东方弧菌荧光酶的分离纯化和性质研究

PURIFICATION AND PROPERTIES OF VIBRIO ORIENTALIS 518 LUCIFERASE

东方弧菌518菌株于20℃摆床液体培养十六小时,菌数达最大,菌体内荧光素酶含量也较高,此时收获菌体.用超声波振动破碎细胞从中提取荧光酶粗液,经DEAE-纤维索和DEAE-sephadex柱层析得到纯化的酶.用葡聚糖凝胶层析法测得该酶分子量为87000道尔顿,用SDS-PAGE法测得该酶两个亚基分子量分别为44000(α)和41000(β). 该酶在pH6.8、18℃时活性最佳,对热不稳定.以FMNH_2为底物催化发光反应,最高发射波长为490nm左右.其光谱特征与文献报道的东方孤菌整体发光的光谱相一致.

Ⅴ.orientalis 518 strain was cultured in liquid medium, with shaking at 20℃ for 16 hours. Cells were harvested at the late log phase by cen-trifugation and lysed by addition of cold distilled water to the cell paste. Then, it Was treated with ultrasonic in an ice bath.The rough luciferase was isolated from the above suspension of cells. Then it was purified by DEAE-Cellulose chromatography as well as DEAE-sephadex chromatograhy. By means of sephadex G-100 gel filtration, it has been determined that the molecular weight of this luciferase is 87000 and by means of SDS-PAGE it has been determined that there are two subunits αand β in luciferase and their molecular weight are 44000 and 41000 respectively.The reaction system contains the compounds with the following concentration: 5.1μuM FMN, 3.4mM sodium dithionite, 2.2μM catalase, 1.9 mM decanal and 85mM H2O2. Some properties of the luciferase were studied. The maximun luciferase activity was obtained at 18℃, pH 6.8. The luciferase was sensitive to heat treatment. It lost its catalititc activity at above 25℃. The results showed that the luciferase lost above 95% of its activities after pre-treatment at 40℃ for 40 minutes.The luciferase had a luminescent peak at 490nm. It was consistent with previous reports.