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.

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.

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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