Cow dung, a cheap and easily available source of energy, was used as the substrate for the production of alkaline protease by solid-state fermentation using the Bacillus subtilis strain VV. In order to achieve the maximum yield of this enzyme, the following optimum process parameters are needed: fermentation period (72 h), pH (10.0), moisture content (140%), inoculum (25%), temperature (30–40°C), carbon source (2% (w/w) maltose) and nitrogen source (1% (w/w) urea). The protease was stable over a broad temperature range (30–50°C) and pH (8.0-10.0), with maximum activity at 50°C and pH 10.0. Among the divalent ions tested, Ca2+ (0.01 M) increased enzyme activity. The purified protease, after being subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis, was found to have a molecular mass of 38.5 kDa. The enzyme was solvent-and surfactant-stable and showed activity even after 24 h incubation along with various commercially available detergents. This enzyme possessed dehairing properties for animal hide after 16 h of incubation at room temperature. From these results it is evident that cow dung is a potential substrate for the production of a detergent-stable, dehairing protease by B. subtilis. This enzyme has a lot of potential applications in the detergent and leather-processing industries.

Bacterial fibrinolytic enzymes find great applications to treat and prevent cardiovascular diseases. The novel fibrinolytic enzymes from food grade organisms are useful for thrombolytic therapy. This study reports fibrinolytic enzyme production by Bacillus sp. IND7 in solid-state fermentation (SSF). In this study, cow dung was used as the cheap substrate for the production of fibrinolytic enzyme. Enzyme production was primarily improved by optimizing the nutrient and physical factors by one-variable-at-a-time approach. A statistical method (two-level full factorial design) was applied to investigate the significant variables. Of the different variables, pH, starch, and beef extract significantly influenced on the production of fibrinolytic enzyme (p < 0.05). The optimum levels of these significant factors were further investigated using response surface methodology. The optimum conditions for enhanced fibrinolytic enzyme production were 1.23% (w/w) starch and 0.3 % (w/w) beef extract with initial medium pH 9.0. Under the optimized conditions, cow dung substrate yielded 8,345 U/g substrate, and an overall 2.5-fold improvement in fibrinolytic enzyme production was achieved due to its optimization. This is the first report of fibrinolytic enzyme production using cow dung substrate from Bacillus sp. in SSF. The crude enzyme displayed potent activity on zymography and digested goat blood clot completely in in vitro condition.

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,Bacillussp. IND12 was selected for fibrinolytic enzyme production.Bacillussp. 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 MgSO₄were 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 showed4143±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%).

Hydrolytic enzymes, such as cellulases and proteases, have various applications, including bioethanol production, extraction of fruit and vegetable juice, detergent formulation, and leather processing. Solid-substrate fermentation has been an emerging method to utilize low-cost agricultural residues for the production of these enzymes. Although the production of carboxy methyl cellulase (CMCase) and protease in solid state fermentation (SSF) have been studied extensively, research investigating multienzyme production in a single fermentation process is limited. The production of multienzymes from a single fermentation system could reduce the overall production cost of enzymes. In order to achieve enhanced production of enzymes, the response surface methodology (RSM) was applied. Bacillus subtilis IND19 utilized cow dung substrates for the production of CMCase and protease. A central composite design and a RSM were used to determine the optimal concentrations of peptone, NaH2PO4, and medium pH. Maximum productions of CMCase and protease were observed at 0.9 % peptone, 0.78 % NaH2PO4, and medium pH of 8.41, and 1 % peptone, 0.72 % NaH2PO4, and medium pH of 8.11, respectively. Under the optimized conditions, the experimental yield of CMCase and protease reached 473.01 and 4643 U/g, which were notably close to the predicted response (485.05 and 4710 U/g). These findings corresponded to an overall increase of 2.1- and 2.5-fold in CMCase and protease productions, respectively. Utilization of cow dung for the production of enzymes is critical to producing multienzymes in a single fermentation step. Cow dung is available in large quantity throughout the year. This report is the first to describe simultaneous production of CMCase and protease using cow dung. This substrate could be directly used as the culture medium without any pretreatment for the production of these enzymes at an industrial scale.

The process parameters governing the production of fibrinolytic enzyme in solid state fermentation employing Bacillus cereus IND5 and using cuttle fish waste and cow dung substrate were optimized. The pH value of the medium, moisture content, sucrose, casein and magnesium sulfate were considered for two-level full factorial design and pH, casein and magnesium sulfate were identified as the important factors for fibrinolytic enzyme production. Central composite design was applied to investigate the interactive effect among variables (pH, casein and magnesium sulfate) and response surface plots were created to find the pinnacle of process response. The optimized levels of factors were pH 7.8, 1.1% casein and 0.1% magnesium sulfate. Enzyme production was increased 2.5-fold after statistical approach. The enzyme was purified up to a specific activity of 364.5 U/g proteins and its molecular weight was 47 kDa. It was stable at pH 8.0 and was highly active at 50 °C. The mixture of cuttle fish waste and cow dung could find great application as solid substrate for the production of fibrinolytic enzyme.