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.

通过CM-Sephadex C-50离子交换色谱法和用Sephadex G-150的Sephadex G-150的连续步骤纯化了来自Pieris rapae crucivcra boisduval p pifter的几丁质酶。然后获得了名为CHI-A和CHI-B的活性分数。对于分数CHI-A和.CHI-B，酶的纯度分别提高到12.4-和2.17倍，酶活性的回收分别为42.4和4.58％。通过SDS页面评估了分离的CHI-A的同质性和分子量。 CHI-A的均匀性被证实为SDS-PAGE上的单个谱带，估计分子量为48,000。当将乙二醇用作底物时，纯化的CHI-A对于水解反应的最佳pH值为5.0。 CHI-A在4.0-8.0的pH范围内保持稳定，并在310K处保留了70％的活性。来自Pieris Rapae Crucivara Boisduval p Pupae的几丁质酶表现出典型的Michaelis-Menten型动力学。我们还发现CHI-A揭示了几丁质的促酶活性。 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凝胶过滤色谱和CM-Sephadex C-50离子交换色谱。这些酶的热稳定性为308-328 K，转移酶活性的最佳pH值为6.5-7.0。每种酶可能与N-乙酰葡萄糖胺反应，并产生不溶性沉积物，表明高度聚合的奇特醇糖类。这些催化特性与属于同一顺序的Pieris rapae crucivora boisduval的Chitintransferase fron pupae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 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

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