by Wang Pui. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 115-128). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abstract --- p.ii / List of Figures --- p.vi / List of Tables --- p.viii / Abbreviations --- p.ix / Chapter 1. --- Introduction Pg no / Chapter 1.1 --- Ligninolytic enzyme systems --- p.1 / Chapter 1.2 --- Three main ligninolytic enzymes --- p.3 / Chapter 1.2.1 --- Lignin peroxidases (LiP) --- p.3 / Chapter 1.2.2 --- Gene structure and Amino acid sequence structure --- p.7 / Chapter 1.2.3 --- Regulation of expression --- p.8 / Chapter 1.3. --- MnP --- p.8 / Chapter 1.3.1 --- General properties --- p.8 / Chapter 1.3.2 --- Gene structure and Amino acid sequence --- p.9 / Chapter 1.3.3 --- Regulation of Expression --- p.12 / Chapter 1.4 --- Laccase --- p.12 / Chapter 1.4.1 --- General Properties --- p.12 / Chapter 1.4.2 --- Gene structure and Amino acid sequence --- p.14 / Chapter 1.5 --- Pentachlorophenol (PCP) --- p.16 / Chapter 1.5.1 --- Production --- p.16 / Chapter 1.5.2 --- Toxicity --- p.15 / Chapter 1.5.3 --- Persistence --- p.19 / Chapter 1.6 --- Oyster mushroom --- p.22 / Chapter 1.7 --- Application of ligninolytic enzymes in bioremediation --- p.23 / Chapter 1.7.1 --- Genetic modification --- p.23 / Chapter 1.7.2 --- Characterization of enzymes properties --- p.25 / Chapter 1.7.3 --- Ligninolytic enzymes Purification and extraction --- p.26 / Chapter 1.7.4 --- Immobilization of ligninolytic enzymes --- p.26 / Chapter 1.8 --- Fermentation --- p.29 / Chapter 1.8.1 --- Different types of fermentation --- p.29 / Chapter 1.8.1.1 --- Submerged fermentation (SF) --- p.29 / Chapter 1.8.1.2 --- Solid State Fermentation (SSF) --- p.30 / Chapter 1.9 --- Proposal and experimental plan of the project --- p.33 / Chapter 1.9.1 --- Objectives --- p.34 / Chapter 2. --- Methods --- p.36 / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Culture maintenance --- p.36 / Chapter 2.1.2 --- Preparation of Pentachlorophenol (PCP) stock solution --- p.36 / Chapter 2.2 --- Optimization of production of ligninolytic enzymes by effective PCP concentration --- p.37 / Chapter 2.2.1 --- Preparation of mycelial homogenate --- p.37 / Chapter 2.2.2 --- Incubation --- p.37 / Chapter 2.2.3 --- Specific enzyme assays --- p.38 / Chapter 2.2.3.1 --- Laccase --- p.38 / Chapter 2.2.3.2 --- Manganese peroxidase (MnP) --- p.39 / Chapter 2.2.3.3 --- Lignin peroxidase (LiP) --- p.39 / Chapter 2.2.3.4 --- Protein --- p.39 / Chapter 2.3 --- Cloning of specific PCP-degradative laccase cDNA --- p.40 / Chapter 2.3.1 --- Isolation of total RNA --- p.41 / Chapter 2.3.2 --- Spectrophotometric quantification and qualification of DNA and RNA --- p.41 / Chapter 2.3.3 --- First strand cDNA synthesis --- p.42 / Chapter 2.3.4 --- Amplification of laccase cDNA --- p.43 / Chapter 2.3.4.1 --- Design of primers for PCR reaction --- p.43 / Chapter 2.3.4.2 --- Polymerase chain reaction --- p.44 / Chapter 2.3.5 --- Agarose gel electrophoresis of DNA --- p.44 / Chapter 2.3.6 --- Purification of PCR products --- p.45 / Chapter 2.3.7 --- TA cloning of PCR products --- p.46 / Chapter 2.3.8 --- Preparation of Escherichia coli competent cells --- p.46 / Chapter 2.3.9 --- Bacterial transformation by heat shock --- p.47 / Chapter 2.3.10 --- Colony screening --- p.48 / Chapter 2.3.11 --- Mini-preparation of plasmid DNA --- p.48 / Chapter 2.3.12 --- Sequencing --- p.49 / Chapter 2.3.13 --- Identification of sequence --- p.51 / Chapter 2.4 --- Study of regulation temporal expression of laccase genes by PCP --- p.51 / Chapter 2.4.1 --- Semi-quantitative PCR --- p.51 / Chapter 2.4.1.1 --- Design of gene-specific primers --- p.51 / Chapter 2.4.1.2 --- Determination of