KR940009047B1 - Method of manufacturing glutamine and iso-ascorbic acid-2-phosphate of ascorbic acid-2-phosphate - Google Patents

Abstract

Iso ascorbic acid-2-phosphoric acid (I) or ascorbic acid-2-phosphoric acid (II) and L-glutamine (III) were synthesized without adenosine triphosphate. The microbes are corynebacterium glutamicum ATCC 13032 and microbacterium ammoniaphium ATCC 15354 etc..

Description

Method for preparing ascorbic acid-2-phosphate or iso ascorbic acid-2-phosphate and glutamine

The present invention relates to a method for preparing L-glutamine and ascorbic acid 2-phosphate or isoascholic acid 2-diacid in the same reaction, more specifically, the genus Coryne bacterium having the ability to produce L- glutamic acid, Microbial cells of the genus Brevibacterium, Micrococcus, Microbacterium, cultures or their treated products are used as enzyme sources and ascorbic acid and L-glutamic acid as substrates. To generate.

L-glutamine is used in large quantities as a major component of anti-ulcer fluids such as gastric ulcer and duodenal ulcer. Ascorbic acid-2-phosphate and iso ascorbic acid-2-phosphate produced in this reaction are vitamin C As a stable compound exhibiting activity, it is widely used in medicine, food and cosmetics.

A method for producing phosphate compounds related to ascorbic acid by enzymatic reaction involves adenosine triphosphate (ATP), which is a bioenergy compound, and known methods of reacting ascorbic acid or iso-ascorbic acid with microorganisms and enzymes. Japanese Unexamined Patent Publication No. 1-199590 and Japanese Agricultural Chemistry Association. Agr. Biol, Chem, vol 54, No. 12, P. 3235-3239 (1990)].

The production method of L-glutamine is a biochemical method in which energy is obtained by glutamine synthetase (六 郞 食 長 바이오: Bioscience of yeast (published by Korean Society for Publication): P.141-154 (1990) and sugars. Direct fermentation by bacteria as a raw material (Middle-Western Fermentation and Industry 40 No. 1 p.15-20 (1982)) is known.

However, in the course of the selective phosphorylation reaction of ascorbic acid-related phosphate compounds by the action of ascorbic acid or iso ascorbic acid with microorganisms or enzymes, a donor such as adenosine triphosphate, an expensive bioenergy compound, must be used as an auxiliary material. There was no known method of producing a mixture of glutamine and ascorbic acid-2-phosphate or isoascholic acid-2-phosphate.

Accordingly, an object of the present invention is to provide a method for producing ascorbic acid-2-phosphate or iso ascorbic acid-2-phosphate without using a bioenergy compound.

It is another object of the present invention to provide a method for simultaneously preparing glutamine and ascorbic acid-2-phosphate or iso ascorbic acid-2-phosphate by a mixed reaction.

In order to achieve the above objects, in the present invention, L-glutamic acid-producing ability of the genus Corynebacterium, Brevibacterium, Micrococcus, Microbacterium microorganisms, cultures or their treatments as enzyme source L-glutamic acid and ascorbic acid or iso-ascorbic acid are used as a substrate to react with adeno acid or adenylic acid (5'-AMP) and a buffer component. Ascorbic acid-2-phosphate and L-glutamine were separately obtained, and thus, ascorbic acid 2-phosphate or isoascholic acid-2-phosphate and L-glutamine could be prepared by a reaction without using a bioenergy compound.

The present invention is described in more detail as follows.

The choice of microorganisms is important in tests in which microorganisms can act on organic compounds to yield more useful derivatives. Therefore, in the present invention, ascorbic acid compounds (ascorbic acid, isoascolvinic acid) among strains classified into the genus Corynebacterium, Brevibacterium, Micrococcus, and Microbacterium known as L-glutamic acid producing bacteria. ) And L-glutamic acid as substrates were selected strains that simultaneously produce the corresponding phosphate compound and L-glutamine.

Selected strains are currently available for general sale and can be purchased commercially, but strains obtained by mutating and isolated from nature can also be used.

The selected strains are then used as a medium commonly used in general bacterial culture methods, for example, carbon sources such as starch, glucose, sucrose, and sucrose, peptone, hexaaxis, yeast extract, C, S, L urea, ammonium salt, etc. It is grown in a medium consisting of phosphate, magnesium sulfate, and other trace metals necessary for the growth of nitrogen sources and other microorganisms. In particular, when cultured using a medium developed for the fermentation of L-glutamic acid, for example, a medium described in Japanese Patent Publication No. 45-110, it is preferable to use a strain having high enzymatic activity in cells in the present invention. Do.

