JP6217323B2 - Method for producing pyrroloquinoline quinone - Google Patents

Description

The present invention relates to a method for producing pyrroloquinoline quinone having a structure represented by the formula (1).

Pyrroloquinoline quinone (hereinafter abbreviated as PQQ) has attracted attention because it has been proposed to be a new vitamin. PQQ exists not only in bacteria but also in eukaryotic molds and yeasts, and plays an important role as a coenzyme. In addition, PQQ has been shown to promote cell proliferation, anti-cataract action, liver disease prevention and treatment action, wound healing action, anti-allergic action, reverse transcriptase inhibitory action and glyoxalase I inhibitory action-anticancer action, nerve fiber. Many physiological activities such as regenerative action have been clarified.

PQQ can be produced by a fermentation method or the like. However, PQQ obtained by these methods has a high content of water and impurities, and a technique for obtaining a stable and highly pure PQQ crystal has been demanded. In particular, since PQQ produced by the fermentation method uses microorganisms, high-molecular impurities derived from cells, crushed cells, and secretions are generated in the culture solution, and the removal thereof is a problem. In particular, there were many protein components, and the removal thereof was a problem.

In addition, heavy metals may be mixed into PQQ due to carry-in from the original medium, dissolution of apparatus materials, or the like. Protein components and heavy metal components cause problems in production such as clogging in column chromatography and life reduction as impurities during purification.

Moreover, when protein is contained as an impurity, its removal as an allergic component is also required. There has been known a method in which proteins are precipitated by lowering the pH and removed by centrifugation. (Patent Document 1). In addition, the PQQ culture solution had a low PQQ concentration and was difficult to handle.

Japanese Patent Publication No.7-131024

Conventional methods for producing PQQ have significant protein residue and are not sufficiently purified. In addition, the packing for column chromatography is expensive and the equipment is expensive. In addition, impurities other than proteins tend to remain. Therefore, there is a demand for a method that can efficiently remove impurities such as protein components and heavy metals. An object of the present invention is to provide high-purity and high-quality pyrroloquinoline quinones or salts thereof from which impurities such as protein components and heavy metals are efficiently removed, and methods for producing them.

As a result of intensive studies to solve the above problems, the present inventor has found that the following items can be solved.
[1] A method for producing high-purity pyrroloquinoline quinones, comprising a step of bringing a solution containing pyrroloquinoline quinones into contact with a resin adsorbent or activated carbon.
[2] The production method according to [1], wherein the resin adsorbent or the activated carbon has a specific surface area of 100 m ² / g or more.
[3] The production method according to [1] or [2], wherein the resin adsorbent has a styrene or acrylic skeleton.
[4] The pyrroloquinoline quinones are oxidized PQQ having the structure of the formula (1), reduced PQQ having the structure of the formula (2), or salts thereof [1] to [3] Any manufacturing method.
[5] The method according to any one of [1] to [4], wherein the solution containing pyrroloquinoline quinones is a pyrroloquinoline quinone culture solution.
[6] The production method according to any one of [1] to [4], wherein the solution containing pyrroloquinoline quinones is a solution obtained by removing a precipitate from a pyrroloquinoline quinone culture solution at pH 2 to 8.
[7] The solution containing pyrroloquinoline quinones is a solution in which the pyrroloquinoline quinone culture solution is adjusted to pH 2 to 8 to remove precipitates, and the resulting crystals are dissolved. [1] The production method according to any one of [4].
[8] The method according to any one of claims 1 to 7, further comprising the step of refining the solution containing pyrroloquinoline quinones by recrystallization or column chromatography after the step of bringing the solution containing pyrroloquinoline quinones into contact with a resin adsorbent or activated carbon. A process for producing the pyrroloquinoline quinone as described above.
[9] The production method according to claim 8, wherein in the step of purification by column chromatography, the pyrroloquinoline quinone is eliminated under basic conditions.
[10] A method for removing heavy metals from a mixed solution containing pyrroloquinoline quinones and heavy metals, which is brought into contact with a resin adsorbent or activated carbon.
[11] A pyrroloquinoline quinone or a salt thereof produced by the method according to any one of [1] to [10].

