Cloning, Purification, and Partial Characterization ofBacillus subtilisUrate Oxidase Expressed inEscherichia coli

Abstract

Urate oxidase (EC 1.7.3.3) is an enzyme involved in purine metabolism which is used in the treatment of gout and as diagnostic reagent for detection of uric acid. In order to produce this enzyme in large quantities for biotechnological purposes, the gene coding for theBacillus subtilisurate oxidase was cloned and heterologously expressed inEscherichia coli. Time course induction inE. colishowed an induced protein with an apparent molecular mass of∼60 kDa. Soluble recombinant enzyme was purified in a single-step procedure using Ni-NTA column. The enzyme was purified 2.1-fold with a yield of 56% compared to the crude extract. MALDI-TOF analysis revealed an ion with a mass of 58675 Da which is in agreement with the expected mass of the recombinant protein. The purified enzyme showed an optimal pH and temperature of 8.0 and37∘C, respectively, and retained 90% of its activity after 72 hours of incubation at −20∘Cand4∘C.