Purification and characterization of an FeII- and ?-ketoglutarate-dependent xanthine hydroxylase from Aspergillus oryzae

XanA is an FeII- and ?-ketoglutarate-dependent enzyme responsible for the conversion of xanthine to uric acid. It is unique to fungi and it was first described in Aspergillus nidulans. In this work, we present the preliminary characterization of the XanA enzyme from Aspergillus oryzae, a relevant fungus in food production in Japan. The XanA protein (GenBank BAE56701.1) was expressed as a recombinant protein in Escherichia coli BL21 (DE3) Arctic cells. Initial purification assays showed low protein solubility; therefore, the buffer composition was optimized using a fluorescence-based thermal shift assay. The protein was stabilized in solution in the presence of either 600 ?M xanthine, 1 M NaCl, 600 ?M ?-ketoglutarate or 20% glycerol, which increases the melting temperature (Tm) by 2, 4, 5 and 6 ?C respectively. The XanA protein was purified by following a three-step purification protocol. The nickel affinity purified protein was subjected to ion-exchange chromatography once the Nterminal 6XHis-tag had been successfully removed, followed by size-exclusion purification. Dynamic light scattering experiments showed that the purified protein was monodisperse and behaved as a monomer in solution. Preliminary activity assays in the presence of xanthine, ?-ketoglutarate, and iron suggest that the enzyme is an iron- and ?-ketoglutarate-dependent xanthine dioxygenase. Furthermore, the enzyme?s optimum activity conditions were determined to be 25 ?C, pH of 7.2, HEPES buffer, and 1% of glycerol. In conclusion, we established the conditions to purify the XanA enzyme from A. oryzae in its active form from E. coli bacteria and determined the optimal activity conditions.

Automated summary

XanA is a unique enzyme in fungi responsible for converting xanthine to uric acid. In this study, the XanA enzyme from Aspergillus oryzae was successfully purified in its active form from E. coli bacteria, and its optimal activity conditions were determined.