Direct recovery of alcohol dehydrogenase from unclarified yeast cell homogenate by IDEBAC using an improved scheme for elution

Procion Red HE-7B was directly immobilized onto STREAMLINE matrix to obtain a moderately substituted dye-ligand matrix in which the dye concentration was 6.72 μmol/ml adsorbent. The adsorption characteristics of alcohol dehydrogenase (ADH) onto the sticky dyed adsorbent in the absence and/or presence of yeast cells and cell debris were investigated. The Langmuir-type isotherm model was reasonably used to describe the adsorption behavior. The results showed that the adsorption characteristics and uptake rate for ADH did not change significantly even in the presence of cells and cell debris. Measurements of adsorption breakthrough curves in expanded beds have shown that the maximum dynamic binding capacity appeared to be significantly affected by the aspect ratio. The dynamic binding capacity of the dyed adsorbent was found to be 50.75 U/ml as the aspect ratio was up to 16. Optimal conditions for elution of the adsorbed ADH were determined in small scale packed bed experiments conducted with clarified yeast cell homogenates. The one-step elution was carried out by injecting a pulse of NAD+ (15 mM, one settled bed volume) with 0.3 M NaCl as a carrier, resulting in the adsorbed ADH eluting completely from the sticky dyed adsorbent. This case study on the adsorption/desorption characteristics of the sticky dyed adsorbent for ADH showed that this enzyme could be recovered with a purification factor of 4.6 and yield of 89% by immobilized dye-ligand expanded bed affinity chromatography (IDEBAC) in a single step.