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Manganese Peroxidase H4 Isozyme Mediated Degradation and Detoxification of Triarylmethane Dye Malachite Green: Optimization of Decolorization by Response Surface Methodology

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Abstract

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.

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Acknowledgments

This research was supported by the Technology Development Program for Agriculture and Forestry, under the Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea. This research was also supported in part by the Basic Science Research Program via the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2010-0015666). We would like to thank the Research Institute of Bioindustry at Chonbuk National University for kindly providing the facilities for which to conduct this research.

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Authors and Affiliations

  1. Division of Biotechnology, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan, 570-752, South Korea

    Thiyagarajan Saravanakumar, Thayumanavan Palvannan & Seung-Moon Park

  2. Department of Molecular Biology, College of Natural Science, Chonbuk National University, Jeonju, 561-756, South Korea

    Dae-Hyuk Kim

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  1. Thiyagarajan Saravanakumar
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  2. Thayumanavan Palvannan
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  3. Dae-Hyuk Kim
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  4. Seung-Moon Park
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Correspondence to Seung-Moon Park.

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Saravanakumar, T., Palvannan, T., Kim, DH. et al. Manganese Peroxidase H4 Isozyme Mediated Degradation and Detoxification of Triarylmethane Dye Malachite Green: Optimization of Decolorization by Response Surface Methodology. Appl Biochem Biotechnol 171, 1178–1193 (2013). https://doi.org/10.1007/s12010-013-0220-4

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