Study On Screening,Identification Of Microbes Producing Fibrinolytic Enzyme And Enzyme Properties

In recent years,thrombotic diseases seriously affect the health of human life,and the incidence and mortality of thrombolytic drugs increased year by year,the increasing demand for thrombolytic drugs,meanwhile found that commercial thrombolytic drug specificity is poor,weak effect time is short,thrombolysis,expensive,plus other side effects after medication.Therefore,it is important for the human health to find or develop high efficiency,low toxicity,rapid,specific and cheap thrombolytic medicine.In this article,the strain Paecilomyces lilacinus secreting high yield of fibrinolytic enzyme were isolated from the plant were studied,and metabolism of fibrinolytic enzymes by the strain were analyzed by us.(1)480 plant endophytes were isolated from 245 plant specimens collected from the Yellow River of Shanxi province,Shaoxing and Changbai Mountain.There are 64 strains having the ability to secreted extracellular protease using skimmed milk powder-agarose slab method;There are 8 strains having the ability to secreted fibrinolytic enzyme using fibrin-agarose slab method,including HS-387 strains produced the highest enzyme activity,screen at the beginning of the fermented liquid of fibrinolytic enzyme activity 266.58U/m L(quite urokinase enzyme activity),The morphological identification and molecular biology of the strain showed that the strain HS-387 for Paecilomyces lilacinus.(2)The influence of the fermentation condition on the fibrinolytic enzyme production of HS-387 strain was argued,when the p H of the medium was 4.0 before sterilization,fungi inoculation amount is 3.0×106CFU,tempreture was 28?,175 rpm,the broth content was 100 m L(250m L conical flask)and incubation time was 7 days.After optimization of enzyme activity of 707.97 U/m L(quite urokinase enzyme activity)the activity of the enzyme was increased up to 2.66 times.(3)By ammonium sulfate precipitation,DEAE-weak anion exchange chromatography and SP-weak cation exchange chromatography,Butyl-hydrophobic chromatography and so on analysis method of purification for the combination of strain HS-387 fibrinolytic enzyme secretion,Native-PAGE gel electrophoresis to verify the enzyme is of high purity(more than 96%)of the enzyme,purification recovery was 37.2%,and ultimately than the enzyme activity of 6461.75 U/mg(urokinase is standard).In addition,the higher purity of the fibrinolytic enzyme was identified by QE mass spectrometry.(4)The enzyme of Isoelectric point about 6.6,the molecular weight between 45 k Da,the p H stability in 5.0 to 9.0,being below p H3.0,It's easy to cause inactivation of enzyme.The enzyme was higher enzyme activity between 30? and 50?,the optimum temperature was at 40?.A typical serine protease inhibitor PMSF on the enzyme rate was 60%,it is speculated that the enzyme may be alkaline serine protease.The fibrinogenolytic and fibrinnolytic activity of enzyme was analyzed by SDS-PAGE.The results indicated that the hydrolytic pattern of enzyme on fibrinogen started from the A?-chain,followed by the B?-chain,and the ?-chain at last,most quickly to the degradation of A?-chain;The hydrolytic pattern of enzyme on fibrin started from the ?-?-chain,followed by the ?-chain,and the ?-chain at last,for the degradation of ?-chain quickly.This hydrolysis pattern is similar to that of the plasmin,the enzyme not only degrade fibrin,but also degrade fibrinogen into fibrin.At the same time,it showed that the way of the fibrinolytic enzyme in vitro could play a role in the degradation of fibrin directly degrade fibrin distinct from the plasminogen activators,it has no or only a small kinase activity.In conclusion,this study screening of high yield and fibrinolytic enzyme strains from which 480 strains of endophytic bacteria isolated strains of Paecilomyces lilacinus is reported for the first one is the fibrinolytic enzyme producing bacteria.It was found that its produce fibrinolytic enzyme for alkaline serine protease,the fibrin and fibrinogen degradation effect,presumably the enzyme in dissolving blood clots can also prevent blood clots to form,and could be used as a potential new way to develop thrombolytic drugs.