定点突变提高羰基还原酶ChKRED03的热稳定性  

Enhancing the thermal stability of ketoreductase ChKRED03 using sitedirected mutagenesis

作　　者：刘艳[1] 杨玉洁 李孜一 李同彪 吴中柳[1] LIU Yan;YANG Yujie;LI Ziyi;LI Tongbiao;WU Zhongliu(CAS Key Laboratory of Environmental and Applied Microbiology&Environmental Microbiology Key Laboratory of Sichuan Province,Chengdu Institute of Biology,Chinese Academy of Sciences,Chengdu 610041,China;University of Chinese Academy of Sciences,Beijing 100049,China)

机构地区：[1]中国科学院成都生物研究所,中国科学院环境与应用微生物重点实验室,环境微生物四川省重点实验室,成都610041 [2]中国科学院大学,北京100049

出　　处：《应用与环境生物学报》2021年第6期1434-1440,共7页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金项目(21708038)资助。

摘　　要：羰基还原酶不对称还原前手性酮生成相应的手性醇,广泛应用于手性药物、农业化学品和液晶材料等结构复杂的化合物的合成.本实验室自有羰基还原酶Ch KRED03催化3,5-二(三氟甲基)苯乙酮、1-苯基-2-丙烯基-1-酮类、N-叔丁氧羰基-3-哌啶酮(NBPO)等底物具有优异的立体选择性和良好的活力,但是该酶较低的热稳定性不利于其进一步应用.因此采用蛋白质工程的方法提高其热稳定性.利用FireProt在线工具和序列一致性原理预测了该酶热稳定相关氨基酸位点,通过理性选择8个位点,构建了单点突变体并识别了4个有益突变位点,进一步将其整合得到了热稳定性大幅度提高的组合突变体M8314(T183V/N188L/A211P/S224P).该突变体最适反应温度为40℃,比野生型Ch KRED03提高了10℃,其催化底物NBPO的活性是野生型的116%. M8314在40℃的热失活半衰期为52 h,是野生型的200倍.本研究成功提高了羰基还原酶Ch KRED03的热稳定性,增加了该酶工业化应用的潜力,所采用的分子进化策略也为其他酶的热稳定性改造提供了思路.Carbonyl reductases(ketoreductases) can catalyze the asymmetric reduction of prochiral ketones to the corresponding chiral alcohols, which are widely used in the synthesis of pharmaceutical intermediates,agricultural chemicals, liquid crystal materials, and other complicated compounds. Ch KRED03, a carbonyl reductase from our laboratory, can catalyze a broad spectrum of ketones, such as 1-[3,5-bis(trifluoromethyl)phenyl] ethanone, acr ylophenone derivatives, and N-Boc-piperidin-3-one(NBPO), with excellent stereoselectivity and good activity. However, exploration of further Ch KRED03 applications is challenging owing to the limitation of low thermostability. To overcome this limitation, we used protein engineering to enhance its thermostability. Herein, the FireProt web server and consensus approach were used to predict potential thermostabilizing amino acid substitutions. Eight mutation sites were objectively selected to generate single-point mutants. Four beneficial substitutions were experimentally identified and were combined to form a quadruple mutant M8314(T183 V/N188 L/A211 P/S224 P) with substantially enhanced thermostability. The mutant M8314 displayed an optimal temperature of 40 ℃, which is 10 ℃ higher than that of the wild type, and a halflife of inactivation of 52 h at 40 ℃, which is approximately 200-fold higher than that of the wild type. Moreover,its catalytic activity toward the substrate NBPO was increased to 116% of that of the wild type. This study demonstrates a significant improvement in the thermostability of carbonyl reductase Ch KRED03 to increase its potential utility in industrial applications. Furthermore, the efficient molecular evolution strategy described here could also be adapted to enhance the thermostability of other enzymes.

关 键 词：羰基还原酶 生物催化 手性醇 热稳定性 FireProt在线服务器 一致性方法 

分 类 号：Q814.9[生物学—生物工程]