Galactooligosaccharide Production Using Immobilized Aspergillus oryzae β-Galactosidase, Part I: Characterization and Influence of Reaction Conditions

The enzymatic production of prebiotic galactooligosaccharides (GOS), functional food ingredients with established health benefits, remains an active research area driven by a rising global demand for GOS. These oligosaccharides are synthesized from lactose via transgalactosylation catalyzed by β-galactosidase, accompanied by hydrolysis of both substrate and products, and the competition between these reactions critically determines the maximum achievable GOS yield. In this study, β-galactosidase from <i>Aspergillus oryzae</i> was immobilized on an anion-exchange resin (Dowex Marathon MSA) using three glutaraldehyde-based crosslinking strategies. The resulting immobilized biocatalysts were characterized and evaluated for GOS synthesis, with product yield as the principal performance indicator. The results demonstrated that the immobilized biocatalysts markedly modulated the balance between transgalactosylation and hydrolytic activities. The biocatalyst prepared by simultaneous resin activation and enzyme crosslinking provided the highest GOS yield and operational stability. This biocatalyst was subsequently used to study the effects of lactose concentration, pH, enzyme loading, and temperature. Among these, lactose concentration most strongly influenced GOS yield, whereas the other factors primarily affected the reaction rate. These findings offer practical insights into enzyme immobilization strategies for optimizing GOS production.