Abstract: The preparative microbial synthesis of amino acids labelled with stable isotopes, including deuterium ( 2 H), suitable for biomedical applications by methylotrophic bacteria was studied using L-phenylalanine as example. This amino acid is secreted by Gram-negative aerobic facultative methylotrophic bacteria Brevibacterium methylicum, assimilating methanol via ribulose-5-monophosphate (RMP) cycle of assimilation of carbon, The data on adaptation of L-phenylalanine secreted by methylotrophic bacterium В. methylicum to the maximal concentration of deuterium in the growth medium with 98% 2 Н 2 O and 2% [ 2 Н]methanol, and biosynthesis of deuterium labelled L-phenylalanine With different levels of enrichment are presented. The strain was adapted by means of plating initial cells on firm (2% agarose) minimal growth media with an increasing gradient of 2 Н 2 O concentration from 0; 24.5; 49.0; 73.5 up to 98% 2 Н 2 O followed by subsequent selection of separate colonies stable to the action of 2 Н 2 O. These colonies were capable to produce L-phenylalanine. L-phenylalanine was extracted from growth medium by extraction with isopropanol with the subsequent cristallization in ethanol (output 0.65 g/l). The developed method of microbial synthesis allows to obtain deuterium labelled L-phenylalanine with different levels of isotopic enrichment, depending on concentration of 2 Н 2 O in growth media, from 17% (on growth medium with 24,5% 2 Н 2 O) up to 75% (on growth medium with 98% 2 Н 2 O) of deuterium in the molecule that is confirmed with the data of the electron impact (EI) mass- spectrometry analysis of methyl ethers of N-dimethylamino(naphthalene)-5-sulfochloride (dansyl) phenylalanine in these experimental conditions.
Reference: Mosin O.V., Shvets V.I., Skladnev D.A., Ignatov I., Microbic synthesis of deuterium labelled L-phenylalanine with different levels of isotopic enrichment by facultative methylotrophic bacterium brevibacterium methylicum with RMP assimilation of carbon, Biomeditsinskaya khimiya, 2014, vol:
2. Vertes A. (2003) in: Elements and isotopes: formation, transformation, distribution (Vertes A. (ed.)) Kluwer Acad. Publ, Dordrecht, pp. 111–112. Scholar google search
3. Crespi H.L. (1986) Biosynthesis and uses of per-deuterated proteins. in: Proceedings of the Second Inter. Symp. On Synthesis and Applications of Isotopically labeled Compounds (Muccino R.R. (ed.)) Elsevier., NY, pp. 111-112. Scholar google search
8. Мосин О.В. (1996) Исследование методов биотехнологического получения аминокислот, белков и нуклеозидов, меченных стабильными изотопами 2 Н и 13 С с высокими уровнями изотопного обогащения. Автореф. дисс. канд. хим. наук. МИТХТ им. М.В. Ломоносова, Москва, с. 1-26. Scholar google search
9. Dewick P.M. (2009) in: Medicinal Natural Products: A Biosynthetic Approach (Derwik P.M. (еd.)) 3-rd Edition, John Wiley & Sons Ltd, Chichester, UK, pp. 137–186. Scholar google search