黑曲霉辅助蛋白AnLPMO9强化木质纤维素降解的分子机制

Lytic polysaccharide monooxygenases (LPMOs) can efficiently improve the degradation of lignocellulose, which makes it to be the most promising accessory protein in lignocellulose biodegradation. However, the action mechanism of LPMOs has been unclear because of its structural diversity, which seriously hinders this accessory protein to play an efficient role in the low-cost conversion and utilization of lignocellulose biomass. LPMOs from Aspergillus niger (AnLPMO9) is the research subject in this project. In order to reveal the molecular mechanism of the oxidation of cellulose chain by AnLPMO9, the modular structure of AnLPMO9 will be studied to analyze the active sites, and the oxydate of AnLPMO9 acting on cellulose will be analyzed and identified. Then, in order to reveal the synergistic mechanism between AnLPMO9 and cellulase, the interaction sites between AnLPMO9 and cellulase will be digged further, and their binding and distribution on cellulose substrates will be analyzed. Finally, based on the above research, the influence of hemicellulose and lignin on the action and synergy of AnLPMO9 will be studied, and the electron transport chain of lignin as the electron donor will also be analyzed to reveal the oxidation process of cellulose chain by AnLPMO9 mediated by lignin. The research findings of this project will not only clearly reveal the molecular mechanism of lignocellulose degradation, but also provide theory evidence and guide the artificial remix of efficient enzyme cocktails for ligonocellulose degradation. It will be of great significance for the low-cost conversion and utilization of lignocellulose biomass.

裂解性多糖单加氧酶（LPMOs）可有效促进木质纤维素的降解，是目前最具应用前景的木质纤维降解辅助蛋白。但由于结构组成多样化，具体作用机制不清楚，严重影响该蛋白在木质纤维素低成本转化中发挥有效作用。本课题拟以黑曲霉LPMOs（AnLPMO9）为研究对象，通过研究AnLPMO9的结构域组成，解析其结合与催化活性位点，构建氧化产物谱，揭示其氧化裂解纤维素多糖链的分子机制；深入挖掘AnLPMO9与纤维素酶相互作用位点，解析它们在纤维素底物上的结合分布规律，揭示纤维素降解中AnLPMO9与纤维素酶的协同机制；探究AnLPMO9催化反应中半纤维素和木质素的影响作用，以及木质素作为电子供体的电子传递链，解析其介导AnLPMO9氧化断裂纤维素多糖链的过程。本项目的研究有助于揭示木质纤维素高效降解的分子机制，同时为高效木质纤维降解酶系复配和辅助蛋白分子改造提供可靠的理论依据，对木质纤维素低成本转化意义重大。

裂解性多糖单加氧酶（LPMOs）可有效促进木质纤维素的降解，是目前最具应用前景的木质纤维降解辅助蛋白。为丰富目前有限的LPMO资源，使该类蛋白在木质纤维素低成本转化中发挥有效的作用，本项目对来源于黑曲霉的两个AA9家族LPMO进行了研究，完成了两个酶的生物信息学分析，结构域组成解析，基因表达及催化特性研究等工作。底物特异性分析表明它们均可作用于微晶纤维素、羧甲基纤维素、木质纤维素（滤纸、草粉、玉米芯等）和木聚糖底物，MALDI-TOF/TOF分析AnLPMO水解微晶纤维素产物谱，并揭示了其氧化裂解纤维素糖苷键的位置在C1位；同源建模预测了AnLPMO具有免疫球蛋白样β-三明治折叠结构，Cu离子活性中心的组氨酸支架结构以及Cu离子结合氨基酸残基（AnLPMO14g中为HIC1、His86和Tyr175，AnLPMO15g中为His1、His78和Tyr164）位点；解析了AnLPMO与糖苷水解酶的相互作用以及它们对底物的竞争性关系，确定了协同度最高时的临界底物竞争点，揭示了纤维素降解中AnLPMO与糖苷水解酶的协同作用机制；探究了木质素对AnLPMO催化活性的影响，验证了木质素作为电子供体的电子传递模型，即可溶性的低分子量木质素小分子提供并传递电子启动AnLPMO的氧化反应，解析了木质素介导AnLPMO氧化断裂纤维素糖苷键的过程。此外，项目团队对AnLPMO15g进行了分子改造，筛选获得3个酶活力以及温度稳定性均得到提高的突变体蛋白。本项目成果既为揭示高效木质纤维降解的分子机制提供可靠的理论依据，又为木质纤维素高效降解酶系的人工复配提供了丰富的LPMO资源，对木质纤维素生物质的低成本生物转化意义重大。

内容获取失败，请点击重试