Methylene blue and azure B oxidation by horseradish peroxidase: a comparative evaluation of class II and class III peroxidases

We have previously shown that the oxidation of methylene blue (MB) or azure B (AB) by Phanerochaete chrysosporium lignin peroxidase (LiP) (class II) occurs via stepwise N-demethylations followed by aromatic ring cleavage under selective reaction conditions related to methylene blue:H2O2 or azure B:H2O2 stoichiometry. In this work, we compare the oxidation of the same dyes by horseradish peroxidase (HRP) of plant origin (class III) and compare LiP and HRP reactions and products. Results show HRP is able to N-demethylate both dyes, but exhibits much slower reaction kinetics than LiP and requires higher H2O2 concentrations. Product yields are also different for HRP, and contrary to LiP, HRP is unable to achieve aromatic ring cleavage. Azure C (AC), which is formed by sequential oxidation of either MB or AB, was a major reaction product in HRP-mediated reactions. Yields of AC up to 60% were obtained, suggesting a potential enzymatic route for AC production that compares quite favorably to the chemical route yield of 35%.