Manganese peroxidase H4 isozyme mediated degradation and detoxification of triarylmethane dye malachite green: Optimization of decolorization by response surface methodology

A cDNA encoding for manganese peroxidase isozyme H4 (MnPH4), isolated from Phanerochaete chrysosporium, was expressed in Pichia pastoris, under the control of alcohol oxidase I promoter. The recombinant MnPH4 was efficiently secreted onto media supplemented with hemin at a maximum concentration of 500 U/L, after which purified rMnPH4 was used to decolorize the triarylmethane dye malachite green (MG). Response surface methodology (RSM) was employed to optimize three different operational parameters for the decolorization of MG. RSM showed that the optimized variables of enzyme (0.662 U), MnSO₄ (448 μM), and hydrogen peroxide (159 μM) decolorized 100 mg/L of MG completely at 3 h. Additionally, UV-VIS spectra, high-performance liquid chromatography, gas chromatography-mass spectrometry, and liquid chromatography-electrospray ionization/mass spectrometry analysis confirmed the degradation of MG by the formation of main metabolites 4-dimethylamino-benzophenone hydrate, N, N-dimethylaniline (N,N-dimethyl-benzenamine), and methylbenzaldehyde. Interestingly, it was found that rMnPH4 mediates hydroxyl radical attack on the central carbon of MG. Finally, rMnPH4 degraded MG resulted in the complete removal of its toxicity, which was checked under in vitro conditions.