title: Purification and characterization of a cell-associated, soluble mannanase from Bacteroides ovatus.
abstract: Bacteroides ovatus, a human colonic anaerobe, utilizes the galactomannan guar gum as a sole source of carbohydrate. Previously, we found that none of the galactomannan-degrading enzymes were extracellular, and we characterized an outer membrane mannanase which hydrolyzes the backbone of guar gum to produce large fragments. We report here the purification and characterization of a second mannanase from B. ovatus. This enzyme is cell-associated and soluble. Using ion-exchange chromatography, gel filtration, and chromatofocusing steps, we have purified the soluble mannanase to apparent homogeneity. The enzyme has a native molecular weight of 190,000 and a monomeric molecular weight of 61,000. It is distinct from the membrane mannanase not only with respect to cellular location but also with respect to stability and isoelectric point (pI of 6.9 for the membrane mannanase and pI of 4.8 for the soluble mannanase). The soluble mannanase, like the membrane mannanase, hydrolyzed guar gum to produce large fragments rather than monosaccharides. However, if galactosyl side chains were removed from the galactomannan fragments by alpha-galactosidase, both the soluble mannanase and the membrane mannanase could degrade guar gum to monosaccharides. Thus either or both of these two enzymes, working together with alpha-galactosidase, appear to be sufficient for the breakdown of guar gum to the level of monosaccharides.
mesh_terms: D001439:Bacteroides / Q000201:enzymology*; D005110:Extracellular Space / Q000201:enzymology; D007526:Isoelectric Point; D008361:Mannosidases / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D012995:Solubility; D013379:Substrate Specificity; D044902:beta-Mannosidase
chemical_list: D008361:Mannosidases; D044902:beta-Mannosidase