Cloning,Expression And Characterization Of Urate Oxidase Gene From Microbacterium Sp.ZZJ4-1 Strain In Escherichia Coli

Urate oxidase (Uox, EC 1.7.3.3) has been widely applied in the clinic detection of uric acid and therapy for its ability of degradation of uric acid. In 2005, a bacterium named Microbacterium sp. ZZJ4-1 strain that produced thermostable Uox was isolated from soil in our laboratory. In this research, the gene (uox) of strain ZZJ4-1 was cloned and expressed in Escherichia coli. After optimization of expression conditions, recombinant Uox was purified and its properties were studied. The results of this research were summarized as follows.1. The uox (GenBank accession no. JQ066818) from Microbacterium sp. ZZJ4-1 strain was cloned and its open reading frame contained 894 base pairs which encoded 297 amino acids. It had no obvious identity to the most of reported uox, with only a 72% identity to most similar uox which from Arthrobacter globiformis. The gene was inserted into the plasmid pET-15b to construct the expression vector pET-15b-uox which expressed the recombinant enzyme in Escherichia coli BL21(DE3).2. Several factors including concentration of sorbitol, biomass, IPTG, induction time and temperature were studied and the optimal fermentation conditions were determined as follows:1% of culture was inoculated in a TB medium with 100 mg/L ampicillin. IPTG was added to the final concentration of 0.5 mmol/L when ODeoo reached approximately 3.5 in a 37℃shaker, and after that it was expressed at 22.5℃for 18 h. After the optimization, the productivity of recombinant Uox was increased more than 30.6 folds.3. With two purification steps, the recombinant Uox was purified 14.5 folds which showed a single band in SDS-PAGE analysis. Its specific activity and yield were 4.6 U/mg and 47.8%, respectively. It was composed of four subunits with a molecular mass of about 35 kDa. The optimal temperature and pH for its activity was 30℃and pH 7.5, respectively. It was stable below 65℃and between the pH from 8.5 to 11.0. With the uric acid as the substrate, the Km was 0.2 mmol/L. Its activity was totally inhibited by Ag+, Zn2+, Cu2+, SDS and slightly inhibited by NaN3,1,10-phenanthroline, EDTA; it was promoted slightly by Tween 20, Tween 80 and Triton X-100. The thermal stability of this enzyme was the most excellent among the reported recombinant Uox.