An ESR assay for alpha-amylase activity toward succinylated starch, amylose and amylopectin

The esterification of the three polysaccharides, starch, amylose and amylopectin was carried out in pyridine–DMSO by succinic anhydride. The carboxylic groups in the succinylated polysaccharides were measured by FT–IR spectroscopy. The succinic derivatives were tested as a- amylase (1,4-a- D -glucan glucano hydrolase, E.C. 3.2.1.1) substrates. A colorimetric assay of the a-amylase activity indicated that this enzyme is active on succinic esters of starch and amylose and that the activity shows a linear decrease with the number of succinic units introduced into the polysaccharide. Since the colorimetric test was not suitable for the detection of the a-amylase activity when succinylated amylopectin was the substrate, we set-up an assay based on the labeling by a paramagnetic probe of the free carboxylic groups of succinylated polysaccharides. The kinetics of the a-amylase reaction were monitored by ESR spectroscopy through the increase of the mobility of the paramagnetic probe. The spin label used was the commercially available 4-amino-tempo. By this method we demonstrated that a-amylase is active on succinylated amylopectin. The utility of the assay for monitoring a-amylase activity when other methods (i.e. colorimetric tests) fail, is discussed.