Sequential fluorometric quantification of γ-aminobutyrate and l-glutamate using a single line flow-injection system with immobilized-enzyme reactors

Abstract

A single line flow-injection system with immobilized-enzyme reactors is proposed for the sequential quantification of γ-aminobutyrate (GABA) and l-glutamate. A co-immobilized l-glutamate oxidase and catalase reactor and an immobilized GABase reactor were introduced into the flow line in series. Sample and reagent were injected into the flow line using an open sandwich method. GABA was selectively detected by GABase when α-ketoglutarate at a high concentration and NADP + were injected as the reagents with a sample. When GABA at a high concentration and NADP + were injected as the reagents with a sample, l-glutamate only was determined by the series of enzymatic reactions. NADPH produced by the immobilized-enzyme reactors was monitored fluorometrically at 455 nm (excitation at 340 nm). Linear relationships between the responses and concentrations of GABA or l-glutamate were observed in the ranges of 5.0 × 10 −6–5.0 × 10 −4 M and 1.0 × 10 −5–5.0 × 10 −4 M, respectively. The relative standard deviations for ten successive injections were less than 2% at the 0.5 mM level. This analytical method was applied to the sequential quantification of GABA and l-glutamate that were produced and consumed, respectively, by lactic acid bacteria, and the results showed good agreement with those obtained using liquid chromatography.