含CBM1结构域的多糖裂解单加氧酶鉴定及其底物结合机制研究

Auxiliary activities family (AA) 9 lytic polysaccharide monooxygenases (LPMO) have the ability to oxidative cleavages the glycosidic bonds of cellulose polysaccharides. The synergy of AA9LPMO and cellulase mixture will great promote the cellulase degradation efficiency. According the AA9 LPMO oxidation selectivity on glucose C1 or C4, AA9 LPMOs could be divided into three sub-families named AA9 LPMO1, AA9 LPMO2 and AA9 LPMO3. The substrate binding mechanism of AA9 LPMO is important to its activity and oxidation selectivity, but lack of in-depth research. Recent research advances suggest that the substrate binding of AA9 LPMO with cellulose is affected by three important factors: the cellulose binding CBM1 domain of LPMO, the glycosylation of the protein, the important amino acids of the protein. In this study, all the CBM1-containing AA9 LPMOs of the efficient cellulose degradation fungus Myceliophthora thermophila will be cloned and charactered. Two of the four CBM1 contained MtLPMOs belong to AA9 LPMO1, one belong to AA9 LPMO2 and one belong to AA9 LPMO3.The effect of the CBM1 domain, N-glycosylation and the important amino acids on substrate binding and catalytic activity of MtLPMOs will be studied carefully. The crystal structures of the AA9 catalytic domain of MtLPMOs (MtLPMO-AA9) and CBM1 domain of MtLPMOs (MtLPMO-CBM1) as well as the MtLPMO-AA9-substrate and MtLPMO-CBM-substrate complex will be obtained and analyzed. Based on these results, the MtLPMOs will be molecular modified to obtain high activity LPMOs. This work will elucidate the substrate binding mechanism of MtLPMOs from several aspects and deepen the knowledge of the LPMO substrate selectivity and catalytic mechanism.

AA9家族多糖裂解单加氧酶(LPMO)可氧化断裂多糖糖苷键助力糖苷酶水解，协同使用可大幅提高纤维素酶效率。依据AA9LPMO对葡萄糖C1和C4位的氧化偏好，可分为1、2、3亚家族。AA9LPMO底物结合方式影响其活性及氧化选择性，但缺乏深入研究，最新研究进展提示纤维素结合域CBM1、糖基化修饰、酶与底物接触平面的芳香族氨基酸及各亚家族保守氨基酸是影响AA9LPMO与底物结合的关键因素。本项目首先获得嗜热毁丝霉中所有含CBM1域的AA9LPMO（共4个，涵盖3个亚家族），明确其功能及性质；重点研究CBM1域、糖基化、关键氨基酸对其与底物结合进而对催化能力的影响；进一步获得其AA9催化域、CBM1域及其与底物共结晶晶体结构，直观解析其催化域、结合域与底物结合机制；利用上述信息指导分子改造，获得高效LPMO。本项目通过从多角度研究LPMO底物结合机制这一科学问题，加深对LPMO催化机制的认识。

AA9家族多糖裂解单加氧酶(LPMO)可氧化断裂多糖糖苷键助力糖苷酶水解，协同使用可大幅提高纤维素酶效率。本项目即通过从多角度研究LPMO底物结合机制这一科学问题，加深对LPMO催化机制的认识。.本项目主要研究内容包括：首先开展嗜热毁丝霉中AA9LPMO酶（MtLPMO）的克隆表达；进而研究氧化能力与酶学性质；底物特异性、活性、氧化位点选择性及与纤维素酶协同降解纤维素能力；底物结合的关键氨基酸；CBM结构域、关键氨基酸对MtLPMO与底物结合、活性、氧化位点选择的影响；利用计算化学的方法探讨LPMO酶与底物的结合模式；利用上述信息，理性设计对MtLPMO进行分子改造。在完成上述计划工作的同时还进行了拓展，开展了LPMO酶与底物结合对过氧化氢产生消耗的影响；光电驱动的LPMO酶催化体系建立等工作。.取得了一系列的科学成果：首先是获得了嗜热毁丝霉中多个AA9LPMO酶并明确其活性与酶学性质、与不同纤维素酶的协同作用效果；确定了影响MtLPMO与底物结合的关键氨基酸，明确结构域、关键氨基酸对MtLPMO与底物结合、活性的影响；进一步解析LPMO与纤维素底物结合机制，提出了新的跨纤维素链的结合机制，还建立了LPMO酶促反应过程中过氧化氢的积累与底物结合之间的关系模型，这也是本项目最有创新性的发现；基于上述结果利用理性设计方法对嗜热毁丝霉LPMO酶进行分子改造并建立了光电驱动LPMO酶催化偶联体系。.上述工作对于揭示AA9家族LPMO底物结合机制具有重要意义，也为从底物结合角度解析LPMO催化机制提供理论依据，同时也有助于高效LPMO的改造与获得。.在Journal of Physical Chemistry Letters、Carbohydrate Polymers、Bioresource Technology、International Journal of Biological Macromolecules等知名期刊发表了SCI论文15篇，目前尚有高水平文章在投稿或撰写；申请了发明专利12项；在国际糖复合物学术会议、中日韩三国酶工程会议、中国生物工程学会年会、全国糖生物学会议等国内外重要学术会议做邀请报告或分会报告多次；项目负责人获得了辽宁省“兴辽英才计划”青年拔尖人才、中国科学院青年创新促进会优秀会员等系列奖励。