JP2507443B2 - Method for producing D-glutamic acid - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for industrially producing D-glutamic acid useful as a pharmaceutical intermediate by a selective assimilation method.

<Prior Art> A method for obtaining D-glutamic acid by hydrolyzing DL-5-carboxyethylhydantoin to N-carbamyl-D-glutamic acid by a microorganism is already known (Japanese Patent Laid-Open No. 61-152291). ).

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

However, the yield of the reaction for obtaining N-carbamyl-D-glutamic acid from DL-5-carboxyethylhydantoin is as low as 20% or less, and when using viable cells, 50 g / l of 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, the present inventors have a method for producing D-glutamic acid by selectively assimilating L-glutamic acid by culturing a microorganism in a medium containing DL-glutamic acid. It was investigated.

According to this method, since L-glutamic acid is consumed as energy for growing microorganisms, only D-glutamic acid is accumulated in the medium when almost all L-glutamic acid is assimilated, and most of the other by-products are present. It doesn't exist.

In addition, by culturing under conditions in which the selected microorganism does not possess racemase or is inactivated even if it does, D- contained in DL-glutamic acid
Glutamic acid will be stored in a substantially logical amount.

The present inventors have arrived at the present invention as a result of extensive investigations aimed at exploring microorganisms that meet such purposes.

That is, in a medium containing DL-glutamic acid as a carbon source and a nitrogen source, only L-glutamic acid was assimilated and D-
As a result of research to find a microorganism having a selective assimilation ability that does not substantially utilize glutamic acid, Flavobacterium, Pseudomonas, Debaryomyces, Hansenula, Pichia, Saccharomycopsis, Candida, Torlopsis The present invention was completed by finding that D-glutamic acid can be obtained at a cumulative concentration of several + g / l or more by culturing a microorganism belonging to the genus Cryptococcus in a medium containing DL-glutamic acid.

That is, the present invention relates to a bacterium of the genus Bacterium, a genus of Flavobacterium, and a pseudomonas (Pseudomona) in a medium containing DL-glutamic acid.
s) genus, Debaryomyces genus, Hansenula genus, Pichia genus, Saccharomycopsis genus, Candida genus, Torulapsis genus, Cryptococcus genus A method for producing D-glutamic acid, which comprises culturing a microorganism belonging to the group and having an ability to assimilate L-glutamic acid and substantially not assimilate D-glutamic acid, and to collect and collect D-glutamic acid from the culture solution. is there.

Here, the microorganism having the ability of not substantially utilizing D-glutamic acid means a microorganism that assimilates only a small amount of D-glutamic acid or L-glutamic acid in the range that does not substantially inhibit the effects of the present invention. After turning into L
-A microorganism that assimilates D-glutamic acid in the absence of glutamic acid is also included.

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

Bacterium cadaveris ATCC9760 Flavobacterium indoltheticum ATCC27950 Pseudomonas putida FERM P-15281 Debalyomyces polymorphisms pastoris) IFO0948 Saccharomycopsis lipolytica ATCC20306 Candida rugosa ATCC10571 Torulopsis candida IFO0380 Cryptococcus CC368 laurentii.

In the present invention, the culture is performed in a medium containing DL-glutamic acid as a carbon source and a nitrogen source, but other carbon sources and / or
Or a nitrogen source, such as glucose, glycerol,
It may contain ammonium chloride or the like. Preferably, the culture is performed in a medium containing DL-glutamic acid as a single carbon source and a nitrogen source.

DL-glutamic acid concentration in the medium is 1 to 150 g in 1
It is preferably 10 to 100 g. When the DL-glutamic 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-glutamic 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 / l and the remaining D
Fed-batch culture in which L-glutamic acid is dividedly added is preferred.

The culturing is preferably carried out acidic. The culture solution is usually adjusted to pH 4 to 6 at the start of the culture, but as the culture progresses, p
H rises. If it is cultured as it is, the recovery rate of D-glutamic acid is lowered and the assimilation rate of L-glutamic acid is slowed, so it is necessary to control the pH 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 L-glutamic acid assimilation rate tends to be slow, and even if it is large, the effect does not change, but rather the concentration of the culture solution becomes high to promote evaporation of the culture solution, and foaming becomes severe. Not preferable.

