Three-phase partitioning constructed by pH-responsive deep eutectic solvents and sugars for purification of radish (Raphanus sativus L.) peroxidase

Ammonium sulfate and t-butanol are widely used in conventional three-phase partitioning (TPP); however, their use is not eco-friendly. In this study, a novel TPP was developed using pH-responsive deep eutectic solvents (DESs) and sugars as substitutes for t-butanol and ammonium sulfate, respectively. This TPP was further used for the separation and purification of radish (Raphanus sativus L.) peroxidase (POD). DES-3 (dodecanoic acid: decanoic acid = 1:1) and sucrose were selected as optimal phase-forming components. Single-factor experiments were performed to investigate the factors influencing the TPP process, and three major factors (sucrose concentration, extraction time, and extraction temperature) were optimized by response surface methodology (RSM). Under the optimized conditions, the POD recovery and purification fold reached 101.66% and 11.56, respectively. Cyclic experiments confirmed the retrievability of DESs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed that the novel TPP had better purification efficiency than the traditional TPP. The method proposed in this study is more efficient than conventional TPP, and it does not require the use of organic solvents and high concentration salt solutions. Therefore, this method is promising in the application of TPP.

Automated summary

In this study, a novel three-phase partitioning (TPP) method was developed using pH-responsive deep eutectic solvents (DESs) and sugars as substitutes for t-butanol and ammonium sulfate. The method was applied to the separation and purification of radish peroxidase (POD). The optimized conditions resulted in high POD recovery rate and purification fold. The method showed better purification efficiency compared to traditional TPP methods, and it does not require the use of organic solvents and high concentration salt solutions.