ABSTRACT:

Objectives: This experiment was conducted to rule out unspecific proteins and enzymes concomitantly extracted during alcohol dehydrogenase enzyme (ADH) purification process in Saccharomyces cerevisiae using affinity chromatography. Method: Cell disruption was performed and the ADH was treated by desalting followed by isolation and purification by affinity chromatography. For the control of the individual purification steps, the enzyme activity and the total protein concentration were measured photometrically. By means of these two values, the specific enzyme activity could be calculated for a statement of the purification quality. Results: The overall purification of ADH was confirmed by the SDS-PAGE visualizing the purification steps. Additionally, the protein size of ADH was determined by comparison with a protein ladder and an ADH standard and confirmed by affinity chromatography. The graphical comparison of values showed a decrease in the enzyme activity and the total protein concentration for all purification steps except for ADH. This is plausible since all proteins except for ADH should be removed during the purification, which causes a lower signal. However, the specific enzyme activity of ADH increased during isolation. Eventually, the desalting of samples resulted in an increase estimated 125% p Conclusion: The desalting treatment step is highly recommended in the process of specific ADH enzyme purification using affinity chromatography