European Journal of Molecular Biotechnology, 2013, Vol.(2),  2 63 UDC 579.871.08 Use of Gram-positive Chemoheterotrophic Bacterium Basillus subtilis В-3157 with HMP-cycle of Carbon Assimilation for Microbiological Synthesis of [ 2 H]riboxine with High Level of Deuterium Enrichment 1 Oleg Mosin 2 Ignat Ignatov 3 Dmitry Skladnev 4 Vitaly Shvets 1 Moscow State University of Applied Biotechnology, Russian Federation Senior research Fellow of Biotechnology Department, Ph. D. (Chemistry) 103316, Moscow, ulitza Talalihina, 33 E-mail: mosin-oleg@yandex.ru 2 T h e Scientific Research Center of Medical Biophysics (SRC MB), Bulgaria Professor, D. Sc., director of SRC MB. 1111, Sofia, N. Kopernik street, 32 E-mail: mbioph@dir.bg 3 Scientific Center of Russian Federation Research Institute for Genetics and Selection of Industrial Microorganisms ―Genetika‖, Russian Federation Professor, D. Sc (Biology), leading scientist of Genetika 117545, Moscow, 1st Dorozhniy proezd, 1 E-mail: genetika@genetika.ru 4 M . V . L o m o n o s o v Moscow University of Fine Chemical Technology, Russian Federation Academician of Russian Academy of Sciences, D. Sc. (Chemistry), head of department of biotechnology and nanobiotechnology. 119571, Moscow, Vernadskogo avenue, 86 E-mail: mitht@mitht.ru Abstract. We studied the growth and biosynthetic properties of a strain of Gram-positive chemoheterotriphic bacterium Bacillus subtilis В-3157 producer of 2 H-labeled purine ribonucleoside riboxine (outcome is 3,9 g/l) in heavy hydrogen (HH) medium with high level of deuterium enrichment (99,8 at.% 2 H2O) with 2 % hydrolysate of deuterated biomass of methylotrophic bacterium Brevibacterium methylicum B-5662 as a source of 2 H-labeled growth substrates, obtained in the minimal М9 growth medium of 98 % 2 Н2О and 2 % [ 2 H]methanol. Isolation of riboxine from liquid culture of riboxine producer strain was carried out by adsorption/resorption on a surface of activated carbon coal, extraction by 0.3 M NH4-formiate buffer (Н = 8.9) with the subsequent crystallization in 80 % of ethanol and column ion exchange chromatography on cation exchanger AG50WX 4, counterbalanced with 0.3 M NH4-formiate with 0.045 M NH4Cl (outcome of riboxine is 3.1 g/l (80 %)). The level of deuterium enrichment of biosynthetically prepared riboxine, analyzed by a method of fast atom bombardment (FAB) mass- spectrometry makes up 5 deuterium atoms with incorporation of 3 deuterium atoms into ribose and 2 deuterium atoms into hypoxantine fragments of the molecule. Keywords: Bacillus subtilis; [ 2 H]riboxine; biosynthesis; heavy water; fast atom bombardment mass spectrometry (FAB).