Microbiological synthesis of [2H]-inosine with a high degree of isotopic enrichment by the gram-positive chemoheterotrophic bacterium Bacillus subtilis

A 2H-labeled purine ribonucleoside inosine was microbiologically synthesized (yield, 3.9 g/L of culture liquid) using a deuterium-adapted strain of the gram-positive chemoheterotrophic bacterium Bacillus subtilis, cultivated in a heavy water medium with a high degree of deuteration (99.8 at % 2H) containing 2% hydrolysate of deuterated biomass of the methylotrophic bacterium Brevibacterium methylicum as a source of 2H-labeled growth substrate produced in an M9 minimal medium with 98% 2H2O and 2% [2H]-methanol. The inosine extracted from the culture liquid of the producer strain was fractionated by adsorption (desorption) on an activated carbon surface, extraction with 0.3 M ammonium-formate buffer (pH 8.9), subsequent crystallization in 80% ethanol, and ion exchange chromatography on a column with AG50WX 4 cation exchange resin equilibrated with 0.3 M ammonium-formate buffer containing 0.045 M NH4Cl. Fast atom bombardment (FAB) mass spectrometry demonstrated incorporation of five deuterium atoms in the inosine molecule (62.5% 2H), three of which were contained in the ribose moiety and two in the hypoxanthine moiety.