by Wong Yuk Hang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 110-114). / Abstract --- p.i / Acknowledgement --- p.iv / Dedication --- p.v / Table of Contents --- p.vi / List of Tables --- p.xi / List of Figures --- p.xii / Chapter Chapter 1 --- Introduction and literature review / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.2 --- Literature review --- p.5 / Chapter 1.2.1 --- General considerations --- p.5 / Chapter 1.2.2 --- Nature of glucosinolate --- p.6 / Chapter 1.2.3 --- Degradation of glucosinolates by myrosinase --- p.7 / Chapter 1.2.4 --- Toxicology of glucosinolate and hydrolysis products --- p.8 / Chapter 1.2.5 --- Plant myrosinase --- p.9 / Chapter 1.2.6 --- Fungal myrosinase --- p.11 / Chapter 1.2.7 --- Purification and properties of fungal myrosinase --- p.11 / Chapter Chapter 2 --- Screening of fungi with myrosinase activity and physiological studies of myrosinase production in Aspergillus oryzae / Chapter 2.1 --- Introduction --- p.15 / Chapter 2.2 --- Materials and methods --- p.16 / Chapter 2.2.1 --- Fungal strains --- p.16 / Chapter 2.2.2 --- Media --- p.16 / Chapter 2.2.3 --- Screening --- p.17 / Chapter 2.2.4 --- Enzyme assay and protein determination --- p.18 / Chapter 2.2.4.1 --- Myrosinase assay --- p.18 / Chapter 2.2.4.2 --- Definition of myrosinase unit and specific activity --- p.19 / Chapter 2.2.4.3 --- Protein determination --- p.19 / Chapter 2.2.5 --- Physiological studies of myrosinase production in Aspergillus oryzae --- p.19 / Chapter 2.2.5.1 --- Incubation time --- p.20 / Chapter 2.2.5.2 --- Inducer concentration --- p.20 / Chapter 2.3 --- Results --- p.21 / Chapter 2.3.1 --- Screening --- p.21 / Chapter 2.3.1.1 --- Degradation of sinigrin in culture medium --- p.21 / Chapter 2.3.1.2 --- Confirmation of myrosinase activity --- p.21 / Chapter 2.3.2 --- Physiological studies of myrosinase production in Aspergillus oryzae --- p.21 / Chapter 2.3.2.1 --- Incubation time --- p.21 / Chapter 2.3.2.2 --- Inducer concentration --- p.22 / Chapter 2.4 --- Discussion --- p.23 / Chapter 2.4.1 --- Fungi selection in screening programme --- p.23 / Chapter 2.4.2 --- Medium composition --- p.23 / Chapter 2.4.3 --- Screening --- p.24 / Chapter 2.4.4 --- Physiological studies of myrosinase production in Aspergillus oryzae --- p.25 / Chapter 2.4.4.1 --- Incubation time --- p.25 / Chapter 2.4.4.2 --- Inducer concentration --- p.25 / Chapter Chapter 3 --- Purification and characterization of myrosinase in Aspergillus oryzae / Chapter 3.1 --- Introduction --- p.33 / Chapter 3.2 --- Materials and methods --- p.35 / Chapter 3.2.1 --- Reagents --- p.35 / Chapter 3.2.2 --- Fungal propagation --- p.35 / Chapter 3.2.3 --- Purification of the fungal myrosinase --- p.36 / Chapter 3.2.3.1 --- Preparation of crude extract --- p.36 / Chapter 3.2.3.2 --- Dialysis --- p.37 / Chapter 3.2.3.3 --- DEAE-Sepharose CL-6B ion-exchange chromatography --- p.37 / Chapter 3.2.3.4 --- Sephacryl S-200 molecular sieving chromatography --- p.37 / Chapter 3.2.3.5 --- FPLC Phenyl Superose hydrophobic interaction chromatography --- p.38 / Chapter 3.2.3.6 --- FPLC Mono P chromatofocusing --- p.38 / Chapter 3.2.4 --- Myrosinase assay and protein concentration determination --- p.39 / Chapter 3.2.4.1 --- Spot test for myrosinase activity --- p.39 / Chapter 3.2.4.2 --- Standard end-point assay --- p.40 / Chapter 3.2.4.3 --- Determination of protein concentration --- p.42 / Chapter 3.2.5 --- Physicochemical characterization of the myrosinase isozymes --- p.42 / Chapter 3.2.5.1 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis --- p.42 / Chapter 3.2.5.2 --- Protein staining and glycoprotein detection --- p.43 / Chapter 3.2.5.3 --- Chromatofocusing --- p.43 / Chapter 3.2.5.4 --- Gel filtration with FPLC Superose 6 --- p.44 / Chapter 3.2.6 --- Enzymatic properties --- p.44 / Chapter 3.2.6.1 --- Effect of pH on crude enzyme stability --- p.44 / Chapter 3.2.6.2 --- Effect of substrate concentration on enzyme activity --- p.45 / Chapter 3.2.6.3 --- Effect of pH on enzyme activity --- p.45 / Chapter 3.2.6.4 --- Effect of temperature on enzyme activity --- p.46 / Chapter 3.2.6.5 --- Effects of metallic ions on enzyme activity --- p.46 / Chapter 3.2.6.6 --- Effects of various compounds on enzyme activity --- p.46 / Chapter 3.2.6.7 --- Effects of various buffers on enzyme activity --- p.47 / Chapter 3.3 --- Results --- p.48 / Chapter 3.3.1 --- Fungal propagation --- p.48 / Chapter 3.3.2 --- Purification of fungal myrosinase in Aspergillus oryzae --- p.48 / Chapter 3.3.2.1 --- Extraction of the enzyme --- p.48 / Chapter 3.3.2.2 --- Dialysis --- p.49 / Chapter 3.3.2.3 --- DEAE-Sepharose ion-exchange chromatography --- p.49 / Chapter 3.3.2.4 --- Sephacryl S-200 molecular sieving chromatography --- p.50 / Chapter 3.3.2.5 --- FPLC Phenyl Superose hydrophobic interaction chromatography --- p.50 / Chapter 3.3.2.6 --- FPLC Mono P chromatofocusing --- p.51 / Chapter 3.3.3 --- Physicochemical characterization --- p.52 / Chapter 3.3.3.1 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis --- p.52 / Chapter 3.3.3.2 --- Chromatofocusing --- p.53 / Chapter 3.3.3.3 --- Gel filtration --- p.53 / Chapter 3.3.4 --- Enzymatic properties --- p.53 / Chapter 3.3.4.1 --- Effect of pH on the crude enzyme stability --- p.53 / Chapter 3.3.4.2 --- Effect of substrate concentration on enzyme activity --- p.54 / Chapter 3.3.4.3 --- Effect of pH on enzyme activity --- p.54 / Chapter 3.3.4.4 --- Effect of temperature on enzyme activity --- p.55 / Chapter 3.3.4.5 --- Effects of metallic ions on enzyme activity --- p.55 / Chapter 3.3.4.6 --- Effects of various compounds on enzyme activity --- p.56 / Chapter 3.3.4.7 --- Effects of various buffers on enzyme activity --- p.57 / Chapter 3.4 --- Discussion --- p.58 / Chapter 3.4.1 --- Purification of Aspergillus oryzae myrosinase --- p.58 / Chapter 3.4.1.1 --- Dialysis --- p.58 / Chapter 3.4.1.2 --- Enzyme purification --- p.58 / Chapter 3.4.2 --- Physicochemical properties --- p.60 / Chapter 3.4.2.1 --- Glycoprotein --- p.60 / Chapter 3.4.2.2 --- Molecular weights --- p.60 / Chapter 3.4.2.3 --- Isoelectric points --- p.61 / Chapter 3.4.3 --- Enzymatic properties --- p.61 / Chapter 3.4.3.1 --- pH and temperature optima --- p.61 / Chapter 3.4.3.2 --- Substrate affinity --- p.62 / Chapter 3.4.3.3 --- Inhibitions by various compounds and metallic ions --- p.63 / Chapter 3.4.3.4 --- Inhibitions by various buffer systems --- p.64 / Chapter Chapter 4 --- Summary --- p.106 / References --- p.110
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_318073 |
| Date | January 1994 |
| Contributors | Wong, Yuk Hung., 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, xiv, 114 leaves : ill. (some mounted col.) ; 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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