ß-Glactosidase from Bifidobacterium adolescentis DSM20083 prefers ß(1,4)-galactosides over lactose

A beta-galactosidase gene (beta-Gal II) from Bifidobacterium 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 degrees C, usingp-nitrophenyl-(beta-D-galactopyranoside as a substrate. The Km and Vmax for Gal(beta1-4)Gal were 60 mM and 1129 U/mg, respectively. The recombinant beta-Gal II was highly active towards Gal(beta1-4)Gal and Gal (beta1-4)Gal-containing oligosaccharides; only low activity was observed towards Gal(beta1-3)Gal, lactose, and Gal (beta1-3)GalOMe. No activity was found towards Gal(beta1-6)Gal, Gal(beta -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)]2Gal peak area ratio was 9:1.