[关键词]
[摘要]
为了开发利用猪血资源,采用猪血为原料,以超氧化物歧化酶的比活力为指标,对热变性提取过程中影响超氧化物歧化酶活力的水浴温度、水浴时间和硫酸铜加入量三个因素进行考察,并在此基础上,采用中心组合(Box-Behnken)试验设计及响应面分析对猪血超氧化物歧化酶(SOD)的热变性提取工艺进行优化,将最优热变性条件下提取的上清液通过氯仿-乙醇处理,丙酮沉淀提取粗酶液,并采用Sephadex G-100对粗酶液进行纯化。最后采用聚丙烯酰胺凝胶电泳对纯化后的酶液进行纯度鉴定。结果表明:水浴温度、硫酸铜加入量对猪血SOD活力影响显著(P<0.05),热处理最佳工艺为:水浴温度为66 ℃,水浴时间为25 min,硫酸铜加入量为3.10%,此时粗酶液的比活力为167.74±0.38 U/mg。经Sephadex G-100纯化后酶液的比活力为6594.55±16.20 U/mg,活力回收率为62.15±0.02%,纯化倍数为39.14±0.36。聚丙烯酰胺凝胶电泳分析酶纯度已到达电泳纯。
[Key word]
[Abstract]
The thermal denaturalization was applied to extract superoxide dismutase from porcine blood. On the basis of single-factor experiments, technological paraments were optimized by Box-Behnken central combination design and response surface methodology (RSM) with the specific activity of SOD as index. The crude enzyme was extracted by ethanol-chloroform and acetone, then purified by Sephadex G-100 gel column chromatography.The purified superoxide dismutase was identified by SDS-PAGE. The results showed that the specific activity of enzyme was significantly affected by bath temperature and copper sulfate content. The optimum conditions were bath temperature 66℃, bath time 25min and copper sulfate 3.10%,under which the special activity of crude enzyme was 167.74?0.38 U/mg. After purification, the special activity was increased to 6594.55?16.20 U/mg, with recovery rate of 62.15?0.02%. The purification of the enzyme was analyzed by polyacrylamide gel electrophoresis and the purified enzyme reached electrophoresis level.
[中图分类号]
[基金项目]
四川省科技厅项目(11ZS2013)