1887

Abstract

The major laccase isoenzyme LAP2 secreted by the white-rot basidiomycete in response to high copper concentrations was purified to apparent electrophoretic homogeneity using anion-exchange chromatography and gel filtration. The monomeric protein has a molecular mass of 65 kDa, of which 18% is glycosylation, and a pI value of 26. The pH optima of the laccase depend on the substrates oxidized and show bell-shaped pH activity profiles with an optimum of 3–45 for phenolic substrates such as 2,6-dimethoxyphenol or syringaldazine, while the non-phenolic substrates ABTS [2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)] and ferrocyanide show a monotonic pH profile with a rate increasing with decreasing pH. The catalytic efficiencies / determined for some of its substrates were 48×10, 47×10, 20×10 and 7×10 M s for ABTS, syringaldazine, ferrocyanide and oxygen, respectively. Furthermore, the gene encoding the purified laccase was cloned and its nucleotide sequence determined. The gene consists of 1997 bp, with the coding sequence interrupted by eight introns and flanked by an upstream region in which putative CAAT, TATA, MRE and CreA consensus sequences were identified. Based on Northern analysis containing total RNA from both induced and uninduced cultures, expression of is highly induced by copper, which is also corroborated by an increase in laccase activity in response to copper. A stimulating effect of various other heavy metal ions on laccase synthesis was also observed. In addition to induction, a second regulatory mechanism seems to be repression of transcription by glucose.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-148-7-2159
2002-07-01
2019-11-16
Loading full text...

Full text loading...

/deliver/fulltext/micro/148/7/1482159a.html?itemId=/content/journal/micro/10.1099/00221287-148-7-2159&mimeType=html&fmt=ahah

