Synthesis of alpha-galactooligosaccharides with alpha-galactosidase from Lactobacillus reuteri of canine origin.
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 degrees C ( K(M) 0.55 mM, V(max) 0.80 micromol 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%.
Tzortzis
G
G
School of Food Biosciences, The University of Reading, Whiteknights, PO Box 226, Reading, RG6 6AP, UK.
Jay
A J
AJ
Baillon
M L A
ML
Gibson
G R
GR
Rastall
R A
RA
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004285:Dogs / Q000382:microbiology*; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology / Q000378:metabolism*; D008553:Melibiose / Q000378:metabolism; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000302:isolation & purification; D011887:Raffinose / Q000378:metabolism; D000519:alpha-Galactosidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D009844:Oligosaccharides; D008553:Melibiose; D000519:alpha-Galactosidase; D011887:Raffinose