suitable PCR cycles --- p.54 / Chapter 2.4.1.3 --- Normalization of the amount of RNA of each sample --- p.54 / Chapter 2.5 --- Quantification of residual PCP concentration --- p.55 / Chapter 2.5.1 --- Extraction of PCP --- p.55 / Chapter 2.5.2 --- High performance liquid chromatography --- p.55 / Chapter 2.5.3 --- Assessment criteria --- p.56 / Chapter 2.6 --- Effect of other componds on laccase activity and laccase expression --- p.56 / Chapter 2.6.1 --- Study of different isoform of laccase --- p.57 / Chapter 2.6.2 --- SDS-PAGE analysis of proteins --- p.58 / Chapter 2.7 --- Study of laccase expression and laccase activity in fruiting process of oyster mushroom --- p.59 / Chapter 2.8 --- Statistical analysis --- p.60 / Chapter 3. --- Results --- p.61 / Chapter 3.1 --- Production of Ligninolytic Enzymes by oyster mushroom / Chapter 3.1.1 --- Optimization of laccase production --- p.62 / Chapter 3.1.2 --- Optimization of MnP production --- p.64 / Chapter 3.1.3 --- Change of Protein content at different PCP concentration and time --- p.64 / Chapter 3.1.4 --- Change of specific activity at different PCP concentration and time --- p.64 / Chapter 3.1.5 --- Toxicity of PCP towards mycelial growth --- p.67 / Chapter 3.1.6 --- Enzyme productivities of laccase and MnP --- p.67 / Chapter 3.1.7 --- Change of % of residual PCP concentrations during 14 days --- p.70 / Chapter 3.2. --- Cloning of PCP-degradative laccase genes --- p.70 / Chapter 3.3 --- Regulation of expression of the laccase genes by PCP --- p.74 / Chapter 3.3.1 --- Determination of suitable PCR cycles --- p.74 / Chapter 3.3.2 --- Normalization of total RNA amount of different samples --- p.74 / Chapter 3.3.3 --- Regulation of temporal expression of the laccase genes by PCP --- p.74 / Chapter 3.4 --- Effect of other compounds and physiological status on laccase activity and expression --- p.81 / Chapter 3.5 --- Study of different forms of laccase --- p.86 / Chapter 4. --- Discussion --- p.93 / Chapter 4.1 --- Production of Ligninolytic enzymes by Pleurotus pulmonarius / Chapter 4.1.1 --- Optimization of laccase and MnP production by PCP --- p.95 / Chapter 4.2 --- Cloning of laccase genes --- p.97 / Chapter 4.2.1 --- Cloning strategy --- p.97 / Chapter 4.2.2 --- Analysis of Nucleotide sequence of Lac1 - Lac3 --- p.99 / Chapter 4.2.3 --- Characterization and comparison of deduced amino acid sequences of Lacl-Lac3 --- p.99 / Chapter 4.3 --- Regulation of expression of the laccase genes by PCP --- p.100 / Chapter 4.3.1 --- Regulation of temporal expression by PCP --- p.100 / Chapter 4.4 --- Effect of the potential inducers on laccase activity and expression --- p.103 / Chapter 4.5 --- Effect of the physiological status on laccase activity and expression --- p.105 / Chapter 4.5.1 --- Production of PCP-degradative laccase by Solid-state fermentation --- p.107 / Chapter 4.5.2 --- Uses of molecular probe in bioremediation --- p.107 / Chapter 4.6 --- Different isoforms of laccase --- p.109 / Chapter 4.7 --- Conclusion --- p.112 / Chapter 4.8 --- Further studies / Chapter 4.8.1 --- Confirmation of PCP-degradation by gene product of Lac1 and Lac2 --- p.114 / Chapter 4.8.2 --- Optimization of PCP-degradative laccases production by solid-state fermentation --- p.114 / Chapter 5. --- References --- p.115
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324062 |
| Date | January 2002 |
| Contributors | Wang, Pui., Chinese University of Hong Kong Graduate School. Division of Environmental Science. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, ix, 128 leaves : ill. (some 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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