Deuterated Methylotrophic Biomass as a Substrate for Microbiological Synthesis of 2H-Labeled Purine Ribonucleoside Inosine by Chemoheterotrophic Bacterium Bacillus Subtilis B-3157

Abstract

We propose to use the hydrolyzed deuterated biomass of the facultative methylotrophic bacterium Brevibacterium methylicum B-5662 as a source of 2 H-labeled growth substrates for microbiological synthesis of 2 H-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) [ 2 H]methanol and increasing gradient of 2 Н 2 O concentration from 0; 24,5; 49,0; 73,5 up to 98% (v/v) 2 Н 2 O. Isolation of 2 H-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 NH 4 Cl. 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% 2 H) with 3 deuterium atoms being included into the ribose and 2 deuterium atoms -into the hypoxanthine residue of the molecule.