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

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...

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Bibliographic Details
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
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Summary: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.
ISSN:0021-9673
DOI:10.1016/S0021-9673(99)00188-0