Methylotrophic biomass as 2H-labeled substrate for biosynthesis of inosine
Methylotrophic biomass as 2H-labeled substrate for biosynthesis of inosine
Methylotrophic biomass as 2H-labeled substrate for
biosynthesis of inosine
Oleg V. Mosin1
1 M. V. Lomonosov State
Academy of Fine Chemical Technology, Vernadskogo Prospect 86, Moscow, 117571
It was proposed to use the 2H-labeled hydrolysate
of RuMP facultative methylotroph Brevibacterium methylicum, obtained
from deuterated salt medium dM9 as a substrate for the growth of inosine
producing bacterium Bacillus subtilis. The growth of the bacterim was
performed via glucose convertion on specially developed medium dHM with 78.5%
(m/m) 2H2O and supplimented with 2.5% (m/m) of 2H-labeled
methylotrophic hydrolysate. To evaluate the level of deuterium enrichment FAB
MS technique was used after the isolation of 2H-labeled inosine. 2H-labeled
inosine obtained from dHM medium represented a mixture of molecular species
containing various number of included deuterium atoms with different
contribution to the enrichment. The level of enrichmet calculated by the
presence of most abandant peak of the molecular ion in cluster ((M+H)+
at m/z 274) was estimated as five deuterium atoms, from which three are
attributed to ribose and two to hypoxantine.
Nucleosides labeled with deuterium (2H) and other
stable isotopes are becoming an indispensable tool for biomedical diagnostic
and the investigation of various aspects of the metabolism [1, 2]. Thus inosine
which is known as an important intermediate in the synthesis of inosine
monophosphate (IMP) is in the focal point of clinical interest in medical
diagnostic of heart deceases and in certain medical cases [3, 4].
There are several
approaches reported for the preparation of 2H- nucleosides.
Chemical synthesis are usually tedious and inefficient. Only by employing
mutant forms of bacteria, which can produce a large quantities of the
nucleosides when growing of an organism on media containing deuterated
substrates, the desired biochemicals can be obtained both with high yields and
enrichments. On the microbial production of inosine, there have been many
studies so far [5-7]. .
For instance, a certain adenine, histidine and tyrosine
auxotrophic mutants derived from Bacillus subtilis have been found to
have a remarkable ability to produce a large amount of inosine in the growth
medium, and at the present it may be produced on an industrial scale.
The major disadvantage
of production of 2H-nuclesides is difficulty in obtaining the
appropriate deuterated growth substrates. One approach to solve this problem is
to use the extracts obtained from microorganisms growing on minimal media with
99,9 at.% 2H2O far . Thus, we recently described a
facultative methylotrophic bacterium Brevibacterium methylicum, which
seems to be an an ideal source for the preparation of uniformelly labeled
growth substrates on the basis of its 2H-biomass prepared from 2H2O
and [U -2H]MetOH [9, 10]. In this article, we demonstrate the
possibility of using the hydrolysates of 2H-labeled biomass of this
bacterium as substrates for growing the inosine producing mutant B. subtillis.
Materials and methods
(99.9 at.% 2H) was obtained from Russian
Scientific Enterprises, Sanct Petersburg and purified by distillation from
alkaline permanganate. [U -2H]methanol (95.7 at.% 2H) was
from Biophysic Center, Pushino. All other chemicals were of reagent grade.
To create a
high isotopic content in growth medium, 2H2O with trade
marked isotopic purity 99.9 at.% 2H, was used. However, the
deuterium content of used 2H2O verified by NMR was found
to be 97 at.% 2H. The water containing salts were several times
preliminarily crystallyzed in pure 2H2O and dried in
vacuum before using (the true content of deuterium in growth media after the
autoclaving was less smaller on 8-10% then isotopic purity of an initial 2H2O.
The bacterial strain
Adenine, tyrosine and hystidine
auxotroph mutant B. subtilis B -3157 capable to produce and accumulate
17 g/liter of inosine during the growth on protonated medium with glucose and
yeast extract was employed. The strain was obtained from Russian State
Scientific Center for Genetics and Selection of Industrial Microorganisms
Preparation of 2H-labeled growth substrates
methylotrophic bacterium B. methylicum # 5662 was grown on salt medium
dM9 with 93.5% (m/m) 2H2O and 2% (m/m) [U -2H]MetOH
in mass culture . Cells were pelleted by centrifugation (2000 g, 10 min),
washed once with 2H2O and stored at -14 0C. Periodically,
10 g (wet weight) portions are thawed, suspended in 0.5 N 2HCl
solution (in 2H2O) and autoclaved at 1200C for
30 min. After adjusting pH till 7.0-7.2 with potassium hydroxide, the
hydrolysate was used as a mixure of 2H-labeled growth substrates for
the growth ofinosine producing strain.
