Innovative three-phase partitioning based on deep-eutectic solvents and sugars (sugaring-out effect) for cucumber peroxidase purification

Abstract

Traditional three-phase partitioning (TPP) systems used for extracting and purifying bioactive substances typically 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 cucumber 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 °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 electrophoresis (SDS-PAGE) was used to analyze cucumber POD, which showed that the TPP system achieved primary 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.