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Cloning of a Laccase Gene from a Novel Basidiomycete Trametes sp. 420 and Its Heterologous Expression in Pichia pastoris

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Abstract

The laccase gene lacD, cloned from a novel laccase-producing basidiomycete Trametes sp. 420, contained 2,052 base pairs (bp) interrupted by 8 introns. lacD displayed a relatively high homology with laccase genes from other white rot fungi, whereas the homology between lacD and laccase genes from plants, insects, or bacteria was less than 25%. A 498–amino acid peptide encoded by the lacD cDNA was heterologously expressed in the Pichia pastoris strain GS115, resulting in the highest yield of laccase (8.3 × 104 U/l) as determined with ABTS (2,2′-azinobis [3-ethylbenzothia-zoline-6-sulfonic acid]) as the substrate. Additionally, the enzyme activity of recombinant laccase on decolorization of some industrial dyes was assessed.

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References

  1. Abadulla E, Tzanov T, Costa S, Robra KH, Cavaco-Paulo A, Gübitz GM (2000) Decolorization and detoxification of textile dyes with a laccase from Trametes hirsute. Appl Environ Microbiol 66:3357–3362

    Article  PubMed  CAS  Google Scholar 

  2. Alexandre G, Zhulin IB (2000) Laccases are widespread in bacteria. Trends Biotechnol 18:41–42

    Article  PubMed  CAS  Google Scholar 

  3. Bulter T, Alcalde M, Sieber V, Meinhold P, Schlachtbauer C, Arnold FH (2003) Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution. Appl Environ Microbiol 69:987–995

    Article  PubMed  CAS  Google Scholar 

  4. Canters GW, Gilardi G (1993) Engineering type 1 copper sites in proteins. FEBS Lett 325:39–48

    Article  PubMed  CAS  Google Scholar 

  5. Chivukula M, Renganathan V (1995) Phenolic azo oxidation by laccase from Pyricularia oryzae. Appl Environ Microbiol 61:4374–4377

    PubMed  CAS  Google Scholar 

  6. Ducros V, Brzozowski AM, Wilson KS, Østergaard P, Schneider P, Svendson A, Davies GJ (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

    Article  PubMed  CAS  Google Scholar 

  7. Eggert C, LaFayette PR, Temp U, Eriksson KEL, Dean JFD (1998) Molecular analysis of a laccase gene from the white rot fungus Pycnoporus cinnabarinus. Appl Environ Microbiol 64:1766–1772

    PubMed  CAS  Google Scholar 

  8. Gavel Y, von Heijine G (1990) Sequence differences between glycosylated and non-glycosylated Asn-X-Ser/Thr acceptor sites: implications for protein engineering. Protein Eng 3:433–442

    Article  PubMed  CAS  Google Scholar 

  9. Gianfreda L, Xu F, Bollag JM (1999) Laccases: a useful group of oxidoreductive enzymes. Biorem J 3:1–25

    Article  CAS  Google Scholar 

  10. Guo M, Lu F, Pu J, Bai D, Du L (2005) Molecular cloning of the cDNA encoding laccase from Trametes versicolor and heterologous expression in Pichia methanolica. Appl Microbiol Biotechnol 69:178–183

    Article  PubMed  CAS  Google Scholar 

  11. Gurr SJ, Unkles SE, Kinghorn JR (1987) The structure and organization of nuclear genes of filamentous fungi. In: Kinghorn JR (ed) Gene structure in eukaryotic microbes. Oxford, IRL Press, pp 93–139

    Google Scholar 

  12. Hong F, Meinander NQ, Jönsson L (2002) Fermentation strategies for improved heterologous expression of Iaccase in Pichaia pastoris. Biotechnol Bioeng 79:438–449

    Article  PubMed  CAS  Google Scholar 

  13. Hoshida H, Nakao M, Kanazawa H, Kubo K, Hakukawa T, Morimasa K, Akada R, Nishizawa Y (2001) Isolation of five laccase gene sequences from the white-rot fungus Trametes sanguinea by PCR, and cloning, characterization and expression of the laccase cDNA in yeasts. J Biosci Bioeng 92:372–380

    Article  PubMed  CAS  Google Scholar 

  14. Hoshida H, Fujita T, Murata K, Kubo K, Akada R (2005) Copper-dependent production of a Pycnoporus coccineus extracellular laccase in Aspergillus oryzae and Saccharomyces cerevisiae. Biosci Biotechnol Biochem 69:1090–1097

    Article  PubMed  CAS  Google Scholar 

  15. Jolivalt C, Madzak C, Brault A, Caminade E, Malosse C, Mougin C (2005) Expression of laccase IIIb from the white-rot fungus Trametes versicolor in the yeast Yarrowia lipolytica for environmental applications. Appl Microbiol Biotechnol 66:450–456

    Article  PubMed  CAS  Google Scholar 

  16. Jönsson LJ, Saloheimo M, Penttila M (1997) Laccase from the white-rot fungus Trametes versicolor: cDNA cloning of lcc1 and expression in Pichia pastoris. Curr Genet 32:425–430

    Article  PubMed  Google Scholar 

  17. Keum YS, Li QX (2004) Fungal laccase-catalyzed degradation of hydroxy polychlorinated biphenyls. Chemosphere 56:23–30

    Article  PubMed  CAS  Google Scholar 

  18. Larsson S, Cassland P, Jönsson LJ (2001). Development of a Saccharomyces cerevisiae strain with enhanced resistance to phenolic fermentation inhibitors in lignocellulose hydrolysates by heterologous expression of laccase. Appl Environ Microbiol 67:1163–1170

