DE1268569B - Process for the biotechnological production of L-aspartic acid - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY

GERMAN

PATENT OFFICE

EDITORIAL

Int. α .:

German class:

Number:
File number:
Registration date:
Display day:

C12d

C07b

6b-16/02

12 q-6/01

P 12 68 569.7-41 May 17, 1963 May 22, 1968

The invention relates to a biotechnological process that can be carried out on an industrial scale Production of L-aspartic acid, which gives high yields.

L-aspartic acid is one of the important amino acids and is used in many fields.

Various methods for the production of L-aspartic acid have already become known, ζ. Β.

the chemical synthesis of DL-aspartic acid by chemical means and the cleavage of the obtained racemic aspartic acid into the optically active isomers,

the isolation of L-aspartic acid from protein hydrolyzate and

the biotechnological or fermentative production of L-aspartic acid from fumaric acid and some suitable amino donors (see French patent specification 1,248,130).

This third method will be for the cheapest and kept most expedient. Even so, it has not yet been possible to produce L-aspartic acid on an industrial scale to manufacture.

According to the process described in French patent specification 1,248,130, L-aspartic acid is produced Microorganisms of the species Pseudomonas fiuorescens and aeruginosa, Escherichia coli, Aerobacter aerogenes, Proteus vulgaris and Bacillus megatherium at almost neutral pH Process for the biotechnological production of L-aspartic acid

Applicant:

Ajinomoto Kabushiki Kaisha, Tokyo representative:

Dr. G. Lotterhos and Dr.-Ing. H. Lotterhos, Patent Attorneys, 6000 Frankfurt, Annastr. 19th

Named as inventor:

Masahiro Takahashi,

Tetsuo Hino,

Yasoji Miura, Tokyo;

Shinji Okumura,

Moriyoshi Ishida, Kanagawa-ken (Japan)

Claimed priority:

Japan May 26, 1962 (21524)

Allowed fumaric acid and / or fumarate to act in the presence of ammonium compounds. the Yields obtained after 4 to 8 days vary between 52 and 87%. They are in the following Table I compiled.

Table I. example

Microorganisms

L-aspartic acid yield For the L-asparagine concentration in the acid formation Reaction mixture (° / o) needed time (g / 100 ecm) 61.0 (Days) 3.6 64.8 8th 4.2 87.0 8th 5.0 55.7 1 + 3 3.2 81.8 9 4.7 83.5 1 + 3 4.8 57.0 1 + 3 3.7 78.3 5 4.5 60.9 1 + 3 3.5 52.2 1 + 3 3.0 1 + 3

1
2
3
4th
5
6th
7th
8th
9
10

Escherichia coli

Escherichia coli

Escherichia coli

Escherichia coli

Aerobacter aerogenes

Ps. Fiuorescens

Ps. Fiuorescens

Ps.aeruginosa

Proteus vulgaris ....
Bacillus megatherium

It has been found that these yields in organisms at a nearly neutral pH value can be reduced in the opposite direction Significantly increase fermentation times from ammonium compounds to fumaric acid can, if in the biotechnological production and / or fumarate according to the invention as a microorganism L-aspartic acid due to the action of micro- nisms Pseudomonas trifolii ATCC 12287, ATCC

809 550/336

14537 or B 26-9-PY-5 (deposited with the Institute of Applied Microbiology, University of Tokyo) in Form of a culture liquid uses.

Those used under the same fermentation conditions as in Table I with those according to the invention Experiments in Table II carried out on Pseudomonas trifolii strains show the tests compared to the previously known Process achieved increase in yield of L-aspartic acid and increase in L-aspartic acid concentration in the culture medium to about two to three times that of the method of the French Patent specification. This makes working up easier.

