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 Km and Vmax 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.
Highlights
Cardiovascular diseases (CVDs) have emanated as one of the leading causes for increase in mortality rate, and account for 31% of deaths world-wide [1]
Plackett–Burman design is an extensively used statistical tool for screening of numerous parameters available to select the most profound ones [45]. This is followed by response surface methodology, which aids in determining the optimal level of the key variables involved in enhanced production of fibrinolytic protease [46]
The protein was eluted at a flow rate of 1.0 mL/min with the same buffer, and collected fractions were examined for fibrinolytic activity
Summary
Cardiovascular diseases (CVDs) have emanated as one of the leading causes for increase in mortality rate, and account for 31% of deaths world-wide [1]. Plackett–Burman design is an extensively used statistical tool for screening of numerous parameters available to select the most profound ones [45] This is followed by response surface methodology, which aids in determining the optimal level of the key variables involved in enhanced production of fibrinolytic protease [46]. These methods assist in reducing the number of experiments, time, and cost involved, and make the use of statistical techniques prominent tasks for the improved production of enzymes. We reveal fibrinolytic protease with high efficacy, specificity, and stability, suggesting it to be a novel and potent fibrinolytic protease
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