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
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324453 |
| Date | January 2003 |
| Contributors | Ho, Yuen Sze., Chinese University of Hong Kong Graduate School. Division of Biology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xiv, 116 leaves : ill. ; 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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