Abstract: Relevant studies have shown that ionic liquid aqueous two-phase extraction of papain gave a high extraction rate through simple operation, and the construction of a suitable ionic liquid aqueous two-phase system is a research hotspot for the extraction of papain. In view of this, this experiment studied the mechanism of the interaction between ionic liquid and papain through fluorescence quenching, molecular docking and the determination of the partition coefficient of papain in ionic liquid aqueous two-phase systems. Fluorescence quenching analysis showed that the ionic liquids [C₄mim]BF₄ and [C₄Py]Cl could bind to papain with one binding site through hydrophobic interaction, forming a complex, and the quenching effect of [C₄Py]Cl was higher than that of [C₄mim]BF₄. Further molecular docking results indicated that the binding site of [C₄mim]BF₄ was located in the hydrophobic pocket of the active region of papain and interacted non-covalently with many surrounding amino acid residues through hydrophobic interaction, van der Waals force and π-π interaction. According to the partition coefficient of papain in ionic liquid aqueous two-phase systems, the extraction efficiency of papain with [C₄Py]Cl was better that with [C₄mim]BF₄ under the same conditions, which was consistent with the results of fluorescence spectroscopy and molecular docking. This study can theoretically guide the selection of suitable ionic liquids for aqueous two-phase extraction of papain.

Key words: ionic liquid aqueous two-phase extraction, papain, fluorescence spectroscopy, molecular docking