Preparation and characterization of magnetic nano structures for protein purification

During the past decade, much attention has been paid toward synthesis of magnetic nano sized particles and their use for immobilization and purification of proteins. In this work, magnetic nano sized mag-nano-p(HEMA-MAPA) polymer was synthesized and used as support for lysozyme immobilization and purification. MAPA comonomer was synthesized from methacryloylchloride and phenylalanine under suitable reaction conditions and was used as ligand. Being a hydrophobic structure, incorporation of MAPA to the polymer renders the polymer hydrophobic and provides a hydrophobic medium for lysozyme adsorption. Additionally, magnetic property of mag-nano-p(HEMA-MAPA) polymer provides several advantages on application. Characterization of mag-nano-p(HEMA-MAPA) polymer was conducted using FTIR, ESR, SEM, AFM, TEM, Zeta potential, Elemental Analysis and MALDI-TOF. Particles synthesized were found to be spherical in shape with approximately 386 nm diameter. Specific surface area of mag-nano-p(HEMA-MAPA) particles was calculated to be 580.0 m2/g. ESR was used for the detection of magnetite existence in the polymeric structure and g factor was measured to be 2.2128. Elemental analysis resulted 4.3 x 10-3 mmol MAPA per gram polymer. Adsorption of lysozyme onto mag-nano-p(HEMA-MAPA) polymer was investigated in batch system under various medium conditions (i.e. pH, ionic strength, lysozyme concentration, temperature). Maximum lysozyme adsorption on mag-nano-p(HEMA-MAPA) polymer was measured to be 517 mg/g polymer at 1.0 mg/mL and 1.0 M concentrations of lysozyme and Na2SO4, respectively. Non-specific adsorption of lysozyme on nano-p(HEMA) was calculated to be 24.0 mg/g polymer. Desorption assays indicated that polymer was reusable. Additionally, activity measurements of free and immobilized lysozyme were conducted in order to determine optimum temperature, optimum pH, storage stability, heat stability and operational stability as well as kinetic parameters. Purification of egg white lysozyme using the polymer synthesized was achieved with a high yield of 78.57 % recovery and 657 purification fold. Purity of enzyme was demonstrated using SDS-PAGE and measured as 96 % using Bio-LC.2008, 153 pagesKey Words:Mag-nano-p(HEMA-MAPA), lysozyme, adsorption, magnetic nano particle, hydrophobic interaction