β-Galactosidase from Lactobacillus pentosus: Purification, characterization and formation of galacto-oligosaccharides

A novel heterodimenc beta-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 U-oNPG/mg protein. The K-m, k(cat) and k(cat)/K-m values for lactose and o-nitrophenyl-beta-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 beta-galactosidase activity was 60-65 degrees C for a 10-min assay, which is considerably higher than the values reported for other lactobacillal beta-galactosidases Mg2- ions enhanced both activity and stability significantly L pentosus beta-galactosidase was used for the production of prebiotic galacto-oligosacchandes (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 beta-(1 -> 3) and beta-(1 -> 6) linkages, and the main transgalactosylation products identified were the disaccharides beta-D-Galp-(1 -> 6)-D-Glc, beta-D-Galp-(1 -> 3)-D-Glc, beta-D-Galp-(1 -> 6)-D-Gal, beta-D-Galp-(1 -> 3)-D-Gal, and the trisaccharides beta-D-Galp-(1 -> 3)-D-Lac, beta-D-Galp-(1 -> 6)-D-Lac