Thrombolytic Potential of Novel Thiol-Dependent Fibrinolytic Protease from Bacillus cereus RSA1

Abstract

The present study demonstrates the production and thrombolytic potential of a novel thermostable thiol-dependent fibrinolytic protease by Bacillus cereus RSA1. Statistical optimization of different parameters was accomplished with Plackett-Burman design and validated further by central composite design with 30.75 U/mL protease production. Precipitation and chromatographic approaches resulted in 33.11% recovery with 2.32-fold purification. The molecular weight of fibrinolytic protease was 40 KDa and it exhibited a broad temperature and pH stability range of 20-80 °C and pH 5-10 with utmost activity at 50 °C and pH 8, respectively. The protease retained its fibrinolytic activity in organic solvents and enhanced the activity in solutions with divalent cations (Mn²⁺, Zn²⁺, and Cu²⁺). The enzyme kinetics revealed K_m and V_max values of 1.093 mg/mL and 52.39 µg/mL/min, respectively, indicating higher affinity of fibrinolytic activity towards fibrin. Also, complete inhibition of fibrinolytic activity with DFP and a 2-fold increase with DTT and β-mercaptoethanol indicates its thiol-dependent serine protease nature. MALDI-TOF analysis showed 56% amino acid sequence homology with Subtilisin NAT OS = Bacillus subtilis subsp. natto. The fibrinolysis activity was compared with a commercial thrombolytic agent for its therapeutic applicability, and fibrinolytic protease was found highly significant with absolute blood clot dissolution within 4 h in in vitro conditions. The isolated fibrinolytic protease of Bacillus cereus RSA1 is novel and different from other known fibrinolytic proteases with high stability and efficacy, which might have wide medicinal and industrial application as a thrombolytic agent and in blood stain removal, respectively.

Keywords: Bacillus cereus RSA1; fibrinolytic protease; thiol-dependent; thrombolytic potential.

Figure 1

Expression of fibrinolytic protease on fibrin plate by bacterial strains: A: Control; B: RSA1 Strain; C: RSA3 Strain, and D: RSE163 Strain.

Figure 2

Neighbor-joining phylogenetic analysis showing the relationship of Bacillus cereus RSA1 (MK288105) with other Bacillus strains. 16S rRNA gene sequences of B. cereus RSA1 and other strains, mainly 10 sub-clusters of Bacillus sensu stricto group were used for analysis. The bootstrap values are provided at nodes.

Figure 3

The three-dimensional (3D) contour plots of fibrinolytic protease activity, showing interactions among peptone, casein, yeast extract, and glucose, keeping the other parameters fixed.

Figure 3

The three-dimensional (3D) contour plots of fibrinolytic protease activity, showing interactions among peptone, casein, yeast extract, and glucose, keeping the other parameters fixed.

Figure 4

SDS-PAGE of purified fibrinolytic protease. (PM) Protein marker; (Lane 1) 7.5 µL dialyzed fibrinolytic protease; (Lane 2) 15 µL dialyzed fibrinolytic protease.

Figure 5

MALDI–TOF mass spectra of purified fibrinolytic protease. The molecular masses of discrete isoforms are designated above the major spectrum peaks.

Figure 6

(A,B) Optimization of temperature and pH, with maximum fibrinolytic protease production at 50 °C and pH 8. (C,D) Stability of enzyme within a wide range of temperature (20–80 °C) and pH (5–10).

Figure 6

(A,B) Optimization of temperature and pH, with maximum fibrinolytic protease production at 50 °C and pH 8. (C,D) Stability of enzyme within a wide range of temperature (20–80 °C) and pH (5–10).

Figure 7

Enzyme kinetics—non-linear regression: Michaelis–Menton plot for kinetic studies.

Figure 8

(A–C) Effect of inhibitors/activators (5 mM and 10 mM), metal ions (2 mM and 5 mM), and organic solvents (10%) on the fibrinolytic activity of the protease.

Figure 8

(A–C) Effect of inhibitors/activators (5 mM and 10 mM), metal ions (2 mM and 5 mM), and organic solvents (10%) on the fibrinolytic activity of the protease.

Figure 9

(A) Effect of incubation time on the dissolution of blood clot. (B) Percentage dissolution of clot in different concentration of fibrinolytic, laboratory and streptokinase. (C) Comparative analysis for the efficacy of fibrinolytic protease, a commercialized thrombolytic agent, and laboratory protease in mammalian blood clot dissolution: (1) Laboratory protease: No blood clot dissolution; (2) commercialized streptokinase: Blood clot dissolves completely within 4 h of incubation; (3) fibrinolytic protease: Complete blood clot dissolution occurs within 4 h of incubation.