Genome Mining Of Carbonyl Reductase And Its Catalytic Performance In Asymmetric Synthesis Of Chiral Alcohols

Enantiometrically pure alcohols are important and valuable chiral synthons for Pharmaceuticals, agrochemicals, flavors, food additives, liquid crystals with high value-added and other fine chemicals. Asymmetric reduction of prochiral ketones with carbonyl reductases is an effective approach to produce chiral alcohols since it can be carried out under mild reaction conditions, results in a high yield, and remarkable enantioselectivity.An NADH-dependent reductase (ScCR1) from Streptomyces coelicolor A3 (2) was discovered by genome mining for carbonyl reductases. ScCRl was overexpressed in E. coli BL21, purified to homogeneity and its catalytic properties were studied. The maximum-activity was observed at pH 6.5 and the optimum temperature was found to be 45℃. The enzyme was quite stable at 30 and 40℃but labile at higher temperatures (50℃). The enzyme displayed a remarkably higher activity upon the addition of Mg2+, indicating that metal ions might be required or beneficial for the enzyme activity. The Km value of ScCR1 for COBE (ethyl 4-chloro-3-oxobutanoate) was 0.49 mM and the corresponding Vmax was 43.7μmol min-1mg-1This enzyme catalyzed the asymmetric reduction of a broad range of prochiral ketones including aryl ketones, a-andβ-ketoesters, with high activity and excellent enantioselectivity (>99%ee) towardsβ-ketoesters. Among them, COBE was efficiently converted to ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE), an important pharmaceutical intermediate, in water/toluene (1:1, v/v) biphasic system. As much as 600 g/L (3.6 M) of COBE was asymmetrically reduced within 22 h using 2-propanol as a cosubstrate for NADH regeneration, resulting in a yield of 93%, an enantioselectivity of>99%ee, and a total turnover number (TTN) of 12,100. These results indicate the potential of ScCR1 for industrial production of valuable chiral alcohols.