Recombinant beta-galactosidase from Lactobacillus plantarum WCFS1, homologously over-expressed in L. plantarum, was purified to apparent homogeneity using p-aminobenzyl 1-thio-beta-d-galactopyranoside affinity chromatography and subsequently characterized. The enzyme is a heterodimer of the LacLM-family type, consisting of a small subunit of 35kDa and a large subunit of 72kDa. The optimum pH for hydrolysis of its preferred substrates o-nitrophenyl-beta-d-galactopyranoside (oNPG) and lactose is 7.5 and 7.0, and optimum temperature for these reactions is 55 and 60 degrees C, respectively. The enzyme is most stable in the pH range of 6.5-8.0. The K(m), k(cat) and k(cat)/K(m) values for oNPG and lactose are 0.9mM, 92s(-1), 130mM(-1)s(-1) and 29mM, 98s(-1), 3.3mM(-1)s(-1), respectively. The L. plantarum beta-galactosidase possesses a high transgalactosylation activity and was used for the synthesis of prebiotic galacto-oligosaccharides (GOS). The resulting GOS mixture was analyzed in detail, and major components were identified by using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as well as capillary electrophoresis. The maximal GOS yield was 41% (w/w) of total sugars at 85% lactose conversion (600mM initial lactose concentration). The enzyme showed a strong preference for the formation of beta-(1-->6) linkages in its transgalactosylation mode, while beta-(1-->3)-linked products were formed to a lesser extent, comprising approximately 80% and 9%, respectively, of the newly formed glycosidic linkages in the oligosaccharide mixture at maximum GOS formation. The main individual products formed were beta-d-Galp-(1-->6)-d-Lac, accounting for 34% of total GOS, and beta-d-Galp-(1-->6)-d-Glc, making up 29% of total GOS. Copyright 2010 Elsevier Ltd. All rights reserved.