JPH07113022B2 - Method for purifying pyrroloquinoline quinone - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for purifying pyrroloquinoline quinone, and more specifically, a method for purifying pyrroloquinoline quinone from a crude aqueous solution of pyrroloquinoline quinone by ion exchange chromatography. Involved in

Pyrroloquinoline quinone (hereinafter referred to as PQQ) is also known as 2,7,9
-Tricarboxy-1H-pyrrolo [2,3-f] quinoline-
Also known as 4,5-dione, it was purified, crystallized, and structurally determined in 1979 as a coenzyme for methanol dehydrogenase of methanol-assimilating bacteria (SASalisbury et al., N.
ature, Volume 280, p.843 (1979)).

Furthermore, it has become clear in recent years that not only bacteria, but also eukaryotic fungi, yeasts, and mammals also have PQQs that act as enzymes. Thus, PQQ is
As a coenzyme, it activates an enzymatic reaction or a substance metabolism system, and is considered to be a substance that plays an important role as a drug.

[Problems to be Solved by Prior Art and Invention]

Conventionally, as a method for purifying PQQ from a crude PQQ aqueous solution, DE
AE-Sephadex (diethylaminoethyl-based anion exchange resin-Cephadex, the same applies below) (Farmasia Co., Ltd., registered trademark) After adsorbing on an A-25 column, elution with an O-1M (mol / the following applies) KCl solution (M. Ameyama et al., Ag
ric.Biol.Chem., Vol. 48, p. 561 to 565 (1984)), and other purification methods are not known at all. However, although PQQ is obtained as a PQQ salt by this method, this PQQ salt-containing fraction contains many impurities such as proteins, sugars and glycolipids in addition to salts such as KCl. The development of a purification method that can remove salts such as KCl and KCl is desired.

[Means and actions for solving problems]

As a result of repeated studies on the PQQ purification method, the present inventors have found that DEA
NaCl obtained by ion exchange chromatography using an anion exchanger such as E-Sephadex A-25 or
By treating the PQQ salt-containing fraction containing salts such as KCl with a specific bead-like gel, it was
The present invention has been completed by finding that salts such as Cl or KCl are also removed, the PQQ salt-containing fraction is concentrated, and the purity of the PQQ salt is significantly improved.

That is, the present invention, when purifying the crude PQQ aqueous solution by ion exchange chromatography, the PQQ salt-containing fraction obtained by ion exchange chromatography, dextran three-dimensionally crosslinked with epichlorohydrin. PQQ was obtained by passing through a column filled with bead-like gel with a bed volume of 2-3 ml per 1 g of dried product (apparent volume after 1 g of dried product was swollen by swelling in 1 g of water). A method for purifying PQQ, which is characterized in that it is recovered as a salt.

Sephadex G-10 is a typical example of a commercially available bead-like gel having a bed volume of 2-3 ml per 1 g of a dried product obtained by three-dimensionally crosslinking dextran used in the present invention with epichlorohydrin. .

As this bead-like gel, for example, Sephadex G
Water was passed through the column packed with -10 to equilibrate the inside of the column, and then the PQQ salt-containing fraction (hereinafter referred to as the fraction containing PQQ salt obtained from the crude PQQ aqueous solution by ion exchange chromatography)
The PQQ salt-containing fraction (hereinafter referred to as the PQQ salt-containing fraction) was further passed through this column to pass water through this column to elute the PQQ salt-containing fraction (hereinafter referred to as the PQQ salt-containing fraction). The latter-stage PQQ salt-containing fraction) is obtained. The PQQ salt contained in the PQQ salt-containing fraction is a salt of PQQ and the alkali metal of the salt added to the buffer solution. This treatment may be repeated once or twice or more, and preferably twice or more. When repeating twice or more, the salt concentration of KCl, NaCl, etc. in the PQQ salt-containing fraction to be passed through the column must be 0.02 M or more. The salt concentration of the PQQ salt-containing fraction of the previous round was 0.02M
When it is less than this, the PQQ salt-containing fraction needs to be concentrated before use.

There are sometimes no restrictions on various conditions such as the flow rate and the column temperature in the column of the PQQ salt-containing fraction of the first stage and the PQQ salt-containing fraction of the previous round and water. For example, the flow rate is normally allowed to flow naturally through the column, but it does not prevent pressurization from the inlet and / or depressurization from the outlet to increase the speed.

The column temperature is usually from room temperature to room temperature,
It does not interfere with cooling or heating as long as the PQQ salt and impurities do not precipitate and the beaded gel used is not destroyed.

The front-stage PQQ salt-containing fraction to which the present invention can be applied is not particularly limited, and may be the PQQ salt-containing fraction obtained by ion exchange chromatography known per se.

