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CHARACTERIZATION AND APPLICATION OF CHITINOLYTIC ENZYME FROM THE LIVER OF SQUID

鱿鱼肝脏几丁质分解酶的表征及应用

基本信息

批准号：
13660208
负责人：
MATSUMIYA Masahiro
金额：
$ 1.22万
依托单位：
Nihon University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13660208/
关键词：
SQUID LIVER CHITINOLYTIC ENZYMES CHITINASE CHARACTERIZATION PURIFICATION APPRICATION ACETYLHEXOSAMINIDASE

项目摘要

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.

1. 对来自日本鱿鱼肝脏的 38 kDa 和 42 kDa 几丁质酶同工酶进行了表征。对于 38 kDa 几丁质酶，观察到胶体几丁质的最佳 pH 为 3.0；对于 42 kDa 几丁质酶，最佳 pH 为 3.0 和 9.0。 38和42kDa几丁质酶对较长底物乙二醇几丁质的K_m和k_<cat>分别为0.071mg/ml和1.22/s，以及0.074mg/ml和0.196/s。两种几丁质酶不仅分解几丁质，还分解壳聚糖（D.A. 95%）。两种几丁质酶均水解 GlcNAc_n (n=4,5, 和 6).2。对日本鱿鱼肝脏酶液的制备及NAHase的一些性质进行了研究。 NAHase的最适pH为4.0，酶活性在pH 4.0~6.5之间稳定。最适温度为60℃，孵育10 min。该酶在60℃、pH 4.5下仍稳定。 pNpGlcNAc 的 K_m 值为 0.227 mM。没有观察到0.1至1.0M NaCl的存在对酶活性的抑制和/或激活。3．使用来自日本鱿鱼和墨鱼肝脏的粗酶研究了从甲壳素酶法生产 N-乙酰基-D-葡萄糖胺 (GlcNAc) 的过程。粗酶中几丁质酶和β-N-乙酰己糖胺酶的活性比，鱿鱼为1:19，墨鱼为1:20。日本鱿鱼的粗酶，相当于2克肝脏重量，在37℃下培养5天时，从50毫克胶体甲壳素中产生26.8毫克还原糖。墨鱼粗酶在相同反应条件下得到还原糖44.4mg。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，鱿鱼肝可能是酶促生产 GlcNAc 的几丁质分解酶的来源。