JP4894673B2 - Large-scale purification method of 2-pyrone-4,6-dicarboxylic acid - Google Patents

Description

  The present invention relates to an industrial purification method for 2-pyrone-4,6-dicarboxylic acid obtained by fermentation production.

  Lignin, a major plant component, is a biomass resource that is universally contained in plant cell walls as an aromatic polymer compound. However, an effective utilization technique has not been developed for aromatic components derived from plants mainly composed of lignin because they are composed of components having various chemical structures and complex polymer structures. As a utilization technique known so far, there is a technique for separating and producing vanillin which is a perfume raw material from a low molecular weight aromatic decomposition product produced by chemical decomposition such as alkali decomposition of the aromatic component. However, at present, there is no known effective method for utilizing a large amount of low-molecular aromatic substances other than vanillin produced by chemical decomposition. For this reason, lignin produced in large quantities in the papermaking process is not effectively used and is burned as a substitute for heavy oil.

  On the other hand, the inventors of the present invention have used plant aromatic components such as lignin by hydrolysis, oxidative decomposition, solvolysis and other chemical decomposition methods, supercritical water and physicochemical decomposition methods using supercritical organic solvents, etc. A single intermediate that can be converted into a low-molecular-weight mixture containing vanillin, syringaldehyde, vanillic acid, syringacic acid, protocatechuic acid, etc., and these five compounds can be used as raw materials for functional plastic materials and chemical products 2 -Found to be converted to pyrone-4,6-dicarboxylic acid.

  In addition, the present inventors also have four types of enzymes (benzaldehyde dehydrogenase, dimethylase, protocatechuate 4,5-dioxygenase) that constitute a multistage reaction process for producing 2-pyrone-4,6-dicarboxylic acid by fermentation. 2-pyrone-4,6-dicarboxylic acid is produced from these five compounds using transformed cells carrying a gene encoding 4-carboxy-2-hydroxymuconic acid-6-semialdehyde dehydrogenase) The method is reported (for example, refer patent document 1).

  However, Patent Document 1 only describes activated carbon treatment as a purification method of 2-pyrone-4,6-dicarboxylic acid, and details are not disclosed. Patent Document 2 discloses a method for producing 2-pyrone-4,6-dicarboxylic acid in the presence of α-hydroxy-γ-carboxymuconic acid-ε-semialdehyde, but does not mention the purification method at all. It has not been.

JP 2005-278549 A JP 2000-32988 A

  Therefore, an object of the present invention is to provide an industrial purification method for 2-pyrone-4,6-dicarboxylic acid obtained by fermentation production, which is useful as a raw material for functional plastics and chemical products.

  As a result of intensive studies in view of the present situation, the present inventors have found that a specific salt is present in a microbial fermentation broth containing 2-pyrone-4,6-dicarboxylic acid, thereby corresponding 2-pyrone-4,6. The inventors have found that dicarboxylates can be isolated with high purity and high yield, and have completed the present invention. Further, the present inventors extract 2-micron-4,6-dicarboxylic acid by extracting the microbial fermentation broth with a specific organic solvent without forming 2-pyrone-4,6-dicarboxylate. Was able to be isolated with high purity and high yield, and the present invention was completed.

That is, (1) the present invention has a cation salt selected from monovalent, divalent, trivalent and tetravalent in a fermentation broth containing 2-pyrone-4,6-dicarboxylic acid produced by a microorganism. A method for purifying 2-pyrone-4,6-dicarboxylic acid is provided.
(2) In the present invention, the salt of the monovalent cation is sodium chloride, potassium chloride, rubidium chloride, silver chloride, sodium bromide, sodium sulfate, disodium phosphate or dipotassium hydrogen phosphate. 1) A method for purifying 2-pyrone-4,6-dicarboxylic acid according to 1) is provided.
(3) The present invention provides the purification of 2-pyrone-4,6-dicarboxylic acid according to (1), wherein the salt of the divalent cation is magnesium chloride, copper sulfate or potassium hexacyanoferrate (II). Provide law.
(4) The present invention provides the purification of 2-pyrone-4,6-dicarboxylic acid according to (1), wherein the salt of the trivalent cation is iron (III) chloride or potassium hexacyanoferrate (III). Provide law.
(5) The present invention provides the method for purifying 2-pyrone-4,6-dicarboxylic acid according to (1), wherein the salt of the tetravalent cation is potassium tin (IV).
(6) The present invention collects 2-pyrone-4,6-dicarboxylate precipitated in the presence of the salt, dissolves in water, and then extracts with ethyl acetate, cyclopentanone or cyclohexanone under acidic conditions The purification method according to any one of (1) to (5), comprising the step of:
(7) The present invention provides the purification method according to (6), wherein an excessive amount of strong acid is added during the extraction step.
(8) The present invention includes a step of recovering 2-pyrone-4,6-dicarboxylate precipitated in the presence of the salt, dissolving it in water, and then treating the solution with a cation exchange resin. The purification method according to any one of 1) to (7) is provided.
(9) The purification method according to any one of (1) to (8), wherein the salt is used in an amount of at least 2 moles with respect to 2-pyrone-4,6-dicarboxylic acid in the fermentation broth. I will provide a.

