Biosynthesis of<sup>2</sup>H-Labeled Phenylalanine by a New Methylotrophic Mutant<i>Brevibacterium methylicum</i>

Mosin, Oleg; Складнев, Д. А.; Shvets, V. I.

doi:10.1271/bbb.62.225

Cited by 35 publications

(18 citation statements)

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“…К 20 мл 40%-ного КОН в 40 мл диэтилового эфира добавляли 3 г нитрозометилмочевины и перемешивали на водяной бане со льдом в течение [15][16][17][18][19][20] мин. После интенсивного газовыделения эфирный слой отделяли и промывали ледяной водой до рН 7,0, сушили безводным NaSO 4 и обрабатывали им N-Dns-производные аминокислот в составе КЖ штамма продуцента.…”

Section: синтез метиловых эфиров N-dns-аминокислотunclassified

Microbic synthesis of deuterium labelled L-phenylalanine with different levels of isotopic enrichment by facultative methylotrophic bacterium brevibacterium methylicum with RMP assimilation of carbon

et al. 2014

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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.

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Section: синтез метиловых эфиров N-dns-аминокислотunclassified

Microbic synthesis of deuterium labelled L-phenylalanine with different levels of isotopic enrichment by facultative methylotrophic bacterium brevibacterium methylicum with RMP assimilation of carbon

et al. 2014

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“…The method of the optional adaptation of the strain of methylotrophic bacteria B. methylicum to grow on 2H2O while maintaining the ability for the biosynthesis of L-phenylalanine was described in article [20]. In this research, were investigated samples of the culture liquids and biomass hydrolysates obtained during the multi-stage adaptation of B. methylicum to heavy water on minimal mineral M9 media with the different content of 2H2O (from 24,5 to 98,0% 2H2O).…”

Section: Resultsmentioning

confidence: 99%

The Development of Biosynthesis of 2Н- and 13С-labeled Amino acids and Proteins with Various Levels of Isotopic Enrichment Using Bacterial Objects

Mosin¹,

Ignatov²,

Складнев³

et al. 2015

European Reviews of Chemical Research

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By the method of microbiological synthesis were obtained and analyzed by electron impact mass-spectrometry 2H, 13C-labeled amino acids of a facultative methylotrophic bacterium Brevibacterium methylicum and an obligate methylotrophic bacterium Methylobacillus flagellatum and 2H, 13C-labeled amino acids of the total protein of biomass obtained on media containing as a source of stable isotopes [2H]methanol, [13C]methanol and 2H2O. It was also performed the incorporation of L- [2,3,4,5,6-2Н]phenylalanine, L-[3,5-2Н]tyrosine and L- [2,4,5,6,7-2Н]tryptopan into the membrane integral protein bacteriorhodopsin synthesised in purple membranes of photoorganotrophic halobacterium Halobacterium halobium ET 1001. For mass-spectrometric analysis the multicomponential mixtures of 2H-and 13C-labeled amino acids, derived from cultural media and protein hydrolysates after hydrolysis in 6 M 2HСl (3% phenol) and 2 M Ва(OH)2 were modified into N-benzyloxycarbonyl-derivatives of amino acids as well as into methyl esters of N-5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl) derivatives of [2H, 13С]amino acids, European Reviews of Chemical Research, 2015, Vol.(5), Is. 3 145 which were preparative separated using a method of reverse-phase HCLP. Biosynthetically obtained 2H-and 13C-labeled amino acids represented the mixtures differing in quantities of isotopes incorporated into molecule. The levels of 2H and 13С enrichment of secreted amino acids and amino acid resigues of protein were found to vary from 20,0 atom.% to L-leucine/isoleucine up to 97,5 atom.% for L-alanine depending on concentration of 2H-and 13C-labelled substrates.Keywords: stable isotopes, methylotrophic bacteria, halobacteria, isotope labeled amino acids, bacteriorhodopsin IntroductionThe enrichment of molecules by stable isotopes (2Н, 13С, 15N, 18O) is an important tool for a variety of biochemical and metabolic studies with amino acids and other biologically active substances (BAS) [1]. The preferential usage of stable isotopes as compared to their counterparts are stipulated by the lack of radioactive radiation hazards and determination of the localization of the label in the molecule by high resolution techniques, including NMR [2], IR spectroscopy [3], and mass spectrometry [4]. The development of these methods for the detection of stable isotopes in biological probes in recent years has significantly increased the efficiency of biological research, as well as studies of the structure and mechanism of action of cellular BAS at the molecular level. In particular, 2H-and 13C-labeled amino acids are used for studying of the spatial structure and conformational changes of proteins, the interaction of protein molecules and in chemical syntheses of some isotope-labeled compounds based on them [5]. For example, isotopically labeled Lphenylalanine is used in the synthesis of peptide hormones and neurotransmitters [6].An important factor in studies using labeled amino acids is their accessibility. 2H and 13C-labeled amino acids can be prepared using ...

