厌氧氨氧化菌特征代谢酶HZO的纯化性质及催化机制研究

The anaerobic ammonium oxidation (anammox), an innovative technology for the removal of nitrogenous contaminants from wastewater, has become a main focus in wastewater treatment. In this process, anammox bacteria can oxidize ammonium under anoxic conditions with nitrite as the electron acceptor to produce nitrogen gas. The molecular mechanism of anammox is not completely known. To fully understand the microbial functionality and mechanisms that control the anammox reaction, key proteins involved in the reaction must be identified. .It has been proposed that the anammox reaction takes place in an intracellular organelle, the anammoxosome. Hydrazine, a unique intermediate of anammox, is formed from hydroxylamine and ammonium by a hydrazine hydrolase and sequentially oxidized to dinitrogen gas by a hydrazine-oxidizing enzyme（HZO） generating four reducing equivalents. HZO is unique for anammox bacteria. In the present project, a novel hydrazine-oxidizing enzyme will be purified and characterized from a membrane aeration and separation single-stage autotrophic denitrification biological reactor in which the anammox bacteria are enriched. The HZO gene will be cloned and expressed in Escherichia coli, and the homology analysis will be carried out. The prediction of secondary structure, homology modeling analysis and nuclear magnetic resonance spectroscopy (NMR) method will reveal the structure–function principle of the hydrazine-oxidizing enzyme. These results will elucidate the catalytic molecular mechanism of HZO. The study will enhance our understanding of metabolic mechanism of anammox bacteria. This will provide a rational basis for selecting metabolic enzymes and building engineering bacteria. The industrial applications of anammox bacteria in pollute control will also be expanded.

厌氧氨氧化（anaerobic ammonium oxidation，Anammox）是去除含氮污染物的新技术，已成为污水处理的研究热点。厌氧氨氧化过程的分子机制还不清楚。联氨是Anammox 菌的中间代谢物，由联氨水解酶催化产生，通过联氨氧化酶（hydrazine-oxidizing enzyme，HZO）将联氨氧化为氮气，HZO为Anammox菌所特有的代谢酶。本项目从膜分离单级自养脱氮生物反应器中富集培养的Anammox菌中分离纯化出HZO，研究环境因子对HZO酶学性质的影响；对HZO基因进行克隆表达，同源性分析；以二级结构预测、同源建模方法、核磁共振技术研究HZO的空间结构与功能关系，阐明HZO催化的分子机制。本研究是Anammox菌代谢机理的补充和深化，为筛选Anammox菌代谢酶、构建脱氮工程菌株奠定理论基础，并拓展Anammox菌在污染物控制及资源化等方面的基础与应用研究。

厌氧氨氧化（Anaerobic ammonium oxidation，Anammox）是去除含氮污染物的新技术，已成为污水处理的研究热点。联氨是Anammox 菌的中间代谢物，由联氨水解酶催化产生，通过联氨氧化酶（hydrazine-oxidizing enzyme，HZO）将联氨氧化为氮气，HZO为Anammox菌所特有的代谢酶。.以模拟高氨氮废水为处理对象，设计了膜分离单级自养脱氮生物反应器，对氨氮及总氮的去除率分别达到了70%及60%。电镜下反应器内大部分细菌细胞聚集成花椰菜状的聚集体，呈现典型的Anammox 细菌特征。对污泥样品中细菌的16S rDNA 进行PCR 扩增、克隆、测序，所得序列提交GenBank。系统发育分析表明，所测的Anammox 菌50%与已知菌Candidates Brocadia 有较近的亲缘关系。.对厌氧氨氧化（Anammox）菌进行实验室扩大培养，制备Anammox 菌的无细胞抽提液，采用超滤、DEAE阴离子层析等方法纯化Anammox菌的特征代谢酶联氨氧化酶HZO，检测酶活性。并研究适宜温度，pH下厌氧氨氧化菌HZO的性质及外源蒽醌类化合物对厌氧氨氧化的催化反应。.富集培养反应器内的效应菌群，提取细菌总DNA，巢式PCR扩增联氨氧化酶（HZO）基因，克隆得到HZO基因，构建质粒表达载体，重组质粒转化到表达宿主菌中进行表达。SDS-PAGE电泳结果表明在pET-28a（+）诱导系统中目的蛋白有表达，但基本为不溶性表达。表达蛋白经TALON树脂纯化、浓缩、透析，得到纯化蛋白，对其进行N端氨基酸序列测定，检测酶活性。.研究可以进一步分析联氨氧化酶结构与功能的关系，说明HZO的催化机制。补充和深化Anammox菌代谢机理，为筛选anammox菌代谢酶、构建脱氮工程菌株奠定理论基础。