纳豆激酶分离纯化与鉴定  

Purification and Characterization of Nattokinase

作　　者：李炳锦[1] 崔京浩[1] 李水林[1] 柳明洙[2] 李英姬[3] 崔然吉[3] 李凤龙[1] 

机构地区：[1]延边大学药学院药剂学教研室,吉林延吉133000 [2]延边大学医学院生物化学与分子生物学教研室,吉林延吉133000 [3]延边大学草仙药业有限公司,吉林龙井133400

出　　处：《药物生物技术》2003年第4期232-237,共6页Pharmaceutical Biotechnology

基　　金：横向课题 (由延边大学草仙药业资助 )

摘　　要：为了提高纳豆激酶的溶栓活性 ,从纳豆粗提物中分离纯化了纳豆激酶。采用Butyl Toyopearl柱层析对纳豆粗提物进行了分离纯化 ,以试管法确定和收集了活性部位 ,用纤维蛋白平板法测定了活力 ,并用十二烷基硫酸钠 聚丙烯酰胺凝胶电泳法 (SDS PAGE)对分离纯化的效果进行了检验。考察了纳豆激酶粗提物在不同温度和湿度条件下的稳定性。结果表明在SDS PAGE中观察到三条明显的蛋白谱带 ,其中相对分子质量为 192 75和 2 710 1的区带具有与文献报道类似的纳豆激酶的相对分子质量和纤溶活性 ,而相对分子质量为 14 4 0 0的蛋白区带与文献报道的纳豆激酶的相对分子质量大有差异。纯化倍数为 5 0倍 ,活性回收率为 75 .5 7%。初步稳定性实验结果表明 :其外观在 4 0℃和 6 0℃保存条件下发生变化 ,纤溶活力在 6 0℃保存条件下发生显著变化。而 4℃条件下活力和外观却很少有变化 ;纳豆粗提物在不同湿度条件下均具有较强吸湿性。纳豆粗提物对高温和高湿稳定性较差。To increase the fibrinolytic activity of nattokinase, nattokinase was purified by using Butyl-Toyopearl chromatography, and fibrinolytic activity fractions were selected through tube test. The fibrinolytic activity of nattokinase was measured by means of fibrin plate method. The relative molecular weight of purified nattokinase were characterized with SDS-PAGE. Preliminary stability experiments of nattokinase were performed under high temperature and relative humidity. Three bands with relative molecular mass of 14 400, 19 275 and 27 101, were observed by SDS-PAGE, respectively. The both bands with relative molecular weight of 19 275 and 27 101 were similar with relative molecular weight of nattokinase,except that of 14 400 one.According to the increase rate of fibrinolytic activity, the purification fold was 54, and the recover rate of fibrinolytic activity was 75.57%. The results of stability experiment indicated that appearance of nattokinase was changed significantly in the condition of 40℃ and 60℃. Fibrinolytic activity of nattokinase was changed significantly in the condition of 60℃.But no significant changes were appeared in 4℃. Crude natto extract could preserve strong fibrinolytic activity under high humidity while the appearance changed during the period of experiment.

关 键 词：纳豆激酶 Butly-Toyopear 柱层析 纤维蛋白平板法 试管法 SDS-聚丙烯酰胺凝胶电泳 

分 类 号：Q55[生物学—生物化学]