Purification, Biochemical Characterization Research Of Douchi Fibrinolytic Enzyme And In Vitro And In Vivo Evaluation Of Thrombolytic Effect

In recent years, the morbility and mortality of thrombotic diseases has been increasing, and has become one of the most serious cardiovascular diseases that threat to human health. For the treatment of thrombosis, the most effective and conventional method is by dissolving fibrin-the major protein component of blood clots orally or intravenously to further cause the collapse of thrombus. However, new thrombolytic agents need to be developed for the various shortages of existing thrombolytic agents, such as:high cost, hemorrhage risk, short high-life and side effects.In this study, we investigated Douchi, a traditional Chinese fermented soybean food, from the following aspects:isolating and identifying a fibrinolytic enzyme-producing strain, optimization of liquid fermention, purifying enzyme and studying its biochemical and molecular structure, in vivo and in vitro evaluating anti-thrombolytic effect. We aimed to isolate a high-fibrinolytic enzyme-producing strain from Douchi and extract fibrinolytic enzyme with heat and alkaline tolerance, high specific activity, high specificity, little risk of hemorrhage, safe and low cost. All of the above will provide scientific evidence for developing new thrombolytic drugs.Part ⅠIsolation, Identification of Douchi Fibrinolytic Enzyme Producing Strain and Optimization of Liquid Fermentation Conditions Objective:This section aimed to isolate a high fibrinolytic enzyme producing strain from different Douchi samples. Then, its species was identified and the fermentation condition and culture medium was optimized to increase enzyme yield.Methods:The fibrin plate method was applied to screen fibrinolytic enzyme strains, then the strain which produced the most amount enzyme was chosed by comparing their enzyme activity. The strain was identified according to physio-biochemical characteristics. The optimal nitrogen and carbon source and fermentation conditions including temperature, primary pH, inoculate volume and medium volume were determined using single factor method. The key components of culture medium for enzyme production were screened by Plackett-Burman design and its concentration were further optimized by response surface method. The optimal culture medium was calculated according to the multiple regression equation that based on the above results. Cell growth and enzyme production curve were drawed following the optimal fermentation condition and culture medium.Results:Eight strains that produce fibrinolytic enzyme were screened, and the strain which produced the most enzyme was named XY-1and selected for further investigation. The enzyme produced by XY-1was name as DFE (Douchi Fibrinolytic Enzyme). Strain XY-1was identified as Bacillus amyloliquefaciens. The optimal fermentation condition were:temperature40℃, medium volume50ml/250ml, inoculate volume4%, primary pH8.0. the optimal nitrogen and carborn source were sucrose and peptone. The optimal culture medium were:peptone1.14%, sucrose0.5%, MgSO40.05%, NaCl0.1%. The actual maximal DFE yield was21.33FU/ml, equal to107.84%of the predicted maximal value, and had increased79.55%comparing to non-optimized culture medium。Intracellular enzyme activity was much higher than extracellular. The growth retardation period of XY-1was short, no more than4hours, then it came to exponential growth phase. After16hours, cell growth came to stationary phase. The production of DFE increased with the growth of bacteria, and reached the maximal value at24hour. Conclusion:The high-fibrinolytic enzyme-producing strain XY-1(Bacillus amyloliquefaciens) was isolated from Douchi. The optimal fermentation condition were:temperature40℃, medium volume50ml/250ml, inoculate volume4%, primary pH8.0. The optimal culture medium were:peptone1.14%, sucrose0.5%, MgSO40.05%, NaCl0.1%. The production of DFE reached the maximal value at24hour. Part IIExtraction and Purification of Douchi Fibrinolytic Enzyme and Its Biochemical CharacteristicsObjective:To research how to extract and purify DFE from the fermentation liquid of XY-1, and further investigate the biochemical characteristics