JPH11269175A - A novel ultraviolet absorbing substance produced by marine bacteria and its production method - Google Patents

Abstract

(57) [Problem] To provide a substance useful as an ultraviolet absorber. SOLUTION: The following formula (I), (II) or (II)
I) Which is a fat-soluble ultraviolet absorbing substance. The compound is produced using a microorganism.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel compound useful as an ultraviolet absorbing material, and a method for producing the same using a microorganism.

[0002]

2. Description of the Related Art Conventionally, benzophenone derivatives, paraaminobenzoic acid derivatives,
Chemically synthesized products such as paramethoxycinnamic acid derivatives and salicylic acid derivatives have been mainly used. However, with the rejection of chemically synthesized products, research is being conducted to search for novel UV-absorbing substances having high safety from natural products, and to apply and utilize them. As a natural ultraviolet absorbing substance, for example, Botanica Marina (Bot. Ma)
r. ) Vol. 16, No. 23 (1974), Marine Biology (Mar. Biol.) Vol. 10
8, No. 157 (1991), Journal of Plankton Research (J. Plankton Res.) V
ol. 12, No. 909 (1990), Plant Daisies Reporter (Plant Dis. Rept)
r. ) Vol. 57, no. As disclosed in 760 (1973), mycosporin-like amino acids produced by seaweeds, marine animals, microalgae, molds and the like are known.

[0003]

However, since these substances are water-soluble, their application range is limited.
For this reason, a fat-soluble ultraviolet absorbing substance derived from a natural product has been demanded.

The present invention has been made under such a technical background, and an object of the present invention is to provide a novel fat-soluble ultraviolet absorbing substance derived from a natural product and a method for producing the same.

[0005]

Means for Solving the Problems The present inventors have found that a compound obtained from a culture of a strain belonging to the genus Paracoccus is a novel compound that has never been known before, and completed the present invention.

That is, the present invention provides the following formulas (I) and (I)
I) or (III)

[0007]

Embedded image

[0008]

Embedded image

[0009]

Embedded image It is a compound represented by these.

[0010] The present invention also belongs to the genus Paracoccus,
A microorganism having the ability to produce the compound described above is cultured in a medium, the compound described above is produced and accumulated in a culture, and the compound described above is collected from the culture. It is a manufacturing method of.

[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.

(1) Properties of the compounds of the present invention A group of novel compounds of the present invention is represented by the following formulas (I) and (I)
It is represented by I) or (III).

[0013]

Embedded image

[0014]

Embedded image

[0015]

Embedded image The physicochemical properties of the compound represented by the formula (I) are shown below. (1) Color of substance: reddish brown (2) Molecular weight: 230 (3) Molecular formula: C ₁₂ H ₁₀ N ₂ OS (4) Mass spectrometry: High-resolution FABMS, measured value 23
1.0606 M + H ⁺ , calculated 231.0592
(C ₁₂ H ₁₁ N ₂ OS) (5) Ultraviolet absorption spectrum (in acetonitrile): λ _max 210 nm (ε 133
00), 258 nm (3400), 269 nm (320
0), 275 nm (3100), 320 nm (230
0) (6) Infrared absorption spectrum (KBr) ν _max 3266, 1605, 1520, 1448, 1
435, 1050, 806, 750 cm ^-1 (7) ¹ H-NMR (measured in deuterated acetone, 500 M
Hz) δ ppm 3.34 (t, 2H, J = 8.7 Hz),
4.62 (t, 2H, J = 8.7 Hz), 7.27
(M, 2H), 7.55 (m, 2H), 8.36 (m, 2H)
1H), 8.80 (s, 1H), 11.1 (s, NH) (8) ¹³ C-NMR (measured in deuterated acetone, 125 M
Hz) δ ppm 32.4 (t), 68.1 (t), 113.
6 (d), 115.1 (s), 123.3 (d), 12
3.9 (d), 124.9 (d), 128.2 (s),
138.1 (s), 128.9 (d), 172.9
(S), 181.0 (s) (9) Solubility: Soluble in DMSO, acetonitrile, methanol, ethanol, acetone, ethyl acetate and chloroform, poorly soluble in water.

