Reaction mechanisms of chitinase and chitosanase produced by bacteria, and application of the enzymes to protoplast fusion of fungi

细菌产生的几丁质酶和壳聚糖酶的反应机制及其在真菌原生质体融合中的应用

• 批准号: 62560094
• 负责人: FUJII Takaaki
• 金额: $ 1.28万
• 依托单位: Department of Agricultural Chemistry, Faculty of Horticulture, Chiba University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1987
• 资助国家: 日本
• 起止时间: 1987 至 1988
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-62560094/
• 关键词: Chitinase; Chitosanase; Protoplasts of fungi; エンド型キトサナーゼ

A chitinase produced in the culture broth by Aeromonas hydrophila subsp. anaerogenes A52 was purified to homogeneity by affinity abso-rption with chitin and ion-exchange chromatography with CM-Sephadex C-50. The isoelectric point of the enzyme was determined to be 4.60. The enzyme showed a molecular weight of about 105,000.A chitosanase was purified from the culture broth of Bacillus circulans MH-Kl to homogeneity by CM-cellulose and gel permeation chromatography. The enzyme has a molecular weight of about 30,000,its Km is 0.63 mg chitosan/ml and its pi is 9.2. The enzyme showed an endo-splitting type of activity, and the end product of chitosan deg-radation contained a mixture of the dimer and trimer of glucosamine. The smallest of the substrate was a tetramer of glucosamine. The N-terminal amino acid sequence of the enzyme was determined to be Ala-Ser-Pro-Asp-Asp-Asn-Phe-Ser-Pro-Glu-Thr-Leu-Gln-Phe-Leu-Arg-Asn-Asn-Cys-Gly-Leu-Asp-. A methionine residue was not found in the enzyme.Protoplasts of aspergillus usami and Aspergillus awamori were efficiently produced by treatment of cells with the above chitinase. The chitosanase was available for preparetion of protoplasts from Mucor javanicus and Rhizopus hangchaoo.

产几丁质酶的气单胞菌Aeromonasseriasubsp.厌氧产气菌A52经几丁质亲和吸附和CM-Sephadex C-50离子交换层析纯化。酶的等电点测定为4.60。该酶的分子量约为105，000。从环状芽孢杆菌（Bacillus circulans）MH-K1的发酵液中分离纯化出一种壳聚糖酶，经CM-纤维素和凝胶渗透色谱分离纯化后，得到一种均一的壳聚糖酶。该酶的分子量约为30，000，其Km为0.63mg壳聚糖/ml，其pi为9.2。该酶具有内切裂解活性，降解产物为氨基葡萄糖二聚体和三聚体的混合物。最小的底物是葡糖胺的四聚体。该酶N端氨基酸序列为Ala-Ser-Pro-Asp-Asp-Asn-Phe-Ser-Pro-Glu-Thr-Leu-Gln-Phe-Leu-Arg-Asn-Asn-Cys-Gly-Leu-Asp-。用上述几丁质酶处理日本曲霉和泡盛曲霉的细胞，可有效地产生原生质体。壳聚糖酶可用于爪哇毛霉和杭州根霉原生质体的制备。

项目成果

M.Yabuki; M.Hirano; A.Ando; T.FUjii; Y.Amemiya: "Isolation and characterzation of a chitosan degrading bacterium and formation of chitosanase by the isolate" The Technical Bulletin of Faculty of Horticulture, Chiba University. 39. 23-27 (1987)

矢吹M.

M.Yabuki,;A.Uchiyama,;K.Suzuki,;A.Ando,;T.Fujii.: Journal of General and Applied Microbiology. 34. 255-270 (1988)

M.Yabuki,;A.Uchiyama,;K.Suzuki,;A.Ando,;T.Fujii.：普通与应用微生物学杂志。

M.Yabuki・A.Uchiyama・K.Suzuki・A.Ando・T.Fujii: J.Gen.Appl.Microbiol.34. 255-270 (1988)

M. Yabuki、A. Uchiyama、K. Suzuki、A. Fujii：J. Gen. Appl。 34. 255-270 (1988)

M. Yabuki, A. Uchiyama, K. Suzuki, A. Ando, and T. Fujii: Journal of General and Applied Microbiology.

M. Yabuki、A. Uchiyama、K. Suzuki、A. Ando 和 T. Fujii：普通与应用微生物学杂志。

M.Yabuki,;M.Hirano,;A.Ando,;T.Fujii,;Y,Amemiya.: The Technical Bulletin of Faculty of Horticulture,Chiba University. 39. 23-27 (1987)

M.Yabuki,;M.Hirano,;A.Ando,;T.Fujii,;Y,Amemiya.：千叶大学园艺学院技术通报。