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Purification, characterization and localization of cellulolytic enzymes produced by the straw mushroom, volvariella volvacea.

by Yijin Cai. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 185-207). / Introduction / Chapter 1.1 --- Biochemistry of cellulose degradation --- p.1 / Chapter 1.1.1 --- "Occurrence, distribution and structure of cellulose" --- p.1 / Chapter 1.1.2 --- Cellulose-degrading microorganisms --- p.3 / Chapter 1.1.2.1 --- Cellulolytic bacteria --- p.4 / Chapter 1.1.2.2 --- Cellulolytic fungi --- p.4 / Chapter 1.1.3 --- An overview of fungal cellulases --- p.4 / Chapter 1.1.3.1 --- Endoglucanase (EG) --- p.7 / Chapter 1.1.3.2 --- Cellobiohydrolase (CBH) --- p.16 / Chapter 1.1.3.3 --- β-Glucosidase (BGL) --- p.25 / Chapter 1.1.4 --- Synergism between the different components of the cellulolytic systems of filamentous fungi --- p.28 / Chapter 1.1.5 --- Molecular genetics of cellulases --- p.31 / Chapter 1.2 --- Secretion of cellulases by filamentous fungi --- p.32 / Chapter 1.2.1. --- Overview of enzyme secretion in filamentous fungi --- p.33 / Chapter 1.2.2 --- Glycosylation --- p.35 / Chapter 1.2.3 --- Protein secretion and the fungal cell wall --- p.37 / Chapter 1.2.4 --- Factors affecting protein secretion --- p.38 / Chapter 1.3 --- Volvariella volvacea --- p.39 / Chapter 1.4 --- Project aims --- p.43 / Materials and methods / Chapter 2.1 --- Organisms and culture conditions --- p.44 / Chapter 2.1.1 --- Basal medium --- p.44 / Chapter 2.1.2 --- Culture conditions for biomass and enzyme production on different carbon sources --- p.45 / Chapter 2.1.3 --- Culture conditions for large-scale enzyme production for purification --- p.45 / Chapter 2.1.4 --- Culture conditions for confocal microscopy --- p.46 / Chapter 2.1.5 --- Culture conditions for electron microscopy --- p.47 / Chapter 2.2 --- Mycelial extracts --- p.47 / Chapter 2.2.1 --- Large scale extraction --- p.47 / Chapter 2.2.2 --- Small scale extraction --- p.48 / Chapter 2.3 --- Enzyme purification --- p.48 / Chapter 2.3.1 --- Cell-associated enzymes (β-glucosidases) --- p.48 / Chapter 2.3.2 --- Extracellular enzymes --- p.50 / Chapter 2.3.2.1 --- Purification of cellulase complex --- p.50 / Chapter 2.3.2.2 --- Purification of CBH --- p.51 / Chapter 2.3.2.3 --- Purification of endoglucanase-III --- p.53 / Chapter 2.3.2.4 --- Partial purification of β-glucosidases --- p.55 / Chapter 2.3.3 --- Other purification methods --- p.56 / Chapter 2.3.3.1 --- FPLC Phenylsuperose hydrophobic interaction chromatography --- p.56 / Chapter 2.3.3.2 --- Affinity gel chromatography --- p.56 / Chapter 2.3.3.3 --- Isoelectric focusing by Rotorfor --- p.57 / Chapter 2.3.3.4 --- Preparative gel electrophoresis --- p.57 / Chapter 2.3.3.5 --- (NH4)2S04 Precipitation --- p.58 / Chapter 2.4 --- Electrophoresis --- p.59 / Chapter 2.4.1 --- Mini Protean-II system (BioRad) --- p.59 / Chapter 2.4.2 --- PhastGel system (Pharmacia) --- p.60 / Chapter 2.5 --- Enzyme assays --- p.61 / Chapter 2.5.1 --- β-Glucosidase --- p.61 / Chapter 2.5.2 --- Endoglucanase --- p.63 / Chapter 2.5.3 --- Cellobiohydrolase --- p.65 / Chapter 2.6 --- β-Glucosidase characterization studies --- p.66 / Chapter 2.6.1 --- pH optimum --- p.66 / Chapter 2.6.2 --- Temperature optimum --- p.66 / Chapter 2.6.3 --- Thermal stability --- p.66 / Chapter 2.6.4 --- Kinetic parameters --- p.67 / Chapter 2.6.5 --- Enzyme inhibitor studies --- p.67 / Chapter 2.6.6 --- Effect of lignin-derived phenolic monomers --- p.67 / Chapter 2.6.7 --- Substrate specificity towards p-nitrophenyl-linked glycosides --- p.67 / Chapter 2.6.8 --- "Substrate specificity towards different cellulosic substrates, mono- and disaccharides, hemicellulose, sugar alcohols and saponins" --- p.68 / Chapter 2.6.9 --- Cellulose-binding