Cloning, purification, and partial characterization of Bacillus subtilis urate oxidase expressed in Escherichia coli.
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 approximately 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 37 degrees C, respectively, and retained 90% of its activity after 72 hours of incubation at -20 degrees C and 4 degrees C.
Pfrimer
Pollyana
P
Laboratório de Biologia Molecular, Universidade de Brasília, 70910-900 Brasília (DF), Brazil.
de Moraes
Lidia Maria Pepe
LM
Galdino
Alexsandro Sobreira
AS
Salles
Loise Pedrosa
LP
Reis
Viviane Castelo Branco
VC
De Marco
Janice Lisboa
JL
Prates
Maura Vianna
MV
Bloch
Carlos
C
Torres
Fernando Araripe Gonçalves
FA
D001412:Bacillus subtilis / Q000201:enzymology*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D013997:Time Factors; D014503:Urate Oxidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*
D011994:Recombinant Proteins; D014503:Urate Oxidase