JP2507465B2 - Method for producing D-aspartic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Field of Application> The present invention relates to a method for industrially producing D-aspartic acid useful as a pharmaceutical intermediate by a selective assimilation method.

<Prior Art> A method of converting DL-5-carboxymethylhydantoin to N-carbamyl-D-aspartic acid by a microorganism and then obtaining D-aspartic acid by hydrolysis is already known (JP-A-61-152291). Issue).

<Problems to be Solved by the Invention> The above method is excellent as a method for obtaining D-aspartic acid from DL-5-carboxymethylhydantoin.

However, the yield of the reaction for obtaining N-carbamyl-D-aspartic acid from DL-5-carboxymethylhydantoin is as low as 20% or less, and when viable cells are used, 50 g / isolated cells can be obtained. Since it is necessary, it cannot be said to be an advantageous method as an industrial manufacturing method.

<Means and Actions for Solving Problems> Therefore, for the purpose of providing a method that can be industrially put to practical use, the present inventors have cultivated a microorganism in a medium containing DL-aspartic acid. A method for producing D-aspartic acid by selectively assimilating only L-aspartic acid was examined.

According to this method, since L-aspartic acid is consumed as energy for the growth of microorganisms, only D-aspartic acid is accumulated in the medium at the time when L-aspartic acid is completely utilized, and other L-aspartic acid is accumulated. There are almost no living things.

In addition, by culturing under conditions in which the selected microorganism does not possess racemase or is inactivated even if it does, D-aspartic acid contained in D
-Aspartic acid will be substantially stoichiometrically accumulated.

The inventors of the present invention have arrived at the present invention as a result of diligent studies in order to search for microorganisms that meet such conditions.

That is, as a result of research to find a microorganism having a selective assimilation ability that assimilates only L-aspartic acid and does not substantially assimilate D-aspartic acid in a medium containing DL-aspartic acid as a carbon source and a nitrogen source. , Bacterium, Flavobacterium, Providencia, Pseudomonas, Debaryomycene, Hansenula, Pichia, Saccharomycopsis, Candida, Torulopsis, Cryptococcus, Rhodotorula, Rhodosporidium It was found that D-aspartic acid can be obtained at an accumulated concentration of several + g / g by culturing the microorganism to which it belongs in a medium containing DL-aspartic acid, and completed the present invention.

That is, the present invention provides a bacterium of the genus Bacterium, a genus of Flavobacterium, and Providencia in a medium containing DL-aspartic acid.
genus encia, genus Pseudomonas, genus Debaryomyces, Hansenul
a) genus, Pichia genus, Saccharomycopsis (S
accharomycopsis genus, Candida genus, Torulopsis genus, Cryptococcus (Cryp
tococcus genus, Rhodotorula genus, Rhodosporidium genus and has the ability to assimilate L-aspartic acid and not substantially assimilate D-aspartic acid. A method for producing D-aspartic acid, which comprises isolating and collecting D-aspartic acid.

Here, the microorganism having the ability to substantially assimilate D-aspartic acid means a microorganism that assimilates only a small amount of D-aspartic acid within a range that does not substantially inhibit the effects of the present invention, or L-asparagine. A microorganism that assimilates D-aspartic acid after assimilating the acid in the absence of L-aspartic acid is also included.

Specific examples of the microorganism used in the present invention include the following.

Bacterium cadaveris ATC
C9760 Flavobacteri
um indoltheticum) ATCC27950 Providencia rettgeri AT
CC9886 Pseudomonas putida FERM P
−15281 Debaryomyces hanseni
i) ATCC10623 Hanseula polymorpha ATC
C26012 Pichia pastoris IFO0948 Saccharomycops Saccharomycops
is liplytica) ATCC20306 Candida succiphila IF
O1911 Candida rugosa ATCC10571 Torulopsis candida IF
O0380 Cryptococcus laure
ntii) ATCC36832 Rhodotorula glutinis IF
O1099 Rhodosporidium tor
uloides) ATCC10788 In the present invention, DL-
Culture can be performed in a medium containing aspartic acid as a carbon source and a nitrogen source. Other carbon sources and / or nitrogen sources, for example, glucose, glycerose, ammonium chloride and the like may be contained, but preferably the culture is carried out in a medium containing DL-aspartic acid as a single carbon source and a nitrogen source. To do.

The DL-aspartic acid concentration in the medium is 1 to 150 in 1
g, preferably 10 to 100 g. When the DL-aspartic acid concentration is low, the production efficiency is poor, and when it is high, the culture time is long and the growth of microorganisms tends to be inhibited.

DL-Aspartic acid may be added to the culture solution from the beginning, but if the concentration becomes high, the growth of microorganisms slows down and the culture time becomes longer, so the initial concentration is set to 20 to 50 g / and the remaining DL-aspartic acid is divided. Fed-batch culture with addition is preferred.

The culturing is preferably carried out acidic. The culture solution is usually adjusted to pH 3.5 to 6 at the start of culture, but the pH increases as the culture progresses. If it is cultured as it is, the recovery rate of D-aspartic acid is lowered and the assimilation rate of L-aspartic acid is slowed down. Therefore, the pH is usually controlled to the acidic side. The pH during culture is usually adjusted to 3 to 7, preferably 4 to 6.5. As the acid for adjustment, for example,
Aqueous inorganic acid solutions such as phosphoric acid, sulfuric acid and hydrochloric acid are preferred.

The culture temperature is usually 20 to 40 ° C, preferably 25 to 35 ° C.

