Methylotrophic biomass as 2H-labeled substrate for biosynthesis of inosine

The method of FAB MS was employed for the evaluation of inosine enrichment. The fragmentation pathways of inosine by FAB

Methylotrophic biomass as 2H-labeled substrate for biosynthesis of inosine

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biomass was performed in mild conditions via its autoclaving (30 min, 08 atm) in 0.5 N solution of 2HCl (in 2H2O). The data on the amino acid composition of hydrolysate and levels of the enrichment are shown in Fig. 2. The contents of tyrosine and histidine in hydrolysate were 1.82 and 3.72% and can ensure the polyauxotrophy of the inosine producing strain. Another important parameter is a high level of amino acid enrichment.

Bacterial growth and production of inosine

Two following media were used for the bacterial growth:

1). FM medium, prepared from ordinary protonated water and yeast extract.

2). dHM medium, prepared from 87.5% (v/v) 2H2O and 2.5% (m/m) of 2H-labeled methylotrophic hydrolisate, obtained accordingly from medium dМ9.

 

Fig.1

 

Curves, reflecting the growth dynamics (a), convercion of glucose (b) and production of inosine (c) are given in Fig. 1. A maximal level of inosine production on ordinary protonated medium was 17 g\liter. When growing on dHM medium the strain produced only 3.9 g/liter of inosine throughout the whole course of the growth. The low level of inosine production was correlated with a degree of glucose conversion in those conditions. 4m/m.% of non-assimilated glucose was detected in medium dHM after the growth, that proved that glucose is metabolized less effectivelly on medium dHM, that is probably a result of non-equvalent replacement of yeast extract by methylotrophic hydrolysate.

The absorption spectra of inosine isolated from medium dHM (a) are shown in Fig. 2 comparatively to the growth medium (b) and commertially available inosine (c). TLC of isolated inosine showed the presence of main spot with Rf=0.5 (inosine) and additional spot with Rf=0.75 (hypoxantine). The output of 2H-labeled inosine was 1 gram from 1 liter of growth medium.

Fig.2

The evaluation of inosine enrichment

The method of FAB MS was employed for the evaluation of inosine enrichment. The fragmentation pathways of inosine by FAB MS are shown in Fig. 3. Two main decomposition processes arised from the molecule: sugar (m/z 133) and hypoxantine (m/z 136) formation. The compounds with a smaller m/z ratio may further to be formed as a result of elitination of HCN and CO from hypoxantine. The level of deuterium enrichment could be evaluated from the FAB mass spectrum of 2H-labeled inosine shown in Fig. 3, b compared with the non-labeled inosine (a). The results, firmely established the labeling of inosine as heterogenious, juging by the presence of clasters of adduct peaks at the molecular ion MH+; the species of molecules with different numbers of deuterium atoms were visualised. The most abundant peak with (M+H)+ at m/z 274 (instead of m/z 269 for non-labeled compound) in the claster was registered by mass spectrometer as a peak with average m/z ratio, from whom the enrichment of inosine was calculated as five deuterium atoms. The presence of peak corresponding to the hypoxantine fragment [C5H4ON4]+ at m/z 138 (instead of m/z 136) and the peak of sugar fragment [C5H9O4]+ at m /z 136 (instead of m/z 133) proved that two deuterium atoms are located in hypoxantine, however, three of them are attributed to the ribose pattern.

Fig.3

Mainly two aspects of the enrichment of inosine were taken into account (scheme). First, because protons in С1-С5 positions of ribose pattern in inosine could be originated from glucose, we assumed, that the character of biosynthetic enrichment of deuterium in sugar pattern of inosine is determined mainly to the functioning of a number of processes of hexose monophosphate shunt of glucose assimilation. But since protonated glucose was added in growth medium, its contribution in the inosine enrichment was minimal. Nevertheless, the results suggested, that ribose contained three deuterium atoms that could not stemp from glucose. Three deuterium atoms probably stemp via some minor reactions of glucose biosynthesis. Secondly, the numerous exchange processes and intermolecular regrouping reactions, occurring with participation of 2H2O could also be resulted in specific labelling of inosine. Such accessible positions are occupied by the easily exchangeable hydrogen (deuterium) atoms both of hydroxylic- and imino groups of inosine. Two protons at C-H positions in inosine could be replaced by deuterium via assimilation of 2H-labeled hydrolysate. The enrichment of inosine was approximately the same as 2H2O content in growth medium (65.5-67.5%).

 

 

LITERATURE.

 

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