Double enzymes coupled screening assisted molecular modification of α-Amino acid ester acyltransferase for L-Alanyl-l-Glutamine biosynthesis

L-Alanyl-l-Glutamine (Ala-Gln) is a kind of functional dipeptide, which is widely used in food and health care. Ala-Gln can be synthesized from l-glutamine and l-alanine methyl ester hydrochloride by α-Amino acid ester acyltransferase (AET). Still, the low catalytic efficiency and conversion rate of AET are key limiting factors in Ala-Gln production. In this study, the mutant library of AET were built based on structural model and semi-rational design. To facilitate the screening of AET mutants, the double enzymes coupled screening method was constructed and optimized. The amino acid residues I115, D203, F211, D300, A298, and R334 were selected as candidate mutation sites by alanine scanning and virtual saturation mutation and the catalytic activity of the optimal mutation A298C increased by 23.7%. Molecular dynamics simulation and molecular docking analysis suggested that the increased flexibility of the catalytic pocket in AET (A298C) and the formation of additional hydrogen bonds with substrate contributed to the improvement of enzyme activity.