JPH02229177A - Novel antibiotic mk1688 substance and production thereof - Google Patents

Abstract

NEW MATERIAL:Antibiotic MK16 substance shown by the formula. This antibiotic is a compound comprising N-methyl-L-valine as a constituent amino acid. USE:Having antibacterial activity against gram-positive bacteria and useful as an antimicrobial agent. PREPARATION:A fungus belonging to the genus Fusarium, capable of producing antibiotic MK1688 substance, is cultured and antibiotic MK1688 substance is collected from the culture mixture thereof. Especially submerged culture is optimum as the culture. The culture temperature is 10-30 deg.C, especially 20-27 deg.C. In the production, the accumulation reaches the optimum both in shaking culture and tank culture usually in 3-10 days, the culture is stopped at that time and the aimed substance is isolated and purified from the culture solution.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a novel enniatin antibiotic MK1688 substance and a method for producing the same.

(Prior Art and Problems to be Solved by the Invention) Various antibiotics produced by microorganisms have been known and have been put to practical use in the fields of pharmaceuticals, veterinary drugs, agricultural chemicals, and the like. However, due to problems such as the emergence of resistant bacteria, there is a constant demand for the emergence of new antibiotics. The present invention aims to solve this problem by providing a new antibiotic and establishing a method for producing the same.

(Means for Solving the Problems) In order to meet the above expectations, the present inventors continued their search for substances with antibacterial activity, and found that a culture of a certain bacterial strain belonging to the genus Fusarium had antibacterial activity. As a result of discovering that a substance was produced, isolating the substance, and measuring its physicochemical properties, it was determined that the substance was an enniatin-based compound.

Niatin A (PI, A, Plattner et al., Helv. Chim, Acta
), vol. 31, p. 2192, published in 1948] ; pseudo-Ennfat in A
) (MM Shemyakin et al., Journal Obshkei Kimii (zh. Obshch, Khim.)
, Vol. 42, pp. 2320-2334, published in 1972]
;cyclo(D2-hydroxy-3-methylpentanoyl-N-methyl-D-valyl-D-2-hydroxy-3
Methylpentanoyl N-methyl-D-valyl D-2-
Hydroxy-3-methylpentanoyl N-methyl-D-
Paril (M.M, Shemyakin,
Twelve Brosiderians of the Yokohama Peptide Symposium (12th. Pro
c. Eur, Pept, Symp. ), 346 ~
352 pages, published in 1972].

However, as a result of further investigation, the antibiotic MK16 of the present invention
It was found that the constituent amino acid of the 88 substance is N-methyl L-valine, and since the constituent amino acid is different from the above-mentioned known compounds, it has been determined that the antibiotic of the present invention is a new compound. Completed the invention.

That is, the gist of the present invention is to cultivate a novel antibiotic MK1688 substance represented by the following formula (I) and a bacteria producing the antibiotic MK1688 substance belonging to the genus Fusarium, and to obtain the antibiotic MK1688 from the culture.
The present invention relates to a method for producing the antibiotic MK1688 substance represented by the above formula (1), which comprises collecting the substance.

To explain the present invention, the novel antibiotic MK1 of the present invention will be described below.
Substance 68B is a novel compound represented by the above formula (I') whose constituent amino acid is N-methyl-L-halin.

The antibiotic MK168B substance of the present invention is produced by the MK1688 substance-producing bacterium belonging to the genus Fusarium.

As such producing bacteria, for example, the Institute of Microbial Technology, Agency of Industrial Science and Technology,
Fusarium oxysporum (P-10564) has been deposited as
rum) D338. Its mycological properties are as follows.

(1) Morphological characteristics Colonies grow vigorously and become felt-like to fimbriae-like on PDA medium, with white on the surface and yellowish-white on the underside.
It has a bright yellow-orange color.

The basal hyphae have septa, are colorless, elongate and branch radially, and reach a width of 8.2 μm.

Aerial hyphae are abundantly formed.

Phialides are formed on aerial hyphae or on conidiophores, and are as short as 3.2 to 43 μm in length, tapering toward the tip, and 1.8 to 2 mm wide at the base. .7μ
It is m.

There are two types of conidia: macroconidia and microconidia, and the conidia formation mode is the phiaro type.

Macroconidia are formed on polyconidiophores and are colorless, with a thin, smooth, crescent-shaped membrane, pointed at both ends.

The macroconidia usually have 3 to 5 septa (rarely 6 to 7 septa)
It has a beak-shaped fusotocell at its base.

