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

Abstract

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 β-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 α-chitin of shrimp and crab shell and β-chitin of squid pen. This coincides well with the feeding habit of the silver croaker, which feeds mainly on these animals.