Characterization of Fibrinolytic Enzymes Produced by the Halophilic Streptomyces Flaveolus and Streptomyces Galtieri Isolated from Wady El-Natron Area in North Egypt

Abstract
Background: Intravascular thrombosis is responsible for an increasing number of deaths every year. Lung blood clots in USA alone affect 1,000,000 patients annually. Clots formed from insoluble fibrin restrict the smooth flow of blood in blood vessels, leading to thrombosis and heart attacks. World-wide, over 29% of the total mortalities are due to thrombosis, thus antithrombotic therapy is of great interest. The three fibrinolytic (thrombolytic) agents that are currently being used for this purpose are urokinase, streptokinase and genet-ically engineered tissue-type Plasminogen Activator (t-PA). However, these enzymes are expensive, thermolabile, with low specificity and stability, large therapeutic doses and can produce undesirable side effects such as gastrointestinal bleeding, allergic reactions, and resistance to repercussion. This warrants the search for novel fibrinolytic enzymes from various sources with higher efficacy, safety, specificity and stability and preferably those direct acting activities. Though new fibrinolytic enzymes are being explored from microbes, microorganisms remain the preferred source due to their biochemical versatility, feasibility of mass culture and ease of genetic manipulation. Hence, many fibrinolytic enzymes have been isolated from a variety of microorganisms, including actinomycetes, bacteria, fungi, and algae.
Aim of Study: To detect the characterization of fibrinolytic enzymes produced by the halophilic Streptomyces flaveolus and Streptomyces galtieri that were isolated from soil of Wady El-Natron region, in North Egypt, as a potential thrombolytic agent.
Material and Methods: Fibrinolytic enzymes, extracted from Streptomyces flaveolus and Streptomyces galtieri, were purified by ammonium sulfate precipitation and gel filtration. By using SDS-Page electrophoresis, determining their molec-ular masses, classified by inhibitory acting materials, charac-terized by determining metal ions influences, anticoagulation clotting time delay with CaCl2, their proteolytic activity in units/mg, the active conc. in μg/ml and the least conc. in μl/ml of thrombolytic activity.
Results: The fibrinolytic enzymes extracted from both Streptomyces flaveolus and Streptomyces galtieri are classified as metallo-protease enzymes, their molecular masses were 1 6kDa and 4 1kDa, their proteolytic activity are 1.4 and 2.3 units/mg of proteins, the anticoagulation clotting time assay showed 20 and 15min delay in clotting time with CaCl2, the active conc. of each enzyme was determined from the standard curve using streptokinase as standard fibrinolytic enzyme as 200 and 300μg/ml, and the least conc. of enzyme thrombolytic activity was measured by performing the modified Holmstrom method, as the crude thrombolytic activity were at concentra-tions of 80-100μl, whereas, precipitated enzymes, showed thrombolytic activity at concentrations 20μl and 30μl/ml respectively. Metal ions exhibited different influences on the fibrinolytic enzyme activity produced by Streptomyces flave-olus, such as Ca2+, Cu2+ and Fe2+ promoted the activity of the enzyme, whereas Mg2+ is strongly promoted it. But on the fibrinolytic enzyme activity produced by Streptomyces galtieri, Cu2+ promoted the activity of the enzyme, whereas Ca2+, partially inhibit the enzyme, but the Fe2+ and Mg2+ were strongly and completely inhibit it.
Conclusion: These results suggest that the extracted enzymes from both isolated Streptomyces flaveolus and Streptomyces galtieri are with potent and potential applications as thrombolytic agent.