JP2505466B2 - Method for producing D-serine - Google Patents

Description

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

<Prior Art> A method is known in which DL-5-hydroxymethylhydantoin is converted to N-carbamyl-D-serine by a microorganism and then hydrolyzed to obtain D-serine.
-152291).

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

However, the reaction for obtaining N-carbamyl-D-serine from DL-5-hydroxymethylhydantoin is as low as 40% or less, and the enzyme activity is low. It cannot be said that it is an advantageous method as an industrial production method because it requires as many as 1 isolated bacterial cell.

<Means and Actions for Solving Problems> Therefore, the present inventors produce D-serine by selectively utilizing L-serine by culturing a microorganism in a medium containing DL-serine. I examined the method.

According to this method, since L-serine is consumed as energy for the growth of microorganisms, only D-serine accumulates in the medium when L-serine is fully utilized, and other by-products. Will almost never exist.

In addition, by culturing under a condition in which the selected microorganism does not carry racemase or is inactivated even if it does, D-serine contained in DL-serine accumulates in a substantially theoretical amount. It will be.

The present inventors have searched for a microorganism that meets such a purpose.

That is, in a medium containing inexpensive DL-serine as a carbon source and a nitrogen source, when L-serine is assimilated and D-serine is not assimilated, a microorganism having a selective assimilation ability is searched, The present invention has been completed by finding that D-serine can be obtained at a cumulative concentration of several + g / l or more by culturing a specific microorganism in a DL-serine medium.

That is, the present invention, in a medium containing DL-serine,
It belongs to yeasts such as Candida, Torulopsis, Cryptococcus, Saccharomycopsis, and Hansenula, or bacteria such as Klebsiella and Providencia and assimilates L-serine, and substantially contributes D-serine. A method for producing D-serine, which comprises culturing a microorganism having an ability not to turn into D-serine, and isolating and collecting D-serine from the culture solution.

 Hereinafter, the configuration of the present invention will be described in detail.

The microorganism used in the present invention includes Candida,
Examples thereof include microorganisms belonging to the yeasts of the genus Torulopsis, the genus Cryptococcus, the genus Saccharomycopsis and the genus Hansenula, or the bacteria of the genus Klebsiella and the genus Providencia.

Among these microorganisms, they can grow substantially in a medium containing DL-serine as a carbon source and a nitrogen source, have L-serine assimilation ability, and substantially utilize D-serine. Microorganisms that do not are used in the present invention.

Here, a microorganism that does not substantially utilize D-serine means D in a range that does not substantially inhibit the effects of the present invention.
-A microorganism that assimilates only a small amount of serine, or a microorganism that assimilates L-serine and then assimilates D-serine in the absence of L-serine is also included.

For example, Candida rugos
a) ATCC10571, Candida repolitica
lipolytica) IFO0717, Torulopsis candida (T
orulopsis candida) IFO0380, Cryptococcus laurentii ATCC36832, Saccharomycopsis l
ipolytica) ATCC20306, Hansenula Anomala
ula anomala) IFO0120, Klebsiella pneumoniae ATCC21316, Providencia rettgeri ATCC9886 and the like.

In the present invention, the culture is performed in a medium containing DL-serine as a carbon source and a nitrogen source, but other carbon source and / or nitrogen source may be contained. Preferably, the culture is performed in a medium containing DL-serine as a single carbon source and a nitrogen source.

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

The total amount of DL-serine may be added to the culture solution from the beginning, but when the concentration becomes high, the growth of microorganisms slows down, or supersaturated DL-serine precipitates, so the initial concentration is 1 to 40 g /
A fed-batch culture method in which the remaining DL-serine is divided and added in a divided manner to 1 is preferable.

The culturing is preferably carried out acidic. The culture solution is usually adjusted to pH 5 to 6 at the start of culture. PH as the culture progresses
Rises. If it is cultured as it is, the growth rate of microorganisms becomes slow and the recovery rate of D-serine tends to be low. Therefore, it is necessary to control the pH to be acidic.

The pH during the culture is usually adjusted to 4 to 7, preferably 4.5 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. Airflow rate is usually 0.5-2.0
vvm, preferably 0.6-1.2 vvm. If the amount of aeration is too small, the growth of microorganisms is inhibited and the assimilation rate of L-serine is also slowed.

Further, even if the amount is large, the effect is not changed, and rather, the concentration of the culture solution is increased to accelerate the evaporation of the culture solution, and the foaming becomes severe, which is not preferable.

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

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

After removing the cells from the thus obtained culture broth by centrifugation, D-serine may be isolated by an ordinary method. For example, ion exchange resin SK-1B (manufactured by Mitsubishi Kasei)
The solution is passed through the column to adsorb serine on the resin and then washed thoroughly. Then elute with aqueous ammonia solution. The eluate may be concentrated. The crude D-serine obtained here is recrystallized with water to obtain purified D-serine.

