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
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_321650 |
| Date | January 1996 |
| Contributors | Cai, Yijin., Chinese University of Hong Kong Graduate School. Division of Biology. |
| Publisher | Chinese University of Hong Kong |
| Source Sets | The Chinese University of Hong Kong |
| Language | English |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, vi, 207, [2] leaves : ill. (some col., some mounted) ; 30 cm. |
| Rights | Use 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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