The Bacillus Subtilis Produce Fibirnolytic Enzyme Got From Marine And Study On Its Fermentation?Enzyme Purification And Enzymatic Properties

At present,the research is focus on finding safe,strong specificity,high efficiency,cheap thrombolytic drugs.Especially the thrombolytic drugs obtained from microorganisms,It has many advantages,such as safety,small molecular weight and To be absorbed easily.The main job of mine was as follows:In the process of rejuvenation,A strain(rHQ-9)was obtained,its enzyme activity was 482U/ml,It was the same as before.They have the same colony morphology,such as yellowish or grey,corrugated on the surface,round,opaque,and wet in the middle of the colony.Through the microscope,it is found that they have the same cell morphology,So we can conclude:it is the Bacillus subtilis.In the process of extraction and purification.The optimum extraction conditions are determined through experiments.The fibrinolytic enzyme was purified by ammonium sulfate fractionation,alkali and dialysis treatment,ion exchange,gel filtration.The activity of enzyme is 9561U/mg,and the recovery rate is 15%.Its molecular weight is 26012D and it is a single chain protein by SDS-PAGE and gel column detection.It was identified by the 4700 plus MALDI TOF/TOF Analyzer,we concluded that this fibrinolytic enzyme is a new finding and it can dissolve the blood clots by dissolving fibrin.This fibrinolytic enzyme is sensitive to temperature,when the temperature is higher than 45?,It will inactivate quickly.It's optimum temperature is between in 45 °C tO 50?.Enzymatic reaction temperature should be located in about 35 °C.This fibrinolytic enzyme stability of the pH is in the range of 6.0?11.0,It will be more stability when pH is greater than 9.0.The optimal pH of reaction is 8.0.PMSF and EDTA(protease inhibitor)can reduce the activity of fibrinolytic enzyme obviously,And the higher the concentration,the stronger the inhibitions.It shows that the fibrinolytic enzyme is a kind of serine protease,it's active center depends on metal ions,It belongs to the metal protease.C1u2+?Zn2+?Fe3+can inhibit the activity of fibrinolytic enzyme,especially the high concentration of Cu2+,Fe3+ can fully inhibit its activity.On the contrary,Mg2+?Ca2+and the low concentration of Mn2+can activate its activity,but the higher concentration of Mn2+ have a little inhibition to the fibrinolytic enzyme.Through the study,the fibrinolytic enzyme is not kinase,it can degrade directly the fibrin.The SDA-PAGE indicates that fibrinolytic enzyme can hydrolyze?-chain of fibrin rapidly,next is ?-chain,the hydrolysis rare of y-y-chain is the slowest.In the security experiments,It is found that the fibrinolytic enzyme is safe,It would not cause hemolytic phenomena.