Facultative Methylotrophic Bacterium ?revibacterium Methylicum in the Process of Preparation of Highly Deuterated Phenylalanine, Alanine, Valine, and Leucine/Isoleucine

摘要

Microbiological preparation of [ 2 H]amino acids was performed by conversion of low molecular weight substrates ([U- 2 H]MeOH and 2 H 2 O) by Gram-positive aerobic facultative methylotrophic bacterium, Brevibacterium methylicum , being L-phenylalanine producer having ribulose-5-monophosphate (RuMP) cycle for carbon assimilation. For this purpose, the cells of the methylotroph with improved growth characteristics were used on minimal salt media M9 supplemented with 2% (v/v) [U- 2 H]MeOH and increasing gradient of 2 ? 2 O concentration from 0; 24.5; 49.0; 73.5 up to 98% (v/v) 2 ? 2 O. L-phenylalanine was isolated from the growth medium after adding 5 M 2 HCl (in 2 ? 2 ?), pH = 2.0 by extraction with isopropanol and subsequent crystallization in ethanol (output 0.65 g/l). Alanine, valine, and leucine/isoleucine were produced and accumulated exogenously in amounts of 5–6 mmol in addition to the main product of biosynthesis. The method allows to obtain [ 2 ?]amino acids with different levels of deuterium enrichment, depending on 2 ? 2 O concentration in growth media, from 17% 2 ? (2 deuterium atoms) (on the growth medium with 24.5% (v/v) 2 ? 2 ?) up to 75% 2 ? (6 deuterium atoms) (on the growth medium with 98% (v/v) 2 ? 2 ?) with introduction of deuterium to benzyl ? 6 ? 5 ?? 2 -fragment of molecule that is confirmed with the data of electron impact (EI) mass spectrometry analysis of methyl ethers of N-5-dimethylamino(naphthalene)-1-sulfochloride [ 2 H]amino acids after the separation by reverse-phase HPLC.