Cereal Chem 68:383-390   |  VIEW ARTICLE

Isolation and Characterization of Alpha-Amylases from Endosperm of Germinating Maize.

D. A. Warner, M. J. Grove, and C. A. Knutson. Copyright 1991 by the American Association of Cereal Chemists, Inc. 

Amylases from germinating maize (cv. B73) were fractionated by affinity chromatography, anion exchange chromatography, and chromatofocusing. Two groups of amylase enzymes were separated by affinity chromatography. About one half of the total amylase activity was bound on a cycloheptaamylose-Sepharose 6B column. Bound proteins were fractionated by anion exchange into four major alpha-amylase fractions, then further separated by chromatofocusing into eight fractions with apparent isoelectric point (pI) values ranging from 5.70 to 4.06. All affinity-bound fractions were confirmed as alpha-amylases by their action on beta-limit dextrin. The affinity-bound alpha-amylases with lowest and highest pI values produced reaction products from soluble starch containing large amounts of dextrins with degrees of polymerization (DP) 2 and 6, with lesser amounts of intermediate oligosaccharides. Intermediate pI fractons produced primarily DP2, large amounts of DP3-5, and little DP6. Enzymes not bound by cycloheptaamylose affinity chromatography were purified by hydroxylapatite chromatography, then resolved by chromatofocusing into four subgroups of alpha-amylase, plus beta-amylase. Among the affinity-unbound fractions, the lowest pI alpha-amylase had a unique action pattern, producing primarily DP7 and 8 oligosaccharides after exhaustive hydrolysis of soluble starch.