beta-galactosidase from Bifidobacterium adolescentis DSM20083 prefers beta(1,4)-galactosides over lactose.
Abstract 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.
Laboratory of Food Chemistry, Wageningen University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands.
Sandra W A
SW
Hinz
Lambertus A M
LA
van den Brock
Gerrit
G
Beldman
Jean-Paul
JP
Vincken
Alphons G J
AG
Voragen
D000595:Amino Acid Sequence; D001644:Bifidobacterium; D005697:Galactosides / Q000378:metabolism*; D007785:Lactose / Q000378:metabolism*; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D005697:Galactosides; D001616:beta-Galactosidase; D007785:Lactose