Novel Sequential Screening and Enhanced Production of Fibrinolytic Enzyme by Bacillus sp IND12 Using Response Surface Methodology in Solid-State Fermentation

Fibrinolytic enzymes have wide applications in clinical and waste treatment. Bacterial isolates were screened for fibrinolytic enzyme producing ability by skimmed milk agar plate using bromocresol green dye, fibrin plate method, zymography analysis, and goat blood clot lysis. After these sequential screenings, Bacillus sp. IND12 was selected for fibrinolytic enzyme production. Bacillus sp. IND12 effectively used cow dung for its growth and enzyme production (687 +/- 6.5U/g substrate). Further, the optimum bioprocess parameters were found out for maximum fibrinolytic enzyme production using cow dung as a low cost substrate under solid-state fermentation. Two-level full-factorial experiments revealed that moisture, pH, sucrose, peptone, and MgSO4 were the vital parameters with statistical significance (p < 0.001). Three factors (moisture, sucrose, and MgSO4) were further studied through experiments of central composite rotational design and response surface methodology. Enzyme production of optimized medium showed 4143 + 12.31 U/g material, which was more than fourfold the initial enzyme production (978 + 36.4 U/g). The analysis of variance showed that the developed response surface model was highly significant (p < 0.001). The fibrinolytic enzyme digested goat blood clot (100%), chicken skin (83 +/- 3.6%), egg white (100%), and bovine serum albumin (29 +/- 4.9%).