Biotechnology and Bioprocess Engineering:BBE

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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Characterization of Membrane-bound Nitrate Reductase from Denitrifying Bacteria Ochrobactrum anthropi SY509

  • Kim Seung-Hwan (Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University) ;
  • Song Seung-Hoon (Bio-MAX Institute, Seoul National University) ;
  • Yoo Young-Je (Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, School of Chemical and Biological Engineering, Seoul National University)
  • Published : 2006.01.01
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Abstract

In this study, we have purified and characterized the membrane bound nitrate reductase obtained from the denitrifying bacteria, Ochrobactrum anthropi SY509, which was isolated from soil samples. O. anthropi SY509 can grow in minimal medium using nitrate as a nitrogen source. We achieved an overall purification rate of 15-fold from the protein extracted from the membrane fraction, with a recovery of approximately 12% of activity. The enzyme exhibited its highest level of activity at pH 5.5, and the activity was increased up to $70^{\circ}C$. Periplasmic and cytochromic proteins, including nitrite and nitrous oxide reductase, were excluded during centrifugation and were verified using enzyme essay. Reduced methyl viologen was determined to be the most efficient electron donor among a variety of anionic and cationic dyestuffs, which could be also used as an electron donor with dimethyl dithionite. The effects of purification and storage conditions on the stability of enzyme were also investigated. The activity of the membranebound nitrate reductase was stably maintained for over 2 weeks in solution. To maintain the stability of enzyme, the cell was disrupted using sonication at low temperatures, and enzyme was extracted by hot water without any surfactant. The purified enzyme was stored in solution with no salt to prevent any significant losses in activity levels.

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References

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