The physicochemical properties of the compound represented by the formula (II) are shown below. (1) Color of the substance: colorless (2) Molecular weight: 314 (3) Molecular formula: C ₂₀ H ₁₄ N ₂ O ₂ (4) Mass spectrometry: High-resolution FABMS, actual measurement 31
5.1125 M + H ⁺ , calculated 315.1134
(C ₂₀ H ₁₅ N ₂ O ₂ ) (5) Ultraviolet absorption spectrum (in DMSO): λ _max 280 nm (ε7500),
349 nm (7000) (6) Infrared absorption spectrum (KBr) ν _max 3326, 3214, 1700, 1539, 1
241, 1207, 743 cm ^-1 (7) ¹ H-NMR (measured in heavy DMSO, 500 M
Hz) δ ppm 5.54 (s, 2H), 6.82
(T, 1H, J = 7.5 Hz), 6.91 (t, 1H,
J = 7.6 Hz), 7.01 (d, 1H, J = 7.8H)
z), 7.06 (t, 1H, J = 8.2 Hz), 7.0
9 (t, 1H, J = 7.6 Hz), 7.26 (d, 1
H, J = 8.1 Hz), 7.39 (d, 1H, J = 8.
3 Hz), 7.39 (s, 1H), 7.43 (d, 1
H, J = 8.3 Hz), 7.50 (d, 1H, J = 2.
4), 11.38 (s, NH), 11.64 (s, N)
H) (8) ¹³ C-NMR (measured in heavy DMSO, 125 M
Hz) δppm 70.6 (t), 105.8 (s), 10
7.3 (s), 111.7 (d), 112.0 (d),
112.9 (s), 118.7 (d), 120.1
(D), 120.3 (d), 120.4 (d), 12
1.1 (d), 121.9 (d), 124.7 (s),
125.0 (s), 126.1 (d), 128.2
(D), 136.0 (s), 136.1 (s), 15
1.7 (s), 174.1 (s) (9) Solubility: soluble in DMSO and acetonitrile, methanol, ethanol, acetone, ethyl acetate,
Poorly soluble in chloroform and water.

The physicochemical properties of the compound represented by the formula (III) are shown below. (1) Color of the substance: colorless (2) Molecular weight: 311 (3) Molecular formula: C ₂₀ H ₁₃ N ₃ O (4) Mass spectrometry: high resolution FABMS actual measurement, 31
2.1144 M + H ⁺ , calculated 312.1137
_{(C 20 H 14 N 3 O} ) (5) UV absorption spectrum (acetonitrile): λ _max 217nm (ε240
00), 289 nm (7900), 310 nm (580
0), 389 nm (6100) (6) Infrared absorption spectrum (KBr) ν _max 3414, 3224, 1601, 1446, 1
139, 735 cm ^-1 (7) ¹ H-NMR (measured in heavy benzene, 500 MHz) δ ppm 6.86 (d, 1 H, J = 8.1 Hz),
6.96 (d, 1H, J = 8.1 Hz), 7.00
(S, NH), 7.21 (t, 1H, J = 7.7H)
z), 7.26 (t, 1H, J = 7.7 Hz), 7.3
9 (t, 1H, J = 7.6 Hz), 7.62 (d, 1
H, J = 5.1 Hz), 7.83 (d, 1H, J = 8.
1 Hz), 8.48 (d, 1H, J = 4.9 Hz),
9.31 (d, 1H, J = 2.9 Hz), 9.32
(D, 1H, J = 7.8 Hz), 10.72 (s, N
H) (8) ¹³ C-NMR (measured in heavy DMSO, 125 M
Hz) δ ppm 112.2 (d), 112.9 (d), 11
4.2 (s), 117.8 (d), 119.7 (d),
120.0 (s), 121.5 (d), 121.5
(D), 121.9 (d), 122.7 (d), 12
7.1 (s), 128.5 (d), 130.6 (s),
134.9 (s), 135.9 (s), 136.8
(D), 137.7 (d), 138.4 (s), 14
1.5 (s), 187.2 (s) (9) Solubility: Soluble in DMSO, acetonitrile, methanol, ethanol, acetone, ethyl acetate, chloroform and benzene, poorly soluble in water.

(2) Production of the compound of the present invention The compound of the present invention is obtained by culturing a microorganism in a medium, producing and accumulating the compound of the present invention in a culture, and collecting the compound of the present invention from the culture. Can be manufactured. The microorganism used herein is not particularly limited as long as it belongs to the genus Paracoccus and has the ability to produce the compound of the present invention. Paracoccus sp. The F-1547 strain can be mentioned. Paracoccus sp. F-15
47 strains are strains newly isolated from nature, and their bacteriological properties are as follows. In addition, a mutant strain of the above strain, which has the ability to produce the compound of the present invention, can also be used.

