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Background: Fibrinolytic enzymes, such as Nattokinases from Bacillus species are known to degrade the fibrin blood clots. They belong to serine protease group having commercial applications, such as therapeutic agents and functional food formulation. Objective: The present study reports some characteristics and fibrinolytic activity of serine protease from B. subtilis C10 strain that was isolated from shrimp shell. Method: Extracellular enzyme from B. subtilis C10 culture was harvested and partially purified by ammonium sulphate precipitation. Fibrinolytic activity of the enzyme was determined by zymography and measured by spectrophotometry with fibrinogen and thrombin used as substrates. The optimal temperature and pH for fibrinolytic activity were studied in the range of 31-43ºC and 5-10, respectively. The thermal and pH stability of enzyme was studied by incubating enzyme for 30 min in the same range of temperature and pH as above. The effect of some metal ions and reagents on fibrinolytic activity of enzyme was evaluated by concentrations of 5 mM and 5%, respectively. Results: Zymogram analysis indicated the presence of four fibrinolytic enzymes with molecular weights of approximately 69, 67, 39 and 36 kDa. The optimal temperature and pH for enzyme activity were 37°C and 9, respectively. The thermal and pH stability ranged from 35-39°C and 8-10, respectively. Fibrinolytic activity reached a maximum value of about 400 U/mg protein after 16 h of C10 strain culture. Enzyme has been drastically inhibited by PMSF and SDS, and partially inhibited by EDTA, while Triton X-100 has significantly increased enzyme activity. Effects of ions such as Mg2+, Ca2+ and Mn2+ on enzyme were negligible, except Cu2+ and Zn2+ have strongly decreased its activity. Conclusion: Results from the present study suggested that enzyme obtained from B. subtilis C10 could be serine protease that has a high fibrinolytic activity up to about 400 U/mg protein at the most appropriate temperature and pH of 37ºC and 9. This activity can be improved up to 142% by incubating enzyme with 5% Triton X-100 for 30 min.

Nattokinase, the fibrinolytic serine protease, has been shown to be beneficial in preventing strokes caused due to blood clots. Therefore, synthesis of recombinant nattokinase in a host system, which can be both safe for human consumption as well as demonstrate good productivity and amenable to the downstream processing, will have a great socioeconomic significance. A nat05 gene encoding nattokinase – the most important serine protease secreted by the Bacillus subtilis natto – was cloned from B. subtilis strain N05 and expressed using pHT43 plasmid in another B. subtilis strain (BD170). Enzymatic assays, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram, were employed to assess the efficiency of the production of nattokinase using a relatively novel recombinant DNA technique. Among the five recombinant bacterial clones (R1–R5), R4 was found to secrete high amounts of nattokinase into the culture medium, as evidenced by the total proteolytic enzyme assay, fibrinolytic enzyme assay, SDS-PAGE, and zymogram. A preliminary analysis of the biochemical properties of the crude enzyme showed characteristics that are typical of nattokinase as reported by other studies. Thus, in this study, we were able to successfully produce nattokinase in B. subtilis strain (BD170) using recombinant DNA approach. The R4 recombinant clone that was found to secrete nattokinase can be used as a source of recombinant protein that can be further purified for various therapeutic applications, or the strain could be used as a probiotic bacterium in functional foods.