Determination of beta-amylase activity by a fluorimetric 2-p-toluidinylnaphthalene-6-sulfonate flow-injection analysis (2,6-TNS-FIA) method, using amylose and amylopectin as substrates

2-p-Toluidinylnaphthalene-6-sulfonate (2,6-TNS) is a -compound which is bar ely fluorescent in pure water. but whose fluorescence can be strongly enhan ced if the environment becomes hydrophobic, i.e. by the addition of suitabl e substrates such as proteins or 1,4-alpha-D-glucans. The enhancement of fl uorescence results from the formation of a 2,6-TNS/substrate complex. For l inear and ramified 1,4-alpha-D-glucans, the fluorescence intensities of the complexes depend linearly on their concentrations but nonlinearly on their average molecular weights (AMW). Thus, the fluorescence detector acts simu ltaneously:as a linear detector concerning the concentration of, 1,4-alpha- D-glucan and as a nonlinear mass-selective detector concerning its AMW. The se properties have been used for the development of a fluorimetric 2,6-TNS- FIA methodology for the determination of beta-amylase activity, using amylo se and amylopectin as substrates. The experimental data points, correspondi ng to the concentration of "detectable" substrate vs depolymerization time, were fitted using a two-parameter exponential decay curve, and the depolym erization rates at time zero were calculated. The depolymerization rates at time zero vs the corresponding initial substrate concentrations;were fitte d using the Michaelis-Menten hyperbola and the enzymic constants k(3) and K -m for amylose (5.93 x 10(-3) g/mu Kat . min and 1.49 g/L, respectively) an d for amylopectin (7.40 x 10(-3) g/mu Kat . min and 1.65 g/L, respectively) were determined. (C) 2000 John Wiley & Sons, Inc.