JP2530662B2 - Method for producing D-asparagine - Google Patents

Description

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

<Prior Art> A method for obtaining D-asparagine by hydrolyzing N-carbamyl-D-asparagine after converting DL-5-carbamoylmethylhydantoin by a microorganism is already known (Japanese Patent Application Laid-Open No. 61-152291). ).

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

However, the yield of the reaction for obtaining N-carbamyl-D-asparagine from DL-5-carbamoylmethylhydantoin is as high as 60% or more, but when using viable cells,
It requires as much as 50 g / separate bacterial cells, and the obtained N
It is difficult to say that carbamoyl-D-asparagine is hydrolyzed to D-asparagine, which is not an advantageous method as an industrial production 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 studied L by culturing a microorganism in a medium containing DL-asparagine. -A method for selectively assimilating asparagine to produce D-asparagine was examined.

According to this method, since L-asparagine is consumed as energy for the growth of microorganisms, only D-asparagine is accumulated in the medium at the time when the entire amount of L-asparagine is assimilated, and most of the other by-products. It doesn't exist.

In addition, by culturing under conditions in which the selected microorganism does not carry racemase, or is inactivated even if it does, D- contained in DL-asparagine
Asparagine will be accumulated in a theoretical amount.

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, in a medium containing DL-asparagine as a carbon source and a nitrogen source, only L-asparagine was assimilated and D-asparagine was used.
As a result of research to find a microorganism having a selective assimilation ability that does not substantially utilize asparagine, the genus Flavobacterium, Klebsiella, Providencia, Pseudomonas, Pichia, Candida, Torlopsis, Cryptococcus It was found that D-asparagine can be obtained at a cumulative concentration of several g / g / or more by culturing microorganisms belonging to the genera Rhodotorula and Rhodosporidium in a medium containing DL-asparagine, and completed the present invention.

That is, the present invention, in a medium containing DL-asparagine, genus Flavobacterium (genus Flavobacterium), Klebsiella (Klebsiella)
Genus, Providencia (Providencia), Pseudomonas (Pseudomonas), Pichia (Pichia), Candida (Candida), Torulopsis (Torulopsis),
Cryptococcus spp., Rhodotorula (Rh
A microorganism belonging to the genus odotorula and the genus Rhodospoudium and having the ability to utilize L-asparagine and substantially not assimilate D-asparagine is cultured, and D-asparagine is isolated and collected from the culture solution. And a method for producing D-asparagine.

Here, the microorganism having the ability of not substantially utilizing D-asparagine means a microorganism that utilizes only a small amount of D-asparagine or L-asparagine within a range that does not substantially impair the effects of the present invention. After turning into L
-In the absence of asparagine, microorganisms that assimilate D-asparagine are also included.

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

Bacterium cadaveris ATCC9760 Flavobacterium indoltheticum ATCC27950 Klebsiella pneumoniae ATCC12658 burtonii) ATCC20279 Candida rugosa ATCC10571 Candida humicola ATCC36992 Torulopsis Candida IFO0380 toruloides) ATCC10788 In the present invention, DL-
The culture can be performed in a medium containing asparagine as a carbon source and a nitrogen source. Other carbon sources and / or nitrogen sources, for example, glucose, glycerose, ammonium chloride, etc. may be contained, but preferably the culture is carried out in a medium containing DL-asparagine as the single carbon source and nitrogen source. .

The concentration of DL-asparagine in the medium is 1 to 150 in 1
g, preferably 10 to 100 g. If the DL-asparagine concentration is low, the production effect will be poor, and conversely, if the DL-asparagine concentration is high, the culture time will be longer and the growth of microorganisms will tend to be inhibited.

DL-asparagine may be added to the culture solution from the beginning, but if the concentration becomes high, the growth of microorganisms will slow down and the culture time will become longer, so the initial concentration is set to 20 to 50 g /
Fed-batch culture in which DL-asparagine is added in portions is preferred.

The culturing is preferably carried out acidic. The culture solution is usually adjusted to pH 4 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-asparagine is lowered and the assimilation rate of L-asparagine is slowed. Therefore, the pH is usually controlled to the acidic side. The pH during culturing is usually 3 to 7, preferably 4
Adjust to ~ 6.5. As the adjusting acid, for example, an aqueous solution of an inorganic acid such as phosphoric acid, sulfuric acid or hydrochloric acid is preferable.

