Single-step purification of ovalbumin from egg white using aqueous biphasic systems

Abstract

The ability of aqueous biphasic systems (ABS) composed of polyethylene glycols of different molecular weights (PEG 400, 600 and 1000) and buffered aqueous solutions of potassium citrate/ citric acid (pH = 5.0 -8.0) to selectively extract ovalbumin from egg white was here investigated. Phase diagrams, tie-lines and tie-line lengths were determined at 25ºC and the partitioning of ovalbumin in these systems was then evaluated. Aiming at optimizing the selective extraction of ovalbumin in the studied ABS, factors such as pH, PEG molecular weight and amount of the phase-forming components were initially investigated with pure commercial ovalbumin. In almost all ABS, it was observed a preferential partitioning of ovalbumin to the polymer-rich phase, with extraction efficiencies higher than 90%. The best ABS were then applied in the purification of ovalbumin from the real egg white matrix. In order to ascertain on the ovalbumin purity and yield, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion high performance liquid chromatography (SE-HPLC) analyses were conducted, confirming that the isolation/purification of ovalbumin from egg white was completely achieved in a single-step with a recovery yield of 65%. The results obtained show that polymer-salt-based ABS allow the selective extraction of ovalbumin from egg white with a simpler approach and better performance than previously reported. Finally, it is shown that ovalbumin can be completely recovered from the PEG-rich phase by an induced precipitation using an inexpensive and sustainable separation platform which can be easily applied on an industrial scale.

Design of stable magnetic hybrid nanoparticles of Si-entrapped HRP

“…Similar to the chemistry used to immobilize proteins on glyoxyl activated supports [40], a further reducing step via Na 2 BH 4 , would transform the first reversible interaction between the enzyme and the matrix into a three dimensional multiple covalent attachment of HRP within the Si particles Fig 1. The strategy was followed in the presence of trehalose, a common additive used to gain protein stability [12]. A direct correlation between the surface tension of trehalose solutions and the thermal stability of various proteins has been established and it is also known that trehalose significantly increases the half-life of HRP [5].…”

“…Apart from industrial large-scale applications, immobilization techniques on nanoscale supports have enabled and amplified the integration of enzymes in biosensors [5–7], nano/microreactors [8,9] or in the generation of hybrid nanostructures for biomedical applications [8,10,11]. Design of tailored strategies for enzyme immobilization has proved essential to achieve better specific activities, robustness and reusability of the material integrated enzymes, addressing the major problems that restrain industrial or therapeutic implementation of enzymatic reactions [12–14].…”

Methods for producing microstructured hydrogels for targeted applications in biology

“…The increase of molecular weight of one or more of the components in the system is a common strategy for inducing the phase separation, and indeed, even low concentrations of PEG of high molecular weight works efficiently for segregation of proteins and other polymers such as poly(vinyl alcohol) [141][142][143][144]. This PEG-induced phase separation has been used to make heterogeneous RLP hydrogels [145] with microscale mechanical properties that vary with the composition of the phase (Fig 4b) [146].…”

Separation of Antioxidant Peptides from Pepsin Hydrolysate of Whey Protein Isolate by ATPS of EOPO Co-polymer (UCON)/Phosphate

“…Although ATPS is a mild extraction and separation technique, there are still some losses during extraction. A thin middle layer was produced due to salting out effects that trapped some impure proteins between the two phases 36 . These impure proteins were likely to be highly polar or hydrophilic peptides.…”