Characterization and Stability Studies on Streptokinase Produced from Streptococcus equinus VIT_VB2 Isolated from Bovine Milk
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Published on: November 4, 2016
B Vaishnavi1, V Mohanasrinivasan1, M Kavin Raja1
1Department of Biotechnology, School of Bio Sciences and Technology, Vellore Institute of Technology, Vellore, 632014, Tamil Nadu, India.
Introduction:
Streptokinase (SK) is a fibrinolytic enzyme with significant clinical applications, particularly in thrombolytic therapy for treating Acute Myocardial Infarction (AMI). The therapeutic potential of SK has prompted extensive research into its biochemical properties, stability, and catalytic efficiency to optimize its use in medical applications. The current study focuses on the characterization and stability of purified streptokinase produced from Streptococcus equinus VIT_VB2 isolated from bovine milk.
Methods:
The effects of pH, temperature, metal ions, organic solvents, surfactants, and inhibitors on the activity and stability of the purified enzyme were evaluated. Purified streptokinase was characterized by fibrin zymography, silver-stained SDS-PAGE, FTIR, and MALDI-TOF.
Results:
The optimum pH and temperature for Streptokinase (SK) activity were 6.0-6.5 and 20°C, respectively. The enzyme retained more than half of its maximal activity between 0°C and 40°C, with peak activity at 20°C and a sharp decline above 60°C. The enzyme showed maximum activity in the presence of CaCl2, Fe2+, and Mn2+ cations. SK activity was inhibited by the tested surfactants and by PMSF and EDTA. The enzyme retained its activity in isoamyl alcohol, whereas activity decreased in all other tested organic solvents. Fibrin zymography showed a clear lytic band at 47 kDa, confirming the fibrinolytic activity of the enzyme. Silver-stained SDS-PAGE showed a single band with a molecular size of 47 kDa, which confirmed the fibrin zymography result. FTIR analysis identified spectral bands consistent with the expected secondary structure of the protein. MALDI-TOF analysis confirmed the expected molecular weight of the protein at 47.4 kDa with three distinct peaks.
Discussion:
SK from S. equinus VIT_VB2 is stable under various physicochemical conditions. The thermal stability of the enzyme at low temperatures, along with its tolerance to isoamyl alcohol, could offer benefits for cold-temperature storage and innovative formulation approaches, especially in low-resource areas. The complete loss of activity in the presence of PMSF and EDTA reflects the assay's dependence on downstream plasmin generation and on divalent-cation cofactors, respectively, rather than an intrinsic catalytic triad in SK itself.
Conclusion:
The streptokinase produced from Streptococcus equinus VIT_VB2 retained high activity under optimal pH and temperature conditions and substantial activity across broader physicochemical ranges. The findings provide preliminary insights into the biochemical and physicochemical properties of the SK produced from Streptococcus equinus VIT_VB2.
