DE1068429B - Process for the microbial production of cobalamines by Nocardia rugosa in the presence of diamines as precursors - Google Patents

Description

GERMAN

The subject of the main patent 1 046 258 is a biosynthetic process for the production of cobalamines, which is characterized by the use of microorganisms of the genus Nocardia, especially the strain Nocardia rugosa, the nutrient fluid optionally containing surface-active agents.

In a further development of the process according to the main patent, it was found that Nocardia rugosa, like other microorganisms, is not only able to form cobalamins from morphological and environmental properties, but also has the ability to act as a recursor additive in biosynthesis in the presence of from cobalamines with other microorganisms known o-phenylenediamines or from its alkyl derivatives to produce so considerable amounts of vitamin B ₁₂ -like cobalamins that their extraction is possible.

The present invention therefore relates to the biosynthetic production of cobalamins with the aid of Nocardia rugosa in the presence of δ-phenylenediamine or its xenyl derivatives.

When using a well-growing culture of Nocardia rugosa, a lot of nutrient broth is added. The culture is then kept under aeration and stirring in a temperature range of 30 to 37 ° C incubated until the microorganisms have grown well, which is generally after about 24 to 30 years Hours is the case. The suspension obtained in this way is used to inoculate the nutrient solution of the actual fermentation process used. The amount of inoculation solution changes from 5 to 10 percent by volume, based on the to inoculating solution.

At the same time, the precursors are added to in such an amount that a final concentration of 50 to 1000 Gamma per cm ³ is obtained, with their sterilization by either Behändem the aqueous suspension in an autoclave or by contact with alcohol at 95 ⁰ C for about 2 hours he follows. The formation of the cobalamin-type substances is conveniently monitored by bioautography and chromatography. The fermentation is considered complete when no further increase in the formation of B ₁₂ -like substance is observed. This is generally the case when the content of substances with a B ₁₂ -like activity against E. choli 113/3 corresponds to a content of approximately 2 gamma vitamin B ₁₂ per cm ³ .

At this point in time, most of the substances with B ₁₂ -like activity are in the mycelium, from which they can be extracted using the usual methods. .

The particularly suitable method for extracting cobalamines from nutrient media consists initially of one Filtration, which achieves two results at the same time: on the one hand, the separation of the active substances, Process for microbial production

of cobalamines by Nocardia rugosa in the presence of diamines

as precursors

Addendum to patent 1,046,258

Applicant:

Societä Farmaceutici Italia,
Milan (Italy)

Representative:

Dipl.-Ing. Dipl.-Chem. Dr. phil. Dr. techn. J. Reitstötter, Patent attorney, Munich 15, Haydnstr. 5

Claimed priority:
Italy 8 May 1956

Aurelio di Marco, Celestino Spalla,
Giulia Boretti and Carlo Giuseppe Alberti, Milan

(Italy),
have been named as inventors

which are in the mycelium mass, on the other hand the removal of the excess still in the solution of additives.

The mycelium is then hydrolyzed by boiling with water, optionally in the presence of small amounts Cyanide.

The hydrolyzed mycelium is removed by centrifugation and the resulting solution is removed by solvent extracted. Among these solvents is e.g. B. Benzyl alcohol into consideration, which after salting out the aqueous phase is used, also phenols or naphthols, halogenated homologues and derivatives thereof, which are used alone or in a mixture with other solvents such as chloroform or carbon tetrachloride which, although they do not improve the extraction, give the mixture higher selectivity and lower Give water solubility.

The active substances can be precipitated from the organic extracts by adding solvents, which reduce their solubility, e.g. B. benzene, acetone, chloroform and ether.

The raw material thus obtained can be prepared according to well-known methods for substances Cobalamin groups are cleaned. Indeed you can

909 647/390

These substances with alcohols of low carbon number, which do not dissolve the inert material, luiert and obtained from such an alcoholic solution of the active substances by adding diethyl ether will.

The solid product obtained can be further purified by exchange absorption with synthetic resins or by absorption on other substances and subsequent elution by repeating the extraction with phenols nd renewed precipitation from the aqueous solution of the solid product. Those present in the cleaned product Substances of the cobalamin group can be separated by processes as they are generally used for separation of substances with a similar chemical structure, e.g. B. by countercurrent distribution between Vasser and a solvent that is immiscible with this, its partition coefficient for the different available fabrics varies. It was observed that this separation ate by chromatography on paper, had or can be carried out satisfactorily in columns: ann.

In the following some exemplary embodiments for ■ ermentations are given using pre- : ursors of the series mentioned.

example 1

Culture nutrient solution:

■ nutrient solution:

4% malt extract, 4% glucose, 1.5% NZ-amine, 0.5% soft grain, 0.5% CaCO ₃ , 0.5%
(NH ₄ ) ₂ SO ₄ , 0.1% NaCl, 10 pM CoCl ₂ .

