title: Purification and characterization of a low-temperature hydroxylamine oxidase from heterotrophic nitrifier Acinetobacter sp. Y16.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors: Institute of Microbiology, Heilongjiang Academy of Sciences, Harbin 150010, Heilongjiang, China.
authors: Shu Mei
authors: SM
authors: Zhang
authors: School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, Heilongjiang, China; State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, Heilongjiang, China.
authors: Wei Guang
authors: WG
authors: Li
authors: School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, Heilongjiang, China.
authors: Duo Ying
authors: DY
authors: Zhang
authors: School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, Heilongjiang, China.
authors: Xiao Fei
authors: XF
authors: Huang
authors: School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, Heilongjiang, China.
authors: Wen
authors: W
authors: Qin
authors: Heilongjiang Academy of Sciences, Harbin 150010, Heilongjiang, China.
authors: Chang Qing
authors: CQ
authors: Sha
mesh_terms: D000150:Acinetobacter / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D003080:Cold Temperature; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D010088:Oxidoreductases; C021401:hydroxylamine dehydrogenase
keywords: Acinetobacter sp. Y16; Heterotrophic nitrifier; Hydroxylamine oxidase; Purification