Production of laccase by the phytopathogenic ... Production of laccase by the phytopathogenic fungus

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  • Production of laccase by the

    phytopathogenic fungus Rhizoctonia solani

    By

    Pranjal Bora

    B.Sc. (Botany Major), Dibrugarh University, India M.Sc. (Biotechnology), Gauhati University, India

    This thesis is presented for the degree of Doctor of Philosophy

    of Murdoch University

    September, 2003

    Division of Science and Engineering School of Biological Sciences and Biotechnology

    Murdoch University, Perth, Western Australia

  • 1

    CONTENTS

    Declaration......................................................................................................................... 8

    Dedication .......................................................................................................................... 9

    ABBREVIATIONS ......................................................................................................... 10

    ABSTRACT..................................................................................................................... 12

    ACKNOWLEDGEMENTS ........................................................................................... 16

    CHAPTER 1 .................................................................................................................... 20

    Literature Survey............................................................................................................ 20

    1 Rhizoctonia solani (Introduction) .............................................................................. 20

    1.1 Rhizoctonia solani- A major plant pathogen in Australia................................... 22

    1.1.1 The infection process ................................................................................... 24

    1.2 Control of Rhizoctonia solani ............................................................................. 25

    1.3 Cell wall degrading enzymes .............................................................................. 28

    1.3.1 Pectinases..................................................................................................... 30

    1.3.2 Cellulases and hemicellulases...................................................................... 32

    1.3.3 Lignolytic enzymes...................................................................................... 34

    1.3.3.1 Laccases ................................................................................................ 35

    1.3.3.2 Laccase genes........................................................................................ 41

    1.4 Thesis Aims ........................................................................................................ 43

    CHAPTER 2. ................................................................................................................... 44

    Materials and general methods...................................................................................... 44

    2.1 Fungal strains, growth media and culture conditions ......................................... 44

    2.2 Measurement of protein concentration (protein assay)................................... 48

  • 2

    2.2.1 Concentration of proteins............................................................................. 48

    2.2.2 Acetone precipitation ............................................................................... 48

    2.2.3 Ammonium sulphate precipitation of supernatant proteins ..................... 49

    2.3 Polyacrylamide gel electrophoresis (PAGE) ...................................................... 49

    2.3.1 Native PAGE ........................................................................................... 49

    2.3.2 SDS-PAGE .............................................................................................. 50

    2.3.3 Coomassie staining .................................................................................. 51

    2.4 Enzyme activity assays ....................................................................................... 51

    2.4.1 Laccase assay ........................................................................................... 51

    2.4.2 Pectinase, xylanase and cellulase assays ................................................. 52

    CHAPTER 3 .................................................................................................................... 54

    Laccase, pectinase, xylanase and cellulase production in Rhizoctonia solani............ 54

    3.1 Introduction......................................................................................................... 54

    3.2 Materials and methods ........................................................................................ 55

    3.2.1 Organism and culture conditions ................................................................. 55

    3.2.2 Growth media............................................................................................... 55

    3.2.2.1 Growth of mycelium for laccase production ........................................ 55

    3.2.3 Protein assay ................................................................................................ 56

    3.2.4 Enzyme activity assays ................................................................................ 56

    3.2.5 Native PAGE and activity staining .............................................................. 56

    3.2.6 Laccase purification ..................................................................................... 56

    3.2.7 Purification by anion exchange chromatography ........................................ 57

    3.2.7.1 Sample preparation ............................................................................... 57

  • 3

    3.2.7.2 Column preparation .............................................................................. 57

    3.2.7.3 Chromatography ................................................................................... 58

    3.2.8 Purification by electroelution from a native polyacrylamide gel................. 58

    3.2.8.1 Sample preparation ............................................................................... 58

    3.2.8.2 Native PAGE ........................................................................................ 59

    3.2.8.3 Elution................................................................................................... 59

    3.2.8.4 Absorption spectra ................................................................................ 60

    3.2.8.5 SDS-PAGE ........................................................................................... 60

    3.3 Results................................................................................................................. 61

    3.3.1 Laccase production ...................................................................................... 61

    3.3.1.1 Time course of laccase production ....................................................... 61

    3.3.1.2 Effect of temperature on the laccase, pectinase, xylanase and cellulase

    activity............................................................................................................... 62

    3.3.1.3 Effect of pH on laccase activity ............................................................ 64

    3.3.1.4 Effect of different growth media and inducers on laccase, pectinase,

    xylanase and cellulase production .................................................................... 65

    3.3.1.5 Effect of inhibitors on laccase activity.................................................. 68

    3.3.2 Laccase purification ..................................................................................... 69

    3.4 Discussion ........................................................................................................... 74

    CHAPTER 4 .................................................................................................................... 79

    Zymogram analysis of laccase........................................................................................ 79

    4.1 Introduction......................................................................................................... 79

    4.2 Materials and methods ........................................................................................ 81

  • 4

    4.2.1 Organism...................................................................................................... 81

    4.2.2 Growth medium and culture conditions....................................................... 81

    4.2.3 Protein assay ................................................................................................ 81

    4.2.4 Laccase assay ............................................................................................... 81

    4.2.5 Protein precipitation..................................................................................... 82

    4.2.6 Native PAGE and activity staining .............................................................. 82

    4.3 Results................................................................................................................. 82

    4.3.1 Zymogram analysis between different anastomosis groups ........................ 82

    4.3.2 Zymogram analysis within the same anastomosis groups ............

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