Isolation Of A Uricase-producing Bacterium And Study On The Characteristics Of The Uricase

Uric acid is a final product of purine catabolism, which is excreted by the renal metabolism. In clinical diagnosis, the concentration of uric acid in serum is an important parameter for evaluation of kidney function. At present the most commonly used assay for uric acid is the enzyme colorimetry method, in which the uricase (Urate Oxidase, EC 1.7.3.3) was used as one most important enzyme. Therefore, the popularization of the method was largely depended on the properties of uricase. Also some research of uricase has been reported, however, most of the reported enzymes do not have good thermal stability.In this study, a strain of bacteria capable of producing uricase was isolated and identified subsequently as a stain of the genus Microbacterium. And the condition for the enzyme fenmentation, and purification, characteristics of the enzyme were carried out in this word. The results were showed as follows:1. The isolation and identification of the uricase producing strain: Five cultures of bacteria capable of producing uricase were isolated from soil, which were strain ZZJ2-1, ZZJ3-4, ZZJ4-1, ZZJ5-2 and ZZJ9-2. Based on the thermal stability and Km value of uricase analysis, the strain ZZJ4-1, which showed the highest thermostability and the lowest Km value, was selected. On the basis of its morphological and physiological characteristics, as well as 16S rDNA sequence and phylogenetic tree analysis, this new isolate belongs to the genus Microbacterium.2. The enzyme fermentation conditions: Based on the studies for the conditions of uricase production, the optimum fermentation conditions were established. The composition of the medium was: uric acid 3g, maize milk 10g, K₂HPO₄· 3H₂O 2g, KH₂PO₄0.5g, MgSO₄·7H₂O 0.5g, NaCl 1g, H₂O 1000ml and pH 7.5. 30ml of this medium was added to a 100 ml flask and incubated on a shaker of 120 rpm at 30℃ for 30-36h. The uricase activity peaked at 0.98U/ml.3. The purification and characterization of uricase: The specific activity of a crude, cell-free extract of uricase was increased 20-fold by a series of purification steps including ammonium sulfate precipitation, DEAE-Cellulose ion-exchange,Toyopearl HW-65C hydrophobic chromatography. The purified enzyme was homogeneous as judged by PAGE. The molecular mass was estimated to be 34kD by SDS-PAGE. Study on the characteristics showed that: the optimum pH for uricase reaction was 8.5 and the optimal temperature was 30°C. Uricase was stable between pH 7.0 to 10.0 and the temperature below 65°C. The Km value for uric acid was 0.3lmmol/L. The enzyme was markedly inactivated by incubation with lmmol/L of Hg2+, Ag2+ and 0.5% of SDS. Tween 20 and Triton X-100 almost had no effect on the uricase activity.