JPH0970296A - Production of imidazopyrroloquinolines - Google Patents

Abstract

PROBLEM TO BE SOLVED: To industrially and advantageously obtain the subject compound useful as a medicine, etc., by culturing a bacterium capable of producing pyrroloquinolinequinone in a medium comprising methanol as a carbon source and reacting the culture solution with various amino acids in the presence of oxygen. SOLUTION: A bacterium (e.g. Methylobacillus glycogenes ATCC29475, etc.) capable of producing pyrroloquinolinequinone in a medium comprising methanol as a carbon source is cultured to give a culture solution containing pyrroloquinolinequinone. The culture solution is mixed with at least one of various amino acids, monomethylamine, etc., and pyrroloquinolinequinone is reacted with at least one of the various amino acids and a compound such as monomethylamine in the presence of an enzyme to readily and stably give the objective imidazopyrroloquinoline of the formula (R is H, methyl, isopropyl, OH, COOH, phenyl, imidazolyl, amino, etc.), etc., having low toxicity, useful as a medicine, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing imidazopyrroloquinolines, and more particularly to a method for producing imidazopyrroloquinolines from pyrroloquinoline quinones obtained by using bacteria. The imidazopyrroloquinolines of the present invention include 7,10-dihydro-7-oxo-imidazo [4,5,1-ij] pyrrolo [2,3-f] quinoline-1,
3,9-Tricarboxylic acid (hereinafter collectively referred to as IPQs)
And the chemical structural formula is as shown in Chemical formula 1.

[0002]

Embedded image [R is a hydrogen atom, methyl group, isopropyl group, 2-methylpropyl group, 1-methylpropyl group, carbamoyl group or hydroxyl group, carboxyl group, carbamoyl group, methylmercapto group, phenyl group, 4-hydroxyphenyl group, mercapto group Represents an alkyl group having 1 to 4 carbon atoms substituted with an imidazolyl group, an amino group, a guanidino group, a methoxy group or a phosphoric acid group.] IPQs are derivatives of pyrroloquinoline quinone (hereinafter referred to as PQQ) , An important substance that can be developed as a drug. PQQ was discovered in 1979 as a coenzyme of methanol dehydrogenase of methanol-assimilating bacteria.

[0003]

2. Description of the Related Art PQQ is present not only in bacteria but also in eukaryotic fungi, yeasts, and mammals, and plays an important role as a coenzyme. Further, until recently, a cell growth promoting action (Japanese Patent Laid-Open No. 61-58584,
No. 63-233783), anti-cataract action (Japanese Patent Laid-Open Nos. 63-41421 and 63-48215,
No. 64-29313), liver disease preventive / therapeutic action (JP-A-63-192717), wound healing action (JP-A-63-152309), antiallergic action (JP-A-63-17493). ), Reverse transcriptase inhibitory action (JP-A-63-156724, JP-A 1-2)
9313) and glyoxalase I inhibitory action-
Anti-cancer effect (JP-A-63-215628, JP-A-1)
-29313), many physiological activities have been clarified. However, PQQ has recently been shown to have nephrotoxicity (Watanabe et al., Hiroshima J. Med. Sc.
i., Vol. 38, No. 1, pp. 49-51 (1989)
), And development of a safe PQQ derivative with low toxicity and nephrotoxicity is desired.

The present inventors have conducted a nephrotoxicity and acute toxicity test on various PQQ derivatives and found that imidazopyrroloquinolines (IPQs) significantly reduce these toxicities. Then IPQ
(Corresponding to R = H in the above chemical formula 1) is PQQ
It is known to be obtained by reacting with glycine.
However, in this method, high-purity PQQ containing no impurities is used. PQQ is usually produced using bacteria. In addition to PQQ, the culture solution obtained at this time contains bacterial cells, bacterial substances such as proteins and sugars, medium components, and contaminants such as by-products. Is contained in a large amount, and it is feared that these contaminants may adversely affect the reaction, and after the fermentation production of PQQ is completed, PQQ rapidly reacts with proteins and is consumed. Therefore, in order to prevent the loss of PQQ, PQQ was rapidly separated and recovered from this culture solution, and PQQ purified as needed was used for the production of IPQ. However, separation / recovery and purification of PQQ from the culture broth require complicated steps, and the accumulated amount of PQQ in the culture broth is extremely low. More complex steps are required. In this way, a high-purity PQQ obtained by trouble
It has been a serious bottleneck in the industrial production of IPQ.

[0005]

Means for Solving the Problems As a result of various studies on methods for culturing IPQs using bacteria, the present inventors have found that PQ
Bacteria capable of producing Q are cultured in a medium containing methanol as a carbon source to accumulate PQQ in the culture solution,
Subsequently, at least one of various amino acids and monomethylamine is added to this culture solution, and PQQ and these compounds are reacted in the presence of oxygen,
The inventors have found that PQQ accumulated in a culture solution is efficiently converted to IPQs, and thus arrived at the present invention. That is, the present invention, a bacterium having the ability to produce pyrroloquinoline quinone in a medium containing methanol as a carbon source is cultured to obtain a culture solution containing pyrroloquinoline quinone, and various amino acids and monomethyl are added to the culture solution. At least one selected from amines is added, and pyrroloquinoline quinone is reacted with at least one selected from various amino acids and monomethylamine in the presence of oxygen to form imidazopyrroloquinolines. It is a method for producing quinolines.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The bacterium used in the present invention may be any bacterium as long as it has the ability to assimilate methanol and has the ability to produce PQQ extracellularly. , Representative strains of these bacteria are: Methylobacillus glycogenes ATCC 29475 (= JCM 2850
= NCIB 11375), ATCC 21275 (= JCM 2841), A
TCC 21452 (= JCM 2842), same ATCC 21961 (= JCM 2843), same
ATCC 21852 (= JCM 2840 = NCIB 11376), ATCC 2136
9 (= JCM 2844), ATCC 21958 (= JCM 2847), ATCC 219
63 (= JCM 2848), ATCC 21370 (= JCM 2849), ATCC 2
1372 (= JCM 2852), ATCC 21959, ATCC 21371 (= JC)
M 2853), ATCC 21957, ATCC 21276 (= JCM 285)
4), ATCC 21453 (= JCM 2855), ATCC 21962 (= JCM)
2856), ATCC 21704 (= JCM 2857), ATCC 21960 (= J)
CM 2858), ATCC 21439, NCIB 10508 (= JCM 285)
9), NCIB 10509, NCIB 10510 (= JCM 2860), N
CIB 10511, NCIB 10512 (= JCM 2861), NCIB 10
513, same NCIB 10514, same NCIB 10592 (= JCM 2862), same
NCIB 10593, NCIB 10594 (= JCM 2863), NCIB 10
595, NCIB 10596 (= JCM 2864), JCM 2851 (= NRRL)
B-5458), JCM 2866, FERM P-1692, FERM P-
1693, FERM P-1694, FERM P-2182, FERM P-21
84, same FERM P-2247, same FERM P-2661, same FERM P-266
2, the same FERM P-2663, the same FERM P-4036, the same FERM P-403
7, the same FERM P-4038, the same FERM P-4039, the same FERM P-404
0, same FERM P-4041, same FERM P-4042 and same FERM P-4
043 (The strain name is International Journal of Systematic
Bacteriology, Vol. 36, pp. 502-511 (19
86)));

