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

摘要

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 (G1DH) and the novel enzyme D-phenylglycine aminotransferase (D-PhgAT).In this system,G1DH 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 (epsilon_(340nm) = 6.22 x 10~3 and 8.90 x 10~3 mol l~(-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 muM,with a detection limit of 0.14 muM.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.