Biochemical characterization and site-directed mutational analysis of the double chitin-binding domain from chitinase 92 ofAeromonas hydrophilaJP101

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Abstract
Chitinase 92 from Aeromonas hydrophila JP101 contains C-terminal repeated chitin-binding domains (ChBDs) which were named ChBDCI and ChBDCII and classified into family 5 carbohydrate-binding modules on the basis of sequence. In this work, we constructed single and double ChBD by use of the pET system, which expressed as isolated ChBDCII or ChBDCICII. Polysaccharide-binding studies revealed that ChBDCICII not only bound to chitin, but also to other insoluble polysaccharides such as cellulose (Avicel) and xylan. In comparison with ChBDCII, the binding affinities of ChBDCICII are about 10- and 12-fold greater toward colloidal and powdered chitin, indicating that a cooperative interaction exists between ChBDCI and ChBDCII. In order to investigate the roles of the highly conserved aromatic amino acids in the interaction of ChBDCICII and chitin, we have performed site-directed mutagenesis. The data showed that W773A, W792A, Y796A and W797A mutant proteins exhibited a much weaker affinity for chitin than wild-type protein, suggesting that these residues play important roles in chitin binding.