β-galactosidase from Bifidobacterium adolescentis DSM20083 prefers β(1,4)-galactosides over lactose

A beta-galactosidase gene (beta-Gal II) from Bifido-bacterium adolescentis DSM 20083 was cloned into a pbluescript SK (-) vector and expressed in Escherichia coli. The recombinant enzyme was purified from the cell extract by anion-exchange and size-exclusion chromatography. beta-Gal II had a native molecular mass of 235 kDa and the subunits had a molecular mass of 81 kDa, indicating that beta-Gal II occurs as a trimer. The enzyme was classified as belonging to glycosyl hydrolase family 42. The optimal pH was 6.0 and the optimal temperature was 50 degreesC, using p-nitrophenyl-beta-D-galactopyranoside as a substrate. The K-m and V-max for Gal(beta1 - 4) Gal were 60 mM and 1,129 U/mg, respectively. The recombinant beta-Gal II was highly active towards Gal( 1 - 4) Gal and Gal (beta1 - 4) Gal-containing oligosaccharides; only low activity was observed towards Gal( 1 - 3) Gal, lactose, and Gal (beta1 - 3) GalOMe. No activity was found towards Gal(beta1-6) Gal, Gal(beta1 - 4) Man, Gal(alpha1 - 4) Gal, Gal(alpha1 - 3) Gal(beta1-4) Gal, cellobiose, maltose and sucrose. beta-Gal II was inhibited at high substrate concentrations ( 100 mg/ml) and no transglycosylation activity was found. At lower substrate concentrations ( 10 mg/ml) only low transglycosylation activity was found; the Gal/[Gal(beta1 - 4)](2)Gal peak area ratio was 9: 1.