Preparation of 15N-labeled l-alanine by immobilized l-alanine dehydrogenase: Differential incorporation of 15N in bacterial proteins

This paper describes a system for continuous synthesis of 15N-labeled l -alanine from lactic acid, 15NH4Cl and NADH, which uses immobilized alanine dehydrogenase and soluble lactate dehydrogenase as enzyme sources. Lactic acid acts both as hydrogen donor for the regeneration of NADH and as pyruvate source, thus providing the carbon skeleton of l -alanine. Citrobacter freundi grown on synthetic media containing 17 unlabeled amino acids and l -(15N)alanine as nitrogen source, incorporated 66% of 15N into alanine found in bacterial proteins. When 15N-labeled glutamic acid, aspartic acid or glycocol were added to the synthetic growth media, their 15N was “diluted” among different amino acids of bacterial proteins. Isotope enrichment of l -(15N)lysine found in newly synthesized proteins of C. freundi was practically unchanged as compared to the isotope content of free amino acid in the growth medium.