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°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 membrane-bound 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
Averill, B. A. and J. M. Tiedje (1982) The chemicals mechanism of microbial denitrification.FEBS Lett. 138: 8–11.
Delgado, M. J., M. Fernandez-Lopez, and E. J. Bedmar (1998) Soluble and membrane-bound nitrate reductase fromBradyrhizobium japonicum bacteroids.Plant Physiol. Biochem. 36: 279–283.
Yoshimatsu, K., T. Sakurai, and T. Fujiwara (2000) Purification and charactreization of dissimilatory nitrate reductase from a denitrifying halophilic archaeon,Haloarcula marismortui.FEBS Lett. 470: 216–220.
Song, S. H., S. H. Yeom, S. S. Choi, and Y. J. Yoo (2003) Effect of oxidation-reduction potential on denitrificationby Ochrobactrum anthropi SY509.J. Microbiol. Biotechnol. 13: 473–476.
Philippot, L. and O. Hojberg (1999) Dissimilatory nitrate reductase in bacteria.Biochim. Biophys. Acta 1446: 1–25.
Kim, S. H., S. H. Song, and Y. J. Yoo (2005) Selection of mediators for bioelectrochemical nitrate reduction.Biotechnol. Bioprocess Eng. 10: 47–51.
Martinez-Espinosa, R. M., F. C. Marhuenda-Egea, and M. J. Bonete (2001) Purification and characterization of a possible assimilatory nitrate reductase from the halophile archaeonHaloferax mediterranei.FEMS Microbiol. Lett. 196: 113–118.
Ozbbek, B. and K. O. Ulgen (2000) Stability of enzymes after sonication.Process Biochem. 35: 1037–1043.
Blumle, S. and W. G. Zumft (1991) Respiratory nitrate reductase from denitrifyingPseudomonas stutzeri, purification, properties and target of proteolysis.Biochim. Biophys. Acta 1057: 102–108.
Sung, D. W., S. H. Song, J. H. Kim, and Y. J. Yoo (2002) Effect of electron donors on nitrate removal by nitrate and nitrite reductases.Biotechnol. Bioprocess Eng. 7: 112–116.
Lee, J. W., T. O. Kwon, and I. S. Moon (2003) Chromatographic separation of maltopentaose from maltooligosaccharides.Biotechnol. Bioprocess Eng. 8: 47–53.
Meller, R. B., J. Ronnenberg, W. H. Campbell, and S. Diekmann (1992) Production of nitrate and nitrite in water by immobilized enzymes.Nature 355: 717–719.
Antipov, A. N., N. N. Lyalikova, T. V. Khiznjak, and N. P. L'vov (1999) Some properties of dissimilatory nitrate reductase lacking molybdenium and molibdenium cofactor.Biochemistry (Moscow). 64: 483–487.
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Kim, S.H., Song, S.H. & Yoo, Y.J. Characterization of membrane-bound nitrate reductase from denitrifying bacteriaOchrobactrum anthropi SY509. Biotechnol. Bioprocess Eng. 11, 32–37 (2006). https://doi.org/10.1007/BF02931865
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DOI: https://doi.org/10.1007/BF02931865