Semi-rational Design To Improve The Application Of Alcohol Dehydrogenase In The Synthesis Of (S)-1-(4-fluorophenyI)ethanol

As a class of chiral building blocks,chiral alcohols can participate in the synthesis of chiral drugs,pesticides,fragrances and other compounds.Compared with chemical synthesis of chiral alcohols,the asymmetric synthesis method utilizing the reductive properties of alcohol dehydrogenase has the advantages of high stereoselectivity and environmental friendliness,therefore,it is widely used in the synthesis of chiral alcohols.The target product of this project,(S)-1-(4-fluorophenyl)ethanol,is an important chiral drug intermediate,which can be used for the synthesis of various drugs such as AIDS treatment drug MLN1251 and Alzheimer’s disease treatment drugs.However,the methods reported in the literature for the synthesis of this chiral alcohol generally have the problem of poor production efficiency and the substrate concentration of the reaction is far less than 100 g/L,which is the standard for large-scale industrial applications.In order to establish a high-efficiency enzymatic synthesis process of(S)-1-(4-fluorophenyl)ethanol,this study firstly screened the alcohol dehydrogenase enzyme library constructed in the laboratory,and selected the alcohol dehydrogenase y45 with high stereoselectivity and relative activity.The catalytic performance of the alcohol dehydrogenase y45 was further improved by semi-rational design,and finally a triple-point mutant N164C/S195W/F157A with a 30.75-fold increase in the catalytic efficiency kcat/Km was obtained.In this study,we used the co-expression technology to achieve the cycle of expensive coenzymes,and finally established an enzymatic synthesis process with a substrate concentration of up to 100 g/L,and the reaction time,conversion rate and stereoselectivity all meet the needs of industrial production.The specific research work and results of this study are as follows:(1)Screening of alcohol dehydrogenase and determination of its enzymatic propertiesUsing 4-fluoroacetophenone as the substrate,the enzyme library containing more than 300 kinds of alcohol dehydrogenases constructed in the laboratory was screened.Finally,a shortchain dehydrogenase y45 from Deinococcus proteolyticus with high stereoselectivity(99.9%)and relative activity was successfully screened.The enzymatic properties showed that the optimum reaction temperature of y45 was 30℃.and the optimum reaction pH was 6.0.Under the optimum conditions,an initial attempt was made to use alcohol dehydrogenase y45 to catalyze 100 g/L 4-fluoroacetophenone,the conversion rate reached 73%in 8 h,and the conversion rate no longer increased when the reaction time was extended to 24 h.Compared with the standard of industrial large-scale preparation route,this process has certain potential for industrial application.(2)Semi-rational design of alcohol dehydrogenaseIn order to improve the catalytic performance,the alcohol dehydrogenase y45 was analyzed by combining the prediction of the site and the amino acids around the substrate.We selected 7 amino acids around the substrate from the 30 hotspots predicted by the website for site-directed mutation.Through the catalytic reaction screening of mutant strains,three sites were found that had a positive effect on the improvement of activity.A triple-point mutant N164C/S195W/F157A with a 30.75-fold increase in the catalytic efficiency kcat/Km was finally obtained by multiple rounds of iterative mutation.Using this mutant to catalyze 4fluoroacetophenone,the reaction can convert 98.5%of the substrate to(S)-1-(4fluorophenyl)ethanol in 9 h with 99.85%stereoselectivity。The substrate concentration of this process is the highest level reported in the literature for the preparation of(S)-1-(4fluoropheny l)ethanol.(3)Optimization and application of catalytic processIn order to improve the coenzyme mass transfer efficiency,the co-expression strain N164C/S195W/F157A-GDH was obtained by co-expressing the mutant N164C/S195 W/F157A and glucose dehydrogenase.Using this strain to catalyze 4-fluoroacetophenone,the time to reach the reaction equilibrium was shortened by 2 h compared with the system using two single expression strains,and the conversion rate was also improved(from 98.5%to 99.6%).The results show that the co-expression system can achieve higher coenzyme transfer efficiency and higher yield.In addition,in order to broaden the application of this catalytic process in the synthesis of chiral alcohols,the co-expression strain was used to catalyze 100 g/L N-bocpiperidone to generate(S)-N-boc-piperidine based on the results of substrate spectrum determination.The reaction can achieve complete conversion within 1 h and the stereoselectivity is greater than 99.9%.Therefore,the application of this catalytic process in the synthesis of chiral alcohols has been broadened,showing a relatively good application prospect.