Separation and Purification of Papain Crude Extract from Papaya Latex Using Quaternary Ammonium Ionic Liquids as Adjuvants in PEG-Based Aqueous Two-Phase Systems

Abstract

In this work, the effect of adding different quaternary ammonium ionic liquids (ILs) on the phase formation behaviour of a PEG400+(NH4)2SO4+H2O aqueous two-phase system (ATPS) and separation and purification of crude papain extract from papaya latex in different ATPSs was studied. Phase diagrams were established for different systems: experimental phase diagram data were fitted by the Merchuk equation, and the tie line (TL), composition and tie line length (TLL) were determined. The papain purification effect of ATPSs with various ILs was systematically scrutinized. The absorption spectrum of papain before and after extraction was characterized by UV-vis absorption spectroscopy. SDS-PAGE electrophoresis was used to verify the purity of papain extracted by the ATPSs. Quaternary ammonium ILs as adjuvants did not extend the biphasic area in the phase diagram, but the partition behaviour of crude papain extract in ATPSs changed, resulting in additional IL and papain interactions that promoted papain purification. The highest papain purity was obtained when tetramethyl ammonium bromide ([N1111]Br) was added to the ATPS, and with 20 wt% (NH4)2SO4 and 20 wt% PEG400 at pH 7.0 and 60 °C, the purification factor of crude papain extract was 2.13 and 13.51 and the papain recovery rate was 64.2% and 96.46% for ATPSs with no IL and 4 wt% [N1111] Br, respectively. The UV spectra of papain before and after extraction were the same. The SDS-PAGE analysis revealed that the ATPS containing 4 wt% [N1111] Br enhanced papain purification relative to the ATPS without IL. The addition of 4 wt% IL greatly improved the purification factor of crude papain extract enriched in the PEG phase, providing a potential method for large-scale industrial production of high-purity papain.