can be written as follows:

H (C5H8O4)nOH + H2OH( C5H8O4)n-pOH + (C5H8O4)P+OH

This is a stoichiometric equation showing hydrolysis of a singles xylan molecule but actually this reaction is occurring at numerous locations in molecule. Classifications of endoxylanases have been done in a number of ways. Endoxylanases have been classified in two types on the basis of end products of the reaction by Wong et al., (1988). One is the non-debranching enzyme that do not cause hydrolysis at 1,3-α-L- arabinofuranosyl branch-points of arabinoxylans, due to which they do not produce arabinose and the other is debranching enzyme that cleaves these points thus producing arabinose. A number of fungal species secrete only one of these two types however some fungi are capable of secreting both of the xylanases, bringing about a more efficient xylan hydrolysis. Wong et al., (1988) suggested another classification of endoxylanases based upon their physicochemical properties such as isoelectric point (pI) and molecular weight (MW). They were classified into two separate groups: acid endoxylanases with molecular weight above than 30 kDa and basic endoxylanase with molecular weight less than 30 kDa. However this classification is only true for only 70% of the endoxylanases and exact opposite is true for many other known endoxylanases. Törrönen and Rouvinen, (1997) suggested classification of endoxylanases into various families. The maximum activity of endoxylanases have been exhibited between 40 and 80°C, and between pH 4.0 and 6.5 however optimal activity has also been found outside these ranges. Multiple endoxylanase activity has

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 13 Chapter 2 Review of Literature been found in individual bacterial and fungal species and in certain cases two or three enzyme activities have been found in the same culture (Rizzatti et al., 2004). Detection of multiple forms is due to a number of factors such as proteolytic digestion, differential processing of mRNA, post-translational modifications such as glycosylation and self-aggregation. Sometimes distinct alleles of one gene or completely separate genes may also express multiple endoxylanase (Sung et al., 1995; de Segura et al., 1998; Chavez et al., 2002).

2.4.2 β-D-Xylosidases

β-D-Xylosidases (1,4-β-D-xylan xylohydrolase; EC 3.2.1.37) are classified on the basis of their substrate affinity to xylobiose and high molecular weight xylooligosaccharides. Xylobiases and exo-1,4-β-xylanases are two different enzyme but still they are known as xylosidases because they both cleave small molecules of xylobiose and xylooligosaccharides producing β-D-xylopyranosyl residues from the non-reducing end. Artificial substrates like o-nitrophenyl- and p-nitrophenyl-β-D- xylopyranoside can also be hydrolyzed by these enzymes. β-xylosidases plays an important role after many successive hydrolysis of xylan by xylanases. These successive hydrolysis steps cause the accumulation of short β-D-xylopyranosyl oligomers. These accumulated oligomers may inhibit the endoxylanase activity. Hydrolysis of these oligomers by β-xylosidase eliminates these inhibitors and increases the efficiency of xylan hydrolysis (de Vargas Andrade et al., 2004; Zanoelo et al., 2004). In yeast and bacteria they are found within cell. β-xylosidases of fungal origin are usually monomeric glycoproteins in nature (Sunna and Antranikian, 1997) and have MW between 60 to 360 kDa. Optimal pH is found to lie between 4.0-5.0 and temperature from 40-80°C however most of them give best assay results at 60°C. Thermostability of enzyme depends upon the organism from which it is isolated. One of the most thermostable β-xylosidases is secreted by Aspergillus phoenicis which kept its 100% activity after 4 hours at 60°C or 21 days at room temperature (Rizzatti et al., 2001).

2.4.3 Acetylxylan esterase

The removal of the O-acetyl groups at positions 2 and/or 3 on the β-D-xylopyranosyl residues of acetyl xylan is catalyzed by the enzyme acetylxylan esterase. Acetylxylan

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 14 Chapter 2 Review of Literature esterase was discovered late may be due to the fact that acetylated xylans are often treated with alkali which removes acetyl from xylan (Shao and Wiegel, 1992; Blum et al., 1999; Caufrier et al., 2003). During xylan hydrolysis these enzymes play an important role, as acetyl side group induces steric hindrance and obstructs endoxylanase from approaching the backbone of polysaccharide. The removal of acetyl side groups allows an efficient endoxylanase activity.

2.4.4 Arabinase

L-arabinose residues which are substituted at 2 or 3 positions of the β-D- xylopyranosyl are cleaved by arabinase. Based upon their mode of action arabinase are classified into two types. Those which digest p-nitrophenyl-α-L- arabinofuranosides and branched arabinose are known as exo-α-L- arabinofuranosidase (EC 3.2.1.55) while those which only degrade linear arabinose are called endo-1,5-α-L-arabinase (EC 3.2.1.99) (Kaneko et al., 1993; de Vries et al., 2000). Most of the arabinase purified and analyzed until now are of exo type.

2.4.5 α-Glucuronidase

The hydrolysis of β-D-xylopyranosyl backbone units found in glucuronoxylan and α- 1,2 bonds between the glucuronic acid residues is governed by the xylanolytic enzyme α-Glucuronidase. The activity of microbial enzyme varies with the nature of substrate. Some are able to hydrolyze only short oligomers of glucuronoxylan while others degrade the whole polymer. The specificity of enzyme for substrate also differs with the microbial source (Puls and Schuseil, 1993; Tenkanen and Siika-aho, 2000). It has also been observed that the presence of acetyl subunits near glucuronosyl subunits hinder the activity of α-glucuronidase.

2.4.6 Ferulic acid esterase (EC 3.1.1.-) and p-coumaric acid esterase (EC 3.1.1.1)

Both of these enzymes are involved in the cleavage of ester bonds present in xylan. Cleavage between arabinose and ferulic acid is brought about by ferulic acid esterase and the cleavage between arabinose and p-coumaric acid is catalyzed by p-coumaric acid esterase (Christov and Prior, 1993; Williamson et al., 1998; Crepin et al., 2004).

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2.5 Synergism between the enzymes of the xylanolytic complex

Xylan, a heteropolymer, can be attacked more efficiently by cooperation among the xylanolytic enzymes (Peij et al., 1997; de Vries et al., 2000). Addition of acetylxylan esterase to xylan releases acetic acid and decrease the amount of acetylated xylan thus allowing the endoxylanase to work more efficiently. Endoxylanase produce small acetylated polymers which are the preferred substrates for esterases. Endoxylanase is unable to easily degrade complex substrates which contain arabinoxylan such as wheat barn. These complex substrates are first treated with α-arabinofuranosidase which enhances the arabinoxylan degradation. Xylooligosaccharides are xylanase inhibitors. They are eliminated by β-xylosidases thus enhancing the xylan hydrolysis. Hence, the exemplary microorganisms for utilization in biotechnology are those which secrete a handsome amount of each type of xylanolytic enzymes. As the multi- complex enzymes present on bacterial surface and are responsible for attaching bacteria to cellulose for a more potent enzymatic attack are called cellulosomes (Bayer et al., 1994; Doi et al., 1994, 1998; Doi and Tamaru, 2001; Doi et al., 2003; Doi and Kosugi, 2004). Analogously, those which are associated with xylan degradation are given the term xylanosome. These multienzyme complexes range in MW from 500-600 kDa and may constitute of more than ten enzymes all having xylanolytic activity. In certain bacteria large complexes are formed by association of cellulosome and xylanosome. These large complexes are associated with degradation of both cellulose and xylan (Sunna and Antranikian, 1997; Beg et al., 2001).

2.6 Xylanase Distribution

Hoppe-Seyler observed the enzymatic degradation of xylan about a hundred year ago. Xylanases are diverse and widespread in nature (Collins et al., 2005; Sharma and Kumar, 2013). They are produced by a variety of microorganisms such as bacteria (Gilbert and Hazlewood, 1993; Sunna and Antranikian, 1997), fungi (Sunna and Antranikian, 1997; Kuhad et al., 1998), yeast (Hrmova et al., 1984; Liu et al., 1998) and actinomycetes (Ball et al., 1989). A number of fungal and bacterial species are known to produce alkalo-thermophilic xylanases for example Streptomyces sp., Dictyoglomus sp., Thermotogales sp., Thermoactinomyces thalophilus, Thermoascus aurantiacus, Fusarium proliferatum, Clostridium abusonum, Arthrobacter,

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Thermomonospora sp, Bacillus sp, Melanocarpus albomyces, Chaetomium thermophilum, Nonomuraea flexuosa etc (Khandeparkar and Bhosle, 2006; Hakulinen et al., 2003; Virupakshi et al., 2005; Ghatora et al., 2007; Garg et al., 2009).

Bacteria became a source of interest for industrial researchers because of their property to produce endo-1,4-β-xylanase. Larger amount of endo-1,4-β-xylanase is produced by Bacillus SSP-34 under optimum nitrogen condition. In optimized medium, it produces this enzyme with the activity of 506 IU/ml (Subramaniyan and Prema, 2002). A cellulose free endo-1,4-β-xylanase was reported to be produced by Bacillus stearothermophilus but it still showed a small cellulolytic activity i.e. 0.021 IU/ml. Dictyoglomus have also been reported to produce endo-1,4-β-xylanase with an optimum pH 5.0 and temperature 90ºC by Mathrani and Ahring (Mathrani and Ahring, 1992).

Endo-1,4-β-xylanases are also produced by fungi with temperature tolerance of 50ºC. These fungi require initial cultivation pH less than 7.0 and the fungal endo-1,4-β- xylanase have an optimal pH lower than those of bacteria. As most of the industries such as paper and pulp industry operate at higher pH thus, bacterial endo-1, 4-β- xylanases are preferred over fungal endo-1, 4-β-xylanase in these industries. Despite of a large number of differences lying in the growth conditions such as temperature, aeration, agitation, pH etc. for endo-1,4-β-xylanase activity, the biochemistry and molecular biology of both fungal and prokaryotic xylanases overlap (Ratto et al., 1992).

2.7 Xylanases: multiplicity and multiple domains

Apart from different varieties of xylanases, multiple xylanases is produced by a number of microorganisms (Gilbert and Hazlewood 1993: Gilbert et al 1988: Yang et al., 1989). A large number of physicochemical properties, yields, specific activities as well as overlapping and dissimilar specifications are related to these enzymes, which not only diversify the complexity of these enzymes but also increase the efficiency and extent of hydrolysis performed by these enzymes. Aspergillus niger and Trichoderma viride produce fifteen and thirteen types of extracellular xylanase respectively (Biely et al., 1985), are considered the typical examples of isoenzymes producing fungi.

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Genetic redundancy may be a cause of this multiplicity (Collin et al., 2005), however, in some cases post-translational processing has also been reported (Biely, 1985). Polycistronic or non-polycistronic copies of isoenzyme genes may be found in the genome of microorganisms. Xylanases may expressed as discrete gene product in some cases for example in Caldocellulosiruptor saccharolyticus the β-xylosidase and acetyl esterase enzymes genes are polycistronic (Luth et al., 1990), while Fibrobacter succinogenes S85 xynC genes encode two different catalytic domains of xylanase (Zhu et al., 1994).

In addition to presence of numerous catalytic domains, xylanases are also categorized on the basis of presence of different supplementary domains for example cellulose binding domain (Black et al., 1997; Millward-Sadler et al., 1995), xylan binding domain (Irwin et al., 1994), thermostabilizing domain (Winterhalter et al., 1995), dockerin domain (supposedly bind to the multidomins complexes produced by certain microorganisms such as Clostridium thermocellum) (Hayashi et al., 1997; Grepinet et al., 1988) and domains whose functions is not yet known. These domains are separated by short hydroxyl enriched junction segments of amino acids (Kulkarni et al., 1999) and may be folded or work independently. As large sized substrate cannot enter into the cell, xylanases are secreted into extracellular environment. Currently it is believed that the products of their own hydrolysis yield the induction of xylanases production (Singh et al., 2003; Biely, 1985; Defaye et al., 1992). It is assumed that constitutively produced small amounts of enzymes produce xylo-oligomers, which are transported into the cell where they are further degraded via intracellular xylanases or β-xylosidases and subsequently result in the induction of xylanase synthesis.

2.8 Classification of xylanases

The complexity and limitation in the classification of xylanases on the basis of substrate specificity only originated due to complexity and heterogeneity of xylanases which lead to diversity not only in specificities but also in folds and primary sequences. Xylanases were classified into two groups on the basis of their physicochemical properties by Wong et al. (Wong et al., 1988), those with acidic pI and high molecular weight (>30 kDa) and those with basic pI and low molecular weight (<30 kDa). However, exceptions are found and roughly 30% of available

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 18 Chapter 2 Review of Literature xylanases exclusively xylanases from fungal origin cannot be categorized on the basis of these classification groups (Sunna and Antranikian, 1997; Matte and Forsberg, 1992).

Another more sophisticated classification system was introduced later on (Henrissat et al., 1989) which classified not only xylanases but also glycosidases in general (EC 3.2.1.x) and it has now become the standard classification system. This system classifies enzymes on the basis of the primary sequence of their catalytic domain and groups them in families having related sequences (Henrissat and Coutinho, 2001). Initially xylanases and cellulases were classified in six families (A-F) (Henrissat et al., 1989). In 1999 they were updated to 77 families and the families continue to increase as new glycosidase are kept on identified. At the time of writing, more than 135 glycoside hydrolase enzyme families exist (see the carbohydrate–active enzyme CAZY server at http://afmb.cnrs-mrs.fr/~cazy/CAZY/), with approximately one-third of these families being poly-specific i.e. contain enzymes with diverse substrate specificities.

As primary structure specifies the molecular mechanism and overall structure of the enzyme, both structural and mechanistic features are reflected in the classification system. Within a same family, all the enzymes share a similar molecular mechanism (Gebler et al., 1992), 3D structure (Henrissat et al., 1989) and it has been observed that for synthetic soluble smaller substrates, they also have same mode of action (Claeyssens and Henrissat, 1992). In addition to this due to divergent evolution certain families also have overlapping 3D structures which lead to the introduction of a higher hierarchal system, known as “clan”. Right now there are about 14 different proposed clans (GH-A – GH-N). Each of these clans includes two to three families except for GH-A, which encompass 17 families.

Xylanases are found to be present in the family 10 (previously F) and 11 (previously G) in this classification system. In addition to that, using the enzyme classification number E.C. 3.2.1.8 to search in an appropriate database like CAZY showed that xylanase enzymes are also present in families 5, 7, 8, 16, 26, 43, 52 and 62. However a deep study of the literature shows that 1,4-β-xylanase activity is depicted by only those sequences which are present in families 5, 7, 8, 10, 11 and 43. Whereas families 16, 52 and 62 comprise of sequences which are bi-functional enzymes having two

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 19 Chapter 2 Review of Literature domains. One domain is family 10 or 11 xylanase whereas the other one is a catalytic glycosidase. For example the enzyme produced by Ruminococcus flavefaciens contains an amino-terminal xylanase of family 11 and a carboxy-terminal lichenase of family 16 and is thus classified in both 11 and 16 families (Flint et al., 1993). Similarly enzymes which are classified under family 26 appeared to be 1,3-β-xylanase rather than 1,4-β-xylanase. Thus the present concept that xylanases are only restricted to families 10 and 11 is not entirely correct and should be expanded in order to include families 5, 7, 8 and 43 as well.

2.8.1 Glycoside hydrolase families 5, 7, 8, 10, 11 and 43

Differences in structure, mode of action, physicochemical properties, and substrate specificities occurs between the members of families 5, 7, 8, 10, 11 and 43. However, similarities also exist such as both families 5 and 10 are confined under GH-A clan which shows that they contain similar 3D folds. In addition to this, families 5, 7, 10 and 11 includes enzymes which carry out hydrolysis while retaining the specific anomeric configuration and two glutamate residues are always utilized in the reaction (Coutinho and Henrissat, 1999). This points out to a double displacement reaction which produces a covalent glycosyl-enzyme intermediate which is further hydrolyzed by oxocarbenium- ion-like transition states (Collin et al., 2005). At the active site two carboxylic acid residues are present (approximately 5.5 A˚ apart) which are responsible for the formation of intermediate. One of these carboxylic acid protonates the substrate and acts as general catalyst while the other causes the departure of leaving group by performing a nucleophilic attack, resulting in the formation of α- glycosyl enzyme intermediate (inversion β to α). In the second step, a proton from nucleophilic water is abstracted by the first carboxylate group that acts as a general base and attacks on the anomeric carbon. In the second substitution stage anomeric carbon undergoes oxocarbenium-ion-like-transition stage and leads to the formation of product with β configuration (inversion α to β). Thus, overall configuration at anomeric center is retained. However, the enzymes present in families 8 and 43 inverts the anomeric center and the catalytic residues are thought to be aspartate and glutamate (Nurizzo et al., 2002). Single displacement reactions are carried out by inverting enzymes in which one of the carboxylate acts as general base and activate a nucleophilic water molecule to attack and cleave the glycosidic bond thus causing

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 20 Chapter 2 Review of Literature inversion of configuration at anomeric center whereas the second carboxylate causes the acid-catalyzed departure of leaving group (Rye and Withers, 2000). The distance between two carboxylate residues is found out to be 9.5 A˚, so that they can accommodate a nucleophilic water molecule in between general base and anomeric carbon (Rye and Withers, 2000,). However 7.5 A˚ distance is suggested by Alzari et al. (1996) and Gue´rin et al. (2002) in the inverting endoglucanase CelA and they have also suggested that the distance between two carboxylate residues is more constrained while retaining the enzyme and less constrained while inverting it.

2.8.2 Glycoside hydrolase family 5

Family 5 (previously family A) of glycoside hydrolase contains more than 537 characterized sequences with different activities at the time of writing. These sequences include: cellulose 1,4-β-cellobiosidase (EC 3.2.1.91), endo-1,6-β- galactanase (EC 3.2.1.164), 1,3-β-mannanase (EC 3.2.1.78), endo-1,4-β-xylanase (EC 3.2.1.8), glucan endo-1,6-β-glucosidase (EC3.2.1.75), endoglycosylceramidase (EC 3.2.1.123), cellulase (EC 3.2.1.4), licheninase (EC 3.2.1.73), β-mannosidase (EC 3.2.1.25), glucan 1,3-β-glucosidase (EC 3.2.1.58). It is one of the largest glycoside hydrolase families. Among this family there are about seven amino acids including the nucleophiles and general acid/base which are strictly conserved in all members. This group of enzymes is rather diverse having alignment of structures in such a way which indicates rms deviation of 1.25 ± 0.12 A˚ in between the equivalent residues among its members (Collin et al., 2005). These enzymes have also been further classified into nine subfamilies (Collin et al., 2005). This family includes eight enzymes which demonstrate xylanolytic activity as well as a number of other enzymes which are thought to be putative xylanase by identifying similarity in sequences by utilizing genome sequencing. These putative xylanase have been reported in Xanthomonas axonopodis pv. citri str. 306 (2 putative xylanases) (Da Silva et al.,2002), Xanthomonas campestris pv. campestris str. ATCC 33913 (Da Silva et al., 2002), Bacillus subtilis str. 168 (Kunst et al., 1997) and Bacteroides thetaiotaomicron VPI-5482 (Xu et al., 2003), Clostridium acetobutylicum ATCC 824 (2 putative xylanases) (Nolling et al., 2001), Leptosphaeria maculans (Coutinho and Henrissat, 1999). Enzymes from Aeromonas puncata (caviae) W-61, Erwinia (Pectobacterium) chrysanthemi P860219, Meloidogyne incognita and Ruminococcus albus 7 (Coutinho

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 21 Chapter 2 Review of Literature and Henrissat, 1999) also depict xylanase activity. Approximately 20 putative xylanase have been proposed. Although these enzymes are not completely characterized but large differences in their catalytic properties are clearly depicted. Some of these enzymes also have poor sequence identity.

2.9 Extremophilic Xylanases

The majority of the studies about bacterial or fungal xylanases reported that these enzymes are usually optimally active at mesophilic or near mesophilic temperatures (40-60°C) (Subramaniyan and Prema, 2002; Sunna and Antranikian, 1997; Collins, 2005) and neutral (in case of bacterial xylanases) or somewhat acidic (in case of fungal xylanases) pH. However, in certain studies xylanases were reported which are both stable and active at extreme temperature and pH. Certainly, xylanases have been reported to be active at pH range of 2-11 (Kulkarni et al., 1999; Fushinobu et al., 1998; Kimura et al., 2000), temperatures range of 5-105°C (Kulkarni et al., 1999; Collins et al., 2002; Collins et al., 2003), and NaCl salts concentration of 30% (Waino and Ingvorsen, 2003; Wejse et al., 2003). It is found, that habitats which were previously thought to be inhospitable for life, enzymes were obtained from microbes which habituated these particular extreme habitats. Among extremophilic xylanases, thermophilic, acidophilic and alkaliphilic xylanases have been comprehensively studied however psychrophilic xylanases have been less examined.

2.10 Thermophiles

Xylanases producing thermophilic (optimally grow at 50-80°C) and hyperthermophilic (optimally grow at >80°C) microbes have been reported from a variety of habitats such as both marine and terrestrial solfataric fields, hot pools, hot springs and organic debris of decaying (Singh et al., 2003; Harris, 1997; Vieille and Zeikus, 2001; Cannio et al., 2004; Sunna and Bergquist, 2003; Sunna et al., 1997). Most of the xylanases belong to families 10 and 11, and no studies confirmed yet the presence of xylanases that are thermophilic in any other glycoside hydrolase families. Amusingly, thermostable xylanases genes (having half-life of 8 min at 100°C) of Thermococcus zilligii a thermophilic archaeon (Uhl et al., 1999) has verified stubborn to cloning with consensus primers of family 10 and 11 (Sunna and Bergquist, 2003), proposing that xylanases may be the member of other less studied families of

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 22 Chapter 2 Review of Literature glycoside hydrolase (such as 5, 7, 8 or 43) or certainly to a new family that is yet unidentified.

Xylanases belong to family 10 have been reported successfully from numerous organisms that are thermophilic and hyperthermophilic in nature, containing Rhodothermus marinus (Abou-Hachem et al., 2003), Caldocellum saccharolyticum sp. (Luthi et al., 1990), Paenibacillus sp. NF1 (Zheng et al., 2014), Thermoascus aurantiacus (Leggio et al., 1999), Bacillus stearothermophilus (Khasin et al., 1993) C. thermocellum (Leggio et al., 1999), Geobacillus thermodenitrificans A333 (Marcolongo et al ., 2015) and Thermotoga sp. (Winterhalter et al., 1995; Zverlov et al., 1996). To date, xylanase, XynA of family 10 obtained from strain FjSS3-B.1 of Thermotoga sp. is one among the highly thermostable xylanases having optimum activity at 105°C temperature and 90 min of half-life at temperature 95°C (Simpson et al., 1991). However, little is reported about family 11 thermophilic xylanases, such as those from Paecilomyces varioti (Kumar et al., 2000), Thermomyces lanuginosus (Singh et al., 2003; Schlacher et al., 1996), Dictyoglomus thermophilum (McCarthy et al., 2000), Caldicellulosiruptor sp Rt69B.1. (Morris et al 1999), Nonomuraea flexuosa (Hakulinen et al., 2003), Thermomyces lanuginosus (Singh et al., 2003; Schlacher et al., 1996), Paecilomyces varioti (Kumar et al., 2000) and strain D3 of Bacillus (Harris et al., 1997; Connerton et al., 1999) being the comprehensively examined. Those reported from Dictyoglomus thermophilum and Nonomuraea flexuosa are in the list of most stable, having optimum temperature of 85°C and 80°C, respectively. Along with the above reported xylanases producing bacteria several of hyperthermophilic archaea capable of xylanases production have been reported in recent times, Pyrococcus furiosus (Uhl and Daniel, 1999), Thermococcus zilligii (Uhl and Daniel, 1999), Pyrodictium abyssi (de Vargas Andrade et al., 1999; Niehaus et al., 1999), Sulfolobus solfataricus (Cannio et al., 2004) and several of Thermofilum strains (Bragger et al., 1989).

Analysis of certain properties of mesophilic and thermophilic xylanases like crystal structure analysis, mutagenesis and sequence alignment have showed that both xylanases are alike and the higher stability is possibly because of an array of minor modification, most xylanases using several distinctive approaches for improved thermal stability nature. These alterations are, high number of hydrogen bonds and

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salt bridges (Hakulinen et al., 2003; Gruber et al., 1998), better interior packing (Hakulinen et al., 2003), high number of charged residues on surface (Luthi et al., 1990), tandem repeats presence of thermostabilising domains (Winterhalter et al.,

1995; Zverlov et al., 1996; Fontes et al., 1995), and disulphide bridge present at C or

N- end or in the helix areas (Kumar et al 2000; Turunen et al., 2001; Wakarchuk et al., 1994). Calcium role in thermostability of family 10 xylanases was confirmed in 2003 (Abou-Hachem et al., 2003), whereas D3 xylanases from Bacillus was also studied to use distinctive adaptation approach. Several of aromatic residues on surface synthesis cluster or some sticky covers between two molecules and hydrophobic interactions between molecules are thought to subsidize thermostability of the xylanase (Harris et al., 1997; Connerton et al., 1999). Together, or one at a time, all of these above alterations might increase the linkage of connections within protein, thus leading to a highly stable and rigid enzyme.

Several inclusive structural studies of xylanases from family 10 and 11, thermal adaptation have permitted exploration of adaptation approaches for every family. Like comparative study of thermophilic xylanases obtained from C. thermocellum and Thermoascus aurantiacus with family 10 xylanases which were mesophilic in nature, showed that the high thermostable property of this family is because of high hydrophobic packing, satisfactory interface of polar side chains with dipoles helix and high proline amount in the N-terminal of helices (Leggio et al., 1999). However, another study of five thermophilic and seven mesophilic enzymes of family 11 structure, proposed that the thermostabilizing ability of this family is because of high number ratio of threonine to serine (threonine has high tendency of β-forming), high amount of β-strand residues and recurrently an extra β-strand B1 at the N-terminal (Hakulinen et al., 2003). Such kind of changes in structure between families is the base for different adaptation tactics, enzymes of family 10 has high contents of a-helix (40%) (Derewenda et al., 1994), on the other hand a high amount of β-sheet content (>50%) are present in family 11 enzymes (Hakulinen et al., 2003).

2.11 Xylanases Producing microorganisms

Microbes, particularly, is a suitable cradle of important enzymes because they divide very rapidly and produce biologically active products that is under human control. In

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 24 Chapter 2 Review of Literature present world, there has been an upsurge in the industrial enzyme usage as a catalysts which offer more advantages over the chemical catalysts for several reasons; the catalytic activity and specificity for substrate is very high, they produce in high amount and highly biodegradable, they are environment friendly and economically feasible (Gote, 2004). For xylan hydrolysis, the microbial-based xylanases are the best option, because of more specificity, slight reaction conditions, very slight loss of substrate and generation of side wise products. Microbes based xylanases have several applications in the feed, paper pulp industries and food industries (Collins et al., 2005; Motta et al., 2013; Kulkarni et al., 1996; Qinnghe et al., 2004). The complete system of xylanolytic enzyme, comprising all these functions, has been widely discovered among bacteria (Motta et al., 2013; Kulkarni et al., 1992), actinomycetes (Elegir et al., 1994) and fungi (Sunna et al., 1997). Some important producers of xylanolytic enzyme comprise Chytridiomycetes, Aspergillus, Streptomyces, Fibrobacteres, Phanerochaetes, Trichoderma, Ruminococcus, Bacillus, Clostridia and Cellvibrio mixtus strain J38 (Kulkarni et al., 1992; Motta et al., 2013; Qinnghe et al., 2004; Wubah et al., 1993; Wu and He, 2015). The habitats of these microbes are widespread and diverse and typically found in environment where there is plenty of plant products gather and deteriorate, also inside the rumen of ruminants (Qinnghe et al., 2004; Motta et al., 2013).

Though, since 1960s there have been numerous studies on xylanases enzymes that are microbial based, the main emphasis has been on plant pathology associated (Lebeda et al., 2001). Throughout the 1980’s, xylanases was used for biobleaching (Viikari et al., 1986). Several of microbes such as bacteria and fungi have been studied to hydrolyze successfully xylan by producing β-xylosidases (EC.3.2.1.37) and 1,4-β-D endoxylanases (E.C. 3.2.18) since 1982 (Esteban et al., 1982). The xylanase production ability of microbe must be enhanced by finding extra effective bacterial and fungal strains or by making the mutant strains to produce high amount of xylanases enzymes. Furthermore, the enzyme production level of microbes is highly effective by several of physiological or nutritional factors, like nitrogen and carbon, chemical and physical conditions.

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2.11.1 Fungi

Main producers of xylanase and some other xylan splitting enzymes are filamentous fungi because it excretes higher level of enzyme into the medium than bacteria and yeast. Along with xylanase, fungi also produce numerous auxiliary enzymes that are obligatory for the substituted xylan degradation (Polizeli et al., 2005).

Some of the fungal genera like Aspergillus, Trichoderma, Pichia and Fusarium are reported high xylanases producers (Adsul et al., 2005). Certain extracellular xylanases have also been isolated from fungus such as white-rot that act upon hemicellulose, are valuable as food and synthesis pharmaceutical metabolites, cosmetics and food items (Buswell et al., 1994). The basidiomycete’s white-rot generally produce high yield of such enzymes to split lignocellulose products. Like, Coriolus versicolor yields complex combination of enzymes that are xylan splitting (Abd El-Nasser et al., 1997) and Phanerochaete chrysosporium yields great amount of α-Glucuronidase (Castanares et al., 1995). Cuninghamella subvermispora also produced xylanases when it grows on cell wall polysaccharide of plants or on wood pieces (de Souza-Cruz et al., 2004). Fungus based xylanases are normally linked with celluloses (Motta et al., 2013). Strains like this can synthesize both cellulase and xylanase when they grow on cellulose, this may be because of the hemicellulose presence in cellulose substrates in trace amount (Gilbert and Hazlewood, 1993), but for selective production of xylanase the carbon source use is only xylan. The mechanisms that run extracellular enzyme production with respects to the presence of carbon source in the medium are highly effect by the precursor’s presence for synthesis of protein. Thus, in certain fungi, the cell growth on xylan without cellulose under lesser nitrogen/carbon ratio can be a probable approach for synthesizing xylanolytic systems without cellulase (Biely, 1991). Additional main issue related with fungi in fermenter studies is the low xylanase. Normally agitation is the only practice to keep the medium homogenized, however, in the fermenter the shearing forces might disturb the fragile fungal biomass, leading to low productivity. The higher agitation can cause the hyphal disruption of fungus, which further minimizes xylanase action (Subramaniyan and Prema, 2000).

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2.11.2 Bacteria

Bacterial based xylanases have been described in Streptomyces, Bacillus and also reported from genera that have no part in pathogenicity of plants (Esteban et al., 1982). The highly thermophilic Rhodothermus marinus have been studied for α-L- arabinofuranosidase (Gomes et al., 2000), and polypeptides of two different types having α-arabinofuranosidase activity reported from Bacillus polymyxa were highly characterized at molecular level for α-arabinofuranosidases production (Morales et al., 1995). Similarly several other industrially important enzymes, bacteria captivated researchers because of their thermostable-alkaline xylanase production feature (Subramaniyan and Prema, 2002). Bacterial based xylanases optimum pH range is generally marginally upper than that of fungal (Khasin et al., 1993), which is an appropriate characteristic in certain industrial uses, such as pulp and paper industries.

Notable makers of best xylanase activity at a high temperature and pH are Bacillus spp. (Subramaniyan and Prema 2002). While in case of temperature only, several xylanases enzymes that exhibit optimum activity at high temperature have been studied from several microbes. These microbes contain Geobacillus thermodenitrificans A333 (Marcolongo et al.,2015), Paenibacillus sp. NF1 (Zheng et al., 2014), Actinomadura sp. strain Cpt20, Geobacillus thermoleovorans, Bacillus firmus, Streptomyces sp. S27 and Saccharopolyspora pathunthaniensis S582 that exhibit optimum activity between 65-90°C (Verma and Satyanarayana, 2012). Xylanase obtained from Thermotoga sp. (Yoon et al., 2004) have been found to show optimum activity between 100-105°C.

2.12 Xylanase production

Xylanases can be produced industrially either on solid substrate or in submerged liquid culture. Around 80-90% of xylanases are synthesized in submerged liquid culture. On the other hand solid substrate culturing, rice and wheat bran are used as an inducers. Several other substrates such as rice husks, sugarcane bagasse and wood pulp are also used as an alternative substrate for enzyme production (Kadowaki et al., 1995; Medeiros et al., 2000; Damaso et al., 2000; Singh et al., 2000; Pandey et al., 2000; Anthony et al., 2003). Production of xylanases in liquid culture is carried out in response to various sources of xylans (Gomes et al., 1994; Liu et al., 1999; Rani and

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Nand, 2000). Some residues of β-D-Xylopyranosyl also acting as inducers of xylanolytic complex (Ghosh and Nanda, 1994; Rizzatti et al., 2001), but it may cause discriminatory control in certain microorganisms, which leads to the endoxylanases catabolite repression (Flores et al., 1996; Mach et al., 1996). β-methyl xyloside is another compound that acts as an effective inducer, which is a low cost structural analogue (non metabolizable) of xylobiose (Morosoli et al., 1987; Simão et al., 1997a,b). Some of the synthetic compounds also induce the xylanolytic system, such as 3-O-β-D-xylopyranosyl D-xylose (Xylβ1-3Xyl), 2-O-β-D-xylopyranosyl D-xylose (Xylβ1- 2Xyl) and 2-O-β-D-glucopyranosil D-xylose (Glcβ1-2Xyl) (Hrmová et al., 1991). The homodisaccharides xylobioses, (Xylβ1-2Xyl e Xylβ1- 3Xyl), are the main inducers of endo-1,4-β-xylanase but unable to carry induction of cellulolytic complex, endo-1,4-β-glucanase. While in the case of heterosaccharide Glcβ1-2Xyl opposite is true. These manmade substrates act as a hybrid inducers which promotes the production of both enzymes complexes. This suggests that there are distinct regulatory systems present for the production of xylanases and cellulases.

Xylanases that are specific in nature are produced when the producer microbes are cultured on xylan, on the other hand on cellulose the producer synthesize cellulases along with xylanases, possibly it is due to the presence of trace amount hemicellulose in cellulose substrate. Xylanases free of cellulase activity is highly important in use of paper and textile industries, it is essential to remove hemicellulose from the fibers without destruction of cellulose. Xylans are polysaccharides which highly induce the enzyme activity in microorganism (Biely 1985, 1993). For some time this effect was asked by several scientists for some time, as these molecules do not contact within the site in the cell, to effect the gene expression regulation. For xylanolytic enzymes induction, there must be physical connection between inducer and regulatory mechanism of the cell, which recommends the actuality of certain recognition position over the cell surface. Comparatively at low level of activity, constitutive xylanases are thought to be accountable for the early xylan hydrolysis, generating small oligosaccharides of β-D-xylopyranosyl like xylotriose and xylobiose among others (Kulkarni et al. 1999a,b; Biely, 1985). Several researchers then study this xylobiose made a true inducer for the synthesis of endoxylanase (Haltrich et al. 1996; Sunna and Antranikian, 1997). These oligosaccharides are conveyed into the cell by β-xyloside permeases where they activate the gene expression of xylanase. Presence of glucose

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 28 Chapter 2 Review of Literature diminishes the permease activity of the induced cells, but this activity is very effective during existence of xylanolytic inducers. The carbon catabolite repression in case of filamentous fungi, is facilitated by the CreA protein (transcription repressor) (Dowzer and Kelly, 1991; Piñaga et al., 1994; Fernández-Espinar et al., 1993; Graaff et al., 1994).

2.13 Cloning and expression of xylanases

An ideal xylanases should have specific properties to achieve some specific industrial needs. These properties includes consistency over a wide range of temperature and pH, strong resistance to chemicals and metal cations and high specific activity (Qiu et al., 2010). Additional qualifications contain eco-friendliness, cost-effectiveness, and easiness of use (Taibi et al., 2012). Consequently, most of the described xylanases do not own the entire features mandatory for industry (Verma and Satyanarayana, 2012).

Natural enzymes are not adequate to fulfill the demand, because of low yield and unsuitability of the industrial standard fermentation processes (Ahmed et al., 2009). So, molecular methodologies must be applied for xylanases designing with obligatory characteristics (Verma and Satyanarayana, 2012). The main tool is heterologous expression for the xylanases production at industrial level (Ahmed et al., 2009). Protein engineering with the help of recombinant DNA technology might be valuable in enhancing the particular characteristics of current xylanases (Verma and Satyanarayana, 2012). Recombinant DNA technology and genetic engineering permit high scale expression of xylanases both in heterologous and homologous protein- expression hosts. Cheaper enzymes are best for industrial application, the raise in expression levels and effective xylanases expression are vital for guaranteeing the process viability (Juturu et al., 2011).

High numbers of publications have described several numbers of xylanases obtained from various sources and the sequencing, cloning, crystallographic and mutagenesis analysis of these enzymes (Kulkarni et al., 1999). X-ray crystallographic data, available amino acid sequence data, molecular dynamics and computational design of xylanases propose information that validates the association between the function and structure of xylanases. Entirely these methods help in the xylanases design that is

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 29 Chapter 2 Review of Literature compulsory for industrial processes like upgrading of the xylanases consistency at alkaline pH and higher temperatures (Verma and Satyanarayana, 2012).

For commercial purposes, these processes have been attempted and various xylanase encoding genes have been successfully cloned in both heterologous and homologous hosts (Kulkarni et al., 1999; Pérez et al., 2002). These recombinant xylanases have exhibited comparable or improved properties than the natural enzymes, and the genes responsible for xylanase in anaerobic microorganisms have been magnificently expressed in hosts that can be active in the fermentation industry (Ahmed et al., 2009).

2.13.1 Expression in bacteria

E.coli is famous for its easy alteration, low-cost growth situations, buildup of high amount of product in the cytoplasm and simple techniques need for transformation; so, this is the organism of choice for expression (Jhamb and Sahoo, 2012). Even though E. coli’s usage as a decent host for recombinant proteins cloning, it does not offer effective and functional expression of several xylanases (Juturu et al., 2011; Belancic et al., 1995) and not entire genes are expressed easily in E. coli (Jhamb and Sahoo, 2012). This issue may be because of the repetition of rare codons and the obligation for particular translational modifications, like disulfide-bond formation and glycosylation (Juturu et al., 2011). Consequently, this organism is valuable for the comprehensive study of xylanase gene construction and for the enhancement of the enzymes through protein engineering (Ahmed et al., 2009). Bacillus subitilis and Lactobacillus species have been better hosts for the heterologous proteins production, gaining advanced expression levels than E. coli (Juturu et al., 2011; Bron et al., 1998). Both of them are gram-positive and perform N-glycosilation (Upreti et al., 2003). Their prime concern in research and industry, is because they are not harmful and are GRAS (generally recognized as safe) (Juturu et al., 2011; Bron et al., 1998). The Bacillus genus, dissimilar of E. coli, do not enclose endotoxins (lipopolysaccharides), which are during purification very hard to eliminate from several proteins. While in industrial production the secretory production could also be helpful (Subramaniyan and Prema, 2002).

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2.14 Molecular engineering strategies

Molecular engineering involves the synthesis and design of novel structures with desirable catalytic properties of certain enzymes (Hida et al., 2007), thus increases the basic understanding of relationships between enzyme physical structure and functionalities.

2.14.1 Directed evolution

Directed evolution is an effective strategy for altering the activity of enzymes for research, therapeutic and industrial applications (Hida et al., 2007). After rounds of random mutations and artificial selection genetic diversity of useful enzymes is created. In order to mimic natural evolution, different attributes of directed mutagenesis are carried out which include combination of random mutagenesis, sequence diversification using error-prone polymerase chain reaction, computer guided mutagenesis, DNA shuffling, non-homologous recombination, simulated alternative splicing, functional assay via staggered extension process and high- throughput screen or selection strategy. It has successfully resulted in mutants with enhanced environmental durability and improved enzymatic activity with novel characteristics (Yang et al., 2014).

Despite their finely tuned architecture and complexity, proteins are extraordinarily evolvable, by adapting the pressure of selection through combinatorial exchange or assembly of their structure and function. The recombined functional sequences may be larger subdomain fragments or simple secondary structural elements with novel combinations, numerous (mostly neutral) mutations, of which can give rise to new protein behaviors and therefore new starting points for optimization of protein function (Urvoas et al., 2012).

2.14.2 Site-directed mutagenesis

Site-directed mutagenesis is an invaluable method for the genetic modification of enzymes causing structural and functional evolution. This is a technique in which single and combinational mutations are analyzed on the basis of catalytic mechanisms, structure, function, and catalytic residues of enzymes, using

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 31 Chapter 2 Review of Literature bioinformatics tools. To simplify and accelerate methods for mutagenesis, multiple mutations and single site-directed mutagenesis have been suggested (Hsieh and Vaisvila, 2013). Wang et al. (2014) employed site directed mutagenesis approach to construct five mutants by replacing the natural residues with proline and glutamic acid and all five mutant enzymes showed enhanced thermal properties.

2.14.3 Saturation mutagenesis

Site-directed saturation mutagenesis is a useful way to cause rapid evolution of proteins in laboratory to replace any amino acid of a protein with other 19 amino acids. It is performed at specific sites on enzymes called “hotspots” with variation of single amino acid resulting in enhanced catalytic efficiency and thermostability (Chen et al., 2012). For Example, in case of cyclodextrin glycosyltransferase from Paenibacillus macerans, after the site saturation mutagenesis of glutamine 265, tyrosine 260 and tyrosine 195, the mutants Q265K, Y260R and Y195S, as compared to wild type, produced higher 2-O- Dglucopyranosyl- L-ascorbic acid yields (Han et al., 2013). In another case, saturation mutagenesis resulted in 24 sites with amino acid replacement in cutinase from Fusarium solani pisi which, in comparison to wild types, caused 2- to 11-fold rise (Brissos et al., 2008). Wang et al. (2012) suggested tochoose the functionally correlated variation at hotspot sites using combinatorial coevolving-site saturation mutagenesis, to construct focused mutant libraries. It resulted in identification of novel beneficial mutation sites with improved thermostability by 8°C of α-amylase from B. subtilis CN7 from wild-type (Wang et al., 2012).

2.14.4 Truncation

The unnecessary domains of enzyme activity in the structures of enzyme proteins are altered using directed or random truncation to enhance improve the enzyme expression. In case of site-directed truncation, truncated enzymes are directly obtained whereas random truncation involves formation of truncation library where optimum properties of mutants are screened. In case of a mutant of Streptococcus ATCC 25175, endo-dextranase mutant TM-NCGΔ, after truncation, showed hydrolytic activity on dextran T2000 (0.4 %) that was same like SmDex90 with 2.0-fold or 1.4- fold or enhanced activity on dextran T10 or T2000 (0.05 %), respectively, and 2.4-

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 32 Chapter 2 Review of Literature fold to 1.6-fold enhanced activity with substrates like pNP-IG4, pNP-IG3 and CI-18 (Kim et al., 2011).

Fig 2.2; Molecular engineering methods and procedures to improve the catalytic performance of industrial enzymes adobted by Yang et al., 2014

2.14.5 Fusion

An effective and novel method of enzyme engineering is the creation of “chimeric enzymes” with enhanced catalytic abilities (i.e. thermostability, catalytic activity and product selectivity or substrate specificity). Construction of chimeric enzymes is carried out by fusion of substrate binding domain with catalytic domain and from various sources. For example, two separate modules, a carbohydrate binding module (CBM) and a catalytic module are present in carbohydrate-active enzymes, which are connected by a flexible linker and act as functional and discrete units (Christiansen et al., 2009). CBMs have 54 families based on amino acid similarities

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 33 Chapter 2 Review of Literature

(http://www.cazy.org/fam/ accCBM.html) (Linke et al., 2012; Han et al., 2013), which vary depending upon the ligand specificity such as crystalline and non- crystalline cellulose, chitin, L-rhamnose, β-1,3–1,4 mixed linkage glucans, β-1,3- glucans, mannan, xylan, starch and galactan (Fujimoto et al., 2013). Enzymes are fused with CBMs to construct new chimeric enzymes so that catalytic quality can be enhanced e.g. to improve the hydrolysis of insoluble xylan, a CBM from Thermotoga neapolitana was fused with xylanase of family-10 from Bacillus halodurans S7 (Mamo et al., 2007).

2.15 Applications of xylanases

Usage of xylans and xylanases in biotechnology has remarkably increased in recent years (Aristidou and Pentillä, 2000; Bhat, 2000; Subramaniyan and Prema, 2000, 2002; Beg et al., 2000, 2001; Techapun et al., 2003; Bibi et al., 2015). Xylitol and furfural are the end products of xylan degradation having commercial application (Parajó et al., 1998). The xylan conversion into β-D-xylopyranosyl and its oligosaccharides is carried out by two types of hydrolysis: enzymatic and acidic. Acidic hydrolysis is faster so it is preferable, but several other toxic compounds are also form during this hydrolysis that can delay subsequent microbial fermentation. Additionally, this contact with the acid it can cause corrosion of metallic equipment. Newly, certain industries have shown keen notice in the advancement of effective enzymatic procedures to be used as a substitute of acid hydrolysis in the treatment of hemicellulose holding material.

Xylanases are commercially formed in industries, such as, in Germany, Finland, Japan, Canada, Denmark, Republic of Ireland and USA. Some well-known microorganism used to get these enzymes is Humicola insolens, Trichoderma sp and Aspergillus niger. However, from bacteria marketable xylanases can also gain. In 1980s xylanase was initially used primarily in the animal feed preparation and after that in the food, paper industries and textile. Presently, cellulase and xylanase, combine with pectinases, is shared 20% of the enzyme market globally (Polizeli et al., 2005).

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2.15.1 Xylanases in animal feed

Enzyme usage in the feed production is a significant field of agribusiness, with improving 600 million tons per annum. In animal feed xylanases are used along with pectinase, glucanases, protease, cellulases, amylases, lipases, phytases and galactosidases. These enzymes reduce viscosity of the raw material by degrading arabinoxylans in the constituents of the feed (Twomey et al., 2003; Passos et al., 2015). The arabinoxylan that are part of grains cell walls has an anti-nutrient influence on poultry. When such constituents exist in soluble form, they increase the viscosity of the consumed feed, and do interfere with absorption and mobility of some other constituents. If xylanase is supplemented in the low viscosity food composed of sorghum and maize, it can increase the nutrient digestion in the primary portion of the digestive tract, causing an enhanced energy use. The combine action of the remaining enzymes listed yields an extra digestible food mixture. Swine and young fowl synthesis endogenous enzymes in minor amounts than adults, so the food supplements having exogenous enzymes must expand their performance as livestock. Furthermore, this type of food reduce undesirable residues in the excreta (nitrogen, phosphorus, zinc, and copper), an influence that could have a part in dropping environmental contamination.

2.15.2 Manufacture of bread, drinks and food

Xylanases can be hired in making bread, along with malting amylase, α-amylase, proteases and glucose oxidase. The xylanases, like other hemicellulases, split the hemicellulose in wheat-flour that help in the water distribution softer the dough and easier to knead. In bread-backing procedure, it stays crumb formation and permitting the dough to grow. An intensification in volume of bread, more water absorption and enhanced fermentation resistance occurred due to the use of xylanases (Maat et al., 1991; Harbak and Thygesen, 2002; Camacho and Aguilar, 2003). Similarly, a higher quantity of arabinoxylooligosaccharides present in bread would be useful to health. Xylanases is highly suggested in biscuit-making, for making cream crackers lighter and texture improvement, uniformity and palatability of the wafers.

The wine and juice industry is also a rich part of the enzyme market. Methods, like extraction, clearing and stabilization are required for fruit and vegetable juice

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 35 Chapter 2 Review of Literature production. In the 1930s, when the citrus fruit juice production started, the production was very low filtration of juice was very difficult, due to its turbidity. As the knowledge about the fruits ingredients and use of microbial enzyme increases, these problems were solved. Currently, xylanases, in combination with cellulases, pectinases and amylases, lead to an enhanced production of juice with liquefaction of vegetables and fruit; the fruit pulp stabilization; improved regaining of aromas, vitamins, essential oils, mineral salts, pigments, edible dyes etc., decrease of viscosity, hydrolysis of substances that delay the chemical or physical clearance of the juice, or that may reason opacity in the concentrate. Xylanase, along with endoglucanase, causes hydrolysis of starch and arabinoxylan, sorting out and detaching the gluten from the wheat flour starch. Also in coffee-bean mucilage, this enzyme is used (Wong et al. 1988; Wong and Saddler, 1993). The chief desired characteristics of xylanases to utilize in the food industry are great stability and optimum activity at acidic pH.

With the advancements in molecular biology techniques, some other uses of xylanases also revealed. A recombinant wine yeast was recently created having xylanases genes of Aspergillus nidulans xlnA resultant in a wine with a more projecting aroma compare to conventional (Ganga et al., 1999). During beer production, the barley cell wall is hydrolyzed and release arabinoxylans long chains, which rise the beer’s viscosity rendering it “muddy” in look. Hence, xylanases are used for arabinoxylans hydroxylation to minimize oligosaccharides lessening the beer’s viscosity and subsequently eradicating its muddy feature (Debyser et al., 1997; Dervilly et al., 2002).

2.15.3 Chemical and Pharmaceutical and applications

The use of both xylan and xylanases is quite limited in pharmacological industry. Xylanases are occasionally mixed in blend with a complex of enzymes, such as protease, hemicellulases, and others, as a nutritional enhancement or to treat reduced digestion, but very limited medicinal goods may be present with this construction. Some of the xylan hydrolytic products, like β-D-xylopyranosyl residues, may be transformed into inflammable liquids such as ethanol, solvents and artificial low- calorie sweeteners. The initial steps are the delignification of hemicellulose of xylan rich material, followed by hemicellulases and xylanases hydrolysis, for sugar production like β-Dxylopyranosyl units. Subsequent, the yields are fermented,

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 36 Chapter 2 Review of Literature generally by yeasts (Candida shehatae and Pichia stipitis), to yield ethanol or xylitol (Slapack et al., 1987; Screenath and Jeffries, 2000). All the sugars that used in the synthesis of ethyl alcohol, β-D-xylopyranosyl residues characterize between 5-20%. Polyalcohol such as xylitol is a sweet powder similar to that of sucrose (Parajó et al., 1998). It is a non-carcinogenic sweetener, appropriate for obese and diabetic persons and suggested for the inhibition of respiratory infections and osteoporosis, lipid metabolic disorder, parenteral and kidney lesions. Several of marketable products having xylitol, like chewing gum, may found in the market. Though, hydrolysis of xylan with enzyme is a suitable method of attaining β-D-xylopyranosyl units, at current time marketable xylitol is synthesized on a large scale by chemical catalysis. This is actually a high-cost process because initial purification of xylose is carried out in several steps. Moreover, several toxic byproducts are frequently produced during fermentation by the chemical reaction. Certainly, splitting of lignocellulosic material results in sugar release which produces toxic compounds such as hydroxymethylfurfural in case of glucose degradation phenolic and aromatic compounds and aldehydes in case of lignin degradation and furfural in case of xylose hydrolysis. Some products released from the lignocellulose structure, like acetic acid, some other liberated materials such as terpenes and its derivatives, phenolic and tropolones like lignans, quinones, stilbenes and flavonoids and tannins or the equipment materials like nickel, chromium, copper and iron, might be the main inhibitors for microbial activity. As advancement occurs in the technology for xylitol synthesis has produced excessive hope for higher and broader use in food, odontological and pharmaceutical industries.

2.15.4 Textiles

Xylanolytic enzyme complex are successfully also used in textile industry to process the linen and hessian plant fibers. Such complex should not have cellulolytic enzymes. One of the famous processes used is the incubation of dried ramee or stems of china grass with xylanase enzymes, to release the cellulose fibers intact. Using this technique, there is no necessity to use the bleaching step, since no oxidation of lignin occurs, which cause the darkness in fibers (Prade 1996; Brühlmann et al., 2000; Csiszár et al., 2001; Fu et al., 2008; Kalim et al., 2015). Comparatively, very slight

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 37 Chapter 2 Review of Literature work has done on enzymatic textile fibers preparation, and yet there are more techniques to be explored and developed.

2.15.5 Cellulose pulp and paper

Bleaching of the cellulose pulp is the chief industrial application of the xylanases. In the last two decades the enzyme is began to be used in this field, ever since the peroxidases were used for the lignin degradation (Viikari et al., 1990; Wong and Saddler, 1993; Tenkanen et al., 1997; Bajpai 1999; Araújo et al., 1999; Christov et al., 1999, 2000; Whitmire and Miti, 2002, Sandrim et al., 2005). Now a days, in several countries, especially in Brazil, chemical process is used in paper construction rather than enzymatic hydrolysis. The method usually used is known as Kraft process, which means strength or force in German. Three famous species such as, Eucalyptus saligna, Eucalyptus. urophylla and Eucalyptus. grandis are generally mainly favoured as the raw material. This method include the initial use of combine form of two reagents, sodium sulphide and sodium hydroxide, for the pretreatment of wood shavings, at 165°C under 8 kgf/cm2, in a digestor. Both these reagents inside the cooking liquor assist to speed up the delignification, and recover a major amount of cellulose fibers. The cellulose pulp at this stage is called brown mass, which is dark in color due to the black liquor. It may be supposed that 90–95% of the lignin and hemicellulose are dissolved and somewhat degraded during the process. Briefly, we have wood shavings such as lignin and fibers plus reagents like cellulose and lignin.

In industrial language: white liquor (NaOH+NaS2) plus wood = black liquor plus cellulose. Lignin that is deposited shows a dark color to the pulp and after washing of brown matter, pre bleaching is done, that contain slight impurities elimination and certain part of lignin that remains from the cooking. Oxygen is the main participant in this process, which minimize the price of additional reagents used in the bleaching process. The color of pre-bleached pulp is pale yellow and found not appropriate for good quality paper making for writing and printing. This is because of the presence of lignin residues on the fibers walls.

In the following step of bleaching, the paste color becomes white, by the elimination of chromophore, a light captivating materials that formed as by product of lignin breakdown. This process of bleaching is divided into three main phases. In first step, chlorine dioxide and ozone are used and in second step, hydrogen peroxide, oxygen

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 38 Chapter 2 Review of Literature and sodium hydroxide. Chlorine dioxide treatment is carried out in the last stage. The Kraft’s process main advantage is the option of chemical product recovery from the black liquor. Still, not the entire industries made recovery of sodium hydroxide and some other organic substances that are present in black liquor. While there are disadvantages of this process present, that include, strong smell of gasses, high initial cost, yield is very low (40–50%) and high bleaching cost. Also in the Kraft process, use of pollutant reagents are in huge amount. The chlorine use results the organochlorines synthesis from the lignin degradation products, which are mostly mutagenic and toxic that require effluents treatment from paper making industry. The protocols of environment have controlled the chlorine compound usage in processes of bleaching of cellulose and paper industries, particularly in North America and Western Europe. Now distinctive consideration in pre-bleaching has been given to xylanase, which could lower the chloride compound usage up to 30%, so 15-20% decrease in organochlorines could be accomplished in the effluents. Xylanases consumption might cause 5-7 kg replacement of chlorine dioxide per ton of Kraft pulp, while an average fall in kappa number of 2–4 units, a lignin content measure in the cellulose pulp. Xylanases do not need to be refined in paper technology, however it must be active at high pH and temperature, and should not have cellulolytic enzyme for conservation of cellulose fibers. The microbial xylanases proficiency in bleaching has been reported as Streptomyces roseiscleroticus (Patel et al., 1993); Streptomyces thermoviolaceus (Garg et al., 1996); Aspergillus niger (Zhao et al. 2002); Streptomyces galbus (Kansoh and Nagieb, 2004); Bacillus circulans (Dhillon et al., 2000); Streptomyces sp. (Beg et al. 2000; Georis et al., 2000); Bacillus pumilus (Bim and Franco, 2000; Duarte et al., 2003); Chaetomium cellulolyticum (Baraznenok et al., 1999); Aspergillus kawachii (Tenkanen et al., 1997); Aspergillus oryzae (Christov et al., 1999); Acrophilophora nainiana and Humicola grisea (Salles et al. 2005); Aspergillus nidulans (Taneja et al., 2002; Shah et al., 1999); Trichoderma reesei (Oksanen et al., 2000); Bacillus sp. (Kulkarni and Rao 1996; Thermomyces lanuginosus (Haarhoff et al., 1999) and Aspergillus fumigatus (Lenartovicz et al., 2002).

Two hypothesis about the role of xylanases in bleaching of cellulose pulp exists. In the first hypothesis, the enzyme xylanases act upon xylan that precipitated on the lignin (Viikari et al., 1994). This precipitation of xylan is because of the dropping of

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 39 Chapter 2 Review of Literature pH at the termination of the cooking stage. Its elimination through xylanases would more expose the lignin i.e., the bleaching compounds of cellulose pulp. Another hypothesis states that, the lignin ability to make complexes with polysaccharides, like xylan and few of the bonds are resistant to alkali condition and that have not been hydrolyzed during the Kraft process (Buchert et al., 1992). Actually, the action of xylanases cleave the remaining connections between xylan and lignin, and opens the assembly of the cellulose pulp and causes the xylan disintegration and following fragments extractions (Paice et al., 1992; Kalim et al., 2015). The xylanases action makes the pulp extra permeable to succeeding chemical extraction of lignin carbohydrate and brown lignin residues from the fibers.

2.15.6 Bioconversion of lignocellulose in biofuels

These days, biofuels of second generation are the main products of lignocellulosic materials bioconversion. Taherzadech and Karimi (Taherzadeh and Karimi, 2008), stated that ethanol is an important fuel that are renewable in terms of market values and volume, and succeeding the crisis of fossil fuel, it has been recognized as a substitute fuel (Pérez et al., 2002). Even though the first-generation ethanol production, from starch and sugar, the second-generation manufacturing of ethanol has only started to be tried in pilot plants (Taherzadeh and Karimi, 2008). Distinct of first-generation biofuels, second generation biofuels not competed with food production and may deliver economic, environmental and strategic aids for fuel production (Viikari et al., 2012). With other hydrolytic enzymes, xylanases might be utilized together for biological fuels production, like from lignocellulosic biomass ethanol production (Beg et al., 2001; Olsson et al., 1996). Though, enzymatic hydrolysis is still a chief cost factor in the alteration of raw lignocellulosic materials to ethanol (Taherzadeh and Karimi, 2008). In biofuel production such as bioethanol, delignification of lignocellulose is the first step, to release hemicellulose and cellulose from their complex with lignin. Depolymerization is the second step, where carbohydrate polymers produce free sugars, and fermentation of mixed hexose and pentose sugars for ethanol production (Beg et al., 2001; Lee et al., 1997). The alternate process is the synchronized saccharification and fermentation, in which mutually fermentative microorganism and hydrolytic enzymes are present in the reaction (Pérez et al., 2002; Chandrakant and Bisaria,1998).

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Material and methods The present study was conducted at Department of Microbiology, Quaid-i-Azam University Islamabad, Pakistan, Molecular Biology laboratory, Karadeniz Technical University, Trabzon, Turkey and Department of Microbiology and Cell Sciences University of Florida, USA. It included cloning, overexpression, purification, characterization, and mutational analysis of enzyme xylanase from bacterial strains.

3.1 Collection of Sampling Hot spring sediments and water samples were collected aseptically from Garam Chashma (Chitral), Tatta Pani (Azad Kashmir), Manghoper hot spring (Karachi) and Chootron hot spring (Shigar, District Sakardu Gilgit Baltistan), aseptically in sterilized bottles. The materials that were used during sampling were sterile bottles, pH strips, thermometer, GPS, ethanol (90%) and cotton. Geographic coordinates, height and atmospheric pressure were measured by GPS. pH and temperature of hot springs was also recorded by means of pH strips and thermometer. These samples were carefully transported to Department of Microbiology, Quaid-i-Azam University Islamabad and stored at 45°C.

3.2 Isolation of bacteria 3.2.1 Viable cell count or colony forming unit CFU In order to find out number and types of bacteria, these samples were diluted eight fold ranging from 10-1 to 10-8. After dilution 100 µL of the sample was transferred to the nutrient agar (NA) medium in sterile way by means of micropipette and was spread by means of sterile glass spreader. These plates were incubated at 50°C for 1 to 2 days. After incubation the number and types of colonies were examined and counted. The distinct bacterial colonies were screened for xylanase production and pure cultures were obtained by streak plate method.

3.2.2 Screening for xylanase producing thermophilic bacteria For isolation of industrially important xylanase enzyme, pure colonies of bacteria isolated from samples, were inoculated on nutrient agar medium supplemented with 0.1% Birchwood xylan (Appendix I) and incubated at 55°C for 24 hours. After incubation these plates were stained with 1% Congo-Red for 30 min and de-stained

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 41 Chapter 3 Materials and methods with 1M NaCl for 30 min and then visualized for zones of hydrolysis appeared around bacterial growth which produce xylanases. Two Positive and better zone producing strains were chosen and stored for further studies.

3.2.3 Screening for cellulase producing thermophilic bacteria For this purpose bacterial strain were directly streaked on the 1% CMC nutrient agar medium (Appendix 2) by means of inoculation loop. The pH of this medium was adjusted to 7.0. After 24 hours of incubation, the plates were stained with 1% Congo- Red solution and left undisturbed for 15 min. To visualize clear zones formed by cellulase positive strains, the plates were de-stained with 1M NaCl solution. Zone producing strains indicated the cellulase activity.

3.2.4 Screening for protease and amylase producing thermophilic bacteria Casein hydrolysis test Bacterial strains were directly streaked on the 1% casein agar medium (Appendix 3). The plates were incubated at specific temperature. After incubation the plates were flooded with 10% glacial acetic acid, clear zones of hydrolysis were observed within 5 min around colonies that produce proteases.

Amylase Test The samples were directly streaked on the 1% starch supplemented nutrient agar plates (Appendix 4). These plated were incubated on specific temperature. After incubation these plates were stained with iodine crystals. Clear zone of hydrolysis appeared around colonies that produce amylases.

3.3 Identification of xylanase producing thermophilic bacteria Xylanase producing strains AK53 and C5 were selected for this study and identified on the basis of morphological, and biochemical characteristics. 16S rRNA gene from xylanase producing strains was also sequenced for their identification.

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3.3.1 Morphological Characteristics Microscopy: Smears from each sample were prepared for staining purpose. Both Gram’s staining and spore staining was performed in order to identify their cellular morphology as well as spore formation. Gram’s staining: Gram’s staining is an auxiliary microscopic technique used to enhance the size and clarity of the microscopic objects. To highlight the morphological features of microbiological specimens, dyes and stains are frequently used. Gram’s staining technique is differential in nature and it is used to differentiate two distinct bacterial groups: gram negative and gram-positives. Gram’s staining reagents:  Primary stain: Crystal violet  Mordant: Gram’s iodine  Decolorizer: Ethanol (95%)  Counter stain: Safranin Gram’s staining was carried out for microscopic examination using fresh bacterial cultures (24 hours). Thin bacterial smear was prepared on glass slide by heat fixation. After heat fixation, crystal violet as primary stain was applied for 1 minute. After removing excessive primary stain with tap water, the smear was washed away with iodine solution for 60 seconds and washed it with water. For decolorizer ethanol (95%) was used for 90 seconds and washed the smear with tap water, and then smear was flooded with safranin solution followed by final wash. Smear was dried and observed under microscope.

Endospore staining: Smear was prepared and fixed by placing over flame for few seconds. A piece of absorbent paper was used to cover the smears by cutting to fit the smear. Slide was placed on ring stand on wire gauze. The paper was saturated with dye (malachite green), slide was heated on flame until steam started rising. Slide was re-heated many times and kept steaming for three min. To keep the paper moist, drops of malachite green were added continuously. The process is not baking but steaming only. With the help of tweezers, the paper was removed and slide was washed with water. After draining the slide, it was counterstained with safranin (0.5%) for 45 seconds, washed, blotted, and observed under the microscope.

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3.3.2 Biochemical Characterization The bacterial strains were subject to various biochemical tests and then characterized using Bergey’s Manual of Determinative Bacteriology. i. Triple sugar iron test Triple sugar iron agar medium was prepared as test tubes slants aseptically. With the help of sterilized needle, pure colony was picked and the butt of slant was stabbed. After that, the surface of slope was streaked out. Slants were incubated at 55oC for 24 hours. After incubation, slants were observed for color change from red to yellow that indicates the fermentation of carbohydrates. ii. Citrate test Simmons citrate agar medium was prepared as test tube slants. With the help of sterilized needle, pure colony was picked and the butt of slant was stabbed and surface of slope was streaked out. Slants were incubated for 24 hours at 55oC. After incubation, the positive result was noted in the form of appearance of blue color from green. iii. Oxidase test N,N-dimethyl-p-phenylenediamine oxalate was used as oxidase reagent in order to determine the activity of oxidase enzyme. Few drops of fresh reagent were poured on a piece of filter paper. Isolated bacterial colony was picked up by sterilized tooth pick and was touched over the surface of filter paper piece. After 20 seconds, color changed from pink to purple to dark purple, which was noted as positive result. iv. Motility test The purpose of this test is to determine the presence of flagella by observing motility. Semisolid sulfide indole motility (SIM) medium was prepared in test tubes; it was a stab inoculated with the help of needle with bacterial inoculum in the center. Incubation was carried out for 24 hours at 55oC and growth pattern was observed along the inoculation line. Motility was indicated by appearance of growth outside the inoculation line.

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v. Catalase test Presence of catalase enzyme was determined by using hydrogen peroxide as agent. A loopful of bacterial culture was mixed in a drop of reagent in the center of a glass slide. Presence of catalase was indicated by formation of bubbles which was noted as positive result. vi. Indole test The purpose of this test is to detect the presence of indole formation by degradation of tryptophan amino acid. Test was performed by preparing tryptophan broth medium in test tubes. The broth was inoculated with test strains and incubated at 55oC for 24 hours. After incubation, Kovac’s reagent was added (few drops), appearance of cherry red color between the layers of reagent and medium was indication of indole formation. vii. Methyl red (MR) and Voges-Proskauer (VP) test Carbohydrrate fermentation (glucose) is determined by Methyl red (MR) and Voges- Proskauer (VP) tests. MR-VP broth media was prepared, sterilized, inoculated with test strain and incubated overnight at 55oC. After incubation, two reagents Barritt’s reagent A and B were added to the medium, which leads to formation of a pink burgundy ring after 20 min indicating 2, 3-butanediol fermentation pathway. viii. Urease test The purpose of urease test is to determine whether bacterial cells can degrade urea or not. Degradation of urea results in production of carbon dioxide and ammonia. 2% urea was added to the urease broth, inoculated with test strain and incubated for 24 hours at 55oC. Phenol red was used as indicator, which was added after incubation. Positive results were based on ammonium carbonate formation which turns the medium alkaline resulting in pink red color in the presence of phenol red indicator.

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ix. Nitrate Reduction test Nitrate broth was used for nitrate reduction test. This test was performed to check the presence of enzyme nitrate reductase that can convert nitrate to nitrite or to end product ammonia. Nitrate broth was inoculated with bacterial sample and incubated for 24 hours at 55°C. After incubation reagent A (N,N-Dimethyl-α-naphthylamine in 5N Acetic acid) and few drops of reagent B (Sulfanilic acid in 5N Acetic acid) was added and color change was observed indicating nitrite production. x. Casein hydrolysis test Bacterial strains were directly streaked on the 1% casein agar medium (Appendix 3). The plates were incubated at specific temperature. After incubation the plates were flooded with 10% glacial acetic acid, clear zones of hydrolysis were observed within 5 min around colonies that produce proteases. xi. Amylase Test The samples were directly streaked on the 1% starch supplemented nutrient agar plates (Appendix 4). These plated were incubated on specific temperature. After incubation these plates were stained with iodine crystals. Clear zone of hydrolysis appeared around colonies that produce amylases. xii. Sugars fermentation test Different sugar broths were prepared for this experiment; phenol red was used as an indicator to observe the color change in case of acid and gas production. To check the ability of a bacterium that can produce gas or not during sugar fermentation, inverted tubes known as Durham Tubes were used. Glucose, lactose and sucrose were the three sugars used in this experiment. After autoclaving all the sugars tubes, bacterial cultures were inoculated with the help of sterile wire loop and incubated for 24 hours at 55oC. After incubation, the color change from pink to yellow in acidic condition was observed and result was noted.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 46 Chapter 3 Materials and methods xiii. Esculin test Bile esculin agar was prepared in test tubes in the form of agar slants. Pure culture was inoculated (streaked), and incubated for 24 hours. Appearance of growth (chocolate brown coloration) on the surface of slant indicates positive result (esculin hydrolysis).

xix. H2S test Slants of triple sugar iron agar (TSIA) were prepared, pure culture was inoculated and incubated for 24 hours at 50°C. TSIA contains ferrous ions (Fe2+), which have high affinity for sulfide ions. As a positive result, iron combines with H2S to produce FeS (black coloration). In tubes of TSIA hydrogen sulfide as bacterial product, turns agar black due to FeS production.

3.3.3 Molecular characterization DNA extraction DNA extraction of bacterial strains AK53 and C5 was carried out. Phenol-chloroform method was used to extract DNA of isolated bacterial strains (Delbe et al., 2000). 3 mL of bacterial culture was centrifuged for 4 min in Eppendorf tubes at 10,000 rpm to collect the pellets (cells). After discarding the supernatant the cells in pellet were washed two times with 600 µL TE buffer (Appendix 5). 3 µL Proteinase-K and 30 µL of 10% SDS (sodium dodecyl sulphate) were added to cell suspension and incubated in water bath for 1 hour at 37°C. After this, CTAB (80 µL) and 5M NaCl (100 µL) were added to suspension, mixed well and again incubated in water bath for 10 min at 65°C. Later on, chloroform-isoamyl alcohol (700-800 µL) was added, and then centrifuged at 10,000rpm for 15 min. The aqueous layer, thus formed was shifted in to another Eppendorf tube. Equal volumes of chloroform, isoamyl alcohol and phenol were added to this aqueous solution, mix well and then centrifuged at 10,000rpm for 15 min. Again the aqueous portion was shifted in to another Eppendorf, where isopropanol (600 µL) was mixed and then centrifuged. A pellet obtained was rinsed with ethanol (70%) and then centrifuged this mixture. The pellet was air dried and dissolved in TE buffer (100 µL). The TE buffer containing purified DNA was stored at -20°C and processed for sequencing afterwards,

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Preparation of the Agarose gel 1% Agarose was dissolved in TBE buffer (1X) (Appendix 6) and heated in microwave oven for 2 min, then cooled and ethidium bromide (3µL) was added. Later, the gel was poured in the gel tray and comb was kept inserted in gel in order to make wells, After solidification, sample (3µL) was mixed with (1µL) of 6X DNA Loading dye and was loaded in wells and electricity was supplied (110V and 400 mA) for 30 min and later it was visualized above ultraviolet rays.

Sequencing-Phylogenetic analysis 16S rRNA gene sequencing was performed for the identification of selected strain. The full length 16S rRNA gene was amplified from extracted DNA using bacterial primers: 27F’ (5-AGAGTTTGATCCTGGCTCAG) and 1494R’ (5'- CTACGGCTACCTTGTTACGA). 20 µL reaction mixture for PCR contained distilled water (10.5 µL), 10x PCR buffer (2 µL), sample DNA (1 µL), forward primer (2 µL), reverse primer (2 µL), deoxynucleotide triphosphate (dNTPs) mixture (2 µL), ex-taq DNA polymerase (0.5 µL) (Takara Shuzo, Otsu). In the first step, the reaction mixture was incubated for 4 min at 96oC. Later, 35 amplification cycles were performed for 45 seconds at 94oC, at 55oC for 60 seconds, and 72oC for 60 seconds. Further incubation was carried out at 72oC for 7 min. In case of bacteria, 1400 bp in DNA fragments were amplified. Genomic DNA of Escherichia coli was used as positive control and a negative control was also run along in PCR reaction. Montage PCR Clean up kit (Millipore) was used to purify PCR product to remove unincorporated dNTPs and PCR primers from final product. The purified PCR products of approximately 1498bp were sequenced by using 2 primers, 518F’ and 800R’. Sequencing was performed by using Big Dye terminator cycle sequencing kit v.3.1 (Applied Bio-Systems, USA). Sequencing products were resolved on an Applied Bio-Systems model 3100 automated DNA sequencing system (Applied Bio-Systems, USA). After assembly of study sequences they were blasted in NCBI and the related sequences were downloaded in FASTA format. All the sequences were aligned by MAFFT software. For the construction of phylogeny used the MEGA 4 software by Clustal W program (Thompson et al., 1994).

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3.4 Optimization Experiments To maximize xylanase production, optimization of different culture conditions was performed. For each parameter, 100 mL of production media was prepared in different Erlenmeyer flasks (500mL) and autoclaved them. After autoclaving inoculate these media with 2% inoculum. Then these flasks were kept in shaking incubator for 72 hours at 150 rpm. After every 24 hours samples were taken from these flaks aseptically and centrifuged for 15 min at 10,000 rpm. Supernatant (crude enzyme) obtained after centrifugation was subjected to enzyme activity assay and protein estimations. At last specific activity of each sample was calculated by using optical densities of enzymes and protein samples.

3.4.1 Inoculum preparation For the optimization trial, a loopful fresh pure bacterial culture was transferred to nutrient broth supplemented with xylan substrate in sterile conditions. The medium was incubated for 16 hours at optimum conditions in shaker incubator at 150 rpm at 55oC. Contents of this broth were dissolved in distilled water and pH was adjusted to 7.

3.4.2 Incubation Period effect on Growth and xylanase Production Incubation time effect on xylanase production was determined by carrying out fermentation at different incubation times (0, 24, 48 and 72 hours) at 55oC. 0.5% xylan containing media was inoculated with 2% inoculum (20 hour old) and its pH was maintained at 7.

3.4.3 Effect of Temperature on Growth and xylanase Production Optimization of temperature was done by carrying out fermentations at different temperatures (45, 50, 55, 60, 65 and 70oC) for maximum xylanase production. The media was inoculated with 2% inoculum and was adjusted at pH 7.

3.4.4 Effect of pH on Growth and xylanase Production For maximal xylanase production, optimization of pH was done by carrying out fermentations at different pH (4, 5, 6, 7, 8, and 9). 1N HCl and 1N NaOH solutions were used to adjust the medias pH. Medias containing xylan concentration of 0.5% was inoculated with 2% inocula (20 hour old).The incubation was done for 72 hours at optimized temperature.

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3.4.5 Effect of substrate concentration on Growth and xylanase Production To determine the effect of substrate concentrations on xylanase production carry out fermentations at different xylan concentrations (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0%). Media was incubated at optimized pH and temperature.

3.4.6 Effect of Size of Inoculum on Growth and xylanase Production For maximal xylanase production, inoculum size was optimized by inoculating the fermentation media with different inoculums sizes (1, 2, 3, 4, 5 and 6%). Media containing 0.5% xylan was adjusted at optimized temperature and pH. For this purpose 20 hour old fresh culture was used.

3.4.7 Effect of Age of Inoculum on Growth and xylanase Production For optimization of the optimal age of inoculum requisite for maximum xylanase production, fermentations were carried out with inocula of optimal size and of ages (20, 48, and 72). Media was incubated at optimized pH and temperature.

3.4.8 Effect of NaCl on Growth and xylanase production To analyze the optimal salt concentration or halophilic range for maximal xylanase production, carry out fermentation at different NaCl concentrations (1 to 10%). Fermentation media containing 0.5% xylan were adjusted at optimized temperature and pH.

3.5 Media, antibiotics, microbial strains and plasmids

3.5.1 Preparation of culture media Luria-Bertani culture media is a favorable medium. It is an appropriate medium mostly for chemoheterotrophic microorganisms. Luria-Bertani (LB) Broth was used for culturing, screening, preservation and storage of the different wild and mutant strains and recombinant clones. Sterilization was performed for 20 min at 121°C in autoclave. The composition of LB medium used in present study was described in Appendix 7.

3.5.2 Preparation of antibiotics All the stock solutions of antibiotics were filtered through 22 μm pore filter paper prior to storage. The cultured media were supplemented with concentrated solution of

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 50 Chapter 3 Materials and methods different antibiotics in sterile condition as seen in Table3.1.1. All antibiotic were used in sterile conditions

Table 3.1.1 Antibiotic used in this study

Antibiotic Mode of action Working Stock solution concentration Ampicillin Stops cell wall synthesis by 50 μg·mL-1 50 mg·mL-1 in

(Amp) inhibiting formation of the H2O Stored at - peptidoglycan cross-link 20°C Kanamycin Causes misreading of 50 μg·mL-1 50 mg·mL-1 in

(Kan) mRNA by binding to 70S H2O Stored at - ribosomes 20°C

3.5.3 Preparation of isopropyl-β-D-thiogalactoside LB medium was supplemented with isopropyl-β-D-thiogalactoside (IPTG) in order to promote the expression of protein of interest by inducible strains. Working solutions were prepared from IM ((238.3) mg/mL) concentration in distilled water, sterilized by filtering it from 22 μm pore sized filter paper, and stored at -20 °C. The final concentration ranging from 0.1 to1.0 mM was used for overexpression of protein.

3.5.4 Selection of Microbial Strains The recombinant bacterial strains for transformation and expression of protein used in this research are listed in table 3.1.2.

Table 3.1.2 E. coli strains used for cloning in the present work Strain Genotype Supplier E. coli JM101 supE thi-1 Δ(lac-proAB) Stratagene Technical [F´ traD36 proAB Services lacIqZΔM15]. E. coli BL21 (DE3) F– ompT hsdSB(rB-, Invitrogen mB-) gal dcm rne131 (DE3)

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3.5.5 Selection of plasmids and Vectors Different plasmids applied in this study are shown in Table 3.1.3.

Table 3.1.3 List of plasmid used in this study plasmid Features Supplier pET28a KanR, , N-terminal Hisx6 fusion Novagen tag, lacI , T7 promoter pGMT AmpR, Tac promoter, Promega p15TV-L AmpR, T7-lacO Novagen

3.5.6 Deigning of primers Primers were designed on sequences of Geobacillus thermodenitrificans xylanase obtained from GenBank containing Nhe1 and HindIII restriction sites in forward and reverse primer respectively (Table 3.1.4). The conserve regions were identified by aligning the sequence by ClustalW (Thompson et al., 1994) program implemented in MEGA 4.0 (Tamura et al., 2007) or MEGA 4.0 (Kumar et al., 2004)

Table 3.1.4 Primer used in this study Primer name Primer sequence XGeoT-F 5’-CTAgCTAgCATgTTgAAAAgATCgCgAAAAg-3’ XGeoT-R 5’CCCAAgCTTTCACTTATgATCgATAATAgCCCA-3’

3.5.7 Isolating Genomic DNA of C5 and AK53 strains through Wizard® Genomic DNA Purification Kit (Promega) Materials Supplied by LAB • Microcentrifuge tubes 1.5mL • Water bath, 37°C and 80°C • Room temperature Isopropanol • Room temperature ethanol 70% • Water bath, 65°C • EDTA (50mM) pH 8.0

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• Lysozyme 10mg/mL • Lysostaphin 10mg/mL Procedure 1. Overnight culture was transferred to microcentrifuge tube (1.5 mL) and centrifuged at 15,000×g for 4 min and supernatant was discarded.

2. Cells pellets was mixed and resuspended in 480 μL EDTA (50mM).

3. Lytic enzyme 120 μL was mixed to the re-suspended cell, and gently pipetted it to mix. The sample was incubated for 1 min at 37°C. Centrifugation was performed for 2 min at 15,000× g and supernatant was removed.

6. The cells were re-suspended in Nuclei Lysis Solution (600 μL) by gentle pipetting. 7. Cells were incubated at 80°C for 5-7 min, and cell suspension solution was cooled at room temperature. 8. RNase solution (3 μL) was added to cell lysate, and mix properly by inverting tube 3–6 times. 9. The sample was incubated for 50 min at 37°C and then cooled at room temperature. 10. 200 μL protein precipitation solution was added to cell lysate, properly mixed it and placed it for 5 min on ice. 12. The cell lysate was centrifugated at 13,000–16,000× g for 3 min. DNA in the supernatant was transferred to a new microcentrifuge tube (1.5 mL) containing isopropanol (600 μL) (room temperature). 14. Sample was mixed gently, by inversion until the DNA appeared as visible thread- like strands. 15. Sample was centrifuged at 13,000–16,000 × g for 2 min. 16. Supernatant was poured off carefully and 70% ethanol (600 μL) was added and mixed by gentle inversion of tube several times. 17. Sample was centrifuged at 13,000–16,000 × g for 2 min. Ethanol was carefully aspirated and pellet was allowed to air-dry for 10–15 min. 19. DNA rehydration solution 100 μL was added to DNA in the tube and incubated for 1 hour at 65°C. The solution was periodically mixed by gentle tapping on tube. Alternatively, DNA can also be rehydrated by overnight incubation at 4°C or room temperature. 20. Finally the DNA was kept at 2–8°C.

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3.5.8 Gel electrophoresis For electrophoresis the DNA samples were prepared by addition of loading dye (Orange/Blue, promega) 6X to final concentration of 1X. The samples were loaded into 0.8% agarose gel in 1X TBE buffer (40 mM Boric acid, 40 mM Tris buffer and 1 mM EDTA). Electrophoreses was carried out in a horizontal wide mini sub ®Cell tray (Bio-Rad) using 120 volt current. DNA bands were estimated by comparing it with DNA ladder (1 kb, GeneRuler™).