The cultured cells themselves proceed with the reaction of the present invention as an enzyme, but in the form or partially purified enzyme protein or immobilized enzyme of a lyophilized product of the washed cells, which is powdered by treating the cells with a solvent such as acetone, ethanol or ether. Since it exhibits sufficient activity, ascorbic acid-2-phosphate or isoascholic acid-2-phosphate and L-glutamine can be produced together by a reaction generating process by a so-called bioreactor including immobilized cells.

Subsequently, L-glutamic acid and ascorbic acid related compounds are used as a substrate, and adeno acid or adenylic acid (5′-AMP), a buffer component, a surfactant, an anhydrous phosphate, a metal salt, and the like are added. By reacting these treatments with the enzyme source, L-glutamine and ascorbic acid or iso ascorbic acid were simultaneously produced.

In particular, it is effective to add a surfactant to the reaction solution to facilitate the reaction progress.

In this case, nonionic surfactants such as polyoxyethylene sorbitan and monostearate and cationic surfactants such as polyoxyethylene stearylamine and cetyltrimethylammonium bromide and anionic surfactant groups such as sodium oleamide sulphate It is preferable to add it to a reaction liquid at the density | concentration of 1-50 mg / ml, selecting suitably according to a characteristic.

In the present invention, adenosine or 5'AMP is converted to ATP by the microorganisms, and plays an auxiliary role in the production of L-glutamine, ascorbic acid-related phosphate compounds, and minerals such as magnesium phosphate and potassium phosphate. Phosphate is used, and a trace amount of manganese, magnesium salt, iron, zinc, copper, etc. is added as a metal salt.

The conditions of the reaction are not particularly limited as long as it is a range in which the enzyme is inactivated, but pH 4.5 to 9.0 and a reaction temperature of 5 to 40 ° C are generally good.

The reaction was carried out under stationary or stirring conditions, but the reaction proceeded in a short time when the dispersion of the enzyme was improved. The addition of magnesium and manganese salts during the course of the reaction as metal salts facilitated ATP production.

In the case where L-glutamic acid is not added as a substrate in this reaction system, asosbinic acid is not phosphorylated at all, the energy supply reaction of 5′-AMP →→ ATP, which is a reaction during the synthesis of L-glutamine, isoisolate. Ascorbic acid-2-phosphate production was involved.

In other words, it was also confirmed that high concentration of ATP of 1.5 g / dl was generated together with glutamine in the reaction solution by removing ascorbic acid from the reaction solution and using only L-glutamic acid as a substrate.

It is assumed that this ATP is used as a phosphate donor for ascorbic acid and that ATP regeneration occurs in the reaction system.

Substrate concentrations in the reaction were 0.5 or 5 g / dl in any of ascorbic acid, isoascholic acid and L-glutamine, and adenosine or 5′-AMP was in the range of 500 mg-1.5 g / dl. The reaction was completed within 10 hours.

The product was recovered from the reaction mixture by using an ion exchange resin for fractionation using the acidity of (iso) ascorbic acid-2-phosphate and the basicity of L-glutamine.

The following examples further illustrate the production methods and effects of the products according to the invention, but are not intended to limit the scope of the invention.

In the following examples, the determination of glutamine was performed by a combination of glutamine dehydrogenase and glutamate decarboxylase, and ascorbic acid-2-phosphate and iso ascorbic acid-2-phosphate were measured in A, yamasan. By high performance liquid chromatography (HPLC) method. Agr. Biol. Chem, 54, 9, p. 2310 (1990)]

Example 1

[Preparation of L-glutamine synthase]

Corynebacterium melassecola ATCC 17965 (see Japanese Patent Publication No. 45-110), a kind of glutamic acid producing strain, was cultured through aeration in a medium having the following composition to obtain cells.

Beet Sorghum 7.0g / dl

Element 1.5g / dl

KH ₂ PO ₄ 0.2g / dl

(NH ₄ ) ₂ SO ₄ 0.1g / dl

MgSO ₄ 7H ₂ O 50mg

Non-ion S-6 (Non-ionic Surfactant: 130 Japan)

pH 7.2

The obtained cells were centrifuged to form L-glutamine synthase as the encapsulated and immobilized cells in the alginate gel by a conventional method as washing cells.

(Enzyme reaction)

5.0 g of sodium L-glutamate

Adenosine 2.5g

Potassium Phosphate 9.0g

Disodium Phosphate 14.1g

Glucose 15.0 g

Ammonium sulfate 15.0 g

Niamine S-215 (Surfactant: general oils and fats) 1.5g

500 ml of water

After adjusting the reaction solution having the composition to pH 6.5 and floating the immobilized mycelium enzyme encapsulated with 10 g of the cells and reacting for 15 hours at 37 ° C. at 15 rpm, the reaction solution was added 3.5 g of sodium ascorbate to the reaction for 3.5 hours. Continued.

