Extracellular urate oxidase from Arthrobacter creatinolyticus SA1: statistical optimization, purification, and...

Urate oxidase (uricase; EC 1.7.3.3) catalyzes the oxidation of uric acid to allantoin, a highly soluble metabolite that is readily excreted. Lack of functional urate oxidase in human's leads to hyperuricemia, which is associated with gout, tumor lysis syndrome and renal complications. The present study reports statistical optimization, purification, and comprehensive characterization of a novel extracellular urate oxidase secreted by Arthrobacter creatinolyticus SA1. Plackett-Burman Design screening followed by Central Composite Design identified inoculum volume, initial pH, peptone, and yeast extract as key determinants for enzyme production. Under optimized conditions, the enzyme yield 36.18 IU/mL was obtained. The enzyme was purified by ammonium sulfate fractionation and DEAE-Sepharose chromatography with an overall purification of 5.8-fold. SDS-PAGE revealed a 30 kDa subunit, while native-PAGE indicated a homo-tetrameric enzyme with an estimated molecular weight of 120 kDa. The purified enzyme displayed optimal activity at pH 9.0 and 30 °C. The K_m and V_max of the enzyme were 14.57 mM and 142.85 µmol/min/mg, respectively. Urate oxidase SA1 showed moderate thermostability, exhibiting half-life of about 2 h at 50 °C. MALDI-TOF/TOF peptide mapping confirmed identity of enzyme with urate oxidase (P78609.1). In vitro assays using hyperuricemic human serum demonstrated dose-dependent uric acid degradation, reducing levels from 7.50 to 2.73 mg/dL within 12 h at 1.3 IU/mg, the observed results were found comparable to commercial uricase. These findings suggest that extracellular urate oxidase SA1 may serve as a promising candidate for further development for the management of hyperuricemia and gout.