Luo, Xiao. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 118-134). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.iv / Abbreviations --- p.v / Contents --- p.vi / List of Figures --- p.ix / List of Tables --- p.xii / Chapter Chapter 1 : --- Literature Review --- p.1 / Chapter 1.1 --- Lentinula edodes --- p.1 / Chapter 1.1.1 --- Introduction and taxonomy --- p.1 / Chapter 1.1.2 --- Nutritional values and medical values --- p.2 / Chapter 1.2 --- Life cycle and morphology --- p.5 / Chapter 1.3 --- Lignocellulolytic system in wood-rotting fungi --- p.9 / Chapter 1.3.1 --- Structures of lignin --- p.9 / Chapter 1.3.2 --- Wood-rotting fungi --- p.11 / Chapter 1.3.3 --- Lignin degradation by white rot fungi --- p.12 / Chapter 1.3.4 --- Ligninolytic enzymes --- p.16 / Chapter 1.3.4.1 --- Lignin peroxidase --- p.16 / Chapter 1.3.4.2 --- Maganese peroxide --- p.16 / Chapter 1.3.4.3 --- Laccases --- p.19 / Chapter 1.3.5 --- Potential Industrial application of liglinolytic enzymes --- p.22 / Chapter 1.3.6 --- Ligninolytic enzymes in L. edodes --- p.23 / Chapter 1.4 --- Expression systems for fungal ligninolytic enzymes --- p.24 / Chapter 1.5 --- Aim of this project --- p.27 / Chapter 1.6 --- Long-term significance --- p.28 / Chapter Chapter 2: --- Sequence analysis of ligninolytic enzymes from Lentinula edodes --- p.29 / Chapter 2.1 --- Introduction --- p.29 / Chapter 2.2 --- Materials and methods --- p.32 / Chapter 2.2.1 --- Phylogenetic study and signal peptide prediction of the decuced ligninolytic enzymes --- p.32 / Chapter 2.2.2 --- Comparison ligninolytic enzymes of L. edodes and other basidiomycetes fungi --- p.32 / Chapter 2.3 --- Results --- p.34 / Chapter 2.3.1 --- Protein sequence analysis and signature sequences identification of L. edodes laccases --- p.34 / Chapter 2.3.2 --- Protein sequence analysis of L. edodes manganese peroxidases --- p.34 / Chapter 2.3.3 --- Phylogenetic study of ligninolytic genes from L.edodes --- p.35 / Chapter 2.4 --- Disscussion --- p.52 / Chapter Chapter 3: --- Transcription profiling of ligninolytic enzymes from Lentinula edodes --- p.56 / Chapter 3.1 --- Introduction --- p.56 / Chapter 3.2 --- Materials and Methods --- p.61 / Chapter 3.2.1 --- Strain cultivation --- p.61 / Chapter 3.2.2 --- "RNA extraction, mRNA isolation and cDNA synthesis" --- p.63 / Chapter 3.2.3 --- RNA Quality Estimation --- p.64 / Chapter 3.2.4 --- cDNA synthesis --- p.65 / Chapter 3.2.5 --- Primer verification --- p.66 / Chapter 3.2.6 --- Quantitative RT-PCR --- p.66 / Chapter 3.3 --- Results --- p.70 / Chapter 3.3.1 --- RNA quality estimation --- p.70 / Chapter 3.3.2 --- Quantification real time PCR --- p.70 / Chapter 3.3.3 --- Transcriptional profiling of laccases during the development of L edodes --- p.70 / Chapter 3.3.4 --- Transcriptional profiling of MnPs during the development of L edodes --- p.71 / Chapter 3.3.5 --- Transcript level analysis of laccases from in mycelia grown on lignocelluloses medium and non -lignocelluloses medium --- p.71 / Chapter 3.3.6 --- Transcript level analysis of MnPs in mycelia grown on lignocelluloses medium and non -lignocelluloses medium --- p.72 / Chapter 3.3.7 --- Differential expression of laccases from L. edodes grownin lignocelluloses medium during mycelia stage --- p.72 / Chapter 3.3.8 --- Differential expression of laccases from L. edodes grownin lignocelluloses medium during mycelia stage --- p.72 / Chapter 3.4 --- Discussion --- p.87 / Chapter 3.4.1 --- Transcriptional profiling of laccases and MnPs during four developmental stages --- p.87 / Chapter 3.4.2 --- Transcriptional profiling of laccases and MnPs in mycelium grown in lignocelluloses and non-lignocelluloses medium --- p.88 / Chapter 3.4.3 --- Temporal differential expression of laccases and manganese peroxidases --- p.90 / Chapter 3.5 --- Conclusion --- p.92 / Chapter Chapter 4: --- "Cloning and heterologous expression of Lentinula edodes laccase, lac1B, in yeast Pichia pastoris" --- p.93 / Chapter 4.1 --- Introduction --- p.93 / Chapter 4.2 --- Materials and Methods --- p.95 / Chapter 4.2.1 --- Strain cultivation --- p.95 / Chapter 4.2.2 --- First strand cDNA synthesis --- p.95 / Chapter 4.2.3 --- Construction of cDNA library --- p.95 / Chapter 4.2.4 --- Signal peptide prediction of Iac1 B --- p.96 / Chapter 4.2.5 --- Cloning of native laccase into Pichia pastoris expression vector --- p.96 / Chapter 4.2.6 --- Screening for positive colonies --- p.97 / Chapter 4.2.7 --- Construction of pool of recombinant vector --- p.97 / Chapter 4.2.8 --- Transformation of P. pastoris --- p.98 / Chapter 4.2.9 --- Screening for expression cassette into Pichia pastoris --- p.98 / Chapter 4.2.10 --- Enzyme Activity assay --- p.99 / Chapter 4.2.11 --- SDS-PAGE --- p.100 / Chapter 4.3 --- Results --- p.103 / Chapter 4.3.1 --- Screening for positive colonies with recombinant vector in TOP10 --- p.103 / Chapter 4.3.2 --- Screening for expression cassette from transform ants of P pastoris --- p.103 / Chapter 4.3.3 --- Enzyme activity assay --- p.103 / Chapter 4.3.4 --- SDS-PAGE --- p.104 / Chapter 4.4 --- Disscussion --- p.109 / Chapter Chapter 5: --- Concluding Remarks --- p.111 / Reference --- p.118

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