INDUSTRIAL APPLICABILITY According to the present invention, impurities such as proteins and heavy metals brought from a culture solution can be removed, and high-purity or high-quality PQQs or salts thereof, and methods for producing them can be provided. In addition, this reduces the load on the resin in column chromatography and enables the purification of more PQQs, which helps to reduce the single cost of the resin.

Example of apparatus used in an embodiment of the present invention

The present invention is a method for producing high-purity or concentrated PQQ having a purity of 60% or more, comprising a step of bringing a solution containing PQQ into contact with a resin adsorbent or activated carbon.

In the present invention, PQQs indicate oxidized PQQ having the structure of formula (1), reduced PQQ having the structure of formula (2), or salts thereof.
Examples of the salt of oxidized PQQ or reduced PQQ include alkali metal salts and ammonium salts. Specific examples of the alkali metal salt include sodium salt, potassium salt, calcium salt, ammonium salt, and lithium salt. When used in an aqueous solution, these cations can also be used in a mixed state.

The solution containing PQQs is obtained by culturing microorganisms that produce PQQs. There are no particular restrictions on the microorganism used, but for example Hyphomicrobum methylobolum, Hyhomicrobum dechitrifficans, Methylobacillus glycogenes, Methylobacterium extroques, Methylobacterium organophyllum, Methylobacterium Rhodium, Methylobacterium, Mesophyllicam, Methylobacterium radiotolerance, Hyphomicrobium bulgare, Hyphomicrobium methylobolum, Ansilobacter Accuticus, Xantobacter autotrophicas, Xantobacter flavum, Acetobacter methanolicus, Methanol-rich resources such as Paracoccus dinitrofincans, Thiobacillus novels, Oligomonas methanolica, Methylofaga marina and Methylofaga sarashica It is preferable to use microorganisms that produce PQQs.

The concentration of the solution containing PQQs is 0.01 to 30 g / L, preferably 0.5 to 10 g / L. By setting this concentration range, good productivity can be maintained without precipitation. If the concentration is low, the solution containing PQQs may be concentrated before use.
Examples of the solvent include water, ethanol, methanol and the like, and water is preferable from the viewpoint of solubility.

As the solution containing PQQs, a liquid containing bacterial cells may be used, but it is preferable to use a liquid from which bacterial cells and protein components have been removed. The amount of protein contained in the solution can be reduced, and the life of the resin adsorbent or activated carbon used in the treatment can be extended.
The protein component can be removed by adjusting the pH to 2-8. More preferably, by adjusting the pH to 3 to 5, the protein component is precipitated, which is effective. The liquid from which the protein component has been removed is preferable for stable operation for a long time because the polymer component contained is low. Furthermore, it is preferable to carry out high purification by recrystallization operation.
Although there is no restriction | limiting in the density | concentration of the protein in the solution containing PQQs, Preferably it is 0.0001 to 10 g / L. If it is this density | concentration range, an insoluble component will not arise and piping will not clog, and it is preferable. If it is lower than this range, it can be easily removed without taking such a method.

The resin adsorbent and the activated carbon preferably have a surface area of 100 m ² / g to 3000 m ² / g. More preferably 2000 m ² / g from 500m ² / g, more preferably 1300 m ² / g from 500m ² / g. As a usage-amount, 0.1-20g per PQQ is preferable. If it is a resin adsorbent, 16-20g is more preferable. If it is activated carbon, 0.1-2g is more preferable, More preferably, it is 0.1-1g. By setting the specific surface area and the amount to be used in this range, the amount of adsorption is suitable and the purification purity is increased. Proteins are removed by adsorbing to the surface. At the same time, fatty acids, amino acids, coloring components and heavy metals contained as impurities are removed. When the surface area is smaller than this range, no adsorption occurs and the purification effect does not appear. If it is larger than this, the removal effect can be expected, but it is not generally sold, and the mechanical strength is lowered. What is necessary is just to choose an optimal shape in manufacturing processes, such as powder and a shaping | molding.

