title: Molecular characterization of a novel family-46 chitosanase from Pseudomonas sp. A-01.
abstract: Pseudomonas sp. A-01, isolated as a strain with chitosan-degrading activity, produced a 28 kDa chitosanase. Following purification of the chitosanase (Cto1) and determination of its N-terminal amino acid sequence, the corresponding gene (cto1) was cloned by a reverse-genetic technique. The gene encoded a protein, composed of 266 amino acids, including a putative signal sequence (1-28), that showed an amino acid sequence similar to known family-46 chitosanases. Cto1 was successfully overproduced and was secreted by a Brevibacillus choshinensis transformant carrying the cto1 gene on expression plasmid vector pNCMO2. The purified recombinant Cto1 protein was stable at pH 5-8 and showed the best chitosan-hydrolyzing activity at pH 5. Replacement of two acidic amino acid residues, Glu23 and Asp41, which correspond to previously identified active centers in Streptomyces sp. N174 chitosanase, with Gln and Asn respectively caused a defect in the hydrolyzing activity of the enzyme.
authors.affiliation: Graduate School of Science and Technology, Chiba University, Chiba, Japan. andnand@faculty.chiba-u.jp
authors.forename: Akikazu
authors.initials: A
authors.lastname: Ando
authors.forename: Akihiro
authors.initials: A
authors.lastname: Saito
authors.forename: Sayaka
authors.initials: S
authors.lastname: Arai
authors.forename: Sakiko
authors.initials: S
authors.lastname: Usuda
authors.forename: Maiko
authors.initials: M
authors.lastname: Furuno
authors.forename: Naomi
authors.initials: N
authors.lastname: Kaneko
authors.forename: Osamu
authors.initials: O
authors.lastname: Shida
authors.forename: Yoshiho
authors.initials: Y
authors.lastname: Nagata
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004789:Enzyme Activation / Q000187:drug effects; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D008670:Metals; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C019868:chitosanase