Preparation,Characterization And Genic Cloning Of The Uricase From Candida Utilis

Uric acid as the end-product of purine metabolism in human body is passed out through kidney. If the production of uric acid surpasses that excreted by kidney, the concentration of serum uric acid will be increased and hyperuricemia will occurr. Hyperuricemia is associated with the development and progress of gout, cardiovascular diseases, tumor lysis syndrome and renal disease. So it is necessary to develop efficient drug for hyperuricemia. Currently, the uricase is considered to be an ideal drug to treat hyperuricemia. Herein, the Candida utilis (C.G.M.C.C. 2.1008) and its uricase was investigated.1. The incubation of the Candida utilis (C.G.M.C.C. 2.1008) and preparation and purification of the uricase.After being induced by uric acid, The cells were harvested through centrifugation and broken with ultrasound. The lysate is fractioned with ammonium sulfate and the uricase specific activity of the product reached to more than 0.32U/mg protein. During the DEAE-cellulose chromatography, the uricase couldn't be absorbed but its specific activity reached to 7.1 U/mg after twice DEAE-cellulose chromatography.2. The characterization of uricase. The native uricase was 134.0 kD by Sephadex G-200 gel filtration. After twice chromatography on DEAE-cellulose 52, the specific activity of the partly purified uricase was more than 7.0 U/mg protein. Only single polypeptide about 33.0 kD was resolved by SDS-PAGE with this sample. Matrix-assisted laser desorption ionization time-of-flight MS analysis resolved a 67.7 kD polypeptide as the principal component and a 131.4 kD polypeptide of slightly lower abundance, but no single polypeptide was detected with the same sample. The optimum pH for this uricase was close to 8.8 at 25±0.5℃. This uricase was stable at 37℃for 4 h and more than 50% activity was reserved at pH 7.4. The Michaelis-Menten constant was (32.8±3.1)μmol/L (n=10). Except that adenosine,guanosine and inosine couldn't inhibit the uricase, the inhibition constant of Oteracil potassium,xanthine,guanine,hypoxanthine,adenine and allopurinol was individually (1.1±0.1)μmol/L (n=3),(4.8±0.2)μmol/L (n=3),(37.9±4.2)μmol/L (n=3),(500.0±60.0)μmol/L (n=3),(748.9±82.0)μmol/L (n=3) and (927.6±95.0)μmol/L (n=3). The activity was decreased by 90% after dialysis against distilled water, which was not restored by treatment with NaCl. 1.0 mmol/L indoacetic acid could make the uricase inactive completely within 30 min. This uricase was inhibited markedly by 1.0 mmol/L Cu2+ and 1.0 mmol/L Fe3+. These results suggested that molecular engineering to this Candida utilis uricase may benefit its use as a drug to treat disease associated with hyperuricemia. 3. The PCR cloning and sequencing of this uricase geneThe genomic DNA was extracted by special kit. Using specific primers, the hypothetical sequence of this uricase gene was cloned by PCR after sequential optimization of the annealing temperature. The final sequence of the uricase gene was obtained by sequencing. Using BLAST by searching in NCBI databases, we found that the bulk of this sequence had highest similarity to Pichia guilliermondii ATCC 6260 hypothetical protein (PGUG₀4255) partial mRNA. So it was concluded that this sequence was the genetic sequence of the uricase from Candida utilis C.G.M.C.C. 2.1008, which is helpful for further investigation.