微生物尿酸氧化酶的筛选、酶学性质及重组表达

Screening, characterization and expression of microbial urate oxidase

尿酸氧化酶(Urate oxidase,Uox)是一种催化尿酸氧化为尿囊素的酶,常用于尿酸的检测以及痛风和高尿酸血症治疗。文中从土壤中筛选出一株Uox高产菌株OUC-1,经16S rRNA部分基因序列分析,与苛求芽孢杆菌Bacillus fastidiosus序列相似度达99%。B.fastidiosus OUC-1的Uox经纯化后,分析表明该酶反应最适pH和温度分别为10.0和40℃;Uox以尿酸为底物反应动力学参数K_m值为(0.15±0.04)mmol/L(n=5)。Mg^(2+)能够提高该酶性活性,而Zn^(2+)和SDS能强烈抑制该酶的酶活。参考GenBank中苛求芽孢杆菌基因组中的uox基因序列,成功扩增出uox基因,通过SWISS-MODEL对Uox空间结构进行预测,推测该酶是同源四聚体,单亚基分子量为35.38 kDa。文中将uox基因克隆并在大肠杆菌中表达,为后续的Uox的性能改造提供条件和技术支持。

Urate oxidase（Uox）, an enzyme catalyzing oxidation of uric acid to allantoin, is widely used as diagnostic reagents and for treatments of uarthritis and hyperuricemia diseases. In our study, a higher Uox producer, bacterial strain OUC-1, was isolated from soil samples. The 16 S rRNA gene sequence of strain OUC-1 showed 99% identity to the homologous fragments of Bacillus fastidiosus. After purification, Uox showed the optimal pH and temperature was 10.0 and 40 °C. The Km value of Uox was（0.15±0.04） mmol/L（n=5） with uric acid as the substrate. Uox activity was enhanced by Mg2＋） and seriously inhibited by Zn2＋ and SDS. Then the uox gene of B. fastidiosus OUC-1 was amplified and sequenced. The 3D structures of Uox, predicted with SWISS-MODEL, showed a homotetramer structure with a subunit molecular weight of 35.38 kDa. Finally, the gene coding for the B. fastidiosus Uox was successfully cloned and heterologously expressed in E. coli,which provides theoretical basis and technical support for improvement of Uox in the future.