Study On Screening Of High-Yielding Fibrinolytic Enzyme Strains, Optimization Of Fermentation Conditions And Enzyme Properties

Thrombus is a kind of disease, which leads to tip top death rate in clinic for the moment. Fibrinolytic therapy is available. Current fibrinolytic enzymes available for clinical use are mostly plasminogen activators. Despite their widespread use, all these agents have undesired side effectsand are very expensive. Therefore, the searches for new fibrinolytic enzymes from various sources are being continued. Microbe has become an important source.The basal medium with skim milk was used as the first screening medium and the fibrin powder was used as the second screening medium. Strain ZLW-2 which had the highest fibrinolytic enzyme activity was isolated from a shamble in Baoding. In this process, a simpler, faster and more precise screening method was established. The fibrinolytic enzyme-producing organism was primarily identified as Bacillus sp. ZLW-2, indicating this strain had a high security. After 10 generations the enzymatic activity was about 165.82 U/mL, verifying that the enzyme production of this strain was stable.A fast, extension and feasible assay procedure of fibrinolytic enzyme activity was put forward. For the method of analysis of fibrinolytic activity, the fibrin plate method is most popular to internal scholars. The fibrin plate method is a most straightforward but long cycle method. Moreover, it has bad relativity result due to the different reagent that is used to preparing plate and the standard sample of urokinase. In our experiment, we use the improved ultraviolet spectrophotometric to measure fibrinolytic enzyme activity, which advance the accuracy. It is very important for increasing the screen efficiency of the production by fibrinolytic enzyme.The fermentation conditions of the Bacillus sp. ZLW-2, including carbon source, nitrogen source, inorganic salt, metal iron, surfactants and so on, were investigated in shake flask level. It was found that the concentrations of Starch soluble, Peptone, NaH₂PO₄·2H₂O, had the more obvious influences. The orthogonal test of the three factors showed that the optimized culture medium was: Starch soluble7.0 %, Peptone7.0%, NaH₂PO₄·2H₂O1.5 %, MgSO₄·7H₂O0.01%, CaCl₂10μmol/L. Through shake flask experiments, it was acquired that the best culture conditions: pH8.0, 32℃, 225r/min, 40 mL/300 mL, and 1%inoculum, for 52h. Under such conditions, the fibrinolytic enzyme activity reached 547.95 U/mL, which was 3.13 times of the initial strain.The test of crude enzyme indicated that the optimal reaction temperature of the Bacillus sp. ZLW-2 was 50℃and the enzyme was thermal lability, 11% of its activity was still remained after 60 min incubation at 70℃. The enzyme optimal pH for activity was pH 8.0. The test of stability in pH indicate that the Bacillus ZLW-2 was more stable in alkaline (pH 7.0～8.0)surrounding. After incubation at 4℃for 30 d, the remnant activity was 91% of its original activity. The crude enzyme activity was 83% of its original activity after incubation at -20℃for 30 d. Metal ions such as pb²⁺and Mn²⁺ inhibited enzyme activity slightly, whereas, Cu²⁺, Ca²⁺and Fe²⁺activated enzyme activity differently. The EDTA test testified that this fibrinolytic enzyme was not metalloproteinase.