DE1926072C - Process for the biochemical production of cyclic adenosine 3,5-monophosphate - Google Patents

Description

The invention relates to a method for biochemical Production of cyclic adenosine 3 ', 5'-monophosphate, hereinafter referred to as 3', 5'-cyclic for short Adenyic acid or A-3 ', 5'-MP.

A-3 ', 5'-MP is known to be participating in its door various chemical reactions in living cells and for its active role in the formation of various Hormones. That is why it is valued as a biochemical reagent. The making of this Acid by chemical means involves many complicated synthesis steps and has not been able to do so until now be replaced by a simpler process, surprisingly it was found according to the invention that the microorganisms Corynebacterium murisepticum ATTC 21 374, Arthrobacter ATCC 21 375 and Microbacterium ATCC 21 376 can produce cyclic A-3 ', 5'-MP when they are grown.

The invention thus relates to a process for the biochemical production of cyclic adenosine 3 ', 5'-monophosphate, which is characterized in that Corynebact-irium murisepticum ATCC 21 374, Arthrobacter ATCC 21 375 or Microbacterium ATCC 21 376 in the presence of adenosine, Adenine, inosine, hypoxanthine, S-amino ^ imidazole carboxamide - riboside, 5 - amino - 4 - imidazole carboxamide, succinyladenosine or succinyladenine in the nutrient medium breeds.

The nutrient medium preferably has a content of 0.1 to 3.0 weight / volume percent of the aforementioned Compounds adenosine, adenine, inosine, hypoxanthine, 5 - amino - 4 - imidazole carboxamide riboside, 5-amino-4-imidazole carboxamide, succinyladenosine or succinyladenine.

From the following the mycological properties of the above mentioned strains emerge:

I. Morphological properties: (Breeding in
Nutrient agar and nutrient broth at 37 or 3O ⁰ C)

Microbacterium. ATCC 21 376
Shape and arrangement:
club-shaped or curved cells were observed in the field of view of the microscope; Occasionally, angular and palisade-like arrangements were observed, which were created by tearing apart.
Cell size (age: 16 hours, 30 C):

0.4 to 0.7 x 1.5 to 3 microns
Gram stain: +
Spore coloring: (spore formation)

Scourges:
Mobility: -

Corynebacterium murisepticum, ATCC 21 374

Shape and arrangement:

Rods, elongated cells at first, branched cells and V-shaped lines were observed; after 2 or 3 days the cultures showed cocoids To form. Cell size (age: 16 hours):

0.5 to 0.8 x 1.5 to 4 microns Gram stain; + Spore color: (spore formation)

Scourges: -

Mobility: -Acid resistance: -

40 Arthrobacter, ATCC 21

Shape and arrangement:

Rods, elongated cells at first, branched cells, V-shaped cells were observed. After 1> or 4 days, the cultures showed coke-like forms. Cell size (age: 16 hours):

0.5 to 0.8 x 2 to 5 micron Gram stain: different Spore stain: (spore formation)

Scourges:

Mobility: - Acid resistance: -

II. Cultivation properties 1. Inclined agar nutrient medium (impact

posed Broth agar, ^30c C. 48 ATCC
21374 Hours) ATcr
21 37b moderate
thread
shaped
glittering
and wet
Milk white
buttery ATCC
21 375 Growth..
shape
shine
color
consistency moderate
prickly
glittering
and wet
yellow
buttery moderate
thread-like
glittering
and wet
Milk white
buttery

2. Agar colonies (bouillon agar, 3O ⁰ C, 48 hours)

ATCC
21 376 ATCC
21 374 ATCC
21375 shape around around around surface
Edge smooth
all
convex
impenetrable
sighted smooth
all
pillow-
shaped
impenetrable
sighted smooth
all
pillow-
shaped
impenetrable
sighted Surface profile
Optical own
societies

3. Nutrient broth: (bouillon broth, 30 ⁰ C, 7 days)

Surface growth ..
Turbidity ....

