JP2001258583A - Method for purifying shikimic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for purifying shikimic acid which is capable of effectively separating shikimic acid from dehydroxyshikimic acid and other analogs chemical structures similar to shikimic acid. SOLUTION: This method for purifying shikimic acid features treating a shikimic acid-containing solution with an alkali and then allowing shikimic acid to be adsorbed by an anion-exchange resin followed by elution. As the anion-exchange resin, a strongly alkaline aqueous solution, particularly a NaOH aqueous solution, is favorable.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to shikimic acid (shikimic acid).
acid) using an ion exchange resin. Shikimic acid is useful as a raw material for synthesis of pharmaceuticals and the like.

[0002]

2. Description of the Related Art Conventionally, shikimic acid has been produced by a synthetic method, and there have been few reports on the production of shikimic acid by a fermentation method, and there is no example relating to a method of producing purified shikimic acid having high purity. Further, in the conventional ion exchange resin, it is often difficult to separate substances having similar chemical structures.

[0003] In 1960, Simonart and Wiau reported that a fermentation broth of shikimic acid was treated with a strongly basic anion exchange resin.
x (Nature, 186, 78-79, 1960).
For the purpose of analyzing shikimic acid and other similar compounds (by-products of fermentation) in fermentation broth, the authors adsorb shikimic acid fermentation broth on strong anion exchange resin and use 0.05N acetic acid / 0.05N sodium acetate mixed solution. Although eluted, separation from the fermentation by-products dehydroshikimic acid and 5-phospho-shikimic acid has not been achieved.

[0004]

SUMMARY OF THE INVENTION The present invention provides a method for purifying shikimic acid capable of efficiently separating shikimic acid from dehydroshikimic acid and other analogous compounds having a chemical structure similar to that of shikimic acid.

[0005]

The present inventors have conducted intensive studies to develop an efficient method for purifying shikimic acid from a liquid containing shikimic acid. As a result, after alkali treatment, the anion exchange resin contained shikimic acid. The present inventors have found a method for purifying shikimic acid that can efficiently remove impurities by adsorbing a liquid and eluting the liquid with an eluent. That is, the present invention relates to a method for purifying shikimic acid, comprising subjecting a shikimic acid-containing liquid to an alkali treatment, and then adsorbing and eluting shikimic acid to an anion exchange resin. As the anion exchange resin, a strongly basic resin is preferable, and as the eluent, a strongly alkaline aqueous solution is preferable, and particularly, an NaOH aqueous solution is preferable. Also,
By using an OH type anion exchange resin for the alkali treatment step and the anion exchange resin adsorption step of the shikimic acid-containing liquid,
It is also possible to do it in one step. Further, as the shikimic acid-containing liquid, a shikimic acid fermentation liquid obtained by culturing a microorganism capable of producing shikimic acid or an aqueous solution obtained by removing microbial cells from the fermentation liquid is preferable.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, the shikimic acid-containing liquid used is a liquid containing shikimic acid and by-products produced by a synthesis method (see JP-A-11-21267) and a fermentation method. The fermentation broth is processed by centrifugation or MF / UF membrane,
A solution containing shikimic acid, such as a bacteria-removing solution from which microorganisms have been removed, a solution decolorized with activated carbon, or a treatment solution during the process, can also be used.

[0007] The shikimate fermentation liquor is prepared from a bacterium belonging to the genus Bacillus deficient in shikimate kinase activity from a carbon source such as glucose, specifically Bacillus subtilis strain 1-118 (Yu
ki, S., Japan J. Genetics, 50 (2), 155-157 (197
5)), and Bacillus subtilis I derived from the same strain
It is obtained by fermentation using a D3 strain (see Reference Example 1 below) in a usual medium and culture method.

It is preferable to remove solids that cause blockage of the resin tower from a solution containing shikimic acid, such as a synthesis reaction solution, a fermentation solution, and a treatment solution during the process.

In the alkali treatment, the concentration of shikimic acid is 1 to 10 g /
L, pH 11-13, preferably 12-13, temperature 10-90 ° C, preferably 20-40 ° C, for 5-30 minutes. FIG. 1 shows the stability of dehydroshikimic acid, a fermentation by-product and an analogous compound, under alkaline conditions. As can be seen from this figure, under alkaline conditions, shikimic acid is stable, whereas dehydroshikimic acid decomposes immediately. Based on this result, it was found that an alkali treatment was effective for separating shikimic acid and dehydroshikimic acid.

The ion exchange resin used in the method of the present invention may be any anion exchange resin such as Diaion ^R (Diaion PA-412), and a strong basic resin is particularly preferred. The amount of resin used is such that the amount of cations in the liquid to be treated, that is, the total molar equivalents of contaminant amino acids, organic acids, chloride ions, etc. contained in the solution in addition to shikimic acid is within the total exchange capacity of the resin. What should be done.

In the present invention, preferred embodiments in the case of treating a fermented solution, a disinfecting solution, a decolorizing solution, and other solutions containing shikimic acid containing shikimic acid subjected to alkali treatment with a resin are as follows.

