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Keywords:

  • Biocatalysis;
  • Food biotechnology;
  • Lactase;
  • Lactic acid bacteria;
  • Transgalactosylation

Abstract

A novel heterodimeric β-galactosidase with a molecular mass of 105 kDa was purified from crude cell extracts of the soil isolate Lactobacillus pentosus KUB-ST10-1 using ammonium sulphate fractionation followed by hydrophobic interaction and affinity chromatography. The electrophoretically homogenous enzyme has a specific activity of 97 UoNPG/mg protein. The Km, kcat and kcat/Km values for lactose and o-nitrophenyl-β-D-galactopyranoside (oNPG) were 38 mM, 20 s-1, 530 M-1·s-1 and 1.67 mM, 540 s-1, 325 000 M-1·s-1, respectively. The temperature optimum of β-galactosidase activity was 60–65°C for a 10-min assay, which is considerably higher than the values reported for other lactobacillal β-galactosidases. Mg2+ ions enhanced both activity and stability significantly. L. pentosus β-galactosidase was used for the production of prebiotic galacto-oligosaccharides (GOS) from lactose. A maximum yield of 31% GOS of total sugars was obtained at 78% lactose conversion. The enzyme showed a strong preference for the formation of β-(1[RIGHTWARDS ARROW]3) and β-(1[RIGHTWARDS ARROW]6) linkages, and the main transgalactosylation products identified were the disaccharides β-D-Galp-(1[RIGHTWARDS ARROW]6)-D-Glc, β-D-Galp-(1[RIGHTWARDS ARROW]3)-D-Glc, β-D-Galp-(1[RIGHTWARDS ARROW]6)-D-Gal, β-D-Galp-(1[RIGHTWARDS ARROW]3)-D-Gal, and the trisaccharides β-D-Galp-(1[RIGHTWARDS ARROW]3)-D-Lac, β-D-Galp-(1[RIGHTWARDS ARROW]6)-D-Lac.