Identification of a chitinase from the hepatopancreas of Chinese black sleeper (Bostrychus sinensis)

Chinese black sleeper (Bostrychus sinensis) is a fish that lives both in seawater and freshwater, feeds on crustaceans, aquatic insects and occasionally shellfish. The existence of digestive enzyme in viscera to act on chitinous exoskeleton of the prey is of interest. In this study, a chitinase was purified to homogeneity using ammonium sulfate precipitation, DEAE-Sephacel ion exchange, Sephacryl S-200 HR and Superdex 200 gel filtration columns. The purified protein presents a molecular mass of 58 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and results in a single band on native PAGE. According to peptide mass fingerprinting, two peptides containing a total of 20 amino acid residues, were 95% identical to a chitinase from yellow perch (Perca flavescens) and 100% identical to the chitinase from greater amberjack (Seriola dumerili). The purified chitinase showed optimum activity at pH 6.0, and was stable at acidic conditions and temperature below 55°C. The enzymatic activity was quite stable in the presence of NaCl, even at 1 mol/L. The chitinase was capable of degrading chitosan into low molecular mass chitooligosaccharides (COS) with sizes in a range of 200–700 Da, and the circular dichroism profile of the COS greatly differed from native chitosan. Full-length cDNA encoding the present chitinase was cloned and the transcript levels of chitinase in various tissues were determined by quantitative real-time PCR. The results showed that the transcript level of chitinase was highest in esophagus and hepatopancreas.