DE2164626A1 - Process for the production of a straight-chain dicarboxylic acid, an omega-hydroxy fatty acid and an omega-1-keto fatty acid - Google Patents

Description

DR. E. WIEGAND DIPL-ING. W. NIFMANN 2164626

DR. M. KÖHLER DIPL-ING. C. GERNHARDT

MÖNCHEN HAMBURG

phone: 55547 "8000 MONCHES 15, 24 December 197"

TELEGRAMS: KARPATENT NUSSBAUMSTRASSE 10

V 40 959/71

T. Hasegawa Company, Ltd. Tokyo (Japan)

Vex ^ drive to the production of a straight-chain Di cart) on acid, an omega-hydroxy fatty acid and an omega-1 keto fatty acid

The invention "relates to a method of manufacture of acids, in which at the same time a straight-chain dicarboxylic acid, an omega-hydroxy fatty acid and an omega-1-keto fatty acid is formed in a culture medium using a microorganism and the obtained fatty acid is won.

It is known that fatty acids are formed from η-paraffins by microorganisms (e.g. yeasts of the genus Pichia, Agr. Biol. Chem. 2 £, 1009 (1965); strain 7EIC (ATCC 13767) of an unknown species belonging to the genus Corynebacterium which differ from the microorganisms used in the method according to the invention in terms of size and shape, and such properties as the fermentability of carbohydrates, J. Bacteriol, 8J3 , 859 (1963); bacteria of the genus Pseudomonas, Nature , 198, 289 (1963); yeasts of the genus Candida, J. Gen. Appl. Microbiol, 12, 119 (1966); molds of the genus Botrytis, J. of Japan Society of Agricultural Chemistry, 40, 364 (1966)) *

209845/1038

ORIGINAL WSPECTEO

In these publications it is only stated that omega-hydroxy fatty acids, dicarboxylic acids or ketocarboxylic acids found as metabolic intermediates during the assimilation of η-paraffins by microorganisms can be.

In J. Gen. Appl. Microbiol, 1 £, 119 (1966) is described in the cultivation of Candida rugosa JF 101 in a medium containing 2.5 ml η-paraffin per 80 ml of an inorganic Saline solution, 16.7 mg of a diacid were formed per liter of culture liquid. It was however, the general view is that the amount of fatty acid produced by microorganisms from η-paraffin is very small. In particular, it is difficult to use an omega-hydroxy fatty acid enrich because it is an unstable metabolic intermediate. It was not previously known that an omega-hydroxy fatty acid, a straight-chain dicarboxylic acid and an omega-1-ketocarboxylic acid are formed in large quantities at the same time will.

η-paraffins are readily available and at low cost and it is therefore desirable to have omega-hydroxy fatty acid, straight-chain dicarboxylic acid and omega-1-ketocarboxylic acid on an industrial scale from η-Par äff inen using of microorganisms.

There were new ones. n-paraffin assimilating strains of the genus Gorynebacterium / Uie have the ability at the same time straight-chain dicarboxylic acids, omega-hydroxy fatty acids and omega-1-keto fatty acids in large quantities

form, found.

The existence of such strains of the genus Corynebacterium, who have this ability was completely unknown.

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_ 3 -

These strains e.g. Corynebacterium dioxydans nov. sp. or Corynebacterium hydrocarbooxydans differ from the previously known η-paraffin assimilating strains ■ and have a pronounced ability to have an oxidizing effect in a certain way (diterminal oxidation) ^ during them Assimilate η-paraffins as a carbon source and give and accumulate large amounts of straight-chain dicarboxylic acids, omega-hydroxy fatty acids and omega-1-ketocarboxylic acids, which have the same number of carbon atoms as the substrate in the culture medium. In particular, these have Strains the unique ability to make omega-hydroxy fatty acids to be enriched in large quantities. This is surprising as they have previously been viewed as very unstable metabolic intermediates and it was difficult to enrich them in substantial amounts.

The present invention is based on the fact that those microorganisms that have this previously unknown ability have, in the form of microorganisms of the genus Corynebacterium be available.

It has also been found that when additives are added that are known to be used alone to obtain other products using microorganisms of other genera, to which in the process Culture medium used according to the invention, the above-mentioned 5 acids formed in large quantities and these acids can be formed simultaneously with increased yield.

It has also been found that the combined use of these additives leads to particularly significant improvements leads.

09845/1038

For example, it was known that dicarboxylic acids corresponding to η-hexane and n-heptane by resting cells of Pseudomonas can be obtained using acrylic acid (Biochim,. Biophys. Acta, 84, 195 (1964-)), It is also known that p-ToL ^ ylic acid from p-xylene by Actynomyces under Using an ion exchange resin it is formed (Biotechnol. Bioeng. -IC), 689j (1968)). It is further known that salicylic acid is formed from naphthalene using a strain of the genus Pseudonomas (Appl. Microbiol. £ 1, 512 (1969 ».

