Spectrophotometric enzymatic cycling method using l-glutamate dehydrogenase and d-phenylglycine aminotransferase for determination of l-glutamate in foods

Abstract

This report describes a new spectrophotometric method capable of determining low levels of l-glutamate. The assay is based on substrate cycling between l-glutamate dehydrogenase (GlDH) and the novel enzyme d-phenylglycine aminotransferase ( d-PhgAT). In this system, GlDH converts l-glutamate to 2-oxoglutarate with concomitant reduction of NAD + to NADH. The 2-oxoglutarate is recycled to l-glutamate in a transamination reaction catalyzed by d-PhgAT using d-4-hydroxyphenylglycine as an amino donor, which is converted to 4-hydroxybenzoylformate. Both NADH and 4-hydroxybenzoylformate strongly absorb UV light at 340 nm ( ε 340 nm =6.22×10 3 and 8.90×10 3 l mol −1 cm −1, respectively). The signal amplification effect of the cycling reactions is thus further enhanced by the combined absorption of the two accumulating reaction products. The standard calibration curve for l-glutamate was linear from 0.2 to 20 μM, with a detection limit of 0.14 μM. Food samples can be significantly diluted before subjected to the assay, thus reducing the effects of interfering substances. Because of the unique substrate specificity of d-PhgAT, l-glutamate could be selectively determined in the presence of other common amino acids at relatively high concentrations. The assay was satisfactorily applied to measure l-glutamate in various kinds of food products. The procedure is simple, rapid, accurate, and should be easily automated.