Directed evolution of a carbonyl reductase LsCR for the enantioselective synthesis of (1S)-2-chloro-1-(3,4-difluorophenyl) ethanol
Traditional screening methods of enzyme engineering often require building large mutant libraries to screen for potentially beneficial sites, which are often time-consuming and labor-intensive with low mining efficiency. In this study, a novel enzyme engineering strategy was established to modify carbonyl reductase Ls CR for the synthesis of (1 S )-2-chloro-1-(3,4-difluorophenyl) ethanol (( S )-CFPL), which is a key intermediate of anticoagulant drug ticagrelor. The strategy was developed by combining HotSpot, FireProt and multiple sequence alignment, resulting in the construction of a “small and smart” mutant library including 10 mutations. Among them, 5 mutations were positive, resulting in a 50% mining accuracy of beneficial sites. Finally, a highly active mutant Ls CR M3 (N101D/A117G/F147L) was obtained by further screening through saturation mutation and iterative mutation. Compared with wild type (WT) Ls CR, the catalytic activity of Ls CR M3 was increased by 4.7 times, the catalytic efficiency k cat / K M value was increased by 2.9 times, and the half-life t 1/2 at 40 °C was increased by 1.3 times. Due to the low aqueous solubility of the substrate 2-chloro-1-(3,4-difluorophenyl) ethanone (CFPO), isopropanol was used as not only the co-substrate but also co-solvent. In the presence of 40% ( v / v ) isopropanol, Ls CR M3 completely reduced 400 g/L CFPO to enantiomerically pure CFPL (99.9%, e.e. ) in 11 h with a space–time yield (STY) as high as 809 g/L∙d.
危险品化学品经营许可证（带存储）