1887

Abstract

Previous studies on laccase have shown that this enzyme is very interesting for both basic research purposes and industrial applications. In order to obtain a reliable and efficient source for this laccase, it was produced in the filamentous fungus . Two approaches were used: production of a non-fused laccase and a hydrophobin–laccase fusion protein. Both proteins were expressed in under the promoter, and significantly higher activities were obtained with the non-fused laccase in shake-flask cultures (corresponding to about 230 mg l). Northern blot analyses showed rather similar mRNA levels from both expression constructs. Western analysis indicated intracellular accumulation and degradation of the hydrophobin–laccase fusion protein, showing that production of the fusion was limited at the post-transcriptional level. No induction of the unfolded protein response pathway by laccase production was detected in the transformants by Northern hybridization. The most promising transformant was grown in a fermenter in batch and fed-batch modes. The highest production level obtained in the fed-batch culture was 920 mg l. The recombinant laccase was purified from the culture supernatant after cleaving the major contaminating protein, cellobiohydrolase I, by papain. The recombinant and wild-type laccases were compared with regard to substrate kinetics, molecular mass, pH optimum, thermostability, and processing of the N- and C-termini, and they showed very similar properties.

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2004-09-01
2024-12-03
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References

  1. Arias M. E., Arenas M., Rodríguez J., Soliveri J., Ball A. S., Hernández M. 2003; Kraft pulp biobleaching and mediated oxidation of a nonphenolic substrate by laccase from Streptomyces cyaneus CECT 3335. Appl Environ Microbiol 69:1953–1958 [CrossRef]
    [Google Scholar]
  2. Bailey M. J., Tähtiharju J. 2003; Efficient cellulase production by Trichoderma reesei in continuous cultivation on lactose medium with a computer-controlled feeding strategy. Appl Microbiol Biotechnol 62:156–162 [CrossRef]
    [Google Scholar]
  3. Berka R. M., Schneider P., Golightly E. J., Brown S. H., Madden M., Brown K. M., Halkier T., Mondorf K., Xu F. 1997; Characterization of the gene encoding an extracellular laccase of Myceliophthora thermophila and analysis of the recombinant enzyme expressed in Aspergillus oryzae. Appl Environ Microbiol 63:3151–3157
    [Google Scholar]
  4. Chefetz B., Chen Y., Hadar Y. 1998; Purification and characterization of laccase from Chaetomium thermophilum and its role in humification. Appl Environ Microbiol 64:3175–3179
    [Google Scholar]
  5. Dawson R. M. C., Elliot D. C., Elliot W. H., Jones K. M. 1959 Data for Biochemical Research London: Oxford University Press;
    [Google Scholar]
  6. 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]
  7. Garzillo A. M. V., Colao M. C., Caruso C., Caporale C., Celletti D., Buonocore V. 1998; Laccase from the white-rot fungus Trametes trogii. Appl Microbiol Biotechnol 49:545–551 [CrossRef]
    [Google Scholar]
  8. Gianfreda L., Xu F., Bollag J.-M. 1999; Laccases: a useful group of oxidoreductive enzymes. Biorem J 3:1–25 [CrossRef]
    [Google Scholar]
  9. Hakulinen N., Kiiskinen L.-L., Kruus K., Saloheimo M., Paananen A., Koivula A., Rouvinen J. 2002; Crystal structure of a laccase from Melanocarpus albomyces with an intact trinuclear copper site. Nat Struct Biol 9:601–605
    [Google Scholar]
  10. IUPAC 1987; Measurement of cellulase activities. Pure Appl Chem 59:257–268
    [Google Scholar]
  11. Kiiskinen L.-L., Saloheimo M. 2004; Molecular cloning and expression in Saccharomyces cerevisiae of a laccase gene from the ascomycete Melanocarpus albomyces. Appl Environ Microbiol 70:137–144 [CrossRef]
    [Google Scholar]
  12. Kiiskinen L.-L., Viikari L., Kruus K. 2002; Purification and characterisation of a novel laccase from the ascomycete Melanocarpus albomyces. Appl Microbiol Biotechnol 59:198–204 [CrossRef]
