BBB : Vol. 72 (2008) , No. 8 pp.2074-2081

Molecular Characterization of a Novel Family-46 Chitosanase from Pseudomonas sp. A-01

Akikazu ANDO¹⁾, Akihiro SAITO²⁾, Sayaka ARAI¹⁾, Sakiko USUDA¹⁾, Maiko FURUNO²⁾, Naomi KANEKO²⁾, Osamu SHIDA³⁾ and Yoshiho NAGATA²⁾

1) Graduate School of Science and Technology, Chiba University
2) Department of Bioresources Chemistry, Faculty of Horticulture, Chiba University
3) R&D Department, Higeta Shoyu, Co., Ltd.

(Received March 18, 2008)
(Accepted May 1, 2008)

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.

Key words:

chitosan; chitosanase; family 46; Pseudomonas

	To cite this article:
	Akikazu ANDO, Akihiro SAITO, Sayaka ARAI, Sakiko USUDA, Maiko FURUNO, Naomi KANEKO, Osamu SHIDA and Yoshiho NAGATA, “Molecular Characterization of a Novel Family-46 Chitosanase from Pseudomonas sp. A-01”, Biosci. Biotechnol. Biochem., Vol. 72, 2074-2081 (2008) .

	doi:10.1271/bbb.80175
	JOI JST.JSTAGE/bbb/80175