CHARACTERIZATION AND APPLICATION OF CHITINOLYTIC ENZYME FROM THE LIVER OF SQUID

1.对日本普通鱿鱼肝脏38 kDa和42 kDa几丁质酶同工酶进行了研究。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 kDa和42 kDa几丁质酶对乙二醇几丁质底物的Km分别为0.071 mg/ml和1.22/S，Km分别为0.074 mg/ml和0.196/S。这两种几丁质酶不仅能分解几丁质，还能分解壳聚糖(D.A.95%)。两种几丁质酶均能降解GlcNAc_n(n=4，5，6)。研究了日本乌贼肝脏NaHase酶溶液的制备及部分性质。NaHase的最适pH为4.0，在pH 4.0~6.5范围内酶活稳定。最适培养温度为60℃，培养时间为10min。该酶在pH值为4.5时仍稳定在60℃。PNpGlcNAc的Km值为0.227 mm。0.1~1.0M的氯化钠对该酶的活性没有抑制和/或激活作用。以日本乌贼和乌贼肝脏粗酶为原料，研究了几丁质酶法生产N-乙酰-D-氨基葡萄糖(GlcNAc)的工艺条件。粗酶中几丁质酶与β-N-乙酰氨基己糖苷酶活性之比，日本乌贼为1：19，乌贼为1：20。日本普通鱿鱼粗酶，相当于2g肝脏重量，在37℃下孵育5天，从50 mg胶体甲壳素中产生26.8 mg还原糖。在相同反应条件下，乌贼粗酶中还原糖含量为44.4 mg。经高效液相色谱分析，其主要产物为GlcNAc。这些结果表明，鱿鱼肝可能是几丁质水解酶的来源，可用于酶法生产GlcNAc。

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.