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

T -butanol is widely used in three-phase partitioning (TPP). This study used deep eutectic solvents (DESs) to substitute t -butanol for tomato peroxidase (POD) purification. DES-5 (menthol and octanoic acid) was screened as the optimal solvent. The extraction process was optimized using single-factor experiments. Thereafter, the major three factors were optimized by response surface methodology (RSM). When the ammonium sulfate ((NH 4 ) 2 SO 4 ) concentration was 42%, DES: crude extract (v/v) was 2:1, pH was 5.7, and extraction temperature was 30 °C, the recovery and purification fold reached a maximum of 104.71% and 9.76, respectively. The obtained tomato POD was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), which showed that this system could effectively purify POD. Finally, recycling studies indicated the good cycle stability of the DES. This TPP based on DESs is greener and more efficient, indicating that DESs can be good alternative solvents for further applications of TPP.