A Strain of a Gram-positive Chemoheterotrophic Bacterium Bacillus Subtilis B-3157 – producer of 2H-Labeled Purine Ribonucleoside Inosine

Abstract

It was carried out the microbiological preparation of ²H-labeled purine ribonucleoside inosine excreted into liquid microbial culture (LC) by Gram-positive chemoheterotrophic bacterium Bacillus subtilis B-3157 while growing of this bacterium on heavy water (HW) medium with 2% (v/v) hydrolysate of deuterated biomass of the methylotrophic bacterium Brevibacterium methylicum B-5662 as a source of ²H-labeled growth substrates. Isolation of ²H-labeled inosine from LC was performed by adsorption/desorption on activated carbon with following extraction by 0.3 M ammonium–formate buffer (pH = 8.9), crystallization in 80% (v/v) EtOH, 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 investigation of deuterium incorporation into the inosine molecule by FAB method demonstrated the incorporation of 5 deuterium atoms into the molecule (the total level of deuterium enrichment – 65.5 atom% ²H) with 3 deuterium atoms included into the ribose and 2 deuterium atoms – into the hypoxanthine residue of the molecule. Three non-exchangeable deuterium atoms were incorporated into the ribose residue owing to reactions of enzymatic izomerization of glucose in ²H₂O-medium. These non-exchangeable deuterium atoms in the ribose residue were originated from glycolysis, associated with the Embden-Meyerhof pathway, while two other deuterium atoms at C2,C8-positions in the hypoxanthine residue were synthesized from [²H]amino acids (glutamine, glycine, aspartate) that originated from the deuterated hydrolysate of the methylotrophic bacterium Brevibacterium methylicum B-5662. However, auxotrophy of this strain in tyrosine, histidine, adenine and uracil, presupposes the branched metabolic pathways, different those indicated above.

Keywords: ²H-labeled inosine, biosynthesis, Embden-Meyerhof pathway, heavy water, Bacillus subtilis