The shikimic acid-containing liquid having been subjected to the alkali treatment is passed through an anion exchange resin. The pH of the liquid to be treated during the passage is from neutral to alkaline at a temperature of 90 ° C. or lower, and SV = 1 to 5, preferably
Adsorb shikimic acid with SV = 1 ~ 3. After adsorption, 1-5 RV of pure water
The solution is passed through to remove impurities such as sugar that do not adsorb to the resin.

Next, for the elution of shikimic acid, an acidic aqueous solution or a strongly alkaline aqueous solution can be used as an eluent. Preferred eluents include a 0.4 to 1.0 N aqueous hydrochloric acid solution, a 1 to 2 N aqueous acetic acid solution, and a 0.2 to 1.0 N caustic soda solution. It is desirable to pass these eluents at SV = 1 to 5, preferably SV = 1 to 3, to elute shikimic acid. However, when separating from decomposed products or related compounds other than dehydroshikimic acid, it is preferable to use acetic acid and a strongly alkaline aqueous solution as an eluent, and particularly preferable to be an aqueous solution of NaOH or KOH. FIG. 2 shows an HPLC analysis chart of the shikimic acid fraction in each eluent. (The analysis method is
Example 1 will be described later. )

When eluting with a strong alkaline aqueous solution such as NaOH or KOH, desalting is preferably performed by passing the solution through a strongly acidic cation exchange resin adjusted to H-form with hydrochloric acid or the like in order to remove cations. The shikimic acid crystal can be obtained by concentrating the shikimic acid-containing flow-through liquid under reduced pressure and then performing cooling crystallization. In addition, a usual crystallization method can be applied.

On the other hand, when elution is carried out with an aqueous acetic acid solution, the eluate is directly concentrated under reduced pressure, and then cooled and crystallized, whereby shikimic acid crystals can be obtained.

The alkali treatment and the adsorption of the anion exchange resin can be performed simultaneously by adsorbing the resin regenerated to the OH type with caustic soda.

That is, a shikimic acid concentration of 1 is added to an anion exchange resin adjusted to OH type using a strong alkaline aqueous solution such as NaOH.
Pass the shikimic acid-containing solution diluted to 1010 g / L. By this liquid passing, alkali treatment and resin adsorption can be performed simultaneously. After passing through the solution, pure water is passed through 1-3 RV, and shikimic acid is eluted using an acidic aqueous solution or a strongly alkaline aqueous solution as an eluent as described above. Processing after elution is as described above.

Hereinafter, a method for preparing a fermentation broth usable in the present invention will be described in detail with reference to Reference Examples. Reference Example (1) Bacterial strain As a shikimic acid-producing bacterium, Bacillus subtilis ID3 strain (aroI116, amy4, amy :: Pr) was used.
_(RpmA) -aroD ^(+)- pheA) was used.
Three Bacillus subtilis ID shares were acquired on March 1, 1999 by the Lucian National Collection of Industrial Microorganisms (VKPM) Depositary GNIIgenetika (Russian National Collection of In
dustrial Microorganisms (VKPM) Depositary, GNIIgen
etika) (Address: 1, Dorozhny Proezd., 1,113545, Mosc
ow, Russia) under the registration number VKPM B-7755.

(2) Seed culture The above strain is L-type containing 10 mg / L erythromycin.
The medium was grown at 37 ° C. for 24 hours on a plate or a slope of medium B agar. Next, cells were incubated in 30 ml of LB medium in a 700 ml flask used as a seed medium. Seed cultures were incubated on a rotary shaker at 37 ° C. for 6-7 hours.

(3) Cultivation in a jar fermenter The culture of the above strain was carried out in a fermenter (V-1-1. 2 L) for "Malvisi" research. Aromatic amino acids (phenylalanine, tryptophan, and tyrosine) were not added to the following medium because they were contained in the yeast extract.

Composition of initial culture medium (in g / L): Glucose: 100 (-150 g per liter is added as a feed solution) (NH ₄ ) ₂ SO ₄ : 2 NH ₄ Cl: 3 KH ₂ PO ₄ : 3 MgSO ₄ : 0.4 FeSO ₄ : 0.02 Yeast extract: 15 (“Sigma” Y4000) Erythromycin: 10 mg / L pH 7.0 Initial volume of medium in fermenter: 500 ml Inoculation size: 6% (Em 10 mg / L) For 6 hours in 30 ml of LB medium containing). The feed solution (200 to 250 ml) has a concentration of 700 g /
Contains L glucose.

