Biochemistry (Moscow)

, Volume 74, Issue 9, pp 1027–1034 | Cite as

Purification and characteristics of an enzyme with both bilirubin oxidase and laccase activities from mycelium of the basidiomycete Pleurotus ostreatus

  • Y. G. PakhadniaEmail author
  • N. I. Malinouski
  • A. G. Lapko


A homogenous enzyme with both bilirubin oxidase and laccase activities was isolated from a submerged culture of the basidiomycete Pleurotus ostreatus mycelium and characterized. The yield of the enzyme was 127 μg/g dry biomass of the mycelium. The specific activity of the enzyme was 21 and 261 U/mg to bilirubin and to a laccase substrate ABTS, respectively. The intracellular phenol oxidase from the P. ostreatus mycelium was identified as bilirubin oxidase with the amino acid sequence highly homologous to that of the pox2 gene-encoded product. The enzyme displayed the maximal laccase activity at 50–55°C to all substrates examined, whereas the pH optimum was substrate-dependent and changed from 3.0 for ABTS to 7.0 for syringaldazine and guaiacol. The enzyme maintained catalytic activity within a broad pH range but was inactivated at pH 4.0. The enzyme was thermostable but very sensitive to metal chelating inhibitors. Trypan Blue (5 mg/liter) was completely decolorizated upon 3 h of incubation with the bilirubin oxidase (20 mU/ml) at room temperature.

Key words

mycelium Pleurotus ostreatus oxidoreductase bilirubin oxidase laccase 



2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)


