Covalent immobilization and characterization of glutathi̇one-S-transferaze enzymes on to magneti̇c i̇ron nanoparti̇cles vi̇a epichlorohydrin i̇ntermedi̇ate spacer arm

In this study ,the synthesis of Fe3O4 magnetic nanoparticles and covalent immobilization of glutathione-S-transferase onto Fe3O4 magnetic nanoparticles via epikolorohydrin spacer arm, the determination of optimal reaction conditions of free and immobilized enzymes and the reusability of immobilized enzyme were investigated. The optimum pH values for free and immobilized enzymes were determined to be 7.0 and 6.0 , respectively. Also the optimum temperature of free and immobilized enzyme were measured as 37 and 40 °C , respectively. Under the optimum conditions, the values of Vmax for glutathione (GSH) and CDNB substrates of free GST enzyme that were measured as 204,8 U/mg prot., and 194,3 U/mg prot. The values of Vmax for the glutathione (GSH) and CDNB substrates of immobilized GST enzyme that were measured as 33,08 U/mg prot., and 33,29 U/mg prot. For the Km values of glutathione and CDNB substrates of free GST enzyme were 0,1873 mM and 0,201 mM, respectively. Similarly, we also measured the values of Km for the substares of immobilized GST enzyme. Km values of glutathione and CDNB were 0,3042 mM and 0,2523 mM, respectively. We compared the thermal stability of free and immobilized GST enzyme at different temperatures. The availability of immobilized GST enzyme was investigated again. After 20 times used, it was reusability that immobilized GST enzyme saved the 50 % activity of itself. It was found that 32 % and 18 % of free GST enzyme activity were saved after 30 days of storage period at 4 °C and 25 °C respectively. For the immobilized enzyme, saved enzyme levels were 41 % and 20 % after 30 days of storage period at 4 °C and 25 °C, respectively.