The culture is agitated with aeration. Aeration rate is usually 0.5-
It is 2.0 vvm, preferably 0.6 to 1.2 vvm. If the amount of aeration is too small, the L-aspartic acid assimilation rate tends to be slow, and even if it is large, the effect remains the same, but rather the concentration of the culture solution is increased to promote evaporation of the culture solution, and foaming is severe. It is not preferable.

The normal culture is terminated when all L-aspartic acid is assimilated. The total utilization of L-aspartic acid was monitored by monitoring DO, and
Can be determined by analyzing the above or by monitoring the addition amount of the acid.

After all L-asparagine is assimilated, further culturing may continue to gradually assimilate D-aspartic acid, so it is preferable to clearly know the end point of the culture.

After removing the bacterial cells from the thus obtained culture broth by centrifugation, D-aspartic acid may be isolated by a usual method. For example, D-aspartic acid can be isolated by treating the culture solution with activated carbon, adjusting the solution to pH 2.8 with hydrochloric acid, and concentrating crystallization. The crude D-aspartic acid obtained here is recrystallized with water to obtain purified D-aspartic acid.

<Examples> Hereinafter, the present invention will be specifically described with reference to Examples.

In the examples, the optical purity of aspartic acid was analyzed by concentrating and drying the powder containing D-aspartic acid into methanol-
It was methyl esterified with hydrochloric acid, reacted with 3,5-dinitrophenylisocyanate, and then analyzed by HPLC under the following conditions.

Column: OA-1000 (Sumitomo Chemical) Mobile phase: n-Hexane: Dichloroethane: Ethanol (6
8: 28: 4) Flow rate: 1 ml / min Detection: UV254 nm Example 1 50 ml of medium containing 30 g of dry broth / (pH 6.0)
It was dispensed into an Erlenmeyer flask and sterilized at 120 ° C. for 20 minutes to obtain a seed culture medium. This is Candida Lugosa ATCC10571
Was inoculated with platinum loops and cultured at 30 ° C. for 1 day with shaking. on the other hand,
DL-aspartic acid 100 g /, phosphoric acid-potassium 2 g /,
A medium (pH 5.0) 1 containing 0.5 g of magnesium sulfate and 3 g of powdered yeast extract was placed in a mini jar fermenter No. 3 and sterilized to obtain a main culture medium. The above seed culture solution was inoculated into this, and cultured at 30 ° C. with aeration and aeration of 1.0 vvm. In addition,
During the culture, the pH was adjusted to 5.0 ± 0.1 with 4N sulfuric acid. About 26
In time, the total amount of L-aspartic acid in the medium was assimilated and D
A culture broth containing 48 g of aspartic acid was obtained.

The culture was centrifuged at 10,000 rpm for 10 minutes to remove the cells, 2 g of activated carbon was added, the mixture was heated at 80 ° C. for 30 minutes, and cooled to room temperature. The solution was adjusted to pH 2.9 with 1N hydrochloric acid and then concentrated and crystallized to obtain 42.8 g of crude D-aspartic acid. 40.1 g of purified D-aspartic acid was obtained by recrystallization from water.
The chemical purity was 99.9% or higher and the optical purity was 99.4% or higher.

Examples 2-14 18 ml of 5 ml of seed medium (pH 5.0) consisting of 30 g of dry broth
It was dispensed into a test tube of × 180 mmφ and sterilized. One platinum loop of the microorganisms shown in Table 1 was inoculated into this and cultured at 30 ° C. for 1 to 2 days with shaking. On the other hand, DL-aspartic acid 10 g /, phosphoric acid-
18 x 180 mm of 5 ml of main culture medium (pH 5.0) consisting of potassium 2 g /, magnesium sulfate 0.5 g /, powdered yeast extract 1 g /
The φ test tube was dispensed and sterilized. Add the above seed culture to 5
% Sheet and inoculated with shaking at 30 ° C. 24 hours later
The cells were centrifuged to remove the cells, concentrated under reduced pressure, dried to dryness, and then dried. With respect to the obtained solid content, the residual ratio of L-form and D-form of aspartic acid was determined by HPLC.

 The results are shown in Table 1.

<Effects of the Invention> The present invention exhibits the following effects.

(1) L-Aspartic acid can be assimilated highly selectively.

(2) D-aspartic acid can be obtained in a short time with a high accumulated concentration.

(3) Furthermore, the microorganism can be cultured using DL-aspartic acid as a substantial carbon source and nitrogen source.

(4) Most of the consumed L-aspartic acid is converted into carbon dioxide gas and water, and by-products other than D-aspartic acid are substantially absent in the culture solution.

(5) Therefore, isolation and purification of D-aspartic acid is easy.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location (C12P 41/00 (C12P 41/00 C12R 1:40) C12R 1:40) (C12P 41/00 (C12P 41/00 C12R 1: 645) C12R 1: 645) (C12P 41/00 (C12P 41/00 C12R 1:78) C12R 1:78) (C12P 41/00 (C12P 41/00 C12R 1:84) C12R 1:84) (C12P 41/00 (C12P 41/00 C12R 1:72) C12R 1:72) (C12P 41/00 (C12P 41/00 C12R 1:88) C12R 1:88)

Claims (1)

(57) [Claims] 1. A medium containing DL-aspartic acid,
Bacterium genus, Flavobacterium (F
genus lavobacterium, Providencia
Genus, Pseudomonas genus, Debaryomyces genus, Hansenula
Genus, Pichia, Saccharomycopsis
haromycopsis genus, Candida genus, Torulopsis genus, Cryptococcus (Crypto)
coccus), Rhodotorula genus, Rhodosporldium genus and has the ability to assimilate L-aspartic acid and not substantially assimilate D-aspartic acid from the culture solution A method for producing D-aspartic acid, which comprises isolating and collecting D-aspartic acid.