bilirubin oxidase








Trypan Blue


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  1. 1.
    Messerschmidt, A., and Huber, R. (1990) Eur. J. Biochem., 187, 341–352.PubMedCrossRefGoogle Scholar
  2. 2.
    Enguita, F., Martins, J. L., Henriques, A. O., and Carrondo, M. A. (2003) J. Biol. Chem., 278, 19416–19425.PubMedCrossRefGoogle Scholar
  3. 3.
    Morozova, O. V., Shumakovich, G. P., Gorbacheva, M. A., Shleev, S. V., and Yaropolov, A. I. (2007) Biochemistry (Moscow), 72, 1136–1150.CrossRefGoogle Scholar
  4. 4.
    Baldrian, P. (2006) FEMS Microbiol. Rev., 20, 1–28.Google Scholar
  5. 5.
    Blaich, R., and Esser, K. (1975) Arch. Microbiol., 103, 271–277.CrossRefGoogle Scholar
  6. 6.
    Hiromi, K., Yamaguchi, Y., Sugiura, Y., Iwamoto, H., and Hirose, J. (1992) Biosci. Biotech. Biochem., 56, 1349–1350.CrossRefGoogle Scholar
  7. 7.
    Matsui, Y., Satoh, T., and Nakajima, K. (1984) Jpn. Patent. Japan Kokai Tokkyo Koho, 59-135886.Google Scholar
  8. 8.
    Uwajima, T., and Hamamatsu, M. (1984) Jpn. Patent. Japan Kokai Tokkyo Koho, 59-198971.Google Scholar
  9. 9.
    Xu, F., Shin, W., Brown, S. H., Wahleitner, J. A., Sundaram, U. M., and Solomon, E. I. (1996) Biochim. Biophys. Acta, 1292, 303–311.PubMedGoogle Scholar
  10. 10.
    Tanaka, N., and Muran, S. (1983) Agric. Biol. Chem., 47, 1627–1628.Google Scholar
  11. 11.
    Coll, P. M., Fernandez-Abalos, J. M., Villanueva, J. R., Santamaria, R., and Perez, P. (1993) Appl. Environ. Microbiol., 59, 2607–2613.PubMedGoogle Scholar
  12. 12.
    Heinzkill, M., Bech, L., Halkier, T., Schneider, P., and Anke, T. (1998) Appl. Environ. Microbiol., 64, 1601–1606.PubMedGoogle Scholar
  13. 13.
    Masuda-Nishimura, I., Ichikawa, K., Hatamoto, O., Abe, K., and Koyama, Y. (1999) J. Gen. Appl. Microbiol., 45, 93–97.PubMedCrossRefGoogle Scholar
  14. 14.
    Palmieri, G., Giardina, P., Bianco, C., Scaloni, A., Capassoi, A., and Sannia, G. (1997) J. Biol. Chem., 272, 31301–31307.PubMedCrossRefGoogle Scholar
  15. 15.
    Bradford, M. M. (1976) Anal. Biochem., 72, 248–254.PubMedCrossRefGoogle Scholar
  16. 16.
    Laemmli, U. K. (1970) Nature, 227, 680–685.PubMedCrossRefGoogle Scholar
  17. 17.
    Dawson, R., Elliott, D., Elliott, W., and Jones, K. (1991) Data for Biochemical Research [Russian translation], Mir, Moscow.Google Scholar
  18. 18.
    McIlvaine, T. C. (1921) J. Biol. Chem., 49, 183–186.Google Scholar
  19. 19.
    Murao, S., and Tanaka, N. (1981) Agric. Biol. Chem., 45, 2383–2384.Google Scholar
  20. 20.
    Garcia, T. A., Santiago, M. F., and Ulhoa, C. J. (2007) Appl. Microbiol. Biotechnol., 75, 311–318.PubMedCrossRefGoogle Scholar
  21. 21.
    Pozdnyakova, N. N., Turkovskaya, O. V., Yudina, E. N., and Rodakevich-Novak, Ya. (2006) Prikl. Biokhim. Mikrobiol., 42, 63–69.Google Scholar
  22. 22.
    Mansur, M., Arias, M., Copa-Patino, J., Flardh, M., and Gonzalez, A. (2003) Mycologia, 95, 1013–1020.CrossRefGoogle Scholar
  23. 23.
    Tanaka, H., and Murao, S. (1982) Agric. Biol. Chem., 46, 2499–2503.Google Scholar
  24. 24.
    Galhaup, C., Goller, S., Peterbauer, C., Strauss, J., and Haltrich, D. (2002) Microbiology, 148, 2159–2169.PubMedGoogle Scholar
  25. 25.
    Kanunfre, C. C., and Zancan, G. T. (1998) FEMS Microbiol. Lett., 161, 151–156.CrossRefGoogle Scholar
  26. 26.
    Rogalski, J., Dawidowicz, A. L., and Leonowicz, A. (1990) Acta Biotechnol., 10, 261–269.CrossRefGoogle Scholar
  27. 27.
    Shleev, S. V., Morozova, O., Nikitina, O., Gorshina, E. S., Rusinova, T., Serezhenkov, V. A., Burbaev, D. S., Gazaryan, I. G., and Yaropolov, A. I. (2004) Biochimie, 86, 693–703.PubMedCrossRefGoogle Scholar
  28. 28.
    Vares, T., and Hatakka, A. (1997) Can. J. Bot., 75, 61–71.CrossRefGoogle Scholar
  29. 29.
    Sakasegawa, S., Ishikawa, H., Imamura, S., Sakuraba, H., Goda, S., and Ohshima, T. (2006) Appl. Microbiol. Biotechnol., 72, 972–975.Google Scholar
  30. 30.
    Pakhadnia, Yu. G., and Lapko, A. G. (2008) Vestsi NAN Belarusi, Ser. Biol. Navuk, 5(Suppl.), 211–215.Google Scholar
  31. 31.
    Nagai, M., Kawata, M., Watanade, H., Ogawa, M., Saito, K., Takesawa, T., Kanda, K., and Sato, T. (2003) Microbiology, 149, 2455–2462.PubMedCrossRefGoogle Scholar
  32. 32.
    Eggert, C., Temp, U., Dean, J. F. D., and Eriksson, K.-E. L. (1995) FEBS Lett., 376, 202–206.PubMedCrossRefGoogle Scholar
  33. 33.
    Galhaup, C., and Haltrich, D. (2001) Appl. Microbiol. Biotechnol., 56, 225–232.PubMedCrossRefGoogle Scholar
  34. 34.
    Kakaoka, K., Tanaka, K., Sakai, Y., and Sakurai, T. (2005) Protein Exp. Purif., 44, 77–83.Google Scholar
  35. 35.
    Zhang, X., Liu, Y., Yan, K., and Wu, H. (2007) J. Biosci. Bioeng., 104, 104–110.PubMedCrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2009

Authors and Affiliations

  • Y. G. Pakhadnia
    • 1
    Email author
  • N. I. Malinouski
    • 2
  • A. G. Lapko
    • 1
  1. 1.Sakharov International Environmental UniversityMinskBelarus
  2. 2.Department of BiochemistryUniversity of NebraskaLincolnUSA

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