DE1695329C - Process for the production of 5-purine nucleotides by fermentation of microorganisms in the presence of antibiotics or surface-active substances. Elimination aas: 1570022 - Google Patents

Description

^{1 2}

Process for the preparation of 5'-purine nucleotides In the context of the invention, antibiotics such as

from purine bases; by fermentation of micro-penicillin, mitomycin, streptomycin, cycloserine,

organisms are from the French patents citracin, tetracycline, hydroxytetracycline, chlorotetra-

849 519 and I 301 052 known. However, the cyclin, carzinophilin (antibiotic from Strep. Saha-

known processes only low yields of the 5 chiroi-KuIturen), Kanamycin or Neomycin used,

desired 5'-purine nucleotide. Mitomycin, penicillin, strepto-

In contrast, the invention is concerned with a mycin and cycloserine.

Fermentation Process for Making 5'-Purine Generally, the antibiotic is used at the beginning nucleotides, such as 5'-inosinic acid, 5'-xanthylic acid, the logarithmic growth penode of micro-5'-guanylic acid and 5'-adenylic acid in economically acceptable organisms, when their increase is usually more favorable Way on an industrial scale from purine bases. taking is strong inflicted. However, there will be another

Suitable bases are e.g. B. hypoxanthias, guanine increase in desired product then achieved and adenine. The bases can be used as such or as if the antibiotic shortly after the above Nucleosides or in natural products, the bases or period in normal growth of the bacteria added nucleosides included. The invention 15 adds, and there is then an excellent applied Microorganisms are brevibacterium enrichment.

ammoniagenes ATCC 6871 and 6872 and Micro- According to the invention, the above can be used with

coccus sodonensis ATCC 15 932. Antibiotics achievable results, i.e. the under-

The invention is concerned with improving the suppression of excessive bacterial growth.

that in the fermentative production of 5'-purine 20 in favor of the formation of 5'-purine nucleotide, instead of by

nucleotides with Brevibacterium ammoniagenes or addition of antibiotics also through the use

Micrococcus sodonensis occurring reduction of surface-active substances in the fermentation

The yield of the desired product can be achieved as a result of excess medium.

There is also a moderate increase in microorganisms due to the formation of the desired products

Presence of too large amounts of growth-promoting »5 is increased by the fact that surfactants are not

Substances. This phenomenon is all the easier to admit only in the case of excessive bacterial growth.

observe when nutrients are added with high content of, but also, similar to when loading

growth-promoting substance, e.g. B. some types of dealing with antibiotics when 5'-purine nucleotide

Corn steeple, rice bran or fish extracts are excellently developed,

be turned. 30 As surface-active agents, those with

Just like the excessive addition of amino-cationic, anionic, amphoteric or not Acid sources cause the addition of small amounts of ionic properties. Cations of Substances, like manganese salt or iron salt, are most effective then in active substances a concentration above a certain value is followed by non-ionic substances, and a little less tration to the culture medium, which pantothenic acid 35 are effective anion-active substances. examples for and contains thiamine, unusual bacterial wax- cation-active substances are polyoxyethylene alkylamine, turn and thus low formation of 5'-purine nucleotide. Cetyl pyridine chloride, cetyl trimethyl ammonium bromide For economic reasons, as a source for and alkyldimethylbenzylammonium chloride. Examples Amino acid appropriately inexpensive natural products for nonionic surfactants are used. However, some of these natural products contain 40 mixtures of 40% polyoxyethylene and 60% polyacrylic significant amounts of iron or manganese salt. propylene or from 10% polyoxyethylene and 90%

The inventive method for producing polypropylene.

of 5'-purine nucleotides by fermentation of In general, it is recommended that the surface

Microorganisms in an aqueous nutrient medium, active agents relatively late in the period of logarithmic

that in addition to the usual amounts of carbon suppliers, 45 mix growth of microorganisms - im

