Optimizing acid stability and catalytic activity of Aspergillus flavus uricase (AfUOX) via surface charge engineering and B-factor guided design.

Uricase with improved acid stability is desirable for biomedical and biotechnological applications, yet enhancing the intrinsic acid tolerance of the enzyme while maintaining high catalytic activity remains a challenge. Here, we employed an integrated rational design strategy combining surface charge optimization and B-factor-guided engineering, followed by iterative combinatorial mutagenesis, to engineer urate oxidase from Aspergillus flavus. The final combinatorial variants retained 67% and 64% of their initial activity after 60 min of incubation at pH 4.5, respectively-substantially higher than the 25% retained by the wild-type enzyme-while also exhibiting enhanced specific activities. Mechanistic analyses combining biophysical characterization, molecular dynamics simulations, residue interaction network analysis, and electrostatic calculations suggested that the enhanced acid tolerance may be associated with surface charge redistribution and strengthened van der Waals interaction networks, which may help alleviate electrostatic-repulsion-driven conformational changes and contribute to conformational stabilization. The enhanced catalytic performance may be linked to improved substrate binding and restructuring of the substrate channel. These variants and the underlying design logic illustrate a practical approach to engineering acid-resistant uricase and other pH-sensitive oligomeric enzymes.