β-galactosidase from Lactobacillus plantarum WCFS1: Biochemical characterization and formation of prebiotic galacto-oligosaccharides

Recombinant β-galactosidase from Lactobacillus plantarum WCFS1, homologously over-expressed in L. plantarum, was purified to apparent homogeneity using p-aminobenzyl 1-thio-β-D-galactopyranoside affinity chromatography and subsequently characterized. The enzyme is a heterodimer of the LacLMfamily type, consisting of a small subunit of 35 kDa and a large subunit of 72 kDa. The optimum pH for hydrolysis of its preferred substrates o-nitrophenyl-β-D-galactopyranoside (oNPG) and lactose is 7.5 and 7.0, and optimum temperature for these reactions is 55 and 60 °C, respectively. The enzyme is most stable in the pH range of 6.5-8.0. The Km, k cat and kcat/Km values for oNPG and lactose are 0.9 mM, 92 s-1, 130 mM-1 s-1 and 29 mM, 98 s-1, 3.3 mM-1 s-1, respectively. The L. plantarum β-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 (600 mM initial lactose concentration). The enzyme showed a strong preference for the formation of β-(1→6) linkages in its transgalactosylation mode, while β-(1→3)-linked products were formed to a lesser extent, comprising ∼80% and 9%, respectively, of the newly formed glycosidic linkages in the oligosaccharide mixture at maximum GOS formation. The main individual products formed were β-DGalp-( 1→6)-D-Lac, accounting for 34% of total GOS, and β-D-Galp-(1→6)-D-Glc, making up 29% of total GOS. © 2010 Elsevier Ltd. All rights reserved.