Synthesis, characterization, and biological impact of PEGylated uricase from Aspergillus flavus: stability, activity, and in vitro immunological evaluation

Despite the commercial success of protein- and peptide-based therapeutics, their clinical application remains limited due to rapid clearance and inactivation via metabolic pathways. In this study, we PEGylated Aspergillus flavus-derived uricase (Uox) using methoxy polyethylene glycol (5 kDa) activated with Succinimidyl Succinamide (mPEG-SS) at a molar ratio of 1:10 (Uox:mPEG-SS) to enhance its pharmacological properties. This study aimed to evaluate and compare the kinetic behavior, thermodynamic stability, and immunogenic potential of native and PEGylated uricase. mPEG-SS was synthesized through a two-step reaction and characterized by FT-IR,1 H-NMR spectroscopy, and TLC. Successful conjugation to Uox (mPEG-SS@Uox) was confirmed using DLS and SDS-PAGE. Kinetic and thermodynamic analyses revealed a 16.44% increase in thermal stability and a 23.37% increase in half-life at 45°C upon PEGylation, with only a modest 27.3% reduction in catalytic efficiency, likely due to steric hindrance. Immunogenicity assays using CFSE-labeled PBMC proliferation and cytokine profiling via ELISA indicated that PEGylated Uox induced significantly lower immune activation compared to the native enzyme (p < 0.0045), with no statistical difference from the control group (p = 0.2426). These findings demonstrate that PEGylation with 5 kDa mPEG-SS substantially improves uricase’s stability and diminishes its in vitro immunogenic potential (as evidenced by reduced PBMC activation), making it a promising candidate for further preclinical development in the treatment of hyperuricemia and gout.