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“…The assimilation rate of methylotrophic biomass by protozoa and eukaryotic cells amounts to 85-98 %, while the productivity calculated on the level of methanol bioconversion into cell components makes up 50-60 % [28]. While developing the composition of growth media on the basis of deutereted biomass of methylotrophic bacteria B. methylicum it was taken into account the ability of methylotrophic bacteria to synthesize large amounts of protein (output, 50 % (w/w) of dry weight), 15-17 % (w/w) of polysaccharides, 10-12 % (w/w) of lipids (mainly, phospholipids), and 18% (w/w) of ash [29].…”

Section: Adaptation To Deuterium the Chemoheterotrophic Bacterium B mentioning

confidence: 99%

Studying Physiological, Morphological, and Cytological Alterations in Prokaryotic and Eukaryotic Cells in Heavy Water

Mosin¹,

Ignatov²,

Складнев³

et al. 2015

Russian Journal of Biological Research

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In this paper the biological influence of deuterium on cells of various taxonomic groups of prokaryotic and eucaryotic microorganisms realizing methylotrophic, chemoheterotrophic, photoorganotrophic, and photosynthetic ways of assimilation of carbon substrates (methylotrophic bacteria Brevibacterium methylicum, chemoheterotrophic bacteria Bacillus subtilis, photoorganotrophic halobacteria Halobacterium halobium, and green algae Chlorella vulgaris) was investigated at the growth on media with heavy water ( 2 H2O). For all investigated microorganisms are submitted data on growth and adaptation on growth media containing as sources of deuterated substrates 2 H2O, [ 2 H]methanol and hydrolysates of deutero-biomass of methylotrophic bacteria B. methylicum, obtained after the multistage adaptation to 2 H2O. The qualitative and quantitative composition of intra-and endocellular amino acids, proteins, carbohydrates and fatty acids in conditions of adaptation to 2 H2O is investigated. It is shown, that the effects observed at adaptation to 2 H2O, possess a complex multifactorial character and connected to physiological, morphological 143Russian Journal of Biological Research, 2015, Vol. (5), Is. 3 and cytological alterations -the magnitude of the lag-period, time of cellular generation, output of biomass, a parity ratio of synthesized amino acids, proteins, carbohydrates and lipids, and also with an evolutionary level of the organization of the investigated object and the pathways of assimilation of carbon substrates.Keywords: deuterium, heavy water, adaptation, isotopic effects, bacteria, micro algae. IntroductionThe most interesting biological phenomenon is the ability of some microorganisms to grow on heavy water ( 2 H2O) media in which all hydrogen atoms are replaced with deuterium [1, 2]. 2 H2O has high environmental potential in biomedical studies due to the absence of radioactivity and poccebility of detecting the deuterium label in the molecule by high-resolution methods as NMR, IR, and mass spectrometry that facilitates its use as a tracer in biochemical and biomedical research [3]. The average ratio of 1 H/ 2 H in nature makes up approximately 1:5700 [4]. In natural waters, the deuterium is distributed irregularly: from 0,02-0,03 mol.% for river water and sea water, to 0,015 mol.% for water of Antarctic ice -the most purified from deuterium natural water containing in 1,5 times less deuterium than that of seawater. O = 1894 . 10 -6 (1894 ppm). In surface waters, the ratio 2 H/ 1 H = ∼(1,32-1,51) . 10 -4 , while in the coastal seawater -∼(1,55-1,56) . 10 -4 . The natural waters of CIS countries are characterized by negative deviations from SMOW standard to (1,0-1,5) . 10 -5 , in some places up to (6,0-6,7) . 10 -5 , but however there are also observed the positive deviations at 2,0 . 10 -5 .The chemical structure of 2 H2O molecule is analogous to that one for Н2O, with small differences in the length of the covalent H-O-bonds and the angles between them. The molecular mass of 2 H2O exceeds on 10% th...

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Biosynthesis of²H-Labeled Phenylalanine by a New Methylotrophic MutantBrevibacterium methylicum

Cited by 35 publications

References 4 publications

Microbic synthesis of deuterium labelled L-phenylalanine with different levels of isotopic enrichment by facultative methylotrophic bacterium brevibacterium methylicum with RMP assimilation of carbon

Microbic synthesis of deuterium labelled L-phenylalanine with different levels of isotopic enrichment by facultative methylotrophic bacterium brevibacterium methylicum with RMP assimilation of carbon

The Development of Biosynthesis of 2Н- and 13С-labeled Amino acids and Proteins with Various Levels of Isotopic Enrichment Using Bacterial Objects

Studying Physiological, Morphological, and Cytological Alterations in Prokaryotic and Eukaryotic Cells in Heavy Water

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Biosynthesis of2H-Labeled Phenylalanine by a New Methylotrophic MutantBrevibacterium methylicum

Cited by 35 publications

References 4 publications

Microbic synthesis of deuterium labelled L-phenylalanine with different levels of isotopic enrichment by facultative methylotrophic bacterium brevibacterium methylicum with RMP assimilation of carbon

Microbic synthesis of deuterium labelled L-phenylalanine with different levels of isotopic enrichment by facultative methylotrophic bacterium brevibacterium methylicum with RMP assimilation of carbon

The Development of Biosynthesis of 2Н- and 13С-labeled Amino acids and Proteins with Various Levels of Isotopic Enrichment Using Bacterial Objects

Studying Physiological, Morphological, and Cytological Alterations in Prokaryotic and Eukaryotic Cells in Heavy Water

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Biosynthesis of²H-Labeled Phenylalanine by a New Methylotrophic MutantBrevibacterium methylicum