DE1695329B1 - Process for the production of 5'-purine nucleotides by fermentation of microorganisms in the presence of antibiotics or surface-active substances - 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 through fermentation of micro-penicillin, mitomycin, streptomycin, cycloserine, organisms are from the French patents citracin, tetracycline, hydroxytetracycline, chlortetra-849 519 and 1 301 052 known. However, the cyclin, Carzinophilin (antibiotic from Strep. Saha known Process only low yields of the 5 chiroi cultures), 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 period 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 ATCC15 932. Antibiotics Achievable Results; d. H. the sub

The invention is concerned with improving the suppression of excessive bacterial growth 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 Yield of the desired product can be achieved as a result of over-medium.

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

The presence of excessive amounts of growth-promoting agents is increased by the fact that surfactants are not substances. This phenomenon is all the more easy to observe only in the case of excessive bacterial growth when nutrients with a high content of are added, but also, similarly to the growth-promoting substance, e.g. B. some types of treatment with antibiotics, if 5'-purine nucleotide corn steep liquor, rice bran or fish extracts is developed excellently,
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, tum 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 logarithmic period in addition to the usual amounts of carbon suppliers, 45 mixed growth of the microorganisms - in the nitrogen swell, inorganic salts and vitamin H in the event of excessive increases - to add larger amounts of growth-promoting substances z. B. the maximum growth 30 mg / ml, zen and iron and manganese salts, based on dried bacterial cells, makes up, in the presence of a 5'-purine nucleotide, so it is useful to use the substance at two thirds of the purine base as a precursor is characterized by the fact that the microorganisms are Brevibacterium, ammoniagenes ATCC 6871 and 6872, or microorganisms, if the surface-active coccus sodonensis ATCC15 is used 932 is used and an antibiotic or a time is added to the substances at a later fermentation medium than the above-mentioned, i.e. shortly afterwards,
Surface-active substance in the period of loga- 55 The following examples explain the invention using rithmic growth or microorganisms or preferred embodiments. If not admit shortly afterwards. otherwise stated, the percentages relate

According to the invention, therefore, the undesirable development in the following examples on the weight per turn of microorganisms without sufficient volume of fermentation medium.
Prevented generation of the 5'-purine nucleotides ^{by adding 60 -R - -Ii}

the fermentation in a fermentation medium with example 1

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

teln, thereby increasing the production of genes (ATCC 6872) for 24 hours in a culture 5'-purine nucleotide. The presence of medium of 2% glucose, 1.5% peptone, 0.2% urinary antibiotics is necessary for a maximum production of 5'-pulp, 0.1% K ₂ HPO ₄ , 0.03% MgSO ₄ -7 H ₂ O, 0.3% rinnucleotide is also useful, even if there are no NaCl, 0.01% FeSO ₄ · 7 H ₂ O and 30 y \\ vitamin H conditions that result in excessive wax (residual percentage = water ), pH = 7.3, cultured. Then favor the growth of microorganisms. are 10 percent by volume of this inoculum on a

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

Table I.

tion and cycloserine added in various concentrations. The other culture conditions remain the same as in Example 1. After 96 hours of culture, 5'-sodium inosinate has formed as in Table III specified.

Table III

Mitomycin C

Addition time
(Hours.)

Added quantities
Ö '/ ml)

15th
30th

20th 40

20th 40

30th 40

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

1.3
2.5

8.82
11.03

3.92
4.03

1.22
1.11

traces

amounts

dried

Bacterial cells

(mg / ml)

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

Arisen

Amounts of

5'-sodium inosinate *)

(mg / ml)

5.3
7.2
9.0

8.1

10.2

9.9

6.4
9.2
6.8

traces

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

12.9 11.0

15.1 14.8

18.0 17.6

21.3 20.7

32.8 ₂ H ₂ O.

35

No encore

*) Determined as 5'-inosinic acid disodium salt

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 shown in Table II.

Table II

*) Determined as 5'-inosinic acid disodium salt · 7 ^χ / ₂ H ₂ 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

Penicillin G Sodium
Addition- I addition time j amount
(Hrs.) (Y / ml)

5
10

5
10
50

10
50

Arisen

Amounts of

5'-sodium inosinate *)

(mg / ml)

2.3
4.1

4.0

6.3

10.2

9.0
7.9

No addition traces

*) Determined as 5'-inosinic acid disodium salt

amounts

dried

Bacterial cells

(mg / ml)

13.3 13.0

15.9 15.9 14.2

19.1 18.3

38.2 ₂ H ₂ O.

