丁酸梭杆菌VPI 3266甘油脱水酶和1,3-丙二醇氧化还原酶的克隆、表达

对丁酸梭杆菌VPI 3266的1,3-丙二醇代谢途径关键酶甘油脱水酶(DhaB)与1,3-丙二醇氧化还原酶(DhaT)进行了研究。甘油脱水酶基因dhaB全长3315bp,编码两个蛋白亚基,分别为甘油脱水酶的核心酶和脱水酶的再激活酶。前者全长2367bp,编码788个氨基酸,后者全长918bp,编码305个氨基酸。通过构建重组质粒,使其在大肠杆菌中实现了活性表达。1,3-丙二醇氧化还原酶基因dhaT全长1166bp,编码388个氨基酸,属于NADP依赖的离子激活的醇脱氢酶家族III。通过IPTG诱导,在大肠杆菌中实现了高效表达,酶活达到5.2U/mL。并通过Ni亲和柱获得了电泳纯的蛋白。蛋白相对分子质量为4.19×10⁴。该酶的最适反应温度为50℃,最适反应pH值为10.0,在pH值8.5~10.0范围内比较稳定,在45℃保温2h,酶活还残存50%。该酶以1,3-丙二醇为底物,在生理条件下的V_max和K_m分别为29.2U/mg和19.8mmol/L。

Cloning and expression of glycerol dehydratase and 1,3-propanediol dehydrogenase fromClostridium butyricum VPI3266

Glycerol dehydratase and 1, 3-propanediol dehydrogenase are the key enzymes for 1, 3-propanediol metabolism in Clostridium butyricum VPI3266. The gene dhaB consisted of a 3315bp fragment encoding two proteins subunits, glycerol dehydratase and its activator protein, respectively. The former consisted of 2367bp encoding 788 amino acids, which belonged to family gly-Radical. The latter consisted of 918bp encoding 305 amino acids, which belonged to family Radical-SAM. The activity of glycerol dehydratase was determined by expressing dhaB in E. coli. 1, 3-PD dehydrogenase gene dhaT consisted of 1166bp encoding 388 amino acids with calculated molecular mass of 4.19×10⁴. The activity of recombinant β-glucosidase was 5.2U/mL in LB medium by IPTG induction. The optimal activity was achieved at pH=10.0 and 50℃. The purified enzyme was stable over pH range of 8.5-10.0, and had a 2h half-life at 45℃. The V_max and K_m for 1, 3-propanediol was 29.2U/mg and 19.8mmol/L, respectively.