References

  1. Arst, H. N.Jr & MacDonald, D. W. ( 1975; ). A gene cluster in Aspergillus nidulans with an internally located cis-acting regulatory region. Nature 254, 26-31.[CrossRef]
    [Google Scholar]
  2. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (1990). Current Protocols in Molecular Biology. New York: Wiley Interscience.
  3. Ballance, D. J. ( 1991; ). Transformation systems for filamentous fungi and an overview of fungal gene structure. In Molecular and Industrial Mycology , pp. 1-19. Edited by S. A. Leong & R. M. Berka. New York: Marcel Dekker.
  4. Baminger, U., Ludwig, R., Galhaup, C., Leitner, C., Kulbe, K. D. & Haltrich, D. ( 2001; ). Continuous enzymatic regeneration of redox mediators used in biotransformation reactions employing flavoproteins. J Mol Catal B Enzymatic 11, 541-550.[CrossRef]
    [Google Scholar]
  5. Bollag, J.-M. & Leonowicz, A. ( 1984; ). Comparative studies of extracellular fungal laccases. Appl Environ Microbiol 48, 849-854.
    [Google Scholar]
  6. Bourbonnais, R. & Paice, M. G. ( 1990; ). Oxidation of non-phenolic substrates: an expanded role of laccase in lignin biodegradation. FEBS Lett 267, 99-102.[CrossRef]
    [Google Scholar]
  7. Canters, G. W. & Gilardi, G. ( 1993; ). Engineering type 1 copper sites in proteins. FEBS Lett 325, 39-48.[CrossRef]
    [Google Scholar]
  8. Collins, P. J. & Dobson, A. D. ( 1997; ). Regulation of laccase gene transcription in Trametes versicolor. Appl Environ Microbiol 63, 3444-3450.
    [Google Scholar]
  9. Dowzer, C. E. & Kelly, J. M. ( 1991; ). Analysis of the creA gene, a regulator of carbon catabolite repression in Aspergillus nidulans. Mol Cell Biol 11, 5701-5709.
    [Google Scholar]
  10. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. & Smith, F. ( 1956; ). Colorimetric method for determination of sugars and related substances. Anal Chem 28, 350-356.[CrossRef]
    [Google Scholar]
  11. Ducros, V., Brzozowski, A. M., Wilson, K. S., Østergaard, P., Schneider, P., Svendson, A. & Davies, G. J. ( 2001; ). Structure of the laccase from Coprinus cinereus at 1·68 Å resolution: evidence for different ‘type 2 Cu-depleted’ isoforms. Acta Crystallogr D Biol Crystallogr 57, 333-336.
    [Google Scholar]
  12. Eggert, C., Temp, U. & Eriksson, K.-E. L. ( 1996; ). Laccase-producing white-rot fungus lacking lignin peroxidase and manganese peroxidase. ACS Symp Ser 655, 130-150.
    [Google Scholar]
  13. Eggert, C., LaFayette, P. R., Temp, U., Eriksson, K.-E. L. & Dean, J. F. D. ( 1998; ). Molecular analysis of a laccase gene from the white rot fungus Pycnoporus cinnabarinus. Appl Environ Microbiol 64, 1766-1772.
    [Google Scholar]
  14. Galhaup, C. & Haltrich, D. ( 2001; ). Enhanced formation of laccase activity by the white-rot fungus Trametes pubescens in the presence of copper. Appl Microbiol Biotechnol 56, 225-232.[CrossRef]
    [Google Scholar]
  15. Galhaup, C., Wagner, H., Hinterstoisser, B. & Haltrich, D. ( 2002; ). Increased production of laccase by the wood-degrading basidiomycete Trametes pubescens. Enzyme Microb Technol 30, 529-536.[CrossRef]
    [Google Scholar]
  16. Garzillo, A. M., Colao, M. C., Buonocore, V. & 10 other authors ( 2001; ). Structural and kinetic characterization of native laccases from Pleurotus ostreatus, Rigidoporus lignosus, and Trametes trogii. J Protein Chem 20, 191–201.[CrossRef]
    [Google Scholar]
  17. Gianfreda, L., Xu, F. & Bollag, J.-M. ( 1999; ). Laccases: a useful group of oxidoreductive enzymes. Biorem J 3, 1-25.[CrossRef]
    [Google Scholar]
  18. Gurr, S. J., Unkles, S. E. & Kinghorn, J. R. ( 1987; ). The structure and organization of nuclear genes of filamentous fungi. In Gene Structure in Eukaryotic Microbes , pp. 93-139. Edited by J. R. Kinghorn. Oxford: IRL Press.
  19. Jovanovic, S. V., Tosic, M. & Simic, M. G. ( 1991; ). Use of the Hammett correlation and σ+ for calculation of one-electron redox potentials of antioxidants. J Phys Chem 95, 10824-10827.[CrossRef]
    [Google Scholar]
  20. Karahanian, E., Corsini, G., Lobos, S. & Vicuña, R. ( 1998; ). Structure and expression of a laccase gene from the ligninolytic basidiomycete Ceriporiopsis subvermispora. Biochim Biophys Acta 1443, 65-74.[CrossRef]
    [Google Scholar]
  21. Karin, M. ( 1985; ). Metallothioneins: proteins in search of function. Cell 41, 9-10.[CrossRef]
    [Google Scholar]
  22. Kojima, Y., Tsukuda, Y., Kawai, Y., Tsukamoto, A., Sugiura, J. & Sakaino, M. ( 1990; ). Cloning, sequence analysis, and expression of ligninolytic phenoloxidase genes of the white-rot basidiomycete Coriolus hirsutus. J Biol Chem 265, 15224-15230.
    [Google Scholar]
  23. Lewin, B. (1997). Genes VI, 6th edn. Oxford: Oxford University Press.
  24. Li, Y. & Jaiswal, A. K. ( 1992; ). Regulation of human NAD(P)H:quinone oxidoreductase gene. Role of AP1 binding site contained within human antioxidant response element. J Biol Chem 267, 15097-15104.
    [Google Scholar]
  25. Mager, W. H. & De Kruijff, A. J. ( 1995; ). Stress-induced transcriptional activation. Microbiol Rev 59, 506-531.
    [Google Scholar]
  26. Mansur, M., Suárez, T. & González, A. E. ( 1998; ). Differential gene expression in the laccase gene family from basidiomycete I-62 (CECT 20197). Appl Environ Microbiol 64, 771-774.
    [Google Scholar]
