O.V. Mosin
Ph.D. (Chem.), Scientific Emploer, Moscow State University of Applied Biotechnology
E-mail: mosin-oleg@yandex.ru
I. Ignatov
Dr.Sc. (Eng.), Professor, Scientific Research Center of Medical Biophysics (Sofia, Bulgaria)
E-mail: mbioph@dir.bg
G. Tyminski
Dr.Sc. (Med.), Chairman of European Scientific Society (Europäische Wissenschaftliche Gesellschaft) (Hannover, Germany)

2H- and 13C-labelled amino acids in composition of secreted into culture media and total protein hydrolysates were synthesized microbiologically by growing methylotrophic bacteria Brevibacterium methylicum and Methylobacillus flagellatum on media containing as a source of stable isotopes [2H]methanol, [13C]methanol and 2H2O, and analyzed by electron impact mass-spectrometry. For mass-spectrometric analysis the multi-componential mixtures of 2H- and 13C-labeled amino acids, derived from culture 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 in methyl esters of N-dansyl-derivatives of amino acids, which were preparative separated using the reverse-phase HCLP. 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 amino acids were varied 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. In all experiments there was a specific increase in the levels of isotopic incorporation of deuterium into molecules of individual [2H]amino acids in the composition of culture liquids with stepwise increasing concentrations of 2H2O in growth medium. The inclusion levels of deuterium into molecules of different [2H] amino acids under the same growing conditions are varied. In all experiments was observed the proportional increase in the levels of isotopic incorporation of 2H into the molecules of metabolically related [2H]amino acids with stepwise increasing concentrations of heavy water in the growth media. This result was recorded in all experiments, where as a source of stable isotopes was used 2H2O. From mass spectrometry data is demonstrated that at other concentrations of 2H2O in growth media deuterium the character of labeling was also specific since the metabolism of the strain of B. methylicum adapted to 2H2O does not undergo significant changes in 2H2O. Under conditions of auxotrophy in L-leucine the levels of inclusion of 2H into the molecules of [2H]leucine/[2H]isoleucine are lower than those for phenylalanine. This feature more clearly manifests in the medium with the highest concentration of 2H2O. Summarizing the data on the level of incorporation of 13C and 2H isotopes into secreted molecules of amino acids, it can be concluded about the maintaining of minor metabolic pathways associated with the biosynthesis of leucine and the metabolically related amino acids de novo. Another logical explanation for the observed effect, if we take into account the origin of leucine and isoleucine due to biosynthesis in various pathways (leucine belongs to the family of pyruvate, while isoleucine – to the family of aspartate could be assimilation by the cell of the unlabeled leucine from the growth media under the background biosynthesis of isotopiclabeled isoleucine de novo. Taking into account these effects it should be emphasized that the use of auxotrophic forms of microorganisms for the production of isotope-labeled amino acids could not be justified practically because of the multiple character of inclusion of isotopes into the molecule.

Key words: stable isotopes
methylotrophic bacteria
isotope labeled amino acids.