Kinetic and redox properties of MnP II, a major manganese peroxidase isoenzyme from Panus tigrinus CBS 577.79

  • Maurizio Petruccioli
  • Marco Frasconi
  • Daniele Quaratino
  • Stefano Covino
  • Gabriele Favero
  • Franco Mazzei
  • Federico Federici
  • Alessandro D’AnnibaleEmail author
Original Paper


A manganese peroxidase (MnP) isoenzyme from Panus tigrinus CBS 577.79 was produced in a benchtop stirred-tank reactor and purified to apparent homogeneity. The purification scheme involving ultrafiltration, affinity chromatography on concanavalin–A Sepharose, and gel filtration led to a purified MnP, termed “MnP II,” with a specific activity of 288 IU mg−1 protein and a final yield of 22%. The enzyme turned out to be a monomeric protein with molecular mass of 50.5 kDa, pI of 4.07, and an extent of N-glycosylation of about 5.3% of the high-mannose type. The temperature and pH optima for the formation of malonate manganic chelates were 45 °C and 5.5, respectively. MnP II proved to be poorly thermostable at 50 and 60 °C, with half-lives of 11 min and 105 s, respectively. K m values for H2O2 and Mn2+ were 16 and 124 μM, respectively. Although MnP II was able to oxidize veratryl alcohol and to catalyze the Mn2+-independent oxidation of several phenols, it cannot be assigned to the versatile peroxidase family. As opposed to versatile peroxidase oxidation, veratryl alcohol oxidation required the simultaneous presence of H2O2 and Mn2+; in addition, low turnover numbers and K m values higher than 300 μM characterized the Mn2+-independent oxidation of substituted phenols. Kinetic properties and the substrate specificity of the enzyme markedly differed from those reported for MnP isoenzymes produced by the reference strain P. tigrinus 8/18. To our knowledge, this study reports for the first time a thorough electrochemical characterization of a MnP from this fungus.


Manganese peroxidase Purification Panus tigrinus Phenols Direct electron transfer 



This work was carried out with the financial support of “Sapienza” University of Rome and of the European Commission under contract number 017350 (BioMedNano).


