中文摘要：

乙醛脱氢酶和乙醇脱氢酶是嗜热厌氧乙醇菌Ther moanaerobacter 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．ethanolicus JW200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1059bp，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pH8．4，最适温度70℃；乙醇脱氢酶活性正、逆反应分别最适pH8．0，最适T55℃；最适pH8．9，最适为T60℃。以乙酰辅酶A（Aceyl—CoA）为底物，该酶的Km为3．32mmol／L，Vmax为12．5μmol／（min·mg）。T．ethanolicus JW200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。

英文摘要：

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.