One-pot simultaneous production and sustainable purification of fibrinolytic protease from Bacillus cereus using natural deep eutectic solvents.

The present study report for the first time on the one-pot production and purification of fibrinolytic protease from Bacillus cereus by extractive fermentation using natural deep eutectic solvents (NADES). Cheese whey was chosen as a sustainable low-cost production alternative yielding a significantly high amount of protease (185.7 U/mg). Five natural deep eutectic solvents with menthol as hydrogen bond donor and sugar molecules as corresponding hydrogen bond acceptors were synthesized and their association was confirmed with H1 NMR. Thermophysical investigation of the synthetic NADES was accomplished as a function of temperature to define their extraction ability. Response surface methodology based optimization of concentration of NADES (77.5% w/w), Na2SO4 (14% w/v) and cheese whey (1% w/w) were accomplished for extractive fermentation. Further, preparative purification using size exclusion chromatography was used to quantify the amount of enzyme obtained in the extraction phase (190 U/ml). On subsequent purification with an anion exchange column, the maximum purity fold (21.2) with enzyme activity (2,607.8 U/ml) was attained. The optimal pH (8.0), temperature (50 °C) were determined and the in-vitro fibrinolytic activity has been confirmed using a fibrin plate assay.

High selective purification of IgY from quail egg: Process design and quantification of deep eutectic solvent based ultrasound assisted liquid phase microextraction coupled with preparative chromatography.

The present research investigates on task-specific deep eutectic solvents (TDES) based aqueous two-phase extraction and purification of immunoglobulins by chromatography from quail egg. The synthesis of TDES was accomplished with quaternary ammonium salt as hydrogen bond acceptor (HBA) and glycerol as hydrogen bond donor (HBD). Aqueous two-phase (ATPS) formation of TDESs with various salts was established and phase partitioning ability was evaluated using standard bovine immunoglobulin. Ultrasound-assisted liquid-liquid microextraction (UA-LLME) was performed with ATPS of better partitioning ability for quail egg yolk. Optimization of influential variables with response surface methodology was accomplished and maximum yield was achieved for 85% (v/v) of NADES for feed of 18 µg/ml of egg yolk isolate with ultrasound temperature of 35 °C and 12 min contact time respectively. The quantification of recovery was accomplished by gel filtration process with the determination of retention time, volume and resolution of separation using marker protein. Ultrapure immunoglobulin-Y (IgY) is achieved with anion exchange chromatography and yield was calculated. The results revealed that the selective concentration of immunoglobulin-Y from quail egg yolk could be performed using environment-friendly TDES based UA-LLME coupled with chromatography.