Development of different deep eutectic solvent aqueous biphasic systems for the separation of proteins

Area of Science:

• Green Chemistry
• Bioseparation Engineering
• Protein Chemistry

Background:

• Aqueous biphasic systems (ABSs) are widely used for protein extraction.
• Deep eutectic solvents (DESs) offer tunable properties for novel separation applications.
• Developing environmentally friendly protein extraction methods is crucial for bioprocessing.

Purpose of the Study:

• To investigate the efficacy of novel aqueous biphasic systems (ABSs) formed by deep eutectic solvents (DESs) for protein extraction.
• To compare the phase-forming abilities of different DES combinations.
• To optimize the extraction of a model protein, chymotrypsin, and assess the integrity of the extracted protein.

Main Methods:

• Preparation of ABSs using combinations of DESs, including [tetrabutylammonium chloride][polypropylene glycol 400] ([TBAC][PPG400]) with [amino acids][polyols].
• Optimization of extraction conditions for chymotrypsin using the [l-proline][xylitol] ([Pro][Xyl]) and [TBAC][PPG400] system.
• Analysis of protein conformation using UV-Vis spectrophotometry, FT-IR, and CD spectroscopy.
• Investigation of the extraction mechanism using conductivity, DLS, and TEM.

Main Results:

• The phase-forming ability of [TBAC][PPG400]/[amino acids][polyols] systems was found to be intermediate between [TBAC][PPG400]/amino acids and [TBAC][PPG400]/polyols.
• Optimal conditions yielded 97.30% extraction of chymotrypsin into the [Pro][Xyl]-rich phase.
• Spectroscopic analyses confirmed that protein conformation remained unchanged during extraction.
• Mechanism studies indicated the formation of DES-protein aggregates.

Conclusions:

• DES/DES-based ABSs are effective for protein extraction, demonstrating tunable phase behavior.
• The [Pro][Xyl]/[TBAC][PPG400] system shows high efficiency for chymotrypsin extraction from a complex matrix (porcine pancreas).
• The extraction process preserves protein structural integrity, highlighting the potential of these systems for green bioseparation.