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

N. A. Willoughby*, T. Kirschner, M. P. Smith, R. Hjorth, Nigel J. Titchener-Hooker

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

32 Citations (Scopus)

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 languageEnglish
Pages (from-to)195-204
Number of pages10
JournalJournal of Chromatography A
Volume840
Issue number2
DOIs
Publication statusPublished - 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

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