The Regulating Blood Lipid And Thrombolytic Effects Of The Fibrinolytic Enzyme From Neurospora Sitophil

The morbidity of thrombosis has increased,fibrinolytic therapy is available. Current fibrinolytic enzymesavailable for clinical use， but all these agents have undesired side effectsand are very expensive.Therefore,researching and developing effective fibrinolytic enzymes play a significant and profound role in thethrombosis therapy. Due to great variety,fast breeding,good security,cheap price,microorganism was animportant source of fibrinolytic enzyme. Getting the new fibrinolytic enzyme from the microorganisms hascertain advantages.The morbidity of thrombosis has increased, fibrinolytic therapy is available. Current fibrinolytic enzymesavailable for clinical use are in effect，but all these agents have undesired side effects and are very expensive.Therefore, researching and developing effective fibrinolytic enzymes are necessary. Microorganism are animportant source of fibrinolytic enzymes due to its great variety, short production time, and lower cost.Neurospora sitophila was screened by our laboratory. The fibrinolytic enzyme was produced through trayfermentation of Neurospora sitophila. The culture medium was composed of bean dregs and wheat bran, and dryweight ratio of them was4:1. The fibrinolytic enzyme was separated using40%-80%fractional salting-out with(NH4)2SO4firstly, and then three fibrinolytic active components were found by Octyl-Sepharose Fast Flowhydrophobic interaction chromatography. The active componentⅡand Ⅲ was further purified using SephadexG-25gel filtration chromatography, SP-Sepharose FF ion exchange chromatography. The specific activity ofpurified fibrinolytic enzyme Ⅲ was301.27U/mg, the purification protocol resulted in25.77-folds purificationand recovery yield was4.03%. The purified enzymes were homogeneous, as examined by SDS-PAGE. Thepurification result of fibrinolytic enzymeⅡwas unsatisfactory. It may be caused by degeneration, therefore wemust do the regeneration and screening of strains. Combined with plate method and Octyl-Sepharose Fast Flowhydrophobic interaction chromatography, a strain which could produce higher activity enzyme was found.Anticoagulant experiments, thrombolytic experiment, toxicological and behavior of mice were investigatedto estimate thrombolysis, anticoagulate and toxicological effects with lyophilized powder of crude fibrinolyticenzyme, fibrinolytic enzyme II and III from Neurospora sitophila. The results of anticoagulant experiments invitro showed that crude fibrinolytic enzyme, fibrinolytic enzyme II, fibrinolytic enzyme II+III displayedanticoagulant effect obviously, and their anticoagulant effects were better than urokinase. Thrombolysisexperiments in vitro showed that blood clots dissolving rate of crude fibrinolytic enzyme and its componentswere more than80%,which were better than that of control group of urokinase. In addition, rythrocyte of thesegroups displayed good dispersion degree and excellent form, while blood cell of urokinase agglutinated intolarge block, which indicated that fibrinolytic enzymes had little injury to cells and could maintain the integrity ofthe form and function of cells. As a result, their thrombolytic effects were superior to urokinase. Fibrinolyticenzymes did not lead to bleeding after hypodermic injection, and general behavior and coordinated movementsof mice were not influenced by fibrinolytic enzymes. Acute toxicity experiment showed that fibrinolyticenzymes was safe. Compared hyperlipemia group with the control group, the content of total cholesterol (TC)、high densitylipoprotein-cholesterol (HDL-C)、 low density lipoprotein-cholesterol (LDL-C) in serum were verysignificantly different(p<0.01), triglyceride (TG) has significantly difference(p<0.05), which showed thathyperlipemia model was built success. The fibrinolytic enzymes can reduce the content of TC, TG, LDL-C andincrease the HDL-C in Hyperlipemia Mice. That can effectively control the formation of Hyperlipidemia. Afterfeeding high fat diet, the weight of mice of hyperlipemia group increased significantly, while the weight of miceof the high and low fibrinolytic enzymes groups were lower compared with those of hyperlipemia group. Thisresult indicated that the fribrinolytic enzyme can accelerate the metabolism of fat, restrain the accumulation offat in the body. From the visceral index, damages of mice were not observed obviously in the high and lowfibrinolytic enzymes groups, compared with hyperlipemia group, which indicated that the enzymes can protectthe internal organs.