文献类型：期刊文献

英文题名：Evolution of Glucose Dehydrogenase for Cofactor Regeneration in Bioredox Processes with Denaturing Agents

作者：Qian, Wen-Zhuo[1];Ou, Ling[1];Li, Chun-Xiu[1];Pan, Jiang[1];Xu, Jian-He[1];Chen, Qi[1];Zheng, Gao-Wei[1]

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

年份：2020

卷号：21

期号：18

起止页码：2680

外文期刊名：CHEMBIOCHEM

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000537504300001)】；

基金：This work was financially supported by the National Natural Science Foundation of China (nos. 21536004, 21672063, 21776085, 21871085 and 31971380), the Natural Science Foundation of Shanghai China (no. 19ZR1472900), the National Key Research and Development Program of China (nos. 2019Y-FA09005000 and 2018YFC1706200), and the Fundamental Research Funds for the Central Universities (no. 22221818014).

语种：英文

外文关键词：biocatalysis; chemical stability; cofactor regeneration; directed evolution; glucose dehydrogenase

摘要：Glucose dehydrogenase (GDH) is a general tool for driving nicotinamide (NAD(P)H) regeneration in synthetic biochemistry. An increasing number of synthetic bioreactions are carried out in media containing high amounts of organic cosolvents or hydrophobic substrates/products, which often denature native enzymes, including those for cofactor regeneration. In this work, we attempted to improve the chemical stability of Bacillus megaterium GDH (BmGDH(M0)) in the presence of large amounts of 1-phenylethanol by directed evolution. Among the resulting mutants, BmGDH(M6) (Q252L/E170K/S100P/K166R/V72I/K137R) exhibited a 9.2-fold increase in tolerance against 10 % (v/v) 1-phenylethanol. Moreover, BmGDH(M6) was also more stable than BmGDH(M0) when exposed to hydrophobic and enzyme-inactivating compounds such as acetophenone, ethyl 2-oxo-4-phenylbutyrate, and ethyl (R)-2-hydroxy-4-phenylbutyrate. Coupled with a Candida glabrata carbonyl reductase, BmGDH(M6) was successfully used for the asymmetric reduction of deactivating ethyl 2-oxo-4-phenylbutyrate with total turnover number of 1800 for the nicotinamide cofactor, thus making it attractive for commercial application. Overall, the evolution of chemically robust GDH facilitates its wider use as a general tool for NAD(P)H regeneration in biocatalysis.