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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Improved catalytic activity and thermostability of Trigonopsis variabilis D-amino acid oxidase mutants.

January 2009 (has links)
Wong, Kin Sing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 86-98). / Abstract also in Chinese. / THESIS COMMITTEE --- p.i / ABSTRACT (ENGLISH) --- p.ii / ABSTRACT (CHINESE) --- p.iv / ACKNOWLEDGEMENTS --- p.v / DECLARATION --- p.vi / ABBREVIATIONS --- p.vii / TABLE OF CONTENTS --- p.x / LIST OF TABLES --- p.xiv / LIST OF FIGURES --- p.xv / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1. --- Antibiotics market and β-lactam antibiotics --- p.1 / Chapter 1.2. --- Semi-synthetic cephems --- p.1 / Chapter 1.3. --- Conversion of CPC to 7-ACA --- p.3 / Chapter 1.4. --- Chemical production versus enzymatic bioconversion --- p.5 / Chapter 1.5. --- Industrial two-step bioconversion of CPC --- p.11 / Chapter 1.6. --- Phylogenetics and physiological roles of DAAO --- p.15 / Chapter 1.7. --- Yeast DAAOs are suitable candidates for enzymatic bioconversion --- p.17 / Chapter 1.8. --- Structural and mechanistic studies of DAAOs --- p.18 / Chapter 1.9. --- "Modifications of pkDAAO, RgDAAO and TvDAAO" --- p.25 / Chapter 1.10. --- Objectives of the study --- p.26 / Chapter CHAPTER 2 --- HOMOLOGY MODELLING / Chapter 2.1. --- Introduction --- p.27 / Chapter 2.2. --- Methods / Chapter 2.2.1. --- Sequence alignment and selection of homologs --- p.28 / Chapter 2.2.2. --- Generation of three-dimensional TvDAAO model --- p.28 / Chapter 2.3. --- Results --- p.29 / Chapter 2.4. --- Discussion --- p.33 / Chapter CHAPTER 3 --- "MUTAGENESIS, EXPRESSION, PURIFICATION AND SCREENING OF MUTANTS" / Chapter 3.1. --- Introduction --- p.38 / Chapter 3.2. --- Materials and methods / Chapter 3.2.1. --- Cloning of TvDAAO mutants / Chapter 3.2.1.1. --- Preparation of competent E. coli --- p.39 / Chapter 3.2.1.2. --- Transformation of E. coli --- p.40 / Chapter 3.2.1.3. --- Agarose gel electrophoresis and gel-purification --- p.41 / Chapter 3.2.1.4. --- Plasmid extraction --- p.42 / Chapter 3.2.1.5. --- Site-directed mutagenesis of TvDAAO --- p.42 / Chapter 3.2.2. --- Heterologous expression and purification of mutants / Chapter 3.2.2.1 --- Shake flask fermentation --- p.45 / Chapter 3.2.2.2. --- Cell harvest and disruption --- p.45 / Chapter 3.2.2.3. --- Purification of WT and mutants --- p.47 / Chapter 3.2.2.4. --- Determination of protein concentration --- p.47 / Chapter 3.2.2.5. --- SDS-PAGE --- p.48 / Chapter 3.2.3. --- Screening of mutants --- p.48 / Chapter 3.3. --- Results / Chapter 3.3.1. --- Preparation of purified TvDAAO mutants --- p.50 / Chapter 3.3.2. --- Evaluation of activity and thermostability --- p.50 / Chapter 3.4. --- Discussion --- p.53 / Chapter CHAPTER 4 --- ENZYME KINETICS / Chapter 4.1. --- Introduction --- p.57 / Chapter 4.2. --- Materials and methods / Chapter 4.2.1. --- Standard assay --- p.58 / Chapter 4.2.2. --- Determination of kinetic parameters --- p.59 / Chapter 4.2.3. --- Inhibitory studies --- p.59 / Chapter 4.2.4. --- Effects of pH --- p.60 / Chapter 4.2.5. --- Heat treatments --- p.60 / Chapter 4.2.6. --- CD measurements --- p.61 / Chapter 4.3. --- Results / Chapter 4.3.1. --- Time progress curve analysis --- p.61 / Chapter 4.3.2. --- Kinetics of WT and mutants --- p.62 / Chapter 4.3.3. --- Temperature-dependent and time-dependent thermostability --- p.67 / Chapter 4.3.4. --- Secondary structure measurements --- p.71 / Chapter 4.4. --- Discussion --- p.71 / Chapter CHAPTER 5 --- CONCLUSIONS AND PERSEPECTIVES --- p.83 / BIBLIOGRAPHY --- p.86

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