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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 H 1  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), Na 2 SO 4  (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.

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