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用途的耐热蛋白质。本项目的主要成果之一是工程化细胞色素C_552（Cyt c_552）获得过氧化物酶活性。Cyt c_552是来源于嗜热栖热菌的血红素蛋白。基于天然过氧化物酶催化机理的基因工程Cyt c552在70 ℃下仍显示出持久的催化活性，这与天然过氧化物酶在常温下工作，在40 ~ 50 ℃下由于变性而失活不同。虽然工程化的Cyt c_552的催化活性仍然低于天然的，但在较高温度下增加的活性显示工程化的Cyt c_552作为耐热人工过氧化物酶的潜力。通过对Cyt c_552基因工程菌催化反应机理的深入研究，提出了通过定点突变提高其催化活性的原理，本项目的研究结果表明，基于分子机制的嗜热菌蛋白质工程是构建人工蛋白质的有效方法其在实际使用中足够稳定和耐用。这可能揭示了在极端微生物中发现的但由于缺乏特异性功能而从未被关注的常见蛋白质，并促进作为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.