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 (Mn 2+ , Zn 2+ , and Cu 2+ ). 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.Biomolecules 2020, 10, 3 2 of 23 (active protease), converting soluble fibrinogen (glycoprotein) into fibrin polymer (insoluble blood clot) [5]. Fibrin clots are hydrolyzed by plasmin [12], which is stimulated from plasminogen by plasminogen activators (PAs) [13,14]. This natural dynamic equilibrium is disturbed when the process of natural fibrin clot hydrolysis undergoes pathophysiological shambles, leading to formation of fibrin clots. Such clots may cause hindrance in the circulation of blood, causing blockage in blood vessels, ultimately leading to cardiac disorders which are life-threatening [15,16].Approaches such as use of anti-coagulant agents, anti-platelet drugs, fibrinolytic enzymes, and surgical operations are employed for the treatment of thrombosis and to dissolve the blood clots [17]. Further, there are numerous side effects which may occur following the administration of the available anti-thrombotic strategies, as well as the high expense, which limit their scope. The effects of reperfusion, urticaria (allergic reaction), and hemorrhage are the major inimical consequences of such thrombolytic approaches on human health [18,19]. Other after effects include headache, dizziness, ulcers, increased clotting time, nausea and vomiting, etc. Management of thrombosis usin...

Computational-approach understanding the structure-function prophecy of Fibrinolytic Protease RFEA1 from Bacillus cereus RSA1

“…The cultures were incubated for 24 h at 37 • C with 1% inoculum and agitation rate of 120 rpm. The enzyme was purified as per our standardized protocol in previous study, employing chilled ethanol precipitation and gel filtration chromatography-Sephadex G75 column (50 × 15 mm; Sigma Aldrich, St. Louis, MO, USA), and identified through MALDI-TOF Mass Spectroscopy (Sharma et al, 2020). Further, statistical compositional amino acid outcome of protein RFEA1 was determined using statistical tool SAP Application Performance Standard (SAPS: http://www.ebi.ac.uk/Tools/seqstats/saps/).…”

“…The effect of calcium ion on efficacy and thermostability of RFEA1 was studied and confirmed in-vitro. RFEA1 was purified as per our standardized protocol in previous study, employing chilled ethanol precipitation and gel filtration chromatography-Sephadex G75 column (50 × 15 mm; Sigma Aldrich, St. Louis, MO, USA) (Sharma et al, 2020). The enzyme was incubated for 2 h at 37 • C with different concentrations of CaCl 2 (0.5-3.0 mM) and then tested for fibrin hydrolysis.…”

“…The enzyme was incubated for 2 h at 37 • C with different concentrations of CaCl 2 (0.5-3.0 mM) and then tested for fibrin hydrolysis. The fibrinolytic activity assay was performed according to Sharma et al (2020). Further, in another set of experiments we have used 2 mM of CaCl 2 and stability of RFEA1 was tested at different temperatures (20-80 • C).…”

“…The protein sequence of RFEA1 derived through MALDI-TOF mass spectrometric analysis in our previous study with Mr 39,483 Da and sequence score of 381 amino acids is mentioned underneath (Sharma et al, 2020).…”

“…Under natural physiological conditions, fibrin deposition is hydrolysed by plasmin, effectuating clot dissolution to avoid thrombotic complications (Sidelmann et al, 2000). However, this constant dynamic equilibrium is disturbed due to numerous pathophysiological shambles leading to life threatening cardiovascular thrombosis (Göbel et al, 2018;Sharma et al, 2020). The World Health Organization delineated that cardiovascular diseases causes 17.9 million deaths per year and projects 31% of global mortality (https://www.who.int/health-topics/cardiovascular-diseases#tab=tab_1).…”