assay --- p.68 / Chapter 2.6.10 --- Effect of purified β-glucosidase on the production of glucose from crystalline cellulose and carboxymethylcellulose by Aspergillus niger cellulase --- p.69 / Chapter 2.7 --- Miscellaneous analytical methods --- p.69 / Chapter 2.7.1 --- Protein determination --- p.69 / Chapter 2.7.2 --- Determination of isoelectric points --- p.69 / Chapter 2.7.3 --- Activity staining of gels for cellulolytic enzyme activity --- p.70 / Chapter 2.7.4 --- Staining for glycoprotein --- p.71 / Chapter 2.7.5 --- Molecular weight determination --- p.71 / Chapter 2.8 --- "Production, purification and specificity of antibodies to β-glucosidases and EG-III" --- p.72 / Chapter 2.8.1 --- Antibodies to β-glucosidases --- p.72 / Chapter 2.8.2 --- Antibodies to EG-III --- p.74 / Chapter 2.9 --- Immunocytochemical studies --- p.75 / Chapter 2.9.1 --- Confocal laser scanning microscopy --- p.75 / Chapter 2.9.1.1 --- β-Glucosidases --- p.75 / Chapter 2.9.1.2 --- Endoglucanase-III --- p.75 / Chapter 2.9.2 --- Transmission electron microscopy --- p.76 / Chapter 2.9.3 --- Scanning electron microscopy --- p.77 / Chapter 2.10 --- Chemicals --- p.77 / Results / Chapter 3.1 --- "Effect of culture conditions on the growth of, and the production of cellulolytic enzymes, by V. volvacea" --- p.79 / Chapter 3.1.1 --- Growth --- p.79 / Chapter 3.1.2 --- Endoglucanases --- p.81 / Chapter 3.1.3 --- Cellobiohydrolase --- p.84 / Chapter 3.1.4 --- β-Glucosidase --- p.87 / Chapter 3.2 --- Purification of cellulolytic enzymes from V. volvacea --- p.92 / Chapter 3.2.1 --- Preliminary purification of V. volvacea extracellular cellulolytic enzymes --- p.92 / Chapter 3.2.1.1. --- (NH4)2SO4 precipitation --- p.92 / Chapter 3.2.1.2 --- Ultrafiltration --- p.94 / Chapter 3.2.1.3 --- Batch adsorption by anion exchanger --- p.94 / Chapter 3.2.1.4 --- Separation by column chromatography --- p.96 / Chapter 3.2.2 --- CBH enzymes …… --- p.102 / Chapter 3.2.3 --- Endoglucanase enzymes --- p.106 / Chapter 3.2.4 --- Cell-associated β-glucosidase enzymes --- p.113 / Chapter 3.2.5 --- Extracellular β-glucosidase enzymes --- p.120 / Chapter 3.3 --- Characterization of cell-associated β-glucosidases from V. volvacea --- p.122 / Chapter 3.3.1 --- Influence of pH and temperature --- p.122 / Chapter 3.3.2 --- Enzyme stability --- p.125 / Chapter 3.3.3 --- Kinetic parameters --- p.128 / Chapter 3.3.4 --- Enzyme inhibitors --- p.131 / Chapter 3.3.5 --- Substrate specificity --- p.137 / Chapter 3.3.6 --- Cellulose-binding and hydrolysing properties --- p.139 / Chapter 3.3.7 --- Molecular weights and isoelectric points --- p.142 / Chapter 3.4 --- Immunocytochemical studies on cellulolytic enzymes from V. volvacea --- p.146 / Chapter 3.4.1 --- Cell-associated β-glucosidase --- p.146 / Chapter 3.4.1.1 --- "Production, specificity and purification of polyclonal antibody" --- p.146 / Chapter 3.4.1.2. --- Localization --- p.151 / Chapter 3.4.1.3. --- Localization of cell-associated β-glucosidases by immuno- labelling --- p.151 / Chapter 3.4.2 --- Endoglucanase-III --- p.161 / Discussion / Chapter 4.1 --- Production --- p.163 / Chapter 4.2 --- Composition of cellulolytic enzyme system of V. volvacea --- p.168 / Chapter 4.3 --- Properties --- p.172 / Chapter 4.4 --- Localization --- p.179 / References --- p.185

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_321650
Date January 1996
ContributorsCai, Yijin., Chinese University of Hong Kong Graduate School. Division of Biology.
PublisherChinese University of Hong Kong
Source SetsThe Chinese University of Hong Kong
LanguageEnglish
Detected LanguageEnglish
TypeText, bibliography
Formatprint, vi, 207, [2] leaves : ill. (some col., some mounted) ; 30 cm.
RightsUse of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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