JPH0740950B2 - Microbial production of nicotianamine - Google Patents

Description

TECHNICAL FIELD The present invention relates to a microbiological method for producing nicotianamine which also has an angiotensin converting enzyme inhibitory action.

[Conventional technology]

Nicotianamine produced by the method of the present invention is a compound represented by the following formula (I).

Nicotianamine was discovered in the leaves of tobacco (Nicotiana tabacum L.) in 1971 by Noma et al. [M.
et al., Tetrahedron Letters, No.22, pp2017-2020 (197
1) However, the structure is Kristensen et al., Phytochemistry,
13 , pp2791-2798 (1974)], followed by isolation from many plants such as rice, wolfberry, beech, and it has been confirmed that this substance is widely distributed in the plant kingdom. It has also been reported to be present in the basidiomycetes (Basidiomycetes) [R. Armin et al., Biochem. Physiol.P].
flanz., 180 (8), 557-63 (1985)]. However, it is not known that nicotianamine accumulates in liquid cultures of Zygomycetes. Regarding the physiological activity of nicotianamine, it is reported that it is Phytosiderophore that plays an important role in intracellular iron migration or metabolism [M. Budensky et al., Phytochemistry, 19 (11),
2295-2297 (1980)], but it has not been reported to inhibit angiotensin converting enzyme. Compounds that inhibit angiotensin converting enzyme are known to be effective in treating hypertension in humans.

[Problems to be solved by the invention]

Nicotianamine is known to be produced by extraction methods from plant leaves and chemical synthesis methods, but it is difficult to consistently secure a large amount of plants by the extraction method from plants. In the chemical synthesis method, a racemate is produced, and a step for the resolution is required, which complicates the production process.

[Means for solving problems]

As a result of continuing the search for angiotensin converting enzyme inhibitors in microbial cultures, the present inventors have found that nicotianamine is accumulated in large amounts in cultures of a strain belonging to the genus Basidiobors, and completed the present invention. did.

That is, the present invention provides a method for producing nicotianamine, which comprises culturing a microorganism belonging to the genus Basidiiobolus and having the ability to produce nicotianamine in a medium, and producing and accumulating and collecting the microorganism in the culture. .

The method for producing nicotianamine will be specifically described below.

(1) Producing Bacteria and Bacteriological Properties of Producing Bacteria The nicotianamine producing bacterium used in the method of the present invention is a microorganism belonging to the genus Basidiobors and produces a sufficient amount of nicotianamine to be collected in the culture. It may be any as long as it has the ability to An example of such a strain is MK3988 strain newly isolated from soil by the present inventors. The mycological properties of the MK3988 strain are as follows.

(1) Culture characteristics and morphological properties on various media 1. On potato-glucose agar medium (PDA), at 30 ℃,
Cultivation for 5 days Colonies spread to a diameter of 4-5 cm in 5 days. The color tone is white, the vegetative mycelium branches, and the septa with a diameter up to 25 μm enter to form a hypha segment. The conidia stalk is derived from vegetative hyphae or segmental cells, is not branched, has a width of 4.6 to 10 μm, and swells in a spear shape at the tip to form a lower sporangial swelling part. The length of the lower sporangial swelling is 21.0
〜28.0μm, width 10.7〜15.0μm; swollen part forms one budding conidia at the tip (primary conidia) Conidia are colorless, pear-shaped, 21.5-65.6 × 20.5〜 It has a size of 60 μm and has fine conical protrusions at the base of the biomolecule. Conidia are actively ejected as the internal pressure in the lower swelling of spores increases. Ejected conidia germinate and emerge hyphae, or secondary conidia or adherent conidia (Adhensive conidia).
dia). The second conidia have almost the same shape and size as the first conidia. Sticky conidia are formed on the tip of a capillary-shaped stem that originates from a first or second conidia, oval, length
40-45 μm, width 13-21 μm, falling off, sticky tip at the tip.

Zygospores are spherical to subspherical in size, formed by fusion of adjacent segmented cells, ranging in diameter from 30.7 to 40 μm. Oil droplets are contained inside the zygospores. 2 mating spore walls
~ 4 μm thick, smooth. Homo tatiku species.

