Biocatalytic characterization of an alcohol dehydrogenase variant deduced from Lactobacillus kefir in asymmetric hydrogen transfer

Hydrogen transfer biocatalysts to prepare optically pure alcohols are in need, especially when it comes to sterically demanding ketones, whereof the bioreduced products are either essential precursors of pharmaceutically relevant compounds or constitute APIs themselves. In this study, we report on the biocatalytic potential of an anti-Prelog (R)-specific Lactobacillus kefir ADH variant (Lk-ADH-E145F-F147L-Y190C, named Lk-ADH Prince) employed as E. coli/ADH whole-cell biocatalyst and its characterization for stereoselective reduction of prochiral carbonyl substrates. Key enzymatic reaction parameters, including the reaction medium, evaluation of cofactor-dependency, organic co-solvent tolerance, and substrate loading, were determined employing the drug pentoxifylline as a model prochiral ketone. Furthermore, to tap the substrate scope of Lk-ADH Prince in hydrogen transfer reactions, a broad range of 34 carbonylic derivatives was screened. Our data demonstrate that E. coli/Lk-ADH Prince exhibits activity toward a variety of structurally different ketones, furnishing optically active alcohol products at the high conversion of 65-99.9% and in moderate-to-high isolated yields (38-91%) with excellent anti-Prelog (R)-stereoselectivity (up to >99% ee) at substrate concentrations up to 100 mM.

Automated summary

There is a need for biocatalysts capable of transferring hydrogen to prepare optically pure alcohols, especially for sterically demanding ketones. This study describes the biocatalytic potential of an anti-Prelog (R)-specific variant of Lactobacillus kefir ADH as a whole-cell biocatalyst for the reduction of prochiral carbonyl substrates. The results show that the biocatalyst exhibits activity towards a variety of ketones, producing optically active alcohol products with high conversion and excellent stereoselectivity.