Semirational Engineering of α-Galactosidase for Improvement of Its Thermostability and Transglycosylation Ability

α-Galactosidase catalyzes the transglycosylation reaction for galactooligosaccharides (GOS) synthesis. However, its strong hydrolytic activity limits the yield of oligosaccharides. Here, semirational design was used to engineer α-galactosidase AglB from Lactobacillus amylolyticus L6, generating three mutants with improved transglycosylation. K480R and H203P exhibited different functional characteristics. The hydrolytic activity of the K480R mutant was significantly reduced by 90.37%, thereby increasing the maximum GOS conversion rate to 34.30%, marking a 3.32-fold increase compared to WT. In contrast, the hydrolytic activity of the H203P mutant was increased by 2.83-fold. Molecular dynamics simulations and substrate-binding pocket analysis revealed that the K480R mutation rendered the active pocket more rigid and shallower, which hindered the access of water molecules through steric hindrance. Conversely, the H203P mutation enhanced the local flexibility at the pocket entrance, thus facilitating nonspecific hydrolysis. This study shifted the functional balance of AglB from hydrolysis toward transglycosylation, providing a high-performance catalyst for GOS production.