3.5.9 Quantification of nucleic acids ND-100 NanoDrop spectrophotometer was used to quantify DNA. This instrument uses sample retention technology and holds the sample at specific point by employing surface tension. About 1-2 µl of DNA sample is placed at the end of optic fiber cable, which is actually a receiving line. The source line, which is also an optic fiber cable, was contacted with liquid sample. This procedure created a gap and also forms a liquid column between both ends. The absorbance of the sample in 0.2 mm path length was measured by utilizing a flash lamp of pulsed xenon. The purity of the sample is determined by calculating the ratio of absorbance at 260nm/280nm, and accepted as approximately equals to 1.8 for DNA, while the concentration is on the basis of OD 260 nm, with a detection limit of 2 ng/μL.

3.5.10 Polymerase Chain Reaction In order to perform PCR reaction to amplify xylanase gene of strain C5 and AK53, their DNA was used as a template (50 ng), 1.5 mM MgCl2, Taq buffer (50 mM) KCl, 1% (v/v) Nonidet P40, Tris-HCl (10 mM), pH 8.8, each dNTPs (200 μM), recombinant Taq DNA polymerase (1.5 units) (Fermentas) and each forward and reverse primer (25 pM). PCR thermal profile for all primer sets include a-pre-PCR melting for 3 min (95°C), unwinding (94°C), 36 cycles (each of 60 seconds), annealing for 60 seconds (61°C), extension for 90 seconds (72°C), and final extension for 7 min (72°C). Electrophoresis was carried out after PCR, using agarose gel 1% (w/v) in TAE buffer (EDTA-1 mM, pH 8.1, Tris-acetate-40 mM).

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3.5.11 QIAquick Gel Extraction Kit Protocol (QIAGEN)

DNA fragment was excised with a sharp, clean scalpel from agarose gel. Excess agarose around band was removed to minimize the gel size. Gel slice was weighted in a clean tube, 3 volume of QG buffer was added to 1 volume of gel slice. Tube containing gel slice was incubated at 50°C for 10-15 min (or until the gel slice has completely dissolved). After dissolving the gel slice, color of mixture was observed as yellow (like Buffer QG in which agarose is not dissolved). Isopropanol equal to volume of 1 gel was added to sample and mixed. QIAquick spin column was kept in a clean collection tube (2 mL). To bind DNA with membrane sample was applied to the column, centrifugation was carried out for 1 min. Flow-through was discarded and column was placed back to same position. Buffer QG (0.5 mL) was added to column and again centrifuged (1 min). To wash, Buffer PE (750 μL) was added to column and again centrifuged for (1 min). Flow-through was discarded and column was centrifuged at ≥10,000 x g (~13,000 rpm) for another 1 min. Column was placed into a new microcentrifuge tube (1.5 mL). For DNA elution, H2O or Buffer EB (50 μL) (pH 8.5, 10 mM Tris·Cl,) was added to center of column membrane and centrifuged at maximum speed for 1 min. Alternatively, to increase the DNA concentration, elution buffer (30 μL) was added to center of the QIAquick membrane, let the column at rest for 1 min, and then centrifugation was performed for 1 min.

3.5.12 Ligation of DNA molecules (PCR Products) The procedure of ligation was performed according to the description provided by manufacturers, with the help of enzyme T4 DNA ligase. The function of T4DNA ligase in a double stranded DNA is to assemble phosphodiester bonds between 3′ OH- and 3′ PO4- ends. A specific tool was used to calculate the amount of components to obtain required ratio to carry out reaction (http://www.gibthon.org/ligate.html). In various ligation experiments, the amount of vector required was not more than their negative control. The ligation experiment was carried out for 16 hours at 16°C. The description of instructor’s manual was followed during the cloning of amplified fragment into vector pGMT.

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T4 DNA ligase Buffer (2X) 5µL pGEM®-T Easy Vectors 1µL T4 Ligase 1µL Insert 3 µL

3.6 Cloning and expression experiments

3.6.1 Transformation of competent cells with gene of interest Transformation of E.coli DH5 strains was carried out after modification of a simple technique proposed by Cohen et al (1972). The preparation of competent E.coli cells was done before carrying out the procedure of transformation.

3.6.2 Preparation of competent cells E.coli was inoculated in LB and the media was incubated at 37°C for 16 hours. After 16 hours, the bacterial culture was shifted into fresh LB (20 mL) and again incubated at 37°C with continuous shaking until the optical density of the cells at 600nm reached to 0.45-0.55, which is considered as the exponential phase of cells. At exponential phase, the culture was transferred to four sterile test tubes (5 mL in each tube) that were already ice cold, and the culture was placed on ice to cool down the tubes up to 0°C. After 10 min, the culture was centrifuged at 3000 x g by maintaining the temperature at 4°C for 5 min. After centrifugation, the harvested cells were suspended in already ice cold 100 mM CaCl2 by carefully stroking the pellet with pen or fingernail. The cells were incubated at 0°C for 20 min and again harvested and suspended with 50 mM CaCl2, which was supplemented with 15% glycerol. The cell suspensions obtained at the end were distributed into 150µL aliquots and stored at 180°C. The suspension can also be used directly for process of transformation as described below.

3.6.3 Transformation of E. coli competent cells Competent cell suspensions (200 µL) were added into sterile 1.5 mL eppendorf tube along with the DNA of interest. In a volume of 10 µL or less, the amount of DNA should not more than 12.5 ng. Then the mixture was placed in ice for 30 min incubation. Later, without any shaking the mixture was heated for 120 seconds at

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42°C and suddenly shifted to the ice for further 10 min. After 10 min, Luria broth (200 µL) was then added to each reaction mixture, which was later incubated at 37°C for 90-120 min, so that the cells may recover and express the antibiotic resistance marker that was encoded by plasmid. When the incubation time was completed, each sample (100 µL) was then transferred in to LB-agar plates supplemented with 50 µg/mL of the antibiotic ampicillin, which helped in selection of recombinant cells. All the plates were then incubated at 37°C where recombinant colonies were appeared in 12-16 hours.

3.6.4 Plasmid isolation of transformed cell Thermo Scientific GeneJET Plasmid Miniprep Kit Pelleted cells were resuspended in resuspension solution (250 μL). Cell suspension was transferred to microcentrifuge tube. To remove the cell clumps, pipetting up and down and vortexing was done to resuspend bacteria. Lysis solution (250 μL) was added in the cell suspension and mixed well by inverting the tube 4-6 times so that solution becomes slightly clear and viscous. Neutralization solution (350 μL) was added and mixed thoroughly. Chromosomal DNA and cell debris was collected as pellet after 5 min centrifugation. Supernatant obtained was pipetted out to the supplied GeneJET spin column. White precipitates were not disturbed during the transfer. The column was centrifuged for 1 min centrifugation, the flow-through was discarded and column was placed back. Wash solution (500 μL) was added to spin column, centrifuged it for 30-60 seconds, the flow-through was discarded and column was placed back. The washing step was repeated using wash solution (500μL). Every time, the flow-through was discarded and centrifuged for 1 min to take out residual wash solution. It was essential to avoid residual ethanol in plasmid preparations. GeneJET spin column was transferred to another microcentrifuge tube (1.5 mL). In order to elute the plasmid DNA, elution buffer (50 μL) was add to the membrane of GeneJET spin column, it was incubated at room temperature for two minutes; centrifugation was carried out for 2 min. Column as discarded and plasmid DNA was stored at -20°C.

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3.6.5 Verification of Cloning The presence of cloned fragment was confirmed by digesting the clones with appropriate restriction endonucleases. The restriction reaction contains plasmid (250 ng), restriction buffer (10X) diluted by nanopure water to 1X concentration and EcoRI enzyme (5.0 U) (Fermentas UAB Lithuania). Plasmid 6 µL Buffer 1 µL EcoRI (10 U/µL) 0.3 µL NP water 2.7 µL

3.6.6 Treatment of DNA with restriction enzymes Cloning and expression plasmid having our cloned xylanase gene product was treated according to specification provide by manufacturers.

Digestion of cloned DNA with HindIII enzyme The most suitable buffer for both types of enzymes was used to carry out the process of double digestions. The purpose of double digestion is to generate adhesive ends on plasmid vector as well as the inserts. In case of PCR amplicons, the quantity of the DNA used in digestion reaction was 1000ng; while 500ng was used for plasmid vectors. The incubation was carried out at 37°C for different time intervals. The isolated cloned plasmid having insert and pET28a plasmid was cut with HindIII restriction enzyme and kept at 37ºC for 2 hour, and then digestion of insert and pET28a plasmid was confirmed with gel electrophoresis. HindIII (New England biolab) (10 U/µL) 3 µL Buffer B 8 µL Plasmid 50 µL Nanopure Water 9 µL

Ethanol Precipitation After digestion with HindIII restriction enzyme, both insert and pET28a plasmids were subjected to ethanol precipitation. 1 to 10 vol of 3 M sodium acetate, pH 5.2 was added to the solution of DNA. Mixed by flicking the tube several times with a finger. 2 to 2.5 vol (calculated after salt addition) of ice-cold 100% ethanol was added.

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Mixed by vortexing and placed in -20°C for 30 min or longer. Centrifuged the mixture for 5 min at maximum speed and remove the supernatant. 1 mL of 70% ethanol was added and inverted the tube several times, then centrifuged. Supernatant was removed; pellet was dried, then re-suspended in in TE buffer, pH 8.0, if it is going to be stored indefinitely.

Digestion of cloned DNA with Nhe1 Enzyme The ethanol precipitated insert and pET28a plasmid was cut with Nhe1 restriction enzyme and kept at 37oC for 2 hour. NheI (New England biolab) (10 U/µL) 2 µL Y+/Tango™ buffer 5 µL Plasmid 43 µL After incubation the digestion of insert and pET28a plasmid was confirmed with gel electrophoresis.

3.6.7 QIAquick Gel Extraction Kit Protocol (QIAGEN) Double digested insert and pET28a plasmid was purified by QIAquick Gel Extraction Kit (QIAGEN) as per manufacturer protocol and then confirmed with gel electrophoresis afterwards.

3.6.8 Ligation of double digested pET28a plasmid and insert The double digested xylanase gene insert was ligated directly into pET28a plasmid as given in instructions. The ligation mixture composition were 10 X T4 DNA Ligase Buffer 1.5 µL Xylanase gene Insert 6 µL T4 DNA Ligase 6 µL pET28a plasmid 2 µL Incubation of ligation mixture was carried out at 14°C overnight.

3.6.9 Transformation of pET28a plasmid + insert in E. coli competent cells

200 μL competent cells prepared with CaCl2 suspensions, was transferred to a clean sterile 2.0 mL Eppendorf tube and 10 μL xylanase ligated product to pET28a plasmid

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 59 Chapter 3 Materials and methods was added. The resulting ligated mixture combined with competent cells was incubated on ice for 30 min, then shifted to 42°C for 120 sec without shaking, and then rapidly shifted to ice again. 100 µL of transformed culture was spread on LB agar. Colonies were selected using kanamycin (50µg/mL). Single colony was selected using sterile toothpicks and was inoculated in LB broth supplemented with kanamycin (50µg/mL). Incubation was carried out in orbital shaker at 37°C

3.6.10 Isolation of plasmid of transformed cell Thermo Scientific GeneJET Plasmid Miniprep Kit The overnight culture was centrifuged to obtain cell pellet. The cells in pellet were re- suspended in Resuspension solution (250 μL). Then the cell suspension was transferred to microcentrifuge tubes. Later, lysis solution (250 μL) was added, mixed well by inverting the tube 4-6 times to make the mixture becomes slightly clear and viscous. Neutralization solution (350 μL) was added to lysis solution, mixed thoroughly by inverting the tube. Centrifugation was carried out for 5 min to shift the chromosomal DNA and cell debris into pellet. Supernatant was transferred by pipetting to GeneJET spin column, centrifuged for 1 min. Flow-through was discarded and column was placed back to the same collection tube. Wash solution (500 μL) was added to spin column, centrifuged for 30-60 seconds and the flow- through was discarded. Column was placed back to the same position. Washing procedure was repeated, discarded flow-through and centrifuged the column for another one min to separate out residues of wash solution. This step is essential as in plasmid preps, it avoids residual ethanol. GeneJET spin column was transferred into another microcentrifuge tube (1.5 mL). Elution Buffer (50 μL) was added to middle of spin column for plasmid DNA elution. Fractions after elution were incubated at room temperature for 2 min and then centrifuged (2 min). Column was discarded and purified plasmid DNA was kept at -20°C.

3.6.11 Verification of xylanase Cloning The presence of xylanase-cloned fragment was confirmed by double digestion using restriction endonucleases. The restriction reaction was containing respective restriction buffer (10X) which was made diluted to 1X final concentration using nanopure water. The mixture was well mixed and incubated for 2 hours at 37°C, after

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 60 Chapter 3 Materials and methods incubation the digestion of cloned plasmid was confirmed by 1% agarose gel electrophoresis as described previously. NheI (New England biolab) (10 U/µL) 0.4 µL Y+/Tango™ buffer 1.5 µL HindIII 0.25 µL Plasmid 6 µL NP Water 1.85 µL

3.6.12 Sequencing of cloned xylanase gene In order to sequence the xylanase cloned pET28a plasmid, purification was carried out using PCR Clean-Up System Kit (Promega Corp., Madison, WI, USA) and Wizard® SV Gel. Xylanase cloned pET28a plasmids were sequenced using the Taq DyeDeoxy Terminator Cycle Sequencing Kit according to the manufacturer’s instructions, and analyzed with an Applied Biosystems (Macrogen, Korea) Model 370A automatic sequencer.

3.7 Bioinformatics analysis BLASTn and BLASTp programs were used for the analysis of nucleotides and their deduced amino acids, respectively (http://www.ncbi.nlm.nih.gov/BLAST/). Multiple sequence alignment of xylanase was carried out using CLUSTALW program (http://www.ebi.ac.uk/clustalW) and phylogenetic analysis and dendrogram construction for the GthAK53Xyl and GthC5Xyl was performed using MEGA 6.0 (with minimum evolution).

3.8 Growth conditions of induced recombinant strains BL21 (DE3) Competent Cell After confirming the DNA sequence of recombinant clone, protein expression was carried out. Luria broth was supplemented with an antibiotic kanamycin and recombinant strain was allowed to grow overnight (16 hours) in this media at 37°C. The overnight culture was transferred to another Erlenmeyer flask that was half-filled with similar constituents of media and antibiotic. However, the inoculum: culture volume ratio at this stage was maintained as 1:20. After almost three hours of incubation, the optical density of culture at 600nm reached from 0.5 to 0.8. At this

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 61 Chapter 3 Materials and methods point, 1mM of IPTG was used to introduce protein expressions, and cells were allowed to grow for 20 hours at 37ºC in shaker incubator.

3.9 Purification 3.9.1 Ammonium sulphate precipitation The crude enzyme was purified from the culture supernatant and lyzed cell by frenh press using ammonium sulphate in a sodium phosphate buffer of pH 7.0. 30-80% ammonium sulphate was used for precipitation of enzymes. The respective levels were mixed in 500 mL of crude enzyme filtrate and kept at 4°C for one to two hours with continuous stirring. The precipitates were collected and analyzed for xylanase activity. The optimum xylanase activity at a specific concentration of ammonium sulphate reflects the best concentration to attain maximum enzyme recovery.

3.9.2 Dialysis of recombinant xylanase enzyme After precipitation, the excessive ammonium sulphate in the enzyme solution was removed by subjecting it to dialysis in a buffer of pH 7.0 at 4°C for 24 hours (Carmona et al., 1998).

3.9.3 DEAE Sepharose chromatography The basic principle in ion exchange chromatography involves the separation of the one component in a mixture from another on the basis of number of charges of appropriate sign carried by each solute molecule for interaction with ion exchanger under the condition used. Procedure The dialyzed enzymes were further purified using an anion-exchange column chromatograph as DEAE Sepharose fast flow. The column of ion-exchange chromatograph (1.5 × 50 cm) of DEAE-Sepharose was used. Sepharose Fast Flow ion exchange media were supplied preswollen in 20% ethanol. Gel slurry was poured into the column. The obtained 60 % precipitates were loaded on a column (1.5 × 50 cm) of DEAE-Sepharose pre-equilibrated with 10 mM sodium phosphate buffer pH 6.0. The column was washed with 1000 mL of the same buffer at flow rate of 0.5 mL/min. and then dialyzed enzyme solution was passed from the column, column was eluted with linear gradient of (0.55 M) NaCl in sodium phosphate buffer. Each fraction (3 mL)

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 62 Chapter 3 Materials and methods was collected. Each fraction was determined for xylanase activity. The active fractions were combined and concentrated by ultrafiltration (Sartorious, 30000 MWCO filters).

3.9.4 Protein electrophoresis SDS - polyacrylamide gel electrophoresis The procedure of protein electrophoresis was done with the help of a Mini-Protean Electrophoresis System. According to supplier instruction, 30% Acryl-bisacrylamide mixture and TEMED were hand cast in the specifically designed apparatus that was provided with electrophoresis system. In order to obtain better definition molecular weight ranges from higher to lower, the acrylamide concentration varied from 8-15% in resolving gel, respectively. Further, 2% of birchwood xylan was supplemented in resolving gel to a final concentration of 0.15% in case of analyzing enzyme activity by zymogram. In stacking gel the concentration of staking gel was maintained as 5% and the buffer were prepared by appropriate procedures as stated in Appendix 8. Before carrying out electrophoresis, proteins were disaggregated by adding 3X loading buffer into all samples and heated at 95°C for 5 minutes. During separation procedure the voltage used for staking gel and resolving gel were 200 and 180 volts respectively (Appendix 9). When the SDS-PAGE separation was completed, the Coomassie solution was used for staining purpose (Appendix 10). Zymogen analysis was also done by the procedure described below (3.9.6). The molecular weight of proteins was assessed by comparing the movement of protein bands with MW standard.

3.9.5 Staining SDS polyacrylamide gels of protein Separated proteins were visualized by Coomassie Brilliant Blue staining on polyacrylamide gel electrophoresis. Gels were placed first in staining then destaining solution for 1 hour each (Appendix 11). The coomassie stain cannot bind to the gel but it intercalates nonspecifically to the proteins, therefore after destaining the proteins appears as blue bands on the gel. The gel was kept for longer time in destaining solution 2 (Appendix 12).

3.9.6 Zymogram analysis After SDS-PAGE, gel was soaked in 2.5 % (v/v) Triton X-100 for 30 min in order to remove SDS and re-nature the proteins. Washed it thoroughly with 50 mM phosphate

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 63 Chapter 3 Materials and methods buffer (pH 6.0) (Appendix 13) and then incubated in 1 % xylan for 30 min at 60°C. Now the gel was placed in 0.1 % (w/v) Congo red solution for 15 min and then destained with l M NaCl until hydrolysis zones appeared against red background. The reaction was stopped by dipping gel into 5 % acetic acid solution.

3.9.7 HPLC of recombinant expressed xylanase The purity of enzyme was checked by High Performance Liquid Chromatography (HPLC System 600 Waters, Waters Corporation, Massachusetts, USA). C-18 column reverse phase (4.6 x 250 mm; E. Merck, Germany) was used for HPLC analysis. The solvent system was acetonitrile-water (70:30) at a flow rate of 0.5 mL/min. Absorbance was read at 280 nm using a highly sensitive photo-diode array (PDA) detector (996 Waters).

3.10 Enzyme assay for xylanase 3.10.1 Reagents Enzyme activity was calculated using quantitative assay by using following reagents. i. Sodium Phosphate Buffer (100 mM) Monosodium phosphate was dissolved in distilled water (1 L). Disodium phosphate was added into it and adjusted its pH up to 7.0. ii. DNS Reagent DNS reagent chemical composition has shown in (Appendix 14). DNS is light sensitive, therefore, an amber brown bottle was used to prepare the solution and kept in refrigerator for long time usage. iii. Xylan Substrate 1.5 gram oat spelt xylan was added in 30 mL of water. Put it at hot plate stirrer until boiling and then shifted to cold plate stirrer for 20 min. It was centrifuged at 5000 rpm for 30 min and supernatant was used as a substrate. iv. Reducing sugar estimation by DNS assay method Enzyme assay was performed by the method described by Bailey et al., (1992) with slight modification. Estimation of reducing sugar was done by a method suggested by Miller (1959) with slight modification. Blanks and assay tubes (enzyme sample) were prepared. For enzyme assay, 100 µL of sample was mixed with 100 µL of 5% oat spelt xylan and 125 µL sodium phosphate buffer in a final volume of 500 µL. The

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 64 Chapter 3 Materials and methods blanks and assay tubes without enzyme samples were incubated together at 60°C in water bath for 10 min. After incubation DNS reagent (500 µL) was added to all the tubes, and then kept in water bath with boiling water for 10 min. Tubes were cooled at room temperature for considerable amount of time. Finally, the color developed in case of each assay tube was read spectrophotometrically against 540nm wavelength. Optical density values of blanks were subtracted from their corresponding enzyme sample values. This was done to get the actual amount of xylose produced from the substrate by the action of enzyme in the sample, during the 10 min incubation period of the assay procedure. An amount of 1 unit of xylanase activity (U) was defined as the amount of enzyme liberating 1 µmol of reducing sugar under assay conditions (Boonchuay et al., 2014). v Standard curve of xylose Xylose standard curve was plotted by preparing xylose stock solution (1mg/mL) in distilled water. Serial dilution was carried out to achieve eleven different dilutions. Reducing sugar content was determined in each dilution. After addition of DNS reagent to each dilution tube, tubes were boiled for 10 min in water bath. After cooling the tubes at room temperature, color development was appeared and measured at 540nm. A graph was plotted between optical densities (absorption spectra) of dilutions against respective concentration using Microsoft OfficeTM Excel. In unknown samples, the xylose concentration was measured by following formula: x= y + 0.0929/0.1291 Here, X= Xylose concentration (µg) (unknown sample) Y= Optical density at 540nm (unknown sample) vi. Protein estimation Bradford (1976) method was used to determine protein concentration in each enzyme sample. Bovine serum albumin was used as a standard in this procedure. The reagents used in protein estimation have shown in Appendix 15. Total protein concentration in a sample was measured using Bradford protein assay. In this assay, protein molecules bind to Coomassie dye at low pH which causes change in color from brown to blue.

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Procedure 10µL of protein sample was added to 0.15 M NaCl (90 µL) solution, while 100 µL of 0.15 M NaCl was added to the control tube. 3 mL Bradford reagent dye was added and kept at room temperature for 10 to 15 min, after that the absorbance was read at 595nm. vii. Protein standard curve 10µL of protein sample was added to 90 µL of 0.15 M NaCl solution, while 100 µL of 0.15 M NaCl was added to the control tube. 3 mL of Bradford reagent dye was added and kept at room temperature for 10 to 15 min, then absorbance was noted at 595nm. A stock of bovine serum albumin (BSA) (1 mg/mL) was prepared. The stock was serially diluted to ten different dilutions to attain final concentration of 1 mg/mL. Bradford (1976) method was used to determine protein content in each dilution by evaluating each dilution quantitatively, and then optical density of each sample (dilution) was calculated spectrophotometrically at 595 nm. By using Microsoft officeTM Excel programme, optical density of each dilution was plotted against their relative concentrations in a graph. Protein content was estimated in unknown samples, by the formula obtained from standard curve graph: x = (y+0.0591)/0.0887

Here, X = Concentration of protein (mg/mL) (unknown sample) Y= Optical density at 595nm (unknown sample) vii. Specific activity calculation In each sample, values of specific activity (U/mg) were obtained by dividing sample enzyme activity (U/mL) to total protein concentration (mg/mL) in each sample.

3.11 Characterization of recombinant xylanase The expressed purified enzyme from the culture supernatant was characterized by various methods:

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3.11.1 Effect of temperature on recombinant xylanase activity and stability The effect of temperature on recombinant purified xylanase was determined by measuring the relative activity at different temperatures ranging from 20-90°C. The maximum activity was expressed as 100% and was used as a reference to determine the relative activity at various temperatures. Thermostability of the xylanase was determined by incubating the enzymes at 50-90°C for 200 min and relative activity was assayed.

3.11.2 Effect of pH on recombinant xylanase activity and stability The relative activity of recombinant xylanase was measured over a wide pH range. The buffers used in the study for different pH ranges include 0.1M Sodium citrate buffer (3.0-6.0), 0.1M Sodium Phosphate buffer (7.0-8.0) and 0.1M Glycine NaOH buffer (9.0-10.0). pH stability of the recombinant xylanase was determined by incubating the enzymes at pH 3.0-10.0 for 200 min. The activity was assayed as described above, and relative activity was determined.

3.11.3 Metal ions effect on purified recombinant xylanase activity The effect of metal ions on activity of recombinant xylanase was determined by measuring the remaining activity after incubation at varying concentrations of metal ions i.e. 1Mm, 5Mm and 10mM at optimum temperature and pH. Metal ions tested were Na, Ca, Mg, Mn, K, Hg, Zn, Fe, Li, Ba, Cu and ZnSO4. The activity was assayed as described above, and relative activity was determined.

3.11.4 Effect of Inhibitors on purified recombinant xylanase activity Effect of inhibitors such as EDTA, β-mercaptoethanol, sodium dodecyl sulphate (SDS) and phenyl methyl sulphonyl fluoride (PMSF) on activity of purified recombinant xylanase was determined. The activity was measured after incubating the enzyme with 1mM, 5mM and 10mM of these inhibitors at optimum pH and temperature. The activity was assayed as described above, and relative activity was determined.

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3.11.5 Effect of organic solvents on purified recombinant xylanase activity The residual activity of enzyme was determined after incubating with different concentration of acetone, isopropanol and ethanol under the standard conditions and relative activity was determined.

3.11.6 Effect of salt concentration on recombinant xylanase activity The residual activity was assayed in the presence of different concentration of NaCl till 1.5 M. The activity was assayed as described above, and relative activity was determined.

3.11.7 Determination of Km value

Km is the characteristic constant of an enzyme catalyzes reaction and is defined as the substrate concentration at which enzyme shows half of its maximum velocity (Vmax).

Km and Vmax was calculated by Lineweaver and Burk plot (1934).

Protocol Purified recombinant GthC5Xyl and GthAK53Xyl xylanase in fixed amount with different amounts of substrate (0 mg/mL to 35mg/mL) was incubated at optimum temperature and pH for 5 min. After calculating enzyme activity, Km was calculated from the graph by plotting reciprocal of V against the reciprocal of substrate concentration (S).

Calculation When 1/V and 1/S were plotted against each other, a straight line was obtained in which 1/Vmax was at intercept on the ordinate and Km/S value was represented at intersect on the abscissa (Lineweaver and Burk 1943).

Following equation was used to determine Km.

Km = Y/X*V Here,

V=Vmax (maximum velocity) X= 1/ S Y= 1/V

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3.11.8 Analysis of hydrolysis product In order to analysis the xylanase hydrolysis products, purified recombinant GthC5Xyl and GthAK53Xyl xylanase were incubated with 1% (w/v) xylan in 100 mM sodium phosphate buffer at pH 6.0 and 5.0, respectively, for 10 hour at 60°C. After incubation, the samples were centrifuged at 3000xg for 12 min to remove insoluble material. Thin layer chromatography (TLC) was performed by ascending method on silica gel 60 F254TLC plates (Merck) with acetic acid, n-butanol and water (2:1:1) containing solvent system. TLC plates were spotted with 3 µL aliquots. At the end of experiment, plates were then sprayed with 5% (v/v) sulfuric acid in ethanol and then heated at 120°C for about 10 min for the sugars detection.

3.11.9 Substrate specificity of recombinant xylanase Recombinant GthC5Xyl and GthAK53Xyl enzyme activity was evaluated at 60 and 70°C and pH 6.0 and 5.0, respectively for 10 min with various substrates for the determination of enzyme specificity. The substrtaes which were tested were Oatspelt xylan, beechwood xylan, breechwood xylan, carboxymethyl cellulose, Avicel, filter paper, pNP-β- Xylopyranoside, pNP-α-L-arabinofuranoside, pNP-α-glucopyranoside, pNP-β-galactopyranoside, pNP-α-D-Xylopyranoside and pNP- acetate.

3.11.10 Shelf life Determination of xylanase Purified recombinant GthC5Xyl and GthAK53Xyl xylanase were kept at room temperature and in refrigerator at 4°C in order to determine their shelf life. Samples were removed at regular intervals and residual activity was determined uptill 24 weeks.

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Mutational analysis of AK53 and C5 recombinant xylanase 3.12.1 Molecular modeling The mimicking of the molecular/atomic behavior of chemical entities can be ascribed as molecular modeling. This procedure is widely applied on large complex biological molecules. A calculation utilized to generate the atomic behavior of large molecule is not suitable for a human mind, therefore, computers are utilized to speed up the calculation. The data utilized for calculation is usually derived from molecular mechanics, which utilizes Newtonian laws and its derivatives to affiliate the energy values to atomic coordinates. These energy values are further utilized to predict the most energetically favorable molecular model.

3.12.2 Homology Modeling The low amount of known crystal structures of proteins relative to known protein sequences becomes a hurdle to scientist working on molecular characterization of these proteins. One solution to this problem is homology modeling, the techniques utilizes the basis of homology to derive a novel structure. The concept of homology usually affiliate in this regard is that if two proteins have similar sequence then their folds will be similar, and if the folding is similar then their structure would also be similar. So, we tend to discover similar structure on the basis of sequence similarity. The rate of accurately determining a structure is high as long as the sequence similarity between the two structures is relatively high.

3.12.3 Fold Recognition Protein modeling is done by fold recognition method which is used to those proteins which possess the same fold as of known proteins but do not have the homologous proteins with known structure. It diverges from the homology modeling technique of structure prediction as protein folding is used for those proteins that do not have their homologous protein arrangements submitted in the Protein Data Bank (PDB), however the proteins which are deposited the homology modeling is being used. Folding operates by using statistical data of the association between the structures deposited in the PDB and the protein sequence which one desires to model.

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Methodology The G. thermodenitrificans strain AK53 and C5 sequences were subjected to similarity search with blast of NCBI server, to find suitable homolog of these sequences. Homolog was then used to construct a 3-D homology model of the sequence with the help of MODELLER 9. Resultant models were analyzed using PROCHECK and ProSA to find steric clashes and energy values of back bone respectively for each model generated. Model of G. thermodenitrificans strain AK53 and C5 was further used to induce the mutation and their sidechain conformations were calculated using scrwl 4.0.

3.12.4 Identification of key residues The 3D structure prediction of given amino acid (aa) sequence for AK53 or C5 was performed by using ESyPred3D Web Server 1.0 (Lambert et al., 2000). After creating the 3D structure of AK53 and C5 proteins, visualized the 3D structure in PyMol software and further analysed the structure manually in order to identify the possible biochemical interactions within the structure. This analysis gives advantage of identifying the weak amino acid (aa) interactions in the structure, the possible targets for rational design-based aa substitutions, which may eventually improve the specific characteristics of the protein. In this case, 3D structure of protein from strain AK53 and C5 was analysed in PyMol software from N terminus to C terminus for its current interaction and related compositions and the specific amino acid residues which could contribute to the overall thermostability were identified through manual inquiry. Later on, the identified amino acid residues were substituted with convenient amino acid, carrying potential to increase stability.

On the basis of bioinformatics tools 11 mutant model W137R, D328R, N84R, D339R, R81P, H82E, W185P, D186E, double mutant W137R/D328R,W185P/D186E and triple mutant H82E/W185P/D186E for C5 xylanase was created. For AK53 Xylanase W127R, D318R, N74R, D329R, double mutant W127R/D318R, R71P/H72E, and W175P/D176E modeled were designed.

3.12.5 Primer Design for mutation Primers were designed on G. thermodenitrificans AK53 and C5 xylanase sequence on specific sites and the codon for amino acid was changed. In G. thermodenitrificans C5

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 71 Chapter 3 Materials and methods xylanase, nucleotide sequence TGG was changed to CGT at 137 position (W137R) to introduce arginine instead of tryptophan amino acid, while GAT was changed to CGA at 328 position to introduce arginine instead of aspartic acid. AAC was changed to CGC at 84 position (N84R) to introduce arginine instead of asparagine, GAT was changed to CGC at 339 position (D339R) to introduce arginine instead of aspartic acid, CGC was changed to CCG at 81 position (R81P) to introduce proline instead of arginine, CAT was changed to GAA at 82 position (H82E) to introduce glutamic acid instead of histidine, TGG was changed to CCC at 185 position (W185P) to introduce proline instead of tryptophan, GAC was changed to GAG at 186 position (D186E) to introduce glutamic acid instead of aspartic acid. Double mutant W185P/D186E primer was also designed, while triple mutant H82E/W185P/D186E was achieved by combining of H82E and DM W185P/D186E mutant model.

In G. thermodenitrificans AK53 xylanase TGG was changed to CGC at 127 positions (W127R) to introduce arginine instead of tryptophan amino acid, GAT was changed to CGA at 318 position (D318R) to introduce arginine instead of aspartic acid. Both W127R and D318R were combined to produce double mutant W127R/D318R. AAC was changed to CGT at 74 positions (N74R) to introduce arginine instead of asparagine amino acid, GAT was changed to CGC at 329 position (D329R) to introduce arginine instead of aspartic acid, CGC was changed to CCT at 71 position (R71P) to introduce proline instead of arginine, CAT was changed to GAG at 72 position (H72E) to introduce glutamic acid instead of histidine, TGG was changed to CCC at 175 position (W175P) to introduce proline instead of tryptophan, while GAC was changed to GAG at 176 position (D176E) to introduce glutamic acid instead of aspartic acid. Forward and reverse primer was synthesized having the altered codon.

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Table 3.2.1 Primer used for C5 xylanase and their mutation

Primer name C5 Sequence XGeoT-F: 5’-CTAgCTAgCATgTTgAAAAgATCgCgAAAAg-3’ XGeoT-R 5’CCCAAgCTTTCACTTATgATCgATAATAgCCCA-3’ p15Xyl-F : 5'- TTgTATTTCCAgggCATggCTAgCATgTTg -3' p15Xyl-R 5'CAAgCTTCgTCATCATCATTgATCgATAATAg-3'

84-F 5’-CGCCATTTTcgcAGCATTGTCGCtg-3’ 84-R 5’-cgACAATGCTgcgAAAATGGCGTTttag-3’ 339-F 5’- AAAAACTCAGCcgcAAAATCAGTAAC-3’ 339-R 5’- GTTACTGATTTTgcgGCTGAGTTTTTC-3’ W137R-F 5’- AGTACCTCAAcgtTTCTTTCTTGACAAG- 3’ W137R-R 5’- CAAGAAAGAAaCgTTGAGGTACTTGG- 3’ D328R-F 5’- GAATCGCTATcgaCGATTGTTTAAGCTG- 3’ D328R-R 5’- TAAACAATCGtcgATAGCGATTCGCTTG- 3’ R81P F 5'- GCTAAAACCGCATTTTAACAGCATTGTCGC -3' R81P R 5'- GCGACAATGCTGTTAAAATGCGGTTTTAGCAT -3' H82E F 5'- GCTAAAACGCGAATTTAACAGCATTGTCGC -3' H82E R 5'- GCTGTTAAATTCGCGTTTTAGCA -3' W185P/D186E F 5'GACATCAAATATCCCGAgGTTGTAAATGAGGTAGTCGGGG -3' W185P/D186E R 5'-CCCCGACTACCTCATTTACAACCTCGGGATATTTGATGTCA -3' W185P-F 5'-GACATCAAATATCCCGACGTTGTAAATGAGGTAGTCGGGG -3' W185P-R 5'-CCCCGACTACCTCATTTACAACGTCGGGATATTTGATGTCA -3' D186E F 5'-GACATCAAATATTGGGAgGTTGTAAATGAGGTAGTCGGGG -3' D186E R 5'-CCCCGACTACCTCATTTACAACCTCCCAATATTTGATGTCA -3'

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Table 3.2.2 Primer used for AK53 xylanase and their mutation

Primer name AK53 Primer sequence XGeoT-F 5’-CTAgCTAgCATgTTgAAAAgATCgCgAAAAg-3’ XGeoT-R 5’CCCAAgCTTTCACTTATgATCgATAATAgCCCA-3’ p15AK53Xyl-F -F 5’ TTGTATTTCCAGGGCATGTTgAAAAgATCgCgAAAAgC- 3’ p15AK53Xyl -R 5’ CAAGCTTCGTCATCATTATgATCgATAATAgCCCA -3’ W127R-F 5'- AGTACCTCAACGCTTCTTTCTTGACAAG -3' W127R-R 5'- AAGAAAGAAGCGTTGAGGTACTTGG -3' D318R-F 5'- GGATCGCTATcgaCGATTGTTTAAGCTG -3' D318R-R 5'- TAAACAATCGtcgATAGCGATCCGCTTG -3' N74R- F 5'- ACGCCATTTTcgtAGCATTGTCGCtgAG-3' N74R-R 5'- cgACAATGCTACGAAAATGGCGTTttag -3' D329R-F 5'- AAAAACTCAGCcgcAAAATCAGTAAC 3' D329R-R 5'- GTTACTGATTTTGcgGCTGAGTTTTTC -3' DM R71P/ H72E-F 5'- CATGCTAAAACctGAGTTTAACAGCATTGTCGC -3' DM R71P/ H72E-F 5'- GCGACAATGCTGTTAAACTCaggTTTTAGCATGG-3' W175P/D176E F 5'-GACATCAAATATcccGAGGTTGTAAATGAGGTAG TCGGGGatg-3' W175P D176E R 5'CATCCCCGACTACCTCATTTACAACCTCGGGATAT TTGATGTCATC-3'

3.13 Plasmid isolation of transformed cell Recombinant cells were centrifuged to obtain cell pellet. The cells were re-suspended in 250 μL buffer P1 containing RNase A and transferred to a microcentrifuge tube. 250 μL buffer P2 was added and mixed thoroughly by inverting the tube 4–6 times. 350 μL buffer N3 was added and mix immediately and thoroughly by inverting the tube 4–6 times. Mixture was centrifuged for 10 min at 13,000 rpm (~17,900 x g) in a table- top microcentrifuge. 800 μL supernatant was applied from above step to the QIAprep 2.0 spin column by pipetting. Centrifuged for 1 minute and discarded the flow-

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 74 Chapter 3 Materials and methods through. Washed the QIAprep 2.0 spin column by adding 0.5 mL buffer PB and centrifuged for 1 min and flow-through was discarded. QIAprep 2.0 spin column was washed by adding 0.75 mL of buffer PE and centrifuged for 1 minute. Flow-through was discarded, and centrifuged at 13,000 rpm for an additional 1 min to remove residual wash buffer. QIAprep 2.0 column was placed in a clean 1.5 mL microcentrifuge tube. To elute DNA, 50 μL buffer EB (10 mM Tris·Cl, pH 8.5) was added to the center of each QIAprep 2.0 spin column, and centrifuged for 1 min. Plasmid was confirmed on agarose gel electrophoresis.

3.14 PCR amplification of G. thermodenitrificans strain C5 xylanase mutants Site-directed mutagenesis was performed by overlap extension PCR in order to replace the specific residues identified in the analyses described above (Ho et al., 1989).

3.14.1 G. thermodenitrificans C5 W137R mutant PCR amplification Two sets of PCR were performed to amplify xylanase gene with mutated nucleotides. In first set, the recombinant plasmid of strain C5 was amplified with XGeoT-R and W137R-F primer and XGeoT-F and W137R-R at a thermal profile of primary denaturation at 94°C for 2 min. after primary denaturation 36 cycles were carried out, denaturation at 94°C for 40 seconds, annealing at 56°C for 40 seconds and extension at 72°C for 60 seconds, and a last extension at 72°C for 6 min. After the first set of PCR, seconds set was carried out with amplified product isolated by QIAquick gel Extraction Kit as a DNA template with XGeoT-F and XGeoT-R primer set in a thermal cycler having PCR condition as initial melting at 95°C for 3 minutes then 36 cycles of denaturing at 94°C for 60 seconds, annealing at 61°C for 60 seconds and extension at 72°C for 80 seconds, and a final extension at 72°C for 7 min. After PCR, the products were analyzed by electrophoresis using 1% (w/v) agarose gel in TAE buffer.

3.14.2 G. thermodenitrificans C5 D328R mutant PCR amplification Two sets of PCR were performed to amplify xylanase gene with mutated nucleotides. In first set, the recombinant plasmid of strain C5 was amplified with XGeoT-R and

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D328R-F primer and XGeoT-F and D328R-R at a thermal profile of denaturation at 94°C for 2 min followed by 35 cycles of denaturing at 94°C for 40 seconds, annealing at 57°C for 40 seconds and extension at 72°C for 60 seconds, and a final extension at 72°C for 8 min. Seconds set of PCR was carried out with amplified product isolated by QIAquick gel Extraction Kit as a DNA template with XGeoT-F and XGeoT-R primer with above PCR conditions and products were analyzed by electrophoresis using 1% (w/v) agarose gel in TAE buffer.

3.14.3 G. thermodenitrificans C5 W137R/D328R mutant PCR amplification The recombinant mutant plasmid of W137R of C5 was amplified with XGeoT-F and D328R-R primer, and D328R was amplified with XGeoT-R and D328R-F primer during first set of PCR. The amplified fragment was isolated from gel by QIAquick Gel Extraction Kit and used as a template in second set of PCR to amplify full double mutant xylanase gene product with XGeoT-F and XGeoT-R primer set in a thermal cycler having PCR condition as primary denaturation at 95°C for 3 min followed by 32 cycles of denaturing at 94°C for 60 seconds, annealing at 61°C for 60 seconds and extension at 72°C for 80 seconds, and a final extension at 72°C for 7 min. After PCR, the products were analyzed by electrophoresis using 1% (w/v) agarose gel in TAE buffer (40 mMTris-acetate, 1 mM EDTA pH 8.1).

3.14.4 G. thermodenitrificans C5 N84R mutant PCR amplification Two sets of PCR were performed to amplify xylanase gene with mutated nucleotides. In first set of PCR reaction, the recombinant plasmid of C5 was amplified with p15Xyl-R and N84R-F primer and p15Xyl-F and N84R-R at a thermal profile of denaturation at 98°C for 30 seconds followed by 36 cycles of denaturing at 94°C for 40 seconds, annealing at 57°C for 40 seconds and extension at 72°C for 60 seconds, and a final extension at 72°C for 6 min. After first set, second set of PCR was carried out with amplified product isolated by QIAquick Gel Extraction Kit as a DNA template with p15Xyl-F and p15Xyl-R primer set in a thermal cycler having PCR condition as primary denaturation at 98°C for 30 seconds followed by 36 cycles of denaturing at 98°C for 10 seconds, annealing at 61°C for 60 seconds and extension at 72°C for 60 seconds, and a final extension at 72°C for 2 min. After PCR, the products were analyzed by electrophoresis using 1% (w/v) agarose gel in TAE buffer (40 mMTris-acetate, 1 mM EDTA pH 8.1) according to Sambrook and Russell (2001).

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3.14.5 G. thermodenitrificans C5 D339R mutant PCR amplification Two sets of PCR were performed to amplify xylanase gene with mutated nucleotides. In first set of PCR reaction, the recombinant plasmid of C5 was amplified with p15Xyl-R and D339R-F primer and p15Xyl-F and D339R-R at a thermal profile of denaturation at 98°C for 30 seconds followed by 35 cycles of denaturing at 98°C for 10 seconds, annealing at 57°C for 45 seconds and extension at 72°C for 45 seconds, and a final extension at 72°C for 2 min. After first set, second set of PCR was carried out with amplified product isolated by QIAquick Gel Extraction Kit as a DNA template with p15Xyl-F and p15Xyl-R primer set in a thermal cycler having PCR condition as primary denaturation at 98°C for 30 seconds followed by 31 cycles of denaturing at 94°C for 10 seconds, annealing at 61°C for 60 seconds and extension at 72°C for 45 seconds, and a final extension at 72°C for 2 min. After PCR, the products were analyzed by electrophoresis using 1% (w/v) agarose gel in TAE buffer.

3.14.6 G. thermodenitrificans C5 R81P and H82E mutant PCR amplification Two sets of PCR were performed to amplify xylanase gene with mutated nucleotides.in first set of PCR reaction. The recombinant plasmid of C5 was amplified with p15Xyl-R and R81P-F primer and p15Xyl-F and R81P-R for R81P mutation and p15Xyl-R and H82E- F primer and p15Xyl-F and H82E-R for H82E mutation at a thermal profile of denaturation at 98°C for 30 seconds followed by 36 cycles of denaturing at 98°C for 10 seconds, annealing at 58°C for 40 seconds and extension at 72°C for 45 seconds, and a final extension at 72°C for 2 min. After first set, second set of PCR was carried out with amplified product isolated by QIAquick Gel Extraction Kit as a DNA template with p15Xyl-F and p15Xyl-R primer set in a thermal cycler having PCR condition as primary denaturation at 98°C for 30 seconds followed by 35 cycles of denaturing at 98°C for 10 seconds, annealing at 61°C for 60 seconds and extension at 72°C for 45 seconds, and a final extension at 72°C for 2 min. After PCR, the products were analyzed by electrophoresis using 1% (w/v) agarose gel in TAE buffer.

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3.14.7 G. thermodenitrificans C5 W185P, D186E, DM W185P/D186E mutant PCR amplification Two sets of PCR were performed to amplify xylanase gene with mutated nucleotides. In first set of PCR reaction, the recombinant plasmid of C5 was amplified with p15Xyl-R and W185P-F primer and p15Xyl-F and W185P-R for W185P mutation, p15Xyl-R and D186E-F primer and p15Xyl-F and D186E-R for D186E mutation, p15Xyl-R and DM W185P/D186E-F and p15Xyl-F and DM W185P/D186E-R primers for W185P/ D186E double mutant at a thermal profile of denaturation at 98°C for 30 seconds followed by 36 cycles of denaturing at 98°C for 10 seconds, annealing at 58°C for 40 seconds and extension at 72°C for 45 seconds, and a final extension at 72°C for 2 min. After first set, second set of PCR was carried out with amplified product isolated by QIAquick Gel Extraction Kit as a DNA template with p15Xyl-F and p15Xyl-R primer set in a thermal cycler having PCR condition as primary denaturation at 98°C for 30 seconds followed by 36 cycles of denaturing at 98°C for 10 seconds, annealing at 61°C for 60 seconds and extension at 72°C for 60 seconds, and a final extension at 72°C for 2 min. After PCR, the products were analyzed by electrophoresis using 1% (w/v) agarose gel in TAE buffer (40 mMTris- acetate, 1 mM EDTA pH 8.1).

H82E mutant and DM W185P/D186E were combined to produce triple mutant H82E/W185P/D186E.

3.15 PCR amplification of G. thermodenitrificans strain AK53 xylanase mutants For G. thermodenitrificans strain AK53 xylanase W127R, D318R, DM W127R/D318R, N74R, D329R, DM R71P/H72E, and W175P/D176E mutations were carried out.

3.15.1 G. thermodenitrificans strain AK53 W127R mutant PCR amplification Two sets of PCR were performed to amplify xylanase gene with mutated nucleotides. In first set of PCR reaction, the recombinant plasmid of AK53 was amplified with XGeoT-R and W127R-F primer and XGeoT-F and W127R-R at a thermal profile of denaturation at 94°C for 2 min. After that 36 cycles having 94°C for 40 seconds

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 78 Chapter 3 Materials and methods denaturation, 57°C for 45 seconds annealing and 72°C for 60 seconds extension, and a final extension of 8 min at 72°C. After the first set, second set of PCR was carried out with amplified product isolated by QIAquick Gel Extraction Kit as a DNA template with XGeoT-F and XGeoT-R primer set in a thermal cycler having PCR condition as primary denaturation at 95°C for 3 min followed by 36 cycles of denaturing at 94°C for 60 seconds, annealing at 61°C for 60 seconds and extension at 72°C for 80 seconds, and a final extension at 72°C for 7 min. After PCR, the products were analyzed by electrophoresis using 1% (w/v) agarose gel in TAE buffer according to Sambrook and Russell (2001).

3.15.2 G. thermodenitrificans AK53 D318R mutant PCR amplification Two sets of PCR were performed to amplify xylanase gene with mutated nucleotides. In first set of PCR reaction, the recombinant plasmid of AK53 was amplified with XGeoT-R and D318R-F primer and XGeoT-F and D318R-R at a thermal profile of denaturation for 2 min at (94°C), followed by 31 cycles, denaturing for 40 seconds at 94°C, annealing at 58°C for 45 seconds and extension for 60 seconds at 72°C, and a final extension of 7 min at 72°C. After first set, second set of PCR was carried out with amplified product isolated by QIAquick Gel Extraction Kit as a DNA template with XGeoT-F and XGeoT-R primer set in a thermal cycler having PCR condition as primary denaturation at 95°C (3 min) followed by 36 cycles of denaturing at 94°C (60 seconds), annealing at 61°C (60 seconds) and extension at 72°C (80 seconds), and a final extension at 72°C (7 min). After PCR, the products were analyzed by electrophoresis using 1% (w/v) agarose gel in TAE buffer according to Sambrook and Russell (2001).

3.15.3 Double mutant W127R/D318R mutant PCR amplification The recombinant mutant plasmid W127R of strain AK53 was amplified with XGeoT- F and D318R-R, and recombinant mutant plasmid of D318R was amplified with XGeoT-R and D318R-F primer during first set of PCR. The obtained fragment was isolated from gel by QIAquick Gel Extraction Kit and used as a template in second set of PCR to amplify full double mutant xylanase gene product with XGeoT-F and XGeoT-R primer set in a thermal cycler having PCR condition as primary denaturation at 95°C for 3 min followed by 32 cycles of denaturing at 94°C for 60

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 79 Chapter 3 Materials and methods seconds, annealing at 61°C for 60 seconds and extension at 72°C for 80 seconds, and a final extension at 72°C for 7 min. After PCR, the products were analyzed by electrophoresis using 1% (w/v) agarose gel in TAE buffer.

3.15.4 G. thermodenitrificans strain AK53 N74R mutant PCR amplification Two sets of PCR were performed to amplify xylanase gene with mutated nucleotides. In first set of PCR reaction, the recombinant plasmid of AK53 was amplified with p15AK53Xyl-R and N74R-F primer and p15AK53Xyl-F and N74R-R at a thermal profile of denaturation at 94°C for 30 seconds followed by 36 cycles of denaturing at 94°C for 10 seconds, annealing at 57°C for 45 seconds and extension at 65°C for 50 seconds, and a final extension at 65°C for 10 min. After first set, second set of PCR was carried out with both amplified product isolated by QIAquick Gel Extraction Kit as a DNA template with p15AK53Xyl-F and p15AK53Xyl-R primer set in a thermal cycler having PCR condition as primary denaturation at 94°C for 30 seconds followed by 36 cycles of denaturing at 94°C for 30 seconds, annealing at 61°C for 60 seconds and extension at 65°C for 60 seconds, and a final extension at 65°C for 10 min. After PCR, the products were analyzed by electrophoresis using 1% (w/v) agarose gel in TAE buffer according to Sambrook and Russell (2001).

3.15.5 G. thermodenitrificans strain AK53 D329R mutant PCR amplification Two sets of PCR were performed to amplify xylanase gene with mutated nucleotides. In first set of PCR reaction, the recombinant plasmid of AK53 was amplified with p15AK53Xyl-R and D329R-F primer and p15AK53Xyl-F and D329R-R at a thermal profile of denaturation at 94°C for 30 seconds followed by 36 cycles of denaturing at 94°C for 20 seconds, annealing at 58°C for 50 seconds and extension at 65°C for 40 seconds, and a final extension at 65°C for 10 min. After first set, second set of PCR was carried out with amplified product isolated by QIAquick Gel Extraction Kit as a DNA template with p15AK53Xyl -F and p15AK53Xyl -R primer set in a thermal cycler having PCR condition as primary denaturation at 94°C for 30 seconds followed by 35 cycles of denaturing at 94°C for 20 seconds, annealing at 61°C for 60 seconds and extension at 65°C for 60 seconds, and a final extension at 65°C for 10 min. After PCR, the products were analyzed by electrophoresis using 1% (w/v) agarose gel in TAE buffer.

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3.15.6 G. thermodenitrificans strain AK53 Double Mutant R71P/H72E PCR amplification Two sets of PCR were performed to amplify xylanase gene with mutated nucleotides. In first set of PCR reaction, the recombinant plasmid of strain AK53 was amplified with DM R71P/H72E-F and p15AK53Xyl-R primer and p15AK53Xyl-F and DM R71P/H72E-R primers for DM R71P/H72E mutation at a thermal profile of denaturation at 94°C for 30 seconds followed by 36 cycles of denaturing at 94°C for 20 seconds, annealing at 58°C for 40 seconds and extension at 65°C for 45 seconds, and a final extension at 65°C for 10 min. After first set, seconds set of PCR was carried out with amplified product isolated by QIAquick Gel Extraction Kit as a DNA template with p15AK53Xyl-F and p15AK53Xyl-R primer set in a thermal cycler having PCR condition as primary denaturation at 94°C for 30 seconds followed by 35 cycles of denaturing at 94°C for 20 seconds, annealing at 61°C for 60 seconds and extension at 65°C for 60 seconds, and a final extension at 65°C for 10 min. After PCR, the products were analyzed by electrophoresis using 1% (w/v) agarose gel in TAE buffer.

3.15.7 G. thermodenitrificans strain AK53 DM W175P/D176E PCR amplification Two sets of PCR were performed to amplify xylanase gene with mutated nucleotides. In first set of PCR reaction, the recombinant plasmid of strain AK53 was amplified with p15AK53Xyl-R and W175P/D176E-F and p15AK53Xyl-F and W175P/D176E- R for W175P/D176E mutation at a thermal profile of denaturation at 94°C for 30 seconds followed by 36 cycles of denaturing at 94°C for 30 seconds, annealing at 58°C for 40 seconds and extension at 65°C for 45 seconds, and a final extension at 65°C for 10 min. After first set, seconds set of PCR was carried out with amplified product isolated by QIAquick Gel Extraction Kit as a DNA template with p15AK53Xyl-F and p15AK53Xyl-R primer set in a thermal cycler having PCR condition as primary denaturation at 94°C for 30 seconds followed by 36 cycles of denaturing at 94°C for 10 seconds, annealing at 61°C for 60 seconds and extension at 65°C for 60 seconds, and a final extension at 65°C for 10 min. After PCR, the products were analyzed by electrophoresis using 1% (w/v) agarose gel in TAE buffer.

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3.16 GeneJET Gel Extraction Kit Thermo Scientific Gel slice containing the DNA was excised with clean razor blade. Gel was cut as close to the DNA to minimize the gel volume contamination. Gel slice was placed into a pre-weighed 1.5 mL tube and weighted. 1:1 volume of binding buffer was added to the gel slice and incubated at 55°C for 15 min. Ensured that the gel was completely dissolved, then loaded on column. 100% isopropanol was added up to 1 gel volume and mixed thoroughly. 800 µL of the solubilized gel solution was transferred to GeneJET purification column and centrifuged for 1 min. Flow through was discarded and the column was kept in collection tube. 100 µL of binding buffer was added to the GeneJET purification column and centrifuged for 60 seconds, flow- through was discarded and the column was kept in collection tube. Wash buffer 700 µL (diluted with ethanol) was added to the GeneJET purification column and centrifuged for 1 min, flow-through was discarded and centrifuged the empty GeneJET purification column for an additional 1 min to completely remove residual wash buffer. GeneJET purification column was transferred to a clean 1.5 mL micro centrifuge tube. 50 µL of elution buffer was added to the center of the purification column membrane. Centrifuged for 1 min and GeneJET purification column gel extracted products were analyzed by electrophoresis using 1% (w/v) agarose gel in TAE buffer and stored the gel extracted products at -20°C.

3.17 Ligation of mutants W137R, D328R and DM W137R/D328R of C5 and W127R, D318R and DM W127R/D318R of strain AK53 xylanase to pGEM®-T Easy Vectors The gel extracted mutated xylanase gene fragments W137R, D328R and DM W137R/D328R of C5 and W127R, D318R, DM W127R/D318R of AK53 were ligated directly into pGEM®-T Easy Vectors according to the manufacturer’s instructions. The ligation mixture was incubated at 16°C overnight and then used for transformation into competent cells. Ligation reactions between vectors and insert molecules were performed with T4 DNA ligase, according to the manufacturer’s specification (Promega). T4 DNA ligase Buffer (10X) (Promega) 1µL pGEM®-T Easy Vectors (Promega) 1µL T4 Ligase (Promega) 1µL

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Insert 2 µL NP water 5µL

3.18 Transformation of bacteria with exogenous DNA E. coli competent cells were transformed using a n easy and quick technique described previously (3.6.3). Before transformation E. coli cells were made competent.

3.18.1 Preparation of E. coli DH5 competent cells (Joerger and Kaenhammer, 1990) E. coli cells DH5 (competent) were inoculated in LB plate supplemented with 10 mM MgCl2 for 20 hours at 37°C. Single colony from this fresh culture was transferred to a 5 mL TYM broth (Appendix 16), and incubated it at 37°C for 2 hours at 300 rpm in shaking incubator. 2.5 mL of cell culture was transferred to 50 mL of fresh TYM broth and incubated it at 37°C until the OD600nm reach to 0.5 (mid-exponential phase). Cells were collected by centrifugation at 4000 rpm for 15 min at 4°C. After this step all cells were kept on ice and suspended in 20 mL of ice-cold TFB-1 solution (Appendix 17) for 1 hour. Cells were centrifuged at 3000 rpm at 37°C for 10 mins and then re-suspended in 1 mL of TFB- 2 (Appendix 18). Cell was aliquoted in 1.5 mL sterile Eppendorf tube and was stored at -80°C until use.

3.18.2 Transformation of E. coli competent cells For transformation in competent cells, 10 µL DNA of interest was added to 50 μL of E. coli DH5 competent cells.

The mixture obtained was placed on ice to incubate for 30 min. Later, without any shaking the mixture was heated for 90 seconds at 42°C and suddenly shifted to the ice for further 10 min. Luria broth (900 µL) was then added to each reaction mixture, which was later incubated at 37°C for 120 min so that the cells may recover and express the antibiotic resistance marker that was previously encoded by plasmid. When the incubation time was completed, each sample (100 µL) was then transferred in to LB- Xgal IPTG agar plates supplemented with an antibiotic ampicillin and the transformed/recombinant cells for W137R, D328R, DM W137R/D328R of strain C5 and W127R, D318R, DM W127R/D318R of strain AK53 were selected. While C5

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 83 Chapter 3 Materials and methods mutants N84R, D339R, R81P, H82E, W185P, D186E, DM W185P/D186E and triple mutant H82E/W185P/D186E and strain AK53 mutants N74R, D329R, DM R71P/H72E and W175P/D176E were spread on LB sucrose agar supplemented with 100 µg/mL of ampicillin (Appendix 19). Plates were kept at 37°C; the transformed colonies will appear in single colony shape after 18 hours of incubation. After incubation blue white screening was performed for W137R, D328R, DM W137R/ D328R of strain C5 and W127R, D318R, DM W127R/D318R of strain AK53 and white colonies were subjected for plasmid isolation. While screening for strain C5 mutants N84R, D339R, R81P, H82E, W185P, D186E, DM W185P/D186E and triple mutant H82E/W185P/ D186E, and strain AK53 mutants N74R, D329R, DM R71P/H72E and W175P/D176E were performed without blue white screening.

3.18.3 Plasmid isolation from pGEM®-T Easy Vectors cloned mutant xylanase of strain C5 and AK53 Plasmids were isolated using mini-preparation protocol according to Sambrook and Russell (2001). Ampicillin (50 µg/mL) supplemented, Lauria-Bertani (LB) liquid media (10 mL) was inoculated by single bacterial colony. After 20 hours incubation, pellet of grown cells was obtained by 3 min centrifugation (at 3300xg) of 3 mL culture. Supernatant was discarded. Pellet obtained was suspended in solution-I (100 µL) having Tris (50 mM), EDTA (1 mM) and RNase (100 mg/mL). After that, solution-II (150 µL) having NaOH (0.2N) and SDS (1% (w/v)), was added to the mixture and mixed well by back and forth inverting. In the next step, solution-III (200 µL), having potassium acetate (3M, pH 4.8 to 5.0) was added and mixed properly. Centrifugation (at 17,950xg) was carried out for 5 min and supernatant was shifted to another eppendorf tube. Using 1:1 volume ratio of mixture and isopropanol, DNA was precipitated. Centrifugation (at 17,950xg) was performed for 5 min and pellet obtained was washed with ethanol (70% (v/v). The isolated plasmid DNA was dissolved in sterilized distilled water (50 µL) and placed at -20°C. Agarose gel electrophoresis was performed to determine the concentration and size of isolated DNA (Sambrook and Russell, 2001).

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3.18.4 Confirmation of Cloning The clones W137R, D328R and DM W137R/ D328R of strain C5 and W127R, D318R, DM W127R/D318R of strain AK53 were confirmed for the presence of cloned fragment was confirmed by digestion the clones with appropriate restriction endonucleases. The restriction reaction containing plasmid (250 ng), required 10X EZ-One™, restriction buffer (10X) which was diluted by nanopure water to 1X concentration , appropriate enzyme EcoRI (Life technologies).

Plasmid 14 µL EZ-One™ 10X buffer 2 µL EcoRI (10 U/µL) 0.6 µL NP water 3.4 µL The presence of digested plasmid and cloned fragment was confirmed by 1 % agarose gel electrophoresis.

3.19 DNA treatment with enzymes The manufacture’s description was followed to perform enzymatic treatment of nucleic acids or DNA. 3.19.1 Digestion of cloned DNA with HindIII enzyme Double digestions of W137R, D328R and DM W137R/ D328R of strain C5 and W127R, D318R, DM W127R/D318R of strain AK53 were performed using the most proper buffer for restriction enzyme. The purpose of digestion was normally to construct adhesive ends on both the gene insert and plasmid vector and incubated at 37ºC for 2 hours. The isolated cloned plasmid having mutated xylanase insert and pET28a plasmid was cut with HindIII restriction enzyme and kept at 37ºC for 2 hours, HindIII Takara 15u/µL 2 µL Y+/Tango™ buffer 5 µL Plasmid 40 µL NP Water 3 µL The digestion of insert W137R, D328R and DM W137R/D328R of strain C5 and W127R, D318R, DM W127R/D318R of strain AK53 and pET28a plasmid was confirmed with gel electrophoresis after 2 hours of incubation period.

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3.19.2 Ethanol Precipitation After digestion with HindIII restriction enzyme mutated xylanase insert W137R, D328R and DM W137R/D328R of strain C5 and W127R, D318R, DM W127R/D318R of strain AK53 and pET28a plasmid were precipitated with ethanol. 1 to 10 vol of 3 M sodium acetate, pH 5.2 was added to the solution of DNA and mixed briefly. 2 to 2.5 vol (calculated after salt addition) of ice-cold 100% ethanol was added and mixed by vortexing and placed in crushed dry ice for 30 min or longer. The mixture was centrifuged for 5 min at maximum speed and supernatant was removed. 1 mL room-temperature 70% ethanol was added. Inverted the tube several times and micro-centrifuged it. Supernatant was removed and dried the pellet. Dry pellet was dissolved in an appropriate volume of TE buffer.

3.19.3 Digestion with NheI and gel extraction The precipitated mutated xylanase insert W137R, D328R and DM W137R/D328R of strain C5 and W127R, D318R, DM W127R/D318R of strain AK53 and pET28a plasmid was digested with NheI restriction enzyme and kept at 37ºC for 2 hours, and then double digestion of insert and pET28a plasmid was confirmed with gel electrophoresis. NheI 2.5 µL Y+/Tango™ buffer 7 µL Plasmid 60 µL

3.19.4 Gel purification through GeneJET Gel Extraction Kit Thermo Scientific The double digested plasmid pET28a and double digestion of mutated xylanase insert W137R, D328R and DM W137R/D328R of strain C5 and W127R, D318R, DM W127R/D318R of strain AK53 were extracted and purified by GeneJET Gel Extraction Kit Thermo Scientific as the manufacturer instruction. Double digestion of mutated xylanase insert and pET28a plasmid gel extraction was confirmed with gel electrophoresis.

3.19.5 Ligation of double digested pET28a plasmid and mutated insert The double digested mutated xylanase gene insert W137R, D328R and DM W137R/D328R of strain C5 and W127R, D318R, DM W127R/D318R of strain AK53

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 86 Chapter 3 Materials and methods were ligated directly into already double digested pET28a plasmid according to the manufacturer’s instructions. A typical ligation mixture recipe were 10X T4 DNA Ligase Buffer 1 µL T4 DNA Ligase 400,000 units/mL NEB 0.2 µL Mutated xylanase Insert 1 µL pET28a plasmid 1.5 µL Nano pure Water 6.3 µL The ligation mixture was incubated at 15°C overnight and then used for transformation into competent cells.

3.20 Preperation of p15TV-L plasmid for Ligase independent ligation p15TV-L plasmid was digested with BseRI restriction enzyme for 1 hour at 37°C, and digested p15TV-L plasmid was isolated from gel by gel extraction kit.

3.21 Ligation with Clontech kit (Ligase independent ligation) One clontech pellet was mixed with 9.5 µL of digested plasmid p15TV-L and allowed it for 10 min to completely dissolve the pellet. 3 µL of p15TV-L digested plasmid was mixed with 2 µL of each mutated gel extracted product N84R, D339R, R81P, H82E, W185P, D186E, DM W185P/D186E and triple mutant H82E/W185P/D186E of strain C5, and N74R, D329R, DM R71P/H72E and W175P/D176E for strain AK53. Incubated the ligation mixture at 37°C for 15 min in PCR machine and then reaction was stopped by incubating the mixture at 50°C for 15 min, after that the entire mixture was incubated on ice for 10 min.

3.22 Transformation of bacteria with insert

Transformation of E.coli DH5 strains was carried out after modification of a simple technique proposed by Cohen et al (1972). The preparation of competent E.coli cells was done before carrying out the procedure of transformation.

3.22.1 Transformation of E. coli DH5 competent cells Competent cell suspensions of E. coli (50 µL) were taken in a sterile eppendorf (1.5 mL). The ligated insert was then transferred in to the same eppendorf. The mixture

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 87 Chapter 3 Materials and methods obtained was placed on ice to incubate for 30 min. Later, without any shaking the mixture was heated for 90 seconds at 42°C and suddenly shifted to the ice for further 10 min. Luria broth (900 µL) was then added to each reaction mixture, which was later incubated at 37°C for 120 min so that the cells may recover and express the antibiotic resistance marker that was previously encoded by plasmid. When the incubation time was completed, each sample (100 µL) was then transferred in to LB- agar plates supplemented with 50 µg/mL of the antibiotic kanamycin for W137R, D328R and DM W137R/ D328R of strain C5 and W127R, D318R, DM W127R/D318R of strain AK53 for selection of the transformed/recombinant cells. While N84R, D339R, R81P, H82E, W185P, D186E, DM W185P/ D186E and triple mutant H82E/W185P/ D186E of strain C5 and N74R, D329R, R71P, DM R71P/H72E and W175P/D176E of strain AK53 samples, 100 μL each was spread on LB agar plates supplemented with the ampicillin (100 µg/mL), plates were incubated at 37°C. After incubation, screening was performed and isolated colonies were subjected for plasmid isolation.

3.22.2 Plasmid isolation of transformed cell Plasmid was isolated from pET28a cloned cell W137R, D328R and DM W137R/D328R of strain C5 and W127R, D318R, DM W127R/D318R of strain AK53 by GeneJET Plasmid Miniprep Kit (Thermo Scientific) and the procedure described by manufacturer was followed. Plasmid was isolated from p15TV-L cloned cell N84R, D339R, R81P, H82E, W185P, D186E, DM W185P/ D186E and triple mutant H82E/W185P/D186E of strain C5 and N74R, D329R, DM R71P/H72E and W175P/D176E of strain AK53 by GeneJET Plasmid Miniprep Kit and the procedure described by manufacturer was followed.

3.22.3 Confirmation of Cloning The clones were confirmed by double digestion with PCR and sequencing. For sequencing, the plasmid DNA was purified by using Wizard® SV Gel and PCR Clean-Up System Kit (Promega Corp., Madison, WI, USA). Sequencing was commercially performed Macrogen Standard Custorm DNA Sequencing Services (Macrogen Inc. Seoul, Korea).

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3.23 Growth conditions of induced recombinant strains BL21 (DE3) Competent Cell After confirming the DNA sequence of protein expression of recombinant mutant xylanase was determined using a suitable inducible strain. Recombinant cells were cultured in antibiotic supplemented LB liquid media for 10 hours at 37ºC. After overnight incubation, the culture was used to prepare an inoculum with freshly prepared media with 1:20 ration. The media contain the same antibiotic that was used for inoculum. When growth turbidity of 0.5 to 0.8 at OD600 was obtained within three hours, at this stage IPTG (1 mM) was used to induce protein expression and kept for 20 hours at 37ºC in shaker incubator. pET28a containing recombinant cell were grown in kanamycin (50µg/mL) while p15TV-L plasmid containing recombinant cell were grown in ampicillin (100µg/mL).

3.24 Purification 3.24.1 Ammonium sulphate precipitation The mutated xylanase enzyme was purified from the culture supernatant fluid and cell lysed mixture using ammonium sulphate in a sodium phosphate buffer of pH 6.0 for W137R, D328R and DM W137R/D328R of strain C5 and buffer of pH 5.0 for W127R, D318R, DM W127R/D318R of strain AK53. The respective levels were mixed in 1000 mL of crude enzyme filtrate and kept at 4°C for 10 hours with continuous stirring. Precipitated xylanase enzymes were collected and activity of enzymes was determined by enzyme assay. Maximum xylanase recovery was achieved on the basis of optimum xylanase activity at respective ammonium sulphate concentration.

3.24.2 Dialysis After precipitation, In order to remove the trace concentration of ammonium sulphate in crude enzyme solution, dialysis was carried out in a buffer of pH 6.0 and pH 5.0 for strain C5 and AK53 mutants, respectively at 4°C for 24 hours (Carmona et al., 1998).

3.24.3 DEAE Sepharose chromatography The dialyzed mutated enzymes W137R, D328R and DM W137R/D328R of strain C5 and W127R, D318R, DM W127R/D318R of strain AK53 were further purified using an anion-exchange column chromatograph (1.5 × 50 cm) as DEAE Sepharose fast

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 89 Chapter 3 Materials and methods flow. Gel slurry was poured into the column in one continuous motion. The obtained 60% precipitates were loaded on a column (1.5 × 50 cm) of DEAE-Sepharose pre- equilibrated with 10 mM sodium phosphate buffer pH 6.0 for W137R, D328R and DM W137R/ D328R of strain C5 and the same buffer at pH 5.0 for W127R, D318R, DM W127R/D318R of strain AK53. The column was washed with 1000 mL of the same buffer at flow rate of 0.5 mL/min, and then dialyzed enzyme solution was passed from the column, column was eluted with linear gradient of (0.55 M) NaCl in sodium phosphate buffer. Each fraction (3 mL) was collected. Each fraction was determined for xylanase activity. The active fractions were combined and concentrated by ultrafiltration (Sartorious, 30000 MWCO filters).

3.24.4 Phenyl Sepharose chromatography The DEAE Sepharose chromatography fractions were further purified using an anion- exchange column chromatograph (1.5 × 50 cm) as phenyl Sepharose fast flow. Phenyl Sepharose Fast Flow ion exchange medium was supplied preswollen in 20% ethanol. Decanted the ethanol solution and replaced it with packing buffer. Slurry was degassed under vacuum. Ensured that there is no air bubbles trapped under the net. Gel slurry was poured into the column in one continuous motion. Phenyl-Sepharose column was washed with 1.3 M ammonium sulphate in 20 mM sodium phosphate buffer pH 6.0 for W137R, D328R and DM W137R/D328R of strain C5 and 10 mM sodium phosphate buffer pH 5.0 for W127R, D318R, DM W127R/D318R of strain AK53. Then equal amount of 2.6 M ammonium sulphate was mixed with equal volume of enzyme and loaded in the column. The column was washed again with 20 mL 1.3 M ammonium sulpahte. Column was eluted with linear gradient of (1.3 M) ammonium sulphate in 20 mM sodium phosphate buffer and fractions (37 drops each) was collected. Each fraction was determined for xylanase activity. The active fractions were combined and concentrated by ultrafiltration (Sartorious, 30000 MWCO filters).

3.24.5 Purification through His60 Ni Superflow Resin 5mL of LB was inoculated with 5µL of 100mg/mL Amp stock (100µg/mL final concentration) and let it grow for 37°C overnight in shaker incubator. 2L of LB medium containing 100 µg/mL Amp was inoculated with 5mL starter culture and allowed the culture to grow in 37°C until OD600 reached to 0.8, culture was induced

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 90 Chapter 3 Materials and methods with 0.5 mM IPTG and allowed the culture to grow overnight at 16°C shaker incubator. Cells were collected by centrifugation at 4°C at 7500rpm for 15 min (Beckmn coulter, JLA 8.100). Supernatant was discarded and cells were collected in 50 mL falcon tube. Cells pellet was re-suspended in 20 mL of binding buffer +10µL TCEP. Cells were lysed via French press 3 times and centrifuged the lysed cell at 17000rpm at 4°C for 30 min. Supernatant was collected in new 50 mL falcon tube.

Column was tested with distilled water to ensure its flow freely and that it is not clogged. Column was washed with ethanol and water. 5 mL of Ni resin was added to the column and suspended in prepared 50 mL binding buffer and allowed it for 10 min. Column was opened and allowed the buffer to flow through until there is only approximately 2 cm remaining above the settled resin. Column was transferred to the cold room for remaining steps.

3.24.6 Protein loading and washing Half of volume of cell free supernatant was loaded to the column. 50 mL binding buffer was added and mixed the column with pipette and allowed to sit for 20 min, opened the column and allowed the buffer to flow through very slowly dropwise. Column was closed when the buffer is about 2 cm above the resin. Half of remaining volume from sample was loaded to the column. 50 mL binding buffer was added and mixed the column with pipette and allowed to sit for 20 min. Column was opened and allowed the buffer to flow through very slowly dropwise. Column was closed when the buffer is about 2 cm above the resin. Ni column was washed with 50 mL wash buffer. Column was opened and buffer was allowed to flow through slowly. Washing step was repeated 2 times. Column was closed when the washing buffer is about 2 cm above the resin.

3.24.7 Elution of protein 35 mL elution buffer was added to the column. Column was allowed to sit for 20 min. 150 µL Bradford reagent was added to some of 96 well plates. Clean falcon tube was placed under the column to collect the protein elution. Open the column and allowed the elution buffer to flow through very slowly. Throughout the elution process 10 µL of eluent was added to well containing Bradford reagent. Elution was stopped when the Bradford reagents no longer turns blue.

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3.24.8 Dialysis 10,000 MWCO dialysis tubing was used to make a dialysis bag for protein dialysis. Elution was added to the dialysis bag closed it with a clip and added a float. Placed in 2L of dialysis buffer +1mL 1M TCEP and allowed to sit overnight in the 4°C on a stirrer plats.

3.24.9 Molecular weight determination by sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS-PAGE)

The molecular weight of all mutants from strain AK53 and C5 was determined by (SDS-PAGE), as described by by Laemmli (1970).

3.25 Characterization of mutant xylanase The expressed purified mutated xylanase enzyme W137R, D328R, DM W137R/D328R, N84R, D339R, R81P, H82E, W185P, D186E, DM W185P/D186E and triple mutant H82E/W185P/D186E for strain C5 xylanase and W127R, D318R, DM W127R/D318R, N74R, D329R, DM R71P/H72E and W175P/D176E for strain AK53 were then characterized.

3.25.1 Effect of temperature on mutated xylanase The optimum temperature of the recombinant mutated purified xylanases of G. thermodenitrificans strain AK53 and C5 was determined. Xylanase activity for mutant strains AK53 and C5 was measured in the form of relative activity from a range of 40-90°C temperatures. 100% enzyme activity was considered as reference using which; relative activity at different temperatures was calculated. The assay for activity was performed as described above, at the respective temperatures. Thermostability of the xylanases was determined by incubating the enzymes at 50- 100°C for 200 min. The activity was assayed as described above, and relative activity was determined.

3.25.2 Effect of pH on mutated xylanase The optimum pH was also determined by measuring the relative activity over a wide range of pH. The buffers used in the study include 0.1M Sodium citrate buffer (3.0-

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6.0), 0.1M Sodium Phosphate buffer (pH 7.0-8.0) and 0.1M Glycine NaOH buffer (pH 9.0-10.0). pH stability was determined by incubating the enzymes at different pH for 200 min. The activity was assayed as described above, and relative activity was determined.

3.25.3 Determination of Km value

Km is the characteristic constant of an enzyme catalyzes reaction and is defined as the concentration of substrate at which enzyme has half of its maximum velocity (Vmax).

Km and Vmax was calculated by Lineweaver and Burk (1934) plot.

3.25.4 Shelf life Determination of xylanase Shelf life was determined by keeping purified recombinant xylanase in refrigerator at 4°C as well as at room temperature. Samples were collected at different intervals and residual activity was determined upto the maximum of 24 weeks.

3.25.5 Half-life of purified mutated xylanase Half-life of all purified mutant enzymes of strain C5 and AK53 were determined from 60-75°C at optimum pH. All mutated xylanases were incubated for different duration at different temperature, and the residual activity was measured under standard conditions.

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3.26 Effect of C terminus tagged proline on strain C5 xylanase 3.26.1 Primer Design Primers for G. thernmodenitrificans and proline were used for tagging proline with xylanase. Proline rich sequence was provided by molecular biology laboratory Karadeniz Technical University, Trabzon, Turkey.

Table 3.3.1 Primer used in this study Primer Name Primer Sequences (5’-3’) XGeoT-F 5’-CTAgCTAgCATgTTgAAAAgATCgCgAAAAg-3’ Xyla-Pro-C5: 5’- TgCCgCCgCTTTCATTgATCgATAATAgCC- 3’ Prol- 5'-cgcggATCCCTATCCAggCTggTTgAgCTCAgAgggCACgA -3' BamHIR

The restriction sites NheI and BamHI are highlighted, respectively.

3.26.2 Plasmid isolation of transformed cell (Roche high pure plasmid isolation Kit) The recombinant cells were grown in LB broth containing kanamycin. Medium was centrifuged and cell pellet was re-suspended in 250 µL suspension buffer containing RNase. Mixed it vigorously and 250 µL of lysis buffer was added. Mixed the suspension gently and left at room temperature for 5 min, afterwards 350 µL chilled binding buffer was added and gently mixed it. After mixing, the suspension was incubated on ice for 5 min and then centrifuged at maximum speed for 10 min. Pellet was discarded and supernatant was collected and transferred to high pure filter tube and again centrifuged for a min at maximum speed. Flow through was discarded and 500 µL of wash buffer 1 was added. Again centrifuged for a min at 13000xg. Discarded the flow through and 700 µL of wash buffer 11 was added, again centrifuged at 14000xg for 60 seconds. Flow-through was discarded and high pure filter tube was again subjected to centrifugation at 14000xg for 1 minute. Flow through was discarded and 100 µL of elution buffer was added at the center of high pure filter tube. A new eppendorf tube was used for collection of elute after 1 minute centrifugation.

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3.26.3 PCR for amplification of proline with xylanase For amplification of xylanase gene via proline, two PCR sets were utilized. In the first set of PCR reactions XGeoT-F and Xyl-Pro primers were used for amplification of recombinant plasmid at thermal profile of denaturation at 94°C for 3 min followed by 36 cycles of denaturing at 94°C for 40 seconds, annealing at 63°C for 40 seconds and extension at 72°C for 90 seconds, and a final extension at 72°C for 5 min. The product was analyzed by electrophoresis 1% (w/v) agarose gel in TAE. The amplified PCR product was extracted using GeneJET Gel Extraction Kit (Thermo Scientific). Amplified product from first set of PCR was used as a template with XGeoT-F and Prol-BamH-R primers with thermal profile of primary denaturation at 94°C for 3 min followed by 36 cycles of denaturing at 94°C for 40 seconds, annealing at 63°C for 50 seconds and extension at 72°C for 100 seconds, and a final extension at 72°C for 7 min. The product analysis after carrying out PCR was done by electrophoresis 1% (w/v) agarose gel in TAE buffer.

3.26.4 GeneJET Gel Extraction Kit (Thermo Scientific) The piece of gel containing DNA fragment was excised with the help of a clean razor blade or scalpel. Transferred the gel piece to a pre-weighed 1.5 mL tube and weighed. The weight of gel slice was recorded. 3:1 volume of binding buffer was added to the slice of gel (volume:weight i.e. added 300 µL of binding buffer for every 100 mg of agarose gel). The gel mixture was then incubated for 10 min at 50-60°C until gel slice was completely dissolved. Tube inversion was done every few min to speed up melting process. Ensuring the complete dissolution of gel and vortexed the mixture briefly before loading it on the column. 1 gel volume of 100% pure isopropanol (HPLC grade) was added to the solubilized gel and thoroughly mixed. About 800 µL of solubilized gel solution was transferred to the GeneJet purification column and centrifuged for 1 min. Column was placed back into the same collection tube after discarding the flow through. About 100 µL binding buffer was transferred to the GeneJet purification column and centrifuged for 1 min. Dicard flow through again and placed the column back on collection tube. 700 µL of wash buffer (diluted with ethanol) was transferred to the GeneJet purification column and centrifuged for 1 min. After discarding the flow through, column was placed back into the same collection tube. The empty GeneJet purification column was centrifuged for an additional 1 min

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 95 Chapter 3 Materials and methods for complete removal of residual wash buffer. GeneJET purificatiom column was then transferred to 1.5 mL micro-centrifuge tube. 50 µL of elution buffer was added to the center of column and centrifuged for 1 min. GeneJET purification column was discarded and the analysis of extracted products was done by electrophoresis using 1% (w/v) agarose gel in TAE buffer and stored the products at -20°C.

