鲍鱼脏器β-葡萄糖苷酶的分离纯化及酶学性质

Purification of β-Glucosidase from Abalone Viscera and Its Enzymological Character

试验研究皱纹盘鲍内脏中β-葡萄糖苷酶的分离纯化技术,以及pH、温度及金属离子和某些抑制剂对β-葡萄糖苷酶活性的影响。结果表明,以硫酸铵分级盐析鲍鱼脏器β-葡萄糖苷酶,选取30%饱和度的硫酸铵初步盐析除杂,80%饱和度的硫酸铵沉淀,能使鲍鱼脏器β-葡萄糖苷酶的初步纯化效果最佳;经SDS-PAGE分析,透析后的鲍鱼脏器β-葡萄糖苷酶纯度较高,分子量约为54.0~76.0 k Da;β-葡萄糖苷酶的最适温度为50℃,最适pH为3.5,在小于50℃、pH 3.0~6.0下均能保持稳定;Zn^(2+)、Cu^(2+)和Ag+对鲍鱼脏器β-葡萄糖苷酶活性有明显的抑制作用;Na^+、K^+、Mg^(2+)、Ca^(2+)和Fe^(2+)对酶活性影响不明显;Ba^(2+)、EDTA和Mn^(2+)对酶活均有激活作用,其中,Mn^(2+)激活作用最强。以p-NPG为底物,β-葡萄糖苷酶动力学常数Km为4.66 mmol/L,Vmax为3.79 U/L。

β-glucosidase extraction and purification technology of abalone viscera, as well as pH, temperature, metallicions and some inhibitor＇s impact on β-glucosidase enzyme activity were studied. The experimental results showed a better separation effect offl-glucosidase could be obtained through adopting the step-by-step salting method which selected ammonium sulphate a saturation of 30%, the second saturation of 80%. The molecular weight was about 54.0-76.0 kDa as determined by SDS-PAGE. The optimum temperature and pH for β-glucosidase were 50 ℃ and 3.5, respectively, β-glucosidase was relatively stable over the pH range of 3.0-6.0 and the temperature under 50 ℃. Zn2＋, Cu2＋ and Ag＋ had a significant inhibitory effect on enzyme activity of abalone viscera fl-glucosidase. Na＋, K＋, Mg2＋, Ca2＋ and Fe2＋ had no significant effect on enzyme activity. Ba2＋, EDTA and Mn2＋ had different degree of activation to the enzyme activity, Mn2＋ strongly increased the enzyme activity. The pNPG as substrate, the kinetic constants were Km=4.66 mmol/L, and Vmax=3.79 U/L.