Feed rate: From the initial stage of culture to 19 hours: 3 to 3.2 ml / L · hour After 19 hours: 4.5 ml / L · hour Temperature: 37 ° C., Agitation: 1000 to 1100 rpm, Air: 0. 6L / min

[0023]

The present invention will be described below in detail with reference to examples. <Example 1> Shikimic acid fermented liquid prepared according to Reference Example
5 g of activated carbon (Wako Pure Chemical Industries, Ltd.) was added to 100 ml of the sterilized solution obtained by separating cells by centrifugation (5000 g) of (shikimic acid 13 g / L), and the mixture was stirred at 60 ° C. for 1 hour with an impeller (200 rpm). Decolorization was performed. The activated carbon was removed with a filter paper (5C, Advantech Toyo) to obtain a decolorized solution. Strongly basic anion exchange resin (Diaion
PA-412) 50 ml was packed in a glass column, and regenerated to the OH form using 2N NaOH. Decolorized solution diluted with pure water (5.1 g
/ L, 100 ml) was passed through a strongly basic anion exchange resin regenerated to the OH form to adsorb shikimic acid. After passing through 100 ml of pure water, elution was carried out sequentially with 100 ml of 0.2N NaOH and 200 ml of 0.4N NaOH to obtain a fraction containing shikimic acid (0.55 m
g). Using a strongly acidic cation exchange resin (Diaion SK1B) adjusted to H-type from this fraction, after removing sodium ions, concentrated, and concentrated with a shikimic acid concentration of 270 g / L (1.7 ml)
I got The resulting concentrated solution was subjected to static crystallization at 4 ° C. to obtain shikimic acid crystals (0.12 g, purity 99%).

The analysis of shikimic acid was carried out using an ODS column (Inerts
il ODS-3 4x250 mm, manufactured by GL Science) in a column oven at 15 ° C, and the moving bed (0.05% acetic acid, 0.11%
Triethylamine) at a flow rate of 0.5 ml / min.
Detected in nm.

<Example 2> In Example 1, caustic soda was added to the prepared decolorizing solution, and 5 g / L of shikimic acid and 0.2 N NaO
The mixture was diluted with pure water so as to obtain H, and shaken at 30 ° C. for 30 minutes to perform an alkali treatment. 100 ml of the solution was passed through a strongly basic anion exchange resin (Diaion PA-412) to adsorb shikimic acid, and elution, desalting, concentration and crystallization were carried out according to the method of Example 1. 99%).

<Example 3> The decolorized solution prepared in Example 1 was diluted so that the concentration of shikimic acid was 5 g / L.
50 ml of the solution was passed through a strongly basic anion exchange resin (Diaion PA-412) regenerated to the OH form to adsorb shikimic acid. Wash with 100 ml of pure water, 100 ml of 0.5 M acetic acid, 1.0 N
Elution was performed sequentially with 100 ml of acetic acid to obtain a fraction containing shikimic acid. According to the method of Example 1, the obtained fraction was concentrated and concentrated.
Crystallization was performed but no desalting was performed (purity 98%).

Example 4 A decolorizing solution prepared by scaling up the above Example 1 was diluted so that the concentration of shikimic acid was 5.0 g / L, and 10 L of the solution was regenerated into an OH type strong base. The solution was passed through 5 L of an anion exchange resin (Diaion PA-412) to adsorb shikimic acid. After passing 10 L of pure water, 0.2N NaO
Elution was carried out sequentially with 10 L of H and 20 L of 0.4N NaOH to obtain a fraction containing shikimic acid (49 g of shikimic acid). According to Example 1 above, using a strongly acidic cation exchange resin (Diaion SK1B) 5L adjusted to H-type, after removing sodium ions, shikimic acid concentration
Concentrated to 37%. The concentrate was transferred to a 300 ml crystallization tank, and the concentrate was stirred at 200 rpm with an impeller (200 rpm).
To 10 ° C, gradually cool at 5 ° C per hour to 10 ° C
For 50 hours (4.3 g, 98%).

[0028]

According to the purification method of the present invention, it is possible to remove dehydroshikimic acid and other by-products and analogous compounds having a similar structure from a shikimic acid-containing solution, and it is possible to produce shikimic acid with high purity.

[Brief description of the drawings]

FIG. 1 is a diagram showing the stability of shikimic acid and dehydroshikimic acid under alkaline conditions.

[Figure 2] HPLC of shikimic acid eluate obtained with various eluents
It is a figure showing an analysis result.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // (C12P 7/42 (C12P 7/42 C12R 1: 125) C12R 1: 125)

Claims (4)

[Claims] 1. A method for purifying shikimic acid, comprising treating a shikimic acid-containing liquid with an alkali, and then adsorbing and eluting shikimic acid on an anion exchange resin. 2. The method according to claim 1, wherein the alkali treatment step of the shikimic acid-containing liquid and the anion exchange resin adsorption step are carried out in one step by using an OH type anion exchange resin. Method. 3. The method according to claim 1, wherein the shikimic acid adsorbed on the anion exchange resin is eluted with an aqueous NaOH solution. 4. The shikimic acid-containing solution is a shikimic acid fermentation solution obtained by culturing a microorganism capable of producing shikimic acid or an aqueous solution obtained by removing microbial cells from the fermentation solution. 2. The method according to claim 1, wherein