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 vi tro thanks to the production of an alpha-galactosidase. The enzyme was puri fied from the cell-free extract of Bi. adolescentis DSM 20083(T). It was fo und to act with retention of configuration (alpha --> alpha), releasing alp ha-galactose from p-nitrophenyl galactoside. This hydrolysis probably opera tes with a double-displacement mechanism, and is consistent with the observ ed glycosyltransferase activity. As alpha-galactosides are interesting subs trates for bifidobacteria, we focused on the production of new types of alp ha-galactosides using the transgalactosylation activity of Bi. adolescentis alpha-galactosides. Starting from melibiose, raffinose and stachyose oligo saccharides could be formed. The transferase activity was highest at pH 7 a nd 40 degrees C. Starting from 300 mM melibiose a maximum yield of 33% olig osaccharides was obtained. The oligosaccharides formed from melibiose were purified by size-exclusion chromatography and their structure was elucidate d by NMR spectroscopy in combination with enzymatic degradation and sugar l inkage 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 hydro xyl 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 intesti nal bacteria, including various bifidobacteria, and might be an interesting pre- and synbiotic substrate.