3.26.5 Ligation of proline tagged xylanase to pGEM®-T Easy Vectors Direct ligation of proline tagged xylanase extracted from gel was performed with pGEM®-T Easy Vectors in accordance with the manufacturer’s manual. Ligation mixture was incubated overnight at 16°C and afterwards used for transformation into competent cells. T4 DNA ligase was used for carrying out ligation reaction between the vectors and insert molecules in accordance with the instructions specified by manufacturer (Pomega). This enzyme is responsible for the catalysis of phosphodiester bond formation between neighboring 3‟-hydroxyl- and 5‟-phosphate ends in double-stranded DNA. Different vector insert ratios were utilized for carrying out ligation reaction i.e. from 1:0 as negative control up to 1:12. To achieve the desired ratios, calculations of appropriate component amounts in reactions were done via tool at (http://www.gibthon.org/ligate.html). In every ligation reaction the amount of vector never exceeded the amount of vector present in negative control. Ligation reactions contained T4 DNA ligase Buffer (10X) 1µL pGEM®-T Easy Vectors 1µL T4 Ligase 1µL Insert 3 µL NP water 4µL Ligation reactions were performed at 16ºC and incubated for 16 hours (overnight). Cloning of amplified fragment into vector pGEM®-T Easy Vectors in accordance with manufacturer’s manual.

3.26.6 Bacterial transformation with exogenous insert E. coli transformation was carried out by modification of a simple technique provided by Cohen et al. (1972). For this purpose, competent cells of E. coli were prepared before carrying out the process of transformation.

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3.26.7 Preparation of E. coli competent cells using calcium chloride LB broth was used to grow E. coli JM 101 strains at 37°C for 12 hours. 0.4 mL of the culture was then transferred to fresh 50 mL LB broth and kept in shaker at 37°C till

OD600 reached 0.45-0.55 (mid-exponential phase). Culture was centrifuged for 5 min at 4500 rpm. Discarded the supernatant and re-suspended the cells in 10 mL 100mM

CaCl2. Cells were placed on ice for 30 min. Again centrifuged at 4500 rpm for 5 mins, discarded supernatant and homogenized the pellet in 2 mL of 100 mM CaCl2. Cells were kept for 2 hours at 4°C.

3.26.8 Transformation of E. coli competent cells

200 μL of CaCl2 treated competent cells were mixed with 10 μL DNA of interest. Transformation was performed by running the controls (positive and negative) alongside samples. After combining competent cells with DNA of interest, the reaction mixtures were kept on ice (30 minutes), warmed (2 minutes at 42°C) statically and transferred to ice again and kept for 10 minutes on ice. 200 μL of LB broth was added to each of the tube and incubated the resulting mixture for 2 hours at 37°C. This allowed the cells to recover from temperature shocks and express the plasmid encoded antibiotic resistant marker gene. 100 μL of each sample was spreaded on LB Xgal IPTG agar plates containing ampicillin for transformed cells selection. Plates were incubated at 37°C for 12–16 hours. Blue and white colonies screening was performed after incubation and selected white colonies for plasmid isolation.

3.26.9 Plasmid isolation from pGEM®-T Easy Vectors cloned proline tagged xylanase Isolation of plasmid was done in accordance with the protocol given by Sambrook and Russel (2001). Single white colonies were inoculated in 10 mL LB broth containing 50 µg/mL ampicillin and incubated overnight. After incubation 3 mL of culture was centrifuged at 3300xg for 3 min and discarded the supernatant. Pellet was re-suspended in 100 µL of solution-I containing Tris 50 mM, EDTA 1 mM, RNase 100 mg/mL. 150 µL of Solution-II containing 0.2N NaOH and 1% (w/v) SDS, was then added to the re-suspended pellet and mixed well by inverting the tube 4 to 5

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 97 Chapter 3 Materials and methods times. Solution-III (200 µL) having potassium acetate (3M, 4.8 to 5.0 pH) was added and mixed well. 5 minutes’ centrifugation (at 17950xg) was carried out and supernatant was shifted to another eppendorf tube. Using 1:1 volume ration of mixture with isopropanol, DNA precipitation was carried out. Sample was centrifuged (at 17950xg) for 5 min and after that, the pellet obtained was rinsed with ethanol (70% (v/v). The isolated plasmid DNA was dissolved in sterilized distilled water (50 µL) and placed at -20°C. Agarose gel electrophoresis was performed to determine the concentration and size of isolated DNA.

3.26.10 Confirmation of Cloning Cloning was confirmed by digestion of cloned plasmid with appropriate restriction endonuclease. The components of the typical restriction reaction were 10X EZ- One™ buffer diluted using nanopure water to 1X final concentration in reaction, appropriate enzyme EcoRI (Life technologies) and about 250 ng plasmid. Plasmid 7 µL EZ-One™ 10X buffer 1 µL EcoRI (10 U/µL) 0.3 µL NP water 1.7 µL Agarose gel (1%) electrophoresis was used for confirming the presence of cloned fragment and digested DNA.

3.27 Enzymatic treatment of DNA Manufacturer’s instructions were followed for all the nucleic acid enzymatic treatments.

3.27.1 Digestion of cloned DNA with HindIII enzyme Most suitable buffer was used for carrying out the double digestions of both enzymes. Enzymatic digestion was generally performed for the creation of adhesive ends on both the plasmid vector and the insert. In digestion reactions the maximum quantity of DNA was not more than1000ng for PCR amplified product and for plasmid vectors not more than 500ng. Incubation temperature was kept 37ºC for different time interval. NheI was the restriction enzyme used to cut the cloned plasmid containing insert and pET28a.

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NheI (New England biolab) (10 U/µL) 2.60 µL Y+/Tango™ buffer 7 µL Plasmid 60 µL NP Water 0.4 µL Gel electrophoresis was used for the confirmation of insert and pET28a digestion after incubation period of 2 hours.

3.27.2 Ethanol Precipitation Ethanol precipitation was performed after digesting the insert and pET28a plasmid with NheI restriction enzyme. 1 to 10 vol of 3M sodium acetate at 5.2 pH was added to DNA solution and mixed via vortexing or flicking the tube with finger several times. 2 to 2.5 vol (calculated after salt addition) of 100% ice-cold ethanol was added. Mixed it via vortexing and then placed it for 30 mins in crushed dry ice. Micro- centrifuged it at maximum speed for 5 min and removed the supernatant. 1 mL of 70% ethanol was added at room temperature. Tubes were mixed several times and micro-centrifuged. Supernatant was removed again and then dried the pellet in a desiccator under vacuum. In order to use pellet for further enzymatic manipulation in buffer, the pellet was dissolved in appropriate volume of water. The pellet was dissolved in TE buffer at pH 8.0 in order to store it for indefinite time.

3.27.3 Digestion with BamHI and gel extraction BamH1 (10 U/µL) restriction enzyme kept at 37°C for 2 hours was used to cut the precipitated insert and pET28a plasmid. Double digestion of insert and pET28a plasmid was confirmed by means of gel electrophoresis. BamHI (10 U/µL) Thermo Scientific 2.5 µL 10 X Buffer BamHI 7 µL Plasmid 60 µL

3.27.4 Purification of digested plasmid and proline tagged xylanse gene product with GeneJET Gel Extraction Kit (Thermo Scientific) The double digested plasmid pET28a and insert was extracted and purified by GeneJET Gel Extraction Kit (Thermo Scientific) in accordance with the manufacturer’s instructions. The double digestion insert and pET28a plasmid gel extraction was then confirmed by gel electrophoresis as described previously.

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3.27.5 Ligation of double digested pET28a plasmid and proline tagged xylanase insert The double digested insert of xylanase gene tagged with proline was directly ligated to the already double digested plasmid pET28a in accordance with the manufacture’s manual. The typical contents of the ligation mixture were as follows: 10 X T4 DNA Ligase Buffer 1 µL T4 DNA Ligase 1 µL Proline tagged Insert 1 µL pET28a plasmid 1 µL NP Water 6 µL Before using the ligation mixture for transformation of competent cells, it was over nightly incubated at 14°C.

3.27.6 Transformation of bacteria with exogenous DNA A modified version of Cohen et al (1972) was used for transforming E. coli cells. Before performing transformation, competent E. coli cells were prepared.

3.27.7 Preparation of E. coli competent cells E.coli JM 101 competent cells were prepared according to the procedure previously described in section (3.26.7).

3.27.8 Transformation of E. coli competent cells 200 µL of competent cells suspension was transferred into a sterile 1.5 mL Eppendorf and 10 µL ligated DNA of interest was added to it. The mixture was further incubated in ice for about 30 mins, 2 mins heat shock was provided at 42°C without shaking and then immediately transferred back to ice. After about 10 min of incubation in ice, transferred 200 µL of LB in each tube and incubated the tubes for 2 hours at 37°C in order to allow the cells to recover from the temperature shocks and express antibiotic genes. After incubation 100 µL of each sample was spread on LB agar plates containing kanamycin and incubated the plates at 37°C for 12-16 hours. Screening of colonies was performed and isolated colonies were then subjected to plasmid extraction.

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3.27.9 Plasmid isolation of transformed cells by GeneJET Plasmid Miniprep Kit (Thermo Scientific) GeneJET Plasmid Miniprep Kit was used for the isolation of plasmid from pET28a cloned cells. Procedure was performed according to manufacturer’s manual.

3.27.10 Confirmation of cloning Double digestion with an appropriate restriction enzyme was performed to confirm cloning. The contents of typical digestion reaction were 10X respective restriction buffer diluted using nanopure water to 1X final concentration in reaction, 5.0 U of appropriate enzyme and about 250 ng plasmid.

3.27.11 Sequencing of Clones Wizard® SV Gel and PCR Clean-Up System Kit (Promega Corp., Madison, WI, USA) were used for plasmid DNA purification. Macrogen Standard Custom DNA Sequencing Services (Macrogen Inc. Seoul, Korea) performed the commercial sequencing.

3.28 Growth conditions of induced recombinant strains BL21 (DE3) Competent Cell Protein expression was assayed using an inducible appropriate strain. LB liquid medium supplemented with the 50µg/mL kanamycin antibiotic was used for growing recombinant strains at 37°C for 16 hour (overnight). With inoculum: culture ratio of 1:20, a new Erlenmeyer flask half filled with culture medium containing the same antibiotic was inoculated and incubated overnight. When growth OD600 0.5-0.8 was achieved, IPTG at 1mM was added to induce the protein expression and again incubated at 37°C for 20 hour.

3.29 Purification 3.29.1 Ammonium sulphate precipitation and Dialysis For the purification of proline tagged crude enzyme it was precipitated from the culture supernatant by means of 60% ammonium sulphate in a sodium phosphate buffer of pH 6.0. The solution was dialyzed in a buffer of pH 6.0 at 4°C for 24 hours in order to remove of ammonium sulphate from enzyme solution after precipitation

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(Carmona et al., 1998). Xylanase activity analysis was then carried out once the precipitates collected.

3.29.2 DEAE Sepharose chromatography DEAE Sepharose fast flow (anion-exchange column chromatograph) was used for further purification of dialyzed enzyme. Loaded the obtained precipitates (from 60% ammonium sulfate) on column (1.5 × 50 cm) of DEAE-Sepharose pre-equilibrated with 10 mM sodium phosphate buffer pH 6.0. Washed the column with 100 mL of same buffer at the flow rate of 0.5 mL/min. Passed the dialyzed enzyme from the column and then eluted the column with linear gradient of (0.55 M) NaCl in sodium phosphate buffer. Each fraction (3 mL) was collected and assayed it for xylanase activity. The active fractions were combined and concentrated by ultrafiltration Sartorious, 30,000 MWCO filters.

3.29.3 Phenyl Sepharose chromatography DEAE Sepharose chromatography fractions were further purified by using an anion- exchange column chromatograph (1.5 × 50 cm) as Phenyl Sepharose fast flow. All material was equilibrated at room temperature. Pre-swollen in 20% ethanol Phenyl Sepharose fast flow exchange medium was obtained. Packing buffer was used to replace the ethanol solution. Degas slurry under vacuum. Column bottom end piece was connected to the pump. End piece was then submerged in buffer and filled with the help of pump. Ensured that there was no air bubbles trapped under net. The tubing was closed with the stopper and end piece was mounted on the column. Column was then flushed with buffer. Few mL of buffer was left at the bottom and kept the column vertically mounted on laboratory stand. In one continuous motion poured the gel slurry inside column. While pouring rod was held against the wall of the column to prevent air bubbles. Remainder of the column was filled with buffer. Loaded the obtained precipitates (from 60% ammonium sulfate) on column (1.5 × 50 cm) pre-equilibrated with 10 mM sodium phosphate buffer pH 6.0. Washed the column with 100 mL of same buffer at the flow rate of 0.5 mL/min. Passed the dialyzed enzyme from the column and then eluted the column with linear gradient of (0.55 M) NaCl in sodium phosphate buffer. Each fraction of about (3 mL) was collected and assayed for xylanase activity. The active fractions were combined and concentrated by ultrafiltration Sartorious, 30000 MWCO filters.

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3.29.4 SDS polyacrylamide gel electrophoresis Polyacrylamide gel contain sodium dodecyl sulfate which is a strongly anionic detergent and act as denaturing agent. (SDSPAGE) was carried out for analytical electrophoresis of proteins (Laemmli 1970).

3.29.5 HPLC of proline tagged C5 xylanase A reverse phase C-18 column (4.6 x 250 mm; E. Merck, Germany) of High Performance Liquid Chromatography (HPLC System 600 Waters, Waters Corporation, Massachusetts, USA) was employed in order to test the purity of enzyme. For the separation of sample components, acetonitrile:water (70:30) solvent system was employed at the flow rate of 0.5 mL/min. Absorbance was read at 280 nm via a highly sensitive photo-diode array (PDA) detector (996 Waters).

3.30 Characterization C terminal proline tagged purified expressed xylanase enzyme was characterized by various methods.

3.30.1 Effect of Temperature Relative activity of recombinant purified proline tagged C5 xylanase at different temperatures ranging from 20-90°C was measured in order to determine the optimum temperature of enzyme. Expressed maximum activity as 100% and used it as a reference for the determination of relative activity of different reactions. The thermostability was determined by incubating enzyme at 50-100°C for 200 min. Assayed the activity as described above and determined the relative activity.

3.30.2 Effect of pH Optimum pH was also determined by measuring the relative activity over a wide range of pH. 0.1M Sodium citrate buffer (pH 3.0-6.0), 0.1M Sodium Phosphate buffer (pH 7.0-8.0) and 0.1M Glycine NaOH buffer (pH 9.0-10.0) are the buffers used for this study. pH stability of C terminal Proline tagged purified expressed xylanase was determined by incubating the enzyme at optimum temperature and pH 3.0-11.0 for 200 min, assayed the activity as described above and determined the relative activity.

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3.30.3 Determination of Km value

The kinetic parameter and maximum rate of metabolism Vmax were determined in sodium phosphate buffer pH 8.0 with xylan concentration from 0.5-35 mg/mL. The kinetics values of proline tagged C5 xylanase were determined by fitting line weaver burk plot.

3.30.4 Analysis of hydrolysis product Hydrolysis product analysis was done by mixing the purified enzyme with 100 mM sodium phosphate buffer (pH 8.0) containing 1% (w/v) xylan and then incubating for 10 hours at 60°C. Insoluble materials were removed from the sample by centrifuging for 12 min at 3000xg, and then spotted TLC plates with 3 µL aliquots.

Chromatography by ascending method was then performed on silica gel 60 F254TLC plates (Merck) with n-butanol, acetic acid and water (2:1:1) containing solvent system. Plates were sprayed with 5% (v/v) sulfuric acid in ethanol. Plates were heated for 10 min at 120°C for sugar detection.

3.30.5 Polysaccharide-binding properties The polysaccharide-binding capacity of the recombinant xylanase was determined by the method described by Tenkanen et al. (1992). Purified GthC5ProXyl xylanase was incubated with different concentrations of avicel, arabinoxylan, Beechwood xylan, Oat spelt xylan and birchwood xylan in 100 mM sodium phosphate buffer (pH 8.0), at 4°C for 1 h with slow shaking. Unbound enzyme was determined by measuring residual activity in the supernatant.

3.30.6 Shelf life determination of xylanase For shelf life determination, the purified recombinant xylanase was kept at 4°C in refrigerator as well as at room temperature. Samples were taken at different intervals and checked residual activity upto 24 weeks.

3.30.7 Enzymatic Treatment of Pulp Oven dried pulp having consistency of 6% was subjected to recombinant enzyme GthC5Xyl, GthC5ProXyl (40 Ugm−1 dry pulp). The sample was incubated in a shaker at 60-70°C and pH 6.0 and pH 8.0 for different time periods. Autoclaved xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 104 Chapter 3 Materials and methods enzyme was used as a control. After the end of enzymatic treatment, the pulp was dried under vacuum. DNS method was used to monitor the reducing sugar in the filtrate. Chromophores and Lignin-Derived Compounds (LDC) after releasing from pulp treated sample with xylanase were determined by monitoring A280 and A465 of the filtrate, respectively.

3.30.8 Kappa number determination Determination of kappa number gives information about the amount of lignin present in pulp. Amount of lignin is measured by determining the pulp’s oxidant demand. For carrying out Kappa number test, pulp samples were first air dried via heated balance and their oven-dried weight was measured. Potassium permanganate treatment was then applied on pulp as indicated in TAPPI Classical Method T 236 cm. Kappa number of pulp. For establishing confidence interval of specimen each sample was subjected to three separate titration.

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4.1.1 Collection of Samples Different samples of water and soil were collected from northern and southern Pakistan including Tatta Pani (Poonch, Azad Kashmir) at N 33.7621° and E 73.7883° (2234 feet), Garam Chashma (Chitral) at N 35.9972° and E 71.5639° (5919 feet), Chotron/Choutron (Shigar, Skardu) at N 35°26' and E 75°44' (7,303 feet) and Manghopir hot spring (Karachi) at N 24°58' and E 67°1'. GPS was used to observe the geographical coordinates. From each hot spring, two samples of hot water and sediments were collected at different locations; their physical characteristics are described in Table 4.1.1.

Table 4.1.1 Physical characteristics of samples isolated from Hot springs of Pakistan

No. Location of hot spring pH Temperature (°C) 1 Tatta Pani (AJK) 5.9 64°C 2 Garam Chashma (Chitral) 6.7 55°C 3 Chotron (Shigar, Skardu) 7 46°C 4 Manghopir hot spring (Karachi) 7.3 42°C

4.1.2 Screening of xylanase producing bacterial isolates Total 22 thermophilic xylanase producers were isolated and 2 were selected on their temperature range for further studies. All these isolates were screened for cellulolytic, proteolytic and amylolytic activity (Fig. 4.1.1a). Xylanase activity was determined on Congo red xylan agar media plates. Congo red has the ability to bind with the complex xylan form but cannot bind to its reduced form. Bacterial strains which showed clear zones (by reducing xylan) around their colonies were considered as xylanase producer strains. Clear zones of hydrolysis were measured after 24 hours of incubation. Two bacteria, designated as strain AK53 and C5, were selected on the basis of maximum size of zone of hydrolysis for further studies (Fig. 4.1.1b).

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4.1.3 Identification of xylanase producing bacterial strains 4.1.3.1 Morphological Characteristics Growth of Bacterial strains Prominent and rapid growth was demonstrated by the strains AK53, C5, S1 and K22 at 60°C, 55°C, 55°C and 37°C respectively.

4.1.3.2 Microscopy The cellular morphology of all the isolates was carried out through Gram’s staining (Appendix 23). Strain AK53 and C5 are Gram-positive, Flat, off-white color colony, rod in shape and are spore former.

4.1.3.3 Biochemical tests Biochemical characteristics of the isolated thermophilic bacteria are shown in the Table 4.1.2. Strain AK53 and C5 were positive for Catalase, Starch hydrolysis and

Glucose test, while negative for H2S formation, Citrate utilization test, Esculin test, Cellulose test and Oxidase test.

4.1.3.4 Molecular identification of bacterial isolates The phylogenetic analysis showed that strain AK53 resembles more than 99% to G. thermodenitrificans SSCT83 followed by Geobacillus and Bacillus species (Fig. 4.1.2). Fig. 4.1.3 shows phylogenetic relation of strain C5 with G. thermodenitrificans subsp. calidus F84b (EU477773) and G. thermodenitrificans subsp. thermodenitrificans NG80-2 (CP000557) having 99% similarity. The sequences of strain AK53 and C5 were deposited in GenBank with accession number KP203955 and KP203956, respectively.

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Table 4.1.2 Physiological and biochemical characteristic of Geobacillus thermodenitrificans AK53 and C5

Tests Results

AK53 C5 Colony morphology Flat ,off-white in color Flat ,off-white in color Gram’s reaction Gram-positive Gram-positive Shape of cells Rod Rod Spore formation + + Temperature range (ºC) 45-65 45-60 pH range 6-8 6-8 Tolerance to NaCI (%) 2.0% 7% Catalase activity + + Oxidase activity - - Starch hydrolysis + + Cellulose test - - Glucose test + + Esculin test - - Nitrate reduction test + + Citrate utilization test - -

H2S formation - -

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25

20

15

10

5

Number isolates of Number 0

A Enzymes

AK53 C5

B

Fig 4.1.1; Screening of thermozymes from hot spring’s isolates A; Screening of thermophilic isolates producing different thermozymes B; Xylan hydrolysis of AK53 and C5 on 0.5% xylan supplemented nutrient agar plate

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Fig 4.1.2; Phylogenetic tree demonstrating the location of G. thermodenitrificans AK53, the evolutionary history was inferred using the Neighbor-Joining method. Evolutionary analyses were conducted in MEGA 5.0

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Fig 4.1.3; Phylogenetic tree of Geobacillus thermodenitrificans C5, the evolutionary history was inferred using the Neighbor-Joining method. Evolutionary analyses were conducted in MEGA 5.0

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4.1.4 DNA Isolation and Gene Expression 4.1.4.1 Isolation of DNA The genomic DNA was isolated by Wizard® Genomic DNA Purification Kit (Promega) and visualized by 1% agarose gel electrophoresis (Fig. 4.1.4). The isolated DNA served as a template for amplification of xylanase gene in PCR reaction.

4.1.4.2 Optimization of PCR amplification Xylanase encoding genes of strain AK53 and C5 amplification were optimized at different temperatures ranging from 50-62°C, optimization clearly indicated that 62°C is the optimum temperature for amplification of xylanase gene shown in (Fig. 4.1.5).

4.1.4.3 PCR amplification of xylanase gene Xylanase encoding genes of strain AK53 and C5 strain were amplified by using G. thermodenitrificans AK53 and G. thermodenitrificans C5 DNA as template with translational initiation codon ATG and termination codon TAG with the newly designed primer in this study. Amplification of approximately 1.2 kb size of xylanase encoding gene for strain C5 and AK53 is shown in lane 1 and 2, while negative control containing all the reagents except the DNA template is shown in lane 3 (Fig. 4.1.6).

4.1.4.4 Gel purification of amplified xylanase gene QIAquick Gel Extraction Kit (QIAGEN) was used to cut and purify the amplified xylanase gene fragment from gel and visualized by 1% agarose gel electrophoresis. 1.2 kb size for xylanase gene was observed under gel documentation system (Fig. 4.1.7).

4.1.4.5 Cloning of xylanase gene in cloning vector Xylanase coding gene was ligated with the pGEM®-T cloning vector. Then, DH5α competent cells were used for transformation of ligated product. For confirmation of cloning, blue-white screening technique was used to select the appropriate transformed cells having ligated xylanase gene product (Fig. 4.1.8). pGEM®-T vector contain the ampicillin resistant gene. Five white and one blue colony for both strains were selected and LB-Amp broth was used for cultivation of inserted cells. The

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Chapter 4 Results plasmids were isolated by GeneJET Plasmid Miniprep Kit. Confirmation of cloning was carried out by sequencing and digestion with restriction enzyme EcoRI. Sequencing clarified the cloning of xylanase gene to pGEM®-T vector. Digestion of pGEM®-T cloned plasmid with restriction enzyme produced the desired band size (approximately 1200 bp) for strain AK 53 and C5 along with 3000 bp of its own size (Fig. 4.1.9).

4.1.4.6 Preparation of pET28a and xylanase gene product for expression pET28a vector and pGEM®-T cloned xylanase gene product of AK53 an C5 were isolated and visualized by gel electrophoresis for their confirmation (Fig. 4.1.10). These plasmids were digested with HindIII enzyme at 37°C for 2 hours and the digestion was confirmed by gel electrophoresis (Fig. 4.1.11). The digested pET28a showed approximately 5300 bp product size of single band while AK53 and C5 showed 4200 and 4230 bp under gel documentation system. After that the single enzyme digested pET28a vector and xylanase gene products were cleaned through ethanol precipitation and the presence of AK53, C5 and pET28a plasmids was again determined by 1% agarose gel electrophoresis.

4.1.4.7 Double digestion and purification of pET28a vector and xylanase gene product Ethanol precipitated pET28a vector and xylanase gene product of strain C5 and AK53 were subjected to second digestion with NheI restriction enzyme for 2 hours at 37°C. This digestion was analyzed through gel electrophoresis, which was confirmed by their size having 5300 bp for pET28a, 1200 bp for strain AK53 and 1230 bp for C5. Strains C5 and AK53 double digested products also released pGEM-T vector of about 3000 bp (Fig. 4.1.12). The double digested xylanase gene products and pET28a vector were obtained from gel by QIAquick Gel Extraction Kit (QIAGEN) as manufacturer protocol. The gel excised purified insert and pET28a plasmid was confirmed through their respective size in agarose gel electrophoresis and was used as insert for ligation (Fig. 4.1.13).

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4.1.4.8 Cloning of xylanase gene in expression vector Coding sequence of xylanase gene from G. thermodenitrificans AK53 (GthAK53Xyl) and G. thermodenitrificans C5 (GthC5Xyl) was used for recombinant expression in E. coli. Xylanase coding gene was ligated with the pET28a expression vector. Then, E.coli JM101 competent cells were used for transformation of ligated product. pET28a vector contained the kanamycin resistant gene. Four white and one blue colonies were selected and LB-kan broth was used for cultivation of inserted cells, after transformation plasmids were isolated from single colonies and transformation was confirmed by sequencing and digestion of plasmid with appropriate restriction enzyme. The digested pET28a plasmid showed the desired xylanase product size of GthAK53Xyl and GthC5Xyl along with the size of pET28a plasmid.

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1 2 3 4

Fig 4.1.4; Gel electrophoresis of isolated genomic DNA 1 and 2 G. thermodenitrificans C5 3 and 4 G. thermodenitrificans AK53

M 1 2 3 4 5 6 7 8

3 kb 1.2 kb 1.2 kb

Fig 4.1.5; Optimization of PCR conditions (55°C to 62°C) M 2 log marker Lane 1-8 temperature from 55°C to 62°C respectively

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M 1 2 M

3 kb

Xylanase gene 1 kb product 1.2 kb 0.5 kb approximately

Fig 4.1.6; PCR amplification of AK53 and C5 xylanase M; 2 log marker, 1; C5 PCR amplified xylanase gene product 1230 bp, 2; AK53 PCR amplified xylanase gene product 1200 bp approximately.

AK53 C5

AK53 C5 xylanase xylanase gene gene product product

Fig. 4.1.7; Gel purification of PCR amplified AK53 and C5 xylanase gene product

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AK53 C5

Fig 4.1.8; Blue/white screening of pGEM®-T transformed cell with strain AK53 and C5 xylanase gene

M AK53 C5

3 kb 3 kb

1.2 kb 1.2 kb 1 kb

Fig 4.1.9; Digestion of strain C5 and AK53 xylanase cloned pGEM®-T vector with EcoR1 restriction enzyme M; 2 log marker

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M 1 2 M 3

pET28a vector 3 kbp

1 kbp

0.5 kbp

Fig 4.1.10; Isolation of xylanase cloned pGEM®-T plasmid and pET28a plasmid M; 2 log marker, 1; AK53 xylanase cloned pGEM®-T plasmid, 2; C5 xylanase cloned pGEM®-T plasmid, 3; pET28a vector

M 1 M 2 M 3

Digested pGEM®-T

plasmid Digested pET28a

plasmid

Fig 4.1.11; Digestion of pGEM®-T cloned plasmid and pET28a plasmid with HindIII restriction enzyme M; 2 log marker, 1; Hind111digested C5 xylanase cloned pGEM®-T plasmid, 2; Hind111 digested AK53 xylanase cloned pGEM®-T plasmid, 3; HindIII digested pET28a plasmid

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M 1 M 2 M 3

5.3 kbp 3 kbp

1.2 kbp 1.2 kbp

Fig 4.1.12; Double digestion of xylanase cloned pGEM®-T plasmid and pET28a plasmid with NheI and HindIII M; 2 log marker, 1; double digested C5 xylanase cloned pGEM®-T plasmid, 2; double digested AK53 xylanase cloned pGEM®-T plasmid, 3; double digested pET28a plasmid

1 2 3

pET28

AK53 C5a

Fig 4.1.13 Gel extraction of double digested pET28a vector, strain AK53 and C5 xylanase gene product 1; double digested purified AK53 xylanase gene product, 2; double digested purified C5 xylanase gene product, 3; double digested purified pET28a vector

C5

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4.1.5 Bioinformatics analysis GthAK53Xyl displayed a high homology with G. thermodenitrificans JK1 (JN209933) and G. thermodenitrificans T-2 (EU599644) followed by other Bacillus and Geobacillus spp. (Fig. 4.1.14). While a wide resemblance with various GH10 family endoxylanases and high homology with other Bacillus sp. and Geobacillus sp. was depicted by amino acid sequence analysis with BLASTp of GthC5Xyl (Fig. 4.1.15). Phylogenetic relationship of GthC5Xyl of G. thermodenitrificans C5 with other xylanases available at NCBI database showed maximum identity with xylanase of Geobacillus sp. TC-W7 (GQ857066) and G. thermodenitrificans strain JK1 (JN209933).

4.1.6 Induction and over production of recombinant xylanase enzyme Induction of recombinant xylanase enzyme was performed through 1 mM IPTG and expression was observed by SDS-PAGE analysis and enzyme activity assay. SDS- PAGE showed clearly expressed band of xylanase enzyme, while xylanase specific activity in expressed supernatant was observed as 151 (U/mg) for strain GthAK53Xyl and 125 (U/mg) for GthC5Xyl (Fig. 4.1.16).

4.1.7 Purification of recombinant enzyme 4.1.7.1 Precipitation with ammonium sulphate Different concentrations of ammonium sulphate were dissolved in particular volume of cell free extracts after lysis by which recombinant xylanase was significantly precipitated. Concentration of xylanase was enhanced till 60% of ammonium sulphate concentration, any further addition of ammonium sulphate decreased enzyme concentration, whereas minimum concentration was achieved at 10, 20, 30 and 40%. 60% concentration of ammonium sulphate was optimum for extraction of targeted enzyme.

4.1.7.2 DEAE Sepharose chromatography After treatment with ammonium sulphate and dialysis, the precipitated dialyzed preparation was fractionated on DEAE Sepharose ion exchange chromatography eluted by 0.9 M NaCl concentration along with 100mM sodium sulphate buffer. 60

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Chapter 4 Results fractions of about 3 mL each were collected, each fraction was assayed for xylanolytic activity and its optical density (OD) was taken at 280 nm for the protein content, one prominent peak was observed for both strain C5 and AK53 recombinant expressed xylanase. DEAE Sepharose chromatography was used to remove the impurities present in the crude enzyme extract, effectively (Fig. 4.1.17; 4.1.18).

4.1.7.3 High-performance liquid chromatography (HPLC) Reverse phase HPLC C-18 column was also applied for enzyme purity. In the HPLC chromatogram, a single peak was revealed for the purified enzyme at a retention time of 2.5 min, which confirmed that strain C5 and AK53 recombinant xylanase were a homogenous enzyme preparation (Fig. 4.1.19; 4.1.20).

4.1.7.4 SDS-PAGE analysis To investigate the purity of the recombinant enzyme, protein size was characterized by SDS-PAGE. The eluted purified fractions of GthAK53Xyl and GthC5Xyl xylanase appeared in the form of single bands on 15% gel indicating that fractions were homogenous. The molecular weight of GthAK53Xyl and GthC5Xyl was determined against standard denaturing marker proteins and found to be approximately 45 and 44 kDa, respectively (Fig 4.1.21; 4.1.22).

The specific activity of the recombinant xylanase GthAK53Xyl was found to be 1113.0357 IU/mg, the yield was 137% and an overall purification fold was observed as 7.34 (Table 4.1.3). While the specific activity of purified xylanase GthC5Xyl was observed as 1243.12 IU/mg, the yield was 176.28 % with an overall purification fold of 9.89 (Table 4.1.4).

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Fig 4.1.14; Phylogenetic relationship of G. thermodenitrificans AK53 xylanase (GthAK53Xyl) with other xylanase available at NCBI database

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Fig 4.1.15; Phylogenetic relationship of xylanase of G. thermodenitrificans C5 with other xylanases available at NCBI database Neighbor-joining tree showed maximum identity with xylanase of Geobacillus sp. TC-W7 (GQ857066) and G. thermodenitrificans strain JK1(JN209933)

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200

150

100

50 (U/mg) Activity 0 AK53 C5 Recombinant xylanase

Fig 4.1.16; Expressed recombinant xylanase of Geobacillus thermodenitrificans C5 and AK53. Error bars show standard deviation among three observations.

GthAK53Xyl Activity 1200 0.2 Protein

1000 0.16

800 0.12 600 0.08 400

Activity (U/mL) Activity 200 0.04

Absorbance nm 280 Absorbance at 0 0 1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 Number of fraction

Fig 4.1.17; DEAE-Sepharose chromatography of the endoxylanase produced by G. thermodenitrificans AK53

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Activity GthC5Xyl 900 Protein 0.35 800

0.3 700 0.25 600 500 0.2 400 0.15

Activity (U/mL) Activity 300 0.1

200 Absorbance nm 280 Absorbance at 100 0.05 0 0 1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 Number of fraction

Fig 4.1.18; DEAE-Sepharose Ion-exchange chromatography of the endoxylanase produced by Geobacillus thermodenitrificans C5

1: 280 nm, 4 nm Ak53 Irfan 60 Retention Time 60

50 50

40 40

AU 30 30

mAU

mAU

20 20

10 10

0 0

0 1 2 3 4 5 6 7 8 9 10 Minutes Minutes

Fig 4.1.19; HPLC profile of the purified recombinant G. thermodenitrificans AK53 xylanase. Minor peaks and noise was due to contamination in sample.

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1: 280 nm, 4 nm C5 Irfan Retention Time 90 90

80 80

70 70

60 60

50 50 mAU 40 40 mAU 30 30 20 20

10 10

0 0

-10 -10

0 1 2 3 4 5 6 7 8 9 10 Minutes

Fig 4.1.20; HPLC profile of the purified xylanase from G. thermodenitrificans C5 using a reverse phase C-18 column (4.6 x 250 mm) HPLC chromatogram of the purified enzyme shows a single peak at a retention time of 2.5 min confirming that it was a pure preparation. Minor peaks and noise was due to contamination in sample.

Fig 4.1.21; SDS-PAGE of crude and purified xylanase from G. thermodenitrificans AK53 (GthAK53Xyl) (an arrow) M; SDS-PAGE molecular mass standards 10–250 kDa, 1; culture supernatant, 2; induced supernatant; 3, ammonium sulphate precipitate, 4; eluate from DEAE Sepharose

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Fig 4.1.22; SDS PAGE showing purified recombinant G. thermodenitrificans C5 Xylanase enzyme obtained from ion-exchange column chromatography by DEAE Sepharose. M, SDS-PAGE molecular mass standards 10–250 kDa New England Biolab; 1, culture supernatant; 2, Induced supernatant; 3, ammonium sulphate precipitation; 4, purified GthC5Xyl.

Table 4.1.3 Summary of GthAK53Xyl purification steps

Total Specific Total Yield Purification Purification step protein activity activity (U) (%) Yield (mg) (U/mg) Cell extract 4.58 695.39 151.51 100 1 Precipitate 2.90 821.75 282.68 118.16 1.8 DEAE- 0.85 952.67 1113.48 137.6812 7.3 Sepharose

Table 4.1.4 Summary of GthC5Xyl purification steps

Total Specific Purification Total Yield Purification Protein activity step activity (U) (%) Yield (mg) (U/mg) Cell extract 4.49 564.1286 125.6411 100 1 Precipitate 3.03 714.4617 235.7959 126.6487 1.876742 DEAE- 0.8 994.5004 1243.125 176.2897 9.894258 Sepharose

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4.1.8 Enzyme assay for xylanase 4.1.8 .1 Determination of enzyme activity Xylanase activity of the purified xylanase was determined by using the dinitrosalicylic acid (DNS) method (Miller 1959) in which the release of hydrolysis products (reducing sugars) from oat spelt xylan substrate was measured. The liberated sugars were estimated using the reagent 3, 5-dinitrosalicylic acid (DNSA). One unit (U) of xylanase is defined as the amount of enzyme that uses oat spelt xylan as the substrate and liberates 1 μmol of reducing sugars as xylose under the assay conditions. By preparing different dilutions of xylose, the standard curve was plotted. Using the equation X=Y+0.0929/0.1291, the concentration of xylanase was calculated indirectly by measurement of concentration of xylose.

4.1.8.2 Determination of protein concentration Bradford protein determination method (Bradford, 1976) was used to monitor concentration of protein. Bovine serum albumin act as a standard in this method. The results of protein estimation were used to calculate the specific activity of recombinant xylanase.

4.1.8.3 Determination of Specific activity Specific activity of enzyme was calculated by using the formula. Specific activity = concentration of xylose (X) of unknown sample/concentration of total protein in unknown sample.

4.1.9 Biochemical Characterization and stability studies of purified enzyme 4.1.9.1 Effect of temperature on purified xylanase activity Temperature effect was analyzed by incubating GthAK53Xyl and GthC5Xyl with xylan substrate at wide range of temperature 40-90°C and the residual activity was calculated. Both the purified GthAK53Xyl and GthC5Xyl exhibited activity over a broad range of temperature with optimum activity at 70°C and 60°C, while retained 85% and 75% of their initial activity from 40-80°C, respectively (Fig. 4.1.23).

4.1.9.2 Study of pH effect on activity of purified xylanase pH effect on activity of GthAK53Xyl and GthC5Xyl was analyzed with xylan substrate by incubating at pH 3.0-11.0 and the residual activity was calculated. The optimum pH of GthAK53Xyl was revealed to be 5 (Fig. 4.1.24). More than 80% of

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Chapter 4 Results activity was retained from pH 4.0-7.0. The optimum pH in case of GthC5Xyl was 6 (Fig. 4.1.24). While more than 90 % of initial activity was retained from pH 4.0-9.0.

4.1.9.3 Temperature effect on stability of purified xylanase Stability of purified fractions of GthAK53Xyl and GthC5Xyl at different temperatures was analyzed with xylan substrate by incubating at 40-100°C for 200 min and the residual activity was calculated. The recombinant enzyme GthAK53Xyl retained more than 90% activity after exposure to 70°C for 200 min, while more than 75% activity was retained for 200 min at 60 and 80°C (Fig. 4.1.25). The recombinant enzyme GthC5Xyl retained more than 80% activity after exposure to 60°C for 200 min, while more than 74% activity was retained for 200 min at 50 and 70°C (Fig. 4.1.26). Half of its activity was lost at 80°C when incubated for 200 min.

4.1.9.4 Effect of pH on stability of purified xylanase pH stability of purified fractions of GthAK53Xyl and GthC5Xyl were analyzed by incubating with xylan substrate from pH 3.0-11.0 for 200 min and the residual activity was calculated. The recombinant enzyme GthAK53Xyl retained 89% activity after exposure to pH 5.0 for 200 min, while more than 70% activity was retained for 200 min at pH 4.0-7.0, and more than 65% at pH 8.0 (Fig. 4.1.27). The recombinant enzyme GthC5Xyl retained more than 93% activity after exposure to pH 6.0 for 200 min, more than 80% at pH 7.0 and 8.0, more than 70% at pH 5.0 and 9.0, and 69% activity was retained at pH 10 for 200 min (Fig. 4.1.28).

4.1.9.5 Effect of substrate specificity of purified recombinant xylanase Specificity of the recombinant GthAK53Xyl and GthC5Xyl enzyme was found towards polymeric xylan sources and attacked no other substrate such as insoluble xylan, carboxymethyl cellulose, avicel, filter paper, pNP-α-L-arabinofuranoside, pNP- β-Xylopyranoside, pNP-α-glucopyranoside, pNP-α-D-Xylopyranoside, pNP-β- galactopyranoside and pNP-acetate (Table 4.1.5). In case of GthAK53Xyl, highest activity was observed with the birchwood xylan (1120 U mg−1) followed by the beechwood xylan (1059 U mg−1) and oatspelt xylan (1005 U mg−1). The enzyme was less active on arabinoxylan (676 U mg−1).

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Whereas GthC5Xyl enzyme activity was evaluated at 60°C, pH 6.0 for 10 min with various substrates for determination of enzyme specificity (Fig. 4.1.29). Different xylan substrates were determined for GthC5Xyl. The highest activity was observed with the beechwood xylan (1203 U mg−1) followed by the oatspelt xylan (1084 U mg−1) and birchwood xylan (1040 U mg−1). The enzyme was less active on arabinoxylan (641 U mg−1).

4.1.9.6 Effects of metal ion In order to verify the effect of metal ions on xylanase activity, the purified enzyme was incubated in the presence of several metallic ions. In general, with the increased concentration of the metal ions, the xylanase GthAK53Xyl activity was enhanced. 2+ 2+ CaSO4, Hg and Cu caused strong inhibition of the xylanase; The GthAK53Xyl has been pointedly inhibited by CoCl2, FeSO4, CuCl2, CaSO4 even at very low concentration of 1 mM (Fig. 4.1.30). AlCl3, ZnSO4, and CuSO4 inhibited the enzyme at higher concentration (5.0 mM) only. With the increased concentration of the metal 2+ ions, the xylanase GthC5Xyl activity was enhanced. FeSO4 and Hg caused strong inhibition of the GthC5Xyl whereas CaSO4 partially inhibit GthC5Xyl at higher concentration. The GthC5Xyl has been inhibited by AlCl3, ZnSO4, MgCl2 and MgSO4 at 10mM concentration (Fig 4.1.31). CsCl inhibited the GthC5Xyl at 5.0 mM concentration only. CoCl2, AgNO3, MnSO4 CaCl2, KCl, CuCl2, MnCl2 and CuSO4 do not show inhibitory effect on GthC5Xyl.

4.1.9.7 Effect of detergents The xylanase GthAK53Xyl is quite stable in the presence of the detergents tested but half of its activity is inhibited in the presence of 1% CTAB concentration and more than 80% activity is retained even at 1% concentration of Tween 20 and Tween 40. Triton X-100 inhibit enzyme at higher concentration (Fig. 4.1.32). The GthC5Xyl is quite stable in the presence of the detergents tested but half of its activity is inhibited in the presence of 1% CTAB concentration and more than 80% activity is retained even at 1% concentration of tween 20. Triton X-100 and tween 40 inhibit enzyme at higher concentration only (Fig. 4.1.33).

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4.1.9.8 Effect of inhibitors TSC increased xylanase activity of GthAK53Xyl at both low and high concentrations. NEM, DEPC, PMSF and NBS inhibited enzyme only at high concentration (10 mM). GthAK53Xyl was found to be resistant to DTT and β-Mercaptoethanol upto 10 mM concentration (Fig. 4.1.34). GthC5Xyl activity was enhanced in the presence of DTT and TSC upto 10 mM. DEPC, NEM, PMSF and NBS inhibited enzyme activity at high concentration (10 mM) (Fig. 4.1.35).

4.1.9.9 Effect of organic solvents The effect of GthAK53Xyl to organic solvents at 15% (v/v) final concentration was evaluated. The enzyme was not substantially influenced by most of the tested organic solvents even after 2 hour of incubation. Slight effects on GthAK53Xyl activity reductions by 12%, and 13%, were detected in presence of acetonitrile and DMSO respectively, while n-butanol enhanced the activity by 5%. Formaldehyde, ethyl acetate and glycerol markedly inhibited the enzyme and the activity was reduced upto 12%, 11% and 42%, after 2 hour of incubation, respectively (Fig. 4.1.36). The enzyme GthC5Xyl was not substantially influenced by most of the tested organic solvents even after 2 hour of incubation. Slight effects on reduction of GthC5Xyl activity were detected by 14%, 7%, and 17% in the presence of acetonitrile, DMSO and DMF, respectively. Formaldehyde, ethyl acetate and glycerol markedly inhibited the enzyme after 2 hours of incubation by 5%, 8% and 25%, respectively, (Fig 4.1.37).

4.1.9.10 Study of hydrolysis product Thin layer chromatography (TLC) was used to study the mode of hydrolysis of purified xylanase. The hydrolyzed products of oat spelt xylan were analyzed by TLC of GthAK53Xyl and GthC5Xyl fractions. Fig. 4.1.38 and 4.1.39 revealed that the enzyme fractions released a range of xylooligosacchrides after hydrolysis of xylan. The main product for both GthAK53Xyl and GthC5Xyl were xylobiose and xylopentose.

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4.1.9.11 Investigation of shelf life of xylanase The purified fractions of recombinant enzyme GthAK53Xyl retained its activity, when stored at 4°C for 12 weeks, but after that, a decline in activity was observed. The enzyme retained more than 90% its activity after 10 weeks which would be important for its application. On the other hand, at room temperature, the recombinant GthAK53Xyl xylanase enzyme was completely stable for 5 weeks but showed 80% and 70% residual activity after storage for 10 and 12 weeks, respectively. No activity was lost by purified GthC5Xyl when stored for 12 weeks at 4°C but after that decline was observed. After 16 weeks, 90% of initial activity was retained by the enzyme which is an important fact for its industrial application. Conversely enzyme remained completely stable for five weeks at room temperature but showed 80% and 70% residual activity after storage for 10 and 12 weeks, respectively.

4.1.9.12 Kinetics of purified recombinant xylanase

In order to study the kinetics of purified recombinant xylanases, Km and Vmax values were calculated by Lineweaver and Burk (1934) plot. Purified recombinant xylanase was incubated with different concentration of substrate and activity was measured. −1 The Km and Vmax of GthAK53Xyl (for oat spelt xylan) was 4.34 mg mL and 2028.94 −1 −1 μmolmg min , respectively (Fig. 4.1.40). The Km and Vmax of GthC5Xyl (for oat spelt xylan) were 3.9084 mgmL−1 and 1839.86 μmolmg−1 min−1, respectively (Fig. 4.1.41). The Km of the enzyme (4.34 mgmL−1 for oat spelt xylan) is within the range for microbial xylanases (0.14–14 mgmL−1).

4.1.9.13 Effect of salt concentration Studies on NaCl effect on activity of GthC5Xyl showed that with increasing concentrations of NaCl till 0.8 M, the activity was enhanced. Relative activity was 101.02% at 1M of NaCl, while at 1.5M of NaCl concentration the activity was sharply decreased to 71% (Fig. 4.1.42). GthAK53Xyl retained more than 90% of its initial activity till 1M NaCl concentration, while at 1.5M NaCl concentration the enzyme is strongly inhibited.

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120

100 C5 80 AK53

60

40

Relative activity (%) activity Relative 20

0 40 50 60 70 80 90 100 Temperature (°C)

Fig 4.1.23; Effect of temperature on the activity of purified recombinant AK53 and C5 xylanase

120

100

80 C5 60 AK53 40

(%) activity Relative 20

0 3 4 5 6 7 8 9 10 pH

Fig 4.1.24; Effect of pH on the activity of purified recombinant AK53 and C5 xylanase

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120 60°C

100 70°C 80

60 80°C

40

(%) activity Relatie 90°C 20 0 0 20 40 60 80 100 120 140 160 180 200 Incubation time (min)

Fig 4.1.25 Effect of temperature on the stability of purified GthAK53Xyl.

120

100

80 50°C

60°C

60 70°C

40 80°C (%) activity Relative 90°C 20

0 0 20 40 60 80 100 120 140 160 180 200 Incubation time (min.)

Fig 4.1.26 Effect of temperature on the stability of purified GthC5Xyl

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120

100 pH 4 80 pH 5

pH 6 60 pH 7 40

pH 8 Relative activity (%) activity Relative 20 pH 9

0 0 20 40 60 80 100 120 140 160 180 200 Incubation time (min.)

Fig 4.1.27; Effect of pH on stability of purified GthAK53Xyl

120

100

pH 6 80 pH 7

pH 8

pH 9

Relative activity (%) activity Relative 60 pH 10

40 0 20 40 60 80 100 120 140 160 180 200 Incubation time (min)

Fig 4.1.28; Effect of pH on stability of purified GthC5Xyl

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Table 4.1.5 Substrate specificity of Geobacillus thermodenitrificans C5 and Geobacillus thermodenitrificans AK53

Xylanase residual activity Substrate (%) AK53 C5 Oalt spelt xylan 89 68 Birchwood xylan 100 65 Beechwood xylan 94 100 Arabinoxylan 57 38 CMC 0 0 Insoluble xylan 0 0 Avicel 0 0 pNP-β- Xylopyranoside 0 0 pNP- β- galactopyranoside 0 0 pNP-α- glucopyranoside 0 0 pNP-α- L- arabinofuranoside 0 0 pNP-α-D- Xylopyranoside 0 0 pNP- acetates 0 0 Laminarin 0 0

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1300 1200

AK53 1100 C5 1000 900 800

Activity Activity (U/mg)

700

600 500 Birchwood Beechwood Oatspelt xylan Arabinoxylan xylan xylan Substrate

Fig 4.1.29; Substrate specificity of Geobacillus thermodenitrificans C5 and GeobacillusthermodenitrificansAK53

1mM 200 5mM 10mM 150

100

50 Relative activity (%) 0 2 3 3 4 4 4 O 4 2 2 4 2 4 Hg 2 Kcl LiCl CsCl AlCl CoCl .2H FeSO CuCl MnCl MgCl AgNO MnSO ZnSO MgSO 2 CaSO CuSO CaCl Control AK53 Metals concentration (mM)

Fig 4.1.30 Effect of various metals ion on GthAK53Xyl activity

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1 mM

200 5 mM

10 mM 150

100

50

Relative activity (%)

0

2 3 3 4 4 4 2 4 2 2 4 2 4 Kcl Hg CsCl LiCl CoCl AlCl CaCl CuCl AgNO MnSO ZnSO MgSO FeSO MnCl CaSO MgCl CuSO

Control C5 Metals concentration (mM)

Fig 4.1.31 Effect of various metals ion on GthC5Xyl activity

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120

100

80

60 0.10%

40 0.50% (%) activity Relative 1% 20 Fig 4.1.32; Effects of various Detergents on GthAK53Xyl

0 Control SDS CTAB TWEEN TWEEN Triton x AK53 20 40 100

Detergents (%)

Fig 4.1.32; Effects of various Detergents on GthAK53Xyl activity

120

100

80

60 0.10% 40 0.50%

Relative activity (%) activity Relative 20 1%

0 Control SDS CTAB TWEEN TWEEN Triton x C5 20 40 100 Detergents (%)

Fig 4.1.33; Effects of various Detergents on GthC5Xyl activity

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140

120 1mM 100 5mM 80 10mM 60 40 Relative activity (%) activity Relative 20 0

Inhibitors (mM)

Fig 4.1.34 Effects of various Inhibitors on recombinant purified AK53 Xylanase activity

140 Inhibitors C5 1mM 120 5mM 100 10mM 80

60 40

Relative activity (%) activity Relative 20

0

Inhibitors (mM)

Fig 4.1.35 Effects of various Inhibitors on recombinant purified C5 Xylanase activity

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120

30 min 100 60 min 80 120 min 60 40

20 Relative activity (%) activity Relative 0

Organic solvent (15%)

Fig 4.1.36; Effects of organic solvent on GthAK53Xyl activity

120

100

80

60

40 30 min 60 min Relative activity (%) activity Relative 20 120 min 0

Organic solvent (15%)

Fig 4.1.37 Effects of organic solvent on GthC5Xyl activity

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1 X1 X2 X3 X4 X5

Fig 4.1.38; TLC analysis for hydrolysis products released from oat spelt xylan by GthAK53Xyl. 1 - sample; X1 - D-xylose; X2 - xylobiose; X3 - xylotriose; X4 - xylotetrose; X5 – xylopentose

X1 X2 X3 X4 X5 1

Fig 4.1.39; TLC analysis for hydrolysis products released from oat spelt xylan by xylanase from Geobacillus thermodenitrificans C5, 1: GthC5Xyl; X5: D-xylose; X4: xylobiose; X3: xylotriose; X2: xylotetrose; X5: xylopentose.

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Table 4.1.6 Shelf life determination of C5 and AK53 recombinant expressed xylanase

Weeks C5 xylanase RA% AK53 xylanase RA% 4°C Room temperature 4°C Room temperature 2 100 100 100 100 4 100 100 100 100 6 100 98 100 96 8 100 91 96 87 10 100 80 95 80 12 99 70 92 70 14 94 66 88 64 16 90 63 86 60

0.004 y = 0.0015x + 0.0006 0.0035 R² = 0.9931 0.003

0.0025

1/V 0.002

0.0015 0.001 0.0005

0 -1 -0.5 0 0.5 1 1.5 2 2.5 -0.0005 1/S

Fig 4.1.40; Lineweaver-burk plot of AK53Xylanase (Km and Vmax values were determined according to Lineweaver-burk plot

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Fig 4.1.41; Lineweaver-burk plot of C5Xylanase (Km and Vmax values were determined according to Lineweaver-burk plot.

110

100

90

80 AK53

70 C5

Relative activity (%) activity Relative 60

50 0 0.2 0.4 0.6 0.8 1 1.2 1.5 NaCl (M)

Fig 4.1.42; Effect of NaCl on purified xylanase GthAK53Xyl and GthC5Xyl activity

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4.2.1 Bioinformatics analysis Deduced amino acid sequence of xylanase from Geobacillus thermodenitrificans C5 and AK53 showed no presence of cysteine amino acid residues but an excess of (Asp+Glu) negatively charged residues. Aliphatic index of 37 was demonstrated by the in silico analysis. A wide resemblance with various GH10 family endoxylanases and high homology with other Bacillus sp. and Geobacillus sp. was depicted by amino acid sequence analysis with BLASTp by strain C5 xylanase while AK53 displayed a high homology with enzymes from G. thermodenitrificans JK1 (JN209933) and G. thermodenitrificans T-2 (EU599644) followed by other Bacillus and Geobacillus spp. Available crystal structure of xylanase (PDB ID, 1HIZ chain A) from G. thermodinitrificans was utilized for proposing GthC5Xyl secondary and tertiary structures. A total of 11 α helices along with 5 sharp turns and 13 β sheets were found in secondary structure. Important catalytic residues Glu was found within the conserved region present inside a “bowl” shaped structure of GH10 xylanase via 3D structure obtained from PyMol PDB viewer.

4.2.2 Homology Modeling of Strain AK53 Xylanase and Prediction of Mutants The sequence obtained for strain AK53 from sequencing was subjected to protein blast on NCBI web server with respect to a PDB database. The result suggested an ortholog template of xylanase in Bacillus stearothermophilus. The template selected for the model construction of AK53 was chain A of crystal structure 1HIZ. The sequence similarity found between template and target sequence of xylanase was 88% with query coverage of 87%. MODELLER 9 was utilized to generate the homology models of target xylanase. The top 5 models were selected on the basis of DOPE score from MODELLER; they were further evaluated on the basis of Ramachandran values and energy plot. These values help in identifying most reliable and stable predicted conformation. The values suggested that the model 3 was the most sterically stable as it has the least number of van der Waal's clashes. The lists of all these values are given in table 4.2.1. The model was subject to energy minimization with 750 steps steepest decent and 750 steps of conjugate gradient. The final model has GLU active site residue represented as sphere and sticks.

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4.2.3 Modeling of Strain C5 Xylanase Mutants Crystal structure for the selected sequence was unavailable. The homology modeling was applied to generate the 3-D structures of the strain C5 sequence of Xylanase. The template for the homology of C5 was same as that of AK53 with increased query coverage of 92% and sequence identity of 88%. Five models were generated using the MODELLER program and ranked using DOPE score. Models were further evaluated on the basis of their Φ and Ψ angle distribution using Ramachandran plot. The results depicted in table 4.2.2 suggested model 4 has least amount of angle distortion and steric discrepancy. The model energy was evaluated using ProSA, and found to be in relative stable conformation and Z-score was accordance with the crystal structure database Z-score spectrum. After determining the energetically stable conformation of strain C5 sequence the side chain of residue to be mutated were trim from the model. The mutant side chain was generated using scrwl on the trimmed model. Individually all the mutant sidechain were calculated and then analyzed using HBOND for novel hydrogen bonds formed by the mutant sidechain.

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Glu66

Glu89 Lys93

Trp347 Trp355

Trp130 Glu296 Glu190 Arg359

Trp304

Tyr234 Gln269

Glu37 Trp272

Fig. 4.2.1; The proposed Three dimensional structure of Geobacillus thermodenitrificans C5 xylanase. The predicted 3D model of C5 xylanase has catalytically important residues (Glu) positioned at the center of the bowl shaped structure

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His343 Glu79 Asn80 Trp337 Lys83 Trp345

His116 Glu286

Trp120 Arg349 Glu180 His257 Gln255 Trp294 Tyr224 Gln259 Asn225 Trp26 2

Fig 4.2.2; The proposed Three dimensional structure of Geobacillus thermodenitrificans AK53 xylanase. The predicted 3D model of AK53 xylanase has catalytically important residues (Glu) positioned at the center of the bowl shaped structure

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Table 4.2.1 Ramachandran values for sequence AK53

Regions Model -1 Model-2 Model-3 Model-4 Model-5

Residues in most favoured 318 317 318 312 316 regions 93.4% 93.2% 93.5% 91.8% 92.6%

Residues in additional 20 22 21 27 22 allowed regions 5.9% 6.5% 6.2% 7.9% 6.5%

Residues in generously 1 0 1 1 1 allowed regions 0.3% 0.0% 0.3% 0.3% 0.3%

Residues in disallowed 1 1 0 0 1 regions 0.3% 0.3% 0.0% 0.0% 0.3%

Number of non-glycine 340 340 340 340 340 and non-proline residues

Number of end-residues 2 2 2 2 2 (excl, Gly and Pro) Number of glycine 17 17 17 17 17 residues (shown as triangles) Number of proline 20 20 20 20 20 residues Total Number of Residues 379 379 379 379 379

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Table 4.2.2 Ramachandran values for sequence C5 Regions Model -1 Model-2 Model-3 Model-4 Model-5

Residues in most favoured 315 315 312 316 312 regions 93.2% 93.2% 92.3% 93.5% 92.3%

Residues in additional 20 21 24 21 24 allowed regions 5.9% 6.2% 7.1% 6.2% 7.1%

Residues in generously 3 2 1 1 2 allowed regions 0.9% 0.6% 0.3% 0.3% 0.6%

Residues in disallowed 0 0 1 0 0 regions 0.0% 0.0% 0.3% 0.0% 0.0%

Number of non-glycine 338 338 338 338 338 and non-proline residues

Number of end-residues 2 2 2 2 2 (excl, Gly and Pro)

Number of glycine 17 17 17 17 17 residues (shown as triangles) Number of proline 21 21 21 21 21 residues Total Number of Residues 378 378 378 378 378

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4.2.4 Isolation of recombinant pET28a Plasmid The recombinant pET28a plasmids of strain AK53 and C5 were isolated by Spin Miniprep Kit and visualized by 1% agarose gel electrophoresis. The isolated pET28a plasmid of AK53 and C5 strains containing xylanase served as a template for mutation and amplification of xylanase gene in PCR reaction.

4.2.5 Overlap extension PCR for mutation Two set of PCR was used to amplify xylanase gene having the desired mutation.

4.2.5.1 W137R mutation in strain C5 Recombinant plasmid of strain C5 showed about approximately 832 bp and approximately 421 bp fragment when amplified with primer XGeoT-R/W137R-F and XGeoT-F/W137R-R respectively for mutation W137R (Fig. 4.2.3). After that both 832 bp and 421 bp purified mutant product was used as a strand to amplify full xylanase gene having mutation of W137R with XGeoT-F/XGeoT-R primer having about 1230 bp product (Fig. 4.2.4). The mutation of xylanase was confirmed by sequencing.

4.2.5.2 Geobacillus thermodenitrificans C5 D328R mutation Recombinant plasmid of strain C5 showed about 260 bp and 994 bp when amplified with mutation primer XGeoT-R/D328R-F, and XGeoT-F/D328R-R under gel documentation system (Fig. 4.2.3). The complete xylanase gene having approximately 1230 bp was amplified with XGeoT-F and XGeoT-R primer with mutated 260 bp and 994 bp as a template and analyzed by gel electrophoresis (Fig. 4.2.4), and mutation was confirmed by sequencing.

4.2.5.3 Geobacillus thermodenitrificans Double mutation W137R/D328R The recombinant plasmid W137R was amplified with XGeoT-F/D328R-R and the amplified product turn out to be about 994 bp, while amplified D328R mutation plasmid with XGeoT-R/D328R-F having 260 bp products was visualized under gel documentation system (Fig. 4.2.3).

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After that both 994 bp and 260 bp purified product was used as a strand to amplify xylanase gene having double mutation with XGeoT-F/XGeoT-R primer having about 1230 bp product (Fig. 4.2.4). The mutation of xylanase was confirmed by sequencing.

4.2.5.4 Geobacillus thermodenitrificans C5 N84R and D339R Recombinant plasmid of strain C5 was amplified with p15Xyl–R/N84R-F primer and showed approximately 262 bp while fragment amplified with p15Xyl–F/N84R-R showed approximately about 990 bp product sizes on 1% agarose gel electrophoresis (Fig. 4.2.5). The complete xylanase gene having approximately 1230 bp was amplified p15Xyl–R and p15Xyl–F primer with mutated 990 bp and 262 bp as a template and complete mutated xylanase gene having 1230 bp was analyzed by gel electrophoresis, and mutation was confirmed by sequencing. Approximately 1029 bp products was obtained when recombinant plasmid was amplified with p15Xyl–R/D339R-F primer, while approximately 228 bp product size was achieved when recombinant plasmid was amplified with p15Xyl–F/D339R-R primer (Fig. 4.2.5). The complete xylanase gene having approximately 1230 bp was amplified with p15Xyl–F and p15Xyl–R primer with mutated 1029 bp and 228 bp as a template and mutated D339R xylanase gene having 1230 bp was analyzed by gel electrophoresis, and mutation was confirmed by sequencing.

4.2.5.5 Geobacillus thermodenitrificans C5 R81P, H82E Approximately 997 bp products was obtained when recombinant plasmid was amplified with p15Xyl–R/R81P-F primer, while approximately 263 bp product size was achieved when recombinant plasmid was amplified with p15Xyl–F/R81P–R primer (Fig. 4.2.6). The complete xylanase gene having approximately 1230 bp was amplified with p15Xyl–F and p15Xyl–R primer with mutated 997 bp and 263 bp as a template and mutated R81P xylanase gene having 1230 bp was analyzed by gel electrophoresis, and mutation was confirmed by sequencing. Approximately 997 bp products obtained when recombinant plasmid was amplified with p15Xyl–R/H82E-F primer, while approximately 255 bp product size was achieved when recombinant plasmid was amplified with p15Xyl–F/H82E -R primer (Fig. 4.2.6). The complete xylanase gene having approximately 1230 bp was amplified with p15Xyl–F and p15Xyl–R primer with mutated 997 bp and 255 bp as a

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 152 Chapter 4 Results template and mutated H82E xylanase gene having 1230 bp was analyzed by gel electrophoresis, and mutation was confirmed by sequencing.

4.2.5.6 Geobacillus thermodenitrificans C5 W185P, D186E, DM W185P/D186E Proline instead of tryptophan mutation at 185 position was achieved when recombinant plasmid was amplified with p15Xyl–R/W185P-F and p15Xyl–F/W185P- R primer having product size of 690 bp and 580 bp respectively (Fig. 4.2.6). The complete proline mutated xylanase gene having approximately 1230 bp was amplified with p15Xyl–F and p15Xyl–R primer with mutated 690 bp and 580 bp as a template, and analyzed by gel electrophoresis. Glutamic acid instead of Aspartic acid mutant at 186 position was achieved when recombinant plasmid was amplified with p15Xyl–R/D186E -F and p15Xyl–F/D186E -R primer having product size of 690 bp and 580 bp respectively (Fig. 4.2.6). The complete proline mutated xylanase gene having approximately 1230 bp was amplified with p15Xyl–F and p15Xyl–R primer with mutated 690 bp and 580 bp as a template, and analyzed by gel electrophoresis. Double mutant W185P/D186E was achieved in similar pattern, while mutant H82E and W185P/D186E were combined to produce triple mutant H82E/W185P/D186E (Fig. 4.2.7 and 4.2.8).

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M 1 2 3 4 5 6

994 bp 832 bp 1000 bp 994 bp 500 bp 421 bp 250 bp 260 bp

260 bp

Fig 4.2.3; Gel electrophoresis of D328R, W137R and W137R/D328R mutant fragment M; 1KD DNA ladder thermoscientific SM0311 1, XGeoT-R/D328R-F; 2, XGeoT-F/D328R-R, 3; XGeoT-R/W137R-F, 4; XGeoT- F/W137R-R, 5; XGeoT-R/D328R-F (W137R mutant plasmid), 6; XGeoT-F/D328R-R (D328R mutant plasmid

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M 1 2 3

1.23 kb

2000 bp

1000 bp

500 bp

Fig 4.2.4; Gel electrophoresis of D328R, W137R and W137R/D328R mutant complete xylanase gene product M 1KD DNA ladder thermoscientific SM0311 1; W137R, 2; D328R, 3; W137R /D328R

M 1 2 3 4

990 bp

1000 bp 1029 bp 500 bp 228 bp 262 bp

Fig 4.2.5; Gel electrophoresis of N84R and D339R mutant xylanase gene product M 2 log ladder 1; p15Xyl–F /N84R-R, 2; p15Xyl–R /N84R-F, 3; p15Xyl–F/D339R-R, 4; p15Xyl– R/D339R-F

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M 1 2 3 4 5 6 M M 7 8 9 10 M

3000 bp 997 bp 690 bp 997 bp 580 bp 255 bp 690 bp 580 bp 263 bp 500 bp

Fig 4.2.6; Gel electrophoresis of R81P, H82E, W185P, D186E, W185P/D186E mutant xylanase fragment product M 2 log marker 1; P15xylR/R81P F, 2; P15xylF/R81P R, 3; P15xylR/H82E F, 4; P15xylF/H82E R, 5; P15xylR/W185P F, 6; P15xylF/W185P R, 7; P15xylRD186E F, 8; P15xylFD186E R, 9; P15xylR/W185P/D186E F, 10; P15xylF/W185P/D186E R

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M 1 2 M

997 bp

400 bp 263 bp

Fig 4.2.7; Gel electrophoresis of strain C5 triple mutant 1; P15xylR/H82E (double mutant 175/176 plasmid), 2; P15xylF/H82E R (Mutant H82E plasmid)

1 M 2 3 4 5 6 7 M 8 9

1.5 kb 1.23 kb 1 kb

0.5 kb

Fig 4.2.8; Gel electrophoresis of mutant R81P, H82E, W185P, D186E, W185P/D186E xylanase gene product M 2 log marker, 1; R81P, 2; H82E, 3; 4; W185P,5; D186E, 6; W185P/D186E, 7; R81P/ W185P/D186E, 8; N84R, 9; D339R

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4.2.6 Generation of strain AK53 mutant models 4.2.6.1 D318R Mutation in AK53 The recombinant plasmid AK53 xylanase was amplified with XGeoT-R and D318R-F showing approximately 280 bp products, while second set was amplified with XGeoT-F and D318R-R and product was viewed under gel documentation system showing approximately 964 bp product sizes (Fig. 4.2.9). The complete xylanase gene having approximately 1.2 kb was amplified with XGeoT- F and XGeoT-R primer with mutated purified product approximately 280 bp and approximately 964 bp as a template and analyzed by gel electrophoresis, and mutation was confirmed by sequencing (Fig. 4.2.10).

4.2.6.2 W127R mutation in strain AK53 The recombinant plasmid of AK53 showing approximately 850 bp and approximately 390 bp when amplified with mutated primer XGeoT-R/W127R-F and XGeoT- F/W127R-R respectively (Fig. 4.2.9). The complete mutated xylanase gene having approximately 1.2 kb was amplified with XGeo T-F and XGeoT-R primer with mutated 850 bp and 390 bp as a template and analyzed by gel electrophoresis and mutation was confirmed by sequencing (Fig. 4.2.10).

4.2.6.3 Double mutation D318R and W127R in strain AK53 The recombinant plasmid D318R was amplified with XGeoT-R/D318R-F and the amplified product turn out to be about 280 bp, while W127R mutated plasmid was amplified with XGeoT-F/D318R-R having 964 bp products and was visualized under gel documentation system (Fig. 4.2.9). After that both 250 bp and 990 bp purified product was used as a strand to amplify complete xylanase gene having double mutation with XGeoT-F/XGeoT-R primer having about 1200 bp product. The mutation of xylanase was confirmed by sequencing (Fig. 4.2.10).

4.2.6.4 Geobacillus thermodenitrificans AK53 N74R and D329R Arginine instead of asparagine at 74 position was achieved when recombinant plasmid was amplified with p15Xyl–R/N74R-F and p15Xyl–F/N74R-R primer having

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 158 Chapter 4 Results product size of 1009 bp and 232 bp, respectively (Fig. 4.2.11). The complete arginine mutated xylanase gene having approximately 1.2 kb was amplified with p15Xyl-F and p15Xyl-R primer with mutated 1009 bp and 232 bp as a template, and analyzed by gel electrophoresis (Fig. 4.2.12). Arginine instead of aspartic acid at 329 position was achieved when recombinant plasmid was amplified with p15Xyl–R/D329R-F and p15Xyl–F/D329R-R primer having product size of 245 bp and 999 bp, respectively (Fig. 4.2.11). The complete arginine mutated xylanase gene having approximately 1.2 kb was amplified with p15Xyl-F and p15Xyl-R primer with mutated 999 bp and 245 bp as a template, and analyzed by gel electrophoresis (Fig. 4.2.12).

4.2.6.5 Geobacillus thermodenitrificans AK53 DM R71P/H72E and W175P/D176E Proline instead of arginine at 71 positions and Glutamic acid instead of histidine at 72 positions was introduced when recombinant plasmid was amplified with p15Xyl– R/R71P: H72E-F and p15Xyl–F/R71P: H72E-R primer having product size of 1018 bp and 233 bp respectively (Fig. 4.2.13). The complete arginine mutated xylanase gene having approximately 1.2 kb was amplified with p15Xyl-F and p15Xyl-R primer with mutated 1018 bp and 233 bp as a template, and analyzed by gel electrophoresis (Fig. 4.2.14). Proline instead of Tryptophan and Glutamic acid instead of Aspartic acid at 175 and 176 positions respectively was achieved when recombinant plasmid was amplified with p15Xyl–W175P/D176E-F and p15Xyl–F/W175P/D176E-R primer having product size of 708 bp and 553 bp respectively (Fig. 4.2.13). The complete proline and glutamic acid mutated xylanase gene having approximately 1.2 kb was amplified with p15Xyl-F and p15Xyl-R primer with mutated 708 bp and 553 bp as a template, and analyzed by gel electrophoresis (Fig. 4.2.14).

4.2.6.6 Geobacillus thermodenitrificans AK53 quadrupole mutant R71P/H72E/ W175P/D176E Both double mutant R71P/H72E and W175P/D176E were combined to produce quadrupole mutant R71P/H72E/W175P/D176E (Fig. 4.2.14)

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M 1 2 3 4 5 M 6 7

850 bp 390 bp

964 bp 964 bp

280 bp 280 bp

Fig 4.2.9; Gel electrophoresis of AK53 D318R, W127R and W127R/D318R mutant fragment M 1KD DNA ladder thermoscientific SM0311 1; XGeoT-R/D318R-F, 2; XGeoT-F/D318R -R, 3; XGeoT-R/W127R -F, 4; XGeoTF/W127R -R, 5; control, 6; XGeoTR/D318R -F (W127R mutant plasmid), 7; XGeoTF/D318R -R (D318R mutant plasmid

M 1 2 M 3

1.2 kb

1 kb 0.5 kb

Fig 4.2.10; Gel electrophoresis of AK53 D318R, W127R and W127R/D318R mutant complete xylanase gene M 2 Log DNA ladder, 1; W127R, 2; D318R, 3; W127R/D318R

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M 1 2 M 3 4

3000 bp

1200 bp

1009 bp 999 bp

232 bp 245 bp

Fig 4.2.11, Gel electrophoresis of AK53 N74R and D329R mutant fragment M 2 log; ladder, 1; p15Xyl–R /N74R-F, 2; p15Xyl–F/N74R-R, 3; p15Xyl– F/D339R-R, 4; p15Xyl–R/D339R-F

1 2 M

1.2 kb 1.5 kb

1 kb

Fig 4.2.12; Gel electrophoresis of mutant AK53 N74R and D329R full xylanase gene amplification M 2 log ladder, 1; N74R, 2; D339R

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 161 Chapter 4 Results

M 1 2 3 4 M M 5 6

1018 bp

1500 bp 708 bp 1000 bp 1018 bp

553 bp 500 bp

233 bp 233 bp

Fig 4.2.13; Fig; 4.2.11, Gel electrophoresis of mutant AK53 R71P/H72E, W175P/D176E and Quadrupole mutant R71P/H72E/ W175P/D176E M; 2 log marker 1; P15xyl-R/R71P: H72E -F, 2; P15xyl-F/R71P: H72E -R, 3; P15xyl- R/W175P; D176E-F, 4; P15xyl-F/W175P; D176E-R, 5; P15xyl-R/R71P; H72E-F (Quadrupole mutant) 6; P15xyl-F/R71P; H72E-R (Quadrupole mutant)

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 162 Chapter 4 Results

1 2 3 4 M 5

3 kb 1.5 kb 1.2 kb 1 kb

Fig 4.2.14; Gel electrophoresis of mutant DM R71P/H72E, W175P/D176E and R71P/H72E/W175P/D176E xylanase gene product M 2 log marker 1, 2; DM R71P/H72E, 3, 4; W175P/D176E, 5; R71P/H72E/W175P/D176E

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4.2.7 Gel purification of amplified mutated xylanase gene GeneJET Gel Extraction Kit (Thermo Scientific) was used to cut and purify the amplified xylanase having single and double mutation of strain AK53 and C5 from gel and visualized by 1% agarose gel electrophoresis. 1230 bp size for strain C5 and 1200 bp for AK53 were observed under gel documentation system indicating the purified gel extracted mutated xylanase.

4.2.8 Cloning of mutated xylanase gene of strain AK53 and C5 in cloning vector Mutated xylanase W127R, D318R and DM D318R/W127R of strain AK53 and W137R, D328R and DM W137R/D328R of strain C5 xylanase was ligated with the pGEM®-T Easy cloning vector. Then, JM 101 competent cells were used for transformation of ligated product, for confirmation of cloning, blue/white screening technique was used to select the appropriate transformed cells with ligated xylanase gene product. pGEM®-T vector contain the ampicillin resistant gene, 5 white and two blue colonies for strain AK53 xylanase mutation W127R, D318R and D318R/W127R double mutant and strain C5 W137R, D328R and W137R/D328R double mutant were selected and LB-Amp broth was used for cultivation of inserted cell, the plasmids were isolated by minipreparation protocol according to Sambrook and Russell (2001). Confirmation of cloning was carried out by sequencing and digestion with restriction enzyme. Sequencing clarified the cloning of mutated xylanase gene to pGEM®-T vector, and digestion of pGEM®-T cloned plasmid with EcoRI restriction enzyme produce the desired band size of 1230 bp size for strain C5 and 1200 bp size for strain AK53 xylanase along with 3000 bp of vector pGEM®-T confirming the cloning of our desired mutated gene of xylanase from strain AK53 W127R, D318R and D318R/W127R and strain C5 W137R, D328R and W137R/D328R.

4.2.9 Preparation of pET28a and mutated xylanase gene product for expression pET28a vector and pGEM®-T cloned mutated xylanase gene product of strain AK53 W127R, D318R and DM D318R/W127R and strain C5 mutated xylanase W137R, D328R and W137R/D328R was isolated and visualized by gel electrophoresis for

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 164 Chapter 4 Results their confirmation and was digested with NheI enzyme at 37°C for 2 hours and the digestion was confirmed by gel electrophoresis. The digested pET28a showed approximately 5300 bp product size of single band while strain AK53 and C5 showed 4.2 kb and 4.23 kb under gel documentation system, respectively. After that the single enzyme digested pET28a vector and mutated xylanase gene product of strain AK53 W127R, D318R and DM D318R/W127R and strain C5 mutated xylanase W137R, D328R and W137R/D328R was cleaned through ethanol precipitation and the presence of strain AK53 W127R, D318R and DM D318R/W127R and strain C5 mutated xylanase W137R, D328R W137R/D328R and pET28a plasmids were again determined by 1% agarose gel electrophoresis.

4.2.10 Double digestion and purification of pET28a vector and mutated xylanase gene product Ethanol precipitated cleaned digested pET28a vector and mutated xylanase gene product of strain AK53 W127R, D318R and DM W127R/D318R and strain C5 mutated xylanase gene product W137R, D328R and W137R/D328R was subjected to second digestion with HindIII Takara restriction enzyme for 2 hours at 37°C. Restriction enzyme digestion was checked through gel electrophoresis, which was confirmed by their size which was 5300 bp for pET28a, 1.23 kb size for strain C5 W137R, D328R and W137R/D328R and 1.2 kb size for strain AK53 W127R, D318R and DM W127R/D318R. Strain C5 W137R, D328R and W137R/D328R and AK53 W127R, D318R and DM D318R/W127R double digested product also released pGEMT vector of about 3000 bp. The double digested mutated xylanase gene product and pET28a vector was isolated from gel by GeneJET Gel Extraction Kit Thermo Scientific as manufacturer protocol. The gel excised purified insert of C5 W137R, D328R, W137R/D328R, AK53 W127R, D318R and DM W127R/D318R and pET28a plasmids were confirmed through their respective size in agarose gel electrophoresis and was used as insert for ligation.

4.2.11 Cloning of mutated xylanase gene in expression vector pET28a Xylanase of G. thermodenitrificans C5 having W137R, D328R W137R/D328R mutation and G. thermodenitrificans AK53 having W127R, D318R and W127R/D318R mutation was used for recombinant expression in E. coli. G.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 165 Chapter 4 Results thermodenitrificans C5 has W137R, D328R and W137R/D328R mutation and G. thermodenitrificans AK53 having W127R, D318R and DM W127R/D318R mutated gene was ligated with the pET28a expression vector. Then, E. coli JM 101 competent cells were used for transformation of ligated product, pET28a vector contain the kanamycin resistant gene, 5 white and 1 blue colonies were selected and LB-kan broth was used for cultivation of inserted cell, after transformation plasmid was isolated from single colonies and transformation was confirmed by sequencing and digestion of plasmid with appropriate restriction enzyme. The digested pET28a plasmid showed the desired gene size product of G. thermodenitrificans C5 mutated xylanase fragment and G. thermodenitrificans AK53 mutated xylanase fragment along with the size of pET28a plasmid.

4.2.12 Cloning of mutated xylanase gene in expression vector p15TV- L Vector (derative of pET15b Novagen) Xylanase of G. thermodenitrificans C5 having N84R, D339R, R81P, H82E, W185P, D186E, DM W185P/D186E and triple mutant H82E/W185P/D186E mutation and G. thermodenitrificans AK53 having N74R, D329R, DM R71P/H72E, W175P/D176E and quadruple mutant R71P/H72E/W175P/D176 mutation was used for recombinant expression in E. coli. G. thermodenitrificans C5 has N84R, D339R, R81P, H82E, W185P, D186E, DM W185P/D186E and triple mutant H82E/W185P/D186E mutation and G. thermodenitrificans AK53 having N74R, D329R, DM R71P/H72E, W175P/D176E and quadruple mutant R71P/H72E/ W175P/D176 mutations were ligated with the p15TV-L expression vector. Then, E. coli JM101 competent cells were used for transformation of ligated product, p15TV-L vector contain the ampicillin resistant gene, LB-Amp broth was used for cultivation of inserted cell, after transformation plasmid was isolated from single colonies and transformation was confirmed by PCR amplification and sequencing.

