摘要
以嗜热脂肪芽孢杆菌CU21的表达型质粒pFDC11为载体,插入来自恶臭假单孢菌的编码邻苯二酚2,3-双加氧酶(CatO_2ase)的xylE基因,构建成重组质粒pFDX1转化入CU21受体,在48℃培养时得到了表达产物,表明常温细菌的xylE基因可以在高温菌启动子的带动下在高温菌宿主中表达。采用提高培养温度后选择卡那毒素抗性突变的方法,得到了在55℃及6O℃的CatO_2ase表达量明显提高的变异菌株CU21-161。本文同时报道一种用完整细胞悬浮液测定CatO_2ase活力的方法。
Using the gene expression vector pF'DCll for Bacillus stearothermophilus CU21, a reco-mbinant plasmid pFDXl was constructed, which carries the Pseudomonas-derived xylE gene coding for Catechol 2,3-dioxygenase (CatOiase) CatO2ase activity can be detected from CU21 (pFDXl) cells grown at 48癈. This result indicates that the promoterless xylE gene can be-expressed in a thermophilic host under thr direction of a promoter from a thermophilic bacterium. By selecting Kmr mutants at elevated growth temperature, a segregant CU21-161 was-obtained, the xylE gene expression of which at 55℃ and 60℃ was much higher than that of the parental strain. A method for CatOiase assay with suspension of intact celles was also reported in this paper.
基金
国家自然科学基金(3870355)
关键词
xYLE基因
嗜热芽孢杆菌
基因表达
Bacillus stearothermophilus, Gene expression vector, xylE gene, Catechol 2,3-dioxygenase assay
