期刊
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY
卷 72, 期 8, 页码 2074-2081出版社
TAYLOR & FRANCIS LTD
DOI: 10.1271/bbb.80175
关键词
chitosan; chitosanase; family 46; Pseudomonas
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 pNCM02. 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 Gin and Asn respectively caused a defect in the hydrolyzing activity of the enzyme.
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