IMPROVED STREPTOKINASE PRODUCTION

Abstract

Several strains of beta-hemolytic Streptococci produce streptokinase enzymethat can bind and activate human plasminogen to plasmin. Streptokinase degrades the fibrinlump by its explicit lysine joining site and so it is applied as a remedy in thrombolytic therapy.The purpose of the study was to subject wild strain of Streptococcus equisimilis to straindevelopment technique, using random mutagenesis by UV irradiation for enhanced productionof streptokinase. Objective: To evaluate the hyper production of streptokinase after mutagenesisof wild Streptococcus equisimilis by means of UV irradiation. Study Design: Randomized study.Period: 2012-2014. Setting: Enzyme Biotechnology Laboratory, Department of Biochemistry,University of Agriculture, Faisalabad-Pakistan. Materials and Methods: UV lamp (TUP 40wlamp which has about 90% of its radiation at 2540-2550 A0) was used for the mutation ofStreptococcus equisimilis cells (1x 107 cells mL-1) for enhanced production of streptokinase. 10mL fresh inoculum was transferred to sterile petri plates, which were exposed to UV light for 30,60, 90, 120, 150, 180, 210, 240 and 270 minutes. The exposure was carried out at distance of20cm from the centre of lamp. A dose producing 87% killing was selected as optimum dose,after preparing kill curve. The kill/ survival curve was prepared and time of exposure giving(210 minutes) 3 log kill was selected for mutation of the Streptococcus equisimilis for hyperproduction of streptokinase enzyme. Results: Enzyme assay was performed for both wildand mutant strains. Dose of 210 minutes was selected as best dose which was followed bythe selection using triton X-100. Finally the selected strain S. equisimilis EBL-UV-210 showed480 U mL-1 of streptokinase activity in quantitative blood clot liquefaction test, which is quitehigher than wild strain (370 U mL-1). This maximum yield of streptokinase was obtained after24h, at CSL 4%, pH 7.5, 37oC, KH2PO4 0.04%, K2HPO4 0.05%, MgSO4. 7H2O 0.04%, NaHCO30.15%, CaCO3 0.004%, CH3COONa. 3H2O 0.10%, FeSO4. 7H2O 0.04%, MnCl2. 4H2O 0.02%,glucose 2%, yeast extract 3% and 5% inoculum size in liquid state fermentation. Conclusions:Results showed that mutated strain gave enhanced streptokinase activity in comparison tothe wild strain. Our current study focused on streptokinase production from this UV mutatedstreptococcus equisimilis species and purification of this enzyme by ammonium sulfateprecipitation, Ion exchange and gel filtration chromatography. The activity of streptokinase wasdetermined by using quantitative blood clot liquefaction method.