Are acrylic acid, its salts or anion exchange resins or mixtures of acrylic acid and / or its salts with an anion exchange resin, which is preferred, added to the culture medium in the method according to the invention, becomes the amount of straight chain dicarboxylic acids, omega-hydroxy fatty acids and omega-1-keto carboxylic acids about 5 fold Value increased compared to the pail that no additives are used.

Accordingly, the object of the invention is to create a method in which at the same time a straight-chain dicarboxylic acid, an omega-hydroxy fatty acid and an omega-1-keto-fatty acid in high yield from η-paraffins using an η-paraffin assimilating strain that has the ability has to form these acids at the same time.

It is also an object of the invention to provide a process for the simultaneous production of these 3 acids in high yield to accomplish.

209845/1038

The normal paraffins, which are used as the carbon source of the 'Culture medium that can be used can be those which adhere at least 10 carbon atoms. They can be used either individually or in combination. The preferred η-paraffins adhere 10 "to 20 carbon atoms and include" for example n-decane, n-dodecane, n-tridecane, n-tetradecane, n-pentadecane, n-hexadecane, n-octadecane and n-eicosan.

The amount of carbon source used is ordinary 10 ft to 100 g, preferably 15 "to 80 g, and most preferably 20 ft to 70 S de liters of culture medium.

In the method according to the invention, an n-paraffin assimilating strain of the genus Corynebactcrium belonging to the Has ability to be a straight-chain dicarboxylic acid, an omega-hydroxy fatty acid and an omega-1-keto fatty acid at the same time to form under aerobic conditions in a medium containing a nitrogen source, minerals and an normal paraffin having at least 10 carbon atoms as a carbon source, grown, and an acid selected from straight-chain Dicarboxylic acid, omega-hydroxy fatty acid and omega-1-keto fatty acid is won.

Any source of nitrogen used to grow microorganisms is suitable, ft ei the method according to the invention as a nitrogen source (or organic nutrient source) can be used. Examples of nitrogen sources are: alkali nitrates such as sodium nitrate or potassium nitrate; Ammonium salts of inorganic acids such as ammonium nitrate, ammonium sulfate, ammonium chloride or ammonium phosphate; Ammonium salts of organic acids, such as ammonium acetate,

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Ammonium lactate or ammonium tartrate; Urea; Ammonia; Yeast extracts; Meat extracts and corn steeping water. These Compounds can be used alone or in the form of mixtures as the nitrogen source. The amount of The nitrogen source is usually between about 0.1 and 10 g and "preferably between 0.5 'and 8 g per liter of culture medium.

Examples of minerals which can be used in the method according to the invention are NaH ₂ PO ₄ Na ₂ HPO ₂ , Na ₅ PO ₄ , KH ₂ PO ₄ K ₂ HPO ₄ , K ₃ PO ₄ , CaHPO ₄ and other phosphates.

They are used either individually or in mixtures. It is also possible to use magnesium, iron, manganese, calcium, copper, zinc, molybdenum and boron salts of inorganic acids which supply ions of these metals. They are also used either individually or in mixtures. Specific examples are: MgSO ₄ . 7H ₂ O, FeSO ₄ . 7H ₂ O _} CaCl ₂ CaCO ₃ , Ca (OH) ₂ , MnSO ₄ . 5H ₂ O, "CuSO ₄ , 5H ₂ O, CuCl ₂ , ZnSO _4. 7H ₂ O, MoO ₅ , H ₅ BO _5, and seawater. The amount of minerals, although depending on the type of mineral used, is usually between about 5 / Ug and 10 g per liter of culture medium,

The yield of the products can in the process according to of the invention by incorporating at least one additive from the group of acrylic acid, the salts thereof, such as the Sodium, potassium, ammonium or calcium salts and anion exchange resins, preferably at least acrylic acid or one of its salts and at least one ^ ion exchange resin, in "the culture medium can be increased.

Any commercially available anion exchange resin including, for example, a quaternary ammonium salt, a primary amine secondary amine, a tertiary amine or a polyamine as an exchange group and the reaction of the anion exchange can be used. The form in which that

209845/1038

Anion exchange resin is introduced into the culture medium is, for example, of the hydroxyl type, chlorine type, weak acid type, such as phosphoric acid, citric acid or tartaric acid.

The microorganism used in the process can use η-paraffin as the only source of carbon

They are microorganisms of the genus Oorynebacterium which have the ability to simultaneously form and accumulate a straight-chain dicarboxylic acid, an omega-hydroxy fatty acid and an omega-1-keto fatty acid in the culture medium. The existence of microorganisms of the genus Corynebacterium that have such abilities was not previously known.