2. Culture on malt agar powder medium (MA) at 27 ° C. for 5 days The characteristics and morphological properties of the culture on this medium are in agreement with those on the PDA.

(2) Physiological properties 1. Optimal growth conditions (PDA medium, 5 days culture) Optimum pH: 5 to 7 Optimum temperature: 30 to 37 ° C 2. Growth range pH: 3 to 9 Temperature: 20 to 37 ° C, Does not grow at 40 ° C (3) Taxonomic device 1. Identification of genus level This strain (MK398) has 1) basal hyphae that form hyphal segments due to the insertion of septa, and 2) conidia. The stalk forms the lower swelling part. 3) One conidium is always formed from the lower swelling part by budding. 4) Conidia are actively ejected. 5) Sexual reproduction is caused by adjacent hyphae. 6) Homotaric species by segmental junction (gametosis junction).

These properties are described by C. Drechsler, Jour. Washington Acad.S.
oi 45: 49-56 (1955) and J. Coremans-Pelseneer, Acta.
It was in good agreement with the characteristics of the genus Basidiobolus described in Zoologica et Pathologica 60: 1-143 (1974).

Therefore, this strain (MK3988) is a subdivision of the zygomycete-the zygomycota-the order of the genus Moth-Basidiobolaceae.
bolus) belonging to the genus.

2. Species level identification C. Drechsler, Jour. Washingtom Acad. Sci 45; 49-56 (19
55), RK Benjamin, Aliso 5 (2), 223-233 (1962),
DLGreer & L. Friedman, Sabouraudia 4; 231-241 (19
66), MCSrinivasan & MTThirumalachar, Mycopath.
Mycologia Applicat a33; 56-64 (1967) and J. Coreman
s−Pelsneer, Acta Zoologica et Pathologica 60; 1-14
According to the taxonomic literature on the genus Basidiobolus of 3 (1974), 5 species (B. ranaru) belong to this genus.
m, B.microsporus, B.magnus, B.haptosporus, B.meristosp
orus) is included. These 5 species are 1) the degree of cell wall thickening of zygospores, 2) whether or not they can grow at 37 ° C, 3) the degree of development of aerial hyphae, and 4) the presence or absence of actinomycete odor (streptomyces odor, Streptomyces odor). 5) Micro Conidi (mi
croconidia).

This strain (MK3988) has a smooth outer wall of 1) zygotes,
2) Good growth at 37 ° C, 3) Remarkable formation of aerial hyphae on various media, 4) Lack of Streptomyces odor, and 5) No formation of microconidia. From that, C. Drechsler, Jou
r.Washington Acad.Sci 45; 49-5 (1955), Basidiobol
(us meristosporus) in good agreement with various properties.

Therefore, this strain is Basidiovor mellisporus (Ba
sidiobolus meristosporus) MK3988 was identified.

The MK3988 strain has been entrusted to the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as Micromachine Research Institute No. 8983 (FERM P-8983).

This strain, that is, the MK3988 strain, is likely to change its properties as seen with other microorganisms. For example, all strains of the genus Vasidiobolus having the ability to produce nicotianamine, whether MK3988 strain or a mutant strain (spontaneous or inducible) derived from this strain, a zygote or a genetically modified strain, can be used in the present invention. Can be used in the method.

(1) Culturing method In the method of the present invention, the above-mentioned bacterium is cultivated in a medium containing nutrients that can be utilized by ordinary microorganisms. As a nutrient source,
Glucose, starch syrup, dextrin, sucrose, starch, molasses, animal and vegetable oils and the like can be used. As the nitrogen source, soybean flour, wheat germ, corn steep liquor, cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea and the like can be used. In addition, if necessary, sodium, potassium, calcium,
It is effective to add magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid, and other inorganic salts capable of forming ions. In addition, organic and inorganic substances that help the growth of bacteria and accelerate the production of nicotianamine can be added appropriately.

