Synthesis Of Unnatural Aliphatic Amino Acids Catalyzed By Transaminase

Unnatural aliphatic amino acids are important chiral pharmaceutical intermediates.L-cyclopropylglycine is a chiral source for treating diabetes,antiviral drugs,protease inhibitors,etc.3-methylnorleucine can be used as a key intermediate for herbicide pesticides.At present,L-cyclopropylglycine and 3-methylnorleucine are mainly synthesized by chemical methods,including catalytic reduction and optical resolution,which require heavy metal catalysts such as palladium and nickel.And the product yield is low,polluting the environment.Existing biosynthesis methods mostly use NADH and other coenzyme reduction systems,but the utilization efficiency is low and the cost is high,which is not suitable for large-scale production.This thesis is based on the construction of an efficient and green biocatalytic synthesis method of non-natural aliphatic amino acid,using transaminase to prepare L-cyclopropylglycine,3-methylnorleucine and improve the conversion rate with enzyme-linked system,and directed evolution optimization.In this study,the recombinant strain of branched-chain aminotransferase and valine pyruvate aminotransferase derived from E.coli were successfully constructed.Methylcyclopropyl ketone sodium and 3-methyl-2-oxohexanoate sodium were used as substrates to detect enzymatic properties:the optimal p H catalyzed by branched-chain aminotransferase was 8.0,the temperature was 40℃,and the substrate molar ratio was1:1.2,the optimal substrate concentration is 50 m M,and the conversion rate is 23%;valine pyruvate aminotransferase:the optimal amino donor is L-alanine,the p H is 8.0,the temperature is 37℃,the substrate molar ratio is 1:1.5,the optimal substrate concentration is 75 m M,and the conversion rate reach 28%.The two have been optimized as follows:In order to comform a combined system,we added aspartate aminotransferase,L-aspartate to the system.Based on the optimal reaction conditions:50 m M methylcyclopropyl ketone sodium,60 m M L-Asp,10 m M L-Glu,the conversion rate increased from 23%to 82.3%,ee%>99%.For the optimization of valine pyruvate aminotransferase,using continuous error-prone PCR,through three rounds of error-prone PCR screening,the forward mutant V₃was obtained,and the conversion rate increased to 2.75 times of wild type,and 4mutation sites were generated:E232K,H370R,L243Q,E200A.Based on these 4 sites,four iterations of saturation mutations were carried out.After screening,the best mutant was were obtained,which contains amino acid combinations at four sites:E200M,H370R,L243T,E232K,and its conversion rate is 88%,3.14 times that of wild-type VPAT,the temperature stability of the mutant is better than the wild-type.This thesis is based on the transaminase-catalyzed synthesis of unnatural aliphatic amino acids,which provides a certain theoretical significance and guidance for the optimization of enzyme catalysis and development of synthetic platforms.