Examples of such microorganisms are Corynebacterium dioxidans nov, sp. (MC-1-1 strain) deposited under PEEM-P No. 690 at the Fermentation Research Institute, Ministry of Trade and Industry, Japan, and Corynebacterium hydrocarbooxydans nov. sp. (JA-1 strain) which is a gram positive Rod bacterium is deposited under FERM-P KO. 800 at above institute.

These microorganisms have already been identified with the American Type Culture Collection under ATCC 21766 and ATCC 21767 deposited.

The microbiological properties of these microorganisms are given below.

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Corynebacterium dioxydans nov.sp. Strain MC-1-1

1. Rods, 0.6-1.2 by 3.0-.9.5 microns. Cell branching and cocci-like forms become established.

No spore formation. Not able to move on its own s.ejself. Gram_positive.

2. Nutrient agarcclony: circular, smooth, undivided, convex / raised, "pale pink Ms orange.

3. Nutrient agar slope: moderate growth, filiform, shiny, rusty pink.

4-. Nutrient broth: fragile membrane, cloudy,
excess sediment.

5. Nutrient gelatin prick, no liquefaction.

6. Litmus milk: alkaline, not peptonized.

7. B.C.P. milk: alkaline, not peptonized.

8. No indole formation

9. No formation of nitrite from nitrate

10. Hydrogen sulfide formation

11. Starch is not hydrolyzed

12. Methyl red test: negative

13. No acetylmethyl carbinol formation

14. Catalase: positive

15 · No ammonia formation

16. GLtrate is used as the sole source of carbon.

17. Aerobic

il

209845/1038

18. Optimal temperature 30 - 35 ° C

19 · Growth at a pH value of 5 * 0 - 9? O

20. Acid formation from glycerine, glucose, JPructose, Mannitol.

Neither acid nor gas formation from xylose, lactose, sucrose, Starch, maltose, galactose, mannose, arabinose, cellulose.

209845/1038

The microbiological properties mentioned above were the classification standard, described according to Bergey's Manual of Determinative Bacteriology, 7 · Circulation examined, and it was found that the part above nailed nn te strain to the genus Corynebacterium. The morphological and physiological properties of the above strain were compared with the descriptions in Bergey's handbook, but no identical strain could be found that would be described in it. The obengena nn te strain differs from Corynebacterium glut ami cum nov. sp., Corynebacterium 1ilium, nov.sp., Gorynebacterium petrophilum

^ nov.sp. and Corynebacterium acetoglutamicum nov.sp. in the size of the microorganisms, various breeding properties, the effect on milk, the formation of Hydrogen sulfide or the fermentation of carbohydrates. Furthermore, the above-mentioned strain is also different from Corynebacterium hydrocarboclastus nov.sp. in the size of the microorganisms, the breeding properties, the Ability to reduce nitrates and ferment carbohydrates. Furthermore differs the above-mentioned strain of Corynebacterium aurantiacum nov. sp '. and Corynebacterium roseum nov.sp. in the size of the Microorganisms, the breeding properties, the ability to reduce nitrate, the ability to

w substance or the ability to ferment carbohydrates. As a result, the MG-1-1 strain (FEEM P No. 690; ATCG 21766) was considered a neme species of the genus Corynebacterium and Corynebacterium dioxidäns nov. sp., called.

209845/1038

ORIGINAL INSPgCTED

Corynebacterium hydrocarbooxydans nov.sp. Strain JA-1.

I. Rods, 0.7-1.2 by 4.0-8.0 microns. Cell branching and cocci-like shapes are found. No spore formation. Not able to move on its own ■. ■ Gram positive.

2. Nutrient agar colonies: circular, smooth, undivided Ms wavy, convex raised, orange "to reddish-orange.

3- nutrient agar slope: moderate growth, filiform, shiny, reddish pink.

4. Nutrient broth: fragile membrane, clear, excess sediment.
5l nutrient gelatinous: no liquefaction

6. Litmus milk: unchanged.

7. B.C.P. milk: unchanged

8. No indole formation

9. No formation of nitrite from nitrate

10. Hydrogen sulfide formation
11 · Starch is not hydrolyzed

12. Methyl red test: negative

13. No acetylmethyl carbinol formation

14. Catalase: positive

15. No ammonia formation

16. Citrate is used as the only source of carbon.

17. Aerobic

18. Optimal temperature: 30 - 35 ° C

19. Growth at a pH of 6.0-9 OK

20. Acid formation from glycerine, glucose, lactose, sucrose, maltose, fructose, mannose. Neither acid nor gas formation from xylose, starch, galactose, mannitol, arabinose, cellulose.

209845/10 38

At

The microbiological properties of the above strain were determined according to Bergey's Manual of Determinative Bacteriology, 7 edition and it was found that the above-mentioned strain (JA-1) belongs to the genus Corynebacterium heard. The morphological physiological properties of the above strain were compared with the descriptions in Bergey's manual, but no identical strain described in it could be found who that.

