Enhancing catalytic efficiency of two microbial uricases making by directed evolution.

Uricase is a key enzyme in purine metabolism that catalyzes the oxidation of uric acid to allantoin, widely used in the treatment of hyperuricemia and gout. In this study, error-prone PCR and one high-throughput screening method were employed to generate uricase mutants with enhanced enzymatic activity from Aspergillus flavus and Candida utilis. After several rounds of mutation and selection, an A. flavus uricase mutant, af-UAM, with activity of 46.21 U/mg, and a C. utilis uricase mutant, cu-UAM, with activity of 31.43 U/mg, were obtained-representing the highest uricase activities reported up to date. Site-directed mutagenesis revealed that the Thr231Ala substitution in A. flavus uricase and the Val234Met substitution in C. utilis uricase were key factors driving their enhanced activities. Furthermore, in vivo experiments demonstrated significant clinical potential of these mutants. These findings offer new insights into the structure-function relationship of uricase and present promising candidates for therapeutic applications in hyperuricemia treatment.