Molecular modification based on site-directed mutagenesis improves the substrate specificity of β-ester acyltransferase for L-carnosine synthesis

Abstract

L-carnosine is a natural β-peptide compound with a wide range of important physiological functions. In this study, β-ester acyltransferase (As DmpA) can catalyze the reaction of β-amino acid methyl ester and β-amino acid methyl ester to form β-peptides and can also catalyze the reaction of β-amino acid methyl ester and L-histidine to form L-carnosine. Therefore, the reaction system will produce a variety of β-peptides, including L-carnosine. To solve this problem, the substrate specificity of As DmpA was improved by molecular modification to further increase the catalytic synthesis yield of L-carnosine. It was found that L341 W, L341K and L341R in saturated mutations increased the ability to catalyze L-carnosine synthesis. It was also found that the high proportion of L-histidine in the reaction system was conducive to the synthesis of L-carnosine. When the concentration ratio of L-histidine to β-alanine methyl ester hydrochloride is 140:10, the conversion rate of L-carnosine can reach more than 90%. When the concentrations of L-histidine and β-alanine methyl ester hydrochloride were 140 mM and 210 mM, respectively, the concentration of L-carnosine reached 105 mM by adding β-alanine methyl ester hydrochloride in batches, which increased the concentration of L-carnosine by 3.28 times compared with the nonbatch feeding. In conclusion, the ability of As DmpA to catalyze L-carnosine synthesis was improved by molecular modification technology and batch feeding methods, which provides an excellent method for the production of L-carnosine.