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

Prikladnaia biokhimiia i mikrobiologiia (2013-07-26)
O V Mosin, V I Shvets, D A Skladnev, I Ignatov
ABSTRACT

A 2H-labeled purine ribonucleoside inosine was microbiologically synthesized (yield, 3.9 g/L of culture liquid) using a deuterium-adapted strain ofthe 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% 2H20 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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Inosine, ≥99% (HPLC)
Sigma-Aldrich
Deuterium, 99.9 atom % D
Sigma-Aldrich
Deuterium, 99.96 atom % D
Sigma-Aldrich
Deuterium, 99.8 atom % D