| Summary: | 碩士 === 慈濟大學 === 微免暨分子醫學研究所 === 95 === UDP-glucose 4'-epimerase (EC 5.1.3.2) is an obligatory enzyme of the galactose metabolic pathway. It catalyses the reversible epimerization of UDP-glucose and UDP-galactose. The protein encoded by galE gene provides UDP-galactose needed for the synthesis of galactose-containing polysaccharides, including lipopolysacchrides (LPS) and extracellular polysacchride (EPS). Mature biofilm structures are encased in an extracellular polymeric matrix. The aim of this study is to identify the relationship between GalE and biofilm formation in the Thermus spp. By comparison of the GalE amino acid sequences from Thermus thermophilus HB8, Thermus thermophilus HB27, Thermus aquaticus NTU103, Thermus aquaticus YT-1 show high-level homology with each other. The amino acid sequences of GalE also contain three highly region conserved NAD-binding domain (GxxGxxG), Ser124, Tyr149-X-X-X-Lys153. The galE genes of four thermophilic bacteria are disrupted by homologous recombination using a thermostable kanamycin-resistant marker. We demonstrate that the galE mutants are reduced in the ability of biofilm formation about 1.5 to 2 folds.The galE gene encodes 35 kDa protein and recombinant GalE expressed in E. coli is purified by Ni2+ affinity chromatography. The recombinant protein is analysed by a coupled enzymatic activity assay to establish kinetic parameters Km and Vmax about 79.3 μM and 0.017 μmol NADH/min-1，respectively.
|
|---|
