title: Purification, characterization, and mode of action of endoxylanases 1 and 2 from Fibrobacter succinogenes S85.
abstract: Two different endoxylanases (1,4-beta-D-xylan xylanohydrolases, EC 3.2.1.8), designated 1 and 2, have been purified by column chromatography to apparent homogeneity from the nonsedimentable extracellular culture fluid of the strictly anaerobic, ruminal bacterium Fibrobacter succinogenes S85 grown on crystalline cellulose. Endoxylanases 1 and 2 were shown to be basic proteins of 53.7 and 66.0 kDa, respectively, with different pH and temperature optima, as well as different substrate hydrolysis characteristics. The Km and Vmax values with water-soluble oat spelts xylan as substrate were 2.6 mg ml-1 and 33.6 mumol min-1 mg-1 for endoxylanase 1 and 1.3 mg ml-1 and 118 mumol min-1 mg-1 for endoxylanase 2. Endoxylanase 1, but not endoxylanase 2, released arabinose from water-soluble oat spelts xylan and rye flour arabinoxylan, but not from arabinan, arabinogalactan, or aryl-alpha-L-arabinofuranosides. With an extended hydrolysis time, endoxylanase 1 released 62.5 and 50% of the available arabinose from water-soluble oat spelts xylan and rye flour arabinoxylan, respectively. Endoxylanase 1 released arabinose directly from the xylan backbone, and this preceded hydrolysis of the xylan to xylooligosaccharides. Endoxylanase 2 showed significant activity against carboxymethyl cellulose but was unable to substantially hydrolyze acid-swollen cellulose. Both enzymes were endo-acting, as revealed by their hydrolysis product profiles on water-soluble xylan and xylooligosaccharides. Because of their unique hydrolytic properties, endoxylanases 1 and 2 appear to have strategic roles in plant cell wall digestion by F. succinogenes in vivo.
authors.affiliation: Department of Microbiology, University of Guelph, Ontario, Canada.
authors.forename: A
authors.initials: A
authors.lastname: Matte
authors.forename: C W
authors.initials: CW
authors.lastname: Forsberg
mesh_terms: D001089:Arabinose / Q000378:metabolism; D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D043364:Endo-1,4-beta Xylanases; D006026:Glycoside Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007525:Isoelectric Focusing; D007700:Kinetics; D009844:Oligosaccharides / Q000378:metabolism; D010084:Oxidation-Reduction; D011134:Polysaccharides / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D009844:Oligosaccharides; D011134:Polysaccharides; D001089:Arabinose; D006026:Glycoside Hydrolases; D043364:Endo-1,4-beta Xylanases; D002266:Carboxymethylcellulose Sodium