RESEARCH ARTICLE Strain improvement, optimization and puri fication studies for enhanced production of streptokinase from Streptococcus uberis TNA-M1 Teetam Ghosal, Nikita Augustine, Ashwini Siddapur, Vaishnavi Babu, Merlyn Keziah Samuel, Subathra Devi Chandrasekaran (✉) School of Biosciences and Technology, VIT University Vellore-632014, TN, India © Higher Education Press and Springer-Verlag Berlin Heidelberg 2017 BACKGROUND: Screening of isolates for their potency to produce streptokinase was an important criterion of this research. The current study emphasizes the strain improvement, optimization and purification studies for enhanced production of streptokinase from Streptococcus uberis TNA-M1 isolated from bovine milk. METHODS: The study was carried out on samples collected from milk sample. Primary screening and characterization is used as an excellent source for the isolation of β-hemolytic organisms. Strain improvement was done by both physical & chemical mutagenesis. The enzyme activity was checked by clot lysis assay and confirmed by fibrin plate method. The partially purified and crude enzyme were analysed by high-performance liquid chromatography. Molecular weight & enzyme purity was checked by SDS –PAGE, further confirmed by fibrin zymography. RESULTS: Out of the 3 isolated strains, only one isolate expressed β-haemolysis with streptokinase (SK) activity. Based on the results of radial caseinolytic assay and blood clot dissolving assay, isolate TNA-M1 demonstrated the highest streptokinase activity. Based on morphological , biochemical and molecular characterization, it was identified as Streptococcus uberis and the strain was named as Streptococcus uberis TNA-M1. The results indicated that ultra-violet (UV) and ethyl methane sulfonate (EMS) were effective mutagenic agents for strain improvement of Streptococcus uberis TNA-M1 and enhanced SK productivity. HPLC analysis was performed in order to confirm the presence of streptokinase with the similar retention time (0.875 min) with its standard (0.854) min. SDS-PAGE of the enzyme showed protein band of approximately 47 kDa and confirmed by fibrin zymography. It exhibited fibrinolytic activity, which was more potent than other fibrinolytic enzymes. Glucose and peptone were recorded to be the optimum carbon and nitrogen sources respectively. CONCLUSION: Thus this study presents its novelty by highlighting the potential of Streptococcus uberis TNA-M1 as a significant source for the production of fibrinolytic enzymes. Keywords Streptokinase, Streptococcus uberis, clot busters, mutagenesis, optimization Introduction Thrombotic diseases are responsible for heavy toll in death and disability worldwide. These are the most common diseases in the United States and in almost all western industrialized countries. Each year cardiovascular disease (CVD) causes 4.3 million deaths in Europe while in United States 2.5 million deaths (Kumar et al., 2011). A blood clot (thrombus) developed in the circulatory system can cause vascular blockage leading to serious consequences including death. A healthy homeostatic system suppresses the devel- opment of blood clots in normal circulation, but reacts extensively in the event of vascular injury to prevent blood loss. Outcomes of a failed homeostasis include stroke, pulmonary embolism, deep vein thrombosis and acute myocardial infarction. Pathologies involving a failure of homeostasis and the development of clot require clinical intervention consisting of intravenous administration of thrombolytic agents Streptokinase is one such agents (Taleb et al., 2005). The clinical importance of streptokinase was first noted by Tillet and Garner (Tillett et al.,1933), who Received June 14, 2017; accepted October 1, 2017 Correspondence: Subathra Devi Chandrasekaran E-mail: csubathradevi@vit.ac.in; subaresearch@rediffmail.com Front. Biol. 2017, 12(5): 376–384 https://doi.org/10.1007/s11515-017-1467-x