The biosynthesis of L-amino acids isotopically labelled with 15N

Full text: In this paper we report a simple enzymatic procedure for preparation of L-[15N]-norvaline, L-[15N]-norleucine and L-[15N]-isoleucine using corresponding α-keto acids as substrate and bacterial NAD-dependent amino acid dehydrogenase, Leucine Dehydrogenase (LeuDH), as a catalyst. The reaction mixtures included Glucose Dehydrogenase (GlucDH) for NADH regeneration with glucose as an electron donor. As isotopically labelled material we used 15NH4Cl 99 at.%15N produced at National Institute for Research and Development of Isotopic and Molecular Technologies, Cluj-Napoca, Romania. The reactions are simple and efficient for producing gram quantities of 15N-labelled amino acids, with high yield (η ∼ 85-90%). The amino acids were purified by ion exchange chromatography. It will be presented also, the biosynthesis of L-[15N] aspartic acid, from fumaric acid and 15NH4Cl 99 at.%15N, under the action of Aspartase. The obtained yield of L-[15N] aspartic acid was 60% with reference to the starting compounds. The structure of L-amino acids labelled with 15N was confirmed by mass spectrometry combined with gas chromatography (GC/MS), and the isotopic label was determined by MS on the molecular compounds. (author)