The resin adsorbent preferably has a styrene or acrylic skeleton. There is no problem even if a component for crosslinking, a halogen atom for controlling the polarity or a substituent is introduced. As commercially available resin adsorbents, Sepa beads SP207, SP700, SP825, HP20 manufactured by Mitsubishi Chemical Corporation as styrene resins, Amberlite XAD4, FPX66 from Organo Corporation, and Amberlite XAD7HP as acrylic resins are used. It can be used. Further, ion exchange resins such as Amberlite IRA958 and IR124 manufactured by Organo, or chelate resins such as Diaion CR20 manufactured by Mitsubishi Chemical Corporation can also be used.

Activated carbon is preferably either coal-based or plant-derived. Specifically, spherical white rabbits, granular white rabbits, carborafine, strong white rabbits, refined white rabbits, special white rabbits, aldenite, etc. from Nippon Enviro Chemicals can be used.

As a method of contacting, for example, a batch method can be performed by adding a resin adsorbent or activated carbon to a PQQ solution containing impurities. Moreover, a resin adsorbent or activated carbon can be packed in a column and continuously removed.
The contact time may be set in consideration of the concentration of impurities and the concentration of PQQs, and can be appropriately determined from 1 second to 1 week.

In the production method of the present invention, the pH at the time of contacting can be 2 to 11, preferably 3 to 7. More preferably, it is acidic conditions, specifically 3 to 6. By setting it within this range, the solubility is appropriate without causing the PQQs to decompose or precipitate. It is desirable to use an aqueous solution having the same pH as the rinsing liquid for extruding PQQs remaining in the voids of the resin grains and activated carbon grains.
The type of acid added for pH adjustment or rinsing is not particularly limited, but inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, perchloric acid, nitric acid, sulfuric acid, formic acid, acetic acid, propionic acid, butyric acid, Examples include organic acids such as trichloroacetic acid, methanesulfonic acid, and benzenesulfonic acid, among which hydrochloric acid is preferable.
The type of base added for pH adjustment is not particularly limited, but sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, ammonia, ammonium carbonate, choline, tetramethyl Ammonium hydroxide can be used.

The amount of protein contained in the solution after the contact is low, and subsequent purification can be performed by general column chromatography or recrystallization.
As column chromatography, for example, DEAE-Sephadex (diethylaminoethyl-based anion exchange resin-Sephadex, hereinafter the same as that of Pharmacia, registered trademark) A-25 column, followed by elution with KCl solution (M. Ameyama et al , Agric.Biol.Chem., Vol. 48, p.561-565 (1984)). The PQQs can be adsorbed using column chromatography, the pyrroloquinoline quinones can be eliminated under basic conditions, and the PQQs can be purified and concentrated. Further, a method of lowering the solubility by salting out using sodium chloride, potassium chloride, sodium sulfate or potassium sulfate, or a method of depositing crystals by adding a poor solvent such as ethanol, methanol, propanol or acetonitrile can be used. Alternatively, a method in which the pH is changed in a suspended or solution state to adjust the solubility for precipitation can also be used.

In the present invention, PQQs can be concentrated by appropriately changing conditions such as pH at the time of contact. PQQs can be adsorbed to a resin or the like by bringing the pH of the PQQ solution into a range of 2-5 and bringing the PQQ solution into contact with the solubility lowered. The acid used for pH adjustment is not particularly limited, but inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, perchloric acid, nitric acid, sulfuric acid, formic acid, acetic acid, propionic acid, butyric acid, trichloroacetic acid, methane Examples thereof include organic acids such as sulfonic acid and benzenesulfonic acid, among which hydrochloric acid is preferable.
Thereafter, it is desorbed by washing under basic conditions using a solution containing an alkaline solution or an organic solvent, and a PQQ concentrate having a concentration of 2 g / L or more can be produced. The alkaline solution used for elution is not particularly limited, but sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, ammonia, ammonium carbonate, choline, tetramethyl Ammonium hydroxide can be used. By performing this process, it is possible to improve crystal purity and process yield in the subsequent crystallization process.

