Characterization of the enzyme system catalyzing Anammox

我们的实验室正在进行厌氧氨氧化（Anammox）生物膜培养物的富集和表征，用于熊本地下水的处理。为了研究成熟生物膜群落的细菌组成，采用PCR扩增16S rDNA序列，并与DNA数据库进行比较分析。只有一个序列与首次发现的厌氧氨氧化植物菌Candidatus Brocadia anammoxidans有显著的相似性（92.2%），新发现的菌株（命名为KSU-1）在生物膜群落的可检测成员中占主导地位。荧光成像显示KSU-1在Zoogloea sp.的薄层中形成球形团簇，我们试图纯化羟胺氧化还原酶（hao），羟胺氧化还原酶被认为是厌氧氨氧化的关键酶。从富集的Anammox生物膜中提取，用DEAE-cellulose Superdex凝胶过滤柱层析纯化，用小于50 kDa分子的纤维素膜进行透析。经SDS-PAGE分析，纯化的郝蛋白具有均匀性，其条带比厌氨氧化候选菌的郝蛋白小110 kDa。虽然与欧洲亚硝基单胞菌的光谱相似，但联氨对hao的光谱变化比羟胺大，表明其对肼的亲和力高于羟胺。

The enrichment and characterization of anaerobic ammonium-oxidizing (Anammox) biofilm cultures for the treatment of groundwater in Kumamoto, are ongoing in our laboratories. To investigate the bacterial composition of the mature biofilm community, 16S rDNA sequences were amplified by PCR and comparative analyses using DNA databases were conducted. Only one sequence had a notable similarity (92.2%) to that of the first discovered anaerobic ammonium-oxidizing planctomycete, Candidatus Brocadia anammoxidans, The newly discovered strain (designated KSU-1) was dominant among detectable members of the biofilm community. By fluorescence imaging, KSU-1 was shown to form spherical clusters wrapped in a thin layer of Zoogloea sp.We attempted to purify the hydroxylamine oxidoreductase(hao) seemed as a critical enzyme in Anammox. The hao was extracted from the enriched Anammox biofilm, purified with DEAE-cellulose Superdex gel filtration column chromatography, and dialyzed with a cellulose membrane passing less than 50 kDa molecules. The purified hao was homogeneous by SDS-PAGE, exhibiting a 110 kDa band smaller than that of hao purified from Candidatus Brocadia anammoxidans. Although the hao showed similar spectra to that from Nitrosomonas europeae, hydrazine gave lager spectral change of the hao than hydroxylamine, suggesting it has higher affinity to hydrazine than hydroxylamine.