基于T1铜离子氧化还原电位的芽孢杆菌漆酶催化活性调控机理的研究

Laccase is useful in food processing, such as beverage clarification, theaflavin biosynthesis, emulsion stability, etc. Compared with fungal laccase, bacterial laccase has the characteristics of thermal and pH stability, but with low catalytic activity. The Type I (T1) copper ion in the active center of laccase is the initial site of the electron capture from the substrate. However, the redox potential of T1 copper ion (T1-RP) of bacterial laccase mostly lower than that of fungal laccase. Lower T1-RP is one of the reasons for the low catalytic activity of bacterial laccase. Why does the same covalent primary coordinating structure of T1 copper ion produce different T1-RPs and different catalytic activity? It is presumed that the reason may be that the distribution of the electron of copper ion was influenced by the non-covalent secondary coordinating structure. In this study, the laccase (fmb-rL103) from the Bacillus vallismortis fmb-103 strain will be grown crystals, and X-ray diffraction techniques will be used to resolve their structure. The three-dimensional structures of fmb-rL103 and fungal laccase will be compared and analyzed. Then, molecular modification based on the crystal structure information, will be applied on the non-covalent secondary coordinating structure of T1 copper ion, to obtain series mutated enzymes with different T1-RP. Ultraviolet spectroscopy and Electron Paramagnetic Resonance techniques will be applied to analyze the differences of T1 copper spectroscopic properties in mutated enzymes. The mutated enzymes will be grown crystals and the structures will be resolved. The secondary coordinating structure characteristics of T1 copper ion in mutated enzymes will be analyzed, such as hydrophobic environment, hydrogen bonds and Van Edward force, etc. The catalytic properties of the mutated enzymes toward to different substrates will be determined, and the catalytic reaction will also be investigated by molecular dynamics simulation. The results will clarify the relationship between the non covalent secondary coordinating structure of T1 copper ion and T1-RP and catalytic activity, and provide a theoretical basis for improving the bacterial laccase activities.

漆酶在饮料澄清、茶黄素合成、乳化液稳定等方面具有应用价值。真菌漆酶稳定性差，易失活，细菌漆酶稳定性虽好，但催化活性低，限制了使用范围。细菌漆酶T1铜离子氧化还原电位（T1-Redox Potential, T1-RP）低于真菌漆酶，是影响酶催化活性的原因之一。细菌漆酶与真菌漆酶具有相同的T1铜离子共价初级定位结构，但为何产生不同的氧化还原电位和催化活性？推测其原因可能是铜离子电子云分布受非共价次级定位结构的影响。本项目以芽孢杆菌漆酶为研究对象，制备并解析其晶体结构，与真菌漆酶结构进行比对分析后，对T1铜离子非共价次级定位结构进行分子改造；测定突变酶T1-RP，及光谱学、波谱学特征；分析突变酶T1铜离子次级定位结构的疏水性、氢键、范德华力等规律特征；比较不同T1-RP突变酶催化特性。研究结果将阐明T1铜离子的非共价次级定位结构与T1-RP、酶催化活性的关系，为改良细菌类漆酶活性提供理论依据。

漆酶在饮料澄清、茶黄素合成、蛋白质交联等方面具有应用价值。真菌漆酶稳定性差，易失活。细菌漆酶稳定性好，但催化活性低，限制了使用范围。针对以上问题，本项目首先实现了Bacillus vallismortis fmb-L103漆酶在E.coli中的高效胞外分泌表达，达到 84916.67±2817 U/L，并从基因转录水平和细胞水平分析了其分泌机理。采用计算机辅助策略，运用ChemBioDraw软件、Pymol软件、AutoDock Vina软件，明确了重组fmb-rL103漆酶T1铜离子5Å范围内，发现氨基酸L386、P419、I494及H497属于双重关键氨基酸，与T1铜离子氧化还原电位、底物结合结合效率均相关。采用丙氨酸扫描、迭代突变、组合突变技术，对R416、T418位点的改造，获得了催化效率（Kcat)提高10倍的漆酶突变体。对突变体的性质进行了表征，首次发现重组fmb-rL103对黄曲霉毒素B1具有催化降解作用，且突变体催化降解效率进一步提高。对链霉菌漆酶T1铜离子非共价次级定位loop结构进行分子改造，获得了突变体漆酶GGN；发现Fe2+对GGN的酶活性具有显著的激活效果，同时GGN对于酚酸类物质（没食子酸，愈创木酚）亲和性显著增强；圆二色谱分析，GGN漆突变酶的柔性结构含量增多。建立并优化了fmb-rL103漆酶的结晶条件，获得了野生型与突变型漆酶的晶体。上述研究结果，揭示了细菌漆酶催化活性与T1铜离子氧化还原电位、底物结合口袋结构的关系，为分子改造提高细菌类漆酶催化活性提供了一定的理论依据。