    [Google Scholar]
  13. Larrondo L. F., 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 [CrossRef]
    [Google Scholar]
  14. Leonowicz A., Grzywnowicz K. 1981; Quantitative estimation of laccase forms in some white-rot-fungi using syringaldazine as a substrate. Enzyme Microb Technol 3:55–58 [CrossRef]
    [Google Scholar]
  15. Linder M., Selber K., Nakari-Setälä T., Qiao M., Kula M.-R., Penttilä M. 2001; The hydrophobins HFBI and HFBII from Trichoderma reesei showing efficient interactions with nonionic surfactants in aqueous two-phase systems. Biomacromolecules 2:511–517 [CrossRef]
    [Google Scholar]
  16. Lomascolo A., Record E., Herpoel-Gimbert I., Delattre M., Robert J. L., Georis J., Dauvrin T., Sigoillot J. C., Asther M. 2003; Overproduction of laccase by a monokaryotic strain of Pycnoporus cinnabarinus using ethanol as inducer. J Appl Microbiol 94:618–624 [CrossRef]
    [Google Scholar]
  17. Mandels M., Weber J. 1969; The production of cellulases. Adv Chem Ser 95:391–414
    [Google Scholar]
  18. Mäntylä A., Paloheimo M., Suominen P. 1998; Industrial mutants and recombinant strains of Trichoderma reesei. In Trichoderma and Gliocladium vol 2 pp. 291–309 Edited by Harman G., Kubicek C. London: Taylor & Francis;
    [Google Scholar]
  19. Martins L. O., Soares C. M., Pereira M. M., Teixeira M., Costa T., Jones G. H., Henriques A. O. 2002; Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillus subtilis endospore coat. J Biol Chem 277:18849–18859 [CrossRef]
    [Google Scholar]
  20. Messerschmidt A. 1997; Copper metalloenzymes. In Comprehensive Biological Catalysis vol III pp. 401–426 Edited by Sinnott M. London: Academic Press;
    [Google Scholar]
  21. Montenecourt B. S., Eveleigh D. E. 1979; Selective screening methods for the isolation of high yielding mutants of Trichoderma reesei. Adv Chem Ser 181:289–301
    [Google Scholar]
  22. Mori K. 2003; Frame switch splicing and regulated intramembrane proteolysis: key words to understand the unfolded protein response. Traffic 4:519–528 [CrossRef]
    [Google Scholar]
  23. Nakari-Setälä T., Aro N., Kalkkinen N., Alatalo E., Penttilä M. 1996; Genetic and biochemical characterization of the Trichoderma reesei hydrophobin HFBI. Eur J Biochem 235:248–255 [CrossRef]
    [Google Scholar]
  24. Niku-Paavola M.-L., Karhunen E., Salola P., Raunio V. 1988; Ligninolytic enzymes of the white-rot fungus Phlebia radiata. Biochem J 254:877–884
    [Google Scholar]
  25. Nyyssönen E., Penttilä M., Harkki A., Saloheimo A., Knowles J., Keränen S. 1993; Efficient production of antibody fragments by the filamentous fungus Trichoderma reesei. Bio/Technology 11:591–595 [CrossRef]
    [Google Scholar]
  26. Otterbein L., Record E., Longhi S., Asther M., Moukha S. 2000; Molecular cloning of the cDNA encoding laccase from Pycnoporus cinnabarinus I-937 and expression in Pichia pastoris. Eur J Biochem 267:1619–1625 [CrossRef]
    [Google Scholar]
  27. Pakula T. M., Laxell M., Huuskonen A., Uusitalo J., Saloheimo M., Penttilä M. 2003; The effects of drugs inhibiting protein secretion in the filamentous fungus Trichoderma reesei. Evidence for down-regulation of genes that encode secreted proteins in the stressed cells. J Biol Chem 278:45011–45020 [CrossRef]
    [Google Scholar]
  28. 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]
  29. Paloheimo M., Mäntylä A., Kallio J., Suominen P. 2003; High-yield production of a bacterial xylanase in the filamentous fungus Trichoderma reesei requires a carrier polypeptide with an intact domain structure. Appl Environ Microbiol 69:7073–7082 [CrossRef]
    [Google Scholar]
  30. Palonen H., Saloheimo M., Viikari L., Kruus K. 2003; Purification, characterization and sequence analysis of a laccase from the ascomycete Mauginiella sp. Enzyme Microb Technol 33:854–862 [CrossRef]
    [Google Scholar]
  31. Paszczynski A., Huynh V.-B., Crawford R. 1985; Enzymatic activities of an extracellular manganese-dependent peroxidase from Phanerochaete chrysosporium. FEMS Microbiol Lett 29:37–41 [CrossRef]
