Open AccessBiochemical characterization and site‐directed mutational analysis of the double chitin‐binding domain from chitinase 92 of Aeromonas hydrophila JP101
Author(s) -
Chang Ming Chung,
Lai Pe Lin,
Wu Mei Li
Publication year - 2004
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1016/s0378-1097(04)00014-x
Subject(s) - chitin , chitinase , biochemistry , carbohydrate binding module , microbiology and biotechnology , aeromonas hydrophila , polysaccharide , chemistry , biology , enzyme , bacteria , glycoside hydrolase , chitosan , genetics
Chitinase 92 from Aeromonas hydrophila JP101 contains C‐terminal repeated chitin‐binding domains (ChBDs) which were named ChBD CI and ChBD CII 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 ChBD CII or ChBD CICII . Polysaccharide‐binding studies revealed that ChBD CICII not only bound to chitin, but also to other insoluble polysaccharides such as cellulose (Avicel) and xylan. In comparison with ChBD CII , the binding affinities of ChBD CICII are about 10‐ and 12‐fold greater toward colloidal and powdered chitin, indicating that a cooperative interaction exists between ChBD CI and ChBD CII . In order to investigate the roles of the highly conserved aromatic amino acids in the interaction of ChBD CICII 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.