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A physiological and biochemical study of selected enzymes involved in central nitrogen and carbon metabolism in Volvariella volvacea.

by Deng Yu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 111-120). / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / List of Abbreviations --- p.viii / List of Tables --- p.ix / List of Figures --- p.x / Chapter 1 --- Introduction / Chapter 1.1 --- Primary nitrogen metabolism in fungi --- p.1 / Chapter 1.1.1 --- Ammonium assimilation --- p.4 / Chapter 1.1.2 --- Regulation of ammonium assimilating enzymes --- p.8 / Chapter 1.2 --- Relevant central carbon metabolism in fungi --- p.11 / Chapter 1.2.1 --- Glyoxylate cycle and isocitrate metabolism --- p.11 / Chapter 1.2.2 --- GABA shunt --- p.15 / Chapter 1.3 --- Relationship between nitrogen metabolism and fungi morphogenesis --- p.15 / Chapter 1.4 --- General background of Volvariella volvacea --- p.17 / Chapter 1.5 --- Objectives of the study --- p.20 / Chapter 2 --- Materials and methods / Chapter 2.1 --- Organism --- p.22 / Chapter 2.2 --- Growth media --- p.22 / Chapter 2.2.1 --- Basal medium --- p.22 / Chapter 2.2.2 --- Solid-state cultivation --- p.23 / Chapter 2.3 --- Effect of different nitrogen sources on the mycelial growth of V volvacea in submerged culture --- p.26 / Chapter 2.4 --- Effect of different carbon and nitrogen sources and concentrations on the production of selected enzymes involved in central carbon and nitrogen metabolism --- p.27 / Chapter 2.5 --- Enzyme extraction --- p.28 / Chapter 2.6 --- Enzyme assays --- p.28 / Chapter 2.6.1 --- NAD-dependent glutamate dehydrogenase --- p.28 / Chapter 2.6.2 --- NADP-dependent glutamate dehydrogenase --- p.29 / Chapter 2.6.3 --- NAD- dependent isocitrate dehydrogenase --- p.29 / Chapter 2.6.4 --- Isocitrate lyase --- p.30 / Chapter 2.7 --- Protein determination --- p.30 / Chapter 2.8 --- Determination of optimum pH for enzyme assays --- p.31 / Chapter 2.9 --- Determination of optimum temperatures for enzyme assays --- p.31 / Chapter 2.10 --- Transfer experiments --- p.31 / Chapter 2.11 --- Enzyme stability --- p.32 / Chapter 2.12 --- Purification of NAD-dependent glutamate dehydrogenase --- p.33 / Chapter 2.12.1 --- Ammonium sulphate precipitation --- p.33 / Chapter 2.12.2 --- Ion exchange chromatography --- p.33 / Chapter 2.12.3 --- Ultrafiltrartion --- p.34 / Chapter 2.12.4 --- Gel filtration chromatography --- p.34 / Chapter 2.12.5 --- Affinity chromatography --- p.34 / Chapter 2.13 --- Electrophoresis --- p.35 / Chapter 2.13.1 --- SDS polyacrylamide gel electrophoresis --- p.35 / Chapter 2.13.2 --- Native polyacrylamide gel electrophoresis --- p.35 / Chapter 2.13.3 --- Activity staining for NAD-dependent glutamate dehydrogenase --- p.36 / Chapter 2.13.4 --- Protein staining --- p.36 / Chapter 2.14 --- NAD-dependent glutamate dehydrogenase characterization studies --- p.37 / Chapter 2.14.1 --- Effect of substrate concentration --- p.37 / Chapter 2.14.2 --- Molecular weight determination --- p.37 / Chapter 2.14.2.1 --- Molecular weight determination by gel filtration chromatography --- p.37 / Chapter 2.14.2.2 --- Molecular weight determination by native PAGE --- p.38 / Chapter 2.14.2.3 --- Protein subunit molecular weight determination by SDS- PAGE --- p.38 / Chapter 3 --- Results / Chapter 3.1 --- Effect of different nitrogen sources on the mycelial growth of V. volvacea in submerged culture --- p.39 / Chapter 3.2 --- Optimum assay conditions for NAD-dependent glutamate dehydrogenase --- p.42 / Chapter 3.3 --- Optimum assay conditions for