Characterization of the enzyme system catalyzing Anammox

我们的实验室正在进行熊本地下水处理的厌氧氨氧化（Anammox）生物膜培养物的富集和表征。为了研究成熟生物膜群落的细菌组成，通过PCR扩增16 S rDNA序列，并使用DNA数据库进行比较分析。只有一个序列与第一个发现的厌氧氨氧化浮游菌-荧光成像显示KSU-1形成球形簇，包裹在一层薄薄的动胶菌中。我们试图纯化羟胺氧化还原酶（hao），它似乎是厌氧氨氧化的关键酶。从富集的厌氧氨氧化生物膜中提取hao，用DEAE-纤维素Superdex凝胶过滤柱层析纯化，并用纤维素膜透析，通过小于50 kDa的分子。SDS-PAGE分析表明，纯化后的hao具有均一性，其分子量约为110 kDa。虽然hao的光谱与欧洲亚硝化单胞菌相似，但肼对hao的光谱变化大于羟胺，表明hao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.