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

本研究项目的最终目标是通过从嗜热菌中提取本构耐热（因此高度稳定）的蛋白质来构建适用于实际使用的耐热蛋白质。该项目的主要成果之一是工程细胞色素C_552 （Cyt C_552）获得过氧化物酶活性。Cyt c_552是一种源自嗜热热菌的血红素蛋白。根据天然过氧化物酶的催化机理而设计的Cyt c552在70℃下也具有持久的催化活性，这与天然过氧化物酶在常温下工作，在40 ~ 50℃时因变性而失活的特点不同。虽然工程Cyt c_552的催化活性仍低于天然Cyt c_552的催化活性，但在较高温度下活性的提高显示了工程Cyt c_552作为耐热人工过氧化物酶的潜力。通过对Cyt c_552的催化反应机理的深入研究，得出了如何通过位点诱变提高其催化活性的一些原理，这将是今后研究开发现有人工过氧化物酶的一个重要方向。该项目的结果表明，基于所需功能的分子机制对嗜热菌蛋白质进行工程设计是一种有效的方法，可以构建在实际使用中足够稳定和持久的人工蛋白质。这可能会揭示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的常见蛋白质，并促进作为具有实际用途的蛋白质工程的多功能蛋白质资源的使用。

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.