Kemal Caglar, M.; Gulesci, Nuri; Bilgin, Ramazan; Yucebilgic, Guzide

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.

Gulesci, Nuri; Yucebilgic, Guzide; Yildirim, Deniz

Objectives: This study aims the covalent immobilization of Jack bean urease on Fe3O4 magnetic nanoparticles via glutaraldehyde (urease@MNPs-Si-Glu) and epichlorohydrin (urease@MNPs-ECH) spacer arms. Methods: The optimum pH and temperature, thermal, storage and reuse stability of free and immobilized urease preparations were investigated. Thermodynamics characterizations of free and immobilized urease preparations were also studied. Results: The free urease and both immobilized urease preparations showed maximal catalytic activity at pH 7.5. The free urease had a maximal catalytic activity at 50 °C, while the both immobilized urease preparations exhibited their maximal catalytic activities at 70 °C. The urease@MNPs-Si-Glu and urease@MNP-ECH showed 2.7- and 1.9-fold higher thermal stability than the free urease at 60 °C, respectively. The free urease remained 30% of their initial activity at 4 °C, while urease@MNPs-Si-Glu and urease@MNPs-ECH retained 72 and 60% of their initial activities at the same conditions. The urease@MNPs-Si-Glu and urease@MNPs-ECH preserved 53 and 52% of their initial activities, respectively for urea hydrolysis after 20 reuses. Conclusions: The urease@MNPs-Si-Glu and urease@MNPsECH may find a potential application area in urea hydrolysis.

Yucebilgic, Guzide; Gulesci, Nuri; Yildirim, Deniz