Growth amount

sediment

ATCC 21 376

ring-shaped moderately cloudy

moderately sparse, sticky

ATCC

21

slightly cloudy ring-shaped

moderately sticky

ATCC

21

ring-shaped slightly trnb

moderately sticky

4. Provided inclined Kartoflel agar (30 ⁰ C, 3 days)

growth

color

ATCC 21376

moderately healthy

ATCC 21374

moderately milk white

ATCC 21375

no growth

5. Optimal growth temperature

6. Thermal resistance when heated in 10% skimmed milk, 15 minutes at 72 ° C and

Minutes at 7O ⁰ C.

ATCC 21 376 ATCC 21,374 ATCC 21,375 37 ° C 37 ^G C 37 ⁰ C

ATCC 21 376

ATCC 21,374

ATCC 21,375

III. Physiological properties:

ATCC 21 376

ATCC 21,374

ATCC 21,375

1. Catalase reaction (3O ⁰ C, oblique

prepared bouillon)

2. Reduction of nitrate (breeding

at 30 ° C)

3. Indole formation (30 ⁰ C, 5 days) ....

4. Hydrogen sulfide formation (30 ⁰ C,

5 days)

5. Exploitation of Cilrat

6. Voges-Proskauer reaction (30 ⁰ C,

5 days)

7. Methyl red test

8. Liquefaction of gelatine (3O ⁰ C,

5 days)

9. Decomposition of cellulose

10. Urease reaction

11. Reduction of methylene blue ....

12. Effect on litmus milk ....

13. Pigment formation

14. Pathogenicity

15. Liquefaction of starch.

is reduced, sour and peptonized

yellow pigment

is reduced, alkaline and peptonized

is reduced, alkaline and peptonized

16. Utilization of sugars

ATCC
Acid picture 21 376
Gas picture ATCC
Acid picture 21 374
Gas picture ATCC
Acid picture 21375
Gas picture glucose _{+ +} Glycerin I I I I I I I I I I I I I I I I + I + I I Xylose + - - - - Sucrose + - - strength + ... Fructose ± Galactose + ± «. Mannose Arabinose Maltose _ Trehalose Dextrin Inulin . ,, ...., .....,. ,, _-

AtCC 21 376

ATCC 21,374

ATCC 21,375

17. Utilization of nitrates

18, utilization of ammonium salts

According to the determination methods in Bcrgeys Manual of Determinative Bacteriology, 7th Edition, the strain Microbacterium ATCC 21 376 described above is pleomorphic catalase-positive, Gram-positive aerobic and therefore belongs to the class Eubacteriales. On further investigation it was found that the strain ATCC 21,376 in 10% skim milk to a temperature of 72 ° C is heat-resistant for 15 minutes and at 7O ⁰ C for 30 minutes, it thus from all genera Listeria, Erysipelothrix, Cellulomonas, Arthrobacter and Corynebacterium, sub-family Corynebacteriaceae different; but it belongs to the genus Microbacterium of this family.

The strain ATCC 21 375 should belong to the genus Arthrobacter as his grief response is different. Organisms belonging to this genus are based on their ability to utilize nitrates, ammoniacal nitrogen and citrates in the essentially divided into two groups; However, this strain uses nitrates and ammoniacal nitrogen, but no citrates, and therefore does not belong to either of the two aforementioned groups. As a result, it is owned this strain belongs to the genus Arthrobacter.

ATCC strain 21 374 catalase-positive, immobile, unable to utilize cellulose, gram-positive and has no heat resistance in 10% skimmed milk; so it belongs to the genus Corynebacterium. In addition, the trunk is immobile, forms nitrite from nitrate and generates acid from glucose, Lactose, sucrose and mannitol. That is why the additional designation »murisepticum« was chosen.

