Studies on development of super-enzymes from marine hyperthermophiles

海洋超嗜热生物超级酶的开发研究

• 批准号: 07556048
• 负责人: SATO Yoshihiko
• 金额: $ 4.1万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07556048/
• 关键词: Hyperthermophiles; Archaea; Hydrothermal vent; Heat stable enzyme; CO_2 fix; protease; DNA polymerase; CO_2固定; PCR

In this study, we firstly isolate the useful marine hyperthermohpilic bacteria for genetic engineering, medicine, food science and chemical engineering. Secondly we purify heat and chemical stable enzymes and clone their genes for the development of unknown genetic resources.The results are as follows ;(1)New anaerobic hyperthermophilic archaeon Pyrococcus sp.growing optimally at 100ﾟC was isolated from a shallow marine hydrothermal vent in Tachibana Bay. The gene for DNA polymerase of this archaeon was cloned and expressed in E.coli. The amino acid sequence of this gene was closely relate to that of pol A from P.furiosus.(2)New aerobic thermophilic bacterium Rhodothermus obamensis OKD7 growing optimally at 80ﾟC was isolated from the same station. CO_2 fixiing enzyme (PEPC) was purified and characterized. The allosteric enzyme PEPC was a homotetramer of 400kDa and showed maximum activity at 75ﾟC.The cloned gene of PEPC was expressed in E.coli. A thermostable recombinant PEPC showed similar property of the wild type.(3)A novel aerobic hyperthermophilic archaeon Aeropyrum pernix K1 growing optimally at 90ﾟC was isolated from a costal soltataric vent at Kodakara Island. Strain K1 produced an extracellular metalloprotease with a molecular mass of 52kDa. The optimum temperature for the enzyme was 100ﾟC.The enzyme was extremely thermostable showing half-life of 1.2h at 125ﾟC.

本研究首次从海洋中分离到了对基因工程、医药、食品、化工等有重要意义的嗜热细菌。研究结果如下：（1）从日本立花湾浅海热液喷口分离到一株最适生长温度为100 ℃的厌氧超嗜热古菌Pyrococcus sp.。克隆了该古细菌的DNA聚合酶基因，并在大肠杆菌中表达。该基因的氨基酸序列与P.furiosus的pol A的氨基酸序列有很高的同源性。（2）从同一站位分离到一株新的好氧嗜热菌Rhodothermus obamensis OKD 7，其最适生长温度为80 ℃。对CO_2固定酶（PEPC）进行了纯化和性质研究。变构酶PEPC为400kDa的同源四聚体，在75 ℃具有最大活性。热稳定的重组PEPC显示出与野生型相似的性质。(3)A从Kodakara岛的海岸盐沼喷口分离到一株新的嗜热古菌Aeropyrum pernix K1，其最适生长温度为90 ℃。菌株K1产生一种胞外金属蛋白酶，分子量为52kDa。该酶的最适反应温度为100 ℃，在125 ℃时半衰期为1.2h，具有极强的热稳定性。

项目成果

石野 良純: "PCR用DNAポリメラーゼの改良「細菌工学」" 秀潤社, 51 (1997)

Yoshizumi Ishino：“用于 PCR‘细菌工程’的 DNA 聚合酶的改进” Shujunsha，51 (1997)

K.Takai: "ppc,the gene for phosphenolpyruvate carboxylase thermophilic bacterium,Rhodothermus obamensis:cloning,sequencing and overexpression in Escherichia coli" Microbiology. (in press). (1998)

K.Takai：“ppc，嗜热细菌，Rhodothermus obamensis 的磷酸酚丙酮酸羧化酶基因：在大肠杆菌中的克隆、测序和过表达”微生物学。

Y.Sako et al.: "Rhodothermus obamensis sp.nov., a modern lineage of extremely thermophilic marine bacteria." Int.J.Syst.Bacteriol. 46. 1099-1104 (1996)

Y.Sako 等人：“Rhodothermus obamensis sp.nov.，极端嗜热海洋细菌的现代谱系。”

K.Takai et al.: "Extremely thermostable phosphoenolpyruvate carboxylase from an extreme thermophile, Rhodothermus obamensis" J.Biochem. 122. 32-40 (1997)

K.Takai 等人：“来自极端嗜热菌，Rhodothermus obamensis 的极其热稳定的磷酸烯醇丙酮酸羧化酶”J.Biochem。

Uemori T. et al.: "The hyperthermophilic archaeon Pyrodictiumoccultum has two a-like DNA polymerase" J. Bacteriol.177. 2164-2177 (1995)

Uemori T. 等人：“超嗜热古菌 Pyrodictium ocultum 有两个 a 样 DNA 聚合酶”J. Bacteriol.177。