title: Molecular characterization and antifungal activity of a family 46 chitosanase from Amycolatopsis sp. CsO-2.
abstract: An actinomycete strain, Amycolatopsis sp. CsO-2, produces a 27-kDa chitosanase. To reveal the molecular characteristics of the enzyme, its corresponding gene ctoA was cloned by a reverse genetic technique, based on the N-terminal amino acid sequence of the protein. The encoded CtoA protein was deduced to be composed of 286 amino acids, including a putative signal peptide (1-48), and exhibited 83% identity in the amino acid sequence with the family 46 chitosanases from Streptomyces sp. N174 or Nocardioides sp. N106. The active recombinant CtoA protein was successfully overproduced in Escherichia coli. The mutant protein E22Q, in which the glutamic acid residue 22 was replaced with glutamine, abolished the chitosanase activity, showing that the Glu22 residue is required for the enzymatic activity. CtoA exhibited antifungal activity against Rhizopus oryzae, which is known to produce chitosan probably as a cell wall component. In contrast, E22Q did not inhibit the growth of the fungus, suggesting that chitosan-hydrolyzing activity is essential for the antifungal activity. It is noteworthy that the antifungal effect of CtoA against R. oryzae was drastically enhanced by the simultaneous addition of the family 19 chitinase ChiC from Streptomyces griseus.
authors.affiliation: Department of Bioresource Chemistry, Faculty of Horticulture, Chiba University, Matsudo, Chiba, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Saito
authors.forename: Takaaki
authors.initials: T
authors.lastname: Ooya
authors.forename: Daisuke
authors.initials: D
authors.lastname: Miyatsuchi
authors.forename: Hiroko
authors.initials: H
authors.lastname: Fuchigami
authors.forename: Kanako
authors.initials: K
authors.lastname: Terakado
authors.forename: Shin-ya
authors.initials: SY
authors.lastname: Nakayama
authors.forename: Takeshi
authors.initials: T
authors.lastname: Watanabe
authors.forename: Yoshiho
authors.initials: Y
authors.lastname: Nagata
authors.forename: Akikazu
authors.initials: A
authors.lastname: Ando
mesh_terms: D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics; D000935:Antifungal Agents / Q000494:pharmacology; D002688:Chitinases / Q000235:genetics / Q000378:metabolism; D048271:Chitosan / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012233:Rhizopus / Q000187:drug effects*; D017422:Sequence Analysis, DNA
chemical_list: D000935:Antifungal Agents; D005656:Fungal Proteins; D011994:Recombinant Proteins; D048271:Chitosan; D006026:Glycoside Hydrolases; C019868:chitosanase; D002688:Chitinases