Improving the Secretion Yield of the β‑Galactosidase Bgal1‑3 in Pichia pastoris for Use as a Potential Catalyst in the Production of Prebiotic-Enriched Milk

In this study, three kinds of milk were treated with the β-galactosidase Bgal1-3 (4 U/mL), resulting in 7.2–9.5 g/L galactooligosaccharides (GOS) at a lactose conversion of 90–95%. Then, Bgal1-3 was secreted from Pichia pastoris X33 under the direction of an α-factor signal peptide. After cultivation for 144 h in a flask culture with shaking, the extracellular activity of Bgal1-3 was 4.4 U/mL. Five more signal peptides (HFBI, apre, INU1A, MF4I, and W1) were employed to direct the secretion, giving rise to a more efficient signal peptide, W1 (11.2 U/mL). To further improve the secretion yield, recombinant strains harboring two copies of the <i>bgal1-3</i> gene were constructed, improving the extracellular activity to 22.6 U/mL (about 440 mg/L). This study successfully constructed an engineered strain for the production of the β-galactosidase Bgal1-3, which is a promising catalyst in the preparation of prebiotic-enriched milk