韭菜酸性磷酸酶的分离纯化及酶学性质

Isolation, Purification and Characterization of Acid Phosphatase from Chinese Chives

取新鲜韭菜,通过匀浆、缓冲液抽提、硫酸铵分级沉淀、CM-Sepharose离子交换层析、Superdex-200凝胶过滤层析等方法纯化,获得韭菜中所含的一种电泳纯的酸性磷酸酶(ACP)。经纯化后的酶溶液比活力为645.83U/mg,纯化倍数达到454.81倍,回收率为10.50%。酶学性质研究表明:该酶的分子质量约为58.97kD;最适反应温度和pH值分别为65℃和5.4;该酶对对硝基苯磷酸二钠(pNPP)的K m值为0.896×10-3mol/L;较高浓度的Mg2+、Mn2+对该酶的激活作用更强;抗坏血酸、甲醇、乙醇、氯仿、异丙醇、Cu2+、Ag+的浓度越大,对该酶的抑制作用越强。

Fresh chives were homogenized, extracted with buffer and precipitated by ammonium sulfate. The precipitant was dissolved in HAc-NaAc and then purified by CM-Sepharose ion-exchange chromatography and superdex-200 gel filtration chromatography. A new type of electrophoretically pure acid phosphatase （ACP） from fresh Chinese chives was then obtained. The enzyme activity of the purified ACP reached 645.83 U/mg. The purification factor was 454.81 and the activity recovery was 10.50%. Characterization studies showed that the molecular mass of this enzyme was approximately 58.97 kD and the optimum reaction temperature and pH value were 65 ℃ and 5.4, respectively. The Km value for this enzyme was 0.896 × 10-3 mol/L when using nitrophenyl phosphate disodium （pNPP） as the substrate. High concentrations of Mg2＋ and Mn2＋ showed stronger activation of the enzyme while the higher concentration of ascorbic acid, methanol, ethanol, chloroform, isopropanol, Cu2＋ and Ag＋, the stronger the inhibition of the enzyme.