点突变对高加索乳杆菌醇脱氢酶酶活的影响  

The Effect of Point Mutation on the Activity of Alcohol Dehydrogenase from Lactobacillus kefir

作　　者：张伟[1] 陈祈磊[1] 朱宝泉[1] 胡又佳[1] 

机构地区：[1]上海医药工业研究院创新药物与制药工艺国家重点实验室,上海200437

出　　处：《生物技术通报》2011年第9期157-162,共6页Biotechnology Bulletin

基　　金："重大新药创制"科技重大专项(2009ZC09301-007)

摘　　要：从高加索乳杆菌基因组中克隆醇脱氢酶基因,构建重组表达菌后发现不同转化子具有不同的活性,测序结果表明在部分位点发生了点突变。结合生物信息学知识通过对醇脱氢酶结构与作用机理分析,认为在酶关键位点的突变对酶的活性影响较大,而非关键位点的突变对酶活的影响虽明显降低,但其突变的数目可能对酶活的影响呈现一定的累加效应。其中活性最高的重组菌表达了一个可将苯乙酮高选择对映还原成(S)-苯乙醇的醇脱氢酶,该研究结果为酶的定向进化研究提供了理论依据。An alcohol dehydrogenase（ADH）gene was cloned from the genome of Lactobacillus kefir DSM 20587.We found that several transformants from this ADH expression recombinants showed different alcohol dehydrogenase activity.Sequencing results confirmed several point mutations occurring in the gene.With the help of bioinformatics,the structure of alcohol dehydrogenase and its molecular mechanism were analyzed.We suggested that the mutation occurring at key motif contributed to the great loss on the enzyme activity,while those occurring at other points revealed minor but accumulative effect on the enzyme activity.The highest enzymatic transformant expressed a recombinant alcohol dehydrogenase which can reduce acetophenone to（S）-phenylethanol with high enantioselectivity.The results of this work provided some theoretical evidences for the directed evolution of enzyme.

关 键 词：高加索乳杆菌 醇脱氢酶 点突变 

分 类 号：Q93[生物学—微生物学]