For the production of cyclic A-3 ', 5'-MP according to the invention, adenosine, adenine, inosine, hypoxanthine, 5-amino-4-imidazole-carboxamide-riboside, 5-amino-4-imidazole-carboxamide, succinyladenosine or succinyiadenine or a mixture of these compounds, solid or liquid material with one or more of the compounds mentioned or fermented mash of the compounds mentioned added to a nutrient medium which contains assimilable carbon sources, nitrogen sources and inorganic phosphate and optionally other inorganic salts and constituents. The microorganism is grown in the nutrient medium at a pH between 5 and 9 at 20 to 40 ° C. until the best possible yield of cyclic Y-3 ', 5'-MP is obtained, e.g. B. 10 to 80 hours. Otherwise, a strain of microorganisms used according to the invention is first inoculated into the medium and grown in this medium at a pH in the range from 5 to 9 at 20 to 40 ^° C. for a suitable period of time, for example 10 to 40 hours. After cultivation, adenosine, adenine, inosine, hypoxanthine, 5-amino-4-imidazole-carboxamide-riboside, 5-amino-4-imidazole-carboxamide, succinyladenosine or succinyladenine or an Oe * are mixed of these compounds of the oärmaische in an amount of 0.1 to 3.0 percent by weight (w / v), based on the total volume of the medium, added. Cultivation is continued until the amount of substance formed in the nutrient medium reaches a maximum, e.g. B. after 10 to 80 hours.

The medium mentioned contains carbohydrates such as glucose, starch hydrolysates or glycerine as carbon sources, Ammonium sulfate, ammonium chloride, urea, amino acids, protein or peptide hydrolysates and cell extracts such as corn steep liquor and yeast extract as nitrogen sources, potassium dihydrogen phosphate or sodium dihydrogen phosphate, dipotassium hydrogen phosphate or disodium hydrogen phosphate or ammonium phosphate as an inorganic phoa sources of phate, as well as other inorganic salts, e.g. magnesium sulfate, magnesium chloride, Iron (III) sulfate or iron (II) sulfate, iron (III) chloride or iron (II) chloride, zinc sulfate or zinc chloride, cobalt sulfate or cobalt chloride. Breeding is applied by any suitable method, e.g. B. with shaking, with stirring, carried out aerobically.

When the formation of cyclic A-3 ', 5'-MP in the nutrient medium reaches its maximum, the cultivation is terminated and the acid is isolated from the fermentation mash and purified. For the isolation and purification of the acid, activated carbon, an anion resin exchanger, or a cyclic A-3 ', 5'-MP non-dissolving solvent can be used. For example, after removing the cells, the fermentation mash is treated with activated charcoal to adsorb the cyclic A-3 ', 5'-MP contained in the fermentation liquid, whereupon the activated charcoal is treated with ammonia-ethanol solution or ammonia-acetone solution to elute the acid mentioned is washed. The eluate obtained is evaporated under reduced pressure to remove excess ammonia and ethanol and further treated with an anion exchange resin, such as a strongly basic anion exchanger with quaternary ammonium groups in the chloride form or formate form, and with a suitable solvent. 7. B. with dilute hydrochloric acid or calcium chloride-containing dilute hydrochloric acid for the anion exchanger in the chloride form or with dilute formic acid containing dilute formic acid or sodium formate for the anion exchanger in the formate form, to elute the cyclic A-3 ', 5'-MP. The eluate is adsorbed again on activated charcoal, washed with ammonia-ethanol solution or ammonia-acetone solution, evaporated under reduced pressure to remove excess ammonia and ethanol, on a cation exchange resin, e.g. B. a strongly acidic cation exchanger with sulfonic acid residues, ^{adsorbed in the H +} form and finally eluted with dilute hydrochloric acid. The eluate obtained in this way is evaporated under reduced pressure, left to stand in the cold or a solvent, such as ethanol or aceion, which does not dissolve cyclic A-3 ', 5'-MP, is added, whereby the acid is obtained in crystalline form.

According to another procedure, the cyclic A-3'.5'-MP formed in the fermentation mash

j5 after removal of the cells from the broth on Akti. money adsorbed and eluted with ammonia-ethanol solution or ammonia-acetone solution. The eluate becomes of excess ammonia after Efitferming and ethanol, e.g. by evaporation under reduced pressure, acidified with hydrochloric acid and with an organic solvent such as alcohol or acetone, added, whereby crude cyclic A-3 ', 5'-MP receives. The crude cyclic A-3 ', 5'-MP obtained in this way can by means of an anion exchanger * resin or a cation exchange resin, as mentioned, are cleaned. The obtained crude cyclic A-3 ', 5' -M P is dissolved in water, acidified with hydrochloric acid or phosphoric acid, with a Ent far-

