JPS6219095A - Racemization of 2-oxo-oxazolidine-4-carboxylic acid - Google Patents

Abstract

PURPOSE:To obtain the L-isomer of the titled compound useful as a raw material for producing L-serine, etc., by racemizing the D-isomer, by reacting a treated material of a microorganism of the genus Rhizopus, etc., with an optically active 2-oxo-oxazoline-4-carboxylic acid to racemize the compound. CONSTITUTION:A treated material of a microorganism, having the ability to racemize 2-oxo-oxazolidine-4-carboxylic acid or a salt thereof and belonging to the genus Rhizopus, Curvularia, Ephelis or Talaromyces, e.g. Rhizopus batatas (FERM-P No.8344), is reacted with optically active 2-oxo-oxazolidine-4-carboxylic acid or a salt thereof to racemize the compound. Examples of the treated material of the microorganism to be used include a culture fluid, separated microbial cells or decomposition products, etc.

Description

Detailed Description of the Invention <Industrial Application Field> The present invention relates to a method for biochemically racemizing 2-oxo-oxazolidine-4-carboxylic acid or a salt thereof (hereinafter simply abbreviated as ooc). . The present inventors have clarified that OOC can be easily supplied in large quantities through chemical synthesis, and that this substance can be used as a starting material for the biochemical production of L-serine. -28133
9 L-serine is an important amino acid as a raw material for pharmaceuticals, food additives, and cosmetics.

<Prior art> OOC can be easily and inexpensively produced by chemical synthesis, but what is synthesized is generally optically inactive or monolithic L, and when an OOC hydrolase-producing bacterium is allowed to act on it. However, only one L can be used as a substrate, and the yield is low.
, not economical. Therefore, in order to improve the yield, it is necessary to racemize D-00C and convert it into L integrally.

<Problems to be solved by the present invention> The problems to be solved by the present invention are that among the DL-integrated OOC obtained by chemical synthesis, when L-integrated OOC is converted to L-serine by hydrolysis etc. The purpose is to effectively utilize the remaining D by racemizing it.

[Structure of the invention]

<Means for Solving the Problems> As a result of various studies on OOC racemization methods, the present inventors have completed the present invention as an OOC racemization method using biochemical techniques.

That is, the present invention provides a culture solution of a microorganism having the ability to racemize OOC, a bacterial cell, a bacterial cell-treated product, a purified enzyme therefrom, or a bacterial cell, a bacterial cell-treated product, or an immobilized product of a purified enzyme, as OOC. The present invention relates to a method for ceramizing OOC by acting on.

<Function> Examples of microorganisms having the ability to ceramize OOC used in the present invention include Zo1 rhaps patatatus AJ 6011 (Rhi
zopua batas ) FER
M-P 1?344 Curvularia gniculata
AJ 6325 (Curvularim gen
iculata) FKRM-PF? 34j
Epheris SP AJ 6338 (Ep
h@lls ep, ) FERM
-P Talaromyces araneus in Fi'34
AJ 7453 (Talaromyem a
FERM-P 83
There are 47 etc.

In addition, any microorganism strain other than those mentioned above can be used in the present invention as long as it has the ability to racemize OOC. For example, Alternaria cucumelina
AJ6262, Cephalocesium SP AJ62
91, Fusarium sp. AJ 6340.

Vestalotia sp. AJ 6454 *Physium sp. AJ 6469, Potrytis renotans AJ 65661) Lycocesium roserum
AJ6574.

Aspergius giganteus AJ 7179, Penicillium citrinum AJ 7014 tEupenicium panens AJ 7430.7 Nixiera reticulata AJ 7504, Gradinospora
longispora AJ 7584 tCorynespora melonis AJ 6315゜Sclerotium glucanilum AJ 6636, 2) Momyces alpine AJ 7572, Microascus longii stris AJ7609, Podospora setra AJ
7625, Monascus Anchor AJ7650, etc.

In the present invention, first, the above-mentioned microorganisms having the ability to racemize OOC are cultured. A conventional culture method may be used.