(10) The present invention provides 2-pyrone-4 without forming 2-pyrone-4,6-dicarboxylate from a fermentation broth containing 2-pyrone-4,6-dicarboxylic acid produced by a microorganism. The present invention provides a method for purifying 2-pyrone-4,6-dicarboxylic acid, characterized by extracting 2,6-dicarboxylic acid.
(11) The present invention provides the purification method according to (10), wherein extraction is performed with ethyl acetate, cyclopentanone, or cyclohexanone.
(12) The present invention provides the purification method according to (10) or (11), which is extracted with ethyl acetate.
(13) The present invention provides the purification method according to any one of (10) to (12), wherein an excessive amount of strong acid is added during the extraction step.
(14) The present invention provides the purification method according to (13), wherein the strong acid is used at least 3% by weight based on the fermentation broth.

  According to the present invention, it is possible to purify 2-pyrone-4,6-dicarboxylic acid in a large amount at a high purity, in a high yield and at low cost from a microbial fermentation broth containing 2-pyrone-4,6-dicarboxylic acid. .

  In the method for purifying PDC of the present invention, a monovalent to tetravalent cation salt is present in a fermentation broth containing 2-pyrone-4,6-dicarboxylic acid (hereinafter referred to as “PDC”) produced by a microorganism. It is characterized in that PDC is used as a salt or PDC salt is obtained in the fermentation broth by preventing PDC salt formation.

  Fermentation liquid containing PDC produced by microorganisms is a plant-derived low molecular weight compound such as vanillin, syringaldehyde, vanillic acid, syringic acid, protocatechuic acid or a mixture thereof for the purpose of obtaining PDC as a single compound. In the presence of, there is no particular limitation as long as it is obtained by culturing a transformed cell containing a gene encoding a suitable enzyme for fermentative production of PDC in multiple stages or in one stage. Examples of such a fermentation broth include those obtained by the method described in JP-A-2005-278549. In the method described in the publication, 10 to 20 g of PDC is usually produced in 1 L of the fermentation broth. Prior to the purification of the following PDC, it is preferable to remove cells from the fermentation broth by centrifugation, activated carbon adsorption, or the like. The activated carbon adsorption is preferably performed by adding activated carbon to the fermentation broth, mixing and stirring, and then removing the activated carbon by filtration or passing the fermentation broth through an activated carbon packed bed.

<Method of forming PDC salt>
A monovalent to tetravalent cation salt is present in a microbial fermentation broth containing PDC to precipitate the corresponding salt of PDC.
Examples of monovalent cations include metal ions such as sodium, potassium, rubidium, silver, lithium, and cesium; ammonium ions; alkylammonium ions such as hexamethylenediamine, ethylenediamine, diethanolamine, and triethanolamine. Among these, sodium ion, potassium ion, rubidium ion or silver ion is preferable.
Monovalent cation salts include inorganic acid salts such as hydrochlorides, nitrates, sulfates and phosphates of the cations; acetic acid, oxalic acid, citric acid, tartaric acid, fumaric acid, maleic acid, malic acid, Examples thereof include organic acid salts such as cyanic acid and thiocyanic acid. Specific examples include potassium chloride, rubidium chloride, silver chloride, sodium bromide, sodium sulfate, disodium phosphate, or dipotassium hydrogen phosphate. Sodium chloride, potassium chloride, rubidium chloride, silver chloride or bromide Sodium is preferred.

