Synthesis of alpha-galactooligosaccharides with alpha-galactosidase from Lactobacillus reuteri of canine origin

Tzortzis, G., Jay, A.J., Baillon, M.L.A., Gibson, G.R. ORCID: https://orcid.org/0000-0002-0566-0476 and Rastall, R.A. (2003) Synthesis of alpha-galactooligosaccharides with alpha-galactosidase from Lactobacillus reuteri of canine origin. Applied Microbiology and Biotechnology, 63 (3). pp. 286-292. ISSN 0175-7598 doi: 10.1007/s00253-003-1426-0

Abstract/Summary

Crude cell-free extracts from Lactobacillus reuteri grown on cellobiose, maltose, lactose and raffinose were assayed for glycosidic activities. When raffinose was used as the carbon source, alpha-galactosidase was produced, showing the highest yield at the beginning of the stationary growth phase. A 64 kDa enzyme was purified by ultra- and gel filtration, and characterized for its hydrolytic and synthetic activity. Highest hydrolytic activity was found at pH 5.0 at 50 degreesC (K-M 0.55 mM, V-max 0.80 mumol min(-1) mg(-1) of protein). The crude cell-free extract was further used in glycosyl transfer reactions to synthesize oligosaccharides from melibiose and raffinose. At a substrate concentration of 23% (w/v) oligosaccharide mixtures were formed with main products being a trisaccharide at 26% (w/w) yield from melibiose after 8 h and a tetrasaccharide at 18% (w/w) yield from raffinose after 7 h. Methylation analysis revealed the trisaccharide to be 6' alpha-galactosyl melibiose and the tetrasaccharide to be stachyose. In both cases synthesis ceased when hydrolysis of the substrate reached 50%.