As a medium for culturing the above-mentioned microorganisms, a normal nutrient medium may be used as appropriate. For example, as a carbon source, sugars such as glucose, sucrose, glycerol, and molasses, organic acids such as acetic acid, ethanol, methanol, etc. nitrogen sources such as ammonium sulfate and ammonium chloride; organic nutritional sources such as yeast extract, peptone, meat extract, and corn staple liquor; and inorganic ions such as magnesium, iron, manganese, potassium,
Ions such as sodium and phosphoric acid, and vitamins such as pyridoxine and pyridoxine phosphate can be used. In addition, by adding a small amount of OOC to the culture medium during culture, a culture with high racemization ability of 00C or
In some cases, bacterial cells can be obtained. Cultivation may be carried out by a conventional method; for example, after inoculating a strain of the present invention in a medium having a volume of 6 to 9, the culture is aerobically cultured at 20 to 40°C for 1 to 3 days.

The treated bacterial cell product used in the present invention refers to a fraction of the culture obtained as described above that has an activity of racemizing OOC. Such processed bacterial cells include the culture solution itself, a liquid obtained by separating the bacterial cells from the culture solution, isolated bacterial cells, a decomposed product of the isolated bacterial cells, or a purified product of the decomposed product. These bacterial cell-treated products may be used as they are, or may be subjected to treatments such as freeze-drying or acetone drying, or may be subjected to treatments such as immobilization.

The OOC concentration in the aqueous medium used in the present invention differs depending on whether it is a patch type or a continuous type, but in a patch type it is generally about 0.1 to 30 g, preferably about 0.5 to 10 g, and in a continuous type it is about 0.1 to 30 g. It is preferable to lower the concentration slightly.

The reaction is usually carried out at 15-60°C, preferably 30°C-40°C.
Around ℃, pH -4~10. Preferably, this is done around 7. The reaction time varies depending on the means of standing, stirring, flowing down, etc., and the form and potency of the object to be treated, so it is not uniform, but in the patch method it is usually about 10 minutes to 72 hours.

While culturing the above microorganisms in an aqueous medium, the microorganisms and 00C
When the microorganism is brought into contact with the microorganism to cause its action, an aqueous medium containing 00C and nutrients such as a carbon source, nitrogen source, and inorganic ions necessary for the growth of microorganisms is used. Furthermore, vitamins
Addition of organic micronutrients such as amino acids often yields desirable results.

As the carbon source, carbohydrates such as glucose and sucrose, organic acids such as acetic acid, alcohols, and others are used as appropriate. As the nitrogen source, ammonia gas, aqueous ammonia, ammonium salt, and others are used. As the inorganic ions, magnesium ions, phosphate ions, potassium ions, iron ions, and others are used as appropriate.

Desired results can be obtained if the culture is carried out under aerobic conditions with pH 4 to 8 and temperature controlled within an appropriate range of 25 to 40°C.

On the other hand, when the above-mentioned microbial cell-treated product is brought into contact with OOC to act, the aqueous medium containing OOC and the microbial cell-treated product is adjusted to an appropriate temperature of 15°C to 60°C.
It is sufficient to let it stand for a while or stir it while keeping the temperature between 4 and 10. Thus, after 10 minutes to 72 hours have elapsed, a large amount of racemized OOC is formed and accumulated in the aqueous medium.

In the present invention, racemization of OOC due to enzymatic reaction was determined by quantifying D-unit and L-unit by liquid chromatography using an optical resolution resin.

By the way, OOC hydrolase production Wi (patent application 1984-
Even if only 281339) acts on D-000, L
-No formation of serine is observed, but 0 as shown in the following example
As shown in the following experimental example, L-serine can be efficiently accumulated from D-00C by coexisting with a microorganism capable of racemizing 0C.

Experimental example: 2% glycerol, 0.5% yeast extract, 0.5% peptone.
5%, NaC10,25%, DL-00CO,2%,
Medium containing 4.0% calcium carbonate (separately sterilized) (pH 7)
,0) into a 500ml flask, 120
℃.

Sterilized for 15 minutes. This was then cultured on Buoy's agar medium at 30°C for 24 hours.
ERM-P P,? 41 cells were inoculated and incubated at 30°C.

After culturing for 16 hours, the bacterial cells were centrifuged, washed, and collected.
Phosphate buffer containing 1% D-00C (0.1M final.

