title: Purification and characterization of an alpha-glucosidase from Rhizobium sp. (Robinia pseudoacacia L.) strain USDA 4280.
abstract: A novel alpha-glucosidase with an apparent subunit mass of 59 +/- 0. 5 kDa was purified from protein extracts of Rhizobium sp. strain USDA 4280, a nodulating strain of black locust (Robinia pseudoacacia L), and characterized. After purification to homogeneity (475-fold; yield, 18%) by ammonium sulfate precipitation, cation-exchange chromatography, hydrophobic chromatography, dye chromatography, and gel filtration, this enzyme had a pI of 4.75 +/- 0.05. The enzyme activity was optimal at pH 6.0 to 6.5 and 35 degrees C. The activity increased in the presence of NH4+ and K+ ions but was inhibited by Cu2+, Ag+, Hg+, and Fe2+ ions and by various phenyl, phenol, and flavonoid derivatives. Native enzyme activity was revealed by native gel electrophoresis and isoelectrofocusing-polyacrylamide gel electrophoresis with fluorescence detection in which 4-methylumbelliferyl alpha-glucoside was the fluorogenic substrate. The enzyme was more active with alpha-glucosides substituted with aromatic aglycones than with oligosaccharides. This alpha-glucosidase exhibited Michaelis-Menten kinetics with 4-methylumbelliferyl alpha-D-glucopyranoside (Km, 0.141 microM; Vmax, 6.79 micromol min-1 mg-1) and with p-nitrophenyl alpha-D-glucopyranoside (Km, 0.037 microM; Vmax, 2.92 micromol min-1 mg-1). Maltose, trehalose, and sucrose were also hydrolyzed by this enzyme.
authors.affiliation: Glycobiologie, Centre de Biophysique Moléculaire, Centre National de la Recherche Scientifique UPR 4301, Université d'Orléans, 45071 Orléans cedex 2, France.
authors.forename: K
authors.initials: K
authors.lastname: Berthelot
authors.forename: F M
authors.initials: FM
authors.lastname: Delmotte
mesh_terms: D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D012231:Rhizobium / Q000201:enzymology* / Q000254:growth & development; D013379:Substrate Specificity; D000520:alpha-Glucosidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D000520:alpha-Glucosidases