细菌漆酶CueO的铜激活机理及应用基础研究

Laccases （EC 1.10.3.2）are a class of blue multi-copper oxidases that catalyze the four-electron reduction of oxygen to water coupled with the oxidation of small organic substrates. The ability of laccases to act on more than 250 organic compounds makes them highly useful green biocatalysts for various biotechnological applications, such as paper bleaching, environment protection, food industry,bioenergy, and so on. Due to the limits in production costs and yields of fungal laccases, the widely used laccases in industry, it is particularly urgent to develop low-cost laccases with prolific quantities production for distinct industrial applications. Bacterial laccases would be good candidates for large scale industrial applications. CueO, a laccase located in E. coli periplasm, has little or no oxidizing activity toward organic substances. However, this activity is enhanced considerbly in the presence of added copper. The mechanism of CueO activated by exogenous copper remains controversial and indecipherable to date, which poses a major impediment to the application of CueO as industrial oxidant.In this proposal,copper-binding unnatural amino acids will be incorporated into the possible sites of copper binding outside the active center of CueO. The activation mechanisms of CueO by copper will be systematically investigated by comparing the relative activity, the reduction potential, and conformation of CueO mutants.The mutants with the highest laccase activity independent of copper will be as the starting point to further improve its activity by engineering of directed evolution. The last variant of the evolution process with high laccase activity and low-cost mass production will be of great significance in industrial applications.

漆酶属于蓝色多铜氧化酶，是一种理想的绿色化学工业催化剂，广泛应用于造纸、环境保护、食品、生物能源等工业领域。由于目前市场应用的真菌漆酶存在诸多产能问题，细菌漆酶的研发尤为迫切。大肠杆菌铜耐受蛋白CueO自身漆酶活性较低，但铜离子可以显著提高其漆酶活性，这种激活机制目前并不清楚，极大的阻碍了CueO的应用研发。本课题将通过基因编码铜离子结合氨基酸：3-吡唑酪氨酸和8-羟基喹啉氨基酸，在CueO的活性中心外可能的铜结合位点整合3-吡唑酪氨酸或8-羟基喹啉氨基酸，比较不同部位铜结合对酶活性、还原电势，分子构象等的影响，阐明铜离子提高CueO漆酶活性的分子机制；并在此不依赖外源铜离子的高漆酶活性CueO基础上进行酶的蛋白质工程改造，进一步提高酶的活性，使之成为工业化应用的理想漆酶。

漆酶是一种理想的绿色化学工业催化剂，广泛应用于造纸，生物能源等领域。目前市场应用的真菌来源漆酶存在严重的产能问题，而细菌漆酶的应用研发还处于初级阶段。阻扰细菌漆酶应用的最大问题是酶活性普遍低于真菌漆酶。大肠杆菌铜耐受蛋白CueO漆酶活性较低，但铜离子可以显著提高其漆酶活性，目前机制不明。为了阐明铜离子激活CueO漆酶活性机制，本项目将铜结合非天然氨基酸通过遗传密码扩展方法插入到CueO表面可能的铜结合位点，比较这些铜结合蛋白的酶活性、氧化还原电势和构象等，期望得到不依赖于环境铜离子的细菌漆酶CueO，在此基础上对CueO进行蛋白质工程改造，为CueO的工业化应用奠定基础。