Cloning, Purification, and Partial Characterization of Bacillus subtilis Urate Oxidase Expressed in Escherichia coli. (28th December 2009)

• Record Type: Journal Article
• Authors: Pfrimer, Pollyana; Moraes, Lidia Maria Pepe de; Galdino, Alexsandro Sobreira; Salles, Loise Pedrosa; Reis, Viviane Castelo Branco; De Marco, Janice Lisboa; Prates, Maura Vianna; Bloch, Carlos; Torres, Fernando Araripe Gonçalves
• Other Names: Mowbray Sherry Academic Editor.
• Is Part Of: Journal of biomedicine and biotechnology. Volume 2010(2010)
• Publication Date: 2009-12-28
• Subjects: Medicine -- Periodicals; Biology -- Periodicals; Biotechnology -- Periodicals; Medicine; Biology; Biotechnology; Médecine; Biologie; Biotechnologie; Biology; Biotechnology; Medicine; Biotechnology; Biomedicine; Electronic journals; Periodical; Periodicals; Electronic journals; 610
• DOI: 10.1155/2010/674908
• Languages: English
• ISSNs: 1110-7243

Abstract

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 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 and37 ∘ C, respectively, and retained 90% of its activity after 72 hours of incubation at −20 ∘ C and4 ∘ C .