UV-breeding Of Fibrinolytic Enzyme-producing Strain And The Thrombolytic Effect Of Fibrinolytic Enzyme

In clinical,thrombolytic drugs are widely used to treat cardiovascular diseases.However,the existing thrombolytic drugs all have some disadvantages.So,it is very essential to exploit a effective,inexpensive and safe fibrinolytic enzyme.In this work,the Bacillus subtilis FA-7 from marine was mutagenesis with UV and a stable mutant strain named Y-22 with high yield of fibrinolytic enzyme was obtained by plate screening,fibrinogen plate re-screening and flask fermentation re-screening.The activity of fibrinolytic enzyme produced by the strain Y-22 was 465 ± 9.7 IU/mL,which was as 1.56 times as the original strain.Fermentation conditions for the strain Y-22 were researched in liquid fermentation.The optimized compositions of fermentation medium and fermentation conditions were as follows:soluble starch 4%,Soybean meal 2.5%,KH2PO4 0.1%,K2HPO4 0.3%,CaCl2 0.025%,MgS04-7H20 0.35%,Tween 80 0.15%,seed age 18 h,inoculating amount 4%,liquid volume 50 mL/250 mL(flask),the initial pH 5.5,temperature 30 ?,180 r/min cultured for 96 h.Under these conditions,the activity of fibrinolytic enzyme could reach 926 ±14.97 IU/mL,which was as 1.99 times as before.The sedimentation effects of ammonium sulfate precipitation and ethanol precipitation on fibrinolytic enzyme was studied.The fibrinolytic enzyme activity recovery and the purification factor was 84.67%and 1.73 times by ammonium sulfate precipitation,compared with 87.98%and 1.88 times by ethanol precipitation.The research of thrombolytic effect of fibrinolytic enzyme showed that this enzyme was not a kinase,it could directly degrade the fibrin.When the fibrinolytic enzyme was added to the fresh clots along with 32 h,it showed 81.02%clot lysis.And this enzyme had a longer potent duration.When blood clots were stored in 4? refrigerator for a month,plasminogen showed 90.92%clot lysis of fresh clot.And it showed 86.94%clot lysis of fresh lot when clots were stored for two months.This fibrinolytic enzyme was destroyed seriously under the action of pepsin,but it was stable in the intestinal environment.The enzyme also had a good effect of anticoagulation and could not destroy blood cells.