As the culture solution, a culture method under aerobic conditions, especially a submerged culture method is most suitable. A suitable temperature for culturing is 15 to 37 ° C, but in many cases, the temperature is around 26 to 30 ° C. Although the production of nicotianamine varies depending on the medium and culture conditions, the maximum accumulation is usually reached within 1-10 days in both shaking culture and tank culture. When the amount of nicotianamine accumulated in the culture reaches a maximum, the culture is stopped and the target substance is isolated and purified from the culture.

(3) Purification When collecting nicotianamine obtained from the culture according to the present invention from a culture, a conventional separation means utilizing its properties, for example, a solvent extraction method, an ion exchange resin method, an adsorption or partition column chromatography is used. Method, gel filtration method, dialysis method, precipitation method and the like can be used alone or in combination as appropriate for extraction and purification. For example, nicotianamine is extracted from the cultured cells with acetone-water or methanol-water. In addition, the nicotianamine accumulated in the culture solution is adsorbed by "Diaion PK208" (manufactured by Mitsubishi Kasei Kogyo Co., Ltd.) which is a strongly basic ion exchange resin.

To further purify nicotianamine, silica gel (“Wako Gel C-200”, manufactured by Wako Pure Chemical Industries, Ltd.), an adsorbent such as alumina, or “Sephadex G-10”
Chromatography using (Falmacia) or the like may be performed.

The nicotianamine thus produced in the culture can be separated in free form, i.e. as nicotianamine itself, and the solution containing nicotianamine or its concentrate can be added to a base such as sodium hydroxide, potassium hydroxide, etc. Alkali metal compounds such as calcium hydroxide, magnesium hydroxide and like alkaline earth metal compounds, ammonia and like inorganic bases such as ethanolamine, triethylamine, dicyclohexylamine and like organic bases, or acids such as hydrochloric acid, sulfuric acid, When treated with an inorganic acid such as phosphoric acid or an organic acid such as formic acid, citric acid, tartaric acid, etc., during the operation of each step, for example during the operation of each step of extraction, separation or purification, nicotianamine is converted into its corresponding salt form. Is changed and separated. Separately, the salt of nicotianamine thus produced can be easily converted into its free form by a conventional method, that is, nicotianamine can be easily converted into itself.

Further, the nicotianamine obtained in a free form may be converted into the corresponding salt thereof by a conventional method with the above base.

Therefore, the method for producing the salts thereof as described above as well as nicotianamine is also included in the scope of the present invention.

The nicotianamine obtained according to the above production method has the following physicochemical properties.

Molecular weight and molecular formula 303, C ₁₂ H ₂₁ N ₃ O ₆ Mass spectrum (FD-MS) m / z 304 (M + H) Comparative luminosity ▲ [α] ²⁵ _D ▼ -48.3 ° (c1.0, H ₂ O) Infrared absorption Spectrum (KBr method, cm ^-1 ) 3420,3030,2870,2600,2370,1580,1500,1470,1410,1370,
1350,1310,1290,1240,1220,1190,1150,1130,1110,1080,
1060,1010,970,940,920,880,840,800,760,670,590,570,
490,410 Ultraviolet absorption spectrum [λ max nm (ε)] In aqueous solution, no characteristic absorption was observed at a concentration of 100 μg / ml.

¹ H NHR spectrum (400 MHz) TSP was measured as an internal standard (0 ppm) in a heavy aqueous solution.

δ (ppm): 4.80 (1H, m), 4.12 (1H, dt), 3.98 (1H, d
d), 3.90 (1H, dd), 3.78 (1H, dd), 3.44 (1H, m), 3.37
(1H, m), 3.25 (2H, m), 2.87 (1H, m), 2.561H, m), 2.25
(2H, m), 2.17 (2H, m), ¹³ C NMR spectrum (100 MHz) Dioxane was measured as an internal standard (67.4 ppm) in a heavy aqueous solution.

δ (ppm): 174.3,174.2,173.6,67.9,60.6,53.8,52.2,5
1.6,45.0,28.3,26.0,22.1 Solubility Soluble in water. Insoluble in methanol, chloroform, ethyl acetate, toluene, and hexane.

Color and properties of substance White fine powder Color reaction Positive for ninhydrin reagent Thin layer chromatography [Examples] Examples of the present invention will be shown below, but the present invention is not limited to the following examples unless it exceeds the gist.

