大孔树脂纯化豆豉溶栓酶的工艺优选

Optimization of Purification Technology of Lobster Sauce Streptokinase by Macroporous Resin

目的:优选豆豉溶栓酶的大孔树脂纯化工艺。方法:以湿比吸附量为指标筛选大孔树脂型号;以豆豉溶栓酶比活性为指标,采用单因素试验考察吸附性能(上样体积、吸附时间)和洗脱性能(洗脱剂种类、洗脱剂pH、洗脱速度、洗脱剂体积),利用SDS-PAGE电泳对纯化效果进行分析。结果:HPD-400型大孔树脂对豆豉溶栓酶具有很好的吸附作用,优选的工艺条件为上样量2 BV,吸附时间2 h,加水2 BV洗去杂质,加pH 8.0的Tris-HCl-30%乙醇缓冲液4 BV以2 BV·h-1流速洗脱;豆豉溶栓酶比活性2 254.29 IU·mg-1,纯化数44.17倍,发酵液20 mL平均可得到蛋白质2.86 mg,电泳检测显示豆豉溶栓酶呈现单一条带,相对分子质量28 kD。结论:HPD-400型大孔树脂可用于豆豉溶栓酶的分离纯化,优选的工艺条件稳定可行。

Objective： To optimize purification technology of lobster sauce streptokinase by macroporous resin. Method： Type of macroporous resin was screened by taking moisture adsorption as index; With specific activity of lobster sauce streptokinase as index, single factor tests were adopted to optimize purification process parameters, such as adsorption performance （loading quantity of sample, adsorption time ） and elution performance （species, pH and volume of eluent, elution rate） , purification effect was analyzed by SDS-PAGE electrophoresis. Result： HPD-400 macroporous resin had a good adsorbabitity to lobster sauce streptokinase, optimum purification conditions were as follows： sample volume of 2 BV, adsorption time of 2 h, removed impurities with 2 BV of water, eluted with 4 BV of Tris HC1-30% ethanol buffer （pH 8.0） with adsorption speed of 2 BV ·h^-1 ; under these conditions, specific activity of lobster sauce streptokinase was 2 254.29 U·mg^-1 purification multiple was 44. 17 times, we could receive 2. 86 mg of protein in 20 mL of fermentation liquor, lobster sauce streptokinase showed a single strip in electrophoretogram whose relative molecular weight was 28 kD. Conclusion： HPD-400 macroporous resin could be adopted to purify lobster sauce streptokinase, this optimized purification technology was stable and feasible.