Purification of papain by metal affinity partitioning in aqueous two‐phase polyethylene glycol/sodium sulfate systems

A simple and inexpensive aqueous two‐phase affinity partitioning system using metal ligands was introduced to improve the selectivity of commercial papain extraction. Polyethylene glycol 4000 was first activated using epichlorohydrin, then it was covalently linked to iminodiacetic acid. Finally, the specific metal ligand Cu²⁺ was attached to the polyethylene glycol‐iminodiacetic acid. The chelated Cu²⁺ content was measured by atomic absorption spectrometry as 0.88 mol/mol (polyethylene glycol). The effects on the purification at different conditions, including polyethylene glycol molecular weight (2000, 4000, and 6000), concentration of phase–forming components (polyethylene glycol 12–20% w/w and sodium sulfate 12–20%, w/w), metal ligand type, and concentration, system pH and the commercial papain loading on papain extraction, were systematically studied. Under optimum conditions of the system, i.e. 18% w/w sodium sulfate, 18% w/w polyethylene glycol 4000, 1% w/w polyethylene glycol‐iminodiacetic acid‐Cu²⁺ and pH 7, a maximum yield of 90.3% and a degree of purification of 3.6‐fold were obtained. Compared to aqueous two phase extraction without ligands, affinity partitioning was found to be an effective technique for the purification of commercial papain with higher extraction efficiency and degree of purification.