甾体雌激素母核A环裂解的酶催化特征及机理研究

Contamination of steroid estrogens in aquatic ecosystems has posed a huge thread to human health. Therefore, the study of biodegradation of steroid estrogens is of great significance. Studies have revealed that nucleus cleavage of ring A and B is the key factor for the biodegradation of steroid estrogens. Although the enzymatic catalysis of ring B cleavage has been revealed, the enzyme participating in the processing of ring A cleavage still remains unknown. In this proposal, we take Mycobacterium neoaurum as model bacteria, screen the whole genome to find the possible key enzymes participating in the nucleus A cleavage by using homology alignment. Following this, key enzymes will be identified by gene knock out and complementation technic. The internal enzymatic catalysis processing of nucleus A cleavage will be confirmed according to the degradation intermediate products detected by UPLC and NMR. After getting the intermediate products and key enzymes, the in-vitro enzymatic catalysis systems will be further established. Characterization of enzymes and catalysis processing will be elucidated in-vitro with the help of ITC, aims to reveal the key factors in the enzymatic catalysis. Based on these studies, the enzymatic characterization and mechanism of nucleus cleavage of steroid estrogens will be elucidated, and application of microbial degradation technic will be more widely used in the field of bioremediation of estrogen pollution in water.

水体环境中甾体雌激素污染严重，危害人类健康，研究其降解方法具有重要意义。甾体雌激素母核B环裂解的酶催化过程已阐明，但鉴于关键酶的未知及中间产物难以富集，A环裂解的酶催化机理尚未报道。本研究拟以模式菌新金分枝杆菌为对象，利用同源比对定向筛选A环裂解过程的关键酶，结合基因敲除/回补等方法揭示体内酶催化过程，锁定关键酶；构建强化菌株和体外酶催化体系，累积关键中间产物，结合UPLC及NMR鉴定中间产物，揭示关键酶的催化功能；通过ITC研究酶催化反应的动力学和热力学，揭示关键步骤及因素，阐明A环裂解的酶催化特性及机理。研究结果将进一步揭示甾体雌激素母核裂解的酶催化特性及机理，为生物修复水体雌激素污染提供理论指导，也为生物降解技术在该领域的推广应用提供依据。

雌激素是一种内分泌干扰物，随人类或动物活动排入水体环境中，对人类健康和生态安全造成了严重的威胁。因此，降解或去除环境中雌激素的污染是全球科研领域的前沿和热点。本项目从复杂的烟草废水环境中筛选获得了多株甾体雌激素降解微生物，选择其中一株高效降解的微生物，经鉴定为假单胞菌（Pseudomonas sp. CO03），其对雌二醇的降解率可达到81.6%。进一步，对其在烟草废水中的应用进行了研究，其对雌二醇的降解率达到了53.1%，对于治理烟草废水有一定的应用前景。采用全基因组测序分析，对假单胞菌Pseudomonas sp. CO03的基因组测序及功能基因注释，发现Pseudomonas sp. CO03基因组较小，但是功能基因极其丰富，且代谢类、催化活性类的基因所占比例较大，种类丰富、数量多，推测该菌株强大催化和代谢能力的基因组特点与其来源于复杂环境有关。通过KEGG数据库分析得：Pseudomonas sp. CO03存在脱氢酶、硫酯酶、单加氧酶、双加氧酶、水合酶等与甾体雌激素相关的降解酶，并推测出一条从甾核B环开始降解的代谢通路、以C19H24O4（3,4-Dihydroxy-9,10- secoandrosta -1,3,5(10)- triene-9,17-dione）为中间产物的代谢通路。选择9种已报道的雌激素降解菌，运用比较基因组学方法进行ANI(平均核苷酸同源性)分析，发现Pseudomonas sp.CO03与假单胞菌P3B5、SJTE-3存在相同甾体雌激素降解酶基因，在分解代谢功能上可能相近。后续研究应进一步分离并克隆相应的甾体雌激素降解酶，并进行体外酶催化验证。