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

Example 3

The same vaccine bacterium and the same fermentation medium as in Example 1 are used, but 0.0005% MnSO ₄ · 4 H ₂ O is added instead of corn steep liquor. Furthermore, instead of mitomycin (Example 1) at different times during the fermentation

55 *) Determined as 5'-guanyl disodium salt.

Example5

The same inoculum as in Example 1 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- j addition
time I crowd
(Std.) J OVmI) 1
2 1
2
5 Arisen
Amounts of
5'-adenylic acid
(mg / ml) amounts
dried
Bacterial cells
(mg / ml) 8th 12: 1
! 2
5 No encore 1.1
2.3 17.1
16.8 17th 2.1
4.1
1.9 18.1
15.3
14.2 0.8
3.8
2.7 13.1
12.9
13.0 traces 40.2

Example 6

The same vaccine bacterium and the same fermentation medium as in Example 1 are used, however, corn steep liquor was replaced by 0.3% of an enzymatic casein 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 Dihydrostreptomycin Sulphate admitted at certain times. Otherwise, the culture conditions are as in the example 1. Table VI gives the amounts found after 120 hours of cultivation in the fermentation medium 5'-sodium inosinate again.

Table VI

Antibiotic
Art

Encore Encore Time lot (Hours.) (r / ml) 24 10 28 20th 30th 50 No 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

Dihydrostreptomycin sulfate

*) Determined as 5'-inosinic acid disodium salt I ¹ I ₂ H ₂ O.

Example 7

Vaccine bacterium: Brevibacterium ammoniagenes (ATCC 6871) fermentation medium according to Example 4. 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

0.01% 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 VIII.

Table VIII

Kanamycin
Addition- j addition time j amount
(Hrs.) I (r / ml)

10

15th

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: from 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,

Penicillin G Potassium
Added amount
OVmI) 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
traces

*) Determined as 5'-inosinic acid disodium salt · 7V ₂ H ₂ O.

Example 9

Vaccine bacterium: Brevibacterium ammoniagenes (ATCC 6872); Culture medium: 2% glucose, 2% peptone, 0.1% urea, 0.1% K ₂ HPO ₄ , 0.03% MgSO ₄ -7H ₂ O, 0.3% NaCl, 0.01% FeSO-7 H ₂ O and 30 μg / ml vitamin H (residual percentage = 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 , 1% KH ₂ PO ₄ , 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 (remaining percent = water). The pH was adjusted to 8.0 with 5N NaOH before sterilization. After the sterilization, 0.6% sterilized urea is added to the permentation medium.

After 20, 24 and 48 hours of culture, polyoxyethylene alkylamine is obtained in different concentrations admitted. The quantities of 5'-sodium inosine * obtained after 120 hours of cultivation are shown in Table IX

Table IX Example 11

The same vaccine bacterium as in Example 11 and the fermentation medium according to Example II are used, which, however, contained _{0.0005% MnSO 4} -4 H _{2 O instead of corn steep liquor.} Cetyltrimethylammonium bromide is used as a surfactant. The other culture conditions are the same as in Example 11. After 96 hours of cultivation, the amounts of 5'-netrium inosinate given in Table XI were obtained.

Polyoxy
alkyl
Encore
Time.
(Hours.) ethylene
irnin
Encore
inengen
(y / ml) 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 traces

Cetyl-trii
ammoniu
Encore
Time ethyl
mbromid
Encore
rn en se Table XI Arisen
Amounts of
5'-sodium inosate *) 15th (Hours.) .L JL JL \ fi JiB ^ \ ^
OVmI) amounts
dried
Bacterial cells (mg / ml) 20th 500
750
1000 (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 25th 28 750
1000
1250 21.3
21.8
19.2 8.2
9.1
8.7 No encore 22.3
22.5
21.5 traces 30th 34.8

35 * -) Determined as 5'-inosinic acid disodium salt

Example 12

H ₂ O.

*) Determined as 5'-inosinic acid disodium salt · 7 ¹ U H ₂ O.

B e i s ρ i e 1 10

The same measures are used as in Example 11; instead of polyoxyethylene alkylamine cetylpyridine chloride is added at different times in different concentrations, Amounts of 5'-sodium inosinate that can be detected after 120 hours of cultivation are in Table X reproduced.

