Study On Fibrinolytic Enzyme Derived From Fusarium Moniliforme CPCC480097

Thrombosis intimidates human’s life and health seriously,and has already become dominating death cause.Therefore the search for new fibrinolytic agents from various sources is being an important area of drug discovery.In this study,a highly active fibrinolytic enzyme which was produced by a plant endophytic fungus CPCC 480097 was studied.The contents of this reseach work are as follow:Firstly the plant endophytic fungus CPCC 480097 was isolated and rejuvenated.Then it was identified as Fusarium sp.by morphological and 18S rDNA,ITS sequence analysis. The fibrinolytic enzyme was named as HCCB00699S1.The optimized fermentation conditions for CPCC 480097 were determined,under which the fibrinolytic activity reached 2×10⁶ U/L（compared to urokinase）,giving a 1 fold increase over the original fermentation conditions.The stability of crude enzyme was investigated,and the results showed that:crude enzyme was stable in a range of temperature and pH,and NaCl solution,（NH₄）₂SO₄ solution and repeated freeze-thaw with several times had little effect on its stability,while acetone and ethanol would cause deactivation.HCCB00699S1 was purified from the culture broth of CPCC 480097 by centrifugation, ammonium sulfate fraction precipitation,microfiltration,ultrafiltration,ion-exchange chromatography on Mono-Q,and gel filtration on Superdex 75.1.8 mg of the purified fibrinolytic enzyme was obtained with a 6.8%yield by these purification steps.The basic parameters of HCCB00699S1 were measured.The specific activity of the final enzyme preparation was 76,111.1 U/mg.The enzyme has a molecular weight of about 28 kDa with no subunits and pI of 8.1.The sequence of the first 15 amino acid at the N-terminal of HCCB00699S1 was determined as QASSGTPATIRVLVV,which is different from those fibrinolytic enzymes produced by other microorganisms,indicating that HCCB00699S1 is a novel fibrinolytic enzyme.The optimal reaction pH and temperature of the enzyme were 8.5 and 45℃, respectively.It was stable in a pH range of 6.0～9.0 as well as a wide temperature range of 4℃～37℃,and drying cryopreservation was preferred.The enzyme was identified as a serine protease and metal protease.