Table II

example Microorganisms L-aspartic acid
concentration in the
Reaction mixture
(g / 100 ecm) yield
(%) For the L-asparagine
acid formation
needed time
(Hours) 1
2
3
4th
5
6th Ps.trifolii ATCC 14537
Ps.trif olii ATCC 14537
Ps.trifolii ATCC 14537
Ps.trifolii ATCC 14537
Ps.trifolii ATCC 12287
Ps.trifolii B 26-9-PY-5 8.6
5.7
10.0
13.5
4.8
8.3 100.3
100.4
87.4
98.0
55.8
99.0 18 + 24
18 + 25
18 + 45
18 +48
18 + 23
18 + 23

To carry out the process, a culture medium of the usual composition is used in each case Pseudomonas trifolii strain mixed with fumaric acid and ammonia, then under about Left to stand under neutral conditions and, after the fermentation has ended, the L-aspartic acid formed isolated.

A good culture of the Pseudomonas trifolii strains used according to the invention can be easily obtained from a nutrient medium of the usual composition (with suppliers of carbon and nitrogen, the essential minerals and some growth factors) in the pH range from 6 to 8 at 15 to 40 ^{Establish 0} C under aerobic conditions.

The actual biotechnological production of L-aspartic acid is an enzyme reaction.

In Table III the ability of the Pseudomonas trifolii strains used according to the invention is ATCC 12287, ATCC 14537 and B 26-9-PY-5, to form L-aspartic acid from fumaric acid, with that of others Compared microorganisms.

The experiments were carried out in the following way: 5 ecm culture medium of the respective bacterium 5 ecm of diammonium fumarate solution containing 5 g / 100 ecm of fumaric acid were added The pH of the mixture was adjusted to 7 and the fermentation was carried out at 37 ° C. for 48 hours. To the Determination of the bacterial growth was the absorption capacity of the culture medium at a Wavelength of 562 ηιμ measured.

Table III

bacteria

share-
growth Remaining fumaric acid % Crowd educated
L-aspartic acid Mole percent CL \ ^ 11O LUJIlX g / 100 ecm 28 g / 100 ecm 1.9 0.449 1.4 68 O ₃ H 2.2 0.361 3.4 68 ' 0.13 20.0 0.127- '- -70 1.15 9.2 0.449 3.5 "" 20th 0.53 7.8 0.650 1.0 10 0.45 54.0 0.418 0.5 10 3.09 39.7 0.283 0.5 34 2.28 3.3 0.388 1.7 34 0.19 0.7 0.310 1.7 18th 0.04 5.7 0.545 0.9 24 0.33 8.7 0.648 1.2 20th 0.50 0.3 0.409 1.0 70 0.02 7.3 0.183 3.5 66 0.42 1.1 0.591 3.3 36 0.06 0.2 0.309 1.8 28 0.01 1.9 0.562 1.4 4th 0.11 100.8 0.458 0.2 4th 5.74 97.9 0.412 0.2 4th 5.56 101.2 0.401 0.2 5.76

Bacillus megatherium B-9-1

Escherichia coli K-12

Aeromonas hydrophila NRRL B-909 ...

Serratia marcesens B 181/1

Pseudomonas aeruginosa IFO 3455

Aerobacter aerogenes ATCC 7256

Proteus vulgaris HX-19

Bacillus subtilis NRRL B-558

Micrococcus flavus ATCC 400

Alcaligenes faecalis ATCC 101

Pseudomonas olevorans ATCC 8062

Staphylococcus aureus 209

Erwinia carotovora IFO 3308

Agrobacterium tumefaciens IFO 3058 ...

Corynebacterium equi B-271-1

Brevibacterium helvolum ATCC 11822 ..

