JPS6212792B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a new substance, cyclic imino acid, which has strong mutation-inhibiting activity, and a method for producing the same. The present inventors have systematically identified mutation inhibitors (hereinafter referred to as inhibitors) in the metabolites of microorganisms in Journal of Antibiotics, Vol. 32, p. 1118 (1979). ), Favolus brasiliensis (Fr.) Fr.
brasiliensis (Fr.) Fr. (Japanese name: Minamia Shigurotake)] From the culture solution of K2013 strain, the general formula (In the formula, n is 2 or 3) We collected two types of inhibitors, and based on their physical and chemical properties and X-ray crystal diffraction, we determined the structure of the substance using the formula 5-cyanoproline (abbreviated below) and the formula 6-cyanopipecolic acid (abbreviated as below)
It was determined that these compounds are new compounds. The cyclic imino acid obtained by the present invention has the property of inhibiting the mutation of microorganisms such as bacteria, and is therefore useful for use in microorganisms such as antibiotics, physiologically active substances, enzymes, etc.
Preventing the decline or loss of production capacity due to mutations in microorganisms that have the ability to produce amino acids, etc.
It is a substance that is effective in retaining useful properties,
Furthermore, it is expected to be useful as a preventive agent for carcinogenesis due to its ability to inhibit tumorigenesis of chicken fetal fibroblasts caused by chicken sarcoma virus. The physicochemical properties of the cyclic imino acid obtained by the present invention are as shown below. is obtained as colorless needle-shaped crystals, which gradually turn brown and decompose at temperatures above 160°C, but do not melt. Soluble in water but almost insoluble in organic solvents. The pKa′ was observed to be 2.3 and 5.5. The specific optical rotation is [α] ²⁶ _D = -93° (C1.0, H ₂ O). It is positive in color reactions such as ninhydrin reaction, Lydon-Smith reaction, and tetrazolium reaction, and also decolorizes potassium permanganate. Ultraviolet absorption shows only terminal absorption. The infrared absorption curve is as shown in Figure 1, with wave numbers of 3450, 3060, 2950, 2250, 2100, 1620,
1500, 1445, 1390, 1320, 1285, 1150, 1060,
The main absorption bands are shown at 1025, 940, 880, 760 and 675. The elemental analysis values were as follows. Calculated value as C ₆ H ₈ N ₂ O ₂ :
C 51.42%, H 5.75%, N 19.99% Actual values:
C 51.54%, H 5.77%, N 19.76% The mass spectrum is as shown in FIG. From these data, the molecular formula C ₆ H ₈ N ₂ O ₂ was determined,
The result of X-ray crystal diffraction determined it to be 5-cyanoproline. is obtained as colorless needle-shaped crystals that gradually turn brown at temperatures above 160°C but do not melt. Soluble in water but almost insoluble in organic solvents. The specific optical rotation is [α] ²⁶ _D
= −45° (C1.0, H ₂ O). In color reactions, it is positive for ninhydrin reaction, Lydon-Smith reaction, and tetrazolium reaction, and also decolorizes potassium permanganese. Ultraviolet absorption shows only terminal absorption. The infrared absorption curve is as shown in Figure 3, with wave numbers of 3430, 2950, 2700, 2500, 2100,
1625, 1590, 1440, 1405, 1330, 1305, 1285,
1195, 1165, 1120, 1090, 1050, 1030, 985,
The main absorption bands are shown at 920, 800 and 780. The elemental analysis values were as follows. Calculated value as C ₇ H ₁₀ N ₂ O ₂ :
C 53.84%, H 6.35%, N 18.12% Actual values:
The mass spectrum of C 54.12%, H 6.45%, N 18.17% is shown in FIG. From these data, the molecular formula C ₇ H ₁₀ N ₂ O ₂ was determined,
The result of X-ray crystal diffraction determined it to be 6-cyanopepicolinic acid. An example of the cyclic imino acid producing bacterium used in the present invention is Fabolus brasiliensis (Fr.) Fr.
(Japanese name: Minamia Shigurotake) There is K2013 strain.
