JP2676808B2 - Method for producing D-phenylalanine - Google Patents

Description

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

<Prior Art> A method for obtaining D-phenylalanine by hydrolyzing DL-5-benzylhydantoin after converting it to N-carbamyl-D-phenylalanine by a microorganism is already known (JP-A 61-152291). .

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

However, from DL-5-benzylhydantoin to N
The yield of the reaction to obtain -carbami-D-phenylalanine is
It is as high as 60% or more, but when using viable cells, 50 g / cell of isolated cells are required, and it is difficult to hydrolyze the obtained N-carbamoyl-D-failalanine to D-phenylalanine. It cannot be said that it is 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-phenylalanine to produce L- A method for producing D-phenylalanine by selectively assimilating only phenylalanine was examined.

According to this method, since L-phenylalanine is consumed as energy for the growth of microorganisms,
-When all the amount of fail-alanine is utilized, D
-Only phenylalanine is accumulated. Moreover, the metabolic intermediate of L-phenylalanine is small in amount, if any, and can be easily separated by the usual isolation operation of D-phenylalanine.

In addition, DL-phenylalanine was added to the selected microorganisms by culturing under conditions in which the microorganism does not possess racemase or is inactivated even if it does.
-Phenylalanine 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, in a medium containing DL-phenylalanine as a carbon source and a nitrogen source, as a result of a study to find a microorganism having a selective assimilation ability that utilizes only L-phenylalanine and does not substantially utilize D-phenylalanine, Rhodotorula sp. By culturing a microorganism belonging to the genus Rhodosporidium, or the genus Providencia in a medium having DL-phenylalanine, at an accumulated concentration of several tens g / or more,
It was found that D-phenylalanine was obtained, and the present invention was completed.

That is, the present invention provides a genus Rhodotorula, a genus Rhodosporidium, or Providencia (Pr) in a medium containing DL-phenylalanine.
a microorganism belonging to the genus Ovidencia) having the ability to assimilate L-phenylalanine and substantially not assimilate D-phenylalanine, and isolating and collecting D-phenylalanine from the culture solution. Is a method for producing D-phenylalanine.

Examples of the microorganism used in the present invention include microorganisms belonging to the genus Rhodotorula, the genus Rhodosporidium or the genus Providencia. Among these microorganisms, microorganisms having an ability to assimilate L-phenylalanine and substantially not assimilating D-phenylalanine are used in the present invention.

Here, the microorganism having the ability of substantially not utilizing D-phenylalanine means a microorganism which assimilates only a small amount of D-phenylalanine within a range that does not substantially inhibit the effects of the present invention, or L-phenylalanine. Microorganisms that assimilate D-phenylalanine in the absence of L-phenylalanine after conversion are also included.

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

Rhodosporidium tor
uloides) ATCC10788 Rhodotorula glutinis JF
O1099 Providencia rettgeri
ATCC2118 In the present invention, DL-
Culture can be performed in a medium containing phenylalanine as a carbon source and a nitrogen source. Other carbon sources and / or nitrogen sources such as glucose, glycerol and ammonium salt may be contained, but preferably the culture is carried out in a medium containing DL-phenylalanine as a single carbon source and a nitrogen source. .

DL-phenylalanine concentration in the medium is 1 to 10 in 1
0g, preferably 10-60g. If the DL-phenylalanine concentration is low, the production efficiency will be poor, while if it is high, the culture time will be long and the growth of microorganisms will tend to be inhibited.

The whole amount of DL-phenylalanine may be added to the culture solution from the beginning, but when the concentration becomes high, the growth of microorganisms slows down and the culture time becomes longer, so the initial concentration is set to 10 to 30 g / and the remaining DL-phenylalanine is dividedly added Fed-batch culture is 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 assimilation rate of L-phenylalanine 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 accelerate the evaporation of the culture solution,
Foaming becomes severe, which is not preferable.

The normal culture is terminated when all L-phenylalanine is assimilated. The total assimilation of L-phenylalanine can be known by monitoring the dissolved oxygen concentration (DO).

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

After removing the cells from the thus obtained culture broth by centrifugation, D-phenylalanine may be isolated by an ordinary method. For example, the culture solution is ion-exchange resin SK
-1B (manufactured by Mitsubishi Kasei Co., Ltd.) to adsorb phenylalanine on the resin, wash it well with water, then elute with an aqueous ammonia solution, and then concentrate the eluate to isolate D-phenylalanine. can do. The crude D-phenylalanine obtained here is recrystallized with water to obtain purified D-phenylalanine.

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

Optical purity analysis of phenylalanine in the examples,
The concentrated and dried D-phenylalanine-containing powder was reacted with 3,5-dinitrophenyl isocyanate methyl esterified with methanol-hydrochloric acid, and this was analyzed by high performance liquid chromatography (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 1 100 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. Rhodosporidium toluroides AT
One platinum loop of CC10788 was inoculated and shake-cultured at 30 ° C for one day. On the other hand, DL-phenylalanine 10 g /, phosphate-potassium 2 g /, magnesium sulfate 0.5 g /, powdered yeast extract
900 ml of a medium (pH 5.0) containing 1.0 g / was charged with 3 in a mini jar fermenter 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. During the culture, the pH was adjusted to 5.0 ± 0.1 with 2N sulfuric acid. After about 20 hours, 20 g of dry heat-sterilized DL-phenylalanine powder was added, and after 3 hours, another 30 g was added, and the culture was continued. About 47 hours after the start of the culture, the total amount of L-phenylalanine in the medium was assimilated, and a culture solution containing 25.8 g of D-phenylalanine 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-phenylalanine.
After thoroughly washing this column with water, add D with 4% ammonia water.
-Phenylalanine was eluted. The eluate was concentrated and dried to obtain 25.0 g of D-phenylalanine. 22.2 g of D-phenylalanine purified by recrystallization from water was obtained, and the chemical purity was 99.9% or more and the optical purity was 99.8% or more.

Examples 2 to 4 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. The microorganisms shown in Table 1 were inoculated with platinum loops and cultured with shaking at 30 ° C for 1 to 2 days. On the other hand, a main culture medium (pH 5.0) 5 ml consisting of DL-phenylalanine 10 g /, phosphate-potassium 2 g /, magnesium sulfate 0.5 g /, powdered yeast extract 0.5 g / was added to 18 × 1.
An 80 mmφ test tube was dispensed 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 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 phenylalanine was determined by HPLC.

 The results are shown in Table 1.

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

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

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

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

(4) Most of the consumed L-phenylalanine is converted into carbon dioxide gas and water, and the accumulation of metabolic intermediates is small. Then, this metabolic intermediate can be completely separated by the usual isolation operation of D-phenylalanine, and the isolation and purification of D-phenylalanine are easy.

Claims (1)

(57) [Claims] 1. A genus Rhodotorula, a genus Rhodosporidium or Providencia in a medium containing DL-phenylalanine.
cia), which has the ability to assimilate L-phenylalanine and does not substantially assimilate D-phenylalanine, and cultivates the microorganism to isolate and collect D-phenylalanine from the culture solution. A method for producing D-phenylalanine.