Functional Dipeptide Production by Immobilized Enzyme on Yeast Cell Surface

L-Alanyl-L-glutamine (Ala-Gln) is a high-value dipeptide with superior stability, solubility, and bioavailability, underscoring its potential for nutritional supplementation. Compared with conventional chemical catalysis, whole-cell biocatalysts offer a more efficient, simpler, and environmentally friendly alternative for peptide synthesis. Among these, enzyme cell-surface immobilization systems enable the stable display of target enzymes on yeast cells, thereby enhancing enzyme stability while simplifying catalyst recovery and reuse, which is particularly advantageous for large-scale industrial applications. In this study, an engineered <i>Saccharomyces cerevisiae</i> strain displaying α-amino acid ester acyltransferase (SsAET) from <i>Sphingobacterium siyangensis</i> SY1 on the cell surface was developed for clean and efficient biocatalytic production of Ala-Gln. Optimal reaction conditions were established (pH 8.0, 3 h, 20 g DCW/l, AlaOMe/Gln = 1:2), resulting in a 6.7-fold increase in Ala-Gln production compared with the pre-optimization conditions. Under these optimal conditions, repeated-batch reactions with an increased reaction volume of 20 ml achieved a maximum Ala-Gln concentration of 14.12 mM, representing the highest yield obtained in this study. The SsAET whole-cell biocatalyst retained more than 60% of its relative Ala-Gln production after three consecutive reaction cycles, while the conversion rate based on Gln consumption decreased only slightly from 32.08% to 29.65%, remaining essentially stable. Overall, this study demonstrates a clean, efficient, and reusable whole-cell biocatalytic system based on enzyme cell-surface immobilization for Ala-Gln production, highlighting its potential for industrial-scale peptide synthesis.