Microbic 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 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.

Nuclear Quantum Effects in Water and Aqueous Systems: Experiment, Theory, and Current Challenges

“…At the outset it is interesting to note that water's seeming ambivalence to quantum effects for some properties is reflected in the fact that prokaryotic organisms such as bacteria can tolerate pure D 2 O conditions (43), while deuterating about 30% of plant's and mammal's water can be lethal (44)(45)(46).…”

“…We have included values for H 2 18 O where available, because it has the same molecular mass as D 2 O, thus allowing one to compare the influence of the molecular mass, which changes upon 16 O/ 18 O substitution, and effects due to the moment of inertia and proton ZPE that change upon H/D substitution. At the outset, it is interesting to note that water’s seeming ambivalence to quantum effects for some properties is reflected in the fact that prokaryotic organisms such as bacteria can tolerate pure-D 2 O conditions, whereas deuterating about 30% of plants’ and mammals’ water can be lethal. − …”

Underrepresented Impurities in 4-Hydroxyphenylpyruvate Affect the Catalytic Activity of Multiple Enzymes

“…Probing the chemical pathway(s) of 4-HPP synthesis, we identified three potential routes (Scheme S1). Pathways 1 and 2 share 4-hydroxybenzaldehyde ( 5 ) as a key intermediate and reach the final product in a fewer steps in comparison to pathway 3 …”

“…Pathways 1 51 and 2 52 share 4-hydroxybenzaldehyde (5) as a key intermediate and reach the final product in a fewer steps in comparison to pathway 3. 53 We employed mass spectrometry to analyze the composition of TCI and Chemodex samples and detect ionizable precursor molecules or other contaminations that interfere with the activity of MIF. These two samples were chosen anticipating they would provide the clearest experimental outcome.…”

Fluorescent probing of biological particles suspensions

“…The assimilation rate of methylotrophic biomass by prokaryotic and eukaryotic cells makes up 85-98 %, and their productivity calculated on the level of methanol bioconversion into cell components reaches 50 % [12]. As we have earlier reported, methylotrophic bacteria are convenient objects capable to grow on minimal salt media containing 2-4 % [ 2 H]methanol, whereon other bacteria are unable to reproduce, and may easily be adapted to maximal 2 Н 2 О concentrations [13]. However, the experiments discovered a bacteriostatic effect of 2 Н 2 О consisted in inhibition of vitally important cell functions of the bacterial producer cells [14].…”