The most effective drugs for treating thrombosis are fibrinolytic enzymes, which can be divided into two types according to their action mode: plasminogen activators (PAs), including tissue plasminogen activator (t-PA), streptokinase (SK, 3.4.99.22) and urokinase (UK, EC 3.4.21.31), which can lyse fibrin through the formation of plasmin from plasminogen 9, 10, and plasmin-like fibrinolytic enzymes, which can degrade the fibrin directly 2. The clots which are not hydrolyzed lead to the occurrence of thrombosis under unstable conditions like myocardial infraction or disorders such as hypertension 8. Additionally, the plasmin also hydrolyzes fibrin clots at the site of injury (EC 3.4.21.7) to prevent thrombosis 2. The fibrinolytic system can not only dissolve fibrin clots resulting from fibrinogen through activated thrombin (EC 3.4.21.5), but also provide a channel for blood flow at the site of vascular injury as hemostatic response 6, 7.
In the various types of CVDs, thrombosis is among the most commonly occurring diseases of modern life and could be responsible for the increasing number of deaths 4, 5. Consistent with the estimation of World Health Organization(WHO), CVDs caused 15.6 million deaths in 2010 worldwide and are expected to cause more than 20 million deaths per year by 2020 2, 3. Overall, the DFE27 enzyme was obviously different from other known fibrinolytic enzymes in the optimum substrate specificity or fibrinolytic action mode, suggesting that it is a novel fibrinolytic enzyme and may have potential applications in the treatment and prevention of thrombosis.Ĭurrently, cardiovascular diseases (CVDs) are dominant causes of death worldwide for people with hypertension, myocardial infarct, coronary heart disease, or stenocardia 1. The enzyme displayed the highest specificity toward the substrate D-Val-Leu-Lys-pNA for plasmin and it could not only directly degrade but also hydrolyze fibrin by activating plasminogen into plasmin. The first 24 amino acid residues of the N-terminal sequence of the enzyme were AQSVPYGVSQIKAPALHSQGFTGS. DFE27 was identified as a serine protease due to its complete inhibition by phenylmethysulfony fluoride. It was 29 kDa in molecular mass and showed the optimal reaction temperature and pH value of 45 ☌ and 7.0, respectively, with a stable fibrinolytic activity below 50 ☌ and within the pH range of 6.0 to 10.0. subtilis DC27 by using UNOsphere Q column chromatography, Sephadex G-75 gel filtration, and high-performance liquid chromatography. The DFE27 enzyme was purified from the fermentation broth of B.
The highly fibrinolytic enzyme-producing bacterium was identified as Bacillus subtilis DC27 and isolated from Douchi, a traditional fermented soybean food.
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