文献类型：期刊文献

英文题名：Increased Catalyst Productivity in α-Hydroxy Acids Resolution by Esterase Mutation and Substrate Modification

作者：Ma, Bao-Di[1];Kong, Xu-Dong[1];Yu, Hui-Lei[1];Zhang, Zhi-Jun[1];Dou, Shuai[1];Xu, Yan-Peng[1];Ni, Yan[1];Xu, Jian-He[1]

机构：[1]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, Lab Biocatalysis & Synthet Biotechnol, Shanghai 200237, Peoples R China

年份：2014

卷号：4

期号：3

起止页码：1026

外文期刊名：ACS CATALYSIS

收录：;EI(收录号：20141217472223);WOS:【SCI-EXPANDED(收录号:WOS:000332756700036)】；

基金：This work was financially supported by the National Natural Science Foundation of China (No. 21276082), Ministry of Science and Technology, People's Republic of China (Nos. 2011CB710800, 2011AA02A210, and 2012AA022201), Shanghai Commission of Science and Technology (No. 11431921600), and Huo Yingdong Education Foundation.

语种：英文

外文关键词：catalyst productivity; deacylation; enzymatic resolution; alpha-hydroxy acid; product:catalyst ratio; semirational design

摘要：Optically pure alpha-hydroxy acids and their derivatives are versatile chiral building blocks in the pharmaceutical industry. In this study, the potential of a recombinant Pseudomonas putida esterase (rPPE01) for the enzymatic resolution of alpha-acetoxy acids was significantly improved by combinatorial engineering of both the biocatalyst and substrate. Semirational design based on homologous modeling and molecular docking provided a single-point variant, W187H, whose K-cat/K-M for sodium 2-acetoxy-2-(2'-chlorophenyl)acetate (Ac-CPA-Na) was increased 100-fold, from 0.0611 to 6.20 mM(-1) s(-1), while retaining its excellent enantioselectivity and broad substrate spectrum. Biocatalyst deactivation under the operating conditions was decreased by using the potassium salt of Ac-CPA instead of Ac-CPA-Na. With 0.5 g L-1 of lyophilized cells containing rPPE01-W187H, 500 mM (R,S)-Ac-CPA-K was selectively deacylated with 49.9% conversion within 15 h, giving satisfactory enantiomeric excesses (ee) for both the S product (>99% cc) and the remaining R substrate (98.7% ee). Consequently, the amount of (S)-2-hydroxy-2-(2'-chlorophenyl)acetate prepared per unit weight of lyophilized cells was improved by a factor of 18.9 compared with the original productivity of the wild-type esterase. Further enzymatic resolution of other important hydroxy acids at the 100 mL scale demonstrated that the rPPE01-W187H-based bioprocess is versatile and practical for the large-scale preparation of chiral alpha-hydroxy acids.