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

The esterification of the three polysaccharides, starch, amylose and amylop ectin was carried out in pyridine-DMSO by succinic anhydride. The carboxyli c groups in the succinylated polysaccharides were measured by FT-IR spectro scopy. The succinic derivatives were tested as alpha-amylase (1,4-alpha-D-g lucan glucano hydrolase, E.C. 3.2.1.1) substrates. A colorimetric assay of the alpha-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. Sinc e the colorimetric test was not suitable for the detection of the alpha-amy lase 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 alpha-amylase reaction were monitored by ESR spectroscopy through the increase of the mob ility of the paramagnetic probe. The spin label used was the commercially a vailable 4-amino-tempo. BY this method we demonstrated that alpha-amylase i s active on succinylated amylopectin. The utility of the assay for monitori ng alpha-amylase activity when other methods (i.e. colorimetric tests) fail , is discussed. (C) 1999 Elsevier Science BN. All rights reserved.