Application of temperature-induced phase partitioning at ambient temperature for enzyme purification

Aqueous two-phase partition and temperature-induced phase separation using a non-ionic, random copolymer composed of 20% ethylene oxide, 80% propylene oxide (EO20 PO80) has been used for purification of glucose-6-phosphate dehydrogenase, hexokinase and 3-phosphoglycerate kinase from bakers' yeast. This EO20PO80 copolymer has a cloud point of 18 degrees C, at which temperature it phase separates from water. Enzymes were first partitioned at 4 degrees C in an initial EO20PO80-dextran T500 aqueous two-phase system. This system had an upper copolymer-rich phase and a lower dextran-rich phase. After phase separation had occurred the upper EO20PO80-rich phase was removed and placed at 24 degrees C. This resulted in formation of a new two-phase system with an upper water phase and a lower phase containing 98% copolymer and 2% water. Enzymes were recovered exclusively in upper water phase leaving a polymer-rich lower phase free of contamination. The phase diagram for the system EO20PO80 and dextran T500 at 4 degrees C has been determined.