Advertisement

Characterization and use of a bacterial lignin peroxidase with an improved manganese-oxidative activity

  • Elisa Vignali
  • Fabio Tonin
  • Loredano Pollegioni
  • Elena Rosini
Biotechnologically relevant enzymes and proteins
  • 52 Downloads

Abstract

Peroxidases are well-known biocatalysts produced by all organisms, especially microorganisms, and used in a number of biotechnological applications. The enzyme DypB from the lignin-degrading bacterium Rhodococcus jostii was recently shown to degrade solvent-obtained fractions of a Kraft lignin. In order to promote the practical use, the N246A variant of DypB, named Rh_DypB, was overexpressed in E. coli using a designed synthetic gene: by employing optimized conditions, the enzyme was fully produced as folded holoenzyme, thus avoiding the need for a further time-consuming and expensive reconstitution step. By a single chromatographic purification step, > 100 mg enzyme/L fermentation broth with a > 90% purity was produced. Rh_DypB shows a classical peroxidase activity which is significantly increased by adding Mn2+ ions: kinetic parameters for H2O2, Mn2+, ABTS, and 2,6-DMP were determined. The recombinant enzyme shows a good thermostability (melting temperature of 63–65 °C), is stable at pH 6–7, and maintains a large part of the starting activity following incubation for 24 h at 25–37 °C. Rh_DypB activity is not affected by 1 M NaCl, 10% DMSO, and 5% Tween-80, i.e., compounds used for dye decolorization or lignin-solubilization processes. The enzyme shows broad dye-decolorization activity, especially in the presence of Mn2+, oxidizes various aromatic monomers from lignin, and cleaves the guaiacylglycerol-β-guaiacyl ether (GGE), i.e., the Cα-Cβ bond of the dimeric lignin model molecule of β-O-4 linkages. Under optimized conditions, 2 mM GGE was fully cleaved by recombinant Rh_DypB, generating guaiacol in only 10 min, at a rate of 12.5 μmol/min mg enzyme.

Keywords

Lignin peroxidase Dye-decolorizing peroxidase Ligninolytic enzymes Lignin valorization Heme incorporation 

Notes

Acknowledgements

E.V. is a PhD student of the “Life Sciences and Biotechnology” course at Università degli studi dell’Insubria.

Funding information

We thank the financial support from CIB, Consorzio Interuniversitario per le Biotecnologie.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

