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Journal of Biomedicine and Biotechnology
Volume 2010, Article ID 674908, 6 pages
Research Article

Cloning, Purification, and Partial Characterization of Bacillus subtilis Urate Oxidase Expressed in Escherichia coli

1Laboratório de Biologia Molecular, Universidade de Brasília, 70910-900 Brasília (DF), Brazil
2Laboratório de Espectrometria de Massa, EMBRAPA Recursos Genéticos e Biotecnologia, 70770-917 Brasília, (DF), Brazil

Received 26 June 2009; Revised 14 October 2009; Accepted 17 November 2009

Academic Editor: Sherry Mowbray

Copyright © 2010 Pollyana Pfrimer et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Urate oxidase (EC 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 the Bacillus subtilis urate oxidase was cloned and heterologously expressed in Escherichia coli. Time course induction in E. coli showed 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 and , respectively, and retained 90% of its activity after 72 hours of incubation at − and .