JP2558485B2 - Process for producing pyrimine - Google Patents

Description

TECHNICAL FIELD The present invention relates to a pyrimine-producing bacterium useful as an intermediate for ferropyrimine which is a natural pigment, and a method for effectively producing pyrimine using the bacterium. .

[Conventional technology]

In recent years, natural pigments have attracted attention from the viewpoint of safety with respect to synthetic pigments. Among them, ferropyrimine, which is a natural red pigment, cultivates a GH strain that conventionally belongs to the genus Pseudomonas, binds iron ions that were present in the culture at the same time as producing pyrimine, to form ferropyrimine, The method is to collect ferropyrimine from the culture. (Biochemistry Vol.4, No.10,
1965, pages 2233 to 2236). Pyrimine has the following structural formula: And has the structural formula described in the above biochemistry.

[Problems to be solved by the invention]

An object of the present invention is to provide a novel method for producing pyrimine useful as, for example, an intermediate of ferropyrimine, by using a bacterium different from the bacterium used in the above conventional method.

[Means for solving problems]

The present invention was made based on the finding that a novel pyrimine-producing bacterium of the genus Pseudomonas was found, and that pyrimine can be efficiently produced by culturing the bacterium with a plant.

That is, the present invention provides a novel pyrimine-producing bacterium of the genus Pseudomonas and provides a method for producing pyrimine, which comprises co-culturing the pyrimine-producing bacterium with a plant and collecting the pyrimine from the culture. .

As the pyrimine-producing bacterium used here, one having the following mycological properties is used.

Mycological properties 1. Morphological properties Morphology: Not forming bacillus spores alone or in chains Motility: Yes, with polar flagella Size: 0.6 × 0.8μ × 1.0-3.5μ Gram stain: Negative 2. Each medium Growth conditions (1) Broth culture Growth: Medium, Skin: Rings formed Sediment: Slightly turbid, Yes (2) Slope culture broth Shape: Filamentous, Surface: Smooth and shiny Edge: Gold edge, Color: Milky white, transparency: opaque (3) Broth agar plate culture Shape: perfect circle, rim: whole edge, surface ridge shape: lenticular, surface: smooth and glossy, color tone: milky white (4) bouillon agar puncture culture surface and Growth along the upper limb 3. Physiological properties (1) Attitude toward oxygen: aerobic (2) Catalase: + (3) Oxidase: + (4) Arginine dehydrolase:-(5) Lipase (addition of Tween 80 moisture ): + (6) Lecithinase (yolk decomposition):-(7) Gelatin hydrolysis:-(8) Starch hydrolysis:-(9) Extracellular poly-β-hydroxybutyric acid (PHB) hydrolysis:- (10) use to independently nutritionally growing H _2: - (11) generated in the dye: King a, B medium, glutamate medium, Pseudomonas F
Fluorescent pigment is not formed on agar, Pseudomonas P agar, TSI agar and potato dextrose agar.

(12) Reducibility of nitrate:-(13) Denitrification reaction:-(14) Decomposition position of protocatechurate: Ortho (15) Cellular accumulation of PHB: + (16) Levan formation from sucrose:-(17) ) Growth temperature 11-35 ℃ Optimal temperature 18 ℃ -35 ℃ (18) Growth pH 4.0-8.5 Optimum pH 4.5-8.0 (19) Carbon source assimilation D-Glucose: + D-Fructose: + D- Arabinose: + Trehalose: ± Mannitol: + Inositol: + Glycerol: + L-valine: ± β-alanine: + Glycolate: + p-Hydroxybenzoate: + Classification of strains of the present invention according to the above mycological properties The academic status of "Bergey's Manual of Systematic Bacteriology"
As a result of obtaining by Volume 1 (1984), this strain was considered to belong to the genus Pseudomonas because it is a Gram-staining negative bacillus, which exhibits motility due to polar hairs, and is aerobic, catalase-positive, and oxidase-positive.

Of the genus Pseudomonas, poly-β-hydroxybutyric acid accumulates intracellularly as a carbon source storage substance, cannot grow at 40 ° C., and Pseudomonas solanaceram (as Pseudomonas solanaceram has the property of decomposing protocatechuic acid at the ortho position). Pseudomonas solanacearum) exists.

