Novel Alcohol Dehydrogenase CgADH from Candida glabrata for Stereocomplementary Reduction of Bulky–Bulky Ketones Featuring Self-Sufficient NADPH Regeneration

Bioreduction of ketones with self-sufficient cofactor regeneration is green and sustainable for the synthesis of chiral secondary alcohols. In this study, a novel alcohol dehydrogenase CgADH was identified from Candida glabrata through genome hunting, exhibiting high oxidation and reduction activities. Conserved motif analysis revealed that CgADH belongs to the extended SDR subfamily. CgADH is NADP(H) dependent and displays the highest activity at pH 5.0 and 65 °C. Substrate spectrum analysis indicated that CgADH exhibited high specificity toward 2-propanol and 2,3-butanediol. Rational engineering of CgADH was performed to modulate its stereoselectivity in the asymmetric reduction of bulky–bulky ketones. Two stereocomplementary mutants, C244A and V222G/C244N, were obtained with e.e. values of 99.6% (R) or 94.5% (S) toward (4-chlorophenyl)-pyridin-2-ylmethanone, respectively. Moreover, the catalytic efficiency (kcat/KM) of C244A and V222G/C244N increased by 70- and 25-fold compared with the wild type (WT), respectively. C244A and V222G/C244N also displayed significantly enhanced catalytic efficiency and stereoselectivity toward diaryl ketones with different substituents. Using isopropanol both as a co-substrate and as a co-solvent, a concise self-sufficient NADPH regeneration system was developed to demonstrate the advantage of CgADH. This study provides evidence of the application potential of the newly identified CgADH in the preparation of enantiopure diaryl alcohols with low byproducts and E factor.