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

Abstract

AbstractCarbonyl 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 LsCRM3, coevolved thermostability, and activity. Compared with LsCRM3, the catalytic efficiency kcat/KM of LsCRM3‐E145A (LsCRM4) was increased from 6.6 to 21.9 s−1 mM−1. Moreover, E145A prolonged the half‐life t1/2 at 40°C from 4.1 to 117 h, was increased by 5°C, was increased by 14.6°C, and Topt was increased by 15°C. Only 1 g/L of lyophilized Escherichia coli cells expressing LsCRM4 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% eeP, with a space‐time yield of 1.0 kg/L d, the substrate to catalyst ratios (S/C) of 600 g/g. Compared with LsCRM3, the substrate loading was increased by 50%, with the S/C increased by 14 times. Compared with LsCRWT, the substrate loading was increased by 6.5 times. In contrast, LsCRM4 completely converted 600 g/L CFPO within 12 h in the neat substrate bioreaction system.