摘要
由嗜热脂肪芽胞杆菌(Bacillus stearothermophilus SIPI1.687)的粗提取液,经液-液双相抽提、DEAE-Toyopearl 650 M 离子交换柱层析及 SIPI-Ⅱ蓝色(?)料配基层析等分离提纯,可得甘油激酶(81U/mg),PAGE 鉴定为单带。用 Sephacryl 400凝胶过滤测得分子量为209,000道尔顿,SDS-PAGE 测定结果表明该酶由四个分子量均为50,000道尔顿的相似亚基组成。酶作用最佳 pH 9.4,pH 5.8~11.2时稳定;最佳温度为50℃,有较好的热稳定性。以甘油为底物时米氏常数为6.25×10M。
Glycerokinase(EC 2.7.1.30)from Bacillus stearothermophilus wasfurified by 35-fold with liquid-liquid two-phasee xtraction,DEAE-toyopearl 650Mcolumn chromatography and SIPI-II blue dye ligand chromatography.Thepurified enzyme showed a single band on PAGE,with molecular weight of about209,000 Daltons and composed of four similar subunits with 50,000 Daltons.Optimum workable pH and temperature are around 9.4 and 50℃ respectively.The enzyme is stable over a wide pH range(5.8~11.2)and possesses goodthermostability.Kvalue for glycerol as substrate is found to be 6.25×10M.
