Magnetic nanoparticles for biocatalysis and bioseparation

Dissertação para obtenção do Grau de Mestre em Biotecnologia

This work aimed to prepare iron oxide magnetic nanoparticles (MNPs) coated with biopolymers and further modified with enzymes and synthetic affinity ligands in order to study their applications in biocatalysis and bioseparation areas. The adsorption of different biopolymers - Gum Arabic (Ga), Dextran (Dex) and Extracellular Polysaccharide (EPS) - was performed during the synthesis of the particles, and the amount of polymer bound was determined and compared to the adsorption profiles of each polymer. Additionally, the storage and chemical modification stability of these supports was also evaluated. All supports showed high stability. The eventual application of these magnetic supports in biocatalysis was evaluated through the immobilization of an enzyme, Enterokinase (EK), through two distinct chemistries. The enzyme was then tested with a synthetic substrate and two fusion proteins. After the enzyme’s immobilization, its activity was observed to decrease, which was compensated by its re-utilization up to ten times. The most promising magnetic support was the MNP_Dex since it presented an activity retention of 35% and a conversion percentage of 0.4% in the first reaction cycle. The utilization of magnetic supports for the purification of IgG was tested through the immobilization of the synthetic affinity ligand 22/8 using three different methods. The MNP_Dex support modified with the ligand synthesized directly on the solid support adsorbed around 130 mg of IgG/g of MNP and presented less nonspecific adsorption. Moreover, this support presented specificity and capability to adsorb IgG and its fragments from crude extracts expressed in yeast and mammalian cells.