Engineering of proteins from thermophilic bacteria as a scaffold for thermally tolerant artificial enzyme

嗜热细菌蛋白质工程作为耐热人工酶的支架

• 批准号: 18560753
• 负责人: NAKAJIMA Hiroshi
• 金额: $ 2.51万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18560753/
• 关键词: Cytochrome c_<552>; Myoglobin; Thermophile; Peroxidase; Heme Proteins; Thermus thermophirus; 機能性タンパク質; 人工酵素

A final goal of the present research project is to construct thermally tolerant proteins applicable to practical use by engineering constitutively heat resistant (and therefore highly stable) proteins from thermophile. One of the major achievements of the project is engineered cytochrome C_552 (Cyt c_552) acquiring the peroxidase activity. Cyt c_552 is a heme protein deriving from Thermus thermophilus. Engineered Cyt c552 on the basis of the catalytic mechanism of natural peroxidases shows durable catalytic activity even at 70 ℃, which is distinctive to natural ones that work at around normal temperature and are deactivated at 40 to 50 ℃ due to denaturation. Although the catalytic activity of the engineered Cyt c_552 is still lower than that of natural ones, increasing activity at the higher temperature shows potential of the engineered Cyt c_552 as a thermally tolerant artificial peroxidase. Close inspection to the catalytic reaction mechanism of the engineered Cyt c_552 gives some principles of how to improve the catalytic activity by site-directed mutagenesis, which will be achieved in future studies to develop the present artificial peroxidase.Results obtained in the project demonstrate that the engineering of proteins from thermophiles based on molecular mechanisms of desired functions is a potent methodology to construct artificial proteins that are enough stable and durable in practical use. This may shed a light on common proteins that have been found in extremophile but never been focused due to lack of specific function, and promote usage as versatile protein resources for protein engineering aspiring practical use.

本研究项目的最终目标是构建适用于工程学的热耐受性蛋白质，可从嗜热剂中组成耐热（因此高度稳定）蛋白质。该项目的主要成就之一是获得过氧化物酶活性的细胞色素C_552（CYT C_552）。 Cyt C_552是一种血红素蛋白，它源自嗜热菌。基于天然过氧化物酶的催化机制，工程性的Cyt C552即使在70℃下也显示出耐用的催化活性，这与天然温度左右的自然作用，并且由于变性而在40至50℃下停用。尽管工程Cyt C_552的催化活性仍然低于天然的催化活性，但在较高温度下的活性升高显示出工程的Cyt C_552作为热耐受的人工过氧化物酶的潜力。 Close inspection to the catalytic reaction mechanism of the engineered Cyt c_552 gives some principles of how to improve the catalytic activity by site-directed mutagenesis, which will be achieved in future studies to develop the present artificial peroxidase.Results obtained in the project demonstrate that the engineering of proteins from thermophiles based on molecular mechanisms of desired functions is a potential methodology to construct artificial proteins that are enough stable and在实际使用方面耐用。这可能揭示了在极端质体中发现的常见蛋白质，但由于缺乏特定功能而从未集中注意力，并促进用作用作多功能蛋白质资源，用于蛋白质工程有抱负的实践用途。

项目成果

シトクロムP450BSβの基質誤認識を利用した非天然基質の水酸化反応

利用细胞色素 P450BSβ 的底物错误识别对非天然底物进行羟基化

• 发表时间: 2006
• 作者: 荘司長三;中島洋;渡辺芳人
• 通讯作者: 渡辺芳人

Watanabe Regulation of catalytic activity of cytochrome P450BSβ by non-natural substrates

Watanabe 非天然底物对细胞色素 P450BSβ 催化活性的调节

• 发表时间: 2006
• 作者: Shoji;O;Nakajima;H
• 通讯作者: H

Creation of thermally tolerant artificial enzymes

耐热人工酶的创建

• 发表时间: 2007
• 作者: Nakajima;H
• 通讯作者: H

Chemical engineering of proteins from Thermophiles

嗜热菌蛋白质的化学工程

• 发表时间: 2008
• 期刊: Gakujyutu Geppou 61
• 作者: Nakajima;H;Watanabe;Y
• 通讯作者: Y

Reactivides of oxo and peroxo intermediates studied by hemoprotein mutants

通过血红素蛋白突变体研究氧化和过氧中间体的反应物

• 发表时间: 2007
• 期刊: Accounts of chemical research 40
• 作者: Watanabe Y;Nakajima H;Ueno T;et. al.
• 通讯作者: et. al.