Identification of a novel copper-activated and halide-tolerant laccase in Geobacillus thermopakistaniensis

Abstract

Genome search of Geobacillus thermopakistaniensis, formerly Geobacillus sp. SBS-4S, revealed the presence of an open reading frame (ESU71923) annotated as laccase. However, the gene product did not display any laccase-like activity against the substrates examined. The laccase activity was, therefore, purified from G. thermopakistaniensis cells and N-terminal amino acid residues of the enzyme were determined. These residues matched the N-terminal sequence of an open reading frame annotated as a copper oxidase (ESU72270). In order to characterize the enzyme, recombinant ESU72270 was prepared in Escherichia coli. The recombinant protein was found to exhibit a negligible amount of laccase activity when produced in the absence of copper in the growth medium. However, the recombinant protein exhibited significantly high laccase activity when produced in the presence of copper. The recombinant enzyme showed highest activity at 60 °C and a pH of 7–7.5. The purified enzyme was highly tolerant to various halides and organic solvents, thus having a potential for various industrial applications. To the best of our knowledge, this is the first characterization of a laccase from genus Geobacillus which identifies a gene responsible for functional laccase in this genus.

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References

  1. Ausec L, Črnigoj M, Šnajder M, Ulrih NP, Mandic-Mulec I (2015) Characterization of a novel high-pH-tolerant laccase-like multicopper oxidase and its sequence diversity in Thioalkalivibrio sp. Appl Microbiol Biotechnol 99:9987–9999

    CAS  Article  PubMed  Google Scholar 

  2. Bourbonnais R, Paice MG, Reid ID, Lanthier P, Yaguchi M (1995) 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 61:1876–1880

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Brander S, Mikkelsen JD, Kepp KP (2015) TtMCO: a highly thermostable laccase-like multicopper oxidase from the thermophilic Thermobaculum terrenum. J Mol Catal B Enzym 112:59–65

    CAS  Article  Google Scholar 

  4. Callejón S, Sendra R, Ferrer S, Pardo I (2016) Cloning and characterization of a new laccase from Lactobacillus plantarum J16 CECT 8944 catalyzing biogenic amines degradation. Appl Microbiol Biotechnol 100:3113–3124

    Article  PubMed  Google Scholar 

  5. Couto SR, Herrera JLT (2006) Industrial and biotechnological applications of laccases: a review. Biotechnol Adv 24:500–513

    Article  Google Scholar 

  6. Enaud E, Trovaslet M, Naveau F, Decristoforo A, Bizet S, Vanhulle S, Jolivalt C (2011) Laccase chloride inhibition reduction by an anthraquinonic substrate. Enzyme Microb Technol 49:517–525

    CAS  Article  PubMed  Google Scholar 

  7. Fang Z, Li T, Wang Q, Zhang X, Peng H, Fang W, Hong Y, Ge H, Xiao Y (2011) A bacterial laccase from marine microbial metagenome exhibiting chloride tolerance and dye decolorization ability. Appl Microbiol Biotechnol 89:1103–1110

    CAS  Article  PubMed  Google Scholar 

  8. Feng H, Zhang D, Sun Y, Zhi Y, Mao L, Luo Y, Xu L, Wang L, Zhou P (2015) Expression and characterization of a recombinant laccase with alkalistable and thermostable properties from Streptomyces griseorubens JSD-1. Appl Biochem Biotechnol 176:547–562

    CAS  Article  PubMed  Google Scholar 

  9. Gochev VK and Krastanov AI (2007) Fungal laccases. Bulg J Agric Sci 13:75–83

    Google Scholar 

  10. Guan ZB, Zhang N, Song CM, Zhou W, Zhou LX, Zhao H, Xu CW, Cai YJ, Liao XR (2014) Molecular cloning, characterization, and dye-decolorizing ability of a temperature- and pH-stable laccase from Bacillus subtilis X1. Appl Biochem Biotechnol 172:1147–1157

    CAS  Article  PubMed  Google Scholar 

  11. Guo X, Zhou S, Wang Y, Song J, Wang H, Kong D, Zhu J, Dong W, He M, Hu G, Ruan Z (2016) Characterization of a highly thermostable and organic solvent-tolerant copper-containing polyphenol oxidase with dye-decolorizing ability from Kurthia huakuii LAM0618T. PLoS One. doi:10.1371/journal.pone.0164810

    Google Scholar 

  12. Hildén K, Hakala TK, Lundell T (2009) Thermotolerant and thermostable laccases. Biotechnol Lett 31:1117–1128

    Article  PubMed  Google Scholar 

  13. Hoegger PJ, Kilaru S, James TY, Thacker JR, Kües U (2006) Phylogenetic comparison and classification of laccase and related multicopper oxidase protein sequences. FEBS J 273:2308–2326

    CAS  Article  PubMed  Google Scholar 

  14. Kalyani DC, Munk L, Mikkelsen JD, Meyer AS (2016) Molecular and biochemical characterization of a new thermostable bacterial laccase from Meiothermus ruber DSM 1279. RSC Adv 6:3910–3918

    CAS  Article  Google Scholar 

  15. Kim HW, Lee SY, Park H, Jeon SJ (2015) Expression, refolding, and characterization of a small laccase from Thermus thermophilus HJ6. Protein Expr Purif 114:37–43

    CAS  Article  PubMed  Google Scholar 

  16. Koschorreck K, Richter SM, Ene AB, Roduner E, Schmid RD, Urlacher VB (2008) Cloning and characterization of a new laccase from Bacillus licheniformis catalyzing dimerization of phenolic acids. Appl Microbiol Biotechnol 79:217–224