This bacterial strain was collected and cultured in Okinawa Prefecture in 1975. The fruiting bodies of this strain are stalked (rarely stalkless), umbrella-shaped, and occur singly or in clusters. Fleshy when fresh, but becomes corky and brittle when dried. The stems are corky, pale yellow-pink to pink-brown, and smooth with no ring marks. The flesh is pink-yellow, corky, and about 2 inches thick.
mm, the surface of the fruiting layer is pale yellowish brown, the pores are large, 1~
3μ×0.5~1.5μ. The pore walls are thin. The basidium is club-shaped, 5-7μ wide. The basidiospores are colorless and transparent, thin film, long oval shape, with 1-2 oil droplets inside, and the size is 8-10μ x 3-4.5μ. Based on the mycological properties mentioned above, this strain has been classified as "Indian Polypores" by Bakshi.
Indian Agriculture Research (1970)
The strain was identified as Phaebolus brasiliensis (Fr.) Fr., which was described on pages 21-22 of [Research], and was given the strain number K2013. This strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology for storage on March 22, 1980, and its deposit number is No. 5458. In carrying out the method for producing a cyclic imino acid of the present invention, the substance-producing bacteria belonging to the genus Fabolus is aerobically cultured using a known cultivation method for mushrooms, and the target substance is produced in the culture. Bye. An example of a producing bacterium is Huabolus brasiliensis (Fr.
) The culture method when using the Fr.K2013 strain is explained below. In the present invention, although a solid culture method can be used as a production culture method, it is advantageous to use a liquid culture method for mass production. As a source of inoculation,
The mycelium grown by the K2013 strain grown on a potato agar medium may be used as is, but it is better to inoculate the production medium, culture it statically for 2 to 20 days, wait for the growth of the mycelium, and then culture it with shaking. It is preferable to use it as an inoculum source. The K2013 strain grows at 15-45°C, but the culture temperature is preferably 25-30°C. As the nutrient source for the medium, any nutrient source known for culturing mushrooms and other microorganisms that can be used by the K2013 strain can be used. As the carbon source, for example, glucose, maltose, sutucarose, dextrin, starch, etc. can be used. Furthermore, vegetable oils such as soybean oil and animal fats such as lard are used not only as antifoaming agents but also as carbon sources. As the nitrogen source, for example, peptone, meat extract, dry yeast, yeast extract, soybean flour, cottonseed flour, corn steep liquor, rice bran, and inorganic nitrogen can be used. In addition, inorganic salts such as potassium dihydrogen phosphate and magnesium sulfate, and trace amounts of metal salts may be used as necessary. The culture conditions may be those normally used for culturing microorganisms, and the culture time may be selected such that the cyclic imino acid is substantially accumulated. For example, when strain K2013 is inoculated into a medium containing 2% glucose, 0.5% peptone, 0.3% yeast extract, 0.3% potassium dihydrogen phosphate, and 0.1% magnesium sulfate heptahydrate, and cultured aerobically at 28°C. The culture time was 3 to 6 days. The bacterial cells are removed from the culture solution by means such as filtration or centrifugation, and the obtained culture solution is used as a material for collecting cyclic imino acids. In order to collect the cyclic imino acid from the culture solution, it is advantageous to use chromatography using an ion exchange resin. A method for isolating and as a crystal will be described below. After adding activated carbon to the K2013 culture solution to adsorb impurities, the activated carbon is separated to obtain a liquid. Dowex the liquid 1x
4 (acetic acid type, manufactured by Dow Chemical Company) to adsorb the inhibitor, and after washing the resin with water,
Elute with 0.5N acetic acid. The active fraction is concentrated to dryness under reduced pressure, and the resulting candy-like substance is dissolved in water. The solution is passed through a column of Amberlite IRA68 (acetic acid type, manufactured by Organo Co., Ltd.) to adsorb the inhibitory substance, and eluted with acetic acid whose concentration is linearly increased from 0 to 0.2 normal. The active fraction is concentrated to dryness under reduced pressure, and the resulting candy-like substance is dissolved in 0.01M sodium acetate. The solution was passed through a Dowex 1×4 column equilibrated with 0.01M sodium acetate to adsorb the inhibitor, the resin was washed with 0.01M sodium acetate, and then eluted with 0.15M sodium acetate. At this time,
The first is eluted and the second is eluted, so separate them into their respective fractions. Fractions containing
The inhibitors are adsorbed through the column and eluted with 0.1N acetic acid. The active fraction is concentrated to dryness under reduced pressure, the resulting candy-like substance is dissolved in warm water, and the crystals that form when left in the refrigerator are collected by filtration and dried to obtain pure crystals of or. High performance liquid chromatography is effective as a purification method other than the above-mentioned ion exchange chromatography, and purification can be carried out more easily and with a higher yield. As described above, according to the present invention, the phthalate is extracted and purified by means such as ion-exchange chromatography, and as a crude solid or solution.