4.2.13 Induction and over production of recombinant mutated xylanase enzyme Induction of recombinant mutated xylanase enzyme of G. thermodenitrificans C5 and AK53 was performed through 1 mm IPTG and expression was observed by SDS-

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 166 Chapter 4 Results

PAGE analysis and enzyme activity assay. SDS PAGE showed clearly expressed band of mutated xylanase enzyme from strain AK53 and C5.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 167 Chapter 4 Results

Fig 4.2.15; Blue white screening of strain AK53 and C5 mutant xylanase

M 1 2 3 4 M 5 6

Fig 4.2.16; Gel electrophoresis of pGEMT cloned mutated xylanase plasmid M; 1kb DNA ladder, 1; C5 W137R, 2; C5 D328R, 3; AK53 W127R, 5; AK53 D318R, 5; C5 W137R/D328R, 6; AK53 D318R/W127R

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 168 Chapter 4 Results

M 1 M 2 3 M 4 5 M 6

3 kb 3 kb

1.2 kb 1.2 kb

Fig 4.2.17; Digestion of pGEM®-T Vector cloned mutant xylanase with EcoRI M; 2 log marker 1; C5 W137R, 2; C5 D328R, 3; AK53 W127R, 5; AK53 D318R, 5; C5 W137R/D328R, 6; AK53 D318R/W127R

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 169 Chapter 4 Results

M 1 2 3 M 4 5 M 6 M 7

Digested pET28a Digested pGEM®-T plasmid plasmid

Fig 4.2.18; Gel electrophoresis of digested pGEMT cloned mutated plasmid and pET28a plasmid M 2log marker,1; C5 W137R, 2; C5 D328R, 3; AK53 W127R, 5; AK53 D318R, 5; C5 W137R/D328R, 6; AK53 W127R/ D318R, 7; pET28a plasmid. C5 W137R, 2; C5 D328R, 3; C5 W137R/D328R, 4; AK53 W127R, 5; AK53 D318R, 6; AK53 W127R/D318R, 7; pET28a plasmid

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 170 Chapter 4 Results

1 M 2 3 M 4 M 5 M 6 M 7

5.3 kb 3 kb 1.2 kb

Fig 4.2.19; Gel electrophoresis of double digestion of pGEM®-T cloned mutant plasmid and pET28a plasmid with Nhe1 and Hind111 M, 2log marker, 1; C5 W137R, 2; C5 D328R, 3; C5 W137R/D328R, 4; AK53 W127R, 5; AK53 D318R, 6; AK53 W127R/D318R, 7; pET28a plasmid.

1 2 3 4 M 5

Fig 4.2.20; Gel electrophoresis of p15TV-L plasmid digested and un digested plasmid M; 2 log marker, 1, 2, 3, 4; undigested p15TV-L plasmid, 5; BseRI digested p15TV-L plasmid

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 171 Chapter 4 Results

4.2.14 Purification of recombinant enzyme 4.2.14.1 Precipitation with ammonium sulphate 60% ammonium sulphate concentration was used for precipitation of W137R, D328R and W137R/D328R of strain C5 and W127R, D318R and W127R/D318R of strain AK53 mutant lyzed xylanase. Precipitates were dissolved in 100mM sodium phosphate buffer and were dialyzed against the same buffer for 24 hours.

4.2.14.2 DEAE Sepharose chromatography After treatment with ammonium sulphate and dialysis tube, the precipitated dialyzed preparation was fractionated on DEAE Sepharose anion exchange chromatography eluted by 0.9 M NaCl concentration along with 100mm sodium sulphate buffer. 60 fractions of about 3 mL each were collected, each fraction was assayed for xylanolytic activity and its optical density (OD) was taken at 280 nm for the protein content, one prominent peak was observed for both strain C5 and AK53 recombinant xylanase, DEAE Sepharose chromatography was used to remove the impurities present in the crude enzyme extract, effectively.

Prominent peak was achieved at 25, 25 and 19 fractions for W137R (Fig 4.2.21), D328R (Fig 4.2.22), and W137R/D328R (Fig 4.2.23) of G. thermodenitrificans C5 xylanases respectively. While for G. thermodenitrificans AK53 maximum and prominent peak was achieved at 25, 19 and 23 fractions for W127R (Fig 4.2.27), D318R (Fig 4.2.28), and W127R/D318R (Fig 4.2.29) mutated xylanase respectively.

4.2.14.3 Phenyl Sepharose chromatography Geobacillus thermodenitrificans C5 maximum and prominent peak was achieved at 23, 27 and 21 fraction for W137R (Fig 4.2.24), D328R (Fig 4.2.25), and W137R- D328R (Fig 4.2.26), mutated xylanase respectively. For Geobacillus thermodenitrificans AK53 mutated xylanases maximum and prominent peak was achieved at 23, 23 and 25 fraction for W127R (Fig 4.2.30), D318R (Fig 4.2.31), and W127R/D318R (Fig 4.2.32).

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 172 Chapter 4 Results

4.2.14.4 Purification through His60 Ni Superflow resin p15TV-L plasmid containing mutant xylanase from AK53 and C5 were purified with His60 Ni Superflow Resin column. p15TV-L vector contained N terminal 6X His followed by TEV cleavage site. Recombinant mutant xylanase containing His were binded to the column and was eluted with elution buffer.

4.2.14.5 SDS-PAGE for mutant proteins Analytical electrophoresis of proteins was carried out by SDS-PAGE as described by Laemmli (1970). SDSPAGE revealed a clear single band of recombinant mutant xylanase of strain C5 (Fig 4.2.33; 4.2.34) and AK53 (Fig 4.2.35; 4.2.36) indicating the purity of xylanase.

The specific activity of purified xylanase of G. thermodenitrificans AK53 W127R, D318R, W127R/D318R was observed as 967 IU/mg, 916 IU/mg and 893 IU/mg respectively, the yield was 264%, 287% and 251% for Geobacillus thermodenitrificans AK53 mutated W127R, D318R, W127R/D318R respectively and an overall purification fold of 18.24, 19.91 and 17.17 for W127R, D318R, W127R- D318R mutated G. thermodenitrificans AK53 xylanase. The specific activity of purified xylanase of G. thermodenitrificans C5 W137R, D328R and W137R/D328R was observed as 1021 IU/mg, 976 IU/mg and 928 IU/mg respectively, the yield was 148%, 176% and 190% for G. thermodenitrificans C5 W137R, D328R and W137R/D328R respectively and an overall purification fold of 7.55, 10.18 and 6.78 for C5 W137R, D328R and W137R/D328R mutated G. thermodenitrificans C5 xylanase.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 173 Chapter 4 Results

DEAE sepharose C5 W137R

800 0.2 Activity

700 0.D 600 0.15 280nm 500 400 0.1 300 Activity (U/mL) Activity 200 0.05

100 nm 280 Absorbance at 0 0 1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 Number of fraction

Fig. 4.2.21; DEAE-Sepharose Ion-exchange chromatography of Geobacillus thermodenitrificans C5 W137R

700 DEAE sepharose C5 D328R 0.3

600 0.25

500 0.2 400 0.15 300

0.1 200 (U/mL) Activity

100 0.05 nm 280 Absorbance at

0 0 1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 Number of fraction Activity O.D at 280 nm

Fig. 4.2.22; DEAE-Sepharose Ion-exchange chromatography of Geobacillus thermodenitrificans C5 D328R

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 174 Chapter 4 Results

700 DEAE sepharose C5 W137R/D328R 0.25

600 0.2 500

400 0.15

300 0.1

Activity (U/mL) Activity 200

0.05 nm 280 Absorbance at 100 Activity 0 0 1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 O.D at 280 nm Number of fraction

Fig. 4.2.23; DEAE-Sepharose Ion-exchange chromatography of Geobacillus thermodenitrificans C5 DM W137R/D328R

Phenyl sepharose C5 W137R Activity OD at 280 nm

700 0.3

600 0.25 500 0.2 400 0.15 300 200 0.1 Activity (U/mL) Activity 100 0.05 0 0 nm 280 Absorbance at 1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 Number of fraction

Fig. 4.2.24; Phenyl-Sepharose Ion-exchange chromatography of Geobacillus thermodenitrificans C5 W137R

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 175 Chapter 4 Results

Phenyl sepharose C5 D328R 700 0.25

600

0.2 500 400 0.15

300 0.1 200 Activity (U/mL) Activity 0.05 100 nm 280 Absorbance at 0 0 1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 Activity Number of fraction O.D at 280 nm

Fig. 4.2.25; Phenyl-Sepharose Ion-exchange chromatography of Geobacillus thermodenitrificans C5 D328R

Phenyl sepharose C5 W137R/D328R

800 0.3

700 0.25

600 500 0.2

400 0.15

300 0.1

Activity (U/mL) Activity 200 100 0.05 nm 280 Absorbance at 0 0 1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 Activity

Number of fraction O.D at 280 nm

Fig. 4.2.26; Phenyl-Sepharose Ion-exchange chromatography of Geobacillus thermodenitrificans C5 DM W137R/D328R

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 176 Chapter 4 Results

DEAE sepharose AK53 W127R Activity 800 0.3

O.D at 280 nm 700 0.25 600

500 0.2 400 0.15

300 0.1

Activity (U/mL) Activity 200 0.05 100 nm 280 Absorbance at 0 0 1 5 9 131721252933374145495357 Number of fraction

Fig. 4.2.27; DEAE-Sepharose Ion-exchange chromatography of Geobacillus thermodenitrificans AK53 W127R

Activity DEAE sepharose AK53 D318R O.D at 280 nm 700 0.3

600 0.25

500 0.2 400 0.15 300 0.1

200 Activity (U/mL) Activity

100 0.05 Absorbance nm 280 Absorbance at 0 0 1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 Number of fraction

Fig. 4.2.28; DEAE-Sepharose Ion-exchange chromatography of Geobacillus thermodenitrificans AK53 D318R

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 177 Chapter 4 Results

800 DEAE sepharose AK53 W127R/D318R 0.35 700 Activity 0.3

600 O.D at 280 nm 0.25 500 0.2 400 0.15 300

Activity (µ/mL) Activity 0.1 200

100 0.05 nm 280 Absorbance at 0 0 1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 Number of fraction

Fig. 4.2.29; DEAE-Sepharose Ion-exchange chromatography of Geobacillus thermodenitrificans AK53 DM W127R/D318R

Activity Phenyl sepharose AK53 W127R O.D at 280 nm

800 0.3

700 0.25 600 500 0.2 400 0.15 300 0.1

Activity (U/mL) Activity 200 Absorbance nm 280 Absorbance at 100 0.05 0 0 1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 Number of fraction

Fig. 4.2.30; Phenyl-Sepharose Ion-exchange chromatography of Geobacillus thermodenitrificans AK53 W127R

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 178 Chapter 4 Results

800 Phenyl sepharose AK53 D318R 0.3 Activity 700 0.25

600 O.D at 280 nm 0.2 500

400 0.15

300

Activity (U/mL) Activity 0.1

200 Absorbance nm 280 Absorbance at 0.05 100

0 0 1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 Number of fraction

Fig. 4.2.31; Phenyl-Sepharose Ion-exchange chromatography of Geobacillus thermodenitrificans AK53 D318R

800 Phenyl sepharose AK53 W127R/D318R 0.3 700

0.25

600 0.2 500 400 0.15 300 0.1 Activity (U/mL) Activity 200 0.05

100 nm 280 at Absorbance 0 0 1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 Activity Number of fraction O.D at 280 nm

Fig. 4.2.32; Phenyl-Sepharose Ion-exchange chromatography of Geobacillus thermodenitrificans AK53 DM W127R/D318R

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1 2 3 4 M 5 6 7 8 9 10 11 12 M

Fig. 4.2.33; SDS-PAGE showing purified recombinant C5 mutated xylanase enzyme obtained from ion-exchange column chromatography by DEAE Sepharose and Phenyl sepharose. M, SDS-PAGE molecular mass standards 10–250 kDa New England biolab 4,5,12, Induced supernatant; 3,6,11, ammonium sulfate precipitation; 2,7,10 DEAE sepharose fractions of W137R, D328R and W137R/D328R respectively 1, 8.9, Purified Phenyl sepharose fraction of W137R, D328R and W137R/D328R respectively

1 2 3 4 5 6 7 8 9

Fig. 4.2.34; SDS-PAGE showing purified recombinant C5 mutated xylanase enzyme obtained from Ni column 1; R81P, 2; H82E, 3; 4; W185P, 5; D186E, 6; DM W185P/ D186E, 7; H82E/ W185P/ D186E, 8; N84R, 9; D339R

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 180 Chapter 4 Results

1 2 3 4 M 5 6 7 8 9 10 11 12 M

Fig. 4.2.35; SDS-PAGE showing purified recombinant AK53 mutated xylanase enzyme obtained from ion-exchange column chromatography by DEAE Sepharose and phenyl Sepharose. M, SDS-PAGE molecular mass standards 10–250 kDa New England Biolab; 4,5,12, Induced supernatant; 3,6,11, ammonium sulfate precipitation; 2,7,10 DEAE Sepharose fraction of W127R, D318R and W127R/D318R respectively 1,8.9, Purified Phenyl Sepharose fraction of W127R, D318R and W127R/D318R respectively.

1 M 2 3 4 5

Fig. 4.2.36; SDS-PAGE showing purified recombinant AK53mutated xylanase enzyme obtained from Ni column 1; N74R, 2; D329R, 3; DM R71P/H72E, 4; W175P/ D176E, 5; R71P/H72E/ W175P/D176E

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4.2.15 Biochemical Characterization and stability studies of purified mutant AK53 and C5 enzyme 4.2.15.1 Effect of temperature on purified C5 mutant xylanase activity Temperature effect was analyzed by incubating C5 mutant xylanase with xylan substrate from 40°C to 100°C and the residual activity was calculated. The purified C5 mutant xylanases exhibits activity over a broad range of temperature (40–80°C), while optimum temperature turned out to be 65°C for W137R, 63°C for D328R, 67°C for DM W137R/D328R, 62°C for D339R and 63°C for N84R, which were 5°C, 3°C, 7°C, 2°C and 3°C higher than the wild type C5 xylanase (60°C) respectively. Optimum temperature for R81P, H82E, W185P, D186E, DM W185P/D186E and triple mutant H82E/W185P/D186E were 57°C, 64°C, 63°C, 62°C, 65°C and 71°C respectively (Fig 4.2.37).

4.2.15.2 Effect of temperature on purified AK53 mutant xylanases’ activity The purified AK53 mutant xylanases optimum temperature was 73°C, 72°C, 75°C, 70°C and 70°C for W127R, D318R, double mutant W127R/D318R, D329R and N74R respectively. The temperature range was 3°C, 2°C, and 5°C higher than that of wild type AK53 xylanase (70°C), respectively. N74R did not change the temperature range of AK53 xylanase. DM R71P/H72E, W175P/D176E and quadruple mutant R71P/H72E/W175P/D176E had optimum temperature of 74°C, 75°C and 78°C respectively, 4 to 8°C higher than that of wild type G. thermodenitrificans AK53 (Fig. 4.2.38).

4.2.15.3 Effect of pH on purified C5 mutant xylanases’activity pH effect on activity of mutants C5 xylanase were analyzed with xylan substrate by incubating from pH 3.0-11.0 and the residual activity was calculated. The pH activity profile of most of the mutants was very similar to the wild type C5 xylanase and a little variation in term of pH was observed with mutation. The purified C5 mutant xylanases had optimum pH of 8.0, 6.5, 8.0, 6.0 and 6.0 for W137R, D328R, W137R/D328R, N84R and D339R, respectively. Optimum pH turned out to be 6.5, 6.0, 6.0, 6.0, 7.0 and 7.0 for R81P, H82E, W185P, D186E, DM W185P/ D186E and triple mutant H82E/W185P/D186E, respectively (Fig. 4.2.39).

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 182 Chapter 4 Results

4.2.15.4 Effect of pH on purified AK53 mutant xylanases’ activity The pH activity profile of most of the mutants was very similar to the wild type AK53 xylanase. The purified AK53 mutant xylanases had optimum pH of 6.0, 5.5, 6.0, 6.0 and 5.0 for W127R, D318R, W127R/D318R, N74R and D329R, respectively. DM R71P/H72E, W175P/D176E and quadruple mutant R71P/H72E/W175P/D176E had optimum pH of 5.5, 6.0 and 6.0, respectively (Fig. 4.2.40).

4.2.15.5 Stability of purified C5 mutant xylanase at different temperatures Stability of purified mutant of GthC5Xyl was analyzed against substrate xylan by incubating at wide temperature ranges from 40-100°C for 200 min and the residual activity was calculated. The recombinant mutant enzyme W137R retained more than 80% activity after exposure to 60°C, 65°C and 70°C for 200 min (Fig. 4.2.41). The recombinant mutant enzyme D328R retained more than 80% activity after exposure to 50°C 60°C, 65°C, and 70°C (Fig. 4.2.42). Double mutant of W137R and D328R retained more than 85% activity at 50°C, 60°C and 65°C while 80% and 75% at 70°C and 75°C, respectively (Fig. 4.2.43). More than 70% activity is retained by N84R (Fig. 4.2.44) and D339R mutant (Fig. 4.2.45) at 50°C, 60°C and 70°C for 200 min. R81P stability was decreased as compared to wild type, while H82E retained more than 80% activity at 60°C and 70°C for 200 min (Fig. 4.2.47). More than 80% activity is retained by W185P (Fig. 4.2.48), more than 75% by D186E (Fig. 4.2.49), and more than 80% by the double mutant DM W185P/D186E at 60°C and 70°C for 200 min (Fig. 4.2.50). More than 85% activity is retained by triple mutant H82E/W185P/D186E at 60°C and 70°C for 200 min (Fig. 4.2.51).

4.2.15.6 Stability of purified AK53 mutant xylanase at different temperatures Stability of purified mutant of GthAK53Xyl at different temperatures was analyzed with xylan substrate by incubating from 50-90°C for 200 min and the residual activity was calculated. The recombinant mutant enzyme W127R and D318R retained more than 80% activity after exposure to 60°C, 70°C and 80°C for 200 min (Fig. 4.2.52; 4.2.53). The double mutant W127R/D318R retained more than 90% activity at 70°C and 80°C and more than 80% at 50°C, 60°C, 65°C and 85°C for 200 min (Fig. 4.2.54). D329R and N74R mutant retained more than 70% activity for 200 min from 50-80°C (Fig. 4.2.56; 4.2.55). Double mutant of R71P/H72E maintained more than

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 183 Chapter 4 Results

80% activity at 70°C and 80°C (Fig. 4.2.57). More than 75% activity was retained by the double mutant W175P/D176E from 60-80°C (Fig. 4.2.58). Quadruple mutant R71P/H72E/W175P/D176E maintained more than 80% activity from 60°C to 80°C for 200 min (Fig. 4.2.59).

4.2.15.7 Stability of purified C5 mutant xylanases at different pH pH stability of purified mutants GthC5Xyl was analyzed by incubating with xylan substrate from pH 3.0-11.0 for 200 min and the residual activity was calculated. More than 85% activity was retained for 200 min at pH 6.0-9.0 by W137R mutant (Fig. 4.2.60). More than 70% activity was retained for 200 min at pH 5.0-9.0 by D328R mutant (Fig. 4.2.61). More than 70% activity was retained for 200 min at pH 5.0-10.0 by double mutant W137R/D328R (Fig. 4.2.62). N84R and D339R mutant retained more than 70% activity from pH 5.0-8.0 for 200 min (Fig. 4.2.63; 4.2.64). More than 70% activity was retained by R81P from pH 6.0-9.0 (Fig. 4.2.65). W185P and D186E retained more than 70% activity at pH 6.0-9.0 (Fig. 4.2.67; 4.2.68). Double mutant W185P/D186E maintained more than 80% activity from pH 5.0-8.0 (Fig. 4.2.69). Triple mutant H72E/W175P/D176E maintained more than 75% activity from pH 4.0-9.0 (Fig. 4.2.70).

4.2.15.8 Stability of purified AK53 mutant xylanases at different pH pH stability of purified mutants GthAK53Xyl was analyzed by incubating with xylan substrate from pH 3.0-11.0 for 200 min and the residual activity was calculated. More than 70% activity was retained by W127R, D318R, D329R and N74R mutant from pH 4.0-8.0 (Fig. 4.2.71; 4.2.72; 4.2.75; 4.2.74). More than 70% initial activity was maintained by the double mutant R71P/H72E from pH 4.0-8.0 (Fig. 4.2.76). More than 80% activity was retained by W127R/D318R from pH 4.0-8.0. More than 60% activity was retained by double mutant W175P/D176E from 3.0-8.0 pH (Fig. 4.2.77). More than 70% activity was retained by Quadruple mutant R71P/H72E/W175P/D176E from pH 4.0-7.0 (Fig. 4.2.78).

4.2.15.9 Determination of kinetic parameters of C5 and its mutants The specific activities and kinetic study of wild type C5 and its mutants were determined with oatspelt xylan as the substrate. All mutant of C5 showed decrease in their specific activities.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 184 Chapter 4 Results

−1 −1 −1 The Km and Vmax (for oat spelt xylan) were 3.94 mgmL and 1307 μmolmg min (Fig. 4.2.79), 3.29 mgmL−1 and 1066 μmolmg−1min−1 (Fig. 4.2.80), 3.02 mgmL−1 and 881 μmolmg−1min−1 (Fig. 4.2.81) for W137R, D328R and W137R/D328R mutant of −1 C5 xylanase, respectively. The Km of N84R and D339R was 4.1 mgmL (Fig. 4.2.82) −1 and 3.26 mgmL (Fig. 4.2.83), respectively while Vmax turned out to be 682 μmolmg−1min−1 and 923 μmolmg−1min−1 for N84R and D339R, respectively. −1 −1 −1 The Km and Vmax was 3.66 mgmL and 1199.98 μmolmg min for R81P (Fig. 4.2.84), 5.13 mgmL−1 and 946.44 μmolmg−1min−1 for H82E (Fig. 4.2.85), 6.5 mgmL−1 and 746 μmolmg−1min−1 for W185P (Fig. 4.2.86), 4.78 mgmL−1 and 889.11 μmolmg−1min−1 for D186E (Fig. 4.2.87), 5.76 mgmL−1 and 386 μmolmg−1min−1 for W185P/D186E (Fig. 4.2.88), and 5.83 mgmL−1 and 754.24 μmolmg−1min−1 for triple mutant H82E/W185P/D186E (Fig. 4.2.89).

4.2.15.10 Determination of kinetic parameters of AK53 mutants −1 −1 −1 The Km and Vmax were 3.94 mgmL and 1589 μmolmg min for W127R (Fig. 4.2.90), 3.54 mgmL−1 and 1464 μmolmg−1min−1 for D318R (Fig. 4.2.91), 3.33 mgmL−1 and 1379 μmolmg−1min−1 for W127R/D318R (Fig 4.2.92), 5.35 mgmL−1 and 806 μmolmg−1min−1 for N74R (Fig. 4.2.93), 3.61 mgmL−1 and 584 μmolmg−1min−1 for D329R (Fig. 4.2.94), 6.08 mgmL−1 and 886 μmolmg−1min−1 for R71P/H72E (Fig. 4.2.95), 4.09 mgmL−1 and 642 μmolmg−1min−1 for W175P/D176E (Fig. 4.2.96), and 5.19 mgmL−1 and 836 μmolmg−1min−1 for Quadruple mutant R71P/H72E/W175P/D176E (Fig. 4.2.97).

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 185 Chapter 4 Results

73 71 69

67 C) ° 65 63 61

59 Temperature ( 57 55

Mutant Xylanases

Fig 4.2.37; Effect of temperature on the activity of purified recombinant mutant C5 xylanases

79

77

C) 75 ° 73 71 69

67 Temperature ( 65

Mutant xylanases

Fig 4.2.38; Effect of temperature on the activity of purified recombinant mutant AK53 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 186 Chapter 4 Results

120 C5 100 W137R

D328R 80 W137R/D328R N84R 60 D339R R81P 40

Relative activity Relative activity (%) H82E 20 W185P D186E 0 W185P/D186E 3 4 5 6 7 8 9 10 H82E/W185P/D186E pH

Fig 4.2.39; Effect of pH on the activity of purified recombinant mutant C5 xylanase

120 AK53 100

W127R

80 D318R

W127R/D318R 60 N74R 40

D329R Relative Relative activity (%) 20 R71P/H72E

0 W175P/ D176E 3 4 5 6 7 8 9 10 11 R71P/H72E/W175P/D176E pH

Fig 4.2.40; Effect of pH on the activity of purified recombinant mutant AK53 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 187 Chapter 4 Results

C5 W137R 120

100 80 50°C 60 60°C 70°C 40

80°C Relative activity (%) activity Relative 20 90°C

0 0 40 80 120 160 200 Incubation time (min)

Fig 4.2.41; Temperature stability of mutant W137R of C5 xylanase

C5 D328R

100 80 50°C 60 60°C 40 70°C

20 80°C Relative activity (%) activity Relative 0 90°C 0 40 80 120 160 200

Incubation time (min)

Fig 4.2.42; Temperature stability of mutant D328R of C5 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 188 Chapter 4 Results

C5 W137R/D328R

100

80 50°C 60°C 60 70°C

40 80°C 90°C

Relative activity (%) activity Relative 20

0 0 40 80 120 160 200 Incubation time (min)

Fig 4.2.43; Temperature stability of mutant W137R/D328R of C5 xylanase

C5 N84R

100

80 50°C

60 60°C 70°C 40 80°C 90°C

20 Relative activity (%) activity Relative

0 0 40 80 120 160 200 Incubation time (min)

Fig 4.2.44; Temperature stability of mutant N84R of C5 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 189 Chapter 4 Results

C5 D339R 50°C 100 60°C

70°C 80 80°C 90°C 60

40

20 (%) activity Relative

0 0 40 80 120 160 200 Incubation time (min)

Fig 4.2.45; Temperature stability of mutant D339R of C5 xylanase

C5 R81P

100

80

60 50°C 60°C 40 70°C Relative activity (%) activity Relative 80°C 20 90°C

0 0 40 80 120 160 200 Incubation time (min)

Fig 4.2.46; Temperature stability of mutant R81P of C5 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 190 Chapter 4 Results

C5 H82E

100

80

60 50°C 40 60°C 70°C 20 Relative activity (%) activity Relative 80°C 0 90°C 0 40 80 120 160 200 Incubation time (min)

Fig 4.2.47; Temperature stability of mutant H82E of C5 xylanase

C5 W185P 100

50°C

80 60°C

60 70°C 40 80°C

20 90°C

Relative activity (%) activity Relative 0 0 50 100 150 200 250 Incubation time (min)

Fig 4.2.48; Temperature stability of mutant W185P of C5 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 191 Chapter 4 Results

C5 D186E

100

80 50°C 60 60°C 70°C 40 80°C

20 90°C Relative activity (%) activity Relative 0 0 40 80 120 160 200 Incubation time (min)

Fig 4.2.49; Temperature stability of mutant D186E of C5 xylanase

C5 W185P/D186E 100 50°C 80 60°C 70°C 60 80°C

40 90°C

20 Relative activity (%) activity Relative 0

0 40 80 120 160 200 Incubation time (min)

Fig 4.2.50; Temperature stability of mutant W185P/D186E of C5 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 192 Chapter 4 Results

C5 H82E/W185P/D186E 100

80

60 50°C 60°C 40 70°C

20 80°C Relative activity (%) activity Relative 90°C 0 0 40 80 120 160 200 Incubation time (min)

Fig 4.2.51; Temperature stability of mutant H82E/W185P/D186E of C5 xylanase

AK53 W127R 120 50ºC

100 60ºC 70ºC 80 80ºC

60 90ºC 40 20

Relative activity (%) activity Relative 0 0 40 80 120 160 200 Incubation time (min)

Fig 4.2.52; Temperature stability of mutant W127R of AK53 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 193 Chapter 4 Results

AK53 D318R 120

100

80 50°C 60 60°C

40 70°C 80°C Relative activity (%) activity Relative 20 90°C 0 0 40 80 120 160 200 Incubation time (min)

Fig 4.2.53; Temperature stability of mutant D318R of AK53 xylanase

AK53 W127R/D318R

120

100

80 50°C 60 60°C 40 70°C

(%) activity Relative 80°C 20 90°C 0

0 40 80 120 160 200 Incubation time (min)

Fig 4.2.54; Temperature stability of mutant W127R/D318R of AK53 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 194 Chapter 4 Results

120 AK53 N74R 50°C 100 60°C

80 70°C

60 80°C 90°C

40

Relative activity (%) activity Relative 20

0 0 40 80 120 160 200 Incubation time (min)

Fig 4.2.55; Temperature stability of mutant N74R of AK53 xylanase

AK53 D329R 120

50°C 100 60°C 80 70°C

60 80°C 90°C 40

Relative activity (%) activity Relative 20 0 0 40 80 120 160 200

Incubation time (min)

Fig 4.2.56; Temperature stability of D329R mutant of AK53 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 195 Chapter 4 Results

AK53 R71P/H72E 120 50°C 60°C 100 70°C 80 80°C

60 90°C

40

Relative activity (%) activity Relative 20

0 0 40 80 120 160 200 Incubation time (min)

Fig 4.2.57; Temperature stability of R71P/H72E mutant of AK53 xylanase

AK53 W175P/D176E

120

100

80 50°C 60 60°C 70°C 40 80°C Relative activity (%) activity Relative 20 90°C

0 0 50 100 150 200 Incubation time (min)

Fig 4.2.58; Temperature stability of W175P/D176E mutant of AK53 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 196 Chapter 4 Results

AK53 R71P/H72E/W175P/D176E 120

80 50°C 60°C

40 70°C 80°C Relative activity (%) activity Relative 0 90°C 0 40 80 120 160 200 Incubation time (min)

Fig 4.2.59; Temperature stability of R71P/H72E/W175P/D176E mutant of AK53 xylanase

C5 W137R 120 pH 3 100 pH 4 80 pH 5 60 pH 6 40 pH 7 20 Relative activity (%) activity Relative pH 8 0 pH 9 0 40 80 120 160 200 Incubation time (min) pH 10

Fig 4.2.60; pH stability of mutant W137R of C5 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 197 Chapter 4 Results

120 C5 D328R 100 pH 3 pH 4 80 pH 5 pH 6 60 pH 7 pH 8 40 pH 9 pH 10

Relative activity (%) activity Relative 20

0 0 50 100 150 200 Incubation time (min)

Fig 4.2.61; pH stability of mutant D328R of C5 xylanase

C5 W137R/D328R

120 pH 4

100 pH 5 pH 6 80 pH 7 60 pH 8 40 pH 9 pH 10

20 Relative activity (%) activity Relative 0 0 50 100 150 200

Incubation time (min)

Fig 4.2.62; pH stability of mutant W137R/D328R of C5 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 198 Chapter 4 Results

C5 N84R pH 3 120 pH 4 100 pH 5 pH 6 80 pH 7 60 pH 8 40 pH 9 pH 10

Relative activity (%) activity Relative 20 0 0 50 100 150 200 Incubation time (min)

Fig 4.2.63; pH stability of mutant N84R of C5 xylanase

120 C5 D339R

pH 3 100 pH 4 80 pH 5

60 pH 6 pH 7 40

pH 8 Relative activity (%) activity Relative 20 pH 9

pH 10 0 0 40 80 120 160 200 Incubation time (min)

Fig 4.2.64; pH stability of mutant D339R of C5 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 199 Chapter 4 Results

C5 R81P

120

100 pH 3 80 pH 4

pH 5 60 pH 6 40

pH 7 Relative activity (%) activity Relative 20 pH 8

pH 9 0 pH 10 0 40 80 120 160 200 Incubation time (min)

Fig 4.2.65; pH stability of mutant R81P of C5 xylanase

H82E 120 pH 3 100 pH 4 80 pH 5 pH 6 60 pH 7 40 pH 8 pH 9 20

Relative activity (%) activity Relative pH 10

0 0 40 80 120 160 200 Incubation time (min)

Fig 4.2.66; pH stability of mutant H82E of C5 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 200 Chapter 4 Results

C5 W185P

100 pH 3 80 pH 4

60 pH 5

40 pH 6 pH 7 20

Relative activity (%) activity Relative pH 8

0 pH 9 0 40 80 120 160 200 pH 10 Incubation time (min)

Fig 4.2.67; pH stability of mutant W185P of C5 xylanase

C5 D186E

100

80 pH 3 60 pH 4 pH 5 40 pH 6 20

Relative activity (%) activity Relative pH 7

0 pH 8 0 40 80 120 160 200 pH 9 Incubation time (min)

Fig 4.2.68; pH stability of mutant D186E of C5 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 201 Chapter 4 Results

C5 W185P/D186E 100 pH 3 80 pH 4 pH 5 60 pH 6 40 pH 7 pH 8

Relative activity (%) activity Relative 20 pH 9 0 pH 10 0 40 80 120 160 200 Incubation time (min)

Fig 4.2.69; pH stability of mutant W185P/D186E of C5 xylanase

C5 H82E/W185P/D186E pH 3 100 pH 4 80 pH 5 60 pH 6 40 pH 7 20 Relative activity (%) activity Relative pH 8 0 0 40 80 120 160 200 Incubation time (min)

Fig 4.2.70; pH stability of mutant H82E/W185P/D186E of C5 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 202 Chapter 4 Results

AK53 W127R

120 100 pH 3

80 pH 4 60 pH 5 40 pH 6

20 pH 7

Relative activity (%) activity Relative 0 pH 8 0 50 100 150 200 250 Incubation time (min) pH 9

Fig 4.2.71; pH stability of mutant W127R of AK53 xylanase

AK53 D318R 120

pH 3 100 pH 4 pH 5 80 60 pH 6 pH 7 40 pH 8

20 Relative activity (%) activity Relative pH 9 0 0 40 80 120 160 200

Incubation time (min)

Fig 4.2.72; pH stability of mutant D318R of AK53 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 203 Chapter 4 Results

AK53 W127R/D318R

120

100 pH 3

80 pH 4 pH 5 60 pH 6 40 pH 7 20 pH 8 (%) activity Relative 0 pH 9 0 40 80 120 160 200 pH 10 Incubation time (min)

Fig 4.2.73; pH stability of mutant W127R/D318R of AK53 xylanase

AK53 N74R

100 pH 3

80 pH 4 60 pH 5

40 pH 6 20 pH 7

Relative activity (%) activity Relative pH 8 0 0 40 80 120 160 200 pH 9 Incubation time (min) pH 10

Fig 4.2.74; pH stability of mutant N74R of AK53 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 204 Chapter 4 Results

AK53 D329R

120 100 pH 3

80 pH 4 60 pH 5 40 pH 6

20 pH 7 Relative activity (%) activity Relative 0 pH 8 0 40 80 120 160 200 pH 9 Incubation time (min)

Fig 4.2.75; pH stability of mutant D329R of AK53 xylanase

AK53 R71P/H72E

120 pH 3 100 pH 4 80 pH 5 60 pH 6 40 pH 7 20 Relative activity (%) activity Relative 0 pH 8

0 40 80 120 160 200 pH 9 Incubation time (min) pH 10

Fig 4.2.76; pH stability of mutant R71P/H72E of AK53 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 205 Chapter 4 Results

AK53 W175P/D176E

120 pH 3

pH 4 100 pH 5 80 pH 6 60 pH 7 40 pH 8

pH 9

Relative activity (%) activity Relative 20 pH 10 0 0 40 80 120 160 200 Incubation time (min)

Fig 4.2.77; pH stability of mutant W175P/D176E of AK53 xylanase

AK53 R71P/H72E/W175P/D176E 120 pH 3

100 pH 4 pH 5 80 pH 6 60 pH 7 40 pH 8

Relative activity (%) activity Relative 20 pH 9

pH 10 0 0 40 80 120 160 200 Incubation time (min)

Fig 4.2.78; pH stability of mutant R71P/H72E/W175P/D176E of AK53 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 206 Chapter 4 Results

C5 W137R 0.006

0.005 y = 0.0021x + 0.0009 R² = 0.9918 0.004

1/V 0.003 0.002

0.001

0 -1 -0.5 0 0.5 1 1.5 2 2.5 1/S

Fig 4.2.79; Kinetic parameters of mutant W137R of C5 xylanase

C5 D328R 0.007 y = 0.0027x + 0.001 R² = 0.9984 0.006 0.005

0.004 1/V 0.003 0.002

0.001 0 -0.5-0.001 0 0.5 1 1.5 2 2.5 1/S

Fig 4.2.80; Kinetic parameters of mutant D328R of C5 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 207 Chapter 4 Results

0.01 C5 W137R/D328R

0.008 y = 0.0032x + 0.0012 R² = 0.9938 0.006

0.004 1/V 0.002

0 -1 0 1 2 3 -0.002 1/S

Fig 4.2.81; Kinetic parameters of mutant W137R/D328R of C5 xylanase

C5 N84R

0.01 y = 0.0038x + 0.0018 R² = 0.9888

0.008

1/V 0.006

0.004

0.002

0 -1 -0.5 0 0.5 1 1.5 2 2.5 -0.002 1/S

Fig 4.2.82; Kinetic parameters of mutant N84R of C5 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 208 Chapter 4 Results

C5 D339R

0.01 y = 0.0033x + 0.0011 R² = 0.9968 0.008

0.006

1/V 0.004

0.002

0 -0.5 0 0.5 1 1.5 2 2.5 1/S

Fig 4.2.83; Kinetic parameters of mutant D339R of C5 xylanase

0.008 C5 R81P 0.007 y = 0.0029x + 0.0009 0.006 R² = 0.996 0.005

0.004

1/V 0.003 0.002 0.001

0 -0.5 0 0.5 1 1.5 2 2.5 -0.001 1/S

Fig 4.2.84; Kinetic parameters of mutant R81P of C5 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 209 Chapter 4 Results

C5 H82E 0.02 y = 0.0082x + 0.0007 R² = 0.9951 0.016

0.012

0.008 1/V 0.004

0 -0.5 0 0.5 1 1.5 2 2.5 -0.004 1/S

Fig 4.2.85; Kinetic parameters of mutant H82E of C5 xylanase

C5 W185P

0.035 y = 0.0157x + 0.0006 0.03 R² = 0.994 0.025 0.02

0.015 1/V 0.01 0.005

0 -0.3 0 0.3 0.6 0.9 1.2 1.5 1.8 2.1 2.4 -0.005 1/S

Fig 4.2.86; Kinetic parameters of mutant W185P of C5 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 210 Chapter 4 Results

C5 D186E 0.016

0.014 y = 0.0071x + 0.0009 R² = 0.9953 0.012 0.01

0.008 1/V 0.006 0.004 0.002

0 -0.2 0.3 0.8 1.3 1.8 2.3 1/S

Fig 4.2.87; Kinetic parameters of mutant D186E of C5 xylanase

C5 W185P/D186E 0.05 y = 0.0188x + 0.002 0.04 R² = 0.9972

0.03

1/V 0.02 0.01 0 -0.25 0.25 0.75 1.25 1.75 2.25

1/S

Fig 4.2.88; Kinetic parameters of mutant W185P/D186E of C5 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 211 Chapter 4 Results

C5 H82E/W185P/D186E 0.025 y = 0.0095x + 0.0011 R² = 0.9985 0.02

0.015

1/V 0.01

0.005

0 -0.5 0 0.5 1 1.5 2 2.5 1/S

Fig 4.2.89; Kinetic parameters of mutant H82E/W185P/D186E of C5 xylanase

AK53 W127R

0.005

0.004 y = 0.0016x + 0.0007 R² = 0.9911

0.003

0.002 1/V 0.001

0 -1 -0.5 0 0.5 1 1.5 2 2.5 -0.001 1/S

Fig 4.2.90; Kinetic parameters of mutant W127R of AK53 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 212 Chapter 4 Results

AK53 D318R

0.005 y = 0.0018x + 0.0007 R² = 0.9931 0.004

0.003

1/V 0.002

0.001

0 -1 -0.5 0 0.5 1 1.5 2 2.5 1/S

Fig 4.2.91; Kinetic parameters of mutant D318R of AK53 xylanase

AK53 W127R/D318R

0.0045

0.004 y = 0.0016x + 0.0008 R² = 0.988 0.0035 0.003

0.0025 1/V 0.002 0.0015 0.001 0.0005

0 -1 -0.5 0 0.5 1 1.5 2 2.5 1/S

Fig 4.2.92; Kinetic parameters of mutant W127R/D318R of AK53 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 213 Chapter 4 Results

AK53 N74R 0.018 y = 0.0071x + 0.0013 0.016 R² = 0.9948

0.014 0.012 0.01

0.008 1/V 0.006 0.004

0.002 0 -0.5 -0.002 0 0.5 1 1.5 2 2.5 1/S

Fig 4.2.93; Kinetic parameters of mutant N74R of AK53 xylanase

AK53 D329R 0.012

0.01 y = 0.0046x + 0.002 0.008 R² = 0.9943

0.006

1/V 0.004 0.002 0

-0.5-0.002 0 0.5 1 1.5 2 2.5 1/S

Fig 4.2.94; Kinetic parameters of mutant D329R of AK53 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 214 Chapter 4 Results

AK53 R71P/H72E

0.02 y = 0.0084x + 0.001 R² = 0.9981 0.015

0.01

1/V 0.005

0 -0.5 0 0.5 1 1.5 2 2.5

-0.005 1/S

Fig 4.2.95; Kinetic parameters of mutant R71P/H72E of AK53 xylanase

AK53 W175P/D176E 0.014 y = 0.0058x + 0.0016 R² = 0.9991 0.012

0.01

0.008

0.006 1/V 0.004

0.002 0 -0.5 0 0.5 1 1.5 2 2.5 -0.002 1/S

Fig 4.2.96; Kinetic parameters of mutant W175P/D176E of AK53 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 215 Chapter 4 Results

AK53 R71P/H72E/W175P/D176E 0.02 y = 0.0073x + 0.0011 R² = 0.9991 0.016

0.012

0.008 1/V

0.004

0 -0.5 0 0.5 1 1.5 2 2.5 -0.004 1/S

Fig 4.2.97; Kinetic parameters of mutant R71P/H72E/W175P/D176E of AK53 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 216 Chapter 4 Results

4.2.16.1 Half-life of C5 mutant and C5 wild type xylanase Half-life of C5 W137R was increased from 18 to 44 min at 60°C, from 14 to 29 min at 65°C, from 8 to 17 min at 70°C and 6 to 8 min at 75°C. Half-life of C5 D328R was increased from 18 to 39 min at 60°C, from 14 to 34 min at 65°C, from 8 to 19 min at 70°C and 6 to 8 min at 75°C. Half-life of C5 W137R/ D328R was increased from 18 to 91 min at 60°C, from 14 to 70 min at 65°C, from 8 to 20 min at 70°C and 6 to 11 min at 75°C. Half-life of C5 N84R was increased from 18 to 27 min at 60°C, from 14 to 19 min at 65°C, from 8 to 12 min at 70°C and 6 to 8 min at 75°C. Half-life of C5 D339R and R81P was decreased at 65, 70 and 75°C. Half-life of C5 H82E was increased from 18 to 39 min at 60°C, from 14 to 34 min at 65°C, from 8 to 16 min at 70°C and 6 to 8 min at 75°C. Half-life of C5 W185P was increased from 18 to 21 min at 60°C, from 14 to 17 min at 65°C, from 8 to 15 min at 70°C. Half-life of C5 D186E was increased from 18 to 25 min at 60°C, from 14 to 20 min at 65°C, from 8 to 15 min at 70°C. Half-life of C5 W185P/D186E was increased from 18 to 29 min at 60°C, from 14 to 23 min at 65°C, from 8 to 15 min at 70°C and 6 to 8 min at 75°C. Half-life of C5 H82E/W185P/D186E was increased from 18 to 240 min at 60°C, from 14 to 211 min at 65°C, from 8 to 70 min at 70°C and 6 to 31 min at 75°C.

4.2.16.2 Half-life of AK53 mutant and AK53 wild type xylanase Half-life of AK53 W127R was increased from 22 to 40 min at 60°C, from 19 to 29 min at 65°C, from 11 to 16 min at 70°C and 9 to 11 min at 75°C. Half-life of AK53 D318R was increased from 22 to 36 min at 60°C, from 19 to 26 min at 65°C, from 11 to 12 min at 70°C and 9 to 10 min at 75°C. Half-life of AK53 W127R/D318R was increased from 22 to 70 min at 60°C, from 19 to 55 min at 65°C, from 11 to 23 min at 70°C and 9 to 13 min at 75°C. Half-life of AK53 N74R and D329R was decreased at high temperature. Half-life of AK53 R71P/H72E was increased from 22 to 97 min at 60°C, from 19 to 91 min at 65°C, from 11 to 31 min at 70°C and 9 to 14 min at 75°C. Half-life of AK53 W175P/D176E was increased from 22 to 33 min at 60°C, from 19 to 22 min at 65°C, from 11 to 13 min at 70°C and 9 to 10 min at 75°C. Half-life of AK53 R71P/H72E/W175P/D176E was increased from 22 to 245 min at 60°C, from 19 to 223 min at 65°C, from 11 to 83 min at 70°C and 9 to 33 min at 75°C.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 217 Chapter 4 Results

Table 4.2.3 Half-life of AK53 and their mutant

Xylanase t1/2 (min) 60°C 65°C 70°C 75°C

AK53 22 19 11 9 W127R 40 29 16 11 D318R 36 26 12 10 W127R/ D318R 70 55 23 13 N74R 19 15 7 3 D329R 19 15 7 3 R71P/H72E 97 91 31 14 W175P/D176E 33 22 13 10 R71P/H72E/W175P/D 245 223 83 33 176E

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 218 Chapter 4 Results

Table 4.2.4 Half-life of C5 and their mutants

Xylanase t1/2 (min) 60°C 65°C 70°C 75°C

C5 18 14 8 6 W137R 44 29 17 8 D328R 39 34 19 8 W137R/ D328R 91 70 20 11 N84R 27 19 12 8 D339R 19 12 6 3 R81P 18 13 6 4 H82E 39 34 16 8 W185P 21 17 15 6 D186E 25 20 15 6 W185P/D186E 29 23 15 8 H82E/W185P/D186E 240 211 70 31

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 219 Chapter 4 Results

4.2.17.1 Hydrogen Bonding Pattern in C5 Mutants Loss of backbone hydrogen bond in the case of mutant R81P can be seen in the figure 4.2.100 that the side chain adaptation blocks the existing hydrogen bond. Bifurcated hydrogen bond formed by the oxygen of the E82 (Fig. 4.2.101) and increase in hydrogen bonding residue could suggest the further stabilizing of the protein by the H82E mutation. There is some slight alteration in the hydrogen bonding of N84R mutant (Fig. 4.2.102). The side chain of D339R mutant lies on the surface of the protein. Proline mutant usually affect the backbone conformation and thus making some conformational changes. D186E mutant side chain elongation helps to extend its hydrogen bond partners. Bifurcated hydrogen bond can also be seen in the E186 (Fig. 4.2.105).

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 220 Chapter 4 Results

Glu101

Arg166

Arg137

Fig 4.2.98; Structural changes in C5 W137R W137Arg substitution initiates a triple ionic interaction within the visualized region of C5 protein. Arg137 is able to create small scale salt bridge between Glu101 and enable us to use Glu101 as joker aa residue in order to establish a small scale ionic interaction between Glu101 –Arg137 and Glu101-Arg166 that are located in the three close lines of the protein structure.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 221 Chapter 4 Results

C terminus

Asp408 Arg328

Fig 4.2.99; Structural changes in C5 D328R D328R substitution of C5 is normally located as close to the C terminus of the protein. It is hypothesized that an ionic interaction between Asp408 and D328R substitution could improve the stability of the protein.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 222 Chapter 4 Results

Fig 4.2.100; Hydrogen bond pattern in C5 R81P model The hydrogen bond pattern (a) in wild type (b) mutant model C5 R81P The picture has been built by VMD

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 223 Chapter 4 Results

Fig 4.2.101; Hydrogen bond pattern in C5 H82E The hydrogen bond pattern (a) in wild type (b) mutant model C5 H82E The picture has been built by VMD. Newly formed hydrogen bonds are encircled.

A, Wild type H82 B, Mutant E82 HIS N ↔ 78 MET O = 2.91 GLU H ↔ 78 MET O = 2.02 HIS ND1 ↔ 51 LEU N = 3.35 GLU O ↔ 52 ASP H = 2.33 HIS NE2 ↔ 45 ALA O = 2.78 GLU O ↔ 52 ASP OD1 = 3.14 HIS O ↔ 52 ASP N = 2.99 GLU OE1 ↔ 78 MET O = 2.26 HIS O ↔ 52 ASP OD1 = 3.20 GLU OE2 ↔ 45 ALA O = 2.93 GLU OE1 ↔ 78 MET SD = 3.61

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Fig 4.2.102; Hydrogen bond pattern in C5 N84R The hydrogen bond pattern (a) in wild type (b) mutant model C5 N84R The picture has been built by VMD

A, Wild type N84 B, Mutant R84 ASN N ↔ 61 ILE O = 3.01 ARG N ↔ 61 ILE O = 2.17 AS N OD1 ↔ 61 ILE N = 2.92 ARG O ↔ 122 ASP H = 2.10 ASN O ↔ 122 ASP N = 2.98 ARG 1HH1 ↔ 59 PHE O = 2.20

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 225 Chapter 4 Results

Fig 4.2.103; Hydrogen bond pattern in C5 W185P The hydrogen bond pattern (a) in wild type (b) mutant model C5 W185P The picture has been built by VMD

A, wild type, W185 B, Mutant P185 TRP N ↔ 228 LYS O = 2.82 PRO N ↔ 228 LYS O = 2.82 TRP O ↔ 230 TYR N = 2.76 PRO O ↔ 230 TYR N = 1.98

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 226 Chapter 4 Results

Wild type

Fig 4.2.104; C5 D186 wild type hydrogen bond pattren ASP N ↔ 125 PHE O = 3.10, ASP OD1 ↔ 124 ARG NH2 = 2.66. ASP OD2 ↔ 124 ARG NH1 = 3.05. ASP O ↔ 1 128 LEU N = 3.0

Mutant C5 186E Fig 4.2.105; Hydrogen bond pattern in C5 D186E mutant The picture has been built by VMD. Newly formed hydrogen bonds are encircled. GLU H ↔ 125 PHE O = 2.23, GLU O ↔ 128 LEU H = 2.14, GLU OE1 ↔ 232 ASN (Asparagine) OD1 = 3.13, GLU OE1 ↔ 126 HIS HD1 = 2.63, GLU OE2 ↔ 124 ARG 2HH2 = 1.92

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4.2.17.2 Hydrogen Bonding Pattern in AK53 Mutants AK53 sequence has little dissimilarity with C5 and the template 1HIZ. According to multiple sequence alignment of AK53 we can see that the there is a deletion of 10 residues in AK53. This deletion slightly alters the orientation of secondary structure. Therefore we observe a slightly different pattern of hydrogen bonding in the mutants as it was observed in the C5. The change in side chain of mutant H72E results in novel hydrogen bonds, adopted by the side chain of E72 (Fig. 4.2.109). The oxygen atom forms a strong hydrogen bond with the backbone oxygen of F49. There was no change of hydrogen bonds for the mutant N74R (Fig. 4.2.110), which could suggest the bulkiness of the sidechain of R and positive charge around the side chain may cause it to adopt some conformation that increased the stability of the enzyme. Proline mutants R71P (Fig. 4.2.108) and W175P (Fig. 4.2.111) might affect the backbone conformation and their effect cannot be depicted by optimization of side chains. They require detailed mechanistic explanation either through crystallography or molecular mechanics. Similarly the mutant residue D329R lies on surface of protein, which suggests that the residue is involved in the interaction with the environment around the enzyme. There was some increase in the hydrogen bond pattern for mutant D176E and H72E. In case of D176E the change in side change resulted in a more bulky side chain, which causes its interaction with other residues in protein. There is bifurcated hydrogen bond by oxygen OE1 of E176 (Fig. 4.2.112).

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 228 Chapter 4 Results

Glu91

Arg156 Arg127

Fig 4.2.106; Structural changes in AK53 W127R W127R replacement overcomes a special task in the 3D structure of AK53, by placing between the two weakly interacting loop and the neighboring alpha helix, it contributes a triple ionic interaction. Asp127Arg substitution is responsible for the creation of a salt bridge at the related position of the protein, which enforces the regional stability.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 229 Chapter 4 Results

C terminus

Arg318

Asp398

Fig 4.2.107; Structural changes in AK53 D318R D318R and 398D visualization of the possible ionic interaction between D318R replacement of recombinant AK53 and neighboring Asp398 residue (the measured distance between is 6.8 A˚).

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 230 Chapter 4 Results

Fig 4.2.108; Hydrogen bond pattern in AK53 R71P The hydrogen bond pattern (a) in wild type (b) mutant model AK53 R71P The picture has been built by VMD

A; 71 ARG O ↔ 50 GLN NE2 = 3.27. Glutamine B; 71 PRO O 50 ↔ GLN HE2 = 2.61 Glutamine

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 231 Chapter 4 Results

Fig 4.2.109; Hydrogen bond pattern in AK53 H72E The hydrogen bond pattern (a) in wild type (b) mutant model AK53 H72E The picture has been built by VMD

A; Wild type HIS N ↔ 68 MET O = 2.78, HIS NE2 ↔ 45 ALA O = 2.76, HIS NE2 ↔ 45 ALA O = 2.76, B; Mutant GLU H ↔ 68 MET O = 1.88GLU O ↔ 52 ASP OD1 = 3.14, GLU O ↔ 52 ASP H = 2.28GLU OE1 ↔ 49 PRO O = 3.07,GLU OE2 ↔ 49 PRO O = 3.47, HIS O ↔ 52 ASP N = 2.96

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 232 Chapter 4 Results

Fig 4.2.110; Hydrogen bond pattern in AK53 N74R The hydrogen bond pattern (a) in wild type (b) mutant model AK53 N74R The picture has been built by VMD

A; Wild type ASN N ↔ 61 ILE O = 2.96, ASN O ↔ 112 ASP N = 2.95, ASN O ↔ 112 ASP OD1 = 2.53 B; Mutant ARG H ↔ 61 ILE O = 2.07, ARG O ↔ 112 ASP H = 2.11, ARG O ↔ 112 ASP OD1 = 2.31

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 233 Chapter 4 Results

Fig 4.2.111; Hydrogen bond pattern in AK53 W175P The hydrogen bond pattern (a) in wild type (b) mutant model AK53 W175P The picture has been built by VMD

A; Wild type TRP N ↔ 218 LYS O = 2.87, TRP NE1 ↔ 172 ILE O = 2.73, TRP O ↔ 220 TYR N = 2.76 B; Mutant PRO N ↔ 218 LYS O = 2.87, PRO O ↔ 220 TYR H = 1.99

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 234 Chapter 4 Results

Fig 4.2.112; Hydrogen bond pattern in AK53 D176E The hydrogen bond pattern (a) in wild type (b) mutant model AK53 D176E The picture has been built by VMD A; Wild type ASP N ↔ 115 PHE O = 3.16, ASP OD1 ↔ 114 ARG NH1 = 3.50, ASP O ↔ 118 LEU N = 2.98 B; Mutant bifurcated hydrogen GLU H ↔ 115 PHE O = 2.29, GLU O ↔ 118 LEU H = 2.12. GLU OE1 ↔ 116 HIS HD1 = 2.60. GLU OE1 ↔ 222 ASN OD1 = 3.27, GLU OE2 ↔ 114 ARG 2HH1 = 1.51

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 235 Chapter 4 Results

4.3.1 Isolation of recombinant pET28a Plasmid

The recombinant C5 pET28a plasmid was isolated by Roche high pure plasmid isolation Kit and visualized by 1% agarose gel electrophoresis. The isolated pET28a plasmid containing xylanase served as a template for amplification of xylanase gene in PCR reaction (Fig. 4.3.1).

4.3.2 PCR amplification of xylanase gene Two sets of PCR were used to amplify xylanase gene with proline, in first set of PCR reaction the recombinant plasmid was amplified with XGeoT-F and Xyl ProC5 for C5 strain xylanase (Fig. 4.3.2). In second set, PCR cycle was performed with XGeoT-F and Prol-BamHR primer, where amplified product of first PCR cycle (Fig. 4.3.3) was served as a template along with proline template. The amplified product was visualized by gel documentation system having approximately 1410 bp for C5 indicating xylanase tagged with proline rich amino acid sequence (Fig. 4.3.4).

4.3.3 Gel purification of amplified proline tagged xylanase gene QIAquick Gel Extraction Kit (QIAGEN) was used to cut and purify the amplified xylanase tagged proline gene fragment of C5 from gel and visualized by 1% agarose gel electrophoresis. Proline tagged C5 xylanase gene product was observed under gel documentation system indicating the purified gel extracted proline tagged xylanase (Fig. 4.3.5).

4.3.4 Cloning of proline tagged xylanase gene in cloning vector Proline tagged Xylanase coding gene was ligated with the pGEM®-T Easy cloning vector while JM 101 competent cells were used for transformation of ligated product. Blue/white screening technique was used to select the appropriate transformed cells that include our proline tagged ligated xylanase gene product (Fig. 4.3.6). pGEM®-T vector contain the ampicillin resistant gene, 10 white and one blue colonies for proline tagged C5 xylanase recombinant strains were selected and LB-Amp broth was used for cultivation of inserted cell, the plasmids were isolated by minipreparation protocol according to Sambrook and Russell (2001) (Fig. 4.3.7). Confirmation of cloning was carried out by sequencing and digestion with restriction enzyme. Sequencing clarified the cloning of xylanase tagged proline gene to pGEM®-T vector, and digestion of

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 236 Chapter 4 Results pGEM®-T cloned plasmid with EcoRI restriction enzyme produced pGEM®-T vector along with proline tagged C5 xylanase gene product confirming the cloning of our gene of interest (Fig. 4.3.8).

4.3.5 Preparation of pET28a and xylanase gene product for expression pET28a vector and pGEM®-T cloned C terminal proline tagged xylanase plasmid of C5 (Fig. 4.3.9) was isolated and visualized by gel electrophoresis for their confirmation. It was digested with NheI enzyme at 37°C for 2 hours, and then run on agarose gel in order to confirm digestion. The digested pET28a showed approximately 5369 bp product size of single band while C5 showed approximately 4410 bp under gel documentation system (Fig. 4.3.10). After that, single enzyme digested pET28a vector and C terminal proline tagged xylanase gene product was cleaned through ethanol precipitation. The presence of proline tagged C5 xylanase gene product and pET28a plasmid was determined by 1% agarose gel electrophoresis (Fig. 4.3.11).

4.3.6 Double digestion and purification of pET28a vector and proline tagged xylanase product Ethanol precipitated cleaned digested pET28a vector and proline tagged xylanase product of C5 were subjected to second digestion with BamHI restriction enzyme for 2 hours at 37°C. Restriction enzyme digestion was analyzed through gel electrophoresis, which confirmed 5369 bp for pET28a and approximately 1410 bp size for proline tagged C5 xylanase (Fig. 4.3.12). C5 double digested product also released pGEMT vector of about 3000 bp. The double digested proline tagged xylanase gene product and pET28a vector was isolated from gel by GeneJET Gel Extraction Kit (Thermo Scientific) as per manufacturer protocol. The gel excised purified insert and pET28a plasmid were confirmed through their respective size in agarose gel electrophoresis (Fig. 4.3.13) and was used as insert for ligation.

4.3.7 Cloning of xylanase gene in expression vector Proline tagged xylanase of G. thermodenitrificans C5 (GthC5ProXyl) was used for recombinant expression in E. coli. Proline tagged xylanase of C5 was ligated with the

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 237 Chapter 4 Results pET28a expression vector. Then, E.coli JM101 competent cells were used for transformation of ligated product (Fig. 4.3.14). pET28a vector contain the kanamycin resistant gene, 6 white and 2 blue colonies were selected and LB-kan broth was used for cultivation of inserted cell, after transformation plasmid was isolated from single colonies and transformation was confirmed by sequencing and digestion of plasmid with appropriate restriction enzyme. The digested pET28a plasmid showed the desired gene size product of proline tagged C5 xylanase along with the size of pET28a plasmid (Fig. 4.3.15).

4.3.8 Induction and over production of recombinant xylanase enzyme Induction of recombinant xylanase enzyme was performed through 1 mM IPTG and expression was observed by SDS-PAGE analysis and enzyme activity assay. SDS PAGE showed clearly expressed band of proline tagged C5 xylanase (GthC5ProXyl).

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 238 Chapter 4 Results

M 1 2

pET28a plasmid

3 kb

Fig 4.3.1; Isolation of pET28a recombinant plasmid of C5 xylanase M 2-Log DNA Ladder (0.1-10.0 kb) 1, 2 xylanase cloned pET28a recombinant plasmid of C5

M 1

3kb

1kb 1.2 kb

Fig 4.3.2; PCR amplification of C5 with amplified with XGeoT-F and Xyl ProC5 M 2-Log DNA Ladder (0.1-10.0 kb) 1 PCR amplified product of C5 xylanase 1230 bp with XGeoT-F and Xyl ProC5

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 239 Chapter 4 Results

1.2 kb

Fig 4.3.3; Gel extraction of C5 xylanase amplified with XGeoT-F and Xyl ProC5 primer

M ProC5Xyl C5Xyl

3000 bp

1410 bp 1000 bp 1230 bp

500 bp

Fig 4.3.4; PCR amplification of wild type (GthC5Xyl) and proline tagged xylanase (GthC5ProXyl) M 2-Log DNA Ladder (0.1-10.0 kb), 1; proline tagged C5 xylanase, 2; wild type C5 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 240 Chapter 4 Results

1410 bp proline tagged C5 xylanase

Fig 4.3.5; Gel extraction of proline tagged C5 xylanase gene product

JM 101 K

Fig 4.3.6; Transformation of pGEM®-T ligated proline tagged xylanase gene product in JM 101

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 241 Chapter 4 Results

M 1 2 3

3 kb

1 kb

Fig 4.3.7; Isolation of proline tagged xylanase cloned pGEM®-T vector M 2-Log DNA Ladder (0.1-10.0 kb) 1, 2, 3 proline tagged xylanase cloned pGEM®-T recombinant vector

M 1 M 2

3 kb 3 kb

1.2 kb 1.4 kb

Fig 4.3.8; Digestion of proline tagged xylanase cloned pGEM®-T vector with EcoRI restriction enzyme M 2-Log DNA Ladder (0.1-10.0 kb) 1, 2 Digested pGEM®-T vector with EcoRI restriction enzyme

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 242 Chapter 4 Results

M 1

10 kb

0.5 kb

Fig 4.3.9; Isolation of pET28a vector M, 2-Log DNA Ladder (0.1-10.0 kb), 1; pET28a plasmid

M 1 2 3 4 M 5 6

4.4 kb

5.369 kb 3 kb

1 kb

Fig 4.3.10; Digestion of proline tagged xylanase cloned pGEM®-T vector and pET28a vector with NheI restriction enzyme M, 2-Log DNA Ladder (0.1-10.0 kb) 1, 2, 3, 4: Digested proline tagged xylanase cloned pGEM®-T vector with NheI restriction enzyme, 5, 6: Digested pET28a vector with NheI restriction enzyme

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 243 Chapter 4 Results

M 1 2 M 3 4

5.369 kb 4 kb 4.4 kb

Fig 4.3.11; Precipitation of proline tagged xylanase cloned pGEM®-T vector and pET28a vector digested with NheI restriction enzyme 1,2, Digested proline tagged xylanase cloned pGEM®-T vector with NheI restriction enzyme, 3, 4, Digested pET28a vector with NheI restriction enzyme

M 1 2 M 3 4 M 5 6

3 kb 3 kb 5.369 kb

1.2 kb 1.4 kb

Fig 4.3.12; Digestion of proline tagged xylanase cloned pGEM®-T vector with BamHI restriction enzyme M, 2-Log DNA Ladder (0.1-10.0 kb) 1, 2, 3, 4: Double digested proline tagged xylanase cloned pGEM®-T vector with NheI and BamHI restriction enzyme 5, 6 : Double digested pET28a vector with NheI and BamHI restriction enzyme

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 244 Chapter 4 Results

M 1 2 M 3

5.369 kb

3 kb 1.4 kb 1 kb

0.5 kb

Fig 4.3.13: Gel extraction of double digested pET28a vector and double digested proline tagged xylanase gene product 1, 2: Gel purified double digested proline tagged xylanase gene product 3: Gel purified double digested pET28a vector with NheI and BamHI restriction enzyme

GthC5ProXyl K

Fig 4.3.14; Transformation of pET28a ligated proline tagged xylanase gene in JM 101 competent cells K; control plate, GthC5ProXyl; proline tagged C5 xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 245 Chapter 4 Results

M 1 M 2 3

5.369 kb

3 kb 1.4 kb

1 kb

Fig 4.3.15; Digestion of pET28a ligated proline tagged xylanase gene with BamHI and NheI restriction enzyme 1, 2 and 3: Double digested proline tagged xylanase cloned pET28a vector with NheI and BamHI restriction enzyme

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 246 Chapter 4 Results

4.3.9 Purification of recombinant enzyme 4.3.9.1 Precipitation with ammonium sulphate Proline tagged C5 recombinant xylanase enzyme was precipitated with 60% of ammonium sulphate concentration and dialyzed against 100 mM sodium phosphate buffer overnight with molecular weight cut-off 10,000 kDa dialysis membrane.

4.3.9.2 DEAE Sepharose chromatography The precipitated and dialyzed preparation of Proline tagged C5 xylanase was fractionated on DEAE Sepharose anion exchange chromatography eluted by 0.9 M NaCl concentration along with 100 mM sodium sulphate buffer. 60 fractions of about 3 mL each were collected, each fraction was assayed for xylanolytic activity and its optical density (OD) was taken at 280 nm for the protein content, one prominent peak was observed for Proline tagged C5 recombinant xylanase (Fig. 4.3.16). DEAE Sepharose chromatography was used to remove the impurities present in the crude enzyme extract, effectively. DEAE Sepharose chromatography purified proline tagged C5 xylanase had a specific activity of 1011 IU/mg and an overall purification fold of 5.42.

4.3.9.3 Phenyl Sepharose chromatography After DEAE Sepharose chromatography the proline tagged C5 xylanase was fractionated on Phenyl Sepharose chromatography (Fig. 4.3.17). The protein eluted was visualized as a single band on 15% SDS-PAGE which indicated its homogeneous nature. Purified proline tagged C5 xylanase yield was 279.45% with a specific activity of 1403.72 IU/mg and an overall purification fold of 7.53.

4.3.9.4 High-performance liquid chromatography (HPLC) Reverse phase HPLC C-18 column was also applied for enzyme purity. In the HPLC chromatogram, a single peak was revealed for the purified enzyme at a retention time of 2.5 min, which confirmed that it was a homogenous preparation (Fig. 4.3.18).

4.3.9.5 SDS-PAGE analysis To investigate the purity of the recombinant enzyme, protein size was characterized by SDS-PAGE. The eluted purified fractions of xylanases GthC5ProXyl appeared in

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 247 Chapter 4 Results the form of single bands on 15% gel indicating that fractions were homogenous. The molecular weight of GthC5ProXyl was determined against standard denaturing marker protein (Fig. 4.3.19).

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 248 Chapter 4 Results

1200 0.4 Activity

0.35 1000 Protein 0.3 800 0.25

A280 600 0.2 0.15 400 0.1

Xylanase Xylanase activity (IU/mL) 200 0.05

0 0 1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 Fraction Number

Fig 4.3.16; DEAE Sepharose chromatography of C5 proline tagged xylanase. Eluted with 0.9 M NaCl - 0.01 M sodium sulphate buffer (pH 6.0) at a flow rate of 0.5 mLmin-1.

1400 Activity 0.25

1200 Protein

0.2

1000

0.15 800

600 A280 0.1

400

Xylanase Xylanase activity (IU/mL) 0.05 200

0 0 1 6 11 16 21 26 31 36 41 46 51 56 Fraction Number

Fig 4.3.17; Phenyl Sepharose chromatography of C5 proline tagged xylanase

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 249 Chapter 4 Results

1: 280 nm, 4 nm C5 Irfan Retention Time 90 90

80 80

70 70

60 60

50 50

mAU

mAU 40 40

30 30

20 20

10 10

0 0

-10 -10

0 1 2 3 4 5 6 7 8 9 10 Minutes

Fig 4.3.18; HPLC profile of the C5 proline tagged purified xylanase from G. thermodenitrificans C5 using a reverse phase C-18 column (4.6 x 250 mm). HPLC chromatogram of the purified enzyme shows a single peak at a retention time of 2.5 min confirming that it was a pure preparation

M 1 2 3 4

80 kDa Purified 40 kDa recombinant Xylanase

25 kDa

Fig 4.3.19; SDS-PAGE showing purified recombinant GthC5ProXyl enzyme M, SDS-PAGE molecular mass standards 10–250 kDa New England Biolab; 1, Induced supernatant; 2, ammonium sulfate precipitation; 3, Purified GthC5ProXyl by DEAE Sepharose; 4, Purified GthC5ProXyl by Phenyl Sepharose

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 250 Chapter 4 Results

Table 4.3.1: Overall purification scheme for recombinant xylanase GthC5ProXyl

Purification Protein content Enzyme activity Specific Purification steps (mg/mL) (U/mL) activity (U/mg) Fold

Crude 2.78 517.97 186.32 0.63 enzyme

(NH4)2SO4 2.163349 727.11 336.10 1.80 precipitates DEAE 1.13221 1145.3 1011.6 5.42

Sepharose fraction Phenyl 1.03117 1447.48 1403.7 7.53

Sepharose fraction

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 251 Chapter 4 Results

4.3.10 Enzyme assay for xylanase 4.3.11 Determination of enzyme activity Xylanase activity of purified proline tagged C5 recombinant xylanase was determined by using the dinitrosalicylic acid (DNS) method (Miller 1959), in which the release of hydrolysis products (reducing sugars) from oat spelt xylan was measured. The liberated sugars were estimated using the reagent 3, 5-dinitrosalicylic acid (DNSA). One unit (U) of xylanase is defined as the amount of enzyme that uses oat spelt xylan as the substrate and liberates 1 μmol of reducing sugars as xylose under the assay conditions. By preparing different dilutions of xylose (2 mg/mL) the standard curve was plotted. Using the equation X = Y- 0.0929/0.129, the concentration of reducing sugar for proline tagged C5 xylanase was calculated indirectly by measurement of concentration of xylose in mg/mL.

4.3.12 Determination of protein concentration Protein concentration was determined by the method of Bradford (Bradford 1976). Bovine serum albumin was used as a standard in this procedure. The results of protein estimation were used to calculate the specific activity of recombinant xylanase.

4.3.13 Determination of Specific activity Specific activity of enzyme was calculated by using the formula. Specific activity = concentration of xylose (X) of unknown sample/concentration of total protein in unknown sample.

4.3.14 Biochemical characterization and stability studies of purified enzyme 4.3.14.1 Effect of temperature on activity of purified proline tagged C5 xylanase Temperature effect was analyzed by incubating proline tagged C5 recombinant xylanase with xylan substrate from 40-100°C and the residual activity was calculated. The purified proline tagged C5 recombinant xylanase exhibits activity over a broad range of temperature (40–80°C) while optimum activity was observed at 70°C (Fig. 4.3.20). Purified proline tagged C5 recombinant xylanase retained more than 85% of its initial activity from 60-80°C. C terminal Proline increases the optimum temperature of xylanase by 10°C from 60°C to 70°C.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 252 Chapter 4 Results

4.3.14.2 Temperature effect on stability of purified proline tagged C5 xylanase Stability of purified fractions of GthC5ProXyl at different temperatures were analyzed with xylan substrate by incubating from 40-100°C for 200 min and the residual activity was calculated. The recombinant enzyme GthC5ProXyl retained more than 90% activity after exposure to 70°C for 200 min, more than 85% activity at 60°C and 80°C (Fig. 4.3.22). More than Half of its activity was lost only at 90°C and 100°C for 200 min. The stability was increased with broader range as compared to its native xylanase C5 which retained more than 80% activity after exposure to 60°C for 200 min, while more than 74% activity was retained for 200 min at 50 and 70°C (Figure 4.3.22). Half of its activity was lost at 80°C for 200 min.

4.3.14.3 Effect of pH on activity of purified xylanase The effect of pH on activity of proline tagged C5 recombinant xylanase was analyzed with xylan substrate by incubating it at pH 3.0-11 and the residual activity was calculated. The optimum pH in case of GthC5ProXyl was 8.0 as compared to its original xylanase, which was found to be 6. Hence 2 degree of pH shift from 6.0 to 8.0 was observed with C terminal proline amino acid. More than 90% of initial activity was retained from pH 6.0-9.0 (Figure 4.3.21).

4.3.14.4 Effect of pH on stability of purified xylanase The effect of pH on stability of purified proline tagged C5 xylanase was analyzed by incubating it with xylan substrate from pH 3.0-11.0 for 200 min and the residual activity was calculated. The recombinant proline tagged C5 xylanase enzyme retained more than 97% activity after exposure to pH 8.0 for 200 min, more than 90% at pH 6.0-8.0 while more than 80% activity was retained for 200 min at pH 9.0, and 75% activity at pH 10 (Figure 4.3.23). pH stability was increased with proline amino acid to xylanase enzyme.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 253 Chapter 4 Results

120

100 GthC5ProXyl 80 GthC5Xyl 60 40

20 Relative activity Relative activity (%)

0 40 50 60 70 80 90 100 Temperature (°C)

Fig 4.3.20; Effect of temperature on activity of proline tagged C5 recombinant xylanase activity

GthC5ProXyl 120

100 GthC5Xyl

80

60 40 20 Relative activity (%) activity Relative 0

3 4 5 6 7 8 9 10 11

pH

Fig 4.3.21; Effect of pH on activity of proline tagged C5 recombinant xylanase activity

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 254 Chapter 4 Results

120 60°C 100 70°C 80 80°C 60 40 90°C

(%) activity Relative 20 100°C

0

0 50 100 150 200 250

Incubation time (min.)

Fig 4.3.22; Effect of temperature on stability of proline tagged C5 recombinant xylanase. Aliquots of varying time intervals were collected and stored at 0°C for determining residual xylanase activity.

120

100

80 pH 5 pH 6 60 pH 7

pH 8 40 (%) activity Relative pH 9 20 pH 10

0 0 20 40 60 80 100 120 140 160 180 200 Incubation time (min.)

Fig 4.3.23; Effect of pH on stability of proline tagged C5 recombinant xylanase. Aliquots of varying time intervals were collected and stored at 0°C for determining residual xylanase activity.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 255 Chapter 4 Results

4.3.14.5 Effect of substrate specificity of purified recombinant xylanase The recombinant proline tagged C5 xylanase enzyme specifically attacked polymeric xylan source while no other substrate such as insoluble xylan, carboxymethyl cellulose, avicel, filter paper, pNP-α- L- arabinofuranoside, pNP-β- Xylopyranoside, pNP-α- glucopyranoside, pNP-α-D- Xylopyranoside pNP- β- galactopyranoside and pNP- acetate. Tagging of proline at C terminus of C5 xylanase did not change its specificitity.

4.3.14.6 Investigation of shelf life of xylanase The purified recombinant proline tagged C5 xylanase enzyme (GthC5ProXyl) retained its activity, when stored at 4°C for 12 weeks, but after that, a decline in activity was observed. The activity was retained by purified GthC5ProXyl when stored for 14 weeks at 4°C but after that decline was observed. After 16 weeks, 93% of initial activity was retained by the enzyme, which is an important fact for its industrial application. Conversely enzyme remained completely stable for five weeks at room temperature but showed 86%, 73% and 70% residual activity after storage for 10, 12 and 13 weeks, respectively (Table 4.3.2).

4.3.14.7 Kinetics of purified recombinant xylanase

In order to study the kinetics of purified recombinant proline tagged C5 xylanase, Km and Vmax values were calculated by Linweaver and Burk (1934) plot. The Km and Vmax of Purified recombinant proline tagged C5 xylanse (GthC5ProXyl) (for oat spelt −1 −1 −1 xylan) were 1.45 mgmL and 1210.166 μmolmg min , respectively). The Km of the enzyme (1.45 mgmL−1 for oat spelt xylan) is within the range for microbial xylanases (0.14–14 mgmL−1) (Fig. 4.3.24).

4.3.15 Polysacchrides Binding capacity GthC5ProXyl and GthC5Xyl The polysaccharide-binding capacity of the purified GthC5ProXyl was carried out by incubating the GthC5ProXyl with avicel, Beechwood xylan, Oatspelt xylan arabinoxylan and birchwood xylan. GthC5ProXyl. showed ability to bind to birchwood xylan, Beechwood xylan, Oatspelt xylan and arabinoxylan, with 68%, 61%, 71% and 83% enzyme activity in the supernatant, respectively (Fig. 4.3.26), while couldn't bind to avicel.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 256 Chapter 4 Results

4.3.16 Enzymatic Treatment of pulp The absorbance of proline tagged C5 xylanase increased at various wavelengths (A280 and A467) clearly specified the liberation of LDCs and chromophores along with the hydrolyzed products of residual Oat spelt xylan, indicating the role of xylanase in bleaching of pulps. Maximum reducing sugars (1.256 mggm−1 oven-dried pulp), 1.233 and 1.37 were recorded during 8 hours of pulp treatment with GthC5ProXyl, GthC5Xyl and C5 W137R/D328R, respectively (Table 4.3.3, 4.3.4). 11.17 mggm−1 oven-dried pulps and 1.15 mggm−1 oven-dried pulps of maximum reducing sugar were released by AK53 and AK53 W127R after 8 hours of pulp treatment (Table 4.3.5). The kappa number efficiency was observed 17%, 32% and 26% for GthC5Xyl, GthC5ProXyl and C5W137R, respectively. The kappa number efficiency for AK53 and AK53 W127R turned out to be 9% and 14.5%, respectively (Table 4.3.6).

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 257 Chapter 4 Results

Table 4.3.2 Shelf-Life of proline tagged C5 xylanase

Weeks Relative activity % 4°C Room temperature

1 100 100

2 100 100 3 100 100 4 100 100 5 100 100 6 100 97 7 100 94 8 100 91 9 100 88 10 100 86 11 100 79 12 100 73 13 100 70 14 100 68 15 96 67 16 93 64

Fig 4.3.24; Lineweaver-burk plot of GthC5ProXyl (Km and Vmax values were determined according to (Linewear-burk plot). The substrate used was oat spelt xylan taken as mg/mL composition. S; Substrate concentration; Initial rate of reaction.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 258 Chapter 4 Results

Fig 4.3.25; TLC analysis for hydrolysis products released from oat spelt xylan by proline tagged C5 xylanase from Geobacillus thermodenitrificans, 1: GthC5ProXyl; 2: D-xylose; 3: xylobiose; 4: xylotriose; 5: xylotetrose; 6: xylopentose.

120

100 Birchwood Xylan

Oatspelt Xylan 80 Beechwood Xylan 60 Arbinoxylan Avicel 40

Unboundxylanase %

20

0 0 5 10 15 20 25 30 Substrate concentration mg/mL

Fig 4.3.26; Binding affinity of GthC5ProXyl xylanase towards various substrates. Purified enzyme was mixed with various substrates 100 mM sodium phosphate buffer (pH 8.0) and incubated at 4ºC for 1h. The relative activity was determined according to the standard assay conditions (pH 8.0 and 70o C).

