深海邻苯二甲酸酯降解真菌的降解机制研究

Plastic products give a lot of convenience in our daily lives. However, their wide utilization also causes serious environmental pollution problems. Phthalate esters (PAEs) are used as plasticizers in plastic products and these compounds have been identified as toxic pollutants due to their endocrine-disrupting functions. With the rapid development of plastic industry, PAEs may pose serious threat in marine ecological system. Marine microorganisms have been demonstrated the potential to degrade PAEs. However, most of current reports focus on coastal microorganisms, including mangrove microorganisms. Degradation study of these compounds by deep sea microorganisms is comparatively scarce, and no reports so far are available for degradation of PAEs by deep sea fungi. In our previous studies, three fungi capable of degrading PAEs have been isolated from deep sea sediments, including Rhodotorula mucilaginosa MAR-Y3, Rhodosporidium paludigenum MAR-Y4, and Aspergillus sp. IR-M4. It was found that Rhodotorula mucilaginosa MAR-Y3 proceeded through different biochemical pathways to degrade three isomers of dimethyl phthalate esters (DMPEs), and there is a new unpublished pathway for degradation of dimethyl phthalate by the fungus. Therefore, this study aims to investigate the biochemical degradation pathways of DMPEs by three deep sea fungi, to assess the general and specific characters of degradation pathways for different deep sea fungal species, and to characterize the molecular and catalytic characteristics of key enzymes (esterase and dioxgenase) involved in the degradation of PAEs. The results will contribute to understanding of the metabolic and enzymatic basis of degradation mechanisms of PAEs by deep sea fungi.

邻苯二甲酸酯（PAEs）作为塑料产品中的增塑剂已被发现具有内分泌干扰毒性。随着塑料工业的发展，这类物质对海洋生态环境造成巨大的威胁。海洋微生物已证明具有降解PAEs的潜力。但是目前的研究主要集中在近海包括红树林环境的微生物。深海微生物特别是深海真菌PAEs降解的研究还未见报道。在前期的研究中，我们从大洋深海底质中分离获得3株高效PAEs降解真菌,并发现其中Rhodotorula mucilaginosa MAR-Y3这株菌对于邻苯二甲酸二甲酯（DMPEs）的三种同分异构体的降解途径有很大的不同，且对于邻位的苯二甲酸二甲酯的降解存在着一条未知的新降解途径。本项研究拟对3株深海真菌的DMPEs降解途径进行分析，探求不同种属的深海真菌PAEs降解途径的共性与特性。同时对降解过程涉及的关键酶--酯酶、双加氧酶进行分子特征与催化活性研究，从化学代谢、酶蛋白两方面探索深海真菌的PAEs降解机制。

本研究对两株深海真菌Rhodotorula mucilaginosa MAR-Y3和Aspergillus versicolor IR-M4降解邻苯二甲酸酯（PAEs）的代谢途径及其降解过程中的关键酶的酶学特征进行研究。发现两株深海真菌均无法完全矿化邻苯二甲酸二甲酯（DMPEs），而是将其转化为各自的单甲酯（MMPEs）或苯二甲酸。两株真菌对于不同同分异构体DMPEs的降解途径均有较大的差异。两株真菌都能降解间位和对位的DMPEs（DMI和DMT）。其可通过两步的酯水解反应将DMT转化为对苯二甲酸（TA），而对于DMI，则只能水解苯环上的一个酯键，将其转化为单甲酯（MMI）。TA和MMI均为菌株代谢的终产物，无法被进一步降解。对于邻位的DMPEs（DMP），R. mucilaginosa MAR-Y3可水解苯环上相邻两个酯键中的一个，将其转化为单甲酯（MMP），但无法继续降解MMP；而A. versicolor IR-M4无法降解DMP。这些研究结果表明两株深海真菌对于DMPEs苯环上两个酯键的水解具有高度的底物选择性。进一步以DMI为底物诱导R. mucilaginosa MAR-Y3菌株产酶并纯化获得一个酯酶蛋白。该酯酶除了能水解其诱导底物DMI外，还能水解DMT，而对于DMP和MMPEs的三种同分异构体均无水解能力，体现了高度的底物选择性与特异性。这些研究结果暗示深海真菌对于DMPEs不同同分异构体的降解途径的差异，并不是由单个酯酶引起的，而是有不同的酯酶参与到不同的酯键水解当中。我们进一步对一株近海真菌Fusarium sp. DMT-5-3的DMI降解酯酶进行纯化，比较深海、近海来源两株真菌DMI降解酯酶的异同。结果表明，两个酯酶在酶学特征（温度、pH、金属离子对酶活影响等）以及底物特异性上高度相似，但在分子特征上有极大的差异。深海真菌R. mucilaginosa MAR-Y3来源的酯酶为单亚基蛋白，分子量只有27kDa；而近海真菌Fusarium sp. DMT-5-3所产的酯酶为二亚基蛋白，分子量为76kDa。这一结果表明，尽管深海、近海来源的真菌DMPEs降解酯酶在分子特征上有很大差异，但都具有高度的底物选择性与特异性。这些研究结果增进了对深海真菌PAEs降解相关机制的认识，也为深海真菌在海洋环境中PAEs污染的生物治理提供了理论基础。