Inmovilización de enzimas en nanoestructuras magnéticas para aplicaciones industriales

La lipasa Candida Rugosa se inmovilizó sobre partículas magnéticas recubiertas vía activación con glutaraldheído. Las partículas magnéticas fueron sintetizadas por el método de co-precipitación de iones Fe+3 y Fe+3 en una solución de hidróxido d sodio. Además las partículas magnéticas fueron recubiertas con APTS-TEOS para lograr tener en la superficie grupos –NH2 y así tener una mejor eficiencia de inmovilización. Los análisis de difracción de rayos X (XRD) mostraron que las partículas magnéticas son magnetita y que ocurrió el recubrimiento de la misma, además de que no ocurre un cambio significativo después de inmovilizar la enzima sobre esta magnetita recubierta, esto fue corroborado con análisis de SEM-EDS y análisis térmico (DSC). Los análisis de espectroscopia infrarroja por transformada de Fourier (FT-IR) sugieren que la partícula magnética ha sido recubierta. Además ocurrió un cambio en cuanto a área superficial, la cual fue obtenida por análisis BET. La cantidad de proteína contenida en 1 gramo de enzima inmovilizada se halló por el método de Biuret y método Kjeldahl, encontrándose que la eficiencia de inmovilización fue del 30%. La actividad enzimática promedio (mmol ácido oleico esterificados / g proteína * h) fue obtenida para la esterificación de ácido oleico con butanol tanto para lipasa libre como para la inmovilizada, para esta última la actividad fue de 0.8 veces la enzima libre. Este sistema biocatalítico fue probado cualitativamente en la transesterificación de aceite de palma con etanol presentado como positiva la prueba de ésteres para un periodo de 72 horas. Finalmente se realizó un análisis teórico de un proceso enzimático extracción reacción en el que se encontró la zona en la que debe ir ubicado el biocatalizador, esto con el fin de que el proceso sea más eficiente. Esta ubicación óptima del biocatalizador puede ser logrado con la aplicación de un campo magnético externo (=11.600 Gauss). / Abstract: Candida Rugosa lipase was immobilized on covered magnetic particles via glutaraldehyde activation. The magnetic particles were synthesized by the method of co- precipitation of Fe+3 y Fe+3 ions in a sodium hydroxide solution. In addition, the magnetic particles were covered with APTS-TEOS to manage to have in the surface group- NH2 and better efficiency of immobilization. The X ray diffraction (XRD) analyses showed that the magnetic particles are magnetite and that the covering (recovering) of them occurred, besides, apparently no significant change after immobilizing the enzyme on this covered magnetite occurred, which was corroborated with SEM-EDS and DSC thermal analyses. The Fourier Transform infrared spectroscopy (FT-IR) analyses suggest that magnetic particle has been covered (recovered), and also a change in the superficial area, which was obtained by BET analysis, was noticed. The amount protein contained in 1 gram of immobilized enzyme was obtained by Biuret and Kjeldahl methods, with an immobilization efficiency being of 30%. The enzymatic activity average (mmol oleic acid esterificated / g protein *h) was obtained using the esterification of oleic acid with butanol for free and immobilized lipases. For the last one, the activity was of 0.8 times the activity of the free enzyme. This biocatalytic system was proven qualitatively in the transesterification of palm oil with ethanol presenting as positive the test of esters for a 72 hours period. Finally, a theoretical analysis of an enzymatic process was carried out for an extraction-reaction system in which the zone where the biocatalyst should be located was found, this in order to make the process more efficient. This optimal location of the biocatalyst can be performed by the application of an external magnetic field (=11600 Gauss).Maestrí

Improved natural melanin production by Aspergillus nidulans after optimization of factors involved in the pigment biosynthesis pathway

Abstract Background Melanin is a natural pigment that can be applied in different fields such as medicine, environment, pharmaceutical, and nanotechnology. Studies carried out previously showed that the melanin produced by the mel1 mutant from Aspergillus nidulans exhibits antioxidant, anti-inflammatory, and antimicrobial activities, without any cytotoxic or mutagenic effect. These results taken together suggest the potential application of melanin from A. nidulans in the pharmaceutical industry. In this context, this study aimed to evaluate the effect of factors L-tyrosine, glucose, glutamic acid, l-DOPA, and copper on melanin production by the mel1 mutant and to establish the optimal concentration of these factors to maximize melanin production. Results The results showed that l-DOPA, glucose, and copper sulfate significantly affected melanin production, where l-DOPA was the only factor that exerted a positive effect on melanin yield. Besides, the tyrosinase activity was higher in the presence of l-DOPA, considered a substrate required for enzyme activation, this would explain the increased production of melanin in this condition. After establishing the optimal concentrations of the analyzed factors, the melanin synthesis was increased by 640% compared to the previous studies. Conclusions This study contributed to elucidating the mechanisms involved in melanin synthesis in A. nidulans as well as to determining the optimal composition of the culture medium for greater melanin production that will make it possible to scale the process for a future biotechnological application