CHARACTERIZATION AND APPLICATION OF CHITINOLYTIC ENZYME FROM THE LIVER OF SQUID

1. 对日本鱿鱼肝脏中38 kDa和42 kDa几丁质酶同工酶进行了鉴定。38 kDa几丁质酶的最适pH为3.0,42 kDa几丁质酶的最适pH为3.0和9.0。38和42 kDa几丁质酶对较长底物乙二醇几丁质的K_m和k_<cat>分别为0.071 mg/ml和1.22/s和0.074 mg/ml和0.196/s。两种几丁质酶均能分解几丁质和壳聚糖（da达95%）。两种几丁质酶都能水解GlcNAc_n (n=4,5和6).2。研究了日本鱿鱼肝脏NAHase酶液的制备及其性质。NAHase的最适pH为4.0，酶活性在4.0 ~ 6.5之间较为稳定。最适温度为60℃，孵育10 min。在pH为4.5的条件下，酶在60℃时仍保持稳定。pNpGlcNAc的K_m值为0.227 mM，在0.1 ~ 1.0M范围内未观察到NaCl对酶活性的抑制和/或激活。以日本鱿鱼和墨鱼肝脏粗酶为原料，研究了几丁质酶法生产n -乙酰- d -氨基葡萄糖（GlcNAc）的工艺。粗酶中几丁质酶活性与β- n -乙酰己糖氨酸酶活性之比，鱿鱼为1:19，墨鱼为1:20。日本鱿鱼粗酶在37℃条件下孵育5 d，从50 mg胶体甲壳素中产生26.8 mg还原糖，相当于2 g肝脏重量。在相同的反应条件下，墨鱼粗酶可得还原糖44.4 mg。所得还原糖的主要产物为GlcNAc。这些结果表明，鱿鱼肝脏可能是GlcNAc酶生产的几丁质水解酶的来源。

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.