Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
69839 | Journal of Molecular Catalysis B: Enzymatic | 2014 | 8 Pages |
•A Bacillus strain with reduction activity toward a set of diverse α,β-unsaturated compounds was identified.•Two NADPH-dependent OYEs, Bac-OYE1 and Bac-OYE2, were cloned and characterized.•These enzymes possessed broad substrate profiles, especially for the less activated alkenes such as methyl acrylate.•Bac-OYE1 catalyzed the reduction of 2-substituted methyl acrylates such as methyl 2-acetamidoacrylate with high conversion and ee value.
By whole cell transformation, 32 out of 71 strains showed OYEs activity toward maleimide in the first round screening. Among them, a Bacillus strain was identified to be active toward a selection of substrates with different electron-withdrawing groups. Two OYE homologous genes, bac-oye1 and bac-oye2 were cloned from this strain and overexpressed in Escherichia coli BL21(DE3). The recombinant enzyme Bac-OYE2 showed a broader pH range (6.0–10.5), while Bac-OYE1 was so sensitive to pH that it lost most of the enzyme activity below pH 6.0 or above pH 9.0. The reaction temperature exerted similar effects on the activities of both enzymes, but the stability of Bac-OYE2 was more sensitive to the temperature than Bac-OYE1. In addition to α,β-unsaturated aldehydes, ketones, nitroalkenes, and the double activated carboxylic acids, esters, nitriles and cyclic imides, Bac-OYE1 and Bac-OYE2 also exhibited activities toward the “borderline” substrates such as unsaturated lactones, mono carboxylic esters, showing their broader substrate scopes. These enzymes also had excellent enantioselectivity as evidenced by the reductions of several α,β-unsaturated cyclic ketones, α-substituted α,β-unsaturated carboxylic esters and 2-methyl maleimide. For example, methyl 2-acetamidoacrylate was reduced by Bac-OYE1 with >99% conversion and >99% ee.
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