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. 1997 Jun;63(6):2166–2174. doi: 10.1128/aem.63.6.2166-2174.1997

Laccase isoenzymes of Pleurotus eryngii: characterization, catalytic properties, and participation in activation of molecular oxygen and Mn2+ oxidation.

C Muñoz 1, F Guillén 1, A T Martínez 1, M J Martínez 1
PMCID: PMC168508  PMID: 9172335

Abstract

Two laccase isoenzymes produced by Pleurotus eryngii were purified to electrophoretic homogeneity (42- and 43-fold) with an overall yield of 56.3%. Laccases I and II from this fungus are monomeric glycoproteins with 7 and 1% carbohydrate content, molecular masses (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of 65 and 61 kDa, and pIs of 4.1 and 4.2, respectively. The highest rate of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) oxidation for laccase I was reached at 65 degrees C and pH 4, and that for laccase II was reached at 55 degrees C and pH 3.5. Both isoenzymes are stable at high pH, retaining 60 to 70% activity after 24 h from pH 8 to 12. Their amino acid compositions and N-terminal sequences were determined, the latter strongly differing from those of laccases of other basidiomycetes. Antibodies against laccase I reacted with laccase II, as well as with laccases from Pleurotus ostreatus, Pleurotus pulmonarius, and Pleurotus floridanus. Different hydroxy- and methoxy-substituted phenols and aromatic amines were oxidized by the two laccase isoenzymes from P. eryngii, and the influence of the nature, number, and disposition of aromatic-ring substituents on kinetic constants is discussed. Although both isoenzymes presented similar substrate affinities, the maximum rates of reactions catalyzed by laccase I were higher than those of laccase II. In reactions with hydroquinones, semiquinones produced by laccase isoenzymes were in part converted into quinones via autoxidation. The superoxide anion radical produced in the latter reaction dismutated, producing hydrogen peroxide. In the presence of manganous ion, the superoxide union was reduced to hydrogen peroxide with the concomitant production of manganic ion. These results confirmed that laccase in the presence of hydroquinones can participate in the production of both reduced oxygen species and manganic ions.

