Expanded Bed Adsorption for Purification of Alcohol Dehydrogenase Using a Dye-Iminodiacetic Acid Matrix

Abstract

Expanded bed adsorption (EBA) was successfully applied for the purification of alcohol dehydrogenase (ADH) from unclarified bakers' yeast homogenate using a new dye-iminodiacetic acid (IDA) matrix carrying immobilized metal ions. The existence of cells and cell debris do not have a significant effect on the adsorption of ADH on the matrix. The presence of imidazole in adsorption buffer was found to be an important factor in reducing the level of the adsorbed contaminant proteins. The optimal imidazole concentration was 5 mM. The dynamic binding capacity of the dye-IDA matrix in EBA was found to be 569 U/ml matrix. A chromatography of one-step elution with imidazole was developed, in which ADH was successfully adsorbed in the presence of 5 mM imidazole; subsequently, the column was washed with 5 mM imidazole containing 1 M NaCl and eluted with 150 mM imidazole containing 1.5 M NaCl. The purification factor and ADH recovery were 8.8 and 93.5%, respectively.