JP2002080421A - Method for producing highly pure shikimic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To isolate highly pure shikimic acid from an aqueous mother liquid in a crystallization process. SOLUTION: This method for producing the highly pure shikimic acid, characterized by washing the crystallization mother liquor with a water-immiscible organic solvent and then recrystallizing the shikimic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing high-purity shikimic acid.

[0002] Shikimic acid is an important compound as a pharmaceutical intermediate, and is expected to be widely used.

[0003]

2. Description of the Related Art Conventionally, as a method for isolating and purifying shikimic acid by a fermentation method, a method is known in which an aqueous solution is heated to reflux and then purified by an activated carbon treatment and an ion exchange resin-acetic acid eluent. (Journal of American
Chemical Society, 121, 1603, 19
Also, a method of purifying with an activated carbon column is known (Journal of Biological Chemistry, 220, 477, 1956).

[0004]

However, the former requires a large amount of energy for heating and refluxing the aqueous solution, and requires the acetic acid to be concentrated and recovered in order to use a large amount of expensive acetic acid. It requires energy and is not suitable for industrial production. In the latter case, the activated carbon used in the column cannot be regenerated and is not suitable for industrial production.

Further, when isolating shikimic acid obtained by fermentation, organic substances such as protocatechuic acid and gallic acid contained in the fermentation liquor, and compounds containing phosphate groups and sulfate groups are precipitated simultaneously with shikimic acid. It is known that isolated shikimic acid cannot be substantially produced as a clay-like solid.
Such a tendency is particularly remarkable in the crystallization mother liquor in which the impurity concentration becomes large.

[0006]

Means for Solving the Problems Accordingly, the present inventors have:
From an aqueous solution containing shikimic acid obtained by fermentation, etc.,
As a result of intensive studies on a method for efficiently isolating high-purity shikimic acid in high yield, the present invention has been achieved.

That is, the present invention is characterized in that in a crystallization process for isolating shikimic acid from an aqueous solution containing shikimic acid, the crystallization mother liquor is washed with an organic solvent immiscible with water and then recrystallized. Method for producing high-purity shikimic acid. "

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.

The aqueous solution containing shikimic acid used in the present invention is not particularly limited. For example, a liquid culture solution cultured using a microorganism capable of producing shikimic acid, a solid culture extraction aqueous solution obtained by extracting a solid culture with hot water, and the like are preferable. When a liquid culture solution is used, it is preferable to remove the cells. Further, the liquid culture solution is a cation exchange resin,
After passing through at least one column selected from anion exchange resin, chelate resin, synthetic adsorbent, chromatographic separation resin, activated carbon, etc. The fraction containing shikimic acid obtained by the above method is also preferable.

Further, an activated carbon treatment may be performed for decolorization during the above-mentioned purification step. P of solution to be treated with activated carbon
Although H is not particularly limited, it is preferable to perform H near neutrality because it is efficient.

The effect of the present invention is particularly remarkable in the mother liquor obtained after isolating shikimic acid by crystallization. The aqueous solution may contain tetrahydrofuran, 1,4-dioxane, methyl ethyl ketone and the like up to a uniform solution amount. Particularly, it is more efficient to reduce the solubility of shikimic acid by adding an organic solvent during crystallization, and in this case, the crystallization mother liquor is an aqueous solution containing an organic solvent. The present invention is also effective for such an aqueous solution. The aqueous layer washed with an organic solvent may be used for crystallization as it is, or may be mixed with another fermentation solution and used for crystallization.

In the present invention, as the organic solvent used for washing the crystallization mother liquor, various organic solvents can be used depending on the crystallization solvent as long as they are immiscible with water.

Here, the organic solvent used for washing the crystallization mother liquor, which does not mix with water, may be any phase as long as it is separated from the crystallization solvent, and specific examples of the organic solvent include benzene,
Toluene, o-xylene, m-xylene, p-xylene, ethylbenzene, mesitylene, hexane, heptane, octane, nonane, decane, ethyl acetate, methyl chloride, methylene chloride, chloroform, 1,2-dichloroethane, chlorobenzene, o-diene Chlorobenzene, m-dichlorobenzene, bromobenzene, hexanol, octanol, diethyl ether, isopropyl ether,
Dibutyl ether and the like.

Of course, it is also possible to use an organic solvent which separates phases with the addition of an inorganic salt such as salt or sodium sulfate. The addition amount of the inorganic salt is preferably the minimum amount that causes phase separation in order to prevent crystals from being precipitated by subsequent concentration or the like. Specific examples of the organic solvent used in this case include butanol, methyl ethyl ketone, methyl isobutyl ketone, tetrahydrofuran, tetrahydropyran, dioxane, dimethoxyethane and the like.

