Selective Synthesis of Galactooligosaccharides Containing β(1→3) Linkages with β‑Galactosidase from Bifidobacterium bifidum (Saphera)

The transglycosylation activity of a novel commercial β-galactosidase from Bifidobacterium bifidum (Saphera) was evaluated. The optimal conditions for the operation of this enzyme, measured with o-nitrophenyl-β-d-galactopyranoside, were 40 °C and pH around 6.0. Although at low lactose concentrations the property of this enzyme was basically hydrolytic, an increase of lactose concentration to 400 g/L resulted in a significant formation (107.2 g/L, 27% yield) of prebiotic galactooligosaccharides (GOS). The maximum amount of GOS was obtained at a lactose conversion of approximately 90%, which contrasts with other β-galactosidases, for which the highest GOS yield is achieved at 40–50% lactose conversion. Using high-performance anion-exchange chromatography with pulsed amperometric detection, semipreparative high-performance liquid chromatography-hydrophilic interaction liquid chromatography, mass spectrometry, and 1D and 2D NMR, we determined the structure of most of the GOS synthesized by this enzyme. The main identified products were Gal-β(1→3)-Gal-β(1→4)-Glc (3′-O-β-galactosyl-lactose), Gal-β(1→6)-Glc (allolactose), Gal-β(1→3)-Glc (3-galactosyl-glucose), Gal-β(1→3)-Gal (3-galactobiose), and the tetrasaccharide Gal-β(1→3)-Gal-β(1→3)-Gal-β(1→4)-Glc. In general, B. bifidum β-galactosidase showed a tendency to form β(1→3) linkages followed by β(1→6) and more scarcely β(1→4).