Synthesis Of Key Chiral Intermediates Of ACEI Catalyzed By Aspartate Transaminase

The unnatural amino acids 3,4-Dimethoxy-L-phenylalanine(3,4-Dimethoxy-L-phenylalanine)and L-Homophenylalanine(L-HPA)are synthetically synthesized Key intermediates for drugs.At present,a large amount of metal catalysts,toxic reactants,cumbersome reaction steps,and high costs are required for the chemical route to prepare 3,4-dimethoxy-L-phenylalanine and L-homophenylalanine.In addition,the enzymatic synthesis process requires the addition of coenzymes such as NADH or the need to use two enzymes for the cascade catalytic reaction,the process is more complex,the enzyme catalytic efficiency is insufficient,the catalytic substrate concentration is low.This experiment focused on how to produce 3,4-dimethoxy-L-phenylalanine and L-homophenylalanine,which are important intermediates for the economical,efficient and green preparation of a series of keto-acid substrates.The method of error-prone PCR was used to modify the wild-type AspAT to obtain mutants 10b,75c,261b,478c,369c with significantly improved 3,4-dimethoxyphenylpyruvate conversion activity to the substrate.The keto acid substrates were catalyzed by 5 mutants obtained above as the result indicated that the catalytic activity of the mutants on other keto acid substrates was generally higher than that of wild-type AspAT.The mutant 75c with the most significant increase in activity was selected and 3,4-dimethoxyphenylpyruvate was used as a substrate to further investigate its related enzymatic properties.The results showed that compared to the wild-type AspAT,the affinity of the mutant 75c to the substrate and the catalytic efficiency were significantly improved.After comparison of the amino acid sequences of the mutants,it was concluded that the arginine at position 316 was important to the Enzyme activity.It is presumed that the aromatic residues can promote the ?-? interaction between the enzyme and the substrate to improve the conversion of the enzyme.active.At the same time,the pre-column derivatization method was used to analyze the optical purity of the enzyme-catalyzed product,and it was found that the mutation site in the mutant 75c did not affect the product configuration.The ee%of all phenylalanine derivatives was>99%.Similarly,the wild-type AspAT was directed to evolve using error-prone PCR method,and the mutant library was screened by using 2-oxo-4-phenylbutyric acid as a substrate to obtain a mutant 22 with 1.73 times increase in catalytic activity.Kinetic studies showed that the Km value of the mutant 22 decreased by 10.2 times,the kcat increased 2.4 times,and the kcat/Km increased by 24 times,indicating that the affinity of the mutant 22 to the substrate and the catalytic efficiency were significantly improved,and the The mutation site which mutant 22 had did not affect the chiral center of the product and the product ee%value was>99%.The whole-cell catalytic reaction conditions of the mutants 75c and 22 were optimized,and pilot scale-up experiments were further performed.The key intermediates L-3,4-dimethoxyphenylalanine and L-homophenylalanine were success-fully prepared.The enzymatic-chemical coupling process for synthesizing the important intermediates of moxipril and quinapril was successfully established.