title: Purification and characterization of a chitinase from Amycolatopsis orientalis with N-acetyllactosamine-repeating unit releasing activity.
abstract: We report a novel enzyme from the culture filtrate of Amycolatopsis orientalis, that endoglycosidically releases an N-acetyllactosamine-repeating unit (Galbeta1,4GlcNAcbeta1,3Galbeta1,4GlcNAc, LN2) from a synthetic chromogenic substrate Galbeta1,4GlcNAcbeta1,3Galbeta1,4GlcNAcbeta-pNP (1). The enzyme activity was purified by 80% saturated ammonium sulfate precipitation followed by gel filtration and affinity chromatography. The enzyme splits 1, Galbeta1,4GlcNAcbeta-pNP (2), GlcNAcbeta1,3Galbeta1,4GlcNAcbeta-pNP (3), and GlcNAcbeta1,4GlcNAcbeta-pNP (4) into the corresponding oligosaccharides and p-nitrophenol. The catalytic efficiencies (k(cat)/K(m)) for compounds 1, 2, and 4 were 0.6, 0.05, and 13, respectively. Compound 4 acts as a fairly good substrate for the enzyme, and LN2-releasing activity was inhibited by 4 and GlcNAcbeta1,4GlcNAcbeta1,4GlcNAcbeta-pNP (7), indicating that this enzyme activity is derived from a kind of chitinase. The enzyme hydrolyzed 1 by a mechanism leading to retention of the anomeric configuration. This is the first report of a N-acetyllactosamine-repeating unit releasing enzyme.
authors.affiliation: Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan. actmura@agr.shizuoka.ac.jp
authors.forename: Takeomi
authors.initials: T
authors.lastname: Murata
authors.forename: Satoshi
authors.initials: S
authors.lastname: Amarume
authors.forename: Takeshi
authors.initials: T
authors.lastname: Hattori
authors.forename: Shinji
authors.initials: S
authors.lastname: Tokuyama
authors.forename: Ken
authors.initials: K
authors.lastname: Tokuyasu
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Kawagishi
authors.forename: Taich
authors.initials: T
authors.lastname: Usui
mesh_terms: D000192:Actinomycetales / Q000145:classification / Q000201:enzymology*; D000606:Amino Sugars / Q000032:analysis* / Q000737:chemistry*; D002384:Catalysis; D002688:Chitinases / Q000032:analysis* / Q000737:chemistry*; D004789:Enzyme Activation; D007536:Isomerism; D007700:Kinetics; D011487:Protein Conformation; D013045:Species Specificity; D013379:Substrate Specificity
chemical_list: D000606:Amino Sugars; C000458:N-acetyllactosamine; D002688:Chitinases