[0007] 2. Methylophilus methylotrophus NCIB 10515 and ATCC 31226 (= NCIB 11809) belonging to the genus Methylophilus (strain name is Intern
ational Journal of Systematic Bacteriology, 37th
Vol., Pp. 446-448 (1987));

[0008] 3. Methylobacterium extroquens DSM 1337 (=
JCM 2802 = NCIB 9399), same JCM 2803 (= NCIB 10409), same
ATCC 8457 (= DSM 1340 = IAM 1081 = JCM 2804 = NCIB2879)
, ATCC 14718 (= DSM 1338 = JCM 2805 = NCIB 9133) and DSM 1339 (= JCM 2806 = NCIB 9686), Methylobacterium rodinum ATCC 14821 (= JCM 2811 = NCIB 942)
1) and NCIB 9145, Methylobacterium rodesianum NCIB 12249, ATCC 21611 (= JCM 2807), ATC
C 21612 (= JCM 2808), same ATCC 21613 (= JCM 2809), same A
TCC 21614 (= JCM 2810), NCIB 10597, NCIB 1059
8 (= JCM 2812), NCIB 10599 (= JCM 2813), NCIB 10
600 (= JCM 2814), NCIB 10601 (= JCM 2817), NCIB
10602 (= JCM2815), NCIB 10611 (= JCM 2816), ATCC
21438 (= JCM 2821) and NCIB9141, Methylobacterium Thatmani NCIB 12243, NCIB 10603 (= JCM)
2818), NCIB 10604 (= JCM 2825), NCIB 10606 (= J
CM 2819), NCIB 10607 (= JCM 2820), NCIB 10608
(= JCM 2821), NCIB 10609 (= JCM 2822), NCIB 106
10 (= JCM 2823) and NCIB 10612 (= JCM 2824), Methylobacterium organophyllum ATCC 27886 (= JCM)
2833) and NRRL B-3449 (= JCM 2834) Methylobacterium mesophyllus ATCC 29983 (= J
CM 2829 = NCIB 11561), Methylobacterium Fujisawa Ensis NCIB 12417 and NCIB 11272, Methylobacterium radiotolerance IAM 12099 (= ATCC 273
29 = JCM 2830 = NCIB 10815), same IAM 12098 (= JCM 2831)
, NCIB 9142 and NCIB 9143, Methylobacterium aminoborans JCM 8240 and Methylobacterium Espi-ATCC 35064, FERM P-4893 and F
ERM P-4894, Same as FERM P- 4895, Same as FERM P-4896, Same as F
ERM P-4897 and FERM P-9466 (strain name is Internat
ional Journal of Systematic Bacteriology, 33rd
Volume, Pages 875-877 (1983), Volume 38, No. 1.
Pp. 24-127 (1988) and 43, 504.
-Based on page 513 (1993));

4. Ansylobacter-genus Ansylobacter acupuncture ATCC 25396 (= CC
M 1786 = DSM 101 = NCIB 9271), ATCC 27068 (= DSM 33)
4), ATCC 27069, ATCC 21373 (= DSM 1106),
NCIB 10516 (= DSM 2457), DSM 2666 (= FERM P-441)
6), DSM 2667 (= FERM P-4417), DSM 2668 (= FERM
P-4418) and DSM 2669 (= FERM P-4419) and ancillobacter-espi DSM 1107, DSM 1108,
DSM 1277, DSM 2455 and DSM 2456 (the strain name is International Journal of Systematic Bacteriolo
gy, 36, 415-421 (1986));

5. Hyphomicrobium vulgare ATCC 27500 belonging to the genus Hyphomicrobium, Hyphomicrobium aesteary NCIB 11052 (= ATCC 274
83), NCIB9698 (= JCM 6889) and DSM 1564 (= AT)
CC 27488) Hyphomicrobium hollandicam ATCC 27498 and ATCC 27497, Hyhomicrobium fasilis
NCIB 10342 (= ATCC 27485 = DSM 1565), NCIB 9699,
NCIB 11053, ATCC 27484, NCIB 9697 (= ATCC
27491) and the same ATCC 27492, Hyphomicrobium
Zavarginii DSM 1566 and NCIB 9696, High Homicobilum Methyloboram IFO 14180 (= JCM 6890) and NCIB 10517 and High Homicobilum Denitroficans DSM 1869 (= NCIB 11706) (Bacterial name is B
ergey's manual of systematic bacteriology, Vol. 3,
p1895-1904, (1989) (The William & Wilkins Co., Bal
timore) and International Journal of Systematic B
acteriology, Vol. 45, pp. 528-532 (199
According to 5)];

6. Xanthobacter, belonging to the genus Xanthobacter, Autotrophicus DSM 432,
DSM 431, DSM 685, DSM 1393, DSM 1618,
DSM 2009 and DSM 2267 and Xantobacter flavus DSM 338 (= NCIB 10071) (the strain name is I
nternational Journal of Systematic Bacteriology,
Volume 28, pp. 573-581 (1978) and ibid.
Vol. 29, pp. 283-287 (1979));

7. Acidomonas methanolica JCM 6891 (= IMET 10945) and JCM 3712 (= FERM P-2664) belonging to the genus Asidomonas (strain name is International
al Journal of Systematic Bacteriology, Volume 39,
50-55 (1989));

8. Paracoccus denitrificans ATCC 17441 (= DSM belonging to the genus Paracoccus
65 = IAM 12479 = NCIB 11627), ATCC 13543 (= CCM 982)
= NRRL B-3784), ATCC 19367 (= DSM 413 = IFO13301 = N
CIB 8944 = NRRL B-3785), CCM 1396 (= DSM 415 = NCIB 9
722) and IFO 12442, and Paracoccus Alkalifils JCM 7364 (= FERM P-9282) and FERM P-
9280 and FERM P-9281 (strain name is International
Journal of Systematic Bacteriology, Volume 39, Volume 1
16-121 (1989));

9. Thiobacillus novels belonging to the genus Thiobacillus ATCC 8093 (= CCM 1077 = DSM 5
06 = IFO 12443 = NCIB 9113) and NCIB10456 (strain name is International Journal of Systematic Bacteriolo
gy, Vol. 30, pp. 225-420 (1980));

10. Methylophaga marina ATCC 35842 (= NCMB 2244) belonging to the genus Methylophaga
Methylophaga moth deer ATCC 33146 (= IAM 12458 = NC
MB 2163), NCMB 2162 (= FERM P-3622) and ATCC
33145 and Methylophaga Gas Esper FERM P-361
9, FERM P-3620, FERM P-3623 and FERM P-
3624 (The strain name is International Journal of Systemati
c Bacteriology, Vol. 37, pp. 402-406 (19
82));

11. Mycobacterium methanolica belonging to the genus Mycobacterium FERM P-8823, FER
M P-8824, same FERM P-8825, same FERM P-8826, same FERM
P-8827, FERM P-9464, FERM P-9465 and FER
MP-9497 (The mycological properties of these strains belonging to the genus Mycobacterium are described in JP-A-63-28385 and 64-64).
-51077 and 64-60371).

All the above-mentioned strains are known. Further, mutant strains obtained from these strains can also be used. The nutrient medium used for culturing these PQQ-producing bacteria needs to contain methanol as a main carbon source. Further, as a medium component, a usual nitrogen source and an appropriate amount of an inorganic substance are used. As the nitrogen source, for example, ammonium sulfate, urea, ammonium nitrate, ammonium phosphate, etc. are usually used, and as the inorganic salts, usually, for example, phosphate, magnesium salt, iron salt, and other trace metals as necessary. Salt is used. Further, when the strain used shows auxotrophy, it is necessary to add the auxotrophic substance to the medium. Further, since Methylophaga genus bacteria require NaCl for growth, it is necessary to add NaCl to the medium so as to be 2 to 4% by weight, or to use seawater as water used for preparing the medium. . Culture temperature is usually 25 to 45 ° C
Within the range, a temperature suitable for growth and proliferation for each strain may be selected. The culture pH is usually in the range of 6 to 8 and a pH suitable for growth and proliferation for each strain is selected. However, the growth pH of the bacterium of the genus Asidomonas is 2.0 to 5.5, and the growth pH of the strain of Paracoccus alcalophilus is
Is 7.0 to 10.0, so that when these strains are used, it is necessary to select a pH within these ranges. If ammonium salt is used as the nitrogen source,
Since the pH of the culture solution decreases as the cells grow, in order to maintain the pH of the culture solution at a predetermined pH during the culture period, ammonia, caustic potash, caustic soda, etc. are added, and the culture is continued. It is necessary to adjust the pH of the liquid. Of these, ammonia is particularly preferred. By carrying out aerobic culture under such conditions by aeration stirring or the like, PQQ is produced in the culture medium.

Next, at least one kind of α-amino acid and monomethylamine is added to this culture medium, and these are reacted with PQQ contained in the culture medium in the presence of oxygen to use the α-amino acid used. Obtain IPQs corresponding to amino acids or monomethylamine. Typical examples of α-amino acids used here are glycine, threonine, proline, tryptophan, alanine, valine, leucine, isoleucine, methionine, glutamic acid, phenylalanine, tyrosine, glutamine, serine, aspartic acid, lysine, histidine, arginine, asparagine. ,
For example, cysteine. Among these compounds, those having an optically active form may be any of D form, L form, or racemate. Further, as the monomethylamine, it is also possible to use monomethylamine salts such as hydrochloride.

When these compounds are added, the PQQ concentration in the culture medium is usually 10 μg to 10 g / l, preferably 10
It was when the amount reached 0 μg to 10 g / l. The amount to be added should be 1 mol times or more, preferably 5 mol times or more, more preferably a large excess, with respect to PQQ in the culture broth. In practice, the addition amount should be 0.1 g / l or more. Is preferred. In the present invention, such a culture broth is usually used as it is, but it is also possible to use it after removing part of the water content of the culture broth by concentration if necessary. The method of addition may be either a method of adding all at once or a method of sequential addition, but the above concentration is required. Generally, the pH of the reaction solution is preferably in the range of 2 to 10, and its optimum pH varies depending on the type of IPQ, but will be described in more detail later.

Practically, the reaction temperature is preferably about 20 to 100 ° C, and particularly preferably 25 to 80 ° C. The reaction time is 48 hours or less, preferably 1 to 30 hours. Also,
The dissolved oxygen concentration in the reaction solution is not particularly limited, but 0.5 to 1 ppm or more is preferable in order to complete the reaction in a short time. For that purpose, means such as aeration with air or oxygen or a mixed gas thereof, stirring, and further increasing the pressure in the reaction tank are used.

By reacting under these conditions, P
IPQs can be obtained using QQ or the like as a raw material. After the fermentation production of PQQ is completed, the time until the addition of various amino acids or monomethylamine to the culture solution is preferably as short as possible, and usually 10 hours or less, preferably 2 hours or less.

IPQ (R = H) is glycine, tryptopha
, Proline, threonine, tyrosine, serine and
Add at least one of the nomethylamines to the culture
The reaction pH is 6 to 9
Preferred. In addition, 1-methylethyl IPQ (R = CH (C
H_Three)₂) Is valine, 1-methylpropyl IPQ (R = CH (C
H_Three) CH₂CH_Three) Is isoleucine, 2-methylpropyl I
PQ (R = CH₂CH (CH_Three)₂) Is leucine, methyl IPQ
(R = CH_Three) Is alanine, 2-carboxyethyl IPQ
(R = CH₂CH₂CO₂H) is glutamic acid, 2-carbomoyl
Ethyl IPQ (R = CH ₂CH₂CONH₂) Is glutamine, 2-
Methylthioethyl IPQ (R = CH₂CH₂SCH_Three) Is methionine
To benzyl IPQ (R = CH₂-C₆H_Five) Is phenylalanine
To carboxymethyl IPQ (R = CH₂CO₂H) is asparagus
Formic acid, carbamoylmethyl IPQ (R = CH₂CONH₂) Is
Asparagine was added to 4-aminobutyl IPQ (R = (CH₂)_FourNH
₂) Is lysine and 3-guanidinopropyl IPQ (R = (C
H₂)_ThreeNHCNH₂) Is arginine, mercaptomethyl IPQ
(R = CH₂SH) is cysteine, methoxymethyl IPQ
(R = CH₂OCH_Three) Is DL-O-methylserine
Roxymethyl IPQ (R = CH ₂OPO_ThreeH₂) Is DL-O-phos
Holylserine was added to carbamoyl IPQ (R = CO₂NH₂) Is D
R is a compound of L-serinamide

[0023]

Embedded image The 4-imidazolylmethyl IPQ represented by is obtained by adding histidine to the culture medium, and the reaction pH is particularly preferably 6 to 9.

On the other hand, hydroxymethyl IPQ (R = CH ₂ OH
) Is obtained by adding serine to the culture medium and setting the reaction pH to 2 to 6, and the optimum pH is 4 to 6. When the reaction pH exceeds 6, IPQ (R = H) can be obtained. 4-hydroxyphenylmethyl IPQ (R = CH ₂ -C ₆
H ₄ OH), the reaction pH is 4 ~ by adding tyrosine to the culture solution.
The optimum pH is 6-8. When the reaction pH exceeds 8, IPQ (R = H) can be obtained.

From the reaction product solution thus obtained,
For example, the solid content such as bacterial cells is removed by an ordinary solid-liquid separation means such as filtration or centrifugation to obtain a supernatant. When IPQs are produced at a low pH such as pH 2 to 5, since the produced IPQs may be present as precipitates in the reaction product solution, the respective pH values of the reaction solution and the reaction product solution should be adjusted. It is necessary to obtain a supernatant after neutralizing the alkaline to alkaline or more and once dissolving the produced IPQs. IPQs are separated and collected from the obtained supernatant. Various methods are used for separating and purifying IPQs from the supernatant. Examples thereof include a precipitation method, a washing method, an ultrafiltration method, an extraction method, and a method using a resin carrier that adsorbs IPQs. These methods can be used alone or in combination to separate and purify IPQs.

