Biotecnología

 

El ABTS^•+ agente oxidante de diversos compuestos químicos y su mecanismo de reciclado entre la lacasa y el sustrato

 

The ABTS^•+ an oxidant agent of different chemical compounds and its recycling process between laccase susbtrate

 

M. Solís–Oba^1*, E. Bárzana², M. García–Garibay³ y G. Viniegra–González³

 

¹ Centro de Investigación en Biotecnología Aplicada del Instituto Politécnico Nacional, Carretera Estatal Santa Inés Tecuexcomac–Tepetitla Km 1.5, Tepetitla de Lardizabal, Tlaxcala, CP. 90700, México. * Autor para la correspondencia: E–mail: msolis@ipn.mx Tel. 57 29 63 00 ext 87810, Fax 57 29 63 00 ext 87826

² Facultad de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, México D.F., México.

³ Departamento de Biotecnología, Universidad Autónoma Metropolitana Unidad Iztapalapa Av. San Rafael Atlixco No. 186, Col. Vicentina, México D.F., México.

 

Recibido 2 de Junio 2007
Aceptado 31 de Octubre 2007

 

Resumen

La lacasa comercial obtenida de Myceliophtera termophila y expresada sobre Aspergillus oryzae se empleó para producir un cation radical estable y muy activo del mediador químico ABTS (ácido 2,2'–azino–bis–(3–etillbenzotiazolin–6–sulfonico) con el fin de probar la factibilidad de emplearlo como un intermediario para la oxidación de una variedad de compuestos aromáticos en ausencia de la enzima. Se demostró que el catión radical ABTS^•+ por si mismo oxidó al azul índigo, ácido tánico, ácido gálico, azul brillante G, azul de Coomassie, naranja 7 y p–cresol. La reacción de oxidación reveló cambios importantes en sus espectros de absorción en la región del espectro visible. Se demostró también que la oxidación de estos compuestos por la mezcla de lacasa y ABTS se puede efectuar de forma cíclica, de tal forma que la velocidad de reacción fue 94 veces mayor para el índigo, 17 veces mayor para el azul brillante G, 34 veces mayor para el naranja 7 y 5 veces mayor para el p–cresol, comparado con usar el mediador o la lacasa en forma individual. Estos resultados muestran la factibilidad de un esquema de reciclado para la oxidación final de una variedad de compuestos orgánicos en los cuales la enzima no este directamente en contacto con el mismo y que se efectúe a través del mediador ABTS^•+.

Palabras clave: ABTS, reciclado, oxidación, colorantes, lacasa.

 

Abstract

The commercial enzyme laccase cloned from Myceliophtera termophila and expressed in Aspergillus oryzae was used to produce an active, stable and oxidized form of the chemical mediator ABTS (2,2'–azino–bis–(3–ethylbenzothiazoline–6–sulphonic acid)) in order to test the feasibility of using the monocation ABTS^•+ as a recyclable intermediate for the oxidation of a variety of aromatic chemicals in the absence of the enzyme. It was shown that ABTS^•+, by itself, oxidized, indigo blue, tannic and galic acids, brilliant blue G, Coomassie blue, orange 7 and p–cresol. Oxidation was assessed by important changes in their visible absorption spectra. It was also shown that oxidation of these compounds by a mixture of ABTS and laccase can be done in a cyclic manner, such a way the oxidation rate was 94 times higher for indigo, 17 times for brilliant blue G, 34 times for orange 7 and 5 times higher for p–cresol compared with using the mediator or the laccase alone. Those results show the feasibility of a recycling scheme for the final oxidation of a wide variety of aromatic compounds in which the enzyme is not directly in contact with the compound but through the intermediate ABTS^•+.

Keywords: ABTS, recycling, oxidation, dyes, lacction.

 

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Referencias

Bourbonnais R., Paice M. (1990). Oxidation of non–phenolic substrates. An expanded role for laccase in lignin biodegradation. FEBS Letters 267, 99–102.         [ Links ]

Bourbonnais R., Paice M., Freiermuth B., Bodie E., Borneman S. (1997). Reactivities of various mediators and laccases with Kraft pulp and lignin model compounds, Applied and Environmental Microbiology, 4627–4632.         [ Links ]

Collins P., Dobson A., Field J. (1998), Reduction of the 2,2 –Azino–bis (3–Ethylbenzthiazoline–6–Sulfonate) Cation Radical by Physiological Organic Acids in the Absence and Presence of Manganese. Applied and Environmental Microbiology 64 (6), 2026–2031.         [ Links ]

Greca M., Costa–Ferreira M., Pessoa M. (2001). Decolorization of an anthraquinone–type dye using a laccase formulation. Bioresource Technology 79, 171–177.         [ Links ]

Ho G. , Liao Ch. (1983). Activation of a siliceous carrier for enzyme immobilization. United States Patent 4,384,045.         [ Links ]

Johannes Ch., Majcherczyk A. (2000a). Natural mediators in the oxidation of polycyclic aromatic hydrocarbons by laccase mediator system. Applied and Environmental Microbiology 66, 524–528.         [ Links ]

Johannes Ch., Majcherczyk A. (2000b). Laccase activity tests and laccase inhibitors. Journal of Biotechnology 78, 193–199.         [ Links ]

Li K., Helm R., Eriksson K. (1998). Mechanistic studies of the oxidation of a non–phenolic lignin model compound by the laccase/I–hidroxibenzotriazole redox system. Biotechnology and Applied Biochemistry 27, 239–243.         [ Links ]

Lonergan G., Mew E., Schliephake K., Baker W., (1997). Phenolic substrate for fluorometric detection of laccase activity. FEMS Microbiology Letters 153, 485–490.         [ Links ]

Majcherczyk A., Johannes C., (2000). Radical mediated indirect oxidation of a PEG–coupled polycyclic aromatic hydrocarbon (PAH) model compound by fungal laccase. Biochimica et Biophysica Acta–General Subjects 1474 (2), 157–162.         [ Links ]

Novo Nordisk, Deni Lite II S Product Sheet, Enzyme Business, Denmark, 1999.         [ Links ]

Potthast A., Rosenau T., Chen C., Gratzl J. (1996). A novel method for the conversion of benzyl alcohols to benzaldehydes by laccase–catalysed oxidation. Journal of Molecular Catalysis A. 108, 5–9.         [ Links ]

Rodríguez E., Pickard M., Vázquez–Duhalt R. (1999). Industrial dye decolorization by laccase from ligninolytic fungi. Current Microbiology 38, 27–32.         [ Links ]

Solís–Oba M., Ugalde–Saldivar V., Gonzalez I., Viniegra–Gonzalez G. (2005). An electrochemical–spectrophotometrical study of the oxidized forms of the mediator 2,2'–azino–bis–(3–ethylbenzothiazoline–6–sulfonic acid) produced by immobilized laccase. Journal of Electroanalytical Chemistry 579, 59–66.         [ Links ]

Thurston Ch. (1994). The structure and function of fungal laccase, review article. Microbiology 140, 19–26.         [ Links ]

Xu F. (1996). Oxidation of phenols, anilines and benzenethiols by fungal laccase: correlation between activity and redox potentials as well as halide inhibition. Biochemistry 35, 7608–7614.         [ Links ]