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The current research work aims at optimization, production, purification and evaluation of fibrinolytic extracellular protease from Bacillus subtilis VITMS2 isolated from fermented milk of Vigna unguiculata. The optimal production was achieved at 4.0% inoculum, pH7.0, 30 °C with (1% w/v) sucrose, (2% w/v) soya bean meal and (2% w/v) malt extract and 10 mM of CaCl 2, MgSO 4, Na 2 HPO 4 and K 2 HPO 4 . The clear cell-free supernatant was purified using conventional ammonium sulphate salt fractionation (75%), ultrafiltration, ion-exchange (DEAE Sepharose FF) and gel filtration (Sephadex G-50). The molecular mass was determined to be 29 kDa using SDS-PAGE analysis. The purified enzyme showed strong fibrinolytic activity with a specific activity of 2418.85 U/mg and has a yield of 12.01%. The enzyme was highly stable up to 60 °C and a pH range of 10.0 until 72 h of incubation. The purified enzyme showed 97.4% in vitro thrombolytic activity. The K m and V max values of the enzyme was determined to be 0.0114 mM and 147.8 µmol min −1 using the chromogenic substrate S-7388. IC 50 of ace inhibition was assessed to be 0.06 mg/mL suggesting anti-hypertensive property of the fibrinolytic enzyme. The above-obtained ace-inhibition results was supported by in silico molecular docking studies which revealed better binding affinity of nattokinase with a HADDOCK score of − 22.0 ± 8.5 confirms affinity towards angiotensin converting enzyme.

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