Immobilization and characterization of human carbonic anhydrase I on amine functionalized magnetic nanoparticles

[Display omitted] •hCA I purified from human erythrocytes.•hCA I covalently immobilized on APTES modified silica coated magnetic nanoparticle by glutaraldehyde.•The structure of modified magnetic nanoparticles was characterized by XRD, FT-IR, VSM, and TEM techniques.•Optimum immobilization conditions, temperature and pH and kinetic parameters of free and immobilized hCA I were determined.•Immobilized enzyme showed 61% of its initial activity after thirteen repeated uses. In this study, we synthesized magnetic nanoparticles (MNPs) by co-precipitation method. After that, silica coating with tetraethyl orthosilicate (TEOS) (SMNPs), amine functionalization of silica coated MNPs (ASMNPs) by using 3-aminopropyltriethoxysilane (APTES) were performed, respectively. After activation with glutaraldehyde (GA) of ASMNPs, human carbonic anhydrase (hCA I) was immobilized on ASMNPs. The characterization of nanoparticles was performed by transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD) and vibrating sample magnetometer (VSM). The immobilization conditions such as GA concentration, activation time of support with GA, enzyme amount, enzyme immobilization time were optimized. In addition of that, optimum conditions for activity, kinetic parameters (Km, Vmax, kcat, kcat/Km), thermal stability, storage stability and reusability of immobilized enzyme were determined. The immobilized enzyme activity was optimum at pH 8.0 and 25°C. The Km value of the immobilized enzyme (1.02mM) was higher than the free hCA I (0.48mM). After 40days incubation at 4°C and 25°C, the immobilized hCA I sustained 89% and 85% of its activity, respectively. Also, it sustained 61% of its initial activity after 13 cycles. Such results revealed good potential of immobilized enzyme for various applications.