Optimization of purification conditions for papain in a polyethylene glycol-phosphate aqueous two-phase system using quaternary ammonium ionic liquids as adjuvants by BBD-RSM

Abstract

This work attempts to study and optimize the conditions for separating and purifying papain in aqueous two-phase systems (ATPSs). Quaternary ammonium ionic liquids (ILs, 4 wt%) were added as adjuvants to a PEG-phosphate ATPS. On the basis of single-factor experiments, a Box-Behnken design with response surface methodology (BBD-RSM) was used to optimize the purification conditions of papain in the ATPS by setting the NaH₂PO₄·2H₂O concentration, PEG concentration and pH as independent variables and the overall desirability (OD) of the recovery rate of papain, the protein recovery rate and the purification factor as dependent variables. The following optimum conditions were determined: PEG4000 16.4 wt%, NaH₂PO₄·2H₂O 13.7 wt%, pH 6.22, temperature 60 °C and enzyme concentration 12.0 mg/ml. Under the optimized conditions, the purification factor for the ATPS supplemented with commercial enzyme increased from 1.331 (no ILs) to 3.380 (containing 4 wt% [N₂₂₂₂]BF₄). The total evaluation OD was 0.9979, the maximum predicted OD was 0.9994, and the deviation rate was -0.15%. Therefore, the model established in this experiment could predict the experimental value well. To verify the practical effect of the model, papain obtained from fresh papaya latex (papain crude extract) was applied to the same ATPS. The results showed that the purification factor of the ATPS with papain crude extract increased from 3.517 (no ILs) to 12.04 (containing 4 wt% [N₂₂₂₂]BF₄). In summary, the addition of 4 wt% ILs to partially replace PEG greatly improved the purification factor for crude papain extract enriched in the phosphate phase, providing a potential method for the large-scale industrial production of papain.

Keywords: ATPS; Adjuvants; BBD-RSM; Ionic liquids; OD value; Papain.