Fluorometric quantification of total D-gluconate by a flow-injection system using an immobilized-enzyme reactor

A method for the quantification of total D-gluconate by flow-injection analysis was developed using an immobilized-enzyme reactor and fluorescence detection. D-Gluconate was quantified using co-immobilized gluconate kinase (GK) and 6-phosphogluconate dehydrogenase (PGDH) reactor. D-Gluconate was phosphorylated to 6-phospho-D-gluconate by GK in the presence of ATP, and then the 6-phospho-D-gluconate produced was oxidized by PGDH with NAD(+). The NADPH produced by the GK-PGDH reactor was monitored fluorometrically at 455 nm (excitation at 340 nm). A linear relationship between the responses and concentrations Of D-gluconate was obtained in the ranges of 1.0 X 10(-6)-1.6 x 10(-4) M. The relative standard deviation for 10 successive injections was 0.57% at the 0.1 mM level. This analytical method was applied to the quantification of D-gluconate in honeys, vinegars and noble rot wines, and the results showed good agreement with those obtained using the conventional F-kit method. (C) 2004 Elsevier B.V. All rights reserved.