5 microorganisms were added as viable cells to 5 microorganisms (pi-17,0), and allowed to stand at 30° C. for 48 hours. After the reaction was completed, Pseudomonas testosteroni ATCC 17409, which had been cultured under the same conditions, was added to the mixture to give a concentration of 5% viable cells, and the mixture was left to react at 30° C. for 48 hours. After the reaction was completed, L-serine was quantified by bioassay, and 142 L-serines were produced. On the other hand, even when only viable cells of Pseudomonas Φtestosteroni ATCC 17409 were allowed to act on D-OCC, no production of L-serine was observed.

Example 1 Using the same medium as in the experimental example, the microorganisms shown in Table 1 were inoculated, cultured at 30°C for 24 hours, and the bacterial bodies were centrifuged, washed and collected, and added to an acetate buffer containing 1% D-00C. (End 0
．． 1 M, final pH 4,0) or phosphate buffer (0.1 M final, final pl-17,0);
Tris buffer (0.1 M final, p final 48.5)
5 cells of viable cells were added to the tube, and the mixture was placed in a reactor at 30° C. for 24 hours. After the reaction was completed, the produced L-00C was quantified by liquid chromatography using an optical resolution resin, and the results are shown in Table 1.

Table 1 Amount of L-000 produced by each bacterial strain at pi-4.0 or pi-17,0 or -8,5 Example 2 16 at 30°C using the same medium 50ynl/500m/flask as in the experimental example D-00C in Talaromyces averaneus FERM-Th'Om nutrient solution cultured for hours.
After aseptically injecting 101 n/ml of an aqueous solution (prepared to a pi of 47.0) containing 500 μl and adjusting the - of the culture solution to 7.0, the culture was continued for a further 10 hours. During culturing, the cells were aseptically adjusted to -7.0 every 2 hours.

When a portion of this culture solution was taken and L-00C was quantified using liquid chromatography using an optical resolution resin, 139 m9/di of L-00C was produced.

Example 3 Talaromyces aperanaeus FERM-'i' was added to the same medium 50yR1/500mA flask as in the experimental example.
fl*mL, after culturing at 30°C for 16 hours, the bacterial cells were centrifuged, washed, collected, and diluted with a phosphate buffer containing L- or D-00C1 (0.1M final, pi17.
0) to a total of 5 cells as viable bacteria, and heated at 30℃ for 18
The time was calm and straight.

After the reaction is completed, liquid chromatography using optical resolution resin is performed.
Quantification of [-1] is shown in Table 2.

Table 2 Amount of L- or D-00C produced when L- or D-00C is used as a substrate Example 4 Talaromyces aperaneus FERM- cultivated at 30°C for 16 hours using the same medium 50d and 1500m flask as in the experimental example. After adding and mixing 4-sodium thyalginate solution 5- to a bacterial solution 5- in which a bacterial strain was suspended in physiological saline to a concentration of 20117 dt, this mixed solution was gradually added dropwise to a calcium chloride solution of 15117 dt. , we created bead-shaped immobilized bacterial cells. The total amount of this immobilized material is D −0
Phosphate buffer containing 0.01% (0.1 M final, p
i-17,0) and reacted at 30°C for 16 hours.

As a result, D-00C was racemized, and 1
22 m9/dl of L-OOC was generated.

Applicant Ajinomoto Co., Ltd. Procedural Amendment 1985 August 1, Case Indication 1985 Patent Application No. 157153 2, Title of Invention 2-Oxo-Ox Amount of Nasiridine-4-Carboxylic Acid
Zeta-minimization method 3, relationship with the case of the person making the amendment Patent applicant address No. 5, Kyobashi-1m, Chuo-ku, Tokyo August 5, Number of inventions increased by amendment None 6, Subject of amendment
Column 7 of Detailed Description of the Invention in the Specification, Contents of Amendment (1) On page 2, line 11 of the specification, ``The yield is low,'' is corrected to ``1 The yield is low.''

Claims (1)

[Claims] Rhizopus having the ability to racemize optically active 2-oxo-oxazolidine-4-carboxylic acid or a salt thereof,
Optically active 2-oxo-oxazolidine-4-carboxylic acid is produced by reacting a processed product of microorganisms belonging to the genus Curvularia, Epheris, and Talaromyces with optically active 2-oxo-oxazolidine-4-carboxylic acid or a salt thereof. A method for racemizing 2-oxo-oxazolidine-4-carboxylic acid, which comprises racemizing 2-oxo-oxazolidine-4-carboxylic acid.