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Selected References

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  1. Archibald F. S., Fridovich I. The scavenging of superoxide radical by manganous complexes: in vitro. Arch Biochem Biophys. 1982 Apr 1;214(2):452–463. doi: 10.1016/0003-9861(82)90049-2. [DOI] [PubMed] [Google Scholar]
  2. Archibald F., Roy B. Production of manganic chelates by laccase from the lignin-degrading fungus Trametes (Coriolus) versicolor. Appl Environ Microbiol. 1992 May;58(5):1496–1499. doi: 10.1128/aem.58.5.1496-1499.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bollag J. M., Leonowicz A. Comparative studies of extracellular fungal laccases. Appl Environ Microbiol. 1984 Oct;48(4):849–854. doi: 10.1128/aem.48.4.849-854.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bono J. J., Goulas P., Boe J. F., Portet N., Seris J. L. Effect of Mn(II) on reactions catalyzed by lignin peroxidase from Phanerochaete chrysosporium. Eur J Biochem. 1990 Aug 28;192(1):189–193. doi: 10.1111/j.1432-1033.1990.tb19213.x. [DOI] [PubMed] [Google Scholar]
  5. Bourbonnais R., Paice M. G. Oxidation of non-phenolic substrates. An expanded role for laccase in lignin biodegradation. FEBS Lett. 1990 Jul 2;267(1):99–102. doi: 10.1016/0014-5793(90)80298-w. [DOI] [PubMed] [Google Scholar]
  6. Bourbonnais R., Paice M. G., Reid I. D., Lanthier P., Yaguchi M. Lignin oxidation by laccase isozymes from Trametes versicolor and role of the mediator 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) in kraft lignin depolymerization. Appl Environ Microbiol. 1995 May;61(5):1876–1880. doi: 10.1128/aem.61.5.1876-1880.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  8. Eggert C., Temp U., Dean J. F., Eriksson K. E. A fungal metabolite mediates degradation of non-phenolic lignin structures and synthetic lignin by laccase. FEBS Lett. 1996 Aug 5;391(1-2):144–148. doi: 10.1016/0014-5793(96)00719-3. [DOI] [PubMed] [Google Scholar]
  9. Eggert C., Temp U., Eriksson K. E. The ligninolytic system of the white rot fungus Pycnoporus cinnabarinus: purification and characterization of the laccase. Appl Environ Microbiol. 1996 Apr;62(4):1151–1158. doi: 10.1128/aem.62.4.1151-1158.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Froehner S. C., Eriksson K. E. Purification and properties of Neurospora crassa laccase. J Bacteriol. 1974 Oct;120(1):458–465. doi: 10.1128/jb.120.1.458-465.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fukushima Y., Kirk T. K. Laccase component of the Ceriporiopsis subvermispora lignin-degrading system. Appl Environ Microbiol. 1995 Mar;61(3):872–876. doi: 10.1128/aem.61.3.872-876.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Giardina P., Aurilia V., Cannio R., Marzullo L., Amoresano A., Siciliano R., Pucci P., Sannia G. The gene, protein and glycan structures of laccase from Pleurotus ostreatus. Eur J Biochem. 1996 Feb 1;235(3):508–515. doi: 10.1111/j.1432-1033.1996.00508.x. [DOI] [PubMed] [Google Scholar]
  13. Giardina P., Cannio R., Martirani L., Marzullo L., Palmieri G., Sannia G. Cloning and sequencing of a laccase gene from the lignin-degrading basidiomycete Pleurotus ostreatus. Appl Environ Microbiol. 1995 Jun;61(6):2408–2413. doi: 10.1128/aem.61.6.2408-2413.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Guillén F., Evans C. S. Anisaldehyde and Veratraldehyde Acting as Redox Cycling Agents for H(2)O(2) Production by Pleurotus eryngii. Appl Environ Microbiol. 1994 Aug;60(8):2811–2817. doi: 10.1128/aem.60.8.2811-2817.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Guillén F., Martínez A. T., Martínez M. J. Substrate specificity and properties of the aryl-alcohol oxidase from the ligninolytic fungus Pleurotus eryngii. Eur J Biochem. 1992 Oct 15;209(2):603–611. doi: 10.1111/j.1432-1033.1992.tb17326.x. [DOI] [PubMed] [Google Scholar]
  16. Guillén F., Martínez M. J., Muñoz C., Martínez A. T. Quinone redox cycling in the ligninolytic fungus Pleurotus eryngii leading to extracellular production of superoxide anion radical. Arch Biochem Biophys. 1997 Mar 1;339(1):190–199. doi: 10.1006/abbi.1996.9834. [DOI] [PubMed] [Google Scholar]
