Coevolving stability and activity of LsCR by a single point mutation and constructing neat substrate bioreaction system

Abstract

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 CR mutant LsCR_M3 , coevolved 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 to 21.9 s^-1 mM^-1 . Moreover, E145A prolonged the half-life t_1/2 at 40°C from 4.1 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 Escherichia 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 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.

Keywords: biocatalysis; carbonyl reductase; coevolution; neat substrate bioreaction system; protein engineering.