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Novel thermotolerant laccases produced by the white-rot fungus Physisporinus rivulosus

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The white-rot basidiomycete Physisporinus rivulosus strain T241i is highly selective for degradation of softwood lignin, which makes this fungus suitable for biopulping. In order to promote laccase production, P. rivulosus was cultivated in nutrient-nitrogen sufficient liquid media containing either charcoal or spruce sawdust as supplements. Two laccases with distinct pI values, Lac-3.5 and Lac-4.8, were purified from peptone-spruce sawdust-charcoal cultures of P. rivulosus. Both laccases showed thermal stability at up to 60°C. Lac-4.8 was thermally activated at 50°C. Surprisingly, both laccases displayed atypically low pH optima (pH 3.0–3.5) in oxidation of the commonly used laccase substrates syringaldazine (4-hydroxy-3,5-dimethoxybenzaldehyde azine), 2,6-dimethoxyphenol and guaiacol (2-methoxyphenol). Steady-state kinetic measurements pointed to unusually low affinity to guaiacol at low pH, whereas the kinetic constants for the methoxyphenols and ABTS were within the ranges reported for other fungal laccases. The combination of thermotolerance with low pH optima for methoxylated phenol substrates suggests that the two P. rivulosus T241i laccases possess potential for use in biotechnological applications.

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  1. Alén R, Kuoppala E, Oesch P (1996) Formation of the main degradation compound groups from wood and its components during pyrolysis. J Anal Appl Pyrolysis 36:137–148

  2. Baldrian P (2006) Fungal laccases - occurence and properties. FEMS Microbiol Rev 30:215–242

  3. Bertrand T, Jolivalt C, Briozzo P, Caminade E, Joly N, Madzak C, Mougin C (2002) Crystal structure of a four-copper laccase compexed with an arylamine: insights into substrate recognition and correlation with kinetics. Biochemistry 41:7325–7333

  4. Bourbonnais R, Paice MG, Freiermuth B, Bodie E, Borneman S (1997) Reactivities of various mediators and laccases with kraft pulp and lignin model compounds. Appl Environ Microbiol 63:4627–4632

  5. Call HP, Mücke I (1997) History, overview and applications of mediated ligninolytic systems, especially laccase-mediator-systems (Lignozym®-process). J Biotechnol 53:163–202

  6. Chefetz B, Chen Y, Hadar Y (1998) Purification and characterization of laccase from Chaetomium thermophilium and its role in humification. Appl Environ Microbiol 64:3175–3179

  7. Coll PM, Fernández-Abalos JM, Villanueva JR, Santamaría R, Pérez P (1993) Purification and characterization of a phenoloxidase (laccase) from the lignin-degrading basidiomycete PM1 (CECT 2971). Appl Environ Microbiol 59:2607–2613

  8. Eggert C, Temp U, Eriksson K-L (1996) The ligninolytic system of the white rot fungus Pycnoporus cinnabarius: purification and characterization of the laccase. Appl Environ Microbiol 62:1151–1158

  9. Enguita FJ, Martins LO, Heniques AO, Carrondo MA (2003) Crystal structure of a bacterial endospore coat component. J Biol Chem 278:19416–19425

  10. Fields A (2001) Review: Protein function at thermal extremes: balancing stability and flexibility. Comp Biochem Physiol Part A 129:417–431

  11. Fu SY, Yu H-S, Buswell JA (1997) Effect of nutrient nitrogen and manganese on manganese-peroxidase and laccase production of Lentinula (Lentinus) edodes. FEMS Microbiol Lett 147:133–137

  12. Fukushima Y, Kirk K (1995) Laccase component of the Ceriporiopsis subvermispora lignin-degrading system. Appl Environ Microbiol 61:872–876

  13. Galhaup C, Goller S, Peterbauer CK, Strauss J, Haltrich D (2002a) Characterization of the major laccase isoenzyme from Trametes pubescens and regulation of its synthesis by metal ions. Microbiology 148:2159–2169

  14. Galhaup C, Wagner H, Hinterstoisser B, Haltrich D (2002b) Increased production of laccase by wood-degrading basidiomycete Trametes pubescens. Enzyme Microb Technol 30:529–536