That is, the crude PQQ is not particularly limited in its source, manufacturing method and extraction method. The crude PQQ is used in the form of an aqueous solution, and the concentration of PQQ in this aqueous solution is not particularly limited, but is usually 0.1 to 10 mg / ml for practical use. As the ion exchanger used in this ion exchange chromatography, an anion exchanger is usually used, and as the anion exchanger, for example, aminoethyl (AE-), diethylamino ( Anion exchange resins having DEAE-) or quaternary aminoethyl (QAE-) and the like are preferable. Typical commercial products of these anion exchange resins include, for example, DEAE-Sephadex.
A-25, DEAE-Sephadex A-50, DEAE-Sephacel, DEAE-Sepharose G
L-6B, QAE-Sefadex A-25, QAE-Sefadex A-50 (above, a product of Pharmacia), DEAE
-Serophane (a product of Chitso Co., Ltd.) and DEAE-Toyopearl 650 (a product of Toyo Soda Co., Ltd.). Of these, DEAE-Sephadex A-25, DEAE-Sepharoin, DEAE-Toyopearl and the like are preferable. Further, the shape of the ion exchange resin may be any of granular, spherical, porous granular, fine granular and film-like.

A buffer solution having a pH of 6 to 8 is allowed to flow down from the top of the column to equilibrate the inside of the column packed with these ion exchangers, and then a crude PQQ aqueous solution is allowed to flow down from the top of the column to PQ.
Adsorb. Next, a buffer solution containing a salt such as NaCl or KCl is allowed to flow down from the top of the column to develop PQQ,
Elute. The salt concentration may be any in the range of 0.1 to 2M, but the point that the developing solution amount can be reduced is 0.
2-1.5M is preferred. Further, a temperature gradient elution method in which a salt is added to a buffer solution and the concentration of the salt is sequentially changed to O-1M is preferable because it has excellent separability from impurities and can reduce the amount of developing solution. The buffer solution used for this ion exchange chromatography is not particularly limited, but, for example, a phosphate buffer solution and a Tris-hydrochloric acid buffer solution are usually used. Further, it is preferable that the buffer used for equilibration and the buffer used for PQQ development and elution have the same kind and pH.

The PQQ salt fraction thus obtained contains a large amount of organic impurities as well as salts. In order to remove this large amount of impurities, it is preferable to apply the method devised by the present inventors. That is, after passing the crude PQQ aqueous solution through the column to adsorb PQQ on the ion exchanger, and before developing and eluting the adsorbed PQQ with a salt-added buffer, the column is buffered. It is a method of washing with a liquid in advance. The buffer used for this washing may be a buffer having a pH of 2 to 10 among ordinary buffers used in ion exchange chromatography, but the same buffer used for equilibration is used. Preferably.

The buffer solution for this washing is usually the same as above.
For example, phosphate buffer and Tris-HCl buffer are used. The amount and supply rate of the buffer solution are not particularly limited, but in practice, the amount used is about 5 to 20 times the apparent volume of the filled anion exchanger, and the supply rate is the buffer solution. Although it may be allowed to flow down naturally in the column, it may be pressurized from the top of the column or sucked from the bottom of the column to accelerate, and in practice, it is usually 200 ml to 3 / hr per 100 ml of apparent volume of the packed anion exchanger. It is said that The temperature at the time of washing is not particularly limited, and may be room temperature to room temperature for practical use, and it is not particularly necessary to cool or heat, but the temperature at which the PQQ salt does not precipitate in the column or the anion exchanger has adsorption ability. As long as the temperature is not lost, cooling and heating can be performed.

〔Example〕

The present invention will be described in more detail with reference to the following examples. The present invention is not limited to these examples.

Example 1 A culture solution obtained by culturing Xanthobacter autotrophicus DSM431 was centrifuged to remove the bacterial cells, and a culture supernatant was obtained. The culture supernatant was purified. DEAE-pre-equilibrated with 2 mM potassium phosphate buffer (pH 7.0)
The culture supernatant 10 was adsorbed to a column (packing amount 2.6 × 10 cm) packed with Toyopearl 650 (Toyo Soda). After that, KCl was added to 2 mM potassium phosphate buffer to add O-1MKC.
Elute with a concentration gradient of l (1.2) to obtain the PQQ salt (PQQ-K
200 ml of a salt-containing fraction was obtained. In the fraction containing PQQ salt
The amount of PQQ (PQQ salt is displayed as PQQ in the analysis is the same below) is 36 mg, KCl concentration is 0.8 M, and P excluding salts is P.
The QQ purity was 68%.

PQQ content and PQQ purity were determined by high performance liquid chromatography (same for other crude PQQ aqueous solutions, other PQQ salt-containing fractions and other Examples).