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

After all the L-glutamine has been assimilated, if the culture is further continued, D-glutamic acid may be gradually assimilated, so it is preferable to clearly know the end point of the culture.

After removing the cells from the thus obtained culture solution by centrifugation, D-glutamic acid may be isolated by a usual method. For example, after treating the culture solution with activated carbon,
The liquid is adjusted to pH 3.2 with hydrochloric acid and concentrated and crystallized to isolate D-glutamic acid. If the crude D-glutamic acid obtained here is recrystallized with water, purified D-glutamic acid can be obtained.

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

In the examples, the optical purity of glutamic acid was analyzed by subjecting concentrated and dried D-glutamic acid-containing powder to methyl esterification with methanol-hydrochloric acid followed by reaction with 3,5-dinitrophenylisocyanate, which was then analyzed by HPLC under the following conditions. It was done by the method.

Column: OA-1000 (Sumitomo Chemical) Mobile phase: n-Hexane: Dichloromethane: Ethanol (20: 8: 1) Flow rate: 1 ml / min Detection: UV254 nm Example 1 Dry broth 30 g / l (pH 6.0) 50 ml of the containing medium was dispensed into one Erlenmeyer flask and sterilized at 120 ° C. for 20 minutes to obtain a seed culture medium. Toltorpsis Candida IFO 0380
Was inoculated with 1 platinum loop and cultured at 30 ° C for 1 day with shaking. on the other hand,
DL-glutamic acid 50g / l, phosphoric acid-potassium 2g / l, magnesium sulfate 0.5g / l, powder yeast extract 1.5g / l containing medium (pH 5.0) 1 into a 3l mini jar fermenter and sterilized It was used as the 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. During the culture, the pH was adjusted to 5.0 ± 0.1 with 2N sulfuric acid. After about 26 hours, the total amount of L-glutamic acid in the medium was assimilated, and a culture solution containing 23 g of D-glutamic 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 liquid was adjusted to pH 3.2 with 1N hydrochloric acid and then concentrated and crystallized to obtain 20.4 g of crude D-glutamic acid. Recrystallization from water gave 19.6 g of purified D-glutamic acid. The chemical purity was 99.9% or higher and the optical purity was 99.4% or higher.

Examples 2 to 10 18 ml of 5 ml of seed medium (pH 5.0) consisting of 30 g / l of dried 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-glutamic acid 10 g / l, phosphate-potassium 2 g / l, magnesium sulfate 0.5 g / l, powdered yeast extract
5 × 5 ml of main culture medium (pH 5.0) consisting of 0.5 g / l is 18 × 180 mmφ
It was dispensed into a test tube and sterilized. Add 5% of the above seed culture
The seeds were inoculated and cultured at 30 ° C with shaking. After 24 hours, the cells were centrifuged to remove cells, concentrated under reduced pressure, dried to dryness, and then dried. With respect to the obtained solid content, the residual ratio of the L-form and the D-form of glutamic 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-glutamic acid can be highly selectively assimilated by culturing a microorganism with DL-glutamic acid as a substantially carbon source and a nitrogen source.

(2) Further, the accumulated concentration is high, and D-glutamic acid can be obtained in a short time.

(3) In addition, most of the consumed L-glutamic acid is converted into carbon dioxide gas and water, and by-products other than D-glutamic acid are substantially absent in the culture solution.

(4) Therefore, isolation and purification of D-glutamic 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)

Claims (1)

(57) [Claims] 1. A bacterium of the genus Bacterium, Flavobacterium (Fla) in a medium containing DL-glutamic acid.
vobacterium), Pseudomonas genus,
The genus Debaryomyces, Hansenula (Ha
nsenula, Pichia, Saccharomycopsis, Candid
a) A microorganism belonging to the genus, Torulopsis genus, Cryptococcus genus and having an ability to assimilate L-glutamic acid and substantially not assimilate D-glutamic acid is cultured, and D-glutamic acid is added from the culture solution. A method for producing D-glutamic acid, which comprises isolating and collecting.