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

Glucose isomerase (GI) catalyzes the conversion of D-glucose to D-fructose in vitro. It is one of the bulkiest commercial enzymes, essential for the mass production of high-fructose corn syrup (HFCS) and crystalline fructose. / In this study, the effects of nitrogen sources, carbon sources, expression vectors, host strains, bacterial (Vitreoscilla) hemoglobin, selective pressure, plasmid stability and fermentation process on the GI production were investigated. The results showed that E. coli could express cloned thermostable GI at high expression level. E. coli transformed with the recombinant plasmid P-lac-GI gave the best result in term of total GI production and expression level. Corn steep liquor could be used as a cheap alternative nitrogen source for what was in LB medium. The concentration of glucose affected the expression level of GI significantly. Replacement of the ampicillin resistance gene by kanamycin resistance gene improved the plasmid stability leading to high productivity of GI in fed-batch fermentation. A suicide system could further improve the plasmid stability resulting in a high productivity of GI. A feeding strategy for fed-batch fermentation with the optimized parameters was developed to result in the production of up to 3g/L recombinant GI, which constituted 50% of the total soluble proteins. The total yield was 5-fold higher than that from flask experiments and 7-fold higher than the highest ever recorded. The expression level was also 100% higher than it was in other reports. / Liu Zhaoming. / "August 2005." / Advisers: J. Wang; W. P. Fong. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3780. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 129-154). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Glucose isomerase (GI) catalyzes the isomerization of glucose to fructose and consequently is one of the bulkiest industrial enzyme for the manufacture of high fructose corn syrup and crystalline fructose. The GI is used in industry mainly in the form of immobilized enzyme. / In this work, the immobilization of GI had been studied by several methods: ion exchange adsorption, covalent binding, alginate cells entrapment and cells cross-linking. Three kinds of carrier support (ion exchange resin, epoxy resin and amino resin) have been used in the immobilization of cells-free enzyme; the whole cells immobilization of GI by cross-linking agents polyethyleneimid and glutaraldehyde were critically examined. The results show that the cells cross-linking is the best method to prepare the immobilized GI products, as it is high in specific activity and thermostability, and low the cost. The method is likely to make significant contribution to the field of immobilization, its application has expanding rapidly in many walks of the society, including environment protection, food and pharmaceutical industries. / Jin, Caike. / "August 2005." / Adviser: Jun Wang. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3521. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 125-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Les aldose-cétose isomérases sont des enzymes catalysant l’isomérisation réversible entre un aldose et un cétose. Nous avons étudiés trois d’entre-elles : les phosphoribose isomérases (RPI), les phosphomannose isomérases (PMI) et les phosphoglucose isomérases (PGI). Ces enzymes interviennent dans différentes voies métaboliques comme la glycolyse, la néoglucogenèse, la voie des pentoses phosphates ou le métabolisme du mannose. Il a été montré qu’elles jouent un rôle important pour assurer la survie et le développement de plusieurs parasites responsables de maladies comme la leishmaniose, la mucoviscidose, la tuberculose, le paludisme ou la maladie du sommeil. Ces enzymes sont donc des cibles thérapeutiques potentielles. Ainsi, les puissants inhibiteurs de ces enzymes peuvent donc être des agents thérapeutiques efficaces pour combattre ces maladies. Les réactions catalysées par ces enzymes impliquent des intermédiaires de haute énergie (IHE) de type 1,2-cis-ènediol(ate). La synthèse d’analogues de ces intermédiaires a permis d’obtenir au laboratoire, les meilleurs inhibiteurs connus de ces enzymes, l’acide 5-phospho-D-arabononohydroxamique (5PAH, meilleur inhibiteur des PMI et PGI) et le 5-phospho-D-ribonate (5PRA, meilleur inhibiteur des RPI). Cependant, ces inhibiteurs possèdent une fonction phosphate facilement hydrolysable en milieu physiologique. Ce qui les rend inactifs in vivo. Au cours de ce travail de thèse, des phosphoanalogues du 5PAH, du 5-phospho-D-ribose (R5P, le substrat des RPI) et du 5PRA possédant une fonction malonate, phosphonate, phosphorothiate, sulfate