The method for separating and purifying IPQs will be described more specifically. As the precipitation method, for example, the reaction product solution is acidified with an inorganic acid such as hydrochloric acid, sulfuric acid and nitric acid and an organic acid such as trichloroacetic acid, trifluoroacetic acid and trifluoromethanesulfonic acid,
An acidic precipitation method for precipitating IPQs, a reaction product solution containing an alkali metal salt such as sodium chloride and potassium chloride,
A salt precipitation method in which an alkaline earth metal salt such as calcium chloride and magnesium chloride is added to precipitate IPQs or a reaction product solution and a solvent precipitation method in which a solvent having a low solubility of IPQs such as acetone is mixed and precipitated. and so on. In these precipitation methods, the lower the liquid temperature,
The recovery rate of IPQs is improved. A washing method is used in which the IPQ powder obtained by the above-mentioned precipitation method or the IPQ-containing precipitate is washed with a solvent in which IPQs such as acetone, diethyl ether and acidic water are difficult to dissolve. By doing so, the purity of IPQs can be further improved. When it is necessary to remove high-molecular contaminants in the culture solution in the production of IPQs by the fermentation method, the ultrafiltration method can be used.

As an ultrafiltration method, Sephadex G-1
0 (trade name, manufactured by Pharmacia Co., Ltd.) and Toyopearl HW series (trade name, manufactured by Tosoh Co., Ltd.), a method of using a resin carrier for gel filtration, or a method of using various ultrafiltration membranes or ultrafiltration hollow fibers. Can be applied. Water, an organic solvent, and the like are used as the extractant used in the extraction method. As the organic solvent, an organic solvent having low compatibility with water is preferable, and for example, an aliphatic alcohol having 4 or more carbon atoms such as n-butanol is preferably used. In the method using a resin carrier that adsorbs IPQs, any resin carrier that can adsorb and desorb IPQs can be used without particular limitation. As a typical example of this resin carrier, in the anion exchange resin carrier, DE of polysaccharide carrier is used.
AE-Sephadex A-25 (trade name, manufactured by Pharmacia Co., Ltd.), DEAE-Toyopearl 650 (trade name, manufactured by Tosoh Corporation), which is a hydrophilic polymer resin, and Amberlyst A-21 (trade name, Rohm and Manufactured by Hearth Co., Ltd., etc. Further, in the adsorption / separation type resin carrier, hydrophobic polymer resin such as Diaion HP series (trade name, manufactured by Mitsubishi Kasei Co., Ltd.) and Amberlite XAD series (trade name, manufactured by Rohm and Haas Co., Ltd.) are used. Others include silica, octadecyl silica and alumina. Resin supports for hydrophobic chromatography include butyl-Toyopearl 650 and phenyl-
Toyopearl 650 (manufactured by Tosoh Corporation) and the like are available. I
To identify PQs, elemental analysis, nuclear magnetic resonance spectrum,
Means such as infrared absorption spectra and mass spectrometry are used. In addition, high-performance liquid chromatography can be used to quantify the IPQs.

[Acute toxicity of PQQ and IPQs and
Nephrotoxicity test] (1) Acute toxicity test for mice 1) SPF-ICR mouse Male, 5 weeks old (Charlesli)
(Made by Bar Co., Ltd.), PQQ ・ Na₂And IPQ
20, 40, 80, 160 and 200 per kg mouse
Inoculated intraperitoneally with each of mg and raised at 25 ° C for 14 days
Was. IPQs include IPQ and 1-methylpropyl I
PQ, 2-methylthioethyl IPQ and benzyl IP
Q was used. It should be noted that there were 8 animals in each group. As a result, PQ
Q ・ Na₂20 mg / kg and 40 mg / kg administration
Usus did not die, but at 80 mg dose, 5 mice, 160 mg
All 8 animals died by administration and administration of 200 mg. PQQ
Na ₂LD50 was about 70 mg / kg mouse. on the other hand,
IPQs did not kill all mice.

2) SPF-ICR mouse Male, 5 weeks old (Charles River Co., Ltd.) and IPQ mouse 1
0.1 g, 0.2 g, 0.4 g, 0.8 g, or 1.2 g per kg was intraperitoneally administered for 14 days at 25 ° C.
Reared in. In addition, one group consisted of 8 animals. IPQ0.1 ~
0.8 g did not kill all mice,
2 animals died and 6 animals died at 1.2 g. The LD50 was about 1.0 g / kg mouse. 3) SPF-ICR mouse Male, 5 weeks old (Charles River Co., Ltd.), and IPQ of 1.0 g per 1 kg of mouse.
, 1.5g or 2.0g orally,
The animals were raised at 25 ° C for 14 days. One group consisted of 8 animals. As a result, all mice did not die. From the results of 1) to 3), it can be seen that the toxicity of IPQs is significantly lower than that of PQQ.

(2) Nephrotoxicity in mice 1) Nephrotoxicity by urinalysis In the same manner as in the acute toxicity test, PQQ.Na ₂ and IP
Qs were intraperitoneally administered and mice were bred. Urine samples are collected daily for clinical examination reagents (trade name: Ulistex I).
I, manufactured by Miles Sankyo Co., Ltd.) was used to examine the glucose concentration. As shown in Table 1, although from the urine of mice treated with PQQ · Na ₂ sugar is detected, from the urine of mice receiving IPQ such sugar was not detected. That is, PQQ showed nephrotoxicity, but IPQs did not. The results are shown in Table 1.

[0031]

[Table 1]

2) Nephrotoxicity by blood test (a) In the same manner as in the acute toxicity test, PQQ.Na ₂ and IPQs were intraperitoneally administered, and mice were bred. One day after administration, the animals were fasted (water was given), and blood was collected 18 hours later to obtain serum. Glucose, urea nitrogen, and creatinine in serum are clinical test reagents (trade name: Fuji Dry Chem Slide, manufactured by Fuji Photo Film Co., Ltd.)
It investigated using. Each value is shown as an average value of 8 animals. The results are shown in Table 2.

Upon administration of PQQ.Na ₂ , a large decrease in glucose and a large increase in urea nitrogen and creatinine were observed, and nephrotoxicity was observed. On the other hand, in the administration of IPQs, the respective contents of glucose, urea nitrogen and creatinine were not so different from the case of "no administration".

[0034]

[Table 2]

(B) In the same manner as in the acute toxicity test, IPQ
150 mg, 300 mg, 400 mg or 600 mg / kg
After intraperitoneal administration, the mice were bred for 1 day. Then, they were fasted (water was given), and blood was collected 18 hours later to obtain serum.
Glucose, urea nitrogen, and creatinine in serum were examined using a clinical test reagent (trade name: Fuji Dry Chem Slide, manufactured by Fuji Photo Film Co., Ltd.). Each value is shown as an average value of 8 animals. The results are shown in Table 3.

[0036]

[Table 3] At each dose of IPQ, the accumulated amounts of glucose, urea nitrogen and creatinine were not significantly different from those in the case of "no administration". From the results of 1) and 2), P
Nephrotoxicity was observed for QQ, but no nephrotoxicity was observed for IPQs.

(3) Nephrotoxicity to rats Wistar rats Male, 5 weeks old (Charles River Co., Ltd.)
Made), PQQ ・ Na ₂And 1 kg of IPQs per rat
40 mg each intraperitoneally administered and raised at 25 ° C for 7 days
Was. In addition, one group consisted of 5 animals. As a result, PQQ ・ Na₂
Then, one animal died on the third day, and two died on the fifth day.
No rat died in feeding. PQQ / Na₂of
The acute toxicity to rats was about 40 mg / kg rat.
Was. Before administration (day 0), 1st day, 3rd day, 5th day after administration
Blood (serum) and urine were collected on day 7 and 7. serum
Urea vs Nitrogen, Creatinine, Oxal Glutamate in
Acetate transaminase (GOT) and glutamate
Fuji pyridate transaminase (GPT)
It was measured using Ichemus light. Also, gluco in urine
Ames urinalysis test strip-Uristex II
(Manufactured by Miles Sankyo Co., Ltd.). result
Is shown in the fourth.

By administration of PQQ, urea nitrogen in serum,
Creatinine and GOT and urinary glucose levels were increased. The amount was particularly high on the 1st to 3rd days of administration, and then tended to decrease. On the other hand, no change was observed in IPQ administration throughout the entire period. That is, PQQ showed renal toxicity but IPQ did not show toxicity in rats.

[0039]

[Table 4]

[0040]

The present invention will be described more specifically with reference to the following examples. Per Example 1 Pure water _{1l, (NH 4) 2 SO} 4 3g, KH 2 PO 4 1.4g
_{, Na 2 HPO 4 2.1g, MgSO} 4 · 7H 2 O 0.2g, CaCl
₂・ 2H ₂ O 30mg, FeC ₆ H ₅ O ₇・ XH ₂ O 30mg, MnCl ₂・
4H ₂ O 5 mg, ZnSO ₄ 7H ₂ O 5 mg, CuSO ₄ 5H ₂ O 0.
5 mg, thiamine hydrochloride 4 mg, calcium pantothenate 4 mg, biotin 20 μg and methanol 12 ml
Was dissolved and 200 ml of the liquid whose pH was adjusted to 7.1 was added to 1
Place in a 1-volume Erlenmeyer flask, sterilize at 120 ° C for 20 minutes,
This was used as the medium. Use the same medium as above for this
1% by volume of each culture solution of various strains (shown in Table 5) pre-cultured at 0 ° C. for 2 days was cultivated at 30 ° C. with rotary shaking to obtain a culture solution 3 days after the start of culture.

Each of these culture broths was divided into two equal parts, and glycine was added to one of them at a concentration of 0.3 g / l to adjust the pH.
Adjust to 8.5 and vortex for 24 hours at 30 ° C to IP
A Q product solution was obtained. On the other hand, 0.4 g of L-serine
The pH was adjusted to 4.0, and the reaction was carried out by shaking at 30 ° C. for 24 hours by rotating to obtain a hydroxymethyl IPQ product solution. These reaction product solutions were centrifuged to obtain a supernatant, and the amount of IPQs contained therein was measured. Table 5 shows the results.

The amount of IPQs was determined by the following high performance liquid chromatograph (the same applies to the following examples). Equipment: Shimadzu high performance liquid chromatograph Column: YMC ODS A-302 (4.6 mmφ x 1)
Developing solution: 0.1M KH ₂ PO ₄ , 0.1M HClO ₄ / CH ₃ CN: H ₂ O = 1: 9 (pH 2.5) Flow rate: 1.5 / min Detector: Shimadzu SPD-6AV UV- VIS detector (259 nm and 420 nm)

[0043]

[Table 5] Table 5 Strains IPQ accumulated amount Hydroxymethyl IPQ accumulated amount (mg / l supernatant) (mg / l supernatant) Additives Glycine (pH8.5) L-serine (pH4.0) Methylobacillus glycogenes ATCC 29475 0.1 0.1 ATCC 21275 0.1 0.1 ATCC 21372 0.1 0.1 ATCC 21371 0.1 0.1 ATCC 21704 0.2 0.1 NCIB 10510 0.4 0.3 FERM P- 4036 0.1 0.1 FERM P-4039 0.3 0.3 FERM P-2661 0.2 0.2 FERM P-2247 0.4 0.4 FERM P-2182 0.1 0.1 Methylfilth methylotrophus NCIB 10515 0.2 0.2 ATCC 31226 0.2 0.2 Methylobacterium extrusion record Enns DSM 1337 0.8 0 7 NCIB 10409 0.7 0.7 Methylobacterium Rodinamu ATCC 14821 0.7 0.7 Methylobacterium Rodeshianamu NCIB 12249 0.6 0.5 Methylobacterium Zattomani NCIB 12243 0.6 0.6 methylol Bacterium organophyllum ATCC 27886 0.5 0.5 Methylobacterium mesophyllica ATCC 29983 0.6 0.6 Methylobacterium Fujisawa ensis NCIB 12417 0.5 0.5 Methylobacterium radiotolerance IAM 12099 0. 6 0.5 Methylobacterium sp FERM P-9466 0.7 0.7 FERM P -4893 0.6 0.6 en white Arthrobacter Akyua Genetics ATCC 25396 1.3 1.2 ATCC 21373 .9 0.9 NCIB 10516 0.6 0.5 DSM 2666 0.7 0.6 Anne White Enterobacter sp. DSM 2455 0.6 0.6 Haiho micro-bi Umm vulgare NCIB 9775 1.4 1.4 Haiho micro-bi Umm Methylovoram IFO 14180 0.5 0.5 NCIB 10517 0.4 0.4 Hyphomicrobium denitroficans DSM 1869 8.2 8.1 Xantobacter Autotrophycus DSM 432 1.4 1.3 Xanthobacter flavus DSM 338 1.2 1.1 Paracoccus denitrificans ATCC 19367 2.3 2.2 IFO 12442 1.8 1.6 Thiobacillus novels NCIB 10456 2.5 2.4 Mycobacterium methanolica FERM P-8823 0.6 0.6 FER MP-9497 0.8 0.6

[0044] Example 2 pure water 1l _{per, (NH 4) 2 SO 4} 3g, KH 2 PO 4 4g, MgSO
_{4 · 7H 2 O 0.2g, CaCl} 2 · 2H 2 O 30mg, FeC 6 H 5 O 7
・ XH ₂ O 30 mg, MnCl ₂・ 4H ₂ O 5 mg, ZnSO ₄・ 7H ₂ O
5 mg, CuSO ₄ .5H ₂ O 0.5 mg, calcium pantothenate 4 mg, and methanol 12 ml were dissolved in the same manner as in Example 1 except that a medium having a pH adjusted to 4.5 was used. Methanolica JCM 6891 was cultured for 3 days to obtain a culture solution. This culture solution was divided into two equal parts, to one of which monomethylamine hydrochloride was added to 0.3 g / l, the pH was adjusted to 8.5, and the mixture was stirred at 30 ° C. for 24 hours to obtain an IPQ reaction product solution. It was To the other one, L-serine was added so as to be 0.4 g /, the pH was adjusted to 4.0, and the reaction was carried out by rotating and shaking at 30 ° C. for 24 hours to obtain a hydroxymethyl IPQ product liquid. These reaction product liquids were centrifuged to obtain a supernatant liquid, and the amount of IPQs contained therein was measured. Table 6 shows the results.

[0045] per Example 3 of pure water _{1l, (NH 4) 2 SO} 4 3g, KH 2 PO 4 1.4g
_{, Na 2 HPO 4 2.1g, MgSO} 4 · 7H 2 O 0.2g, CaCl
₂・ 2H ₂ O 30mg, FeC ₆ H ₅ O ₇・ XH ₂ O 30mg, MnCl ₂・
4H ₂ O 5 mg, ZnSO ₄ 7H ₂ O 5 mg, CuSO ₄ 5H ₂ O 0.
5 mg, 20 μg of biotin and 12 ml of methanol were dissolved and sterilized at 120 ° C. for 20 minutes, after which Na ₂ CO ₃ 1 was added.
Paracoccus Alcalophilus JCM 7364 was cultured for 3 days in the same manner as in Example 1 except that a 0% by weight aqueous solution was added aseptically and the pH was adjusted to 9.0, to obtain a culture solution. This culture solution was divided into two equal parts, and L-serine was added to one of them at 0.4 g / l to adjust the pH.
It was adjusted to 8.5 and stirred at 30 ° C. for 24 hours to obtain an IPQ reaction product liquid. On the other hand, 0.4 g / L of serine
It was added to adjust the pH to 4.0, and the pH was adjusted to 4.0.
After stirring for 4 hours, a hydroxymethyl IPQ product liquid was obtained.
These reaction product liquids were centrifuged to obtain a supernatant liquid, and IPQs contained therein were measured. Table 6 shows the results.

Example 4 (NH ₄ ) ₂ SO ₄ 3.0 g, KH ₂ PO ₄ 1.
4 g, Na ₂ HPO ₄ 2.1 g, MgSO ₄ .7H ₂ O 0.2 g, Ca
Cl ₂ · 2H ₂ O 30mg, FeC ₆ H ₅ O ₇ · XH ₂ O 30mg, MnCl ₂
4H ₂ O 5 mg, ZnSO ₄ 7H ₂ O 5 mg, CuSO ₄ 5H ₂ O 0.
In the same manner as in Example 1 except that 5 mg and 12 ml of methanol were dissolved and a medium whose pH was adjusted to 7.1 was used.
Bacteria belonging to the genus Methylophaga were cultured for 3 days to obtain a culture solution.
This culture solution was divided into two equal parts, and L-threonine was added to one of them.
0.5 g / l was added to adjust the pH to 8.5,
It stirred at 30 degreeC for 24 hours, and obtained the IPQ reaction product liquid. To the other one, D-serine was added so as to be 0.4 g / l, the pH was adjusted to 4.0, and the mixture was stirred at 30 ° C. for 24 hours to obtain a hydroxymethyl IPQ product liquid. These reaction product liquids are centrifuged to obtain a supernatant liquid, which contains IP
Qs were measured. Table 6 shows the results.

[0047]

[Table 6]

Example 5 3.0 g of (NH ₄ ) ₂ SO ₄ and KH ₂ PO ₄ were added per liter of pure water.
4 g, Na ₂ HPO ₄ 2.1 g, MgSO ₄ .7H ₂ O 0.2 g, Ca
Cl ₂ · 2H ₂ O 30mg, FeC ₆ H ₅ O ₇ · XH ₂ O 30mg, MnCl ₂
・ 4H ₂ O 5 mg, ZnSO ₄・ 7H ₂ O 5 mg, CuSO ₄・ 5H ₂ O
Dissolve 0.5 mg and 8 ml methanol, pH 7.1
Put 200 ml of the solution adjusted to 1 into a 1-liter Erlenmeyer flask,
It was sterilized at 120 ° C. for 20 minutes and used as a medium. This was inoculated with Hyphomicrobium bulgare NCIB 9775,
It was obtained by rotary shaking culture at a rotating speed of 220 rpm for 30 minutes at a rotary shaker. This culture solution was used as the seed mother liquor.

For 1 liter of pure water, (NH_Four)₂SO_Four1.0 g,
MgSO_Four・ 7H₂O 1.0g, KH₂PO_FourCulture in which 1.4g was dissolved
15 l of the ground was placed in a 30 l culture tank and sterilized. Pure water 10
FeSO per ml_Four・ 7H₂O 75mg, ZnSO_Four・ 7H₂O 1
50mg, CaCl₂・ 2H₂O 150mg, NaCl 150mg, Mn
SO_Four・ 4 ~ 5H₂O 45mg, H_ThreeBO_Three3mg, CuSO_Four・ 5H ₂O
1.5 mg, CoCl₂・ 2H₂O 1.5mg, KI 1.5mg, (N
H_Four)₆Mo₇O_{twenty four}・ 4H₂A mineral solution containing 1.5 mg O
Sterilized. The temperature of the 30-liter culture tank dropped to 30 ° C.
Then, aseptically add 10 ml of this mineral solution to the medium in the tank.
In addition, add ammonia water aseptically to add the culture solution.
The pH was adjusted to 6.8. In this culture tank, methanol
Aseptically add 150 ml and 200 ml of the seed mother liquor described above.
Aeration rate of 10 l / min and rotation speed of 300 times / min.
While keeping the temperature at 30 ° C and the pH of the culture solution at 6.8,
The culture was performed while adding ammonia water. For bacteria to grow
Therefore, although the concentration of methanol in the culture solution decreased,
Analysis of methanol in gas by gas chromatography
The concentration of methanol in the culture broth was determined to be 0.
The methanol was replenished so as to be 1 to 0.5% by weight.

By this method, the cells were cultured in 7 30-liter culture tanks for 10 days, and (1) 90 g of glycine and (2) 120 g of L-serine were added to the culture solution in each culture tank.
, (3) D-serine 120 g, (4) L-threonine 150 g, (5) D-threonine 150 g,
(6) L-proline 150 g, (7) D-proline
150 g, (8) L-tyrosine 210 g, (9) D-
Tyrosine 210 g, (10) L-tryptophan 2
40 g, (11) D-tryptophan (240 g) and (12) monomethylamine hydrochloride (75 g) were added, and the reaction was continued for 24 hours while adjusting the pH of the reaction solution to 8.5 to obtain a reaction product solution. It was Table 7 shows the amount of IPQ accumulated in the reaction product solution.

[0051]

[Table 7]

Example 6 Glycine was added to a culture solution cultivated for 10 days in 7 30-liter culture tanks in the same manner as in Example 5 except that Hyphomicrobium denitroficans DSM 1869 was used as the strain. (1) 4.5 g, (2) 9.0 g,
(3) 15.0 g, (4) 45 g, (5) 75 g,
150 g of (6) and 225 g of (7) were added respectively, and the reaction solution was obtained by further culturing for 5 hours while adjusting the pH of the reaction solution to 8.0. Table 8 shows the amount of IPQ accumulated in the reaction product solution.

[0053]

[Table 8]

Example 7 In the same manner as in Example 6, the culture of Hyphomicrobium denitrophykans DSM1869 was carried out for 10 days in eight 30-liter culture tanks. Then, the pH of the culture solution in each culture tank was adjusted to (1) pH 3.0, (2) pH 4.0, (3).
pH 5.0, (4) pH 6.0, (5) pH 7.0,
(6) pH 8.0, (7) pH 9.0 and (8) p
H1 was adjusted to 30 and glycine (30 g) was added to the culture solution in each culture tank, and the reaction was continued for 3 hours to obtain a reaction product solution. Regarding the IPQ amount in the supernatant obtained by directly centrifuging the reaction product solution in each culture tank and the above (1) to (5), the pH of the reaction product solution was adjusted to 8.5 and then centrifuged. Table 9 shows the IPQ amounts in the resulting supernatants.

[0055]

[Table 9] Table 9 IPQ accumulation amount pH (mg / l reaction product solution) (A) Centrifugation as it is (B) Centrifugation after adjusting to pH 8.5 (1) pH 3.0 12 202 (2) pH 4.0 179 235 (3 ) PH 5.0 298 296 (4) pH 6.0 293 304 (5) pH 7.0 300 300 301 (6) pH 8.0 298- (7) pH 9.0 294- (8) pH 10.0 260-

Example 8 In the same manner as in Example 6, the culture of Hyphomicrobium denitrophykans DSM1869 was carried out in eight 30-liter culture tanks for 10 days. Then, the pH of the reaction solution in each culture tank was adjusted to (1) pH 2.0, (2) pH 3.0, (3) p.
H4.0, (4) pH 5.0, (5) pH 6.0,
(6) pH 7.0, (7) pH 8.0, (8) pH 9.
(9) pH was adjusted to 10.0 and L-serine (42 g) was added to the culture solution in each culture tank, and the reaction was carried out for 5 hours to obtain a reaction product solution. The reaction product in each culture tank was centrifuged as it was, and the amount of IPQ and hydroxymethyl IPQ in the separated liquid and the pH of the reaction product were adjusted to 8.5, and then IPQ and The amounts of hydroxymethyl IPQ are shown in Tables 10 and 11.

[0057]

[Table 10] Table 10 IPQ accumulated amount pH (mg / l reaction product solution) (A) Centrifugation as it is (B) Centrifugation after adjusting to pH 8.5 (1) pH 2.0 3 20 (2) pH 3.0 5 49 (3) ) PH 4.0 751 (4) pH 5.0 11 55 (5) pH 6.0 146 149 (6) pH 7.0 258 260 (7) pH 8.0 283 − (8) pH 9.0 280 − (9) pH 10.0 258 −

[0058]

[Table 11] Table 11 Hydroxymethyl IPQ accumulation pH (mg / l reaction product solution) (A) Centrifugation as it is (B) Centrifugation after adjusting to pH 8.5 (1) pH 2.0 10 120 (2) pH 3.0 15 187 (3) pH 4.0 149 281 (4) pH 5.0 272 270 (5) pH 6.0 253 243 (6) pH 7.0 98 98 101 (7) pH 8.0 50 50 49

Example 9 In the same manner as in Example 6, the culture of Hyphomicrobium denitrophykans DSM1869 was carried out for 10 days in four 30-liter culture tanks. Then, the temperature of the culture solution in each culture tank was (1) 20 ° C, (2) 30 ° C, (3) 40 ° C,
(4) 50 ° C., (5) 70 ° C., L-threonine 4
Add 8 g to the culture solution in each culture tank to adjust the pH of the culture solution.
The reaction was carried out for 5 hours while adjusting to 8.0 to obtain a reaction product liquid. Table 12 shows the IPQ accumulation amount of the reaction product solution.

[0060]

[Table 12]

Example 10 Methylobacillus glycogenes was prepared in the same manner as in Example 6.
FERM P-2247 was cultured. 27 g of monomethylamine hydrochloride was added to the culture medium on the 4th day from the start of the culture, and the culture was further continued for 6 hours while adjusting the pH of the culture medium to 8.0 to obtain a reaction product solution. The amount of IPQ accumulated in the reaction product is
It was 220 mg / l.

Example 11 Paracoccus denitrificans ATCC 19367 was cultured in the same manner as in Example 6. 42 g of D-serine was added to the culture medium 10 days after the start of culture to adjust the pH of the culture medium to 8.
The reaction was carried out for 6 hours while adjusting to 0 to obtain a reaction product liquid. The accumulated amount of IPQ in the reaction product solution was 140 mg / l.

Example 12 Example 6 was repeated except that seawater was used as the water used for the medium.
In the same manner as in Methylofaga moth deer ATCC 33146
Was cultured. D-threonine (48 g) was added to the culture medium 10 days after the start of the culture, and the reaction was carried out for 6 hours while adjusting the pH of the culture medium to 8.0 to obtain a reaction product solution. The amount of IPQ accumulated in the reaction product solution was 163 mg / l.

[0064] per Example 13 pure water _{1l, (NH 4) 2 SO} 4 3g, KH 2 PO 4 1.4g
_{, Na 2 HPO 4 2.1g, MgSO} 4 · 7H 2 O 0.2g, CaCl
₂・ 2H ₂ O 30mg, FeC ₆ H ₅ O ₇・ XH ₂ O 30mg, MnCl ₂・ 4H ₂
O 5mg, ZnSO ₄ / 7H ₂ O 5mg, CuSO ₄ / 5H ₂ O 0.5m
g and 12 ml of methanol were dissolved, and 200 ml of the solution whose pH was adjusted to 7.1 was placed in a 1-liter Erlenmeyer flask, and 12
It was sterilized at 0 ° C. for 20 minutes and used as a medium. This was inoculated with Hyphomicrobium bulgare NCIB 9775, and
The culture broth obtained by rotary shaking culture at a rotation speed of 220 rpm with a rotary shaker at ℃ was used as a seed mother liquor.

Per liter of pure water, 1 g of (NH ₄ ) ₂ SO ₄ and MgSO _4
4 · 7H ₂ O 1g, medium 15 with the addition of KH ₂ PO ₄ 1.4 g
1 was placed in a 30-liter culture tank and sterilized. Per pure water _{10ml, FeSO 4 · 7H 2 O} 75mg, ZnSO 4 · 7H 2 O 150m
g, CaCl ₂ · 2H ₂ O 150 mg, NaCl 150 mg, MnSO _{4
· 4 ~5H 2 O 45mg, H} 3 BO 3 3mg, CuSO 4 · 5H 2 O 1.
5 mg, CoCl ₂ .2H ₂ O 1.5 mg, KI 1.5 mg, (NH ₄ ) ₆
Mineral solution of _{Mo 7 O 24 · 4H 2 O} 1.5mg were sterilized. After the temperature of the 30-liter culture tank was lowered to 30 ° C., 10 ml of this mineral solution was aseptically added to the medium in the above-mentioned tank, and then ammonia water was aseptically added to adjust the pH of the culture solution to 6.8. Adjusted to. 150 ml of methanol and 200 ml of the above seed mother liquor were aseptically added to this culture tank, and the temperature was 30 ° C. while adding ammonia water while stirring at an aeration rate of 10 l / min and a rotation speed of 300 times / min. Culture was performed while maintaining the pH of the culture solution at 6.8. As the bacteria grew, the concentration of methanol in the culture solution decreased.
The concentration of methanol in the culture solution was maintained at 0.1 to 0.5% by weight by supplementing with methanol in an amount corresponding to this decrease.

By such a method, the cells were cultured in two 30 l culture tanks for 10 days, and the p
After adjusting H to 4.0 with hydrochloric acid, (1) L-serine
120 g / 15 l and (2) D-serine 120 g / 1
While adding 5 liters respectively, and aerating and stirring with air, while adjusting the pH of the culture solution to 4.0,
The reaction was carried out at 30 ° C. for 24 hours to obtain a reaction product liquid. After adjusting the pH of the reaction product solution to 7.0, centrifugation was performed to obtain a supernatant. Hydroxymethyl IP contained in the supernatant
Table 13 shows the accumulated amount of Q.

[0067]

[Table 13]

Example 14 As a strain, hyphomicrobium denitroficans
In the same manner as in Example 13 except that DSM 1869 was used, the cells were cultured in a 30-liter culture tank for 10 days, then the pH of the culture solution was adjusted to 4.0, and 42 g of L-serine was added,
The reaction was performed. After adding L-serine, the reaction product solution was collected every time, adjusted to pH 7, and then centrifuged.
The amount of hydroxymethyl IPQ contained in the reaction product solution was measured. The results are shown in Table 14.

[0069]

[Table 14] Table 14 Hydroxymethyl IPQ accumulation amount after addition of L-serine Time (mg / l reaction product solution) 1 hour 35 2 hours 109 4 hours 177 10 hours 240 14 hours 262

Example 15 Methylobacillus glycogenes FERM P-2247 was cultured in the same manner as in Example 13 except that the strain was changed.
The culture was performed for 4 days in a 1-volume culture tank. Then, the pH of the culture solution in the culture tank was adjusted to 4.5, 42 g of L-serine was added, and the reaction was carried out for 6 hours to obtain a reaction product solution. After adjusting the pH of the reaction product solution to 7.0, centrifugation was performed to obtain a supernatant. The accumulated amount of hydroxymethyl IPQ contained in the supernatant was 200 mg per liter.

Example 16 Paracoccus denitrificans ATCC 19367 was cultured in the same manner as in Example 13 except that the strain was changed to 30.
The cells were cultured in an 1-volume culture tank for 10 days. Then, the pH of the culture solution in the culture tank was adjusted to 6.0, 42 g of D-serine was added, and the reaction was carried out for 6 hours to obtain a reaction product solution. The accumulated amount of hydroxymethyl IPQ is 11 per 1 l of the reaction product solution.
It was 0 mg.

Example 17 Methylofaga-gasa larica ATCC 33146 was cultured in a 30-liter culture tank for 10 days in the same manner as in Example 13 except that the strain was changed and seawater was used as the water for the medium.
Then, the pH of the reaction solution in the culture tank was adjusted to 4.0, and D
L-serine (42 g) was added, and the reaction was carried out at 30 ° C. for 6 hours while aerating and stirring with air to obtain a reaction product liquid. After adjusting the pH of the reaction product solution to 7.0, centrifugation was performed to obtain a supernatant. The accumulated amount of hydroxymethyl IPQ contained in the supernatant was 142 mg per liter.

Example 18 In the same manner as in Example 14, culture of Hyphomicrobium denitroficans DSM 1869 was carried out in a 30-liter culture tank to obtain 15 liter of culture solution. 200 times each of this culture solution
Add 1 ml of each to a 1-liter Erlenmeyer flask, add 0.6 g of each amino acid, adjust to pH 4, 7 or 9 with NaOH or HCl and shake at 30 ° C for 2
The reaction was carried out for 4 hours to obtain a reaction product liquid. Table 15 shows the amount of IPQs accumulated in the supernatant obtained by centrifugation after adjusting the pH of the reaction product solution in each flask to 7.0.

[0074]

[Table 15] X-IPQ: Shows IPQs corresponding to each added amino acid, and specific correspondences are as shown in Table 16 below.

[0075]

Table 16 X-IPQ added amino acids corresponding to the added amino acids X-IPQ glycine IPQ L-alanine methyl IPQ L-valine 1-methylethyl IPQ L-leucine 2-methylpropyl IPQ L-isoleucine 1-methylpropyl IPQ L-methionine 2-methylthioethyl IPQ L-glutamic acid 2-carboxyethyl IPQ L-glutamine 2-carbamoylethyl IPQ L-phenylalanine benzyl IPQ L-aspartic acid carboxymethyl IPQ L-asparagine carbamoylmethyl IPQ L-arginine 3-guanidinopropyl IPQ L-histidine 4-imidazolylmethyl IPQ L-lysine 4-aminobutyl IPQ

Example 19 The same culture broth as the PQQ-containing culture broth used in Example 18 was placed in 12 1-liter Erlenmeyer flasks, each containing 200 ml.
L-threonine, L-tyrosine, L
-0.6 g each of serine or L-cysteine was added, the pH was adjusted to 4, 7 or 9 using NaOH or HCl, and the reaction was carried out for 24 hours while shaking at 30 ° C to obtain a reaction product solution. PH of reaction product in each flask
Table 17 shows the amount of accumulated IPQs in the supernatant obtained by centrifugation after adjusting the pH to 7.0. In each reaction solution, IPQ was present in addition to IPQs corresponding to each amino acid.

[0077]

[Table 17] Table 17 No-added amino acids Reaction pH generation IPQs (mg / l) IPQ X-IPQ 1 L-threonine 4 115 342 L-threonine 7 291 13 3 L-threonine 9 311 3 4 L-tyrosine 4 11 91 5 L-tyrosine 7 45 209 6 L-tyrosine 9 157 59 7 L-serine 4 13 13 182 8 L-serine 7 262 669 L-serine 9 309 10 10 L-cysteine 45 5 5 11 L-cysteine 7 45 58 12 L-Cysteine 9 26 10 X-IPQ: IPQs corresponding to each added amino acid are shown, and specific correspondences are as shown in Table 18 below.

[0078]

Table 18 Table 18 Added amino acids and corresponding X-IPQ added amino acids X-IPQ L-threonine 1-hydroxyethyl IPQ L-tyrosine 4-hydroxybenzyl IPQ L-serine hydroxymethyl IPQ L-cysteine mercaptomethyl IPQ

Example 20 The IPQ-containing reaction solution obtained in Example 9 with the temperature of the culture solution in the culture tank set to 30 ° C. was centrifuged at 12,000 × g for 20 minutes, and IPQ was recovered using the supernatant. An experiment was conducted.
50 ml each, using HCl, pH 7.2, 6.
5, 5.7, 5.0, 4.7, 4.2, 3.7, 3.
It was adjusted to 1, 2.6, 2.1, 1.5 and 1.0, left at room temperature for 4 hours, and then centrifuged at 12,000 × g for 20 minutes to recover IPQ as a precipitate. Table 19 shows the results of high-performance liquid chromatography analysis of IPQ remaining in the supernatant. It can be seen that IPQ can be recovered as a precipitate at a pH of 4 or less.

[0080]

[Table 19]

[0081]

INDUSTRIAL APPLICABILITY According to the present invention, imidazopyrroloquinolines can be easily and stably obtained from a culture broth containing PQQ obtained by using bacteria without separating, recovering or purifying PQQ. is there.

Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location (C12P 17/18 C12R 1:32)

Claims (1)

[Claims] 1. A bacterium having the ability to produce pyrroloquinoline quinone in a medium containing methanol as a carbon source is cultured to obtain a culture broth containing pyrroloquinoline quinone, and various amino acids and monomethylamine are added to the culture broth. An imidazopyrroloquinoline is formed by reacting pyrroloquinoline quinone with at least one selected from various amino acids and monomethylamine in the presence of oxygen. Method for producing quinolines.