DE1276583B - Process for the preparation of 5'-inosic acid - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY

GERMAN

PATENT OFFICE

EDITORIAL

Int. α .:

German KL:

Number:
File number:
Registration date:
Display day:

C12d

C07d

6b-16/03

12 ρ-7/10

P 12 76 583.2-41 (K 58208)

January 21, 1966

5th September 1968

The present invention relates to a process for the preparation of 5'-inosinic acid.

5'-inosinic acid, chemically called hypoxanthine riboside-5-phosphoric acid is a well-known compound. So far was 5'-inosinic acid from meat extract, from muscles through enzymatic deamination of muscle adenylic acid and through Hydrolysis of inosine triphosphate produced. The compound is somewhat in water and formic acid soluble, but only slightly soluble in alcohol and ether. Hydrolyzed with an acid when boiled 5'-inosinic acid to 1 mole of phosphoric acid, 1 mole of hypoxanthine and 1 mole of D-ribose.

It is already known from US Pat. No. 3,152,966, in a nutrient medium containing biotin Mutants of Micrococifcus glutamicus and Brevibacterium ammoniagenes for the production of 5'-inosinic acid to breed.

The present invention has the task of producing 5'-inosinic acid in high purity and with produce increased yield on an industrial scale.

Based on the prior art for the production of 5'-inosinic acid by growing a Biotin-requiring mutants of Brevibacterium ammoniagenes or Micrococcus glutamicus im According to the invention, this object is achieved in the customary aqueous nutrient medium and under the conditions customary for this purpose solved by Brevibacterium ammoniagenes ATCC 19 183, ATCC 19 184, Micrococcus glutamicus ATCC 19 185 or ATCC 19 186 begins.

The microorganisms listed above which are used according to the invention are Mutant strains caused by repeated treatment of Brevibacterium ammoniagenes ATCC 6871 and Micrococcus glutamicus ATCC 13 032 with rays (UV rays, X-rays) or chemical Agents (diethyl sulfate) were obtained.

Process for the preparation of 5'-inosinic acid

Applicant:

Kyowa Hakko Kogyo Co., Ltd., Tokyo

Representative:

Dr.-Ing.H. Ruschke

and Dipl.-Ing. H. Agular, patent attorneys,

8000 Munich 27, Pienzenauer Str. 2

Named as inventor:

Shigeo Abe, Asagaya, Suginami-ku; Tokyo;

Akira Furuya, Machida-shi (Japan)

Claimed priority:
Japan January 23, 1965 (3376),
dated ^ January 1965 (3798)

The organisms used as the original strains Brevibacterium ammoniagenes ATCC 6871 and Micrococcus glutamicus ATCC 13 032 grow in the area described in footnote 1 of the table Minimal nutrient medium good. Also the mutant strains used according to the present invention grow in this minimal nutrient medium. However, the mutant strains will grow through Significantly accelerates the use of a nutrient medium, which by adding amino acids, listed in footnote 2 of Table I, or a nucleic acid component, either individually or as a mixture of two or more of these components, prepared for the specified minimal nutrient medium became. The influence of these substances on the growth of the mutant strains according to the invention is shown in the table below.

Growth rate of Brevibacterium
ammoniagenes
Stem of origin
ATCC 6871 dried cell bodies g / ml ³ ) Mutants
tribe
ATCC 19184 Micrococcus
glutamicus
Stem of origin
ATCC 13032 Mutants
tribe
ATCC 19185 Mutants
tribe
ATCC 19186 6.2
8.3
6.4 Mutant »
tribe
ATCC 19183 2.8
5.1
5.4 6.3
8.2
6.3 2.4
4.8
5.2 2.9
5.2
5.6 Minimal culture medium ¹ )
Minimal culture medium
+ Amino acids ² )
Minimal culture medium
+ Adenine. 20 mg 1 2.3
4.7
5.1