1 92θ 072

exercise resin (see, Ullmatins Bneyeloeadte der tech * see Cheitiig, Vol, 8 [1957] »S, 813) decolorized and mixed with ethanol, acetone or other diacylic A'3 ', 5'-MP solvents which dissolve quickly, wouföh one eyetlsehes Α4 \ 5 '· ΜΡ Sn crystalline Gets shape,

ÖernäÖ vain further separation * and cleaning * rtrfahrcn the Öärmaische is flat removal of the Cells directly with an anion or cation exchange resin to adsorb the cyclic A-3.5-MP treated. After treating the obtained eluate with activated charcoal or a decolorization resin gives cyclic «A-.V, 5'-MP in crystalline form by adding a non-dissolving solvent.

The product prepared by the process of the invention showed, with respect to elemental analysis, R ibose- or phosphorus content and UV spectrum and IR spectrum the same results as an authentic sample of cyclic! A-3.5-MP

The process of the invention is a one-step process. So far, cyclic »A-3'.3'-MP could only are produced chemically in a complicated multi-step process.

The examples illustrate the invention. Example I.

Microbacterium ATCC 21 376 was an inclined nutrient medium containing 0.5% ammonium sulfate, 0.5% potassium dihydrogen phosphate, 0.05% magnesium sulfate. 1.0% amino acids from casein. 0.3% Yeast extract. 1.0% glucose and 10% agar-agar and its pH value with 3N potassium lye 7.0 was set, inoculated and grown on.

Separately from this, 40 ml of a nutrient medium was added each time. the 0.01% Zmksuhat. 5.0% ohtcose, 0.5% urea, 0.5% ammonram sulfate, 1.0% potassium dihydrogen phosphate. 1.0% dicariurepphate, 1.0% polypeptone. 0.1 ml per liter of maize quench water. Contained 0.3% inosine and 1.0% magnesium sulfet and its pH value was adjusted with 3 e-Ral9aege aaf 8.0, filled in SOtKml-Schütteau-kurflaachen. After 12mieutigem Aatoklavieren at 115 ° C the above-obtained Anzuchtkufter Oermatsche was inoculated and cultured 32 hours with shaking at 30 ⁰ C. 3.1 mg per milliliter of cycfoches A-3 \ 5'-MP were bathed in the oätwaeche.

The fermentation mash obtained was used to remove the cells Astigr and the Obetstaod adjusted to pH 2.0 with concentrated hydrochloric acid and then with activated charcoal for adsorption of the cyclic A-3 \ 5'-MP. The acid adsorbed on the activated carbon was with a 1.4% ammonia-ethanol solution duiert, and the budget was for the removal of ammonia and of the ethanol was evaporated under reduced pressure. Then the eluate thereby obtained was in a pH value of 8.0 on a strongly basic anionic oil * in the form (grain size 044 constricted. The solution obtained was adjusted to an eleventh pH value of 2.0 with diluted Sälüsaufe and put on a strongly acidic Katlöfienausöner in the H ⁺ 'form (grain size 0.14 Ws 0.074 mm)

chfomaiögfaphieft, IeI of the leaves with 0.1 n * sala acid, a fraction with eyeliwhem Μ'4'ΉΡ was obtained out

to 300 ml fermentation mash 920 mg crystalline cyclic « A-3.5-MP

Example 2 Corynebacterium murisepticum ATCC 21 374

i) an inclined nutrient medium containing 0.3% Ammonium sulfate. 0.5% potassium dihydrogen phosphate, 0.005% magnesium sulfate, 1% amino acids from casein. 0.3% yeast extract. 1.0% glucose and 2.0% Agar-agar contained and with 3 η-potassium hydroxide solution on the

w pH was adjusted to 7.0, inoculated and bred on it.

