Purification and characterization of a low-temperature hydroxylamine oxidase from heterotrophic nitrifier Acinetobacter sp. Y16.
Purification and characterization of a low-temperature hydroxylamine oxidase from heterotrophic nitrifier Acinetobacter sp. Y16.
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DOI：
10.3967/bes2014.004
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发表时间：
2014-07
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影响因子：
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通讯作者：
Shu Mei Zhang;W. Li;Duoying Zhang;Xiaofei Huang;W. Qin;Changqing Sha
中科院分区：
文献类型：
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作者：
Shu Mei Zhang;W. Li;Duoying Zhang;Xiaofei Huang;W. Qin;Changqing Sha
目的从一株异养硝化细菌不动杆菌中分离纯化一种低温羟胺氧化酶。Y16，并对其酶学性质进行了研究。方法采用阴离子交换层析和凝胶过滤层析的方法从Y16菌株中分离纯化AHO。用反相高效液相色谱和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法测定其纯度和相对分子质量。以羟胺为底物，以铁氰化钾为电子受体，通过监测高铁氰化钾还原反应来检测高铁氰化钾的活性。用质谱仪测定其部分氨基酸序列。结果通过阴离子交换层析和凝胶过滤层析，从Y16菌株中分离纯化出一种相对分子质量为61 kDa的低温HAO。该酶在体外以羟胺为底物，以铁氰化物为电子受体，在较宽的温度范围(4~40℃)内表现出氧化羟胺的能力。该酶在4~15℃，pH 6.0~8.5范围内稳定，活力变化小于30%。最适温度为15℃，最适pH为7.5。用质谱仪测定了三种多肽，它们与其他报道的HAOS不完全相同。结论本研究首次从一株异养硝化菌不动杆菌中分离纯化出一株低温HAO。它在分子质量和酶性质上不同于其他已报道的HAOS。本研究的结果表明，Y16菌株经历了低温HAO催化的羟胺氧化脱氨过程。
OBJECTIVE To purify a low-temperature hydroxylamine oxidase (HAO) from a heterotrophic nitrifying bacterium Acinetobacter sp. Y16 and investigate the enzyme property. METHODS A HAO was purified by an anion-exchange and gel-filtration chromatography from strain Y16. The purity and molecular mass were determined by RP-HPLC and SDS-PAGE. The HAO activity was detected by monitoring the reduction of potassium ferricyanide using hydroxylamine as substrate and ferricyanide as electron acceptor. The partial amino acid sequence was determined by mass spectrometry. RESULTS The low-temperature HAO with a molecular mass of 61 kDa was purified from strain Y16 by an anion-exchange and gel-filtration chromatography. The enzyme exhibited an ability to oxidize hydroxylamine in wide temperature range (4-40 °C) in vitro using hydroxylamine as substrate and ferricyanide as electron acceptor. It was stable in the temperature range of 4 to 15 °C and pH range of 6.0 to 8.5 with less than 30% change in its activity. The optimal temperature and pH were 15 °C and 7.5, respectively. Three peptides were determined by mass spectrometry which were shown to be not identical to other reported HAOs. CONCLUSION This is the first study to purify a low-temperature HAO from a heterotrophic nitrifier Acinetobacter sp. It differs from other reported HAOs in molecular mass and enzyme properties. The findings of the present study have suggested that the strain Y16 passes through a hydroxylamine-oxidizing process catalyzed by a low-temperature HAO for ammonium removal.