title: [Microbial synthesis of deuterium labelled L-phenylalanine with different levels of isotopic enrichment by facultative methylotrophic bacterium Brevibacterium methylicum with RMP assimilation of carbon].
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 crystallization 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.
authors.forename: O V
authors.initials: OV
authors.lastname: Mosin
authors.forename: V I
authors.initials: VI
authors.lastname: Shvets
authors.forename: D A
authors.initials: DA
authors.lastname: Skladnev
authors.forename: I
authors.initials: I
authors.lastname: Ignatov
mesh_terms: D019840:2-Propanol; D000222:Adaptation, Physiological; D001951:Brevibacterium / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D002244:Carbon / Q000378:metabolism*; D003619:Dansyl Compounds / Q000737:chemistry; D003903:Deuterium / Q000378:metabolism* / Q000494:pharmacology; D007553:Isotope Labeling / Q000379:methods; D059625:Liquid-Liquid Extraction; D013058:Mass Spectrometry; D000432:Methanol / Q000378:metabolism; D010649:Phenylalanine / Q000096:biosynthesis* / Q000302:isolation & purification; D012274:Ribulosephosphates / Q000378:metabolism*; D012997:Solvents
chemical_list: D003619:Dansyl Compounds; D012274:Ribulosephosphates; D012997:Solvents; C031524:ribulose 5-phosphate; D010649:Phenylalanine; D002244:Carbon; D003903:Deuterium; D019840:2-Propanol; C029038:dansyl chloride; D000432:Methanol
keywords: Brevibacterium methylicum; L-phenylalanine; biosynthesis; electron impact mass spectrometry; heavy water