Selective α-Amylase Determination with Cross-Linked Substrate Tablets

Abstract

The method is amyloclastic in nature and use cross-linked (CL)-amylose and CL-starch as specific insoluble substrates for α-amylase only and not for exoamylases. α-Amylase is allowed to act on the CL-substrate liberating soluble polysaccharide chains, large enough and in a suitable conformation to allow the formation of iodine inclusion complexes. Unlike the classical iodometric methods, the reaction is followed by an Increase In iodine color. The method has much in common with the well known chromogenic (Phadebas^R) methods, Both, use insoluble substrates (ß-limit amyloses) which are not susceptible to attack by exoamylases and in both cases the enzymatic reaction is followed by the release of soluble polysaccharide chains. These soluble polysaccharide released chains are in linear dependence with the enzyme concentrations. The CL-amylose and CL-starch tablets are prepared such as to allow a quick disintegration. One tablet in two ml water and 0.1 ml α-amylase sample should be incubated for 3 min. The reaction is stopped with 1 ml of 1N HC1. 6 ml of 2mM iodine reagent is added and the absorbance is measured at 570 nm, The new method was calibrated against the usual methods for -amylase determination, as reference tests.

Key Words:

• selective α-amylase determination
• amyloclastic method
• cross-linked anylose tablets
• cross-linked starch tablets