Abstract
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 Zn2+, Ni2+ and Cu2+ and eluted using 0-50 mM EDTA gradient found that charging with Zn2+ 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 mM 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.
Original language | English |
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Pages (from-to) | 195-204 |
Number of pages | 10 |
Journal | Journal of Chromatography A |
Volume | 840 |
Issue number | 2 |
DOIs | |
Publication status | Published - 30 Apr 1999 |
Keywords
- Alcohol dehydrogenase
- Enzymes
- Expanded bed adsorption
- Immobilised metal ion affinity chromatography
- Yeast
ASJC Scopus subject areas
- Analytical Chemistry
- Biochemistry
- Organic Chemistry