Biochemical characterization of a novel NAD+-dependent alcohol dehydrogenase BaADH9 from Bacillus amyloliquefaciens with C2-selective catalytic sites

Alcohol dehydrogenases (ADHs) are key biocatalysts for the biosynthesis of food flavor additives, but most natural ADHs lack strict substrate selectivity, limiting their industrial application. In this study, a novel NAD + -dependent alcohol dehydrogenase (BaADH9) from Bacillus amyloliquefaciens was cloned and heterologously expressed. Biochemical characterization, substrate specificity assays, and molecular docking analyses were performed to evaluate its potential for food additive synthesis. The results showed that BaADH9 exhibits optimal activity at pH 9.5 and 55 °C, with Fe 2+ and Zn 2+ significantly enhancing its enzymatic activity. Kinetic analysis revealed a K m value of 6.75 mM for isopropanol. Substrate specificity assays confirmed that BaADH9 preferentially catalyzes C2-hydroxylated secondary alcohols with negligible activity toward short-chain C1-hydroxylated primary alcohols. Molecular docking and site frequency analysis identified SER81 and ALA83 as core catalytic sites, which mediate substrate activation and proton transfer via hydrogen bond interactions. While ASP265 was characterized as a C2-substrate-specific recognition site that exclusively binds to secondary alcohols with hydroxyl groups at the C2 position. This study clarifies the molecular basis of C2-selective catalysis of BaADH9 in short-chain primary alcohols, providing a promising biocatalyst for the regio-selective synthesis of food flavor additives derived from C2-hydroxylated secondary alcohol precursors.