Examples of the divalent cation include metal ions such as magnesium, calcium, iron (II), copper (II), zinc, barium, cobalt, nickel, manganese, and chromium (II). The divalent cation salt is the same as described above. Specific examples of the divalent cation salt include magnesium chloride, copper sulfate, and potassium hexacyanoferrate (II).
Examples of the trivalent cation include iron (III), aluminum, gallium and the like, and specific examples of the salt of the trivalent cation include iron (III) chloride or potassium hexacyanoferrate (III). It is.
Tetravalent cations include ions such as tin (IV), and a specific example of a tetravalent cation salt is potassium tin (IV).
These monovalent to tetravalent cation salts may be used in combination of two or more.

In addition to the above monovalent to tetravalent cations, ionic compounds including vanadyl ion (VO ²⁺ ), titanate (TiO ₂ ) ion, cyan ion, thiocyan ion, and thiocarbonyl can also be used. .

  PDC has carboxylic acid groups at the 2 and 4 positions, but these carboxylic acid groups do not form mere PDC carboxylates upon contact with the above salts, but as shown in FIG. A hydrogen bond is formed between them to form a double salt having an octahedral 6-coordinate structure in which two carbonyl groups are coordinated around a metal ion (a sodium ion in FIG. 1). Various double salt structures formed around the carbonyl group are reported in various documents (for example, Acta. Cryst. (1992) C48, 460-465). FIG. 2 shows the aqueous solubility of a double salt formed by PDC and a monovalent cation. As apparent from FIG. 2, among the double salts formed with PDC and monovalent cations, in particular, the sodium salt, potassium salt, rubidium salt or silver salt of PDC is free PDC, microbial culture medium. It was found that the solubility in water was very low compared to the components, water-soluble components extracted from various plants (water solubility of free PDC: 182 mM). Therefore, in particular, the sodium salt, potassium salt, rubidium salt or silver salt of PDC can easily separate PDC from many medium components and plant-extracted water-soluble components for salting out. It has been found suitable.

  The salt is preferably used in a large excess amount with respect to PDC present in the microbial fermentation broth, for example, preferably at least 2-fold mol, particularly 2-fold to 10-fold mol. In order to enhance the salting-out effect, the fermentation broth in which a monovalent to tetravalent cation salt is present may be cooled or concentrated. When cooling, the fermentation broth is allowed to stand at about 0 to 4 ° C. for 12 to 24 hours. The salted out PDC salt can be recovered by filtration.

  The above PDC salt can be obtained as free PDC with higher purity by further carrying out the following two different purification steps.

  (1) The PDC salt can be dissolved in water, for example, pure water, and extracted with an organic solvent under acidic conditions (about pH 1-2), and the PDC salt can be extracted as free PDC. Examples of the extraction solvent include ethyl acetate, cyclohexanone, cyclopentanone, hexane, heptane, toluene, benzene, diethyl ether, tetrahydrofuran, chloroform and dichloromethane. Ethyl acetate, cyclohexanone or cyclopentanone is preferable, and the boiling point is low. Therefore, ethyl acetate is particularly preferable. In order to make the acidic condition, an excessive amount of an aqueous strong acid solution, preferably 3% or more of the aqueous layer, more preferably 3% to 7% of the aqueous layer may be used. Examples of strong acids include hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, and hydrochloric acid is particularly preferable. If necessary, the obtained organic layer may be treated with a strong acid aqueous solution several times. The purity of the obtained PDC can be further increased by repeating recrystallization as necessary.

  (2) Free PDC can also be obtained by dissolving the above PDC salt in water, for example, pure water, and treating this solution with an H-type cation exchange resin. As the H-type cation exchange resin, for example, Amberlite (registered trademark) IR120B having a ion exchange capacity of about 2.0 meq / ml, which is a gel type polystyrene / sulfonic acid type ion exchange resin, DIAION (DIAION) ( (Registered trademark) SK1B etc. can be used.

  From the obtained solvent concentrate of PDC or the concentrate of the effluent of the ion exchange resin, PDC can be obtained as crystals by recrystallization. By repeating recrystallization as necessary, the purity of the crystal can be further increased. This purification method to form PDC salt does not require extraction of large-capacity microbial fermentation broth, and PDC can be easily purified as a salt by salting out.At the same time, organic molecules with higher water solubility than PDC salt can be obtained. It can be easily removed.