The present invention can further improve the purity or concentrate PQQs by combining a plurality of activated carbons or resin adsorbents. The type of resin used in the second stage is not limited, and the same resin or different resins may be used in the first stage and the second stage.

The present invention can also remove heavy metals. Examples of heavy metals include cadmium, chromium, copper, iron, manganese, and nickel. Since these metals are toxic when they enter the body, it is desirable to remove them. If the heavy metal concentration before treatment is in the range of 20 to 5000 ppb, it is easy to remove. The removal of heavy metals can be carried out in the same manner as in the production method for improving or concentrating the purity of PQQs. In particular, the pH when contacting with activated carbon or a resin adsorbent is preferably 4 to 8.

The high-purity PQQs obtained by the present invention can be used as active ingredients such as pharmaceuticals, functional foods, cosmetics, and feeds. That is, it can be provided in the form of external preparations for skin, injections, oral preparations, suppositories, etc., or in the form of foods and drinks for daily consumption, nutrition-enhanced foods, various hospital foods and the like. In addition, as an additive used in the preparation, saccharides such as water, fructose and glucose, oils such as falling raw oil, soybean oil and olive oil, and glycols such as polyethylene glycol and polypropylene glycol are used as liquid agents. be able to. As an excipient for solid preparations such as tablets, capsules, granules, etc., sugars such as lactose, sucrose, mannitol, etc., kaolin, talc, magnesium stearate etc. as lubricants, starch, sodium alginate as disintegrants, Examples of the binder include polyvinyl alcohol, cellulose, and gelatin, examples of the surfactant include fatty acid esters, and examples of the plasticizer include glycerin and the like, but are not limited thereto. You may add a dissolution accelerator, a filler, etc. as needed.

PQQs can be used alone or in combination with other materials. Materials that can be combined include vitamin B group, vitamins such as vitamin C and vitamin E, amino acids, astaxanthin, carotenoids such as α-carotene and β-carotene, ω3 fatty acids such as docosahexaenoic acid and eicosapentaenoic acid, Although omega-6 fatty acids, such as arachidonic acid, are illustrated, it is not limited to these.

Hereinafter, the present invention will be described in more detail with reference to examples and reference examples. However, the present invention is not limited to these examples and reference examples.

In the examples, Wako reagents were used unless otherwise specified.
In this example and comparative examples, UV measurement was performed using a HITACHI U-2000 SpectropHometer.
[Analysis of PQQs]
Apparatus: Shimadzu Corporation, high performance liquid chromatography, LC-20A
Column: YMC-Pack ODS-TMS (5 μm), 150 × 4.6 mm D.
Measurement temperature: 40 ° C
Detection: Absorbance at 260 nm Eluent: 100 mM CH ₃ COOH / 100 mM CH ₃ COONH ₄ (30/70, pH 5.1)
Elution rate: 1.5 mL / min
[Protein analysis]
Bradford method The absorbance at 595 nm was measured using the Bradford reagent of Bio-rad, and the protein content was determined from the value. The calibration curve was prepared with a dilution series solution of γ-globulin.
[Calculation of PQQ purity]
The PQQ purity in the sample was calculated as follows.
PQQ purity (%) = 100 × PQQ concentration / total organic solids concentration PQQ concentration: PQQ concentration (g / L) measured by HPLC
Total organic solids concentration: sum of protein concentration (g / L) measured by Bradford method and PQQ concentration (g / L) divided by HPLC purity (%) of PQQ (g / L) [heavy metal concentration Measurement]
ICP method equipment: Varian, ICP equipment, VISTA-PRO
The cadmium concentration was determined based on the intensity of 227 nm.
The chromium concentration was determined based on the intensity of 267 nm.
The copper concentration was determined based on the intensity of 327 nm.
The iron concentration was determined based on the intensity of 238 nm.
The manganese concentration was determined based on the intensity of 258 nm.
The nickel concentration was determined based on the intensity of 230 nm.

Reference Example 1 Preparation of PQQs-Containing Solution Based on Example 1 of Japanese Patent No. 2692167, the DSM strain was cultured, and a culture solution was obtained using a 3 L jar. The obtained culture broth was centrifuged at 5000 G to remove the cells and obtain a culture broth containing PQQ.

