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.
Alred
P A
PA
Department of Biochemistry, University of Lund, Sweden.
Kozlowski
A
A
Harris
J M
JM
Tjerneld
F
F
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002845:Chromatography / Q000379:methods*; D004798:Enzymes / Q000302:isolation & purification*; D004852:Epoxy Compounds; D005027:Ethylene Oxide; D005954:Glucosephosphate Dehydrogenase / Q000302:isolation & purification; D006593:Hexokinase / Q000302:isolation & purification; D010735:Phosphoglycerate Kinase / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013696:Temperature
D004798:Enzymes; D004852:Epoxy Compounds; D005954:Glucosephosphate Dehydrogenase; D006593:Hexokinase; D010735:Phosphoglycerate Kinase; D005027:Ethylene Oxide; C009068:propylene oxide