Enhancing stability and catalytic activity of urate oxidase using natural deep eutectic solvent: insights from experimental and computational approaches.

Abstract

This study investigated the impact of natural deep eutectic solvents (DES) based on sucrose, fructose and glycerol on the recombinant uricase enzyme. The plasmid recombinant pET-28a⁺ containing uricase coding sequence was extracted from the DH5α strain and transferred into the BL21 expration strain. Subsequently, overnight culture, induction with IPTG, and purification of the recombinant uricase using Ni-NTA affinity chromatography methods were carried out. The effect of DES containing sucrose, fructose and glycerol was assessed and 5% DES concentration verified for subsequent experiment. Thermodynamic parameters were analyzed using thermal inactivation and intrinsic fluorescence methods at temperatures of 35, 45, 55, and 65 °C. The results demonstrated lengthened enzyme half-life by approximately 61 min, higher activation energy and T_m, indicating improved thermal stability compared to the free enzyme. Kinetic tests revealed a reduction in the k_m value from 0.16 mM in the free enzyme to 0.09 mM in the treated enzyme, suggesting enhanced substrate binding affinity. Moreover, the K_cat/K_m ratio, reflecting enzyme specificity towards the substrate, was enhanced. In the molecular dynamics simulation section, the root mean square deviation (RMSD), root mean square fluctuation (RMSF), and solvent accessible surface area (SASA) were analyzed. Lower RMSD and RMSF values indicate that the structure is more stable in the presence of the eutectic solvent compared to the free enzyme.