This problem differs from Corynebacterium pe "t3 ?? philum nov. sp., Corynebacterium acetogutamicum nov.sp., Coryrebacterium lilium nov.sp. and Corynebacterium acetoacidophilum nov.sp. in the fermentation of carbohydrates and the formation of hydrogen sulfide. He differentiates furthermore from Corynebacterium hydrocarboqlmstus nov.sp., Corynebacterium paraaldehydium nov.sp. and Corynebacterium flavo-aurantiacum nov.sp. in the fermentation of carbohydrates, the effect on milk, the reduction of nitrate, etc., and of Corynebacterium aurantiacum nov.sp., Corynebacterium acetophilum nov.sp. and Corynebacterium roseum nov. sp. in. the fermentation of carbohydrates, the Decomposition of starch or the reduction of nitrate. The above-mentioned strain JA-1 (FERM P Ho. 800 "'; ATCC 21767) regarded as a new species of the genus Corynebacterium and Corynebacteri'Um hydrocarbooxydans nov.sp. called.

In the method according to the invention, an η-paraffin assimilating strain of the genus Corynebacterium, as described above, which has the ability to produce a straight-chain dicarboxylic acid, an omega-hydroxy fatty acid and to form an omega-1 keto fatty acid, below aerobic conditions in a culture medium that contains the nitrogen source and the minerals specified above.

209845/103 8

holds, using η-paraffin with at least 10 Carbon atoms grown as a carbon source, with * at the same time the straight-chain dicarboxylic acid, the omega-hydroxy fatty acid and the omega-1-keta fatty acid is formed in the culture liquid.

The pH of the medium is usually adjusted to 3 to 9, preferably auiä to 8. The culture temperature is usually 20 ° to 40 ⁰ C and preferably 20 ° to 37 ⁰ O.wird it is especially from 5 to 8.5 and in particular 6 desired, the omega-hydroxy fatty acid to obtain in large quantities, a temperature zwisehen approximately and preferably from 30 ⁰ C. The cultivation time is usually around 2 days. If desired, the cultivation can be carried out for more than 8 days, for example 10 days to 2 weeks, but this is not necessary. Usually, with a cultivation time of about 2 to 10 days, the goal, ie the formation and accumulation, of these 3 acids can be fully achieved.

The breeding can be carried out on any Wemse known per se. A culture medium which contains the above-mentioned carbon source, nitrogen source and minerals is adjusted to the desired pH value and then ^{sterilized by compressed water vapor at a temperature of, for example, about 120 °} C. for a period of, for example, 10 to 30 minutes. The sterilization can, for example, also be carried out in such a way that ^{intermittent sterilization is carried out at 100 °} C. for 1 hour each time. The η-paraffin-assimilating strain of the genus Corynebacterium is then inoculated into the culture medium and at the temperature given above under aerobic conditions (for example as a shaking culture or as a culture which is carried out by

209845/1038

Air supply is stirred).

As stated above, the joint use of at least one of the compounds from the group of acrylic acid and its salts and at least one anion exchange resin in the culture medium is preferred. Cease breeding. It is possible to sterilize these additives and add them to the culture medium before or after inoculating the above-mentioned n-paraffin assimilating strain or during the cultivation. The preferred concentration of these additives is about 0.1 to 5 g as acrylic acid per liter of culture medium, and a concentration between about 0.5 and 1.5 g as acrylic acid per liter of culture medium is particularly preferred. In a special embodiment of the method according to the invention, in which only acrylic acid or its salts are added, a suitable amount of acrylic acid or its salts is ^{sterilized for 10 minutes at 120 °} C. and after the start of cultivation, for example 1 to 48 hours after the start of breeding, admittedly.

Will only be an anionic exchange resin or acrylic acid or its salts and an anionic exchange resin admittedly, the cultivation can be carried out in the same manner. For example, after the Culturing the culture liquid made acidic, and using the obtained fatty acids from the culture liquid an organic solvent, warm ether or benzene, extracted. The extracted fatty acids are then with a sodium hydroxide solution or a extracted from another alkali solution, acidified with a strong acid, such as hydrochloric acid or sulfuric acid, and then acidified with an organic solvent, such as ether or benzene extract, t. After that, the solvent is removed by evaporation

209845/1038

removed, whereby the above-mentioned three fatty acids are obtained.

Becomes an ^ ion exchange resin. used alone or together with acrylic acid or its salts, the culture medium is separated from the ion exchange resin by filtration after the end of the cultivation. The replacement resin is then mixed with a mixture of an aqueous solution of a strong acid such as hydrochloric acid or sulfuric acid, with a water-miscible organic solvent such as methanol or acetone, treated to leach out the fatty acids adsorbed on the ion exchange resin. After removing it of the organic solvent from this liquid by evaporation, the fatty acids become in a similar manner with an organic solvent, ether or benzene, extracted and after removing the solvent, the three fatty acids are obtained in the raw state. Crude fatty acids can also be filtered off of fatty acids can be obtained from the aqueous phase after removal of the organic solvent remains behind.