    CAS  Article  PubMed  Google Scholar 

  17. Lee SY (1996) High cell density culture of Escherichia coli. Trends Biotechnol 14:98–105

    CAS  Article  PubMed  Google Scholar 

  18. Liu H, Cheng Y, Du B, Tong C, Liang S, Han S, Zheng S, Lin Y (2015) Overexpression of a novel thermostable and chloride-tolerant laccase from Thermus thermophilus SG0. 5JP17-16 in Pichia pastoris and its application in synthetic dye decolorization. PLoS One. doi:10.1371/journal.pone.0119833

    Article  Google Scholar 

  19. Lu L, Wang TN, Xu TF, Wang JY, Wang CL, Zhao M (2013) Cloning and expression of thermo-alkali-stable laccase of Bacillus licheniformis in Pichia pastoris and its characterization. Bioresour Technol 134:81–86

    CAS  Article  PubMed  Google Scholar 

  20. Machczynski MC, Vijgenboom E, Samyn B, Canters GW (2004) Characterization of SLAC: a small laccase from Streptomyces coelicolor with unprecedented activity. Protein Sci 13:2388–2397

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. Martins LO, Soares CM, Pereira MM, Teixera M, Costa T, Jones GH, Henriques AO (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

    CAS  Article  PubMed  Google Scholar 

  22. Miyazaki K (2005) A hyperthermophilic laccase from Thermus thermophilus HB27. Extremophiles 9:415–425

    CAS  Article  PubMed  Google Scholar 

  23. Reiss R, Ihssen J, Thony-Meyer L (2011) Bacillus pumilus laccase: a heat stable enzyme with a wide substrate spectrum. BMC Biotechnol. doi:10.1186/1472-6750-11-9

    PubMed  PubMed Central  Google Scholar 

  24. Riva S (2006) Laccases: blue enzymes for green chemistry. Trends Biotechnol 24:219–226

    CAS  Article  PubMed  Google Scholar 

  25. Rivera-Hoyos CM, Morales-Álvarez ED, Poutou-Piñales RA, Pedroza-Rodríguez AM, RodrÍguez-Vázquez R, Delgado-Boada JM (2013) Fungal laccases. Fungal Biol Rev 27:67–82

    Article  Google Scholar 

  26. Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front microbiol 5:172

    PubMed  PubMed Central  Google Scholar 

  27. Sanchez-Amat A, Lucas-Elío P, Fernández E, García-Borrón JC, Solano F (2001) Molecular cloning and functional characterization of a unique multipotent polyphenol oxidase from Marinomonas mediterranea. Biochim Biophys Acta 1547:104–116

    CAS  Article  PubMed  Google Scholar 

  28. Sharma P, Goel R, Capalash N (2007) Bacterial laccases. World J Microbiol Biotechnol 23:823–832

    CAS  Article  Google Scholar 

  29. Siddiqui MA, Rashid N, Ayyampalayam S, Whitman WB (2014) Draft genome sequence of Geobacillus thermopakistaniensis strain MAS1. Genome Announc. doi:10.1128/genomeA.00559-14

    Google Scholar 

  30. Sondhi S, Sharma P, Saini S, Puri N, Gupta N (2014) Purification and characterization of an extracellular, thermo-alkali-stable, metal tolerant laccase from Bacillus tequilensis SN4. PLoS One. doi:10.1371/journal.pone.0096951

    PubMed  PubMed Central  Google Scholar 

  31. Sørensen HP, Mortensen KK (2005) Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microb Cell Fact 4:1. doi:10.1186/1475-2859-4-1

    PubMed  PubMed Central  Google Scholar 

  32. Suzuki T, Endo K, Ito M, Tsujibo H, Miyamoto K, Inamori Y (2003) A thermostable laccase from Streptomyces lavendulae REN-7: purification, characterization, nucleotide sequence, and expression. Biosci Biotechnol Biochem 67:2167–2175

    CAS  Article  PubMed  Google Scholar 

  33. Tayyab M, Rashid N, Akhtar M (2010) Isolation and identification of lipase producing thermophilic Geobacillus sp. SBS-4S: cloning and characterization of the lipase. J Biosci Bioeng 111:272–278

    Article  PubMed  Google Scholar 

  34. Verma A, Shikot P (2014) Purification and characterization of thermostable laccase from thermophilic Geobacillus thermocatenulatus MS5 and its applications in removal of textile dyes. Sch Acad J Biosci 2:479–485

    Google Scholar 

  35. Vieille C, Zeikus GJ (2001) Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol Mol Biol Rev 65:1–43

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. Wang TN, Zhao M (2017) A simple strategy for extracellular production of CotA laccase in Escherichia coli and decolorization of simulated textile effluent by recombinant laccase. Appl Microbiol Biotechnol 101:685–696

    CAS  Article  PubMed  Google Scholar 

  37. 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

    CAS  Article  PubMed  Google Scholar 

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Correspondence to Naeem Rashid.

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Communicated by A. Driessen.

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Basheer, S., Rashid, N., Ashraf, R. et al. Identification of a novel copper-activated and halide-tolerant laccase in Geobacillus thermopakistaniensis . Extremophiles 21, 563–571 (2017). https://doi.org/10.1007/s00792-017-0925-3

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Keywords

  • Geobacillus thermopakistaniensis
  • Laccase
  • Copper oxidase
  • Thermostable
  • Halide-tolerant