Engineering β‑Galactosidase with Enhanced Catalytic and Transglycosylation Activity for GOS Production

Lactases are important glycoside hydrolases that have widespread applications in the food and pharmaceutical industries. However, the application of lactase is limited by its poor stability and transglycosylation activity. In this study, a Kluyveromyces lactis-derived lactase was engineered to improve its hydrolytic activity and transglycosylation. L764T and M2–3(L764T/V842G) had increased catalytic performance (kcat/KM) compared to Kl-β-Gal (2.9 and 4.8-fold increases, respectively) by multistrategy engineering including sequence alignment, flexible regions modification, and model prediction. The M2–3 mutant achieved a galactooligosaccharide (GOS) yield of 47.9%, which is higher than that of the wild type (35.2%). Molecular dynamics (MD) simulations suggested that the improvement of catalytic ability can be attributed to optimized substrate binding affinity and increased rigidity of specific domains. This enhancement is further supported by reinforced intersubunit interfacial interactions and enhanced compactness of the tetrameric structure. These modifications function synergistically, rendering the mutant enzymes highly promising as efficient biocatalysts for industrial applications.