The crude fatty acids are alkyl-esterified with a suitable alcohol such as methanol and through a Separation process, the distillation under reduced pressure into the dimethyl ester of a straight-chain dicarboxylic acid, a methyl ester, an omega-hydroxy fatty acid and a methyl ester of an omega-1-keto fatty acid.

The addition of acrylic acid or its salts and an anion exchange resin to the culture medium the method according to the invention is preferred. Compared with the case that these compounds are not added the amounts of crude fatty acids are around 1.5 2 times higher for the case that acrylic acid or its salts are added, about 2-3 times higher for the

• 209845/1038

/ ft

that an anion exchange resin is added and more than about 5 times higher in the case that acrylic acid or their salts and an anion exchange resin are added together; verden.

The invention is explained in more detail below with the aid of examples.

Examples 1-4 and Comparative Example 1

1 g ammonium nitrate, 0.6 g monopotassium phosphate, 4 g dipotassium phosphate, 0.5 g magnesium sulfate, 10 mg iron CEI) sulfate, 30 mg galpium chloride, 10 / ug manganese chloride and 100 mg yeast extract were dissolved in one liter of tap water and the solution adjusted to a pH of 7 °. 50 ml portions of the resulting solution were each poured into 500 ml shake flasks. In öeden piston 5 ml of n-tridecane as a source of carbon was 1 is introduced and the solution sterilized for 20 minutes at 120 ⁰ C.

A weakly basic anion exchange resin (Amberlite IR-4-5) was used as the anion exchange resin which was added to the culture medium. The resin was first converted to a hydroxyl group type by using 5 times the volume of 1N sodium hydroxide aqueous solution and then washing with 10 times the volume of distilled water. It warde 5-fsche amount of a 5% ISS © ⁿ Phosphatpuffaiösung (pH 7io) consisting of monopotassium phosphate and Dinatriuuiphosphat, added to convert the resin in one of the Phosphorsäureart. The resin was then washed with 10 times its volume of distilled water. Parts, 10 ml each, of the> resin were placed separately in a 100 ml Erilenraeyer flask and

2 Ü 9 BZ ₁ B / 1 0 3 8

2164628

3 days intermittently at 100 ° C for each Sterilized for 30 minutes. The sterilized solutions were aseptically added to the culture medium prior to inoculation.

Subsequently, Corynebacterium dioxydans MC-1-1 (EEEM-P No. 690 ATCC 21766) was cultured in the above-mentioned culture medium for 20 hours, and the obtained sperm cell solution was aseptically inoculated in an amount of 5% each.

It became a shake culture at 30 ° C. with 98 changes (Amplidude 70mia). carried out, and after 24 hours 50 ml of 10 minutes at 120 ⁰ C sterilized sodium acrylate were added aseptically to 50 ml of the culture medium.

The culture was stopped 120 hours after the sperm inoculation. The culture medium to which neither the ion exchange resin, sodium acrylate had been added (Example 1) and the culture medium to the only sodium acrylate had been added (Example 2), were made strongly acidic with concentrated sulfuric acid and 3 times with the one corresponding to the culture medium Amount of ether extracted. The extracts were combined and extracted 3 times with a 1N sodium hydroxide solution. The solution containing sodium hydroxide was then made acidic with concentrated sulfuric acid and then extracted with ether. The ether was dehydrated with anhydrous sodium sulfate and then the ether is removed by evaporation. Crude acids were obtained.

On the other hand, the culture medium to which only the ion exchange resin had been added, and the culture medium to which both the ion exchange resin and the sodium acylate had also been added (Example 4 ·) ^ through Abriut already in the KuI door liquid and that

2 0 9 8 4 5 / I ü 3 8

At

Ion exchange resin separated. The resin was placed in a column and 40 times its volume a mixture of equal amounts of 2N hydrochloric acid and Methanol was allowed to flow through the column to elute the fatty acids absorbed on the resin. If the methanol was removed from this eluate by evaporation, the eluted fatty acids became insoluble. Extraction with Ither yielded crude acids.

For comparison purposes, the operation of the Example 4 repeated, but Corynebacterium strain? EIC (ATCC 13767) was used. The results are given under Comparative Example 1.

The crude acids thus obtained were weighed / and the results are summarized in Table I “Me Acids were methylated with diazomethane. and the content of undecane-1, H-diaarboxylic acid, 13-hydroxytridecanoic acid and 12-ketotridekanoic acid was measured. The results are given in Table I. The proportion of MonoO: carboxylic acid (tridecanoic acid) was very low.

Dimethylundecane-1,11-dicarboxylate, methyl-13-hydroxytridecanate and methyl 12-ketotridekanate were obtained by gas chromatography, and the melting points, Mass spectra and infrared absorption spectra are determined. These esters were saponified to give undecane-1,11-di-carbonic acid, Obtain 13-Hydroxytridkansäure and 12-Ketotridekansäure. The melting points, mass spectra and infrared spectra were measured. These measured values corresponded to those of standard products.

