Enhancing Acetophenone Tolerance of Anti-Prelog Short-Chain Dehydrogenase/Reductase EbSDR8 Using a Whole-Cell Catalyst by Directed Evolution

The short-chain dehydrogenase/reductase (SDR) from Empedobacter brevis ZJUY-1401 (EbSDR8, GenBank: ALZ42979.1) is a promising biocatalyst for the reduction of acetophenone to (R)-1-phenylethanol, but its industrial application restricted by insufficient tolerance acetophenone. In this paper, we developed chromogenic reaction-based high-throughput screening method and employed directed evolution enhance EbSDR8. resulting variant, M190V, showed 74.8% improvement over wild-type in specific activity when catalyzing 200 mM Kinetic analysis revealed 70% enhancement catalytic efficiency (kcat/Km). Molecular docking was conducted reveal possible mechanism behind improved tolerance, result implied that M190V mutation conducive binding release coenzyme. Aside performance dealing with high concentration acetophenone, other features such as broad pH range (6.0 10.5), low optimal cosubstrate (1% isopropanol), temperature optimum close E. coli cells (35 °C), also contribute practical whole-cell catalyst. study, first designed means engineer enzyme obtained positive variant which has under concentrations After that, optimized adapt applications.