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【目的】确立蛹拟青霉深层培养液中高纯度、高纤溶活性纤溶酶的分离纯化方法并测定其酶学性质。【方法】采用硫酸铵盐析、Sephadex G-25凝胶色谱、Phenyl-Sepharose HP疏水相互作用色谱、CM-Sepharose FF弱阳离子交换色谱和Superdex 75凝胶色谱对蛹拟青霉纤溶酶进行分离。用Lowry法测定蛋白质浓度,纤维蛋白平板法测定其纤溶活性,SDS-PAGE鉴定其纯度并确定其分子量,IEF法测定其等电点。【结果】研究发现,以蔗糖和豆饼为培养基主要基质时,蛹拟青霉深层培养可以产生至少两种纤溶酶。提纯后的纤溶酶Ⅱ比活力达到800.46 U/mg,总纯化倍数为30.07倍。纤溶酶Ⅱ的相对分子量和等电点分别为32 kD和9.3±0.2。纤溶酶Ⅱ是一种糖蛋白,总含糖量为0.98%(W/V)。该酶可以顺次降解人血纤维蛋白(原)的α、β和γ链。其最适作用pH及温度分别为7.4和41°C。Aprotinine与PMSF对该纤溶酶的活性完全抑制,推测此纤溶酶可能是一种丝氨酸蛋白酶。【结论】单一的高纤溶活性纤溶酶的获得和酶学性质的确定,为该酶开发成为新型溶栓药物提供了理论依据。
【Objective】 To establish a method for isolation and purification of high-purity, high-fibrinolytic activity fibrinolytic enzyme in Paecilomyces pupae culture medium and determine its enzymatic properties. 【Method】 Paecilomyces pupa was isolated by ammonium sulfate salting-out, Sephadex G-25 gel permeation chromatography, Phenyl-Sepharose HP hydrophobic interaction chromatography, CM-Sepharose FF weak cation exchange chromatography and Superdex 75 gel chromatography . The protein concentration was determined by Lowry method. The fibrinolytic activity was determined by fibrin plate method. The purity was determined by SDS-PAGE and its molecular weight was determined. The isoelectric point was determined by IEF method. 【Result】 The results showed that when sucrose and soybean cake were the main substrates, the culture of Paecilomyces pupae could produce at least two kinds of plasmin. The specific activity of purified plasmin II reached 800.46 U / mg, the total purification fold was 30.07 times. The relative molecular weight and isoelectric point of plasmin II were 32 kD and 9.3 ± 0.2, respectively. Plasmin II is a glycoprotein with a total sugar content of 0.98% (W / V). This enzyme can sequentially degrade the alpha, beta and gamma chains of human fibrin (prot). Its optimum pH and temperature were 7.4 and 41 ° C, respectively. Aprotinine and PMSF completely inhibited the activity of this plasmin, presumably this plasmin may be a serine protease. 【Conclusion】 The determination of single fibrinolytic activity and enzymatic properties of the fibrinolytic enzyme provides a theoretical basis for the development of this enzyme into a novel thrombolytic drug.