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

基本信息

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

项目摘要

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工程化组成型耐热（因此高度稳定）蛋白质来构建适用于实际应用的耐热蛋白质。该项目的主要成就之一是获得工程细胞色素C_552（Cyt c_552）。 Cyt c_552 是一种基于嗜热栖热菌的工程化 Cyt c552 的血红素蛋白。天然过氧化物酶的催化机制即使在70℃下也显示出持久的催化活性，这与在常温附近工作并在40至50℃时因变性而失活的天然过氧化物酶不同，尽管工程化的Cyt c_552的催化活性仍然较低。与天然细胞相比，在较高温度下活性增加显示了工程化 Cyt c_552 作为耐热人造细胞的潜力仔细研究工程化Cyt c_552的催化反应机制，给出了如何通过定点诱变提高催化活性的一些原则，这将在未来开发现有的人工过氧化物酶的研究中实现。该项目获得的结果表明基于所需功能的分子机制对嗜热菌进行蛋白质工程是构建在实际使用中足够稳定和耐用的人工蛋白质的有效方法，这可能有助于了解具有以下性质的常见蛋白质。已在极端微生物中发现，但由于缺乏特定功能而从未受到关注，并促进其作为多功能蛋白质资源用于有志于实际应用的蛋白质工程。