2 0 <■) 8 A ^l »/ 1 Π 'J 8

Table I.

J ί
j Kenge to educated
¹ raw acids
Cg / I culture medium) T-indecane-1,11-
dicarboxylic acid
(p / l culture medium) at
game 1 May be-
cpiei 2 at
game 3 Example! Comparison--
4 [for example 1 üTatrium
acrylate added
j _ninfl " _niic , give
exchange resin ■ 4J C3 13-H7droxytridec: an-
acid (g / l culture
medium) acrylate not
lonenaus- ^ _o ? _o ~
exchange resin ⁵ "" ^ ITatrium
acrylate
Trains
give Ion off
exchange
resin too
given Sodium
acrylate ^ e-
Ion output
exchange resin 2.43 C ^ 05 12-ketetridecanoic acid
(g / l culture medium) 3.32 5.05 9.60 16.20 0.73 ¹ ^ -3 -5 -ρ 0.72 1.01 2.04 3.85 0.24 1.48 2.08 4.20 7.24 \
i
0.44
■ 0.68 1.16 1.80

CD K) CD

Examples 3 "to 8

3 g ammonium chloride, 1 g monopotassium phosphate, 3> 5 g disodium phosphate, 0.5 g magnesium sulfate, 10 mg iron (II) sulfate, 30 mg calcium chloride and 100 mg meat extract were dissolved in one liter of tap water to form a culture medium . the pH of the culture medium ■ was adjusted to 7>. 5 Subsequently, 100 ml portions were placed in 500 ml shake flasks. to each of these piston 3 nil n-octadecane was added and at 120 ⁰ sterilized C 15 Hinuten. Subsequently, 5g of peptone were , 2.5 g meat extract, 2.5 g yeast extract and 1 liter of tap water mixed to form a culture medium with a pH value of 7 »0. Corynebacterium dioxydans MC-1-1 strain (FEEM-P No. 690; ATCC 21? 66) was cultured for 10 hours, and the obtained sam-island liquid was inoculated into the flasks in an amount of 2% each.

A strongly basic anion exchange resin (Amberlite) IRA-400) was made in the same way ^ as in example 3 described ^ sterilized and a total of 15 ml of the resin per 100 ml of culture medium were in 5 ml portions added after 24, 48 or 2 hours. K & lium aerylate, in 'the same way as Batr, iumacrvlat in Example 2 ! was sterilized in an amount of 150 mg per 100 ml Culture medium in 50 mg parts simultaneously with the addition des ΰΐοηεηβμΒΐβ ^ οηϊΐΒΓζεΒ added. Overall was 150 hours at 125 ° C with shaking with 98 changes (Amplitude 70 mm) cultivated.

After the completion of the cultivation, the same treatment as described in Examples 1-4 was carried out, carried out and obtained the crude acids. These acids were weighed. The results obtained are in Table II shown. The acids were metliylicrt with diazomethane,

209845/1038

and then the content of hexadecane-1,16-dicarboxylic acid, '18 -hydroxystearic acid and 17-ketoste.arinsäure measured. The results are given in Table II. The proportion of monocarboxylic acid (stearic acid)
was very low. Dimethylnexadekan-i ^ ö-dicarboxylate, methyl-18-hydroxystearate and methyl-17-ketostear.at
were obtained separately by gas chromatography
and the melting points, mass spectra and infrared spectra are measured. These esters were saponified to form hexadecane-1,16-dicarboxylic acid, 18-hydroxystearic acid, and 17-keto-stearic acid _; and the melting points, mass spectra and infrared spectra of these acids were determined. These values corresponded to those of synthesized standard products.

^209845/1 038

Table II

Amount of educated
raw acids
(g / 1 culture medium ") Hexadecane-1,16-
dicarboxylic acid
(g / l culture
medium) Example 5 At-
game6. Example7 Example 8 • IVl mmt through
r aphie 18-hydroxystea
ric acid (g / l
Culture medium) ^Kali r; not
acrylate ⁿ ^
Ion Aus- ~ οξο _η
exchange resin ₍ S ^ oen Potassium-
acrylate
Trains
give Ion off
exchange resin
admitted potassium
acrylate
Ion release
exchange resin ^ε . CD
-Ο-
cn
IVT Salary determinedii
Gas chromatog 17-ketostearixn-
acid (g / l
Culture medium) 3.04 4.56 9.46 1,572 O 0.66 1.02 2.10 3.14 98A5 / 10 1.38 2.10 4.40 7.10 approx
αο
O
33
Ώ
I.
53 0.38 0.54 0.98 1.80 Ä
a

