蕹菜过氧化物酶的分离纯化及理化性质

Isolation, Purification and Characterization of Peroxidase from Ipomoea aquatica Forsk.

新鲜蕹菜经硫酸铵分级沉淀、DEAE-Sepharose离子交换层析和Superdex-200凝胶过滤层析后获得电泳纯的过氧化物酶(peroxidase,POD),该酶的比活力、回收率、纯化倍数和产率分别为35 972.96 U/mg、12.21%、168.48和211.07 U/g。该酶的亚基分子质量为42.7 kD,最适温度为40 ℃,最适pH值为 6,并且在20～50 ℃及pH 5～8的范围内具有良好的稳定性。以不同浓度的H2O2为底物,测得该酶的Km值为18.32 mmol/L。NaCl、尿素(Urea)、Zn2+、Mg2+对该酶都具有较强的激活作用,十二烷基硫酸钠(sodium dodecyl sulfate,SDS)、硫氰化钾(potassium thiocyanate,KSCN)、抗坏血酸(ascorbic acid,AsA)、Ba2+、Mn2+ 、Fe2+、甲醇、乙醇、正丁醇和异丙醇对蕹菜POD活力均有抑制作用,其中抗坏血酸对POD有极强的抑制作用,当浓度为0.01 mol/L时,酶活力接近于0。

Electrophoresis-purity peroxidase (POD) was extracted and purified from Ipomoea aquatica Forsk. through ammonium sulfate precipitation, DEAE-Sepharose and Superdex-200 chromatography. The specific activity of the purified peroxidase was 35 972.96 U/mg and the yield was 211.07 U/g with a recovery rate of 12.21% and a purification fold of 168.48. Its molecular mass was around 42.7 kD as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). Besides, the POD with the optimal temperature and pH of 40 ℃ and 6, respectively, was relatively stable in the temperature range of 20–50 ℃ and pH range of 5–8. This enzyme showed a Km value of 18.32 mmol/L towards the substrate hydrogen peroxide. In addition, the POD was found to be activated by NaCl, urea, Zn2+and Mg2+ but inhibited by SDS, KSCN, ascorbic acid (AsA), Ba2+, Mn2+, Fe2+, methanol, ethanol, n-butyl alcohol and isopropanol, and completely inactivated by 0.01 mmol/L AsA.