1. Characterization and use of a bacterial lignin peroxidase with an improved manganese-oxidative activity.
- Author
-
Vignali E, Tonin F, Pollegioni L, and Rosini E
- Subjects
- Anthraquinones chemistry, Anthraquinones metabolism, Azure Stains chemistry, Azure Stains metabolism, Bacterial Proteins genetics, Coloring Agents metabolism, Dimethyl Sulfoxide chemistry, Escherichia coli genetics, Guaifenesin metabolism, Hydrogen Peroxide metabolism, Hydrogen-Ion Concentration, Kinetics, Naphthalenesulfonates chemistry, Naphthalenesulfonates metabolism, Oxidation-Reduction, Peroxidases genetics, Polysorbates chemistry, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Rhodococcus genetics, Temperature, Bacterial Proteins metabolism, Coloring Agents chemistry, Guaifenesin analogs & derivatives, Manganese metabolism, Peroxidases metabolism
- 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 Mn
2+ ions: kinetic parameters for H2 O2 , 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.- Published
- 2018
- Full Text
- View/download PDF