title: Purification and characterization of extracellular chin deacetylase from Colletotrichum lindemuthianum.
abstract: Chitin deacetylase, active in the presence of acetate (96% of the enzymatic activity was retained in the presence of 100 mM sodium acetate), was purified to electrophoretic homogeneity from a culture filtrate of Colletotrichum lindemuthianum (944-fold with a recovery of 4.05%). The enzyme was induced in the medium after the eighth day of incubation simultaneously with the blackening of the medium. The molecular mass of the enzyme was 31.5 kDa and 33 kDa as judged by SDS-PAGE and gel filtration, respectively, suggesting that the enzyme is a single polypeptide. The optimum temperature was 60 degrees C and the optimum pH was 11.5-12.0 when glycol chitin was used as substrate. The enzyme was active toward glycol chitin, partially N-deacetylated water soluble chitin, and chitin oligomers the degrees of polymerization of which were more than four, but was less active with chitin trimer and dimer, and inactive with N-acetylglucosamine. The Km and kcat for glycol chitin were 2.55 mM and 27.1 s-1, respectively, and those for chitin pentamer were 414 microM and 83.2 s-1, respectively. The reaction rates of the enzyme toward glycol chitin and chitin oligomers seemed to follow the Michaelis-Menten kinetics.
authors.affiliation: Biomaterials Conversion Laboratory, National Food Research Institute, Ibaraki, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Tokuyasu
authors.forename: M
authors.initials: M
authors.lastname: Ohnishi-Kameyama
authors.forename: K
authors.initials: K
authors.lastname: Hayashi
mesh_terms: D000581:Amidohydrolases / Q000096:biosynthesis / Q000302:isolation & purification*; D002686:Chitin / Q000031:analogs & derivatives* / Q000737:chemistry; D048271:Chitosan; D004790:Enzyme Induction; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D003904:Mitosporic Fungi / Q000201:enzymology*; D015394:Molecular Structure; D013696:Temperature
chemical_list: D002686:Chitin; D048271:Chitosan; C031610:glycolchitin; D000581:Amidohydrolases; C019866:chitin deacetylase