title: Purification of endo-N-acetyl-beta-D-glucosaminidase H by substrate-affinity chromatography.
abstract: Endo-N-acetyl-beta-D-glucosaminidase H (Endo H) was purified to homogeneity (3000-fold) from a culture filtrate to Streptomyces plicatus. The key step was substrate-affinity chromatography, which afforded a 1000-fold purification and yielded a protease- and exoglycosidase-free preparation of Endo H. Proteins from the crude sample were applied to the substrate-affinity column, consisting of yeast-invertase glycopeptides bound to Sepharose-immobilized concanavalin A. After washing off the unbound proteins, Endo H was quantitatively eluted by methyl alpha-D-mannopyranoside. Various conditions were tested to achieve an optimal binding of Endo H to this substrate-affinity gel. After substrate-affinity chromatography, Endo H was separated from the coeluted gylcopeptide substrate and some protein impurities by gel filtration and hydrophobic chromatography.
authors.affiliation: Institute of Biotechnology, ETH-Hönggerberg HPT, Zürich, Switzerland.
authors.forename: U F
authors.initials: UF
authors.lastname: Greber
authors.forename: B
authors.initials: B
authors.lastname: Kozulić
authors.forename: K
authors.initials: K
authors.lastname: Mosbach
mesh_terms: D000118:Acetylglucosaminidase / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D006596:Hexosaminidases / Q000302:isolation & purification*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D013302:Streptomyces / Q000201:enzymology
chemical_list: D006596:Hexosaminidases; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase