JPH0479890A - Recovery and purification of pyrroloquinolinequinones - Google Patents

Abstract

PURPOSE:To recover and purify the title high-purity quinolines useful for drugs, etc., readily and on an industrial scale by prescribing pH of a culture solution or a supernatant of culture in a specific range and removing impurities. CONSTITUTION:In using a bacterium capable of assimilating methanol and producing pyrroloquinolinequinone, the bacterium is multiplied in a medium containing methanol as a main carbon source, pH of the culture solution having formed pyrroloquinolinequinone or a supernatant liquid of culture prepared by wholly or partially removing bacterium cells from the culture solution is made 3.0 to 4.5 and impurities are removed to recover and purify the objective quinolines.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for producing pyrroloquinoline quinones, and more particularly to a method for recovering and purifying pyrroloquinoline quinones produced using bacteria. Involved.

Pyrroloquinoline quinones (hereinafter referred to as PQQs) are:
It is present not only in bacteria but also in the tissues of eukaryotic molds and yeasts, as well as mammals, and plays an important role as a coenzyme for dehydrogenases and oxidases. Furthermore, in recent years, cell proliferation promoting effects (for example, JP-A No. 61-58584, JP-A No. 63-233783)
(Japanese Patent Application Laid-Open No. 63-41421, Japanese Patent Application Laid-open No. 63-41421,
-48215 Publication, Japanese Patent Application Laid-Open No. 1988-6429313),
Preventive and therapeutic effects on liver diseases (e.g. JP-A-63-1927)
No. 17), wound healing effect (for example, JP-A-63-1
52309), anti-allergic action (for example, JP-A-63-174931), reverse transcriptase inhibitory action (
For example, JP-A-63-156724, JP-A-01
Many physiological activities have been revealed, including glyoxalase (1) inhibitory action and anticancer action (for example, JP-A-63-215628 and JP-A-01-239313). It has also been shown that it has a vitamin effect, and is considered to be a substance that plays an important role as a medicine.

[Prior art, problems to be solved by the invention] Conventionally,
As a method for recovering and purifying PQQs from a culture solution containing PQQs, bacterial cells are removed from the culture solution by a method such as centrifugation, and the culture supernatant is purified using DEAE-3epha.
A method of passing through a dex (diethylaminoethyl anion exchange resin - Sephadex - A25 Pharmacia, trade name) column to adsorb PQQs on this column, and then eluting the PQQs with a 0 to IM KCI solution ( M., Ameyama et al., Agric
Biol, Chem, Volume 48, Page 561~
565 pages (1984) is known.

However, with this method, the purity of the PQQs obtained is low due to impurities such as proteins contained in the culture supernatant, and in order to obtain PQQs with high purity, a large amount of DEAE-3 ephadex resin is required. Therefore, there is a need to develop an effective method for removing organic substances other than PQQ from the culture solution.

[Means and actions for solving problems]

As a result of repeated research on methods for recovering and purifying PQQs from culture fluids, the present inventors found that the pH of culture fluids or culture supernatants from which all or part of the bacterial cells have been removed is
By setting the value to 3.0 to 4.5, impurities such as proteins contained in the culture solution or culture supernatant become precipitates.
The present invention has been completed based on the discovery that impurities such as proteins can be removed by removing this by centrifugation or filtration, making it easy to recover and purify PQQs.

That is, the present invention has methanol assimilation ability, and
In a method for producing pyrroloquinoline quinones using bacteria capable of producing pyrroloquinoline quinone,
A culture solution in which the bacteria are grown in a medium containing methanol as a main carbon source to produce pyrroloquinoline quinones,
Alternatively, the collection of pyrroloquinoline quinones is characterized by removing all or part of the bacterial cells from the culture solution, adjusting the pH of the culture supernatant to 3.0 to 4.5, and removing impurities.
This is a purification method.

PQQ in the present invention means PQQ salts such as PQQ, sodium salt of PQQ, and potassium salt of PQQ.

Examples of bacteria that have the ability to assimilate methanol and produce PQQ include Methylobacillus glycogenes, Methylobacterium extroquens,
Methylobacterium organophyllum, Methylobacterium robium, Methylobacterium mesophilicum, Methylobacterium radiotolerance, Hyphomicrobium pulgare, Hyphomicrobium methyloborum, Ancylobacter acuaticus, Xanthobacter autotrophicus , Xanthobacter flavus, Acidomonas methanolica, Paracotcus denitrificans, Baracotcus alkalinephyllus, Thiobacillus novelus, Methylophaga marina, and Methylophaga salicica, and these bacteria can be cultured in a medium using methanol as a carbon source. In addition to PQQ, the culture solution contains a relatively large amount of impurities consisting of organic substances such as proteins.

The pH of the culture solution itself containing bacterial cells is 3.0-4°5.
By doing so, impurities such as contained proteins will precipitate together with the bacterial cells, so they can be removed by means such as centrifugation or filtration, and a PQQ-containing liquid free of impurities such as bacterial cells and proteins can be obtained. I can do it. However, if the culture solution containing bacterial cells itself is used, a large amount of precipitate will be produced, and PQQs will also be mixed into the precipitate.
There are problems such as a decline in the QQ collection rate. Therefore, it is particularly preferable to obtain a culture supernatant from which all or part of the bacterial cells have been removed in advance, adjust its pH to 3.0 to 4.5, and remove impurities such as proteins.

The concentration of PQQs in the culture solution or culture supernatant is not particularly limited, but if the concentration of PQQs is high, the recovery rate of PQQs will be low, so it is preferably 1 g/Il or less,
Particularly preferred is 800 mg/V or less. When using a culture solution or culture supernatant containing PQQs in a larger amount than these values, it is necessary to add water so that the concentration of PQQs is below these values.

Conversely, if the PQQ concentration is below these values, concentration processing or the like may be performed.

If the pH of the culture supernatant exceeds 4.5, the amount of protein removed will decrease, while if the pH is less than 3.0, the protein removal rate will be good, but the PQQ recovery rate will be poor. Therefore, the pH of the culture supernatant is set between 3.0 and 4.5.From the viewpoint of protein removal rate and PQQ recovery rate, pH 3.5 to 4.0 is particularly preferred.

In order to adjust the pH of the culture solution or culture supernatant to 3.0 to 4.5, acidic substances such as acids such as hydrochloric acid and sulfuric acid and inorganic acid salts such as sodium bicarbonate or acidic calcium phosphate are added. Among these, acids are preferred, and hydrochloric acid is particularly preferred.

The PQQ-containing liquid obtained according to the present invention was applied to DEAR-3e
phadex column treatment, 5ephadex G-10
Column treatment, high porous polymer column treatment (Japanese patent application No. 62-232296), salting-out treatment (Japanese patent application No. 63-2
16317>, etc., it is possible to easily obtain PQQs with good purity at a high recovery rate.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example l Hyphomicrobium methyloborum DSM 186
9 (Urakami and Komagata, J.
, Gen, Appl, Microbiol, Vol. 33, pp. 521-542, (1987)), the resulting culture solution was centrifuged to remove bacterial cells,
A culture supernatant containing PQQ was obtained. This culture supernatant contained about 600 μg/m of PQQ and about 2 g/l of protein.

This culture supernatant was divided into 10 groups of 100 ml each, and the pH of each group was adjusted to 6.0 to 1.5 by adding hydrochloric acid. After 4 hours of adjustment, the mixture was centrifuged at 12,000 rpm for 20 minutes to remove the precipitate, and the PQQ concentration and protein concentration in the supernatant were analyzed. PQQ was analyzed by high performance liquid chromatography, and protein was analyzed by the Lowry method. The results are shown in Table 1.

Table Example 2 Hyphomicrobium Methyloborum DSM 1869
The culture solution obtained by culturing was centrifuged to remove the bacterial cells and obtain a culture supernatant containing PQQ. This culture supernatant contained 433 μg/l of PQQ and 1.350 g of protein.
g#' was contained.

HCI was added to this culture supernatant 21 to adjust the pH to 3.5.
Adjusted to. 12. After 4 hours of adjustment. OOOrpm 2
The mixture was centrifuged for 0 minutes, the precipitate was removed, and a PQQ-containing solution was obtained. This PQQ-containing solution contained 348 μg/l of PQQ,
After adding 120 g of NaCl to the PQQ-containing solution containing 38 μg/l of protein and dissolving it,
HCI was added to adjust the pH to 2.5, and after standing at 5°C for 24 hours, centrifugation was performed at 12.00 rpm for 20 minutes to obtain a precipitate. This precipitate was freeze-dried to obtain 1.42 g of PQQ-containing powder. This powder contains 697m of PQQ・2Na.
g, 587 mg of NaCl, 9 mg of protein,
It contained 114 mg of water and 13 mg of other substances.

Comparative Example 1 Culture supernatant 21 containing 433 μg/l of PQQ and 1.350 gg/V of protein obtained in Example 2
After adding 120g of NaCl and dissolving it, HCI was added to adjust the pH to 2.5, and after leaving it at 5°C for 24 hours,
2. The mixture was centrifuged at OOOrpm for 20 minutes to obtain a precipitate.

This precipitate was freeze-dried to obtain 4.27 g of PQQ-containing powder.
Obtained. This powder contains 750 mg of PQQ 2Na,
700mg of NaCl, 2.5g of protein, 220g of water
mg, and 100 mg of others.

〔Effect of the invention〕

The present invention makes it possible to easily recover and purify PQQ, which has an extremely large industrial advantage.

Claims (1)

[Claims] In a method for producing pyrroloquinoline quinones using bacteria that have the ability to assimilate methanol and produce pyrroloquinoline quinone, the bacteria are grown in a medium containing methanol as the main carbon source, pH of the culture solution that produced quinoline quinones or the culture supernatant after removing all or part of the bacterial cells from the culture solution
3.0 to 4.5 and removing impurities.