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通过从嗜热菌中设计出组成耐热(因此高度稳定)的蛋白质来构建适用于实际应用的耐热蛋白质。该项目的主要成果之一是工程细胞色素C_552(Cyt C_552)获得了过氧化物酶活性。细胞色素C_(552)是一种来源于嗜热嗜热菌的血红素蛋白。基于天然过氧化物酶催化机理的工程细胞色素C552即使在70℃也显示出持久的催化活性，这与天然过氧化物酶在大约常温下工作，并在40至50℃由于变性而失活是不同的。尽管工程细胞色素c_552的催化活性仍低于天然细胞色素c_552，但在较高温度下活性增加显示了工程细胞色素c_552作为一种耐热人工过氧化物酶的潜力。通过对工程细胞Cyt c_552催化反应机理的深入研究，提出了通过定点突变提高催化活性的一些原理，这将在未来的人工过氧化物酶研究中实现。本项目的结果表明，基于预期功能的分子机制从嗜热菌中工程蛋白质是一种有效的方法，可以在实际应用中构建足够稳定和持久的人造蛋白质。这可能会揭示在极端细菌中发现的但由于缺乏特定功能而从未被关注的常见蛋白质，并促进其作为多功能蛋白质资源的用途，为有志于实际应用的蛋白质工程提供参考。

项目成果

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

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

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.