| Along with the population aging phenomenon becomes more and more serious,older recurrent disease is extremely common in thromboembolic disease. This diseaseis currently one of the causes of mortality rate.The principal means of treatment ofthis kind of disease is thrombolysis therapy. Fibrinolytic enzyme is primarily the mainingredient in catalytic thrombus fibrous protein hydrolysis, thus achieve the purposeof dissolving thrombus. Fibrinolytic enzyme can be derived from animals, plants,microorganisms, and microorganism is the main source of fibrinolytic enzyme.Bacillus subtilis is the earliest bacteria that humans found. Because of its variousgrowing environment, its unpathogenic, the endurance of high temperature and highheat, the strong resistance of electromagnetic radiation and ultraviolet ray, it can usevarious nutrients, and can directly to produce many proteins into the medium, produceall kinds of enzymes. It always is a hot spot in the biological world.In this paper, we optimized the fermentation culture medium composition andthe fermentation conditions of the strains of Bacillus subtilis in our laboratory. Andthen we purified the enzyme in the fermentation liquor. We found the Bacillussubtilis-protease has fibrinolytic activity. We studied some physical and chemicalproperties, and the characteristics of in vitro thrombolysis of the enzyme. It laid thefoundation for the follow-up work.1. In order to improve the yield and the activity of the Bacillus subtilis-protease, weused the one-factor-at-a-time methodology design. Through shake flask culture ofbacillus subtilis, the medium and condition of fermentation are optimized. The resultsshows that the optimized medium (w/v) as follows: maltose1%, peptone2%,0.05%MgSO47H2O,0.02%CaCl2, KH2PO40.4%; and optimal cultivation conditions weresettled as follows: initial pH was7and incubated at the temperature of37℃for48h.2. We ferment the Bacillus subtilis according to the optimal fermentation medium described above. Then we centrifuge the fermented liquid of4℃,4000RPM for20min. The enzyme is prepared from the fermented broth by adding the ammoniumsulfate to the fermentation supernatant of60%saturation and then dialysis into water.Then we get the crude enzyme powder after freeze drying. Next we use the CM-sepharose Fast Flow to purificate Bacillus subtilis-protease. The purified enzymemoves as a single electrophoretic band in SDS-PAGE and the molecular weight is lessthan14.3kDa. And it has fibrinolytic enzyme activity.3. In order to know the properties of Bacillus subtilis-protease, temperature, pH,metal ions were studied on the influence of its stability. The results showed thatoptimum reaction temperature、temperature stability range、 optimum pH and pHstability range of3bacillus subtilis-protease were60-70℃, below40℃,7and6-8,and we didn’t find metal ion which can enhance its activity.4. The thrombolysis characteristics in vitro of Bacillus subtilis-protease werestudied. The results showed that the protease from Bacillus subtilis in vitro has a goodability of thrombolysis. And red blood cells were morphologically intact and did notcause hemolysis in the thrombolysis process. The enzyme has a concentrationdependence, it has a strong ability of fibrin decomposition, and it has the ability ofdecomposition fibrin directly.In this paper, it is of great significance to research the separation and purificationof Bacillus subtilis-protease with fibrinolytic and thrombolysis ability in vitro. Thespecific properties and the thrombolysis ability of in vivo will be the next experiment.That lays the foundation for the study of clinical thrombolytic medicine. |
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