Directed Evolution Of Anti-prelog Short-chain Dehydrogenase/reductase EbSDR8 For Enhanced Acetophenone Tolerance

Chiral alcohols are one of the most important intermediates for the synthesis of chiral pharmaceuticals,agrochemicals and fine chemicals.Compared with the chemical methods,biocatalytic asymmetric reduction of prochiral ketones is an important technology for the green synthesis of chiral alcohols attribute to its mild reaction conditions,few by-products,high theoretical yields and environmentally friendliness.Nowadays,most of prochiral ketone-reducing enzymes normally follow the Prelog's rule,and the biocatalysts follow the anti-Prelog's rule are relatively scarce.The short-chain dehydrogenase/reductase EbSDR8 which following the anti-Prelog's rule is a promising biocatalyst for reduction of acetophenone to?R?-1-Phenylethanol.However,there are no research on the enzyme asymmetric reduction of high concentrations of acetophenone.Hence,in this study,we firstly designed a directed evolution means to engineering the enzyme,and obtained the positive variant which has a high activity under high concentrations of acetophenone.After that,we optimized the catalytic performance of the variant for the purpose of adapting to industrial applications.The short-chain dehydrogenase gene Ebsdr8 was successfully cloned from the Empedobacter Brevis ZJUY-1401 and the corresponding protein EbSDR8 was heterologously expressed in E.coli BL21?DE3?by means of molecular biology.The short-chain dehydrogenase EbSDR8 following the anti-Prelog's rule can asymmetric reduction acetophenone to?R?-1-Phenylethanol.Though the EbSDR8 crystal structure is not available,error-prone PCR using the EbSDR8 gene as the template has been adopted to construct the variant libraries with the template diluted 1000 times and the manganese ion concentration of 0.5 mM,containing one or two mutant sites.Then developed a chromogenic reaction-based high-throughput screening method.The resulting variant M190V showed74.8%improvement over the wild type in specific activity when catalyzing the reduction of 200 mM acetophenone.Kinetic analysis revealed a 70%enhancement in its catalytic efficiency(k_cat/k_m).Molecular docking was conducted to reveal the possible mechanism behind the improved acetophenone tolerance,and the result implied that the M190V mutation is conducive to the binding and release of coenzyme.Aside from the improved catalytic performance when dealing with high concentration of acetophenone,other features of M190V such as the optimal pH is 7.5,low optimal cosubstrate concentration?1%isopropanol?,and a temperature optimum close to that of E.coli cells?35??,also contribute to its practical application as a whole-cell catalyst.What's more,under the optimal reaction condition,the conversion of M190V reached 87.52%by 135 min.