高甘露糖型N-糖基化修饰对灰盖鬼伞菌漆酶Lcc9酶学性质的影响

Fungal laccase is a kind of polyphenol oxidase containing copper. It has important application value in the development of new drugs, the bioenergy refining and pulping wastewater treatment field. Most of the sugar chains of fungal laccase belong to high mannose glycosylation modification, which might affect on the structure and catalytic activity of laccase. However, at present, there is no systematic study on the effect of glycosylation modification on the structure and function of fungal laccase. Our previous study found that enzyme activity of Coprinus cinereus laccase Lcc9 was greatly increased after heterologously expressed in Pichia pastoris, and the stability and kinetic constants of Lcc9 were changed after removal of glycosylation, which indicated that glycosylation might play an important role in the enzymatic properties of Lcc9. Therefore, we took rLcc9 and several glycosylated mutants as the research objects, analyzed the effects of the position of glycosylation modification, the length of sugar chain on enzymatic properties of Lcc9, explored the effect of glycosylated modification on the structure of Lcc9 by circular two chromatography or ultraviolet fluorescence. Our results provide a theoretical basis for the molecular mechanism of N- glycosylation modification affecting the catalytic activity and stability of fungal laccase，and also lay a foundation for the engineering fungal laccase to improve its catalytic efficiency.

真菌漆酶是一类含铜的多酚氧化酶，在新型药物开发、生物能源炼制和纸浆废水处理等领域有重要潜在的应用价值。真菌漆酶糖链大多属于高甘露糖型N-糖基化修饰，糖链的差异对漆酶的结构与功能有重要影响。然而，目前并没有系统的研究糖基化修饰对真菌漆酶结构和功能影响的报道。前期，我们发现灰盖鬼伞菌漆酶Lcc9在毕赤酵母中异源表达后，rLcc9比酶活远高于野生型Lcc9，去除糖基化修饰后rLcc9稳定性及动力学常数发生变化，暗示糖基化修饰可能对Lcc9的酶学性质有重要作用。因此，本项目拟以毕赤酵母中表达的rLcc9及去糖基化突变体为研究对象，分析糖基化修饰位置、糖链长度对Lcc9酶学性质的影响，确定糖链位置、糖链长短的改变对漆酶Lcc9整体结构的影响。研究结果为明晰N-糖基化修饰影响真菌漆酶催化活性和稳定性的分子机制提供理论依据，也为开展真菌漆酶的工程改造进而提高其催化应用效率奠定一定基础。

本项目以灰盖鬼伞菌中漆酶Lcc9和在毕赤酵母中表达的rLcc9及去糖基化突变体为研究对象，发现灰盖鬼伞菌漆酶Lcc9在毕赤酵母中异源表达后 ，rLcc9比酶活远高于野生型Lcc9，去除糖基化修饰后rLcc9稳定性及动力学常数发生变化 ，暗示糖基化修饰可能对Lcc9的酶学性质有重要作用。进一步分析了糖基化修饰位置对Lcc9酶学性质的影响，确定糖链位置的改变对漆酶Lcc9整体结构的影响。相同的蛋白在不同的宿主菌中表达，rLcc9较Lcc9而言，提高了分子量大小、比酶活以及稳定性。利用Endo H糖苷水解酶去除rLcc9的N-糖基化修饰获得drLcc9。脱糖基化修饰后drLcc9的比酶活明显下降；且通过生物信息学方法预测了Lcc9中含有3个潜在的N-糖基化位点（N293、N313和N454），并通过定点突变、同源建模深入研究了不同位置的糖基化修饰对rLcc9比酶活的影响，进一步阐明不同的糖基化位点对于rLcc9比酶活的具体作用。该项研究结果将有助于了解糖基化对真菌漆酶生化特性的影响，为基于N-糖基化修饰提高真菌漆酶活性的酶工程改造提供理论依据.