The purification of thermostable enzymes from a thermophilic hydrogen oxidizing bacterium and the development of a energy conversion system with those enzymes

从嗜热氢氧化细菌中纯化热稳定性酶并利用这些酶开发能量转换系统

• 批准号: 03453084
• 负责人: OKURA Ichiro
• 金额: $ 4.22万
• 依托单位: Tokyo Institute of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03453084/
• 关键词: Thermophilic Hydrogen; Hydrogenase; Ferredoxin Oxidizing Bacterium; Iron-Sulfur Cluster; 水素発生反応; 好熱性細菌; ヒドロゲナ-ゼ

In this work, we purified a ferredoxin and a hydrogenase from a thermophilic hydrogen oxidizing bacterium, Bacillus schlegelii and studied their biochemical and biophysical properties. The ferredoxin contains one [3Fe-4S] and one [4Fe-4S] cluster in a molecule. The amino acid sequence of the ferredoxin is homologous to other 7Fe type ferredoxin. This ferredoxin is active as an electron transfer agent in a reaction system for the reduction of cytochrome c consisting of NADPH, FNR, and cytochrome c. Only [3Fe-4S] cluster acts as the active site for the electron transfer reaction in the reduction of cytochrome c. This is caused by too low redox potential of the [4Fe-4S] cluster to be reduced in the reaction system.The hydrogenase purified from the bacterium is an up-take type hydrogenase that catalyzes the oxidation of hydrogen predominantly. This hydrogenase possesses 50% of the initial activity after 5 and 2.5 hours of heat treatment at 70 and 80ﾟC, respectively showing is very thermostable.

本论文从嗜热氢氧化细菌施氏芽孢杆菌（Bacillus schlegelii）中分离纯化了铁氧还蛋白和氢酶，并对其生物化学和生物物理性质进行了研究。铁氧还蛋白分子中含有一个[3Fe-4S]和一个[4Fe-4S]簇。该蛋白的氨基酸序列与其它7 Fe型铁氧还蛋白的氨基酸序列同源。该铁氧还蛋白在由NADPH、FNR和细胞色素c组成的细胞色素c还原反应体系中作为电子转移剂具有活性。在细胞色素c还原反应中，只有[3Fe-4S]原子簇是电子转移反应的活性中心。这是由于[4Fe-4S]团簇的氧化还原电位太低而不能在反应体系中被还原所造成的。从该菌中纯化的氢化酶是一种主要催化氢氧化的摄取型氢化酶。在70 ℃和80 ℃下分别热处理5和2.5小时后，该氢化酶具有50%的初始活性，显示出非常耐热。

项目成果

中田 伸治: "ヒドロゲナ-ゼを用いたNADH再生系によるオキサロ酢酸の還元" 日本化学会誌. 12号. 1688-1690 (1991)

Shinji Nakata：“使用氢化酶通过 NADH 再生系统还原草酰乙酸”，日本化学学会杂志第 12 期。1688-1690 (1991)。

M.Shimoda: "Selective inhibition of methanol dehydrogenase from Methylosinus trichosporium(OB3b)by cyclopropanol" J.Mol.Catal.64. L23-L25 (1991)

M.Shimoda：“环丙醇对毛孢甲基窦菌 (OB3b) 的甲醇脱氢酶的选择性抑制”J.Mol.Catal.64。

S.Aono: "Characterization and thermostability of a membrane-bound hydrogenase from a thermophilic hydrogen oxidizing bacterium,Bacillus schlegelii" Biosci.Biotech.Biochem.57. 1177-1179 (1993)

S.Aono：“来自嗜热氢氧化细菌，Bacillus schlegelii 的膜结合氢化酶的表征和热稳定性”Biosci.Biotech.Biochem.57。

M.Shimoda, Y.Seki, I.Okura: "Oxidation of halogenated propanes with Methylosinus trichosporium (OB3b)" J.Mol. Catal.83. L5-L10 (1993)

M.Shimoda、Y.Seki、I.Okura：“用毛孢甲基窦 (OB3b) 氧化卤化丙烷”J.Mol。

M.Shimoda: "Oxidation of allyl compounds with Methlosinus trichosporium(OB3b)" J.Mol.Catal.78. 27-30 (1993)

M.Shimoda：“用毛孢甲藻 (OB3b) 氧化烯丙基化合物”J.Mol.Catal.78。