应用于河湖底泥难降解有机物污染原位修复的固定化酶活性、构象变化及与底物交互作用的关联机制研究

Numbers of recalcitrant organics accumulated in the sediment of rivers and lakes pose a long term threat to the aqueous ecological system. The addition of immobilized enzymes into the sediment can efficiently degrade some recalcitrant organics, which will initiate or enhance the microbial degradation process. However, the conformational change of immobilized enzymes and its internal correlation with enzyme activity and interaction with sediment substrate of the immobilized enzyme are still unclear, hindering the application of immobilized enzymes in the remediation of sediment recalcitrant organics contamination and the promotion of their catalytic degradation ability. This project will select the mesoporous carbon material with adjustable pore size as the representing carrier for immobilizing several functional enzymes to be applied in the remediation of sediment contamination by phenolic recalcitrant organics in rivers and lakes. Confocal laser scanning microscope, high-resolution electron microscope, Raman spectrometry, nuclear magnetic resonance, time-resolved fluorescence spectrometry, atomic force microscope as well as molecular dynamics simulation will be used to profoundly study the spatial distribution, aggregation, activity and conformational change of the immobilized enzymes, and their interaction mechanism with the sediment substrate. The effect of surfactants on the remediation process will also be explored. The reaction mechanisms and the environmental impact of the immobilized enzyme for in-situ remediation of sediment recalcitrant organics contamination in rivers and lakes will be revealed on the micro-scale level. The research results of this project will provide theoretical evidence and technical support for profound study of in-situ remediation of sediment recalcitrant organics contamination and effective control of sediment pollution in rivers and lakes.

河流湖泊底泥中积累的大量难降解有机物对水生态系统构成长期威胁。向底泥中添加固定化酶可以高效降解某些难降解有机物，启动或强化微生物降解进程。然而，固定化酶的构象变化及其与酶活性、与底泥底物交互作用的内在关联机制仍未得到阐明，一定程度上制约了固定化酶在底泥难降解有机物污染修复中的应用及其催化降解效率的提高。本项目选择变孔径介孔碳为代表性载体，固定化多种功能酶，用于河湖底泥酚类难降解有机物污染修复。拟运用激光共聚焦显微镜、高分辨电镜、拉曼光谱、核磁共振、时间分辨荧光光谱、原子力显微镜等技术结合分子动力学模拟来深入研究固定化酶的空间分布及团聚、活性及构象变化以及与底泥底物的交互作用机制，并探究表面活性剂对污染修复过程的影响，从微观尺度阐明固定化酶应用于河湖底泥难降解有机物污染原位修复的作用机制及环境影响。项目成果将为深入研究底泥难降解有机物污染原位修复、有效控制河湖底泥污染提供理论依据和技术支撑。

项目主要针对河湖底泥中难降解有机物污染修复的焦点和难点，建立基于介孔碳载生物复合材料的吸附催化降解难降解有机物的新方法，研究其应用于河湖底泥难降解有机物污染修复的作用机制及环境影响。本项目以介孔碳为代表性载体，固定化多种污染催化降解功能酶去除河湖底泥难降解有机物机理，及其碳载体协同微生物、催化活化过硫酸盐降解转化难降解有机物的过程机制。主要研究结果如下：（1）介孔碳固定化多种降解功能酶的性能评估及其对难降解有机污染物的降解、作用机制与快速检测。固定化辣根过氧化物酶（HRP）对苯酚的催化去除可达77.83%，且酶的二级结构并没有发生明显的变化；采用物理吸附法时，较小孔径（10nm）的碳载体更有利于酶活性的保留；漆酶在载体及底泥多孔介质中均可均匀分布，无团聚；固定化HRP对底泥中2,4-二氯苯酚的降解效率可达64.93%，优于游离酶；表面活性剂促进了固定酶对苯酚的降解效果；固定化乙酰胆碱酯酶可快速测定实际样品中的有机磷农药。（2）介孔碳和介孔生物炭催化降解酚类等难降解有机物的性能与机制。研究发现不同结构的介孔碳和介孔生物炭活化过硫酸盐降解酚类污染物的机理不同，存在自由基和非自由基这两个过程的共同作用。（3）重金属共存对介孔碳材料修复酚类难降解有机物污染底泥具有影响。较低浓度的铅和对硝基酚能分别促进载铁锌生物炭对这两种物质的吸附。高浓度的铅和对硝基酚则会抑制载铁锌生物炭对这两种物质的吸附。（4）最后还探讨了碳材料协同微生物对底泥多孔环境介质中难降解有机污染物的降解转化与可利用性的影响。生物炭协同硫还原地杆菌对硝基苯去除率可提高到99%。总之，本项目通过开展系统性、多方位的研究，从微观尺度阐明以介孔碳为载体的固定化酶生物复合材料应用于河湖难降解有机物污染底泥修复的作用机制，对推动固定化酶用于河湖底泥难降解有机物污染修复技术的进步有显著的指导意义，为有效控制河湖底泥难降解有机物污染对生态环境造成的不利影响提供理论依据和技术支撑。