Nitrogen sources, inorganic salts and vitamin H in case of excessive increase - to add,

also larger amounts of growth-promoting substances. B. the maximum growth 30 mg / ml,

zen as well as iron and manganese salts, based on dried bacterial cells, makes up

in the presence of one of the 5'-purine nucleotide, it is advisable to reduce the substance to two-thirds

the purine base as a precursor is characterized by this size, i. H. at about 20 mg / ml, to add

net that one becomes Brevibacterium as microorganisms The increase in the product to be obtained

ammoniagenes ATCC 6871 and 6872 or micro- further increased if one uses the surface-active

coccus sodonensis ATCC 15 932 and the substances at a later date than the above

Fermentation medium adds an antibiotic or a point in time, i.e. shortly afterwards,

Surface-active substance in the period of the loga 55 The following examples illustrate the invention

rithmic growth or microorganisms or hand of preferred embodiments. If not

shortly thereafter admits. otherwise stated, the percentages relate

According to the invention, the undesired Ent- in the following examples is thus based on the weight prc

development of microorganisms without sufficient volume of fermentation medium.

Production of the 5'-purine nucleotides prevented by 60

the fermentation in a fermentation medium with example 1

added antibiotics or surface-active co- As a vaccine, Brevibacterium ammonia is used

is carried out, whereby the generation of genes (ATCC 6872) 24 hours in a culture

5'-purine nucleotide is increased. The presence of medium of 2% glucose, 1.5% peptone, 0.2% urine

Antibiotics are for a maximum production of 5'-Pu- 6 ₅ substance, 0. Γ '/ ₀ K ₂ HPO ₄ , 0.03% MgSO ₄ · 7 H ₂ O, 0.3 "/,

Rinnucleotide is also useful, even if no NaCl, 0.01% FeSO ₁ · 7 H ₂ O and 30 μ / l vitamin H

Conditions exist that an excessive wax (residual percent - water), pH = 7.3, cultivated. Dam

favor the turn of microorganisms. 10 percent by volume of this inoculation culture are placed on eir

Transfer fermentation medium. Both media have been sterilized beforehand. The cultivation is carried out at 30 ⁰ C with shaking in a fermentation medium which aufweißt the following composition: 10 "/" of glucose, 1% K ₈ HPO _4, 1.3% KH ₂ PO _4, 1.1% MgSO ₄ · 7 H ₂ O, 0.01% / ₀ CaCl ₂ · 2 H ₈ O, 30 y / 1 vitamin H, 5 y / ml calcium pantothenate, 3 y / ml thiamine hydrochloride, 0.5% corn steep water, 3 mg / ml hypoxanthine ( Remaining percent = water). Before sterilization, the pH is adjusted to 8.0 with 5N NaOH. After sterilization, previously sterilized urea is added to the culture medium up to a content of 0.6%. The amounts of mitomycin listed below C. were added in individual doses after a certain culture period.Table I shows the amounts of 5'-sodium ininosinate that have accumulated in the fermentation liquid after cultivation for 96 hours.

Table 1 Mitome
Encore
time
(Hours.) ycin C
Encore
amounts
(V / ml) Arisen
Amounts of
5'-sodium inosinate *)
(mg / ml) amounts
dried
Bacterial cells
(mg / ml) 8th 15th
30th 1.3
2.5 12.9
11.0 12th 20th
40 8.82
11.03 15.1
14.8 16 20th
40 3.92
4.03 18.0
17.6 20th 30th
40 1.22
1.11 21.3
20.7 No encore sense 32.8

Penicillin G Sodium Encore Encore narrow time (r / ml) (Hours.) 5 8th 10 5 12th 10 50 10 18th 50 No encore

Arisen

Amounts of

5'-sodium inosinate *)

(mg / ml)

2.3
4.1

4.0
6.3

10.2

9.0
7.9

sense

amounts

dried

Bacterial cells

(mg / ml)

13.3
13.0

15.9
15.9

14.2

19.1
18.3

38.2 tion and cycloserine in various concentrations added. The other culture conditions remain the same as in Example 1. After 96 continuous culture 5'-sodium inosinate formed as indicated in Table III.

