Immobilization of enzymes on iron oxide magnetic nanoparticles: Synthesis, characterization, kinetics and...

Immobilization of enzymes on matrices like iron oxide magnetic nanoparticles provides a new technique against the stagnant conventional approaches for stabilization of enzymes. Along with ease in synthesis and modification, the advantages of using these iron oxide nano-metallic matrices as enzyme support matrices are three dimensional viz. structural and thermal stability, storage stability, and reusability. This book chapter details the protocol(s) for synthesis of iron oxide magnetic nanoparticles via chemical co-precipitation method, its surface modification and functionalization with 3-aminopropyl-triethoxysilane (APTES), followed by immobilization of enzymes using glutaraldehyde as a cross linker. A detailed protocol for instrumental characterization is also described. Additionally, the chapter also elucidates the basic characterization of enzyme(s), effect of operating conditions (pH, temperature) on the enzyme activity, thermal inactivation kinetics, thermodynamic parameters, storage stability, and reusability of the free and immobilized enzymes. Further, the results of all the indicated protocols and stability studies are thoroughly discussed with the findings on immobilization of cellulase in a tri-enzyme mixture on iron oxide magnetic nanoparticles. The optimum parameters for immobilization were 30mM glutaraldehyde with 3h incubation and 3:1 (w/w) ratio of nanoparticles:protein content. Immobilization altered the kinetic constants (K_m and V_max) marginally but enhanced the thermal stability as evident from inactivation kinetic constants (k_d, t_1/2 and D-value) and thermodynamic parameters (E_d, ΔH°, ΔG° and ΔS°) within 55-75°C. The immobilized cellulase retained 71.68±3.48% activity during 21-day storage, and 81.15±5.27% activity till fifth reusability cycle.