DE1442259A1 - Process for the production of α-ketoglutaric acid by fermentation - Google Patents

Description

- "-" "Pef enf afiwSile",

Dr.-Ing. HANS RU5CHKE
Dipi.-lng. HESNZ AGULAR

8 Munich 27, PienzenauerSlr. 2

P t4 42 259.4

1 Mov. 1968

Kyowa Baldco Kogyo Co., Ltd., Tokyo, Japan

Process for the production of α-ketoglutaric acid by

fermentation

The present invention relates to a method of manufacture of -ketoglutaric acid from hydrocarbons using of microorganisms *:

It has been known for some time that a-ketoglutaric acid is used as a Intermediate product used in the production of glutamic acid can be. However, the production of α-ketoglutaric acid has been made So far not carried out on a technical scale, as glutamic acid is produced using a direct fermentation method became.

However, developments in recent years have made it possible that α-ketoglutaric acid as a starting material for at least one Organic synthesis can be used except for the production of glutamic acid. Hence there is a need for one cheap process for the production of α-ketoglutaric acid, the Among other things, it can be used as a valuable compound for organic synthesis.

909828/0393
New documents (Art 7, Paragraph 1, Paragraph 2, No. 1 satr 3 of the amendment «. _V. 4.9. \ J

The ability * α-ketoglutaric acid from carbohydrates as a starting material has already been observed in various microorganisms. However, many are carbohydrate materials very expensive. Even if a cheap raw material «, How, for example, a molasses "is used: if there are certain other disadvantages, for example a low product concentration" 'the formation of by-products or the like', and still having difficulty separating them the ^ -ketoglutaric acid can be counted from the fermentation medium muH ». .- .. /

The invention has set itself the goal of developing a method for the production of -ketoglutaric acid from hydrocarbon materials, which method should be permissible on an industrial scale. This goal is achieved by using bacteria that produce a very high amount of ketoglutaric acid from certain n-paraffins. Such paraffins are the only source of carbon.

The erfindungsgemäSe process for producing a-ketoglutaric by fermentation is ά & _ΈΊη Ρ UMB a microorganism of a strain ^ sr € © "KeteglutaFsliss ⁵ © au © rzeugen: can (with ea © I tm ®iaen Stam® act --teMxti, - · the to the 'Brevibacteriaceae' the Microeoeoaoeae or the Corynebaoteriaoeae) is cultivated aerobically in a nutrient medium which contains a C ₁₀ - to C ₃₅ -Ji paraffin or a C ₁₀ - to C ₂ Rn paraffin containing petroleum fraction, this medium is stirred the pH is kept within a range of 4.0 to 9.0, the (»ketoglutaric acid is accumulated in the medium and is then separated off.

The bacteria contained in the specified n-paraffin Culture media are able to grow and are able to to accumulate considerable slopes in α-ketoglutaric acid, find

909828/0393

% vi *

«O oo ro oo

• tan in many species and genera. Strains that special good Possess a-ketoglutaric acid generating properties, include for example, see the Genera Corynebaeterium, Brevibaoterium, Arthrobaoter (this genus belongs to the Corynebacteriaceae) and Miorooocoue. The strains in imprint produce excess amounts of en a-ketoglutaric acid and are capable of a-ketoglu tareic acid can be produced from many η-paraffins with 10 to 25 carbon atoms in the molecule KU.

As a result of Klassifikationsuntereuohung it does look out that the three erttndungegeaXfl special suitable StKaae are new insulated Stlbme whose bakterlologl- • before properties are given below * These look here \ xm new Iü3croorgani8iien "in accordance with their Wentifi cation ice Brevibacteriun ketoglutamiciun, Mleroooeous paraff inolytious and Arthrobaoter paraf f ineus.

Brevibaoteriun ketoglutacloua Mr. 2 »73 UTCC-75588) Stube, 0, f - 0.9 x 0 * 8 ->#Oyu.

Bs are chewed "long cells observed" spores do not become educated. No hotness. Oraen-positive.

Hgr§gjrko | on | ens fleokenSimilar, smooth, complete, impenetrable sighted »shining

2 moderate watch do ", mycoid-like, moist" glossy

zend, pale orange-red, similar to butter

Nutrient broth ^ somewhat cloudy, merabran-like on the surface,

compact sediment

25 - 37 ⁹ C, extremely good growth at 37 ^e C 6.0 - 8 _Α 0 aerobic or optionally anaerobic not changed or alkaline not liquefied not generated

1. Optimal temperature

2. Optimal pH:

3 Oxygen demand:

4. Laocraus milk:

5. Gelatin:

6. Hydrogen sulfide:

8 * 9.

10 »11. 12.

Xndol:

Strong nitrate Catalase:

Urease:

13th

Usability of carbohydrates;

not generated
not hydrolyzed, also reduces nitrite positively
positive

The trunk grows under well Consumption of n «paraffin as. sole carbon source »where ^ Ketoglutaric acid, glutamic acid and alanine is produced.

weak production of acids from glucose »fructose and Cane sugar, maltose cannot be used.

