Characterization of a β-Galactosidase from Kosakonia oryzendophytica and Its Heterologous Expression in Bacillus subtilis for Galactooligosaccharides Production

Galactooligosaccharides (GOS) typically consist of 2-8 D-galactose units linked together, terminating in a D-glucose unit. GOS are commonly used in dairy products, infant formulas, and functional foods. GOS offer beneficial properties for food processing, such as low caloric value, mild clean taste, and excellent solubility in water. Additionally, GOS function as non-digestible prebiotics, supporting microbiota balance and offering benefits such as promoting infant health, immune modulation, laxative effects, and potential metabolic advantages. β-galactosidase plays a key role in GOS production, catalyzing both hydrolysis and transglycosylation reactions. In this study, a putative GH2 family β-galactosidase from <i>Kosakonia oryzendophytica</i> (Koor β-gal) was identified. The enzyme exhibited optimal activity at pH 7.0 and 45-50 °C with the addition of 1 mM Mg²⁺, showing a specific activity of approximately 288.6 U/mg towards <i>o</i>-nitrophenyl-β-D-galactopyranoside (ONPG). After optimizing the reaction conditions, Koor β-gal successfully produced 124.7 g/L of GOS from 300 g/L D-lactose, achieving a GOS yield of 41.6%. LC-MS analysis revealed that the primary products consisted of GOS with degrees of polymerization (DP) ranging from 2 to 4. Additionally, Koor β-gal was heterologously expressed in <i>Bacillus subtilis</i> following comprehensive optimization of the promoter and 5'-UTR, resulting in an enzyme activity in culture filtrate of 106.2 U/mL after 60 h.