Switchable deep eutectic solvent driven micellar extractive fermentation of ultrapure fibrin digesting enzyme from Bacillus subtilis

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.

Simultaneous production and sustainable eutectic mixture based purification of narringinase with Bacillus amyloliquefaciens by valorization of tofu wastewater

“…2 (B). Also, NADES with high density was observed to exhibit low phase partitioning due to the densely packed top phase infiltrating water and target analyte to the bottom phase 37 , 38 . The phase formation mainly depends on various factors of NADESs including their moderate density and corresponding saturation level.…”

Advances of responsive deep eutectic solvents and application in extraction and separation of bioactive compounds

“…The reason for the high viscosity of DESs can be the strong interaction between the DESs components. Muniasamy et al determined the dependence of viscosity on temperature for all RDESs in the range 293 to 323 K [61]. It was found that the viscosity of all RDESs decreased nonlinearly with increasing temperature.…”