| Summary: | 碩士 === 國立屏東科技大學 === 生物科技研究所 === 91 === Chitinases (EC 3.2.1.14) are present in a wide range of organisms, including bacteria, insects, plants and animals. Chitinase is a glycosyl hydrolase that catalyzes the degradation of chitin, an insoluble linear -1,4-linked polymer of N-acetylglucosamine. The Chi92 is the chitinase secreted by Aeromonas hydrophila JP101when induced with chitin and it is encoded by an open reading frame of 2,598 nucleotides coding for 865 amino acids. The Chi92 is a modular enzyme comprised of a family 18 catalytic domain, an unknown functional domain (A domain) and triple chitin binding domain (Chi92-N, ChBDCI and ChBDCII)。
In our experiment, we use site-directed mutagenesis technology to investigate the roles of the four highly conserved aromatic amino acids in the interaction of ChBDCICII with chitin. These aromatic amino acids were independently mutated to alanine or phenylalanine, to create W773F, W773A, W792A, Y796A and W797A, and proceded to analysis of binding ability. The ChBDCICII exhibited binding activity over a wide range of pH, and highest binding activity of ChBDCICII was observed at pH 8.0. The the binding activity was enhanced in the presence of NaCl, suggesting that the binding of ChBDCICII is mediated mainly by hydrophobic interactions. The ChBDCICII not only bound to colloidal chitin but also cellulose. Additionally, these mutant proteins exhibited much weaker affinity for colloidal and powdered chitin than wild type in adsorption-affinity expriments. These data indicate that all four aromatic residues are important for binding to chitin.
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