迭代饱和突变提高卤醇脱卤酶HheA_(AM)催化制备6-氰基-3,5-二羟基己酸叔丁酯的酶活  

Enhancement of HheA_(AM) Activity via Iterative Saturation Mutagenesis for tert-Butyl 6-Cyano-3,5-Dihydroxyhexanoate Synthesis

作　　者：王露 徐刚[1] 杨立荣[1] 吴坚平[1] WANG Lu;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,浙江杭州310027

出　　处：《高校化学工程学报》2018年第5期1127-1133,共7页Journal of Chemical Engineering of Chinese Universities

基　　金：国家自然科学基金(21076187);国家"973"计划资助项目(2011CB710800)

摘　　要：(3R,5R)-6-氰基-3,5-二羟基己酸叔丁酯(A7)是合成降血脂药物阿托伐他汀钙的关键手性中间体,利用卤醇脱卤酶催化(3R,5S)-6-氯-3,5-二羟基己酸叔丁酯(D3)的脱卤氰化反应制备A7是很有潜力的合成方法。现有研究存在的主要问题是卤醇脱卤酶种类少,对底物D3的催化活性很低,且鲜见有关脱卤酶分子改造提高其对D3催化活性的报道。本研究利用基因挖掘技术获得了一个对D3表现出较高催化活力的卤醇脱卤酶HheA_(AM) (来源于Agromycesmediolanus sp.),运用定向进化和半理性设计方法确定了4个影响酶活的关键位点(84、88、136、144位),然后使用迭代饱和突变(ISM)策略对这4个位点进行组合突变,突变株M4-HheA_(AM)(84S-88L-136T-144C)的比酶活为0.89U·mg-1,是野生型HheA_(AM)(Wt-HheA_(AM))的13倍,对D3的催化活性得到了显著的提高。tert-butyl（3 R,5 R）-6-cyano-3,5-dihydroxyhexanoate（A7） is a key chiral synthon of the cholesterollowering drug atorvastatin. Application of halohydrin dehalogenases for dehalogenation and cyanogenation of tert-butyl（3 R,5 S）-6-chloro-3,5-dihydroxyhexanoate（D3） to prepare A7 is a promising approach. However, the main problems are lack of halohydrin dehalogenases, low catalytic activity and not enough research on improving catalytic activity towards D3. In this study, a halohydrin dehalogenase from Agromyces mediolanus sp. was obtained by gene mining, which showed relatively high activity to D3. Four key sites affecting enzyme activity are found（site 84, 88, 136 and 144） using directed evolution and semi-rational design methods. M4-HheAAM（84 S-88 L-136 T-144 C） is obtained（specific activity of M4-HheAAM is 0.89 U·mg^-1） with 13-fold activity enhancement to wild type（Wt-HheAAM） after applying iterative saturation mutagenesis at these four sites.

关 键 词：卤醇脱卤酶 脱卤氰化反应 酶活 迭代饱和突变 (3R 5R)-6-氰基-3 5-二羟基己酸叔丁酯 

分 类 号：TQ465.92[化学工程—制药化工]