<Method not to form PDC salt>
By extracting a microbial fermentation broth containing PDC with an organic solvent under acidic conditions, PDC can be isolated as free PDC. Examples of the organic solvent used in the present invention include ethyl acetate, cyclohexanone, cyclopentanone, hexane, heptane, toluene, benzene, diethyl ether, tetrahydrofuran, chloroform and dichloromethane, and ethyl acetate, cyclopentanone or cyclohexanone is preferred. Of these, ethyl acetate is particularly preferred because of its low boiling point. In order to prevent extraction of the PDC salt before extraction with the organic solvent, it is preferable to make the fermentation solution strongly acidic and wash the organic solvent after extraction with a strong acid aqueous solution. As the strong acid, hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid and the like can be used, and hydrochloric acid is particularly preferable. The strong acid used before and after extraction with the organic solvent is preferably 3 to 7%, particularly preferably 3% to 5%, based on the microbial fermentation broth.

  In order to remove the protein in the fermentation broth, a polar solvent having a relatively low boiling point such as acetone; alcohols such as methanol and ethanol may be further present in the fermentation broth before the extraction step. When alcohols are present, the acid is preferably added after the alcohol is removed.

  PDC can be obtained as a crystal by concentrating the organic layer under reduced pressure by a conventional method and recrystallizing the concentrated dried product in pure water. This PDC crystal can be further improved in purity by repeating recrystallization.

  According to this purification method which does not involve the formation of PDC salt, PDC is obtained as a complete educt with high PDC purity (99.5% to 99.9%) by one extraction operation. PDC obtained by this purification method does not contain its salt, so it can be easily dissolved in various solvents, and can be expected to be widely used as a synthetic material for polymers and chemical products.

  EXAMPLES Next, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these Examples at all.

(material)
A microbial fermentation broth containing PDC was obtained by the method described in JP-A-2005-278549.

Example 1 Purification Method to Form PDC Sodium Salt
The microorganism culture solution containing PDC (1.5 L) was centrifuged (4000 rpm, 1 hour, 4 ° C.) to remove the cells. To the obtained supernatant, 15 g of sodium chloride was added and left at 4 ° C. for 12 hours to precipitate a PDC sodium salt, which was collected by filtration to obtain a crude PDC sodium salt (22 g).
Next, 10 g of this crude PDC sodium salt was dissolved in 600 ml of pure water, adjusted to pH 1.5 with 3N HCl, and extracted with ethyl acetate (100 ml × 3 times). The ethyl acetate layer was washed with 100 mL of pH 1.0 hydrochloric acid, concentrated under reduced pressure, and crystallized at 4 ° C. The crystals were collected by filtration, dissolved in pure water, recrystallized and dried under reduced pressure at 60 ° C. to obtain 5.7 g of PDC (recovery rate 70%). The purity was 98.5% as determined by HPLC.
HPLC analysis conditions: apparatus: Waters; flow rate: 0.2 ml / min; inj .: 50 μl; column: 4.6φ × 250 mm (manufactured by Senshu Kagaku Co., Ltd .: ODS-1251-SS); mobile phase: water: acetonitrile: acetic acid = 74: 25: 1; Detection wavelength: 294 nm.

Examples 2-11
To 25 ml of pure water, 0.25 g (1.4 mmol) of PDC (free form) was added to obtain a PDC aqueous solution (0.054 mol / L). Next, the metal salts listed in Table 1 below were added to the PDC aqueous solution in the amounts shown in Table 1. Depending on the type of metal salt, some precipitated immediately after addition, some precipitated after heating, and some precipitated after concentration of water. The precipitate (or precipitate) is filtered, dried at 60 ° C overnight, and the weight of the precipitate determines the PDC based on the reported composition of sodium chloride (Na ₂ PDC ₂ H ₂ O). The precipitation rate (recovery rate) was calculated. The results are shown in Table 1.

Example 12 Purification Method for Forming PDC Sodium Salt 10 g of crude PDC sodium salt obtained in the same manner as in Example 1 was dissolved in 600 mL of pure water, and this was converted into an H-type cation having an ion exchange capacity of 2.2 meq / ml. It was passed through a column (40 mmφ) made from 100 g of exchange resin (Amberlite). The extract was washed with pure water, developed with hydrochloric acid having a pH of 1, and an effluent fraction was collected, concentrated under reduced pressure, and crystallized at 4 ° C. The crystals were collected by filtration, dissolved in pure water, recrystallized and dried under reduced pressure at 60 ° C. to obtain 7.2 g of PDC (recovery rate 88%, purity 98.5%).

