Clarification of the Organic Solvent-Stability of Enzymes Based on Protein Engineering and Development of Organic Solvent-Stable Enzymes

基于蛋白质工程的酶的有机溶剂稳定性的阐明和有机溶剂稳定性酶的开发

• 批准号: 11450317
• 负责人: ISHIKAWA Haruo
• 金额: $ 9.28万
• 依托单位: Osaka Prefecture University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11450317/
• 关键词: organic solvent-stable enzyme; organic solvent-tolerant microorganism; lipase; protease; nonaqueous bioprocess; タンパク質工学; Pseudomonas aeruginosa; 酵素精製

Enzymes exhibit high substrate and reaction specificities as catalysts under mild conditions. Therefore, if enzymes are stable in the presence of organic solvents, they can be applied to develop environmentally friendly synthetic processes of fine-chemicals. In this research project, the clarification of the organic solvent-stability of enzymes based on protein engineering and molecular evolution engineering and fundamental research to develop organic solvent-stable enzymes were performed. The examined item in this research is as follows.1) Growth of organic solvent-tolerant Pseudomonas aeruginosa LST-03 in the presence of an organic solvent and production of organic solvent-stable enzymes2) Purification and characterization of organic solvent-stable LST-03 lipase3) Cloning and sequence of the gene of organic solvent-stable LST-03 lipase4) Cloning and sequence of the lipase genes of Pseudomonas aeruginosa LST-035) Purification and characterization of organic solvent-stable PST-01 protease6) Peptide synthesis catalyzed by PST-01 protease7) Conformational change of enzymes in the presence of an organic solvent8) Cloning and sequence of the gene of organic solvent-stable PST-01 protease9) Role of disulfide bonds of organic solvent-stable PST-01 protease10) Clarification of the organic solvent-stability of enzymes based on molecular evolution engineering

酶在温和条件下作为催化剂表现出高底物特异性和反应特异性。因此，如果酶在有机溶剂存在下是稳定的，则它们可以应用于开发环境友好的精细化学品合成工艺。本研究以蛋白质工程和分子进化工程为基础，对酶的有机溶剂稳定性进行了澄清，并对开发有机溶剂稳定酶进行了基础研究。本研究的研究项目如下：1）耐有机溶剂铜绿假单胞菌LST-03在有机溶剂存在下的生长和有机溶剂稳定酶的产生2）有机溶剂稳定LST-03脂肪酶的纯化和特性3）有机溶剂稳定LST-03脂肪酶基因的克隆和序列4）铜绿假单胞菌LST-03脂肪酶基因的克隆和序列035）有机溶剂稳定的PST-01蛋白酶的纯化和表征6）由PST-01蛋白酶催化的肽合成7）在有机溶剂存在下酶的构象变化8）有机溶剂稳定的PST-01蛋白酶的基因的克隆和序列9）有机溶剂稳定的PST-01蛋白酶的二硫键的作用10）有机溶剂的澄清-基于分子进化工程的酶稳定性研究

项目成果

H.Ogino et al.: "Cloning and Sequencing of a Gene of Organic Solvent-Stable Protease Secreted from Pseudomonas aeruginosa PST-01 and Its Expression in Escherichia coli"Biochemical Engineering Journal. 5・3. 191-200 (2000)

H.Ogino 等：“铜绿假单胞菌 PST-01 分泌的有机溶剂稳定蛋白酶基因的克隆和测序及其在大肠杆菌中的表达”《生化工程杂志》191-200 (2000)。

H. Ogino and H. Ishikawa: "Enzymes Which Are Stable in the Presence of Organic Solvents (Review)"Journal of Bioscience and Bioengineering. 91(2). 109-116 (2001)

H. Ogino 和 H. Ishikawa：“有机溶剂存在下稳定的酶（评论）”生物科学与生物工程杂志。

H.Ogino et al.: "Purification and Characterization of Organic Solvent-Stable Lipase from Organic Solvent-Tolerant Pseudomonas aeruginosa LST-03"Biochemical Engineering Journal. 89・5. 451-457 (2000)

H.Ogino 等：“来自有机溶剂耐受铜绿假单胞菌 LST-03 的有机溶剂稳定脂肪酶的纯化和表征”生物化学工程杂志 89·5（2000）。

T. Ito, H. Kikuta, E. Nagamori, H. Honda, H. Ogino, H. Ishikawa, and T. Kobayashi: "Lipase Production in Two-Step Fed-Batch Culture of Organic Solvent-Tolerant Pseudomonas aeruginosa LST-03"Journal of Bioscience and Bioengineering. 91(3). 245-250 (2001)

T. Ito、H. Kikuta、E. Nagamori、H. Honda、H. Ogino、H. Ishikawa 和 T. Kobayashi：“有机溶剂耐受铜绿假单胞菌 LST-03 两步补料分批培养中的脂肪酶生产

H. Ogino, M. Yasuda, and H. Ishikawa: "Role of Disulfide Bonds of a Protease from Pseudomonas aeruginosa PST-01 in the Organic Solvent-Stability"Proceeding of the Sixth Japan-China Joint Symposium on Enzyme Engineering. 65 (2000)

H. Ogino、M. Yasuda 和 H. Ishikawa：“铜绿假单胞菌 PST-01 蛋白酶的二硫键在有机溶剂稳定性中的作用”第六届中日酶工程联合研讨会论文集。