Title : Laccase immobilization on natural zeolites and commercial supports: Production and application in fixed-bed column
Abstract:
The enzyme laccase has been widely explored in the food industry due to its ability to catalyze oxidation reactions without the need for additional cofactors, using only molecular oxygen. Its use is promising in the removal of undesirable phenolic compounds in juices and wines, reducing astringency and improving sensory stability. Additionally, laccase can be applied in the modification of proteins and carbohydrates, contributing to the improvement of texture and functionality in food products. Another relevant application is in the degradation of contaminants, such as dyes and pesticide residues, promoting food safety and reducing environmental impacts in food processing. The standard laccase activity was determined by the oxidation of ABTS (2,2'-azino bis(3-ethylbenzothiazoline-6-sulfonic acid)) at 25°C. The substrate solution consisted of 2.5 mL of ABTS (1 mM) in sodium phosphate buffer (5 mM, pH 6.0). A suitable amount of soluble or immobilized enzyme was then added, and the oxidation of ABTS was monitored by measuring the increase in absorbance at 430 nm in a spectrophotometric cell with a 1 cm path length (εABTS = 36,000 M⁻¹ cm⁻¹). One international unit (U) of laccase activity corresponds to the oxidation of 1 μmol of ABTS per minute under these conditions. The thermal and pH stabilities were determined for the free and immobilized enzymes at temperatures of 40 and 50°C and pH values of 7.0, 8.5, and 10. For the thermal stability assays, the reactions were carried out in phosphate buffer (pH 7.0, 5 mM) until the activity dropped below 50% of the initial activity. After heat treatment, the samples were immediately cooled in an ice bath to stop the inactivation reaction, and the residual activity was then determined at room temperature as previously described. For the pH stability assays, room temperature was used.In the thermal stability tests of the derivatives produced with various supports and enzymes, the respective half-life times were determined for the derivatives Zeolite Filtrocel-PEI, Zeolite ZI-PEI, and Zeolite Watercel-PEI with L. Aspergillus enzyme, which were 3.3 h, 2.9 h, and 0.8 h, respectively. The column containing Aspergillus laccase immobilized on Zeolite ZI showed a slight decrease in its ability to oxidize the substrate (ABTS) as the number of cycles increased. However, after nine operation cycles, the column maintained 90% oxidation capacity (more than 8 mg of ABTS per g of derivative), demonstrating a high potential for operation even after multiple cycles. On the other hand, the derivative obtained by immobilizing Aspergillus laccase on the commercial Purolite support showed a reduction in oxidation efficiency from 35% to 5% in just four cycles.
