N-Demethylation of Methylene Blue by Lignin Peroxidase from Phanerochaete chrysosporium

Abstract

Phanerochaete chrysosporium lignin peroxidase (LiP) can degrade synthetic dyes such as heterocyclic, azo, and triphenylmethane on its activation by H₂O₂. Analysis of the reaction products indicated that N-demethylation reactions are involved in the degradation of crystal violet and methylene blue (MB). We studied LiP oxidation of methylène blue and azure B (AB) in reaction mixtures containing different dye:H₂O₂ stoichiometric relations aiming at the selective formation of _N-demethylated derivatives. High yields, about 70%, of the mono- and didemethylated derivatives, azure B and azure A, were obtained with the use of 1: 1 and 1: 2 MB:H₂O₂, respectively. Using azure B as substrate in reaction mixtures containing 1: 1 AB:H₂O₂, a yield of 70% was also observed in azure A. Reaction mixtures containing 1: 3 MB:H₂O₂ and 1: 2 AB:H₂O₂, originated several oxidation products in similar proportions. These results indicated that the process of enzymatic degradation of methylene blue and azure B initiates via N(CH₃)₂ oxidation. According to the yields that were obtained for azure B and azure A, this enzymatic route can be used for the synthesis of these dyes since these data compare favorably to the chemical route that has a yield of 35%. The use of a dye:H₂O₂relation of 1: 10 resulted in a decoloration level of about 85%, showing the usefulness of this procedure for wastewater treatment. The reaction products were followed by spectrophotometric analysis within the wavelength of 500–700 nm. The product identifications were performed using a reverse-phase high-performance liquid chromatography (HPLC) C–18 column and thin-layer chromatography.

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