文献类型：期刊文献

英文题名：Enhancing the Activity of an Alcohol Dehydrogenase by Using "Aromatic Residue Scanning" at Potential Plasticity Sites

作者：Ye, Wen-Jie[1];Xie, Jing-Wen[1];Liu, Yan[1];Wang, Yi-Lin[2];Zhang, Yu-Xin[1];Yang, Xiao-Ying[1];Yang, Lin[1];Wang, Hua-Lei[1];Wei, Dong-Zhi[1]

机构：[1]East China Univ Sci & Technol, Inst Biotechnol, State Key Lab Bioreactor Engn New World, 130 Meilong Rd, Shanghai 200237, Peoples R China;[2]Georgetown Preparatory Sch, North Bethesda, MD 20852 USA

年份：2023

卷号：29

期号：25

外文期刊名：CHEMISTRY-A EUROPEAN JOURNAL

收录：;EI(收录号：20231313796958);WOS:【SCI-EXPANDED(收录号:WOS:000951699400001)】；

基金：Acknowledgments This work is supported by the grant from the National Natural Science Foundation of China (No.21776084), the National Key Research and Development Program of China (2021YFC2100300), and the Fundamental Research Funds for the Central Universities.

语种：英文

外文关键词：alcohol dehydrogenase; aromatic residue scanning; plasticity sites; protein engineering; (S)-2-chloro-1-(3; 4-difluorophenyl)ethanol

摘要：An alcohol dehydrogenase LkADH was successfully engineered to exhibit improved activity and substrate tolerance for the production of (S)-2-chloro-1-(3,4-difluorophenyl)ethanol, an important precursor of ticagrelor. Five potential hotspots were identified for enzyme mutagenesis by using natural residue abundance as an indicator to evaluate their potential plasticity. A semi-rational strategy named "aromatic residue scanning" was applied to randomly mutate these five sites simultaneously by using tyrosine, tryptophan, and phenylalanine as "exploratory residues" to introduce steric hindrance or potential pi-pi interactions. The best variant Lk-S96Y/L199W identified with 17.2-fold improvement in catalytic efficiency could completely reduce up to 600 g/L (3.1 M) 2-chloro-1-(3,4-difluorophenyl)ethenone in 12 h with >99.5 % ee, giving the highest space-time yield ever reported. This study, therefore, offers a strategy for mutating alcohol dehydrogenase to reduce aromatic substrates and provides an efficient variant for the efficient synthesis of (S)-2-chloro-1-(3,4-difluorophenyl)ethanol.