Instrument: Shimadzu High Performance Liquid Chromatograph Column: Nucleosil 5C ₁₈ , 4.0 x 15
0mm Developing solution: H ₃ PO ₄ (85wt% aqueous solution) / H ₂ O / CH ₃ OH ＝ 0.4 / 72.6 / 2
7 (Volume ratio) Flow rate: 0.7 ml / min Detector: Shimadzu SPD-6A UV spectrophotometer (259 nm) Sephadex G-10 column (packing amount 2.6 x 100 cm) previously equilibrated with pure water 200 ml of PQQ salt-containing fraction
Is adsorbed, eluted with pure water, and the latter PQQ salt-containing fraction 32 ml
Got The amount of PQQ in this latter-stage PQQ salt-containing fraction was 37 mg, and the KCl concentration was 0.07M. The PQQ purity was 96%.

Further, 32 ml of the latter-stage PQQ salt-containing fraction was adsorbed to Sephadex G-10 (manufactured by Pharmacia) Kara (2.6 × 100 cm) that had been equilibrated with pure water, and eluted with pure water to contain PQQ salt. A fraction of 25 ml was obtained. The amount of PQQ in this fraction was 37 mg, and the KCl concentration was 0.02M. The PQQ purity was 99.0%.

Example 2 A culture supernatant was purified from a culture solution obtained by culturing Ancylobacter aquatex ATCC 25396. 2m
DE equilibrated with M potassium phosphate buffer (pH 7.0)
AE-cellophane AL (Chitsuso) column (packing amount 2.
The culture supernatant 10 was adsorbed on 6 × 10 cm). Then 2mM
Add NaCl to the potassium phosphate buffer to add O-0.5M NaCl.
Elute with the concentration gradient of (1.2), and use the PQQ salt (PQQ-Na
300 ml of a salt-containing fraction was obtained. In the fraction containing PQQ salt
The PQQ amount is 32 mg, the NaCl concentration is 0.4 M, and the PQQ purity is
It was 66%.

Add the above-mentioned PQQ to the Sephadex G-10 (manufactured by Pharmacia) column (packing amount 2.6 x 100 cm) equilibrated with pure water.
300 ml of the salt-containing fraction was adsorbed and eluted with pure water to obtain 40 ml of the latter-stage PQQ salt-containing fraction. The amount of PQQ in this latter-stage PQQ salt-containing fraction was 31 mg, and the NaCl concentration was 0.04M. The PQQ purity was 97%.

Example 3 The culture supernatant obtained from the culture obtained by culturing Hyphomicrobium sp. DSM 1869 was purified. DEAE-S equilibrated with 2 mM potassium phosphate buffer (pH 7.0)
ephadex A-25 (made by Pharmacia) column (packing amount
The culture supernatant 10 was adsorbed on 2.6 × 10 cm).

After that, 600 ml of 2 mM potassium phosphate buffer (pH 7.0) was applied to the column to wash the inside of the column, and then KCl was added to the 2 mM potassium phosphate buffer to elute with a concentration gradient of o-1M KCl (1.2). Then, 300 ml of a fraction containing the former PQQ salt (PQQ-K salt) was obtained. The amount of PQQ in this pre-stage PQQ salt-containing fraction was 58 mg,
The KCl concentration was 0.8M, and the PQQ purity was 91%.

The Sephadex G-10 (manufactured by Pharmacia) column equilibrated with pure water (packing amount: 2.6 x 100 cm) was added to the above-mentioned PQ.
Adsorb 300 ml of Q salt-containing fraction and elute with pure water.
A 42 ml fraction was obtained. The amount of PQQ in this fraction was 57 mg, and KCl
The concentration was 0.056M and the PQQ purity was 99%.

Furthermore, the Sephadex G-10 (manufactured by Pharmacia) column (packing amount 2.6 × 100 cm) that had been equilibrated with pure was adsorbed with 42 ml of the fraction containing the PQQ salt of the last stage of the previous step and eluted with pure to obtain 35 ml of the PQQ fraction. . The amount of PQQ in this fraction was 57 mg, and KCl
The concentration was 0.02M. The PQQ purity was 99.5%.

Effects of the Invention According to the present invention, not only organic impurities but also NaCl and
Salts such as KCl are also removed, and a fraction containing PQQ salt in which the concentration of both organic impurities and salts is low is obtained, and the burden of subsequent purification is significantly reduced.

However, this PQQ salt has the same drug efficacy as PQQ.

Claims (1)

[Claims] 1. When a crude pyrroloquinoline quinone aqueous solution is purified by ion exchange chromatography, the pyrroloquinoline quinone salt-containing fraction obtained by ion exchange chromatography is dextran three-dimensionally with epichlorohydrin. Pyrroloquinoline quinone is recovered as a pyrroloquinoline quinone salt by passing through a column packed with beaded gel having a bed volume of 2-3 ml per 1 g of dried product, which is obtained by cross-linking How to purify quinone.