嗜热菌漆酶LacTT催化染料脱色作用机理的NMR解析

我国环境污染日益严重，染料废水是主要污染源之一，研发高效、环境友好的处理技术成为热点。我们前期研究表明：嗜热菌漆酶能较好地对染料废水进行脱色脱毒处理，然而，存在着不同染料该漆酶作用效率的差异。由此本项目提出漆酶催化染料分解作用机理的研究，期待进一步提升或拓展漆酶对染料废水的脱色作用。项目基于NMR这一表征蛋白结构和运动的有效工具、结合酶反应动力学等方法，研究漆酶在催化氧化染料过程中结构和运动变化，了解催化循环过程中各个态的结构及态之间的转化过程；进而解析漆酶结合底物的多样性与专一性，探索影响漆酶氧化还原势、电子转移途径扰动的关键因素，揭示促进和影响漆酶催化作用的机制；从而建立漆酶结构、运动与功能的关系。此外，拟构建漆酶突变子，筛选提高催化效率和/或增加氧化还原势的正突变，分析验证其催化染料脱色的机制。研究结果将为拓展漆酶催化氧化底物、有效定向改造漆酶以提升其处理染料废水的能力提供理论基础。

The environmental pollution in our country becomes more and more severe. Dyestuff wastewaters from the textile industry are the major pollution source and very difficult to process. Developing new processing technology of high efficiency and environment-benign nature thus arouses wide interest. Our previous work showed that the laccase from thermus thermophiles SG0.5JP17-16 （LacTT）can effectively decolorize and detoxicify dyestuff wastewaters. However, there exists evident difference at effectiveness when using this laccase to process different dyestuffs. We therefore propose to investigate the underlied mechanisms with the expectation of enhancing the processing capability of laccase as applied for decolorization and detoxicification of dyestuff wastewaters. This project will take dye compounds as substrates, and employ NMR―the powerful tool for characterizing structure and dynamics of proteins, in combination with other suitable methods such as the kinetics study method for enzyme, to detect structural and dynamic changes of the laccase during a single catalytic cycle, and to characterize the structures of states and the transition between states. Further, we will analyze the diversity and specificity of the substrate catalyzed by the laccase, find the structural and dynamic determinants that affect the redox of the laccase and/or disrupt the pathway of the electric transfer, and reveal the mechanisms for improving and/or reducing the catalytic functions of the laccase. Based on these, we will establish relationships between the dynamics, structure and function of the laccase. In addition, we will design and screen some mutants that can increase the redox of the laccase and keep the catalytic efficiency high, and verify the mechanisms that these mutants decolorize and detoxify dyestuff wastewaters. This project is expected to provide novel ideas to enlarge the family of substrates catalyzed by the laccase and to modify the laccase for improving its capability for processing dyestuff wastewaters.