PURIFICATION OF BARLET MALT Α-Amylase BY IMMUNOAFFINITY CHROMATOGRAPHY

Abstract

Immunoaffinity chromatography was used to purify the high pi a-amylase (a-amylase II) in a one step procedure after fractionation of the whole barley malt extract on Sephadex G25. The immunoglobulin G (IgG) fraction of an immune serum specific for the malt a-amylase II was immobilized on Ultrogel. A mild desorption procedure was used, combining distilled water elution with an interrupted elution. The purification was achieved within half a day including kernel extraction. The quality of the purification was assayed by SDS polyacrylamide gel electrophoresis, crossed immunoelectrophoresis and isoelectric focusing. For the second technique, an immune serum was used which was polyspecific for malt proteins including the high pi a-amylase (a-amylase II). The effect of this procedure on the specific activity of the enzyme and on its antigenicity was evaluated. The results underline the efficiency of the purification procedure and indicate that a-amylase II accounts for a few percent of the total soluble pro tein in malts. However, the a-amylase II fraction was not completely free from a-amylase I. The pro cedure resulted in a partial loss of the enzymatic activity but not of the antigenicity.

Some methods for isolation and assays of enzymes occurring in cereals and legumes

“…However, the low dissociation constant of many antigen antibody complexes requires drastic conditions for desorbing the antigen (low pH, chao tropic agents) which may be detrimental to the activity of the enzyme (123). Nevertheless, with the usual desorption procedures or with less denaturating procedures, several plant enzymes can be purified (124)(125)(126)(127)(128)(129)(130). The difficulty of desorption lies, in part, in the fact that the antigen is retained by several epitopes to immobilized antibodies that have differing affinities with the epitopes.…”