Purification of soybean peroxidase (Glycine max) by metal affinity partitioning in aqueous two-phase systems.

Combining two concepts in downstream processing, this work investigates the partitioning of a crude soybean peroxidase (Glycine max) in an aqueous two-phase system by metal affinity. A liquid-liquid extraction process using metal ligands was developed in two steps with the aim of purifying the enzyme peroxidase. PEG 4000 was activated using thionyl chloride, covalently linked to iminodiacetic acid (IDA), and the specific metal ligand Cu2+ was attached to the PEG 4000-IDA. In the first step, the system was composed of 14% (w/w) PEG 4000-IDA-Cu2+ and 8% (w/w) Na2SO4, and the peroxidase partitioned mainly to the top phase (K = 24). In the second step, a system formed by 14% PEG 4000 and 10% phosphate was used to revert the value of the partition coefficient of peroxidase to the bottom salt-rich phase (K = 0.05), thereby achieving a recovery of 64% of the purified enzyme.