Transglycosidase activity of Bifidobacterium adolescentis DSM 20083 alpha-galactosidase.
Bifidobacterium adolescentis, a gram-positive saccharolytic bacterium found in the human colon, can, alongside other bacteria, utilise stachyose in vitro thanks to the production of an alpha-galactosidase. The enzyme was purified from the cell-free extract of Bi. adolescentis DSM 20083T. It was found to act with retention of configuration (alpha-->alpha), releasing alpha-galactose from p-nitrophenyl galactoside. This hydrolysis probably operates with a double-displacement mechanism, and is consistent with the observed glycosyltransferase activity. As alpha-galactosides are interesting substrates for bifidobacteria, we focused on the production of new types of alpha-galactosides using the transgalactosylation activity of Bi. adolescentis alpha-galactosides. Starting from melibiose, raffinose and stachyose oligosaccharides could be formed. The transferase activity was highest at pH 7 and 40 degrees C. Starting from 300 mM melibiose a maximum yield of 33% oligosaccharides was obtained. The oligosaccharides formed from melibiose were purified by size-exclusion chromatography and their structure was elucidated by NMR spectroscopy in combination with enzymatic degradation and sugar linkage analysis. The trisaccharide alpha-D-Galp-(1-->6)-alpha-D-Galp-(1-->6)-D-Glcp and tetrasaccharide alpha-D-Galp-(1-->6)-alpha-D-Galp- (1-->6)-alpha-D-Galp-(1-->6)-D-Glcp were identified, and this indicates that the transgalactosylation to melibiose occurred selectively at the C-6 hydroxyl group of the galactosyl residue. The trisaccaride alpha-D-Galp-(1-->6)-alpha- D-Galp-(1-->6)-D-Glcp formed could be utilised by various intestinal bacteria, including various bifidobacteria, and might be an interesting pre- and synbiotic substrate.
Van Laere
K M
KM
Wageningen Agricultural University, Department of Food Technology and Nutritional Sciences, Food Science Group, The Netherlands.
Hartemink
R
R
Beldman
G
G
Pitson
S
S
Dijkema
C
C
Schols
H A
HA
Voragen
A G
AG
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001644:Bifidobacterium / Q000201:enzymology*; D002845:Chromatography; D005697:Galactosides / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D008553:Melibiose / Q000378:metabolism; D009844:Oligosaccharides / Q000096:biosynthesis / Q000302:isolation & purification; D013552:Swine; D013696:Temperature; D000519:alpha-Galactosidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D005697:Galactosides; D009844:Oligosaccharides; D008553:Melibiose; D000519:alpha-Galactosidase