References

  1. Ahmad M, Roberts JN, Hardiman EM, Singh R, Eltis LD, Bugg TD (2011) Identification of DypB from Rhodococcus jostii RHA1 as a lignin peroxidase. Biochemistry 50:5096–5107.  https://doi.org/10.1021/bi101892z CrossRefPubMedGoogle Scholar
  2. Boerjan W, Ralph J, Baucher M (2003) Lignin biosynthesis. Annu Rev Plant Biol 54:519–546.  https://doi.org/10.1146/annurev.arplant.54.031902.134938 CrossRefPubMedGoogle Scholar
  3. Bugg TDH, Rahmanpour R (2015) Enzymatic conversion of lignin to renewable chemicals. Curr Opin Chem Biol 29:10–17.  https://doi.org/10.1016/j.cbpa.2015.06.009 CrossRefPubMedGoogle Scholar
  4. Bugg TDH, Ahmad M, Hardiman EM, Singh R (2011) The emerging role for bacteria in lignin degradation and bio-product formation. Curr Opin Biotechnol 22:394–400.  https://doi.org/10.1016/j.copbio.2010.10.009 CrossRefPubMedGoogle Scholar
  5. Caldinelli L, Iametti S, Barbiroli A, Bonomi F, Fessas D, Molla G, Pilone MS, Pollegioni L (2005) Dissecting the structural determinants of the stability of cholesterol oxidase containing covalently bound flavin. J Biol Chem 280:22572–22581.  https://doi.org/10.1074/jbc.M500549200 CrossRefPubMedGoogle Scholar
  6. Caldinelli L, Molla G, Sacchi S, Pilone MS, Pollegioni L (2009) Relevance of weak flavin binding in human D-amino acid oxidase. Protein Sci 18:801–810.  https://doi.org/10.1002/pro.86 CrossRefPubMedPubMedCentralGoogle Scholar
  7. Cheng K, Sorek H, Zimmermann H, Wemmer DE, Pauly M (2013) Solution-state 2D NMR spectroscopy of plant cell walls enabled by a dimethylsulfoxide-d 6/1-ethyl-3-methylimidazolium acetate solvent. Anal Chem 85:3213–3221.  https://doi.org/10.1021/ac303529v CrossRefPubMedGoogle Scholar
  8. Cleland W (1983) Contemporary enzyme kinetics and mechanism. Academic Press, New York, pp 253–266Google Scholar
  9. de Gonzalo G, Colpa DI, Habib MH, Fraaije MW (2016) Bacterial enzymes involved in lignin degradation. J Biotechnol 236:110–119.  https://doi.org/10.1016/j.jbiotec.2016.08.011 CrossRefPubMedGoogle Scholar
  10. Durão P, Chen Z, Fernandes AT, Hildebrandt P, Murgida DH, Todorovic S, Pereira MM, Melo EP, Martins LO (2008) Copper incorporation into recombinant CotA laccase from Bacillus subtilis: characterization of fully copper loaded enzymes. J Biol Inorg Chem 13:183–193.  https://doi.org/10.1007/s00775-007-0312-0 CrossRefPubMedGoogle Scholar
  11. Eggert C, Temp U, Eriksson KEL (1997) Laccase is essential for lignin degradation by the white-rot fungus Pycnoporus cinnabarinus. FEBS Lett 407:89–92CrossRefGoogle Scholar
  12. Fernandez-Fueyo E, Castanera R, Ruiz-Dueñas FJ, López-Lucendo MF, Ramírez L, Pisabarro AG, Martínez AT (2014) Ligninolytic peroxidase gene expression by Pleurotus ostreatus: differential regulation in lignocellulose medium and effect of temperature and pH. Fungal Genet Biol 72:150–161.  https://doi.org/10.1016/j.fgb.2014.02.003 CrossRefPubMedGoogle Scholar
  13. Glenn JK, Gold MH (1985) Purification and characterization of an extracellular Mn(II)-dependent peroxidase from the lignin degrading basidiomycete Phanerochaete chrysosporium. Archiv Biochem Biophy 242:329–341CrossRefGoogle Scholar
  14. Glenn JK, Akileswaran L, Gold MH (1986) Mn (II) oxidation is the principal function of the extracellular Mn-peroxidase from Phanerochaete chrysosporium. Arch Biochem Biophys 251:688–696.  https://doi.org/10.1016/0003-9861(86)90378-4 CrossRefPubMedGoogle Scholar
  15. Gupta VK, Kubicek CP, Berrin JG, Wilson DW, Couturier M, Berlin A, Filho EXF, Ezeji T (2016) Fungal enzymes for bio-products from sustainable and waste biomass. Trends Biochem Sci 41:633–645.  https://doi.org/10.1016/j.tibs.2016.04.006 CrossRefPubMedGoogle Scholar
  16. Harris CM, Pollegioni L, Ghisla S (2001) pH and kinetic isotope effects in D-amino acid oxidase catalysis. Eur J Biochem 268:5504–5520.  https://doi.org/10.1046/j.1432-1033.2001.02462.x CrossRefPubMedGoogle Scholar
  17. Hong CY, Park SY, Kim SH, Lee SY, Choi WS, Choi IG (2016) Degradation and polymerization of monolignols by Abortiporus biennis, and induction of its degradation with a reducing agent. J Microbiol 54:675–685.  https://doi.org/10.1007/s12275-016-6158-9 CrossRefPubMedGoogle Scholar
  18. Leonowicz A, Matuszewska A, Luterek J, Ziegenhagen D, Wojtaś-Wasilewska M, Cho NS, Hofrichter M, Rogalski J (1999) Biodegradation of lignin by white rot fungi: review. Fungal Genet Biol 2:175–185.  https://doi.org/10.1006/fgbi.1999.1150 CrossRefGoogle Scholar
  19. Longe L, Garnier G, Saito K (2016) Lignin biodegradation with fungi, bacteria and enzymes for producing chemicals and increasing process efficiency. In: Fang Z, Smith RL (eds) Production of biofuels and chemicals from lignin. Springer, Singapore, pp 147–179CrossRefGoogle Scholar
