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

A β-galactosidase gene (β-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. β-Gal II had a native molecular mass of 235 kDa and the subunits had a molecular mass of 81 kDa, indicating that β-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°C, using p-nitrophenyl-β-D-galactopyranoside as a substrate. The K(m) and V(max) for Gal(β1-4)Gal were 60 mM and 1,129 U/mg, respectively. The recombinant β-Gal II was highly active towards Gal(β1-4)Gal and Gal(β1-4)Gal-containing oligosaccharides; only low activity was observed towards Gal(β1-3)Gal, lactose, and Gal(β1-3)GalOMe. No activity was found towards Gal(β1-6)Gal, Gal(β1-4)Man, Gal(β1-4)Gal, Gal(β1-3)Gal(β1-4)Gal, cellobiose, maltose and sucrose. β-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(β1-4)](2)Gal peak area ratio was 9:1.