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 259 Chapter 4 Results

Table 4.3.3 Biobleaching of C5 and proline tagged C5 xylanase

Time Chromophores (A465) LDC (A280) Reducing sugars (mg g-1 (hours) oven-dried pulp GthC5Xy GthC5ProXyl GthC5Xyl GthC5Pro GthC5Xyl GthC5ProXyl l Xyl 0 0.006 ± 0.006 ± 0.002 0.060± 0.056 ± 0.23 ± 0.10 0.223 ± 0.068 0.004 0.0090 0.041

2 0.057 ± 0.069 ± 0.018 0.15 ± 0.174 ± 0.47 ± 0.37 0.366 ± 0.090 0.010 0.052 0.045

4 0.072 ± 0.081 ± 0.014 0.16 ± 0.198± 0.54 ± 0.11 0.6 ± 0.08 0.034 0.03 0.093

6 0.075 ± 0.084 ± 0.021 0.23 ± 0.231 ± 0.84 ± 0.27 0.78 ± 0.166 0.014 0.1587 0.130

7 0.073 ± 0.086 ± 0.011 0.22 ± 0.261 ± 1.33 ± 0.75 1.13 ± 0.211 0.0090 0.070 0.114

8 0.07 ± 0.081±0.02 0.20 ± 0.255 ± 1.23 ± 0.32 1.25 ± 0.53 0.026 0.099 0.083

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 260 Chapter 4 Results

Table 4.3.4 Biobleaching of C5 W137R/D328R xylanase

Time Chromophores (A465) LDC (A280) Reducing sugars (mg g-1 (hours) oven-dried pulp C5 W137R/D328R C5 W137R/D328R C5 W137R/D328R

0 0.005 ± 0.006 0.050 ± 0.01 0.20 ± 0.09

2 0.069 ± 0.08 0.14 ± 0.09 0.28 ± 0.1

4 0.087 ± 0.010 0.17± 0.17 0.56 ± 0.23

6 0.080 ± 0.015 0.23 ± 0.10 0.74 ± 0.27

7 0.084 ± 0.016 0.28 ± 0.15 1.17 ± 0.45

8 0.082±0.020 0.25 ± 0.09 1.37 ± 0.30

Table 4.3.5 Biobleaching of AK53 and AK53 W127R xylanase

Time Chromophores (A465) LDC (A280) Reducing sugars (mg g- 1 oven-dried pulp AK53 AK53 W127R AK53 AK53 AK53 AK53 W127R W127R 0 0.002±0.001 0.003±0.002 0.056±0.0 0.063±0.04 0.22±0.06 0.24±0.10 45 7 2 0.05±0.032 0.059±0.006 0.19±0.07 0.21±0.05 0.33±0.15 0.38±0.11 5 4 0.069±0.044 0.072±0.021 0.21±0.09 0.23±0.09 0.56±0.13 0.6±0.08 0 6 0.067±0.023 0.075±0.015 0.23±0.10 0.27±0.11 0.68±0.15 0.78±0.22 7 0.072±0.031 0.078±0.021 0.27±0.04 0.29±0.03 1.07±0.18 1.10±0.18 8 0.07±0.01 0.073±0.041 0.25±0.11 0.27±0.11 1.17±0.55 1.15±0.58

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 261 Chapter 4 Results

Table 4.3.6 Kappa number determination of xylanase

Parameters C5 GthC5ProXyl W137R/D328R AK53 AK53 W127R Control 45.6 44.8 45.2 44.4 43.2 Kappa 38.2 30.7 33.8 40.4 36.9 number

Kappa 17% 32% 26% 9% 14.5% efficiency (%)

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 262 Chapter 5 Discussion

Some of the unique properties associated with enzymes make them able to use for various industrial applications. Some major properties associated with enzymes are their high selectivity, ease of disposal and environment friendly nature. The most interesting property associated with these biological catalysts is their ability to work under mild reaction condition. The major commercial exploitations of enzymes are detergent, food, diagnostics, pharmaceuticals and fine chemical industries. The primary sources of these enzymes are from mesophilic microorganisms and their application hence restricted due to their limited stability under adverse conditions, e.g. extreme pH and temperature. Extremophiles are a source of extremozymes (i.e. enzymes functional under more extreme conditions) which offers new opportunities for bio catalysis and biotransformation as a result of their extreme stability (Karan et al., 2012). Extremophiles are the microorganisms having the ability to survive under extreme environmental conditions like high temperature, pH, pressure, salt concentration etc. Their ability to survive at extreme conditions gives them a unique property to produce highly stable biocatalyst. These enzymes have wide industrial applications due to their stability at highly adverse conditions, which are more demanded characteristics to utilize these enzymes in various industries. The advent of recombinant DNA technology has further boosted the industrial production of useful enzymes and many enzymes are now being produced by recombinant microorganisms, animals and plant cells. Moreover, developments in genetic and protein engineering have improved the stability, economy, specificity and overall application potential of industrial enzymes (Xu et al., 2016). Thus, it is not surprising that together with the increasing number of biotechnological endeavor’s the industrial market continues to grow rapidly and has an ever increasing demand for additional biocatalysts.

Hemicelluloses are considered as an important constituent of renewable biomass, which can be exploited for various purposes. Annually, a huge amount of hemicelluloses are being wasted, which should be utilized properly for various commercial purposes to overcome economic crises. Microbes have the tendency to produce extracellular enzymes which degrade complex macromolecules to release simple soluble monomers which are later taken by enzyme producing microbes to support their growth. These microbes are adapted to the environment where it is required to release extracellular enzymes to carry out the process of degradation.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 263 Chapter 5 Discussion

Xylanase is considered as an important enzyme that can have great industrial application due to its unique property of carrying out reaction in an environment friendly way with higher yield under less severe reaction conditions thus providing a better alternative to energy intensive processes. Various industrial processes require more stable enzyme to carry out the process with more efficiency under extreme conditions. For example, ethanol tolerant xylanase is required for enhanced production of biofuels while using xylan as a substrate. Similarly, salt and solvent tolerant xylanase are required for the bioremediation of water contaminated with salt or solvents, and solvent and surfactant tolerant xylanase helps in deinking in paper recycling industry. Moreover, solvent tolerance improves the recovery, precipitation and repeated utilization of enzymes. For industrial enzymes thermostability is one of the most valued properties, thus investigating the tactics utilized by thermophilic enzymes is an intense area of research.

In the present study, it was aimed to isolate the best thermophilic xylanase-producing bacteria from hot springs, and qualitative test was performed for the initial screening of xylanase production. Two strains of Geobacillus thermodenitrificans (strain C5 from Chitral hotspring and AK53 from Azad Kashmir hotspring) were found to be the most potent xylanase producers, which are the first ever, reported from hot springs in Pakistan. Most of the G. thermodenitrificans were isolated from compost (Verma et al., 2013; Coleri et al., 2009). Isolates were inoculated on nutrient agar, supplemented with xylan as a substrate; zone of hydrolysis was measured after 24 hours of incubation and maximum zone correlated to the maximum xylanase producing isolate. A wide variety of thermophilic xylanase producing microorganisms has been isolated. These microbes were categorized as either thermophiles or hyper thermophiles. Thermophiles have the ability to grow at 50°C to 80°C, whereas hyper thermophiles can grow optimally at above 80°C. All thermophiles and hyperthermophiles are commonly found in thermal springs, solfataric fields, self-heating decaying organic debris, hot pools and other terrestrial and marine environments (Vieille et al., 2001; Sunna et al., 2003; Marcolongo et al., 2015).

In this study, enzyme activity was determined on the basis of amount of D-Xylose as assay product. High amount of colorful product (violet product) was a clear indication of presence of high amount of D-xylose in the reaction mixture. The solution was

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 264 Chapter 5 Discussion diluted at least 30, even 50 times so that Beer-Lambert Law was not violated after the enzyme reaction. Xylanase gene from G. thermodenitrificans C5 and AK53 was cloned and expressed in E. coli BL21 host. It is interesting to note that the xylanase activity in the E. coli host was found to be higher than that of native G. thermodenitrificans C5 and AK53 strains, possibly due to higher level of protein expression. The enzyme produced by E. coli was functionally active and capable of degrading oat spelt xylan even in SDS-PAGE gel also. Various studies reported that the xylanase genes from G. thermodenitrificans and other gram-positive bacteria have also been cloned and expressed in E. coli host (Verma et al., 2013; Verma and Satyanarayana, 2012; Mamo et al., 2007; Jalal et al., 2009). Therefore, It was determined that the bacterium exhibited stability and optimal activity at slightly acidic to neutral pH i.e. 5.0-7.0 and at higher temperature conditions i.e. 50-80°C.The cloned xylanase gene from G. thermodenitrificans is of 1,200 bp like those of Bacillus and Geobacillus sp. (Verma and Satyanarayana, 2012; Gerasimova and Kuisiene, 2012; Canakci et al., 2012; Verma et al., 2013).

The deduced amino acid sequence of strain AK53 xylanase (GthAK53Xyl) contained more number of negatively charged residues (Asp+Glu), no cysteine amino acid residues were found in the xylanase. In silico analysis demonstrated the aliphatic index of 37. BLASTp analysis of amino acid sequence exposed extensive similarity with various endoxylanases of GH10 family and displayed a high homology with G. thermodenitrificans JK1 (JN209933) and G. thermodenitrificans T-2 (EU599644) followed by other Bacillus and Geobacillus spp. By using the available crystalline structure of xylanase (PDB ID, 1HIZ chain A) from G. thermodenitrificans, various secondary and tertiary structures have been proposed. The secondary structure constitutes of 11 α helices, 13 β sheets and 5 sharp turns. PyMol PDB viewer was used to get three-dimensional structure that showed Glu in a conserved region which is catalytically very important residues and found in “bowl” shaped structure of AK53 and C5 xylanase. Similar Glu catalytic structures have been reported for xylanases (Verma and Satyanarayana, 2013). Almost all known xylanases of Geobacilli (Saksono and Sukmarini, 2010), including G. thermodenitrificans AK53 and C5 belong to the GH10 family. The purity of enzyme was determined by reverse phase HPLC on C-18 column and SDS-PAGE. The molecular weight of recombinant xylanase of strain C5 and AK53 was 44 and 45 kDa, respectively, which is lower than

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 265 Chapter 5 Discussion

50 KDa of G. thermodenitrificans TSAA1 (Verma et al., 2013). Xylanolytic enzymes of G. thermodenitrificans, partially characterized by Canakci et al., (2007), were reported to be only 30 kDa in size. Characterization of xylanase molecular weight of other Geobacilli was found to be 35 kDa, 36 kDa (Canakci et al., 2007) and 40 kDa (Wu et al., 2006). 45 kDa of G. thermodenitrificans JK1 (Gerasimova and Kuisiene, 2012) and 47 kDa G. thermodenitrificans TSAA1 xylanases (Verma et al., 2013) were the most similar in size with strain AK53 and C5 xylanase.

Both, the activity and stability of GthAK53Xyl were tested in the range from pH 3.0 to 10.0. Verma et al. (2013) and Gerasimova and Kuisiene (2012) reported an optimum activity at pH 9 from G. thermodenitrificans TSAA1 and pH 7 from G. thermodenitrificans JK1. It was observed that GthAK53Xyl was active and stable at broad pH range from 4.0-8.0. It is quite important to determine the pH stability of GthAK53Xyl to prevent it from activity loss, which might be possible due to its storage at various pH conditions. Recombinant xylanase from G. thermodenitrificans C5 (GthC5Xyl) exhibits remarkably high specific activity, which is appreciated for a thermophilic enzyme and is interesting candidate for further investigations. The purified GthC5Xyl showed the maximum activity at pH 6.0, which is lower than that of XynG1-1 (7.5) from Paenibacillus. campinasensis G1-1 (Zheng et al., 2012) and retained 52% of its maximum activity at pH 3.0. It was stable between pH 5.0 and 8.0 for 200 min, retaining at least 73% of its activity, manifesting much greater pH stability at the condition of lower pH than those of most bacterial xylanases reviewed (Beg et al., 2001; Subramaniyan and Prema, 2000; Zhao et al., 2011).

Temperature dependent activity and stability of GthAK53Xyl and GthC5Xyl recombinantely expressed xylanases were tested between 50-90°C. GthAK53Xyl exhibited optimum activity at 70°C. Most of the previously studied G. thermodenitrificans xylanases have been reported to operate at an optimum temperature of 70°C (Marcolongo et al., 2015, Verma et al., 2013; Gerasimova and Kuisiene, 2012). Xylanase from other microorganisms showed activity at higher temperature range e.g. (85°C of Streptomyces sp. and Bacillus sp.; 95°C for Thermotoga neapolitana) (Bhosale et al., 1996; Karaoglu et al., 2013). While xylanases from Paenibacillus sp.DG-22, Geobacillus sp. 70PC53, Bacillus firmus, Thermobifida fusca, and Bacillus sp.NG-27 were optimally active at the temperature

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 266 Chapter 5 Discussion range from 60°C to 70°C (Hyeong et al., 2007; Ng et al., 2009; Chang et al., 2004; Cheng et al., 2005). G. thermodenitrificans A333 retained 50% of its initial activity at 80°C while GthAK53Xyl retained more than 85% its initial activity at 80°C confirming its stability at high temperature as compared to other xylanases (Marcolongo et al., 2015). GthAK53Xyl enzyme retained more than 90% activity after exposure to 70°C for 200 min and more than 77% activity was retained at 60°C and 80°C for 200 min. This stability is far better than that of G. thermodenitrificans TSAA1 (85% initial activity at 70°C for 180 min) (Verma et al., 2013) and Paenibacillus sp. NF1 (46.88% of its initial activity at 70°C) (Zheng et al., 2014). The optimal reaction temperature of GthC5Xyl was found to be 60ºC, similar to Streptomyces sp. CS628 (Rahman et al., 2014), and higher than that of Paenibacillus sp. 12-11 (55ºC) (Zhao et al., 2011). GthC5Xyl retained 82% of its maximum activity at 70ºC and 77% at 80ºC. The GthC5Xyl retained more than 70% of the original activity after incubation at 40-60ºC, and still retained about 79% and 45% after incubation at 70ºC and 80ºC, respectively. This thermal stability is better than earlier reports on most of the bacterial xylanases (Shi et al., 2013, Subramaniyan and Prema, 2000, Zhao et al., 2011). C5 xylanase stability at high temperature (60ºC-80ºC) and activity at lower pH (pH 4.0-7.0) make GthC5Xyl valuable for various industrial applications, such as animal feeding, bioenergy conversion and food industry.

Impurities such as metal ions, which are present in the agro-industrial wastes, can potentially inhibit the activity of xylanases (Marcolongo et al., 2015). The activity of GthC5Xyl was not significantly inhibited by the presence of different metal ions. 2+ However, it was strongly inhibited by Hg , partially inhibited by AlCl3 and ZnSO4. It was found different from Geobacillus sp. 71 which showed resistance to ZnSO4 (Canakcı et al., 2012). Hg2+, can oxidize the indole ring, it can likely interact with tryptophan residues of the enzyme (Zhang et al. 2007). The GthC5Xyl showed no inhibition by MnCl2 and CoCl2, similarly to Geobacillus sp. 71 (Canakcı et al., 2012), while Bacillus halodurans xylanase was strongly inhibited by Mn (Mamo et al. 2006).

The GthC5Xyl was not inhibited by CuCl2 and differed to those of G. thermodenitrificans TSAA1, Plectosphaerella cucumerina, G. thermoleovorans, Thermotoga thermarum, Penicillium glabrum, Penicillium sclerotiorum, Aspergillus ficuum AF- 98 (Verma et al., 2012; Zhang et al., 2007; Shi et al., 2013; Knob et al., 2013; Knob and Carmona, 2010; Lu et al., 2008) that were inhibited by Cu2+.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 267 Chapter 5 Discussion

2+ 2+ However, the xylanase activity was greatly stimulated by Mn and Co , AgNO3, and

CuCl2. Xylanases from Streptomyces olivaceoviridis A1 (Wang et al., 2007), and Bacillus subtilis strain R5 (Jalal et al., 2009) had been reported to be stimulated by Fe2+ while recombinant xylanase GthC5Xyl is inhibited by Fe2+. The effect of different metal ions on GthAK53Xyl xylanase activity was unique among all the characterized xylanases of Geobacilli. Xylanases of the other Geobacilli were inhibited by Mn2+ or it had no effect on the activity (Knob et al., 2013; Sharma et al., 2007; Khasin et al., 1993; Gerasimova and Kuisiene, 2012). While GthAK53Xyl from G. thermodenitrificans AK53 was stimulated by Mn2+ ion. Similar stimulation of G. thermodenitrificans JK1 xylanase by Mn2+ ion was also observed by Gerasimova and Kuisiene (2012). The inhibition of GthAK53Xyl by Hg2+ confirmed the presence of tryptophan residues in the binding site, since oxidation of indole ring disrupts the interaction (Cheng et al., 2005; Gupta et al., 1992; Zhang et al., 2007; Liu et al., 2010; Verma et al., 2013). The inhibition of GthAK53Xyl by Cu2+ and Pb2+ is similar to those of Bacillus sp. YJ6, Staphylococcus sp. SG-13, Plectosphaerella cucumerina, Cellulosimicrobium sp. MTCC 10645, Aspergillus versicolor, Aspergillus ficuum AF- 98 and G. thermodenitrificans TSAA1 (Yin et al., 2009; Zhang et al., 2007; Gupta et al., 2000; Kamble and Jadhav, 2012; Salama et al., 2008; Lu et al., 2008; Verma et al., 2013). At low concentration of Cu2+, xylanases from Bacillus and Geobacillus species were strongly inhibited whereas GthAK53Xyl was partially inhibited. Xylanase of 2+ strain JK1 was inhibited even at low concentration of Zn , while GthAK53Xyl was inhibited only at higher concentration. The stimulation of enzyme GthAK53Xyl in the presence of K+ can be attributed to its effect in stabilizing the molecular structure of protein. It is also expected that this metal ion act as cofactor during enzyme-substrate reaction (Vieille and Zeikus, 2001: Marcolongo et al., 2015).

The xylanase is quite stable in the presence of most of the detergents tested as in Geobacillus sp and G. thermodenitrificans (Zhang et al., 2007; Verma et al., 2013). Sodium dodecyl sulphate (SDS) is a strong protein denaturant that inactivates most xylanases even at low concentration. There are few enzymes in the literature that can retain their activity in the presence of a high concentration of SDS. For example, xylanase BSX from alkalophilic Bacillus sp. NG-27, GH11 xylanase from symbiotic Streptomyces sp. TN119 and GH 10 xylanase XynAHJ3 from Lechevalieria sp. HJ3, have been reported to retain over 100% activity in the presence of 100 mM SDS

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 268 Chapter 5 Discussion

(Bhardwaj et al., 2010; Zhou et al., 2012; Zhou et al., 2011; Zheng et al., 2013). The GthC5Xyl retained more than 80% its activity even at 0.5% of SDS. The chelating agent EDTA did not affect the activity of recombinant xylanase, suggesting that this enzyme does not require metallic cations for its activity. Likewise G. stearothermophilus and Bacillus halodurans xylanases were not inhibited by EDTA, while Geobacillus sp. 71 was inhibited by EDTA. CTAB and triton X inhibited the enzyme only at high concentration similar to G. thermodenitrificans TSAA1 xylanase (Verma et al., 2013). GthAK53Xyl was inhibited by N-bromosuccinimide (N-BS) only at high concentration (5 and 10 mM), whereas xylanases from other Geobacillus species were inhibited at low concentration. N-BS, a tryptophan modifier, reduced xylanase activity, which is an evidence for the role of aromatic amino acid residues at active site (Cardoso and Filho, 2003). GthAK53Xyl is quite stable in the presence of most of the detergents tested similar to those of Geobacillus sp., G. thermodenitrificans TSAA1 (Verma et al., 2013), G. thermoleovorans, sp. MT-1 and Dictyoglomus thermophilum Rt46B.1 (Wu et al., 2006; Zhang et al., 2007). The slight inhibitory effect of these detergents on GthAK53Xyl is similar to that of Geobacillus sp. (Zhang et al., 2007). Tween 40 and 60 partially inhibited the GthAK53Xyl only at high concentration and is resistant at low concentration, similar result was also observed in case of Aspergillus awamori xylanase. Even with 1% SDS the enzyme retained more than 80% residual activity, while xylanases from Bacillus, Geobacillus (Verma et al., 2013) and Paenibacillus sp. NF1 were strongly inhibited at this concentration (Zheng et al., 2014).

As cellulase activity may result in poor fiber mechanical strength, for paper and pulp treatment, xylanase should be free from cellulolytic activity (Baba et al., 1994; Subramaniyan and Prema, 2000), which is one of the major properties as a bio- bleaching agent for paper and pulp industries. C5 and AK53 xylanase did not show any cellulase activity when assayed with artificial substrates like avicel, carboxymethyl cellulose, p-nitrophenyl glucopyranoside (pNPG), and p-nitrophenyl - β-D-cellobioside (pNPC). These results demonstrated that C5 and AK53 xylanase are cellulase-free xylanase, similar to those reported in Streptomyces sp. (Li et al., 2010; Kim et al., 2011; Rahman et al., 2014). Thus the cellulase free xylanase has an advantage in the production of high quality pulp. Our results indicated that

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 269 Chapter 5 Discussion

GthAK53Xyl has a cellulase free nature and thus can be used in the production of high quality pulp.

Different xylan substrates were analyzed for GthAK53Xyl specificity, which indicated that GthAK53Xyl could hydrolyze all the xylans, tested. The highest activity was observed with the birchwood xylan followed by the beechwood and oat spelt xylan. The enzyme was less active on arabinoxylan. Zheng et al. (2014) reported a xylanase from Paenibacillus sp. NF1 had more specificity towards oat spelt xylan than beechwood and birchwood xylan. C5 xylanase hydrolyzed all the selected xylans. The highest activity was observed with the beechwood xylan followed by the oat spelt and birchwood xylan. The substrate specificity was different from GthAK53Xyl which hydrolyzed birchwood xylan more efficiently. C5 xylanase specificity towards different xylan substrates was also different from A. niger US368 xylanase (Elgharbi et al., 2015) which proficiently hydrolyzed birchwood xylan followed by oat spelt and beechwood xylan. Malbranchea pulchella (Ribeiro et al., 2014) hydrolyzed oat spelt and birchwood xylan. The pattern of specificity of C5 xylanase was similar to that of G. thermodenitrificans A333 (Marcolongo et al., 2015). Among three natural xylans, C5 xylanase showed slight specificity towards beechwood xylan, whereas GthAK53Xyl showed preference towards birchwood xylan. GthAK53Xyl and GthC5Xyl were less active on arabinoxylan similar to other xylanases (Elgharbi et al., 2015; Zheng et al., 2014). These results clearly highlight the strict specificity of GthAK53Xyl and GthC5Xyl towards polysaccharides containing 1,4-β-xylan bonds.

The result of thin layer chromatography analysis of oat spelt xylan hydrolysis indicated that GthC5Xyl and GthAK53Xyl xylanase are endoxylanase in nature, as xylobiose and xylopentose were the major end products of hydrolysis. Based on the substrate specificity and kinetic parameter determination, no other activities were exhibited by GthC5Xyl and GthAK53Xyl xylanase. Cellulase-free xylanase has an advantage in the production of high-quality pulp. Our results indicated that recombinant GthC5Xyl and GthAK53Xyl xylanase has a cellulase free nature.

The Km values of GthAK53Xyl xylanase (4.34 mgmL−1 for oat spelt xylan) and GthC5Xyl (3.9084 mgmL−1) are within the range for microbial xylanases (0.14–14 mgmL−1) (Verma et al., 2013), and closely resemble to the Km of Pichia stipites

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 270 Chapter 5 Discussion xylanase (4.2 mgmL−1) (Basaran et al., 2000) and halophilic Bacterium-OKH (Sanghvi et al., 2014). These low Km values towards oat spelt xylan were found to be better than those of other microorganisms, such as Aspergillus niger SCTCC 400264 (Li et al., 2014), Scytalidium acidophilum (Balaa et al., 2009), Bacillus sp. (Kamble and Jadhav 2012), Aspergillus usamii E001 (Zhang et al., 2007), Paenibacillus sp. NF1 (Zheng et al., 2014), Bacillus licheniformis (Lee et al., 2008) and Bacillus circulans (Kazuyo et al., 2014).

GthC5Xyl also tolerated salt concentration up to 1.2M with maximum activity at 0.8M. Similar result was observed in case of Gordonia sp. (Kashyap et al., 2014). NaCl tolerant and cold active GH10 family xylanase produced by Glaecicola mesophila KMM241 was reported by (Guo et al., 2009), retaining at least 90% of its initial activity at 2.5M NaCl concentration, while the optimum concentration turned out to be 0.5M NaCl. A marine bacterium Bacillus pumilus produce xylanase having optimum activity at 1.2M NaCl, while at 1.75 and 2.5M NaCl concentration, its retain more than 87% and 73% activity, respectively. Xylanase isolated from Bacillus subtilis cho40 had optimum xylanase activity at 0.5M NaCl (Khandeparker et al., 2011). Solvent and detergent tolerance of xylanase isolated from different marine microorganisms has not been characterized. These explanations pointed out that xylanase isolated from G. thermodenitrificans C5 is a good candidates at harsh conditions at industrial level.

The effect of 15% (v/v) organic solvents was investigated by pre-incubating the enzyme in the presence of different solvents for 30, 60 and 120 min. The C5 and AK53 xylanases were not substantially influenced by most of the selected solvents even after 2 hours of incubation. Formaldehyde, ethyl acetate and glycerol inhibited both AK53 and C5 xylanases within 30 min of incubation. Formaldehyde and ethyl acetate inhibition of xylanase from G. thermodenitrificans A333 was also reported by Marcolongo et al. (2015). Both AK53 and C5 xylanases were quite stable in the presence of ethanol, methanol, acetone, acetonitrile, 2-propanol, DMSO, DMF and n- butanol for 120 min at 15% (v/v) concentrations. The high resistance, particularly with ethanol, makes AK53 and C5 xylanases suitable for different applications such as in production of the alcoholic beverages. As reported by Sato et al. (2010) and Marcolongo et al. (2015), the addition of xylan-degrading enzymes during the

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 271 Chapter 5 Discussion simultaneous hydrolysis and fermentation process of rice starch, the alcohol yield was significantly enhanced in the sake preparation. AK53 and C5 xylanases can also be regarded as promising candidates for bioethanol production in consolidated bioprocessing due to the enzyme capability to remain fully active at high ethanol concentrations.

C5 xylanase also addresses the property of xylanase such as stability at broad pH range, temperature and NaCl concentration and resistant to SDS. Thus, this strain could be good contender for different biotechnological applications under extreme conditions. Due to AK53 xylanase broad pH range and stability, high thermal stability, high activity, profile of xylooligosaccharides, and resistant to some detergent and some metals, GthAK53Xyl is an attractive candidate for the production of xylooligosaccharides in the paper and pulp and food industries as compared to other xylanase from G. thermodenitrificans. The use of this enzyme in biopulping and biobleaching is subject for further investigation.

Thermophilic enzymes are preferable over their mesophilic counterparts as high temperature reduces mass transfer limitation, contamination hazard and substrate viscosity (Wang et al., 2014). For example, thermophilic Pulp bleaching and bioconversion processes involve xylanases. In these processes a number of high temperature processes are applied either before or along with enzymatic treatment. In the preparation of animal feed addition of xylanase is done before pelleting process which is carried out at about 70 to 95°C. Different strategies can be acquired to hunt for thermophilic xylanases. One of them involves the search of extremophilic microorganisms that produce novel xylanases. GH 10 thermophilic xylanases have been reported from Thermotoga maritima MSB8 (90°C) (Ihsanawati et al., 2005), Thermotoga petrophila RKU-1 (90°C) (Santos et al., 2010), Thermobifida alba UL JB1 (80°C) (Blanco et al., 1997) and acidomesophilic Bispora sp. strain MEY-1 (85°C) (Luo et al., 2009). The available sequences and structures of these thermophilic xylanases i.e., 1VBU and 3NIY (Ihsanawati et al., 2005; Santos et al., 2010) provide valuable information for better understanding of the structural and functional characters of thermostable enzymes.

It is nothing short of astonishing to witness the amazing array of enzymes that nature has offered us. Over many centuries, mutation have been collecting in enzyme

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 272 Chapter 5 Discussion allowing them to bind to countless substrates perform a different type of functions. High throughput technology has allowed millions of enzymes to be screened. To look for their variants with enhanced performance. However the time of simple screening for variants is being replaced by a new way of thinking. Instead of looking to nature, man is taking it upon himself to design the enzyme himself. By doing this man can fill in the gap between what natures has given us, and what is actually needed from an enzyme to retain function under the different industrial conditions (Johannes and Zhao, 2006). Another approach for obtaining thermophilic enzymes is to mutate mesophilic enzymes and increase their thermal tolerance. It is usually done by mutating their N terminus and introduction of disulfide bridges, hydrogen bonds and salt bridges (Wang et al., 2014). Examples of increased thermophilicity of enzymes by successful protein engineering includes development of a mutant protease by DNA shuffling and attaining increased melting temperature of 25°C and half-life at 60°C (1200-fold) (Wintrode et al 2001). The efficiency of this method is increased by aiding it with computerized prediction via rational and random design. Fenel et al. (2004) and Wang et al. (2012) increased the number of disulfide bridges in the xylanase of Trichoderma reesei and Thermomyces languinosus and increased their temperature optima by 10°C.Wang et al. (2014) increased temperature range by 17°C of Streptomyces sp. strain S9 xylanase by site directed mutagenesis. Joo et al. (2011) improved the Tm of Bacillus circulans xylanase by 4.2°C based on thermal fluctuation analysis.

In the present study glutamic acid, arginine and proline amino acid were introduced in C5 and AK53 xylanase. Thermostability of the enzyme can be attributed to the helix forming tendency of glutamic acid and also the hydrogen bond and salt bridges formed by its anionic carboxylates. Another amino acid proline is also commonly found in thermostable proteins, which helps to improve stability due to the backbone of its distinctive cyclic structure that involves in providing extra conformational rigidity in turns and loops. Previously, it has been investigated that proline improves the thermostability of various enzymes when it was introduced at specific positions (Tian et al., 2010; Suzuki et al., 1991; Watanabe et al., 1994; Allen et al., 1998; Zhu et al., 1999; Goihberg et al 2007; Wang et al., 2014). AK53 and C5 xylanase from G. thermodenitrificans contains α and β barrel sheet and has weak thermostability for industrial application (Irfan et al., 2016). Escherichia coli was used to express the

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 273 Chapter 5 Discussion xylanase enzyme of G. thermodenitrificans C5 and AK53 strain, and interestingly it exhibited better properties in terms of wide range pH adaptation as well as stability. However, it was observed that the thermostability was adversely affected at temperatures above 60°C and 70°C for the strains C5 and AK53, respectively. Extensive information already exists regarding the factors involved in stability of α and β barrel enzymes that involve the packing of the hydrophobic core efficiently, the loop stabilization, cavity fillings and the presence of amino acid prolines at the N termini of α-helices (Wang et al., 2014).

To widen its application spectrum in industries, it is necessary to improve the thermal properties of AK53 and C5 xylanase. In this study, we developed several highly thermostable mutants of AK53 and C5 xylanase by site-directed mutagenesis techniques. AK53 and C5 mutants produced in Escherichia coli showed an increased temperature optimum (71°C) and (78°C) for C5 and AK53 mutants, respectively. In this study, an effort was made to develop a mutant xylanase with comparatively more thermostability with the help of bioinformatics, rational engineering. It was expected that the replacement of some important residues will improve the thermostability of C5 and AK53 xylanase. Computational designing helped to make the mutation more effective by providing detailed guidance to engineer enzyme. The results obtained after computational design illustrated that at high temperature, the enzyme stability is greatly affected by key residues, thus different mutations were designed accordingly. Compared with wild-type, C5 xylanase mutants W137R, D328R, W137R/D328R, N84R, D339R, H82E, W185P, D186E, W185P/D186E and triple mutant H82E/W185P/D186E showed enhanced resistance to high temperatures, and the W137R/D328R and H82E/W185P/D186E double and triple mutants showed the greatest shift, 7°C and 11°C respectively in temperature optima. AK53 xylanase mutants W175P/D176E, W127R/D318R and R71P/H72E/W175P/D176E showed a temperature shift of 5°C, 5°C and 8°C respectively in optimum temperature. DM R71P/H72E and W175P/D176E had optimum temperature of 74°C and 75°C respectively, 4 and 5°C higher than that of wild type G. thermodenitrificans AK53. The pH activity profile for most of the mutants was very similar to the wild type C5 and AK53 xylanase and a little variation in term of pH was observed with mutation. The purified C5 mutant xylanases had optimum pH of 8, 6.5, 8, 6 and 6 for W137R, D328R, W137R/D328R, N84R and D339R respectively. Optimum pH turned out to

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 274 Chapter 5 Discussion be 6.5, 6, 6, 6, 7 and 7 for R81P, H82E, W185P, D186E, DM W185P/D186E and triple mutant H82E/W185P/D186E respectively. Similarly 0.5 to 1 degree shift in pH was observed with AK53 mutant models. Similar result in change of pH 1.2 degree was observed when the Asp48 of XynC-C Aspergillus kawachii was replaced with Asn (Qiu et al., 2016).

The comparative investigation of protein structure among mesophiles and thermophiles have shown more arginine content on the surface of thermophilic proteins. Moreover, these thermophilic proteins were comparatively more stable at higher temperature which might be attributed to more content of arginine present on their surface. This higher stability may be due: (1) the resonance stability and high pKa value of arginine δ-guanido moiety involves in reduction of its chemical reactivity. (2) The positive charge on arginine δ-guanido moiety helps it to precipitate in multiple non covalent interactions by providing a surface area for charge to charge interactions. (3) Normally, the increase in temperature drops pKa values; however, due to high pKa (approximately 12) of arginine residue; it is possible to maintain net positive charge ion pair at higher temperature conditions (Sriprang et al., 2006). Arginine substitution increased thermal stability. The recombinant mutant enzyme C5 W137R retained more than 80% activity after exposure to 60°C, 65°C and 70°C for 200 min, while more than 70% and 65% activity is retained at 50°C and 75°C, respectively for 200 min. The recombinant mutant enzyme C5 D328R retained more than 80% activity after exposure to 50°C 60°C, 65°C, and 70°C, while more than 70% at 75°C respectively for 200 min. Double mutant of W137R and D328R retained more than 85% activity at 50°C, 60°C and 65°C respectively and more than 80% and 75% at 70°C and 75°C, respectively. Sriprang et al. (2006) investigated that numerous threonine and serine amino acid was replaced with 4-5 arginine amino acid of xylanase produced by Aspergillus niger BCC14405 successfully increased stability by 18–20-fold of Aspergillus niger BCC14405 (Sriprang et al., 2006). More than 80% activity is retained by W185P at 60°C and 70°C, more than 75% activity is retained by D186E at 60°C and 70°C and more than 80% activity is retained by the double mutant DM W185P/D186E for 200 min. More than 85% activity is retained by triple mutant H82E/W185P/D186E at 60°C and 70°C while more than 80% and 60% activity is retained at 50°C and 80°C respectively for 200 min. R81P stability was

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 275 Chapter 5 Discussion decreased as compare to wild type while H82E retained more than 80% activity at 60°C and 70°C respectively for 200 min.

The recombinant mutant enzyme AK53 W127R and D318R retained more than 80% activity after exposure to 60°C, 70°C and 80°C for 200 min. The double mutant AK53 W127R/D318R retained more than 90% activity at 70°C and 80°C and more than 80% at 50°C, 60°C, 65°C and 85°C for 200 min. AK53 D329R and AK53 N74R mutant retained more than 70% activity for 200 min from 50°C to 80°C. Both AK53 and C5 mutant model stability was improved with arginine, proline or glutamic acid substitution. Thermostability of T. reesei xylanase II was also improved when five arginine residues had been engineered into the Ser/Thr surface (Turunen et al., 2002). Double mutant of R71P/H72E maintained more than 80% activity at 70°C and 80°C. More than 75% activity was retained by the double mutant W175P/D176E from 60°C to 80°C. Quadruple mutant R71P/H72E/W175P/D176E maintained more than 80% activity from 60°C to 80°C for 200 min. This temperature stability pattern was far better than that of wild type AK53 and C5 xylanase. Replacement of aspartic acid with glutamic acid made mesophilic Aspergillus awamori glucoamylase thermostable up to 70°C (Chen et al., 1995). Introduction of proline and glutamic acid substitution in Streptomyces xylanase improved its thermal stability profile by 20% with combination of mutants (Wang et al., 2014).

Compared with the high specific activity of C5 xylanase (1243U/mg), all mutants showed decreased specific activities, ranging from 433 of C5 W185P/D186E to 1021 U/mg of C5 W137R. Moreover, these residue substitutions had profound impacts on the kinetics of all mutants. All C5 mutants were found to have increased Km value indicating their low affinity toward substrate, except W137R/D328R, D328R, D339R and R81P which affinity toward substrate was increased. Similar result relating to low affinity toward substrate and decrease in specific activity with mutant model of Streptomyces sp. strain S9 xylanase was also observed by Wang et al., (2014). Sriprang et al., (2006) revealed that introduction of arginine instead of threonine altered the specific activity of Aspergillus niger BCC14405. Compared with the high specific activity of AK53 xylanase (1113U/mg), all mutants showed decreased specific activities, Km value was decreased for AK53 W127R, D318R,

W127R/D318R and D339R, while all other AK53 mutant have increased Km value

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 276 Chapter 5 Discussion

indicating its low affinity toward xylan substrate. A slight reduction in Vmax and specific activity was observed in arginine rich environment due to large guanidinium group (Borders et al., 1994).

Half-life of W137R and D328R and H82E mutant models of C5 xylanase was enhanced more than 2 times at 60°C, 65°C and 70°C, respectively while the double mutant W137R/D328R enhanced the half-life 5 times at 60°C and 65°C. H82E/W185P/D186E enhanced half-life by 13 times at 60°C, 15 times at 65°C and 8 times at 70°C. The engineered Aspergillus niger BCC14405 xylanase was not stable in the absence of substrate. The ST4 mutant Aspergillus niger BCC14405 enzyme had half-life of 45 min, which was similar to that of wild-type enzyme. The half-life of ST5 in the absence of substrate was only 17 min, which was lower than that of wild- type. In context of this half-life our enzyme half-life is far better from the reported one (Sriprang et al., 2006). Half-life of quadrupole mutant R71P/H72E/W175P/D176E of AK53 xylanase was enhanced more than 11 times at 60°C and 65°C and 7 times at 70°C. A thermostable xylan mutant from Bacillus subtilis has been obtained successfully by applying combined approach (site saturation, DNA shuffling and random point mutation) thus producing three different amino acid mutations in the enzyme increasing its half inactivation temperature from 58°C to 68°C. These mutations filled a cavity around Gln-7/Asn-8 with hydrophobic proteins which were absent in wild type enzyme (Miyazaki et al., 2006).

Hydrogen bonds are defined when the distance between the donor and acceptor is less than 3.5 Å, which can be larger upto 4 Å if the donor is sulphur. The angle between the donor and acceptor should be larger than 90º (Hadfield and Mulholland, 1999). Introduction of amino acid bring some structural changes in wild type xylanase structure of C5 and AK53.The backbone hydrogen bonds are similar to their wildtype counterpart in each mutant. Gain in temperature can be explained in-terms of increased hydrogen bonding network, which is the clear case in C5 H82E, C5 N84R and C5 D186E mutant models. Bifurcated hydrogen bond formed by the oxygen of the E82 and increase in hydrogen bonding residue could suggest the further stabilizing of the protein by the H82E mutation. Similarly the C5 D186E mutant side chain elongation helps to extend its hydrogen bond partners. Bifurcated hydrogen bond can also be seen in the E186. There is some slight alteration in the hydrogen bonding of

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 277 Chapter 5 Discussion

N84R mutant. The only explanation for increase in temperature can be explained in term of the electrostatic repulsion created by the nitrogen atoms of neighboring residue of R84 side chain which may compel the side chain to strongly bind to backbone oxygen of F59. The side chain of D339R mutant lies on the surface of the protein. The possible cause of the increased stability could be interaction of the residue with other entities in the environment, which depict the structural significance of this residue. Proline mutant usually affect the backbone conformation and thus making some conformational changes. Loss of backbone hydrogen bond in case of mutant R81P can be seen in the figure 4.2.100, side chain adaptation blocks the existing hydrogen bond. Lack of novel hydrogen bonds suggest that there are some alteration in backbone conformation in the mutant which cannot be comprehended by mere calibration of side chain, it requires a deeper mechanistic explanation through crystallography or molecular dynamic simulation. Loss of hydrogen bond in C5 R81P decreased the stability and thermal profile of R81P mutant model.

AK53 xylanase sequence has little dissimilarity with C5 and the template 1HIZ. According to multiple sequence alignment of AK53, C5 and 1HIZ, it was observed that the there is a deletion of 10 residues in AK53. This deletion slightly alters the orientation of secondary structure. Therefore we observed a slightly different pattern of hydrogen bonding in the mutants. Proline mutants R71P and W175P might affect the backbone conformation and their effect cannot be depicted by optimization of side chains. They require detailed mechanistic explanation either through crystallography or molecular mechanics. Similarly the mutant residue D329R lies on surface of protein, which suggests that the residue is involved in the interaction with the environment around the enzyme. Hydrogen bonding pattern was not changed in N74R mutant model, but some conformational changes were introduced by bulkiness of the sidechain of arginine and positive charge around the side chain, which could possibly increase the thermal stability of enzyme. There was some increase in the hydrogen bond pattern for mutant D176E and H72E. In case of D176E the change in side change resulted in a more bulky side chain, which causes it to interact with other residues in the protein. There is bifurcated hydrogen bond by oxygen OE1 of E176. H72E model result in formation of novel hydrogen bond, which is due to the side chain of glutamic acid at 72 position. The oxygen atom forms a strong hydrogen bond

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 278 Chapter 5 Discussion with the backbone oxygen of F49, which is the reason for thermostability of AK53 xylanase.

The alteration of natural enzyme conformation is likely to affect the physical and catalytic properties of enzymes. To understand and identify the factors contributing to the thermostability of protein which helps extremophilic organisms to maintain their activities at extreme condition in industries is an active area of research (Cheng et al., 2014). The extraordinarily high specific activity depicted by the recombinant proline tagged xylanase obtained from G. thermodenitrificans C5 makes it a worthy candidate for further research. Proline has a side chain of typical cyclic formation that locks its backbone and leads to surprising conformation. Proline rich sequence may rigidify main chain, stabilize the mutants and thus improve their thermostability. Proline rich sequence also contains glutamic acid and arginine. Arginine has stronger ability towards formation of salt bridges and hydrogen bond. Thermostability of the enzyme can be attributed to the helix forming tendency of glutamic acid and also the hydrogen bond and salt bridges formed by its anionic carboxylates.

Proline rich regions are involved in binding processes (Williamson, 1994). The 57 residue long tag may provide an interface between the protein and the binding surface. A linker between the tag and the protein may provide “wag the dog” motion. Thus, the protein may give full function as opposed to unmodified form. Binding of unmodified wild type protein to a surface or other macromolecules may lead it to lose function and/or stability as well as may cause aggregation. Prediction of secondary structure of the tag indicates that the sequence forms two helices, which give stability and coiled structures between these two helices. The coiled structure may adopt the protein to bind a receptor without altering xylanase conformation or stability. Further, the coiled structure provides flexibility and facilitates binding. Proline structure restricts most of the conformational changes but proline rich amino acid site has smooth hydrophobic surface with charged amino acids at the periphery. This provides selectivity resulting from the periphery amino acids. Proline residues have lower conformational flexibility and the tag is designed to provide convenient environment for proline residues to form a hydrophobic surface with charged amino acids at the binding pocket conveniently. Number of proline residues and the current sequence is derived from known sequence motifs and provides an optimum interaction. Alteration

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 279 Chapter 5 Discussion of the sequence may either decrease the stability or alter-perturb conformational flexibility. Additionally, alteration of the sequence may perturb secondary structure of the protein and the hydrophobic nature of the tag may lead the tagged protein to aggregate. Thus, maintain this optimized sequence will be beneficial for the sake of stability and optimal xylanase activity (Irfan et al., 2016).

In the present study, C-Terminal Proline-Rich Sequence was tagged to xylanase of G. thermodenitrificans C5 and then cloned and expressed in E. coli BL21 (DE3) host. The enzyme produced by E. coli functionally active and proficiently degrades oat spelt xylan even in SDS-PAGE gel. Proline is highly predominant in thermophilic proteins because it has a side chain of typical cyclic structure that mops its backbone and central to an extraordinary conformational rigidity in the turns and loops. Introduction of proline residues at specific positions has been used successfully to advance the thermostability of many enzymes (Tenkanen et al., 1992; Cheng et al., 2014; Watanabe et al., 1994; Allen et al., 1998; Zhu et al., 1999; Goihberg et al., 2007; Wang et al., 2014). Li et al. (2014) reported that C-terminus modification with proline residues led to 5°C rise in temperature (35 to 40°C) of ruminal xylanases XynB-Fu (Li et al., 2014). Optimum temperature of Streptomyces xylanase was increased from 73°C to 90°C by the introduction of proline and glutamic acid amino acid in the long secondary-structure elements (Wang et al., 2014). We can conclude that proline tagging at C terminus of GthC5Xyl is one of the key factors responsible for the improved thermal properties of the GthC5ProXyl. GthC5ProXyl optimal reaction temperature was found to be 70ºC that is found 10ºC higher than that of G. thermodenitrificans C5 wild type xylanase, and 15ºC than Paenibacillus sp. 12-11 (Zhao et al., 2011). GthC5ProXyl also retained more than 80% of its activity at 80ºC, which is far better than that of its wild type xylanase. This increase in thermal stability is far better than most of the bacterial xylanases that are previously reported (Zhao et al., 2011; Li et al., 2014; Shi et al., 2013; Subramaniyan and Prema, 2000).

C-terminus modification with proline rich amino acid sequence also resulted a shift in optimum pH of purified GthC5ProXyl from 6.0 to 8.0 that was found similar to proline fused XynB-Fu of ruminal xylanases (Li et al., 2014). The GthC5ProXyl retained more than 90% of its activity at pH 6.0-8.0, seemed much higher stability than that of wild type xylanase at wide pH range. Beg et al. (2001) revealed xylanase,

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 280 Chapter 5 Discussion which showed stability at low pH (Beg et al., 2001). The stability of this GthC5ProXyl at higher temperature (60-80ºC) and alkaline pH renders it worthy for countless industrial applications such as bioenergy conversion, food industry, bio- bleaching and animal feed.

Catalytic efficiency is an extent of the enzyme-substrate reaction scheme. Substrate specificity and kinetic parameter determination of GthC5ProXyl showed that it only exhibits xylanolytic activity related to the wild type. The enzymatic hydrolysis products were identified as xylobiose and xylopentose, hence confirmed that it is an endo-xylanase. The Km and Vmax of GthC5Xyl for oat spelt xylan was slightly higher than GthC5ProXyl. Proline increases the specificity toward oat spelt xylan. The polysaccharide-binding capacity of the purified GthC5ProXyl was carried out by incubating it with avicel, beechwood. The enzyme efficiently binds to these substrates except Avicel. GthC5ProXyl exhibited a high activity on beechwood xylan, but no activity was detected for cellulosic substrates, that indicate the absence of cellulose- binding sites on enzyme. It was confirmed by polysaccharide-binding analysis where more than 96% unbound enzyme was detected in the supernatant when incubated with avicel. Shelf life of GthC5ProXyl increased as compared to GthC5Xyl when incubated at 4°C. More than 90% of initial activity was retained by GthC5ProXyl after 16 weeks which is an important fact for its industrial application. Conversely, proline tagged enzyme retained more than 80% of activity at room temperature after storage for 10 weeks as compared to wild type enzyme.

The use of xylanases in the paper pulp industries has a progressive impact on environmental sustainability and on the quality of the final product. Since thermostability is required property for this and other industrial applications of xylanase enzymes. Much effort has been directed in the identification or production of novel and modified thermostable enzymes. Traditional methods were based on the screening of naturally occurring enzymes from microorganisms, and commercial xylanases have been mainly produced by fungus and bacteria. New strategies have been the prospection within metagenomics libraries, the use of molecular engineering techniques to stabilize critical structural motifs and the immobilization on nanoparticles. The combination of these approaches is a promising roadmap for the

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 281 Chapter 5 Discussion generation of xylanases with optimum performance under the harsh environmental conditions required for some industrial applications (Kumar et al., 2016).

In order to reduce the utilization of chlorine in paper and pulp industries, various enzymes are preferred over chlorine requirement for pre-bleaching purposes. It is considered that an increase in release of sugars along with an increase in absorbance at 280 and 465 nm is due to hydrolytic actions exhibited by xylanase and proves its usefulness during the process of bio-bleaching in paper and pulp industry. GthC5ProXyl exhibited profound effect on pulp samples (under high temperature and alkaline conditions) as compared to wild type xylanase. Similar effects of xylanases from B. licheniformis (Damiano et al., 2003), B. pumilus (Kaur et al., 2010) and B. coagulans (Choudhury et al., 2006) have been reported. An increase in the absorbance at 280 and 465 nm and release of reducing sugars by AK53 and mutant AK53 W127R indicated the hydrolytic action of xylanase on pulp fibers and its applicability in bio- bleaching of pulps. The Release of lignin, phenolic compounds and reducing sugars is an inter-related phenomenon. Xylose is pressed between the layers of lignin and phenolic compounds in the hemicellulose layer of pulp. When xylan is hydrolyzed, xylose in hemicellulose and reducing sugars are released, lignin and phenolic compounds are also subsequently with reducing sugars released thereby increasing the absorbance (Beg et al., 2000).

Kappa number is the measurement of the amount of lignin present in the pulp. Amount of reducing sugars released from Kraft pulp by AK53 and mutant AK53 W127R xylanase was significantly greater with increasing time. Kappa number efficiency was enhanced from 9% to 14.5% with mutant AK53 W127R xylanase. Kappa number efficiency was enhanced from 17% to 32% and 26% with C terminal proline rich sequence C5 xylanase and C5 double mutant W137R/D328R xylanase. Raj et al. (2013) reported 28.5% kappa number efficiency by Providencia sp. strain X1 xylanase (Raj et al., 2013). Bacillus halodurans C-125 kappa number efficiency was found to be 5% (Lin et al., 2013). Commercial xylanases decreases 19.3% in kappa number for AU-PE89 xylanase and a reduction of 27.2% in kappa number of Pulpzyme HC xylanase (Lin et al., 2013). This proved that C terminal proline rich C5 xylanase exerted at least as good a bleach boosting effect as the commercial enzymes and Providencia sp. strain X1 xylanase. After tagging the proline at C terminal, its

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 282 Chapter 5 Discussion alkali stability and thermostability was enhanced thus making the proline tagged xylanase as a biocatalyst choice for bio-bleaching in paper pulp. The improvement of release of chromophores and reducing sugars from residual lignin and xylan of pulp samples clearly suggests the applicability of the enzyme in pulp pre-bleaching for minimizing chlorine/chlorine dioxide (Irfan et al., 2016).

Pulp treatment with commercially available xylanases such as pulpzyme (Novo Nordisk a/s, Denmark at 50°C and 0.75 IU/g dose) and Cartazyme (Sandoz, USA at pH 4.8, 50°C, and 0.5– 2.0 IU/g dose,) have been reported (Bajpai et al., 1994). External fibrillations due to xylanase treatment gives strength to pulp fiber thus cause an increased bust factor and tear index in case of S. cyaneus SN32. During pulp formation, enzymatic bleaching actions are enhanced due to factors like increased pulp fibrillation, restoration of fiber bonding, increased freeness and water retention (Bajpai, 1999). When pulp was treated with xylanase obtained from Streptomyces sp. QG-11-3, it resulted in enhanced swelling up, increased porosity and separation of pulp fibers and pulp microfibrils as compared to untreated pulp fiber with smooth surfaces (Beg et al., 2000). Ninawe et al. (2006) also reported alkalistable endoxylanase from S. cyaneus SN32 having the ability to reduce kappa number by 8.7%, enhanced the brightness index by 3.56% of pulp and also reduced hypochlorite by 6%.

Research has been actively underway to categorize and extract enzymes from extremophilic bacteria of hot springs (Tolan et. al.,1995; Demuner et al., 2011). Tolan et al. (1995) also described that enzymes promise to be more resilient to pH and temperature conditions, allowing for greater delignification capabilities. Van der Burgt (2002), Demuner et al. (2011) and Li et al. (2015) pointed out that there are indications that most of the xylanase applied are engineered and they are more effective than before. One mutant models and proline tagged C5 xylanase result revealed that one of the major interests in the field of enzyme engineering is to introduce more thermostable enzymes. Moreover, recent studies in field of enzymes mainly focused on production, optimization and designing of enzymes to make them more favorable at higher temperature conditions and also more tolerable to alkaline pH (Azeri et al., 2010; Kaur and Mahajan, 2011; Zhang et al., 2010; Joshi and Khare, 2011; Mishra and Thakur, 2011; Demuner et al., 2011; Liu et al., 2013; Li et al.,

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 283 Chapter 5 Discussion

2015; Wang et al., 2014; Bai et al. 2010; Kapilan and Arasaratnam, 2015; Nagar et al., 2010).

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 284 Conclusions

 Due to its high activity, production of xylooligosaccharides as well as resistance to some detergents and metals, GthAK53Xyl is an attractive candidate for its application in paper and pulp and food industries as compared to other xylanases from G. thermodenitrificans.

 Geobacillus thermodenitrificans C5 xylanase has shown stability at broad pH and temperature range, high NaCl concentration as well as resistance to SDS. Thus, this strain could be good contender for different biotechnological applications under extreme conditions.

 C-terminal proline-rich sequence simultaneously increased the optimal temperature, enhanced the catalytic efficiency, broadened the pH and temperature ranges, and completely degraded the xylan substrate. Our results therefore advocate that the modified GthC5ProXyl could suitably meet the concert of commercially available xylanase preparations. The industrially significant characteristics of the GthC5ProXyl, signpost potential for its cost- effective application in the pulp and paper industry as a bio-bleaching agent.

 Modified AK53 and C5 with single, double, triple and Quadruple mutations revealed a significant increase in enzymatic thermostability under neutral as well as alkaline conditions. The enhanced stability of xylanase enzyme makes it more favorable to be utilized for various industrial applications. One of the major application that is directly associated with this property is bleaching of Kraft pulp. Although, these stabilizing mutations improve the thermostability and optimum temperature conditions of enzyme; however, it could cause reduction in its catalytic activity.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 285 Future Prospects

• More thermophilic habitats need to be explored for different industrially important enzymes.

• One of the major advantages relates with the modern researches on discovery of novel microbial enzymes is their vast applications to solve various environmental issues and aids in providing various new synthetic processes.

• Need deep metagenomics studies for the detection of more potent xylanase producing microbes.

• Search for novel isolates by formulation of new culturing media.

• Application of thermophilic enzymes in different industrial fields.

• CD measurement to analyze change in the secondary structure of protein.

• DSC analysis to determine the Tm value of mutant’s enzyme.

• Industrial application of mutant’s enzyme.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 286 References

1. Abd El-Nasser NH, Helmy SM, El-Gammal AA. 1997. Formation of enzymes by biodegradation of agricultural wastes with white rot fungi. Polymer Degradation and Stability 55:249-255. 2. Abou-Hachem M, Olsson F, Karlsson EN. 2003. Probing the stability of the modular family 10 xylanase from Rhodothermus marinus. Extremophiles 7:483- 491. 3. Adsul MG, Ghule JE, Shaikh H, Singh R, Bastawde KB, Gokhale DV, Varma AJ. 2005. Enzymatic hydrolysis of delignified bagasse polysaccharides. Carbohydrate Polymers 62:6-10. 4. Agnihotri S, Dutt D, Tyagi CH, Kumar A, Upadhyaya JS. 2010. Production and biochemical characterization of a novel cellulase-poor alkali-thermo- tolerant xylanase from Coprinellus disseminatus SW-1 NTCC 1165. World Journal of Microbiology and Biotechnology 26:1349-1359. 5. Ahmed S, Riaz S, Jamil A. 2009. Molecular cloning of fungal xylanases: an overview. Applied Microbiology and Biotechnology 84:19-35. 6. Al Balaa B, Brijs K, Gebruers K, Vandenhaute J, Wouters J, Housen I. 2009. Xylanase XYL1p from Scytalidium acidophilum: site-directed mutagenesis and acidophilic adaptation. Bioresource technology 100:6465- 6471. 7. Allen MJ, Coutinho PM, Ford CF. 1998. Stabilization of Aspergillus awamori glucoamylase by proline substitution and combining stabilizing mutations. Protein engineering 11:783-788. 8. Allen MJ, Coutinho PM, Ford CF. 1998. Stabilization of Aspergillus awamori glucoamylase by proline substitution and combining stabilizing mutations. Protein engineering 11:783-788. 9. Alzari PM, ne Souchon H, Dominguez R. 1996. The crystal structure of endoglucanase CelA, a family 8 glycosyl hydrolase from Clostridium thermocellum. Structure 4:265-275. 10. Andrade CM, Pereira Jr N, Antranikian G. 1999. Extremely thermophilic microorganisms and their polymer-hidrolytic enzymes. Revista de Microbiologia 30:287-298. 11. Aristidou A, Penttilä M. 2000. Metabolic engineering applications to renewable resource utilization. Current Opinion in Biotechnology 11:187-198.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 287 References

12. Azeri C, Tamer U, Oskay M. 2010. Thermoactive cellulase-free xylanase production from alkaliphilic Bacillus strains using various agro-residues and their potential in biobleaching of kraft pulp. African Journal of Biotechnology 9. 13. Baba T, Shinke R, Nanmori T. 1994. Identification and characterization of clustered genes for thermostable xylan-degrading enzymes, beta-xylosidase and xylanase, of Bacillus stearothermophilus 21. Applied and environmental microbiology 60:2252-2258. 14. Baba T, Shinke R, Nanmori T. 1994. Identification and characterization of clustered genes for thermostable xylan-degrading enzymes, beta-xylosidase and xylanase, of Bacillus stearothermophilus 21. Applied and Environmental Microbiology 60:2252-2258. 15. Bai W, Zhou C, Xue Y, Huang C-H, Guo R-T, Ma Y. 2014. Three- dimensional structure of an alkaline xylanase Xyn11A-LC from alkalophilic Bacillus sp. SN5 and improvement of its thermal performance by introducing arginines substitutions. Biotechnology letters 36:1495-1501. 16. Bai W, Zhou C, Xue Y, Huang CH, Guo RT, Ma Y. 2014. Three-dimensional structure of an alkaline xylanase Xyn11A-LC from alkalophilic Bacillus sp. SN5 and improvement of its thermal performance by introducing arginines substitutions. Biotechnology Letters 36:1495-1501. 17. Bai Y, Wang J, Zhang Z, Yang P, Shi P, Luo H, Meng K, Huang H, Yao B. 2010. A new xylanase from thermoacidophilic Alicyclobacillus sp. A4 with broad-range pH activity and pH stability. Journal of industrial microbiology & biotechnology 37:187-194. 18. Bailey M, Biely P, Poutanen K. 1992. Interlaboratory testing of methods for assay of xylanase activity. Journal of Biotechnology 23:257-270. 19. Bajpai P, Bhardwaj N, Bajpai P, Jauhari M. 1994. The impact of xylanases on bleaching of eucalyptus kraft pulp. Journal of biotechnology 38:1-6. 20. Bajpai P. 1999. Application of enzymes in the pulp and paper industry. Biotechnology progress 15:147-157. 21. Bajpai P. 1999. Application of enzymes in the pulp and paper industry. Biotechnology progress 15:147-157. 22. Ball A, Betts W, McCarthy A. 1989. Degradation of lignin-related compounds by actinomycetes. Applied and Environmental Microbiology 55:1642-1644.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 288 References

23. Baraznenok V, Becker E, Ankudimova N, Okunev N. 1999. Characterization of neutral xylanases from Chaetomium cellulolyticum and their biobleaching effect on eucalyptus pulp. Enzyme and Microbial Technology 25:651-659. 24. Basaran P, Basaran N, Hang YD. 2000. Isolation and characterization of Pichia stipitis mutants with enhanced xylanase activity. World Journal of Microbiology and Biotechnology 16:545-550. 25. Bayer EA, Morag E, Lamed R. 1994. The cellulosome—a treasure-trove for biotechnology. Trends in biotechnology 12:379-386. 26. Beg Q, Kapoor M, Mahajan L, Hoondal G. 2001. Microbial xylanases and their industrial applications: a review. Applied microbiology and biotechnology 56:326-338. 27. Beg QK, Bhushan B, Kapoor M, Hoondal G. 2000. Enhanced production of a thermostable xylanase from Streptomyces sp. QG-11-3 and its application in biobleaching of eucalyptus kraft pulp. Enzyme and Microbial Technology 27:459-466. 28. Belancic A, Scarpa J, Peirano A, Díaz R, Steiner J, Eyzaguirre J. 1995. Penicillium purpurogenum produces several xylanases: purification and properties of two of the enzymes. Journal of Biotechnology 41:71-79. 29. Bhardwaj A, Leelavathi S, Mazumdar-Leighton S, Ghosh A, Ramakumar S, Reddy VS. 2010. The critical role of N-and C-terminal contact in protein stability and folding of a family 10 xylanase under extreme conditions. PloS one 5:e11347. 30. Bhat M. 2000. Cellulases and related enzymes in biotechnology. Biotechnology advances 18:355-383. 31. Bhosale SH, Rao MB, Deshpande VV. 1996. Molecular and industrial aspects of glucose isomerase. Microbiological reviews 60:280-300. 32. Bibi Z, Qader SAU, Aman A. 2015. Calcium alginate matrix increases the stability and recycling capability of immobilized endo-β-1, 4-xylanase from Geobacillus stearothermophilus KIBGE-IB29. Extremophiles 19:819-827. 33. Biely P, Markovič O, Mislovičová D. 1985. Sensitive detection of endo-1, 4-β- glucanases and endo-1, 4-β-xylanases in gels. Analytical biochemistry 144:147- 151. 34. Biely P. 1985. Microbial xylanolytic systems. Trends in Biotechnology 3:286- 290.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 289 References

35. Biely P. 1991. Biotechnological Potential and Production of Xylanolytic Systems Free of Cellulases, p. 408-416, Enzymes in Biomass Conversion, vol. 460. American Chemical Society. 36. Biely P. 1993. Biochemical aspects of the production of microbial hemicellulases, Hemicelluloses and hemicellulases. Cougland, MP, Hazlewood, GP, Portland Press, London. 37. Bim MA, Franco TT. 2000. Extraction in aqueous two-phase systems of alkaline xylanase produced by Bacillus pumilus and its application in kraft pulp bleaching. Journal of Chromatography B: Biomedical Sciences and Applications 743:349-356. 38. Black GW, Rixon JE, Clarke JH, Hazlewood GP, Ferreira LM, Bolam DN, Gilbert HJ. 1997. Cellulose binding domains and linker sequences potentiate the activity of hemicellulases against complex substrates. Journal of biotechnology 57:59-69. 39. Blackburn J. 2000. Engineering and design: Rational versus combinatorial approaches. Current Opinion in Structural Biology 10:399-400. 40. Blanco J, Coque J, Velasco J, Martin J. 1997. Cloning, expression in Streptomyces lividans and biochemical characterization of a thermostable endo- β-1, 4-xylanase of Thermomonospora alba UL JB1 with cellulose-binding ability. Applied microbiology and biotechnology 48:208-217. 41. Blum DL, Li X-L, Chen H, Ljungdahl LG. 1999. Characterization of an acetyl xylan esterase from the anaerobic fungus Orpinomyces sp. strain PC-2. Applied and environmental microbiology 65:3990-3995. 42. Boonchuay P, Techapun C, Seesuriyachan P, Chaiyaso T. 2014. Production of xylooligosaccharides from corncob using a crude thermostable endo-xylanase from Streptomyces thermovulgaris TISTR1948 and prebiotic properties. Food Science and Biotechnology 23:1515-1523. 43. Borders C, Broadwater JA, Bekeny PA, Salmon JE, Lee AS, Eldridge AM, Pett VB. 1994. A structural role for arginine in proteins: multiple hydrogen bonds to backbone carbonyl oxygens. Protein Science 3:541-548. 44. Bowers KJ, Mesbah NM, Wiegel J. 2009. Biodiversity of poly-extremophilic Bacteria: does combining the extremes of high salt, alkaline pH and elevated temperature approach a physico-chemical boundary for life? Saline Systems 5:9.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 290 References

45. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry 72:248-254. 46. Bragger J, Daniel RM, Coolbear T, Morgan H. 1989. Very stable enzymes from extremely thermophilic archaebacteria and eubacteria. Applied Microbiology and Biotechnology 31:556-561. 47. Brissos V, Eggert T, Cabral J, Jaeger K-E. 2008. Improving activity and stability of cutinase towards the anionic detergent AOT by complete saturation mutagenesis. Protein Engineering Design and Selection 21:387-393. 48. Bron S, Bolhuis A, Tjalsma H, Holsappel S, Venema G, van Dijl JM. 1998. Protein secretion and possible roles for multiple signal peptidases for precursor processing in bacilli. Journal of biotechnology 64:3-13. 49. Brühlmann F, Leupin M, Erismann KH, Fiechter A. 2000. Enzymatic degumming of ramie bast fibers. Journal of biotechnology 76:43-50. 50. Buchert J, Ranua M, Kantelinen A, Viikari L. 1992. The role of two Trichoderma reesei xylanases in the bleaching of pine kraft pulp. Applied microbiology and biotechnology 37:825-829. 51. Bull AT. 2001. Biotechnology for industrial sustainability. Korean Journal of Chemical Engineering 18:137-148. 52. Buswell JA, Chang S-t. 1994. Biomass and extracellular hydrolytic enzyme production by six mushroom species grown on soybean waste. Biotechnology Letters 16:1317-1322. 53. Butt MS, Tahir-Nadeem M, Ahmad Z, Sultan MT. 2008. Xylanases and their applications in baking industry. Food Technology and Biotechnology 46:22-31. 54. Camacho NA, Aguilar G. 2003. Production, purification, and characterization of a low-molecular-mass xylanase from Aspergillus sp. and its application in baking. Applied biochemistry and biotechnology 104:159-171. 55. Canakcı S, Cevher Z, Inan K, Tokgoz M, Bahar F, Kacagan M, Sal FA, Belduz AO. 2012. Cloning, purification and characterization of an alkali-stable endoxylanase from thermophilic Geobacillus sp. 71. World Journal of Microbiology and Biotechnology 28:1981-1988. 56. Canakci S, Inan K, Kacagan M, Belduz AO. 2007. Evaluation of arabinofuranosidase and xylanase activities of Geobacillus spp. isolated from

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 291 References

some hot springs in Turkey. Journal of microbiology and biotechnology 17:1262. 57. Cannio R, Di Prizito N, Rossi M, Morana A. 2004. A xylan-degrading strain of Sulfolobus solfataricus: isolation and characterization of the xylanase activity. Extremophiles 8:117-124. 58. Cardoso OAV, Ferreira Filho EX. 2003. Purification and characterization of a novel cellulase-free xylanase from Acrophialophora nainiana. FEMS microbiology letters 223:309-314. 59. Castanares A, Hay AJ, Gordon AH, McCrae SI, Wood TM. 1995. d-Xylan- degrading enzyme system from the fungus Phanerochaete chrysosporium. isolation and partial characterisation of an α-(4-O-methyl)-d-glucuronidase. Journal of Biotechnology 43:183-194. 60. Caufrier F, Martinou A, Dupont C, Bouriotis V. 2003. Carbohydrate esterase family 4 enzymes: substrate specificity. Carbohydrate research 338:687-692. 61. Chan CH, Yu TH, Wong KB. 2011. Stabilizing salt-bridge enhances protein thermostability by reducing the heat capacity change of unfolding. PLoS One 6:e21624. 62. Chandrakant P, Bisaria V. 1998. Simultaneous bioconversion of cellulose and hemicellulose to ethanol. Critical reviews in biotechnology 18:295-331. 63. Chang P, Tsai W-S, Tsai C-L, Tseng M-J. 2004. Cloning and characterization of two thermostable xylanases from an alkaliphilic Bacillus firmus. Biochemical and biophysical research communications 319:1017-1025. 64. Chávez R, Schachter K, Navarro C, Peirano A, Aguirre C, Bull P, Eyzaguirre J. 2002. Differences in expression of two endoxylanase genes (xynA and xynB) from Penicillium purpurogenum. Gene 293:161-168. 65. Chen H-m, Ford C, Reilly PJ. 1995. Identification and elimination by site- directed mutagenesis of thermolabile aspartyl bonds in Aspergillus awamori glucoamylase. Protein engineering 8:575-582. 66. Chen K, Liu S, Ma J, Zhang D, Shi Z, Du G, Chen J. 2012. Deletion combined with saturation mutagenesis of N-terminal residues in transglutaminase from Streptomyces hygroscopicus results in enhanced activity and thermostability. Process Biochemistry 47:2329-2334.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 292 References

67. Cheng Y-F, Yang C-H, Liu W-H. 2005. Cloning and expression of Thermobifida xylanase gene in the methylotrophic yeast Pichia pastoris. Enzyme and microbial technology 37:541-546. 68. Cheng Y-S, Chen C-C, Huang C-H, Ko T-P, Luo W, Huang J-W, Liu J-R, Guo R-T. 2014. Structural Analysis of a Glycoside Hydrolase Family 11 Xylanase from Neocallimastix patriciarum insights into the molecular basis of a thermophilic enzyme. Journal of Biological Chemistry 289:11020-11028. 69. Cherry JR, Fidantsef AL. 2003. Directed evolution of industrial enzymes: an update. Current Opinion in Biotechnology 14:438-443. 70. Choudhury B, Chauhan S, Singh S, Ghosh P. 2006. Production of Xylanase of Bacillus coagulans and its bleaching potential. World Journal of Microbiology and Biotechnology 22:283-288. 71. Christiansen C, Abou Hachem M, Janeček Š, Viksø‐Nielsen A, Blennow A, Svensson B. 2009. The carbohydrate‐binding module family 20–diversity, structure, and function. Febs Journal 276:5006-5029. 72. Christov L, Biely P, Kalogeris E, Christakopoulos P, Prior B, Bhat M. 2000. Effects of purified endo-β-1, 4-xylanases of family 10 and 11 and acetyl xylan esterases on eucalypt sulfite dissolving pulp. Journal of biotechnology 83:231- 244. 73. Christov L, Szakacs G, Balakrishnan H. 1999. Production, partial characterization and use of fungal cellulase-free xylanases in pulp bleaching. Process Biochemistry 34:511-517. 74. Christov LP, Prior BA. 1993. Esterases of xylan-degrading microorganisms: production, properties, and significance. Enzyme and microbial technology 15:460-475. 75. Claeyssens M, Henrissat B. 1992. Specificity mapping of cellulolytic enzymes: classification into families of structurally related proteins confirmed by biochemical analysis. Protein Science 1:1293-1297. 76. Cohen SN, Chang AC, Hsu L. 1972. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A 69:2110-2114. 77. Coleri A, Cokmus C, Ozcan B, Akkoc N, Akcelik M. 2009. Isolation of alpha- glucosidase-producing thermophilic bacilli from hot springs of Turkey. Mikrobiologiia 78:68-78.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 293 References

78. Collins T, Gerday C, Feller G. 2005. Xylanases, xylanase families and extremophilic xylanases. FEMS microbiology reviews 29:3-23. 79. Collins T, Meuwis M-A, Gerday C, Feller G. 2003. Activity, stability and flexibility in glycosidases adapted to extreme thermal environments. Journal of molecular biology 328:419-428. 80. Connerton I, Cummings N, Harris GW, Debeire P, Breton C. 1999. A single domain thermophilic xylanase can bind insoluble xylan: evidence for surface aromatic clusters. Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology 1433:110-121. 81. Corral OL, Villaseñor-Ortega F. 2006. Xylanases. Advances in Agricultural and Food Biotechnology 305-322. 82. Coutinho P, Henrissat B. 1999. Carbohydrate-active enzymes server at URL: http://afmb. cnrs-mrs. fr/~ pedro. CAZY/db. html. 83. Crepin V, Faulds C, Connerton I. 2004. Functional classification of the microbial feruloyl esterases. Applied Microbiology and Biotechnology 63:647- 652. 84. Csiszar E, Urbánszki K, Szakacs G. 2001. Biotreatment of desized cotton fabric by commercial cellulase and xylanase enzymes. Journal of Molecular Catalysis B: Enzymatic 11:1065-1072. 85. da Silva AR, Ferro JA, Reinach F, Farah C, Furlan L, Quaggio R, Monteiro-Vitorello C, Van Sluys M, Almeida Na, Alves L. 2002. Comparison of the genomes of two Xanthomonas pathogens with differing host specificities. Nature 417:459-463. 86. Dalbøge H. 1997. Expression cloning of fungal enzyme genes; a novel approach for efficient isolation of enzyme genes of industrial relevance. FEMS microbiology reviews 21:29-42. 87. Damiano V, Bocchini D, Gomes E, Da Silva R. 2003. Application of crude xylanase from Bacillus licheniformis 77-2 to the bleaching of eucalyptus Kraft pulp. World Journal of Microbiology and Biotechnology 19:139-144. 88. De Araújo JH, De Moraes FF, Zanin GM. 1999. Twentieth Symposium on Biotechnology for Fuels and Chemicals: 713-722 89. de Segura BG, Durand R, Fèvre M. 1998. Multiplicity and expression of xylanases in the rumen fungus Neocallimastix frontalis. FEMS microbiology letters 164:47-53.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 294 References

90. de Souza-Cruz PB, Freer J, Siika-Aho M, Ferraz A. 2004. Extraction and determination of enzymes produced by Ceriporiopsis subvermispora during biopulping of Pinus taeda wood chips. Enzyme and Microbial Technology 34:228-234. 91. de Vargas Andrade S, de Moraes MdLT, Terenzi HF, Jorge JAl. 2004. Effect of carbon source on the biochemical properties of β-xylosidases produced by Aspergillus versicolor. Process biochemistry 39:1931-1938. 92. de Vries RP, Kester HC, Poulsen CH, Benen JA, Visser J. 2000. Synergy between enzymes from Aspergillus involved in the degradation of plant cell wall polysaccharides. Carbohydrate research 327:401-410. 93. Debyser W, Derdelinckx G, Delcour J. 1997. Arabinoxylan solubilization and inhibition of the barley malt xylanolytic system by wheat during mashing with wheat wholemeal adjunct: evidence for a new class of enzyme inhibitors in wheat. Journal of the American Society of Brewing Chemists 55:153-156. 94. Defaye J, Guillot J-M, Biely P, Vršanská M. 1992. Positional isomers of thioxylobiose, their synthesis and inducing ability for D-xylan-degrading enzymes in the yeast Cryptococcus albidus. Carbohydrate research 228:47-64. 95. Dekker RF, Richards GN. 1976. Hemicellulases: their occurrence, purification, properties, and mode of action. Advances in Carbohydrate Chemistry and Biochemistry 32:277-352. 96. Delbes C, Moletta R, Godon JJ. 2000. Monitoring of activity dynamics of an anaerobic digester bacterial community using 16S rRNA polymerase chain reaction--single-strand conformation polymorphism analysis. Environmental Microbiology 2:506-515. 97. Demuner BJ, Pereira Junior N, Antunes A. 2011. Technology prospecting on enzymes for the pulp and paper industry. Journal of technology management & innovation 6:148-158. 98. Derewenda U, Swenson L, Green R, Wei Y, Morosoli R, Shareck F, Kluepfel D, Derewenda ZS. 1994. Crystal structure, at 2.6-A resolution, of the Streptomyces lividans xylanase A, a member of the F family of beta-1, 4-D- glycanases. Journal of Biological Chemistry 269:20811-20814. 99. Dervilly G, Leclercq C, Zimmermann D, Roue C, Thibault J-F, Saulnier L. 2002. Isolation and characterization of high molar mass water-soluble arabinoxylans from barley and barley malt. Carbohydrate polymers 47:143-149.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 295 References

100. Dhillon A, Gupta J, Jauhari B, Khanna S. 2000. A cellulase-poor, thermostable, alkalitolerant xylanase produced by Bacillus circulans AB 16 grown on rice straw and its application in biobleaching of eucalyptus pulp. Bioresource Technology 73:273-277. 101. Doi RH, Goldstein M, Hashida S, Park J-S, Takagi M. 1994. The Clostridium cellulovorans cellulosome. Critical reviews in microbiology 20:87- 93. 102. Doi RH, Kosugi A, Murashima K, Tamaru Y, Han SO. 2003. Cellulosomes from mesophilic bacteria. Journal of bacteriology 185:5907-5914. 103. Doi RH, Kosugi A. 2004. Cellulosomes: plant-cell-wall-degrading enzyme complexes. Nature Reviews Microbiology 2:541-551. 104. Doi RH, Park J-S, Liu C-c, Malburg Jr LM, Tamaru Y, Ichiishi A, Ibrahim A. 1998. Cellulosome and noncellulosomal cellulases of Clostridium cellulovorans. Extremophiles 2:53-60. 105. Doi RH, Tamaru Y. 2001. The Clostridium cellulovorans cellulosome: an enzyme complex with plant cell wall degrading activity. Chem Rec 1:24-32. 106. Dowzer C, Kelly JM. 1991. Analysis of the creA gene, a regulator of carbon catabolite repression in Aspergillus nidulans. Molecular and Cellular Biology 11:5701-5709. 107. Duarte MCT, da Silva EC, Ponezi AN, Portugal EP, Vicente JR, Davanzo E. 2003. Xylan-hydrolyzing enzyme system from Bacillus pumilus CBMAI 0008 and its effects on Eucalyptus grandis kraft pulp for pulp bleaching improvement. Bioresource technology 88:9-15. 108. Elegir G, Szakacs G, Jeffries TW. 1994. Purification, Characterization, and Substrate Specificities of Multiple Xylanases from Streptomyces sp. Strain B- 12-2. Applied and Environmental Microbiology 60:2609-2615. 109. Elgharbi F, Hlima HB, Farhat-Khemakhem A, Ayadi-Zouari D, Bejar S, Hmida-Sayari A. 2015. Expression of A. niger US368 xylanase in E. coli: purification, characterization and copper activation. International Journal of Biological Macromolecules 74:263-270. 110. Esteban R, Villanueva JR, Villa TG. 1982. β-D-Xylanases of Bacillus circulans WL-12. Canadian Journal of Microbiology 28:733-739. 111. F. L. Motta CCPA, M. H. A. Santana. 2013. A Review of Xylanase Production by the Fermentation of Xylan: Classification, Characterization and

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 296 References

Applications, Sustainable Degradation of Lignocellulosic Biomass-Techniques, Applications and Commercialization. Anuj K. Chandel. 112. Fenel F, Leisola M, Jänis J, Turunen O. 2004. A de novo designed N-terminal disulphide bridge stabilizes the Trichoderma reesei endo-1, 4-β-xylanase II. Journal of biotechnology 108:137-143. 113. Fernández-Espinar MT, Piñaga F, Sanz P, Ramón D, Vallés S. 1993. Purification and characterization of a neutral endoxylanase from Aspergillus nidulans. FEMS microbiology letters 113:223-228. 114. Flint H, Martin J, McPherson C, Daniel A, Zhang J. 1993. A bifunctional enzyme, with separate xylanase and beta (1, 3-1, 4)-glucanase domains, encoded by the xynD gene of Ruminococcus flavefaciens. Journal of bacteriology 175:2943-2951. 115. Flores ME, Perea M, Rodríguez O, Malváez A. 1996. Physiological studies on induction and catabolite repression of β-xylosidase and endoxylanase in Streptomyces sp. CH-M-1035. Journal of biotechnology 49:179-187. 116. Fontes C, Hall J, Hirst B, Hazlewood G, Gilbert H. 1995. The resistance of cellulases and xylanases to proteolytic inactivation. Applied microbiology and biotechnology 43:52-57. 117. Fu J, Yang X, Yu C. 2008. Preliminary research on bamboo degumming with xylanase. Biocatalysis and Biotransformation 26:450-454. 118. Fujimoto Z, Jackson A, Michikawa M, Maehara T, Momma M, Henrissat B, Gilbert HJ, Kaneko S. 2013. The structure of a Streptomyces avermitilis α- L-rhamnosidase reveals a novel carbohydrate-binding module CBM67 within the six-domain arrangement. Journal of Biological Chemistry 288:12376-12385. 119. Fushinobu S, Ito K, Konno M, Wakagi T, Matsuzawa H. 1998. Crystallographic and mutational analyses of an extremely acidophilic and acid- stable xylanase: biased distribution of acidic residues and importance of Asp37 for catalysis at low pH. Protein Engineering 11:1121-1128. 120. Ganga M, Pinaga F, Valles S, Ramón D, Querol A. 1999. Aroma improving in microvinification processes by the use of a recombinant wine yeast strain expressing the Aspergillus nidulans xlnA gene. International journal of food microbiology 47:171-178.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 297 References

121. Garg A, McCarthy A, Roberts J. 1996. Biobleaching effect of Streptomyces thermoviolaceus xylanase preparations on birchwood kraft pulp. Enzyme and microbial technology 18:261-267. 122. Garg S, Gupta R, Jain K. 2009. Sublethal effects of heavy metals on biochemical composition and their recovery in Indian major carps. Journal of hazardous materials 163:1369-1384. 123. Gebler J, Gilkes NR, Claeyssens M, Wilson DB, Béguin P, Wakarchuk WW, Kilburn DG, Miller RC, Warren R, Withers SG. 1992. Stereoselective hydrolysis catalyzed by related beta-1, 4-glucanases and beta-1, 4-xylanases. Journal of Biological Chemistry 267:12559-12561. 124. Georis J, Giannotta F, De Buyl E, Granier Bt, Frère J-M. 2000. Purification and properties of three endo-β-1, 4-xylanases produced by Streptomyces sp. strain S38 which differ in their ability to enhance the bleaching of kraft pulps. Enzyme and microbial technology 26:178-186. 125. Gerasimova J, Kuisiene N. 2012. Characterization of the novel xylanase from the thermophilic Geobacillus thermodenitrificans JK1. Microbiology 81:418- 424. 126. Gessesse A, Mamo G. 1998. Purification and characterization of an alkaline xylanase from alkaliphilic Micrococcus sp. AR-135. Journal of Industrial Microbiology and Biotechnology 20:210-214. 127. Ghatora SK, Chadha BS, Badhan A, Saini H, Bhat M. 2007. Identification and characterization of diverse xylanases from thermophilic and thermotolerant fungi. BioResources 1:18-33. 128. Ghosh M, Nanda G. 1994. Physiological studies on xylose induction and glucose repression of xylanolytic enzymes in Aspergillus sydowii MG49. FEMS microbiology letters 117:151-156. 129. Gilbert HJ, Hazlewood GP. 1993. Bacterial cellulases and xylanases. Microbiology 139:187-194. 130. Gilbert HJ, Sullivan DA, Jenkins G, Kellett LE, Minton NP, Hall J. 1988. Molecular cloning of multiple xylanase genes from Pseudomonas fluorescens subsp. cellulosa. Microbiology 134:3239-3247. 131. Goihberg E, Dym O, Tel‐Or S, Levin I, Peretz M, Burstein Y. 2007. A single proline substitution is critical for the thermostabilization of Clostridium

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 298 References

beijerinckii alcohol dehydrogenase. Proteins: Structure, Function, and Bioinformatics 66:196-204. 132. Goihberg E, Dym O, Tel‐Or S, Levin I, Peretz M, Burstein Y. 2007. A single proline substitution is critical for the thermostabilization of Clostridium beijerinckii alcohol dehydrogenase. Proteins: Structure, Function, and Bioinformatics 66:196-204. 133. Gomes D, Gomes J, Steiner W. 1994. Factors influencing the induction of endo-xylanase by Thermoascus aurantiacus. Journal of biotechnology 33:87-94. 134. Gomes J, Gomes I, Steiner W. 2000. Thermolabile xylanase of the Antarctic yeast Cryptococcus adeliae: production and properties. Extremophiles 4:227- 235. 135. Gote MM. 2004. Isolation, purification and characterization of thermostable α- galactosidase from Bacillus stearothermophilus (NCIM-5146). CSIR-National Chemical Laboratory, Pune, India. 136. Graaff LK, Broeck HC, Ooijen AJ, Visser J. 1994. Regulation of the xylanase‐encoding xlnA gene of Aspergilius tubigensis. Molecular microbiology 12:479-490. 137. Grépinet O, Chebrou M-C, Béguin P. 1988. Purification of Clostridium thermocellum xylanase Z expressed in Escherichia coli and identification of the corresponding product in the culture medium of C. thermocellum. Journal of bacteriology 170:4576-4581. 138. Gruber K, Klintschar G, Hayn M, Schlacher A, Steiner W, Kratky C. 1998. Thermophilic xylanase from Thermomyces lanuginosus: high-resolution X-ray structure and modeling studies. Biochemistry 37:13475-13485. 139. Guérin DM, Lascombe M-B, Costabel M, Souchon H, Lamzin V, Béguin P, Alzari PM. 2002. Atomic (0.94 Å) resolution structure of an inverting glycosidase in complex with substrate. Journal of molecular biology 316:1061- 1069. 140. Guo B, Chen XL, Sun CY, Zhou BC, Zhang YZ. 2009. Gene cloning, expression and characterization of a new cold-active and salt-tolerant endo-beta- 1,4-xylanase from marine Glaciecola mesophila KMM 241. Appl Microbiol Biotechnol 84:1107-1115. 141. Gupta N, Vohra R, Hoondal G. 1992. A thermostable extracellular xylanase from alkalophilic Bacillus sp. NG-27. Biotechnology letters 14:1045-1046.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 299 References

142. Gupta S, Bhushan B, Hoondal G. 2000. Isolation, purification and characterization of xylanase from Staphylococcus sp. SG‐13 and its application in biobleaching of kraft pulp. Journal of Applied Microbiology 88:325-334. 143. Haarhoff J, Moes C, Cerff C, Van Wyk W, Gerischer G, Janse B. 1999. Characterization and biobleaching effect of hemicellulases produced by thermophilic fungi. Biotechnology letters 21:415-420. 144. Hadfield AT, Mulholland AJ. 1999. Active‐site dynamics of ASADH—A bacterial biosynthetic enzyme. International journal of quantum chemistry 73:137-146. 145. Hakulinen N, Turunen O, Jänis J, Leisola M, Rouvinen J. 2003. Three‐ dimensional structures of thermophilic β‐1, 4‐xylanases from Chaetomium thermophilum and Nonomuraea flexuosa. European journal of biochemistry 270:1399-1412. 146. Haltrich D, Nidetzky B, Kulbe KD, Steiner W, Župančič S. 1996. Production of fungal xylanases. Bioresource Technology 58:137-161. 147. Han R, Liu L, Shin H-d, Chen RR, Li J, Du G, Chen J. 2013. Systems engineering of tyrosine 195, tyrosine 260, and glutamine 265 in cyclodextrin glycosyltransferase from Paenibacillus macerans to enhance maltodextrin specificity for 2-Od-glucopyranosyl-l-ascorbic acid synthesis. Applied and environmental microbiology 79:672-677. 148. Harbak L, Thygesen H. 2002. Safety evaluation of a xylanase expressed in Bacillus subtilis. Food and chemical toxicology 40:1-8. 149. Harris AD, Ramalingam C. 2010. Xylanases and its application in food industry: a review. Journal of Experimental Sciences 1 (7):1-11. 150. Harris GW, Pickersgill RW, Connerton I, Debeire P, Touzel J-P, Breton C, Perez S. 1997. Structural basis of the properties of an industrially relevant thermophilic xylanase. Proteins Structure Function and Genetics 29:77-86. 151. Hayashi H, Takagi K-I, Fukumura M, Kimura T, Karita S, Sakka K, Ohmiya K. 1997. Sequence of xynC and properties of XynC, a major component of the Clostridium thermocellum cellulosome. Journal of bacteriology 179:4246-4253. 152. Headon DR, Walsh G. 1994. The industrial production of enzymes. Biotechnol Adv 12:635-646.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 300 References

153. Henrissat B, Claeyssens M, Tomme P, Lemesle L, Mornon J-P. 1989. Cellulase families revealed by hydrophobic cluster analysi. Gene 81:83-95. 154. Henrissat B, Coutinho PM. 2001. Classification of glycoside hydrolases and glycosyltransferases from hyperthermophiles. Methods in enzymology 330:183- 201. 155. Hida K, Hanes J, Ostermeier M. 2007. Directed evolution for drug and nucleic acid delivery. Advanced drug delivery reviews 59:1562-1578. 156. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR. 1989. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51-59. 157. Hoppe-Seyler E. 1889. Hemicellulose degrading microorganisms from river mud. Hoppe-Seylers Zeitschrift für Physiologische Chemie 3:65-101. 158. Horikoshi K. 1996. Alkaliphiles—from an industrial point of view. FEMS Microbiology Reviews 18:259-270. 159. Hrmová M, Biely P, Vršanská M, Petráková E. 1984. Induction of cellulose- and xylan-degrading enzyme complex in the yeast Trichosporon cutaneum. Archives of microbiology 138:371-376. 160. Hrmová M, Petráková E, Biely P. 1991. Induction of cellulose-and xylan- degrading enzyme systems in Aspergillus terreus by homo-and heterodisaccharides composed of glucose and xylose. Microbiology 137:541- 547. 161. Hsieh P-C, Vaisvila R. 2013. Protein engineering: single or multiple site- directed mutagenesis. Enzyme Engineering: Methods and Protocols:173-186. 162. Ihsanawati, Kumasaka T, Kaneko T, Morokuma C, Yatsunami R, Sato T, Nakamura S, Tanaka N. 2005. Structural basis of the substrate subsite and the highly thermal stability of xylanase 10B from Thermotoga maritima MSB8. Proteins 61:999-1009. 163. Irfan M, Guler HI, Aysegul O, Sapmaz MT, Belduz AO, Hasan F, Shah AA. C-Terminal Proline-Rich Sequence Broadens the Optimal Temperature and pH Ranges of Recombinant Xylanase from Geobacillus thermodenitrificans C5. Enzyme and Microbial Technology. 164. Irfan M, Guler HI, Aysegul O, Sapmaz MT, Belduz AO, Hasan F, Shah AA. C-Terminal Proline-Rich Sequence Broadens the Optimal Temperature and

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 301 References

pH Ranges of Recombinant Xylanase from Geobacillus thermodenitrificans C5. Enzyme and Microbial Technology. 165. Irfan M, Guler HI, Belduz AO, Shah AA, Canakci S. 2016. Cloning, purification and characterization of a cellulase-free xylanase from Geobacillus thermodenitrificans AK53. Applied Biochemistry and Microbiology 52:277- 286. 166. Irfan M, Guler HI, Shah AA, Sal FA, Inan K, Belduz AO. 2016. Cloning, purification and characterization of halotolerant xylanase from Geobacillus Thermodenitrificans C5. Journal of Microbiology, Biotechnology and Food Sciences 5:523-529.

