Secretion of β-Galactosidase in Lactic Acid Bacteria Mediated by Enzyme-Active Reconstitution and Its Potential for GOS Formation During Milk Fermentation

This work constructed a recombinant lactic acid bacterium secreting β-galactosidase for GOS formation during milk fermentation. First, GalINF, a β-galactosidase derived from infant feces, was characterized to effectively produce GOS in milk, reaching a content of 10.03 g/L. To export GalINF in Lactococcus lactis, six signal peptide candidates were employed, resulting in extracellular activities as low as 52.83-85.65 U/L. Then, GalINF (114.6 kDa) was split into two complementary modules, M₁-P₇₂₃ and A₇₂₄-I₁₀₂₃, which could be independently secreted and actively reconstituted with the help of the protein scaffold SpyCatcher/SpyTag. The resultant Lc. lactis B1RG exhibited an extracellular β-galactosidase activity of 544.22 U/L. Fermentation of pasteurized milk with Lc. lactis B1RG and the traditional yogurt starters reduced lactose to 19.67 g/L and yielded 7.17 g/L GOS. This work established an effective strategy to export large-sized proteins extracellularly and demonstrated the applicability of LAB secreting β-galactosidase for GOS-enriched fermented dairy products.