809 599/171

continuation

Brevibacterium

ammoniagenes

Stem of origin

ATCC 6871

ATCC 19183 mutant strain

Mutant strain
ATCC 19184

Micrococcus

glutamicus

Stem of origin

ATCC 13032

ATCC 19185 mutant strain

ATCC 19186 mutant strain

Minimal culture medium

+ Guanine, 20 mg / 1

Minimal culture medium

+ Adenine and guanine, respectively
20 mg / 1

Minimal culture medium
+ Adenine, 20 mg / 1,
+ Amino acids ² )

Minimal culture medium
+ Guanine, 20 mg / 1,
+ Amino acids ² )

Minimal culture medium

-F- adenine and guanine, respectively

20 mg / 1,

+ Amino acids ² )

6.5
6.7
9.0
9.2

9.4

5.2
6.3
8.1
7.2

8.5

6.6
6.8
9.0
9.3

9.6

4.5 6.4 7.9

7.4

8.6

5.4 6.4 8.2 7.6

8.7

¹ J The miniinal nutrient medium had the following composition (per liter of deionized water):

Glucose; 50 g

KH ₂ PO ₄ . 1.0g

K ₂ HPO ₄ 3.0g

MgSO ₄ · 7 H ₂ O 0.3 g

FeSO ₄ -7H ₂ O 10mg

CaCl ₂ · 2 H ₂ O 10 mg

MnCl ₂ -4H ₂ O, 3μξ

Calcium pantothenate 10 mg

Biotin 30 µg

Urea 5 g

The pH of the solution was adjusted to 7.2 before sterilization. The urea was sterilized separately from the other ingredients and then added.

² ) The amino acids that can be used include the following: L-alanine, L-arginine, L-aspartic acid, L-cysteine, L-cystine, L-giutamic acid, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tyrosine, L-tryptophan and L-valine. These amino acids were added to the nutrient medium in an amount of 20 mg / l.

³⁾ The growth of each strain was off in the following manner 'out: sterilized conical flask of 25OmI content under pressure at a temperature of 120 ⁰ C during 15 minutes 20 ml of the nutrient medium. Then the urea solution which had been sterilized separately was added. To the nutrient medium prepared in this way, 0.2 ml of the cell suspension, which had been obtained by washing the respective pre-cultured strains to be free of spores, was added to each flask. The culture was then shaken at 30 ^° C. for 94 hours. After this time, the amount of cell bodies obtained was determined

The S'-inosinic acid is not obtained when the original strain is grown in the described minimal nutrient medium ^1). However, 5'-inosinic acid is obtained using the mutant strains of the invention. In addition, this production of S'-inosmic acid is further increased by adding amino acids, adenine or guanine, and a further increase occurs when these compounds are added in combination of two or more substances.

The known synthetic nutrient media or natural nutrient media are in the method of present invention suitable if they have the essential nutrients for the growth of the applied Tribal included. Such nutrients are known in the art. These include appropriate amounts of substances such as a carbon source, a nitrogen source, inorganic compounds and the like used by the applied microorganism. For example, carbohydrates, such as glucose, fructose ,. Maltose, cane sugar, starch, starch hydrolysates, molasses and the like, or any other suitable carbon sources, such as glycerin, mannitol, sorbitol, organic acids, Glutamic acid and the like can be used.

These substances can be used either alone or in mixtures of two or more compounds. As the nitrogen source, various kinds of inorganic or organic ⁵ salts or compounds, such as urea or ammonium salts such as ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium phosphate and the like, or one or more amino acids, in combination, or natural substances containing nitrogen such as corn steep liquor , Yeast extract, meat extract, peptone, fish meal, casein hydrolyzate, soluble fish matter, rice husk extract, ribonucleic acid and the like can be used. These substances can also be used individually or in combination of two or more substances. The inorganic compounds which can be added to the nutrient medium include e.g. B. magnesium sulfate, sodium phosphate, potassium dihydrogen phosphate, potassium monohydrogen phosphate, iron sulfate or other iron salts, manganese chloride, calcium chloride and the like. The here applied for the production of 5'-inosinic acid fermentation is performed under aerobic conditions Be *, for example, aerobic shaking culture or agitation submerged culture, at a temperature of about 20 to 40 ⁰ C and

