title: Characterization of a Thermostable Uricase Isolated from Bacillus firmus DWD-33 and its Application for Uric Acid Quantification in Human Serum.
abstract: A thermostable uricase identified in Bacillus firmus DWD-33, which was isolated for the first time from soil, with an apparent molecular weight of 33.5 kDa was stable against oxidants and SDS. The highest expression yields were obtained in medium containing 0.8% maltose and 1.2% soybean powder as carbon and nitrogen sources, respectively. Enzyme purification increased the specific activity about 24-fold with 27% recovery. As compared with other microbial uricases, the pure enzyme showed a high thermostability. The Vmax was 387 µmol/L/min, the turnover number (Kcat) was 21.8x10(3) s(-1) and the catalytic efficiency (Kcat /Km) was 2.76x10(8) s(-1)M(-1). The enzyme was stable from pH 7.0 to 10.0 and up to 70 °C and the optimal conditions were 50 °C and pH 8.0. Mg(2+) significantly enhanced the enzymatic activity, while Hg(2+), EDTA, and o-phenanthroline greatly suppressed the activity. Mg(2+) might be the uricase cofactor, as the enzyme activity was restored after its addition to EDTA-chelated enzyme. Inhibition of the enzyme by the copper- chelating agent 2,9-dimethyl-1,10-phenanthroline suggests that this enzyme belongs to the cuprouricase-type. The purified uricase retained 72% and 82% of its original activity after incubation with 0.5% H2O2 and 0.5% SDS for 6 h, respectively. It was possible to determine uric acid in human sera with the enzyme with none of the tested uric acid analogs being a competitive substrate, indicating a high specificity of uricase with respect to uric acid measurement in vitro for uric acid concentration up to 500 µmol/L.
authors.affiliation: Research Laboratory of Bacteriology, Department of Microbiology, Faculty of Science, Zagazig University, Zagazig, Egypt 44519. ekotb@hotmail.com.
authors.forename: Essam
authors.initials: E
authors.lastname: Kotb
mesh_terms: D001407:Bacillus / Q000737:chemistry / Q000201:enzymology*; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D004492:Edetic Acid / Q000378:metabolism; D004795:Enzyme Stability; D004867:Equipment Design; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D008628:Mercury / Q000378:metabolism; D010618:Phenanthrolines / Q000378:metabolism; D014503:Urate Oxidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014527:Uric Acid / Q000097:blood*
chemical_list: D010618:Phenanthrolines; D014527:Uric Acid; D004492:Edetic Acid; D014503:Urate Oxidase; D008628:Mercury; D008274:Magnesium; C025205:1,10-phenanthroline