Chemicals and reagents
All chemicals and reagents of analytical grade were used in this study; purchased from Sigma Aldrich. All experiments were conducted in triplicates and the mean values were considered.

Isolation and cultivation of bacterial isolates
Samples were collected by scraping off the soil from the various locations near slaughter house, Vazhayoor, Malappuram, Kerala, India. Isolation was performed by the soil dilution plate technique³⁵ on minimal agar medium supplemented with bovine fibrin. Nutrient broth was used to maintain cultures, supplemented with glycerol (70%), and preserved at -20°C²⁷. A stock suspension was prepared and adjusted to 7×10³ cell/ml^{10, 33}.

Determination of proteolytic activity
The proteolytic activity of the organism was determined by a modified method¹¹. Casein agar was prepared by using 1% casein. The organisms were spot inoculated on the medium and incubated at room temperature for 48 hr, the colonies were observed, proteolytic organisms showed a clear hallow around the colony, which indicated the casein hydrolysis by the organism.

Preparation of blood clot for nitrogen source
Buffalo blood was collected from the slaughter house and clot was formed by incubating at room temperature. Then the clots were shredded by using the surgical blade and were placed in 1L of distilled water in a beaker. The beaker was placed on the magnetic stirrer for further shredding and RBC removal. Water was replaced every half an hour, this procedure was repeated for 2-3 days, until the clot became grey in colour. This clot was grinded into fine powder by using mortar and pestle, sterilized using ethanol and dehydrated by acetone. This powder was used as a source of fibrin and stored at 4°C in refrigerator.

Screening of fibrinolytic organism
Fibrinolytic organisms were identified by using clot agar with mild modifications²⁸. The caseinolytic organisms were spot inoculated on the clot agar medium which contained powdered fibrin. The plates were incubated at 37°C for 48hr. Fibrinolytic organisms showed clear zone on fibrin plate agar after adding congo red as an indicator and inoculated into minimal media with 0.5% buffalo fibrin as a substrate, and incubated at 37°C for 48hr until the substrate got disappeared, centrifuged and the supernatant was used to check enzyme activity against the substrate fibrin.

Identification of organism
Bacterial colonies were identified macroscopically and microscopically by using staining methods, and by 16s rRNA sequencing, sequence comparison with the databases were performed using BLAST through the NCBI server²⁹.

Enzyme assay and protein content
Fibrinolytic enzyme activity was estimated^5,8 using 0.5% bovine fibrin in 0.1 M carbonate buffer with an equal volume of extracted enzyme solution and incubated for 30 min at 37 °C. After 30 min, the reaction was stopped by adding 3 mL of 10% cold tri chloroacetic acid (TCA); centrifuged at 5,000 rpm for 10 min. To 0.5 mL supernatant, 2.5 mL of 0.5 M Na₂CO₃ was added, followed by 0.5mL of two-fold diluted Folin-Ciocalteaure agent; incubated for 30 min at 37°C and colour developed was read at 660 nm against a reagent blank. Tyrosine served as the reference standard. The optical density was measured in a UV-Vis (Shimadzu) spectrophotometer. For obtaining the quantitative estimation of the protein content of individual fraction different steps was done systematically¹⁷. One unit of fibrinolytic activity was defined as the amount of enzyme required to catalyze the release 1 µg of tyrosine per mL under the reaction conditions.

Submerged fermentation using different nitrogen sources
Various nitrogen sources such as casein, fibrin, peptone, yeast extract, skim milk powder were tested for fibrinolytic enzyme production. The enzyme activity and protein content were determined along with the specific activity.

Production and extraction of fibrinolytic enzyme
The organism was inoculated in production media containing minimal media with beef extract (0.3%), casein (1%), buffalo fibrin (0.5%) and peptone (0.5%); pH 7.0, incubated at 37°C in orbital shaker for 48hr at 200rpm. After 76hr of incubation, the contents of the flasks were filtered through Whatman No.1 filter paper, centrifuged at 5,000 rpm for 10 min and the supernatant was used as the crude enzyme⁵.

Statistical optimization of cultural conditions using RSM
A three variable Box-Behnken design is used to study the combined effect of the substrate concentration, broth pH and fermentation temperature on fibrinolytic enzyme production over three levels. The range and levels of the variables optimized are shown in the Table 1. The Box-Behnken design is suitable for the exploration of quadratic response surfaces and generates a second degree polynomial model, which in turn is used in optimizing a process using a small number of experimental runs. This design requires an experimental number of runs according to:  N = k² + k + C_p, where k is the factor number which is 3 in this case and C_p is the number of replications at the center point which is also 3 in this case.
Table (1):
Actual levels for the factors for three factors Box-Behnken design.

The design which is developed using the Design Expert 10.0.3.1 resulted in 17 experimental runs. The second degree polynomial is expressed as follows:

Y= b₀ + b₁A + b₂B + b₃C + b₁₂AB + b₁₃AC+ b₂₃BC + b₁₁A² + b₂₂B² + b₃₃C² (equation 1), where Y is predicted response, A, B and C are independent variables, b₀ is the offset term, b_1, b_2, b₃ are linear effects, and b_11, b_22, and b₃₃ are interaction terms. Regression analysis and analysis of variance (ANOVA) are used to fit the model according to the equation1. Adequacy of models is checked by model analysis and R² analysis; F value is checked to find out the significance of all the fitted equation at 5% level of significance. To visualize relationship between response and experimental levels of each factor and to find out optimum conditions, the fitted equations are expressed as contour plots, which is explained by statistical software, Design Expert 10.0.3.1.

Application studies
Blood clot degradation method was used to determine in-vitro fibrinolytic activity^{19, 7}. Blood was collected from male human volunteer in capillary tube and blood clot was formed by incubating for 1hr at room temperature. After 1hr, artificial blood clot in capillary tube was rinsed thoroughly and dipped in 3mL of carbonate buffer (pH 9.0) containing enzyme solution for clot dissolution, incubated at room temperature and 37°C, normal saline was kept as control.