RU2100434C1 - Method of preparing the uniformly isotope-modified natural l-$$$-amino acids - Google Patents

Abstract

FIELD: biotechnology, organic chemistry, biochemistry. SUBSTANCE: uniformly isotope-modified natural amino acids were prepared by incubation of strains - producers of the corresponding amino acid. Firstly, cells were grown on medium with glucose and acetate as carbon sources. Then cells were separated, washed out and used for preparing a suspension on the salt medium with a single carbon source as isotope-modified acetate as sodium acetate and/or ammonium acetate modified with isotopes $$$ or $$$ and/or $$$. New compounds modified with $$$ and $$$ were obtained first. EFFECT: improved method of preparing. 5 cl, 1 tbl

Description

 The invention relates to the field of biotechnology and organic chemistry, to methods for producing isotopically modified natural compounds, namely: L-α-amino acids, and may find application in experimental biology, medicine, veterinary medicine, and agriculture.

A known method for producing uniformly isotopically modified natural La-amino acids by incubating microorganism cells on a medium containing a single carbon source in the form of isotopodified salts of acetic acid with subsequent isolation of the target product. As an isotopically modified salt in this known method, ¹⁴ C sodium acetate is used. Cells of the microorganism Bacillus pumilus, when grown in the presence of this carbon source, incorporate ¹⁴ C into their body matter. After the incubation is completed, the cell protein is hydrolyzed and the target products are isolated and homogeneous isotopically modified natural La amino acids are isolated from the hydrolyzate.

The disadvantage of this method is the complicated procedure for separating the hydrolyzate of the protein of the cells of the microorganism into individual isotopically modified La-amino acids. In addition, La-amino acids modified only with the ¹⁴ C isotope are obtained by this known method. Recently, interest in natural compounds, including amino acids labeled with the stable ¹³ C isotope, has increased. But such compounds are required in large quantities to work with them. inaccessible to receive them in a known manner. Most of the natural La amino acids labeled with the stable ¹³ C isotope have not yet been obtained and been described. Not described and natural La-amino acids that are uniformly modified by two isotopes: ¹⁴ C and ¹⁵ N or ¹³ C and ¹⁵ N.

The technical result achieved by the implementation of the present invention is to obtain new compounds uniformly isotopically modified with the ¹³ C isotope or ¹³ C and ¹⁵ N isotopes, as well as simplifying the production of natural La-amino acids uniformly modified with the ¹⁴ C isotope or ¹⁴ C and ¹⁵ N isotopes.

 This is achieved by the fact that in the method of producing uniformly isotopically modified natural La-amino acids by incubating microorganism cells on a medium containing a single carbon source in the form of isotopodified salts of acetic acid, followed by isolation of the target product, the distinctive feature is that cells of microorganism strains of the producers of the target product are used that are pre-incubated on a medium containing carbon sources in the form of salts of acetic acid in combination with carbohydrates odes, followed by separation and washing of the obtained cells, and upon completion of incubation on a medium with uniformly isotopically modified acetic acid salts, the cells are separated and discarded, and the target product is isolated from the medium.

 In this case, the strains of La-amino acid producers are the lysine producer Corynebacterium glutamicum VKPM B-38, the glutamic acid producer Corynebacterium glutamicum VKPM B-832, the producer of Leucine Brevibacterium flavum VKPM B-2736, the producer of isoleucine Breavumacterium Brevibacterium Brevibacterium Brevivacterium Breviberumium Brevibacterium Breviberumium Brevivacterium Breviverium bacteriumprevent flavum VKPM B-1280, a serine producer Brevibacterium flavum VKPM B-3559, a histidine producer of Corynebacterium glutamicum VKPM B-4380, and the producer of alanine is a producer of glutamic acid Corynebacterium glutamicum VKPM B-8iderum glutamicum VKPM Bistidum gistamidum bistum gumbistum gumbistum gumbistum

In the method, sodium acetate and / or ammonium acetate, unmodified or uniformly modified with ¹³ C or ¹⁴ C isotopes, are used as acetic acid salts, moreover, ammonium acetate can be further modified with the ¹⁵ N isotope.