IS

Examples 9 Ms 12

0.5 g urea, 1 g monopotassium phosphate, 3 g disodium phosphate, 0.5 S magnesium sulfate, 10 mg iron (II) sulfate and 100 mg meat extract were dissolved in one liter of tap water and the pH of the solution was adjusted to 6 , 5 set. Subsequently, 50 ml portions each were placed in 500 ml shake flasks and 1.5 ml n-pentadecane was added to each of these flasks as a carbon source. Then 20 minutes at 120 ⁰ G was sterilized. Then a spoon (one lobpful) Öoryneba'c'-terium Hydrocarbooxydans nov. sp., JA-1 (FEEM-P No. 800, ATCC 21767) /. which had previously been cultivated for 24 hours in a slant culture (pH 7.0) consisting of 10 g peptone, 5 g meat extract, 5 g yeast extract, 20 g agar and 1 liter tap water, inoculated into the flasks.

As the ion exchange resin, a strongly basic anion exchange resin (Amberlite IRA 900) which had been converted into one of the hydroxyl group type and sterilized in the manner described in Example 3 was used. The addition to the culture medium was carried out in such a way that after 12 hours after the inoculation of the cells, 10 ml of the ion exchange resin was added to each shake flask. Acrylic acid was sterilized for 10 minutes at 120 ^° C. and an amount of 25 mg per 50 ml of culture medium was added to the culture medium 18 hours after the inoculation of the sperm cells. It was carried out by shaking culture during 120 hours under SchütteD.bedingungen 120 changes per minute at 30 ⁰ C.

2098 k 5/1038

After completion of the cultivation, the products were in the same manner as described in Examples 1 "to 4, treated. The crude acids obtained were weighed and the results are shown in Table III reproduced.

These products were methylated with diacomethane, and by gas chromatography the content of tridekan-1,3-Ä.i.i.carbonsäur'e, 15-hydroxyp ent ad ekan acid and 14— Ketopentadecanoic acid was measured. And the results are listed in Table III. The proportion of monocarboxylic acid (Pentadecanoic acid) was very little. Dimethyl tridecane-1,13-dicarboxylate, methyl-15-hydroxypentadecanate and methyl 14-ketopentadecanate obtained by gas chromatography and measured the melting points, mass spectra and infrared spectra. !Further these products were formed with the formation of tridecane-1,13-dicarboxylic acid, 15-hydrocypentadecanoic acid and 14-ketopentadecanoic acid saponified. The melting points, mass spectra and infrared spectra of these acids were determined measured. The fourth corresponded to those of the synthesized Standard products.

5 /

Table III

CD CO OO

Tridecane-1,13-
decarboxylic acid
(g / l culture »
medium) at
game 9 at
game 10 at
game 11 at
game 12 13-hydroxypenta
decinic acid (g / l '
Culture medium) sour _draws _
Ionic give
exchange
resin acrylic
acid
admitted
ben Ion off
exchange resin
Trains
give Acrylic acid
Ion Aus- TSt ~
exchange resin o ^^ = · " 12-ket opentade
canic acid (g / l
Culture medium) 2.04 3.15 6.20 10.34 Amount of educated
raw acids
(g / l culture medium) 1.04 1.42 3.00 5.40 Salary determined by
Gas chromatography 0.04 0.08 0.12 0.16 0.32 0.48 1.05 1.48 '

Examples 13 and 14

2 g ammonium acetate, 0.6 g monopotassium phosphate, 4 g disodium phosphate, 0.5 g magnesium phosphate ·, 10 mg Iron (EI) sulfate, 30 mg C-alc.ium chloride, 10 / u g Manganese chloride, 100 mg of yeast extract and 1 liter of tap water were mixed to form a culture medium. In each case 15 liters of the culture medium were poured into

given two 30 liter fermentation jugs. After adjusting the pH to 7> 0, 450 ml of a mixture of 70% n-pentadecane were added around 30% n-hexadecane. It was then sterilized with water at 120 ^{° C. for 20 minutes.} Then 5% by volume of a sperm cell fluid obtained by preculturing Corynebacterium dioxydans nov.sp. for 15 hours. Strain MC-1-1 had been obtained, aseptically insert the two 30 liter fermentation jars

3 liters of a weakly basic anion exchange resin (Amberlite) IRA ~ 93) were prepared in the same manner wie.in Example 3, treated, and added after 20 minutes of sterilization at 120 ⁰ C to a Fermentierkrug. No anion exchange resin was added to the other fermentation jar. It became a breed

P which was stirred by the supply of air, at 30 ^° C

• carried out, the air flow rate being 10 liters per minute and the stirring speed being 250 rpm. Was 24 hours after the inoculation, 15 g Hatriumacrylat that 10 minutes at 120 ⁰ C sterilized been the! Fermentierkrug by the ion exchange resin was located, added. The cultivation was continued for another 9½ hours. The fermentation jar containing no ion exchange resin and sodium acrylate was subjected to cultivation for 120 hours.

