Glycoprotein enzymes secreted by Aspergillus niger: purification and properties of alpha-glaactosidase.
An alpha-galactosidase (alpha-D-galactoside galactohydrolase [EC 3.2.1.22]) was purified to homogeneity from the culture filtrate of Aspergillus niger. The enzyme had an apparent molecular weight of 45,000 and was a glycoprotein. Radioactive enzyme was prepared by growing cells in [14C]fructose and this enzyme was used to prepare 14C-labeled glycopeptides. The glycopeptides emerged from Sephadex G-50 between stachyose and the glycopeptide from ovalbumin. Based on calibration of the column with various-sized dextran oligosaccharides, the glycopeptides appeared to have a molecular weight of 1,200 to 1,400. Analysis of the glycopeptide(s) indicated that it contained mannose and N-acetylglucosamine (GlcNAc) in an approximate ratio of 3 or 4 to 1. Assuming that there are two GlcNAc residues in the oligosaccharide and based on the molecular weight of the glycopeptide, the oligosaccharide probably contains eight to nine sugar residues. Alks probably attached to the protein by a GlcNAc leads to asparagine linkage. The purified alpha-galactosidase was most active on raffinose (Km = 5 x 10--4 M, Vmax = 3 mumol/min per mg of protein), but also showed good activity on p-nitrophenyl-alpha-D-galactoside ans somewhat less activity on stachyose and melibitol. The enzyme also hydrolyzed guar flour and locust bean gum, but did not attack the p-nitrophenyl glycosides of beta-galactose, alpha- or beta-glucose, or alpha- or beta-mannose.
D000117:Acetylglucosamine / Q000032:analysis; D001230:Aspergillus / Q000201:enzymology*; D001234:Aspergillus niger / Q000201:enzymology*; D011232:Chemical Precipitation; D002845:Chromatography; D005696:Galactosidases / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D006020:Glycopeptides / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008358:Mannose / Q000032:analysis; D008970:Molecular Weight; D009598:Nitrophenylgalactosides / Q000378:metabolism; D011887:Raffinose / Q000378:metabolism
D006020:Glycopeptides; D009598:Nitrophenylgalactosides; D005696:Galactosidases; D011887:Raffinose; D008358:Mannose; D000117:Acetylglucosamine