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Prevalence, Antimicrobial Susceptibility and Molecular 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.
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