Abstract
The highly fibrinolytic enzyme-producing bacterium was identified as Bacillus subtilis DC27 and isolated from Douchi, a traditional fermented soybean food. The DFE27 enzyme was purified from the fermentation broth of B. subtilis DC27 by using UNOsphere Q column chromatography, Sephadex G-75 gel filtration, and high-performance liquid chromatography. It was 29 kDa in molecular mass and showed the optimal reaction temperature and pH value of 45 °C and 7.0, respectively, with a stable fibrinolytic activity below 50 °C and within the pH range of 6.0 to 10.0. DFE27 was identified as a serine protease due to its complete inhibition by phenylmethysulfony fluoride. The first 24 amino acid residues of the N-terminal sequence of the enzyme were AQSVPYGVSQIKAPALHSQGFTGS. 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. 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.
Highlights
Cardiovascular diseases (CVDs) are dominant causes of death worldwide for people with hypertension, myocardial infarct, coronary heart disease, or stenocardia[1]
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 results showed that the strain DC27 was Gram positive, rod shaped and endospore forming, with a typical characteristic of B. subtilis
Summary
Cardiovascular diseases (CVDs) are dominant causes of death worldwide for people with hypertension, myocardial infarct, coronary heart disease, or stenocardia[1]. The fibrinolytic system can dissolve fibrin clots resulting from fibrinogen through activated thrombin (EC 3.4.21.5), and provide a channel for blood flow at the site of vascular injury as hemostatic response[6,7]. Microorganisms contribute to the production and use of the fibrinolytic enzymes since ancient times, and researchers have paid www.nature.com/scientificreports/. Www.nature.com/scientificreports increasing attention to the production of fibrinolytic enzymes by microorganisms, those from the food sources. It has been confirmed that Nattokinase (NK) can directly lyse thrombi in vivo and its oral administration can improve plasma fibrinolytic activity as well as t-PA production[22,23,24]. Similar fibrinolytic enzymes from Douchi were confirmed to degrade fibrin directly and efficiently in vitro and in vivo[5,25]. A novel enzyme with potential fibrinolytic activity was isolated, purified and characterized from the bacterial strain B. subtilis DC27 in Douchi, a fermented soybean food popular in China since ancient times
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