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

doi:10.16085/j.issn.1000-6613.2016.01.028

化工进展 ›› 2016, Vol. 35 ›› Issue (01): 210-215.DOI: 10.16085/j.issn.1000-6613.2016.01.028

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

裴建军^1,2, 屈依然^1,2, 殷冉^1,2, 陈安娜^1,2, 赵林果^1,2

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

收稿日期:2015-05-04 修回日期:2015-05-15 出版日期:2016-01-05 发布日期:2016-01-05
通讯作者: 赵林果,博士,教授。E-mail:lg.zhao@163.com。
作者简介:裴建军(1979-),男,副教授。E-mail:peijj2000@sina.com.cn;屈依然(1993-),女,本科。
基金资助:
国家自然科学青年基金(30900008),江苏省自然科学基金(BK20131423)和江苏省优势学科建设工程项目(PAPD)。

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

PEI Jianjun^1,2, QU Yiran^1,2, YIN Ran^1,2, CHEN Anna^1,2, ZHAO Linguo^1,2

1 College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, Jiangsu, China;
2 Jiangsu Key Lab of Biomass Based Green Fuels and Chemicals, Nanjing 210037, Jiangsu, China

Received:2015-05-04 Revised:2015-05-15 Online:2016-01-05 Published:2016-01-05

摘要/Abstract

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(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。

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

Abstract: 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.

Key words: Clostridium butyricum, glycerol dehydratase, 1,3-propanediol dehydrogenase, recombinant expression

中图分类号:

Q814

裴建军, 屈依然, 殷冉, 陈安娜, 赵林果. 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[J]. 化工进展, 2016, 35(01): 210-215.

PEI Jianjun, QU Yiran, YIN Ran, CHEN Anna, ZHAO Linguo. Cloning and expression of glycerol dehydratase and 1,3-propanediol dehydrogenase from Clostridium butyricum VPI3266[J]. Chemical Industry and Engineering Progree, 2016, 35(01): 210-215.

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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

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

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