Determination of the Depolymerisation Kinetics of Amylose,Amylopectin and Maltodextrin by Aspergillus niger Glucoamylase Using a 2-p-toluidinylnaphthalene-6-sulfonate/Flow-injection Analysis System with Fluorimetric Detection

Abstract

This work describes the determination of depolymerisation kinetics of amylose, amylopectin and maltodextrin by Aspergillus niger glucoamylase using a flow-injection analysis system with fluorimetric detection and 2-p-toluidinylnaphthalene-2-sulfonate as the fluorescent probe. Experimental data corresponding to the time evolution of the concentration of detectable substrate were fitted to a single exponential decay curve in the case of amylose (linear substrate) and to a double exponential decay curve in the case of amylopectin and maltodextrin (ramified substrates). For all the substrates assayed, the depolymerisation rates at time zero correlated well with the initial substrate and enzyme concentrations through the Michaelis-Menten hyperbola. Therefore, this methodology allowed the determination of glucoamylase activity using any of these substrates. The determined value of the enzymic constant K _m was lower for amylose than for amylopectin and maltodextrin, thus reflecting the higher difficulty of glucoamylase to hydrolyse the (1,6) when compared to the (1,4) linkages. In contrast, the values obtained for the rate constant k ₃ were very similar for all the substrates assayed.