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 assimilation rate of L-asparagine will tend to be slow, and even if it is large, the effect will not change, but rather the concentration of the culture solution will be high to promote evaporation of the culture solution, and foaming will become violent. Not preferable.

Normal culture is terminated when L-asparagine is completely assimilated. The total utilization of L-asparagine can be known by monitoring DO, or by analyzing D and L of asparagine or by monitoring the amount of acid added.

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

After removing the bacterial cells from the thus obtained culture solution by centrifugation, D-asparagine may be isolated by a usual method. For example, the culture solution is ion exchange resin SK-1B.
(Made by Mitsubishi Kasei Co., Ltd.) to adsorb asparagine on the resin, wash it well with water, then elute with an aqueous ammonia solution, and then concentrate the eluate to isolate D-asparagine. You can Coarse D-obtained here
Recrystallization of asparagine with water gives purified D-asparagine.

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

In the examples, the optical purity analysis of asparagine was carried out by concentrating and drying the D-asparagine-containing powder after methyl esterification with methanol-hydrochloric acid and then reacting it with 3,5-dinitrophenylisocyanate.
It was carried out by the method of analyzing by LC.

Column: OA-1000 (Sumitomo Chemical) Mobile phase: n-Hexane: Dichloroethane: Ethanol (6
8: 28: 4) Flow rate: 1 ml / min Detection: UV254 nm Example 1 100 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 1 platinum loop and cultured at 30 ° C for 1 day with shaking. on the other hand,
A medium (pH 5.0) 900 ml containing DL-asparagine 60 g /, phosphate-potassium 2 g /, magnesium sulfate 0.5 g /, powdered yeast extract 1.0 g / was charged into 3 mini jar fermenters and sterilized to obtain a main culture medium. did. 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 2N sulfuric acid. About 15
The total amount of L-asparagine in the medium was assimilated and D-
A culture solution containing 29.3 g of asparagine was obtained.

The culture was centrifuged at 10,000 rpm for 10 minutes to remove the cells, and then passed through a column packed with ion exchange resin SK-1B (H type) to adsorb D-asparagine. After thoroughly washing this column with water, D-asparagine was eluted with 4% aqueous ammonia. Concentrate and dry the eluate to D
-Obtained 28.4 g of asparagine. Recrystallization from water gave 25.2 g of purified D-asparagine. The chemical purity was 99.9% or higher and the optical purity was 99.8% or higher.

Examples 2 to 12 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, 5 ml of a main culture medium (pH 5.0) consisting of DL-asparagine 10 g /, phosphate-potassium 2 g /, magnesium sulfate 0.5 g /, powdered yeast extract 0.5 g / 18 × 180 mm
It was dispensed into a φ test tube and sterilized. Add the above seed culture to 5
% Seeds and shake culture at 30 ° C. 24 hours later
The cells were centrifuged to remove the cells, concentrated under reduced pressure, dried to dryness, and then dried. The abundance of asparagine L-form and D-form of the obtained solid content was determined by HPLC.

 The results are shown in Table 1.

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

(1) L-asparagine can be assimilated highly selectively.

(2) The accumulated concentration is high, and D-asparagine can be obtained in a short time.

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

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

(5) Therefore, isolation and purification of D-asparagine 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 1:22) C12R 1:22) (C12P 41/00 (C12P 41/00 C12R 1:40) C12R 1:40) (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) (C12P 41/00 (C12P 41/00 C12R 1: 645) C12R 1: 645)

Claims (1)

(57) [Claims] 1. A bacterium of the genus Bacterium, Flavobacterium (Fla) in a medium containing DL-asparagine.
vobacterium genus, Klebsiella genus, Providencia genus, Pseudomonas (Pse)
udomonas genus, Pichia genus, Candida (Ca
genus ndida, genus Torulopsis, genus Cryptococcus, Rhodotorul
a) A microorganism belonging to the genus Rhodosporldium and having the ability to utilize L-asparagine and substantially not utilize D-asparagine is cultured, and D-asparagine is isolated and collected from the culture solution. A method for producing D-asparagine characterized by the following.