title: A new peptide-N4-(acetyl-beta-glucosaminyl)asparagine amidase from soybean (Glycine max) seeds: purification and substrate specificity.
abstract: We report here the isolation and characterization of a peptide-N4-(acetyl-beta-glucosaminyl) asparagine amidase (peptide: N-glycanase) from soybean (Glycine max) seeds. The enzyme was purified to homogeneity with 6.5% yield from defatted soybean meal extract by ion-exchange chromatography, gel filtration, hydroxyapatite chromatography, and hydrophobic chromatography. The purified enzyme, designated PNGase-GM, had the apparent molecular mass of 93 kDa by SDS-PAGE and 90 kDa by gel filtration, indicating this PNGase is a monomeric protein. The enzyme showed maximal activity at pH 4.5-5.0. PNGase-GM was capable of hydrolyzing the beta-aspartylglycosylamine linkage (GlcNAc beta 1-->Asn) of various glycopeptide substrates bearing high-mannose type, hybrid type, and xylose/fucose-containing plant complex type N-glycan units, while this amidase was far less active on the glycopeptides bearing sialylated animal complex-type glycans.
authors.affiliation: Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Kimura
authors.forename: A
authors.initials: A
authors.lastname: Ohno
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002240:Carbohydrate Sequence; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D017886:Durapatite; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D012639:Seeds / Q000201:enzymology*; D013025:Glycine max / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D006020:Glycopeptides; D017886:Durapatite; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase