Novel Sequential Screening and Enhanced Production of Fibrinolytic Enzyme byBacillussp. IND12 Using Response Surface Methodology in Solid-State Fermentation

Abstract

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 ± 6.5 U/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%).

Cost-effective fibrinolytic enzyme production by Bacillus subtilis WR350 using medium supplemented with corn steep powder and sucrose

“…Similar results were obtained in a study by Vijayaraghavan et al . 28 , where sucrose was identified as the optimal carbon source for fibrinolytic enzyme production by Bacillus sp. IND12.…”

The Application of Solid State Fermentation for Obtaining Substances Useful in Healthcare

“…When authors used two-level factorial design in the case of Bacillus sp. IND12 examining the impact of moisture, sucrose, and MgSO 4 levels added to the cow dung, maximum enzyme activity reached 4,143 U/g [54]. Further, orthogonal design (corn steep powder, sucrose and MgSO 4 • 7H 2 O) provided even greater enzyme activity (5,865 U/ml) in 100 l fermenter when applying Bacillus subtilis WR350 [57].…”

“…The majority of research was focused on fibrinolytic enzymes (FE , Table 1) -subtilisin, however, in one case it was nattokinase [55]. In the majority of cited papers authors verified anticoagulant properties of obtained enzymes and they used different methods -some of them applied spectrophotometric method which measured the increase of turbidity at 275 nm caused by added enzyme [53,54,58,60,62,65] and others measured zones of clearance in solid media containing fibrinogen [64,66,67].…”

“…It was demonstrated that the representatives of the Bacillus genus were able to produce fibrinolytic enzymes by SSF [52][53][54][55][56][57][58]60]. That ability is mostly assigned to the expression of fibE gene which encodes enzyme called subtilisin [88] which solubilises blood clots.…”

Optimizing the fermentation conditions and enhanced production of keratinase from Bacillus cereus isolated from halophilic environment

“…The statistical experimental designs such as, fractional experimental design and RSM involve large number of factors and enhancement on enzyme production (Senthilkumar et al, 2005). RSM was previously studied using solid substrate (Vijayaraghavan et al,2016, Vijayaraghavan et al, 2017). There is no specific culture medium that has been proposed to increase the production of keratinolytic proteases for any bacterium.…”