Media and growth conditions
bacterial growth was carried out on FM medium (m/m.%): glucose 12; yeast
extract 2.5; ammonium nitrate 3; magnium sulphate 2; chalk 2. The composition
of dHM was as the same as FM except dHM was prepared from 2H2O
and the hydrolysate of 2H-labeled methylotrophic biomass was added.
The media were sterilized by autoclaving at 1200C for 30 min and
cooled. Glucose was sterilized separetely in 2H2O
solution, and after that added in growth medium. рН was adjusted till 6.5-6.7 with potassium hydroxide. The
bacterium was grown in 250 ml Erlenmeyer flasks containing 20 ml of the medium
at 32-34 0С and vigorously
aerated on an orbital shaker. After 7 days the cells were pelleted by centrifugation
(2000 g, 10 min). The supernatant was separated, lyophilized and used for the
isolation of 2H-labeled inosine.
Isolation of inosine
solution in H2O (50 v/v %, 20 ml) was added to a lyophilized growth
medium. The mixture was allowed to - 4 0C and after 10 h the total
protein was precipitated and removed by centrifugation (1200 g, 10 min). MetOH
was evaporated under reduced pressure. The resulting mixture was dissolved in 2H2O
(30 ml) and 5 g of activated carbon was added. After keeping for 24 h at -4 0C,
the inosine, eluting with ammonia, was concentrated and twice recrystallized
from MetOH (nd20 = 1.33). The purity of the product was
judged by using controls of normal nucleosides, and running mixed TLC with
graded amounts of the neighboring nucleosides.
the growth inosine was separated by TLC on Silufol UV-254 plates with mobile
phases: n -ButOH - AcOH - water (2:1:1, v/v) using pure commercial available
inosine as a standard. The amount of inosine was determined for 10 ml aliquots
of liquid growth medium by TLC. The sports were eluted by 0.1 N solution of
HCl (10 ml). The absorbance of the eluates was measured at 249 nm and the
content of inosine was determined using a standard curve.
convertion of glucose was estimated enzymatically with glucoseoxydenase method
was measured with a spectrophotometer Beckman DU-6 (USA).
analysis of protein hydrolisates was carried out using a Biotronic LC 50001
chromatograph (Germany), 230 x 3.2 mm, working pressure 50-60 atm, flow-rate
levels of deuterium enrichment of amino acids were investigated with the aid of
EI MS after derivatization to methyl esters of N-Dns-amino acids .
MS was performed on Hitachi MBA spectrometer (Japan) on glyserol template at
potential 5 кV and an ion current of 0.6-0.8 мА.
RESULTS AND DISCUSSION
Production of 2H-labeled inosine
biosynthesis of 2H- labeled inosine we employed bacterium
Bacillus subtillis, which could produce and accumulate a conciderable
amount of inosine exogeniously due to an altered nucleoside metabolism. This
strain displayed the maximum productivity on FM medium, containing as a source
of carbon and energy glucose (12 m/m.%), and as a source of growth factors and
additional source of nitrogen the yeast extract. Since the small availability
of commercial available 2H-labeled biomass prepared from yeast, it
was necessary to find the more suitable microbial source, from which the 2H-labeled
growth substrates could be obtained. For this purpose we employed the available
RuMP facultative methylotroph Brevibacterium methylicum  with the
content of the total protein and polycarbohydrates in biomass 53 and 10%
The content of amino acids in biomass of B. methylicum
and the deuterium enrichment are shown in Table.
The content of amino acids in biomass of B. methylicum
and the deuterium enrichment.
content in biomass, %
enrichment , %
hydrolysis of 2H-labeled 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
Bacterial growth and production of inosine
following media were used for the bacterial growth:
FM medium, prepared from ordinary protonated water and yeast extract.
dHM medium, prepared from 87.5% (v/v) 2H2O and 2.5% (m/m)
of 2H-labeled methylotrophic hydrolisate, obtained accordingly from
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.
The evaluation of inosine enrichment
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.
two aspects of the enrichment of inosine were taken into account (scheme).
First, because protons in С’1-С’5 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%).
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