Table X

The same inoculum bacteria and fermentation medium are used as in Example 11, but without hypoxanthine used. After cultivation for 50 hours, 2.5 mg / ml guanine are added. As a surface active The agent used is alkyldimethylbenzylammonium chloride. The rest, culture conditions correspond to those of Example 11. After 96 hours of culture, those given in Table XII are found Amounts of 5'-sodium guanylate.

Table XII

Alkyl dimethyl benzyl ammonium chloride

Addition time
(Hours.)

Cetyl pyridine chloride Encore Encore amounts Time (r / ml) (Hours.) 250 21 500 750 250 25th 500 750 500 29 750 No encore

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) 22

55

3.5
4.9
4.1

8.1
12.9
10.2

6.3

7.1

Tracks 26

30th

6o added quantities
(r / 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

traces

*) Determined as 5'-inosinic acid disodium salt · 7 ¹ J ₂ H ₂ O.

*) Determined as 5'-guanyl disodium salt.

Example 13

The same inoculum and fermentation medium are used as in Example 11, however

109 552/433

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

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

Encore Encore Time lot (Hours.) (r / ml) 38 400 36 200 38 400 No encore

amounts

dried

Bacterial cells

(mg / ml)

Arisen

Amounts of

5'-sodium inosate *)

(mg / ml)

Polyoxyethylene alkylamine

Cetyl pyridine chloride

Alkyl dimethyl benzyl ammonium chloride

*) Determined as 5'-inosinic acid disodium salt · 7Va H ₂ O.

18.6
18.9
19.0
22.9

15.8

15.0

14.7

8.9

Example 14

Vaccine bacterium: Brevibaczerium ammoniagenes (ATCC 6871); Fermentation medium according to Example 11, but with only 2 mg / ml hypoxanthine. Furthermore same culture conditions as in Example 11. A mixture of 40% polyoxyethylene and 60% polypropylene are 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
(yimYi dried
Bacterial cells Amounts of
5'-sodium inosinate * "> UtIQ
60% polypropylene 500 Encore 1000 (mg / ml) (mg / ml) Time
(Hours.-) 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 traces

30th

35

Polyoxyethylene amounts Amounts created alkylamine dried of 5'-sodium Added quantities Bacterial cells adenylate *) OVmI) (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 traces

45

*) Determined as 5'-inosinic acid disodium salt · 77a H ₂ O.

Example 15

Vaccine bacterium: Micrococcus sodonensis (ATCC 15932); Culture medium: 2% glucose, 0.5 ^fl / ₀ 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% KH ₂ PO ₄ , 0.6% MgSO ₄ · 7H ₂ O, 0.01% CaCl · 2H ₂ O, 5 y / ml Calcium pantothenate, 1 μg / ml thiamine, 30 μg / ml vitamin H, 0.1% FeSO ₄ · 7H ₂ O, 1% casein amino acids and 0.6% urea (residual 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'-sodium adenylate given in Table XV could be obtained.

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

Example 16

The same vaccine bacterium and culture medium were used as used in Example 19; instead of adenine will be in the medium after 48 hours however 2.0 mg / ml guanine given. Furthermore, after 30 hours of cultivation, cetylpyridine chloride is used in different Concentrations added. The other culture conditions correspond to those of Example 11. The amounts of 5′r sodium guanylate obtained after 96 hours of cultivation are shown in the table XVI.

Table XVI

Cetyl pyridine chloride
Added quantities amounts
dried
Bacterial cells Amounts created
of 5'-sodium
guanylate *) (y / ml) (mg / ml) (mg / ml) 250 22.8 2.1 500 22.0 4.3 750 20.1 4.7 1000 19.6 1.8 No encore 28.7 traces

*) Determined as S'-guanyl disodium salt.

Claims (1)

Claim: Process for the production of 5'-purine nucleotides by fermentation of microorganisms in an aqueous nutrient medium which, in addition to the usual amounts of carbon suppliers, nitrogen sources, inorganic salts and vitamin H too 11 12 larger amounts of growth-promoting substances 6871 and 6872 or Micrococcus sodonensis ATCC as well as iron and manganese salts, used in 15 932 and the fermentation medium Presence of an antibiotic or a surface-active sub- corresponding purine base as a precursor, thereby punching in the period of the logarithmic wax characterized in that one as micro- 5 turns of the microorganisms or shortly thereafter Organisms Brevibacterium ammoniagenes ATCC admits.