Separately, 30 ml of a nutrient medium containing 3.0% glucose, 0.3% ammonium chloride, 1.0% dipotassium hydrogen phosphate, 1.0% Katiunv

i) contained dihydrogen phosphate, 1.0% polypeptone, 0.5% yeast extract and 0.0027% zinc sulfate and its iH value was emgertellt to 8.0 with 3 η potassium hydroxide solution. 5TOmI- ^ huttel culture bottles filled. To Autoclave this nutrient medium for 10 minutes 115 "C and after the addition of 3% separately sterilized calcium carbonate, the seed culture became this Inoculated fermentation mash and cultivated under gravel at 30 ° C for 10 hours. Then 60 mg of inosine and breeding was carried out with it

Temperature continued with shaking for 70 hours cycise A-3 \ 5'-MP was formed in an amount of 21 mg / ml.

The obtained company mixture was used for removal the ZeBen ru, which was survived with

Φ 3 n-8aYes set to pH 4.0 and rarely adsorption of the cyclic A-3 \ 5'-MP contained in the intestine was treated with activated charcoal -Ldsaag determined. The

4 $ Eleat will be used to remove ammo »staks as a weakly snuien tCattoMsaustaescbcr β oer ti -roon cBroBimS) _t riipnicn βοα <wnn enter changed discs. etagedaiQpfl. Addition of alcohol to the residue gave matt 830 mg of critical

so s ^ uliniescTsicK ^^ A ^ '^ lliPaelxn

Example 3

Microbacterium ATCC 21 376 was a slanted nutrient, the 0 \ 5% ammonram sutfate,

up to 0.074 mm). When eluting with a mixture of 0.1 n-hydrous acid and 0.1 sodium formrate became a fraction with Cydic Α-3% 5'-ΜΊ> receive,

The FraktiOB obtained is re-adsorbed on activated carbon; oils activated charcoal was mixed with water n and with a t, 4% ipB ammonia-ethanol solution flat using the method described above The eloat was hardened under reduced pressure ^ ygip ,, g

sulfate, 1.0% ammo acids aas casern, 0.3% yeast extract, 1.0% glucose and 2.0% agar-agar and whose pH value was adjusted to 3 normal amounts and 1.0 %, Received and then ignited

Getrenöt davoo wtttdett each 40 ml of a nutrient medium containing 4.0% glycerine, (. 0.5% Kafioiadihyd'ogerJpBosp ^ at, 0.5%

xantlriti uwi 0j002 /% Snksaifet eät6ielt nod dteseo pH-value with t n ^ Kahiaege Anf f, 0 eägg «äft WBide, m 500-mi-Sdschen ab ^ efelL After Jti Aatoklavierefj des NahftifedHHns

115 ° C and addition of 3% separately sterilized Caiciutnearbonat was the Ansucht obtained above 'culture inoculated by suspension and uflder Schiit * stuffs for 30 hours at 3O grown ⁰ C, wherein cyclic * A * 3', 5 '* MP in an amount Vöfi 1.5 mg / ftit was formed.

The Öärniaische obtained was centrifuged to remove the cells and treated in the same manner as described in Example 1. 350 mg of cyclic * A-.V.5-MP were obtained from 500 ml of fermentation mash. to

Example 4

Arihrobacter AT (V 21 .175 was grown on an inclined nutrient medium of the same composition as in Example 2 at W Γ 24 hours. The culture culture obtained was inoculated into 50O ml shake culture flasks in 40 ml nutrient medium each, the 5.0% glucose. 0.5% ammonium sulfate. 1% dipotassium hydrogen phosphate. 0.5% urea. 1.0% potassium dihydrogen phosphate. 1.0% polypeptone. 1.0% Magnesium sulfate. 0.5% yeast extract. 0.2% 5-amino-4-imida? Olcarboxamid-riboside and 0.001% cobalt sulfate, the pH of which with 3 η potassium hydroxide solution 7.0 was set, which was 12 minutes at 115 ° C autoclaved and treated with 3% separately sterilized calcium carbonate. One bred 40 hours at 30 "Γ with shaking and received 1.8 mg / ml cyclic« A-3'.5'-MP.

The obtained fermentation mash was centrifuged to remove the cells and in the same manner. as described in Example 1, treated. One received 415 mg cyclic * A-3'.5'-MP from 500 ml garma mixture.

