两种来自苏云金芽孢杆菌的多糖裂解单加氧酶的结构与催化机制研究

Lytic polysaccharide monooxygenases (LPMOs), a key enzyme involved in the biodegradation of biomass in nature, can catalyze the oxidation of biomass with the help of oxygen and electron donor. As a catalysis, LPMOs can boost bioconversion of chitin into chitin oligosaccharides or N-acetylglucosamine by chitinases, which is economically significant. The main bottleneck for this bioconversion is the low catalytic efficiency of LPMO and, the less studies on the structure and catalytic mechanism of LPMO, leading to our poor understandings on biocoversion..We have in our former work obtained two AA10 family LPMOs (BtLPMO10A and BtLPMO10B) from Bacillus thuringiensis with clearly distict sequence structure between them. Our studies indicated that BtLPMO10A and BtLPMO10B might have new catalytic mechanism in oxidatively degrade chitin. In this program, we would like to investigate the whole 3-D structures, cooperation structures of copper ion located in their active site, stucture of reaction products, and kinetics of BtLPMO10A and BtLPMO10B, harnessing some advanced instruments, like XRD, EPR, NMR and SEM, among others. The aim of our work is to illustrate the synergistic mechanism of BtLPMO10A and BtLPMO10B and their structural basis, which will provide a theoratical basis and new pathway for large scale production of chitin oligosaccharides or N-acetylglucosamine.

多糖裂解单加氧酶（LPMO）是自然界中参与生物质降解的重要的一类酶，该酶可辅助几丁质水解酶促成几丁质的高效降解，一旦被规模放大，可提升几丁寡糖或N-乙酰氨基葡萄糖的产业化生产，经济意义重大。规模放大的瓶颈是LPMO的酶解效率低，对LPMO结构与催化机制的研究较少，将其用于大规模生物转化的理论基础不足。申请者前期从苏云金芽孢杆菌中发现两个结构与功能未知的LPMO酶，后续研究表明两个酶均可氧化水解几丁质，但表现出不同的催化能力和底物选择性，更为重要的是，两者可协同氧化降解几丁质，基于此，我们推测这两个酶具有不同的结构与催化机制。本申请拟利用X射线衍射、电子顺磁共振、核磁共振等技术手段，从酶的分子结构、反应动力学和反应机制等方面进行研究，最终，在分子水平上阐明两个LPMO酶的催化机制及其结构基础，为提高LPMO的酶解效率，实现几丁质产业大规模绿色转型提供理论依据。

多糖裂解单加氧酶（LPMO）是自然界中参与生物质降解的重要的一类酶，该酶可辅助几丁质水解酶促成几丁质的高效降解，一旦被规模放大，可提升几丁寡糖或N-乙酰氨基葡萄糖的产业化生产，经济意义重大。规模放大的瓶颈是LPMO的酶解效率低，对LPMO结构与催化机制的研究较少，将其用于大规模生物转化的理论基础不足。本项目获得苏云金芽孢杆菌中BtLPMO10A、BtLPMO10B以及BtLPMO10B-N，并明确其底物结合偏好性及氧化活性；明确CBM结构域对LPMO活性功能的影响，同时揭示BtLPMO10A和BtLPMO10B与几丁质降解酶的协同作用机制；利用XRD解析BtLPMO10A和BtLPMO10B的晶体结构；利用ITC和EPR等技术方法，明确BtLPMO10A和BtLPMO10B的活性中心铜离子的结构特点，明确BtLPMO10A和BtLPMO10B氧化降解几丁质的催化机制；BtLPMO与几丁质酶协同降解南极磷虾壳，将晶体几丁质高效转化为几丁寡糖或N-乙酰氨基葡萄糖，为实现几丁质的高效生物转化提供重要的理论基础。上述工作对于揭示AA10家族LPMO氧化降解机制具有重要意义。

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