Efficient asymmetric synthesis of chiral alcohols using high 2-propanol-tolerant alcohol dehydrogenase SmADH2 via an environmental friendly TBCR system
Alcohol dehydrogenases (ADHs) together with the economical substrate-coupled cofactor regeneration system play a pivotal role in the asymmetric synthesis of chiral alcohols; however, severe challenges concerning the poor tolerance to 2-propanol of enzymes and adverse effects of the byproduct acetone, limit its applications, causing this strategy to lapse. Herein, a novel ADH gene smadh2 was identified from Stenotrophomonas maltophilia by traditional genome mining technology. The gene was cloned into Escherichia coli cells and then expressed to yield SmADH2. SmADH2 exhibits excellent tolerance and superb activity to 2-propanol even at 10.5 M (80%, v/v) concentration. Moreover, a new thermostatic bubble column reactor (TBCR) system is successfully designed to alleviate the inhibition of byproduct acetone by gas flow and continuously supplement 2-propanol. The organic waste can be simultaneously recovered for the purpose of green synthesis. In the sustainable system, structurally diverse chiral alcohols are synthesised at a high substrate loading (>150 g L−1) without adding external coenzymes. Among these, about 780 g L−1 (6 M) ethyl acetoacetate is completely converted into ethyl (R)-3-hydroxybutyrate in only 2.5 h with 99.9% ee and 7488 g L−1 d−1 space-time yield. Molecular dynamic simulation results shed light on the high catalytic activity toward the substrate. Therefore, the high 2-propanol-tolerant SmADH2 with the TBCR system proves to be a potent biocatalytic strategy for the synthesis of chiral alcohols on an industrial scale.