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目的分离筛选及鉴定产耐热尿酸氧化酶的菌株,优化发酵条件并研究酶学性质。方法利用透明圈法从高温酒曲中筛选产耐热尿酸氧化酶的菌株,扩增菌株16S rDNA序列并构建系统发育进化树进行鉴定,优化菌株产尿酸氧化酶的发酵条件并研究该酶的酶学性质。结果筛选获得一株产耐热尿酸氧化酶的菌株,经分子鉴定命名为Bacillus subtilis ZX-6。于优化后的最佳发酵产酶条件下(30℃、初始p H 8.1、4 m L/L玉米浆)尿酸氧化酶水平达135.9 U/L,比优化前提高了133.7%。该酶最适反应温度及p H分别为45℃和7.6,于37℃保温48 h后,其剩余酶活仍为17.2%,具备良好的热稳定性。结论产耐热尿酸氧化酶菌株筛选及发酵条件优化的成功为进一步的研究奠定了良好的基础。
Objective To isolate and identify strains producing heat-resistant uric acid oxidase, optimize the fermentation conditions and study the enzymatic properties. Methods The strains of heat-resistant uricase oxidase were screened from the high-temperature wine yeast by the transparent circle method. The 16S rDNA sequence of the strain was amplified and phylogenetic tree was constructed for identification. The fermentation conditions of uric acid oxidase were optimized and the enzymology nature. Results A strain of heat-resistant uricase was screened and identified as Bacillus subtilis ZX-6. The uricase oxidase level reached 135.9 U / L in the optimal fermentation conditions (30 ℃, initial p H 8.1, 4 m L / L corn steep liquor), which was 133.7% higher than that before optimization. The optimum reaction temperature and p H were 45 ℃ and 7.6, respectively. After incubation at 37 ℃ for 48 h, the remaining enzyme activity was still 17.2% with good thermal stability. Conclusion The successful screening of heat-resistant uricase oxidase strains and the optimization of fermentation conditions have laid a good foundation for further research.