Preparation of a new generation polymeric nanostructure and usage for enzyme immobilization

IMAC has become a widespread analytical and preparative separation method for therapeutic proteins, peptides, nucleic acids, hormones, and enzymes. The progress of proteomics is closely related with the development of advanced and sensitive protein separation technologies. In proteomics, Immobilized metal affinity chromatography (IMAC) has been widely employed as a prefractionation method to increase the resolution in protein separation. The combination of IMAC with other protein analytical technologies has been successfully utilized to characterize metalloproteome and post-translational modifications. Reduction in the size of enzyme-carrier materials can generally improve the efficiency of adsorbed enzymes. In the case of surface attachment, smaller particles can provide a larger surface area for the attachment of enzymes, leading to higher enzyme loading per unit mass of particles. Cu(II) attached poly(ethylene glycol dimethacrylate-co-methacryloly amidotryptophan) [nano-poly(EGDMA-MAT)] nanospheres with a size of 100 nm utilizing N-methacryloly-(L)-tryptophan methyl ester (MAT) as an aminoacid monomer was prepared via surfactant free emulsion polymerization method. MAT was synthesized using methacryloyl chloride and L-tryptophan methyl ester. L-Tryptophan groups of the nano-poly(EGDMA-MAT) nanospheres were then chelated with Cu2+ ions (nano-poly(EGDMA-MAT)?Cu2+). Laccase (benzendiol: oxygen oxydoreductase; EC 1.10.3.2) immobilization onto the nano-poly(EGDMA-MAT) nanospheres and nano-poly(EGDMA-MAT)?Cu2+ nanospheres from aqueous solutions was investigated in a batch system. Specific surface area of the nonporous nanospheres was found to be 1902.3 m2/g. Nano-poly(EGDMA-MAT) nanospheres were characterized by fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). Elemental analysis of MAT for nitrogen was estimated as 1.36 mmol/g polymer. The maximum Laccase adsorption on the nano-poly(EGDMA-MAT)-Cu2+ was observed at pH 5.0. Higher adsorption values (up to 1144 mg/g) were obtained when the nano-poly(EGDMA-MAT)-Cu2+ were used from aqueous solutions. Laccase molecules could be repeatedly adsorbed and desorbed with the nano-poly(EGDMA-MAT)-Cu2+ without noticeable loss in the Laccase adsorption capacity.