209845/1038

After breeding was over, the crude acids were weighed. The acids were methylated and the Amounts of tridecane-1,13-dicarboxylic acid, tetradecane-1,14-dicarboxylic acid, 15-hydroxypentadecanoic acid, 16-hydroxypalmitic acid, 14-ketopentadecanoic acid and 15-ketopalmitic acid were measured in a similar manner. The results are given in Table IV. the Shares of monocarsonic acids (pentadecanoic acid and palmitic acid) were very low.

Dimethyltridecane-1, Λ 3-dic: ar "boxylate, dimethyltetradecane-1,14 ~ dicarbo: xylate, 15-hydroxypentadecanate, methyl

16-hydroxypalmitate,, I ^ -Methylketopentadekanat and Methyldur cn
15-ketopalmitate, gas chromatography was obtained and the melting points, mass spectra and infrared spectra were measured. These esters were saponified to obtain tridecane-1,13-dicarboxylic acid, tetradecane-1, i ^ -dicarboxylic acid, 15-hydroxypentadecanoic acid, 16-hydroxypalmitic acid, 14-etopentadecanoic acid and 15-ketopalmitic acid. The melting points, mass spectra and infrared spectra were measured. These values corresponded to those of synthesized standard products.

2098A5 / 1038

Tabel

O GG cn

I. Amount of formed raw
Acids
(g / 151 culture medium) Tridecane-1,13-dicarbon-
acid (g / 15l culture medium) Example 13 Example 14 j
(D
? £
Ph P Tetradecane-1,14-dicarboxylic acid
^ e (g / l5l ^ culture medium) Sodium acrylate _{does not}
Ion Exchange _Z ri _ee _ Sodium acrylate _toee _
Ion exchange _ _Q ^ _OT ,
resin · ■ ^geDen Salary determined c
Gas chromatography Ka 15-hydroxypentadecanoic acid
(g / 15 1 culture medium) 45.1 231.4 16-hydroxypalmitic acid
(g / 15 lb of culture medium 6.8 33.8 14-ketopentadecanoic acid
(g / 151 culture medium) 2.9 1> »5 15-ketopalmitic acid
(s / 15! culture medium) 14.1 70.4 6.1 30.5. 21.5 1.8 8.5

Claims (12)

Claims 1. Process for the preparation of a straight-chain dicarboxylic acid, 'an omega-hydroxy fatty acid and a omega-1-kdto-fatty acid, characterized by the fact that one an η-paraffin assimilating strain of the genus Corynebacteriunijder has the ability at the same time straight chain dicarboxylic acid, omega-hydroxy fatty acid and to form omega-1-keto fatty acid in a culture medium, one. "Nitrogen source, minerals and an n-paraffin with at least 10 carbon atoms as carbon source contains, under. aerobic conditions and cultivates an acid from the group of straight-chain dicarboxylic acid, omega-hydroxy fatty acid and omega-1-keto fatty acid, which are formed in the culture liquid wins. 2. The method according to claim 1, characterized in that the culture medium further comprises an additive from the Acrylic acid group, its salts and anion exchange resins contains. 3. The method according to claim 1 or 2, characterized in that the η-paraffin assimilating strain Corynebawterium dioxydans nov. sp · is. 4-, method according to claim 1 or 2, characterized in, that the n-paraffin assimilating strain Corynebacterium hydrocarbooxydans nov. sp. is. 5. The method according to any one of claims 1 to 4, characterized characterized in that the pH of the culture medium is 3 to 9. 209845/1038 6. The method according to any one of claims 1 Ms 5, characterized in that "at a temperature between 20 ° and 40 ⁰ C is grown. 7. The method according to any one of claims 1 to 6, characterized characterized in that the cultivation time is at least 2 days. 8. The method according to any one of claims 1 to 7, characterized characterized in that the n-paraffin 10 to 20 Has carbon atoms. 9. The method according to any one of claims 1 to 8, characterized in that the η-paraffin n-decane, n-dodecane, n-tridecane, n-tetradecane, n-pentadecane, n-hexadecane, n ~ octadecane or n-eicosane. 10. The method according to any one of claims 1 to 9? through this characterized in that the nitrogen source consists of alkali nitrates, ammonium salts of inorganic acids, Ammonium salts of organic acids, urea, ammonia, yeast extracts, meat extracts or corn steeping water consists. 11. The method according to any one of claims 1 to 10, characterized in that the mineral is an inorganic one Is acid salt that supplies magnesium, iron, calcium, manganese, copper, zinc, molybdenum or boron ions. 12. The method according to any one of claims 1 to 11, characterized in that the mineral NaH ₂ PO ₄ , Na _{5 is} PO ₄ , KH ₂ PO ₄ , K ₂ HPO ₄ , K ₅ PO _4, or CaHPO ₄ . 209845/1038.