Enhanced synthesis of alanyl-glutamine dipeptide via molecular modification and process optimization of α-amino acid ester acyltransferase EAET

Abstract

This study presents a significant advancement in the enzymatic synthesis of alanyl-glutamine (Ala-Gln) through the development of a high-performance α-ester acyltransferase (EAET) using molecular engineering. We successfully cloned EAET from Bacillus pumilus, which exhibits low sequence identity (<30 %) compared to other reported acyltransferases (AETs). Using AlphaFold3, we constructed a highly reliable structural model of EAET, validated by metrics such as pLDDT, ipTM, and PAE. Through semi-rational design, we introduced the F330Y mutation, which enhanced enzymatic activity by 1.5-fold and extended the half-life at 30°C by 1.3-fold. Molecular dynamics simulations revealed increased flexibility around residue 330 and additional hydrogen bonds in the F330Y mutant, both of which contribute to its improved catalytic efficiency and thermostability. Response surface methodology was employed to optimize enzyme production conditions, resulting in a maximum enzyme activity of 218.20 U/mL[91.3 U/(mL·OD600)]. Systematic optimization of catalytic parameters, including pH, temperature, substrate ratio, concentration, enzyme dosage, and reaction time, enabled an 82.59 % conversion yield at 300 mM substrate concentration. These results not only surpass previous benchmarks but also demonstrate the industrial potential of engineered EAET variants for Ala-Gln synthesis. This study highlights the successful integration of computational tools with molecular engineering to enhance enzyme performance, offering a greener and more sustainable approach for Ala-Gln production.