Purification of glycoside hydrolases from Bacteroides fragilis.
Six glycoside hydrolases in the culture medium of Bacteroides fragilis--alpha-glucosidase, beta-glucosidase, alpha-galactosidase, beta-galactosidase, beta-N-acetylglucosaminidase, and alpha-L-fucosidase-were systematically purified by ammonium sulfate precipitation, gel filtration chromatography, and density gradient isoelectric focusing. The isoelectric focusing resolved the glycosidases into distinct, well-separated fractions and revealed three differently charged forms of beta-N-acetylglucosaminidase and of alpha-L-fucosidase. Furthermore, alpha-glucosidase and beta-N-acetylglucosaminidase were shown to possess dual affinities for the respective galactoside substrates, and beta-galactosidase also hydrolyzed beta-D-fucoside. alpha-Glucosidase was purified to homogeneity, as indicated by a thin-layer isoelectric focusing zymogram technique. The glycosidases, with exception of beta-glucosidase and the acid alpha-L-fucosidase, were each separated from other glycosidic activities to 99%. The molecular weights varied between 58,000 and 125,000. The pH optima ranged from 4.8 to 6.9.
D000118:Acetylglucosaminidase / Q000302:isolation & purification; D001441:Bacteroides fragilis / Q000201:enzymology*; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D006026:Glycoside Hydrolases / Q000302:isolation & purification*; D007525:Isoelectric Focusing; D008722:Methods; D000519:alpha-Galactosidase / Q000302:isolation & purification; D000520:alpha-Glucosidases / Q000302:isolation & purification; D005644:alpha-L-Fucosidase / Q000302:isolation & purification; D001616:beta-Galactosidase / Q000302:isolation & purification; D001617:beta-Glucosidase / Q000302:isolation & purification
D006026:Glycoside Hydrolases; D000520:alpha-Glucosidases; D001617:beta-Glucosidase; D000519:alpha-Galactosidase; D001616:beta-Galactosidase; D005644:alpha-L-Fucosidase; D000118:Acetylglucosaminidase