Menthol-based hydrophobic deep eutectic solvents for the purification of tea peroxidase using a three-phase partition method

Traditional three-phase partitioning (TPP) systems use t -butanol as an organic solvent. However, owing to its volatility and flammability, t -butanol cannot be applied in the context of the large-scale application of TPP. Therefore, hydrophobic deep eutectic solvent (HDES) with menthol as the hydrogen bond acceptor has been investigated as a potential replacement for t -butanol in TPP systems. The properties of eight synthesized HDESs were characterized in the present study. Based on its recovery and purification fold, HDES-2 (menthol: hexanoic acid) was evaluated as a potential reagent for the purification of tea peroxidase (POD). Single-factor and response surface methodology experiments revealed that the optimum HDES-based TPP system comprised 56% (NH 4 ) 2 SO 4 , had an extract-to-HDES volume ratio of 1:1, and functioned best pH 6.0. Additionally, this system had a recovery rate of 70.26% and a purification fold of 8.25 regarding tea POD. Compared to that produced by the t -butanol-based TPP control, the tea POD purified using HDES-based TPP was purer and more stable, as shown through SDS-PAGE and scanning electron microscopy. Moreover, recycling studies indicated that the HDES had good cycle stability. The HDES-based TPP method for extracting enzymatic proteins is “green”, efficient, and inexpensive, pointing to its excellent applicability in large-scale production. • Eight types of hydrophobic deep eutectic solvent (HDES) were characterized. • The HDES were constructed for three-phase partitioning (TPP). • Menthol-based HDES was used to substitute volatile and flammable t -butanol in TPP. • The HDES-based TPP system improved the purity and stability of the tea peroxidase. • The HDES is easily recycled and reused.