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Purification and characterization of the NAD(P)H-nitroreductase for the catabolism of 2,4,6-trinitrotoluene (TNT) inPseudomonas sp. HK-6

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Abstract

The NAD(P)H-nitroreductase of thePseudomonas sp. HK-6 which is capable of catabolizing 2,4,6-trinitrotoluene (TNT), was purified and biochemically characterized. The specific activity of the purified TNT nitroreductase was approximately 1.47 units/mg, and was concentrated to 10.1-fold compared to the crude extract. The optimal temperature and pH of the highest nitroreductase activity was 30°C and 7.5, respectively. The substrate specificity test revealed that the nitroreductase exhibited the highest enzyme activity for the TNT substrate of the nitroaromatic compounds tested in this study. Moreover, the molecular weight of the TNT nitroreductase was approximately 27 kDa on the SDS-PAGE. The N-terminal amino acid sequence of the purified protein was 5′-MDTVSLAKRRYTTKAYDASR, which is identical topnrB ofPseudomonas putida JLR11, and is capable of TNT reduction. The molecular analysis of the approximately 650-bp PCR product, orginating from the HK-6, revealed that the oxygen-insensitive NAD(P)H-nitroreductase gene, which transforms TNT in strain HK-6 with five unique amino acid sequences and diverges from the nitroreductases identified so far inPseudomonas, Burkholderia, andRalstonia, is frequently found amidst the powerful degraders of aromatic compounds.

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References

  1. Robertson, B. K. and P. K. Jjemba (2005) Enhanced bioavailability of sorbed 2,4,6-trinitrotoluene (TNT) by a bacterial consortium.Chemosphere 58: 263–270.

    Article  CAS  Google Scholar 

  2. Bryant, C. and M. DeLuca (1991) Purification and characterization of an oxygen-insensitive NAD(P)H-nitroreductase fromEnterobacter cloacae.J. Biol. Chem. 266: 4119–4125.

    CAS  Google Scholar 

  3. Kim, S. H., S. H. Song, and Y. J. Yoo (2006) Characterization of membrane-bound nitrate reductase from denitrifying bacteriaOchrobactrum anthropi SY509,Biotechnol. Bioprocess Eng. 11: 32–37.

    Article  CAS  Google Scholar 

  4. McCormick, N. G., F. E. Feeherry, and H. S. Levinson (1976) Microbial transformation of 2,4,6-trinitrotoluene and other nitroaromatic compounds.Appl. Environ. Microbiol. 31: 949–958.

    CAS  Google Scholar 

  5. Vorbeck, C., H. Lenke, P. Fischer, J. C. Spain, and H. J. Knackmuss (1988) Initial reductive reactions in aerobic microbial metabolism of 2,4,6-trinitrotoluene.Appl. Environ. Microbiol. 64: 246–252.

    Google Scholar 

  6. Fiorella, P. D. and J. C. Spain (1997) Transformation of 2,4,6-trinitrotoluene byPseudomonas pseudoalcaligenes JS52.Appl. Environ. Microbiol. 63: 2007–2015.

    CAS  Google Scholar 

  7. Chang, H. W., H. Y. Kahng, S. I. Kim, J. W. Chun, and K. H. Oh (2004) Characterization ofPseudomonas sp. HK-6 cells responding to explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine).Appl. Microbiol. Biotechnol. 65: 323–329.

    Article  CAS  Google Scholar 

  8. Haidour, A. and J. L. Ramos (1996) Identification of products resulting from the biological reduction of 2,4,6-trinitrotoluene, 2,4-dinitrotoluene and 2,6-dinitrotoluene byPseudomonas sp.Environ. Sci. Technol. 30: 2365–2370.

    Article  CAS  Google Scholar 

  9. Riefler, R. G. and B. F. Smets (2000) Enzymatic reduction of 2,4,6-trinitrotoluence and related nitroarenes: Kinetics linked to one-electron redox potentials.Environ. Sci. Technol. 34: 3900–3906.

    Article  CAS  Google Scholar 

  10. Oh, B. T., G. Sarath, and P. J. Shea (2001) TNT nitroreductase from aPseudomonas aeruginosa strain isolated from TNT-contaminated soil.Soil Biol. Biochem. 33: 875–881.

    Article  CAS  Google Scholar 

  11. Haynes, C. A., R. L. Koder, A. F. Miller, and D. W. Rodgers (2002) Structure of nitroreductase in three states.J. Biol. Chem. 277: 11513–11520.

    Article  CAS  Google Scholar 

  12. Wang, C. J., S. Thiele, and J. M. Bollag (2002) Interaction of 2,4,6-trinitrotoluene (TNT) and 4-amino-2,6-dinitrotoluene with humic monomers in the presence of oxidative enzymes.Arch. Environ. Contam. Toxicol. 42: 1–8.

    Article  CAS  Google Scholar 

  13. Perez-Reinado, E., R. Blasco, F. Castillo, C. Moreno-Vivian, and M. D. Roldan (2005) Regulation and characterization of two nitroreductase genes,nprA andmprB, ofRhodobacter capsulatus.Appl. Environ. Microbiol. 71: 7643–7649.