  15. Garzillo AM et al (2001) Structural and kinetic characterization of native laccases from Pleurotus ostreatus, Rigidoporus lignosus, and Trametes trogii. J Prot Chem 20:191–201

  16. Gianfreda L, Xu F, Bollag J-M (1999) Laccases: a useful group of oxidoreductive enzymes. Bioremed J 3:1–25

  17. Hakala TK, Maijala P, Konn J, Hatakka A (2004) Evaluation of novel wood-rotting polypores and corticioid fungi for the decay and biopulping of Norway spruce (Picea abies) wood. Enzyme Microb Technol 34:255–263

  18. Hakala TK, Lundell T, Galkin S, Maijala P, Kalkkinen N, Hatakka A (2005) Manganese peroxidases, laccases and oxalic acid from the selective white-rot fungus Physisporinus rivulosus grown on spruce wood chips. Enzyme Microb Technol 36:461–468

  19. Hakala TK, Hildén K, Maijala P, Olsson C, Hatakka A (2006) Differential regulation of manganese peroxidases and characterization of two variable MnP encoding genes in the white-rot fungus Physisporinus rivulosus. Appl Microbiol Biotechnol 73:839–849

  20. Hatakka A, Uusi-Rauva AK (1983) Degradation of 14C-labelled poplar wood lignin by selected white-rot fungi. Eur J Appl Microbiol Biotechnol 17:235–242

  21. Hatakka A, Maijala P, Hakala TK, Hauhio L, Ellmén J (2003) Novel white-rot fungus and use thereof in wood pretreatment. Finnish patent FI112248

  22. Kotiranta H, Penttilä R (1996) Short-term effects of prescribed burning on wood-rotting fungi. Silva Fennica 30:399–419

  23. Lankinen P, Hildén K, Aro N, Salkinoja-Salonen M, Hatakka A (2005) Manganese peroxidase of Agaricus bisporus: grain bran-promoted production and gene characterization. Appl Microbiol Biotechnol 66:401–407

  24. Larrondo LF, Avila M, Salas L, Cullen D, Vicuña R (2003) Heterologous expression of laccase cDNA from Ceriporiopsis subvermispora yields copper-activated apoprotein and complex isoform patterns. Microbiology 149:1177–1182

  25. Leonowicz A et al (2001) Fungal laccase: properties and activity on lignin. J Basic Microbiol 3–4:185–227

  26. Lorenzo M, Moldes D, Rodríquez Couto S, Sanromán A (2002) Improving laccase production by employing different lignocellulosic wastes in submerged cultures of Trametes versicolor. Biores Technol 82:109–113

  27. Lundell T, Hatakka A (1994) Participation of Mn(II) in the catalysis of laccase, manganese peroxidase and lignin peroxidase from Phlebia radiata. FEBS Lett 348:291–296

  28. Mayer AM, Staples RC (2002) Laccase: new functions for an old enzyme. Phytochemistry 60:551–565

  29. Palmieri G, Giardina P, Bianco C, Scaloni A, Capasso A, Sannia G (1997) A novel white laccase from Pleurotus ostreatus. J Biol Chem 272:31301–31307

  30. Pickard MA, Vandertol H, Roman R, Vasquez-Duhalt R (1999) High production of ligninolytic enzymes from white rot fungi in cereal bran liquid medium. Can J Microbiol 45:627–631

  31. Pointing SB, Jones EBG, Vrijmoed LLP (2000) Optimization of laccase production by Pycnoporus sanguineus in submerged liquid culture. Mycologia 92:139–144

  32. Rodakiewicz-Nowak J, Haber J, Pozdnyakova N, Leontievsky A, Golovleva LA (1999) Effect of ethanol on enzymatic activity of fungal laccases. Biosci Reports 19:589–600

  33. Rodakiewicz-Nowak J, Jarosz-Wilkolazka A (2007) Catalytic activity of Cerrena unicolor laccase in aqueous solutions of water-miscible organic solvents-experimental and numerical description. J Mol Catal B 44:53–59

  34. Rodríquez E, Pickard MA, Vasquez-Duhalt R (1999) Industrial dye decolorization by laccases from ligninolytic fungi. Curr Microbiol 38:27–32