Example 1 Batch-type activated carbon treatment of culture broth supernatant The supernatant obtained by removing cells by centrifugation of the culture solution obtained in Reference Example 1 (PQQ concentration 1.6 g / L, purity 54%, protein concentration 1.0 g / L) was added with activated carbon carborafin R (specific surface area 1000 m ² / g) of Nippon Enviro Chemicals so as to be 0.2 g / L, stirred at pH 6 to 8 for 2 hours at room temperature, and centrifuged. The PQQ concentration of the supernatant was 1.7 g / L (purity 61%), and the protein concentration was 0.78 g / L. The protein removal rate was 22%.

Example 2 Batch Type Activated Carbon Treatment of Culture Broth Supernatant As in Example 1, supernatant obtained by removing cells by centrifugation (PQQ concentration 1.6 g / L, purity 54%, protein concentration 1.0 g / L) was added with activated carbon carborafin R (specific surface area 1000 m ² / g) manufactured by Nippon Enviro Chemicals so as to be 0.9 g / L, stirred at pH 6 to 8 for 2 hours at room temperature, and centrifuged. The PQQ concentration of the supernatant was 1.6 g / L (purity 64%), and the protein concentration was 0.64 g / L. The protein removal rate was 36%.

Comparative Example 1
For the supernatant containing PQQ obtained by removing cells by centrifugation of the culture solution obtained by the same method as in Reference Example 1, the result when no activated carbon was used was Comparative Example 1. The results are shown in Table 1.
As a result, a high-purity PQQ solution with a low protein content could be prepared.

Example 3 FIG. 1 shows an apparatus for purification using a resin adsorbent. Column 1 contains protein-containing liquid from tank 3 from pump 2. The liquid that has passed through the column 1 is collected in the tank 4 as a purified liquid. Sepa beads RSP207 (manufactured by Mitsubishi Chemical, specific surface area 600 m ² / g) 20 ml (resin weight is 16 g) was used as a resin adsorbent for the column.
Sulfuric acid was added to the liquid containing PQQ obtained in Reference Example 1 to adjust the pH to 4, and the mixture was stored at room temperature for 1 day. This was filtered to remove precipitates, and diluted with pure water so that the PQQ concentration became 1.0 g / L. At this time, the protein amount was 0.13 g / L with a PQQ purity of 75%. In addition, the reason made acidic here is that the refinement | purification by a resin contact method has a higher purity on the weakly acidic conditions of pH.
A solution prepared to have an elution amount of 160 ml / h (the contact time of the liquid and the resin was 7.5 minutes) was passed through a column filled with the resin adsorbent described above at 400 ml at pH 4. The first 20 ml of the liquid eluted from the column was discarded, and thereafter recovered as a purified liquid. Finally, 40 ml of 85 ppm phosphoric acid aqueous solution was passed therethrough, and the solution eluted from the column was also combined with the purified solution.
The results are shown in Table 2. The amount of impurities was calculated by (100−purity after column treatment (%)) / (100−purity immediately before column treatment (%)) × 100.
The protein concentration was 0.005 g / L with a PQQ recovery rate of 100%. The protein removal rate was 96%, and a PQQ solution having a purity higher than that of the raw material and a much lower protein content could be produced. The total amount of impurities was also cut by 44%.

Examples 4 and 5 Purification by resin adsorbent A PQQ solution having a PQQ purity of 75% and a protein amount of 0.13 g / L was purified in the same manner as in Example 3 except that each resin shown in Table 2 was used. The results are shown in Table 2.
In Examples 3 to 5, the stock solution having a PQQ purity of 75% before treatment was improved to a purity of 80% or more. The protein most required to be removed as an impurity component has a removal rate of 80% or more, which is a very effective means.

