Determination of depolymerization kinetics of amylose, amylopectin, and soluble starch by Aspergillus oryzae alpha-amylase using a fluorimetric 2-p-toluidinylnaphthalene-6-sulfonate/flow-injection analysis system.

Abstract

This study reports on the determination of the depolymerization kinetics of amylose, amylopectin, and soluble starch by Aspergillus oryzae alpha-amylase using flow-injection analysis with fluorescence detection and 2-p-toluidinylnaphthalene-6-sulfonate as the fluorescent probe. The experimental data points, corresponding to the evolution of the concentration of "detectable" substrate with depolymerization time, were fit to a single exponential decay curve in the case of amylose and to a double exponential decay curve in the cases of amylopectin and soluble starch. For all the assayed substrates, the determined depolymerization rates at time zero correlated well with the initial enzyme and substrate concentrations through the usual Michaelis-Menten hyperbola. Therefore, this methodology allows the determination of alpha-amylase activity using any of these substrates. For amylopectin and soluble starch, the value of the total depolymerization rate at any depolymerization time was the result of the additive contribution of two partial depolymerization rates. In contrast, the total depolymerization rate for amylose was always a single value. These results, in conjunction with the relative time evolution of the two partial depolymerization rates (for amylopectin and soluble starch), are in good agreement with a linear molecular structure for amylose, a "grape-like" cluster molecular structure for amylopectin, and an extensively degraded grape-like cluster structure for soluble starch.