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 Ls CR M3 , coevolved thermostability, and activity. Compared with Ls CR M3 , the catalytic efficiency k cat / K M of Ls CR M3 ‐E145A ( Ls CR 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, was increased by 5°C, was increased by 14.6°C, and T opt was increased by 15°C. Only 1 g/L of lyophilized Escherichia coli cells expressing Ls CR 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 (1 S )‐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 Ls CR M3 , the substrate loading was increased by 50%, with the S/C increased by 14 times. Compared with Ls CR WT , the substrate loading was increased by 6.5 times. In contrast, Ls CR M4 completely converted 600 g/L CFPO within 12 h in the neat substrate bioreaction system.