Multi-enzyme Pyruvate Removal System And Its Application In Asymmetric Synthesis Of Chiral Amine By ?-aminotransferase

With the increasing proportion of chiral drugs in the market,the demand for chiral amine compounds has also become larger.At present,the main methods for the preparation of chiral amines are chemical method,biological resolution method and biological asymmetric synthesis method.Chiral amines synthesized by chemical method have many shortcomings,such as the need to use expensive metal catalysts,high production costs,low enantioselectivity,and environmental pollution.The maximum theoretical conversion of chiral amine prepared by biological resolution is only 50%.Thus,chemical method and biological resolution could not meet the needs of industrial production.Biological asymmetric synthesis method becomes the preferred strategy for the production of chiral amines because of its theoretical conversion of up to 100%.Therefore,the synthesis of chiral amines by biological asymmetric synthesis has been increasing interest.However,due to the reversible reaction of asymmetric synthesis of chiral amine by ?-aminotransferase,the removal of by-product pyruvate has become the most important factor affecting conversion rate.Here,we designed a novel multi-enzyme system to promote the conversion of the amination reaction.Firstly,we constructed the ArR-?TA/TdcE/FDH/LDH multi-enzyme system,by combination of(R)-selective ?-transaminase derived from Arthrobacter sp.(ArR-?TA),formate dehydrogenase(FDH)derived from Candida boidinii,formate acetyltransferase(TdcE)and lactate dehydrogenase(LDH)derived from E.coli MG1655.This multi-enzyme system was used to remove the by-product pyruvate by TdcE and LDH to facilitate the transamination reaction.Secondly,we optimized the reaction conditions,including D-alanine,DMSO,and pyridoxal phosphate(PLP)with different concentration of 2-pentanone(as a model substrate).Thirdly,by using the ArR-?TA/TdcE/FDH/LDH system,conversions of 2-pentanone,4-phenyl-2-butanone and cyclohexanone were 84.5%,98.2% and 79.3%,respectively.Furthermore,the TdcE/FDH pathway dominates the by-product pyruvate removal in the transamination reaction.