After sterilization and cooling to 33 ° C., 01 nutrient solution is ^{inoculated by adding 100 cm 3 of} a suspension which is obtained by washing a culture on a Roux nutrient agar. The main amount is added over a period of 25 to 40 hours at 33 ° C., with a ventilation of 30 to 50 liters of blown air per knuckle and, with stirring, of 150 to 300 revolutions per minute. When the thickness of the culture corresponds to a dry weight of 3 to 5 mg / cm ³ , the culture is used to inoculate the medium of a larger volume fermentation vessel.

In this stage, a nutrient solution is used which has the above-mentioned composition and to which 00 to 400 gamma o-phenylenediamine per cm ^{3 is} added after it is sterilized in aqueous suspension or by contact with 95% alcohol for two hours; at.

Samples are taken every 20 hours. ) The mycelium is then removed by centrifugation, washed with water, taken up again in a water acidified with hydrochloric acid in the same volume as the nutrient liquid, mixed with 5% KCN and heated ^{to 120 ° C. in the autoclave for 5 minutes.}

The presence of B ₁₂ -active substances compared to i. choli is determined by paper chromatography with see. Sutyl alcohol, acidified with acetic acid, as a mobile phase (according to J. Ford, ES HoldsworthundS. Kon: Siochem. J., 59, 86, 1955), followed by bioautography on an agar layer containing E. choli 113 _{, which requires vitamin B 12} / 3 is inoculated.

Under standard conditions, chromatography without the use of precursors in the first samples shows, in addition to the presence of vitamin B _12, two further growth factors for E. choli, one of which has an Rf value of approximately 0 (the Rf value; the ratio between the rate of migration of the substance and the solvent), the other has an Rf value of 1 '3 d "s Rf-W ^ rt <\ s of vitamin B _1,.

In contrast, in the presence of o-phenylenediamine, the presence of two factors with a low Rf value is not yet encountered, whereas a growth factor with an only slightly lower Rf value than that of vitamin B ₁₂ can be detected. _{It is not possible to separate this factor from vitamin B 12} by electrophoresis in acetic acid according to ES Holdsworth (Nature, 171, 148, 1955).

_Lo example 2

Culture broth:

4% glucose, 2% lactose, 1% peptone, 0.25% yeast extract, 0.2% KH ₂ PO ₄ , 0.5% Na ₂ HPO ₄ , 1% NaCl, 10 pM CoCl ₂ .

The process is carried out as described in Example 1 using 100 to 200 gamma / cm ^{3 of} 4,5-diethyl-1,2-phenylenediamine as a precursor. The bioautography shows the presence of a factor with a higher Rf value than vitamin B ₁₂ , which factor may be different from factor B because it cannot be separated from vitamin B ₁₂ by electrophoresis.

Example 3

Culture broth:

nutrient broth:

4% malt extract, 4% glucose, 1.75% NZ-amine, 0.7% soft grain, 0.5% CaCO ₃ , 0.5% (NHJ ₂ SO ₄ , 0.15% NaCl, 10 pM CoCl ₂ .

The process is carried out as described in Example 1 using 100 gamma / cm ^{3 of} 4-ethyl-5-propyl-1,2-phenylenediamine as precursors. The bioautography shows the presence of a growth factor with a slightly larger Rf value than vitamin B ₁₂ and smaller than factor B, which cannot be separated from vitamin B ₁₂ by electrophoresis. The separation of B ₁₂ -like vitamins (obtained according to the preceding examples) from vitamin B ₁₂ can be achieved on columns filled with cellulose powder using suitable solvents, e.g. B. from lake saturated with water and acidified with acetic acid. Butyl alcohol.

The substances obtained using the above-mentioned precursors can be used for the therapy of Addison's pernicious anemia or another macrocytic anemia and also for all forms of disease which are preferably influenced _{by the vitamin B 12 action.}

The same substances can also be used as growth factors for humans and animals.

Claims (3)

Claims; 1. Process for the microbiological production of Cobalamines using microorganisms of the strain Nocardia rugosa according to patent 1046258, characterized in that the nutrient fluid is used as a precursor additive in the biosynthesis of cobalamines o-phenylenediamine or its alkyl derivatives known from other microorganisms are added will. 2. The method according to claim 1, characterized in that 4,5-diethyl-1,2-phenylenediamine is added will. 3. The method according to claim 1, characterized in that that 4-ethyl-5-propyl-1,2-phenylenediamine is added. Documents considered: Biochem. Journal (Cambridge), 1955, No. 1, p. 79 to 81. 647/390 10.59