In the method of isolation, along with the target isotopically modified amino acid of its accompanying amino acids, the degree of conversion of carbon isotopes from isotopically modified salts of acetic acid to the resulting isotopically modified amino acids is increased. To do this, after passing the incubation mixture supernatant through Dowex and eluting with ammonia solution, the eluate is additionally passed through Amberlite GC-50 II in copper form, the amino acid fractions are eluted with ammonia solution and passed through carboxyl methyl cellulose in H ⁺ form.

New compounds of natural La amino acids were obtained: alanine, lysine, glutamic acid, leucine, isoleucine, threonine, serine and histidine, uniformly modified with the ¹³ C isotope or ¹³ C and ¹⁵ N isotope.

Example 1: An overnight culture of strains of cells grown in complete medium, grown on salt medium containing 1% sodium acetate, 0.2% glucose and mineral salts: KH ₂ PO _4, NaCl, / NH _4/2 SO ₄ and MgSO ₄ ; the medium has a neutral pH value. As the culture consumes acetate ion, the pH of the medium rises, and therefore the medium is periodically neutralized by the addition of acetic acid. When the culture reaches the late logarithmic phase of growth, the biomass is precipitated by centrifugation and poured with the same volume of the same medium, but without glucose. In this case, the fresh medium contains uniformly modified sodium acetate and / or ammonium acetate. The concentration of cells in fresh medium is high and therefore, during the incubation of the resulting cell suspension, they practically do not grow, but they catalyze the biosynthesis of uniformly labeled amino acids from uniformly labeled salts of acetic acid, which is accompanied by an increase in the pH of the incubation mixture. The mixture is periodically neutralized by the addition of hydrochloric acid. The incubation time is 1-2.5 days at 30-37 ^o C depending on the strain used.

 In cases where the cells accumulate only one amino acid, the incubation mixture is centrifuged after incubation, the supernatant is passed through a Dowex ion exchange resin, and after elution with an ammonia solution, the desired amino acid is isolated from the eluate. If the used producer strain accumulates in the incubation mixture not only the target, but also other amino acids accompanying it, then they are also isolated.

The separation of the basic and associated amino acids is carried out using ligand exchange chromatography on a carboxylic sorbent filled with copper ions. The degree of charge is 100, 70, 30%, depending on the set of amino acids in the incubation medium. Amberlite GC-50 II (particles with a size of 200-400 mesh /, passes through it an amino acid eluate obtained after passing the supernatant through Dowex 50 and removing ammonia. The elution with ammonia solution collects fractions of amino acids in the form of their copper complexes. Copper is removed. passing the eluate fractions through carboxymethyl cellulose in H ⁺ form.

 Example 2. Obtaining isotopically modified lysine.

An overnight culture of cells of Corynebacterium glutamicum strain VKPM B-38 lysine producer, inoculated on a medium containing 0,2% glucose, 0,1% KH ₂ PO _4, 1% / NH _4/2 SO _4, 0,04% MgSO ₄ , 1.5% sodium acetate and 0.05% NaCl and incubated at 30 ^o C for a day. The resulting cells are separated by centrifugation, washed, poured with the same volume of the same salt medium, but without glucose containing uniformly isotopically modified sodium acetate and / or ammonium acetate. The cell suspension is incubated at 30 ^o C for 8 hours. The incubation mixture is centrifuged, the supernatant is passed through Dowex 50 • 8 in NH $\genfrac{}{}{0ex}{}{\text{+}}{\text{4}}$ form. Elute lysine with 2N ammonia solution. Ammonia is removed by evaporation, lysine is recrystallized from ethanol (see table).

Example 3. Obtaining isotopically modified glutamic acid
The culture of Corynebacterium glutamicum VKPM B-832 was used as the producer strain. When incubating a suspension of washed cells under the conditions of Example 2, but for 16 hours, along with glutamic acid, the formation of small amounts of alanine is observed. The incubation mixture is centrifuged, the supernatant is passed through Dowex 1 • 8 in formate form. Elute 0.1 N. formic acid solution. Alanine is purified on a Dowex 50 • 8 in the H ⁺ form, eluting with 0.5 n. ammonia solution (see table).