of DFE.Methods:The purified DFE was acquired by ammonium sulfate segmenting salting-out, dialysis desalination and SP sepharose fast flow chromatography. The change of enzyme activity and protein concentration during the process was calculated. The effect of pH, temperature, metal ions and chemical inhibitors on enzyme activity and stability were evaluated. The fibrinolytic manner of DFE was identified according to the difference of dissolving circle on fibrin plate with or without plasminogen. Following the method of Lineweaver-Burk, the K's constant(Km, Vmax) of DFE was determined with fibrin as substrate. DFE was identified by HPLC-MS/MS, and the fragmented structure was compared with mass spectral information of NCBI non-redundant protein database.Results:The samples purified by above extraction steps were identified by SDS-PAGE, and only one band existing indicated it was pure with molecular weight of27000Da. DFE had been purified24.3-folds with recovery rate of19.9%and specific activity of47.3FU/mg. It was stable at pH7.0-10.0and the optimal pH was 9.0. DFE showed the highest activity at40℃and was stable below40℃. Ba2+and Co2+promoted DFE activity, while Pb2+, Fe3+and Hg+inhibited its activity. Ca2+and Cu2+inhibited its activity slightly, and Na+, K+and Mg2+didn't exhibit significant promotion and inhibition. EDTA and PMSF inhibited the enzyme activity completely, while P-mercaptoethanol almost had no effect. DFE could degrade fibrin directly, but not activate plasminogen first. Km and Vmax of DFE was6.98e-08mmol and232.5FU ml-1. The first15N-terminal amino acids of DFE was APALHSQGYTGSNVK, which is consistent with nattokinase.Conclusion:Pure DFE was acquired after steps of extraction methods. According to its biochemical property, the enzyme was identified as a serine protease. It could tolerate high temperature and alkaline and had high afinity with fibrin. Part ⅢIn vitro and vivo Anti-thrombolytic Evaluation of Douchi Fibrinolytic EnzymeObjective:To evaluate the thrombolytic effect of DFE by in vitro blood clot dissolving test and in vivo anti-thrombolytic activity on a rat thrombus model.Methods:DFE was purified following the methods in part II and prepared in different concentrations. Blood was drawed from a rat and clotted spontaneously. High, middle and low dose DFE solution, UK solution and physiology saline were added in blood clots, then the clots were incubated at37℃for16hours. The weight of blood clots before and after incubation were determined. S-D rats with tails more than15cm were selected for thrombus modeling. Agents for testing were injected through left caudal vein at first. After30min, the tails were ligated and lmg/ml κ-carrageenan were injected through right caudal vein. The tails were immersed in icy bath for1min, and after20min, the ligation was removed. The tail thrombosis length were measured6hours after κ-carrageenan were injected. Rat blood were collected from abdominal aorta under anesthesia and the plasma level of APTT, TT, PT and D-dimer were determined.Results:in normal saline group, the dissolution rate of blood clots was6.5%, while UK group dissolved about47.9%. In middle and high dose DFE group, the dissolution rate of blood clots increased significantly with a dose-response manner(59%vs.6.5%, p<0.01;71.7%vs.6.5%,p<0.01). Compared to negative control group(14.19±0.51cm), the average tail thrombosis length of UK group were significantly short(10.45±3.02cm,/p<0.01), so were the middle and high dose DFE group(10.25±3.24cm,/p<0.05;8.31±3.32cm,p<0.01). besides, the red swelling and inflammation degree of thrombosis in agents treated groups were also not obvious as negative control group. Only DFE middle group showed significant increase for APTT value(p<0.01). PT value in UK and DFE group were prolonged significantly. No differences were observed between groups for TT value. Compared with blank group, the level of D-dimer in negative control group increased significantly(p<0.01). In UK(p<0.05) and high dose DFE group(p<0.01), the D-dimer level were significantly higher than negeative control group.Conclusion:In vitro dissolving effect of DFE on blood clots was obviouse and it could also inhibit the formation of thrombus in vivo significantly.