Example 6 Concentration and purification of PQQ by two-stage purification Sepa beads RSP207 (Mitsubishi Chemical, specific surface area 600 m ² / g) was packed in a column. Next, the PQQ purified solution (PQQ concentration 1.0 g / L) obtained in Example 3 was adjusted to pH 2.5 using sulfuric acid, and the rate was 60 ml / h (the contact time between the solution and the resin was 10 minutes). 150 ml was passed through the column. When passing through the stock solution, the entire eluate was discarded. After the application, 0.4% caustic soda aqueous solution (pH 13) was passed through the column at 20 ml / h (the contact time between the liquid and the resin was 30 minutes) for 1 hour, and the first 10 ml eluate containing no PQQ was discarded. Thereafter, 43 ml of a high PQQ concentration portion of the column eluate was recovered as a purified solution. As a result, a PQQ solution having a PQQ concentration of 3.5 g / L (purity 88%) was obtained. Succeeded in improving and concentrating the purity of PQQ.
Thereafter, 100 ml of a concentrated solution (PQQ concentration 3.5 g / L, purity 88%) obtained by the same operation was taken, 6 g of sodium chloride was added, and then sulfuric acid was added to adjust the pH to 3.5. After stirring for 3 hours, PQQ precipitated. This operation resulted in a PQQ purity of 93%. Further, the PQQ reduction in this crystallization was 3%. Moreover, the quantity of the salt used was 17g with respect to 1g of obtained PQQ quantities. When salting out as described above without concentrating the PQQ concentration at 1.0 g / L, 62 g of salt is required for 1 g of PQQ. Concentration significantly reduces the amount of salt used, leading to cost reduction. Further, when salting out at the same salt concentration and pH, the amount of PQQ remaining in the salting-out supernatant does not change, so concentrating the stock solution results in an improvement in the PQQ recovery rate in the salting-out step.

Examples 7 to 11 and Comparative Example 2 Removal of heavy metal An aqueous solution (PQQ concentration 4.5 g / L, cadmium concentration 2) prepared by dissolving PQQ disodium mixed with heavy metal from equipment piping or the like and adjusting to pH 6 to 8 with 25% caustic soda. .2 ppb, chromium concentration 180 ppb, copper concentration 25 ppb, iron concentration 370 ppb, manganese concentration 210 ppb, nickel concentration 16 ppb), activated carbon carborafin R (Example 7) of Nippon Envirochemicals, synthetic adsorbent Sepabead RSP207 (manufactured by Mitsubishi Chemical) Example 8), chelate resin Diaion RCR20 (Example 9), strong basic anion exchange resin Amberlite RIRA958 (Example 10) manufactured by Organo, strong acid cation exchange resin Amberlite RIR124 (Example 11) Mix as shown in Table 3 and let stand for 5 hours. It was. Moreover, the result when it was left as it was without using activated carbon etc. was made into the comparative example 2. The results are shown in Tables 3 and 4.
From this, it was found that heavy metals could be removed from the PQQ solution containing heavy metals.

The present invention is effective in fields such as foods, pharmaceuticals, cosmetics, and feeds.

1: Column 2: Pump 3: Tank (raw material)
4: Tank (filtrate)

Claims (6)

The pyrroloquinoline quinone culture solution is adjusted to pH 2 to 8 to remove precipitates, and selected from the group consisting of oxidized PQQ having the structure of formula (1), reduced PQQ having the structure of formula (2), and salts thereof obtaining a solution containing pyrroloquinoline quinones being,
Contacting the solution with the resin adsorbent and,
Recovering the solution after contact with the resin adsorbent as a purified solution;
A process for producing high purity pyrroloquinoline quinones.
The production method according to claim 1, wherein the specific surface area of the resin adsorbent is 100 m ² / g or more.   3. The production method according to claim 1, wherein the resin adsorbent has a styrene or acrylic skeleton. The solution containing pyrroloquinoline quinones is a solution in which the pyrroloquinoline quinone culture solution is adjusted to pH 2 to 8 to remove precipitates, and the resulting crystals are dissolved. 3. The production method according to any one of 3 . Manufacturing method 4 according claim 1 comprising the step of purifying the purified solution by recrystallization or column chromatography. 6. The process according to claim 5 , wherein in the step of purification by column chromatography, the pyrroloquinoline quinone is eliminated under basic conditions.