Characterization and use of a bacterial lignin peroxidase with an improved manganese-oxidative activity
- 216 Downloads
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.
KeywordsLignin peroxidase Dye-decolorizing peroxidase Ligninolytic enzymes Lignin valorization Heme incorporation
E.V. is a PhD student of the “Life Sciences and Biotechnology” course at Università degli studi dell’Insubria.
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.
This article does not contain any studies with human participants or animals performed by any of the authors.
- 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
- 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
- Cleland W (1983) Contemporary enzyme kinetics and mechanism. Academic Press, New York, pp 253–266Google Scholar
- 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
- 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
- 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
- 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
- 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
- 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