The size of the above macroconidia is 35 to 60μ for 3-septate conidia.
mX4-5.6μm, 51.6-57μ with 5 septate conidia
m x 4-5.6 μm, 54-7 with 6-7 septate conidia
The size is 3 μm×4 to 5.6 μm.

Microconidia grow on conidiophores that arise laterally from hyphae, and are colorless, smooth, and cylindrical to oval in shape with a thin membrane.

Moreover, the microconidia consist of 1 to 2 cells and have a size of 7.6 to 27 μm×2 to 4 μm.

Note that no complete generation was observed.

(2) Physiological conditions ■ Optimum growth conditions ■ Growth range pu: pH 2 to 9 (cultured for 2 weeks in LCA liquid medium) Temperature = 10°C to 30°C (cultured for 2 weeks on PDA medium) ? 3) Taxonomic considerations ■Identification of the genus Leher This strain (D338) 1) has a phiaro-type conidiation mode, 2) the macroconidia are crescent-shaped and have septa, and the base has clear huntocells (F (,■t Cell).

Deuterofilamentous fungi class D{yphom having the above mycological properties
ycetes) by H. L. Barnett.
．． and B. B. Hunter's “Illustrated Sienera of Imperfect Funghi”
rated genera of Imper
As a result of searching according to the search table described in "Fect Fungi", it was found that this strain (D 3 3.8) belongs to the genus Fusarium.

■ Species level identification This strain (D338) has 1) macroconidia and microconidia;
2) Microconidia occur on simple phialite-type conidiophores, and the conidiophores are short. 3) Microconidia are not formed in chains. 4) Microconidia are cylindrical to oblong. It is.

“The genus Fusarium” by Booth (1971), 1-
According to the description on page 237', there are 47 Fusarium bacteria.
species are known.

Morphological properties of this strain (D338) and 4 described in the above literature
When seven bacterial species were compared, the properties of this bacterial strain almost matched those of Fusarium oxysborum described in the above-mentioned literature. Among them, it was distinguished from the closely related species Fusarium solani (F. solani) by the difference in the length of the microconidiophore. Therefore, this strain (D338) is Fusarium oxysorum (F.oxys orum).
was identified.

In general, the properties of Fusarium genus bacteria tend to change as seen in the case of other fungi, but in the present invention, for example, the D338 strain or a mutant strain derived from this strain (natural Any type of MK1688 that has the ability to produce the novel constituent MK1688 substance can be used in the method of the present invention, whether it is a transgenic or transgenic form (genetically or inducibly produced), or a transzygote or genetically recombinant thereof.

In the present invention, the above bacteria are cultured in a medium containing nutrients that can be used by ordinary microorganisms. As a nutrient source, glucose, starch syrup, dextrin, sucrose, starch, molasses, animal/vegetable oil, etc. can be used. Also, soybean flour, wheat germ, corn stew liquor, cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea, etc. can be used as the nitrogen source. In addition, it is effective to add inorganic salts capable of producing sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid, and other ions as necessary. In addition, organic and inorganic substances that help the growth of bacteria and promote the production of the antibiotic MK1688 substance can be appropriately added.

The most popular culture methods are those under aerobic conditions, especially deep culture methods. The appropriate temperature for culturing is 1.
The temperature is 0 to 30°C, but in most cases it is cultured at around 20 to 27°C. Production of the antibiotic MK1688 substance varies depending on the medium and culture conditions, but the accumulation usually reaches its maximum within 3 to 10 days in both shaking culture and tank culture. When the amount of MK1688 substance accumulated in the culture reaches the maximum, the culture is stopped, and the target substance is isolated and purified from the culture solution.

In the present invention, when collecting the MK1688 substance from a culture, conventional separation methods utilizing its properties are used, such as solvent extraction method, ion exchange resin method, adsorption or distribution force chromatography method, gel filtration method, and dialysis method. Extraction and purification can be carried out using , precipitation methods, etc. alone or in appropriate combinations. For example, the MK168B substance is extracted from cultured bacterial cells with acetone water or methanol-water. Furthermore, if the MK1688 substance accumulated in the culture solution is extracted with an organic solvent that does not mix with water, such as butanol or ethyl acetate, the MK1688 substance will be extracted into the organic solvent layer. M.K.
To further purify the 1688 substance, it is recommended to perform chromatography using an adsorbent such as silica gel (Wako Gel C-200, manufactured by Wako Pure Chemical Industries, Ltd.), alumina, or Sephadex LH-20 (manufactured by Pharmacia). .

