CHARACTERIZATION AND APPLICATION OF CHITINOLYTIC ENZYME FROM THE LIVER OF SQUID

1.研究了日本鱿鱼肝脏38 kDa和42 kDa几丁质酶同工酶的特性。对胶体几丁质的最佳pH值为3.0的38 kDa的几丁质酶，和3.0和9.0的42 kDa的几丁质酶。38<cat>和42 kDa几丁质酶对长底物乙二醇几丁质的Km和k2分别为0.071 mg/ml和1.22/s，0.074 mg/ml和0.196/s。这两种几丁质酶不仅降解几丁质，而且降解壳聚糖（D.A.95%）。两种几丁质酶均水解GlcNAc_n（n= 4，5和6）.以日本鱿鱼肝脏为原料，研究了酶液的制备及酶的性质。NAHase的最适pH为4.0，在pH4.0 ~ 6.5范围内酶活稳定。最适培养温度为60℃，培养时间为10 min。该酶在pH4.5的温度下60℃仍稳定。pNpGlcNAc的K_m值为0.227 mM，在0.1 ~ 1.0M的NaCl浓度范围内，对酶活性的抑制和/或激活作用均不明显.以日本鱿鱼和墨鱼肝脏粗酶为原料，研究了甲壳素酶法合成N-乙酰-D-氨基葡萄糖（GlcNAc）的工艺条件。粗酶液中几丁质酶和β-N-乙酰氨基己糖苷酶的活性比，日本鱿鱼为1：19，墨鱼为1：20。日本鱿鱼粗酶液（相当于2g肝重）在37℃下孵育5d，从50 mg胶态甲壳素中可产生26.8mg还原糖。在相同的反应条件下，在乌贼粗酶中得到44.4mg还原糖。通过HPLC分析，所产生的还原糖的主要产物是GlcNAc。这些结果表明，鱿鱼肝脏可能是酶促生产GlcNAc的几丁质分解酶的来源。

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.