Purification and characterization of a novel fibrinolytic enzyme from Paecilomyces militaris

Abstract:

[Objective] Establish purification methods of fibrinolytic enzyme with high purity and fibrinolytic activity from Paecilomyces militaris submerged culture liquid and determine its characterization. [Methods] The fibrinolytic enzyme Ⅱ of Paecilomyces militaris was purified by ammonium sulfate precipitation, Sephadex G-25 gel filtration chromatography, Phenyl-Sepharose HP hydrophobic interaction chromatography, CM-Sepharose FF ion exchange chromatography and Superdex 75 gel filtration chromatography. The protein concentration of enzyme Ⅱwas determined by Lowry method. The fibrinolytic activity was determined by Fibrin plate method. The purity and molecular weight was estimated by SDS-PAGE. The isoelectric point was estimated by isoelectric focusing electrophoresis. [Results] We found that Paecilomyces militaris produced at least two kinds of fibrinolytic enzymes by submerged cultivation, sucrose and bean cake as major materials. The specific activity of purified fibrinolytic enzyme Ⅱ was 800.46 U/mg and the purification protocol resulted in 30.07-folds purification of the enzyme Ⅱ. The molecular weight and isoelectric point of the enzyme Ⅱ was 32 kD and 9.3±0.2, respectively. The enzyme Ⅱwas a glycoprotein, the total sugar content of it was 0.98% (w/v). The enzyme Ⅱ could degrade α, β and γ chains of human fibrinogen. The optimal pH and temperature of the enzyme were 7.4 and 41°C. The fibrinolytic activity was completely inhabited by Aprotinine and PMSF, which indicated that it might be a serine protease. [Conclution] The acquisition of enzyme with single and high fibrinolytic activity and the determination of its characterization, which provides theoretical basis for the enzyme to become a new thrombolysis drug.