167. Irwin D, Jung ED, Wilson DB. 1994. Characterization and sequence of a Thermomonospora fusca xylanase. Applied and Environmental Microbiology 60:763-770. 168. Jalal A, Rashid N, Rasool N, Akhtar M. 2009. Gene cloning and characterization of a xylanase from a newly isolated Bacillus subtilis strain R5. Journal of bioscience and bioengineering 107:360-365. 169. Jhamb K, Sahoo DK. 2012. Production of soluble recombinant proteins in Escherichia coli: effects of process conditions and chaperone co-expression on cell growth and production of xylanase. Bioresource technology 123:135-143. 170. Joerger M, Klaenhammer TR. 1990. Cloning, expression, and nucleotide sequence of the Lactobacillus helveticus 481 gene encoding the bacteriocin helveticin J. Journal of bacteriology 172:6339-6347.

171. Johannes TW, Zhao H. 2006. Directed evolution of enzymes and biosynthetic pathways. Current opinion in microbiology 9:261-267. 172. Joo JC, Pack SP, Kim YH, Yoo YJ. 2011. Thermostabilization of Bacillus circulans xylanase: computational optimization of unstable residues based on thermal fluctuation analysis. Journal of biotechnology 151:56-65. 173. Joshi C, Khare S. 2011. Utilization of deoiled Jatropha curcas seed cake for production of xylanase from thermophilic Scytalidium thermophilum. Bioresource technology 102:1722-1726. 174. Kadowaki M, Pacheco M, Peralta R. 1995. Xylanase production by Aspergillus isolates grown on corn cob. Revista de Microbiologia (Brazil) 263:219-223.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 302 References

175. Kalim B, Böhringer N, Ali N, Schäberle TF. 2015. Xylanases–from microbial origin to Industrial application. British Biotechnology. J 7:1-20. 176. Kamble RD, Jadhav AR. 2012. Properties and Application of a Partially Purified Thermoalkali Stable Xylanase from Cellulosimicrobium sp. MTCC 10645 in Kraft Pulp Bleaching. ISRN biotechnology 2013. 177. Kaneko S, Shimasaki T, Kusakabe I. 1993. Purification and some properties of intracellular α-L-arabinofuranosidase from Aspergillus niger 5–16. Bioscience, biotechnology, and biochemistry 57:1161-1165. 178. Kansoh AL, Nagieb ZA. 2004. Xylanase and mannanase enzymes from Streptomyces galbus NR and their use in biobleaching of softwood kraft pulp. Antonie Van Leeuwenhoek 85:103-114. 179. Kapilan R, Arasaratnam V. 2015. Improvement of Bacillus pumilus for Higher Xylanase Production by Mutation. Jacobs Journal of Enzymology and Enzyme Engineering 1:003. 180. Kar S, Mandal A, Mohapatra PKD, Samanta S, Pati BR, Mondal KC. 2008. Production of xylanase by immobilized Trichoderma reesei SAF3 in Ca-alginate beads. Journal of industrial microbiology & biotechnology 35:245-249. 181. Karan R, Capes MD, DasSarma S. 2012. Function and biotechnology of extremophilic enzymes in low water activity. Aquatic Biosystems 8:1-15. 182. Karaoglu H, Yanmis D, Sal FA, Celik A, Canakci S, Belduz AO. 2013. Biochemical characterization of a novel glucose isomerase from Anoxybacillus gonensis G2 T that displays a high level of activity and thermal stability. Journal of Molecular Catalysis B: Enzymatic 97:215-224. 183. Kashyap R, Subudhi E. 2014. A novel thermoalkaliphilic xylanase from Gordonia sp. is salt, solvent and surfactant tolerant. Journal of basic microbiology 54:1342-1349. 184. Kaur A, Mahajan R, Singh A, Garg G, Sharma J. 2010. Application of cellulase-free xylano-pectinolytic enzymes from the same bacterial isolate in biobleaching of kraft pulp. Bioresource technology 101:9150-9155. 185. Kaur A, Mahajan R, Singh A, Garg G, Sharma J. 2011. A novel and cost effective methodology for qualitative screening of alkalo-thermophilic cellulase free xylano-pectinolytic microorganisms using agricultural wastes. World Journal of Microbiology and Biotechnology 27:459-463.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 303 References

186. Kavya V, Padmavathi T. 2009. Optimization of growth conditions for xylanase production by Aspergillus niger in solid state fermentation. Polish Journal of Microbiology 58 (2):125-130. 187. Kazuyo F, Hong SY, Yeon YJ, Joo JC, Yoo YJ. 2014. Enhancing the activity of Bacillus circulans xylanase by modulating the flexibility of the hinge region. Journal of industrial microbiology & biotechnology 41:1181-1190. 188. Khandeparkar R, Bhosle NB. 2006. Purification and characterization of thermoalkalophilic xylanase isolated from the Enterobacter sp. MTCC 5112. Research in Microbiology 157:315-325. 189. Khandeparker R, Verma P, Deobagkar D. 2011. A novel halotolerant xylanase from marine isolate Bacillus subtilis cho40: gene cloning and sequencing. New Biotechnology 28:814-821. 190. Khasin A, Alchanati I, Shoham Y. 1993. Purification and characterization of a thermostable xylanase from Bacillus stearothermophilus T-6. Applied and Environmental Microbiology 59:1725-1730. 191. Kim Y-M, Shimizu R, Nakai H, Mori H, Okuyama M, Kang M-S, Fujimoto Z, Funane K, Kim D, Kimura A. 2011. Truncation of N-and C-terminal regions of Streptococcus mutans dextranase enhances catalytic activity. Applied microbiology and biotechnology 91:329-339. 192. Kimura T, Ito J, Kawano A, Makino T, Kondo H, Karita S, Sakka K, Ohmiya K. 2000. Purification, characterization, and molecular cloning of acidophilic xylanase from Penicillium sp. 40. Bioscience, biotechnology, and biochemistry 64:1230-1237. 193. Knob A, Beitel SM, Fortkamp D, Terrasan CRF, Almeida AFd. 2013. Production, purification, and characterization of a major Penicillium glabrum xylanase using Brewer's spent grain as substrate. BioMed research international 2013. 194. Knob A, Carmona EC. 2010. Purification and characterization of two extracellular xylanases from Penicillium sclerotiorum: a novel acidophilic xylanase. Applied Biochemistry and Biotechnology 162:429-443. 195. Kuhad RC, Singh A, Eriksson KE. 1997. Microorganisms and enzymes involved in the degradation of plant fiber cell walls. Advances in Biochemical Engineering/Biotechnology 57:45-125.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 304 References

196. Kulkarni N, Lakshmikumaran M, Rao M. 1999. Xylanase II from an alkaliphilic thermophilic Bacillus with a distinctly different structure from other xylanases: evolutionary relationship to alkaliphilic xylanases. Biochemical and biophysical research communications 263:640-645. 197. Kulkarni N, Rao M. 1996. Application of xylanase from alkaliphilic thermophilic Bacillus sp. NCIM 59 in biobleaching of bagasse pulp. Journal of biotechnology 51:167-173. 198. Kulkarni N, Shendye A, Rao M. 1999. Molecular and biotechnological aspects of xylanases. FEMS microbiology reviews 23:411-456. 199. Kumar PR, Eswaramoorthy S, Vithayathil PJ, Viswamitra M. 2000. The tertiary structure at 1.59 Å resolution and the proposed amino acid sequence of a family-11 xylanase from the thermophilic fungus Paecilomyces varioti Bainier. Journal of molecular biology 295:581-593. 200. Kumar S, Tamura K, Nei M. 2004. MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform 5:150-163. 201. Kumar V, Marin-Navarro J, Shukla P. 2016. Thermostable microbial xylanases for pulp and paper industries: trends, applications and further perspectives. World J Microbiol Biotechnol 32:34. 202. Kunst F, Ogasawara N, Moszer I, Albertini A, Alloni G, Azevedo V, Bertero M, Bessieres P, Bolotin A, Borchert S. 1997. The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390:249-256. 203. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. nature 227:680-685. 204. Lam SY, Yeung RC, Yu TH, Sze KH, Wong KB. 2011. A rigidifying salt- bridge favors the activity of thermophilic enzyme at high temperatures at the expense of low-temperature activity. PLoS Biol 9:e1001027. 205. Lambert C, Leonard N, De Bolle X, Depiereux E. 2002. ESyPred3D: Prediction of proteins 3D structures. Bioinformatics 18:1250-1256. 206. Lebeda A, Luhová L, Sedlářová M, Jančová D. 2001. The role of enzymes in plant-fungal pathogens interactions/Die Rolle der Enzyme in den Beziehungen zwischen Pflanzen und pilzlichen Erregern. Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz Journal of Plant Diseases and Protection 108:89-111.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 305 References

207. Lee CC, Kibblewhite-Accinelli RE, Smith MR, Wagschal K, Orts WJ, Wong DW. 2008. Cloning of Bacillus licheniformis xylanase gene and characterization of recombinant enzyme. Current Microbiology 57:301-305. 208. Lee J. 1997. Biological conversion of lignocellulosic biomass to ethanol. Journal of biotechnology 56:1-24. 209. Leggio LL, Kalogiannis S, Bhat M, Pickersgill R. 1999. High resolution structure and sequence of T. aurantiacus Xylanase I: Implications for the evolution of thermostability in family 10 xylanases and enzymes with βα‐barrel architecture. Proteins: Structure, Function, and Bioinformatics 36:295-306. 210. Lenartovicz V, Marques De Souza CG, Moreira FG, Peralta RM. 2002. Temperature effect in the production of multiple xylanases by Aspergillus fumigatus. Journal of basic microbiology 42:388-395. 211. Li F, Xie J, Zhang X, Zhao L. 2015. Improvement of the Optimum pH of Aspergillus niger Xylanase towards an Alkaline pH by Site-Directed Mutagenesis. Journal of microbiology and biotechnology 25:11-17. 212. Li X, Jiang Z, Li L, Yang S, Feng W, Fan J, Kusakabe I. 2005. Characterization of a cellulase-free, neutral xylanase from Thermomyces lanuginosus CBS 288.54 and its biobleaching effect on wheat straw pulp. Bioresource Technology 96:1370-1379. 213. Li X, She Y, Sun B, Song H, Zhu Y, Lv Y, Song H. 2010. Purification and characterization of a cellulase-free, thermostable xylanase from Streptomyces rameus L2001 and its biobleaching effect on wheat straw pulp. Biochemical Engineering Journal 52:71-78. 214. Li XR, Xu H, Xie J, Yi QF, Li W, Qiao DR, Cao Y, Cao Y. 2014. Thermostable sites and catalytic characterization of xylanase XYNB of Aspergillus niger SCTCC 400264. Journal of Microbiology and Biotechnology 24:483-488. 215. Liab K, Azadi P, Collins R, Tolan J, Kim JS, Eriksson KL. 2000. Relationships between activities of xylanases and xylan structures. Enzyme and Microbial Technology 27:89-94. 216. Liao H, Sun S, Wang P, Bi W, Tan S, Wei Z, Mei X, Liu D, Raza W, Shen Q. 2014. A new acidophilic endo-β-1, 4-xylanase from Penicillium oxalicum: cloning, purification, and insights into the influence of metal ions on xylanase activity. Journal of industrial microbiology & biotechnology 41:1071-1083.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 306 References

217. Liao H, Xu C, Tan S, Wei Z, Ling N, Yu G, Raza W, Zhang R, Shen Q, Xu Y. 2012. Production and characterization of acidophilic xylanolytic enzymes from Penicillium oxalicum GZ-2. Bioresource technology 123:117-124. 218. Lin X-q, Han S-y, Zhang N, Hu H, Zheng S-p, Ye Y-r, Lin Y. 2013. Bleach boosting effect of xylanase A from Bacillus halodurans C-125 in ECF bleaching of wheat straw pulp. Enzyme and microbial technology 52:91-98. 219. Lineweaver H, Burk D. 1934. The Determination of Enzyme Dissociation Constants. Journal of the American Chemical Society 56:658-666. 220. Linke C, Siemens N, Oehmcke S, Radjainia M, Law RH, Whisstock JC, Baker EN, Kreikemeyer B. 2012. The extracellular protein factor Epf from Streptococcus pyogenes is a cell surface adhesin that binds to cells through an N-terminal domain containing a carbohydrate-binding module. Journal of Biological Chemistry 287:38178-38189. 221. Liu J-H, Selinger BL, Tsai C-F, Cheng K-J. 1999. Characterization of a Neocallimastix patriciarum xylanase gene and its product. Canadian journal of microbiology 45:970-974. 222. Liu W, Shi P, Chen Q, Yang P, Wang G, Wang Y, Luo H, Yao B. 2010. Gene cloning, overexpression, and characterization of a xylanase from Penicillium sp. CGMCC 1669. Applied biochemistry and biotechnology 162:1- 12. 223. Liu Z, Feng T, Ji Q, Cong P, Chen Y, He Z. 2013. Introduction of silent mutations in a codon-optimized xylanase (xynB) results in enhanced protein expression in HEK293A cells. Biotechnology letters 35:2105-2111.

224. Lu F, Lu M, Lu Z, Bie X, Zhao H, Wang Y. 2008. Purification and characterization of xylanase from Aspergillus ficuum AF-98. Bioresource Technology 99:5938-5941. 225. Luo H, Li J, Yang J, Wang H, Yang Y, Huang H, Shi P, Yuan T, Fan Y, Yao B. 2009. A thermophilic and acid stable family-10 xylanase from the acidophilic fungus Bispora sp. MEY-1. Extremophiles 13:849-857. 226. Luthi E, Jasmat NB, Bergquist PL. 1990. Xylanase from the extremely thermophilic bacterium "Caldocellum saccharolyticum": overexpression of the gene in Escherichia coli and characterization of the gene product. Applied and Environmental Microbiology 56:2677-2683.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 307 References

227. Lüthi E, Jasmat NB, Bergquist PL. 1990. Xylanase from the extremely thermophilic bacterium" Caldocellum saccharolyticum": overexpression of the gene in Escherichia coli and characterization of the gene product. Applied and environmental microbiology 56:2677-2683. 228. Luthi E, Love DR, McAnulty J, Wallace C, Caughey PA, Saul D, Bergquist PL. 1990. Cloning, sequence analysis, and expression of genes encoding xylan- degrading enzymes from the thermophile "Caldocellum saccharolyticum". Applied and Environmental Microbiology 56:1017-1024. 229. Maalej-Achouri I, Guerfali M, Gargouri A, Belghith H. 2009. Production of xylo-oligosaccharides from agro-industrial residues using immobilized Talaromyces thermophilus xylanase. Journal of Molecular Catalysis B: Enzymatic 59:145-152. 230. Maat J RM, Verbakel J, Stam H, Santos da Silva MJ, Bosse M, Egmond MR, Hagemans MLD, van Gorcom RFM, Hessing JGM, van den Hondel CAMJJ, van Rotterdam C 1991. Xylanases and their application in bakery, Xylans and xylanases. Visser J, Beldman G, Someren Kusters-van MA, Voragem AGJ, Elsevier Amsterdam.

231. Mach RL, Strauss J, Zeilinger S, Schindler M, Kubicek CP. 1996. Carbon catabolite repression of xylanase I (xyn1) gene expression in Trichoderma reesei. Molecular microbiology 21:1273-1281. 232. Mamo G, Hatti-Kaul R, Mattiasson B. 2007. Fusion of carbohydrate binding modules from Thermotoga neapolitana with a family 10 xylanase from Bacillus halodurans S7. Extremophiles 11:169-177. 233. Marcolongo L, La Cara F, Morana A, Di Salle A, del Monaco G, Paixão SM, Alves L, Ionata E. 2015. Properties of an alkali-thermo stable xylanase from Geobacillus thermodenitrificans A333 and applicability in xylooligosaccharides generation. World Journal of Microbiology and Biotechnology 31:633-648. 234. Mathrani IM, Ahring BK. 1992. Thermophilic and alkalophilic xylanases from several Dictyoglomus isolates. Applied microbiology and biotechnology 38:23-27. 235. Matsutani M, Hirakawa H, Nishikura M, Soemphol W, Ali IA, Yakushi T, Matsushita K. 2011. Increased number of Arginine-based salt bridges

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 308 References

contributes to the thermotolerance of thermotolerant acetic acid bacteria, Acetobacter tropicalis SKU1100. Biochem Biophys Res Commun 409:120-124. 236. Matte A, Forsberg CW. 1992. Purification, characterization, and mode of action of endoxylanases 1 and 2 from Fibrobacter succinogenes S85. Applied and Environmental Microbiology 58:157-168. 237. McCarthy AA, Morris DD, Bergquist PL, Baker EN. 2000. Structure of XynB, a highly thermostable β-1, 4-xylanase from Dictyoglomus thermophilum Rt46B. 1, at 1.8 Å resolution. Acta Crystallographica Section D: Biological Crystallography 56:1367-1375. 238. McDonough T. 1992. Bleaching agents (pulp and paper). In: Kirk-Othmer Encyclopedia of Chemical Technology; Grayson, M., Ed., 4th ed.; John Wiley and Sons: New York, 301–311. 239. Medeiros R, Soffner M, Thomé J, Cacais A, Estelles R, Salles B, Ferreira H, Neto S, Silva F. 2000. The production of hemicellulases by aerobic fungi on medium containing residues of banana plant as substrate. Biotechnology progress 16:522-524. 240. Menon G, Mody K, Keshri J, Jha B. 2010. Isolation, purification, and characterization of haloalkaline xylanase from a marine Bacillus pumilus strain, GESF-1. Biotechnology and Bioprocess Engineering 15:998-1005. 241. Miller GL. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical chemistry 31:426-428. 242. Millward-Sadler S, Davidson K, Hazlewood G, Black G, Gilbert H, Clarke J. 1995. Novel cellulose-binding domains, NodB homologues and conserved modular architecture in xylanases from the aerobic soil bacteria Pseudomonas fluorescens subsp. cellulosa and Cellvibrio mixtus. Biochemical journal 312:39- 48. 243. Mishra M, Thakur IS. 2011. Purification, characterization and mass spectroscopic analysis of thermo-alkalotolerant β-1, 4 endoxylanase from Bacillus sp. and its potential for dye decolorization. International Biodeterioration & Biodegradation 65:301-308. 244. Miyazaki K, Takenouchi M, Kondo H, Noro N, Suzuki M, Tsuda S. 2006. Thermal stabilization of Bacillus subtilis family-11 xylanase by directed evolution. Journal of Biological Chemistry 281:10236-10242.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 309 References

245. Morales P, Sendra JM, Perez-Gonzalez JA. 1995. Purification and characterization of an arabinofuranosidase from Bacillus polymyxa expressed in Bacillus subtilis. Appl Microbiology and Biotechnology 44:112-117. 246. Morosoli R, Durand S, Letendre ED. 1987. Induction of xylanase by β- methylxyloside in Cryptococcus albidus. FEMS microbiology letters 48:261- 266. 247. Morris DD, Gibbs MD, Ford M, Thomas J, Bergquist PL. 1999. Family 10 and 11 xylanase genes from Caldicellulosiruptor sp. strain Rt69B. 1. Extremophiles 3:103-111. 248. Mrabet NT, Van den Broeck A, Van den brande I, Stanssens P, Laroche Y, Lambeir AM, Matthijssens G, Jenkins J, Chiadmi M, van Tilbeurgh H, et al. 1992. Arginine residues as stabilizing elements in proteins. Biochemistry 31:2239-2253. 249. Nagar S, Gupta VK, Kumar D, Kumar L, Kuhad RC. 2010. Production and optimization of cellulase-free, alkali-stable xylanase by Bacillus pumilus SV- 85S in submerged fermentation. Journal of industrial microbiology & biotechnology 37:71-83. 250. Nakamura S, Wakabayashi K, Nakai R, Aono R, Horikoshi K. 1993. Purification and some properties of an alkaline xylanase from alkaliphilic Bacillus sp. strain 41M-1. Applied and Environmental Microbiology 59:2311- 2316. 251. Ng I-S, Li C-W, Yeh Y-F, Chen PT, Chir J-L, Ma C-H, Yu S-M, Ho T-hD, Tong C-G. 2009. A novel endo-glucanase from the thermophilic bacterium Geobacillus sp. 70PC53 with high activity and stability over a broad range of temperatures. Extremophiles 13:425-435. 252. Niehaus F, Bertoldo C, Kähler M, Antranikian G. 1999. Extremophiles as a source of novel enzymes for industrial application. Applied microbiology and biotechnology 51:711-729. 253. Ninawe S, Kapoor M, Kuhad RC. 2008. Purification and characterization of extracellular xylanase from Streptomyces cyaneus SN32. Bioresource Technology 99:1252-1258. 254. Ninawe S, Kuhad RC. 2006. Bleaching of wheat straw-rich soda pulp with xylanase from a thermoalkalophilic Streptomyces cyaneus SN32. Bioresource technology 97:2291-2295.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 310 References

255. Nölling J, Breton G, Omelchenko MV, Makarova KS, Zeng Q, Gibson R, Lee HM, Dubois J, Qiu D, Hitti J. 2001. Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum. Journal of bacteriology 183:4823-4838. 256. Nurizzo D, Turkenburg JP, Charnock SJ, Roberts SM, Dodson EJ, McKie VA, Taylor EJ, Gilbert HJ, Davies GJ. 2002. Cellvibrio japonicus α-L- arabinanase 43A has a novel five-blade β-propeller fold. Nature Structural & Molecular Biology 9:665-668. 257. Nuyens F, Van Zyl WH, Iserentant D, Verachtert H, Michiels C. 2001. Heterologous expression of the Bacillus pumilus endo-β-xylanase (xynA) gene in the yeast Saccharomyces cerevisiae. Applied microbiology and biotechnology 56:431-434. 258. Oksanen T, Pere J, Paavilainen L, Buchert J, Viikari L. 2000. Treatment of recycled kraft pulps with Trichoderma reesei hemicellulases and cellulases. Journal of Biotechnology 78:39-48. 259. Olsson L, Hahn-Hägerdal B. 1996. Fermentation of lignocellulosic hydrolysates for ethanol production. Enzyme and Microbial technology 18:312- 331. 260. Paice M, Gurnagul N, Page D, Jurasek L. 1992. Mechanism of hemicellulose- directed prebleaching of kraft pulps. Enzyme and microbial technology 14:272- 276. 261. Parajó JC, Domínguez H, Domínguez J. 1998. Biotechnological production of xylitol. Part 1: Interest of xylitol and fundamentals of its biosynthesis. Bioresource Technology 65:191-201. 262. Passos AA, Park I, Ferket P, von Heimendahl E, Kim SW. 2015. Effect of dietary supplementation of xylanase on apparent ileal digestibility of nutrients, viscosity of digesta, and intestinal morphology of growing pigs fed corn and soybean meal based diet. Animal Nutrition 1:19-23. 263. Patel R, Grabski A, Jeffries T. 1993. Chromophore release from kraft pulp by purified Streptomyces roseiscleroticus xylanases. Applied microbiology and biotechnology 39:405-412. 264. Peij NN, Brinkmann J, Vršanská M, Visser J, Graaff LH. 1997. β‐ Xylosidase activity, encoded by xlnD, is essential for complete hydrolysis of

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 311 References

xylan by Aspergillus niger but not for Induction of the xylanolytic enzyme spectrum. European Journal of Biochemistry 245:164-173. 265. Penstone K. 1996. Zooming in on enzymes. Food Review-Cape Town 23:36- 42. 266. Pérez J, Munoz-Dorado J, de la Rubia T, Martinez J. 2002. Biodegradation and biological treatments of cellulose, hemicellulose and lignin: an overview. International Microbiology 5:53-63. 267. Piñaga F, Fernández-Espinar M, Vallés S, Ramón D. 1994. Xylanase production in Aspergillus nidulans: induction and carbon catabolite repression. FEMS microbiology letters 115:319-323. 268. Polizeli M, Rizzatti A, Monti R, Terenzi H, Jorge JA, Amorim D. 2005. Xylanases from fungi: properties and industrial applications. Applied microbiology and biotechnology 67:577-591. 269. Polizeli ML, Rizzatti AC, Monti R, Terenzi HF, Jorge JA, Amorim DS. 2005. Xylanases from fungi: properties and industrial applications. Applied Microbiology and Biotechnology 67:577-591. 270. Prade RA. 1996. Xylanases: from biology to biotechnology. Biotechnology and Genetic Engineering Reviews 13:101-132. 271. Puls J, Poutanen K. 1989. Mechanisms of enzymatic hydrolysis of hemicelluloses (xylan) and procedures for determination of the enzyme activities involved. In: “Enzyme system for lignocellulose degradation, Coughlan M. P.(Ed).”, Elsevier Applied Science, London, pp. 151–165. 272. Puls J, Schuseil J. 1993. Chemistry of hemicelluloses: relationship between hemicellulose structure and enzymes required for hy- drolysis. In: Coughlsn, M. P.; Hazlewood, G. P. (ed.). Hemicellulose and hemicellulases, London: Portland Press, chapter 1,1993. p.1-27. 273. Puls J. 1993. Chemistry of hemicelluloses: relationship between hemicellulose structure and enzymes required for hydrolysis, p. 1-27. In Coughlan MP, Hazlewood GP (ed.), Hemicellulose and hemicellulases. Portland Press, London. 274. Qinnghe C, Xiaoyu Y, Tiangui N, Cheng J, Qiugang M. 2004. The screening of culture condition and properties of xylanase by white-rot fungus Pleurotus ostreatus. Process Biochemistry 39:1561-1566.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 312 References

275. Qiu J, Han H, Sun B, Chen L, Yu C, Peng R, Yao Q. 2016. Residue mutations of xylanase in Aspergillus kawachii alter its optimum pH. Microbiological research 182:1-7.

276. Qiu Z, Shi P, Luo H, Bai Y, Yuan T, Yang P, Liu S, Yao B. 2010. A xylanase with broad pH and temperature adaptability from Streptomyces megasporus DSM 41476, and its potential application in brewing industry. Enzyme and microbial technology 46:506-512. 277. Rahman MA, Choi YH, Pradeep GC, Choi YS, Choi EJ, Cho SS, Yoo JC. 2014. A novel low molecular weight endo-xylanase from Streptomyces sp. CS628 cultivated in wheat bran. Applied Biochemistry and Biotechnology 173:1469-1480. 278. Raj A, Kumar S, Singh SK. 2013. A Highly Thermostable Xylanase from Stenotrophomonas maltophilia: Purification and Partial Characterization. Enzyme Research 2013:8. 279. Ramchuran SO, Mateus B, Holst O, Karlsson EN. 2005. The methylotrophic yeast Pichia pastoris as a host for the expression and production of thermostable xylanase from the bacterium Rhodothermus marinus. FEMS Yeast Res 5:839- 850. 280. Ramchuran SO, Vargas V, Hatti-Kaul R, Nordberg Karlsson E. 2005. The methylotrophic yeast Pichia pastoris as a host for the expression of a lipolytic enzyme from Bacillus halodurans LBB2. 281. Ramchuran SO, Vargas VA, Hatti-Kaul R, Karlsson EN. 2006. Production of a lipolytic enzyme originating from Bacillus halodurans LBB2 in the methylotrophic yeast Pichia pastoris. Applied Microbiology and Biotechnology 71:463-472. 282. Rani DS, Nand K. 2000. Production of thermostable cellulase-free xylanase by Clostridium absonum CFR-702. Process Biochemistry 36:355-362. 283. Rättö M, Poutanen K, Viikari L. 1992. Production of xylanolytic enzymes by an alkalitolerant Bacillus circulans strain. Applied microbiology and biotechnology 37:470-473. 284. Reilly PJ. 1981. Xylanases: structure and function, p. 111-129, Trends in the Biology of Fermentations for Fuels and Chemicals. Springer.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 313 References

285. Ribeiro LF, De Lucas RC, Vitcosque GL, Ribeiro LF, Ward RJ, Rubio MV, Damásio AR, Squina FM, Gregory RC, Walton PH, Jorge JA, Prade RA, Buckeridge MS, Polizeli MdLT. 2014. A novel thermostable xylanase GH10 from Malbranchea pulchella expressed in Aspergillus nidulans with potential applications in biotechnology. Biotechnology for biofuels 7:115. 286. Rizzatti A, Jorge J, Terenzi H, Rechia C, Polizeli M. 2001. Purification and properties of a thermostable extracellular β-D-xylosidase produced by a thermotolerant Aspergillus phoenicis. Journal of Industrial Microbiology and Biotechnology 26:156-160. 287. Rizzatti AC, Sandrim VC, Jorge JA, Terenzi HF, Maria de Lourdes T. 2004. Influence of temperature on the properties of the xylanolytic enzymes of the thermotolerant fungus Aspergillus phoenicis. Journal of Industrial Microbiology and Biotechnology 31:88-93. 288. Rye CS, Withers SG. 2000. Glycosidase mechanisms. Current opinion in chemical biology 4:573-580. 289. Saitou N, Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular biology and evolution 4:406-425. 290. Saksono B, Sukmarini L. 2010. Structural Analysis of Xylanase from Marine Thermophilic Geobacillus stearothermophilus in Tanjung Api, Poso, Indonesia. Hayati Journal of Biosciences 17:189. 291. Salama M, Ismail K, Amany H, El-Lill A, Geweely N. 2008. Biochemical studies of purified extracellular xylanases from Aspergillus versicolor. International Journal of Botany 4:41-48. 292. Salleh HM, Mullegger J, Reid SP, Chan WY, Hwang J, Warren RA, Withers SG. 2006. Cloning and characterization of Thermotoga maritima beta- glucuronidase. Carbohydrate Res 341:49-59. 293. Salles BC, Medeiros RG, Báo SN, Silva FG, Edivaldo Filho X. 2005. Effect of cellulase-free xylanases from Acrophialophora nainiana and Humicola grisea var. thermoidea on eucalyptus kraft pulp. Process biochemistry 40:343- 349. 294. Sambrook J. 2001. Russel., W.: Molecular cloning: a laboratory manual 3rd ed. Harbor Laboratory Press, Cold Spring Harbor, NY. 295. Sandrim V, Rizzatti A, Terenzi H, Jorge J, Milagres A, Polizeli M. 2005. Purification and biochemical characterization of two xylanases produced by

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 314 References

Aspergillus caespitosus and their potential for kraft pulp bleaching. Process Biochemistry 40:1823-1828. 296. Sanghi A, Garg N, Kuhar K, Kuhad RC, Gupta VK. 2009. Enhanced production of cellulase-free xylanase by alkalophilic Bacillus subtilis ASH and its application in biobleaching of kraft pulp. BioResources 4:1109-1129. 297. Sanghvi G, Jivrajani M, Patel N, Jivrajani H, Bhaskara GB, Patel S. 2014. Purification and characterization of haloalkaline, organic solvent stable xylanase from newly isolated halophilic bacterium-OKH. International Scholarly Research Notices 2014. 298. Santos CR, Meza AN, Hoffmam ZB, Silva JC, Alvarez TM, Ruller R, Giesel GM, Verli H, Squina FM, Prade RA. 2010. Thermal-induced conformational changes in the product release area drive the enzymatic activity of xylanases 10B: Crystal structure, conformational stability and functional characterization of the xylanase 10B from Thermotoga petrophila RKU-1. Biochemical and biophysical research communications 403:214-219. 299. Santos CR, Meza AN, Hoffmam ZB, Silva JC, Alvarez TM, Ruller R, Giesel GM, Verli H, Squina FM, Prade RA. 2010. Thermal-induced conformational changes in the product release area drive the enzymatic activity of xylanases 10B: Crystal structure, conformational stability and functional characterization of the xylanase 10B from Thermotoga petrophila RKU-1. Biochemical and biophysical research communications 403:214-219. 300. Scheller HV, Ulvskov P. 2010. Hemicelluloses. Annual Reviewof Plant Biology 61:263-289. 301. Schlacher A, Holzmann K, Hayn M, Steiner W, Schwab H. 1996. Cloning and characterization of the gene for the thermostable xylanase XynA from Thermomyces lanuginosus. Journal of biotechnology 49:211-218. 302. Schulze E. 1891. Information regarding chemical composition of plant cell membrane. Ber Dtsch Chem Ges 24:2277-2287. 303. Shah A, Sidid S, Ahmad A, Rele M. 1999. Treatment of bagasse pulp with cellulase-free xylanases from an alkalophilic Bacillus sp. Sam-3. Bioresource technology 68:133-140. 304. Shallom D, Shoham Y. 2003. Microbial hemicellulases. Current opinion in microbiology 6:219-228.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 315 References

305. Shao W, Wiegel J. 1992. Purification and characterization of a thermostable beta-xylosidase from Thermoanaerobacter ethanolicus. Journal of bacteriology 174:5848-5853. 306. Sharma A, Adhikari S, Satyanarayana T. 2007. Alkali-thermostable and cellulase-free xylanase production by an extreme thermophile Geobacillus thermoleovorans. World Journal of Microbiology and Biotechnology 23:483- 490. 307. Sharma M, Kumar A. 2013. Xylanases: an overview. British Biotechnology Journal 3:1-28. 308. Shi H, Zhang Y, Li X, Huang Y, Wang L, Wang Y, Ding H, Wang F. 2013. A novel highly thermostable xylanase stimulated by Ca2+ from Thermotoga thermarum: cloning, expression and characterization. Biotechnology for biofuels 6:1. 309. Shi P, Chen X, Meng K, Huang H, Bai Y, Luo H, Yang P, Yao B. 2013. Distinct actions by Paenibacillus sp. strain E18 α-l-arabinofuranosidases and xylanase in xylan degradation. Applied and environmental microbiology 79:1990-1995. 310. Simao R, Souza C, Peralta R. 1997. Induction of xylanase in Aspergillus tamarii by methyl β-D-xyloside. Applied microbiology and biotechnology 47:267-271. 311. Simāo R, Souza C, Peralta R. 1997. The use of methyl β-D-xyloside as a substrate for xylanase production by Aspergillus tamarii. Canadian journal of microbiology 43:56-60. 312. Simpson HD, Haufler UR, Daniel RM. 1991. An extremely thermostable xylanase from the thermophilic eubacterium Thermotoga. Biochemical Journal 277:413-417. 313. Singh S, Madlala AM, Prior BA. 2003. Thermomyces lanuginosus: properties of strains and their hemicellulases. FEMS Microbiology Reviews 27:3-16. 314. Slapack GE, Russell I, Stewart GG. 1987. Thermophilic microbes in ethanol production. CRC Press Inc. 315. Sreenath H, Jeffries T. 2000. Production of ethanol from wood hydrolyzate by yeasts. Bioresource Technology 72:253-260.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 316 References

316. Sriprang R, Asano K, Gobsuk J, Tanapongpipat S, Champreda V, Eurwilaichitr L. 2006. Improvement of thermostability of fungal xylanase by using site-directed mutagenesis. Journal of biotechnology 126:454-462. 317. Strub C, Alies C, Lougarre A, Ladurantie C, Czaplicki J, Fournier D. 2004. Mutation of exposed hydrophobic amino acids to arginine to increase protein stability. BMC biochemistry 5:9. 318. Subramaniyan S, Prema P. 2000. Cellulase-free xylanases from Bacillus and other microorganisms. FEMS microbiology Letters 183:1-7. 319. Subramaniyan S, Prema P. 2002. Biotechnology of microbial xylanases: enzymology, molecular biology, and application. Critical reviews in biotechnology 22:33-64. 320. Sung WL, Luk CK, Chan B, Wakarchuk W, Yaguchi M, Campbell R, Willick G, Ishikawa K, Zahab DM. 1995. Expression of Trichoderma reesei and Trichoderma viride xylanases in Escherichia coli. Biochemistry and cell biology 73:253-259. 321. Sunna A, Antranikian G. 1997. Xylanolytic enzymes from fungi and bacteria. Critical reviews in biotechnology 17:39-67. 322. Sunna A, Bergquist PL. 2003. A gene encoding a novel extremely thermostable 1, 4-β-xylanase isolated directly from an environmental DNA sample. Extremophiles 7:63-70. 323. Sunna A, Moracci M, Rossi M, Antranikian G. 1997. Glycosyl hydrolases from hyperthermophiles. Extremophiles 1:2-13. 324. Suzuki Y, Hatagaki K, Oda H. 1991. A hyperthermostable pullulanase produced by an extreme thermophile, Bacillus flavocaldarius KP 1228, and evidence for the proline theory of increasing protein thermostability. Applied microbiology and biotechnology 34:707-714. 325. Taherzadeh MJ, Karimi K. 2008. Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: a review. International journal of molecular sciences 9:1621-1651. 326. Taibi Z, Saoudi B, Boudelaa M, Trigui H, Belghith H, Gargouri A, Ladjama A. 2012. Purification and biochemical characterization of a highly thermostable xylanase from Actinomadura sp. strain Cpt20 isolated from poultry compost. Applied biochemistry and biotechnology 166:663-679.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 317 References

327. Tamura K, Dudley J, Nei M, Kumar S. 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24:1596-1599. 328. Taneja K, Gupta S, Kuhad RC. 2002. Properties and application of a partially purified alkaline xylanase from an alkalophilic fungus Aspergillus nidulans KK- 99. Bioresource Technology 85:39-42. 329. Techapun C, Poosaran N, Watanabe M, Sasaki K. 2003. Thermostable and alkaline-tolerant microbial cellulase-free xylanases produced from agricultural wastes and the properties required for use in pulp bleaching bioprocesses: a review. Process Biochemistry 38:1327-1340. 330. Tenkanen M, Puls J, Poutanen K. 1992. Two major xylanases of Trichoderma reesei. Enzyme and Microbial Technology 14:566-574. 331. Tenkanen M, Siika-aho M. 2000. An α-glucuronidase of Schizophyllum commune acting on polymeric xylan. Journal of biotechnology 78:149-161. 332. Tenkanen M, Viikari L, Buchert J. 1997. Use of acid-tolerant xylanase for bleaching of kraft pulps. Biotechnology techniques 11:935-938. 333. Thompson JD, Higgins DG, Gibson TJ. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22:4673-4680. 334. Tian J, Wang P, Gao S, Chu X, Wu N, Fan Y. 2010. Enhanced thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 by rational engineering of a glycine to proline mutation. Febs Journal 277:4901- 4908. 335. Tolan J, Olson D, Dines R. 1995. Survey of xylanase enzyme usage in bleaching in Canada: results highlight benefits and problems. Pulp & Paper Canada 96:107-110. 336. Törrönen A, Rouvinen J. 1997. Structural and functional properties of low molecular weight endo-1, 4-β-xylanases. Journal of biotechnology 57:137-149. 337. Turunen O, Etuaho K, Fenel F, Vehmaanperä J, Wu X, Rouvinen J, Leisola M. 2001. A combination of weakly stabilizing mutations with a disulfide bridge in the α-helix region of Trichoderma reesei endo-1, 4-β- xylanase II increases the thermal stability through synergism. Journal of biotechnology 88:37-46.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 318 References

338. Turunen O, Vuorio M, Fenel F, Leisola M. 2002. Engineering of multiple arginines into the Ser/Thr surface of Trichoderma reesei endo-1, 4-β-xylanase II increases the thermotolerance and shifts the pH optimum towards alkaline pH. Protein engineering 15:141-145. 339. Twomey L, Pluske J, Rowe J, Choct M, Brown W, McConnell M, Pethick D. 2003. The effects of increasing levels of soluble non-starch polysaccharides and inclusion of feed enzymes in dog diets on faecal quality and digestibility. Animal Feed Science and Technology 108:71-82. 340. Uhl AM, Daniel RM. 1999. The first description of an archaeal hemicellulase: the xylanase from Thermococcus zilligii strain AN1. Extremophiles 3:263-267. 341. Umemoto H, Ihsanawati, Inami M, Yatsunami R, Fukui T, Kumasaka T, Tanaka N, Nakamura S. 2009. Improvement of alkaliphily of Bacillus alkaline xylanase by introducing amino acid substitutions both on catalytic cleft and protein surface. Bioscience, biotechnology, and biochemistry 73:965-967. 342. Upreti RK, Kumar M, Shankar V. 2003. Bacterial glycoproteins: functions, biosynthesis and applications. Proteomics 3:363-379. 343. Urvoas A, Valerio-Lepiniec M, Minard P. 2012. Artificial proteins from combinatorial approaches. Trends in biotechnology 30:512-520. 344. Van der Burgt T, Tolan JS, Thibault LC. 2002. Tappi pulping conference, San Diego, CA. 345. Vena PF, García-Aparicio MP, Brienzo M, Görgens JF, Rypstra T. 2013. Effect of Alkaline Hemicellulose Extraction on Kraft Pulp Fibers from Eucalyptus Grandis. Journal of Wood Chemistry and Technology 33:157-173. 346. Verma D, Anand A, Satyanarayana T. 2013. Thermostable and alkalistable endoxylanase of the extremely thermophilic bacterium Geobacillus thermodenitrificans TSAA1: cloning, expression, characteristics and its applicability in generating xylooligosaccharides and fermentable sugars. Applied biochemistry and biotechnology 170:119-130. 347. Verma D, Satyanarayana T. 2012. Cloning, expression and applicability of thermo-alkali-stable xylanase of Geobacillus thermoleovorans in generating xylooligosaccharides from agro-residues. Bioresource Technology 107:333-338. 348. Verma D, Satyanarayana T. 2012. Molecular approaches for ameliorating microbial xylanases. Bioresource technology 117:360-367.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 319 References

349. Vieille C, Zeikus GJ. 2001. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiology and Molecular Biology Reviews 65:1-43. 350. Viikari L RM, Kantelinen A, Sundiquist J, Linko M. 1986. 3rd Int. Con. Stockholm, Stockholm. 351. Viikari L, Kantelinen A, Poutanen K, Ranua M. 1990. Characterization of pulps treated with hemicellulolytic enzymes prior to bleaching. Biotechnology in pulp and paper manufacture:145-151. 352. Viikari L, Kantelinen A, Sundquist J, Linko M. 1994. Xylanases in bleaching: from an idea to the industry. FEMS Microbiology Reviews 13:335- 350. 353. Viikari L, Vehmaanperä J, Koivula A. 2012. Lignocellulosic ethanol: from science to industry. Biomass and Bioenergy 46:13-24. 354. Virupakshi S, Babu KG, Gaikwad SR, Naik G. 2005. Production of a xylanolytic enzyme by a thermoalkaliphilic Bacillus sp. JB-99 in solid state fermentation. Process Biochemistry 40:431-435. 355. Vogt G, Woell S, Argos P. 1997. Protein thermal stability, hydrogen bonds, and ion pairs. Journal of Molecular Biology 269:631-643. 356. Wakarchuk WW, Sung WL, Campbell RL, Cunningham A, Watson DC, Yaguchi M. 1994. Thermostabilization of the Bacillus circulansxylanase by the introduction of disulfide bonds. Protein engineering 7:1379-1386. 357. Wang C, Huang R, He B, Du Q. 2012. Improving the thermostability of alpha- amylase by combinatorial coevolving-site saturation mutagenesis. BMC bioinformatics 13:263. 358. Wang K, Luo H, Tian J, Turunen O, Huang H, Shi P, Hua H, Wang C, Wang S, Yao B. 2014. Thermostability improvement of a Streptomyces xylanase by introducing proline and glutamic acid residues. Applied and environmental microbiology 80:2158-2165. 359. Wang K, Luo H, Tian J, Turunen O, Huang H, Shi P, Hua H, Wang C, Wang S, Yao B. 2014. Thermostability Improvement of a Streptomyces Xylanase by Introducing Proline and Glutamic Acid Residues. Applied and Environmental Microbiology 80:2158-2165. 360. Wang Q, Du W, Weng X-Y, Liu M-Q, Wang J-K, Liu J-X. 2015. Recombination of thermo-alkalistable, high xylooligosaccharides producing

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 320 References

endo-xylanase from Thermobifida fusca and expression in Pichia pastoris. Applied biochemistry and biotechnology 175:1318-1329. 361. Wang Y, Fu Z, Huang H, Zhang H, Yao B, Xiong H, Turunen O. 2012. Improved thermal performance of Thermomyces lanuginosus GH11 xylanase by engineering of an N-terminal disulfide bridge. Bioresource technology 112:275- 279. 362. Wang Y, Zhang H, He Y, Luo H, Yao B. 2007. Characterization, gene cloning, and expression of a novel xylanase XYNB from Streptomyces olivaceoviridis A1. Aquaculture 267:328-334. 363. Watanabe K, Masuda T, Ohashi H, Mihara H, Suzuki Y. 1994. Multiple Proline Substitutions Cumulatively Thermostabilize Bacillus Cereus ATCC7064 Oligo‐1, 6‐Glucosidase. European Journal of Biochemistry 226:277-283. 364. Watanabe K, Masuda T, Ohashi H, Mihara H, Suzuki Y. 1994. Multiple Proline Substitutions Cumulatively Thermostabilize Bacillus Cereus ATCC7064 Oligo‐1, 6‐Glucosidase. European Journal of Biochemistry 226:277-283. 365. Whistler RL, Masak Jr E. 1955. Enzymatic Hydrolysis of Xylan1. Journal of the american chemical society 77:1241-1243. 366. Whitmire D, Maiti B. 2002. Xylanase effects on pulp delignification. Chemical Engineering Communications 189:608-622. 367. Williamson G, Faulds C, Kroon P. 1998. Specificity of ferulic acid (feruloyl) esterases. Biochemical Society Transactions 26:205-210. 368. Williamson MP. 1994. The structure and function of proline-rich regions in proteins. Biochemical Journal 297:249-260.

369. Winterhalter C, Heinrich P, Candussio A, Wich G, Liebl W. 1995. Identification of a novel cellulose‐binding domain the multidomain 120 kDa xylanase XynA of the hyperthermophilic bacterium Thermotoga maritima. Molecular microbiology 15:431-444. 370. Wintrode PL, Miyazaki K, Arnold FH. 2001. Patterns of adaptation in a laboratory evolved thermophilic enzyme. Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology 1549:1-8. 371. Wiseman A. 1995. Introduction to principles. In: Wiseman, A (ed.,) Handbook of enzyme biotechnology 3rd edition. Padstow, Cornwall, U.K:Ellis Horwood Ltd., T.J Press Ltd pp 3 -8.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 321 References

372. Wong K, Tan L, Saddler JN. 1988. Multiplicity of beta-1, 4-xylanase in microorganisms: functions and applications. Microbiological Reviews 52:305- 317. 373. Wong KK, Saddler JN. 1993. Applications of hemicellulases in the food, feed, and pulp and paper industries. Hemicellulose and hemicellulases 4:127-143. 374. Wu S, Liu B, Zhang X. 2006. Characterization of a recombinant thermostable xylanase from deep-sea thermophilic Geobacillus sp. MT-1 in East Pacific. Applied microbiology and biotechnology 72:1210-1216. 375. Wu Y-R, He J. 2015. Characterization of a xylanase-producing Cellvibrio mixtus strain J3-8 and its genome analysis. Scientific reports 5. 376. Wubah DA, Akin DE, Borneman WS. 1993. Biology, fiber-degradation, and enzymology of anaerobic zoosporic fungi. Crit Rev Microbiol 19:99-115. 377. Xu J, Bjursell MK, Himrod J, Deng S, Carmichael LK, Chiang HC, Hooper LV, Gordon JI. 2003. A genomic view of the human-Bacteroides thetaiotaomicron symbiosis. Science 299:2074-2076. 378. Xu X, Liu M-q, Huo W-k, Dai X-j. 2016. Obtaining a mutant of Bacillus amyloliquefaciens xylanase A with improved catalytic activity by directed evolution. Enzyme and Microbial Technology 86:59-66. 379. Yang H, Li J, Shin H-d, Du G, Liu L, Chen J. 2014. Molecular engineering of industrial enzymes: recent advances and future prospects. Applied microbiology and biotechnology 98:23-29. 380. Yang R, MAcKENZIE CR, Bilous D, Narang S. 1989. Identification of two distinct Bacillus circulans xylanases by molecular cloning of the genes and expression in Escherichia coli. Applied and environmental microbiology 55:568-572. 381. Yin L-J, Lin H-H, Chiang Y-I, Jiang S-T. 2009. Bioproperties and purification of xylanase from Bacillus sp. YJ6. Journal of agricultural and food chemistry 58:557-562. 382. Yoon H-S, Han NS, Kim CH. 2004. Expression of Thermotoga maritima Endo-beta-1, 4-xylanase Gene in E. coli and Characterization of the Recombinant Enzyme. Agricultural Chemistry and Biotechnology 47:157-166. 383. Zanoelo FF, de Moraes MdLT, Terenzi HF, Jorge JA. 2004. Purification and biochemical properties of a thermostable xylose-tolerant β-D-xylosidase from

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 322 References

Scytalidium thermophilum. Journal of Industrial Microbiology and Biotechnology 31:170-176. 384. Zhang GM, Huang J, Huang GR, Ma LX, Zhang XE. 2007. Molecular cloning and heterologous expression of a new xylanase gene from Plectosphaerella cucumerina. Applied Microbiology and Biotechnology 74:339-346. 385. Zhang W, Lou K, Li G. 2010. Expression and characterization of the Dictyoglomus thermophilum Rt46B. 1 xylanase gene (xynB) in Bacillus subtilis. Applied biochemistry and biotechnology 160:1484-1495. 386. Zhao J, Li X, Qu Y, Gao P. 2002. Xylanase pretreatment leads to enhanced soda pulping of wheat straw. Enzyme and Microbial Technology 30:734-740. 387. Zhao YY, Luo HY, Shi PJ, Bai YG. 2011. Cloning, expression, and characterization of a new xylanase from alkalophilic Paenibacillus sp. 12-11. Journal of microbiology and biotechnology 21:861-868. 388. Zheng F, Huang J, Yin Y, Ding S. 2013. A novel neutral xylanase with high SDS resistance from Volvariella volvacea: characterization and its synergistic hydrolysis of wheat bran with acetyl xylan esterase. Journal of industrial microbiology & biotechnology 40:1083-1093. 389. Zheng H, Liu Y, Liu X, Wang J, Han Y, Lu F. 2012. Iisolation Purification an d Characterization of a Thermostable Xylanase from a Novel Strain, Paenibacill us campinasensis G1-1. Journal of Microbiology and Biotechnology 22(7): 930- 938. 390. Zheng H-c, Sun M-z, Meng L-c, Pei H-s, Zhang X-q, Yan Z, Zeng W-h, Zhang J-s, Hu J-r, Lu F-p. 2014. Purification and characterization of a thermostable xylanase from Paenibacillus sp. NF1 and its application in xylooligosaccharides production. Journal of Microbiology and Biotechnology 24:489-496. 391. Zhou J, Gao Y, Dong Y, Tang X, Li J, Xu B, Mu Y, Wu Q, Huang Z. 2012. A novel xylanase with tolerance to ethanol, salt, protease, SDS, heat, and alkali from actinomycete Lechevalieria sp. HJ3. Journal of industrial microbiology & biotechnology 39:965-975. 392. Zhou J, Shi P, Zhang R, Huang H, Meng K, Yang P, Yao B. 2011. Symbiotic Streptomyces sp. TN119 GH 11 xylanase: a new pH-stable, protease-and SDS-

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 323 References

resistant xylanase. Journal of industrial microbiology & biotechnology 38:523- 530. 393. Zhu GP, Xu C, Teng MK, Tao LM, Zhu XY, Wu CJ, Hang J, Niu LW, Wang YZ. 1999. Increasing the thermostability of D-xylose isomerase by introduction of a proline into the turn of a random coil. Protein engineering 12:635-638. 394. Zhu H, Paradis F, Krell P, Phillips J, Forsberg C. 1994. Enzymatic specificities and modes of action of the two catalytic domains of the XynC xylanase from Fibrobacter succinogenes S85. Journal of bacteriology 176:3885- 3894. 395. Zverlov V, Piotukh K, Dakhova O, Velikodvorskaya G, Borriss R. 1996. The multidomain xylanase A of the hyperthermophilic bacterium Thermotoga neapolitana is extremely thermoresistant. Applied microbiology and biotechnology 45:245-247.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 324 Appendices

Appendix 1: Composition of xylan nutrient agar

Enzymatic Digest of Gelatin 0.5 g

Beef Extract 0.3 g

Agar 1.5 g

Birchwood xylan 0.1 g

Water 100 mL

Appendix 2: CMC nutrient agar

Enzymatic Digest of Gelatin 0.5 g

Beef Extract 0.3 g

Agar 1.5 g

CMC 1 g

Water 100 mL

Appendix 3: Casein nutrient agar

Enzymatic Digest of Gelatin 0.5 g

Beef Extract 0.3 g

Agar 1.5 g

Casein 1 g

Water 100 mL

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 325 Appendices

Appendix 4: Starch nutrient agar

Enzymatic Digest of Gelatin 0.5 g

Beef Extract 0.3 g

Agar 1.5 g

Starch soluble 1 g

Water 100 mL

Appendix 5(A): TE buffer

10 mM Tris 0.1211 g

1 mM EDTA 0.029 g pH 8.0 With HCl

Water 100 mL

Appendix 5 (B): TE buffer

1M Tris 1 mL

0.5M EDTA 200 µL pH 8.0 With HCl

Water 98.8 mL

Appendix 6: TBE Buffer 5X 1 litre

Tris base 54 g

Boric acid 27.5 g

0.5 M EDTA 20 mL

Total volume 1000 mL

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 326 Appendices

Appendix 7: LB Broth

bacterial tryptone 10 g yeast extract 5 g NaCl 5 g Total volume 1000 mL

Appendix 8: Running gel

Serial Gel % 15% 12.5% Number 1 30% and 8% Acrylamide/bis 4.9 mL 4.2 mL 2 Tris(1.5M pH 8.8) 2.5 mL 2.5 mL 3 SDS 10% 100 µL 100 µL 4 Water 2.4mL 3.4 mL 5 TEMED 20 µL 20 µL 6 10% APS 50 µL 50 µL

Appendix 9: Stacking gel

Serial Gel % Volume Number 1 30% and 8% Acrylamide/bis 696 µL 2 Tris(1.5M pH 6.8) 650 µL 3 SDS 10% 100 µL 4 Water 3.65mL 5 TEMED 10 µL 6 10% APS 50 µL

Appendix 10: Protein dye solution

Water 40 mL Methanol 50 mL Acetic acid 10 mL Commasi brilliant blue R-250 0.125 g

Appendix 11: Distaining solution 1

Water 40 mL Methanol 50 mL Acetic acid 10 mL

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 327 Appendices

Appendix12: Distaining solution 2

Water 132 mL Methanol 7.5 mL Acetic acid 10.5 mL

Appendix 13: Sodium phosphate buffer

Components 1M 100MM 50MM 20MM NaH2PO4.2H2O 156 g 15.601 g 7.8005 g 3.12 g Na2HPO4.7H2O 268 g 26.807 g 13.40 g 5.36 g Water 1000 mL 1000 mL 1000 mL 1000 mL

Appendix 14: DNS

Potassium sodium tartarate 36 g NaOH 1 g DNS 1 g phenol 200 mg Na2SO3 50 mg Water 100 mL

Appendix 15: Bradford reagent

Coomassie Brilliant Blue G-250 50 mg methano 50 mL 85% (w/v) phosphoric acid (H3PO4) 100 mL Water 850 mL

Appendix 16: TYM Broth

S.No Components Gram 1 2 % Tryptone 10 g 2 0.5 % Yeast Extract 2.5 g 3 100 mM NaCl 0.292 g 4 10 mM MgCl2 0.47g 5 Water 500 mL

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 328 Appendices

Appendix 17: TFB-1solution

S.No Components Gram 1 Potassium acetate 1.47 g 2 MnCl2 4.95 g 3 KCl 0.137 g 4 CaCl2 0.74 g 5 Glycerol 75 mL 6 Total volume 500 mL

Appendix 18: TFB-2 solution

S.No Components Gram 1 10 mM Na-MOPS 1.045 g 2 75 mM CaCl2 4.16 g 3 10 mM KCl 0.37 g 4 15% Glycerol 75 mL 5 Total volume 500 mL

Appendix 19: LB Sucrose

Agar 7.5 g Tryptone 5 g NaCl 5 g Yeast extract 2.5 g Sucrose 25 g Water 500 mL

Appendix 20: solution-I plasmid

Tris, 50 mM, RNase 100 mg/mL EDTA 1 mM,

Appendix 21: solution-I1 plasmid

0.2N NaOH, 1% (w/v) SDS

Appendix 22: Solution III solution-III containing Potassium acetate 3 M, pH 4.8-5.0

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 329 Appendices

Appendix 23: Cellular morphology by Gram’s staining

Isolate Gram’s reaction Arrangement Shape AK 53 +ve Chain Rod shape C5 +ve Chain Rod shape K22 +ve Chain Rod shape S1 +ve Chain Rod shape

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 330 Appendices

Appendix 24: Geobacillus thermodenitrificans strain AK53 16S ribosomal RNA gene (KP203955)

TAATTAATACCGGATAACACCAAAGACCGCATGGTCTTTGGTTGAAAGGC GGCTTCGGCTGTCACTTGCGGATGGGCCCGCGGCGCATTAGCTAGTTGGT GAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGGCCTGAGAGGGTG ACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAG CAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCGACGCCGCG TGAGCGAAGAAGGCCTTCGGGTCGTAAAGCTCTGTTGTGAGGGACGAAGG AGCGCCGTTTGAATAAGGCGGCGCGGTGACGGTACCTCACGAGAAAGCCC CGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCGAGCGTTG TCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTCCTTTAAGTCTGAT GTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGGACTT GAGTGCAGGAGAGGAGAGCGGAATTCCACGTGTAGCGGTGAAATGCGTA GAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGCCTGTAACTG ACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGT AGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGGTCACACCCTT TAGTGCTGTAGCTAACGCGATAAGCACTCCGCCTGGGGAGTACGGCCGCA AGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCA TGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCC CCTGACAACCCAAGAGATTGGGCGTTCCCCCTTCGGGGGGACAGGGTGAC AGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTC CCGCAACGAGCGCAACCCTTGCCTCTAGTTGCCAGCATTCAGTTGGGCAC TCTAGAGGGACTGCCGGCTAAAAGTCGGAGGAAGGTGGGGATGACGTCA AATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCGG TACAAAGGGCTGCGAACCCGCGAGGGGGAGCGAATCCCAAAAAGCCGCT CTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAATCGCT AGTAATCGCGGATCACATGCCGGGGGAAAAAAATTTTCCCGGGGCCTTTG TAAAACCCGGCCCGGTCACACCACGAAGAGCTTGCAACACCCGAAAGTCG GTGAGGTAACCCTTACGGGAGCCAGCCGCCGAAGGTGGGGCAAGTGATTG GGGTGAATCT

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 331 Appendices

Appendix 25: Geobacillus thermodenitrificans strain C5 16S ribosomal RNA gene, partial sequence (KP203956)

AATACCGGATAACACCAAAGACCGCATGGTCTTTGGTTGAAAGGCGGCTT CGGCTGTCACTTGCGGATGGGCCCGCGGCGCATTAGCTAGTTGGTGAGGT AACGGCTCACCAAGGCGACGATGCGTAGCCGGCCTGAGAGGGTGACCGG CCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTA GGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCGACGCCGCGTGAGC GAAGAAGGCCTTCGGGTCGTAAAGCTCTGTTGTGAGGGACGAAGGAGCGC CGTTTGAATAAGGCGGCGCGGTGACGGTACCTCACGAGAAAGCCCCGGCT AACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCGAGCGTTGTCCGG AATTATTGGGCGTAAAGCGCGCGCAGGCGGTCCTTTAAGTCTGATGTGAA AGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGGACTTGAGTG CAGGAGAGGAGAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGAT GTGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGCCTGTAACTGACGCT GAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCC ACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGGTCACACCCTTTAGTG CTGCAGCTAACGCGATAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCT GAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGG TTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCCTGA CAACCCAAGAGATTGGGCGTTCCCCCTTCGGGGGGACAGGGTGACAGGTG GTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCA ACGAGCGCAACCCTTGCCTCTAGTTGCCAGCATTCAGTTGGGCACTCTAGA GGGACTGCCGGCTAAAAGTCGGAGGAAGGTGGGGATGACGTCAAATCAT CATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCGGTACAAA GGGCTGCGAACCCGCGAGGGGGAGCGAATCCCAAAAAGCCGCTCTCAGT TCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAATCGCTAGTAAT CGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCG CCCGTCACACCACGAGAGCTTGCAACACCCGAAGTCGGTGAGGTAACCCT TACGGGAGCCAGCCGCCGAAGGTGGGGCAAGTGATTGGGGTGAA

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 332 Appendices

Appendix 26: Xylanase gene Geobacillus thermodenitrificans strain AK53

ATGGCTAGCATGTTGAAAAGATCGCGAAAAGCGATAATAGTTGGATTCTC ATTTATGCTGTTGCTCCCTTTAGGGATGACGAATGCATTGGCAAAAACTGA ACAATCATACGCTAAAAAGCCTCAAATCAGCGCATTGCATGCCCCACAAT TGGACCAGCGCTACAAAGATTCATTCACTATTGGTGCGGCATGGCTAGCC ATGCTAAAACGCCATTTTAACAGCATTGTCGCTGAGAACGTTATGAAGCC GATTAATATTCAACCGGAAGAAGGAAAATTCAATTTTGCTGAGGCGGATC AAATCGTTCGGTTTGCTAAAAAACACCATATGGATATCCGTTTCCACACGC TCGTTTGGCATAGCCAAGTACCTCAATGGTTCTTTCTTGACAAGGAAGGCC AACCAATGGTCAATGAAACGGACCCTGTGAAACGCGAACAAAATAAACA GCTGTTATTAAAACGGATCGAAACCCATATTAAAACGATTGTCGAGCGGT ATAAAGATGACATCAAATATTGGGACGTTGTAAATGAGGTAGTCGGGGAT GATGGAGAATTGCGCGATTCCCCATGGTATCAAATCGCTGGCATCGATTA TATTAAGGTAGCGTTCCAAACAGCAAGAAAATATGGCGGCAACAAGATTA AACTTTACATTAATGATTACAATACGGAAGTTGAACCAAAGCGAAGCGCT CTTTATAACTTGGTGAAACAATTAAAAGAAGAGGGCATTCCCATTGATGG TATTGGCCATCAGTCCCACATCCAAATTGACTGGCCTTCTGAAGAGGAAA TCGAAAAAACGATTATCATGTTTGCCGATCTAGGGTTAGACAACCAAATT ACTGAGCTGGATGTGAGCATGTACGGCTGGCCGCCGCGGGCTTACCTGTC GTATGACGCCATTCCGGAGCAAAAGTTTTTGGACCAAGCGGATCGCTATG ATCGATTGTTTAAGCTGTATGAAAAACTCAGCGATAAAATCAGTAACGTC ACCTTCTGGGGCATCGCCGACAACCATACGTGGCTCGACAGTCGAGCGGA TGTTTACTATGATGCTGATGGGAATGTGATTGTAGACCCGAAAGCCCCAT ATACGAGAGTGGAAAAAGGGAATGGAAAAGATGCGCCATTTGTGTTCGA CCCCGAATACAACGTAAAACCTGCGTATTGGGCTATTATCGATCATAAGG CTATTATCGATCAATGA

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 333 Appendices

Appendix 27: Geobacillus thermodenitrificans strain C5 extracellular xylanase gene KP202694 ATGGCTAGCATGTTGAAAAGATCGCGAAAAGCGATAATAGTTGGATTCTC ATTTATGCTGTTGCTCCCTTTAGGGATGACGAATGCATTGGCAAAAACTGA ACAATCATACGCTAAAAAGCCTCAAATCAGCGCATTGCATGCCCCACAAT TGGACCAGCGCTACAAAGATTCATTCACTATTGGTGCGGCTGTTGAACCTT ATCAGCTACTAAACGAGAAAGACGCACAAATGCTAAAACGCCATTTTAAC AGCATTGTCGCTGAGAACGTTATGAAGCCGATTAATATTCAACCGGAAGA AGGAAAATTCAATTTTGCTGAGGCGGATCAAATCGTTCGGTTTGCTAAAA AACACCATATGGATATCCGTTTCCACACGCTCGTTTGGCATAGCCAAGTAC CTCAATGGTTCTTTCTTGACAAGGAAGGCCAACCAATGGTCAATGAAACG GACCCTGTGAAACGCGAACAAAATAAACAGCTGTTATTAAAACGGATCGA AACCCATATTAAAACGATTGTCGAGCGGTATAAAGATGACATCAAATATT GGGACGTTGTAAATGAGGTAGTCGGGGATGATGGAGAATTGCGCGATTCC CCATGGTATCAAATCGCTGGCATCGATTATATTAAGGTAGCGTTCCAAAC AGCGAGAAAATATGGCGGCAACAAGATTAAACTTTACATTAATGATTACA ATACGGAAGTTGAACCAAAGCGAAGCGCTCTTTATAACTTGGTGAAACAA TTAAAAGAAGAGGGCATTCCCATTGATGGTATTGGCCATCAGTCCCACAT CCAAATTGACTGGCCTTCTGAAGAGGAAATCGAAAAAACGATTATCATGT TTGCCGATCTAGGGTTAGACAACCAAATTACTGAGCTGGATGTGAGCATG TACGGCTGGCCGCCGCGGGCCTACCCGTCGTATGACGCCATTCCGGAGCA AAAGTTTTTGGACCAAGCGAATCGCTATGATCGATTGTTTAAGCTGTATGA AAAACTCAGCGATAAAATCAGTAACGTCACCTTCTGGGGCATCGCCGACA ACCATACGTGGCTCGACAGTCGAGCGGATGTTTACTATGATGCTGATGGG AATGTGATTGTAGACCCGAAAGCCCCATACACGAGAGTGGAAAAAGGGA ATGGAAAAGATGCGCCATTTGTGTTCGACCCCGAATACAACGTAAAACCT GCGTATTGGGCTATTATCGATCAATGA

Appendix 28: Geobacillus thermodenitrificans C5 xylanase protein Amino acid

MASMLKRSRKAIIVGFSFMLLLPLGMTNALAKTEQSYAKKPQISALHAPQLD QRYKDSFTIGAAVEPYQLLNEKDAQMLKRHFNSIVAENVMKPINIQPEEGKFN FAEADQIVRFAKKHHMDIRFHTLVWHSQVPQWFFLDKEGQPMVNETDPVKR EQNKQLLLKRIETHIKTIVERYKDDIKYWDVVNEVVGDDGELRDSPWYQIAG IDYIKVAFQTARKYGGNKIKLYINDYNTEVEPKRSALYNLVKQLKEEGIPIDGI GHQSHIQIDWPSEEEIEKTIIMFADLGLDNQITELDVSMYGWPPRAYPSYDAIP EQKFLDQANRYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDAD GNVIVDPKAPYTRVEKGNGKDAPFVFDPEYNVKPAYWAIIDQ

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 334 Appendices

Appendix 29: p15TV-L Vector

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 335 Appendices

Appendix 30: pGEM®-T Easy Vectors

Appendix 31: Mutant model C5 R81P amino acid sequence

MASMLKRSRKAIIVGFSFMLLLPLGMTNALAKTEQSYAKKPQISALHAPQLD QRYKDSFTIGAAVEPYQLLNEKDAQMLKPHFNSIVAENVMKPINIQPEEGKFN FAEADQIVRFAKKHHMDIRFHTLVWHSQVPQWFFLDKEGQPMVNETDPVKR EQNKQLLLKRIETHIKTIVERYKDDIKYWDVVNEVVGDDGELRDSPWYQIAG IDYIKVAFQTARKYGGNKIKLYINDYNTEVEPKRSALYNLVKQLKEEGIPIDGI GHQSHIQIDWPSEEEIEKTIIMFADLGLDNQITELDVSMYGWPPRAYPSYDAIP EQKFLDQANRYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDAD GNVIVDPKAPYTRVEKGNGKDAPFVFDPEYNVKPAYWAIIDQ

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 336 Appendices

Appendix 32: C5 H82E amino acid sequence

MASMLKRSRKAIIVGFSFMLLLPLGMTNALAKTEQSYAKKPQISALHAPQLD QRYKDSFTIGAAVEPYQLLNEKDAQMLKREFNSIVAENVMKPINIQPEEGKFN FAEADQIVRFAKKHHMDIRFHTLVWHSQVPQWFFLDKEGQPMVNETDPVKR EQNKQLLLKRIETHIKTIVERYKDDIKYWDVVNEVVGDDGELRDSPWYQIAG IDYIKVAFQTARKYGGNKIKLYINDYNTEVEPKRSALYNLVKQLKEEGIPIDGI GHQSHIQIDWPSEEEIEKTIIMFADLGLDNQITELDVSMYGWPPRAYPSYDAIP EQKFLDQANRYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDAD GNVIVDPKAPYTRVEKGNGKDAPFVFDPEYNVKPAYWAIIDQ

Appendix 33: C5 N84R amino acid sequence

MASMLKRSRKAIIVGFSFMLLLPLGMTNALAKTEQSYAKKPQISALHAPQLD QRYKDSFTIGAAVEPYQLLNEKDAQMLKRHFRSIVAENVMKPINIQPEEGKFN FAEADQIVRFAKKHHMDIRFHTLVWHSQVPQWFFLDKEGQPMVNETDPVKR EQNKQLLLKRIETHIKTIVERYKDDIKYWDVVNEVVGDDGELRDSPWYQIAG IDYIKVAFQTARKYGGNKIKLYINDYNTEVEPKRSALYNLVKQLKEEGIPIDGI GHQSHIQIDWPSEEEIEKTIIMFADLGLDNQITELDVSMYGWPPRAYPSYDAIP EQKFLDQANRYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDAD GNVIVDPKAPYTRVEKGNGKDAPFVFDPEYNVKPAYWAIIDQ

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 337 Appendices

Appendix 34: C5 W185P amino acid sequence

MASMLKRSRKAIIVGFSFMLLLPLGMTNALAKTEQSYAKKPQISALHAPQLD QRYKDSFTIGAAVEPYQLLNEKDAQMLKRHFNSIVAENVMKPINIQPEEGKFN FAEADQIVRFAKKHHMDIRFHTLVWHSQVPQWFFLDKEGQPMVNETDPVKR EQNKQLLLKRIETHIKTIVERYKDDIKYPDVVNEVVGDDGELRDSPWYQIAGI DYIKVAFQTARKYGGNKIKLYINDYNTEVEPKRSALYNLVKQLKEEGIPIDGI GHQSHIQIDWPSEEEIEKTIIMFADLGLDNQITELDVSMYGWPPRAYPSYDAIP EQKFLDQANRYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDAD GNVIVDPKAPYTRVEKGNGKDAPFVFDPEYNVKPAYWAIIDQ

Appendix 35: C5 D186E amino acid sequence

MASMLKRSRKAIIVGFSFMLLLPLGMTNALAKTEQSYAKKPQISALHAPQLD QRYKDSTIGAAVEPYQLLNEKDAQMLKRHFNSIVAENVMKPINIQPEEGKFNF AEADQIVRFAKKMDIRFHTLVWHSQVPQWFFLDKEGQPMVNETDPVKREQN KQLLLKRIETHIKTIVERYDDIKYWEVVNEVVGDDGELRDSPWYQIAGIDYIK VAFQTARKYGGNKIKLYINDYNTEVEPKRSALYNLVKQLKEEGIPIDGIGHQS HIQIDWPSEEEIEKTIIMFADLGLDNQITELDVSMGWPPRAYPSYDAIPEQKFL DQANRYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRAVYYDADGNVIVD PKAPYTRVEKGNGKDAPFVFDPEYNVKPAYWAIIDQ

Appendix 36: C5 D339R amino acid sequence

MASMLKRSRKAIIVGFSFMLLLPLGMTNALAKTEQSYAKKPQISALHAPQLD QRYKDSFTIGAAVEPYQLLNEKDAQMLKRHFNSIVAENVMKPINIQPEEGKFN FAEADQIVRFAKKHHMDIRFHTLVWHSQVPQWFFLDKEGQPMVNETDPVKR EQNKQLLLKRIETHIKTIVERYKDDIKYWDVVNEVVGDDGELRDSPWYQIAG IDYIKVAFQTARKYGGNKIKLYINDYNTEVEPKRSALYNLVKQLKEEGIPIDGI GHQSHIQIDWPSEEEIEKTIIMFADLGLDNQITELDVSMYGWPPRAYPSYDAIP EQKFLDQANRYDRLFKLYEKLSRKISNVTFWGIADNHTWLDSRADVYYDAD GNVIVDPKAPYTRVEKGNGKDAPFVFDPEYNVKPAYWAIIDQ

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 338 Appendices

Appendix 37: Geobacillus thermodenitrificans C5 xylanase proline tagged Amino acid (in this 60 amino acid sequence having proline was attached to c terminal in yelllow)

MASMLKRSRKAIIVGFSFMLLLPLGMTNALAKTEQSYAKKPQISALHAPQLD QRYKDSFTIGAAVEPYQLLNEKDAQMLKRHFNSIVAENVMKPINIQPEEGKFN FAEADQIVRFAKKHHMDIRFHTLVWHSQVPQWFFLDKEGQPMVNETDPVKR EQNKQLLLKRIETHIKTIVERYKDDIKYWDVVNEVVGDDGELRDSPWYQIAG IDYIKVAFQTARKYGGNKIKLYINDYNTEVEPKRSALYNLVKQLKEEGIPIDGI GHQSHIQIDWPSEEEIEKTIIMFADLGLDNQITELDVSMYGWPPRAYPSYDAIP EQKFLDQANRYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDAD GNVIVDPKAPYTRVEKGNGKDAPFVFDPEYNVKPAYWAIIDQMKAAAWPIPE KPKPNPNQQRQRRRGGFGGPQRPPFNPALAFAEQPGVKEDFVPSELNQPG