Example 5

Arthrobacter ATCC 21 375 was placed in an oblique · J5 Provided nutrient medium of the same composition as in Example 2 at 30 ° C for 24 hours. The seed culture obtained was in ftO ml shake culture flasks are inoculated into 40 ml culture medium each, the 5% glucose. 0.5% ammonium sulfate. 1% dipotassium hydrogen phosphate, 0.5% urea. 1% potassium dihydrogen phosphate, 1% polypeptone. 0.5% magnesium sulfate. 0.5% yeast extract. 02% 5 * Amino-4-imtdazolcarbo * amide and 0.001% cobalt sulfate contained, with 3 N KaKtaoge to the pH 7.0 and autoclaved, and cultured at 30 ° C for 72 hours with shaking. It formed sHi 1.4 mg / ml cycsc A-3'.5'-M P in the nutrient medium. The garma mix was treated in the same way as described in Example 1 332 mg cyclic A-3 ', 5'-MP from 500 ml garma mixture.

Example 6

Corynebacterhim murisepticum ATGC 21374 was grown in a seeding medium in the same manner as in Example 5, and a nutrient medium was inoculated which had the same composition as in Example 4, except that the 5-ammidazoIcarboxamidribosid 0.2 % Succinyladenosiri were added. Cultivation was carried out at 30 ° C. for 40 hours with shaking. 1.2 mg / ml cycfiscfieVA> 3 ^ 5'-MP were formed. The garma mixture was centrifuged to remove the cells and treated as in Example 1; ma * received 180 mg of cyclic "A'3", 5 "MP from SÖÖfti OMrmaische.

Example 1

Midrobactorium ATCC 21 376 was laboriously the same cultivation procedure as in Example 4 and inoculated into a suturing medium which had the same composition as in Example 5, with one exception that 0.2% succinyladenine was added instead of VAmino-4-imiJazolcarboxamid . After growing for 70 hours with shaking at 30 ° C, 0.7 mg / ml cyclic A-3.5 MP was produced. The fermentation mash was centrifuged to remove the cells and treated as in Example I; 121 mg of cyclic A-3.5-MP were obtained from 500 ml of fermentation mix.

Example 8

Microbacterium ATCC 21 376 was found in the same Cultivation medium grown as in Example 5, inoculated into a nutrient medium which had the same components as in Example 6, with the exception that no 0.2% adenosine was added instead of succinyladenosine, and under rubble for 40 hours Bred at 30 ° C. 2.0 mg / ml of cyclic was obtained A-3'.5'-MP. The fermentation mash was centrifuged to remove the cells and treated as in Example 2: 750 mg of cyclic A-3'.5'-MP were obtained from 500 ml fermentation mash.

Example 9

Microbacterium ATCC 2t 376 was m similar Share culture medium grown as in Example 4, inoculated into a nutrient medium containing the had the same composition as in Example 5, with the exception that 0.1% adenine was added to the side of 5-ammo-4-rmidazole carboxamide, and grown with shaking for 70 hours at 3 (TC. U mg / ml cyclic A-3 \ 5'-MP were obtained. The Fermentation mash was centrifuged to remove cells and aaf in the same manner as in Example I. treated. 230 mg of cyclic A-3'.5'-MP were obtained from 500 ml of Girmatsche

Claims (2)

Patentae: 1. Process for biochemical production, ung of cyclic adenosine - 3'4'- fBcmophosphate, characterized in that one in compliance with the requirements of cultivation Corynebacterium murisepticum ATCC 21 374, Arthrobacter ATCC 21 375 or Microbacterium ATCC 21 376 in the presence of adenosine, adenine, inösine, hypoxanthme, S-Amino ^ imidazolcarboxamid-ribosid, S-Äiümo ^ -nnidazoIcarboxafflid, Succmyfadenosin or Succmyladenm breeds in the nutrient medium 2. The method according to claim I, characterized in that the content of the nutrient medium Adenosine, adenine, isosra ^ hypgxanthin, 5-amino-4-imidazole caiboxamid-nT3ösM, 5-amino-4-imide-; azolearboxainid, succinyladenosm or succmyladeninO ,! to 3.0 weight / volume percent