Deep eutectic solvents-based three-phase partitioning for tomato peroxidase purification: A promising method for...

Area of Science:

• Biochemistry
• Green Chemistry
• Separation Science

Background:

• T-butanol is a conventional solvent in three-phase partitioning (TPP) for protein purification.
• There is a growing need for environmentally friendly alternatives in biochemical separation processes.

Purpose of the Study:

• To investigate the use of deep eutectic solvents (DESs) as a substitute for t-butanol in tomato peroxidase (POD) purification via TPP.
• To optimize the TPP process using DESs for enhanced efficiency and environmental sustainability.

Main Methods:

• Screening of various DESs to identify the optimal solvent (DES-5: menthol and octanoic acid).
• Optimization of extraction parameters including ammonium sulfate concentration, DES to crude extract ratio, pH, and temperature using single-factor experiments and response surface methodology (RSM).
• Analysis of purified POD using SDS-PAGE to confirm purity and assessment of DES recyclability.

Main Results:

• DES-5 (menthol and octanoic acid) was identified as the optimal DES for POD purification.
• Optimized conditions (42% (NH4)2SO4, 2:1 DES:crude extract, pH 5.7, 30°C) yielded a maximum recovery of 104.71% and a purification fold of 9.76.
• SDS-PAGE confirmed effective purification of tomato POD, and recycling studies demonstrated the good cycle stability of the DES.

Conclusions:

• Deep eutectic solvents provide a greener and more efficient alternative to t-butanol for TPP-based protein purification.
• The developed DES-based TPP method is effective for tomato POD purification and shows potential for broader applications in TPP.