Bacterial endophytes are a promising source of novel fibrinolytic enzymes with unique metabolic pathways and desirable characteristics that may not be present from conventionally explored sources. This study aimed to characterize fibrinolytic enzymes from selected endophytic bacteria isolated from papaya (Carica papaya L.) leaves and the genes encoding the enzymes. A strain BFP1 (InaCC-B1657) that showed the highest fibrinolytic activity was identified as Bacillus cereus based on the phylogenetic analysis of the 16S rRNA sequence. The enzyme exhibited optimum activity at 50°C and pH 7.0, and remained stable until 80°C and pH 6 - 10 for 24 h. The assay of metal ions and inhibitors on the fibrinolytic enzyme activity found that adding Cu2+ stimulated, while Fe2+ reduced the activity. PMSF and TPCK inhibited the enzyme activity, while adding EDTA and EGTA increased the activity. These suggest that the fibrinolytic enzymes belong to the serine protease group. Of the 21 proteases/peptidases determined from the 5,257,484 base pairs (bp) genome, minor extracellular protease Vpr and S8 family peptidase genes were found related to the fibrinolytic enzyme activity. The Vpr gene has a molecular weight of 98.5 kDa. The subtilase domain (peptidase S8 family) and the catalytic triad subtilase active sites (Asp204, His237, and Ser531) were detected. A prediction of physicochemical characteristics of the Vpr gene showed that the enzyme is hydrophilic and exhibited alkali-halo tolerant and thermo tolerant over a broad range of temperatures.

Fibrinogen supplies the primary building block of the blood clot or thrombus after α-thrombin converts this fibrinogen to fibrin during the final phases of coagulation. When the homeostasis system is disrupted, blood clots that aggregate in the blood vessels can lead to thrombosis. Fibrin degrading enzyme from Bacillus subtilis K2 (Subtilisin K2) has many excellent characteristics with strong fibrinolytic activity. Bioinformatic analysis indicated that the enzyme molecule appeared to share conserved domain, with peptidase s8 super family also known as the subtilase family with its motif: Asp subtilase (motif: VAVIDSGIDSsH), His subtilase (motif: HGTHVAGTIAA) and Ser subtilase (motif: GTSMATPHVAG). Study on molecular docking between this fibrin degrading enzymes and the specific substrates, fibrin and fibrinogen was aimed to predict the mechanism of action. This analysis, revealed no productive interaction between Subtilisin K2 and fibrinogen. However, hydrolysis reaction is indicated strongly between Subtilisin K2 and fibrin substrate. Amino acid Asp19, His51, and Ser208 in the Subtilisin K2’s active site interacted with Leu168, Ile171, and Leu172 of the fibrin substrate with ∆G of –19.4 kcal/mol that showed suitable substrate specificity. The fibrin degrading enzyme Subtilisin K2 tend to act more as fibrin degrading enzyme than as fibrinogen degrading enzyme.