丁酸梭杆菌VPI3266甘油脱水酶和1,3-丙二醇氧化还原酶的克隆、表达  

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

作　　者：裴建军[1,2] 屈依然 殷冉[1,2] 陈安娜[1,2] 赵林果[1,2] 

机构地区：[1]南京林业大学化学工程学院,江苏南京210037 [2]江苏省生物质绿色燃料与化学品重点实验室,江苏南京210037

出　　处：《化工进展》2016年第1期210-215,共6页Chemical Industry and Engineering Progress

基　　金：国家自然科学青年基金(30900008);江苏省自然科学基金(BK20131423);江苏省优势学科建设工程项目(PAPD)

摘　　要：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（Dha B）与1,3-丙二醇氧化还原酶（Dha T）进行了研究。甘油脱水酶基因dha B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dha T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/m L。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p H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Vmax和Km分别为29.2U/mg和19.8mmol/L。Glycerol dehydratase and 1,3-propanediol dehydrogenase are the key enzymes for 1,3-propanediol metabolism in Clostridium butyricum VPI3266. The gene dha B consisted of a 3315 bp fragment encoding two proteins subunits,glycerol dehydratase and its activator protein,respectively. The former consisted of 2367 bp encoding 788 amino acids,which belonged to family gly-Radical. The latter consisted of 918 bp encoding 305 amino acids,which belonged to family Radical-SAM. The activity of glycerol dehydratase was determined by expressing dha B in E. coli. 1,3-PD dehydrogenase gene dha T consisted of 1166 bp encoding 388 amino acids with calculated molecular mass of 4.19×104. The activity of recombinant β-glucosidase was 5.2U/m L in LB medium by IPTG induction. The optimal activity was achieved at p H=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.

关 键 词：丁酸梭杆菌 甘油脱水酶 1 3-丙二醇氧化还原酶 重组表达 

分 类 号：Q814[生物学—生物工程]