溶解性多糖单加氧酶与糖苷水解酶的底物协同竞争机制研究

Non-starch polysaccharides are known to act as antinutrient factors in plant-derived feed diet, which can be effectively alleviated by enzyme additives. However, traditional glycosyl hydrolase (GH) showed negligible effects on highly crystalline regions among polysaccharide chains. Recently, the lytic polysaccharide monooxygenase (LPMO) is reported as novel lignocellulose-degrading enzymes that break glycosidic bonds of polysaccharides via oxidation. The enzymes boost lignocellulose deconstruction, by introducing new chain breaks that allow hydrolases to initiate further degradation. Notably, possible competitive substrate-binding between LPMO and GH is observed at early phase of catalysis, resulting in decreased release velocity of reducing sugars. In this study, we employed transcriptome technique to screen key genes involved in polysaccharide-degradation pathways, especially novel LPMOs with multisubstrates-active and desired properties, from lignocellulose induced Phanerochaete chrysosporium. The enzymatic properties and substrate-degradation pattern, as well as relationships between key amino acid residues and catalysis will be characterized. After that, competition mode and inhibition-type between LPMO and GH will be studied. Furthermore, molecular docking and molecular dynamics will be used to simulate movement locus and conformational alternation of proteins, and the substrate synergic competition will be finally elucidated.

非淀粉多糖是植物性饲料中的一种主要抗营养因子，在饲料中添加酶制剂能有效缓解这个问题。然而，传统的糖苷水解酶很难降解多糖链之间致密有序的结晶区。溶解性多糖单加氧酶（LPMO）是一种全新的多聚糖降解酶。它以氧化形式切断多聚糖长链，释放出更多还原端利于糖苷水解酶进一步催化，发挥协同增效降解作用。值得注意的是，LPMO与糖苷水解酶在协同催化多聚糖时，反应前期存在底物竞争结合现象，限制了还原糖的释放速率。基于以上现象，本研究利用富含木质纤维素的底物诱导培养黄孢原毛平革菌，采用转录组学技术研究多聚糖降解途径中差异表达的关键功能基因，挖掘具备多底物催化活性或优良酶学性质的新型LPMO基因；分析其酶学性质、底物降解模式和关键氨基酸残基与其功能的关系，进一步采用抑制动力学法系统研究酶的竞争方式和抑制类型，采用分子对接和分子动力学法研究蛋白运动轨迹与构象偏差，阐明LPMO与糖苷水解酶的底物协同竞争机制。

裂解多糖单加氧酶（LPMO）是一种全新的多聚糖降解酶，以氧化形式切断多聚糖长链，释放出更多还原端利于糖苷水解酶（GHs）进一步催化。本研究筛选了多个LPMO与GHs基因，研究其催化活性和酶学性质，进一步通过生化试验、光谱与质谱技术、分子模拟等手段研究LPMO与GHs的协同竞争机制。主要结果如下：.（1）基因挖掘与性质表征：获得5条LPMO基因，其中BsLPMO10A具有广泛的活性底物谱，高效催化β-1，4糖苷键连接的多糖，如β-（Man1→4Man），β-（Glc1→4Glc）和β-（Xyl1→4Xyl），主要释放聚合度为3-6的天然低聚寡糖和氧化寡糖；获得并表征2个葡聚糖酶、2个果胶酶和1个几丁质酶的催化性质。.（2）LPMO与GHs的协同催化作用：BsLPMO10A以C1氧化方式打断糖苷键与葡聚糖酶、木聚糖酶、纤维素酶和几丁质酶等GHs发挥协同催化作用，显著提高底物的糖化效率；此外，BsLPMO10A可以提高商业化复合酶制剂Celluclast®1.5 L对滤纸、花生秸秆和水稻秸秆的降解能力。.（3）LPMO与GHs的竞争抑制作用：提出并验证了LPMO与GHs催化过程中的三种竞争作用机制，即（I）游离LPMO释放H2O2对GHs产生氧化损伤；（II）LPMO通过“占位”方式与GHs发生底物性竞争结合；（III）LPMO生成的氧化产物抑制GHs的催化效率。.综上所述，本项目深入研究LPMO的酶学性质和催化模式并阐明了LPMO与糖苷水解酶的协同竞争机制，为畜牧生产实践中合理使用复合酶制剂、降低生产成本建立了有力基础。