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

Author:
Vignali, Elisa, Tonin, Fabio, Pollegioni, Loredano, Rosini, Elena
Source:
Applied microbiology and biotechnology 2018 v.102 no.24 pp. 10579-10588
ISSN:
0175-7598
Subject:
Escherichia coli, Rhodococcus jostii, biocatalysts, chromatography, culture media, decolorization, dimethyl sulfoxide, dyes, enzyme activity, gene overexpression, guaiacol, hydrogen peroxide, ions, lignin, lignin peroxidase, manganese, melting point, microorganisms, models, pH, peroxidase, polysorbates, sodium chloride, synthetic genes, thermal stability
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.
Agid:
6231151