Table III

*) Determined as S'-inosinic acid disodium salt · 7 ¹ / »H _a O.
Example 2

The procedure corresponds to that given in Example 1, except that instead of Mitomycin C here too Penicillin G sodium was added at various times. The resulting amounts of 5'-sodium inosinate after 120 hours of cultivation are given in Table II.

Table Il

*) Determined as disodium 5'-inosinate · 7 ¹ / »H _a O.
Example 3

The same inoculating bacterium and the same fermentation medium as in Example 1 are used, but 0.0005% MnSO ₄ · 4 H ₁₁ O is added instead of corn steep liquor. Furthermore, instead of mitomycin (Example 1) at different times during the Fermen

Cycloserine Encore Encore crowd time (y / ml) (Hours.) 400 7th 800 1600 800 10 1600 2000 800 13th 1600 2000 No encore

Arisen

Quantities of '

5'-sodium inosinate *)

(mg / ml)

5.3

• 7.2

9.0

3.1

10.2

9.9

6.4 9.2 6.8

sense

amounts

dried

Bacterial cells

(mg / ml)

13.8 12.0 12.2

• 14.2 14.0 13.6

18.2 17.3 17.5

39.1

*) Determined as S'-inosinic acid disodium salt - 7 ¹ / * H ₁ O.

Example 4

The same vaccine bacterium as in example 1 and the fermentation medium as in example are used 3, but used without hypoxanthine. After 48 hours of cultivation, guanine at 3.0 mg / ml added. Dihydrostreptomycin sulfate is used as an antibiotic in different concentrations and admitted at different times. The other culture conditions correspond to those of Example 1.

Table IV shows the formation of 5'-sodium guanylate after 96 hours of culture.

Table IV.

Dihydrostrepto- sulfate
Encore
crowd Arisen Menffcn mycin
Encore
time (WmI) Amounts of
5'-sodium
guanylate *) dried
Bacterial cells (Hours.) 5 (mg / ml) (mg / ml) 8th 10 1.3 15.2 50 2.6 14.9 5 2.7 14.1 14th 10 2.3 19.2 50 4.4 18.1 5 5.1 18.8 20th 10 1.2 22.5 50 1.2 23.0 No encore 2.0 21.6 sense 38.9

·) Determined as S'-guanyl disodium salt.

Example 5

The same inoculum as in Example I and the fermentation medium as in Example 4 are used used. After 48 hours of cultivation, 2.5 mg / ml adenine are added. Tetracycline is called Antibiotic added in different concentrations at different times. The remaining culture conditions correspond to those of Example 1. After 96 hours, the amounts listed in Table V are To detect 5'-adenylic acid.

Table V

Tetracycline additive Addition time amount (Std.) I (WmI)

1
2

1
2
5

1
2
5

No encore

Arisen

Amounts of

5'-adenylic acid

(mg / ml)

1.1 2.3

2.1 4.1 1.9

0.8 3.8 2.7

sense

amounts

dried

Bacterial cells

(mg / ml)

17.1 16.8

18.1 15.3 14.2

13.1 12.9 13.0

40.2 At game 6

The same vaccine bacterium and the same fermentation medium as in Example 1 are used, however, corn steep water is replaced by 0.3% of an enzymatic gas hydrolyzate and the addition of hypoxanthine increased to 4 mg / ml. During the culture period, certain concentrations of

ίο Penicillin G Potassium, Mitomycin C and Dihydrostreptoiriycin Sulphate admitted at certain times. Otherwise, the culture conditions are as in the example. Table VI gives those after 120 hours of culture amounts of 5'-sodium inosinate found in the fermentation medium.

Table VI

Antibiotic Art

Encore Encore time crowd (Hours.) (WmI) 24 10 28 20th 30th 50 None Encore

Arisen amounts Amounts of dried 5'-sodium inosinate *) Bacterial cells (mg / ml) (mg / ml) 15.1 15.7 16.0 16.3 13.8 17.0 9.9 19.8

Penicillin G Potassium

Mitomycin C ... χ. ί Dihydrostreptomycihsulfate

*) Determined as S'-inosinic acid dinacrium salt ■ 7 Vt H ₁ O.

