Characterization Of An Anti-Prelog's Short-Chain Dehydrogenase/Reductase LcSDR And Its Application In Chiral Alcohol Synthesis

Enantiometrically pure alcohols have become gradually significant for the preparation of fine chemicals during the last two decades.Enzyme-catalyzed biosysnthesis of optically active alcohols with carbonyl reductases has drawn more and more attention due to its advantages of remarkable enantioselectivity,high atom utilization and mild reaction conditions.However,most of microorganisms and reported enzymes asymmetrically reduce ketones mainly following the Prelog's rule forming S-configuration products.Moreover the limited amount of the anti-Prelog's specific biocatalysts can not satisfy the high demand for R-alcohol.In this work,a new anti-Prelog short-chain dehydrogenase LcSDR possessing excellent activity toward acetophenone was derived from Lactobacillus composti based on the homology driven“gene mining”.It was heterologously expressed in E.coli.Cultivation condition optimization for E.coli BL21?DE3?/pET28b-lcsdr was conducted.After culvatited at 37°C for 3 h,2%inoculation size?seed age 7 h?,brothes were induced by 9.0 g?L^-1 lactose,after induced in 28°C for 10 h,it obtained a highest enzyme activity of 3511.6 U?g^-1,and the cell density of 3.82 g?DCW?L^-1.The recombinant enzyme had a molecular mass of 30 kDa.The optimal pH and temperature were 6.5 and 30°C respectively.EDTA had insignificant influence on LcSDR activity,suggesting that it is not a metalloenzyme.For acetophenone its maximal reaction rate(V_max)was 133.9?mol??mg·min?^-1,The Michaelis–Menten constant K_m of LcSDR were0.345 mmol?L^-1 for acetophenone and 0.085 mmol?L^-1 for NADPH.An LcSDR-biocatalyzed?R?-phenylethanol synthesis method with outstanding time-space yield was developed via gene mining,LcSDR was found to be active to acetophenone with strict stereoselectivity.Through detailed opertimization,an ideal acetophenone bioconversion process was established.At 50 g?L^-1 acetophenone,a?R?-phenylethanol was formed in yield 93.8%,e.e._p?99%and space-time yield of 562.8 g?L^-1?d^-1.At 100 g?L^-1 acetophenone,30°C,pH6.5 and converted for 3.5 h,a yield of 78.3%and e.e._p value over 99%and space-time yield of 536.8 g?L^-1?d^-1 was attained.With the substrate concentration increased to 150 g?L^-1 and 200 g?L^-1,the yield reduced to 63.8%and 48.4%respectively.The data indicated that LcSDR could produce?R?-phenylethanol in high concentration and enantioselectivity,which could be potentially applied in industrial production.