Glutamine (L-Gln) is a conditionally essential amino acid that plays a crucial role in various physiological processes, particularly under stress conditions such as major surgery, sepsis or trauma [1]. Clinical studies have demonstrated that intravenous glutamine supplementation in intensive care unit ICU patients improves regional vascular resilience and reduces the risk of intestinal bacterial translocation [2]. Additionally, research indicates that L-Gln can induce autophagy in intestinal epithelial cells, promoting cell survival under stress by regulating pathways like mTOR and p38 MAP kinase. This autophagic response is likely a key factor in its protective effects in the gut, potentially reducing complications in critically ill patients [3]. However, its therapeutic potential is limited by poor aqueous solubility and chemical instability. To overcome these challenges, L- alanyl-L-glutamine (Ala-Gln), a dipeptide synthesized through the dehydration-condensation of alanine and glutamine, has been developed [4]. This dipeptide rapidly undergoes enzymatic hydrolysis in vivo, releasing bioactive L-Gln to modulate intracellular pools [5], maintain cellular homeostasis [6], [7], [8], [9], [10], enhance intestinal cell viability [11], [12], [13], and support anabolic pathways, including protein and lipid biosynthesis [14], [15], [16]

Compared to conventional chemical synthesis, which employs toxic reagents and has low atom economy [17], enzymatic catalysis offers a more environmental friendly and efficient alternative for dipeptide production. As consumer demand for natural and sustainable food additives increases, enzymatic peptide synthesis is emerging as a greener option. Various enzymatic systems facilitate dipeptide biosynthesis, including cyanidin synthase [18], non-ribosomal peptide synthase (NRPS) [19], D-alanine-alanine ligase [20], L-amino acid α-lyase (Lal) [21], glutathione synthetase [22], and α-amino acid ester acyltransferases (AETs) [23]. Among these, AETs have shown superior industrial applicability characterized by rapid reaction kinetics(kcat > 2.5 s⁻¹), minimal byproduct formation(<5 % side products) and ATP-independent catalysis [23]. Despite these merits, the industrial use of AETs remains currently limited by suboptimal catalytic efficiency (Km > 300 mM) and thermal denaturation susceptibility (T½ < 40 min at 30°C). Current research predominantly focuses on fermentation parameter optimization and host strain engineering [24], [25], with limited exploration of structure-function relationships or rational protein engineering approaches. Recent research has explored novel AETs through sequence similarity network studies and phylogenetic trees. Additionally, the GRASP tool has been used for ancestral sequence reconstruction to predict variants with improved thermostability and broader substrate specificity. These efforts aim to enhance stability and catalytic efficiency of AET for broader dipeptide production applications.

In our laboratory, we successfully cloned an AET homolog, designated as EAET, from Bacillus sp. SH-1. EAET exhibits low sequence identity (<30 %) to other AET sequences deposited in the NCBI database. -Given its unresolved crystal structure and unique catalytic properties, we employed a structure-guided semi-rational design strategy to enhance its performance. Through comparative sequence analysis, molecular dynamics simulations, and automated docking, we identified potential mutational hotspots within the substrate-binding cleft of EAET. Subsequent site-saturation mutagenesis of EAET yielded thermostable variants with enhanced catalytic efficiency. To gain deeper insights into the catalytic mechanism of the engineered EAET variants, we conducted stopped-flow kinetics and hydrogen-deuterium exchange mass spectrometry analyses. These studies provided valuable information on the enzyme's catalytic process. By establishing optimal reaction parameters through this systematic engineering approach, we achieved gram-scale enzymatic synthesis of Ala-Gln with a conversion efficiency exceeding 80 %. This accomplishment highlights the potential of our strategy for the practical application of EAET in dipeptide production.