  35. Rogalski J, Lundell T, Leonowicz A, Hatakka A (1991) Influence of aromatic componds and lignin production of ligninolytic enzymes by Phlebia radiata. Phytochemistry 30:2869–2872

  36. Rosales E, Couto R, Sanromán A (2002) New uses of food waste: application to laccase production by Trametes hirsuta. Biotechnol Lett 24:701–704

  37. Ryan S, Schnizhofer W, Tzanov T, Cavaco-Paulo A, Gübitz GM (2003) An acid-stable laccase from Sclerotium rolfsii with potential for wool dye decolourization. Enzyme Microb Technol 33:766–774

  38. Rüttimann-Johnson C, Salas L, Vicuña R, Kirk K (1993) Extracellular enzyme production and synthetic lignin mineralization by Ceriporiopsis subvermispora. Appl Environ Microbiol 59:1792–1797

  39. Salas C, Lobos S, Larraín J, Salas L, Cullen D, Vicuña R (1995) Properties of laccase isoenzymes produced by the basidiomycete Ceriporiopsis subvermispora. Biotechnol Appl Biochem 21:323–333

  40. Somero GN (2004) Adaptation of enzymes to temperatures: searching for basic “strategies". Comp Biochem Physiol Part B 139:312–333

  41. Swamy J, Ramsay JA (1999) Effects of Mn2+ and \(NH_4^ + \) concentrations on laccase and manganese peroxidase production and Amaranth decolorization of Trametes versicolor. Appl Microbiol Biotechnol 51:391–396

  42. Šušla M, Novotný C, Svobodová K (2007) The implication of Dichomitus squalens laccase isoenzymes in dye decolorization by immobilized fungal cultures. Biores Technol 98:2109–2115

  43. Thurston CF (1994) The structure and function of fungal laccases. Microbiology 140:19– 26

  44. Tinoco R, Pickard MA, Vasquez-Duhalt R (2001) Kinetic differences of purified laccases from six Pleurotus ostreatus strains. Lett Appl Microbiol 32:331–335

  45. Tomšovský M, Kolarík M, Pažoutová S, Homolka L (2006) Molecular phylogeny of European Trametes (Basidiomycetes, Polyporales) species based on LSU and ITS (nrDNA) sequences. Nova Hedwigia 82:269–280

  46. Trovaslet M, Enaud E, Guiavarc'h Y, Corbisier A-M, Vanhulle S (2007) Potential of a Pycnoporus sanguineus laccase in bioremediation of wastewater and kinetic activation in the presence of an anthraquinonic acid dye. Enzyme Microb Technol 41:368–376

  47. Tzanov T, Basto C, Gübitz GM, Cavaco-Paulo A (2003) Laccases to improve the whiteness in a conventional bleaching of cotton. Macromol Mater Eng 288:807–810

  48. Vares T, Kalsi M, Hatakka A (1995) Lignin peroxidases, manganese peroxidases, and other ligninolytic enzymes produced by Phlebia radiata during solid-state fermentation of wheat straw. Appl Environ Microbiol 61:3515–3520

  49. Xu F, Shin W, Brown SH, Wahleithner JA, Sundaram UM, Solomon EI (1996) A study of a series of recombinant fungal laccases and bilirubin oxidase that exhibit significant differences in redox potential, substrate specificity, and stability. Biochim Biophys Acta 1292:303–311

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The work was supported by the Academy of Finland (Center of Excellence “Microbial Resources Research Unit” grant no: 53305 and no: 205027, research grant no: 118993 to T.H. and no: 205027 to K.H.). Terhi K. Hakala was supported by Viikki Graduate School of Biosciences, University of Helsinki. Dr. P. Lankinen is acknowledged for critical reading of the manuscript.

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Correspondence to Kristiina Hildén.

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Hildén, K., Hakala, T.K., Maijala, P. et al. Novel thermotolerant laccases produced by the white-rot fungus Physisporinus rivulosus . Appl Microbiol Biotechnol 77, 301–309 (2007). https://doi.org/10.1007/s00253-007-1155-x

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  • Laccase
  • White-rot fungi
  • Physisporinus rivulosus
  • Thermostability
  • Thermal activation
  • Lignin degradation