鄂尔多斯高原碱湖钝顶螺旋藻SOD的纯化与性质研究

Purification and Properties of SOD in Spirulina platensis from Erdos Plateau Alkaline Lake

以鄂尔多斯高原碱湖特有的钝顶螺旋藻为材料,经超声破碎、硫酸铵分步盐析、离子交换层析及凝胶过滤,纯化得到SOD酶,经PAGE检测为1条带,所得酶液平均比活为2 078 U/mg,(总活力)回收率为19.9%.对SOD酶的PAGE电泳条带进行活性染色,亮斑清晰整齐;H2O2处理对照显示该酶活性明显受到抑制,KCN处理对照显示酶活性不受影响,初步判断为Fe-SOD;又经金属元素分析,确认为Fe-SOD.经SDS-PAGE测定,亚基相对分子量为20 kD,经PG-PAGE测定,全酶分子量为54 kD,据分子量推断全酶应为三聚体分子.该酶在紫外区275.5 nm有最大光吸收.在磷酸缓冲液中酶活力的最适pH为8.1,最适温度为25℃;酶活性的pH稳定范围为5～10,40℃以上酶活力开始明显下降.酶在室温下存放9 d后活力仍保持不变.

Spirulina platensis from alkaline lake in Erdos Plateau are broken with ultrasonic waves and then salted out fractionally by ammonium sulfate. The crude SOD enzyme in platensis is purified with the steps of ion exchange chromatography and sephadex G-100 gel chromatography. The specific activity of the purified SOD is 2 078 U/rag and the recovery efficiency 19.9%. It is determined as electrophoresis purity and identified as Fe-SOD because its activity can be inhibited by H2O2 but unaffected by KCN. Metal analysis also shows that it contains the atom of iron. The molecular weight of the enzyme is 54 kD and its subunit molecular mass 20 kD. The enzyme exhibits one absorption peak at 275.5 nm. Its activity remains stable in the range of pH=5~10 and below 40 ℃. The optimum pH in Phosphate Saline buffer is 8.1 and the optimum temperature 25 ℃. Its activity remains stable within 9 days although it is kept at room temperature.