Genetic engineering improvement of glucose isomerase.

Abstract

Shen Dong.Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.Includes bibliographical references (leaves 97-104).Abstracts in English and Chinese.Chapter Chapter 1 --- Introduction --- p.1Chapter 1.1 --- High fructose corn syrup (HFCS) --- p.2Chapter 1.1.1 --- Status quo and prospect of HFCS --- p.2Chapter 1.1.2 --- Industrial process for HFCS production --- p.3Chapter 1.1.3 --- Glucose (xylose) isomerase in industrial application --- p.5Chapter 1.2 --- Glucose (xylose) isomerase --- p.9Chapter 1.2.1 --- Source organisms --- p.9Chapter 1.2.2 --- Functions of glucose (xylose) isomerase --- p.12Chapter 1.2.3 --- Structure of glucose isomerase --- p.13Chapter 1.2.4 --- Catalytic mechanism of glucose isomerase --- p.17Chapter 1.2.5 --- Biochemical properties of glucose isomerase --- p.18Chapter 1.2.6 --- Immobilization studies --- p.22Chapter 1.3 --- Aims of my study --- p.25Chapter Chapter 2 --- Materials and Methods --- p.26Chapter 2.1 --- Cloning of parental glucose isomerase gene --- p.27Chapter 2.1.1 --- Materials --- p.27Chapter 2.1.1.1 --- Bacterial strain --- p.27Chapter 2.1.1.2 --- Growth media --- p.29Chapter 2.1.1.3 --- Antibiotics --- p.29Chapter 2.1.1.4 --- Reagents for isolation of chromosomal DNA --- p.30Chapter 2.1.1.5 --- Reagents for PCR reaction --- p.30Chapter 2.1.1.6 --- Reagents for agarose gel electrophoresis --- p.30Chapter 2.1.1.7 --- Reagents for DNA recovery from agarose gel --- p.31Chapter 2.1.1.8 --- Vector and enzyme for ligation --- p.31Chapter 2.1.1.9 --- Reagents for preparation of competent cells --- p.32Chapter 2.1.1.10 --- Reagents for extraction of plasmid DNA --- p.32Chapter 2.1.1.11 --- Reagents for DNA sequencing --- p.32Chapter 2.1.2 --- Methods --- p.32Chapter 2.1.2.1 --- Isolation of chromosomal DNA --- p.32Chapter 2.1.2.2 --- Preparation of primers --- p.33Chapter 2.1.2.3 --- Amplification of parental glucose isomerase gene --- p.33Chapter 2.1.2.4 --- Agarose gel electrophoresis of DNA --- p.35Chapter 2.1.2.5 --- DNA recovery from agarose gel --- p.35Chapter 2.1.2.6 --- Ligation of purified DNA fragment into vector --- p.36Chapter 2.1.2.7 --- Making competent cells --- p.37Chapter 2.1.2.8 --- TransformationChapter 2.1.2.9 --- Plasmid DNA preparation --- p.38Chapter 2.1.2.10 --- DNA sequencing --- p.39Chapter 2.2 --- Mutagenesis of glucose isomerase --- p.40Chapter 2.2.1 --- Materials --- p.40Chapter 2.2.2 --- Methods --- p.40Chapter 2.2.2.1 --- Preparation of primers --- p.40Chapter 2.2.2.2 --- Introduction of point mutations --- p.42Chapter 2.2.2.3 --- Assembly of DNA fragments --- p.44Chapter 2.2.2.4 --- Amplification of full-length genes --- p.45Chapter 2.2.2.5 --- Agarose gel electrophoresis of DNA --- p.46Chapter 2.2.2.6 --- DNA recovery from agarose gel --- p.46Chapter 2.2.2.7 --- Ligation of purified DNA fragment into vector --- p.46Chapter 2.2.2.8 --- Transformation --- p.46Chapter 2.2.2.9 --- Plasmid DNA preparation --- p.46Chapter 2.2.2.10 --- DNA sequencing --- p.46Chapter 2.3 --- Expression and purification of glucose isomerase --- p.47Chapter 2.3.1 --- Materials --- p.47Chapter 2.3.1.1 --- Phosphate buffer preparation --- p.47Chapter 2.3.1.2 --- Reagents for SDS-PAGE --- p.48Chapter 2.3.2 --- Methods --- p.48Chapter 2.3.2.1 --- Incubation of bacteria --- p.48Chapter 2.3.2.2 --- Extraction of crude protein --- p.49Chapter 2.3.2.3 --- Partial purification of glucose isomerase --- p.49Chapter 2.3.2.4 --- Further purification of glucose isomerase --- p.50Chapter 2.3.2.5 --- SDS-PAGE --- p.51Chapter 2.4 --- Enzyme assays --- p.52Chapter 2.4.1 --- Materials --- p.52Chapter 2.4.1.1 --- Substrate for activity assay --- p.52Chapter 2.4.1.2 --- Buffer and bivalent metal cations --- p.52Chapter 2.4.1.3 --- Reagents for protein concentration determination --- p.53Chapter 2.4.1.4 --- Reagents for activity determination --- p.54Chapter 2.4.2 --- Methods --- p.54Chapter 2.4.2.1 --- Protein concentration determination --- p.54Chapter 2.4.2.2 --- Specific activity assay --- p.55Chapter 2.4.2.3 --- Thermostability assay --- p.57Chapter 2.4.2.4 --- Temperature curve of activity --- p.57Chapter 2.4.2.5 --- pH effects --- p.57Chapter 2.4.2.6 --- pH stability assay --- p.58Chapter 2.4.2.7 --- Bivalent metal cations --- p.58Chapter 2.4.2.8 --- Conversion rate of isomerization --- p.59Chapter Chapter 3 --- Results --- p.61Chapter 3.1 --- Cloning of parental glucose isomerase gene --- p.62Chapter 3.2 --- Mutagenesis of glucose isomerase --- p.64Chapter 3.3 --- Expression and purification of glucose isomerase --- p.65Chapter 3.4 --- Enzyme assays of glucose isomerase --- p.70Chapter 3.4.1 --- Specific activity --- p.70Chapter 3.4.2 --- Thermostability --- p.72Chapter 3.4.3 --- Activity at different temperatures --- p.76Chapter 3.4.4 --- pH effects --- p.77Chapter 3.4.5 --- pH stability --- p.78Chapter 3.4.6 --- Bivalent metal cations --- p.79Chapter 3.4.7 --- Conversion rate of isomerization --- p.84Chapter Chapter 4 --- Discussions --- p.87Chapter 4.1 --- Different glucose isomerase mutants --- p.88Chapter 4.2 --- Enzymatic physicochemical and catalytic properties --- p.94Chapter 4.3 --- Future work --- p.95References --- p.9