Optimization Of Catalytic Conditions And Molecular Modification Of α-amino Acid Ester Acyltransferase

α-amino acid ester acyltransferase（SAET）is a dipeptide synthetase,which usesamino acid methyl ester as acyl donor and amino acid as nucleophilic substance.It can catalyze the synthesis of L-alanyl-L-glutamine（Ala-Gln）from alanine methyl ester and glutamine.Ala-Gln has excellent stability,water solubility,fast metabolism and no side effects.It is the best substitute for glutamine nutritional supplement.We have constructed a high expression engineering strain QC01 and obtained high activity SAET.On this basis,the following studies are carried out.（1）The conditions for the preparation of Ala-Gln catalyzed by SAET free enzyme were optimized.Through a series of experiments,the optimal catalytic conditions of the enzyme were obtained as follows:the p H value of the reaction system was maintained at 8.5,the reaction temperature was 27℃,the substrate ratio（methyl alanine:glutamine）was 1:0.8,the substrate concentration was 200 m M,the enzyme dosage was 10 U/ml,and the reaction time was 40 min.Under these conditions,the molar conversion of Ala-Gln was 84.22%.Although SAET free enzyme can efficiently synthesize Ala-Gln,and the conversion rate can reach the expected,but in the experimental process,the enzyme production process is more complicated,the enzyme is easy to inactivate,and the p H value of the reaction system needs to be maintained,which is not conducive to industrial production andapplication.（2）The conditions of whole cell catalytic synthesis of Ala-Gln by engineering strain QC01 were optimized.Through response surface experiments,the optimal catalytic conditions were obtained as follows:cell concentration of 3 g/L,substrate ratio of 1:0.6,reaction temperature of 30℃,substrate concentration of 300 m M,initial p H value of9.0 and reaction time of 40 min.Under these conditions,the molar conversion of Ala-Gln can reach 73.85%,which is 51.10%higher than that under the initial reaction conditions.（3）In order to improve the stability of SAET,we also studied the immobilization of SAET free enzyme and the whole cell of expression bacteria QC01.The results showed that the stability of SAET free enzyme was not good,and the immobilization effect was poor.But in the whole cell immobilization,the recovery of enzyme activity,the mechanical strength and five round reaction performance of immobilized enzyme were compared with the different embedding materials.The results showed that the agar embedding method was finally determined.The specific operation was as follows:1.5%agar was embedded 2g（wet weight）bacteria.After 10 rounds of reaction,the retention rate of enzyme activity was still 57.03%.The stability of immobilized enzyme was better than that of free enzyme and whole cell enzyme.The catalytic mechanism of SAET was studied.By means of sequence alignment,molecular modeling,molecular docking and site directed mutagenesis.It was confirmed that SAET belongs to serine hydrolase family,and its catalytic triplets are Ser¹⁵⁸,Asp²⁹⁷and His³²⁹.The mechanism of synthesis of Ala-Gln catalyzed by SAET was reasonably inferred.On this basis,a high-throughput screening method for SAET mutants was established,which laid a foundation for the molecular modification and directed evolution of SAET.