Construction of System for Formation of Chitin Oligomer Having Highly Physiological Activity by Enzymatic Hydrolysis Reaction

酶解反应形成高生理活性甲壳素低聚物体系的构建

• 批准号: 13650832
• 负责人: KONDO Kazuo
• 金额: $ 2.11万
• 依托单位: DOSHISHA 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 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13650832/
• 关键词: Enzyme; Purification; Physiological Activity; Chitin; Chitin Oligomer; Hydrolysis Reaction; 結晶化

Chitinase from the extract of pupae of Pieris rapae crucivcra Boisduval was purified through the successive steps of CM-Sephadex C-50 ion exchange chromatography and gel filtration chromatography with Sephadex G-150 from crude enzyme extract. Then the active fractions named Chi-A and Chi-B were obtained. The purity of the enzyme increased up to 12.4- and 2.17-fold and the recovery of the enzyme activity were 42.4 and 4.58%, for the fraction Chi-A and.Chi-B, respectively. The homogeneity and molecular weight of isolated Chi-A were evaluated by SDS PAGE. The homogeneity of Chi-A was confirmed as a single band on SDS-PAGE and the molecular weight was estimated to be 48,000. The purified Chi-A had an optimal pH of 5.0 for the hydrolysis reaction when glycol chitin was used as a substrate. Chi-A was stable in the pH range of 4.0-8.0 and retained its 70% activity at 310K. Chitinase from pupae of Pieris rapae crucivara Boisduval exhibited typical Michaelis-Menten type kinetics. We also found that Chi-A revealed a chitin synfase activity. A large amount of N-acetylchitopentaose was effectively formed by the transglycosylation from N-acetylglucosamine with Chi-A.On the other hand, three kinds of N-acetylglucosamine transferase were purified from the crude extract of pupae of Papilio xuthus Linne through Sephadex G-25 gel filtration chromatography and CM-Sephadex C-50 ion exchange chromatography. Thermal stability of these enzymes was 308-328 K and optimal pH for transferase activity appeared at 6.5-7.0. Each enzyme could react with N-acetylglucosamine, and produced insoluble deposit suggesting highly polymerized chitooligosaccharides. These catalytic properties somewhat differ from those of chitintransferase fron pupae of Pieris rapae crucivora Boisduval belonging to the same order.

从十字花科粉蝶（Pieris raiscrucivcra Boisduval）蛹中提取几丁质酶，粗酶液经CM-SephadexC-50离子交换层析和SephadexG-150凝胶过滤层析纯化。然后得到活性组分Chi-A和Chi-B。酶的纯度分别提高到12.4倍和2.17倍，酶活回收率分别为42.4%和4.58%。SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析纯化的Chi-A的均一性和分子量。在SDS-PAGE上确认Chi-A的均一性为单一条带，估计分子量为48，000。以乙二醇甲壳素为底物时，纯化的Chi-A的最适pH为5.0。Chi-A在pH4.0 -8.0范围内稳定，在310 K时仍保持70%的活性。十字粉蝶（Pieris raiscrucivara Boisduval）蛹几丁质酶具有典型的米氏动力学。我们还发现Chi-A具有几丁质合成酶活性。N-乙酰氨基葡萄糖经Chi-A转糖基化后，可有效地生成大量的N-乙酰壳五糖。另一方面，通过Sephadex G-25凝胶过滤层析和CM-Sephadex C-50离子交换层析，从柑橘凤蝶蛹粗提物中分离纯化了3种N-乙酰氨基葡萄糖转移酶。这些酶的热稳定性为308-328 K，转移酶活性的最适pH出现在6.5-7.0。每种酶都能与N-乙酰氨基葡萄糖反应，并产生不溶性存款，表明壳寡糖高度聚合。这些催化性质与同一目的短十字粉蝶（Pieris raiscrucivora Boisduval）腐皮几丁质转移酶的催化性质有所不同。

项目成果

近藤和生: "Purification and characterization of chitinase from pupae of Pieris rapae crucivora Boisduval"Journal of Chemical Engineering of Japan. 35・3(印刷中). (2002)

近藤和夫：“菜青虫蛹的几丁质酶的纯化和特性”日本化学工程学报35・3（出版中）。

Purification an Characterization of N-Acetyl-D-Glucosamine Transferase from Pupae of Papilio xuthus Linne

凤蝶蛹 N-乙酰基-D-葡萄糖胺转移酶的纯化及表征

• 发表时间: 2005
• 期刊: Journal of Chemical Engineering of Japan 38巻6号(印刷中)
• 作者: 前田 良輔

Purification and Characterization of N-A cetyl-D-Glucosamine Transferase from Pupae of Papilio xuthus Linne

凤蝶蛹 N-A 十六烷基-D-葡萄糖胺转移酶的纯化和表征

• 发表时间: 2005
• 期刊: Journal of Chemical Engineering of Japan Vol.38, No.6(in press)
• 作者: Ryousuke Maeda;Akihiro Nozawa;Michiaki Matsumoto;Kazuo Kondo
• 通讯作者: Kazuo Kondo

Purification an Characterization of Chitinase from Pupae of Pieris rapae crucivora Boisduval

菜青虫蛹几丁质酶的纯化及表征

• 发表时间: 2002
• 期刊: Journal of Chemical Engineering of Japan 35巻3号
• 作者: Ryousuke Maeda;Akihiro Nozawa;Michiaki Matsumoto;Kazuo Kondo;近藤 和生
• 通讯作者: 近藤 和生

Purification and Characterization of Chitinase from Pupae of Pieris rapae cruclvora Boisduval

菜青虫蛹几丁质酶的纯化及表征

• 发表时间: 2002
• 期刊: Journal of Chemical Engineering of Japan Vol.35, No3
• 作者: Kazuo Kondo;Michiaki Matsumoto;Akane Kojo;Ryousuke Maeda
• 通讯作者: Ryousuke Maeda