Example 7

Kanamycin

Addition time (Hours.)

Added amount
WI

10
20th
50

10 20th 50

amounts

dried '

Bacterial cells

(mg / ml)

12.0 11.5 10.1

14.5 13.1 13.3

Amounts created

of 5'-sodium

guanylate *)

(mg / ml)

0.9 2.9 1.3

2.0 3.9

5.2

No addition 28.3 tracks

*) Determined as S'-guanyl disodium salt.

Example 8

Vaccine bacterium: Micrococcus sodonensis (ATCC 15932); Culture medium: 2% glucose, 0.5% yeast extract, 1.5% peptone and 0.25% NaCl (residual percentage = water), pH = 7.3; Fermentation medium: 10% glucose, 0.6% K ₂ HPO ₄ , 0.6% MgSO ₄ · 7H ₂ O,

65 0.01 »/,

Vaccine bacterium: Brevibacterium ammoniagenes (ATCC 6871), fermentation medium according to the example After a culture time of 50 hours, 3.0 mg / ml guanine are added. Kanamycin sulfate is used in different ways Concentration added after 10 or 15 hours. The other culture conditions correspond those of Example 1. Table VII gives the amounts of 5'-sodium guanylate obtained after 96 hours again.

Table VII

ο CaCl ₂ · 2 H ₂ O, 5 y / ml calcium pantothenate, 1 y / ml thiamine, 30 y / vitamin H, 0.1% FeSO ₄ 7 H ₂ O, 1% casein amino acids, 0.6% urea and 2, 5 mg / ml hypoxanthine (residual percentage = water); pH = 8.0 before sterilization. The other culture conditions correspond to those of Example 1. After 10 hours of cultivation, penicillin G-potassium is added in different concentrations. The amounts of 5'-sodium inosinate found after 96 hours of culture are shown in Table V3II.

Table VIIJ

Penicillin G Potassium
Added amount
(WmI) amounts
dried
Bacterial cells
(mg / ml) Arisen
Amounts of
5'-sodium inosinate *]
(mg / ml) 5
10
20th
50
No encore 19.3
18.2
14.3
14.1
30.8 4.1
5.8
7.2
7.0
sense

*) Determined as S'-inosinic acid disodium salt · 7Vi H ₂ O.

Example 9

Vaccine bacterium: Brevibacterium
(ATCC 6872); Culture medium: 2%

ammoniacal glucose, 2%

Peptone, 0.1% urea, 0.1% K ₂ HPO ₄ , 0.03% MgSO ₄ -7 H ₂ O, 0.3% NaCl, 0.01% FeSO-7 H ₂ O and 30 µg / ml vitamin H (remainder percent = water); Culture time 24 hours; pH = 7.3. 10 percent by volume of this inoculation medium is poured into a fermentation

tion medium given. Both culture media are sterilized beforehand. The fermentation medium has the composition given below. The aerobic cultivation is ^{carried out at 30 °} C. with shaking.

Composition of the fermentation medium: 10% glucose, 1% K ₂ HPO _4, l ° / ₀ KH ₂ PO _1, 1% MgSO ₄ -7 H ₂ O, 0.1% CaCl ₂ · 2 H ₂ O, 30 y / ml Vitamin H, 5 y / ml calcium pantothenate, 3 y / ml thiamine hydrochloride, 4 mg / ml hypoxanthine (residual percentage = water). The pH is adjusted to 8.0 with 5N NaOH before sterilization. After sterilization, 0.6% of sterilized urea is added to the fermentation medium.

After 20, 24 and 48 hours of culture, polyoxyethylene alkylamine is added in different concentrations . The amounts of 5'-sodium inosinate obtained after culturing for 120 hours are shown in Table IX.

Table IX Example 11

The same vaccine bacterium as in Example 1 and the fermentation medium according to Example 1 are used, which, however, contained 0.0005 _{% MnSO 4} -4 H ₂ O instead of corn steep liquor. Cctyl trimethylammonium bromide is used as a surfactant. The other culture conditions are the same as in Example 1. After 96 hours of cultivation, the amounts of 5'-sodium inosinate given in Table Xl were obtained.

