AFFINITY THERMOPRECIPITATION OF YEAST ALCOHOL-DEHYDROGENASE THROUGH METAL ION-PROMOTED BINDING WITH EUDRAGIT-BOUND CIBACRON-BLUE-3GA

Metal ion-promoted binding of Saccharomyces cerevisiae alcohol dehydrogenase to Cibacron Blue 3GA was used for its isolation by affinity precipitation with Eudragit-bound dye. The yeast cells were broken and the cell debris was separated, by flocculation with poly(ethylene imine). The supernatant containing the enzyme activity was mixed with Eudragit-Cibacron Blue in the presence:of zinc ions. The precipitation of the affinity complex was induced by the addition of 50 mM CaCl2 and a subsequent increase in temperature to 40 degrees C. The enzyme was desorbed by treating the precipitate with iminodiacetic acid solution. The procedure resulted in about 66% recovery of enzyme activity with more than 20-fold purification. Recycling of Eudragit-dye for enzyme purification was also shown to be possible.