科研之友
微信
新浪微博
Facebook
分享链接
摘要
Carbonyl reductases (ketoreductases) can catalyze the asymmetric reduction of prochiral ketones to the corresponding chiral alcohols, which are widely used in the synthesis of pharmaceutical intermediates, agricultural chemicals, liquid crystal materials, and other complicated compounds. ChKRED03, a carbonyl reductase from our laboratory, can catalyze a broad spectrum of ketones, such as 1-[3,5-bis(trifluoromethyl) phenyl] ethanone, acrylophenone derivatives, and N-Boc-piperidin-3-one (NBPO), with excellent stereoselectivity and good activity. However, exploration of further ChKRED03 applications is challenging owing to the limitation of low thermostability. To overcome this limitation, we used protein engineering to enhance its thermostability. Herein, the FireProt web server and consensus approach were used to predict potential thermostabilizing amino acid substitutions. Eight mutation sites were objectively selected to generate single-point mutants. Four beneficial substitutions were experimentally identified and were combined to form a quadruple mutant M8314 (T183V/N188L/A211P/S224P) with substantially enhanced thermostability. The mutant M8314 displayed an optimal temperature of 40 ℃, which is 10 ℃ higher than that of the wild type, and a half-life of inactivation of 52 h at 40 ℃, which is approximately 200-fold higher than that of the wild type. Moreover, its catalytic activity toward the substrate NBPO was increased to 116% of that of the wild type. This study demonstrates a significant improvement in the thermostability of carbonyl reductase ChKRED03 to increase its potential utility in industrial applications. Furthermore, the efficient molecular evolution strategy described here could also be adapted to enhance the thermostability of other enzymes.
-
单位中国科学院; 中国科学院大学