NADP-dependent glutamate dehydrogenase --- p.46 / Chapter 3.4 --- Optimum assay conditions for NAD-dependent isocitrate dehydrogenase --- p.50 / Chapter 3.5 --- Optimum assay conditions for isocitrate lyase --- p.54 / Chapter 3.6 --- Biomass production and enzyme activities in extracts of in vegetative mycelia grown with different nitrogen and carbon sources provided at different concentrations --- p.58 / Chapter 3.6.1 --- Mycelia growth under different conditions --- p.58 / Chapter 3.6.2 --- NAD- and NADP-dependent glutamate dehydrogenases in extracts of vegetative mycelia grown with different nitrogen and carbon sources provided at different conditions --- p.58 / Chapter 3.6.3 --- NAD-dependent isocitrate dehydrogenase and isocitrate lyase in vegetative mycelia grown with different nitrogen and carbon sources provided at different conditions --- p.64 / Chapter 3.7 --- Transfer experiments --- p.67 / Chapter 3.7.1 --- Activities of glutamate dehydrogenases in extracts of myceila transferred to media containing different carbon sources --- p.67 / Chapter 3.7.2 --- Effect of different carbon sources on the glutamate dehydrogenases in submerged cultures --- p.67 / Chapter 3.8 --- Glutamate dehydrogenase activity in various parts of the fruit body during different stages of fruit body development --- p.70 / Chapter 3.9 --- Stabilization of NAD-dependent glutamate dehydrogenase activity --- p.75 / Chapter 3.10 --- Purification of NAD-dependent glutamate dehydrogenase --- p.77 / Chapter 3.10.1 --- Ammonium sulphate precipitation --- p.77 / Chapter 3.10.2 --- Partial purification by column chromatography --- p.78 / Chapter 3.10.3 --- Electrophoretic determination of the protein profiles of crude extract and partially purified samples --- p.83 / Chapter 3.11 --- Characterization of partially purified NAD-dependent glutamate dehydrogenase from V. volvacea --- p.86 / Chapter 3.11.1 --- Optimum pH and temperature --- p.86 / Chapter 3.11.2 --- Kinetic parameters --- p.86 / Chapter 3.11.3 --- Molecular weight --- p.92 / Chapter 3.11.3.1 --- Molecular weight determination by gel filtration chromatography --- p.92 / Chapter 3.11.3.2 --- Molecular weight determination by native PAGE --- p.92 / Chapter 3.11.3.3 --- Subunit molecular weight determination by SDS-PAGE --- p.92 / Chapter 4 --- Discussion / Chapter 4.1 --- Nutrient nitrogen for the growth of Volvariella volvacea --- p.97 / Chapter 4.1.1 --- Mycelial growth on simple nitrogen compounds --- p.97 / Chapter 4.1.2 --- Nutrient nitrogen in mushroom compost --- p.98 / Chapter 4.2 --- Production and regulation of selected enzymes in vegetative mycelia --- p.98 / Chapter 4.2.1 --- Production and regulation of glutamate dehydrogenases --- p.98 / Chapter 4.2.2 --- Production and regulation of isocitrate dehydrogenase and isocitrate lyase --- p.103 / Chapter 4.3 --- Glutamate dehydrogenases and fruit body development --- p.104 / Chapter 4.4 --- Purification and characterization of NAD-dependent glutamate dehydrogenase --- p.105 / Chapter 4.4.1 --- Enzyme purification --- p.105 / Chapter 4.4.2 --- Enzyme stability --- p.106 / Chapter 4.4.3 --- Enzyme properties --- p.107 / Chapter 4.5 --- Future works: nitrogen metabolism and the growth of Vohariella volvacea --- p.109 / References --- p.111

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_322432
Date January 1999
ContributorsDeng, Yu., Chinese University of Hong Kong Graduate School. Division of Biology.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xi, 120 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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