假诺卡氏菌CB1190降解四氢呋喃的代谢机理及关键酶研究

The cyclic ether pollutant tetrahydrofuran (THF) is used in large amounts for various industrial purposes. Pseudonocardia dioxanivorans strain CB1190 has been identified to utilize THF as a sole source of carbon and energy. It was predicted that the four-gene cluster thmADBC of strain CB1190 is involved in assembly of a new member of multi-component diiron monooxygenases. Transformation of THF was observed by whole cells of Rhodococcus jostii RHA1 harboring thmADBC, but the oxidation product has not yet been identified and no biochemical properties of this enzyme have been characterized. In addition, no metabolic intermediate or catabolic gene (except for thmADBC) involved in THF biodegradation by strain CB1190 has ever been identified. This study will present the first purification of ThmADBC. Based on this, the product of oxidized THF will be identified and the characteristics of the multi-component enzyme, such as the biochemical and physical properties of each component, substrate range and kinetic parameters, will be examined. Besides that, based on the reported genomic and transcriptomic data of strain CB1190, the complete THF catabolic genes will be obtained by bioinformatics approaches and genomic library construction. The function of each gene will be identified through methods of gene knockdown, metabolic intermediates identification, enzyme expression in vitro, and enzyme assay. And in this study, the pathway and mechanisms of THF metabolism in strain CB1190 will be elucidated at molecular, biochemical, and genetic levels. The data present here will be greatly helpful to understand the diverse capabilities for the microbial degradation mechanisms of organic pollutants and the functional characterization of thmADBC genes will certainly promote the theoretical study of this family of multi-component oxygenases. Meanwhile, it will provide a theoretical basis and technical instruction for bioremediation of tetrahydrofuran in the contaminated environment.

四氢呋喃（THF）是一种大宗工业环醚类污染物。假诺卡氏菌CB1190能以THF为唯一碳源能源进行生长。基因簇thmADBC被推测编码一种新的多组分含双核铁单加氧酶，全细胞生物转化证明其能转化THF，但产物未鉴定，更无酶学性质研究的报道。该菌株THF代谢中的中间产物及除thmADBC外的代谢基因也从未被鉴定。本研究将首次纯化ThmADBC，鉴定THF催化产物，研究其理化特征、底物范围及酶学动力学参数等性质；同时，基于该菌株已有的基因组和转录组数据，本研究将通过生物信息学分析和文库构建来获得完整的THF降解基因；基因敲低、代谢产物积累分析结合基因体外表达、酶活测定等方法将用于分析这些基因的功能，从而在分子、生化和遗传学水平上阐明THF完整的代谢途径。该研究将有助于理解微生物代谢有机污染物的多样性，推动此多组分加氧酶家族的理论研究，同时为THF的生物修复提供理论基础和技术指导。

四氢呋喃（THF）是一种大宗工业环醚类污染物。假诺卡氏菌上基因簇thmADBC被推测编码一种新的多组分含双核铁单加氧酶，但无酶学性质研究的报道。本研究检测到ThmADBC的粗酶活性，并尝试了不同的蛋白表达体系和亲和标签，分别成功纯化出ThmADBC的氧化酶组分ThmAB、偶联蛋白ThmC和还原酶组分ThmD，并对后者进行了酶学性质测定。由于可能需要一些辅因子或分子伴侣蛋白，目前纯化后的ThmADBC的酶活检测体系在研究中。由于氧化反应常在微生物代谢途径中发挥重要功能，我们同时对其它一些有机物代谢的氧化酶进行了功能鉴定和酶学研究。在2，4-二甲苯酚代谢中氧化代谢中间产物4-羟基间苯二甲酸生成原儿茶酸的基因序列之前没有被发现，本研究通过转座子突变的方法在Pseudomonas putida NCIMB 9866中发现了该关键酶4-羟基间苯二甲酸羟化酶的基因序列hipH并对其编码的重组蛋白HipH-His6的酶学性质进行研究；通过基因文库的构建和生化分析，本研究同时在Arthrobacter sp. strain YF-06中筛选出了新的具有潜在工业应用价值的黄嘌呤氧化酶XodAB，其大小亚基的序列进化与已知的黄嘌呤氧化酶有明显区别，同时具有强的热稳定性。本部分取得的成果将为研究四氢呋喃代谢提供研究基础，有助于了深入了解氧化酶在不同有机物的微生物代谢途径中的功能多样性。另外，研究基于李斯特菌的笼形蛋白Dps，在蛋白内腔进行了超小铈纳米颗粒的生物矿化，构建出具有高超氧化物歧化酶活性的人造模拟酶Ce-Dps。相较于天然酶，模拟酶更易于设计、制备且稳定性更强，因此模拟酶的研究对于传统的微生物酶学研究及应用是一个有力的补充，也有助于学科交叉融合。