a pH of about 4 to 9. Adjustment of the pH of the culture medium during fermentation can by using neutralizing agents such as aqueous ammonia, sodium hydroxide and the like, or urea. After 2 to 8 days of cultivation they collect remarkably large amounts of 5'-inosinic acid in the culture medium and in the cell bodies. At the same time, small amounts of hypoxanthine are also formed.

After the fermentation has ended, the cell bodies are removed and the 5'-inosinic acid is used as usual Way won, e.g. B. by an ion exchange resin treatment as described in Example 1 below or by any other conventional ion exchange process, extraction with solvents, Precipitation with metal salts, chromatography and the like.

The following examples serve to further illustrate the invention. Unless otherwise stated the percentages are by weight.

example 1

The strain Brevibacterium ammoniagenes ATCC 19183 was used as the microorganism and incubated at 30 ⁰ C for 24 hours in a culture medium of 2 ° / o glucose, 1% peptone, 1% meat extract, 0.3% NaCl, and 20 .mu.g / l biotin pre-cultured.

The fermentation medium was made by dissolving 70 g of glucose, 6.0 g of urea, 1 g of KH ₂ PO _4, 3.0 g of K2HPO4, 0.3 g of MgSO ₄ · 7H ₂ O, 0.01 g of FeSO ₄ · 7H ₂ O, 5.0 g casamino acid, 30 μg biotin, 5 mg vitamin Bi and 10 mg calcium pantothenate in 11 water. The pH of the resulting solution was adjusted to 7.5 before sterilization. The urea was sterilized separately in the form of a 40% solution and added in such an amount that 6.0 g urea per liter of the nutrient medium resulted.

10 percent by volume of the above-listed inoculum were in the fermentation medium described vaccinated. 30 ml 'quantities of the mixture of both media were poured into sterile conicals 250 ml flask. Shaking of the culture was then carried out at 30 ° C.

After culturing for 110 hours, 16.4 mg / ml 5'-inosinic acid was found in the fermentation liquid. 11 of the supernatant solution after Removal of the cell bodies obtained from the fermentation liquor was carried out on one Adjusted pH value of 1.4 with hydrochloric acid and through a column with a Dia-Ion-SK-Nr.-1 resin (Η-form) given. Immediately afterwards, distilled water was passed through the column, and an aliquot of the run-through used during the early stage of washing with distilled Water was obtained with the subsequent eluate containing 5'-inosinic acid mixed. The aliquot mixture of wash liquid and eluate was then brought to pH of 7.2 adjusted with sodium hydroxide. By cooling the resulting solution after concentration under reduced pressure, crystals of sodium 5'-inosinate were obtained. The yield was 11.6 g. Some 5'-inosinic acid also accumulated in the cell bodies.

Example 2

The same strain as in Example 1 and the pH of the culture medium were used maintained at about 7.0 during the fermentation by adding the urea solution described.

The fermentation medium used in this example contained 100 g glucose, 2.0 g KH ₂ PO ₄ , 4.0 g K ₂ HPO ₄ , 0.3 g MgSO ₄ -7H ₂ O, 5.0 g meat extract, 5.0 g Urea (added as a separately sterilized 40% solution in such an amount that there were 5.0 g urea per liter nutrient medium), 30 μg biotin, 5 mg vitamin Bi and 10 mg calcium pantothenate, dissolved in 11 water. The pH of the solution was adjusted to 7.5 before sterilization.