    [Google Scholar]
  32. Penttilä, Nevalainen H., Rättö M, Salminen E., Knowles J. M. 1987; A versatile transformation system for cellulolytic filamentous fungus Trichoderma reesei . Gene 61:155–164 [CrossRef]
    [Google Scholar]
  33. Record E., Punt P. J., Chamkha M., Labat M., van den Hondel C. A. M. J. J., Asther M. 2002; Expression of the Pycnoporus cinnabarinus laccase gene in Aspergillus niger and characterization of the recombinant enzyme. Eur J Biochem 269:602–609 [CrossRef]
    [Google Scholar]
  34. Saloheimo M., Niku-Paavola M.-L. 1991; Heterologous production of a ligninolytic enzyme: expression of the Phlebia radiata laccase gene in Trichoderma reesei. Bio/Technology 9:987–990 [CrossRef]
    [Google Scholar]
  35. Saloheimo M., Barajas V., Niku-Paavola M.-L., Knowles J. 1989; A lignin peroxidase-encoding cDNA from the white-rot fungus Phlebia radiata: characterization and expression in Trichoderma reesei. Gene 85:343–351 [CrossRef]
    [Google Scholar]
  36. Saloheimo M., Lund M., Penttilä M. 1999; The protein disulphide isomerase gene of the fungus Trichoderma reesei is induced by endoplasmic reticulum stress and regulated by the carbon source. Mol Gen Genet 262:35–45 [CrossRef]
    [Google Scholar]
  37. Saloheimo M., Valkonen M., Penttilä M. 2003; Activation mechanisms of the HACI-mediated unfolded protein response in filamentous fungi. Mol Microbiol 47:1149–1161 [CrossRef]
    [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;
    [Google Scholar]
  39. Schneider P., Caspersen M. B., Mondorf K., Halkier T., Skov L. K., Østergaard P. R., Brown K. M., Brown S. H., Xu F. 1999; Characterization of a Coprinus cinereus laccase. Enzyme Microb Technol 25:502–508 [CrossRef]
    [Google Scholar]
  40. Selber K., Collén A., Hyytiä T., Penttilä M., Tjerneld F., Kula M.-R. 2001; Parameters influencing protein extraction for whole broths in detergent based aqueous two-phase systems. Bioseparation 10:229–236 [CrossRef]
    [Google Scholar]
  41. Soden D. M., O'Callaghan J., Dobson A. D. W. 2002; Molecular cloning of a laccase isozyme gene from Pleurotus sajor-caju and expression in the heterologous Pichia pastoris host. Microbiology 148:4003–4014
    [Google Scholar]
  42. Suominen P. L., Mäntylä A. L., Karhunen T., Hakola S., Nevalainen H. 1993; High frequency one-step gene replacement in Trichoderma reesei. II. Effects of deletions of individual cellulase genes. Mol Gen Genet 241:523–530
    [Google Scholar]
  43. Thurston C. 1994; The structure and function of fungal laccases. Microbiology 140:19–26 [CrossRef]
    [Google Scholar]
  44. Uldschmid A., Dombi R., Marbach K. 2003; Identification and functional expression of ctaA, a P-type ATPase gene involved in copper trafficking in Trametes versicolor. Microbiology 149:2039–2048 [CrossRef]
    [Google Scholar]
  45. van Tilbeurgh H., Tomme P., Claeyssens M., Bhikhabhai R., Pettersson G. 1986; Limited proteolysis of the cellobiohydrolase I from Trichoderma reesei: separation of functional domains. FEBS Lett 204:223–227 [CrossRef]
    [Google Scholar]
  46. Wariishi H., Valli K., Gold M. H. 1992; Manganese(II) oxidation by manganese peroxidase from the basidiomycete Phanerochaete chrysosporium. J Biol Chem 267:23688–23695
    [Google Scholar]
  47. Xu F. 1999; Recent progress in laccase study: properties, enzymology, production, and applications. In The Encyclopedia of Bioprocessing Technology: Fermentation, Biocatalysis and Bioseparation pp. 1545–1554 Edited by Flickinger M. C., Drew S. W. New York: Wiley;
    [Google Scholar]
  48. Xu F., Shin W., Brown S., 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. 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]
  50. Yaver D. S., Overjero M. J., 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]
  51. Yoshida H. 1883; Chemistry of lacquer (Urushi), part 1. J Chem Soc 43:472–486 [CrossRef]
    [Google Scholar]
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