Cereal Chem 65:413-416   |  VIEW ARTICLE

Immobilized Metal Affinity Chromatography of Wheat Alpha-Amylases.

U. Zawistowska, K. Sangster, J. Zawistowski, J. Langstaff, and A. D. Friesen. Copyright 1988 by the American Association of Cereal Chemists, Inc. 

An immobilized metal affinity chromatography technique for the purification of alpha-amylases from germinated wheat is described. Wheat alpha-amylases were retained on a column with Cu-iminodiacetic acid-epoxy-Sepharose 6B equilibrated with a 0.05M Tris-HCl buffer, pH 7.5, containing 0.15M NaCl and 10(-4)M CaCl2. A major portion of the contaminating proteins passed through in the equilibration buffer. Of the bound proteins, two groups of alpha-amylases were eluted from the column by the addition of increasing concentrations of glycine to the equilibration buffer. The group of alpha-amylases exhibiting low isoelectric points (pI) was eluted by the buffer containing 20 mM glycine, whereas the predominant group of high-pI alpha-amylases was eluted by the buffer containing 100 mM glycine. For the fraction eluted by the buffer containing 100 mM glycine, an increase is specific activity of approximately 20-fold with a recovery of 51.3% of the enzyme activity in the crude extract was obtained. The purity of the wheat alpha- amylase fractions was determined by isoelectric focusing followed by the beta-limit dextrin plate technique used for the detection of alpha-amylase activity and protein staining.