초록
<P>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 of enzymes to 2-propanol and the adverse effects of the by-product, acetone, limit its applications, causing this strategy to lapse. Herein, a novel ADH gene <I>smadh</I>2 was identified from <I>Stenotrophomonas maltophilia</I> by traditional genome mining technology. The gene was cloned into <I>Escherichia coli</I> cells and then expressed to yield <I>Sm</I>ADH2. <I>Sm</I>ADH2 has a broad substrate spectrum and 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 the by-product 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<SUP>−1</SUP>) without adding external coenzymes. Among these, about 780 g L<SUP>−1</SUP> (6 M) ethyl acetoacetate is completely converted into ethyl (<I>R</I>)-3-hydroxybutyrate in only 2.5 h with 99.9% ee and 7488 g L<SUP>−1</SUP> d<SUP>−1</SUP> space-time yield. Molecular dynamics simulation results shed light on the high catalytic activity toward the substrate. Therefore, the high 2-propanol tolerance <I>Sm</I>ADH2 with the TBCR system proves to be a potent biocatalytic strategy for the synthesis of chiral alcohols on an industrial scale.</P>