Separation and purification of horseradish peroxidase from horseradish roots using a novel integrated method

The aim of the present work was to develop a novel method integrating two-step aqueous two-phase extraction and temperature-controlled affinity precipitation for the separation and purification of horseradish peroxidase (HRP) from horseradish roots. First, a two-step aqueous two-phase extraction (ATPE) process was used as a primary separation process to remove sugars and pigments, and then a temperature-controlled affinity precipitation process based on a thermosensitive triblock copolymer (PEG113-b-PVBA49-b-PNIPAM105) was exploited as a further purification process to selectively isolate HRP from other impurity proteins. The optimum PEG4000 (0.20, w/w) − (NH4)2SO4 (0.18, w/w) aqueous two-phase system (ATPS) was obtained by studying the liquid–liquid equilibrium of PEG (400/1000/2000/4000) + (NH4)2SO4/K3C6H5O7/K2HPO4 ATPSs and the distribution behaviors of 9 kinds of model compounds in these PEG-salt ATPSs. The successful isolation of HRP from horseradish roots was achieved by the integrated method without having a negative effect on the structure of HRP, and the extraction efficiency and purification factor of HRP reached 84.26 ± 0.15% and 9.15 ± 0.10, respectively. The integrated method shows outstanding separation and purification performances and has good prospects for the industrial production of plant glycoprotein.