Rapid separation of α-amylases from barley by ion-exchange high-performance liquid chromatography on non-porous columns

Abstract

x-Amylase (EC 3.2.1.1) is an important enzyme in biology and industry because of its central role in the hydrolysis of starch. Multiple forms of a-amylase are found in many organisms due to the presence of isoenzymes and possible post-translational modifications of the proteins. The different forms of a-amylase in plants, especially the cereals wheat and barley, have been extensively studied because of their importance in hydrolysis of starch during germination, and the functional significance of these enzymes in the industrial utilisation of the grain.
lsoelectric focusing is widely used to separate x-amylases but is difficult to quantify. X-Amylases from wheat and barley have been separated by ion-exchange chromatography on DEAE-cellulose CM-cellulose and CM-Sepharose and more recently by chromatofocusing. However, these separation techniques have been generally less resolving than electrophoretic methods and have been very slow. Omichi and Ikenaka recently reported high-performance liquid chromatography (HPLC) of human salivary a-amylases using gel permeation, ion-exchange and chromatofocusing columns. However their study used columns containing porous media. Ion-exchange columns based upon non-porous packing materials have become available for protein separation. These materials allow much faster flow-rates permitting more rapid separations.
This paper reports rapid separation of a-amylases from barley by high-performance ion-exchange chromatography on non-porous media.