Polyoxy
alkyl
Encore
time
(StQ.) ethylene-
imin
Encore
amounts
(y / tnl) amounts
dried
Bacterial cells
(mg / ml) Arisen
Amounts of
5'-sodium inosinate *)
(mg / ml) 20th 500
750
1000 24.3
21.2
19.9 7.6
9.0
6.3 24 750
1000
1300 22.0
20.1
21.0 10.1
15.9
13.6 28 750
1000
1300 23.2
22.0
21.7 9.8
12.2
12.0 No encore 30.8 sense

Cetyl trir
ammoniu
Encore
time
(Hours.) tiethyl
mbromid
Encore
crowd
O / ml) Table XI Arisen
Amounts of
S'-sodium inosinate *]
(mg / ml). 15th 20th 500
750
1000 amounts
dried
Bacterial cells
(mg / ml) 3.6
5.2
6.5 20th 24 750
1000
1250 24.0
20.2
16.5 10.6
14.9
15.1 «5 28 750
1000
1250 21.3
21.8,
19.2 8.2
9.1
8.7 No encore 22.3
22.5
21.5 sense 30th 34.8

35

·) Determined as S'-inosinic acid disodium salt · I ¹ I, H, O.

Example 10

The same measures are used as in Example 1; instead of Polyoxyäthylenaikylamin is at different times in under-i Cetylpyridine chloride added to different concentrations, amounts of 5'-sodium inosinate that can be detected after 120 hours of cultivation are in Table X reproduced.

Table X

*) Determined as S'-inosinic acid disodium salt · 7 ¹ / «H ₁ O.

Example 12

The same inoculum and fermentation medium as in Example 1, but without hypoxanthine, are used. After 50 hours of cultivation 2.5 mg / ml guanine are added. Alkyldimethylbenzylammonium chloride is used as the surfactant. The remaining culture conditions correspond to those of Example 1. After 96 hours' culture, those given in Table XII are found Amounts of 5'-sodium guanyiate.

Table XlI

Alkyldimethylbenzylaramonium moride

Addition time (SId.)

Cetyl pyridine chloride

Addition time (Hours.)

250 500 750

500 750

No encore

Added quantities (y / ml)

250 500 750

amounts

dried

Bacterial cells

(mg / ml)

25.0 22.3 19.1

25.9 23.2 20.4

24.8 23.2

32.9

Arisen Amounts of 5'-sodium inosinate *) (mg / ml) 22nd

3.5 4.9 4.1

8.1 12.9 10.2

6.3 7.1

sense 26th

30th

6o Added quantities (V / ml)

500
1000

500
1000
1250

500
1000
1250

No encore

amounts

dried

Bacterial cells

(mg / ml)

19.5 17.1

20.3 19.3 20.1

21.5 21.3 22.7

36.8

Amounts of

5'-sodium

guanylate *)

(mg / ml)

1.1 2.0

1.9 5.9 4.7

2.3 4.0 3.1

sense

*) Determined as S'-inosinic acid disodium salt · 7 '/ t H, O.

·> Determined as S'-guanyl dinatriam salt.

Example 13

The same vaccine bacterium and infection medium as in Example 1 are used, however

209632/285

0.4% peptone was used in place of corn steep liquor. In certain concentrations, polyoxyethylene alkylamine, cetylpyridine chloride and alkyldimethylbenzylammonium chloride are used at certain times

admitted. The other culture conditions are the same as in Example 11. After 120 hours In cultivation, the amounts of 5'-sodium inosinate given in Table XIII are to be detected.

Table XIII

Surface active substance Art

amounts

dried

Bacterial cells

(mg / ml)

Arisen

Amounts of

5'-sodium inosinate *)

(mg / ml)

Polyoxyethylene alkylamine Cetyl pyridine chloride Alkyl dimethyl benzyl ammonium chloride

*) Determined as 5'-inosinic acid disodium salt · 7 '/ i H ₈ O.

