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 comm on squid Todarodes pactficus by ammonium sulfate fractionation and col umn chromatographies with Chitopearl Basic BL-03, CM-Toyopearl 650S, a nd Bio-Gel HTP. The purified enzyme gave a single protein band on poly acrylamide gel electrophoresis (PAGE), and its molecular weight was es timated to be 38 kDa by SDS-PAGE. The isoelectric point was 8.3. The e nzyme 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 incubat ion at 50 degrees C for 10 min. The optimum temperature was 50 degrees C. The chitinase hydrolyzed glycol chitin and colloidal chitin, but n ot p-nitrophenyl N-acetyl-beta-D-glucosaminide and Micrococcus lysodei kticus. The cleavage pattern was investigated using N-acetylchitooligo saccharides (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 GlcNA c(3) (16%). The substrate specificity of the enzyme was that of endo-t ype chitinase.