Research Specifications

Aspergillus flavus Urate oxidase (AFUOX) is promising for potential therapeutic applications, particularly in gout treatment. However, the enzyme’s low thermostability and solubility limit its efficacy. A targeted mutation, substituting Gln with Leu at position 269 (Q269L) has been proposed to enhance its stability. The turnover number, catalytic efficiency, and specific activity of Q269L were 3.7 (s-1), 53.2 (mM-1. s-1), and 3.926 U/mg, respectively. In comparison, for the wild type, these were 3.1 (S-1), 35.1 (mM-1. s-1), and 4.018 U/mg, respectively. Notably, the wild type exhibited maximum activity at pH 9 and 25 °C, whereas the activity of Q269L was obtained at pH 9.5 and 30 °C. Furthermore, the half-life of Q269L at 40 °C is significantly longer (85.55 min) compared to the wild-type (49.85 min). The thermodynamic parameters ΔH≠, ΔS≠, and ΔG≠ at 40 °C for Q269L were 60.9 kJ.mol-1, -276 J.mol-1, and 147.3 kJ.mol-1, respectively. Intrinsic fluorescence reductions and ANS fluorescence increases suggest that tryptophan resides in a polar environment with augmented hydrophobic pockets. FTIR analysis of Q269L reveals a decrease in β-sheet and an increase in α-helix structures, supporting molecular dynamics simulations. Collectively, MD and experimental results underscore Q269L’s enhanced thermostability and localized structural alterations, advancing AFUOX’s therapeutic potential.