Screening And Identification Of Fibrinolytic Enzyme Producing Strain And Studies On Its Liquid Fermentation Conditions

The diseases related with thrombus formation are damaging human's health and life severely. Thrombolytic therapy has been thought as the effective alternative. In present, the thrombolytic medicines such as urokinase , streptokinase and tissue plasminogen activator have been widely used in the clinical therapy, but these enzymes exist a range of disadvantages such as short circulating half life , potential bleeding tendency and high cost. Therefore, it has enormous social and economic value to find a novel thrombolytic medicine that is safer, more effective and cheaper. Microorganism is a kind of important resource that can produce fibrinolytic enzymes. Besides streptokinase discovered from Streptococcus hemolyticus and staphylokinase discovered from Staphulococcus aureus in the beginning, many strains such as Actinomyces thermovulgaris, Streptomyces megasporus and Bacillus subtilis that show fibrinolytic activity have been discovered in recent years. Fibrinolytic enzymes produced by means of microorganism technology have many advantages such as short cycle time, simple procedure, low cost and high production, so it stands for a tendency to develop the biological medicine. Because the ability of producing fibrinolytic enzyme from different strain is different each other, acquiring new fibrinolytic agents through screening of novel strains with the highest fibrinolytic activity or changing of former strains by means of genetic engineering is the main task in the future , and that is , the aim of our study.Crude fibrinolytic enzyme was extracted from fermented soybean with saline in our study. The strains C1A-01 and C1A-03 with strong fibrinolytic activities were screened successfully by the fibrin plate method and identified as Bacillussubtitis by their morphological, physiological and biological characters. The fibrinolytic activities of the supernatant by ClA-01 and ClA-03 solid fementation are 378 urokinase units per milliliter and 324 urokinase units per milliliter. Because the fibrinolytic activity of the supernatant is higher than activity of bacterium lysate, we concluded that the fibrinolytic enzyme is extracellular product.Except the effect of the strain itself, the effect of fermentation conditions is very important on the activity of the fibrinolytic enzyme from microorganism. According to the optimization of liquid fermentation, the optimal fermentation conditions for ClA-01 producing fibrinolytic enzyme were determined and the results are as follows. The best nitrogen source is soya flour, the concentration is 6%;the best carbon source is glucose, the concentration is 2%;the bacteria grew fast under pH9.0, but the most suitable pH is 7.0 for the production of the fibrinolytic enzyme;the optimum content of inoculation is 10%;the optimal fermentation time is 72h. We choosed the cultivation temperature to be 37°C , the shaking speed to be 150rpm, when 500ml flask contains 100ml. Under these conditions, the activity of the fibrinolytic enzyme can reach 394 urokinase units per milliliter broth.Both anti-thrombotic substances and thrombolytic agents take effect by interfering some procedure of thrombus formation. The essence of thrombolytic therapy is that the fibrinolytic enzyme digests fibrin to soluble degradation products, which is accomplished by hydrolyzing fibrin directly and by activating plasminogen indirectly. At present, the thrombolytic agents such as urokinase and streptokinase take effect by activating plasminogen indirectly. To study the fibrinolytic mechanism of fibrinolytic enzyme from ClA-01 , we heated up the fibrin plate in order to inactivate plasminogen mixed in purchased fibrinogenreagent. The plasminogen activator can not show fibrinolytic activity, but the fibrinolytic enzyme hydrolyzing fibrin directly can show the activity on the pyrogenetic fibrin plate. By means of this method, the fibrinolytic mechanism of the enzyme can be investigated. The results is as follows: urokinase had no dissolving circle on the pyrogenetic fibrin plate, wheareas it possesed fibrinolytic activity on the fibrin plate mixed with plasminogen, which showed urokinase take effect by activating plasminogen;the fibrinolytic enzymes from C1A-01 and C1A-03 formed dissolving circle of the same diameter on the heated fibrin plate and on the fibrin plate, which showed they lysated thromobus by hydrolyzing fibrin directly. Since they did not activate plasminogen in the thrombolytic way, it maybe have less potential for bleeding than the present thrombolytic medicines and its safety have been elevated.The thrombolytic experiment of crude fibrinolytic enzyme in vitro showed that fibrinolytic enzyme from C1A-01 could prolong thrombus forming time and decrease the formation of thrombus, which indicated that it has anticoagulant effect;Moreover, it could dissolve the thrombus in a short time and keep this fibrin state for more than eight hours, which indicated it has a strong thrombolytic effect and longer circulating half life.The criteria to evaluate thrombolytic drug is primarily as follows: 1) High fibrinolytic ability. Ideal thrombolytic agent should dissolve new and old thrombus quickly and completely, decrease the reocclusion of vessel. 2) High fibrin-specific. The drug can hydrate fibrin directly in clots. 3) Immunity effect problem. The medicine can not induce allergic reaction and maintain fibrinolytic activity without antigen. 4) Long circulating half life. Thrombolytic agent is not easy to be cleared or hydrated and its activity can not be decreased by inhibitors. 5) Less side-effect. The drug should not lead to sympotom such as bleeding andhypotension. In addition, it can not affect the normal coagulation ability of body. The fibrinolytic enzyme from C1A-01 found in our study posses strong fibrinolytic activity;Furthmore, it can dissolve fibrin in specific way, hydrate thrombus directly and inactivate the plasminogen pathway in vivo, so the enzyme has high safety and less side-effect;it has strong anticoagulant and thrombolytic effect with longer circulating half life;it can be produced on a large scale by bacteria fermentation, acquired in quantity in a short time and extracted easily with low costs. From the characters, The fibrinolytic enzyme from C1A-01 is represented as an ideal medicine candidate for prevention and treatment of thrombus.