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- Title
ß-Galactosidase fromBifidobacterium adolescentisDSM20083 prefers ß(1,4)-galactosides over lactose.
- Authors
Hinz, Sandra W. A.; Van den Broek, Lambertus A. M.; Beldman, Gerrit; Vincken, Jean-Paul; Voragen, Alphons G. J.
- Abstract
A ß-galactosidase gene (ß-Gal II) fromBifidobacterium adolescentisDSM 20083 was cloned into apbluescript SK (-) vector and expressed inEscherichia coli. The recombinant enzyme was purified from the cell extract by anion-exchange and size-exclusion chromatography. ß-Gal II had a native molecular mass of 235 kDa and the subunits had a molecular mass of 81 kDa, indicating that ß-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, usingp-nitrophenyl-ß-d-galactopyranoside as a substrate. TheKm andVmax for Gal(ß1-4)Gal were 60 mM and 1,129 U/mg, respectively. The recombinant ß-Gal II was highly active towards Gal(ß1-4)Gal and Gal(ß1-4)Gal-containing oligosaccharides; only low activity was observed towards Gal(ß1-3)Gal, lactose, and Gal(ß1-3)GalOMe. No activity was found towards Gal(ß1-6)Gal, Gal(ß1-4)Man, Gal(a1-4)Gal, Gal(a1-3)Gal(ß1-4)Gal, cellobiose, maltose and sucrose. ß-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(ß1-4)]2Gal peak area ratio was 9:1.
- Publication
Applied Microbiology & Biotechnology, 2005, Vol 66, Issue 3, p276
- ISSN
0175-7598
- Publication type
Academic Journal
- DOI
10.1007/s00253-004-1745-9