    Article  PubMed  CAS  Google Scholar 

  19. Li JF, Hong YZ, Xiao YZ (2007) Cloning and efficient expression of the gene of laccase C from Trametes sp. 420 & potential of the recombinant laccase in dye decolorization. Acta Microbiol Sinica 27:54–58

    Google Scholar 

  20. Liu W, Chao Y, Liu S, Bao H, Qian S (2003) Molecular cloning and characterization of a laccase gene from the basidiomycete Fome lignosus and expression in Pichia pastoris. Appl Microbiol Biotechnol 63:174–181

    Article  PubMed  CAS  Google Scholar 

  21. Necochea R, Valderrama B, Diaz-Sandoval S, Folch-Mallol JL, Vazquez-Duhalt R, Iturriaga G (2005) Phylogenetic and biochemical characterisation of a recombinant laccase from Trametes versicolor. FEMS Microbiol Lett 244:235–241

    Article  PubMed  CAS  Google Scholar 

  22. O’Callaghan J, O’Brien MM, McClean K, Dobson ADW (2002) Optimisation of the expression of a Trametes versicolor laccase gene in Pichia pastoris. J Ind Microbiol Biotechnol 29:55–59

    Article  PubMed  CAS  Google Scholar 

  23. Padgett RA, Konarska MM, Grabowski PJ, Hardy SF, Sharp PA (1984) Lariat RNAs as intermediates and products in the splicing of messenger RNA precursors. Science 255:898–903

    Article  Google Scholar 

  24. Record E, Punt PJ, Chamkha M, Labat M, van Den Hondel CAMJJ, 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

    Article  PubMed  CAS  Google Scholar 

  25. Soden DM, Dobson ADW (2001) Differential regulation of laccase gene expression in Pleurotus sajor-caju. Microbiology 147:1755–1763

    PubMed  CAS  Google Scholar 

  26. Soden DM, O’Callaghan J, Dobson ADW (2002) Molecular cloning of a laccase isozyme gene from Pleurotus sajor-caju and expression in the heterologous Pichia pastoris host. Microbiology 148:4003–4014

    PubMed  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  28. Ullah MA, Bedford CT, Evans CS (2000) Reactions of pentachlorophenol with laccase from Coriolus versicolor. Appl Microbiol Biotechnol 53:230–234

    Article  PubMed  CAS  Google Scholar 

  29. Vianello F, Cambria A, Ragusa S, Cambria MT, Zennaro L, Rigo A (2004) A high sensitivity amperometric biosensor using a monomolecular layer of laccase as biorecognition element. Biosens Bioelectron 20:315–321

    Article  PubMed  CAS  Google Scholar 

  30. Xiao YZ, Tu XM, Wang J, Zhang M, Cheng Q, Zeng WY, Shi YY (2003) Purification, molecular characterization and reactivity with aromatic compounds of a laccase from basidiomycete Trametes sp. strain AH28-2. Appl Microbiol Biotechnol 60:700–707

    CAS  Google Scholar 

  31. Xiao YZ, Chen Q, Wu J, Hang J, Hong YZ, Shi YY, Wang YP (2004) Selective induction, purification and characterization of a laccase isozyme from the basidiomycete Trametes sp. AH28-2. Mycologia 96:26–35

    CAS  Google Scholar 

  32. Xiao YZ, Zhou T, Hong YZ, Zhang M, Ge HH (2005) Dye decolorization and heterologous expression of laccase A from Trametes sp. AH28-2. In: Li SP (ed) The 8th internal academic seminar on environmental microbes. Beijing, CI Press, pp 618–625

    Google Scholar 

  33. Xiao YZ, Hong YZ, Li JF, Hang J, Tong PG, Fang W, Zhou CZ (2006) Cloning of novel laccase isozyme genes from Trametes sp. AH28-2 and analyses of their differential expression. Appl Microbiol Biotechnol 71:493–501

    Article  PubMed  CAS  Google Scholar 

  34. 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

    PubMed  Google Scholar 

  35. Xu F, Berka RM, Wahleithner JA, Nelson BA, Shuster JR, Brown SH, Palmer AE, Solomon EI (1998) Site-directed mutagenesis in fungal laccases: effect on redox potential, activity and pH profile. J Biochem 334:63–70

    CAS  Google Scholar 

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Acknowledgments

We are grateful to Dr. Weixin Wang for reviewing this manuscript. This work was supported by grants from the National Natural Sciences Foundation of China (30370045, 30470056, 30670069), the Key Programs for Science and Technology of Chinese Ministry of Education (204064), the Science and Technology Foundation of Distinguished Young Scholars of Anhui Province (04043048, 050430302), and the Innovative Research Team of 211 Project of Anhui University (02203109).

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Authors and Affiliations

  1. School of Life Sciences and Modern Experiment Technology Center, Anhui University, Hefei, 230039, P.R. China

    Yu-zhi Hong, Hong-min Zhou, Jian-feng Li & Ya-zhong Xiao

  2. School of Life Sciences, University of Science and Technology of China, Hefei, 230026, P.R. China

    Xiao-ming Tu

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  1. Yu-zhi Hong
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  2. Hong-min Zhou
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Correspondence to Xiao-ming Tu.

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Hong, Yz., Zhou, Hm., Tu, Xm. et al. Cloning of a Laccase Gene from a Novel Basidiomycete Trametes sp. 420 and Its Heterologous Expression in Pichia pastoris . Curr Microbiol 54, 260–265 (2007). https://doi.org/10.1007/s00284-006-0068-8

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