INFLUENCE OF DIFFERENT IMMOBILIZATION TECHNIQUES ON IMMOBILIZED LACCASE ACTIVITY

In this work, immobilization of laccase from Trametes versicolor using different immobilization techniques was investigated and obtained results regarding immobilized enzyme activity were compared. The enzyme was successfully immobilized onto different nanoparticles such as magnetic (MNPs) as well as cellulose (CNP) and silica nanoparticles (SNP). Functionalized MNPs were prepared by co-precipitation of Fe2+ and Fe3+ ions, which were initially coated with a layer of citric acid to prevent particle agglomeration. Further, they were coated with sodium silicate and functionalized with aminosilane. MNPs were activated with the crosslinking reagent glutaraldehyde and laccase was immobilized onto functionalized MNPs. On the other hand, CNPs were prepared by ultrasonic treatment of microcellulose while the SNPs were obtained from rice. Functionalization of these nanoparticles was performed by introducing amino groups onto their surface, by modification of CNPs with poly(ethyleneimine) and with organosilane in case of SNPs. Additionally, the immobilized laccase in the form of cross-linked enzyme aggregates (Lac-CLEAs) was prepared by simple immobilization method involving precipitation of the enzyme from aqueous buffer using ethanol as precipitation solvent followed by cross-linking of aggregates of enzyme molecules by glutaraldehyde. When the magnetic cross-linked enzyme aggregates from laccase (Lac-mCLEAs) were synthesized, magnetic nanoparticles were added to the synthesis process. The immobilization yield of laccase immobilized onto nanoparticles was 95%, 83% and 84% for MNPs, CNPs and SNPs, respectively, followed by activity yield of 77%, 73% and 66%, respectively. Some lower immobilization yield was achieved for the Lac-CLEAs and Lac-mCLEAs, 74% and 75%, respectively. Lac-mCLEAs showed the highest activity yield (85%) while activity yield of 76% was achieved for Lac-CLEAs when BSA, as proteic feeder was used. Obtained results suggest that laccase could be successfully