Co-evolving Stability and Activity of LsCR by A Single Point Mutation and Constructing Neat Substrate Bioreaction System.

Carbonyl reductase (CR)-catalyzed bioreduction in the organic phase and the neat substrate reaction system is a lasting challenge, placing higher requirements on the performance of enzymes. Protein engineering is an effective method to enhance the properties of enzymes for industrial applications. In the present work, a single point mutation E145A on our previously constructed carbonyl reductase mutant LsCR M3 , co-evolved thermostability, and activity. Compared with LsCR M3 , the catalytic efficiency k cat /K M of LsCR M3 -E145A (LsCR M4 ) was increased from 6.6 s -1 ∙mM -1 to 21.9 s -1 ∙mM -1 . Moreover, E145A prolonged the half-life t 1/2 at 40 °C from 4.1 h to 117 h, T m was increased by 5 °C, T 50 30 was increased by 14.6 °C, and T opt was increased by 15 °C. Only 1 g/L of lyophilized E. coli cells expressing LsCR M4 completely reduced up to 600 g/L 2-chloro-1-(3,4-difluorophenyl)ethanone (CFPO) within 13 h at 45°C, yielding the corresponding (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol ((S)-CFPL) in 99.5% ee P , with a space-time yield (STY) of 1.0 kg/L∙d, the substrate to catalyst ratios (S/C) of 600 g/g. Compared with LsCR M3 , the substrate loading was increased by 50%, with the S/C increased by 14 times. Compared with LsCR WT , the substrate loading was increased by 6.5 times. In contrast, LsCR M4 completely converted 600 g/L CFPO within 12 h in the neat substrate bioreaction system (NSBRS). This article is protected by copyright. All rights reserved.