Immobilised metal ion affinity chromatography purification of alcohol dehydrogenase from baker’s yeast using an expanded bed adsorption system

• Published in: Journal of Chromatography A 1999-04, Vol.840 (2), p.195-204
• Main Authors: Willoughby, N.A., Kirschner, T., Smith, M.P., Hjorth, R., Titchener-Hooker, N.J.
• Format: Article
• Language: English
• Subjects: Adsorption; Alcohol dehydrogenase; Alcohol Dehydrogenase - isolation & purification; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Biotechnology; Chromatography, Affinity; Edetic Acid; Enzyme engineering; Enzymes; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Histidine - analysis; Imidazoles - analysis; Improved methods for extraction and purification of enzymes; Metals - chemistry; Methods. Procedures. Technologies; Oxidoreductases; Saccharomyces cerevisiae - chemistry; Solvents; Spectrophotometry, Ultraviolet
• ISSN: 0021-9673
• DOI: 10.1016/S0021-9673(99)00188-0

Alcohol dehydrogenase (ADH) from solutions of homogenised packed bakers’ yeast has been successfully purified using immobilised metal-ion affinity chromatography in an expanded bed. Method scouting carried out using pure ADH solutions loaded onto 5-ml HiTrap columns charged with Zn 2+, Ni 2+ and Cu 2+ and eluted using 0–50 m M EDTA gradient found that charging with Zn 2+ gave the highest recovery and the lowest EDTA concentration required for elution. These results were used to develop a protocol for the expanded bed system and further tested using clarified yeast homogenate loaded onto XK16/20 packed beds (approximately 30 ml) packed with Chelating Sepharose FastFlow matrix in order to determine the optimum elution conditions using EDTA. The ADH was found to elute at 5 m M EDTA and the dynamic and total binding capacities of Streamline chelating for ADH were found to be 235 U/ml and 1075 U/ml matrix, respectively. Expanded bed work based on a step EDTA elution protocol demonstrated that ADH could be successfully eluted from unclarified homogenised bakers’ yeast diluted to 10 mg/ml total protein content with a recovery of 80–100% that was maintained over five consecutive runs with a vigorous clean-in-place procedure between each run.