Nattokinase (NK) is a potent fibrinolytic enzyme with the potential for fighting against cardiovascular diseases. In this study, UV mutated Pseudomonas aeruginosa CMSS was used for production, purification and to study the stability of the enzyme. The enzyme was subjected to step by step purification by ammonium sulphate precipitation, dialysis, ion-exchange chromatography and gel filtration chromatography. The purified NK showed 91.84 % of clot lysis, comparable to standard streptokinase. The stability of the purified enzyme was analysed by different parameters such as pH, temperature, metal ions, surfactants and organic solvents. The molecular weight of the enzyme was determined as 27 kDa by SDS-PAGE and confirmed by fibrin zymography. The enzyme obtained its highest activity at pH 5 and at 45 °C. The present study showed the presence of fibrinolytic enzyme by its specificity. Further analysis of the biochemical properties and the precise mechanism of fibrinolytic enzymes will expand the scope of research for development of therapeutic agents to treat thrombosis. © 2015, Springer Science+Business Media New York.