Purification And Fibrinolytic Activity Analysis For The Production Of Fibrinolytic Enzyme By Strain Bacillus Aerius S-7

Thrombotic disease is a seriously harmful disease, which severely impaired human body and health, especially the old ones. It contains acute myocardial infarction, cerebral thrombosis, type embolization, arterial thrombus and ischemic shock etc, in which the mortality of acute myocardial infarction attains up to 30%. In recent years, the research of thrombolysis drug mainly concentrated in two aspects, one is screening of new sources to thrombolysis drug, and the other is reforming existing thrombolysis drug through genetic engineering and monoclonal antibodies technology, aiming to reduce the antigenicity of fibrinolytic enzyme, and enhance its security as well as vitality. Most of efficient thrombolytics were discovered from organisms, which are the main source. There are many advantages in microorganism originated fibrinolytic enzyme, as a result it makes up one of the most primary sources. For a multiple of fibrinolytic activity strain have been isolated so far, consequently international researchers paid a highly attention on it.A higher fibrinolytic activity strain S-7 was screened from the fermented soybean through fibrin plate method. We studied the S-7 stain by strain identification and optimization of liquid fermentation conditions, and purified the fibrinolytic enzyme from fermentation broth of S-7 stain, discussed the characterization of enzyme, as well as thrombolysis characteristics in vitro and security, The results were as follows:The morphological, physiological, biochemical characteristics and 16S rDNA sequence analysis of the stain demonstrated that these characteristics of S-7 stain are similar to those of Bacillus aerius, and got a 99.60% identity with Bacillus aerius through the nucleotide sequences test. The results suggested that S-7 strain as a kind of Bacillus aerius.The flask fermentation conditions of Bacillus aerius S-7 was studied by single factor experiments and orthogonal experiments. We made a conclution that the optimal components of fermentation culture medium were as follows:corn flour 5 g/L, glucose 5 g/L, soybean powder 20 g/L, K2HPO4·12H₂O 2 g/L, KH₂PO4·2H₂O 1g/L, MgSO4·7H₂O 1.5 g/L, CaCl2·2H₂O 0.5 g/L,NaCl 1 g/L, Besides, in 250 mL sharking flask the optimum fermentation conditions were:fermentation time is 48 hours, medUm initial pH is 7.5, inoculation volume2%, medUm volume 30 mL, temperature 37℃, rotate speed 200 r/min. The activity of fibrinolytic enzyme （as urokinas） reached to 942.09±4.18 U/mL, which increased 4.7 times than the initial fermentation conditions.The fibrinolytic enzyme from strain Bacillus aerius S-7 was extracted by the ammonium sulfate fractional precipitation, dialysis and DEAE-Sepharose Fast Flow anion exchange method. A single protein fraction with fibrinolytic activity was obtained, whose relative molecularweight of purified protein was 55 kDa. The N-terminal amino acid sequence of the S-7FE-1 were ANLNGTLGGM, which appeared no homology from other micro organism that have been reported, that means it is a new type of fibrinolytic enzyme. The optimum temperature for S-7FE-1 is 30℃,pH at 8.0, and the activity of this fibrinolytic enzyme showed a stabilities when the pH at 4 to 9, and temperature at 4 to 50℃. Fe3+ could make the S-7FE-1 completely inactivated, however the other ions showed on significant difference. The EDTA has a obvious inhibiting effect on it, as well, the PMSF put up a absolutely suppression activity, that demonstrate this fibrinolytic enzyme could actually be a kind of serine proteases contains metallic ion.Thrombolysis characteristics in vitro and safety test indicated that S-7FE-1 did have a favorable effect on thrombolysis and anticoagulation, without hemolysis phenomenon; what’s more it could not cause bleeding allergic reaction in mouse. A preliminary conclusion would be made as the S-7FE-1 is safty.