|Khampha, W.;Meevootisom, V.; Wiyakrutta, S,
|Spectrophotometric enzymatic cycling method using L-glutamate dehydrogenase and D-phenylglycine aminotransferase for determination of L-glutamate in foods
|Anal Chim Acta
|Phenylglycine aminotransferase; Enzymatic substrate cycling assay; Food; Glutamate
|This report describes a new spectrophotometric method capable of determining low levels of -glutamate. The assay is based on substrate cycling between -glutamate dehydrogenase (GlDH) and the novel enzyme -phenylglycine aminotransferase (-PhgAT). In this system, GlDH converts -glutamate to 2-oxoglutarate with concomitant reduction of NAD+ to NADH. The 2-oxoglutarate is recycled to -glutamate in a transamination reaction catalyzed by -PhgAT using -4-hydroxyphenylglycine as an amino donor, which is converted to 4-hydroxybenzoylformate. Both NADH and 4-hydroxybenzoylformate strongly absorb UV light at 340 nm (340nm=6.22×103 and 8.90×103 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 -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 -PhgAT, -glutamate could be selectively determined in the presence of other common amino acids at relatively high concentrations. The assay was satisfactorily applied to measure -glutamate in various kinds of food products. The procedure is simple, rapid, accurate, and should be easily automated.