As a result of quantification of the product in the reaction solution, L-glutamine was 4.6 g / dl, and ascorbic acid-2-phosphate was 3.8 g / dl.

The reaction solution was superheated and the precipitate was removed by centrifugation, and then the reaction solution adjusted to pH 4.5 was adsorbed onto Amberlite 1RA-900 (cl type) to separate ascorbic acid-2-phosphate. L-glutamine was adsorbed through Dow's 50 (H type) and each fraction was crystallized by a conventional method. As primary crystals, 3.8 g of L-glutamine and 3.4 g of ascorbic acid-2-calcium phosphate were recovered.

Example 2

As enzyme source microorganisms, penicillin resistant mutant RIIBS-1039 strain of Brevibacterium lactofermen tam ATCC 13655 strain was used, and sodium isocholate acid instead of ascorbic acid was reacted with cetylpyrididium as a surfactant. The reaction was carried out in the same manner as in Example 1 except that 1.0 g of chromide was used.

It was confirmed that 4.1 g / dl of L-glutamine and 3.2 g / dl of isoascholic acid 2-phosphate were produced in the obtained reaction liquid.

Example 3

After culturing Corynebacterium glutamicum ATCC 13032 strain in the same manner as in Example 1, lyophilized cells were obtained without washing and immobilizing the cells, except that 5 g of the cells were added to the reaction solution. As a result of the enzymatic reaction in the same manner as in Example 1, 4.0 g / dl of L-glutamine and 2.8 g of ascorbic acid-2-phosphate were obtained from the reaction solution.

Example 4

After incubating the microbacterium ammoniaphium ATCC 15354 strain in the same manner as in Example 1, the dried cells treated with cooled acetone were the enzyme source, and Na-5′-AMR was used instead of adeno acid in the reaction solution. As a result of the enzymatic reaction in the same manner as in Example 1 except for the use, L-glutamine 3.9 g / dl and 3.5 g ascorbic acid-2-phosphate were obtained from the reaction solution.

Comparative Example 1

As a result of the enzymatic reaction in the same manner as in Example 1, except that sodium L-glutamate was not added to the enzyme reaction composition, phosphorylation products of ascorbic acid were not produced.

Comparative Example 2

The reaction was carried out in the same manner as in Example 1 except that no additional ascorbic acid was added. As a result, ATP having a concentration of 2.3 g / dl was generated in the reaction solution.

As described above, the reaction solution containing L-glutamic acid and ascorbic acid or isoascholic acid as a substrate reacts and reacts cells or enzymes selected from a bacterial group having the ability to produce L-glutamine from L-glutamic acid. By separating the liquid using an ion exchange resin, ascorbic acid-2-phosphate or isoascholic acid-2-phosphate and glutamine were easily produced at the same time.

Claims (9)

Corynebacterium merracera ATCC 17965, Brevibacterium lactofermentam ATCC, having the ability to produce L-glutamine from L-glutamic acid in a reaction solution containing L-glutamic acid and ascolic acid or iso ascolic acid as a substrate 13655 penicillin resistant mutant RIIBS-1039, Corynebacterium gritamicam ATCC 13032 or Microbacterium ammoniapyram ATCC 15354, with ascorbic acid-2-phosphate or iso ascorbic acid-2-phosphate Method for preparing glutamine. The method of claim 1, wherein the reaction solution contains a surfactant, adeno acid or adenylic acid, an inorganic phosphate, a metal salt, and a buffer component. According to claim 2, wherein the surfactant is selected from the group consisting of polyoxyethylene sorbitan, monostearate polyoxyethylene stearylamine, cetyltrimethyl ammonium bromide, sodium oleamide sulphate concentration of 1 to 50 mg / ㎖ Characterized in that the addition. The method of claim 2 wherein the inorganic phosphate is magnesium phosphate, potassium phosphate. The method of claim 2, wherein the metal salt is manganese, magnesium, iron, zinc, copper salt. The method of claim 1, wherein the cells are immobilized cells. The method according to claim 1, wherein the enzymatic reaction is performed at a pH of 4.5 to 90 and a reaction temperature of 5 to 40 ° C. The method of claim 2, wherein adenosine or adenylic acid is added at a concentration of 50 mg to 1.5 g / dl. The method of claim 1, wherein the separation of the product is characterized in that the separation using an ion exchange resin.