ABSTRACT
Several strains of beta-hemolytic Streptococci produce streptokinase enzyme that can bind and activate human plasminogen to plasmin. Streptokinase degrades the fibrin lump 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 strain development technique, using random mutagenesis by UV irradiation for enhanced production of streptokinase. To evaluate the hyper production of streptokinase after mutagenesis of wild Streptococcus equisimilis by means of UV irradiation. Randomized study. 2012-2014. Enzyme Biotechnology Laboratory, Department of Biochemistry, University of Agriculture, Faisalabad-Pakistan. UV lamp [TUP 40w lamp which has about 90% of its radiation at 2540-2550 A[0]] was used for the mutation of Streptococcus equisimilis cells [1x 10[7] cells mL[-1]] for enhanced production of streptokinase. 10 mL 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 of 20cm 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 hyper production of streptokinase enzyme. Enzyme assay was performed for both wild and mutant strains. Dose of 210 minutes was selected as best dose which was followed by the selection using triton X-100. Finally the selected strain S. equisimilis EBL-UV-210 showed 480 U mL[-1] of streptokinase activity in quantitative blood clot liquefaction test, which is quite higher than wild strain [370 U mL[-1]]. This maximum yield of streptokinase was obtained after 24h, at CSL 4%, pH 7.5, 37[degree sign] C, KH[2]PO[4] 0.04%, K[2]HPO[4] 0.05%, MgSO[4]. 7H[2]O 0.04%, NaHCO[3] 0.15%, CaCO[3] 0.004%, CH[3]COONa. CH[3]CO 0.10%, FeSO[4]. 7H[2]O 0.04%, MnCl[2]. 4H[2]O 0.02%, glucose 2%, yeast extract 3% and 5% inoculum size in liquid state fermentation. Results showed that mutated strain gave enhanced streptokinase activity in comparison to the wild strain. Our current study focused on streptokinase production from this UV mutated streptococcus equisimilis species and purification of this enzyme by ammonium sulfate precipitation, Ion exchange and gel filtration chromatography. The activity of streptokinase was determined by using quantitative blood clot liquefaction method