Abstract
Fibrinolytic protease (FLP) is a therapeutic enzyme used in the treatment of thrombolytic diseases. The present study proposed the concept of pH-driven swappable micellar two-phase extraction for the concurrent production and purification of FLP from Bacillus subtilis at cloud point extraction. Extractive fermentation was carried out with a pH swap mechanism and FLP was extracted to the top phase by surfactant deep eutectic solvents (SDES). Shrimp waste was chosen as a sustainable low-cost substrate that yielded a maximum protease of 185 U/mg. Six SDESs were synthesized with nonionic surfactants as hydrogen bond donors and quaternary ammonium salts as hydrogen bond acceptors and their association was confirmed by H1 NMR. Thermophysical investigation of the synthetic SDES was accomplished as a function of temperature. Response surface methodology for extractive fermentation was performed with the concentration of SADES (35% w/v), Na2SO4 (15% w/v) and pH (6.3) as variables and the enzyme activity (248 IU/mg) as a response. Furthermore, purification using gel filtration chromatography was used to quantify the amount of enzyme obtained in the extraction phase (849 IU/ml). After final purification with an anion exchange column, the maximum purity fold (22.32) with enzyme activity (1172 IU/ml) was achieved. The in-vitro fibrinolytic activity has been confirmed using a fibrin plate assay.
Highlights
Fibrinolytic protease (FLP) is a therapeutic enzyme used in the treatment of thrombolytic diseases
B. subtilis is the most promising bacteria for the production of fibrinolytic protease with the optimal yield varying with the type of complex substrate supplied[33]
Among the various nitrogen sources, the maximum yield of protease (185 U/ mg) was observed to occur with shrimp waste as the primary nitrogen source compared to groundnut cake and cottonseed cake with moderate enzyme production of 100 U/mg and 110 U/mg respectively (Table 2)
Summary
Fibrinolytic protease (FLP) is a therapeutic enzyme used in the treatment of thrombolytic diseases. Recent investigations have identified Bacillus subtilis as a promising source for the production of specific Fibrinolytic Proteases (FLPs) that are reported to be safer for therapeutic use[5,6]. Purification of these therapeutically significant enzymes was accomplished with a sequence of downstream operations such as ammonium sulfate precipitation, ultrafiltration, ion exchange c hromatography[7] and affinity chromatography[8].
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