Abstract
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).
Highlights
Beta-Galactosidases (Beta-D-galactoside galactohydrolases, EC 3.2.1.23) catalyze the hydrolysis of different oligosaccharides with a galactosyl moiety located at the nonreducing end.[1,2] The dairy industry is taking advantage of this reaction to remove lactose from milk to obtain lactose-free products.[3,4] Due to the high amount of people suffering from lactose intolerance, the demand for lactose-free products is steadily increasing.[5]
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
It has been demonstrated that GOS produced by Betagalactosidases from Bifidobacterium species have a different structure and significantly higher prebiotic potential compared with commercial mixtures of GOS,[28,32] probably due to the prevalence of β(1 3) and β(1 6) linkages,[25] whilst β(1 4) and β(1 6) bonds prevail in commercial GOS.[6,33] β-galactosidases from Bifidobacterium species typically belong to families GH2 and GH42.1,25 Interestingly, GOS synthesis catalyzed by βgalactosidases from bifidobacteria is not inhibited by the presence of glucose or galactose,[34,35] which are commonly recognized as inhibitors of other Beta-galactosidases.[36]
Summary
Beta-Galactosidases (Beta-D-galactoside galactohydrolases, EC 3.2.1.23) catalyze the hydrolysis of different oligosaccharides with a galactosyl moiety located at the nonreducing end.[1,2] The dairy industry is taking advantage of this reaction to remove lactose from milk to obtain lactose-free products.[3,4] Due to the high amount of people suffering from lactose intolerance, the demand for lactose-free products is steadily increasing.[5]. Using HPAEC-PAD, semipreparative HPLC-HILIC, MS, 1D and 2D NMR, we determined the structure of most of the GOS synthesized by this enzyme.
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