MicrococcuB paraffinolyticus No. 4ΐ (ATCC-I5589) spheres or short rods, 0.5 - 1 # 2 χ 0.8 - I Au * no motility, gram-positive.

i? §iH: §S2 ££ 2i25it-i sparse growth · KStoeohrä ^ agart sparse growth, like myeoid, moist, shiny, bright red or orange-red, butteräbnlioh

Nutrient broth! slightly cloudy, compact sediment

? ÖiSi2i2Sii2Öf-IiSfS§2 ^

1. Optimal temperature j 25 -

2. Optimal pH: ^& 6.0-8.0,

>. Oxygen demand: aerobic or optionally anaerobic

4. Litmus milkί not changed

5- Oilate: not liquefied

6; Hydrogen sulfide: not generated

7. ~ 3iidols not produced

8. Starch: "^ not hydrolyzed

9. Nitrate: ■ '■■ _; ''"'■ / "- ^-i^ueiW^' .^ ^{1 ^".:} - -... '■ -

10. Catalase: positive

12; The trunk grows up well under

Consumption of n-paraffin as 9 Q g g 2 §7 0 3 ^ jnzigör KOlilenstoffquelle, where-

15.Usability of carbohydrates:

in amino acids and organic satires, especially a-ketoglutaric acid.

cheap production of acids from fructose, weak production of acids from alukose and maltose, and no generation of acid from sugar cane ·

Arthrobacter paraffineus No. 2411 (ATCC-1559D rods, generally 0.8 - 1.2 χ I - 2 Ai. The cells vary in size and shape. Irregular shapes (curved, swollen or branched) are observed. Elongated Cells and spherical cells are found to be x gram-variable In general, the elongated Graan-negative and the spherical cells are gram-positive.

N | iH! ÄSäi ^ 2i22iSSi excessive growth, ring-shaped, smooth on the whole surface, pale yellowish-brown, opaque, moist, shiny -

£! ££! 32ΐ £ § £ ^& β3 £ 1 overgrowth, mycoid-like, bloated or flat, shiny, light yellow to grayish brown, butter-like

Nutrient broth ^ slightly cloudy, slightly flaky sediment

1. Optimal temperature: 20 - 30 ⁹ G, easy growth at

5t ° C

2. Optimal pH: 6.0-8.0

J. Oxygen demand: aerobic or optionally anaerobic

4. Litmus milk: unchanged or alkaline

5. Gelatin: not liquefied

6. Hydrogen sulfide: not generated 7 * Indole: not generated

8. Starch: not hydrolyzed

9. Nitrate: not reduced to nitride

10. Catalase: positive

11. Urease: positive

909828/0393

12. The trunk grows well, whereby .. . · N-paraffin is used as the only carbon source «Large amounts of glutamic acid are produced from n-paraffin or Acetic acid generated

13. Use of coal «Production of acids from fructose hydrates:. and mannitol.

- Will acetic acid or paraffin

used as a carbon source in a Hooker's medium then becomes NH ₂₁ H ₂ FO ₂ . not used as a nitrogen source.

The hydrocarbon materials which can be used _{according to the invention can consist of individual C 10} - to Cgc-n paraffins or of mixtures of such paraffins or of a crude oil fraction which contains such n «paraffins.

In addition to the C ₁₀ to Cgc-n paraffins, other constituents can also be used in the fermentation medium to carry out the fermentation. The following may be mentioned of such constituents: ammonium salts, such as, for example, ammonium sulfate, ammonium chloride or ammonium nitrate, or urea, these compounds being used as a nitrogen source. In addition, inorganic salts or organic nutrients can be added if necessary. Organic nutrients include peptones, corn liquor, meat extracts, hydrolyzates of soybean meal and yeast extracts.

a-Ketoglutaric acid can be produced in high yield by adding various * dener vitamins, amino acids or organic nucleotide bases are produced instead of the organic nutrients just mentioned promote are able to be added.

90 9828/039 3

-T-

We also prefer calciuracarbonate to fermentation medium to increase the accumulation of «! -ketoglutaric acid. It is also important to control the supply of oxygen, i. to control the ventilation and stirring conditions of the heating medium, with the pH within a range from 4.0 to 9 * 0 should be held *

Under suitable fermentation conditions, according to the invention, 40 to 50 g per liter can be enriched in α-ketoglutaric acid, ie more than 50 % of the paraffin used is converted into α-ketoglutaric acid and enriched in the fermentation medium.