The morphological observation was performed by using
sp. F-1547 strain was inoculated at 30 ° C. for 24 to 48 hours.
After culturing for an hour, the measurement was performed using an optical microscope and a transmission electron microscope. The medium was marine agar (Bacto M)
aline Agar 2216, manufactured by Difco) or marine broth (Bacto Marine Br)
oth 2216, manufactured by Difco).

A. Form 1) Cell shape and size: short rod to cocci, 1.30 to
0.63 × 0.90-0.56 μm or 0.42-
0.97 μm 2) Cell polymorphism: No 3) Motility, flagellar formation: Yes, polar hair 4) Spores: No 5) Gram stain: Negative 6) Acid-fast Presence: None.

B. Growth condition in each medium 1) Marine agar plate culture: good growth, colony is circular, convex, whole edge, wet light, yellow 2) Marine agar slope culture: good growth, filamentous, yellow 3) Marine broth culture: good 4) Marine broth gelatin stab culture: does not liquefy 5) Litmus milk: does not change.

C. Physiological properties 1) Nitrate reduction: not reduced 2) MR test: negative 3) VP reaction: negative 4) Indole formation: not generated 5) Starch hydrolysis: not degraded 6) Use of citric acid: Simmons medium: Use: Christensen: Use 7) Use of inorganic nitrogen source: Use 8) Pigment generation: Yes, yellow (zeaxanthin) 9) Urease activity: Negative 10) Oxidase activity: Positive 11) Catalase activity: Positive 12) Growth Range (temperature, pH): temperature 10 to 45
℃, 20-35 ° C., best growth, pH 5-10, optimal growth pH range 6-9 13) Attitude to oxygen: aerobic 14) OF test: oxidatively degrade glucose 15) Acids from saccharides and Whether gas is generated

[0023]

[Table 1] 16) Decomposition of esculin: not decomposed 17) Decomposition of arginine: not decomposed 18) Decomposition of DNA: not decomposed 19) Nitrate respiration: none 20) Halophilicity: none, growth salt concentration range 0-15% 21) β -Galactosidase: positive 22) PHB accumulation: accumulates 23) GC content (mol%): 65.8 24) isoprenoid quinone: Q-10.

Based on the mycological properties described above, a comparison was made with known bacterial species in accordance with the classification standard of the Virgie's Manual of Deterministic Bacteriology. This strain is an aerobic gram-negative bacillus with polar flagella. It is considered to belong to the genus Paracoccus from the fact that glucose is oxidatively degraded, oxidase and catalase are positive, main quinone is Q-10, GC content is 65.8 mol%, and PHB is accumulated. (Paracoccus sp.). Then, this strain was transformed into Paracoccus species.
coccus sp. ) F-1547
Deposited on February 27, 2012 under the accession number FERM P-16675 at the National Institute of Bioscience and Human-Technology.

For culturing the microorganism used in the present invention, a usual culturing method of the microorganism is used. As the medium, any of a synthetic medium and a natural medium can be used as long as the medium contains assimilable carbon sources, nitrogen sources, inorganic substances, and necessary growth and production promoting substances. As a carbon source, glucose, starch, dextrin, mannose, fructose,
Sucrose, lactose, xylose, arabinose, mannitol, molasses and the like are used alone or in combination. Further, if necessary, hydrocarbons, alcohols, organic acids and amino acids such as tryptophan are used. As the nitrogen source, ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate, urea, peptone, meat extract, yeast extract, dried yeast, corn steep liquor, soybean powder, casamino acid and the like are used alone or in combination. In addition, if necessary, inorganic salts such as salt, potassium chloride, magnesium sulfate, calcium carbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ferrous sulfate, calcium chloride, manganese sulfate and zinc sulfate are added. Further, a trace component which promotes the growth of the used bacterium and the production of the compound of the present invention can be appropriately added. The most suitable culture method is a liquid culture method.
A cultivation temperature of 30 to 35 ° C. is appropriate, and the P
It is desirable to maintain H at 7-8. When liquid culture is performed for 3 to 5 days, the target substance is produced and accumulated in the culture solution. The culture is stopped when the production in the culture reaches a maximum.

