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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 the macroporous resin purification technology of bean thrombolytic enzyme. Methods: The macroporous resin model was screened with wet specific adsorption capacity as an index. The thrombolytic enzyme specific activity of bean curd was used as an index. The adsorption performance (loading volume, adsorption time) and elution performance (eluent type, Eluent pH, elution speed, eluent volume), and the purification effect was analyzed by SDS-PAGE electrophoresis. Results: HPD-400 macroporous resin had good adsorption on thrombolytic enzyme. The optimum conditions were as follows: sample volume 2 BV, adsorption time 2 h, water 2 BV to remove impurities, pH 8.0 Tris- HCl-30% ethanol buffer 4 BV at a flow rate of 2 BV · h-1; Thrombolytic enzyme activity of bean curd was 2 254.29 IU · mg-1 and the number of purified was 44.17-fold. Electrophoresis showed that the thrombolytic enzyme showed a single band, the relative molecular mass of 28 kD. Conclusion: HPD-400 macroporous resin can be used for the isolation and purification of thrombolytic enzyme from bean curd. The optimal process conditions are stable and feasible.