| Abstract |
Owning to its unique properties of breaking down proteins into soluble small peptides, pepsin has been advantageously applied in many different industries, such as food, feed, or pharmaceuticals. However, industrial production of enzymes is still considered a major bottleneck because of the lack of robust, cost-effective, and sustainable purification methods, which can account for up to 65\% of the total manufacturing cost. In this work, the potential of betaine hydrochloride (BeHCI)- and BeHCI-based deep eutectic solvents (DESs) as a phase-forming compound of aqueous biphasic systems (ABSs) for the simultaneous extraction and recovery of pepsin is investigated as a sustainable integrated ABS platform for enzyme production. Liquid-liquid equilibrium (LLE) of ABSs composed of polypropylene glycol and DES composed of BeHCl, as a hydrogen-bond acceptor (HBA), and fructose, glucose, sucrose, and urea, as hydrogen-bond donors (HBDs), was measured at a temperature of 25 degrees C and atmospheric pressure and is presented here. It is shown that the salting-out ability of the HBA is a driving force for ABS formation, and the HBDs act as additives, leading to either increase or decrease in the biphasic region, depending on their chemical structure. The biocompatibility of the prepared DESs and their components was studied using two different human cell lines, HEK 293 and Huh-7. It is shown that these DESs are less toxic at higher concentrations than their separate components. The partitioning studies revealed high affinity of pepsin to the BeHCl-enriched phase in all the studied systems. Very high extraction efficiencies and pepsin recovery yield, above 96 and 141.9\%, respectively, were obtained and it was possible to significantly increase the pepsin activity recovery yield up to 141.9\% in the BeHCl:glucose-based ABSs. In conclusion, the ABSs formed with DESs allow the development of a promising platform to simultaneously extract, recover, and formulate the BeHCl-pepsin drug, without the need to remove the phase-forming compounds or recover the enzyme from the BeHCl-enriched phase, thus improving sustainability in pharmaceutical manufacturing. |