Preferred are benzene, toluene, o-xylene, m-xylene, p-xylene, ethylbenzene, methyl acetate, ethyl acetate, n-propyl acetate, and more preferably methyl acetate, ethyl acetate, n-acetate. -Propyl and the like. In addition, the organic solvent does not need to be used alone, and may be a mixed solvent. For example, toluene-ethyl acetate and the like can be mentioned.

The organic solvent used in the present invention preferably has a mutual solubility with water at 20 ° C. of 10% or less.

Although the amount of the organic solvent used in the present invention is not particularly limited, it is generally 0.1 to the weight of the aqueous solution containing shikimic acid.
1 to 5 times, 0.1 to achieve the present invention particularly effectively.
Three to two times are used. The method of contacting the aqueous solution containing shikimic acid with the organic solvent is arbitrary, and may be a batch method or a continuous method.

The aqueous solution containing shikimic acid is preferably concentrated for efficient extraction and for improving the pot efficiency. The concentration is preferably such that the inorganic salt and shikimic acid do not precipitate as crystals. Although the degree of concentration varies depending on the concentration of shikimic acid and salt in the aqueous solution, the concentration is preferably reduced to 1/4 or more, more preferably reduced to 1/10 or more on a weight basis.

The purification method of the present invention is particularly effective for purification when an aqueous solution containing shikimic acid contains protocatechuic acid and / or gallic acid as impurities.

As described above, impurities are selectively extracted from the aqueous solution containing shikimic acid to obtain an aqueous solution containing shikimic acid with high purity. High-purity shikimic acid can be isolated from this aqueous solution by a usual method. The method of recovering shikimic acid of high purity after washing with an organic solvent is not particularly limited. For example, when an aqueous solution containing shikimic acid is concentrated to an appropriate concentration and then crystallized, high-purity shikimic acid is precipitated. The degree of concentration varies depending on the concentration of shikimic acid in the aqueous solution, but is preferably reduced to 1/4 or more, more preferably reduced to 1/10 or more on a weight basis. At the time of crystallization, the concentrated solution may be cooled as it is, or may be cooled after adding a necessary amount of an organic solvent. The concentration method may be either normal pressure concentration or reduced pressure concentration, but the liquid temperature during concentration is usually lower than the boiling point of the organic solvent used. With such a liquid temperature, there is no possibility that shikimic acid is decomposed. High purity shikimic acid can be obtained by isolating the crystals precipitated by crystallization by a usual solid-liquid separation method such as centrifugation.

The organic solvent separated from shikimic acid in the concentration step is preferably recycled after distillation.

Next, the organic phase containing shikimic acid concentrated in the concentration step is separated by crystallization to isolate shikimic acid. The method for isolating shikimic acid from the organic phase containing shikimic acid is not particularly limited. For example, a method of crystallizing shikimic acid by cooling the concentrated liquid as it is, followed by solid-liquid separation, or a method of adding a solvent azeotropic with water to the concentrated liquid, concentrating the liquid, cooling and crystallizing, and then performing solid-liquid separation. And the like.

The shikimic acid thus isolated can be purified by a known crystallization purification method, that is, after forming a homogeneous solution with an organic solvent, cooling the homogeneous solution, or cooling the hot-filtrated filtrate,
Alternatively, the solution can be concentrated or, after concentration, an organic solvent can be added to crystallize shikimic acid to obtain a highly purified product.

The extraction tank in the extraction step is not particularly limited.
A general extraction tank and an extraction tower or a culture tank are used. The extraction tank is preferably stirred. The rotation speed depends on the type of the tank, but is preferably 100 to 1,000 rpm. Further, the temperature of the extraction tank is not particularly limited, but may be 0 to 50.
It is preferred that the temperature be controlled at ° C.

[0025]

The present invention will be specifically described below with reference to examples.

The strain BPR-3 (FERM BP-6723) was added to a medium (Bactotryptone 10 g / L, Bacto yeast extract).
5g / L, NaCl 5g / L adjusted to pH 7.5 with NaOH) at 30 ° C
After pre-incubation by shaking for 4 hours,
0 minutes The above sterilized medium (glucose 50g / L, phosphoric acid 1
Potassium 1g / L, Ammonium sulfate 25g / L, Magnesium sulfate heptahydrate 0.4g / L, L-tyrosine 0.1g / L, L
-Phenylalanine 0.1 g / L, L-tryptophan 0.
1g / L, p-aminobenzoic acid 1mg / L, ferrous sulfate heptahydrate 9.9g / L, manganese sulfate tetrahydrate 7.2mg / L, zinc chloride 25mg / L, copper sulfate hydrate 0.5mg / L, calcium carbonate
20g / L) Inoculate in a 30L dissolution flask containing 3L, 1
The cells were cultured for 144 hours under the conditions of 80 rpm and an amplitude of 30 cm.

