Biotecnología

 

Influence of the type of support and immobilization on the activity and stability of laccase enzyme (Trametes versicolor)

 

Influencia del tipo de soporte y la inmovilización sobre la actividad y estabilidad de la enzima lacasa (Trametes versicolor)

 

A. Castro¹, I. González²*, F. Tzompantzi³ and G. Viniegra-González¹

 

¹ Departamento de Biotecnología, Universidad Autónoma Metropolitana-Iztapalapa. Avenida San Rafael Atlixco No. 186, Colonia Vicentina, México D. F. 09340, México.

² Departamento de Química, área de Electroquímica, Universidad Autónoma Metropolitana Unidad Iztapalapa. Avenida San Rafael Atlixco No. 186, Colonia Vicentina, México D. F. 09340, México. *Corresponding author. E-mail: igm@xanum.uam.mx Tel. 52 55 58044671-12; Fax: 52 55 58044666.

³ Departamento de Química, área de catálisis, Universidad Autónoma Metropolitana Unidad Iztapalapa. Avenida San Rafael Atlixco No. 186, Colonia Vicentina, México DF 09340, México.

 

Received January 19, 2013
Accepted April 23, 2013

 

Abstract

This work presents a comparative study of the influence of the nature of the substrate and the immobilization mechanism on laccase enzyme activity and stability. The supports employed were: hydrotalcite-like particles (ZnAl₂), amorphous silica crystals, and glassy carbon surface. Two immobilization mechanisms were applied: one physical, by adsorption, and the other chemical, with two versions of covalent bonding. In the first, using silanes and glutaraldehyde (GA), in the second, thiols were used as anchoring reagents. Hydrotalcite and silica supports were characterized before and after immobilizing the enzyme by X-ray diffraction analysis (XRD), while in the case of glassy carbon supports electrochemical characterization was performed. The catalytic properties K_m and K_cat/K_m of every enzymatic system were evaluated in a complementary fashion, as well as the free enzyme. The kinetic characterizations were done using ABTS (ammonium 2,2' azino-bis-(3 -ethylbenzothiazoline-6-sulfonate acid)), as a reagent typical of laccase in 0.1 M acetate buffer solution pH 3.7. Efficiency of the enzyme adsorbed on each support it was demonstrated that the method of immobilization is decisive in the catalysis. Greater efficiency was observed in the system that employed glassy carbon with either immobilization mechanism and these results are analogous with the tree enzyme.

Keywords: laccase, immobilization, thiol, glassy carbon, hydrotalcite.

 

Resumen

Este trabajo presenta un estudio comparativo de la influencia de la naturaleza del sustrato y del mecanismo de inmovilización en la actividad de la enzima y estabilidad. Los soportes empleados fueron: partículas de hidrotalcita (ZnAl₂), cristales amorfos de sílica y superficie de carbón vítreo. Se aplicaron dos mecanismos de inmovilización: uno físico por adsorción y el otro químico con dos versiones de enlace covalente. En el primero, utilizando silanos y glutaraldehído (GA) y en el segundo tioles. La hidrotalcita y la sílica se caracterizaron antes y después de la inmovilización de la enzima por análisis de difracción de rayos X, en el caso de carbono vítreo se realizó la caracterización electroquímica. Se evaluaron las propiedades catalíticas K_m y K_cat/K_m de cada sistema enzimático así como de la ennzima libre. Se realizaron las caracterizaciones cinéticas usando ABTS (ácido 2,2' azino-bis-(3-etil benzotiazolin-6-sulfonato de amonio)) en 0.1 M buffer de acetatos pH 3.7. La eficiencia de la enzima adsorbida en cada soporte demostró que el método de inmovilización es decisivo en la catálisis, además hay mayor eficiencia en el sistema que emplea carbón vítreo con cualquier mecanismo de inmovilización; estos resultados son análogos a los datos obtenidos con la enzima libre.

Palabras clave: lacasa, inmovilización, tiol, carbón vítreo, hidrotalcita.

 

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References

Acunzo F. and Galli C. (2003). First evidence of catalytic mediation by phenolic compounds in the lacase-induced oxidation of lignin models. European Journal Biochemical 270, 3634-3640.         [ Links ]

Chiacchierini E, Restuccia D. and Vinci G. (2004). Bioremediation of food industry effluents: recent applications of free and immobilized polyphenoloxidases. Food Science and Technological International 10, 373-382.         [ Links ]

D'Annibale A., Stazi S. R., Vinciguerra V., Mattia E. D. and Sermanni G. G. (1999). Characterization of immobilized laccase from Lentinus edodes and its use in olive-mill wastewater treatment. Process Biochemical 34, 697-706.         [ Links ]

Durán N., Rosa M. A., D'annibale A. and Gianfreda L. (2002). Applications of laccase and tyrosinase (phenoloxidases) immobilized on different supports: a review. Enzyme Microbiology Technology 31, 907-931.         [ Links ]

Fernández-Fernández M., Sanromán M. A. and Moldes D. (2013). Recent development and applications of immobilized laccase. Biotechnology Advances. Article in Press.         [ Links ]

Freyre R. S., Duran N. and Kubota T. (2001). Effects of fungal laccase immobilization procedures for the development of a biosensor for phenol compounds. Talanta 54, 681-686.         [ Links ]

Ho G. and Liao C. United States patent (1983) 4,384,045.         [ Links ]

