定点突变提高醇脱氢酶LkTADH催化制备他汀关键手性砌块的酶活  

Enhancing the Activity of LkTADH by Site-Directed Mutagenesis to Prepare Key Chiral Block of Statins

作　　者：陈方[1] 徐刚[1] 杨立荣[1] 吴坚平[1] CHEN Fang,XU Gang,YANG Li-rong,WU Jian-ping(College of Chemical and Biological Engineering,Zhejiang University, Hangzhou 310027, China)

机构地区：[1]浙江大学化学工程与生物工程学院生物工程研究所

出　　处：《中国生物工程杂志》2018年第9期59-64,共6页China Biotechnology

基　　金：国家自然科学基金面上项目(21676240);国家973计划(2011CB710800)资助项目

摘　　要：（S）-6-氯-3-羰基-5-羟基己酸叔丁酯[（S）-CHOH]是他汀类药物合成的关键手性中间体。利用醇脱氢酶催化6-氯-3,5-二羰基己酸叔丁酯不对称合成（S）-CHOH是很有潜力的制备路线,目前存在的主要问题是醇脱氢酶催化活性较低。首先对来源于Lactobacillus kefir DSM 20587的醇脱氢酶的四点突变体LkTADH（A94T/F147L/A202L/L199H）进行回复突变,确定了关键位点147和202,并获得比酶活提高1倍的突变体MF147L-A202L。对这两个位点进行饱和突变,获得比酶活比LkTADH提高1.47倍的突变体MF147I-A202L。其比酶活为10.17U/mg,为目前文献报道最高水平。通过动力学分析和分子对接,分析了突变位点对酶活影响的机制,为后续研究奠定了良好的基础。（ S）-tert-butyl-6-chloro-5-hydroxyl-3-oxohexanoate [（ S）-CHOH] is the key chiral intermediate of statins. Asymmetric reduction of tert-butyl-6-chloro-3,5-dioxohexanoate（ CDOH） to（ S）-CHOH catalyzed by alcohol dehydrogenases is a promising method. Nevertheless,the main problems is the low catalytic activity towards CDOH. First an alcohol dehydrogenase LkTADH（ A94 T/F147 L/L199 H/A202 L） was further studied by reverse mutation and key sites（ 147,202） had been identified. MF147 L-A202 Lwas obtained,which demonstrated 1-fold improvement in specific activity over LkTADH. After applying saturation mutagenesis at these two sites,MF147 I-A202 Lwas obtained with 1. 47-fold improvement in specific activity over LkTADH. The specific activity reached 10. 17 U/mg,which is the highest level as reported. Through dynamic analysis and molecular docking,the effect of mutation sites on enzyme activity was further analyzed.

关 键 词：醇脱氢酶 (S)-6-氯-3-羰基-5-羟基己酸叔丁酯 不对称合成 分子改造 饱和突变 

分 类 号：Q819[生物学—生物工程]