The fermentation was carried out under the same cultivation conditions as in Example 1, except the pH control is carried out. After 96 hours of cultivation there was 18.6 mg / ml 5'-inosinic acid and 0.9 mg / ml hypoxanthine accumulated in the fermentation liquid. *

Example 3

The strain Brevibacterium ammoniagenes ATCC

19 184 was used as a microorganism. The pre-growth and the fermentation media and conditions used were as in Example 2. After 96 hours of cultivation, 17.8 mg / ml 5'-inosinic acid and 0.6 mg / ml hypoxanthine had each other educated.

Example 4

The strain Micrococcus glutamicus ATCC 19 185 was in a nutrient medium of 2% glucose, 1% peptone, 1% meat extract, 0.3% NaCl and

Biotin 20 ug per liter of water cultured at 3O ⁰ C for 24 hours. The fermentation medium used in this example contained 70 g glucose, 6.0 g urea, 1 g KH ₂ PO ₄ , 3.5 g K ₂ HPO ₄ , 0.3 g MgSO ₄ -7H ₂ O, 0.01 g FeSO ₄ . 7H ₂ O, 5.0 g casamino acid, 30 μg biotin, 5 mg vitamin Bi and 10 mg calcium pantothenate, dissolved in 11 water. The pH of the fermentation liquor was adjusted to 7.5 before sterilization. The urea was added as a separately sterilized 40% solution in such an amount that the amount of urea was 6 g per liter of nutrient medium.

10 percent by volume of the inoculum was inoculated into the fermentation medium. Then 30 ml of the mixture of these media was placed in sterile 250 ml conical flasks. The culture was then shaken at 30 ^° C.

After culturing for 110 hours, there were 15.6 mg / ml of 5'-inosinic acid in the fermentation liquid. After removing the cell body from the fermentation liquor became 11 of the supernatants Solution adjusted to a pH value of 1.4 with hydrochloric acid and through a column with Dia-Ion SK no. 1 resin (Η shape) given. Direct then distilled water was passed through the column and an aliquot of the run-through, which had been obtained during the early stage of washing was taken along with the 5'-Inosinic acid-containing eluate mixed and the pH of this mixture to 7.2 with sodium hydroxide set. Crystals of sodium 5'-inosinate were then obtained by cooling the

Solution obtained after concentration under reduced pressure. The yield was 11.2 g. Additional Inosinic acid was also present in the cell bodies.

Example

The same strain as in Example 4 was used as the microorganism and the pH of the Fermentation medium adjusted to about 7 "by adding urea solution.

The fermentation medium contained 100 g glucose, 2.0 g KHaPO ₄ , 4.0 g K ₂ HPO ₄ , 0.3 g MgSO ₄ · 7H ₂ O, 5.0 g meat extract, 5.0 g urea (a separately sterilized 40 % solution was added in such an amount that 5.0 g of urea per liter of fermentation medium resulted), 30 μg of biotin, 5 mg of vitamin Bi and 10 mg of calcium pantothenate, dissolved in 1 liter of water. The pH was adjusted to 7.5 before sterilization.

The cultivation conditions used in Example 4 were exclusively for pH regulation applied. After 96 hours of cultivation, 18.9 mg / ml 5'-inosinic acid and 0.9 mg / ml hypoxanthine became obtain.

Example 6

The fermentation was carried out with the strain Micrococcus glutamicus ATCC 19186 as the microorganism executed. The preculture, inoculation medium and fermentation medium were like in Example 5. After 96 hours of cultivation there were 19.6 mg / ml 5'-inosinic acid and 0.9 mg / ml hypoxanthine present in the fermentation liquid.

Claims (1)

Claim: Process for the production of 5'-inosinic acid by culturing a mutant requiring biotin of Brevibacterium ammoniagenes or Micrococcus glutamicus in the usual aqueous Nutrient medium and under the usual conditions for this, characterized in that that Brevibacterium ammoniagenes ATCC 19 183, ATCC 19 184, Micrococcus glutamicus ATCC 19 185 or ATCC 19 186 uses. Considered publications:
U.S. Patent No. 3,152,966.