Ivnitski D. and Atanassov P. (2007). Electrochemical studies of intramolecular electron transfer in laccase from Trametes versicolor. Electroanalysis 19, 2307-2313.         [ Links ]

Johnson D. L., Thompson J. L., BrinK_mann S. M., Schuller K. A. and Martin L. L. (2003). Electrochemical characterization of purified Rhus vernicifera laccase: voltammetric evidence for a sequential four-electron transfer. Bioelectrochemistry 42, 10229-10237.         [ Links ]

López-Salinas E. and Pedraza Archiva F. (2006). Hidrotalcitas: precursores de materiales adsorbentes de SOx. IMP, 91-96.         [ Links ]

Mateo C., Palomo J. M., Fernández-Lorente G., Guisan J. M. and Fernández-Lafuente R. (2007). Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microbiology and Technology 40, 1451-1463.         [ Links ]

Mayer, A. M. and Staples R. C. (2002). Laccase: new functions for an old enzyme, review. Phytochemistry 60, 551-565.         [ Links ]

Mousty C., Vieille L. and Cosnier S. (2007). Laccase immobilization in redox active layered double hydroxides: A reagentless amperometric biosensor. Biosensors and Bioelectronics 22, 1733-1738.         [ Links ]

Onda A., Ochi T., Kajiyoski K. and Yanagisawa K. (2008). Lactic acid production from glucose over activated hydrotalcites as solid base catalysts in water. Catalysis Communications 9, 1050-1053.         [ Links ]

Palmieri G., Giardina P., Desiderio B., Marzullo L., Giamberini M. and Sannia G. (1994). A new enzyme immobilization procedure using copper alginate gel: application to a fungal phenol oxidase. Enzyme Microbiology and Technology 16, 151-158.         [ Links ]

Piontek K., Antorini M. and Choinowski T. (2002). Crystal structure of a laccase from the fungus Trametes versicolor at 1.90-Å resolution containing a full complement of coppers. Biological Chemistry 277, 37663-37669.         [ Links ]

Quan D., Kim Y., Byung K. and Shin W. (2002). Assembly of laccase over platinum oxide surface and application as an amperometric biosensor. Bulletin Korean Chemical Society 23, 385-390.         [ Links ]

Ren L., He J., Evans J. D., Duan X. and Ma R. (2001). Some factors affecting the immobilization of penicillin G acylase on calcined layered double hydroxides. Journal Molecular Catalysis B: Enzyme 16, 65-71.         [ Links ]

Ren L., He J., Zhang S., Evans D. G. and Duan X. (2002). Immobilization of penicillin G acylase in layered double hydroxides pillared by glutamate ions. Journal of Molecular Catalysis B: Enzyme 18, 3-11.         [ Links ]

Rodríguez Cuoto S., Osma J. F., Saravia V., Gübitz G. M. and Toca Herrera J. L. (2007). Coating of immobilized laccase for stability enhancement: A novel approach. Applied Catalysis A: Chemical 329, 156-160.         [ Links ]

Sachdev D., Dubey A. and Mishra B. G. (2008). Enviromentally bening liquid phase oxidation of vanillin over copper containing ternary hydrotalcites. Catalysis Communications 9, 391-394.         [ Links ]

Shleev S., Christenson A., Serezhenkov V., Burbaev D., Yaropolov A., Gordon L. and Ruzgas T. (2005). Electrochemical redox transformations of T1 and T2 copper sites in native Trametes hirsute laccase at gold electrode. Biochemical Journal 385, 745-754.         [ Links ]

Solís-Oba M., Baírzana E., García-Garibay M. and Viniegra-González G. (2007). The ABTS^•+ and oxidant agent of different chemical compounds and its recycling process between laccase and substrate. Revista Mexicana de Ingeniería Química 6, 275-281.         [ Links ]

Solís-Oba M., Eloy-Juárez M., Teutli M., Nava J. L. and González I. (2009). Comparison of advanced techniques for the treatment of an indigo model solution: Electro incineration, chemical coagulation and enzymatic. Revista Mexicana de Ingeniería Química 8, 275-282.         [ Links ]

Solís-Oba M., Ugalde V. M., González I. and Viniegra-González 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 Electroanalysis Chemical 579, 59-66.         [ Links ]

Tavares P. M. A., Rodriguez O., Fernández-Fernández M., Domínguez A., Moldes D., Sanromán M. A. and Macedo A. (2013). Immobilization of laccase on modified silica: stabilization, thermal inactivation and kinetic behavior in 1-ethyl-3-methylimidazolium ethyl sulfate ionic liquid. Bioresource Technology 131, 405-412.         [ Links ]

Téllez-Téllez M., Sánchez C., Díaz R. and Díaz-Godínez G. (2012). Zymogram patterns of extracellular laccases of Pleurotus Species grown on non inducer agar medium. Revista Mexicana de Ingeniería Química 11, 383-388.         [ Links ]

Torres-Salas P., Del Monte-Martínez A., Cutido-Avila B., Rodríguez-Colinas B., Alcalde M., Ballesteros O. A. and Plou J. F. (2011). Immobilized Biocatalysts: Novel approaches and tools for binding enzymes to supports. Advanced Materials 23, 5275-5282.         [ Links ]

Voet D. and Voet J. G., (1992). Bioquímica, Ediciones Omega S. A. Barcelona España.         [ Links ]

Zaborsk O. R. (1974). Immobilized enzymes. OH Cleveland, USA. CRC Press.         [ Links ]