It was proposed to use the ² H-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) ² H₂ O and supplimented with 2.5% (m/m) of ² H-labeled methylotrophic hydrolysate. To evaluate the level of deuterium enrichment FAB MS technique was used after the isolation of ² H-labeled inosine. ² H-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.

Keywords: ² H-labeled growth substrates - Bacillus subtilis - Biosynthesis - ² H-labeled inosine

Introduction

Nucleosides labeled with deuterium (² H) 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 ² H-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 ² H-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.% ² H₂ O far [8]. 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 ² H-biomass prepared from ² H₂ O and [U -² H]MetOH [9, 10]. In this article, we demonstrate the possibility of using the hydrolysates of ² H-labeled biomass of this bacterium as substrates for growing the inosine producing mutant B. subtillis .

Materials and methods

Chemicals

² H₂ O (99.9 at.% ² H[1] ) was obtained from Russian Scientific Enterprises, Sanct Petersburg and purified by distillation from alkaline permanganate. [U -² H]methanol (95.7 at.% ² H) was from Biophysic Center, Pushino. All other chemicals were of reagent grade.

To create a high isotopic content in growth medium, ² H₂ O with trade marked isotopic purity 99.9 at.% ² H, was used. However, the deuterium content of used ² H₂ O verified by NMR was found to be 97 at.% ² H. The water containing salts were several times preliminarily crystallyzed in pure ² H₂ O 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 ² H₂ O.

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 GNIIGENETIKA.

Preparation of ² H-labeled growth substrates

The methylotrophic bacterium B. methylicum # 5662 was grown on salt medium dM9 with 93.5% (m/m) ² H₂ O and 2% (m/m) [U -² H]MetOH in mass culture [11]. Cells were pelleted by centrifugation (2000 g, 10 min), washed once with ² H₂ O and stored at -14 ⁰ C. Periodically, 10 g (wet weight) portions are thawed, suspended in 0.5 N ² HCl solution (in ² H₂ O) and autoclaved at 120⁰ C for 30 min. After adjusting pH till 7.0-7.2 with potassium hydroxide, the hydrolysate was used as a mixure of ² H-labeled growth substrates for the growth ofinosine producing strain.

Media and growth conditions

The 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 ² H₂ O and the hydrolysate of ² H-labeled methylotrophic biomass was added. The media were sterilized by autoclaving at 120⁰ C for 30 min and cooled. Glucose was sterilized separetely in ² H₂ O 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 ⁰ С 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 ² H-labeled inosine.

Isolation of inosine

MetOH solution in H₂ O (50 v/v %, 20 ml) was added to a lyophilized growth medium. The mixture was allowed to - 4 ⁰ C 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 ² H₂ O (30 ml) and 5 g of activated carbon was added. After keeping for 24 h at -4 ⁰ C, the inosine, eluting with ammonia, was concentrated and twice recrystallized from MetOH (n^d ₂₀ = 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.

Quantitative determination

During 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.

The convertion of glucose was estimated enzymatically with glucoseoxydenase method [].

Equipment

Absorbance was measured with a spectrophotometer Beckman DU-6 (USA).

The 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 18.5 ml/h.

The 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 [].

FAB 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 ² H-labeled inosine

For biosynthesis of ² H-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 ² H-labeled biomass prepared from yeast, it was necessary to find the more suitable microbial source, from which the ² H-labeled growth substrates could be obtained. For this purpose we employed the available RuMP facultative methylotroph Brevibacterium methylicum [5] with the content of the total protein and polycarbohydrates in biomass 53 and 10% respectively [6].

The content of amino acids in biomass of B. methylicum and the deuterium enrichment are shown in Table.

Table:

The content of amino acids in biomass of B. methylicum and the deuterium enrichment.

Amino Acids	The content in biomass, %	Deuterium enrichment , %
Glycine	9,69	90,0
Alanine	13,98	97,5
Valine	3,74	50,0
Leucine/Isoleucine	7,33/3,64	49,0
Phenylalanine	3,94	95,0
Tyrosine	1,82	92,8
Serine	4,90	90,0
Threonine	5,51	not determined
Methionine	2,25	not determined
Aspartic Acid	9,59	66,6
Glytamic Acid	10,38	70,0
Lysine	3,98	58,9
Arginine	5,27	not determined
Histidine	3,72	not determined

The hydrolysis of ² H-labeled biomass was performed in mild conditions via its autoclaving (30 min, 08 atm) in 0.5 N solution of ² HCl (in ² H₂ O). 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.

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