A study on ligninolytic enzyme coding genes of Pleurotus pulmonarius for degrading pentachlorophenol (PCP).

Abstract

Yau Sze-nga.Thesis (M.Phil.)--Chinese University of Hong Kong, 2005.Includes bibliographical references (leaves 155-177).Abstracts in English and Chinese.Acknowledgement --- p.iAbstract --- p.ii摘要 --- p.vTable of Contents --- p.viiList of Figures --- p.xiList of Tables --- p.xivChapter 1 --- INTRODUCTION --- p.1Chapter 1.1 --- Organopollutants and environment --- p.1Chapter 1.2 --- Pentachlorophenol --- p.3Chapter 1.2.1 --- Application of pentachlorophenol --- p.3Chapter 1.2.2 --- Characteristics of PCP --- p.4Chapter 1.2.3 --- Toxicity of PCP --- p.5Chapter 1.2.4 --- Environmental exposure of PCP --- p.6Chapter 1.3 --- Wastewater treatments of organopollutants --- p.9Chapter 1.3.1 --- Physical treatment --- p.10Chapter 1.3.2 --- Chemical treatment --- p.10Chapter 1.3.3 --- Bioremediation --- p.11Chapter 1.4 --- Biodegradation of PCP --- p.13Chapter 1.4.1 --- Biodegradation of PCP by bacteria --- p.13Chapter 1.4.2 --- Biodegradation of PCP by fungi --- p.14Chapter 1.5 --- Ligninolytic enzyme --- p.16Chapter 1.5.1 --- Lignin peroxidase --- p.16Chapter 1.5.2 --- Manganese peroxidase --- p.19Chapter 1.5.3 --- Laccase --- p.21Chapter 1.5.4 --- Biodegradation of PCP and other organopollutants by ligninolytic enzymes --- p.25Chapter 1.6 --- Structure and gene regulation --- p.27Chapter 1.6.1 --- MnP gene and structure --- p.27Chapter 1.6.1.1 --- Structure of MnP --- p.27Chapter 1.6.1.2 --- MnP gene regulation --- p.30Chapter 1.6.2 --- Laccase gene and structure --- p.31Chapter 1.6.2.1 --- Structure of laccase --- p.31Chapter 1.6.2.2 --- Laccase gene regulation --- p.32Chapter 1.7 --- Pleurotus pulmonarius --- p.36Chapter 1.8 --- Aims of study --- p.37Chapter 2 --- MATERIALS & METHOD --- p.39Chapter 2.1 --- Optimization of PCP induction in broth system --- p.39Chapter 2.1.1 --- Specific enzyme assays --- p.41Chapter 2.1.1.1 --- Assay for laccase activity --- p.41Chapter 2.1.1.2 --- Assay for manganese peroxidase (MnP) activity --- p.41Chapter 2.1.1.3 --- Assay for protein assay --- p.41Chapter 2.1.2 --- PCP effect on biomass gain --- p.42Chapter 2.1.3 --- Extraction of PCP --- p.42Chapter 2.1.3.1 --- Preparation of PCP stock solution --- p.43Chapter 2.1.3.2 --- Extraction efficiency of PCP --- p.43Chapter 2.1.3.3 --- Quantification of PCP by HPLC --- p.43Chapter 2.1.3.4 --- Study of PCP degradation pathway using GC-MS --- p.44Chapter 2.2 --- Isolation of laccase and manganese peroxidase coding genes --- p.46Chapter 2.2.1 --- Preparation of ribonuclease free reagents and apparatus --- p.46Chapter 2.2.2 --- Isolation of RNA --- p.46Chapter 2.2.3 --- Quantification of total RNA --- p.47Chapter 2.2.4 --- First strand cDNA synthesis --- p.47Chapter 2.2.5 --- Polymerase Chain Reaction (PCR) --- p.48Chapter 2.2.6 --- Gel electrophoresis --- p.50Chapter 2.2.7 --- Purification of PCR products --- p.50Chapter 2.2.8 --- Preparation of Escherichia coli competent cells --- p.51Chapter 2.2.9 --- Ligation and E. coli transformation --- p.51Chapter 2.2.10 --- PCR screening of E. coli transformation --- p.52Chapter 2.2.11 --- Isolation of recombinant plasmid --- p.52Chapter 2.2.12 --- Sequence analysis --- p.53Chapter 2.2.13 --- Construction of dendrogram for Pleurotus sp. laccase and manganese peroxidase dendrogram --- p.54Chapter 2.2.13.1 --- Dendrogram of laccase genes --- p.55Chapter 2.2.13.2 --- Dendrogram of manganese genes --- p.55Chapter 2.3 --- Differential regulation profiles of laccase and manganese peroxidase genes --- p.57Chapter 2.3.1 --- Time course of the effects of PCP on levels of laccase and manganese peroxidase mRNAs --- p.57Chapter 2.3.1.1 --- Isolation of RNA --- p.57Chapter 2.3.1.2 --- RT-PCR --- p.57Chapter 