DFT-guided sustainable extractive fermentation and partial purification of fibrinolytic protease from agro-industrial waste

Abstract

In recent years, there has been a growing interest in employing greener solvents like deep eutectic mixture in biphasic systems for the separation of proteins from agricultural waste. This study leverages Density Functional Theory-based optimal identification of greener solvents for extracting fibrinolytic protease from agro-industrial residues. An in-depth analysis was conducted on nine quaternary ammonium salt-based eutectic solvents and five salts, emphasizing their phase behavior and interactions with the protease. B:BG and Na2CO3 were identified as the optimal biphasic system for protease extraction, based on their interaction energies of 22.11 kcal/mol and 3.02 kcal/mol, respectively. Using vegetable waste as a substrate, extractive fermentation yielded a protease activity of 987.45 U/mL in the solvent-rich phase. The enzyme was then partially purified through size exclusion chromatography, achieving an activity of 759.23 U/mL. The in-vitro fibrinolytic potential of the purified protease was further validated using fibrin plate assay.