Example 4. Obtaining isopmodified leucine and isoleucine
As producer strains, cultures of Brevibacterium flavum VKPM B-2736 and Brevibacterium flavum VKPM B-1507, respectively, are used. The washed cell suspensions of Example 2 were incubated in a medium with 2% isotopically modified sodium acetate for 1.5 days. After passing the supernatant of the incubation mixture through Dowex 50, amberlite resin GC-50 II in copper form with a degree of charge of copper of 70% Eluent 0.2 N is used to isolate the amino acids. ammonia solution. After ammonia was removed from the obtained fractions, they were passed through carboxymethyl cellulose in the H ⁺ form. The results are shown in the table.

Example 5. Obtaining isotopically modified threonine
As a producer strain, a Brevibacterium flavum VKPM B-1280 culture was used (the suspension of washed cells was incubated as in Example 2, but in a medium with 1% isotopically modified sodium acetate and 0.05% glucose). In addition to threonine, cells form leucine and alanine. The separation of the mixture of amino acids is carried out on Amberlite GC-50 II resin with 100% copper charge. Eluent 0.2 N. ammonia solution (see table).

Example 6. Obtaining istopmodified serine
As a producer strain, a Brevibacterium flavum VKPM B-3559 culture is used. The biomass of cells was grown in accordance with Example 2, but for 2 days (the suspension of washed cells was incubated in Example 2, but on a medium with 1% isotopically modified sodium acetate and 0.05% glucose). In addition to serine, the culture forms alanine and glutamic acid. The separation of amino acids in accordance with example 5. The results are shown in the table.

Example 7. Obtaining isotopically modified histidine and alanine
The histidine producing strain Corynebacterium glutamicum VKPM B-4380, which additionally synthesizes alanine, was used. Cell biomass is obtained in saline with 0.1% glucose. For the separation of amino acids, Amberlite GC-50 II, charged with copper at 30%, is eluted with a 0.5 N ammonia solution. The remaining operations in examples 1-2. The results are shown in the table.

Claims (5)

 1. The method of producing uniformly isotopically modified natural L-α-amino acids by incubating microorganism cells on a medium containing a single carbon source in the form of isotopodified salts of acetic acid, followed by isolation of the target product, characterized in that cells of microorganism strains of the producers of the target product are used, which are previously incubated in a medium containing carbon sources in the form of salts of acetic acid in combination with carbohydrates, followed by separation and washing according to irradiated cells, and upon completion of incubation on a medium with uniformly isotopically modified acetic acid salts, the cells are separated and discarded, and the target product is isolated from the medium.  2. The method according to claim 1, characterized in that the producer strains use the lysine producer Corynebacterium glutamicum VKPM B-38, the glutamic acid producer Corynebacterium glutamicum VKPM B-832, the producer of Brevibacterium flavum leucine VKPM B-2736, the producer of isoleucine flumvav Brev B-1507, a producer of Brevibacterium flavum threonine VKPM B-1280, a serine producer of Brevibacterium flavum VKPM B-3559, histidine producer Corynebacterium glutamicum VKPM B-4380, and a glutamic acid producer Corynebacterium 8 or glutamic acid producer Corynebumerumium glutamicum VKPM B-4380. 3. The method according to claim 1, characterized in that sodium acetate and / or ammonium acetate uniformly modified with ¹³ C or ¹⁴ C isotopes are used as acetic acid salts. 4. The method according to claim 1, characterized in that they use uniformly modified isotopes ¹³ C or ¹⁴ C ammonium acetate, additionally modified with the isotope ¹⁵ N. 5. The method according to claim 1, characterized in that after isolation of the target isotopically modified L-α-amino acid, if necessary, additionally uniformly isotopically modified L-α-amino acids accompanying it are further isolated, while the target L-α-amino acids are isolated by passing the incubation mixture supernatant through Dowex and elution with an ammonia solution, and the concomitant L-α-amino acids are isolated by additional passing the obtained eluate through Amberlite GS-50P in copper form, eluting with an ammonia solution and passing this eluate of carboxymethylcellulose in H ⁺ form.

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