嗜热厌氧乙醇杆菌组氨酸融合双活性乙醇脱氢酶的克隆、表达和酶学性质  

Cloning,Expression and Characterization of Histag Fusion Biofunctional

作　　者：蒋宇[1] 邵蔚蓝[1] 

机构地区：[1]江南大学工业生物技术教育部重点实验室,江苏无锡210046

出　　处：《食品与生物技术学报》2006年第5期15-19,共5页Journal of Food Science and Biotechnology

基　　金：国家"973"计划子项目(2004CB719600);国家自然科学基金项目(30170511)

摘　　要：乙醛脱氢酶和乙醇脱氢酶是嗜热厌氧乙醇菌Thermoanaerobacter ethanolicus乙醇代谢途径中的关键酶。根据高同源性的NADP(H)依赖型Ⅱ型乙醇脱氢酶(S-ADH)的序列设计引物,从T.ethanolicusJW200基因组中PCR扩增出编码S-ADH的基因,插入带组氨酸标签的pET-20(b),测序获得基因大小为1 059 bp,并在大肠杆菌JM109(DE3)中表达。表达产物经Ni亲和柱提纯达电泳纯。带组氨酸标签的重组NADP(H)依赖型乙醇脱氢酶具乙醇脱氢酶和乙醛脱氢酶双活性,能将乙酰辅酶A转化成乙醇.带组氨酸标签的重组酶酶学性质为乙醛脱氢酶活性最适值为pH 8.4,最适温度70℃;乙醇脱氢酶活性正、逆反应分别最适pH 8.0,最适T 55℃;最适pH8.9,最适为T 60℃。以乙酰辅酶A(Aceyl-CoA)为底物,该酶的Km为3.32 mmol/L,Vmax为12.5μmol/(min.mg)。T.ethanolicusJW200中双活性S-ADH的首次发现,建立了该菌乙醇代谢途径中从乙酰辅酶A到的乙醇的另一条通路。Thermoanaerobacter ethanolicus JW200, ethanol and carbon dioxide, production microorganism which is regulated by several key enzymes involved in ethanol formation, including aldehyde dehydrogenase and alcohol dehydrogenases. The 1059bp adhB encoding S- ADH was amplified by P＇CR by primers based on high conserved sequences, and the recombinant S-ADH was expressed in E. coli with histag. The fusion protein was purified by affinity chromatography and characterized as a bifunctional aldehyde-alcohol dehydrogenase. The temperature for the maximum initial activity in a 10-min assay of the purified histag fusion S- ADH, was observed around 70 ℃, 55 ℃ and 60 ℃, with acetyl-CoA, aldehyde and ethanol as substrate respectively. The pH optimum was 8.0 and 8. 9 with aldehyde and ethanol as substrate respectively, and 8.4 with acetyl-CoA as substrate. The histag fusion S-ADH has Km of 3.32 mM, and V of 12.5 mol/（min · mg） towards acetyl-CoA. The results suggested that a new pathway in ethanol formation in T. ethanolicus JW200.

关 键 词：嗜热厌氧乙醇菌 双活性 乙醛脱氢酶 乙醇脱氢酶 

分 类 号：Q789[生物学—分子生物学]