Methylotrophic Biomass as Substrate for Cultivation of Chemoheterotrophic Bacterium Bacillus Subtilis B-3157

Abstract

We propose to use a hydrolyzed deuterated biomass of the facultative methylotrophic bacterium Brevibacterium methylicum B-5662 as a source of ²H-labeled growth substrates for microbiological preparation of ²H-labeled purine ribonucleoside inosine, excreted into the liquid microbial culture (LC) by a Gram-positive chemoheterotrophic bacterium Bacillus subtilis B-3157. The bacterium was grown on heavy water (HW) medium with 2% (v/v) hydrolysate of deuterated biomass of the methylotrophic bacterium Brevibacterium methylicum B-5662 obtained on minimal salt media M9 supplemented with 2% (v/v) [U-²H]MeOH and increasing gradient of ²Н₂O concentration from 0; 24.5; 49.0; 73.5 up to 98% (v/v) ²Н₂O. 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 incorporation of 5 deuterium atoms into the molecule (the total level of deuterium enrichment – 65.5 atom% ²H) with 3 deuterium atoms being included into the ribose and 2 deuterium atoms – into the hypoxanthine residue of the molecule.

Key words: ²H-labeled inosine, biosynthesis, biosynthetic pathways, heavy water, Bacillus subtilis, Brevibacterium methylicum