  17. Gutiérrez A., Caramelo L., Prieto A., Martínez M. J., Martínez A. T. Anisaldehyde production and aryl-alcohol oxidase and dehydrogenase activities in ligninolytic fungi of the genus Pleurotus. Appl Environ Microbiol. 1994 Jun;60(6):1783–1788. doi: 10.1128/aem.60.6.1783-1788.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hanna P. M., McMillin D. R., Pasenkiewicz-Gierula M., Antholine W. E., Reinhammar B. Type 2-depleted fungal laccase. Biochem J. 1988 Jul 15;253(2):561–568. doi: 10.1042/bj2530561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kappus H., Sies H. Toxic drug effects associated with oxygen metabolism: redox cycling and lipid peroxidation. Experientia. 1981 Dec 15;37(12):1233–1241. doi: 10.1007/BF01948335. [DOI] [PubMed] [Google Scholar]
  20. Kirk T. K., Farrell R. L. Enzymatic "combustion": the microbial degradation of lignin. Annu Rev Microbiol. 1987;41:465–505. doi: 10.1146/annurev.mi.41.100187.002341. [DOI] [PubMed] [Google Scholar]
  21. Kojima Y., Tsukuda Y., Kawai Y., Tsukamoto A., Sugiura J., Sakaino M., Kita Y. Cloning, sequence analysis, and expression of ligninolytic phenoloxidase genes of the white-rot basidiomycete Coriolus hirsutus. J Biol Chem. 1990 Sep 5;265(25):15224–15230. [PubMed] [Google Scholar]
  22. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. Martínez M. J., Ruiz-Dueñas F. J., Guillén F., Martínez A. T. Purification and catalytic properties of two manganese peroxidase isoenzymes from Pleurotus eryngii. Eur J Biochem. 1996 Apr 15;237(2):424–432. doi: 10.1111/j.1432-1033.1996.0424k.x. [DOI] [PubMed] [Google Scholar]
  24. Munoz C, Guillen F, Martinez AT, Martinez MJ. Induction and Characterization of Laccase in the Ligninolytic Fungus Pleurotus eryngii. Curr Microbiol. 1997 Jan;34(1):1–5. doi: 10.1007/s002849900134. [DOI] [PubMed] [Google Scholar]
  25. Ollinger K., Buffinton G. D., Ernster L., Cadenas E. Effect of superoxide dismutase on the autoxidation of substituted hydro- and semi-naphthoquinones. Chem Biol Interact. 1990;73(1):53–76. doi: 10.1016/0009-2797(90)90108-y. [DOI] [PubMed] [Google Scholar]
  26. Perry C. R., Smith M., Britnell C. H., Wood D. A., Thurston C. F. Identification of two laccase genes in the cultivated mushroom Agaricus bisporus. J Gen Microbiol. 1993 Jun;139(Pt 6):1209–1218. doi: 10.1099/00221287-139-6-1209. [DOI] [PubMed] [Google Scholar]
  27. Popp J. L., Kalyanaraman B., Kirk T. K. Lignin peroxidase oxidation of Mn2+ in the presence of veratryl alcohol, malonic or oxalic acid, and oxygen. Biochemistry. 1990 Nov 20;29(46):10475–10480. doi: 10.1021/bi00498a008. [DOI] [PubMed] [Google Scholar]
  28. Saloheimo M., Niku-Paavola M. L., Knowles J. K. Isolation and structural analysis of the laccase gene from the lignin-degrading fungus Phlebia radiata. J Gen Microbiol. 1991 Jul;137(7):1537–1544. doi: 10.1099/00221287-137-7-1537. [DOI] [PubMed] [Google Scholar]
  29. Srinivasan C., Dsouza T. M., Boominathan K., Reddy C. A. Demonstration of Laccase in the White Rot Basidiomycete Phanerochaete chrysosporium BKM-F1767. Appl Environ Microbiol. 1995 Dec;61(12):4274–4277. doi: 10.1128/aem.61.12.4274-4277.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. TREVELYAN W. E., HARRISON J. S. Studies on yeast metabolism. I. Fractionation and microdetermination of cell carbohydrates. Biochem J. 1952 Jan;50(3):298–303. doi: 10.1042/bj0500298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tien M., Kirk T. K. Lignin-Degrading Enzyme from the Hymenomycete Phanerochaete chrysosporium Burds. Science. 1983 Aug 12;221(4611):661–663. doi: 10.1126/science.221.4611.661. [DOI] [PubMed] [Google Scholar]
  32. Wariishi H., Akileswaran L., Gold M. H. Manganese peroxidase from the basidiomycete Phanerochaete chrysosporium: spectral characterization of the oxidized states and the catalytic cycle. Biochemistry. 1988 Jul 12;27(14):5365–5370. doi: 10.1021/bi00414a061. [DOI] [PubMed] [Google Scholar]
  33. Winterbourn C. C. Cytochrome c reduction by semiquinone radicals can be indirectly inhibited by superoxide dismutase. Arch Biochem Biophys. 1981 Jun;209(1):159–167. doi: 10.1016/0003-9861(81)90268-x. [DOI] [PubMed] [Google Scholar]
  34. Xu F., Shin W., Brown S. H., Wahleithner J. A., Sundaram U. M., Solomon E. I. 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. 1996 Feb 8;1292(2):303–311. doi: 10.1016/0167-4838(95)00210-3. [DOI] [PubMed] [Google Scholar]
  35. Yaver D. S., Xu F., Golightly E. J., Brown K. M., Brown S. H., Rey M. W., Schneider P., Halkier T., Mondorf K., Dalboge H. Purification, characterization, molecular cloning, and expression of two laccase genes from the white rot basidiomycete Trametes villosa. Appl Environ Microbiol. 1996 Mar;62(3):834–841. doi: 10.1128/aem.62.3.834-841.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]

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