FIBRINOLYTIC ENZYME FROM BACILLUS AMYLOLIQUEFACIENS: OPTIMISATION AND SCALE UP STUDIES
Keywords:Fibrinolytic enzyme, Bacillus sp, Optimisation, Scale up studies, Commercial prospects
Objective: This research work was carried out to identify a potent microorganism, which produced the fibrinolytic enzyme and to optimise the media and growth parameters to achieve the maximal enzyme production for commercial application.
Methods: Microorganisms were isolated from different sources and assayed for fibrinolytic activity. The shortlisted cultures after preliminary screening (casein hydrolysis, blood plate assay and blood clot dissolution) were identified using 16S rRNA amplification method. The media and growth parameters were optimized to achieve the maximal enzyme activity. In-silico studies were carried out to identify the activators and inhibitors of the enzyme.
Results: Two species of Bacillus, namely, Bacillus amyloliquefaciens and Bacillus licheniformis, isolated from spoilt milk and soy flour, respectively, exhibited fibrinolytic activity. In the laboratory scale studies, of these two cultures, B. amyloliquefaciens produced the Fibrinolytic enzyme in higher quantities, 28.98 FU/mL, compared to 26.63 FU/mL in B. licheniformis. The maximal activities were obtained after 72 h. The optimum conditions at laboratory scale for the maximal production of the fibrinolytic enzyme were: pH 7.2, temperature 37 C and agitation 200 rpm. When scale up studies with B. amyloliquefaciens in a 7 L Fermentor were undertaken. The maximal activity obtained was 55.60 FU/mL in 72 h, compared to that of 28.98 FU/mL in shake flask studies. The molecular weight of the enzyme was estimated to be about 38 kDa. In in-silico studies, it was observed that PMSF inhibited the fibrinolytic activity, thereby, confirming this fibrinolytic enzyme is a serine protease (Nattokinase).
Conclusion: The enzyme had exhibited excellent blood clot dissolving property and therefore may be considered for further scale up and commercial exploitation.
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