The isolation and purification of the compound of the present invention from the culture
It is performed according to a method commonly used for isolating and purifying a microbial metabolite from its culture. For example, the culture is separated into a culture filtrate and cells by filtration or centrifugation, and the cells are extracted with aqueous methanol, aqueous ethanol, or the like. Subsequently, the extract and the culture filtrate or the extract obtained by extracting the culture filtrate with ethyl acetate, chloroform and the like are combined, concentrated, and subjected to column chromatography on silica gel, gel filtration, high performance liquid chromatography, and the like to give the compound of the present invention. Get.

The trend of the compound of the present invention during the culturing and purification operations can be traced by using the ultraviolet ray absorption by high performance liquid chromatography as an index, but it can be traced using a multi-wavelength detector (photodiode array detector (manufactured by Shimadzu Corporation). It is preferable to use SPD-M6A) for purification using the UV spectrum as an index.

(3) Use of the compound of the present invention The compound of the present invention has a strong ultraviolet absorbing ability as shown by the absorption ability at the absorption maximum wavelength in the ultraviolet absorption spectrum of physicochemical properties, and therefore, it is used in cosmetics or pharmaceuticals. Can be used as an ultraviolet absorbing component.

Hereinafter, the present invention will be described specifically with reference to examples. However, the technical scope of the present invention is not limited by these examples.

[0030]

EXAMPLES Paracoccus sp. F-15
Forty-seven strains were used. The strain was placed in a 1 l Erlenmeyer flask containing 300 ml of marine broth (manufactured by Difco, 37 g / l) and 1 g of tryptophan in a liquid medium.
C. for 5 days with shaking (100 rpm). During the culture, the pH of the medium was not particularly controlled.

3 L of the thus obtained culture was centrifuged. The supernatant obtained by centrifugation was extracted with ethyl acetate (3 L). The cells were added with ethanol (100 ml), sonicated for 10 minutes, and centrifuged to obtain an ethanol extract. An ethanol extract of the cells was added to the ethyl acetate extract of the supernatant, and concentrated under reduced pressure at 40 ° C or lower. The obtained concentrated liquid was purified by HPLC using an acetonitrile-water (4: 6, v / v) as a mobile phase and an ODS column (manufactured by Nomura Chemical Co., Ltd., DEVELOSIL ODS-HG-5) to obtain a compound of the present invention. 1.0 mg and 2.5 mg of (I) and (II) were obtained, respectively.

Next, the compound (II) was immediately purified by HPLC after stepwise addition to acetonitrile-water (6: 4, v / v) immediately after the compound (II) was discharged.
1.0 mg of II) was obtained.

The compounds (I), (II) and (III) obtained here exhibited the above-mentioned physicochemical properties.

The conditions in this purification step were as follows. ODS column size: 20 mmφ × 250 mm Mobile phase flow rate: 14 ml / min Compound (I) retention time: 16.8 minutes, solvent: acetonitrile-water (4: 6, v / v) Compound (II) retention time: 24 2 minutes, solvent: acetonitrile-water (4: 6, v / v) Compound (III) Retention time: 41.6 minutes, solvent: 1-26 minutes is acetonitrile-water (4: 6, v / v), 26-41.6 minutes for acetonitrile-water (6: 4, v / v)

[0035]

The present invention provides a novel compound and a method for producing the substance using a microorganism. The compounds of the present invention
Since it is fat-soluble and has a strong ultraviolet absorbing ability, it can be used as a component of cosmetics or pharmaceuticals.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12P 17/18 C12P 17/18 B // (C12P 17/16 C12R 1:01) (C12P 17/18 C12R 1:01) ( 72) Inventor Miyuki Nishijima 1900 Sodesoshi-cho, Shimizu City, Shizuoka Prefecture Inside the Kaiyo Biotechnology Research Institute, Shimizu Research Laboratories (72) Inventor Kenichi Mochida 1900 Sodesoshi-cho, Shimizu City, Shizuoka Prefecture Shimizu Biotechnology Research Institute, Inc. Inside

Claims (4)

[Claims] 1. A compound represented by the following formula (I). Embedded image 2. A compound represented by the following formula (II). Embedded image 3. A compound represented by the following formula (III). Embedded image 4. A microorganism belonging to the genus Paracoccus and capable of producing the compound according to claims 1 to 3 is cultured in a medium, and the compound according to claims 1 to 3 is produced and accumulated in a culture, and the culture is performed. The method for producing a compound according to any one of claims 1 to 3, wherein the compound is collected from the substance.