文献类型：期刊文献

英文题名：Double substituted variant of Bacillus amyloliquefaciens esterase with enhanced enantioselectivity and high activity towards 1-(3′,4′-methylenedioxyphenyl)ethyl acetate

作者：Liu, Jia-Yan[1];Bian, Han-Ping[2];Tang, Yun[2];Bai, Yun-Peng[1];Xu, Jian-He[1]

机构：[1]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]E China Univ Sci & Technol, Sch Pharm, Shanghai Key Lab New Drug Design, Shanghai 200237, Peoples R China

年份：2015

卷号：99

期号：4

起止页码：1701

外文期刊名：APPLIED MICROBIOLOGY AND BIOTECHNOLOGY

收录：;EI(收录号：20143600010788);WOS:【SCI-EXPANDED(收录号:WOS:000350028600014)】；

基金：This work was financially supported by National Natural Science Foundation of China (Nos. 31200050 & 21276082), Ministry of Science and Technology, People's Republic of China (Nos. 2011AA02A210 & 2011CB710800), and the Fundamental Research Funds for the Central Universities, Ministry of Education, People's Republic China.

语种：英文

外文关键词：Bacillus amyloliquefaciens; Esterase; Enantioselectivity; Iterative saturation mutagenesis; 1-(3 ',4 '-Methylenedioxyphenyl)ethanol

摘要：Bacillus amyloliquefaciens esterase (BAE) was applied to produce (R)-1-(3',4'-methylenedioxyphenyl) ethanol, a chiral drug intermediate. In this study, we improved the enantioselectivity of BAE by protein engineering instead of process engineering as used in our previous work. Saturation mutagenesis was carried out on eight positions of BAE based on structure modeling and substrate docking. A double substituted variant V10 (K358D/A396C) showed an excellent enantioselectivity without decreasing the activity. The functions of these two mutations (K358D and A396C) were investigated, revealing a synergic effect on the BAE enantioselectivity. Using the variant V10, enantiopure (R)-1-(3',4'-methylenedioxyphenyl) ethanol could be readily prepared in >97 % ee, affording a high space-time yield (123 g L(-1)day(-1)) and a high ratio of substrate/catalyst (40 g g(-1)) in 1-L reaction.