Example 1 (Culture) 4.0% starch syrup, 0.3% soybean oil, 2.0% soybean powder, 1.0% cottonseed oil,
Sun grain 0.5%, CaCO ₃ 0.3%, FeSO ₄ / 7H ₂ O 0.001
%, COCl ₂・ 6H ₂ O 0.0001% and NiCl ₂・ 6H ₂ O 0.0001% containing medium (pH 6.0) 40 ml each 200 ml Erlenmeyer flask 10
Dispense into books and autoclave at 121 ° C for 20 minutes.

To this, Basidiob Melistopols (Basidiob)
olus meristosporus) MK3988 strain was inoculated with 1 platinum loop each,
Incubate with shaking at 210 rpm for 4 days at 26 ° C. Separately, corn starch 2.0%, glucose 0.5%, soybean oil 5
%, Soybean flour 6.25%, Cottonseed oil 2.0%, Staminol 0.2% and CaCO ₃ 1.0% (pH 6.0)
l Dispense into 50 Erlenmeyer flasks and autoclave at 121 ℃ for 20 minutes. Each flask is inoculated with 4 ml of the preculture obtained by the above method, and cultured at 26 ° C. for 6 days with shaking at 210 rpm. The resulting culture is centrifuged and the supernatant 3.2
Got

Example 2 3.2 L of the culture filtrate obtained in Example 1 was added to "Diaion PK-
The solution was passed through a column of 208 "(H ⁺ type 300 ml), and 2 L of 3.2 L of the passing solution was further passed through a column of" Dowex 1x2 "(300 ml of Cl ^- type) and a column of activated carbon (300 ml) successively. When this passing solution was concentrated and dried, 39 kg of brown powder was obtained.

Example 3 28 g of the brown powder obtained in Example 2 was dissolved in a small amount of water, sprinkled with 20 g of silica gel and dried sufficiently under reduced pressure. This was packed in the upper part of a column of silica gel (1000 ml) and the developing solvent propanol-pyridine-acetic acid-water (50: 10: 3: 6) 4
00 ml, then propanol-pyridine-acetic acid-water (15: 1
(0: 3: 12) When developed with 6000 ml, the fraction mainly containing nicotianamine in Fr150-270 was eluted and concentrated to dryness.
53.4 g of white powder was obtained.

Dissolve 18 g of this white powder in a small amount of water, put it on the top of the column of "Sefadex G-10" (800 ml), develop with water and collect the fractions mainly containing nicotianamine.
9 mg of white powder was obtained.

Example 4 104 mg of the white powder finally obtained in Example 3 was dissolved in a small amount of water, adsorbed on a column of "Diaion PK-208" (H ⁺ , 50 ml), washed with water and washed with 0.5N ammonium hydroxide. Eluted. Concentrate this eluent and concentrate it as "Kirex 100".
(Na ⁺ , 5 ml, manufactured by Bio-Rad) was passed through. The pH of the flow-through was adjusted to 6.5 with "CM-Sephadex C-25" (H ⁺ ), and concentrated to dryness to give 42 mg of nicotianamine (1C ₅₀ = 0.82 µg / ml).

〔The invention's effect〕

According to the method of the present invention, nicotianamine can be efficiently obtained.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Takashi Mikawa, Takashi Mikawa, 1000, Kamoshida-cho, Midori-ku, Yokohama Sanryo Kasei Kogyo Co., Ltd. (72) Inventor Haruyuki Ogishi 1000 Kamoshida-cho, Midori-ku, Yokohama, Kanagawa Sanryo Kasei Co., Ltd.

Claims (2)

[Claims] 1. A microorganism belonging to the genus Basidiobolus and having a capability of producing Nicotianamine (Nicotianamine) is cultivated in a nutrient medium, and nicotianamine is produced and accumulated in the culture, and the microorganism is collected. Method for producing nicotianamine by microorganisms. 2. A production strain belonging to the genus Basidiobors is Basidiobolus meris.
tosporus) MK 3988 (Microtechnology Research Institute, Microbial Research No. 8983).