|Budget Amount *help
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 2003: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2002: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2001: ¥900,000 (Direct Cost: ¥900,000)
1. Characterization was investigated on the 38 kDa and 42 kDa chitinase isozymes from the liver of Japanese common squid. Optimum pH toward colloidal chitin was observed at pH 3.0 for the 38 kDa chitinase, and 3.0 and 9.0 for the 42 kDa chitinase. K_m and k_<cat> of the 38 and 42 kDa chitinases toward a longer substrate, glycol chitin, were 0.071 mg/ml and 1.22/s, and 0.074 mg/ml and 0.196/s, respectively. Both the chitinases decomposed not only chitin but also chitosan (D.A. 95%). Both the chitinases hydrolyzed GlcNAc_n (n=4,5, and 6).
2. Preparation of enzyme solution and some properties of NAHase from the liver of Japanese common squid was investigated. The optimal pH of NAHase was 4.0, and the enzyme activity was stable between pH 4.0 and 6.5. The optimum temperature was 60℃ at 10 min incubation. The enzyme was stable still 60℃ at pH 4.5. The K_m value for pNpGlcNAc was 0.227 mM. Inhibition and/or activation of the enzyme activity by presence of NaCl from 0.1 to 1.0M were not observed.
3. Enzymatic production of N-acetyl-D-glucosamine (GlcNAc) from chitin was investigated using crude enzyme from the liver of Japanese common squid and cuttlefish. The ratio for the activities of chitinase and β-N-acetylhexosaminidase in the crude enzyme was 1:19 for Japanese common squid and 1:20 for cuttlefish. Crude enzyme of Japanese common squid, corresponding to 2 g of liver weight, produced 26.8 mg of reducing sugar from 50 mg of colloidal chitin during 5 days of incubation at 37℃. In the crude enzyme from cuttlefish, 44.4 mg of reducing sugar was obtained under the same reaction conditions. The main product of the produced reducing sugar, analyzed by HPLC, was GlcNAc. These results suggest that squid liver could be a source of chitinolytic enzyme for the enzymatic production of GlcNAc.