The antibiotic MK168B substance of the present invention represented by the formula (r) thus obtained has antibacterial activity against Durham-positive bacteria, as shown in Table 1 below.

15%, sungrain 0.25%, cottonseed meal 0.5%, F
eSOa H 7HzO O. O O O 5%
, NiC1z ・68zO O. 00005%, C
OCl2 ・6H20 0. 0 0 0 0 5%
and CaCOz 0. The main fermentation medium containing 1% (
p}l 6. 0) and its 80 m j+
was dispensed into 100 500 mA Erlenmeyer flasks, and 12
Autoclave for 20 minutes at 1°C. This main fermentation medium is inoculated with 4 mR of the above-mentioned seed culture solution, and cultured with shaking at 210 rpm for 6 days at 26°C. The resulting culture was centrifuged to obtain a culture supernatant at 4°C and cultured bacterial cells.

■ Purification of culture Approximately 500 g (wet weight) of bacterial cells obtained in
After adding % acetone-water (4β) and stirring for 30 minutes,
The bacterial cells were filtered and the acetone was distilled off under reduced pressure to obtain the bacterial cell extract (
2.0β) was obtained. This bacterial cell extract was mixed with the culture supernatant (4E) obtained in step ① above, and ethyl acetate (6
The active ingredient was extracted in step 1), and the solvent layer was concentrated to dryness. The obtained oily substance was added to a silica gel column (wakoge) filled with chloroform-methanol (20:1) in advance.
An acute toxicity test was conducted by intraperitoneal administration to ICL mice (3 male mice weighing 25 g), but at 100 mg/kg.
There were no deaths at this dose.

The present invention will be described in more detail with reference to Examples below, but the present invention is not limited to the following Examples unless it exceeds the gist of the present invention.

Example 1 ■ Cultured cornstarch 3.0%, soybean flour 3.5%, soybean oil 0.
2%, wheat germ 1.5%, KJPOt0. 2%, Ca
Seed medium containing 0.5% CO3 (pH 6.0)
4. The mixture was dispensed into 10 Erlenmeyer flasks at 200 mA each, and sterilized under high pressure at 121°C for 20 minutes.

Next, one platinum loopful of Fusarium oxysvorum D338 (Feikoken Bibori No. 10564) was inoculated and cultured at 26° C. for 3 days with shaking at 210 rpm.

Separately, 2.0% starch syrup, 1.0% soybean flour, 0.0% soybean oil. C-200, 100 g) and developed with tetrachloroform-methanol (20:1), and the eluate was diluted with 15 g.
It was fractionated by g. The active fraction (lkl25143) was concentrated to dryness to yield 796 mg of oil. This oily substance was transferred to a Cefpadesox L Hj No 20 (870 mm) column (Φ3 5 mm x l
The eluate was applied to the upper end of a tube (packed with Sephadesox LH-20 to a height of 9 Q cm), developed with methanol, and the eluate was fractionated into 10 g portions. The active fraction (IIkl212B) was concentrated to dryness to obtain 387 mg of MK1688 substance.

The antibacterial activity in each step was measured using B. subtilis AT as the test bacterium.
Performed using CC 6633.

The physicochemical properties of the MK1688 substance thus obtained were as follows.

(1) Color and shape: colorless powder 12) Molecular formula: C 3b H b 3 N 30, (
3) Elemental analysis value: C 63.02 H 9.5
8N6.08 (4) Mass spectrum (El-MS): m/z681 (
M1) (5) Melting point: Does not show a clear melting point due to hygroscopicity.

(7) Ultraviolet absorption spectrum: Terminal absorption (8) Infrared absorption spectrum (KBr cm-'): 3430, 2950, 293
0 2860.1735.166'0,1460,1
415,1375,[295,1190.1125,1
100.1040, 1010.87'O, . 770 (9)'HNMR spectrum (400MHz, CD3
0D) δ (ppm): 0.92 (3H, d, 6.5Hz),
0.95 (3H.t, 7.4Hz), 0.99 <3
H, d, 6.7Hz), 1.0 7 (3H, d,
6.7Hz). 1.2 3 (I H, m) 1.4 7 (I H, m) , 1.9 6 (L H, m) , 2.28
(IH. d septet, 10.
0, 6. 7 Hz)3.15 (3H,
s), 4.76 (IH, d, 10.OH2), 5
．． 38 (LH, d, 6.5Hz).