  20. Louis-Jeune C, Andrade-Navarro MA, Perez-Iratxeta C (2012) Prediction of protein secondary structure from circular dichroism using theoretically derived spectra. Proteins 80:374–381.  https://doi.org/10.1002/prot.23188 CrossRefPubMedGoogle Scholar
  21. Machczynski MC, Vijgenboom E, Samyn B, Canters GW (2004) Characterization of SLAC: a small laccase from Streptomyces coelicolor with unprecedented activity. Protein Sci 13:2388–2397CrossRefGoogle Scholar
  22. Miki K, Renganathan V, Gold MH (1986) Mechanism of beta-aryl ether dimeric lignin model compound oxidation by lignin peroxidase by Phanerochaete chrysosporium. Biochemistry 25:4790–4796.  https://doi.org/10.1021/bi00365a011 CrossRefGoogle Scholar
  23. Partridge JD, Sanguinetti G, Dibden DP, Roberts RE, Poole RK, Green J (2007) Transition of Escherichia coli from aerobic to micro-aerobic conditions involves fast and slow reacting regulatory components. J Biol Chem 282:11230–11237.  https://doi.org/10.1074/jbc.M700728200 CrossRefPubMedGoogle Scholar
  24. Pollegioni L, Tonin F, Rosini E (2015) Lignin-degrading enzymes. FEBS J 282:1190–1213.  https://doi.org/10.1111/febs.13224 CrossRefPubMedGoogle Scholar
  25. Qing Q, Yang B, Wyman CE (2010) Impact of surfactants on pretreatment of corn stover. Bioresour Technol 101:5941–5951.  https://doi.org/10.1016/j.biortech.2010.03.003 CrossRefPubMedGoogle Scholar
  26. Rosini E, Monelli CS, Pollegioni L, Riva S, Monti D (2012) On the substrate preference of glutaryl acylases. J Mol Catal B Enzym 76:52–58.  https://doi.org/10.1016/j.molcatb.2011.12.001 CrossRefGoogle Scholar
  27. Sánchez C (2009) Lignocellulosic residues: biodegradation and bioconversion by fungi. Biotechnol Adv 27:185–194.  https://doi.org/10.1016/j.biotechadv.2008.11.001 CrossRefPubMedGoogle Scholar
  28. Singh R, Grigg JC, Armstrong Z, Murphy ME, Eltis LD (2012) Distal heme pocket residues of B-type dye-decolorizing peroxidase arginine but not aspartate is essential for peroxidase activity. J Biol Chem 287:10623–10630.  https://doi.org/10.1074/jbc.M111.332171 CrossRefPubMedPubMedCentralGoogle Scholar
  29. Singh R, Grigg JC, Qin W, Kadla JF, Murphy ME, Eltis LD (2013) Improved manganese-oxidizing activity of DypB, a peroxidase from a lignolytic bacterium. ACS Chem Biol 8:700–706.  https://doi.org/10.1021/cb300608x CrossRefPubMedPubMedCentralGoogle Scholar
  30. Sugano Y, Muramatsu R, Ichiyanagi A, Sato T, Shoda M (2007) DyP, a unique dye-decolorizing peroxidase, represents a novel heme peroxidase family ASP171 replaces the distal histidine of classical peroxidases. J Biol Chem 282:36652–36658.  https://doi.org/10.1074/jbc.M706996200 CrossRefPubMedGoogle Scholar
  31. Tessaro D, Pollegioni L, Piubelli L, D’Arrigo P, Servi S (2015) Systems biocatalysis: an artificial metabolism for interconversion of functional groups. ACS Catal 5:1604–1608.  https://doi.org/10.1021/cs502064s CrossRefGoogle Scholar
  32. Tien M, Kirk T (1983) Lignin-degrading enzyme from the hymenomycete Phanerochaete chrysosporium. Science 221:661–663.  https://doi.org/10.1126/science.221.4611.661 CrossRefPubMedGoogle Scholar
  33. Tonin F, Melis R, Cordes A, Sanchez-Amat A, Pollegioni L, Rosini E (2016) Comparison of different microbial laccases as tools for industrial uses. New Biotechnol 33:387–398.  https://doi.org/10.1016/j.nbt.2016.01.007 CrossRefGoogle Scholar
  34. Tonin F, Vignali E, Pollegioni L, D’Arrigo P, Rosini E (2017) A novel, simple screening method for investigating the properties of lignin oxidative activity. Enzym Microb Technol 96:143–150.  https://doi.org/10.1016/j.enzmictec.2016.10.013 CrossRefGoogle Scholar
  35. Wong DWS (2009) Structure and action mechanism of ligninolytic enzymes. Appl Biochem Biotechnol 157:174–209.  https://doi.org/10.1007/s12010-008-8279-z CrossRefPubMedGoogle Scholar
  36. Xu F (1999) Recent progress in laccase study: properties, enzymology, production, and applications. In: Flickinger MC, Grew SW (eds) Encyclopedia of bioprocess technology: fermentation, biocatalysis, and bioseparation. John Wiley & Sons, New York, pp 1545–1554Google Scholar
  37. Zakzeski J, Jongerius AL, Bruijnincx PC, Weckhuysen BM (2012) Catalytic lignin valorization process for the production of aromatic chemicals and hydrogen. ChemSusChem 5:1602–1609.  https://doi.org/10.1002/cssc.201100699 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Biotechnology and Life SciencesUniversity of InsubriaVareseItaly
  2. 2.Department of BiotechnologyDelft University of TechnologyDelftThe Netherlands

Personalised recommendations