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.