Example 13 Method of not forming PDC sodium salt
1.5 L of acetone and 45 ml of concentrated hydrochloric acid were added to 1.5 L of PDC stock solution (filtrate after microbial culture) and stirred and mixed. After adding 20 g of activated carbon (037-02115, Wako Pure Chemical Industries, Ltd.) and stirring for 15 minutes, Celite (08003-02, Celite 503, Kanto Chemical Co., Inc.) was spread on # 131 filter paper and suctioned. Filtration was performed to remove activated carbon. Acetone was distilled off from the filtrate under reduced pressure, and the amount of the solution was concentrated to 1.5 L. Concentrated hydrochloric acid (18 ml) was added to the concentrated solution, and extraction was performed with ethyl acetate (230 ml x 3 times + 150 ml x 2 times). 36 g of anhydrous magnesium sulfate was added to the ethyl acetate extract, dried, suction filtered and evaporated to dryness. The obtained solid was dried under reduced pressure (55 ° C.) for about 3 hours, dissolved in 15 ml of distilled water at 60 ° C., added with 2 ml of concentrated hydrochloric acid, and left in a refrigerator for recrystallization. The solid was collected by filtration and dried under reduced pressure (55 ° C.) to obtain about 13.2 g of PDC (recovery: 68%, purity: 98.5%).
Moreover, even when the amount of the PDC stock solution was scaled up (PDC stock solution 50 L), a similar recovery rate was obtained.

FIG. 1 is a diagram showing the structure of PDC-Na ⁺ double salt. FIG. 2 is a diagram showing the water solubility of various PDC salts.

Claims (12)

A cation salt selected from monovalent, divalent, trivalent and tetravalent is present in a fermentation broth containing 2-pyrone-4,6-dicarboxylic acid produced by a microorganism. A method for purifying pyrone-4,6-dicarboxylic acid , wherein 2-pyrone-4,6-dicarboxylate precipitated in the presence of the salt is recovered, dissolved in water, and then ethyl acetate under acidic conditions. , A purification method comprising a step of extracting with cyclopentanone or cyclohexanone .   The 2-pyrone according to claim 1, wherein the salt of the monovalent cation is sodium chloride, potassium chloride, rubidium chloride, silver chloride, sodium bromide, sodium sulfate, disodium phosphate or dipotassium hydrogen phosphate. -4,6-dicarboxylic acid purification method.   The method for purifying 2-pyrone-4,6-dicarboxylic acid according to claim 1, wherein the salt of the divalent cation is magnesium chloride, copper sulfate or potassium hexacyanoferrate (II).   The method for purifying 2-pyrone-4,6-dicarboxylic acid according to claim 1, wherein the salt of the trivalent cation is iron (III) chloride or potassium hexacyanoferrate (III).   The method for purifying 2-pyrone-4,6-dicarboxylic acid according to claim 1, wherein the salt of the tetravalent cation is potassium tin (IV). The purification method according to any one of claims 1 to 5, wherein an excessive amount of strong acid is added during the extraction step. Recovered pyrone-4,6-dicarboxylate deposited by the presence of the salt was dissolved in water and then comprising the step of treating the solution with a cation exchange resin, claim 1-6 2. The purification method according to item 1. The purification method according to any one of claims 1 to 7 , wherein the salt is used in an amount of at least 2 mol per mol of 2-pyrone-4,6-dicarboxylic acid in the fermentation broth. 2-pyrone-4,6-dicarboxylic acid is converted to acetic acid from the fermentation broth containing 2-pyrone-4,6-dicarboxylic acid produced by microorganisms without forming 2-pyrone-4,6-dicarboxylate. A method for purifying 2-pyrone-4,6-dicarboxylic acid, characterized by extracting with ethyl, cyclopentanone or cyclohexanone . The purification method according to claim 9 , wherein the extraction is performed with ethyl acetate. The purification method according to claim 9 or 10 , wherein an excessive amount of strong acid is added during the extraction step. The purification method according to claim 11 , wherein the strong acid is used in an amount of at least 3% by weight based on the fermentation broth.

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