文献类型：期刊文献

英文题名：Improved o-chlorobenzoylformate bioreduction by stabilizing aldo-keto reductase YtbE with additives

作者：Xu, Yan-Peng[1];Guan, Yue Hugh[1];Yu, Hui-Lei[1];Ni, Yan[1];Ma, Bao-Di[1];Xu, Jian-He[1]

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

年份：2014

卷号：104

起止页码：108

外文期刊名：JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC

收录：;EI(收录号：20141817646053);WOS:【SCI-EXPANDED(收录号:WOS:000336779700017)】；

基金：Thanks to financial supports from National Natural Science Foundation of China (Nos. 31200050 & 21276082), Ministry of Science and Technology, P.R. China (Nos. 2011AA02A210 & 2011CB710800), and Huo Yingdong Education Foundation.

语种：英文

外文关键词：Aldo-keto reductase; Enzyme stability; Enzyme inactivation; Inactivation mechanism; Bioprocess enhancement

摘要：Asymmetric reduction of methyl o-chlorobenzoylformate (CBFM) using aldo-keto reductase YtbE is a potentially cost-effective and green technology in manufacturing methyl (R)-o-chloromandelate which is a key intermediate for synthesizing (S)-clopidogrel (a popular medicine for treating atherosclerosis). At the moment, large scale application of YtbE has been complicated by uncertain thermal and operational stabilities. Consequently, we endeavored possible enzyme inactivation mechanism, and showed that (a) unfolding of YtbE explains enzyme activity loss, and (b) YtbE dimerization has a less significant effect owing to a small quantity detected. The effects of substrate and temperature on YtbE are mostly upheld by a one-step inactivation model, whereas the effect of product by a 2-step activity reduction modality. Partially based on these new understandings, a multi-factor experimental strategy was rationalized for improving the YtbE stability. For instance, glycerol was introduced to reduce enzyme unfolding whilst dithiothreitol to suppress its dimerization. This improved substrate conversion from 62.9% to 98.7%, and from 70.5% to 96.6% at 0.1 M and 1.0 M CBFM, respectively, with YtbE half-life being increased from 46.6 min to 159 min. (C) 2014 Elsevier B.V. All rights reserved.