Specific detection of l-glutamate in food using flow-injection analysis and enzymatic recycling of substrate Original Research Article

A flow injection analysis (FIA) system for specific determination of l-glutamate in food samples based on a bi-enzymatic amplification system has been developed. The content of l-glutamate in the sample was amplified by cycling between l-glutamate dehydrogenase (GlDH) and a novel enzyme, d-phenylglycine aminotransferase (d-PhgAT). In this system, GlDH converts l-glutamate to 2-oxoglutarate with concomitant reduction of NAD+ to NADH. d-PhgAT transfers an amino group from d-4-hydroxyphenylglycine to 2-oxoglutarate, thus recycling l-glutamate. Accumulation of NADH in the course of the enzymatic recycling was monitored both by fluorescence and UV absorbance and used for quantification of l-glutamate. The assay was characterized by high long-term stability (at least 70 days) and good reproducibility (within-day and between-day RSDs were 4.3–7.3% and 8.9%). The fluorimetric assay was slightly more sensitive with a l-glutamate detection limit of 0.4 μM and linear range of 2.5–50 μM. The assay was specific for l-glutamate, with recoveries between 95–103% in the presence of 17 different amino acids tested one by one. The method was applied to analysis of real food samples and results were correlated with a commercial Boehringer Mannheim assay kit.