LAUSR

Abstract This work used ionic liquid aqueous two-phase (ILATP) extraction technology to obtain high-purity and high-activity papain. Papain was extracted using [CnPy]Cl(n=2,4,6)-K2HPO4 ILATP systems. Response surface methodology (RSM) was developed to optimize the conditions. Firstly, the effects of the reagent, temperature and pH on papain activity were investigated. Secondly, the influences of the salt concentration, ionic liquid concentration, papain dosage, pH and temperature on the partition behaviour of papain were investigated. The overall desirability (OD) of the enzyme activity recovery and partition coefficient was response indicator. Furthermore, the absorption spectrum of papain before and after extraction was characterized by UV–vis absorption spectroscopy. Finally, the purity of papain extracted by ILATP was analysed by SDS-PAGE electrophoresis. The order of the effect of various factors on papain extraction was: K2HPO4 concentration, [C4Py]Cl concentration, pH and papain dosage. A high temperature is not conducive to the extraction of papain from an ILATP system. The extraction efficiency of the [C4Py]Cl-K2HPO4 system is superior to that of the [C2Py]Cl and [C6Py]Cl systems. The maximum OD value 0.8985 was obtained under the conditions of 0.35 g/mL K2HPO4, 0.25 g/mL [C4Py]Cl, pH of 7.87, papain dosage of 2.17 mg/mL at 30℃. The UV spectra of papain before and after extraction were the same. The specific activity of papain extracted by ILATP was 4120.17 U/mg, which was 1.88-fold the purity of commercial papain. This work provides theoretical guidance for optimized extraction process parameters and the preparation of high-activity enzymes.