文献类型：期刊文献

英文题名：Preparation of Structurally Diverse Chiral Alcohols by Engineering Ketoreductase CgKR1

作者：Zheng, Gao-Wei[1];Liu, Yuan-Yang[1];Chen, Qi[1];Huang, Lei[1];Yu, Hui-Lei[1];Lou, Wen-Yong[2];Li, Chun-Xiu[1];Bai, Yun-Peng[1];Li, Ai-Tao[3];Xu, Jian-He[1]

机构：[1]East China Univ Sci & Technol, Shanghai Collaborat Innovat Ctr Biomfg, State Key Lab Bioreactor Engn, 130 Meilong Rd, Shanghai 200237, Peoples R China;[2]South China Univ Technol, Sch Food Sci & Engn, Lab Appl Biocatalysis, Guangzhou 510640, Guangdong, Peoples R China;[3]Max Planck Inst Kohlenforsch, Dept Biocatalysis, Kaiser Wilhelm Pl 1, D-45470 Mulheim, Germany

年份：2017

卷号：7

期号：10

起止页码：7174

外文期刊名：ACS CATALYSIS

收录：;EI(收录号：20174504362316);WOS:【SCI-EXPANDED(收录号:WOS:000412795700088)】；

基金：This work was financially supported by the National Natural Science Foundation of China (Nos. 21472045, 21536004, and 21776085), the Fundamental Research Funds for the Central Universities (22A201514043), and Shanghai Pujiang Program (15PJ1401200).

语种：英文

外文关键词：asymmetric reduction; ketoreductase; chiral alcohol; protein engineering; substrate specificity; biocatalysis

摘要：Ketoreductases are tools for the synthesis of chiral alcohols in industry. However, the low activity of natural enzymes often restricts their use in industrial applications. On the basis of computational analysis and previous reports, two residues (F92 and F94) probably affecting the activity of ketoreductase CgKR1 were identified. By tuning these two residues, the CgKR1-F92C/F94W variant was obtained that exhibited higher activity toward all 28 structurally diverse substrates examined than the wild-type enzyme. Among them, 13 substrates have a specific activity over 50 U mg(-1) (54-775 U mg(-1)). Using CgKR1-F92C/F94W as a catalyst, five substrates at high loading (>100 g(-1) L-1) were reduced completely in gram scale preparative reactions. This approach provides accesses to pharmaceutically relevant chiral alcohols with high enantioselectivity (up to 99.0% ee) and high space-time yield (up to 583 g(-1) L-1 day(-1)). Molecular dynamics simulations highlighted the crucial role of residues 92 and 94 in activity improvement. Our findings provide useful guidance for engineering other ketoreductases, especially those possessing a similar active pocket to that in CgKR1.