Immobilization of urate oxidase enzyme on the surface of graphene oxide and evaluation of its stability

Enzymes are used as biocatalysts in diagnostic and large-scale industrial processes. Urate oxidase or uricase (UOX) is a medicinal enzyme that is used in the treatments of patients with hyperuricemia and belongs to the family of oxidoreductases (EC ۱.۷.۳.۳). UOX converts the uric acid to ۵-hydroxyisourate and H۲O۲ and reduces the amount of uric acid in the blood. Enzyme immobilization using nanomaterials, as a novel approach, can improve the half-life, stability, catalytic activity, and reusability of enzymes. In this work, the immobilization of UOX on the surface of graphene oxide was examined. For this purpose, the gene encoding uricase was cloned in E. coli and purified after expression. Then, ۱ ml enzyme solution (۰.۴ mg/ml) was mixed with GO in the buffer and mixture was shaken at ۵C for ۲ h at ۱۵۰ rpm. The GO-enzyme suspensions are centrifuged at ۱۳,۰۰۰ rpm and the supernatant is removed. The residue is washed with buffer ۶ times to ensure the thorough removal of free enzyme. The enzyme concentration in the supernatant after immobilization was measured, using Bradford method to determine the enzyme bound to the GO surface .The specific activity of free and immobilized states of UOX was measured. Furthermore, the kinetic and thermodynamic parameters, optimum temperature and pH, and the intrinsic fluorescence of free and immobilized enzymes were examined. The obtained results showed that the immobilized enzyme retained ۴۸% of its activity after one hour at ۳۷℃ while, free enzyme had only ۲۸% of its original activity at this temperature. Furthermore, immobilization of UOX on the surface of GO increased the half-life of the enzyme at ۴۵℃ from ۱۳ minutes (free enzyme) to about ۲۱ minutes (immobilized enzyme).

Keywords:

Uricase , Thermal stabilization , Enzyme immobilization , Graphene oxide nanosheets