Studying of the Biosynthesis of 2H-Labeled Inosine by a Gram-positive Chemoheterotrophic Bacterium Bacillus Subtilis B-3157 on Heavy Water (2H2O) Medium

Abstract

We studied the biosynthesis of ²H-labeled purine ribonucleoside inosine (output, 3.9 g/l of liquid microbial culture (LC)) using a strain of the Gram-positive chemoheterotrophic bacterium Bacillus subtilis B-3157, adapted to deuterium. The strain was grown on heavy water (HW) medium with high degree of isotopic purity (99.9 atom% ²H) containing 2% (v/v) hydrolysate of deuterated biomass of the methylotrophic bacterium Brevibacterium methylicum B-5662 as a source of ²H-labeled growth substrates, obtained in minimal M9 growth medium with 98% (v/v) ²H₂O and 2% (v/v) [²H]methanol. Inosine was isolated from LC by adsorption/desorption on activated carbon with following extraction by 0.3 M ammonium–formate buffer (pH = 8.9), subsequent crystallization in 80% (v/v) ethanol, and ion exchange chromatography (IEC) on a column with AG50WX 4 cation exchange resin equilibrated with 0.3 M ammonium–formate buffer and 0.045 M NH₄Cl. The evaluation of the level of deuterium enrichment performed by fast atom bombardment (FAB) mass spectrometry demonstrated incorporation of 5 deuterium atoms into the inosine molecule (the total level of deuterium enrichment – 65.5 atom% ²H); 3 deuterium atoms were included into the ribose and 2 deuterium atoms – into the hypoxanthine residue of the molecule.

Key words: ²H-labeled inosine, biosynthesis, heavy water, Bacillus subtilis