Purification and characterization of chitinase from the stomach of silver croaker Pennahia argentatus.

A chitinase was purified from the stomach of a fish, the silver croaker Pennahia argentatus, by ammonium sulfate fractionation and column chromatography using Chitopearl Basic BL-03, CM-Toyopearl 650S, and Butyl-Toyopearl 650S. The molecular mass and isoelectric point were estimated at 42 kDa and 6.7, respectively. The N-terminal amino acid sequence showed a high level of homology with family 18 chitinases. The optimum pH of silver croaker chitinase toward p-nitrophenyl N-acetylchitobioside (pNp-(GlcNAc)2) and colloidal chitin were observed to be pH 2.5 and 4.0, respectively, while chitinase activity increased about 1.5- to 3-fold with the presence of NaCl. N-Acetylchitooligosaccharide ((GlcNAc)n, n = 2-6) hydrolysis products and their anomer formation ratios were analyzed by HPLC using a TSK-GEL Amide-80 column. Since the silver croaker chitinase hydrolyzed (GlcNAc)4-6 and produced (GlcNAc)2-4, it was judged to be an endo-type chitinase. Meanwhile, an increase in beta-anomers was recognized in the hydrolysis products, the same as with family 18 chitinases. This enzyme hydrolyzed (GlcNAc)5 to produce (GlcNAc)2 (79.2%) and (GlcNAc)3 (20.8%). Chitinase activity towards various substrates in the order pNp-(GlcNAc)n (n = 2-4) was pNp-(GlcNAc)2 >> pNp-(GlcNAc)4 > pNp-(GlcNAc)3. From these results, silver croaker chitinase was judged to be an enzyme that preferentially hydrolyzes the 2nd glycosidic link from the non-reducing end of (GlcNAc)n. The chitinase also showed wide substrate specificity for degrading alpha-chitin of shrimp and crab shell and beta-chitin of squid pen. This coincides well with the feeding habit of the silver croaker, which feeds mainly on these animals.