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目的从可口革囊星虫中分离纯化纤溶酶,并初步研究其酶学性质。方法实验以纤维蛋白平板法检测纤溶活性,采用匀浆、抽提离心、G-25脱盐、DEAE梯度洗脱等方法从可口革囊星虫中分离纯化出纤溶酶PFE,并进一步研究pH、温度、金属离子及抑制剂对该酶活性的影响。结果分离纯化得到纤溶酶PFE,SDS-PAGE法测得相对分子质量为35kDa左右。此外,其酶学性质研究结果表明:PFE的最适反应pH为7.4,酶活力在pH6.6～9.0时相对稳定;PFE在20～40℃条件下,酶活力保持相对稳定。Fe3+对酶活性有强烈的抑制作用,Ca2+、Mg2+、Fe2+、K+、EDTA和β-巯基乙醇对酶活性均具有一定的抑制作用。结论可口革囊星虫中存在纤溶酶PFE,此酶可直接降解纤维蛋白,具有重要的临床开发价值。
OBJECTIVE To isolate and purify plasmin from the fungus, and to study its enzymatic properties. Methods The fibrinolytic activity was detected by fibrin plate assay. Plasmin PFE was isolated and purified by homogenization, centrifugation, G-25 desalting and DEAE gradient elution. , Temperature, metal ions and inhibitors on the enzyme activity. Results The plasmin PFE was isolated and purified. The relative molecular mass was about 35 kDa by SDS-PAGE. In addition, its enzymatic properties showed that the optimal reaction pH of PFE was 7.4 and the enzyme activity was relatively stable at pH6.6-9.0. The activity of PFE kept relatively stable at 20-40 ℃. Fe3 + has a strong inhibitory effect on the enzyme activity. Ca2 +, Mg2 +, Fe2 +, K +, EDTA and β-mercaptoethanol have certain inhibitory effects on the enzyme activity. Conclusion Plasmodium spp. Exists in the plasmin PFE, this enzyme can directly degrade fibrin, has important clinical development value.