2.3.2 --- The effect of different stresses --- p.65Chapter 2.3.2.1 --- Pollutant removal analysis --- p.66Chapter 2.3.2.2 --- Differential gene expression under different stresses --- p.69Chapter 2.4 --- Construction of full-length cDNA --- p.69Chapter 2.4.1 --- Primer design --- p.69Chapter 2.4.2 --- First-strand cDNA synthesis --- p.71Chapter 2.4.3 --- RACE PCR reactions --- p.71Chapter 2.5 --- Statistical analysis --- p.73Chapter 3 --- RESULT --- p.74Chapter 3.1 --- Optimization of PCP induction in broth system --- p.74Chapter 3.1.1 --- Enzyme Assay --- p.74Chapter 3.1.1.1 --- Protein content --- p.74Chapter 3.1.1.2 --- Specific laccase activity --- p.74Chapter 3.1.1.3 --- Specific MnP activity --- p.76Chapter 3.1.1.4 --- Laccase productivity --- p.78Chapter 3.1.1.5 --- MnP productivity --- p.78Chapter 3.1.2 --- PCP effect on biomass development --- p.80Chapter 3.1.3 --- PCP removal --- p.80Chapter 3.2 --- isolation of laccase and manganese peroxidase coding genes --- p.83Chapter 3.2.1 --- Dendrogram construction for heterologous MnP and laccase coding genes --- p.83Chapter 3.2.2 --- Phylogeny of ligninolytic enzyme coding genes of P. pulmonarius --- p.85Chapter 3.2.2.1 --- Phylogeny of MnP coding genes --- p.88Chapter 3.2.2.2 --- Phylogeny of laccase coding genes --- p.88Chapter 3.3 --- differential regulation profiles of laccase and MnP genes --- p.91Chapter 3.3.1 --- Time course of the effects of PCP on levels of MnP and laccase mRNAs --- p.91Chapter 3.3.1.1 --- Time course of the effects of PCP on levels of MnP mRNAs --- p.91Chapter 3.3.1.2 --- Time course of the effects of PCP on levels of laccase mRNAs --- p.97Chapter 3.3.2 --- The effects of different stresses and two lignocellulosic substrates --- p.99Chapter 3.3.2.1 --- The effect on laccase and MnP enzyme activities --- p.99Chapter 3.3.2.1.1 --- Protein content --- p.99Chapter 3.3.2.1.2 --- Specific laccase activity --- p.100Chapter 3.3.2.1.3 --- Specific MnP activity --- p.102Chapter 3.3.2.1.4 --- Dry weight of P. pulmonarius --- p.102Chapter 3.3.2.1.5 --- Laccase productivity --- p.105Chapter 3.3.2.1.6 --- MnP productivity --- p.105Chapter 3.3.2.2 --- Organopollutant removal --- p.107Chapter 3.3.2.3 --- Differential gene expression under different stresses --- p.107Chapter 3.3.2.3.1 --- The effect on MnP mRNAs --- p.107Chapter 3.3.2.3.2 --- The effect on laccase mRNAs --- p.115Chapter 3.4 --- Construction of full-length cDNA --- p.116Chapter 3.4.1 --- PPMnP5 --- p.117Chapter 3.4.2 --- PPlac2 --- p.120Chapter 3.4.3 --- PPlac6 --- p.120Chapter 4 --- DISCUSSION --- p.123Chapter 4.1 --- Optimization of PCP induction in broth system --- p.123Chapter 4.2 --- Isolation of MnP and laccase coding genes --- p.126Chapter 4.3 --- Differential regulation profiles of MnP and laccase genes --- p.128Chapter 4.3.1 --- The effects incubation time and PCP on levels of MnP and laccase mRNAs --- p.128Chapter 4.3.1.1 --- MnP --- p.129Chapter 4.3.1.2 --- Laccase --- p.129Chapter 4.3.2 --- Regulation of MnP and laccase by different substrates --- p.130Chapter 4.3.2.1 --- Regulation of MnP and laccase activities --- p.131Chapter 4.3.2.2 --- Organopollutant removal --- p.132Chapter 4.3.2.3 --- Regulation of MnP coding genes --- p.136Chapter 4.3.2.4 --- Regulation of laccase coding genes --- p.137Chapter 4.4 --- "Characterization of full length cDNAs of PPMnP5, PPlac2 and PPLAC6" --- p.140Chapter 4.4.1 --- PPMnP5 --- p.140Chapter 4.4.2 --- PPlac2 and PPlac6 --- p.144Chapter 4.4.3 --- Real-time PCR --- p.146Chapter 4.4.3.1 --- Methodology for SYBR-Green real-time PCR --- p.146Chapter 4.4.3.2 --- Comparison of conventional PCR and real-time PCR --- p.148Chapter 4.5 --- APPLICATION AND FURTHER INVESTIGATION --- p.150Chapter 5 --- CONCLUSION --- p.152Chapter 6 --- REFERENCES --- p.15