It is collected in pure or crude form as pure crystals or solutions, or as its salts. Next, the biological activities of and revealed by the present invention will be described. The activity was measured by the method described in the aforementioned Journal of Antibiotics. and Salmonella typhimurium TA1535 or Salmonella typhimurium by chemical mutagens.
typhimurium) strongly inhibits the mutation of TA100,
Furthermore, it is a compound that inhibits tumorigenesis of chicken fetal fibroblasts caused by chicken sarcoma virus.

[Table] As shown in Table 1, and are N-ethyl-
N'-nitro-N-nitrosoguanidine (ENNG)
It inhibits the mutation caused by Salmonella typhimurium TA1535, while at a concentration of 1mcg/ml it hardly inhibits the growth of the bacteria. In addition, and are not only substitutional mutations caused by alkylating agents, but also
4-nitroquinoline-1-oxide or 2(2
-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2)-induced flameshift mutation is also inhibited. That is, at 8 mcg/ml, it inhibited the mutation of Salmonella typhimurium TA100 by 4-nitroquinoline-1-oxide and AF-2 by 55% and 66%, respectively, and the growth of the fungus was hardly inhibited at this concentration. Moreover, and inhibit not only bacterial mutations but also tumorigenesis of chicken fetal fibroblasts by chicken sarcoma virus.

[Table] As shown in Table 2, and inhibit chicken sarcoma virus-induced tumorigenesis of chicken fetal fibroblasts at concentrations that hardly inhibit growth. Toxicity to mice with intravenous injection of 25
No abnormality was observed in mg/Kg. As described above, and these related compounds are effective substances for stably retaining the useful properties of microorganisms such as bacteria by inhibiting their mutation. Examples of the production method of and will be shown below, but since the properties, production method, extraction and purification method of and have been clarified according to the present invention, a modified method of the method described in this specification can be easily established. The present invention includes all modifications thereof and is not limited to the examples. Example 1 Huabolus brasiliensis (Fr.) Fr.
Agar slope of K2013 strain (potato-glucose medium)
Scrape off about 0.2 to 0.3 ^cm2 of the mycelia that have grown in the culture, and add 2.0% glucose, 0.5% peptone, 0.3% yeast extract, 0.3% potassium dihydrogen phosphate, and 0.1% magnesium sulfate heptahydrate (PH should not be corrected). No, usually 5.6
- 5.8) was inoculated into a liquid medium (125 ml of medium placed in a 500 ml Sakaguchi flask), statically cultured at 28°C for 4 days, and then cultured with shaking (130 revolutions/minute, amplitude 8 cm) for an additional 4 days. was used as the inoculation source.
125ml of liquid medium with the above composition in a 500ml Sakaguchi flask
The mixture was sterilized at 120°C for 20 minutes, inoculated with 5 ml of the above inoculum, and cultured with shaking at 28°C for 6 days in the same manner as above. After culturing, the bacterial cells were separated, 100 g of activated carbon was added to this solution 10, and the mixture was left to stand for 30 minutes with gentle stirring. After separating the activated carbon, the resulting liquid was passed through a column packed with strongly basic anion exchange resin DOWEX 1×4 (acetic acid type, 50-100 mesh, manufactured by Dow Chemical Company) to adsorb and. After washing with water, it was eluted with 0.5N acetic acid, and the active fraction was concentrated under reduced pressure and dried. Dissolve 1.4 g of the obtained candy-like crude substance in 200 ml of water, adjust the pH to 6.0, and add medium basic anion exchange resin Amberlite.
The inhibitor was adsorbed through a column packed with 200 ml of IRA68 (acetic acid type, manufactured by Organo Co., Ltd.).
