白腐菌木质素降解酶系交联多酶聚集体的制备及其降解酚类EDCs 的研究

Endocrine disrupting chemicals (EDCs) are pollutants with estrogenic or androgenic activity at very low concentrations and are emerging as a major concern for water quality. This growing environmental problem requires the development of cost-competitive processes capable of efficiently eliminating these EDCs and the associated hormonal disruptions. Among the bioprocesses developed, white rot fungi (WRF) and their lignin modifying enzymes (LMEs) such as laccase (Lac), Mn peroxidase (MnP) and lignin peroxidase (LiP) have been successfully used for the treatment of several EDCs. However, the detail mechanisms on the detoxification of these compounds by lignin modifying enzymes in vitro are still unknown. We make an assumption that LMEs as a whole will be organically immobilized in order to improve LMEs’ stability and use their synergistic effect on biodegradation. In our previous studies, it has been shown that the white rot fungus Trametes sp.LH-3 can degrade two phenolic EDCs and simultaneously secrete extracellular oxidases including Lac, MnP and LiP. In this research project, the three oxidases will be purified and combinedly immobilized as cross linked enzyme aggregates (combi-CLEAs). Characteristic features, activity and stability of these combi-CLEAs are investigated. The oxidative degradation of some phenolic EDCs by these combi-CLEAs will be studied. Removal of EDCs is monitored by high performance liquid chromatography (HPLC) or gas chromatography (GC).The putative metabolites of the EDCs and their possible structure are analyzed by LC–MS and hypothetical pathways of the EDCs conversion are proposed. The estrogenic activlty of EDCs is evaluated by an in vitro screening test for chemicals with hormonal activities using yeast two-hybrid system. Removal mechanisms for EDCs by combi-CLEAs will be discussed. Based on our results, it will be helpful to the development of bioprocesses capable of efficiently eliminating EDCs.

环境内分泌干扰物（EDCs）具有污染广、浓度低和危害大等特点，研究高效、新型的去除技术具有重要意义。文献表明，白腐菌的木质素降解酶系（LMEs）能降解环境异生物质，但体外应用LMEs多酶协同降解作用的研究还鲜见报道。我们设想将白腐菌LMEs中主要降解酶看成一个整体，用酶固定化技术使它们有机地共固定，以提高酶的稳定性和充分发挥多酶协同作用，进而提高降解效率。在前期研究中，发现白腐菌Trametes sp.能分泌木质素过氧化物酶、锰过氧化物酶和漆酶等主要木质素降解酶，高效降解酚类EDCs。本项目拟纯化出这些酶，用交联聚集体固定化技术将它们共固定，分析固定多酶体的特征和性质；研究固定酶降解典型酚类EDCs，应用HPLC、LC-MS等鉴定降解中间产物，分析降解途径，评估EDCs降解前后的雌激素效应，阐明共固定的LMEs协同降解作用机理。本研究结果为发展新型、高效降解EDCs技术提供理论和实践基础。

多数环境污染物包括内分泌干扰物（EDCs）和染料等具有污染广和危害大等特点，研究高效、新型的去除技术具有重要意义。文献表明，白腐菌的木质素降解酶系（LMEs）能降解环境异生物质，但体外应用LMEs多酶协同降解作用的研究还鲜见报道。我们设想将白腐菌LMEs中主要降解酶看成一个整体，用酶固定化技术使它们有机地共固定，以提高酶的稳定性和充分发挥多酶协同作用，进而提高降解效率。本课题通过层析和三相分离等方法从白腐菌中分离出的漆酶（Lac），木质素过氧化物酶（Lip）和锰过氧化物酶（MnP）等主要木质素降解酶，并制备出不同组合的多酶聚集体，其中发现Lac和MnP组合的酶聚集体协同作用最强、活性最高且性质稳定。而以磁性纳米颗粒为载体的酶聚集体有利于保证酶聚集体结构的完整性，且易于回收和重复使用。Lac和MnP的多酶聚集体主要通过聚合作用对典型EDCs双酚A进行去除，而主要通过去甲基化等方式对染料孔雀绿进行逐步分解。而通过细胞彗星电泳、植物种子发芽率、微生物抑制率等实验证实了目标化合物降解后，其分解产物对环境生物的毒性显著降低。研究结果表明，白腐菌木质素降解酶系组成的交联多酶聚集体是一种去除环境EDCs和其它污染物的有效工具，值得在环境污染处理的实际应用中进一步研究和探索。