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 339 Appendices

Appendix 38: pET28a vector

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 340 Appendices

Appendix 39: AK53 xylanase temperature profile

AK53 xylanase Relative Temperature (°C) O.D Activity U/mL Activity % 40 0.7667 665.8404 81.86511 50 0.7799 676.0651 83.12223 60 0.8786 752.5174 92.52205 70 0.95712 813.3385 100 80 0.82472 710.7823 87.39072 90 0.5828 523.3927 64.35116

Appendix 40: C5 xylanase temperature profile

C5 xylanase

Temperature (°C) O.D Activity U/mL Relative Activity % 40 0.6787 597.6762 73.443048 50 0.83479 718.5825 88.3001312 60 0.95771 813.7955 100.000001 70 0.77703 673.842 82.8023727 80 0.72056 630.1007 77.4274 90 0.40709 387.2889 47.5904479

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 341 Appendices

Appendix 41: C5 xylanase pH profile

C5

Relative pH O.D Activity U/mL Activity % 3 0.412 391.0922 52.13378 4 0.62666 557.3664 74.29864 5 0.81599 704.0201 93.84803 6 0.87557 750.1704 100 7 0.83561 719.2177 95.87391 8 0.81879 706.189 94.13715 9 0.632 561.5027 74.85002 10 0.38442 369.7289 49.28599

Appendix 42: AK53 xylanase pH profile

AK53

pH O.D Activity U/mL RA % 3 0.78329 606.7312 83.69556

4 0.83885 649.7676 89.63222 5 0.93588 724.9264 100 6 0.91428 708.1952 97.69201 7 0.89168 690.6894 95.27717 8 0.78589 608.7452 83.97337 9 0.58565 453.6406 62.57747 10 0.502 388.8459 53.63936

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 342 Appendices

Appendix 43: C5 xylanase pH stability profile

C5 Relative activity %

Time pH (minutes) 5 6 7 8 9 10 0 100 100 100 100 100 100 20 88.00436 100 99.60206 96.93413 83.54187 73.97597 40 86.60296 98.84731 98.84731 96.93413 83.54187 73.97597 60 85.64637 97.89072 96.93413 96.93413 81.62869 72.06279 80 85.55071 97.89072 95.02095 96.93413 80.6721 72.06279 100 83.54187 95.97754 92.15118 94.06436 78.75892 72.06279 120 80.6721 95.97754 89.28141 93.10777 78.75892 70.14961 140 78.75892 95.02095 88.32482 93.10777 78.75892 70.14961 160 75.88915 95.02095 86.41164 91.19459 76.84574 72.06279 180 74.93256 94.06436 85.45505 91.19459 74.93256 69.19302 200 73.97597 93.10777 85.45505 89.28141 73.97597 69.19302

Appendix 44: AK53 xylanase pH stability profile

AK53 Relative activity % pH Time (minutes) 3 4 5 6 7 8 9 0 100 100 100 100 100 100 100 20 77.52496 78.8416 100 88.6089 88.3889 81.12963 57.1078 40 77.41581 78.26197 99.41615 86.72364 87.56981 80.80047 55.41547 60 75.72347 78.26197 90.10831 86.72364 86.72364 80.80047 54.5693 80 77.41581 76.56964 89.26214 85.87748 85.87748 79.10814 48.64613 100 74.03114 76.56964 89.26214 85.87748 85.87748 79.10814 48.64613 120 70.64647 77.41581 88.41598 85.03131 85.87748 75.72347 46.9538 140 65.56947 75.72347 89.26214 85.03131 84.18514 75.72347 48.64613 160 61.33864 74.87731 89.26214 83.33898 84.18514 75.72347 40.18446 180 61.33864 73.18497 89.26214 83.33898 84.18514 65.56947 40.18446 200 58.89321 71.75918 89.59046 84.30022 83.48282 65.56947 40.18446

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 343 Appendices

Appendix 45: AK53 xylanase temperature stability profile

AK53 Relative activity %

Temperature (°C) Time (minutes) 50 60 70 80 90 0 100 100 100 100 100 20 85.93984 87.70397 100 91.14404 77.78865 40 84.1757 85.93984 94.76052 85.93984 74.47296 60 84.1757 85.05777 94.76052 85.93984 71.65035 80 81.5295 84.1757 94.48707 82.41157 69.00414 100 81.5295 81.5295 93.59442 80.64743 66.88718 120 80.64743 81.5295 92.81026 80.64743 63.1825 140 77.11916 80.64743 92.29337 80.64743 59.47781 160 77.11916 80.64743 91.84969 78.8833 55.06748 180 77.11916 78.8833 91.49687 77.11916 50.65714 200 68.29849 77.11916 91.23225 77.11916 42.48566

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 344 Appendices

Appendix 46: C5 xylanase temperature stability profile

C5 Relative activity % Temperature (°C) Time (minutes) 50 60 70 80 90 0 100 100 100 100 100 20 93.7855 100 88.00994 74.53363 50.37253 40 92.82291 97.14591 86.08475 74.53363 50.27627 60 91.86031 95.71069 83.19697 71.64585 48.25482 80 91.86031 95.71069 81.27178 67.79547 47.581 100 88.00994 95.71069 81.27178 64.90769 46.61841 120 84.15956 93.7855 80.30919 60.09472 45.65582 140 80.30919 93.7855 80.30919 60.09472 45.65582 160 78.384 84.15956 80.30919 57.20694 42.76804 180 76.45881 81.27178 79.34659 52.39397 38.91766 200 74.53363 80.30919 79.08381 45.65582 40.6484

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 345 Appendices

Appendix 47: DEAE Sepharose profile of AK53 xylanase

DEAE Sepharose of AK53 % Fraction O.D Activity U/mL 0.D at 280 1 0.001 72.73431 0.0011 3 0.0058 76.45236 0.0013 5 0.0077 77.92409 0.00248 7 0.01 79.70565 0.0256

9 0.1 149.4191 0.0355 11 0.31 312.0837 0.058 13 0.33 327.5755 0.076

15 0.51 467.0023 0.08 17 0.8 691.6344 0.09 19 1 846.5531 0.11 21 1.3161 1091.402 0.13 23 1.2 1001.472 0.1 25 0.98295 833.3462 0.13 27 0.56462 509.3106 0.1397 29 0.54 490.2401 0.13 31 0.3 304.3377 0.135 33 0.22937 249.6282 0.135 35 0.2 226.8784 0.131

37 0.17 203.6406 0.123 39 0.16 195.8947 0.124 41 0.13 172.6569 0.101

43 0.1 149.4191 0.082 45 0.05 110.6894 0.054 47 0.04 102.9435 0.0106 49 0.03 95.19752 0.0094 51 0.03 95.19752 0.008 53 0.02 87.45159 0.008 55 0.01 79.70565 0.013 57 0.0018 73.35399 0.006

59 0.0016 73.19907 0.007

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 346 Appendices

Appendix 48: DEAE Sepharose profile of C5 xylanase

DEAE Sepharose of C5 Absorbance at Fraction 280 0.D 540 Activity U/mL 1 0.011 0.001 72.73431 3 0.013 0.003 74.2835 5 0.0148 0.043 105.2672 7 0.018 0.005 75.83269 9 0.019 0.014 82.80403 11 0.06 0.09 141.6731 13 0.09 0.35 343.0674 15 0.14 0.55154 499.1789 17 0.12 0.7 614.1751 19 0.14 0.9 769.0937 21 0.157 0.93 792.3315 23 0.174 0.84 722.6181 25 0.17 0.8 691.6344 27 0.14 0.6 536.7157 29 0.17 0.57 513.4779 31 0.13 0.5 459.2564 33 0.15 0.27 281.0999 35 0.25 0.17 203.6406 37 0.24 0.16 195.8947 39 0.2 0.1 149.4191 41 0.2 0.04 102.9435 43 0.17 0.02 87.45159 45 0.16 0.016 84.35321 47 0.095 0.012 81.25484 49 0.06 0.009 78.93106 51 0.04 0.007 77.38187 53 0.008 0.004 75.05809 55 0.007 0.004 75.05809 57 0.005 0.005 75.83269 59 0.001 0.005 75.83269

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 347 Appendices

Appendix 49: AK53 xylanase Kinetics profile

AK53 Kinetics Specific Absorbance Activity activity at 540 nm (U/mL) (U/mg) 1/s 1/v Substrate -0.4 0 0.5 0.01 239.117 279.4043 2 0.003579 1 0.078332 397.9055 464.9461 1 0.002151 2 0.16 587.684 686.6991 0.5 0.001456 3 0.21024 704.4307 823.1157 0.333333 0.001215 4 0.26 820.062 958.229 0.25 0.001044 5 0.29096 892.0062 1042.295 0.2 0.000959 7.5 0.35 1029.202 1202.606 0.133333 0.000832 10 0.42522 1203.997 1406.851 0.1 0.000711 15 0.50232 1383.16 1616.2 0.066667 0.000619 20 0.51235 1406.468 1643.435 0.05 0.000608 25 0.52921 1445.647 1689.215 0.04 0.000592 30 0.556 1507.901 1761.958 0.033333 0.000568 35 0.605 1621.766 1895.007 0.028571 0.000528

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 348 Appendices

Appendix 50: C5 xylanase Kinetics profile

C5 Kinetics Absorbance Activity at 540 nm (U/mL) SA (U/mg) 1/s 1/v Substrate (mg/ml) -0.4 0

0.5 0.00069 217.4826 271.8532 2 0.003678 1 0.0758 392.0217 490.0271 1 0.002041 2 0.15311 571.6731 714.5914 0.5 0.001399 3 0.17945 632.8815 791.1019 0.333333 0.001264 4 0.21311 711.0999 888.8749 0.25 0.001125 5 0.24713 790.1549 987.6936 0.2 0.001012 7.5 0.30311 920.2401 1150.3 0.133333 0.000869 10 0.34377 1014.725 1268.406 0.1 0.000788 15 0.39201 1126.824 1408.53 0.066667 0.00071 20 0.42311 1199.094 1498.867 0.05 0.000667 25 0.45223 1266.762 1583.453 0.04 0.000632 30 0.52463 1435.004 1793.755 0.033333 0.000557

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 349 Appendices

Appendix 51: BSA standard curve C5 proline

C5 proline Curve C5 W137R and D328R mutation curve BSA BSA Concentration 0.D at 540 nm Concentration 0.D at 540 nm 5 0.0433 5 0.032 10 0.19296 10 0.199769 20 0.38888 20 0.409 40 0.60788 40 0.61 50 0.78 50 0.82 70 0.99 70 1.05 90 1.21 90 1.251

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 350 Appendices

Appendix 52: Temperature profile of C5 proline

Activity Temperature O.D U/mL *30 *10 RA % 40 0.13 1.726569 51.79706 517.9706 42 50 0.2191 2.416731 72.50194 725.0194 59

60 0.39916 3.811464 114.3439 1143.439 93 70 0.4336 4.078234 122.347 1223.47 100 80 0.3657 3.552285 106.5686 1065.686 87 90 0.1632 1.983734 59.51201 595.1201 48 100 0.1098 1.570101 47.10302 471.0302 38

Appendix 53: pH profile of C5 proline

Activity C5 Proline pH O.D U/mL *30 *10 RA % 2 0.011 0.804802 24.14407 241.4407 20.25736 3 0.04 1.029435 30.88304 308.8304 25.91148 4 0.19 2.191325 65.73974 657.3974 55.15694 5 0.33 3.275755 98.27266 982.7266 82.4527 6 0.39 3.740511 112.2153 1122.153 94.15089 7 0.4 3.817971 114.5391 1145.391 96.10059 8 0.42 3.972889 119.1867 1191.867 99.99998 9 0.38 3.663052 109.8916 1098.916 92.20119 10 0.33 3.275755 98.27266 982.7266 82.4527 11 0.16 1.958947 58.7684 587.684 49.30785

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 351 Appendices

Appendix 54: C5 proline temperature stability profile

C5 proline RA %

Time Temperature (°C) (Minutes) 60 70 80 90 100 0 100 100 100 100 100

20 90.80443 100.0003 88.3803 38.10589 32.48033 40 90.80443 100.0003 88.3803 35.47097 30.20111 60 90.80443 97.3654 88.3803 35.47097 30.20111 80 90.80443 97.3654 88.3803 32.83604 24.93126 100 90.80443 97.3654 85.74537 32.83604 22.29633

120 90.80443 97.3654 85.74537 32.83604 19.66141 140 90.80443 94.73047 85.74537 32.83604 17.02648 160 88.1695 94.73047 85.74537 32.83604 17.02648 180 88.1695 94.73047 85.74537 30.20111 17.02648 200 88.1695 92.09554 85.74537 30.20111 17.02648

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 352 Appendices

Appendix 55: C5 proline pH stability profile

C5 proline RA % Time pH (Minutes) 5 6 7 8 9 10 0 100 100 100 100 100 100

20 77.16402 94.66031 97.57636 100.4924 94.66031 88.82821 40 77.16402 94.66031 94.66031 100.4924 94.66031 88.82821 60 74.24797 94.66031 94.66031 100.4924 94.66031 91.74426 80 71.33192 94.66031 94.66031 100.4924 88.82821 88.82821 100 65.49983 94.66031 94.66031 100.4924 85.91217 82.99612

120 65.49983 94.66031 91.74426 100.4924 85.91217 82.99612 140 59.66773 91.74426 91.74426 100.4924 85.91217 82.99612 160 59.66773 91.74426 91.74426 97.57636 82.99612 77.16402 180 53.83563 91.74426 91.74426 97.57636 82.99612 77.16402 200 53.83563 91.74426 91.74426 97.57636 82.99612 77.16402

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 353 Appendices

Appendix 56: DEAE Sepharose of C5 proline

0.D Activity Fraction 280nm 0.D 540 U/mL *10 *10 1 0.011 0.001 0.727343 7.273431 72.73431 3 0.013 0.003 0.742835 7.42835 74.2835 5 0.0148 0.006 0.766073 7.660728 76.60728 7 0.015 0.009 0.789311 7.893106 78.93106 9 0.019 0.1 1.494191 14.94191 149.4191 11 0.02 0.21 2.346243 23.46243 234.6243 13 0.06 0.34 3.353215 33.53215 335.3215 15 0.08 0.44154 4.139737 41.39737 413.9737 17 0.1 0.6 5.367157 53.67157 536.7157 19 0.18 0.93 7.923315 79.23315 792.3315 21 0.2 1 8.465531 84.65531 846.5531 23 0.26 1.2 10.01472 100.1472 1001.472 25 0.3 1.36 11.25407 112.5407 1125.407 27 0.28 1.1 9.240124 92.40124 924.0124 29 0.34 0.67 5.909373 59.09373 590.9373 31 0.18 0.42 3.972889 39.72889 397.2889 33 0.161 0.38 3.663052 36.63052 366.3052 35 0.1801 0.35 3.430674 34.30674 343.0674 37 0.204 0.3 3.043377 30.43377 304.3377 39 0.1681 0.25 2.656081 26.56081 265.6081 41 0.113 0.2 2.268784 22.68784 226.8784 43 0.0812 0.15 1.881487 18.81487 188.1487 45 0.071 0.1 1.494191 14.94191 149.4191 47 0.065 0.08 1.339272 13.39272 133.9272 49 0.05 0.01 0.797057 7.970565 79.70565 51 0.04 0.001 0.727343 7.273431 72.73431 53 0.008 0.004 0.750581 7.505809 75.05809 55 0.007 0.004 0.750581 7.505809 75.05809 57 0.005 0.005 0.758327 7.583269 75.83269 59 0.001 0.005 0.758327 7.583269 75.83269

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 354 Appendices

Appendix 57: Phenyl Sepharose of C5 proline

0.D Activity Fraction 280nm 0.D 540 U/mL *10 *10 1 0.00214 0.0002 0.721146 7.211464 72.11464 3 0.0032 0.0002 0.721146 7.211464 72.11464 5 0.0078 0.0005 0.72347 7.234702 72.34702 7 0.012 0.00088 0.726414 7.264136 72.64136 9 0.025 0.012 0.812548 8.125484 81.25484 11 0.019 0.191 2.19907 21.9907 219.907 13 0.045 0.36 3.508133 35.08133 350.8133 15 0.071 0.484 4.468629 44.68629 446.8629 17 0.099 0.665 5.870643 58.70643 587.0643 19 0.13 0.87 7.458559 74.58559 745.8559 21 0.15 1.1 9.240124 92.40124 924.0124 23 0.16 1.3 10.78931 107.8931 1078.931 25 0.17 1.41 11.64136 116.4136 1164.136 27 0.21 1.3 10.78931 107.8931 1078.931 29 0.2 0.8 6.916344 69.16344 691.6344 31 0.17 0.56 5.05732 50.5732 505.732 33 0.21 0.43 4.050349 40.50349 405.0349 35 0.16 0.4 3.817971 38.17971 381.7971 37 0.17 0.38 3.663052 36.63052 366.3052 39 0.163 0.32 3.198296 31.98296 319.8296 41 0.15 0.27 2.810999 28.10999 281.0999 43 0.12 0.22 2.423703 24.23703 242.3703 45 0.09 0.17 2.036406 20.36406 203.6406 47 0.06 0.1 1.494191 14.94191 149.4191 49 0.01 0.01 0.797057 7.970565 79.70565 51 0.004 0.001 0.727343 7.273431 72.73431 53 0.0008 0.0009 0.726569 7.265686 72.65686 55 0.0007 0.0004 0.722696 7.226956 72.26956 57 0.0005 0.0005 0.72347 7.234702 72.34702 59 0.0001 0.0005 0.72347 7.234702 72.34702

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 355 Appendices

Appendix 58: Kinetic of C5 proline

Activity SA Substrate O.D U/mL *30 *10 U/mg 1/S 1/V -0.66667 0 0.5 0.043 1.052672 31.58017 315.8017 306.6036 2 0.003262 1 0.1229 1.671572 50.14717 501.4717 486.8658 1 0.002054 3 0.28 2.888459 86.65376 866.5376 841.2986 0.333333 0.001189 5 0.33 3.275755 98.27266 982.7266 954.1035 0.2 0.001048 7.5 0.35 3.430674 102.9202 1029.202 999.2254 0.133333 0.001001 10 0.36 3.508133 105.244 1052.44 1021.786 0.1 0.000979 15 0.37 3.585593 107.5678 1075.678 1044.347 0.066667 0.000958 20 0.4 3.817971 114.5391 1145.391 1112.03 0.05 0.000899 25 0.43 4.050349 121.5105 1215.105 1179.713 0.04 0.000848 30 0.44 4.127808 123.8342 1238.342 1202.274 0.033333 0.000832

Appendix 59: Xylose standard curve of AK53 mutant model

Xylose curve AK53 W127R

O.D Xylose substrate 50 0.02 75 0.14 100 0.28 200 0.44 300 0.57 400 0.72 500 0.88 600 1.021 700 1.17 800 1.3

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 356 Appendices

Appendix 60: Xylose standard curve of C5 N84R mutant model and D339R

Xylose standard curve C5 N84R and D339R

Xylose O.D O.D O.D O.D Concentration ug/mL 1 2 3 average 100 0.197 0.26 0.1 0.185667 200 0.475 0.47 0.479 0.474667 300 0.816 0.808 0.825 0.816333 400 1.107 1.1 1.36 1.189 500 1.609 1.67 1.56 1.613 600 1.975 2 1.95 1.975 700 2.38 2.5 2.27 2.383333 800 2.78 2.82 2.69 2.763333 900 3.09 2.98 3.21 3.093333 100 3.47 3.6 3.32 3.463333

Appendix 61: Xylose standard curve of AK53 mutant model

Xylose standard curve AK53 N74R D329R Xylose Conc O.D ug/mL O.D1 O.D 2 O.D 3 average 100 0.181 0.19 0.21 0.193667 200 0.475 0.51 0.489 0.491333 300 0.816 0.89 0.91 0.872 400 1.107 1.37 1.19 1.222333 500 1.609 1.65 1.7 1.653 600 1.975 2 1.99 1.988333 700 2.3 2.28 2.37 2.316667 800 2.78 2.83 2.7 2.77 900 3.09 3.21 3 3.1 100 3.47 3.57 3.3 3.446667

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 357 Appendices

Appendix 62: Xylose standard curve

Xylose standard curve C5 Xylose standard curve AK53 Concentration O.D Concentration O.D

100 0.11 100 0.112 200 0.17 200 0.21 300 0.26 300 0.29 400 0.35 400 0.38 500 0.45 500 0.48

600 0.53 600 0.55 700 0.6 700 0.63 800 0.7 800 0.73 900 0.78 900 0.8 1000 0.87 1000 0.9

Appendix 63: BSA standard curve of AK53 mutant model

AK53 mutation BSA curve BSA conc 0.D 1 0.D 2 0.D 3 0.D average 100 0.129 0.159 0.133 0.140333

150 0.245 0.289 0.14 0.224667 200 0.257 0.325 0.252 0.278 250 0.343 0.38 0.3 0.341 300 0.401 0.402 0.41 0.404333 350 0.427 0.54 0.43 0.465667

400 0.477 0.573 0.52 0.523333 450 0.544 0.647 0.57 0.587 500 0.58 0.73 0.63 0.646667 600 0.677 0.78 0.69 0.715667 700 0.775 0.856 0.71 0.780333

800 0.841 0.921 0.78 0.847333

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 358 Appendices

Appendix 64: Transformation of mutant AK53 and C5 in competent cell

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 359 Appendices

Appendix 65: The effect of pH on the stability of purified GthC5Xyl

RA % on different pH Xylanase 5 6 7 8 9

C5 wild type 73 93 85 89 73

W137R 75 86 91 93 94

D328R 79 89 90 94 75

W137R/D328R 81 93 92 97 78

N84R 74 86 82 73 68

D339R 71 93 81 74 68

R81P 69 89 87 83 73

H82E 73 92 84 74 70

W185P 77 91 84 86 72

D186E 79 90 83 82 75

W185P/D186E 85 91 88 83 66

H82E/ W185P/D186E 89 91 93 88 77

pH stability of GthC5Xyl and its mutant were performed for 200 minutes and the relative activity was calculated.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 360 Appendices

Appendix 66: Optimum pH of GthC5Xyl and its mutants

Xylanase pH

Wild type C5 6

W137R 8

D328R 6.5

W137R/D328R 8

N84R 6

D339R 6

R81P 6.5

H82E 6

W185P 6

D186E 6

W185P/D186E 7

H82E/W185P/D186E 7

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 361 Appendices

Appendix 67: Optimum pH of GthAK53Xyl and its mutants

Xylanase pH

Wild type AK53 5

W127R 6

D318R 5.5

W127R/D318R 6

N74R 6

D329R 5

R71P/H72E 5.5

W175P/ D176E 6

R71P/H72E/W175P/D176E 6

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 362 Appendices

Appendix 68: The effect of pH on the stability of purified GthAK53Xyl and its mutants

RA % on different pH Xylanase 4 5 6 7 8

Wild type AK53 71.7 89.5 84 83 65.5

W127R 77 90 96 93 76

D318R 78 93 97 91 77

W127R/D318R 89 95 97 94 82

N74R 78.50 84.57 89 87.85 79.90

D329R 76 91.66 79 74 70

R71P/H72E 76.68 85 83 82.5 72

W175P/ D176E 72 88 86 85 67

R71P/H72E/W175P/D176E 75.6 84.6 87 83 67.9

pH stability of GthAK53Xyl and its mutant were performed for 200 minutes at different pH, and the relative activity was calculated.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 363 Appendices

Appendix 69: Optimum temperature of GthC5Xyl and its mutants

Xylanase Temperature (°C)

Wild type C5 60

W137R 65

D328R 63

W137R/D328R 67

N84R 63

D339R 62

H82E 64

R81P 57

W185P 63

D186E 62

W185P/D186E 65

H82E/W185P/D186E 71

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 364 Appendices

Appendix 70: Optimum temperature of GthAK53Xyl and its mutants

Xylanase Temperature (°C)

Wild type AK53 70

W127R 73

D318R 72

W127R/D318R 75

N74R 70

D329R 70

R71P/H72E 74

W175P/ D176E 75

R71P/H72E/W175P/D176E 78

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 365 Appendices

Appendix 71: The effect of temperature on the stability of purified GthC5Xyl and its mutants

RA % on different temperature (°C) Xylanase 50 (°C) 60 (°C) 70 (°C) 80 (°C) 90(°C)

C5 wild type 74.53 80.3 79.08 45.65 40.64 W137R 75.46 85.88 82.6 49.6 46

D328R 80 86 83.85 54.6 44.6

W137R/D328R 87.9 87 84.87 61.5 50.4

N84R 78.74 78.7 81 50.65 39

D339R 71 82 74.5 47.5 28.8

R81P 52.9 60.68 52.9 31.6 21.3

H82E 76.5 82 82 55 45.6

W185P 75 84.7 81 45.6 40.5

D186E 77 87.8 78 48.78 42.9

W185P/D186E 81.8 89.9 83.7 51.6 46.8

H82E/ W185P/D186E 81 85.8 90 61 50.5

Temperature stability of GthC5Xyl and its mutant were performed for 200 minutes at different temperature, and the relative activity was calculated.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 366 Appendices

Appendix 72: The effect of temperature on the stability of purified GthAK53Xyl and its mutants

RA % on different temperature (°C) Xylanase 50 (°C) 60 (°C) 70 (°C) 80 (°C) 90(°C)

Wild type AK53 68 77 91 77 42 W127R 81 85.7 91 82.8 51

D318R 83 82 93.5 82 54

W127R/D318R 84.5 87 94 91 57

N74R 77 81.7 95 75.6 41

D329R 70.7 76 87.6 76 33

R71P/H72E 74.5 79.6 88 80 47

W175P/ D176E 69.6 79 88.6 78.5 47

R71P/H72E/W175P/D176E 73.5 80 83.9 81.6 51.7

Temperature stability of GthAK53Xyl and its mutant were performed for 200 minutes at different temperature, and the relative activity was calculated.

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 367 Appendices

Appendix 73: Bradford protein assay

The Bradford assay is a protein determination method that involves the binding of Coomassie Brilliant Blue G-250 dye to proteins (Bradford 1976).

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 368 Appendices

Appendix 74: HIS60 NI resin and gravity column

Expression vector p15TV -L

Purification of his-tagged proteins using His60 Ni Superflow Resin

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 369 Appendices

Appendix 75: kinetic parameters of C5 and its mutants

Xylanase Vmax μmolmg−1 min−1 Km mg ml−1

C5 wild type 1839.86 3.9084

W137R/D328R 881 3.02

D328R 1066 3.29

D339R 923 3.26

R81P 1199 3.66

W137R 1307 3.94

N84R 682 4.1

H82E 946 5.13

W185P 746 6.5

D186E 889 4.78

W185P/D186E 386 5.76

H82E/ 754 5.83 W185P/D186E

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 370 Appendices

Appendix 76: kinetic parameters of AK53and its mutants

Xylanase Vmax μmolmg−1 Km mg ml−1 min−1

AK53 wild type 2028.94 4.34

W127R 1589 3.94

D318R 1464 3.54

W127R/D318R 1379 3.33

N74R 806 5.35

D329R 584 3.61

R71P/H72E 886 6.08

W175P/D176E 642 4.09

R71P/H72E/W175P/D176E 836 5.19

Cloning functional expression and site-directed mutagenesis of thermostable xylanases from Geobacillus thermodenitrificans 371 ISSN 0003-6838, Applied Biochemistry and Microbiology, 2016, Vol. 52, No. 3, pp. 277–286. © Pleiades Publishing, Inc., 2016.

Cloning, Purification and Characterization of a Cellulase-Free Xylanase from Geobacillus thermodenitrificans AK531 M. Irfana, H. I. Gulerb, A. O. Belduzc, A. A. Shaha, and S. Canakcic aDepartmet of Microbiology, Faculty of Biological Sciences, Quaid I Azam University, Islamabad, Pakistan bDepartment Departmet of Molecular Biology and Genetic, Faculty of Sciences, Karadeniz Technical University, 61080 Trabzon, Turkey cDepartment of Biology, Faculty of Sciences, Karadeniz Technical University, 61080 Trabzon, Turkey e-mail: [email protected] Received October 26, 2015

Abstract—Geobacillus thermodenitrificans AK53 xyl gene encoding xylanase was isolated, cloned and expressed in Escherichia coli. After purifying recombinant xylanase from G. thermodenitrificans AK53 (GthAK53Xyl) to homogeneity by ammonium sulfate precipitation and ion exchange chromatography, bio- chemical properties of the enzyme were determined. The kinetic studies for GthAK53Xyl showed KM value –1 –1 to be 4.34 mg/mL (for D-xylose) and Vmax value to be 2028.9 μmoles mg min . The optimal temperature and pH for enzyme activity were found out to be 70°C and 5.0, respectively. The expressed protein showed the highest sequence similarity with the xylanases of G. thermodenitrificans JK1 (JN209933) and G. thermo- denitrificans T-2 (EU599644). Metal cations Mg2+ and Mn2+ were found to be required for the enzyme activ- ity, however, Co2+, Hg2+, Fe2+ and Cu2+ ions caused inhibitor effect on it. GthAK53Xyl had no cellulolytic activity and degraded xylan in an endo-fashion. The action of the enzyme on xylan from oat spelt produced xylobiose and xylopentose. The reported results are suggestive of a xylanase exhibiting desirable kinetics, sta- bility parameters and metal resistance required for the efficient production of xylobiose at industrial scale.

Keywords: xylanase, Geobacillus thermodinitrificans, thermostable, GthAK53Xyl (recombinant xylanase) DOI: 10.1134/S0003683816030066

One of the most important sources of renewable attention because of their potential applications in energy is hemicellulose biomass. It is chiefly com- agro-industrial processes such as bioconversion of posed of xylan, mannan and galactan [1] and is the hemicellulosic biomass into fermentative sugars, pulp second most abundant natural polysaccharide. Xylans bleaching, bleach boosting, improvement of baking are heteropolymers made up of a linear chain of β-D- properties in bread making, enhancement of the xylopyranose residues with β-(1,4) linkages substi- digestibility of animal feed, clarification of fruit juices tuted with sugars (arabinose, xylose, galactose, etc.), and xylitol production [5]. glucuronic acids, and other groups (e.g., acetyl, feru- Large scale industrial production of enzymes is loyl, p-coumaryl). Xylan is among the most important associated mainly with the substrate as well as depends hemicellulose components of the plant cell wall [2]. It upon the conditions prevailing in that particular is generally known that the complete hydrolysis of industry where enzyme is required. The industrial xylan requires the synergistic activities of several demand of thermostable xylanases has not yet been hydrolytic enzymes, mainly xylanase, β-xylosidase met adequately. Moreover, xylanases are also required and additional enzymes such as α-l-arabinofuranosi- for hydrolyzing abundantly available and renewable dase, α-d-glucuronidase, and acetyl xylan esterase [3]. ligniocellulosic biomass in order to produce xylooligo- Among these enzymes, the most important one is saccharides and fermentable sugars that are used as endo-β-1,4-xylanase (EC 3.2.1.8) which cleaves inter- probiotics and for bioethanol production [6]. nal glycosidic bonds in xylan to create short xylo-oli- In consideration with the facts mentioned above, gosaccharides [4]. Majority of identified xylanases the present study aims to clone, purify and character- belong to glycoside hydrolase (GH) families 10 and 11 ize extracellular thermophilic xylanase produced by while others belong to GH families 5, 7, 8, 16, 26, 43, Geobacillus thermodenitrificans AK53 (GthAK53Xyl) 52 and 62 [5]. Xylanases have captured much of the and to test it for cellulolytic activity. This xylanase was found to have the ability to degrade xylan into xylooli- 1 The article is published in the original. gosaccharides.

277 278 IRFAN et al.

MATERIALS AND METHODS D-galactopyranoside, isopropyl β-D-1-thiogalactopy- Substrates and chemicals. The chemicals were pur- ranoside, and LB-ampicillin agar plates. The white col- chased from Fluka Chemie AG (Switzerland), Merck onies were picked up and confirmed for the insert anal- (Germany), Sigma-Aldrich (USA) and Acumedia ysis by double digestion. Three clones having the insert Manufacturers, Inc. (USA). The Wizard genomic were processed for sequencing. DNA purification kit, Wizard plus SV minipreps DNA Bioinformatics analysis. BLASTn and BLASTp pro- purification system, Taq DNA polymerase, dNTP, and grams were used for the analysis of nucleotides and their all restriction enzymes were obtained from Promega deduced amino acids, respectively (http://www.ncbi. Corp. (USA). All chemicals were reagent grade. SDS- nlm.nih.gov/BLAST/). Multiple sequence alignment PAGE molecular mass standards (10–250 kDa) were of xylanase was carried out using CLUSTALW pro- purchased from New England Biolabs (UK). gram (http://www.ebi.ac.uk/clustalW) and phyloge- Strains, vectors and media. Escherichia coli BL21 netic analysis and dendrogram construction for the (DE3), E. coli JM101 and pET28a (+) vector were GthAK53Xyl was performed using MEGA 6.0 (with gently supplied from Karadeniz Technical University, minimum evolution). Molecular Biology Laboratory (Turkey). E. coli con- taining recombinant plasmids was cultured according Construction and expression of the recombinant to the method of Karaoglu [7]. vector pET_GthAK53Xyl. Recombinant vector Screening of xylanolytic strains. Water and soil pET_GthAK53Xyl was constructed using the above samples were collected from the hot springs of Azad primers having NheI and HindIII restriction sites Kashmir (Pakistan). Enrichment was performed using compatible to pET28a (+) vector. The full-length oat spelt xylan (Sigma-Aldrich, USA) as a sole carbon xylanase gene was first amplified and then digested source. Twenty five bacterial strains were screened for with NheI and HindIII. The digested product was xylanolytic ability. purified by gel extraction and ligated into already digested and purified pET28a (+) vector using Phenotypic characteristics. One prominent isolate T4 DNA ligase overnight at 16°C. The ligated prod- with high xylanolytic activity was selected and then ucts were transformed into E. coli JM101 competent identified on the basis of morphological, cultural and cells using heat shock method [8]. The positive clones biochemical properties [8] along with 16S rRNA were picked from LB agar plates with kanamycin sequencing. (50 μg/mL) and confirmed by double digestion of the Phylogenic analysis. The partial 16S rRNA putative plasmids with respective restriction enzymes. sequences were recovered on NCBI server (http:// Three plasmids having xylanase gene was sequenced blast.ncbi.nlm.nih.gov/Blast.cgi) using BLAST tool. using the Taq DyeDeoxy terminator cycle sequencing Similar sequences were downloaded in FASTA format. kit according to the manufacturer’s instructions, and ClustalW2 program was utilized to perform multiple analyzed with Model 370A automated sequencer alignment of sequences and calculations of levels of (Applied Biosystems, Inc., USA). Plasmids having the sequence similarity. The obtained phylogenetic tree was same xylanase sequences were processed for expres- analyzed for closely related organism. Neighbor-joining sion. The recombinant vector pET_GthAK53Xyl was method [6] was used to deduce evolutionary history. transformed into E. coli BL21 (DE3) cells. Five clones Amplification and sequencing of xylanase gene. were grown in LB broth overnight and 1% (vol/vol) of Genomic DNA isolation was performed using the Wiz- this was used as inoculum to cultivate E. coli BL21 ard genomic DNA purification kit according to the man- (DE3) cells. The expression of xylanase gene was ufacturer’s directions. The extracted DNA was used for induced by 1 mM IPTG at OD600 of 0.5–0.6. The the amplification of the xylanase gene using XyGeoT-F: induced cells were further cultivated at 37°C for 20 h (5'CTAgCTAgCATgTTgAAAAgATCgCgAAAAg-3') for higher expression of the protein. and XyGeoT-R: (5'-CCCAAgCTTTCACTTATgATC- gATAATAgCCCA-3') primers having NheI and HindIII Purification of the recombinant xylanase from sites in forward and reversed primers, respectively. The G. thermodenitrificans AK53. After 20 h incubation at primers were designed using the conserved domain 37°C, the culture was reaped by centrifugation at 1300 g sequence of GthAK53Xyl available in NCBI database. for 15 min and suspended in 100 mM Na-phosphate The xylanase encoding gene was amplified using buffer (pH 7.0). Culture supernatant was precipitated defined PCR conditions in a thermal cycler (Bio-Rad, using 60% ammonium sulphate. The obtained precip- USA). Initial denaturation was performed at 95°C for itate was dissolved in 100 mM Na-phosphate buffer 3 min followed by 36 cycles of denaturation (94°C for (pH 7.0). Precipitate was dialyzed against the same 1 min), annealing (62°C for 1 min), and extension buffer overnight and loaded on a column (1.5 × 50 cm) (72°C for 1 min 30 s) in a PCR vial containing 50 μL of of DEAE-Sepharose pre-equilibrated with 20 mM reaction volume. Final extension was done at 72°C for Na-phosphate buffer (pH 7.0). The column was 7 min. The amplified gene was cloned into pGEM®-T washed with 250 mL of the same buffer at flow rate of easy vector and positive clones were selected using blue 1 mL/min. The enzyme was eluted with linear gradi- white selection on 5-bromo-4-chloro-3-indolyl-beta- ent of (0–0.5 M) NaCl in same buffer. The active frac-

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 52 No. 3 2016 CLONING, PURIFICATION AND CHARACTERIZATION 279 tions were collected and concentrated by ultrafiltra- tion of temperature stability of GthAK53Xyl the resid- tion (30000 MWCO filters, Sartorious, Germany). ual activity was measured after incubating enzyme for Homogeneous enzyme test. To test the GthAK53Xyl 200 min at 40, 50, 60, 70, 80, 90 and 100°C. purity a reverse phase C-18 column (4.6 × 250 mm; Effect of pH on GthAK53Xyl activity and stability. Merck, Germany) of HPLC System 600 Waters (Waters Enzyme’s pH optimum was measured at 70°C using the Corp. USA) was employed. The solvent system acetoni- buffer solutions of different pH values. The following trile-water (70 : 30 vol/vol) at a flow rate of 0.5 mL/min buffers (100 mM) were used: Na-acetate (pH 5.0–6.0), was utilized for the separation of sample components. K-phosphate (pH 6.0–7.0), Tris-HCl (pH 7.0–9.0) OD OD280 was read using a highly sensitive photo- and glycine-NaOH (pH 9.0–10.0). The results were diode array detector (996 Waters). expressed as relative activity (%). For determination of the pH stability of enzyme, it was incubated at each SDS–PAGE, zymogram analysis and protein identi- ° fication. The fractions containing GthAK53Xyl activ- pH value and 70 C temperature for 200 min, and then ity were analyzed by SDS-PAGE and zymography as the residual activities were measured. described by Liao et al. [7]. SDS-PAGE was per- Effect of some metal ions on GthAK53Xyl activity. formed using an 11% polyacrylamide gel with a Bivalent metal ions Zn2+, Mn2+ and Mg2+ were 5% stacking gel with the mini-protean II system (Bio- reported to be activators of xylanase [12]. The effect of Rad, USA) according to Laemmli [9]. The protein various metal ions was assayed on GthAK53Xyl activ- bands were visualized by staining with Coomassie ity under optimum reaction conditions. To find acti- Brilliant Blue R-250 (Bio-Rad, USA). For the zymo- vator effect the enzyme solution was pre-incubated gram analysis, briefly, after the separation of the with 1, 5 or 10 mM Zn2+, Mn2+ or Mg2+. To reveal the enzyme samples by SDS-PAGE, the zymogram gel inhibitor effect of various metal ions on GthAK53Xyl was soaked in 0.1% oat spelt xylan at 37°C for 1 h in activity enzyme was made devoid of its own metal ions 2.5% (vol/vol) Triton X-100 to remove the SDS and by dialysis and then enzyme solution was pre-incu- re-nature the proteins in the gel, which was then bated with 1, 5 or 10 mM Cd2+, Ca2+, Hg2+, Ni2+, Fe2+ washed thoroughly in MilliQ water (Millipore, USA) or Cu2+. 100% xylanase activity was defined for and incubated at 70°C for 20 min in 100 mM Na- enzyme without metal ions and residual (%) activity phosphate buffer (pH 7.0). The gel was submerged in was assayed. ° 0.1% (wt/vol) Congo red solution at 37 C for 30 min Analysis of hydrolysis product from oat spelt xylan. and destained with l.0 M NaCl until pale-red hydrolysis GthAK53Xyl after purification was mixed with 100 mM zones appeared against a red background. The reaction Na-phosphate buffer (pH 5.0) containing 1% (wt/vol) was stopped by dipping the gel into 5% acetic acid. To xylan and incubated at 70°C for 12 h. Samples were identify the protein sequence, a homology search was then taken and centrifuged at 3000 g for 10 min to performed using Mascot (http://www.matrixscience. remove insoluble materials. Aliquots of 3 μL were com). The partial amino acid sequence was used to iden- spotted on the TLC plates. Chromatography was then tify analogous proteins through a BLAST search of the performed by the ascending method on silica gel 60 NCBI protein sequence database. Amino acid homol- F TLC plates (20 cm × 20 cm), Merck, Germany) ogy alignment of the predicted XYN11A with other 254 highly homologous known xylanases was carried out. with a solvent system consisting of n-butanol, acetic acid and water (2 : 1 : 1, vol/vol/vol). For detection of sugars, Xylanase activity assay. The activity of plates were sprayed with 5% (vol/vol) sulfuric acid in GthAK53Xyl was measured by the release of reducing ethanol and then heated for about 10 min at 120°C. sugars from oat spelt xylan following the dinitrosali- Determination of the GthAK53Xyl shelf life. For the cylic acid method [10]. The xylanase assay was carried determination of shelf life, enzyme was kept both at out by incubating suitably diluted enzyme with room temperature and at 4°C. For 12 weeks, samples 1% xylan in 100 mM Na-phosphate buffer (pH 5.0) at ° were withdrawn at different intervals and residual 60 C for 20 min. The liberated sugars were estimated activity of xylanase was determined. using 3,5-dinitrosalicylic acid reagent. One unit of xylanase was defined as the amount of enzyme that liberates 1 μmol of reducing sugar as xylose under the RESULTS assay conditions using oat spelt xylan as the substrate. Isolation and identification of bacteria. Twenty f ive Determination of protein concentration. Protein bacterial strains isolated from the hot springs of Azad concentration was determined by the method of Brad- Kashmir (Pakistan) were screened for xylanolytic abil- ford [11]. BSA was used as a standard. ity. Bacterial strains, which formed clear halos around Effect of the temperature on GthAK53Xyl activity and their colonies on xylan agar plates, were picked up for stability. Using xylan as substrate the effect of tempera- further studies. The strain that showed the largest zone ture (over the range from 40 to 100°C) on GthAK53Xyl of hydrolysis around the colony proved its xylanolytic activity was determined spectrophotometrically. The ability. It was identified as G. thermodenitrificans AK53 results were then expressed as relative activity (%) on the basis of various morphological and biochemical obtained at optimum temperature. For the determina- characteristics along with 16S rRNA sequencing.

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Geobacillus thermodenitrificans BGSC W9A26 (AY608963) 2 0 Geobacillus thermodenitrificans R-32500 (FN428639)

0 Bacillus sp. BGSC W9A54 (AY608967)

0 Bacillus sp. BGSC W9A66 (AY608969) 0 Bacillus sp. BGSC W9A87 (AY608978) 0 Geobacillus thermodenitrificans SSCT85 (AB210952) 0 Geobacillus thermodenitrificans NG80-2 (CP000557) 9 Geobacillus thermodenitrificans R-32511 (FN428647)

14 Geobacillus thermodenitrificans M62 (AB116111)

Geobacillus sp. TC-W7 (GQ866911)

25 29 Geobacillus thermodenitrificans R-32623 (FN428666) 11 Geobacillus thermodenitrificans R-32618 (FN428662)

Geobacillus thermodenitrificans SSCT83 (AB210956)

Geobacillus thermodenitrificans AK 53 (KP203955)

Acetobacter pasteurianus 386B (529230092)

Fig. 1. Phylogenetic tree demonstrating the location of G. thermodenitrificans AK53.

Amplification and sequencing of xylanase gene. The modenitrificans JK1 (JN209933) and G. thermodenitri- gene encoding xylanase was amplified with previously ficans T-2 (EU599644) followed by other Bacillus and described primer having 1200 bp from G. thermode- Geobacillus spp. (Fig. 2). Secondary and tertiary struc- nitrificans DNA as a template having translational ini- ture of GthAK53Xyl has been proposed using the tiation code ATG and termination code TGA. available crystal structure of xylanase (PDB ID, 1HIZ Construction and expression of the recombinant vec- chain A) from G. thermodinitrificans. The secondary tor pET_GthAK53Xyl. The cloning of xylanase gene structure contained a total of 13 β- sheets and 11-α into pET28a (+) vector was confirmed by double diges- helices along with 5 sharp turns. Three-dimensional tion of the recombinant vector with respective restric- structure, obtained from PyMol PDB viewer, showed tion enzymes. The construct pET_GthAK53Xyl was catalytically important residues, Glu1 in conserved transformed and successfully expressed in E. coli BL21 regions in “bowl” shaped structure of GH10 xylanase (DE3) cells. A higher GthAK53Xyl production was (Fig. 3). achieved by inducing the expression of xylanase with Purification of the GthAK53Xyl. GthAK53Xyl was 1mM IPTG at 37°C. purified by ammonium sulfate precipitation and ion Bioinformatics analysis. The isolate was confirmed exchange chromatography on DEAE-Sepharose as G. thermodinitrificans AK53 strain. The sequence of (Table 1). The yield of the purified xylanase was 138% 16S rRNA gene of G. thermodinitrificans AK53 strain with a specific activity of 1113.5 U/mg protein and an was deposited in GenBank with accession number overall purification fold of 7.3. The GthAK53Xyl was (KP203955). Comparative 16S rRNA gene sequence eluted from DEAE-Sepharose column by 0.6 M NaCl analysis showed that the strain studied is phylogeneti- (Fig. 4). The eluted protein appeared as a single band cally most closely affiliated to the genus Geobacillus. on 15% SDS-PAGE. The molecular weight of The phylogenetic tree revealed that G. thermodinitrifi- GthAK53Xyl was determined against denaturing pro- cans AK53 strain is closely associated (99.0%) with tein markers and found to be of 45 kDa. The purity of G. thermodenitrificans SSCT83 (AB210956) as shown the purified enzyme was confirmed by SDS-PAGE in Fig. 1. and reverse phase HPLC on C-18 column. The puri- The deduced amino acid sequence contained more fied enzyme showed a single band in SDS-PAGE gel (Asp+Glu) negatively charged residues and no Cys (Fig. 5a) and revealed a single peak at a retention time residues were found in the xylanase. In silico analysis of 2.5 min on HPLC chromatogram (Fig. 5b) indicat- demonstrated the aliphatic index of 37. BLASTp anal- ing that it was homogeneous. ysis of amino acid sequence exposed extensive similar- Effects of temperature and pH on GthAK53Xyl activ- ity with various endoxylanases of GH10 family and ity. The purified GthAK53Xyl exhibited activity over a displayed a high homology with enzymes from G. ther- broad range of temperatures (40–100°C) and pH (3.0–

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Bacillus halodurans MIR32 (AF534180) 58 Bacillus sp. C-125 (D00087) 79 Bacillus sp. 31 clone BH-G10 (JF912896) 67 Bacillus halodurans TSEV1 (KC342810) 100 Bacillus sp. JB 99 (JN039035) Bacillus sp. N16-5 (HM046619) Geobacillus sp. WBI (EU931582) 53 Geobacillus thermoleowrans (JN680872) Geobacillus stearothermophilus 21 (D28122) Geobacillus sp. 7 (JF792185) Geobacillus sp. TC-W7 (GQ857066) Geobacillus thermodenitrificans JK1 (JN209933) Geobacillus thermodenitrificans AK53 (KP202693) Geobacillus thermodenitrificans T-2 (EU599644) Thermotoga thermarum DSM 5069 (CP002351)

0.2

Fig. 2. Phylogenetic relationship of GthAK53Xyl with other xylanases available at NCBI database.

10.0) with optima at 70°C (Fig. 6a) and pH 5.0 (Fig. 7a), respectively. The recombinant enzyme retained more than 90% activity after exposure to 70°C for 200 min (Fig. 6b). On the other hand, GthAK53Xyl retained more than 80% activity after 3 h incubation at various pH values (5.0, 6.0 and 7.0) (Fig. 7b). Effects of metal ions, inhibitors and detergents on

GthAK53Xyl activity. The activity of the purified GLUGLU 159159 GGLULU 265265 GthAK53Xyl was tested in the presence of several metal ions, inhibitors and detergents at different con- centrations (Table 2). Metal cations Mg2+ and Mn2+ were found to be required for the enzyme activity. On the other hand, Co2+, Hg2+, Fe2+ and Cu2 were reveled as strong inhibitors of the xylanase. The GthAK53Xyl has been pointedly inhibited by CoCl2, FeSO4, CuCl2 and CaSO4 even at very low concentration of 1 mM. Fig. 3. The proposed structure of GthAK53Xyl. AlCl3, ZnSO4, and CuSO4 inhibited the enzyme at higher concentration (5.0 mM) only. Trisodium citrate (TSC) enhanced the enzymatic activity. β-mer- Activity, µM/mL OD280 captoethanol, DTT and EDTA did not have adverse 1200 0.16 effect on enzyme activity, while diethylpyrocarbonate 0.14 (DEPC), N-ethylmaleimide (NEM) and phenyl- 1000 0.12 methylsulfonyl fluoride (PMSF) caused inhibitory 800 effect only at higher concentration (5.0 mM) (Table 2). 0.10 The xylanase is quite stable in the presence of the 600 0.08 detergents tested but half of its activity was inhibited in 2 0.06 the presence of 1% cetyltrimethylammonium bromide 400 1 (CTAB) concentration and more than 80% activity is 0.04 retained even at 1% Tween 20 and 1% Tween 40. 200 0.02 Substrate specificity and kinetic parameters. The 0 0 xylanase activity of GthAK53Xyl was evaluated with 1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 various substrates at 70°C (pH 5.0) for 20 min to deter- Fractions mine the enzyme specificity. GthAK53Xyl showed specificity toward polymeric xylan, but not to other Fig. 4. DEAE-Sepharose chromatography of the endox- substrates such as insoluble xylan, carboxymethyl cel- ylanase produced by G. thermodenitrificans AK53. 1—activ- lulose, filter paper, avicel, pNP-α-glucopyranoside, ity; 2—protein.

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Table 1. Purification of GthAK53Xyl Specific activity, Purification step Total protein, mg Total activity, U Yield, % Purification, -fold U/mg protein Cell extract 4.58 695.4 151.5 100 1 Ammonium sulfate 2.90 821.8 282.7 118 1.8 precipitation DEAE-Sepharose 0.85 952.7 1113.5 138 7.3 pNP-β-xylopyranoside, pNP-β- galactopyranoside, lyzed product of oat spelt xylan. As shown in Fig. 8, pNP-α-L-arabinofuranoside, pNP-acetate and pNP- the enzyme released a range of xylooligosacchrides α-D- xylopyranoside. from xylan. The main products were xylobiose and xylopentose. The KM and Vmax of GthAK53Xyl (for oat spelt xylan) were found to be 4.34 mg/mL and Determination of shelf life of xylanase. The purified ° 2028.9 μmoles mg–1 min–1, respectively. enzyme did not lose any activity when stored at 4 C for 12 weeks but thereafter, a decline was observed. The Mode of hydrolysis. The mode of action of puri- GthAK53Xyl retained more than 90% of initial activ- fied GthAK53Xyl was analyzed by TLC of the hydro- ity after 10 weeks which would be important for its

(a) M 1234

50 kDa 40 kDa 45 kDa

25 kDa

mAU (b) 60 50 40 30 20 10 0

0 12345678910 min

Fig. 5. SDS-PAGE (a) of crude and purified GthAK53Xyl (an arrow). M—SDS-PAGE molecular mass standards 10–250 kDa; 1—culture supernatant; 2—induced supernatant; 3—ammonium sulfate precipitate; 4—eluate from DEAE-Sepharose. (b)—HPLC profile of the purified GthAK53Xyl.

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Relative activity, % (a) Relative activity, % (a) 120 120 100 100 80 80 60 60 40 40 20 20 0 4050 6070 80 90 100 °C 0 345pH 679 8 10 Relative activity, % (b) 120 Relative activity, % (b) 120 100 100 1 3 2 80 2 80 3 4 4 5 1 60 60 40 6 5 40 20 20 0 0 20 40 60 80 100 120 140 160 180 200 min 0 0 20 40 60 80 100 120 140 160 180 200 min Fig. 6. The effect of temperature on the activity (a) and sta- Fig. 7. The effect of pH on the activity (a) and stability (b) bility (b) of purified GthAK53Xyl. b: 1—50°C; 2—60°C; of purified GthAK53Xyl. b: 1—5.0; 2—6.0; 3—7.0; 4—4.0; 3—70°C; 4—80°C; 5—90°C. 5—8.0; 6—pH 9.0. application. On the other hand, at room temperature, according to the revealed D-xylose amount. after the the enzyme was completely stable for 5 weeks but enzyme reaction. showed 80 and 70% residual activity after storage for Both, the activity and stability of GthAK53Xyl 10 and 12 weeks, respectively. were tested in the range pH 3.0–10.0. The enzyme exhibited the highest activity around pH 5.0. It was observed that GthAK53Xyl was active and stable at DISCUSSION 70°C in a broad pH range from 4.0 to 8.0. The pH sta- Microorganisms are well known to have adapted to bility of the enzyme is very important for the predic- the environment by producing extracellular enzymes tion of its storage conditions. As GthAK53Xyl did not which are expected to form nutrients by degrading lose activity after being stored at different pH values, it complex macromolecules into soluble monomers for supporting the growth of the producing microbes. Iso- late AK53 from the Tattapani hot spring of Azad 1X1X2X3X4X5 Kashmir (Pakistan) was revealed to be a strain of G. thermodenitrificans based on biochemical analysis and 16S rRNA sequencing. Xylanase produced by the bacterium displayed optimal activity and stability at elevated temperatures (50–80°C) and under neutral to slightly acidic conditions (pH 5.0–7.0). The xylanase gene cloned from G. thermodenitrificans is of 1200 bp like those of Bacillus and Geobacillus spp. [13, 14]. In this study, GthAK53Xyl gene was cloned to Fig. 8. TLC analysis for hydrolysis products released from pET-28a (+) expression vector and expression, purifi- oat spelt xylan by GthAK53Xyl. 1—sample; X1— cation and characterization of recombinant xylanase D-xylose; X2—xylobiose; X3—xylotriose; X4—xylo- was carried out. Enzyme activity was determined tetraose; X5—xylopentose.

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Table 2. The effect of various metal ions, reagents and bacillus sp. DG-22, Geobacillus sp. 70PC53, Bacillus detergents on GthAK53Xyl acitivity firmus, Thermobifida fusca, and Bacillus sp. NG-27 Metal Ions* 1 mM 5 mM 10 mM were optimally active in the temperature range between 60 and 70°C [16, 17]. 2+ 88 60 51 Co The effect of different metal ions on GthAK53Xyl Ag+ 108 101 80 activity was unique among all the characterized Cs+ 117 107 10 4 xylanases of Geobacilli [13]. Xylanases of the other Geo- 2+ 3+ bacilli were inhibited by Mn or it had no effect on the Al 99 84 48 activity, while xylanase of G. thermodenitrificans JK1 2+ 2+ Mn (SO4) 144 109 100 was slightly stimulated by these ions. In contrast, Mn ions at low concentration stimulated GthAK53Xyl in Zn2+ 116 82 68 2− 2+ 2+ 2+ 2 Mg2+ (SO ) 110 99 97 the presence of SO4 ions. Co , Hg , Fe and Cu 4 were reveled as strong inhibitors of the enzyme studied. 2+ Ca (Cl2) 108 98 89 Similarly, Penicillium sclerotiorum and Aspergillus ficuum xylanases were introverted by these ions [18– Fe2+ 89 46 27 20]. The inhibition by Hg2+ seems to be a universal K+ 108 10 9 111 property of xylanases, demonstrating the presence of Li+ 108 111 124 thiol groups of Cys residues in active sites or around 2+ 2+ 88 72 65 them [20]. The inhibition of GthAK53Xyl by Hg con- Cu (Cl2) firmed the presence of Trp residues in the active site, 2+ Mn (Cl2) 99 94 86 since oxidation of indole ring disrupts the interaction [14]. The inhibition of GthAK53Xyl by Cu2+ is similar Ca2+ (SO ) 89 71 60 4 to those of Bacillus sp. YJ6, Staphylococcus sp. SG-13, 2+ Mg (Cl2) 124 99 94 Plectosphaerella cucumerina, Cellulosimicrobium sp. 2+ 99 86 62 MTCC 10645, Aspergillus versicolor, A. ficuum AF-98 Cu (SO4) and G. thermodenitrificans TSAA1 [14, 21–26]. Hg2+ 54 27 Xylanase of G. thermodenitrificans JK1 was inhibited 2+ Reagents even at low concentration of Zn while GthAK53Xyl DTT 98 96 89 was inhibited only at higher concentration of this ion. TSC 118 115 114 The GthAK53Xyl was inhibited by N-bromosuc- EDTA 89 80 70 cinimide (N-BS) only at high concentration (10 mM), where xylanase from other Geobacillus species was β-mercaptoethanol 99 98 98 inhibited even at low concentration [14]. N-BS, Trp DEPC 94 89 68 modifier, reduced xylanase activity at 5 and 10 mM; NEM 90 89 61 this is an evidence for the role of aromatic amino acid PMSF 80 71 58 residues in the active site [27]. The xylanases were quite stable in the presence of the detergents tested in Detergents 0.1% 0.5% 1% Geobacillus sp., G. thermodenitrificans TSAA1 [14], SDS 91 86 84 G. thermoleovorans, sp. MT-1 and Dictyoglomus ther- CTAB 85 53 49 mophilum Rt46B.1 [21, 28]. The slight inhibitory effect Tween 20 96 89 82 of different detergents (besides CTAB) on GthAK53Xyl (Table 2) is similar to other microbe Tween 40 90 86 80 xylanases [21]. Tweens (40 and 60) and Triton X-100 Triton X-100 107 84 74 inhibited GthAK53Xyl only at high concentration and * Control—100%, values are given in% of control. these results are comparable to those in Aspergillus awamori [22]. Even at 1% SDS the enzyme retained more than 80% residual activity while xylanase from can be stored under conditions with a broad range of Bacillus, Geobacillus [14] and Paenibacillus sp. NF1 pH for a long period of time. was strongly inhibited in presence of this detergent [5]. GthAK53Xyl exhibited optimum activity at 70°C. Almost all known xylanases of Geobacilli belong to Most of the previously studied G. thermodenitrificans the GH10 family [29]. GthAK53Xyl was also found to xylanases have been reported to operate at an optimum belong to this family. It was ~45 kDa in size, while temperature of 70°C [13, 14]. Xylanases from some xylanolytic enzymes of other Geobacilli were 30, 35, other microorganisms have higher temeprature 36, and 40 kDa [30, 28]. 43 kDa xylanase of G. ther- optima, e.g 85°C for enzymes of Streptomyces sp. and modenitrificans JK1 [13] and 47 kDa enzyme of Bacillus sp, 95°C for enzyme of Thermotoga neapoli- G. thermodenitrificans TSAA1 [14] were the most sim- tana [15, 7]. On the other hand, xylanases from Paeni- ilar in size to GthAK53Xyl. However, the xylanase

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Table 3. Comparison of properties of recombinant xylanases from 3 different strains of G. thermodenitrificans pH Strains Tem. optima, °C Thermostability pH stability, min References optima G.thermodenitrificans 6.0 70 T1/2 (70°C), 0.5 h Not determined [13] JK1 G. thermodenitrificans 9.0 70 >85% activity retained >90% activity [14] TSAA1 at 70°C for 3 h; retained for 3 h T 1/2 (80°C), 10 min at pH 5.0 G.thermodenitrificans 5.0 70 >90% activity retained >85% activity Present investigation AK53 at 70°C for 3 h retained for 3 h at pH 5.0 studied differed from these enzymes in their pH and 2. Ravalason, H., Gwénaël, J., Daniel, M., Maryvonne, P., temperature optima as well as pH stability and ther- and Coutinho, P.M., Appl. Microbiol. Biotechnol., 2008, mostability (Table 3). vol. 80, no. 4, pp. 719–733. As cellulase activity may result in poor fiber 3. Wong, K., Tan, L., and Saddler, J.N., Microbiol. Mol. mechanical strength, xylanase should be free from cel- Biol. Rev., 1988, vol. 52, no. 3, pp. 305–317. lulolytic activity for paper and pulp treatment [31]. 4. Beg, Q., Kapoor, M., Mahajan L., and Hoondal, G., Thus, the cellulase-free xylanase has an advantage in Appl. Microbiol. Biotechnol., 2001, vol. 56, no. 3, the production of high quality pulp. Our result indi- pp. 326–338. cated that GthAK53Xyl had a cellulase-free nature 5. Zheng, H.C., Sun, M.Z., Meng, L.C., Pei, H.S., and can be used in the production of high quality pulp. Zhang, X.Q., et al., J. Microbiol. Biotechnol., 2014, vol. 24, no. 4, pp. 489–496. Substrate specificity and kinetic parameters of 6. Saitou, N. and Nei, M., Mol. Biol. Evol., 1987, vol. 4, GthAK53Xyl revealed that the enzyme contained no no. 4, pp. 406–425. other enzyme activity. The result of TLC analysis of the 7. Karaoglu, H., Yanmi, D., Sal, F.A., Celik, A., Canakci, S., hydrolysis products of oat spelt xylan indicated the and Belduz, A.O., J. Mol. Catal. B: Enzym., 2013, vol. 97, enzyme as endoxylanase (Fig. 8). Xylobiose and pp. 215–224. xylopentose were the major end products of hydrolysis. 8. Bergey’s Manual of Systematic Bacteriology, 9th ed., The KM of the enzyme was within the range for KM of Hensyl, W.M., Ed., Philadelphia, USA: Williams and other microbial xylanases (0.14–14 mg/mL) [14] and Wilkins, 1994. closely resemble to the KM of Pichia stipites xylanase 9. Laemmli, U.K., Nature, 1970, vol. 227, no. 5259, (4.2 mg/mL [32]). The KM value of GthAK53Xyl with pp. 680–685. oat spelt xylan as substrate was lower than those for the 10. Miller, G.R., Anal. Chem., 1959, vol. 3, pp. 426–428. xylanases isolated from other microorganisms, such as 11. Bradford, M.M., Anal. Biochem., 1976, vol. 72, no. 2, Aspergillus niger SCTCC 400264 [33], Scytalidium aci- pp. 248–254. dophilum [34], Bacillus sp [24], Aspergillus usamii E001 12. Sharma, A., Adhikari, S., and Satyanarayana, T., World [35], Paenibacillus sp. NF1 [5]. J. Microbiol. Biotechnol., 2007, vol. 23, no. 4, pp. 483– In summary, due to biochemical properties, high 490. activity, profile of produced xylooligosaccharides and 13. Gerasimova, J., and Kuisiene, N., Microbiology, 2012, resistance to some detergents and metals, GthAK53Xyl vol. 8, no. 4, pp. 418–424. is an attractive candidate for the production of xylooli- 14. Verma, D., Anand, A., and Satyanarayana, T., Appl. gosaccharides from the paper and pulp and food indus- Biochem. Biotechnol., 2013, vol. 170, no. 1, pp. 119–130. tries as compared to other xylanases from G. thermode- 15. Bhosale, S.H., Rao, M.B., and Deshpande, V.V., nitrificans. Microbiol. Rev., 1996, vol. 60, pp. 280–300. 16. Son-Ng, I., Li, C.W., Yeh, Y., Chen, P.T., Chir, J.L., and Ma, C.H., Extremophiles, 2009, vol. 13, no. 3, ACKNOWLEDGMENT pp. 425–435. This study was financially supported by TUBITAK 17. Cheng, Y.F., Yang, C.H., and Liu, W.H., Enzyme Scientific and Research Council of Turkey and Higher Microbiol. Technol., 2005, vol. 37, no. 5, pp. 541–546. Education Commission of Pakistan. 18. Polizeli, M.L., Rizzatti, A.C., Monti, R., Terenzi, H.F, Jorge, J.A., and Amorim, D.S., Appl. Microbiol. Biotechnol., 2005, vol. 67, no. 5, pp. 577–591. REFERENCES 19. Knob, A., Beitel, S.M., Fortkamp, D., Terrasan, C.R.F., 1. Collins, T., Gerday, C., and Feller, G., FEMS Microbiol. Fernando de Almeida, A., Biomed. Res. Int., 2013, vol. 8, Rev., 2005, vol. 29, no. 1, pp. 3–23. pp. 728–735.

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20. Bastawde, K.B., World. J. Microbiol. Biotechnol., 1992, 28. Wu, S., Liu, B., and Zhang, X., Appl. Microbiol. vol. 8, no. 4, pp. 353–368. Biotechnol., 2006, vol. 72, no. 6, pp. 1210–1216. 21. Zhang, G.M., Huang, J., Huang, G.R, Ma, L.X, and 29. Saksono, B. and Sukmarini, L., J. Biosci., 2010, vol. 17, Zhang, X.E., App. Microbiol. Biotechnol., 2007, vol. 74, no. 4, pp. 189–197. no. 2, pp. 339–346. 30. Canakci, S., Inan, K., Kacagan, M., and Belduz, A.O., J. Microbiol. Biotechnol., 2007, vol. 17, no. 8, pp. 1262– 22. Yin, L., Lin, H., Chiang, Y., and Jiang, S., J. Agricult. 1270. Food Chem., 2010, vol. 58, no. 1, pp. 557–562. 31. Subramaniyan, S. and Prema, P., FEMS Microbiol. 23. Gupta, S., Bhushan, B., and Hoondal, G.S., J. Appl. Lett., 2000, vol. 183, no. 1, pp. 1–7. Microbiol., 2000, vol. 88, no. 2, pp. 325–334. 32. Basaran, P., Basaran, N., and Hang, Y.D., World J. 24. Kamble, R.D. and Jadhav, A.R., Asian Pacific J. Trop. Microbiol. Biotechnol., 2000, vol. 16, no. 6, pp. 545– Biomed., 2012, vol. 2, pp. 1790–1797. 550. 25. Salama, M.A., Ismail, K.M.I., Amany, H.A, Abo, E.L., 33. Li, X.R., Xu, H., Xie, J., Yi, Q.F., Li, W., Qiao, D.R., and Genweely, N.S.I., Int. J. Bot., 2008, vol. 4, no. 1, Cao,Y., and Cao, Y., J. Microbiol. Biotechnol., 2014, pp. 41–48. vol. 24, no. 4, pp. 483–488. 34. Balaa, B.A., Brijs, K., Gebruers, K., Vandenhaute, J., 26. Lu, F., Lu, M.M., Lu, Z., Bie, X., Zhao, H., and Wouters, J., and Housen, I., Bioresour. Technol., 2009, Wang, Y., Bioresour. Technol., 2008, vol. 99, no. 13, vol. 100, no. 24, pp. 6465–6471. pp. 5938–5941. 35. Zhou, C., Zhang, M., Wang, Y., Guo, W., Liu, Z., 27. Cardoso, O.A.V. and Filho, E.X.F., FEMS Microbiol. Wang, Y., and Wang, W., African J. Microbiol.Res., Lett., 2003, vol. 223, no. 2, pp. 309–314. 2013, vol. 7, no.16, pp. 1535–1542.

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 52 No. 3 2016

CLONING, PURIFICATION AND CHARACTERIZATION OF HALOTOLERANT XYLANASE FROM Geobacillus Thermodenitrificans C5

1Muhammad Irfan, 2Halil Ibrahim Guler, 1Aamer Ali Shah, 3Fulya Ay Sal, 2Kadriye Inan, 3*Ali Osman Belduz

Address(es): 1Departmet of Microbiology, Faculty of Biological Sciences, Quaid I Azam University, Islamabad, Pakistan. 2Department of Molecular Biology and Genetic, Faculty of Sciences, Karadeniz Technical University, 61080 Trabzon, Turkey. 3Department of Biology, Faculty of Sciences, Karadeniz Technical University, 61080 Trabzon, Turkey.

*Corresponding author: [email protected] doi: 10.15414/jmbfs.2016.5.6.523-529

ARTICLE INFO ABSTRACT

Received 18. 2. 2015 High levels of extracellular xylanase activity (994.50 IU/ml) produced by Geobacillus thermodenitrificans C5 originated gene was Revised 13. 1. 2016 detected when it was expressed in E. coli BL21 host. Thermostable xylanase (GthC5Xyl) was purified to homogeneity and showed a Accepted 14. 1. 2016 molecular mass of approximately 44 kDa according to SDS-PAGE. The specific activity of the purified GthC5Xyl was up to Published 1. 6. 2016 1243.125IU/mg with a 9.89-fold purification. The activity of GthC5Xyl was stimulated by CoCl2, MnSO4, CuSO4, MnCl2 but was inhibited by FeSO4, Hg, CaSO4. GthC5Xyl showed resistant to SDS, Tween 20, Triton X-100, β- Mercaptoethanol, PMSF, DTT, NEM and DEPC, SDS, and EDTA. A greater affinity for oat spelt xylan was exhibited by GthC5Xyl with maximum enzymatic activity at Regular article 60°C and 6.0 pH. The activity portrayed by GthC5Xyl was found to be acellulytic with stability at high temperature (70°C-80°C) and low pH (4.0 to 8.0). Xylobiose and xylopentose were the end products of proficient oat spelt xylanase hydrolysis by GthC5Xyl. High SDS resistance and broader stability of GthC5Xyl proves it to be worthy of impending application in numerous industrial processes especially textile, detergents and animal feed industry.

Keywords: Geobacillus thermodenitrificans, thermostable GthC5Xyl enzyme, recombinant xylanase

INTRODUCTION For application in industry, it is necessary for xylanase to be acellulolytic (Goswami et al., 2014) and requiring minimum downstream processing for its Hemicellulose in plant cell walls contain xylan as the major component which production. The aim of this study was to carry out the heterologous expression of accounts for one third of the earth’s total renewable organic carbon source Geobacillus thermodenitrificans xylanase gene in E. coli for increased xylanase (Cheng et al., 2014). These hetero-polysaccharides are primarily composed of production as well as to secrete this expressed enzyme via E. coli in the medium xylose subunits linked by β-1, 4-glycosidic bonds which forms the backbone. to minimize its downstream processing for industrial application. Purification and This backbone further contains different side groups such as acetyl groups, biochemical characteristic’s analysis was further carried out. methyl groups and other sugar molecules (Collins et al., 2005). As xylan has a complex and intricate structure, it requires a combination of enzymes for its MATERIALS AND METHODS complete breakdown. These enzymes include endo-1, 4-β-xylanase, β-xylosidase, acetylxylan esterase, arabinose and glucuronidase (Khandeparker and Numan, Bacterial Strains, Substrates, Vectors and Chemicals 2008) out of which the most potent and thus most important one are the endo-1, 4-b-xylanases (Li et al., 2014). Xylan is degraded to short xylo-oligosaccharides The chemicals were obtained commercially from Merck A.G. (Darmstadt, of various lengths by the action of these enzymes. Xylanases conjoin with β - Germany), Sigma Chem. Co. (St. Louis, MO, USA), Fluka Chemie A.G. (Buchs, xylosidases (EC 3.2.1.37) for completely hydrolyzing xylan to xylose monomers. Switzerland), and Acumedia Manufacturers, Inc. (Baltimore, Maryland, USA), There are various benefits of carrying out xylan hydrolysis at high temperatures Aldrich-Chemie (Steinheim, Germany). The, Wizard Plus SV Minipreps DNA by utilizing thermostable enzyme. These advantages include increased reactant Purification System, Wizard Genomic DNA Purification Kit, dNTP, restriction and products solubility due to low viscosity, a higher mass transport rate, a enzymes and Taq DNA Polymerase were obtained from Promega Corp. decreased hazard of contamination by mesophilic microorganism and half-lives (Madison, WI, USA). All chemicals were reagent grade and all solutions were at elevated temperatures leads to better hydrolysis (Bhalla et al., 2014). One of made with deionized and double distilled water. E. coli JM101: E. coli BL21 the important concern that still persists is the enzyme stability while thermal (DE3): pET28 (a) + were gently provided by Karadeniz Technical University, processing. The changes in the 3D structural confirmation of enzymes at high Molecular Biology Laboratory. The method of Karaoglu was used for culturing temperature lead to enzyme inactivation (Bankeeree et al., 2014). of recombinant E. coli (Karaoglu et al., 2013). There is a frequent isolation and identification of Geobacillus from various sources because of its ability to produce thermostable endo-xylanases for xylan Screening and Phylogenic Analysis for Xylanase Producing Bacteria hydrolysis. Examples of Geobacillus strains producing xylanases with potential industrial applications are novel thermostable endo-xylanase from Geobacillus Water and soil samples were collected from the Garam Chashma hot springs of sp. WSUCF1(Bhalla et al., 2014), Geobacillus stearothermophilus 1A05583 Chitral KPK Pakistan. Enrichment was done using oat spelt xylan (Sigma (Yan Wang et al., 2013) producing xylanase, Geobacillus thermodenitrificans Chemicals, Germany) as a sole carbon source. Twenty bacterial strains were TSAA1 producing Thermostable and Alkalistable Endoxylanase (Verma et al., screened for xylanolytic ability. One prominent isolate was selected and then 2013), novel thermophilic xylanase from Geobacillus thermodenitrificans JK1 identified on the basis of cultural, morphological and biochemical properties (Gerasimova and Kuisiene, 2012), G. thermoleovorans producing highly (Sneath, 1994) along with 16S rRNA sequencing (Supplementary Table 1). thermophilic endoxylanase (Verma and Satyanarayana, 2012), thermophilic NCBI server (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was utilized to recover the Geobacillus sp. 7 1(Canakci et al., 2012) producing an alkali-stable partial 16S rRNA via BLAST tool. Similar sequences were then downloaded in endoxylanase. FASTA format. Multiple alignment of sequences and calculations of levels of sequence similarity were performed by using ClustalW2 program. Analysis for

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closely related organisms was carried out by using obtained phylogenetic tree. re-nature the proteins in the gel, which was then washed thoroughly in 50 mM Evolutionary history was deduced by Neighbor joining method (Saitou and Nei, Phosphate buffer (pH 6) and incubated at 60 °C for 30 min in 1% xylan. The gel 1987). was flooded in 0.1% (w/v) Congo red solution for 15 min and destained with l M NaCl until hydrolysis zones appeared against a red background. The reaction Amplification, Sequencing and Bioinformatics Analysis of Xylanase was then stopped by dipping the gel into 5% acetic acid solution. To classify the protein sequence, a homology search was performed using Mascot Wizard Genomic DNA Purification Kit was used to isolate Genomic DNA. (http://www.matrixscience.com). The partial amino acid sequence was used to Xylanase gene was amplified by using XyGeoT-F: identify analogous proteins through a BLAST search of the NCBI protein (5’CTAgCTAgCATgTTgAAAAgATCgCgAAAAg-3’) having NheI and sequence database. Amino acid homology alignment of the predicted GthC5Xyl XyGeoT-R: (5’- CCCAAgCTTTCACTTATgATCgATAATAgCCCA-3’) having with other highly homologous known xylanases was carried out. HindIII restriction sites in defined PCR conditions. Thermo Cycler (Bio-Rad, Hercules, CA) was used for amplification of xylanase encoding gene. Initial Activity Assay, Determination of Protein Concentration and Kinetics of denaturation was at 94°C for 3 minutes followed by 36 cycles (denaturation Xylanase (94°C for 1 min), annealing (62°C for 40 sec), and extension (72°C for 90 sec) were performed in a PCR vial containing 50 μl of reaction volume, and final Dinitrosalicylic acid (DNS) method (Miller, 1959) was used for studying the extension was done at 72°C for 8 min. The amplified gene was cloned into activity of purified GthC5Xyl via measuring the reducing sugar release from oat pGEM®-T Easy vector and positive clones were selected using blue white spelt xylan. All xylanase assays was performed with 100 mM sodium phosphate selection on 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside, Isopropyl β- buffer pH. 6.0 (unless otherwise specified). The xylanase assay was carried out D-1-thiogalactopyranoside, and ampicillin plate. The white colonies were picked by incubating suitable diluted enzyme with 1% xylan in 100 mM sodium up and confirmed for the insert by double digestion. Two clones having the insert phosphate buffer for 20 minutes at 60ºC. 3,5-dinitrosalicylic acid reagent were processed for sequencing. (DNSA) was used for the estimation of liberated sugars. One unit of xylanase is Nucleotides and their deduced amino acids were analyzed by using BLASTn and defined as the amount of enzyme that liberates 1 μmol of reducing sugar under BLASTp programmers respectively (http://www.ncbi.nlm.nih.gov/BLAST). the assay conditions using oat spelt xylan as the substrate. Bradford method CLUSTALW program was used for carrying out multiple sequence alignment of (1976) was used for determining protein concentration. In this procedure, Bovine xylanase (http://www.ebi.ac.uk/clustalW) and MEGA 4.0 (with minimum serum albumin was used as standard (Bradford, 1976). Kinetic parameters Vmax evolution) was used for phylogenetic analysis and dendogram construction of (µmol/min/mg) and Km were determined by Michaelis-Menten plots of specific xylanase. activities at multiple xylan concentrations varying between 0.5 mg/ml to 30 mg/ml. Construction and Expression of the Recombinant Vector pET_GthC5Xyl Determination of pH and Temperature Effects on Activity and Stability of Complete xylanase gene was amplified and purified by gel extraction and ligation GthC5Xyl was done into pGEM®-T Easy Vector. Heat shock method (Belduz et al., 1997) was used to transformed the ligated product in E. coli JM101 competent cells. The effect of temperature on activity of GthC5Xyl was determined Recombinant vector pET_GthC5Xyl having NheI and HindIII restriction sites spectrophotometrically using xylan as substrate. By using the method previously compatible to pET28a (+) vector was constructed. The xylanase gene cloned described enzyme activity was assayed over a range of temperatures from 40°C pGEM®-T Easy Vector was isolated and digested with respective restriction to 100°C. Results were expressed as relative activity (%) obtained at optimum enzymes. Xylanase gene product was purified by gel extraction and ligated into temperature. GthC5Xyl temperature stability was determined by incubating already purified and digested pET28a (+) vector using T4 DNA ligase overnight enzyme at 40, 50, 60, 70, 80, 90 and 100°C for 200 min and then measuring the at 16°C. Heat shock method (Belduz et al., 1997) was used to transform the residual activity. ligated product in E. coli JM101 competent cells. The positive clones were pH optimum was determined at 540nm and 60°C by using buffer solutions of established by double digestion of the recombinant plasmid with specific different pH. Results were expressed as relative activity (%) obtained at optimum restriction enzymes. Two plasmids having xylanase gene was sequenced using pH. pH stability of enzyme was determined by incubating enzyme at each pH the Taq DyeDeoxy Terminator Cycle Sequencing Kit according to the value for 200 min at 60°C and then measuring the residual activity. manufacturer’s instructions, and analyzed with an Applied Biosystems (Macrogen, Korea) Model 370A automatic sequencer. The recombinant vector Activator and Inhibitor Effects of Metal Ions on GthC5Xyl Activity pET_GthC5Xyl was transformed into E. coli BL21 (DE3) cells. Four clones were grown in LB broth overnight and 1% (v/v) of this was used as inoculum to For the determination of activation and inhibition effect of various metal ions, the cultivate E. coli BL21 (DE3) cells. 1 mM IPTG was used for induction of the enzyme was incubated for 20 min with 1, 5 and 10 mM of bivalent metal ions 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ recombinant xylanase at OD600nm of 0.5–0.6. For higher expression of xylanase such as, Zn , Mn , Mg , Cd , Ca , Hg , Ni , Fe and Cu at optimum the induced cell were cultivated for 20 h. reaction conditions. GthC5Xyl activity was defined 100% without metal ions and residual activity (%) was determined spectrophotometrically (Table 2). Purification of the Recombinant GthC5Xyl Analysis of Hydrolysis Products and Shelf life Determination of Xylanase After higher expression of recombinant xylanase the culture was centrifuged for 15 minutes at 10,000 rpm and suspended in 50 mM sodium phosphate buffer. For analysis of xylanase hydrolysis product purified GthC5Xyl was mixed with 60% ammonium sulphate was used for precipitation of enzyme and then DEAE 100 mM sodium phosphate buffer (pH 6.0) containing 1% (w/v) xylan and affinity chromatography was used to purify the enzyme. The obtained 60% incubated for 10 h at 60°C. For the removal of insoluble materials samples were precipitates were loaded on a column (1.5 × 50 cm) of DEAE-Sepharose pre- centrifuged for 12 min at 3000 g. TLC pates were then spotted with 3 µl aliquots. equilibrated with 10 mM sodium phosphate buffer pH 6.0. The column was Chromatography by ascending method was then performed on silica gel 60 washed with 1000 ml of the same buffer at flow rate of 0.5 ml/min. After F254TLC plates (Merck) with n-butanol, acetic acid and water (2:1:1) containing washing, column was eluted with linear gradient of (0.55 M) NaCl in sodium solvent system. Plates were then sprayed with 5% (v/v) sulfuric acid in ethanol phosphate buffer. The active fractions were combined and concentrated by and then heated at 120°C for about 10 min for the sugar detection. Shelf life ultrafiltration (Sartorious, 30000 MWCO filters). determination was done by keeping GthC5Xyl in refrigerator at 4°C as well as at GthC5Xyl enzyme purity was checked by reverse phase C-18 column (4.6 x 250 room temperature. Samples were then removed at different intervals and residual mm; E. Merck, Germany) of High Performance Liquid Chromatography (HPLC activity was determined for 16 weeks. System 600 Waters, Waters Corporation, Massachusetts, USA).The solvent system acetonitrile-water (70:30) at a flow rate of 0.5 ml/min was employed for RESULTS the separation of sample components. Absorbance was read at 280 nm using a highly sensitive photo-diode array (PDA) detector (996 Waters). Isolation and Identification of Bacteria

SDS-PAGE, Zymogram Analysis and Protein Identification Bacterial strains, which formed clear halos around their colonies on xylan agar plates, were picked up for further studies. The isolate was confirmed as The fractions containing xylanase activity were analyzed by SDS-PAGE and Geobacillus thermodenitrificans C5 with partial 16S rRNA sequencing having a zymography as described by Liao et al. (Karaoglu et al., 2013; Liao et al., length of 1419 bp nucleotide. The sequence was deposited in GenBank 2012). SDS-PAGE was performed using an 11% (w/v) polyacrylamide gel with (Accession No. KP203956). The Fig. 1 shows phylogenetic relation of isolate. It a 5% stacking gel with the Mini-Protean II system (Bio-Rad, Hercules, CA) shows a very close relation with Geobacillus thermodenitrificans subsp.calidus according to Laemmli (1970). The protein bands were visualized by staining F84b (EU477773) and Geobacillus thermodenitrificans subsp. with Coomassie Brilliant Blue R-250 (Bio-Rad, Hercules, USA). For the thermodenitrificans NG80-2 (CP000557) (Fig. 1) having 99% similarity. zymogram analysis, briefly, after performing SDS-PAGE the zymogram gel was soaked for 30 minutes in 2.5% (v/v) Triton X-100 to remove the SDS and

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Purification of the GthC5Xyl

Elution of recombinant GthC5Xyl was done by DEAE affinity chromatography column with 0.55 M of NaCl (Supplementary Fig. 2). The protein eluted was visualized as a single band on 15% SDS- PAGE which indicated its homogeneous nature (Fig. 3). GthC5Xyl’s molecular weight was found out to be approximately 44 kDa. This was confirmed by denaturing protein markers. SDS- PAGE and reverse phase HPLC on C-18 column were used to check the purity of purified protein. A single peak was revealed at retention time of 2.5 min by HPLC chromatogram confirming the purity of preparation (Fig 4) with control of Bacillus subtilis (Supplementary Fig. 3). Purified xylanase yield was 176.28% with a specific activity of 1243.12 IU/mg and an overall purification fold of 9.89 (Table 1).

