水体剧毒有机锡微生物降解的分子机制与速效降解调控

Organotins are highly toxic pollutants persistently existed in the global environment for their worldwide use. To accelerate their biodegradation, information regarding the biodegradation mechanisms, including degradation reaction,functional genes, key enzymes and regulation method of degradation, should be revealed. Therefore, the study on the behavior and mechanism of triphenyltin biodegradation by Rhodopseudomonos spheroids will be conducted. To explore the rate-limiting reaction and biodegradation pathway,the concentrations of metabolic intermediates, such as diphenyltin, monophenyltin and small molecule organic compounds will be detected by chromatography, mass-spectrography, nuclear magnetic resonance, X-ray photoelectron spectroscopy and stable isotope label technique. The protein electrophoresis, mass spectrum identification, bioinformatics analysis and gene clone will be performed to identify the proteomics characteristics of R. spheroids, the expression of differentially expressed proteins and the functional genes. Moreover, functional verification will reveal the key enzymes of triphenyltin degradation and the catalytic reactions, which are catalyzed by them. After then, to determine the relations among influencing factors, degradation enzymes, degradation reactions of triphenyltin and production rate of intermediates, the effects of some key factors on proteomics and enzymes expression of R. spheroids during triphenyltin biodegradation will be investigated. Through the study of the current project, the mechanisms of organotins biodegradation will be revealed, and the results can offer theoretical guide for organotins bioremediation.

有机锡是持久性剧毒污染物，为加速水体有机锡的微生物降解，需深入探究有机锡烃基降解和碳锡键断裂的反应规律、关键酶、功能基因及降解调控的科学问题。为此，本项目将利用色谱、质谱、能谱、核磁共振和稳定性同位素示踪等技术，定量分析高效降解菌（球形红假单胞菌）降解三苯基锡过程产生的各种苯基锡和小分子有机产物；确定有机锡降解的限速反应并阐明其降解途径。通过蛋白质分离、质谱鉴定、生物信息学分析、功能基因克隆和降解酶功能验证技术，分析降解菌的蛋白质组学特征、差异蛋白的表达规律，并确定有机锡降解的功能基因、关键酶及其催化的反应步骤，揭示有机锡降解的分子机制；分析关键因素在促进有机锡降解过程对菌体蛋白质组和关键酶的影响，从而明确有机锡速效降解的调控技术，探明关键影响因素、降解酶表达水平、有机锡降解速率以及产物生成速率的内在关系，最终阐明有机锡的微生物降解机理，为指导水体有机锡的微生物修复提供理论依据与技术支持。

有机锡是持久性剧毒污染物，为加速水体有机锡的微生物降解，需深入探究有机锡烃基降解和碳锡键断裂的反应规律、关键酶、功能基因及降解调控的科学问题。为此，本项目利用色谱、质谱、能谱等技术，定量分析了高效降解菌降解三苯基锡过程产生的各种苯基锡和小分子有机产物；确定了三苯基锡的降解始于碳锡键断裂。降解途径为三苯基锡→二苯基锡→一苯基锡→无机锡。通过蛋白质分离、质谱鉴定、生物信息学分析、功能基因克隆和降解酶功能验证技术，探明了细胞色素CYP450是三苯基锡的降解酶。该酶基因片段全长1214 bp，具有一个完整的开放阅读框，编码404个氨基酸残基，等电点为5.57，理论蛋白分子量为46.87 kDa；表达了该酶的大肠杆菌E. coli BL21 CYP450对三苯基锡的降解率可达到54.6%。CYP450的亚铁血红素中心与三苯基锡的锡结合，启动三苯基锡的降解。三苯基锡被CYP450催化过程需要NADH为整个反应过程提供电子。三苯基锡中碳锡键的断裂会产生苯酚和二苯基锡，二苯基锡再以三苯基锡相同的降解路径进一步降解生成一苯基锡和无机锡。代谢物从酶反应中心释放后，CYP450酶又返回到它的最初状态。蛋白质组学研究结果表明，三苯基锡降解过程会诱导丙酮酸及氨基酸代谢、能量代谢、糖酵解、磷酸戊糖途径、三羧酸循环、核糖体代谢、蛋白合成相关蛋白的上调。项目研究成果对指导有机锡污染水体的修复，有效地降低其环境危害和生态风险具有重要的科学意义和应用价值。研究成果对其他种类有机金属污染物的微生物治理也具有借鉴作用。课题组共8人参与了项目的研究，3人晋升高一级职称，4名研究生顺利毕业；项目负责人晋升教授，并以第6完成人获得了2017年度教育部高等学校科学研究优秀成果一等奖；发表SCI论文11篇、中文核心期刊论文1篇；获授权发明专利1项。