After completion of the culture, the concentration of shikimic acid in the filtrate from which the cells and calcium carbonate were removed was determined by an HPLC method (column: Shimadzu SCR-
101H, mobile phase: 0.1 M phosphoric acid 1 mL / min., Detection: 254 n
As determined by m), 7 g / L of shikimic acid was accumulated.

The supernatant of the culture solution was passed through a cation exchange resin Livatit S100 (manufactured by Rohm and Haas),
The flow-through fractions were collected, passed through an anion exchange resin Diaion WA30 (manufactured by Mitsubishi Chemical Corporation), and subjected to 0.1N NaOH
And the fractions containing shikimic acid were collected. The solution was concentrated under reduced pressure to about 1/2 by weight. Thereafter, the solution was further passed through a cation exchange resin Livatit S100 (manufactured by Rohm and Haas), and concentrated under reduced pressure (HPLC analysis value: shikimic acid concentration: 34.6 wt%).

Next, 1312.4 g of this concentrated solution was placed in a 2 L three-necked flask, and water was distilled off under reduced pressure. Concentration was terminated when the viscosity of the slurry increased and the stirring operability began to deteriorate (shikimic acid concentration: 51.8%). Quickly 90
8.2 g of tetrahydrofuran (hereinafter abbreviated as THF) was added, and the mixture was cooled to 20 ° C and aged. This was subjected to solid-liquid separation (using 365.5 g of THF rinse) to obtain 193 g of liquid-containing crystals and about 2 L of mother liquor (hereinafter, this operation is referred to as one crystallization).

Example 1 500 g of mother liquor (63.7 g of shikimic acid) was placed in a 1 L four-necked flask equipped with a thermometer, a condenser and a stirrer, and 300 g of ethyl acetate was added thereto, followed by stirring for 10 minutes. The lower layer was placed in a 1 L four-necked flask equipped with a thermometer, a condenser, and a stirrer, and allowed to stand overnight (a small amount of crystals had already precipitated).
The water was distilled off under reduced pressure and concentrated. The concentration of shikimic acid was 51.0% when the concentration was completed due to an increase in slurry viscosity. After adding THF in the same manner as described above, crystallization, solid-liquid separation and drying were performed to obtain 26.1 g of shikimic acid white crystals. The shikimic acid isolation yield based on the charged shikimic acid was 40.9%, purity 9
It was 4.5%.

Example 2 500 g of mother liquor (63.7 g of shikimic acid) was placed in a 1 L four-necked flask equipped with a thermometer, a condenser and a stirrer, 300 g of ethyl acetate was added, and the mixture was stirred for 10 minutes. The lower layer was placed in a 1-L four-necked flask equipped with a thermometer, a condenser, and a stirrer, and concentration was immediately started in the same manner as in the comparative example. The concentration of shikimic acid was 52.5% when the concentration was completed due to the increase in slurry viscosity. After adding THF in the same manner as above, crystallization, solid-liquid separation, and drying were performed to obtain 30.6 g of white crystals of shikimic acid.
I got The shikimic acid isolation yield based on the charged shikimic acid is 4
It was 8.0% and the purity was 95.9%.

Comparative Example 500 g of the obtained mother liquor (63.7 g of shikimic acid) was placed in a 1 L four-necked flask equipped with a thermometer, a condenser and a stirrer, and concentrated under reduced pressure to distill off water. During the concentration, when the shikimic acid concentration became 45.1%, the viscosity of the slurry increased, and the whole system became viscous and could not be stirred. Immediately, THF of the same weight as the concentrated liquid was added, and the same treatment as in the above-mentioned crystallization was carried out to obtain 35.0 g of shikimic acid crystals. The shikimic acid isolation yield based on the charged shikimic acid was 55.0%, and the purity was 83.0%.
8%, clay-like.

[0033]

According to the present invention, in a crystallization process for isolating shikimic acid from an aqueous solution, the crystallization mother liquor is washed with an organic solvent immiscible with water, and then recrystallized to obtain a high yield from the crystallization mother liquor. Pure shikimic acid can be isolated.

Claims (3)

[Claims] In a crystallization process for isolating shikimic acid from an aqueous solution containing shikimic acid, the crystallization mother liquor is washed with an organic solvent immiscible with water and then recrystallized. Production method. 2. The method for producing high-purity shikimic acid according to claim 1, wherein the mutual solubility of the organic solvent with water at 20 ° C. is 10% or less. 3. The method according to claim 1, wherein the aqueous solution containing shikimic acid contains protocatechuic acid and / or gallic acid.