CONICET | Buscador de Institutos y Recursos Humanos

Aqueous two-phase systems (ATPSs) formed by a polymer and a kosmotropic salt provide a powerful method for separating biomaterials. Those ATPSs containing biodegradable anions such as citrate are particularly interesting due to their eco-friendly character. Extraction with ATPSs exhibits several advantages compared to other purification methods such a high capacity, short processing times and low cost. However, its utilization has been hampered by the poor understanding of the mechanism governing the solute partitioning equilibrium. Papain (PAP) and bromelain (BR) are two well-known cisteine-proteases with multiple applications in food, leather and pharmaceutical industries. PAP is obtained from the latex of Carica papaya while BR is purified from the stem of Anana Comosus. Their purification involves precipitation with salts, solvents and successive chromatographic steps that are time-consuming and expensive. The aim of this work was to study those variables that affect the partitioning equilibrium of PAP and BR in ATPSs formed by polyethyleneglycol (PEG) and sodium citrate (NaCit) in order to contribute to a better understanding of the molecular features involved in the partitioning process, and therefore, to a rational design of a extraction protocol with ATPSs. ATPSs formed by NaCit pH 5.20 and PEGs of different molecular weights (MWs): 600; 1,000; 2,000; 4,600 and 8,000 at compositions corresponding to several tie line lengths were assayed. Partition coefficients of PAP and BR (KpPAP and KpBR) were calculated from their enzymatic activity at the top and bottom phases. Protein surface hydrophobicity (fsurface) and the accessible superficial area (ASA) of tryptophan rests -amino acids reported to be participants in PEG-protein interaction- were calculated by computational procedures in an attempt to localize the structural features responsible for partitioning behavior. Partition profiles quite different were obtained for PAP and BR. Partitioning equilibrium for PAP was observed to be displaced to the salt-enriched phase in all the assayed systems with KpPAP values between 0.3 and 0.9. On the other hand, BR exhibited a higher affinity for the polymer phase in systems formed by PEGs of low MW (600 and 1,000) with KpBR values up to 6. The increase in PEG molecular weight decreased both KpPAP and KpBR. This could be attributed to a reduction of the space available for proteins when the polymer chain length increased. The highest changes in the Kp values (up to six times) were observed when the PEG MW varied from 600 to 2,000 in agreement with the behavior predicted by the Flory Huggins´ theory. At increasing tie line lengths, BR and PAP displaced to the bottom phase. Exceptionally, BR evidenced the opposite behavior in ATPSs formed by PEG of MW 600, thus suggesting a favorable PEG-BR interaction. KpBR resulted higher than KpPAP in all the cases. This difference could be assigned neither to the charge nor to the size of the partitioned biomolecule since PAP and BR possess similar molecular weights ( 25,000) and isoelectric points ( 9.60). According to the calculated fsurface, BR showed to possess hydrophobic character slightly higher than PAP. The significant difference observed in the ASA of tryptophan rests (353 and 194 Å2 for BR and PAP respectively) seemed to be determinant of higher partition coefficient (for BR) due to a selective charge transfer interaction between tryptophan and PEG molecule. We conclude that the mechanism involved in BR and PAP partitioning in the assayed ATPSs comprises general effects observed for other protein-ATPS such as the exclusion due to the polymer size and the hydrophobic character of protein. However, a relevant role must be assigned to the content of highly exposed tryptophan, amino acid rests responsible for a specific PEG-protein interaction.

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