et sulfonate à la place de la fonction phosphate ont été obtenus par des voies de synthèse multi-étapes faisant intervenir le D-arabinose ou le D-ribose comme produit de départ. Les propriétés inhibitrices de ces composés ont ensuite été déterminées et leur stabilité en milieu physiologique évaluée. Le phosphoanalogue du 5PAH de type malonate, l’acide 5-désoxy-5-dicarboxyméthyl-D-arabinonohydroxamique (5DCAH) est un inhibiteur moyen et stable de la PMI d’Escherichia Coli. Parmi les phosphoanalogues du R5P, les composés de type sulfate et sulfonate, respectivement, le 5-sulfate-D-ribose (5SR) et 5-désoxy-5-sulfonométhyl-D-ribose (5SMR) sont de bons inhibiteurs de trois RPI (la RPI d’épinard, la RPI d’Escherichia Coli et la RPI de Micobacterium tuberculosis). Seul le composé de type sulfonate est stable en milieu physiologique. Le phosphoanalogue de type malonate, le 5-désoxy-5-dicarboxyméthyl-D-ribose (5DCR) est un inhibiteur moyen de ces trois RPI. En revanche, les phosphoanalogues de type phosphorothioate et phosphonate, respectivement, le 5-désoxy-5-phosphorothioate-D-ribose (5PTR) et 5-désoxy-5-phosphonométhyl-D-ribose (5PMR) sont de mauvais inhibiteurs. Le phosphoanalogue de type phosphonate du 5PRA, le 5-désoxy-5-phosphonométhyl-D-ribonate (5PMRA) est un bon inhibiteur de la RPI de Micobacterium tuberculosis. De plus, ce composé est stable en milieu physiologique. Il est en revanche un mauvais inhibiteur de la RPI d’épinard et d’Escherichia Coli. Ces résultats sont particulièrement prometteurs puisque le 5PMRA est à ce jour le meilleur inhibiteur stable et spécifique de la RPI de Micobacterium tuberculosis. / Aldose-ketose isomerases are enzymes which catalyze the interconversion of an aldose and a ketose. We have studied three of them: phosphoribose isomerase (RPI), phosphomannose isomerase (PMI) and phosphoglucose isomerase (PGI). These enzymes play a major role in various metabolic pathways as glycolysis, neoglucogenesis, the pentoses phosphates pathways or the mannose metabolism. It has been shown to have a crucial role for the survival and development of several microorganisms responsible for diseases as the leishmaniose, the cystic fibrosis, the tuberculosis, the malaria or the insomnia. These enzymes are thus potential therapeutic targets. Consequently, strong inhibitors of these enzymes could provide efficient therapeutic tools against these deseases. The reactions catalyzed by these enzymes involve intermediaries of high energy (IHE) of 1,2-cis-enediol(ate) type. The synthesis of analogues of these intermediaries allowed to obtain in the laboratory, the best inhibitors known for these enzymes, the acid 5-phospho-D-arabononohydroxamique (5PAH, the best inhibitor of the PMI and PGI) and the 5-phospho-D-ribonate (5PRA, the best inhibitor of the RPI). However, these inhibitors possess a phosphate group which is easily hydrolysable in physiological environment, what makes them inactive in vivo. During this work of thesis, phosphoanalogues of the 5PAH, the 5-phospho-D-ribose (R5P, the substrate of the RPI) and of the 5PRA possessing a malonate, phosphonate, phosphorothiate, sulphate and sulfonate were obtained by multi-steps synthesis bringing in D-arabinose or D-ribose as starting product. The inhibitive properties of these compounds were then determined and their stability in physiological environment evaluated. The phosphoanalogue of the 5PAH of malonate type, the acid 5-desoxy-5-dicarboxyméthyl-D-arabinonohydroxamique (5DCAH) is a modest and stable inhibitor of the PMI of Escherichia Coli. Among the phosphoanalogues of the R5P, the compounds of sulphate and sulfonate types, respectively, the 5-sulfate-D-ribose (5SR) and 5-desoxy-sulfonomethyl-D-ribose (5SMR), are good inhibitors of three RPI (the RPI of spinach, the RPI of Escherichia Coli and the RPI of Micobacterium tuberculosis). Only the compound of sulfonate type is stable in physiological environment. The phosphoanalogue of malonate type, the 5-desoxy-5-dicarboxymethyl-D-ribose (5DCR) is a modest inhibitor of this three RPI. On the other hand, the phosphoanalogues of phosphorothioate and phosphonate types, respectively, the 5-desoxy-5-phosphorothioate-D-ribose (5PTR) and the 5-desoxy-5-phosphonomethyl-D-ribose (5PMR), are bad inhibitors. The phosphoanalogue of phosphonate type of the 5PRA, the 5-desoxy-5-phosphonomethyl-D-ribonate (5PMRA), is a good inhibitor of the RPI of Micobacterium tuberculosis. Furthermore, this compound is stable in physiological environment. It is on the other hand a bad inhibitor of the RPI of spinach and Escherichia Coli. These results are particularly promising because the 5PMRA is this day the best stable and specific inhibitor of the RPI of Micobacterium tuberculosis.