漆酶介导腐殖化反应中雌激素-腐殖酸单电子氧化的偶联机制

Estrogens from anthropogenic and natural sources are a category of hydrophobic organics widespread in the aquatic environments, which have been linked to reproductive health problems reported in humans and wild animals. It is well documented that the extracellular fungal laccase is promising for the oxidative coupling of estrogen and humic acid (HA) to form macromolecule polymers by laccase-mediated humification reactions (L-MHRs), thus reducing the biotoxicity of estrogen. However, little information is available in the literature on the oxidative coupling mechanism between estrogen and HA during L-MHRs. The primary objective of this study project is to illuminate the oxidative coupling mechanism of estrogen and humic molecules in aqueous solution by L-MHRs. To achieve the goal, a series of experiments will be conducted. Initially, batch balance experiments will be performed to explore the transformation dynamic process, regulation, and influence factor of estrogen in HA solution by L-MHRs, whereby the change of molecular conformation, functional group, size distribution, and chemical structure of HA will be investigated. Then, the chemical structure and distribution characteristic of oxidative coupling products between estrogen and humic molecules will be probed in L-MHRs based on our previous work, particularly using high-resolution mass spectrometry (HRMS) combined with 13C-labeling and nuclear magnetic resonance (NMR) spectroscopy. Finally, the binding site, formation mechanism, and environmental stability of covalent bonds into the oxidative coupling products will be characterized. These findings will have significant ramifications in the accurate assessment of the dynamic process, product distribution, and coupling mechanism for single-electron oxidation of estrogen and humic molecules in L-MHRs. This may benefit the ecological restoration measures, based on the humification reactions to lower the biotoxicity of estrogen in natural aquatic environments.

水体中雌激素能够诱发生物体内分泌失调和生殖发育异常。漆酶介导腐殖化反应（L-MHRs）可促进雌激素-腐殖酸共价偶联形成大分子聚合物，降低雌激素生物毒性，但其偶联机制仍不清楚。项目在申请人课题组前期建立高分辨质谱（HRMS）结合13C-同位素比值识别未知偶联产物的基础上，针对L-MHRs中雌激素-腐殖酸单电子氧化的偶联机制问题，研究雌激素转化的基本动力学过程、规律和影响因素，探讨反应体系中腐殖酸分子动态变化与雌激素转化的内在相关性；主要利用HRMS结合13C-同位素比值和核磁共振（NMR）技术，明确雌激素与腐殖酸分子之间形成共价偶联产物的化学结构及分布特征，揭示偶联产物的共价键结合位点、形成机理及其在环境中的稳定性。研究结果有望阐明L-MHRs中雌激素-腐殖酸单电子氧化的动力学过程、产物分布和作用机制，为基于腐殖化反应降低雌激素生物毒性的水生态修复技术提供理论依据。

水体中雌激素能够诱发生物体内分泌失调和生殖发育异常。漆酶介导腐殖化反应（L-MHRs）可促进雌激素-腐殖酸共价偶联，但其偶联机制仍不清楚。本项目采用批量平衡试验、高分辨质谱结合稳定性同位素比值、谱学分析和计算化学等方法，研究了L-MHRs中雌激素-腐殖酸单电子氧化的动力学过程、产物分布和偶联机制。主要研究结果包括：（1）通过假一级动力学和米氏方程双倒数方法，明确了L-MHRs中雌激素转化的基本动力学过程和规律；揭示了pH、酶剂量和腐殖酸浓度等关键因素对L-MHRs中雌激素转化的影响；阐明了L-MHRs中腐殖酸分子动态变化与雌激素转化的内在关联。（2）基于精确分子质量、同位素标记差值和产物相对强度比值等理论基础，鉴定了L-MHRs中雌激素-腐殖酸偶联产物；解析了L-MHRs中雌激素-腐殖酸偶联产物的形貌特征、官能团组成和化学结构；揭示了L-MHRs中雌激素-腐殖酸偶联产物的分布特征和变化规律。（3）明确了L-MHRs中腐殖酸前体物的酚羟基和醌基是影响雌激素转化和偶联的关键功能团；阐明了雌激素-腐殖酸偶联产物是以自由基介导的C−C、C−O和C−N共价结合而生成；证实了雌激素-腐殖酸偶联产物的共价键具有pH和温度稳定性，仅含有极少量松散结合的雌激素单体（< 1%）。研究成果为基于酶促腐殖化反应规避雌激素污染风险的水生态修复技术提供了理论依据和科学指导。依托项目，发表JCR Q1区SCI论文15篇（含iScience 1篇、Environ. Sci. Technol. 1篇和Water Res. 1篇）、CSCD-中文核心论文6篇；申请或授权国家发明专利4件（含授权1件）；出版学术专著1部；开展国际合作与交流2人次；培养博士/硕士研究生3名。