Innovative Three-Phase Partitioning Based on Deep-Eutectic Solvents and Sugars (Sugaring-out Effect) for Cucumber Peroxidase Purification

Traditional three-phase partitioning (TPP) systems used for extracting and purifying bioactive substances typi-cally consist of t-butanol and ammonium sulfate. However, the use of volatile and flammable organic solvents and high-concentration inorganic salts results in environmental pollution. Therefore, an innovative TPP based on deep eutectic solvents (DESs) and sugars (sugaring-out effect) was used to extract and preliminarily purify cu-cumber peroxidase (POD). Various factors affecting the extraction were optimized using response surface methodology (RSM). DES-8 (menthol: octanoic acid = 1:1) and sucrose were selected as the optimal constituents of TPP. Under these conditions, the sucrose concentration was 36% (w/v), DES: crude extract (v/v) was 1.4:1, system pH was not adjusted, extraction time was 6 min, and extraction temperature was 31 degrees C; the enzyme recovery and purification fold reached 84.53% and 5.28, respectively. Recycling experiments demonstrated that the DESs can be well recycled and reused after five recycles. Sodium dodecyl sulfate polyacrylamide gel elec-trophoresis (SDS-PAGE) was used to analyze cucumber POD, which showed that the TPP system achieved pri-mary purification of cucumber POD. This TPP is much greener and more efficient than traditional TPP systems and shows great application potential in the enzymes and proteins separation field.