链霉菌C-3662产生的纤溶活性蛋白酶的纯化与理化性质

Purification and Characterization of a Fibrinolytic Enzyme from Streptomyces sp.C-3662

从链霉菌 C- 3662发酵上清液中 ,通过硫酸铵沉淀 ,CM- Sepharose Fast Flow和 Phenyl-Sepharose Fast Flow等层析色谱 ,分离纯化得到了具有纤溶活性的蛋白酶 CGW- 3,反向 HPLC鉴定纯度为 90 % ;每立升发酵上清液可得到 8mg纯品 ,活性回收率 46% ,CGW- 3为一单肽链蛋白 ,分子量 2 2 72 1 ,对丝氨酸蛋白酶抑制剂 PMSF敏感 ,对 EDTA不敏感 ;其 N端 1 5个氨基酸的顺序为 VVGGTRAAQGEFPFM,与微生物来源的胰蛋白酶类丝氨酸蛋白酶有较高的同源性 . CGW- 3的等电点 p I9.0 ,纤溶活性的最适 p H为 7.5～ 8.0 ,对温度比较敏感 .CGW- 3不仅具有直接降解纤维蛋白作用 。

A novel protease with fibrinolytic activity designated as CGW\|3 was isolated and purified from Streptomyces sp.C\|3662 by ammonium sulfate precipitation,DEAE\|Sepharose FF chromatography,CM\|Sepharose FF chromatography and phenyl\|Sepharose FF chromatography.CGW\|3 was a single chain polypeptide with a molecular weight of 22 721 determined by MALDI\|MS.The N\|terminal 15 amino acid sequence was VVGGTRAAQGEFPFM,which showed high homology to trypsin\|like serine proteases from different microorganisms.The fibrinolytic activity of CGW\|3 was inhibited by PMSF but not by EDTA.The p I of CGW\|3 was 9.0 and the optimum pH of fibrinolytic activity was 7.5～8 0.CGW\|3 could not only degrade firbrin but also activate plasminogen.