Biotechnology Bulletin ›› 2014, Vol. 0 ›› Issue (7): 69-73.

• Research Report • Previous Articles     Next Articles

Separation and Purification of Hydroxylamine Oxidase from Agrobacterium tumefaciens LAD9

Chen Qian, Ma Tao, Wang Ting   

  1. The Key Laboratory of Water and Sediment Sciences, Ministry of Education, Department of Environmental Engineering, Peking University, Beijing 100871
  • Received:2013-12-02 Online:2014-07-15 Published:2014-07-16

Abstract: The metabolic pathway of heterotrophic nitrification-aerobic denitrification bacteria was directly determined by the actions of their hydroxylamine oxidase(HAO). Isolating high-purity HAO from this kind of bacteria has become particularly important to explain the mechanism of nitrogen removal. In this study, the separation and purification technic of HAO from a novel heterotrophic nitrification-aerobic denitrification strain Agrobacterium tumefaciens LAD9 was established. Electrophoretic purity of HAO could be sucessfully purified through DEAE Sepharose CL-6B ion-exchange chromatography and Sephacryl S-100 gel filtration from its periplasm. The final purification fold was 5.79 and the yield was 39.71%. SDS-PAGE eclectrophoresis results revealed that the molecular weight of HAO in the strain LAD9 was 18.8 kD. Studies on enzymatic properties showed that the purified enzyme could oxidize hadroxylamine to nitrite and its activity could be enhanced by the addition of Fe2+.

Key words: Nitrogen removal, Hydroxylamine oxidase, Purification, Agrobacterium tumefaciens, LAD9 Heterotrophic nitrification-aerobic denitrification

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