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Biodegradation of 2,4,6-TCA by the white-rot fungus Phlebia radiata is initiated by a phase I (O-demethylation)–phase II (O-conjugation) reactions system: implications for the chlorine cycle

Authors

  • Sonia Campoy,

    1. Instituto de Biotecnología INBIOTEC, Parque Científico de León, Avenida Real, 24006- León, Spain.
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  • María Luisa Álvarez-Rodríguez,

    1. Instituto de Biotecnología INBIOTEC, Parque Científico de León, Avenida Real, 24006- León, Spain.
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  • Eliseo Recio,

    1. Instituto de Biotecnología INBIOTEC, Parque Científico de León, Avenida Real, 24006- León, Spain.
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  • Angel Rumbero,

    1. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Química Orgánica, Cantoblanco, 28049- Madrid, Spain.
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  • Juan-José R. Coque

    Corresponding author
    1. Instituto de Biotecnología INBIOTEC, Parque Científico de León, Avenida Real, 24006- León, Spain.
    2. Área de Microbiología, Departamento de Biología Molecular, Universidad de León, 24071- León, Spain.
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*E-mail jjrubc@unileon.es; Tel. (+34) 987 291 435; Fax (+34) 987 291 409.

Summary

Thirteen species of white-rot fungi tested have been shown to efficiently biodegrade 1 mM 2,4,6-trichloroanisole (2,4,6-TCA) in liquid cultures. The maximum biodegradation rate (94.5% in 10-day incubations) was exhibited by a Phlebia radiata strain. The enzymes of the ligninolytic complex, laccase, lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP) were not able to transform 2,4,6-TCA in in vitro reactions, indicating that the ligninolytic complex was not involved in the initial attack to 2,4,6-TCA. Instead, the first biodegradative steps were carried out by a phase I and phase II reactions system. Phase I reaction consisted on a O-demethylation catalysed by a microsomal cytochrome P-450 monooxygenase to produce 2,4,6-trichlorophenol (2,4,6-TCP). Later, in a phase II reaction catalysed by a microsomal UDP-glucosyltransferase, 2,4,6-TCP was detoxified by O-conjugation with d-glucose to produce 2,4,6-TCP-1-O-d-glucoside (TCPG). This compound accumulated in culture supernatants, reaching its maximum concentration between 48 and 72 h of growth. TCPG levels decreased constantly by the end of fermentation, indicating that it was subsequently metabolized. A catalase activity was able to break in vitro the glycosidic link to produce 2,4,6-TCP, whereas ligninolytic enzymes did not have a significant effect on the biotransformation of that compound. Once formed, 2,4,6-TCP was further degraded as detected by a concomitant release of 2.6 mol of chloride ions by 1 mol of initial 2,4,6-TCA, indicating that this compound underwent almost a complete dehalogenation and biodegradation. It was concluded that P. radiata combines two different degradative mechanisms in order to biodegrade 2,4,6-TCA. The significance of the capability of white-rot fungi to O-demethylate chloroanisoles for the global chlorine cycle is discussed.

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