Production of magnetic nanoparticles coated with polysaccharides and their utilization in enzyme immobilization

In this study, -amylase immobilization on magnetic nanoparticles synthesized by co-precipitation method at room temperature and under inert atmosphere was studied. In addition to bare magnetic nanoparticles, polysaccharide-coated magnetic nanoparticles were produced by the simultaneous synthesis-coating method in media containing agar, pectin, or carboxymethyl cellulose (CMC) at different concentrations (0.5%, 1% w/v). The synthesized nanoparticles were characterized by X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA), vibrating sample magnetometry (VSM) and scanning electron microscope (SEM). -amylase was immobilized on the synthesized bare, 0.5%- and 1%- agar-coated magnetic nanoparticles by adsorption and covalent bonding. In the immobilization by adsorption method, the effects of parameters such as sonication, immobilization time (0.5; 1; 2; 4; 8; 16 h), presence and concentration of surface coating material (0.5% and 1% w/v agar), immobilization pH (pH 3,4,5,6,7) on the amount of bonded protein, enzyme activity and specific activity were investigated. In the immobilization by covalent bonding, the surface activation of the particles was made with glutaraldehyde (GA) and then enzyme immobilization processes was carried out. The effects of concentration of surface-coating material (0.5% and 1% w/v agar), immobilization pH (pH 4.5,6,7,8) and glutaraldehyde concentration (0.5%; 1%; 3%; 5% v/v) on the amount of bonded protein, enzyme activity and specific activity were investigated. The amount of immobilized protein was calculated by Bradford method. Enzyme activity was determined by starch-iodine method. As a result of the studies, enzymes that showed a specific activity increase of 666.6 % compared to the free enzyme and which were immobilized by covalent bonding on 1% agar-coated magnetic nanoparticles activated with 3% GA at pH 8 were selected. As a result of the temperature stability experiments carried out between 45°-60°C, it was revealed that the immobilization process did not change the temperature optimum (55°C), but increased the enzyme activity 4 times. Storage stability experiments were performed at +4ºC and after 77 days, the activity of free -amylase reduced to 88%, while 99% of the activity of the immobilized enzyme was preserved. In multiple use studies, immobilized -amylase retained approximately 55% of its specific activity after 6 uses. This result revealed that covalent immobilization of -amylase on magnetic nanoparticles is suitable for repeated use and has high potential for many industrial applications.