Table 1 Summary of GthC5Xyl purification steps Total Total Specific Purification Yield Purificati Protein activity activity step (%) on Yield (mg) (U) (U/mg)

Cell extract 4.49 564.1286 125.6411 100 1

Precipitate 3.03 714.4617 235.7959 126.6487 1.876742 DEAE- 0.8 994.5004 1243.125 176.2897 9.894258 Sepharose

Figure 1 Phylogenetic tree of Geobacillus thermodenitrificans C5. The evolutionary history was inferred using the Neighbor-Joining method. Evolutionary analyses were conducted in MEGA 5.0.

Cloning and Sequence Analysis of Xylanase

Xylanase encoding gene having 1230 bp was amplified by using G. thermodenitrificans DNA as template with translational initiation codon ATG and termination codon TGA. Confirmation of xylanase gene cloning in pET28 (+) vector was done by recombinant vector double digestion with NheI and HindIII restriction enzymes. A successful transformation and expression of the construct pET_GthC5Xyl was carried out in E. coli BL21 (DE3) cells. Xylanase expression was induced with 1 mM IPTG at 37°C and higher production of recombinant xylanase was achieved.

Bioinformatics Analysis

Deduced amino acid sequence of xylanase from Geobacillus thermodenitrificans C5 having showed no presence of cysteine amino acid residues but an excess of (Asp+Glu) negatively charged residues. Aliphatic index of 37 was demonstrated by the in silico analysis. A wide resemblance with various GH10 family endoxylanases and high homology with other Bacillus sp and Geobacillus sp was depicted by amino acid sequence analysis with BLASTp (Fig. 2). Available crystal structure of xylanase (PDB ID, 1HIZ chain A) from G. thermodinitrificans was utilized for proposing GthC5Xyl secondary and tertiary structures. A total of 11 α helices along with 5 sharp turns and 13 β sheets were found in secondary structure. Important catalytic residues Glu was found within Figure 3 SDS-PAGE showing purified recombinant Xyl enzyme obtained from ion-exchange the conserved region present inside a “bowl” shaped structure of GH10 xylanase column chromatography by DEAE Sepharose. M, SDS-PAGE molecular mass standards 10– 250 kDa New England Biolab; 1, culture supernatant; 2, Induced supernatant; 3, ammonium via 3D structure obtained from PyMol PDB viewer. Phylogenetic relationship of sulfate precipitation; 4, Purified GthC5Xyl GthC5Xyl of G. thermodenitrificans C5 with other xylanases available at NCBI database showed maximum identity with xylanase of Geobacillus sp. TC-W7 1: 280 nm, 4 nm C5 Irfan Retention Time (GQ857066) and G. thermodenitrificans strain JK1 (JN209933). 90 90

80 80

70 70

60 60

50 50 mAU 40 40 mAU

30 30

20 20

10 10

0 0

-10 -10

0 1 2 3 4 5 6 7 8 9 10 Minutes Figure 4 HPLC profile of the purified xylanase from Geobacillus thermodenitrificans C5 using a reverse phase C-18 column (4.6 x 250 mm) HPLC chromatogram of the purified enzyme shows a single peak at a retention time of 2.5 min confirming that it was a pure preparation.

Biochemical Characterization of Recombinant Xylanase

The purified GthC5Xyl exhibits activity over a broad range of temperature (40– Figure 2 Phylogenetic relationship of xyl of G. thermodenitrificans C5 with other 90°C) and pH (3.0–9.0) with optimum temperature at 60°C (Fig. 5) while xylanases available at NCBI database. Neighbor-joining tree showed maximum optimum pH was 6.0 (Fig. 6). The recombinant GthC5Xyl retained more than identity with xylanase of Geobacillus sp. TC-W7 (GQ857066) and G. 80 % activity after exposure to 60°C for 200 min, and retained more than 70% thermodenitrificans strain JK1 (JN209933)

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activity after exposure to 70°C and 50°C for 200 min (Fig. 7). The enzyme retained more than 80% activity after 3 h at various pH values (6.0, 7.0 and 8.0) (Fig. 8). In order to verify the effect of substances on GthC5Xyl activity, the purified GthC5Xyl was incubated in the presence of several metallic ions, and detergents such as, sodium dodecyl sulfate (SDS), tetrasodium ethylenediaminetetraacetate (EDTA), dithiothreitol (DTT), phenylmethylsulfonyl fluoride (PMSF), and 훽- mercaptoethanol, at 1 mM, 5 mM and 10 mM concentrations (Table 2). In general, the GthC5Xyl activity was enhanced with increased concentration of the 2+ substances used. Hg and FeSO4 were strong inhibitors of the GthC5Xyl while CaSO4 partially inhibit GthC5Xyl at higher concentration. The GthC5Xyl has been inhibited by AlCl3, ZnSO4, MgCl2 and MgSO4 at 10 mM concentration (Table 3). CsCl inhibited the GthC5Xyl at 5.0 mM concentration only. CoCl2, AgNO3, MnSO4, CaCl2, KCl, CuCl2, MnCl2 and CuSO4 does not show inhibitory effect on GthC5Xyl. The GthC5Xyl is quite stable in the presence of the detergents tested but half of its activity is inhibited in the presence of 1% CTAB concentration and more than 80% activity is retained even at 1% concentration of Tween 20. Triton X-100 and Figure 5 The effect of temperature on the activity of purified xylanase. Tween 40 inhibit enzyme at higher concentration only. The effect of NaCl on activity of GthC5Xyl revealed that the activity was increased with increasing concentrations till 0.8 M. At 1 M of NaCl, relative activity was 101% which sharply decreased to 71 % at 1.5 M of NaCl (Table 3).

Table 2 The effects of various metal ions, detergents and inhibitors on GthC5Xyl Control 100% 1 mM 0.1% 5 mM 0.5% 10 mM 1%

(v/v) (v/v) (v/v) Metal Ions

AgNO3 91.24 126.81 111.35 CsCl 105.16 85.05 89.69 MnSO4 135.43 157.75 107.48 AlCl3 107.71 110.57 81.96 CoCl2 125.27 120.63 116.76 ZnSO4 93.83 91.24 81.96 MgSO4·7H2O 107.48 98.20 71.13 Figure 6 The effect of pH on the activity of purified xylanase. CuSO4 115 105 92

FeSO4 88 74 66 MnCl2 139 124 125 LiCl 101 97 93 CaCl2 105 101 91 MgCl2 115 98 77 CaSO4 94 88 81 Hg 57 30 KCl 106 92 91 CuCl2 105 130 101 Inhibitors EDTA 104 90 79 TSC 103 103 102 β- Mercaptoethanol 96 97 97 NEM 102 100 74 DEPC 104 102 79 DTT 106 107 113 PMSF 100 86 76 NBS 100 73 54 Detergents Figure 7 The effect of temperature on the stability of purified xylanase.

Tween 20 99 96 84 Tween 40 90 81 63 CTAB 84 46 43 SDS 96 91 85 Triton X-100 98 87 73

Table 3 Effect of NaCl on purified xylanase GthC5Xyl Salt Concentration Xylanase activity % 0 100±0.08 0.2 M 101 ±0.07 0.4 M 104±0.10 0.6 M 105±0.20 0.8 M 106±0.15 1 M 101±0.22

1.2 M 98±0.30 Figure 8 The effect of pH on the stability of purified xylanase 1.5 M 71±0.24

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Substrate Specificity, Kinetic Parameters, Mode of Hydrolysis and pH 6.0 which is lower than that of XynG1-1 (7.5) from P. campinasensis G1-1 Determination of Shelf Life of Xylanase (Zheng et al., 2012). Purified enzyme retained 52 % of its activity at pH 3.0 (Fig. 6) and was found stable for 180 min at pH between 5.0 and 8.0 with Recombinant GthC5Xyl enzyme activity was evaluated at 60°C and pH 6.0 for retaining about 73% activity (Fig. 8). These facts revealed that GthC5Xyl has 20 min with various substrates for the determination of enzyme specificity much higher stability at lower pH than most of the bacterial xylanases reported (Supplementary Table 2). Specificity of the recombinant enzyme was towards (Beg et al., 2001; Subramaniyan and Prema, 2000; Zhao et al., 2011). The polymeric xylan source and attacked no other substrate such as insoluble xylan, stability of this enzyme at higher temperature (60ºC-80ºC) and lower pH renders carboxymethyl cellulose, Avicel, filter paper, pNP-β- Xylopyranoside, pNP-α- L- it worthy for countless industrial applications such as bioenergy conversion, arabinofuranoside, pNP-α- glucopyranoside, pNP- β- galactopyranoside, pNP-α- food industry and animal feed. D- Xylopyranoside and pNP- acetate. No significant inhibition of GthC5Xyl activity occurred in the presence of 2+ The Km and Vmax of GthC5Xyl (for oat spelt xylan) were 3.9084 mgml−1 and different metallic ions however, it was prudently inhibited by Hg and partially 2+ 1839.86 μmolmg−1min−1, respectively (Supplementary Fig. 1). TLC of the oat by AlCl3 and ZnSO4. As oxidation of indole ring occurs by Hg it is possible that spelt xylan hydrolyzed product was done for the analysis of purified GthC5Xyl the enzyme gets inhibited by the reaction of Hg2+ with tryptophan residues mode of action (Fig. 9). A range of xylooligosacharides were released by the (Zhang et al., 2007). MnCl2 and CoCl2, showed no inhibitory effect on the action of GthC5Xyl on xylan. Xylopentose and xylobiose were the main products enzyme which is similar to Geobacillus sp. 71 (Canakci et al., 2012). However released. Mn strongly inhibited xylanase isolated from Bacillus halodurans (Mamo et al. No activity was lost by purified GthC5Xyl when stored for 12 weeks at 4°C but 2006/31) but it differs from Geobacillus sp. 71 as it gets inhibited by ZnSO4 after that decline was observed. After 16 weeks, 90% of initial activity was whereas Geobacillus sp. 71 is resistant to it. retained by the enzyme which is an important fact for its industrial application. CuCl2 also showed no inhibitory effect on GthC5Xyl activity. This property Conversely enzyme remained completely stable for five weeks at room differed it from xylanases isolated from Geobacillus thermodenitrificans TSAA1 temperature but showed 80% and 70% residual activity after storage for 10 and (Verma et al., 2013), Plectosphaerella cucumerina (Zhang et al., 2007), 12 weeks, respectively. Geobacillus thermoleovorans (Verma and Satyanarayana, 2012), Thermotoga thermarum (Shi et al., 2013), Penicillium glabrum (Knob et al., 2013), Penicillium sclerotiorum,( Knob and Carmona, 2010), Aspergillus ficuum AF- 2+ 2+ 2+ X1 X2 X3 X4 98 (Lu et al., 2008) which are inhibited by Cu . Mn , Co , AgNO3, and CuCl2 were found to be activity stimulators of GthC5Xyl. Xylanases isolated from Streptomyces olivaceoviridis A1 (Wang et al., 2007) and Bacillus subtilis strain R5 (Jalal et al., 2009) had been reported to be stimulated by Fe2+. Recombinant xylanase GthC5Xyl differed from them as it is inhibited by Fe2+. Sodium dodecyl sulfate (SDS) is a known protein denaturant which strongly inactivates most of the proteins. Even low concentration of SDS deactivates most of the xylanases. Only a few enzymes are reported in literature which show resistance against high concentrations of SDS. For instance xylanase BSX from alkalophilic Bacillus sp. NG-27, GH11 xylanase from symbiotic Streptomyces sp. TN119 and GH 10 xylanase XynAHJ3 from Lechevalieria sp. HJ3, have been reported to retain over 100% of their activities in the presence of SDS Figure 9 TLC analysis for hydrolysis products released from oat spelt xylan by xylanase (Bhardwaj et al., 2010; Zhou et al., 2012; Zhou et al., 2011; Zheng et al., from Geobacillus thermodenitrificans C5, 1: GthC5Xyl; X5: D-xylose; X4: xylobiose; X3: 2013). More than 85% of activity is retained by GthC5Xyl even at 1% SDS xylotriose; X2: xylotetraose; X1: xylopentose concentration and 96% and 91% at 0.1% and 0.5% of SDS concentration respectively which is far better than that of Geobacillus thermodenitrificans DISCUSSION TSAA1 showing 91% at 0.1% and 72% at 0.5% of SDS concentration (Verma et al., 2013). While xylanase from Geobacillus sp. MT-1(Wu et al., 2006), Bacillus Hemicellulose is a promising renewable raw material source with immense thermantarcticus (Lama et al., 2004) and Paenibacillus sp. NF1 (Zheng et al., potential. Its annual production and wastage is humongous and if utilized 2014) is significantly inhibited even at 0.02% concentration of SDS. While sensibly it has numerous uses to offer. Xylanase, being regarded as industrially xylanase from Melanocarpus albomyces (Gupta et al., 2014) is partially most important enzyme, if employed prudently in industry it can yield clean inhibited even at low concentration 0.02% of SDS having 85% of its residual processes, low energy consumption and higher yields (Kashyap et al., 2014). activity. Moreover, GthC5Xyl’s activity was also not affected by the presence of High concentration of salts, surfactants and solvents are required in most of the the chelating agent EDTA which proposed that metallic cations are not required industrial processes. For instance biofuel production requires ethanol tolerant for its activity. Similar to this Geobacillus stearothermophilus and Bacillus xylanase, for the bioremediation of industrial waste water contaminated with halodurans xylanases were not inhibited by EDTA, while Geobacillus sp. 71 was solvents requires solvent and salt resistant xylanase and deinking of recycled inhibited by EDTA. CTAB and Triton X can only inhibit the activity of paper requires solvent and surfactant resistant xylanase. Moreover, solvent GthC5Xyl at high concentrations. tolerance enables the enzyme to be recovered, recycled and reused (Shafiei et Substrate specificity and kinetic parameter determination of GthC5Xyl showed al., 2011; Ibara et al., 2012; Shin et al., 2004; Juturu and Wu, 2012; that it only exhibits xylanolytic activity. The thin layer chromatography analysis Kashyap et al., 2014). of oat spelt xylan hydrolysis product specified it to be endo-xylanase. Like many In industry, the most treasured property of an enzyme is considered to be its other xylanases, the major end products of GthC5Xyl hydrolysis of oat spelt thermostability (Cheng et al., 2014). So, to study the strategies that an enzyme xylan were xylobiose and xylopentose. Acellulytic xylan has added advantages in employs to become thermophilic is an intense area of research. The high quality pulp production. Our results showed that GthC5Xyl has cellulase- extraordinarily high specific activity depicted by the recombinant xylanase free nature. obtained from Geobacillus thermodenitrificans C5 makes it a worthy candidate Up to 1.2 M salt concentration is tolerated by GthC5Xyl with maximum activity for further research. of 0.8 M. Similar results were also seen with Gordonia sp (Kashyap et al., In the presently conducted study xylanase gene was isolated from Geobacillus 2014). Glaecicola mesophila KMM241 has been reported to secrete xylanase that thermodenitrificans C5 and then cloned and expressed in E. coli BL21 (DE3) is active at low temperatures and is salt tolerant as well (Guo et al., 2009). host. The expression of this cloned gene of xylanase into the extracellular Glaecicola mesophila retained 90% residual activity at 2.5 M NaCl where as its medium of E. coli is the most substantial part of this study which can be optimal activity was described at 0.5 M NaCl. Whereas Bacillus pumilus employed industrially for commercial xylanase production. One of the interesting xylanase has optimum activity at 1.2 M NaCl (Menon et al., 2010). Bacillus facts is the greater xylanase activity of clone in E.coli over that of Geobacillus subtilis cho40, a marine bacterium has highest activity at at 0.5 M NaCl thermodinitrificans C5 strain which may be is the result of higher protein (Khandeparker et al., 2011). However marine bacteria generally secrete halo expression. The enzyme produced by E. coli functionally active and proficiently tolerant enzymes. Xylanase produced by these bacteria have not been degrades oat spelt xylan even in SDS-PAGE gel. characterized for their tolerance to solvents or detergents. All these observations GthC5Xyl optimal reaction temperature was found to be 60ºC which is found to indicated to the fact that xylanase obtained from Geobacillus thermodenitrificans be even higher than that of Paenibacillus sp. 12-11 (55ºC) (Zhao et al., 2011). C5 has the utmost conditions to resist the harsh conditions of industrial processes GthC5Xyl also retained 77% of its maximum activity at 80ºC and 82 % of its and meet the industrial demands. maximum activity at 70ºC (Fig. 5). GthC5Xyl thermal stability was assessed by incubating the enzyme for 200 min at temperatures from 40ºC to 100ºC. The CONCLUSION enzyme retained more than 70% of its original activity when incubated for 200 min at temperatures ranging from 40ºC to 60ºC and still retained 79% and 45% The present work reports the expression, characterization of halo thermostable of its original activity when incubated for 200 min at 70ºC and 80ºC respectively xylanase from bacterium Geobacillus thermodenitrificans C5. It also addresses (Fig. 7). This thermal stability is far better than most of the bacterial xylanases the property of xylanase such as stability in broad pH range, temperature, NaCl that are previously reported (Shi et al., 2013; Subramaniyan and Prema, concentration and resistant to SDS. Thus, this strain could be good contender for 2000; Zhao et al., 2011). Maximum activity of purified GthC5Xyl was found at

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different biotechnological applications under extreme conditions. Further, KARAOGLU, H., YANMI, D., FULYA A.S., CELIK, A., CANAKCI, S., improvements in enzyme production using optimization parameters by statistical BELDUZ, A.O. 2013. Biochemical characterization of a novel glucose isomerase approach and use in biobleaching are in progress. from Anoxybacillus gonensis G2 T that displays a high level of activity and thermal stability. J Mol Catal B: Enzym, 97, 215-224. Acknowledgement: Authors are thankful to TUBITAK (2216 Research http://dx.doi.org/10.1016/j.molcatb.2013.08.019 Fellowship Programme) and Karadeniz Technical University, Turkey to support KASHYAP, R., MONIKA., SUBUDHI, E. 2014. A novel thermoalkaliphilic the project and provide necessary infrastructure to carry out the present research xylanase from Gordonia sp. is salt, solvent and surfactant tolerant. J Basic work. Microbiol, 54, 1–8. http://dx.doi.org/10.1002/jobm.201400097 KHANDEPARKER, R., NUMAN, M.T. 2008. Bifunctional xylanases and their REFERENCES potential use in biotechnology. J Ind Microbiol Biotechnol, 35, 635-644. http://dx.doi.org/10.1007/s10295-008-0342-9 BAI, Y., WANG, J., ZHANG, Z., YANG, P., SHI, P., LUO, H., MENG, K., KHANDEPARKER, R., VERMA, P., DEOBAGKAR, D. 2011. A novel HUANG, H., YAO, B. 2010. A new xylanase from thermoacidophilic halotolerant xylanase from marine isolate Bacillus subtilis cho40: gene cloning Alicyclobacillus sp. A4 with broad-range pH activity and pH stability. J Ind and sequencing. N Biotechnol, 28(6), 814–821. Microbiol Biotechnol, 37(2), 187–194. http://dx.doi.org/10.1007/s10295-009- http://dx.doi.org/10.1016/j.nbt.2011.08.001 0662-4 KNOB, A., BEITEL, S.M., FORTKAMP, D., TERRASAN, C.R.F., BANKEEREE, W., LOTRAKUL, P., PRASONGSUK, S., CHAIAREEKIJ, S., FERNANDO DE ALMEIDA, A. 2013. Production, Purification, and EVELEIGH, D.E., KIM, S.W., PUNNAPAYAK, H. 2014. Effect of polyols on Characterization of a Major Penicillium glabrum Xylanase Using Brewer’s Spent thermostability of xylanase from a tropical isolate of Aureobasidium pullulans Grain as Substrate. BioMed Research Int, 10, 11-55. and its application in prebleaching of rice straw pulp. SpringerPlus, 3, 1-37. http://dx.doi.org/10.1155/2013/728735 http://dx.doi.org/10.1186/2193-1801-3-37 KNOB, A., CARMONA, E.C. 2010. Purification and characterization of two BEG, Q.K., KAPOOR, M., MAHAJAN, L., HOONDAL, G.S. 2001. Microbial extracellular xylanases from Penicillium sclerotiorum: a novel acidophilic xylanases and their industrial applications: a review. Appl Microbiol Biotechnol, xylanase. Appl Biochem Biotechnol, 162(2), 429–443. 56, 326-338. http://dx.doi.org/10.1007/s002530100704 http://dx.doi.org/10.1007/s12010-009-8731-8 BELDUZ, A.O., DEMIRBAG, Z., DULGER, S., COSKUNCELEBI, H. 1997. LAEMMLI, U.K. 1970. Cleavage of structural proteins during the assembly of The Effect of a CO2-Saturated CaCl2 Solution on the Transformation Efficiency the head of bacteriophage T4. Nature, 227(5259), 680–685. of Escherichia coli JM 109 with an M 13 Derivative. Turk J Biol, 21, 133-139. http://dx.doi.org/10.1038/227680a0 BERGE, A.C.B., ATWILL, E.R., SISCHO, W.M. 2005. Animal and farm LAMA, L., CALANDRELLI, V., GAMBACORTA, A., NICOLAUS, B. 2004. influences on the dynamic of antibiotic resistance in faecal Eschrichia coli in Purification and characterization of thermostable xylanase and β-xylosidase by young dairy calves. Preventive Veterinary Medicine, 69, 25-38. the thermophilic bacterium Bacillus thermantarcticus. Res Microbiol, 155, 283– http://dx.doi.org/10.1016/j.prevetmed.2005.01.013 289. http://dx.doi.org/10.1016/j.resmic.2004.02.001 BHALLA, A., BISCHOFF, K.M., UPPUGUNDLA, N., BALAN, V., SANI, LI, X.R., XU, X., XIE, J., YI, Q.F., LI, W., DAI, R., QIAO, D.R, CAO, Y., R.K. 2014. Novel thermostable endo-xylanase cloned and expressed from CAO, Y. 2014. Thermostable Sites and Catalytic Characterization of Xylanase bacterium Geobacillus sp. WSUCF1. Bioresour Technol, 165, 314–318. XYNB of Aspergillus niger SCTCC 400264. J. Microbiol Biotechnol, 24(4), http://dx.doi.org/10.1016/j.biortech.2014.03.112 483–488. http://dx.doi.org/10.4014/jmb.1307.07086 BHARDWAJ, A., LEELAVATHI, S., MAZUMDAR-LEIGHTON, S., GHOSH, LIAO, H., XU, C., TAN, S., WEI, Z., LING, N., YU, G., RAZA, W., ZHANG, A., RAMAKUMAR, S., REDDY, VS. 2010. The critical role of N- and C- R., SHEN, Q., XU, Y. 2012. Production and characterization of acidophilic terminal contact in protein stability and folding of a family 10 xylanase under xylanolytic enzymes from Penicillium oxalicum GZ-2. Bioresour Technol, extreme conditions. PLoS One, 5(6), 13-47. 123(2), 117–124. http://dx.doi.org/10.1016/j.biortech.2012.07.051 http://dx.doi.org/10.1371/journal.pone.0011347 LU, F., LU, M., LU, Z., BIE, X., ZHAO, H.,WANG, Y. 2008. Purification and BRADFORD, M.M. 1976. A rapid and sensitive method for the quantification of characterization of xylanase from Aspergillus ficuum AF- 98. Bioresour Technol, microgram quantities of protein utilizing the principle of protein dye binding. 99(13), 5938–5941. http://dx.doi.org/10.1016/j.biortech.2007.10.051 Anal Biochem, 72, 248–254. http://dx.doi.org/10.1016/0003-2697(76)90527-3 MAMO, G., HATTI-KAUL, R., MATTIASSON, B. 2006. A thermostable CANAKCI, S., CEVHER, Z., INAN, K., TOKGOZ, M., BAHAR, F., alkaline active endo-b-1-4-xylanase from Bacillus halodurans S7: Purification KACAGAN, M., FULYA A.S., BELDUZ, AO. 2012. Cloning, purification and and characterization. Enzyme Microb Technol, 39, 1492–1498. characterization of an alkali-stable endoxylanase from thermophilic Geobacillus http://dx.doi.org/10.1016/j.enzmictec.2006.03.040 sp. 71. World J Microbiol Biotechnol, 28,1981–1988. MENON, G., MODY, K., KESHRI, J., JHA, B. 2010. Isolation, purification, and http://dx.doi.org/10.1007/s11274-011-1000-3 characterization of haloalkaline xylanase from a marine Bacillus pumilus strain. CHENG, Y.S., CHEN, C.C., HUANG, C.H., KO, T.P., LUO, W., HUANG, Biotechnol Bio-process Eng, 5, 998–1005. http://dx.doi.org/10.1007/s12257-010- J.W., LIU, J.R., GUO, R.T. 2014. Structural analysis of a glycoside hydrolase 0116-x family 11 xylanase from Neocallimastix patriciarum: insights into the molecular MILLER, G.R. 1959. Use of dinitrosalicylic acid reagent for determination of basis of a thermophilic enzyme. J Biol Chem, 289, 11020-11028. reducing sugars. Anal Chem, 31, 426–428. http://dx.doi.org/10.1074/jbc.m114.550905 http://dx.doi.org/10.1021/ac60147a030 COLLINS, T., GERDAY, C., FELLER, G. 2005. Xylanases, xylanase families SAITOU, NEI, M. 1987. The neighbor-joining method: a new method for and extremophilic xylanases. FEMS Microbiol Rev, 29, 3-23. reconstructing phylogenetic trees. Mol Biol Evol, 4, 406–425. Gerasimova, J., Kuisiene, N. 2012. Characterization of the Novel Xylanase from http://dx.doi.org/10.1016/j.ympev.2008.01.019 the Thermophilic Geobacillus thermodenitrificans JK1. Microbiol, 81, 418–424. SHAFIEI, M., ZIAEE, A.A., AMOOZEGAR, M.A. 2011. Purification and http://dx.doi.org/10.1016/j.femsre.2004.06.005 characterization of an organic-solvent-tolerant halophilic a-amylase from the GOSWAMI, G.K., KRISHNAMOHAN, M., NAIN., V., AGGARWAL, C., moderately halophilic Nesterenkonia sp. Strain. F. J Ind Microbiol Biotechnol, RAMESH, B. 2014. Cloning and heterologous expression of cellulose free 38, 275–281. http://dx.doi.org/10.1007/s10295-010-0770-1 thermostable xylanase from Bacillus brevis. SpringerPlus, 3, 1-20. SHI, H., ZHANG, Y., LI, X., HUANG, Y., WANG, L., WANG, Y., DING, H., GUO, B., CHEN, X.L., SUN, C.Y, ZHOU, B.C, ZHANG, Y.Z. 2009. Gene WANG, F. 2013. A novel highly thermostable xylanase stimulated by Ca2+ from cloning, expression and characterization of a new cold-active and salt-tolerant Thermotoga thermarum: cloning, expression and characterization. Biotechnol endo-b-1, 4-xylanase from marine Glaciecola mesophila KMM 241. Appl Biofuels, 6, 26-30. http://dx.doi.org/10.1186/1754-6834-6-26 Microbiol Biotechnol, 84, 1107–1115. http://dx.doi.org/10.1007/s00253-009- SHI, P., CHEN, X., MENG, K., HUANG, H., BAI, Y., LUO, H. 2013. Distinct 2056-y actions by Paenibacillus sp. strain E18 alpha-Larabinofuranosidases and xylanase GUPTA, G., SAHAI, V., GUPTA, R.K. 2014.Thermal stability and in xylan degradation. Appl Environ Microbiol, 79, 1990-1995. thermodynamic of xylanase from melanocarpus albomyces in presence of polyols http://dx.doi.org/10.1128/aem.03276-12 and salts. Bioresour, 9(4), 5801-5816. SHIN, YO., WAHNON, D., WEBER, M.E., VERA, J.H. 2004. Precipitation and http://dx.doi.org/10.15376/biores.9.4.5801-5816 recovery of xylanase using surfactant and organic solvent. Biotechnol Bioeng, 86, IBARA, D., MONTE, M.C, BLANCO, A., MARTINEZ, A.T, MARTINEZ, M.J. 698–705. http://dx.doi.org/10.1002/bit.20080 2012. Enzymatic deinking of secondary fibers: cellulases/hemicellulases versus SNEATH, P.H.A 1994. Endospore forming Gram positive rods and cocci,” in laccase-mediator system. J Ind Microbiol Biotechnol, 39, 1–9. Bergey’s Manual of Systematic Bacteriology, Hensyl WM Ed, Williams & http://dx.doi.org/10.1007/s10295-011-0991-y Wilkins, Philadelphia, Pa, USA, 9th edition, 1994. JALAL, A., RASHID, N., RASOOL, N., AKHTAR, M. 2009. Gene cloning and http://dx.doi.org/10.1016/s0723-2020(11)80095-0 characterization of a xylanase from a newly isolated Bacillus subtilis strain R5. J SUBRAMANIYAN, S., PREMA, P. 2000. Cellulase-free xylanases from Biosci Bioeng, 107 (4), 360–365. http://dx.doi.org/10.1016/j.jbiosc.2008.12.005 Bacillus and other microorganisms. FEMS Microbiol Lett, 183, 1-7. JUTURU, V., WU, J.C. 2012. Microbial xylanases: engineering, production and http://dx.doi.org/10.1111/j.1574-6968.2000.tb08925.x industrial applications. Biotechnol Adv, 30(6), 1219–1227. VERMA, D., ANAND, A., SATYANARAYANA, T. 2013. Thermostable and http://dx.doi.org/10.1016/j.biotechadv.2011.11.006 Alkalistable Endoxylanase of the Extremely Thermophilic Bacterium

528

J Microbiol Biotech Food Sci / Irfan et al. 2016 : 5 (6) 523-529

Geobacillus thermodenitrificans TSAA1: Cloning, Expression, Characteristics and Its Applicability in Generating Xylooligosaccharides and Fermentable Sugars. Appl Biochem Biotechnol, 170, 119–130. http://dx.doi.org/10.1007/s12010-013-0174-6 VERMA, D., SATYANARAYANA, T. 2012. Cloning, expression and applicability of thermoalkali-stable xylanase of Geobacillus thermoleovorans in generating xylooligosaccharides from agro-residues. Bioresour Technol, 107, 333–338. http://dx.doi.org/10.1016/j.biortech.2011.12.055 WANG, S-Y., HU, W., LIN, X-Y., WU, Z-H., LI, Y-Z. 2012. A novel cold- active xylanase from the cellulolytic myxobacterium Sorangium cellulosum So9733-1: gene cloning, expression, and enzymatic characterization. Appl Microbiol Biotechnol, 93(4), 1503–1512. http://dx.doi.org/10.1007/s00253-011- 3480-3 WANG, Y., FENG, S., ZHAN, T., HUANG, Z., WU, G., LIU, Z. 2013. Improving catalytic efficiency of endo 1, 4-xylanase from Geobacillus stearothermophilus by directed evolution and H179saturation mutagenesis. J Biotechnol, 168, 341– 347. http://dx.doi.org/10.1016/j.jbiotec.2013.09.014 WANG, Y., ZHANG, H., HE, Y., LUO, H., YAO, B. 2007. Characterization, gene cloning, and expression of a novel xylanase XYNB from Streptomyces olivaceoviridis A1. Aquaculture, 267 328–334. http://dx.doi.org/10.1016/j.aquaculture.2007.03.005 WU, S., LIU, B., ZHANG, X. 2006. Characterization of a recombinant thermostable xylanase from deep-sea thermophilic Geobacillus sp. MT-1 in East Pacific. Appl Microbiol Biotechnol, 72, 1210–1216. http://dx.doi.org/10.1007/s00253-006-0416-4 ZHANG, G.M., HUANG, J., HUANG, G.R, MA, L.X, ZHANG, X.E. 2007. Molecular cloning and heterologous expression of a new xylanase gene from Plectosphaerella cucumeria. Appl Microbiol Biotechnol, 74, 339–346. http://dx.doi.org/10.1007/s00253-006-0648-3 ZHAO, Y., MENG, K., LUO, H., YANG, P., SHI, P., HUANG, H. 2011. Cloning, expression, and characterization of a new xylanase from alkalophilic Paenibacillus sp. J Microbiol Biotechnol, 21, 861-868. http://dx.doi.org/10.4014/jmb.1102.02024 ZHENG, F., HUANG, J., YIN, Y., DING, S. 2013. A novel neutral xylanase with high SDS resistance from Volvariella volvacea: characterization and its synergistic hydrolysis of wheat bran with acetyl xylan esterase. J Ind Microbiol Biotechnol, 40, 1083–1093. http://dx.doi.org/10.1007/s10295-013-1312-4 ZHENG, H.C., SUN, M.Z., MENG, L.C., PEI, H.S., ZHANG, XQ., YAN, Z., ZENG, W.H., ZHANG, J.S., HU, J.R., LU, F.P., SUN, J.H. 2014. Purification and Characterization of a Thermostable Xylanase from Paenibacillussp. NF1 and its Applicationin Xylooligosaccharides Production. J Microbiol Biotechnol, 24(4), 489–496. http://dx.doi.org/10.4014/jmb.1312.12072 ZHENG, HC., LIU, Y.H., LIU, X.G., HAN, Y., WANG, J.L., LU, F.P. 2012. Overexpression of a Paenibacillus campinasensis xylanase in Bacillus megaterium and its applications to biobleaching of cotton stalk pulp and saccharification of recycled paper sludge. Bioresour Technol, 125, 182-187. http://dx.doi.org/10.1016/j.biortech.2012.08.101 ZHOU, J., GAO, Y., DONG, Y., TANG, X., LI, J., XU, B., MU, Y., WU, Q., HUANG, Z. 2012. A novel xylanase with tolerance to ethanol, salt, protease, SDS, heat, and alkali from actinomycete Lechevalieria sp. HJ3. J Ind Microbiol Biotechnol, 39(7), 965–975. http://dx.doi.org/10.1007/s10295-012-1113-1 ZHOU, J., SHI, P., ZHANG, R., HUANG, H., MENG, K., YANG, P., YAO, B. 2011. Symbiotic Streptomyces sp. TN119 GH 11 xylanase: a new pH-stable, protease- and SDS-resistant xylanase. J Ind Microbiol Biotechnol, 38(4), 523– 530. http://dx.doi.org/10.1007/s10295-010-0795-5

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Enzyme and Microbial Technology 91 (2016) 34–41

Contents lists available at ScienceDirect

Enzyme and Microbial Technology

journal homepage: www.elsevier.com/locate/emt

C-Terminal proline-rich sequence broadens the optimal temperature

and pH ranges of recombinant xylanase from Geobacillus

thermodenitrificans C5

a b c c

Muhammad Irfan , Halil Ibrahim Guler , Aysegul Ozer , Merve Tuncel Sapmaz ,

c,∗ a a

Ali Osman Belduz , Fariha Hasan , Aamer Ali Shah

a

Department of Microbiology, Faculty of Biological Sciences, Quaid-I-Azam University, Islamabad, Pakistan

b

Department of Molecular Biology and Genetic, Faculty of Sciences, Karadeniz Technical University, 61080 Trabzon, Turkey

c

Department of Biology, Faculty of Sciences, Karadeniz Technical University, 61080 Trabzon, Turkey

a r t i c l e i n f o a b s t r a c t

Article history: Efficient utilization of hemicellulose entails high catalytic capacity containing xylanases. In this study,

Received 24 February 2016

proline rich sequence was fused together with a C-terminal of xylanase gene from Geobacillus ther-

Received in revised form 27 May 2016

modenitrificans C5 and designated as GthC5ProXyl. Both GthC5Xyl and GthC5ProXyl were expressed in

Accepted 31 May 2016

Escherichia coli BL21 host in order to determine effect of this modification. The C-terminal oligopep-

Available online 2 June 2016

tide had noteworthy effects and instantaneously extended the optimal temperature and pH ranges and

progressed the specific activity of GthC5Xyl. Compared with GthC5Xyl, GthC5ProXyl revealed improved

Keywords: ◦ ◦

specific activity, a higher temperature (70 C versus 60 C) and pH (8 versus 6) optimum, with broad

Xylanase ◦ ◦

ranges of temperature and pH (60–80 C and 6.0–9.0 versus 40–60 C and 5.0–8.0, respectively). The

Geobacillus thermodinitrificans C5 ◦

modified enzyme retained more than 80% activity after incubating in xylan for 3 h at 80 C as compared

recombinant xylanase (GthC5Xyl)

−

Proline tagged xylanase (GthC5ProXyl) to wild type with only 45% residual activity. Our study demonstrated that proper introduction of proline

Characterization residues on C-terminal surface of xylanase family might be very effective in improvement of enzyme ther-

Thermostability mostability. Moreover, this study reveals an engineering strategy to improve the catalytic performance

of enzymes.

© 2016 Elsevier Inc. All rights reserved.

1. Introduction GH 5, 8, 10, 11, 30 and 43. This classification is based upon cat-

alytic domains and among these families GH10 and GH11 are the

The second most abundant polysaccharide that occurs in nature most well studied [7]. Usually thermophilic enzymes are preferred

is hemicellulose, first being cellulose. Hemicellulose contains xylan over their mesophilic counterparts as high temperatures increases

as the major carbohydrate component constituting about 30–35% the rate of mass transfer and decreases contamination risk and

of lignocellulosic biomass [1]. In order to convert hemicellulose viscosity of substrate [8,9]. For example bioconversion processes

into other value added compounds, it is necessary to effectively and pulp bleaching processes utilize xylanases but temperature

utilize xylan. However, xylan has a complex structure and for its at which these processes are carried out is quite high. Similarly,

complete degradation, an enzyme system comprising of several in preparing animal feed high temperature tolerant xylanase are

hydrolytic system is required. Amidst these hydrolytic enzymes mixed into the feed before pelleting process which is carried out at

◦

xylanase is the most essential as it hydrolyzes 1,4-␤-d-xylosidic 70–95 C. To acquire such thermophilic xylanases one approach is

linkages in xylan converting it to short xylooligosaccharides. At to hunt for novel xylanases from extremophiles. A variety of high

present xylan has become focus of attention due to its biotechno- temperature tolerant GH10 xylanases have been reported from

◦

logical applications in pulp and paper industries, animal feed, food, thermophilic Thermotoga petrophila RKU-1 (90 C); [10], Thermobi-

◦ ◦

cellulosic deconstruction to generate biofuels and textile indus- fida alba UL JB1 (80 C); [11], Thermotoga maritima MSB8 (90 C);

◦

try [2–6]. Xylanases are classified into glycoside hydrolase families [12], and Acidomesophilic Bispora sp. MEY-1 (85 C); [13]. The

sequence and structures of these thermostable xylanases are avail-

able (i.e. 1VBU and 3NIY) [14,10], and hence provide opportunity

∗ for better understanding the functional and structural aspects of

Corresponding author.

thermostable enzymes.

E-mail address: [email protected] (A.O. Belduz).

http://dx.doi.org/10.1016/j.enzmictec.2016.05.012

0141-0229/© 2016 Elsevier Inc. All rights reserved.

M. Irfan et al. / Enzyme and Microbial Technology 91 (2016) 34–41 35

Another approach for obtaining thermostable enzymes is by 2.4. Purification of the recombinant GthC5ProXyl

utilizing protein engineering to improve mesophilic enzymes prop-

◦

All purification steps were carried out at 4 C unless otherwise specified. The

erties. The generally used techniques are N-terminus replacement,

crude extract was concentrated and dialyzed (against 20 mM sodium phosphate

disulfide bridges introduction and adding salt bridges or hydrogen

buffer, pH 8.0) by ultrafiltration using an Amicon system with a 10 kDa cut-off point

bonds [2]. Many publications quoting improvement of stability via membrane (PM 10).

successful protein engineering are present. Wintrode et al. [15] cre- The dialyzed retentate was subjected to DEAE affinity. The obtained 60% precip-

◦ itates were loaded on a column (1.5 × 50 cm) of DEAE-Sepharose pre-equilibrated

ated a protease mutant having increased half-life at 60 C (1200

◦ with 10 mM sodium phosphate buffer pH 8.0. The column was washed with 1000 ml

folds) and melting temperature (Tm) of 25 C using DNA shuffling −1

of the same buffer at flow rate of 0.5 mlmin . After washing, column was eluted

technique. This method proves to be more effective when utilized in

with linear gradient of (0.50 M) NaCl in sodium phosphate buffer. The active frac-

combination with rational and random design and computer aided tions were combined and concentrated by ultrafiltration (Sartorious, 30000 MWCO

filters).

prediction. Fenel et al. [16] and Wang et al. [17] lifted the optimum

The fractions having the maximum xylanase activity were collected and applied

temperature of Trichoderma reesei and Thermomyces lanuginosus

◦ to hydrophobic interaction chromatography in a Phenyl-Sepharose column equi-

xylanase 10 C higher by introducing disulphide bridges into it. Joo −1

librated with the above buffer at a flow rate of 100 mlh . The residual protein

et al. [18] utilized thermal fluctuation analysis to upgrade the Tm was eluted with linear gradient of (1.3 M) ammonium sulphate in 20 mM sodium

◦

of Bacillus circulans xylanase by 4.2 C. phosphate buffer. Fractions of 1.5 ml, corresponding to xylanase activity were

◦

pooled, concentrated by ultrafiltration and stored for later use at 4 C. GthC5ProXyl

A GH10 xylanase GthC5Xyl obtained from Geobacillus ther-

enzyme purity was checked by reverse phase C-18 column (4.6 × 250 mm; E. Merck,

modenitrificans C5 when expressed in Escherichia coli, depicted

Germany) of High Performance Liquid Chromatography (HPLC System 600 Waters,

improved properties like adaptability and stability at a broad

Waters Corporation, Massachusetts, USA). The solvent system acetonitrile-water

−1

range of pH. However, its ability to remain stable at temper- (70:30) at a flow rate of 0.5 mlmin was employed for the separation of sample

◦

components. Absorbance was read at 280 nm using a highly sensitive photo-diode

ature above 60 C was poor. In order to amplify its industrial

array (PDA) detector (996 Waters).

utilization, it was essential to enhance GthC5Xyl’s thermal proper-

ties. In our study, a thermostable recombinant xylanase GthC5Xyl

2.5. SDS-PAGE, analysis and protein identification

was the developed by introduction of proline at its C terminus

through rational engineering. Recombinant xylanase GthC5ProXyl The fractions containing xylanase activity were analyzed by SDS-PAGE as

expressed in Escherichia coli depicted higher temperature optima described by Liao et al. [19,23]. SDS-PAGE was performed using 15% (w/v) poly-

◦

acrylamide gel with the Mini-Protein II system (Bio-Rad, Hercules, CA) according

(70 C). To the best of our knowledge, this is the first report where

to Laemmli [24]. The protein bands were visualized after staining with Coomassie

C-terminus modification of recombinant xylanase from G. ther-

Brilliant Blue R-250 (Bio-Rad, Hercules, USA).

modenitrificans with proline and its effect on thermostability was

studied.

2.6. Characterization of purified recombinant GthC5ProXyl

2. Materials and methods

2.6.1. Activity assay and kinetics of xylanase

The activity of purified GthC5ProXyl was checked by the method of Miller [25]

2.1. Strains, vectors and media

via measuring the reducing sugar release from oat spelt xylan. All xylanase assays

were performed with 100 mM sodium phosphate buffer pH. 8.0, unless otherwise

E. coli JM101; E. coli BL21 (DE3); pET28a(+) were gently supplied from Molec-

specified. The xylanase assay was carried out by incubating diluted enzyme with 1%

◦

ular Biology Laboratory, Karadeniz Technical University, Turkey. E. coli containing

xylan in 100 mM sodium phosphate buffer for 10 min at 70 C. 3,5-dinitrosalicylic

recombinant plasmids was cultured according to the method of Karaoglu [19]. The

acid (DNSA) reagent was used for the estimation of liberated sugars. One unit of

Wizard Genomic DNA Purification Kit, Wizard plus SV Minipreps DNA Purification

xylanase is defined as the amount of enzyme that liberates 1 ␮mol of reducing sugar

System, Taq DNA polymerase, dNTP’s, and all of the restriction enzymes were pur-

under the assay conditions using oat spelt xylan as the substrate. Bradford method

chased from Promega Corp. (Madison, WI, USA). All chemicals were reagent grade

[26] was used for determining protein concentration, using bovine serum albumin as

− −

and all solutions were made with double distilled and deionized water. 1 1

standard [26]. Kinetic parameters Vmax (␮molmin mg ) and Km were determined

by Michaelis-Menten plots of specific activities at multiple xylan concentrations

−1 −1

2.2. Amplification and sequencing of proline tagged xylanase gene varying between 0.5 mgml to 30 mgml .

Specific C-terminal proline rich sequence [20] was attached to the xylanase

2.6.2. Determination of pH and temperature effects on activity and stability of

of Geobacillus thermodenitrificans C5 by overlap extension PCR method [21]

GthC5ProXyl

and the overlapping ends of the fragments were annealed to each other

  The effect of temperature on activity of GthC5ProXyl was determined by

by Xyla-Pro-C5 5 –TgCCgCCgCTTTCATTgATCgATAATAgCC–3 and Prol-BamHIR ◦

  conducting assays under different temperatures from 40 to 100 C. Results were

5 –cgcggATCCCTATCCAggCTggTTgAgCTCAgAgggCACgA–3 primer (Supplementary

expressed as relative activity (%) obtained at optimum temperature. The thermosta-

Table 1). Final products approximately 1400 bp (Supplementary Fig. 1) was purified

bility of the enzyme was determined by pre-incubating the enzyme sample at

using the QIAquick Gel Extraction Kit. The products were analyzed by electrophore-

various temperatures for 200 mins and the residual activity was measured under

sis and used in ligation as insert.

standard assay conditions. In order to investigate the effect of pH, the enzyme activ-

ity was measured over a pH range 1.0–11.0. The pH stability of the enzymes was

2.3. Construction and expression of the recombinant vector pET GthC5ProXyl ◦

determined by incubation with different buffer systems at 70 C for 200 min and

finally the residual activity was calculated.

Complete xylanase gene was amplified and purified by gel extraction and lig-

®

ation was done into pGEM -T Easy Vector. Heat shock method [22] was used to

2.6.3. Enzymatic treatment of pulp

transform the ligated product in E. coli JM101 competent cells. Recombinant vector

Oven dried pulp having consistency of 6% was subjected to recombinant enzyme

pET GthC5XylPro, having BamHI and NheI restriction sites compatible to pET28a

−1 ◦

® GthC5ProXyl (40 Ugm dry pulp). The sample was incubated in a shaker at 70 C,

(+) vector was constructed. The proline tagged xylanase gene cloned pGEM -T

pH 8.0 and 200 rpm for 8 h. A control was run in separate where autoclaved xylanase

Easy Vector was isolated and digested with respective restriction enzymes. Pro-

enzyme was used in reaction. After the end of enzymatic treatment, the pulp was

line tagged xylanase gene product was purified by gel extraction and ligated into

dried under vacuum. DNS method [25] was used to monitor the reducing sugar

already purified and digested pET28a (+) vector using T4 DNA ligase overnight at

◦ in the filtrate. Chromophores and Lignin-derived compounds (LDC) released as a

15 C. Heat shock method [22] was used to transform the ligated product in E. coli

result of enzymatic hydrolysis of pulp by monitoring A280 and A465 of the filtrate,

JM101 competent cells. The positive clones were established by double digestion

respectively [27,28].

of the recombinant plasmid with specific restriction enzymes. Five plasmids having

xylanase gene was sequenced using the Taq DyeDeoxy Terminator Cycle Sequenc-

ing Kit according to the manufacturer’s instructions, and analyzed with an Applied 2.6.4. Polysaccharide-binding properties

Biosystems (Macrogen, Korea) Model 370A automatic sequencer. The recombinant The polysaccharide-binding capacity of the recombinant xylanase was deter-

vector pET GthC5ProXyl was transformed into E. coli BL21 (DE3) cells. Five clones mined by the method described by Tenkanen et al. [29] Purified GthC5ProXyl

were grown in LB broth overnight and 1% (v/v) of this was used as inoculum to cul- xylanase was incubated with different concentrations of Avicel, arabinoxylan,

tivate E. coli BL21 (DE3) cells. 1 mM IPTG was used for induction of the recombinant beechwood xylan, oat spelt xylan and birchwood xylan in 100 mM sodium phos-

◦

xylanase at OD600nm of 0.5–0.6. For higher expression of xylanase the induced cell phate buffer (pH 8), at 4 C for 1 h with slow shaking 50 rpm. Unbound enzyme was

were cultivated for 22 h. determined by measuring residual activity in the supernatant.

36 M. Irfan et al. / Enzyme and Microbial Technology 91 (2016) 34–41

1200 0.4 Activity 1100 1400 0.25 Activity 0.35 Protein

) 1000 -1 Protein 1200 900 0.3 ) -1 0.2 800

(IUml 0.25 1000 700 ty (IUml 0 0.15 600 0.2 ty 28 800

500 A

0.15 280 400 600 0.1 A 300 0.1 400

Xylanase activi 200 0.05 0.05 100 Xylanase activi 0 0 200 1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 0 0 Fraction Number 1 4 7 1013161922252831343740434649525558

Fraction Number

Fig. 1. DEAE sepharose chromatography of proline tagged xylanase GthC5ProXyl.

Fig. 2. Phenyl sepharose chromatography of proline tagged xylanase GthC5ProXyl.

2.6.5. Analysis of hydrolysis products and shelf life determination of xylanase

The purified enzyme GthC5ProXyl mixed with 100 mM sodium phosphate buffer

◦

(pH 8.0) containing 1% (w/v) xylan was incubated at 70 C for 10 h and products of

enzymatic hydrolysis were determined by thin layer chromatography (TLC). After

incubation, the sample was centrifuged for 12 min at 3000xg in order to remove

insoluble material. 3 ␮l of sample was spotted on silica gel 60 F254TLC plates (Merck)

and then placed in n-butanol, acetic acid and water (2:1:1) containing solvent system

that facilitate the flow of samples in ascending order. Plates were then sprayed with

◦

5% (v/v) sulfuric acid in ethanol and then heated at 120 C for about 10 min for the

sugars detection. The shelf life of enzyme was determined after incubation at both

◦

room and refrigerator temperature (4 C) for maximum up to 16 weeks and residual

activity was measured at different intervals.

3. Result

3.1. Cloning and sequence analysis of xylanase

Proline was attached to C terminus of xylanase by overlap exten-

sion PCR method by using G. thermodenitrificans DNA as template.

Confirmation of amplified proline tagged xylanase gene cloning in

pET28a (+) vector was done by recombinant vector double digestion

with BamHIand NheI restriction enzymes. A successful transforma-

tion and expression of the construct pET GthC5ProXyl was carried

out in E. coli BL21 (DE3) cells. Xylanase expression was induced with

◦

1 mM IPTG at 30 C and higher production of recombinant xylanase

Fig. 3. SDS-PAGE showing purified recombinant GthC5ProXyl enzyme M, SDS-PAGE

was achieved.

molecular mass standards 10–250 kDa New England Biolab; 1, Induced supernatant;

2, ammonium sulphate precipitation; 3, Purified GthC5ProXyl by DEAE Sepharose;

3.2. Purification of the GthC5ProXyl 4, Purified GthC5ProXyl by Phenyl Sepharose.

Elution of recombinant GthC5ProXyl was done by DEAE affinity

(8.0 instead of 6.0) (Fig. 4b) were observed with respect to the wild

chromatography (Fig. 1) column with 0.50 M of NaCl and phenyl

type enzyme. The recombinant GthC5ProXyl retained more than

sepharose chromatography (Fig. 2). The protein eluted was visu-

◦

90% activity at 70 C for 200 min (Fig. 5a), while 80% activity at pH

alized as a single band on by SDS-PAGE, which indicated its

6.0-8.0 for 3 h (Fig. 5b).

homogeneous nature (Fig. 3). A single peak was detected by reverse

phase HPLC at retention time of 2.5 min confirming the purity of

recombinant enzyme. Purified xylanase yield was 279.45% with a 3.4. Substrate specificity, kinetic parameters of proline tagged

−1

specific activity of 1403.72 IUmg and an overall purification fold xylanase

of 7.53 (Table 1).

The activity of recombinant GthC5ProXyl enzyme against var-

◦

3.3. Biochemical characterization of recombinant xylanase ious substrates was evaluated at 70 C and pH 8.0. The enzyme

was highly specific as indicated by activity against polymeric

The purified GthC5ProXyl exhibited activity over a broad range xylan only, whereas no activity was observed against other

◦ ◦

of temperature (40–100 C) and pH (3.0–10.0). A 10 rise in opti- substrates such as insoluble xylan, carboxymethyl cellulose,

◦

mum temperature (70 instead of 60 C) (Fig. 4a) and 2 digits in pH Avicel, filter paper, pNP-␤-xylopyranoside, pNP-␣-l- arabinofu-

Table 1

Steps of GthC5ProXyl purification from G. thermodenitrificans C5.

Purification step TotalProtein (mg) Total activity (U) Specific activity (U/mg) Yield(%) Purification Yield

Cell extract 2.78 517 186 100 1

Precipitate 2.16 727 336 140 1.80

DEAE-Sepharose 1.13 1145 1011 221 5.42

Phenyl sepharose 1.03 1447 1403 279 7.53

M. Irfan et al. / Enzyme and Microbial Technology 91 (2016) 34–41 37 a a

120

GthC5ProXyl 120

C5 Proline temp stability 100 GthC5Xyl 100 80 80 60°C 60 60 70°C activity (%) 40 80°C 40 (%) activity xylanase al 20 90°C 100°C Relative 20

0

Residu

0 50 100 150 200 250

0

Incub ation time (min.)

40 50 60 70 80 90 100 Temperature (°C) b pH Stability of C5 Proline b 120 GthC5ProXyl 100 GthC5Xyl 120 80 pH 5 pH 6 100 60 pH 7 80 40 pH 8 xylanase activity (%) activity xylanase 60 pH 9

dial 20 40 pH 10

20 Resu 0

0 20 40 60 80 100 120 140 160 180 200

Relative activity (%) activity Relative 0

Incub ation time (min.)

3 4 5 6 7 8 9 10 11

pH

Fig. 5. Effect of temperature (A) and pH (B) on stability of the recombinant

GthC5ProXyl, Residual activity was defined as the percentage of activity detected

with respect to the maximum enzyme activity. For determining the stability, the

Fig. 4. (a, b). Effect of temperature (B) and pH on activity (A) of the recombinant

activity of the enzyme without any treatment was taken as 100%.

GthC5Xyl and GthC5ProXyl, respectively. Relative activity was defined as the per-

centage of activity detected with respect to the maximum enzyme activity. For

determining the stability, the activity of the enzyme without any treatment was

by the action of GthC5ProXyl on xylan. Xylopentose and xylo-

taken as 100%.

biose were the main products released similar to that by wild type

xylanase GthC5Xyl.

ranoside, pNP-␣-glucopyranoside, pNP-␤-galactopyranoside, pNP-

␣

-d-xylopyranoside and pNP- acetate.

3.6. Determination of shelf life of GthC5ProXyl and wild type

The Km and Vmax of GthC5ProXyl (for oat spelt xylan) was 1.45 GthC5Xyl xylanase

−1

mgml and 1210.166 as compared to wild type that was 3.9084

−1 −1 −1

mgml and 1839.86 ␮molmg min , respectively (Fig. 6).

No activity was lost by purified GthC5ProXyl when stored for 12

◦

weeks at 4 C but after that a gradual decline was observed. After

3.5. Analysis of products of xylan hydrolysis 16 weeks, 93% of initial activity was retained by the enzyme that

confirms its industrial application. Wild type GthC5Xyl xylanase

The products released as a result of xylan hydrolysis were retained 90% of initial activity for 16 weeks. Conversely enzyme

analyzed by TLC that represents mechanism of action of purified remained completely stable for 5 weeks at room temperature but

GthC5ProXyl (Fig. 7). A range of xylo-oligosacharides were released a decline in residual activity up to 86% and 73% was observed after

Table 2

Effect of GthC5Xyl and GthC5XylPro on −bleaching of kraft pulp. lignin derived compound.

Time (h) Chromophores (A465) LDC (A280) Reducing sugars -1

released (mgg

oven-dried pulp)

GthC5Xyl GthC5ProXyl GthC5Xyl GthC5ProXyl GthC5Xyl GthC5ProXyl

0 0.006 ± 0.004 0.006 ± 0.002 0.060 ± 0.0090 0.056 ± 0.041 0.23 ± 0.10 0.223 ± 0.068

2 0.057 ± 0.010 0.0693 ± 0.018 0.15 ± 0.052 0.1743 ± 0.045 0.47 ± 0.37 0.366 ± 0.090

4 0.072 ± 0.034 0.081 ± 0.014 0.16 ± 0.03 0.198 ± 0.093 0.54 ± 0.11 0.6 ± 0.08

6 0.075 ± 0.014 0.084 ± 0.021 0.23 ± 0.1587 0.2316 ± 0.130 0.84 ± 0.27 0.78 ± 0.166

7 0.073 ± 0.0090 0.086 ± 0.011 0.22 ± 0.070 0.2613 ± 0.114 1.33 ± 0.75 1.13 ± 0.211

8 0.07 ± 0.026 0.081 ± 0.024 0.20 ± 0.099 0.2553 ± 0.083 1.23 ± 0.32 1.25 ± 0.53

38 M. Irfan et al. / Enzyme and Microbial Technology 91 (2016) 34–41

Fig. 6. Lineweaver-burk plot of GthC5ProXyl (Km and Vmax values were determined according to Linewear-burk plot).

oatspelt xylan, beechwood xylan and Avicel. The GthC5ProXyl

showed binding affinity towards birchwood xylan, beechwood

xylan, oatspelt xylan and arabinoxylan, except Avicel (Fig. 8).

4. Discussion

The alteration of natural enzyme conformation is likely to affect

the physical and catalytic properties of enzymes. To understand

and identify the factors contributing to the thermostability of

protein which helps extremophilic organisms to maintain their

activities at extreme condition in industries is an active area of

research [30]. The extraordinarily high specific activity depicted by

the recombinant proline tagged xylanase obtained from Geobacil-

lus thermodenitrificans C5 makes it a worthy candidate for further

research. Proline has a side chain of typical cyclic formation that

locks its backbone and leads to surprising conformation. Proline

rich sequence may rigidify main chain, stabilize the mutants and

thus improve their thermostability. Proline rich sequence also con-

tains glutamic acid and arginine. Arginine has stronger ability

Fig. 7. TLC analysis for hydrolysis products released from oat spelt xylan by xylanase towards formation of salt bridges and hydrogen bond. Glutamic

from Geobacillus thermodenitrificans C5, 1: GthC5ProXyl; 2: D-xylose; 3: xylobiose;

acid has an anionic carboxylate that easily forms hydrogen bonds

4: xylotriose; 5: xylotetraose; 6: xylopentose.

and salt bridges and has helix-forming liability which contributes

to thermostability.

storage for 10 and 12 weeks, respectively, while wild type GthC5Xyl

Proline rich regions are involved in binding processes [44]. The

xylanase showed 80% and 70% residual activity after storage for 10

57 residue long tag may provide an interface between the protein

and 12 weeks, respectively.

and the binding surface. A linker between the tag and the protein

may provide “wag the dog” motion. Thus, the protein may give full

3.7. Enzymatic treatment of pulp with GthC5ProXyl and wild type function as opposed to unmodified form. Binding of unmodified

GthC5Xyl wild type protein to a surface or other macromolecules may lead it

to lose function and/or stability as well as may cause aggregation.

LDCs and chromophores were detected at A280 and A465 wave- Prediction of secondary structure of the tag by indicates that the

length, respectively, besides hydrolyzed products of oat spelt xylan sequence forms two helixes which give stability and coiled struc-

indicates the role of xylanase in bleaching of pulps. Maximum tures between these two helices. The coiled structrure may adopt

reducing sugars were recorded during 8 h of pulp treatment with the protein to bind a receptor without altering xylase conforma-

GthC5ProXyl and GthC5Xyl, as shown in Table 2. tion or stability. Further, the coiled structure provides flexibility

and facilitates binding. Proline structure restricts most of the con-

3.8. Polysacchrides binding capacity of GthC5ProXyl and formational changes but proline rich amino acid site has smooth

GthC5Xyl hydrophobic surface with charged amino acids at the perifery. This

provides selectivity resulting from the periferic amino acids.

Purified GthC5ProXyl binding capacity to polysaccharides was Proline residues have lower conformational flexibility and the

performed by incubating it with birchwood xylan, arabinoxylan, tag is designed to provide convenient environment for proline

M. Irfan et al. / Enzyme and Microbial Technology 91 (2016) 34–41 39

Fig. 8. Binding affinity of GthC5ProXyl xylanase towards various substrates. Purified enzyme was mixed with Birchwood xylan, Oatspelt xylan, Beechwood xylan, Arbinoxylan

◦

and Avicel substrates 100 mM sodium phosphate buffer (pH 8.0) and incubated at 4 C for 1 h. The relative activity was determined according to the standard assay conditions

◦

(pH 8.0 and 70 C).

residues to form a hydrophobic surface with charged amino acids its activity at pH 6.0-8.0, seemed much higher stability than that

at the binding pocket conveniently. Number of proline residues of wild type xylanase at wide pH range. Beg et al., 2001 revealed

and the current sequence is derived from known sequence motifs xylanase, which showed stability at low pH [40]. The stability of

◦ ◦

and provides an optimum interaction. Alteration of the sequence this GthC5ProXyl at higher temperature (60 C–80 C) and alka-

may either decrease the stability or alter-perturb conformational line pH renders it worthy for countless industrial applications such

flexibility. Additionaly, alteration of the sequence may perturb sec- as bioenergy conversion, food industry, bio-bleaching and animal

ondary structure of the protein and the hydrophobic nature of the feed.

tag may lead the tagged protein to aggregate. Thus, maintaing this Catalytic efficiency is an extent of the enzyme-substrate

optimised sequence will be beneficial for the sake of stability and reaction scheme. Substrate specificity and kinetic parameter deter-

optimal xylanase activity. mination of GthC5ProXyl showed that it only exhibits xylanolytic

In the presently conducted study, C-Terminal Proline-Rich activity related to the wild type. The enzymatic hydrolysis prod-

Sequence was tagged to xylanase of Geobacillus thermodenitrifi- ucts were identified as xylobiose and xylopentose, hence confirmed

cans C5 and then cloned and expressed in E. coli BL21 (DE3) host. that it is an endo-xylanase. The Km and Vmax of GthC5Xyl for oat

The enzyme produced by E. coli functionally active and proficiently spelt xylan was slightly higher than GthC5ProXyl. Proline increases

degrades oat spelt xylan even in SDS-PAGE gel. the specificity toward oat spelt xylan. The polysaccharide-binding

Proline is highly predominant in thermophilic proteins because capacity of the purified GthC5ProXyl was carried out by incu-

it has a side chain of typical cyclic structure that mops its back- bating it with Avicel, Beechwood. GthC5ProXyl did not bind to

bone and central to an extraordinary conformational rigidity in Avicel. The enzyme efficiently bind to these substrate except Avi-

the turns and loops. Introduction of proline residues at specific cel. GthC5ProXyl exhibited a high activity on beechwood xylan, but

positions has been used successfully to advance the thermostabil- no activity was detected for cellulosic substrates, that indicates the

ities of many enzymes [29–35]. Li et al. reported that C-terminus absence of cellulose-binding sites on enzyme. It was confirmed by

◦

modification with proline residues led to 5 C rise in temperature polysaccharide-binding analysis where more than 96% unbound

◦

(35–40 C) of ruminal xylanases XynB-Fu [20]. Optimum temper- enzyme was detected in the supernatant when incubated with

◦ ◦

ature of Streptomyces Xylanase was increased from 73 C to 90 C Avicel.

by the introduction of Proline and glutamic acid amino acid in the Shelf life of GthC5ProXyl increased as compared to GthC5Xyl

◦

long secondary-structure elements [35]. when incubated at 4 C. More than 90% of initial activity was

We can conclude that proline tagging at C terminus of GthC5Xyl retained by GthC5ProXyl after 16 weeks which is an important fact

is one of the key factors responsible for the improved thermal for its industrial application. Conversely, proline tagged enzyme

properties of the GthC5ProXyl. GthC5ProXyl optimal reaction tem- retained more than 80% of activity at room temperature after stor-

◦ ◦

perature was found to be 70 C that is found 10 C higher than age for 10 weeks as compared to wild type enzyme. Paper and pulp

◦

that of G. thermodenitrificans C5 wild type xylanase, and 15 C than industries need such enzymes in pre-bleaching of pulps for mit-

Paenibacillus sp. 12-11 [36]. igating the chlorine requirement. An increase in the absorbance

◦

GthC5ProXyl also retained more than 80% of its activity at 80 C at 280 and 465 nm and release of reducing sugars indicated the

(Fig. 4), which is far better than that of its wild type xylanase. This hydrolytic action of xylanase on pulp fibers and its applicability in

increase in thermal stability is far better than most of the bacterial bio-bleaching of pulps. GthC5ProXyl displayed better and longer

xylanases that are previously reported [36–39]. effects on the hot and alkaline pulp samples than the wild type

C-terminus modification with proline rich amino acid sequence xylanase (Table 2). Similar effects of xylanases from B. licheniformis

also resulted a shift in optimum pH of purified GthC5ProXyl from [41] B. pumilus [42] and B. coagulans [43] have been reported. After

6.0 to 8.0 that was found similar to proline fused XynB-Fu of rumi- tagging the proline at C terminal, its alkalistability and thermosta-

nal xylanases [20]. The GthC5ProXyl retained more than 90% of bility was enhanced thus making the proline tagged xylanase as a

40 M. Irfan et al. / Enzyme and Microbial Technology 91 (2016) 34–41

biocatalyst choice for bio-bleaching in paper pulps. The improve- changes in the product release area drive the enzymatic activity of xylanases

10B: Crystal structure, conformational stability and functional

ment of release of chromophores and reducing sugars from residual

characterization of the xylanase 10 B from Thermotoga petrophila RKU-1,

lignin and xylan of pulp samples clearly suggests the applicability of

Biochem. Biophys. Res. Commun. 403 (2010) 214–219.

the enzyme in pulp pre-bleaching for minimizing chlorine/chlorine [11] J. Blanco, J. Coque, J. Velasco, J. Martin, Cloning expression in Streptomyces

dioxide. lividans and biochemical characterization of a thermostable

endo-␤-1,4-xylanase of Thermomonospora alba UL JB1 with cellulose-binding

ability, Appl. Microbiol. Biotechnol. 48 (1997) 208–217.

5. Conclusion [12] Ihsanawati, T. Kumasaka, T. Kaneko, C. Morokuma, R. Yatsunami, T. Sato, S.

Nakamura, N. Tanaka, Structural basis of the substrate subsite and the highly

thermal stability of xylanase 10 B from Thermotoga maritima MSB8, Proteins

In summary, we fused a C-terminal proline-rich sequence to

61 (2005) 999–1009.

GthC5Xyl of Geobacillus thermodenitrificans C5. This oligopeptide [13] H. Luo, J. Li, J. Yang, H. Wang, Y. Yang, H. Huang, P. Shi, T. Yuan, Y. Fan, B. Yao,

A thermophilic and acid stable family-10 xylanase from the acidophilic

simultaneously increased the optimal temperature, enhanced the

fungus Bispora sp. MEY-1, Extremophiles 13 (2009) 849–857.

catalytic efficiency, broadened the pH and temperature ranges,

[14] T. Kumasaka, T. Kaneko, C. Morokuma, R. Yatsunami, T. Sato, S. Nakamura, N.

and completely degraded the xylan substrate. Our results there- Tanaka, Structural basis of the substrate subsite and the highly thermal

stability of xylanase 10 B from Thermotoga maritima MSB8, Proteins 61 (2005)

fore advocate that the modified GthC5ProXyl could suitably meet

999–1009.

the concert of commercially available xylanase preparations. The

[15] P.L. Wintrode, K. Miyazaki, F.H. Arnold, Patterns of adaptation in a laboratory

industrially significant characteristics of the GthC5ProXyl, signpost evolved thermophilic enzyme, Biochim. Biophys. Acta 1549 (2001) 1–8.

[16] F. Fenel, M. Leisola, J. Janis, O. Turunen, A de novo designed N-terminal

potential for its cost-effective application in the pulp and paper

disulphide bridge stabilizes the Trichoderma reesei endo-1,4-␤-xylanase II, J.

industry as a bio-bleaching agent.

Biotechnol. 108 (2004) 137–143.

[17] Y. Wang, Z. Fu, H. Huang, H. Zhang, B. Yao, H. Xiong, O. Turune, Improved

thermal performance of Thermomyces lanuginosus GH11 xylanase by

Author agreement

engineering of an N-terminal disulfide bridge, Bioresour. Technol. 112 (2012)

275–279.

The manuscript with substantially the same content has not [18] J.C. Joo, S.P. Pack, Y.H. Kim, Y.J. Yoo, Thermostabilization of Bacillus circulans

xylanase: computational optimization of unstable residues based on thermal

been published elsewhere. All authors (Muhammad IRFAN, Halil

fluctuation analysis, J. Biotechnol. 151 (2011) 56–65.

Ibrahim GULER, Aysegul OZER, Merve Tuncel SAPMAZ, Ali Osman

[19] H. Karaoglu, D. Yanmis, F.A. Sal, A. Celik, S. Canakci, A.O. Beldu, Biochemical

BELDUZ, Fariha HASAN, Aamer Ali SHAH) and have read and characterization of a novel glucose isomerase from Anoxybacillus gonensis G2

approved the version of the manuscript and are responsible for its T that displays a high level of activity and thermal stability, J. Mol. Catal. B:

content. Enzym. 97 (2013) 215–224.

[20] Z. Li, X. Xue, H. Zhao, P. Yang, H. Luo, J. Zhao, H. Huang, B. Yao, A C-terminal

proline-rich sequence simultaneously broadens the optimal temperature and

Acknowledgements pH ranges and improves the catalytic efficiency of glycosyl hydrolase family

10 ruminal xylanases, Appl. Environ. Microbiol. 80 (11) (2014) 3426–3432.

[21] S.N. Ho, H.D. Hunt, R.M. Horton, J.K. Pullen, L.R. Pease, Site-directed

Authors are thankful to TUBITAK (2216 Research Fellow- mutagenesis by overlap extension using the polymerase chain reaction, Gene

77 (1989) 51–59.

ship Programme) and Karadeniz Technical University, Turkey and

[22] A.O. Belduz, Z. Demirbag, S. Dulger, H. Coskuncelebi, The effect of a

Higher Education Commission of Pakistan to support the project

CO2-Saturated CaCl2 solution on the transformation efficiency of escherichia

and provide necessary infrastructure to carry out the present coli JM 109 with an M 13 derivative, Turk. J. Biol. 21 (1997) 133–139.

research work. [23] H. Liao, C. Xu, S. Tan, Z. Wei, N. Ling, G. Yu, W. Raza, R. Zhang, Q. Shen, Y. Xu,

Production and characterization of acidophilic xylanolytic enzymes from

Penicillium oxalicum GZ-2, Bioresour. Technol. 123 (2) (2012) 117–124.

Appendix A. Supplementary data [24] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head

of bacteriophage T4, Nature 227 (1970) 680–685.

[25] G.L. Miller, Use of dinitrosalicylic acid reagent for determination of reducing

Supplementary data associated with this article can be found, in

sugars, Anal. Chem. 31 (1959) 426–428.

the online version, at http://dx.doi.org/10.1016/j.enzmictec.2016. [26] M.M. Bradford, A rapid and sensitive method for the quantification of

05.012. microgram quantities of protein utilizing the principle of protein dye binding,

Anal. Biochem. 72 (1976) 248–254.

[27] R.N. Patel, A.C. Grabski, T.W. Jeffries, Chromophore release from kraft pulp by

References purified Streptomyces roseiscleroticus xylanases, Appl. Microbiol. Biotechnol.

39 (1993) 405–412.

[28] D. Verma, A. Anand, T. Satyanarayana, Thermostable and alkalistable

[1] T. Collins, C. Gerday, G. Feller, Xylanases, xylanase families and extremophilic

endoxylanase of the extremely thermophilic bacterium Geobacillus

xylanases, FEMS Microbiol. Rev. 29 (2005) 3–23.

thermodenitrificans TSAA1: cloning expression, characteristics and its

[2] X. Li, Z. Jiang, L. Li, S. Yang, W. Feng, J. Fan, I. Kusakabe, Characterization of a

applicability in generating xylooligosaccharides and fermentable sugars,

cellulase-free, neutral xylanase from Thermomyces lanuginosus CBS 288.54

Appl. Biochem. Biotechnol. 170 (2013) 119–130.

and its biobleaching effect on wheat straw pulp, Bioresour. Technol. 96 (2005)

1370–1379. [29] M. Tenkanen, J. Puls, K. Potanen, Two major xylanases of Trichoderma reesei,

Enzyme Microb. Technol. 14 (1992) 566–574.

[3] M. Polizeli, A. Rizzatti, R. Monti, H. Terenzi, J. Jorge, D. Amorim, Xylanases

[30] Y.S. Cheng, C.C. Chen, C.H. Huang, T.P. Ko, W. Luo, J.W. Huang, J.R. Liu, R.T. Guo,

from fungi: properties and industrial applications, Appl. Microbiol.

Structural analysis of a glycoside hydrolase family 11 xylanase from

Biotechnol. 67 (2005) 577–591.

Neocallimastix patriciarum: insights into the molecular basis of a thermophilic

[4] H. Xiong, N. von Weymarn, O. Turunen, M. Leisola, O. Pastinen, Xylanase

enzyme, J. Biol. Chem. 289 (2014) 11020–11028.

production by Trichoderma reesei Rut C-30 grown on L-arabinose-rich plant

[31] K. Watanabe, T. Masuda, H. Ohashi, H. Mihara, Y. Suzuki, Multiple proline

hydrolysates, Bioresour. Technol. 96 (2005) 753–759.

substitutions cumulatively thermostabilize Bacillus cereus ATCC7064

[5] Y. Huang, Y. Chen, C. Chen, W. Chen, Y. Ciou, W. Liu, C. Yang, Production of

oligo-1,6-glucosidase, Eur. J. Biochem. 226 (1994) 277–283.

ferulic acid from lignocellulolytic agricultural biomass by Thermobifida fusca

[32] M.J. Allen, P.M. Coutinho, C.F. Ford, Stabilization of Aspergillus awamori

thermostable esterase produced in Yarrowia lipolytica transformant,

glucoamylase by proline substitution and combining stabilizing mutations,

Bioresour. Technol. 102 (2011) 8117–8122.

Protein Eng. 11 (1998) 783–788.

[6] J. Zhang, P. Tuomainen, M. Siika-Aho, L. Viikari, Comparison of the synergistic

[33] G. Zhu, C. Xu, M. Teng, L. Tao, X. Zhu, C. Wu, J. Hang, L. Niu, Y.Z. Wang,

action of two thermostable xylanases from GH families 10 and 11 with

Increasing the thermostability of D xylose isomerase by introduction of a

thermostable cellulases in lignocellulose hydrolysis, Bioresour. Technol. 102

proline into the turn of a random coil, Protein Eng. 12 (1999) 635–638.

(2011) 9090–9095.

[34] E. Goihberg, O. Dym, S. Telr, I. Levin, M. Peretz, Y. Burstein, A single proline

[7] B. Henrissat, A classification of glycosyl hydrolases based on amino acid

substitution is critical for the thermostabilization of Clostridium beijerinckii

sequence similarities, Biochem. J 280 (1991) 309–316.

alcohol dehydrogenase, Proteins 66 (2007) 196–204.

[8] S. Badieyan, D.R. Bevan, C. Zhang, Study and design of stability in GH5

[35] K. Wang, H. Luo, J. Tian, O. Turunen, H. Huang, P. Shi, H. Hua, C. Wang, S.

cellulases, Biotechnol. Bioeng. 109 (2012) 31–44.

Wang, B. Bin Yao, Thermostablility improvement of a 1 Streptomyces xylanase

[9] P. Turner, G. Mamo, E.N. Karlsson, Potential and utilization of thermophiles

by introducing proline and glutamic acid residues, Appl. Environ. Microbiol.

and thermostable enzymes in biorefining, Microb. Cell Fact. 6 (2007) 1–23.

80 (2014) 2158–2165.

[10] C.R. Santos, A.N. Meza, Z.B. Hoffmam, J.C. Silva, T.M. Alvarez, R. Ruller, G.M.

Giesel, H. Verli, F.M. Squina, R.A. Prade, Thermal-induced conformational

M. Irfan et al. / Enzyme and Microbial Technology 91 (2016) 34–41 41

[36] Y. Zhao, K. Meng, H. Luo, P. Yang, P. Shi, H. Huang, Cloning, expression, and [41] V.B. Damiano, D.A. Bocchini, E. Gomes, R.D. Silva, Application of crude

characterization of a new xylanase from alkalophilic Paenibacillus sp, J. xylanase from Bacillus licheniformis 77-2 to the bleaching of eucalyptus Kraft

Microbiol. Biotechnol. 21 (2011) 861–868. pulp, World J. Microbiol. Biotechnol. 19 (2003) 139–144.

[37] X.R. Li, X. Xu, J. Xie, Q.F. Yi, W. Li, R. Dai, D.R. Qiao, Y. Cao, Thermostable sites [42] A. Kaur, R. Mahajan, A. Singh, G. Garg, J. Sharma, Application of cellulase-free

and catalytic characterization of xylanase XYNB of Aspergillus niger SCTCC xylano-pectinolytic enzymes from the same bacterial isolate in biobleaching

400264, J. Microbiol. Biotechnol. 24 (4) (2014) 483–488. of kraft pulp, Bioresour. Technol. 101 (2010) 9150–9155.

[38] H. Shi, Y. Zhang, X. Li, Y.J. Huang, L. Wang, Y. Wang, H. Ding, F. Wang, A novel [43] B. Choudhury, C. Sunita, S.N. Singh, P. Ghosh, Production of xylanase enzyme

2+

highly thermostable xylanase stimulated by Ca from Thermotoga of Bacillus coagulans and its bleaching potential, World J. Microbiol.

thermarum: Cloning, expression and characterization, Biotechnol. Biofuels 6 Biotechnol. 22 (2006) 283–288.

(2013) 1–9. [44] M.P. Williamson, The structure and function of proline-rich regions in

[39] S. Subramaniyan, P. Prema, Cellulase-free xylanases from Bacillus and other proteins, Biochem. J. 297 (1994) 249–260.

microorganisms, FEMS Microbiol. Lett. 183 (2000) 1–7.

[40] Q.K. Beg, M. Kapoor, L. Mahajan, G.S. Hoondal, Microbial xylanases and their

industrial applications: a review, Appl. Microbiol. Biotechnol. 156 (2000) 326–338.