After washing with water, elution was carried out while linearly increasing the concentration of acetic acid within the normal range of 0(1) to 0.2(1). The active fraction was concentrated under reduced pressure to dryness, and a mixture of 595 and
I got mg. This was dissolved in 100 ml of water, adjusted to pH 6.0, passed through a 200 ml column of Dowex 1x4 (100-200 mesh) equilibrated with 0.01 molar sodium acetate to adsorb the inhibitor, and eluted with 0.15 molar sodium acetate. . The active fraction eluted first contains;
The active fraction, which is eluted later, contains. Both fractions were separately passed through a column packed with 500 ml of Dowex 1×4 (OH type, 50-100 mesh) to adsorb or. After washing with water, the column was eluted with 0.1N acetic acid. The active fraction was concentrated under reduced pressure to dryness, and the resulting candy-like substance was dissolved in warm water and left in a refrigerator to obtain 117 mg and 10 mg of needle-like crystals, respectively. Example 2 200 g of activated carbon was added to the culture solution 40 obtained in the same manner as in Example 1, and after standing for 30 minutes, the activated carbon was separated and the liquid was poured into Dowex 1×4 (acetic acid type, 50 to
100 mesh) was passed through a column packed with 4.
Elution was performed with 0.5N acetic acid, and the active fraction was concentrated under reduced pressure and dried. 6 g of the obtained candy-like substance was dissolved in 1.0 m water, and after adjusting the pH to 6.0, it was passed through a column packed with 600 ml of Dowex 1 x 4 (acetic acid type, 100-200 mesh) and eluted with 0.2N acetic acid. The active fraction was concentrated under reduced pressure and the resulting pale yellow candy-like substance was mixed with water for 10 minutes.
ml and subjected to high performance liquid chromatography (Waters System 500, reverse phase silica gel, developing solvent was water) to obtain 810 mg and 885 mg of needle-like crystals of and, respectively. Example 3 10 of the liquid medium having the composition of Example 1 was placed in a 30-volume jar fermenter, and after sterilization at 120°C for 20 minutes,
This was inoculated with 600 ml of the inoculum obtained in the same manner as in Example 1, and the temperature was 28°C, the aeration rate was 5/min, and the stirring speed was 250.
After culturing at rotation/min for 4 days, the same operation as in Example 1 was carried out to obtain 105 mg of crystals and 7 mg of crystals.

[Brief explanation of the drawing]

Figure 1 shows the infrared absorption spectrum curve of 5-cyanoproline taken as a potash bromide tablet;
The figure shows the mass spectrum of a mixture of 1-acetyl-5-cyanoproline and 1-acetyl-5-cyanoproline methyl ester, and Figure 3 shows the infrared absorption spectrum curve of 6-cyanopipecolic acid as a potash bromide tablet. Figure 4 shows 1-acetyl-6-cyanopipecolic acid and 1-acetyl-6-
1 shows a mass spectrum of a mixture of cyanopipecolic acid methyl ester.

Claims (1)

[Claims] 1. General formula (wherein n is 2 or 3) A cyclic imino acid represented by: 2 formulas The cyclic imino acid according to claim 1, which is represented by: 3 formulas The cyclic imino acid according to claim 1, which is represented by: 4 Belongs to the genus Fabolus, general formula: A general formula characterized by culturing aerobically a bacterium capable of producing a cyclic imino acid represented by (wherein n is 2 or 3) and collecting the cyclic imino acid from the culture. (In the formula, n is 2 or 3.) A method for producing a cyclic imino acid represented by the following formula. 5. The production method according to claim 4, wherein the cyclic imino acid producing bacterium is Phaebolus brasiliensis (Fr.) Fr. (Japanese name: Minamia Shigurotake) strain K2013. 6. The manufacturing method according to claim 4 or 5, wherein the cyclic imino acid is 5-cyanoproline. 7. The manufacturing method according to claim 4 or 5, wherein the cyclic imino acid is 6-cyanopipecolic acid. 8. A method for collecting cyclic imino acids from a culture of a cyclic imino acid-producing bacterium belonging to the genus Fabolus, in which 5-cyanoproline and 6-cyanopipecolic acid are separated using a basic anion exchange resin.