PASylated Urate Oxidase Enzyme: Enhancing Biocatalytic Activity, Physicochemical Properties, and Plasma Half-Life

Recombinant urate oxidase (UOX, E.C.1.7.3.3) is an important therapeutic enzyme used in preventing and treating chemotherapy-induced hyperuricemia and severe gout. However, UOX use is limited due to the poor stability and short plasma half-life. To solve this problem, we designed three PASylated variants of Aspergillus flavus UOX with different PAS sequences at the C- or N-terminus. The genes of native and PASylated variants (UOX-PAS₂₀, PAS₂₄-UOX, and UOX-PAS₁₀₀) were designed and produced in Escherichia coli strain BL21 (DE3). The expressed recombinant native and PASylated enzymes were compared in terms of biophysical properties, kinetics parameters, and pharmacokinetics behavior using standard methods. PASylation of UOX with PAS₁₀₀ polymer caused a 1.24-fold reduction in K _m to 52.61 μM, and a 3.87-fold increase in K _cat/K _m for uric acid compared to the native variant. UOX-PAS₁₀₀ retained its activity in different temperatures (20-55 °C); however, other variants lost nearly 50% of their original activity at 55 °C. UOX-PAS₁₀₀ exhibited a 1.78-fold increase in hydrodynamic radius and a 1.64-fold larger apparent molecular size in comparison to the native UOX. Circular dichroism (CD) spectroscopy demonstrated that the addition of the PAS tag does not change the secondary structure of the fusion enzyme. The tryptophan fluorescence emission spectra for PASylated enzymes showed a significant modification in the conformational state of UOX by the PAS polymer presence. UOX-PAS₁₀₀ retained 89.0% of the original activity following 72 h incubation in the presence of plasma at 37 °C. However, only about 61.0%, 57.0%, 50.0%, and 52.0% of activity from PAS₂₄-UOX, UOX-PAS₂₀, native UOX, and rasburicase (Fasturtec, Italy) remained, respectively, at the identical time. UOX-PAS₁₀₀ had an increased biological half-life (8.21 h) when compared with the rasburicase (3.12 h) and native UOX (2.87 h) after being injected into a rat. Having considering everything, our results suggest that the UOX-PAS₁₀₀, an A. flavus UOX fused with a C-terminally 100 amino acid PAS-residue, is a proper candidate with enhanced biological activity and extended plasma half-life for clinical therapy in patients suffering from hyperuricemia.