The results of tests using dodeoan as a carbon source, the enriched concentrations being duroh different bacterial strains are generated are summarized in the following table

Name of the bacteria fclfc

Arthrobaoter paraffineus Mr. 2411 (ATCC 1559t)

Corynebaoterium hydrocarbo- -olastus No. 2438 (ATCC 15592)

Brevibaaterium ketoglutamioura No. 2473 (ATCC 15588)

Microcoocue paraffinolytious No. 4.1 (ATCC 15589)

Enriched
Concentrations
in a-ketoglutar
acid (g per 1) Exploit,
based on
n-dodecane 85.0 50 11.0 22nd 8.0 16 9 * 0 18th

n-dodecane 5.0 % » K ₂ HPO ^ 0.2 %, MgSO ^ H ₃ O 0.1 fc ftoS0 ₄ .4l ^ 0

6 %

0.002 #, PeSO ^ .THgO 0.02 Ji, corn liquor 0.01 % and pH 7 # 2

2.0 j6 _r CaCO ₅ 2 _Ä 0

909828/0 3

20 πα portions of the fermentation medium are each in 500 ml. Sakaguchi flask was poured and grown in a reciprocating shaker at JO ⁹ C for a period of 72 hours. Before inoculation, 1 to 2 ml portions of n-dodeoan, sterilized beforehand by heating with steam, are added to an Ia culture. This inoculation culture is produced in a meat broth at 30 ° C. for a period of 24 hours.

a Sakaguchi flask is a modified round-bottomed flask "where the upper part of the flask is lowered to the" equator * ^{1 of} the ball. The flask can therefore be connected to a round beaker with a lid "that has a neck" ·

Example 1 of the invention is illustrated by the following examples

3 1 of a fermentation medium of 0.2 # KgHPO ^ * 0.1 # MgSh ^. 0.002 % MnSO ₁ ^ H ₂ O, 0.02 % PeSO ₄ .7HgO * 2.0 Jg NH ^ NO ^,>, Q $ CaCO, and 0.01 μl corn liquor (with a pH of 7 # 2) are in poured a 5 liter fermentation vessel and then sterilized.

200 g of a mixture of ^C 12 ** ⁰ I ^ "" ¹ ^ ¹ * C ^ -n-paraffins are added to the sterilized medium, whereupon the combined mixture is inoculated with an inoculum of Arthrobaoter paraffineus No. 2411. This culture is previously prepared by shaking in a meat broth medium for a period of 24 hours. The mixture is added in an amount of 10% by weight, based on the weight of the fermentation medium. The whole is grown with stirring at 700 Upni. The increase is achieved by passing sterilized air through the medium

90 ^ 028/0393

30 ° C for a period of 72 hours.

After a 24-hour cultivation time, 100 g of the mixture of C ₁₂ -C ₁₅ - and C ₁ ^ -n paraffins are added. After 72 hours of cultivation, the accumulated concentration of (»ketoglutaric acid is 60 g per 1.

The «ketoglutaric acid produced in this way is converted by« send separated by an ion exchange resin. In this way 120 g of α-ketoglutaric acid are obtained.

Example 2

It is grown in a manner similar to that described in Example 1, using 100 g of a mixture of n-paraffins having 12 to 16 carbon atoms in the molecule can be added at the beginning of the cultivation. In this case, however, paraffin is not added after culturing for 24 hours. The enriched concentration at α-Ketoglutaric acid is 15 g per 1.

Example 3

It is grown in a manner similar to that described in Example 2, using 100 g of a crude n-paraffin material. This material contains 90 wt. ~% Of n-paraffins having 11 to 18 carbon atoms in the molecule * 50 g a-Ketoglutarsäurekrlstalle be obtained in this way.

Example 4

72 hours of cultivation is carried out "with the cultivation the cultivation method described in Example 3 is similar. 200 g of kerosene are used. The accumulated concentration an «-ketoglutaric acid is 18 g per 1 *

90 9 8 28/0393

ORIGINAL INSPECTiD

Claims (5)

V 10 P a ten t a η β ρ rÜο h e 1. Procedure for. Production of cs-ketoglutaric acid by fermentation, characterized in that «aerobically a microorganism of a strain which ^; "Ketoglutaric acid is able to generate" is cultivated aerobically in a nutrient medium which contains a C ₁₀ to Cgc-n paraffin or a _{petroleum fraction containing C 10} to Cgg-n paraffins, this medium is stirred * whereby the pH between 4.0 and 9 * 0 is kept «the α-ketoglutaric acid is enriched in the medium and then separated * 2. The method according to claim 1, characterized in that the strain used to the Gtenus Gorynebacterium, Mlcroooccus, Belongs to Brevlbacterium or Arthrobacter. 3. The method according to claim 1 or 2, characterized in that that the strain used is from Corynebacterium hjdrooarboclastus No. 2438 (ATCC 15592), Brevlbaofcerium ketoglutamicuni No. 2473 (ATTCC 15588), Micrococcus paraffinelyticus No. 41 (ATCC 15589) or Arthrobacter paraffia © us No. 241.1 (ATCC 15591). 4. The method according to claim 1, 2 or 3, characterized in that the cooking medium used also contains thiamine * and / or one or more other substances capable of promoting the growth of the microorganism used « 5. The method according to claim 1, 2, 3 or 4, characterized in that the fermentation medium used also calcium carbonate contains. Documents (Art 7, Paragraph 1, Paragraph 2, No. 1, Clause 3 of the Amendment Act v. 4.1.1- 909 828/0 39 3