Purification and characterization of chitinase from the liver of Japanese common squid Todarodes pacificus

A chitinase (EC 3.2.1.14) was purified from the liver of Japanese common squid Todarodes pactficus by ammonium sulfate fractionation and column chromatographies with Chitopearl Basic BL-03, CM-Toyopearl 650S, and Bio-Gel HTP. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis (PAGE), and its molecular weight was estimated to be 38 kDa by SDS-PAGE. The isoelectric point was 8.3. The enzyme showed two optimum pHs at 1.5 and 8.5 using glycol chitin as the substrate. The enzyme was stable between pH 4.0 and 6.0 after incubation at 50 degrees C for 10 min. The optimum temperature was 50 degrees C. The chitinase hydrolyzed glycol chitin and colloidal chitin, but not p-nitrophenyl N-acetyl-beta-D-glucosaminide and Micrococcus lysodeikticus. The cleavage pattern was investigated using N-acetylchitooligosaccharides (GlcNAc(n), n=2 to 6). The enzyme hydrolyzed GlcNAc(4) to two molecules of GlcNA(2), GlcNAc(5) to GlcNAc(2) plus GlcNAc(3), and GlcNAc(6) to GlcNAc(2) plus GlcNAc(4) (84%) and two molecules of GlcNAc(3) (16%). The substrate specificity of the enzyme was that of endo-type chitinase.