A novel heterodimeric 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. Mg(2+) ions enhanced both activity and stability significantly. L. pentosus beta-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 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.