Strengthening core-region hydrogen-bond networks and rigidifying surface loop to enhance thermostability of an (R)-selective transaminase converting chiral hydroxyl amines.

IF 4.1 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Yuwen Wei, Fulong Li, Yukun Zheng, Youxiang Liang, Yan Du, Huimin Yu
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引用次数: 0

Abstract

Transaminases have important applications in the synthesis of drug intermediates such as chiral amines. However, natural transaminases exhibit suboptimal thermal stability, limiting their further applications. Building upon an Rhodobacter sp.-derived (R)-selective transaminase (RbTA), we report a dual-region coupling engineering approach to improve thermostability of RbTA by strengthening the core hydrogen-bond networks and rigidifying the flexible surface loop. Through single strategy, we identified 4 thermostability improved single mutations, among which I249Q demonstrated the most substantial improvement, achieving a 18-fold increase in half-life (t1/240) and a 11.2 ℃ increase in T5010. Then in strategic coupling, the synergistic effect of dual-region modification was observed in both thermal stability and activity enhancement, as mutant with the best high-temperature catalytic performance, R136P/F228Y, had its T5010 improved by 7.1℃ and exhibited a 4.2-fold increase in kcat/Km towards (R)-3-amino-1-butanol. Finally, R136P/F228Y achieved a 20.5% improvement in conversion over WT in an analytical-scale synthesis in 72h at a 5 ℃ elevated catalytic temperature. Molecular dynamics simulations demonstrated that the synergy of the formation of new hydrogen bonds and decrease in flexibility accounted for the thermostability improvements. This study provides guidance for enhancing thermostability of similar fold-type enzymes without impairing enzymatic activity in an efficient manner.

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来源期刊
Journal of biotechnology
Journal of biotechnology 工程技术-生物工程与应用微生物
CiteScore
8.90
自引率
2.40%
发文量
190
审稿时长
45 days
期刊介绍: The Journal of Biotechnology has an open access mirror journal, the Journal of Biotechnology: X, sharing the same aims and scope, editorial team, submission system and rigorous peer review. The Journal provides a medium for the rapid publication of both full-length articles and short communications on novel and innovative aspects of biotechnology. The Journal will accept papers ranging from genetic or molecular biological positions to those covering biochemical, chemical or bioprocess engineering aspects as well as computer application of new software concepts, provided that in each case the material is directly relevant to biotechnological systems. Papers presenting information of a multidisciplinary nature that would not be suitable for publication in a journal devoted to a single discipline, are particularly welcome.
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