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The biochemical study of lipases from Meiothermus spp.

Ho Yuen Sze. / Thesis submitted in: December 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 103-116). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Table of Contents --- p.vi / List of Figures and Tables --- p.x / Abbreviations --- p.xiv / Chapter 1. --- Introduction / Chapter 1.1 --- General --- p.1 / Chapter 1.2 --- Mechanism of lipolysis --- p.3 / Chapter 1.3. --- Distinction between lipases and esterases --- p.5 / Chapter 1.4. --- Interface activation --- p.7 / Chapter 1.5 --- Substrate specificity of lipases --- p.10 / Chapter 1.6 --- Classification and properties of bacterial lipolytic enzymes --- p.13 / Chapter 1.7 --- Assays used to measure lipase activity --- p.15 / Chapter 1.7.1 --- Titrimetric method --- p.15 / Chapter 1.7.2 --- Colorimetric methods --- p.16 / Chapter 1.7.2.1 --- p-Nitrophenyl ester assay --- p.16 / Chapter 1.7.2.2 --- Chain reaction assay --- p.18 / Chapter 1.8. --- Factors affecting lipase synthesis and secretion --- p.20 / Chapter 1.9. --- Biotechnological applications of lipases --- p.21 / Chapter 1.9.1 --- Lipases in the dairy industry --- p.21 / Chapter 1.9.2 --- Lipases in household detergents --- p.23 / Chapter 1.9.3 --- Lipases in the oleochemical industry --- p.24 / Chapter 1.9.4 --- Synthesis of pharmaceuticals and agrochemicals --- p.25 / Chapter 1.10 --- Meiothermus --- p.26 / Chapter 1.11 --- Aims of study --- p.29 / Chapter 2. --- Methods and Materials / Chapter 2.1. --- Microbial strains --- p.30 / Chapter 2.2. --- Cultivation --- p.32 / Chapter 2.3. --- Preparation of samples for lipase assay --- p.33 / Chapter 2.4. --- Enzyme assays --- p.33 / Chapter 2.5. --- Characterization of lipases from selected strains --- p.35 / Chapter 2.5.1 --- Optimal pH for lipase activity --- p.35 / Chapter 2.5.2 --- Optimal temperature for lipase activity --- p.36 / Chapter 2.5.3 --- Effect of temperature on lipase stability --- p.36 / Chapter 2.5.4 --- Effect of pH on lipase stability --- p.36 / Chapter 2.5.5 --- Substrate specificity --- p.36 / Chapter 2.6. --- Purification of lipases I and II from Meiothermus strain 17R --- p.37 / Chapter 2.6.1. --- Pigment removal --- p.37 / Chapter 2.6.2. --- DEAE-Sepharose column chromatography --- p.38 / Chapter 2.6.3. --- Gel filtration chromatography --- p.38 / Chapter 2.6.4. --- FPLC Mono P chromatography --- p.38 / Chapter 2.6.5. --- Preparative polyaerylamide gel electrophoresis --- p.39 / Chapter 2.7. --- Purification of lipase III from Meiothermus strain 17R --- p.40 / Chapter 2.7.1 --- Phenyl-Sepharose chromatography --- p.40 / Chapter 2.8. --- Other enzyme purification methods used in this study --- p.40 / Chapter 2.8.1 --- Ammonium sulphate fractionation --- p.40 / Chapter 2.8.2 --- Cation exchange chromatography --- p.41 / Chapter 2.8.3 --- Heparin-Sepharose affinity chromatography --- p.41 / Chapter 2.9. --- Partial characterization of lipases I and II from Meiothermus strain 17R --- p.41 / Chapter 2.9.1 --- Molecular mass determination --- p.41 / Chapter 2.9.2 --- Isoelectric point determinations --- p.42 / Chapter 2.10. --- N-terminal amino acid sequencing of lipases I and II --- p.43 / Chapter 2.11. --- Molecular cloning of lipase II --- p.43 / Chapter 2.11.1 --- Isolation of genomic DNA --- p.43 / Chapter 2.11.2 --- Design of degenerate primers --- p.44 / Chapter 2.11.3 --- PCR amplification of genomic DNA of lipase II --- p.44 / Chapter 3. --- Results / Chapter 3.1 --- Screening of Meiothermus spp. and Thermus spp --- p.46 / Chapter 3.2 --- Characterization of selected strains --- p.50 / Chapter 3.2.1 --- Optimum pH --- p.50 / Chapter 3.2.2 --- Optimum temperature --- p.50 / Chapter 3.2.3 --- Relationship between the release of p-nitrophenol from p- nitrophenyl caprate and enzyme concentration under standard assay conditions --- p.50 / Chapter 3.2.4 --- Effect of pH on lipase stability --- p.58 / Chapter 3.2.5. --- "Temperature stability of lipases from Meiothermus strains 11R, 17Rand 12RB" --- p.58 / Chapter 3.2.6 --- Substrate specificity --- p.59 / Chapter 3.2.7 --- Ejfect of culture supplementation on lipase production --- p.69 / Chapter 3.3. --- Purification of cell-associated lipase activity from Meiothermus strain 17R --- p.71 / Chapter 3.3.1 --- Ammonium sulphate fractionation --- p.71 / Chapter 3.3.2 --- Purificationof lipase I and lipase II using column chromatography --- p.72 / Chapter 3.3.3 --- Purification of lipase III --- p.81 / Chapter 3.4. --- N-terminal amino acid sequencing of lipase I and lipase II from Meiothermus strain 17R --- p.85 / Chapter 3.5. --- Molecular studies of lipase II from Meiothermus strain 17R --- p.86 / Chapter 3.5.1 --- Extraction of genomic DNA --- p.86 / Chapter 3.5.2 --- Cloning of a fragment encoding part of lip II --- p.86 / Chapter 4. --- Discussion / Chapter 4.1 --- Screening of Meiothermus spp. and Thermus spp --- p.89 / Chapter 4.2 --- "Characterizations of lipase from Meiothermus 11R, 17R and 12RB" --- p.90 / Chapter 4.3. --- Biochemical and molecular studies of lipases from Meiothermus strain 17R --- p.94 / Chapter 4.3.1 --- Production of lipases --- p.94 / Chapter 4.3.2 --- Purification of lipases --- p.96 / Chapter 4.3.3 --- Characterization of purified lipases --- p.97 / Chapter 4.3.4 --- Amino acid sequencing of lipase --- p.100 / Chapter 5. --- Summary and suggestions --- p.101 / Chapter 6. --- Appendix --- p.102 / Chapter 7. --- References --- p.103

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324453
Date January 2003
ContributorsHo, Yuen Sze., Chinese University of Hong Kong Graduate School. Division of Biology.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xiv, 116 leaves : ill. ; 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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