Significantly enhanced specific activity of Bacillus subtilis (2,3)-butanediol dehydrogenase through computer-aided refinement of its substrate-binding pocket

(2,3)-Butanediol dehydrogenases (BDHs) are widely utilized for the stereoselective interconversion between α-hydroxy ketones and vicinal diols to produce various functional building blocks. In this study, to enhance the specific activity towards (R)-phenyl-1,2-ethanediol (1a) for 2-hydroxyacetophenone (1b), the substrate-binding pocket of a Bacillus subtilis BDH (BsBDHA) was refined through site-directed mutagenesis. Based on molecular docking simulations, 14 residues were identified and subjected to alanine scanning mutagenesis. After screening, two residues, His