纤维素协同氧化体系LPMO-CDH的适配性研究

Lytic polysaccharide monooxygenase (LPMO)-cellobiose dehydrogenase (CDH) are redox partners for oxidation of oxidative cleavage of glycoside bonds of celluloses. Synergistic LPMO-CDH system offer great promise for further process improvements owing to their ability to boost the activity of biomass-degrading enzyme consortia. LPMO-CDH synergistic redox system provide celluloses with better accessible oligosaccharides by oxidative breaking cleavage of glycoside bonds of celluloses. The adaptability of LPMO and CDH determines the oxidative activity of LPMO-CDH redox system. However, there is a lack of research on this aspect at the moment. This project is focus on optimizing the structure adaptability of LPMO-CDH system through rational design to optimize the protein-protein interface for improved electron transfer and activity adaptability through co-evolution analysis. Through computational analysis, we will study the molecular reasons of the key amino acid substitutions in protein-protein interface influencing electron transfer and amino acid substitutions influencing the catalytic activity compatibility. This study will generate basic principles and new method to rationally design the synergistic system of LPMO-CDH for efficient cellulose deconstruction.

裂解多糖单加氧酶(LPMO)与纤维二糖脱氢酶(CDH)互为电子受体与供体，二者组成的协同氧化体系能够氧化断裂纤维素的糖苷键使其结构趋于松散，为纤维素水解酶提供更多作用位点，可显著提高纤维素水解酶对结晶纤维素的降解效率。高效的LPMO-CDH协同氧化体系对纤维素降解至关重要，而LPMO与CDH的结构适配性和催化活性适配性是决定其协同氧化活性的关键因素。本课题拟进行LPMO-CDH的结构适配性和催化活性适配性研究，借助计算机辅助设计手段和共进化分析，分别获得二者接触界面上影响结构适配性和各个结构域影响催化活性适配性的关键氨基酸位点；通过筛选获得结构适配性和催化活性适配性提高的突变体；结合分子动力学模拟、自由能分析和电子传递速率测定等手段分析并阐明氨基酸位点影响结构适配性和催化适配性的分子机制，为进一步优化改造LPMO-CDH氧化还原体系的纤维素糖苷键断裂能力奠定理论基础并提供新方法。

裂解多糖单加氧酶(LPMO)与纤维二糖脱氢酶(CDH)互为电子受体与供体，二者组成的协同 氧化体系能够氧化断裂纤维素的糖苷键使其结构趋于松散，为纤维素水解酶提供更多作用位点 ，可显著提高纤维素水解酶对结晶纤维素的降解效率。高效的LPMO-CDH协同氧化体系对纤维素 降解至关重要，而LPMO与CDH的结构适配性和催化活性适配性是决定其协同氧化活性的关键因素。我们开展了LPMO-CDH的结构适配性和催化活性适配性研究，借助蛋白质-蛋白质分子对接，分别获得二者接触界面上影响结构适配性和各个结构域影响催化活性适配性的关键氨基酸位点，发现NcLPMO有19个氨基酸位点与CDH的24个氨基酸位点有相互作用，其中的极性接触有五对氨基酸对；通过筛选获得催化活性提高的LPMO突变体, 其对纤维素的氧化活性高出2.7倍，在与纤维素酶协同作用的结果中，M1对结晶纤维素降解的活性高2.1倍；结合分子动力学模拟、自由能分析等手段分析关键氨基酸位点影响LPMO结构分子机制，发现其CBM结构域对催化活性有重要影响。进而优化了LPMO-CDM的催化活性适配性，发现摩尔比为2:0.4为最佳。以上工作为进一步优化改造LPMO-CDH氧化还原体系的纤维素糖苷键断裂能力奠定理论基础并提供新方法。

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