Pseudomonas trifolii ATCC 14537

Pseudomonas trifolii ATCC 12287

Pseudomonas trifolii ATCC B 26-9-PY-5

5 6

Table III can be seen that the according to Erfin-Glucose 20 g

Pseudomonas trifolii strains used in Mieki * 10 ecm

to an excellent degree have the ability to j ^ tt dq ι

Reason of the used by the Pseudomonas trifolii- * ⁴ '""' ^

Strains developed aspartase activity Fumar- 5 MgbU ₄ -7H ₂ U ü, lg

acid into! - aspartic acid. Topped up with water to 11

The enzymatic reaction, ie the stage that consists of *> _{Mieki, is the} trade name for soybean hydrolyses in the culture medium of the Pseudomonas- sat used and contains 2.4 g of total nitrogen in 100 ecm.
trifolii strains are let fumaric acid and ammonia mixture consisting, in the pH range is made io a culture medium and sterilized after adjustment of 7 to 9 and in the temperature range of 25 to the pH to 7.0 at 110 ⁰ C.
50 ⁰ C carried out. The reaction system consists of then the medium was inoculated with the strain fumaric acid or its salts, ammonia or Pseudomonas trifolii ATCC 14537, the ammonium salt and the culture medium of the pseudo-24 hours in a bouillon-agar medium kultimonas trifolii strains. Shaking or aeration is 15 fourth, then cultivation is not necessary for 18 hours. carried out at 3O ⁰ C, the pH through

In the case of the enzymatic reaction, the optimal automatic supply of ammonia gas is 7.0

male fumaric acid concentration was kept about 4 to 40 g /. After 18 hours the bakery reached

100 ecm of the entire reaction mixture. Adding serial growth to its climax. The one in the medium

excess fumaric acid prolongs the reac- 20 contained glucose was completely consumed,

duration. If the fumaric acid is added to the culture medium, the preparation of the culture medium followed

the Pseudomonas enzymatic reaction used according to the invention to form the L-asparagine

Trifolii strains added, the enzyme activity goes acidic. An aqueous solution of diammonium

lost due to the lowering of the pH. fumarate, made from 150 g of fumaric acid and

Therefore, the fumaric acid should carefully and together 25 175 ecm 28% ^r aqueous ammonia solution, the

had been diluted to 11 with basic reagents such as ammonia or alkali, 1 1 des

added to the appropriate pH of the culture medium of Pseudomonas trifolii ATCC

Maintain the reaction system. Diammonium-14537 mixed, which is given in the above

fumarate is preferred for the response. Way had been won. The pH of the

The amino donors obtained in the enzymatic solution were set to 7.0 and the Reaction ammonia or ammonium compounds, solution without shaking or aeration for 24 hours such as aqueous ammonia solution, ammonium chloride, incubated. The accumulated during the incubation Ammonium sulfate and ammonium nitrate, used, L-aspartic acid was 8.6 g / 100 ecm in the end. of which ammonia gas or aqueous ammonia- The formed L-aspartic acid was-like micro-solutions preferred because they did not reveal anions 35 biological studies - 100.3 moles, the isolation of the formed asparagine percent of the supplied diammonium fumarate equic acid prevent. Aqueous ammonia solutions and valent.

Ammonia gas can be used as a neutralizing agent. The reaction solution was adjusted to pH 4 with hydrochloric acid

acidified as a beneficial amino donor, decolorized with activated charcoal, which

be turned. 40 tene clear filtrate concentrated in vacuo to pH 2.8

It has proven to be useful to set the enzy-up and leave it to stand in the ice overnight,

matic reaction under anaerobic conditions whereby crystals separate out, which are filtered off and

to be carried out as shaking and venting were dried. They were 126 g (73.4% of the

Reduce L-aspartic acid yield. Theory). The elemental analysis and determination of the

The enzymatic reaction takes about 20 to 45 optical rotation revealed that it is L-asparagine

100 hours. The reaction solution contains the L-Aspa acid acted,

raginic acid in a concentration of 5 to 40 g /

100 ecm.

The isolation of L-aspartic acid from the reaction

tion mixture can be carried out by known methods 50 after nitrogen
gen. B. the solution for separating the Kjedahl.

Centrifuged bacterial cells and the clear,

Found 10.51%

24.5 °

Calculated

10.53% 24.6 °

concentrated supernatant liquid.

Then the concentrate becomes isoelectric. .

