文献类型：期刊文献

英文题名：Protein engineering of thioether monooxygenase to improve its thermostability for enzymatic synthesis of chiral sulfoxide

作者：Zhao, Peng[1];Ren, Shi-Miao[1];Liu, Feng[1];Zheng, Yu-Cong[1];Xu, Na[1];Pan, Jiang[1];Yu, Hui-Lei[1];Xu, Jian-He[1]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, 130 Meilong Rd, Shanghai 200237, Peoples R China

年份：2021

卷号：509

外文期刊名：MOLECULAR CATALYSIS

收录：;EI(收录号：20212110401325);WOS:【SCI-EXPANDED(收录号:WOS:000670356600009)】；

基金：This work was financially supported by The National Key Research and Development Program of China (2019YFA09005000), The National Natural Science Foundation of China (21922804 & 21871085 & 21776085), and the Fundamental Research Funds for the Central Universities (22221818014).

语种：英文

外文关键词：Monooxygenase; Esomeprazole; Directed evolution; Thermostability; Enzymatic asymmetric oxidation

摘要：Esomeprazole, the S-enantiomer of omeprazole, is the best-selling proton pump inhibitor. In our previous work, a mutant of cyclohexanone monooxygenase from the Acinetobacter calcoaceticus (named AcCHMO-M6) was successfully obtained through protein engineering which could catalyze the oxidation of omeprazole sulfide. However, its practical application is still hindered by the poor thermostability, especially in the up-scaled reaction process. In this work, site mutagenesis based on consensus analysis and directed evolution were used to engineer this enzyme in order to improve the stability of AcCHMO-M6. The half-lives of the resultant mutants AcCHMO-M9 (F29L/R444E) and AcCHMO-M10 (F29L/R444E/A145S/G430T) at 40 degrees C were increased from 2.2 h to 8.5 h and 5.9 h respectively, while the corresponding T-m values were increased by 7 degrees C and 5.3 degrees C in comparison to AcCHMO-M6. The specific activity of AcCHMO-M9 was comparable to that of AcCHMO-M6, and the specific activity of AcCHMO-M10 was about 4-fold that of AcCHMO-M6. The AcCHMO-M10 catalyzed sulfide oxidation reaction reached 100% conversion after 16 hours at 30 degrees C, in contrast to 39.4% conversion in the case of AcCHMO-M6. These results show that the potential of this thioether monooxygenase can be significantly improved by protein engineering.