38 36 38

400
200
400

No encore

18.6 18.9 19.0 22.9

15.8

15.0

14.7

8.9

Example 14

Vaccine bacterium: Brevibacterium ammoniagenes (ATCC 6871), fermentation medium according to Example 11, but with only 2 mg / ml hypoxanthine. Otherwise the same culture conditions as in Example 11. As As a surfactant, a mixture of 40% polyoxyethylene and 60% polypropylene is used. After 120 hours, the values in Table XIV indicated amounts of 5'-sodium inosinate were formed.

Table XIV Table XV

A mixture of Encore amounts Arisen 40% polyoxyethylene amounts
(ylrnW dried
Bacterial cells Amounts of
5'-sodium minosinate · '» and
60% polypropylene 500 Encore 1000 (mg / ml) (mg / ml) time
(StcU 1000 25.4 1.3 16 1500 23.2 '2.2 1000 23.9 7.5 20th 1500 21.0 6.1 No encore 24.3 4.0 24 22.0 2.6 37.2 sense

33

Polyoxyethylene amounts Amounts created alkylamine dried of 5'-sodium Added quantities Bacterial cells adenylate) (WmI) (mg / ml) (mg / ml) 500 24.9 1.08 750 22.1 3.93 1000 20.4 4.08 1250 18.2 2.99 No encore 29.3 sense

*) Determined as S'-adenylic acid disodium salt.

*) Determined as S'-inosinic acid disodium salt ■ 7 '/ i H ₁ O.

Example 15

Vaccine bacterium: Micrococcus sodonensis (ATCC 15932); Culture medium: 2% glucose, 0.5% yeast extract, 1.5% peptone and 0.25% NaCl (residual percentage = water); pH = 7.3. The fermentation medium: 10% glucose, 0.6% K ₂ HPO ₄ , 0.6% KHgPO ₄ , 0.6% MgSO ₄ · 7H ₈ O, 0.01% CaCl · 2 H ₂ O, 5 y / ml calcium pantothenate , 1 y / ml thiamine, 30 y / ml vitamin H, 0.1% FeSO ₄ · 7 H ₂ O, 1% casein amino acids and 0.6% urea (remaining percentage = water); pH = 8.0 before sterilization. After 48 hours of cultivation, 20 mg / ml of adenine and, after 30 hours of cultivation, polyoxyethylene alkylamine in various concentrations are added. The other culture conditions correspond to those of Example 11. After 96 hours of cultivation, the amounts of 5'-netrium adenylate given in Table XV could be obtained.

Example 16

The same vaccine bacterium and culture medium as in Example 15 were used; instead of Adenine will be in the medium after 48 hours, however 2.0 mg / ml guanine given. Furthermore, cetylpyridine chloride is added in various concentrations after culturing for 30 hours. The others Culture conditions correspond to those of Example 11. The men obtained after 96 hours of cultivation genes of 5'-sodium guanylate result from Ta belle XVI.

Table XVI

Cetyl pyridine chloride Added quantities

(y / ml)

55

250

500

750

1000

No encore

amounts

dried

Bacterial cells

(mg / ml)

22.8 22.0 20.1 19.6 28.7

Amounts created

. of 5'-sodium

guanylate *)

(mg / ml)

2.1 4.3 4.7 1.8 sense

*) Determined as 5'-guanyl acid: disodium salt.

Claims (1)

Claim: Process for the production of 5'-purianucleotides by fermentation of microorganisms in an aqueous nutrient medium that has the usual amounts of carbon supplies, nitrogen sources, inorganic salts and vitamin H too may contain larger amounts of growth-promoting substances as well as iron and manganese salts, in Presence of a purine base corresponding to the 5'-purine nucleotide as a precursor, thereby characterized in that the microorganisms Brevibacterium ammoniagenes ATCC 6871 and 6872 or Micrococcus sodonensis ATCC 15 932 and the fermentation medium an antibiotic or a surface-active substance in the period of logarithmic growth the microorganisms or briefly danacr admits.