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

In the examples, the optical purity of serine was analyzed by concentrating and drying the D-serine-containing powder to methyl ester with methanol-hydrochloric acid, reacting it with 3,5-dinitrophenylisocyanate, and then analyzing it by HPLC under the following conditions. By the way.

Column: OA-1000 (Sumitomo Chemical) Mobile phase: n-Hexane: Dichloromethane: Ethanol (4: 2: 1) Flow rate: 0.7 ml / min Detection: UV254nm Example 1 Dry broth 30 g / l (pH 6.0) 50 ml of the culture medium containing the above was dispensed into one Erlenmeyer flask and sterilized at 120 ° C. for 20 minutes to obtain a seed culture medium. One platinum loop of Candida rugosa ATCC10571 was inoculated into this and cultured at 30 ° C for one day with shaking. On the other hand, DL-
A medium (pH 5.6) containing 80 g / l of serine, 2 g / l of phosphoric acid-potassium, 0.5 g / l of magnesium sulfate, and 1.0 g / l of powdered yeast extract.
1 was placed in a 3 liter mini jar fermenter and sterilized to obtain a main culture medium. Inoculate this with the above seed culture, and
C., and 1.0 vvm aeration stirring culture was performed. During the culture, the pH was adjusted to 5.5 ± 0.1 with 4N sulfuric acid. After about 70 hours, the entire amount of L-serine in the medium was assimilated and about 1.1 of a culture solution containing 37 g of D-serine and 25 g of ammonium sulfate 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-serine. After thoroughly washing this column with water, D-serine was eluted with 4% aqueous ammonia. The eluate was concentrated under reduced pressure and dried to dryness, and D-serine 35
got g. When analyzed by HPLC, the optical purity was 99.6.
% Ee or more. The chemical purity was 99.4%.

Example 2 DL-serine 10 g / l, glucose 5 g / l, phosphate-potassium 2 g / l, magnesium sulfate 0.5 g / l, powdered yeast extract 0.5 g
50 ml of a medium (pH 5.0) containing / l was dispensed into one Erlenmeyer flask and sterilized to obtain a seed culture medium. This is Candida
Lugoza ATCC 10571 was inoculated with 1 platinum loop and cultured at 30 ° C for about 24 hours with shaking.

On the other hand, of the above medium composition, DL-serine was set to 30 g / l, and glucose-free medium 1 was placed in a 3 l mini jar fermenter and sterilized to obtain a main culture medium. This was inoculated with the seed main culture solution, and aerated and stirred at 30 ° C. and 1.0 vvm. During the culturing, the pH was adjusted to 5.6 ± 0.1 with 2N sulfuric acid. After about 20 hours, when L-serine in the medium was assimilated, 30 g of DL-serine was added and the cells were further cultured for 30 hours.

Thereafter, in the same manner as in Example 1, 23.3 g of D-serine was obtained. The optical purity and the chemical purity were almost the same as in Example 1.

Examples 3 to 9 18 ml of 5 ml of seed medium (pH 5.0) consisting of 30 g / l of dried broth
It was dispensed into a 180 mm test tube 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-serine 10 g / l, phosphoric acid-potassium 2 g /
5 ml of a main culture medium (pH 5.6) consisting of 1, magnesium sulfate 0.5 g / l and powdered yeast extract 1.0 g / l was dispensed into an 18 × 180 mmφ test tube and sterilized. The above seed culture solution was inoculated with 5% seed and cultivated with shaking at 30 ° C. After 24 hours, the cells were centrifuged to remove bacterial cells, concentrated under reduced pressure to dryness, and then dried. The residual ratio of the L-form and the D-form of serine remaining in the obtained solid content was determined by HPLC.

 Table 1 shows the results.

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

(1) L-serine can be highly selectively assimilated by culturing a microorganism using DL-serine as a carbon source and a nitrogen source.

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

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

(4) Therefore, isolation and purification of D-serine is easy.

Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location (C12P 41/00 (C12P 41/00 C12R 1:78) C12R 1:78) (C12P 41/00 (C12P 41 / 00 C12R 1:22) C12R 1:22) (C12P 41/00 (C12P 41/00 C12R 1: 645) C12R 1: 645) (C12P 41/00 (C12P 41/00 C12R 1:01) C12R 1: 01)

Claims (1)

(57) [Claims] 1. In a medium containing DL-serine, the genus Candida, the genus Torulopsis,
Cryptococcus genus, Saccharomycopsis genus, Hansenul
a) yeasts such as genus or Klebsieil
a) A microorganism that belongs to a bacterium such as a genus or a genus Providencia and has the ability to utilize L-serine and substantially not utilize D-serine is cultured, and D-serine is isolated from the culture solution. A method for producing D-serine, which comprises separating and collecting.