  27. Muñoz, C., Guillén, F., Martı́nez, A. T. & Martı́nez, M. J. ( 1997; ). Laccase isoenzymes of Pleurotus eryngii: characterization, catalytic properties, and participation in activation of molecular oxygen and Mn2+ oxidation. Appl Environ Microbiol 63, 2166-2174.
    [Google Scholar]
  28. Naqui, A. & Varfolomeev, S. D. ( 1980; ). Inhibition mechanism of Polyporus laccase by fluoride ion. FEBS Lett 113, 157-160.[CrossRef]
    [Google Scholar]
  29. Padgett, R. A., Konarska, M. M., Grabowski, P. J., Hardy, S. F. & Sharp, P. A. ( 1984; ). Lariat RNAs as intermediates and products in the splicing of messenger RNA precursors. Science 225, 898-903.[CrossRef]
    [Google Scholar]
  30. Palmer, A. E., Randall, D. W., Xu, F. & Solomon, E. I. ( 1999; ). Spectroscopic studies and electronic structure description of the high potential type 1 copper site in fungal lacase: insight into the effect of the axial ligand. J Am Chem Soc 121, 7138-7149.[CrossRef]
    [Google Scholar]
  31. 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.[CrossRef]
    [Google Scholar]
  32. Palmieri, G., Giardina, P., Bianco, C., Fontanella, B. & Sannia, G. ( 2000; ). Copper induction of laccase isoenzymes in the ligninolytic fungus Pleurotus ostreatus. Appl Environ Microbiol 66, 920-924.[CrossRef]
    [Google Scholar]
  33. Palmieri, G., Bianco, C., Cennamo, G., Giardina, P., Marino, G., Monti, M. & Sannia, G. ( 2001; ). Purification, characterization, and functional role of a novel extracellular protease from Pleurotus ostreatus. Appl Environ Microbiol 67, 2754-2759.[CrossRef]
    [Google Scholar]
  34. Proudfoot, N. ( 1991; ). Poly(A) signals. Cell 64, 671-674.[CrossRef]
    [Google Scholar]
  35. Reinhammar, B. R. M. ( 1984; ). Laccase. In Copper Proteins and Copper Enzymes , pp. 1-35. Edited by R. Lontie. Boca Raton, FL: CRC Press.
  36. Ronne, H. ( 1995; ). Glucose repression in fungi. Trends Genet 11, 12-17.[CrossRef]
    [Google Scholar]
  37. Rushmore, T. H., Morton, M. R. & Pickett, C. B. ( 1991; ). The antioxidant responsive element: activation by oxidative stress and identification of the DNA consensus sequence required for functional activity. J Biol Chem 266, 11632-11639.
    [Google Scholar]
  38. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  39. Soden, D. M. & Dobson, A. D. W. ( 2001; ). Differential regulation of laccase gene expression in Pleurotus sajor-caju. Microbiology 147, 1755-1763.
    [Google Scholar]
  40. Solomon, E. I., Sundaram, U. M. & Machonkin, T. E. ( 1996; ). Multicopper oxidases and oxygenases. Chem Rev 96, 2563-2605.[CrossRef]
    [Google Scholar]
  41. Strauss, J., Horvath, H. K., Abdallah, B. M., Kindermann, J., Mach, R. L. & Kubicek, C. P. ( 1999; ). The function of CreA, the carbon catabolite repressor of Aspergillus nidulans, is regulated at the transcriptional and post-transcriptional level. Mol Microbiol 32, 169-178.[CrossRef]
    [Google Scholar]
  42. Tamai, K. T., Liu, X., Silar, P., Sosinowski, T. & Thiele, D. J. ( 1994; ). Heat shock transcription factor activates yeast metallothionein gene expression in response to heat and glucose starvation via distinct signalling pathways. Mol Cell Biol 14, 8155-8165.
    [Google Scholar]
  43. Thiele, D. J. ( 1992; ). Metal-regulated transcription in eukaryotes. Nucleic Acids Res 20, 1183-1191.[CrossRef]
    [Google Scholar]
  44. Thurston, C. F. ( 1994; ). The structure and function of fungal laccases. Microbiology 140, 19-26.[CrossRef]
    [Google Scholar]
  45. Xu, F. ( 1996; ). Oxidation of phenols, anilines, and benzenethiols by fungal laccases: correlation between activity and redox potentials as well as halide inhibition. Biochemistry 35, 7608-7614.[CrossRef]
    [Google Scholar]
  46. Xu, F. ( 1997; ). Effect of redox potential and hydroxide inhibition on the pH activity profile of fungal laccases. J Biol Chem 272, 924-928.[CrossRef]
    [Google Scholar]
  47. Xu, F. ( 1999; ). Laccase. In Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis, and Bioseparation , pp. 1545-1554. Edited by M. C. Flickinger & S. W. Drew. New York: Wiley.
  48. Xu, F., Shin, W., Brown, S. H., Wahleithner, J. A., Sundaram, U. M. & Solomon, E. I. ( 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.[CrossRef]
    [Google Scholar]
  49. Yaropolov, A. I., Skorobogat’ko, O. V., Vartanov, S. S. & Varfolomeyev, S. D. ( 1994; ). Laccase: properties, catalytic mechanism, and applicability. Appl Biochem Biotechnol 49, 257-280.[CrossRef]
    [Google Scholar]
  50. Yaver, D. S. & Golightly, E. J. ( 1996; ). Cloning and characterization of three laccase genes from the white-rot basidiomycete Trametes villosa: genomic organization of the laccase gene family. Gene 181, 95-102.[CrossRef]
    [Google Scholar]
  51. Yaver, D. S., Xu, F., Golightly, E. J. & 7 other authors ( 1996; ). Purification, characterization, molecular cloning, and expression of two laccase genes from the white rot basidiomycete Trametes villosa. Appl Environ Microbiol 62, 834–841.
    [Google Scholar]
  52. Yaver, D. S., Del Carmen Overjero, M., Xu, F., Nelson, B. A., Brown, K. M., Halkier, T., Bernauer, S., Brown, S. H. & Kauppinen, S. ( 1999; ). Molecular characterization of laccase genes from the basidiomycete Coprinus cinereus and heterologous expression of the laccase lcc1. Appl Environ Microbiol 65, 4943-4948.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-148-7-2159
Loading
/content/journal/micro/10.1099/00221287-148-7-2159
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error