Covalent Immobilization of Glutathione-S-Transferase onto Magnetic Iron Nanoparticles via Epichlorohydrin Spacer Arm

This study examined the synthesis of Fe₃O₄ magnetic nanoparticles (MNPs) and the covalent immobilization of glutathione-S-transferase (GSTs) onto these nanoparticles using an epichlorohydrin (ECH) spacer arm, as well as the optimal reaction conditions for both free and immobilized enzymes and the reusability of the immobilized enzyme. The optimal pH values for free and immobilized enzymes were established as 7.0 and 6.0, respectively. It was found that the ideal temperature for both free and immobilized enzymes was 37 and 40°C, respectively. Under the optimum conditions, the values of V_max for glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) substrates of free GSTs enzyme were measured as 204.8 U/mg prot. and 194.3 U/mg prot., respectively. The immobilized GSTs enzyme's CDNB and GSH substrates had V_max values of 33.29 U/mg prot. and 33.08 U/mg prot., respectively. The free GSTs enzyme's CDNB and GSH substrates have K_m values of 0.201 mM and 0.1873 mM, respectively. GSH and CDNB have K_m values of 0.3042 mM and 0.2523 mM, respectively. At various temperatures, we contrasted the thermal stability of free and immobilized GSTs. The immobilized GSTs enzyme retained 50% of its activity after 20 reuses. After 30 days of storage at 25°C and 4°C, it was discovered that 18% and 32% of the free GSTs enzyme activity were preserved, and 30 days of storage at 25°C and 4°C resulted in 20% and 41% preserved enzyme activity for the immobilized enzyme, respectively.