Q(II' 3C NMR spectrum (100
MHz, C D3 0 D) δ (ppm): 12.5 Q. 15.9
q, 20.9 q. 21.3
q, 27.2 t, 30.1d, 33
．． 7q. 3B. 5 d, 64.5
d76. O d. 172.41 s,
172.44SO Qll Solubility: Soluble in methanol, chloroform, ethyl acetate, acetone, insoluble in water.

αku Distinction between basic, acidic, and neutral: Neutral substance MK-1 obtained from the above physical and chemical properties and the results of Reference Example 1 below
It was confirmed that substance 688 had a structure represented by the above formula (I).

l8 Reference Example I MK1688 substance (50 mg) was dissolved in 6N HCI (3
ml) and heated under reflux at 11'0°C for 16 hours.

After cooling, the reaction solution was concentrated to dryness. The oily substance obtained is 5
Dissolve in mit water and adjust to pH 4 with IN NaOH.
After adjusting to 0, it was passed through a Dow Sox 50Wx2 (H"") (manufactured by Dow Chemical Company, 5 ml) column.

The passing liquid and the washing liquid (10 ml) were mixed and concentrated to dryness to obtain a crude material. This crude material was prepared using Sephadex G-10 (manufactured by Pharmacia, 440 m
m! ) column chromatography, and the eluate was
It was fractionated by g.

1-butanol-methanol-water (4:1:2
) as a developing solvent, containing a substance detected at an Rf value of 0.51 on silica gel thin layer chromatography (Nl
30-35) was concentrated to dryness to obtain 2-hydroxy-3-methylpentanoic acid (20.2 mg).

The components adsorbed on the DOWEX 50Wx2 (H''') column were eluted with 0.5N HCI and concentrated to dryness to obtain a crude material. After column chromatography of 440 ml!), it was purified by preparative TLC (manufactured by Merck & Co., Art. 5744, developing solvent: 1-butanol-methanol-water, 4:1:2). A crude powder that was positive for ninhydrin was obtained.
0mm2) (71 column chromatography was performed,
The ninhydrin-positive fraction was concentrated to dryness to give N-methyl-L-
Valine (11.9 mg) was obtained.

From these results, it was found that the MK1688 substance consists of 2-hydroxy-3-methylbentanoic acid and N-methyl L-valine.

<2-Hydroxy-3-methylpentanoic acid> Mass spectrum (El-MS): mlz 132 (M4) 'H NMR spectrum (400 MHz
, D zO)δ (1)I)m): 0. '8
6 (3H, d, ?.OH2') ,0.
92 (3H, t, 7; 4Hz), 1.3 1 (I
H. ddq. 14.0.7. 0 Hz) (LH, ddq, 14.0. 7.0Hz), (IH, ddq, 7.0, 7.0, 3.3Hz), (IH, d, 3.3Hz).

(100MHz, DzO) q. 13.6 q. 26. d, 73.5 d, 178.7.4, 1.44 7.4, 1.86 7,0, 4.30 13C NMR spectrum δ (p pm): 1 1.7 1t, 38.6 9 S6 <N-methyl-valine> Mass spectrum (EI-MS) (M+) Specific optical rotation: [α). = +26.9 6N HCI) : m/z 131 6 (co.7 NMR spectrum (p pm): 1.0 1 1.04 2.23 t, (4 0 0 MHz, DzO) (3H,
d, 7.0Hz), (3H, d. 7.0Hz) (IH.d septe 4.5, 7.0 Hz)2.7 1
(3H, s) ,3.47 (LH, d
, 4.5Hz).

I3C NMR spectrum (100MHz, D2
0) δ (ppm): 18. Oq, 1
8.5 q. 29.8 d, 33.3
q, 70. O d. 173. (Effects of the Invention) The novel antibiotic substance MK168B of the present invention has antibacterial activity against Durham-positive bacteria, so it can be expected to be useful as an antibacterial agent.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the infrared absorption spectrum of MK1688 substance in potassium bromide tablets. FIG. 2 is a diagram showing a 400 MHz 'H NMR spectrum of MK 1 6 8 8 substance in a heavy methanol solution. FIG. 3 is a diagram showing a 100 MHz 13C NMR spectrum of MK168B substance in a heavy methanol solution.

Claims (2)

[Claims] (1) Novel antibiotic MK16 represented by the following formula (I)
88 substances ▲ Contains mathematical formulas, chemical formulas, tables, etc. ▼ (I) (2) Cultivate bacteria that produce the antibiotic MK1688 belonging to the genus Fusarium, and use the culture to produce the antibiotic MK168.
The method for producing the antibiotic MK1688 substance according to claim 1, characterized in that eight substances are collected.