    Article  CAS  Google Scholar 

  14. Peterson, F. J., R. P. Mason, J. Hovsepian, and J. L. Holtzman (1979) Oxygen-sensitive and-insensitive nitroreduction byEscherichia coli and rat hepatic microsomes.J. Biol. Chem. 254: 4009–4014.

    CAS  Google Scholar 

  15. Watanabe, M., T. Nishino, K. Takio, T. Sofuni, and T. Nohmi (1998) Purification and characterization of wildtype and mutant “classical” nitroreductase ofSalmonella typhimurium. L33R mutation greatly diminishes binding of FMN to the nitroreductase ofS. typhimurium.J. Biol. Chem. 273: 23922–23928.

    Article  CAS  Google Scholar 

  16. Nivinskas, H., R. L. Koder, Z. Anusevicius, J. Sarlauskas, A. F. Miller, and N. Cenas (2001) Quantitative structure-activity relationships in two-electron reduction of nitroaromatic compounds byEnterobacter cloacae NAD(P)H: nitroreductase.Arch. Biochem. Biophys. 385: 170–178.

    Article  CAS  Google Scholar 

  17. Nokhbeh, M. R., S. Boroumandi, N. Pokorny, P. Koziarz, E. S. Paterson, and I. B. Lambert (2002) Identification and characterization of SnrA, an inducible oxygen-insensitive nitroreductase inSalmonella enterica serovar Typhimurium TA1535.Mutat. Res. 508: 59–70.

    CAS  Google Scholar 

  18. Rafii, F., R. Wynne, T. M. Heinze, and D. D. Paine (2003) Mechanism of metronidazole-resistance by isolates of nitroreductase-producingEnterococcus gallinarum andEnterococcus casseliflavus from the human intestinal tract.FEMS Microbiol. Lett. 225: 195–200.

    Article  CAS  Google Scholar 

  19. Caballero, A., J. J. Lazaro, J. L. Ramos, and A. Esteve-Nunez (2005) PnrA, a new nitroreductase-family enzyme in the TNT-degrading strainPseudomonas putida JLR11.Environ. Microbiol. 7: 1211–1219.

    Article  CAS  Google Scholar 

  20. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 72: 248–254.

    Article  CAS  Google Scholar 

  21. Kim, H. G., Y. Kim, H. M. Lim, H. J. Shin, and S. W. Kim (2006) Purification, characterization, and cloning of trimethylamine dehydrogenase fromMethylophaga sp. strain SKI.Biotechnol. Bioprocess Eng. 11: 337–343.

    Article  CAS  Google Scholar 

  22. French, C. E., S. Nicklin, and N. C. Bruce (1998) Aerobic degradation of 2,4,6-trinitrotoluene byEnterobacter cloacae PB2 and by pentaerythritol tetranitrate reductase.Appl. Environ. Microbiol. 64: 2864–2868.

    CAS  Google Scholar 

  23. Bollag, D. M., M. D. Rozycki, and S. J. Edelstein (1996)Protein Method. 2nd ed., Wiley-Liss, Inc., New York, NY, USA.

    Google Scholar 

  24. Yoon, Y. C., H. J. Park, N. K. Lee, and H. D. Paik (2005) Characterization and enhanced production of enterocin HJ35 byEnterococcus faecium HJ35 isolated from human skin.Biotechnol. Bioprocess Eng. 10: 296–303.

    Article  CAS  Google Scholar 

  25. Yun, S. H., C. Y. Yun, and S. I. Kim (2004) Characterization of protocatechuate 4,5-dioxygenase induced fromp-hydroxybenzoate-culturedPseudomonas sp. K82.J. Microbiol. 42: 152–155.

    CAS  Google Scholar 

  26. Kahng, H. Y. (2002) Cellular responses ofPseudomonas sp. KK1 to two-ring polycyclic aromatic hydrocarbon, naphthalene.J. Microbiol. 40: 38–42.

    CAS  Google Scholar 

  27. Blasco, R. and F. Castillo (1993) Characterization of a nitrophenol reductase from the phototrophic bacteriumRhodobacter capsulatus E1F1.Appl. Environ. Microbiol. 59: 1774–1778.

    CAS  Google Scholar 

  28. Rau, J. and A. Stolz (2003) Oxygen-insensitive nitroreductases NfsA and NfsB ofEscherichia coli function under anaerobic conditions as lawsone-dependent azo reductases.Appl. Environ. Microbiol. 69: 3448–3455.

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Environmental Education, College of Education, Sunchon National University, 540-742, Sunchon, Korea

    Hyung-Yeel Kahng

  2. Department of Biological Sciences, Kosin University, 606-701, Busan, Korea

    Bheong-Uk Lee

  3. Department of Biotechnology, Soonchunhyang University, 336-600, Asan, Korea

    Yun-Seok Cho & Kye-Heon Oh

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  1. Hyung-Yeel Kahng
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  2. Bheong-Uk Lee
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  4. Kye-Heon Oh
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Correspondence to Kye-Heon Oh.

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Kahng, HY., Lee, BU., Cho, YS. et al. Purification and characterization of the NAD(P)H-nitroreductase for the catabolism of 2,4,6-trinitrotoluene (TNT) inPseudomonas sp. HK-6. Biotechnol. Bioprocess Eng. 12, 433–440 (2007). https://doi.org/10.1007/BF02931067

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  • DOI: https://doi.org/10.1007/BF02931067

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