Mutagenesis of Indigenous Streptococcus equisimilis isolates for Enhanced Production of Streptokinase

Abstract

Acute myocardial infarction and ischemic stroke are caused by thrombosis or the obstruction of blood vessels with clots and this is the leading causes of death. The single handling accessible is the use of thrombolytic agent to liquefy the blood lump. Hyper production of streptokinase was carried out in this research work. Beta hemolytic Streptococcus equisimilis was isolated from indigenous sources and then mutagenesis of this isolate was carried out by means of chemicals as well as radiations. To seek the optimum activity, different kinetic and thermodynamic parameters like pH, temperature, Km, Vmax, molecular weight, melting temperature, half-life, enthalpy and entropy etc. were applied on the purified enzyme. UV irradiated strain resulted in 335 U/mL activity with 1116.66 U/mg specific activity, 0.30 mg/mL protein, 41.92 fold purification and 69.79% recovery whereas Sodium azide derived mutant resulted in 400 U/mL activity with 2000 U/mg specific activity, 0.20 mg/mL protein, 71.94 fold purification and 64.51% recovery of the finally purified enzyme. Gamma irradiated strain exhibited 300 U/mL activity with 1428.57 U/mg specific activity, 0.21 mg/mL protein, 59.52 fold purification and 75.94% recovery whereas ethidium bromide derived mutant showed 365 U/mL activity with 1659.09 U/mg specific activity, 0.22 mg/mL protein, 66.52 fold purification and 85.08% recovery. Optimum pH and temperature of the finally purified enzyme was 7 and 45oC. Enthalpy of denaturation (ΔH*) of streptokinase at 450C was 43.67 kJ/mole. The Energy of thermal denaturation ΔG* was 101.14 kJ/mole and entropy of inactivation ΔS* was -197.32 kJ/mole at 45oC. The negative value of ΔS* indicated that streptokinase was thermodynamically stable. Km and Vmax values of streptokinase were 26.31 mM and 50 MS-1. Streptokinase produced from sodium azide derived mutant exhibited activity within the pH range of 6 to 8 while it presented its best performance at pH 7. Thermal stability between 45oC to 80oC was shown by the streptokinase along with half-life of 244 minutes while less stability was shown at 80oC along with 45 minutes of half-life and 40.41 kJ/mole as enthalpy of denaturation (ΔH*).