IMPROVED STREPTOKINASE PRODUCTION

UV IRRADIATION OF STREPTOCOCCUS EQUISIMILIS

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. Objective: To evaluate the hyper production of streptokinase after mutagenesis
of 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 40w
lamp which has about 90% of its radiation at 2540-2550 A0) was used for the mutation of
Streptococcus equisimilis cells (1x 107 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. Results: 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, 37oC, KH2PO4 0.04%, K2HPO4 0.05%, MgSO4. 7H2O 0.04%, NaHCO3
0.15%, 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 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.