Point of L-Aspartic Acid acidified (on a 55 example

pH value of about 2.5 to 3.5) and the precipitated 660 ecm of a culture medium of Pseudomonas

crystalline L-aspartic acid collected. More than trifolii ATCC 14537, which is shown in the example 1

85% of the total amount obtained in the reaction mixture was mixed

Tenen L-aspartic acid can be first precipitated with 1340 ecm aqueous diammonium fumarate solution,

to be isolated. The remaining L-aspartic acid can 60 which contained 100 g of fumaric acid, made the mixture

from the mother liquor by concentration and cooling to pH 7.4 and carried out at 37 ⁰ C for 25 hours

ment can be gained. Incubation through. The enriched at the end of the reaction L-aspartic acid was 100.4 mole percent

Example 1 added fumaric acid equivalent.

To cultivate the pseudo-example 3 according to the invention

monas trifolii strains were obtained from the following sub-11 of a culture medium of Pseudomonas trifolii

punching: ATCC 14537, manufactured in the example 1

given manner, it was mixed with 11% aqueous diammonium fumarate solution which contained 200 g of fumaric acid, the mixture was adjusted to pH 7.4 and ^{the incubation was carried out at 37 °} C. for 45 hours. The L-aspartic acid yield was equivalent to 87.4 mole percent of the added fumaric acid.

Example 4

120 g of fumaric acid were suspended in 400 cc of water and ig ^he contacted with 140 cc of 28% aqueous ammonia solution in solution. Then 350 ecm of a culture medium of Pseudomonas trifolii ATCC 14537, which had been prepared in the manner indicated in Example 1, was added to the diammonium fumarate solution, the mixture was incubated for 16 hours at 37 ° C. and the mixture was adjusted to pH 7.5 one, diluted to 11 with water and continued the incubation at 37 ^° C. for 48 hours. All of the fumaric acid was converted into L-aspartic acid, and the reaction solution contained L-aspartic acid in an amount which corresponded to 98 mol percent, based on the fumaric acid used. The mixture was concentrated to 0.51 and acidified to pH 2.8 with concentrated hydrochloric acid to give 150 g of crude crystalline aspartic acid containing 83% ^ -aspartic acid, the main impurities of which were ammonium chloride, bacterial cells and their debris. However, no amino acids other than L-aspartic acid were found. 115 g of pure L-aspartic acid were obtained from the crude crystals by recrystallization, which was equivalent to 84 mol percent based on the added fumaric acid. The analytical data and the optical rotation value of the obtained crystals were satisfactorily agreed with those of L-aspartic acid.

Example 5

11 of a culture medium of Pseudomonas trifolii ATCC 12287 was prepared in the manner described in Example 1, 150 g of fumaric acid and 175 ecm of 28% strength ammonium hydroxide solution were mixed and made up to 11 with water. The culture medium was added to the mixture, the pH was adjusted to 7.4 and the reaction was carried out ^{at 37 ° C. for 23 hours.} The content of L-aspartic acid in the reaction solution was 4.8 g / 100 ecm, as the biological determination showed.

Example 6

A culture medium of Pseudomonas trifolii, strain No. B 26-9-PY-5, manufactured, 11 of the medium with 11 Diammonium fumarate solution mixed, which had been prepared as indicated in Example 1, the The pH of the mixture was adjusted to 7.0 and the incubation was carried out at 37 ° C. for 23 hours. Man received 8.3 g / 100 ecm of L-aspartic acid.

Claims (1)

Claim: Process for the biotechnological production of L-aspartic acid, in which microorganisms at almost neutral pH in the presence of ammonium compounds on fumaric acid and / or fumarate is allowed to act, characterized in that the microorganism Pseudomonas trifolii ATCC 12287, ATCC 14537 or Pseudomonas trifolii B 26-9-PY-5 deposited with the Institute of Applied Microbiology of the University of Tokyo, in the form of a culture liquid begins. Considered publications:
French patent specification No. 1 248 130. aO9 550/386 5.68 © Bundesdruckerei Berlin