黄曲霉毒素B1降解酶的分离纯化及其酶学特性

施氏假单胞菌（Pseudomonas stutzeri）F4能产生黄曲霉毒素B1的降解酶（aflatoxin B1-degrading enzyme，ADE），本实验通过3 步法提取纯化ADE，即包括硫酸铵分级沉淀、DEAE-Sepharose阴离子交换柱层析和SephadexG-100凝胶过滤层析。结果表明，通过以上纯化方法，ADE的回收率为56%，纯化的ADE比活力为6.4×104 U/mg，纯化了123 倍。利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测得ADE分子质量约为24 kD；ADE与AFB1反应的最适温度和pH值分别为30 ℃和6.0；Cu2+对ADE酶活性有抑制作用；利用双倒数作图法测得ADE表观Km值为5.61×10-5 mol/L。

Purification and Characteristics of an Aflatoxin B1-Degrading Enzyme from Pseudomonas stutzeri F4

Aflatoxin B1 (AFB1)-degrading enzyme (ADE) was purified from Pseudomonas stutzeri F4 by a three-step
procedure including ammonium sulfate fractional precipitation and chromatography on DEAE-Sepharose and Sephadex
G-100. A total of 123-fold purification of ADE with a recovery of 56% and an ultimate specific activity of 6.4 × 104 U/mg
were achieved. The apparent molecular mass of ADE was estimated to be about 24 kD by SDS-PAGE. The ADE displayed
the highest degradation activity for AFB1 at 30 ℃ and pH 6.0. Cu2+ exhibited strong inhibitory activity with an apparent Km
of 5.61 × 10-5 mol/L as indicated by the double-reciprocal plot.