  1. 1.
    López C, Moreira MT, Feijoo G, Lema JM (2008) Biotechnol Prog 20:74–81CrossRefGoogle Scholar
  2. 2.
    Moreira MT, Feijoo G, Canaval J, Lema JM (2003) Wood Sci Technol 37:117–123CrossRefGoogle Scholar
  3. 3.
    Hofrichter M (2002) Enzyme Microb Technol 30:454–466CrossRefGoogle Scholar
  4. 4.
    Waarishi H, Akileswaran L, Gold MH (1988) Biochemistry 27:5365–5370CrossRefGoogle Scholar
  5. 5.
    Waarishi H, Valli K, Gold MH (1992) J Biol Chem 267:23688–23695Google Scholar
  6. 6.
    D’Annibale A, Crestini C, Di Mattia E, Giovannozzi Sermanni G (1996) J Biotechnol 48:231–239CrossRefGoogle Scholar
  7. 7.
    Forrester IT, Grabsky AC, Mishra C, Burgess RR, Leatham GF (1988) Biochem Biophys Res Commun 157:992–999CrossRefPubMedGoogle Scholar
  8. 8.
    Fenice M, Giovannozzi Sermanni G, Federici F, D’Annibale A (2003) J Biotechnol 100:77–85CrossRefPubMedGoogle Scholar
  9. 9.
    Quaratino D, Fenice M, Federici F, D’Annibale A (2006) J Chem Technol Biotechnol 81:832–840CrossRefGoogle Scholar
  10. 10.
    Quaratino D, D’Annibale A, Federici F, Cereti CF, Rossini F (2007) Chemosphere 66:1627–1633CrossRefPubMedGoogle Scholar
  11. 11.
    D’Annibale A, Ricci M, Quaratino D, Federici F, Fenice M (2004) Res Microbiol 155:596–603CrossRefPubMedGoogle Scholar
  12. 12.
    Leontievsky AA, Myasoedova NM, Golovleva LA (1994) J Biotechnol 32:299–307CrossRefGoogle Scholar
  13. 13.
    Maltseva OV, Niku-Paavola ML, Leontievsky AA, Myasoedova NM, Golovleva LA (1991) Biotechnol Appl Biochem 13:291–302Google Scholar
  14. 14.
    Golovleva LA, Leontievsky AA, Maltseva OV, Myasoedova NM (1993) J Biotechnol 30:71–77CrossRefGoogle Scholar
  15. 15.
    Lisov AV, Leontievsky AA, Golovleva LA (2003) Biochemistry (Mosc) 68:1027–1035CrossRefGoogle Scholar
  16. 16.
    Lisov AV, Leontievsky AA, Golovleva LA, Evans CS (2004) J Mol Catal B Enzym 31:1–8CrossRefGoogle Scholar
  17. 17.
    Lisov AV, Leontievsky AA, Golovleva LA (2005) Biochemistry (Mosc) 70:467–472CrossRefGoogle Scholar
  18. 18.
    Neuhoff V, Arold N, Taube D, Ehrhardt W (1988) Electrophoresis 9:255–262CrossRefPubMedGoogle Scholar
  19. 19.
    Kuan IC, Johnson K, Tien M (1993) J Biol Chem 268:20064–20070PubMedGoogle Scholar
  20. 20.
    Schlosser D, Höfer C (2002) Appl Environ Microbiol 68:3514–3521CrossRefPubMedGoogle Scholar
  21. 21.
    Bard AJ, Faulkner LR (eds) (2002) Electrochemical method, 2nd edn. Wiley, New YorkGoogle Scholar
  22. 22.
    Presnova G, Grigorenko V, Egorov A, Ruzgas T, Lindgren A, Gorton L, Börchers T (2000) Faraday Discuss 116:281–289CrossRefPubMedGoogle Scholar
  23. 23.
    Nassar A-EF, Zhang Z, Hu N, Rusling JF, Kumosinski TF (1997) J Phys Chem B 101:2224–2231CrossRefGoogle Scholar
  24. 24.
    Léger C, Bertrand P (2008) Chem Rev 108:2379–2438CrossRefPubMedGoogle Scholar
  25. 25.
    Aitken MD, Irvine RL (1990) Arch Biochem Biophys 276:405–414CrossRefPubMedGoogle Scholar
  26. 26.
    Hirai H, Sugiura M, Kawai S, Nishida T (2006) FEMS Microbiol Lett 246:19–24CrossRefGoogle Scholar
  27. 27.
    Ruiz-Dueñas FJ, Camarero S, Pérez-Boada M, Martínez MJ, Martínez AT (2001) Biochem Soc Trans 29:116–122CrossRefPubMedGoogle Scholar
  28. 28.
    Ruiz-Dueñas FJ, Morales M, García E, Miki Y, Martínez MJ, Martínez AT (2009) J Exp Bot 60:441–452CrossRefPubMedGoogle Scholar
  29. 29.
    Martìnez MJ, Ruiz-Duenas FJ, Guillèn F, Martinez AT (1996) Eur J Biochem 237:424–432CrossRefPubMedGoogle Scholar
  30. 30.
    Giardina P, Palmieri G, Fontanella B, Rivieccio V, Sannia G (2000) Arch Biochem Biophys 376:171–179CrossRefPubMedGoogle Scholar
  31. 31.
    Gelpke MDS, Youngs HL, Gold MH (2000) Eur J Biochem 267:7038–7045CrossRefPubMedGoogle Scholar
  32. 32.
    Heinfling A, Ruiz-Dueñas FJ, Martínez MJ, Bergbauer M, Szewzyk U, Martínez AT (1998) FEBS Lett 428:141–146CrossRefPubMedGoogle Scholar
  33. 33.
    Camarero S, Sarkar S, Ruiz-Dueñas FJ, Martínez MJ, Martínez AT (1999) J Biol Chem 274:10324–10330CrossRefPubMedGoogle Scholar
  34. 34.
    Pérez-Boada M, Ruiz-Dueñas FJ, Pogni R, Basosi R, Choinowski T, Martínez MJ, Piontek K, Martínez AT (2005) J Mol Biol 354:385–402CrossRefPubMedGoogle Scholar
  35. 35.
    Mc Eldoon JP, Pokora AR, Dordick JS (1995) Enzyme Microb Technol 17:359–365CrossRefGoogle Scholar
  36. 36.
    Armstrong FA (2002) J Chem Soc Dalton Trans 661–671Google Scholar
  37. 37.
    Krishtalik LI (2000) Biochim Biophys Acta 1458:6–13CrossRefPubMedGoogle Scholar
  38. 38.
    Hirst J (2006) Biochim Biophys Acta 1757:225–239CrossRefPubMedGoogle Scholar
  39. 39.
    Ferapontova EE, Castillo J, Gorton L (2006) Biochim Biophys Acta 1760:1343–1354PubMedGoogle Scholar
  40. 40.
    Santucci R, Bongiovanni C, Marini S, Del Conte R, Tien M, Banci L, Coletta M (2000) Biochem J 349:85–90CrossRefPubMedGoogle Scholar
  41. 41.
    Oyadomari M, Shinohara H, Johjima T, Wariishi H, Tanaka H (2003) J Mol Catal B Enzym 21:291–297CrossRefGoogle Scholar
  42. 42.
    Munteanu F-D, Ruiz-Dueñas FJ, Martínez MJ, Cavaco-Paulo A (2005) J Electroanal Chem 580:35–40CrossRefGoogle Scholar

Copyright information

© SBIC 2009

Authors and Affiliations

  • Maurizio Petruccioli
    • 1
  • Marco Frasconi
    • 2
  • Daniele Quaratino
    • 1
    • 3
  • Stefano Covino
    • 1
  • Gabriele Favero
    • 2
  • Franco Mazzei
    • 2
  • Federico Federici
    • 1
  • Alessandro D’Annibale
    • 1
    Email author
  1. 1.Dipartimento di Agrobiologia and AgrochimicaUniversità degli Studi della TusciaViterboItaly
  2. 2.Dipartimento di Chimica e Tecnologie del FarmacoUniversità La SapienzaRomeItaly
  3. 3.Consorzio Interuniversitario della Chimica per l’AmbienteVeniceItaly

Personalised recommendations