Development of Reaction Element for Nonaquous Bioprocess

非水生物过程反应元件的开发

• 批准号: 11555209
• 负责人: ISHIKAWA Haruo
• 金额: $ 8.77万
• 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-11555209/
• 关键词: nonaqueous Bioprocess; organic solvent-tolerant enzyme; organic solvent-tolerant immobilized enzyme; modified enzyme material; organic solvent-tolerant microorganism; peptide synthesis; amphiphilic polymer particle; surfactant; 非水系バイオ; β構造; プロテア

An organic solvent-tolerant protease (PST-01 protease) from Pseudomonas aeruginosa PST-01 was purified and characterized. The PST-01 protease was stable in the presence of various organic solvents. Peptide syntheses were performed in monophasic aqueous solution containing an organic solvent using PST-01 protease. The initial reaction rate and the equilibrium yield of the peptide in the presence of 60%(v/v) DMSO were 5.1 times and 7.0 times higher than that in the absence of an organic solvent, respectively.Amphiphilic polymer particles which have hydrophilic guanidino groups and hydrophobic stearoyl groups were the best particles for the immobilization of Rhizopus delemar lipase. The hydrolytic activity of the immobilized lipase prepared with the amphiphilic polymer particles was 150-8700 times higher than those of the immobilized lipases prepared by previous investigators using Dowex MWA-1, porous glass beads, and Sepharose 4B. The stability and the transesterification activity of lipase were both enhanced by immobilizing lipase on the amphiphilic polymer particles. The specific transesterification activity of the immobilized lipase prepared with the amphiphilic polymer particles was 93.4 times higher than that of the lyophilized lipase.Surfactant-coated protease was prepared with a synthesized surfactant. The peptide synthetic activity and transeste rification activity of the surfactant-coated protease in organic solvents were higher than that of the powder protease. The activity of the surfactant-coated protease was 260 times higher that of the powder protease.

从铜绿假单胞菌PST-01中分离纯化了一种耐有机溶剂的蛋白酶（PST-01蛋白酶），并对其进行了性质研究。PST-01蛋白酶在各种有机溶剂中稳定。使用PST-01蛋白酶在含有有机溶剂的单相水溶液中进行肽合成。结果表明，在60%（v/v）DMSO存在下，反应的起始速率和肽的平衡产率分别是无有机溶剂时的5.1倍和7.0倍，具有亲水性胍基和疏水性硬脂酰基的两亲性聚合物颗粒是固定化德氏根霉脂肪酶的最佳颗粒。用两亲性聚合物颗粒制备的固定化脂肪酶的水解活性比以前的研究者使用Dowex MWA-1、多孔玻璃珠和Sepharose 4 B制备的固定化脂肪酶高150-8700倍。将脂肪酶固定在两亲性聚合物粒子上，提高了脂肪酶的稳定性和酯交换活性。用两亲性聚合物颗粒制备的固定化脂肪酶比酯交换活性是冻干脂肪酶的93.4倍。在有机溶剂中，包被蛋白酶的肽合成活性和转酯化活性均高于粉末蛋白酶。该蛋白酶的酶活是粉末蛋白酶的260倍。

项目成果

M.Goto et al.: "Surfactant-HRP Complex Catalytically Active in Organic Media"Biotechnology Progress. 16・1. 52-58 (2000)

M.Goto 等：“有机介质中的表面活性剂-HRP 复合物的催化活性”16・1（2000 年）。

M.Goto et al.: "Preparation and Catalytic Performance of Surfactant-Manganese Peroxidase-MnII Ternary Comple in Organic Media"Enzyme and Microbial Technology. 28. 329-332 (2001)

M.Goto等：“有机培养基中表面活性剂-锰过氧化物酶-MnII三元复合物的制备及其催化性能”酶与微生物技术。

H. Ogino, Y. Gemba, M. Yamada, M. Shizuka, M. Yasuda, H. Ishikawa: "The Synthetic Rate of Dipeptide Catalyzed by Organic Solvent-Stable Protease from Pseudomonas aeruginosa PST-01 in the Presence of Water-Soluble Organic Solvents"Biochem. Eng. J.. 5(3). 2

H. Ogino、Y. Gemba、M. Yamada、M. Shizuka、M. Yasuda、H. Ishikawa：“水溶性铜绿假单胞菌 PST-01 有机溶剂稳定蛋白酶催化二肽的合成率

H. Ogino et al.: "Peptide Synthesis Catalyzed by Organic Solvent-Stable Protease from Pseudomonas aeruginosa PST-01 in Monophasic Aqueous-Organic Solvent Systems"Journal of Bioscience and Bioengineering. 88・5. 513-518 (1999)

H. Ogino 等人：“单相水性有机溶剂系统中铜绿假单胞菌 PST-01 的有机溶剂稳定蛋白酶催化的肽合成”《生物科学与生物工程杂志》88·5 (1999)。

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)。