However, as shown below, D as a denitrification reaction and nitrogen source
-The property is different from this strain in that arabinose cannot be used.

Therefore, this strain was judged to be appropriate as a new strain belonging to the genus Pseudomonas, and was named Pseudomonas sp. K strain. This strain has been deposited as Microindustrial Research Institute No. 9628.

Preferable pre-culture conditions for the Pseudomonas sp. K strain used in the present invention include a method of directly inoculating the LS medium with culturing in a broth agar medium at 25 ° C for 2 to 3 days. It can also be cultured in LS medium.

In the present invention, the cultivation of the fungus is characterized by culturing with the plant. The plants used here include lilies such as green onions, cruciferous plants such as lettuce, cabbage, wasabi, radish, and watercress, licorice leaves, basil, sorel, mint and other plants, and green beans. Leguminous plants and St. Paulia plants such as St. Paulaceae can be preferably used.

Here, as the plant, α-naphthyl acetic acid, the mesophyll of the plant on an LS agar medium containing hormones such as kinetin,
It is preferable to use those obtained by aseptically planting sections of stems, rhizomes, etc. and then inducing differentiation.

Further, the callus and plant cells (tobacco etc.) obtained by sterilizing the plant body and aseptically implanting the explant into the medium (callus induction) can also be used. In addition, the number of bacteria in the medium
It is better to make it exist at 10 ⁵ to 10 ⁷ / ml.

The medium composition used for the mixed culture of the above-mentioned fungus and plant in the present invention includes a carbon source ... Sugars such as glucose and fructose or sugar alcohols such as glycerol Nitrogen source ... Inorganic salts such as ammonium salts and nitrates. Nitrogen-containing compound Inorganic salt: Uses components such as phosphates and sulfates of metals such as potassium and magnesium that are usually added to the medium. In order to directly obtain ferropyrimine from pyrimine, it is preferable that divalent iron ions be present in the medium. Such iron ion can be supplied in the form of a salt such as iron sulfate and iron citrate, and it is preferable that the salt is present in the medium at 10 ppm or more, preferably 20 to 100 ppm.

The medium for producing pyrimine may be solid or liquid, and can be cultured by various methods, but rotary shaking culture and aeration and stirring culture are preferable. Furthermore, it is preferable to carry out the culture under aerobic conditions such as culture under light irradiation.
Culture conditions are appropriately selected and adjusted so as to maximize the target amount of accumulated ferropyrimine, and the following conditions are preferable.

Medium pH 4.0-7.0, more preferably pH 5.0-6.0 Culture temperature 15-35 ℃, More preferably 20-25 ℃ Culture time 14-20 days To obtain the pyrimine produced in the medium by the above method, Separation and purification means can be adopted by a conventional method.

EFFECTS OF THE INVENTION The present invention provides a novel pyrimine-producing bacterium and a novel method for producing pyrimine. Therefore, the red natural pigment ferropyrimine can be easily obtained by adding an iron salt to the obtained pyrimine in an aqueous solution.

 Next, the present invention will be described with reference to examples.

Example 1 Linsmaier-Skoog basal medium (NH ₄ No ₃ 1650 mg, KNO ₃ 190
0 mg, CaCl ₂ .2H ₂ O 440 mg, MgSO ₄ 7H ₂ O 370 mg, KH ₂ PO ₄
170 mg, H ₃ BO ₃ 6.2 mg, MnSO ₄ / 4H ₂ O 22.3 mg, ZnSO ₄ / 7H ₂
O 8.6mg, KI 0.83mg, Na ₂ MoO ₄・ 2H ₂ O 0.25mg, CoCl ₂・ 6
_{H 2 O 0.025mg, CuSO 4 ·} 5H 2 O 0.025mg, Na 2 -EDTA 37.3m
g, FeSO ₄ .7H ₂ O 27.8 mg, myo-inositol 0.4 mg, thiamine hydrochloride 0.1 mg, sucrose 3000 mg, distilled water 1000 ml)
Add 0.1 mg each of naphthyl acetic acid and kinetin, and adjust the pH.
The medium adjusted to 5.8 (hereinafter referred to as LS medium) 100 ml 300 m
Ten pieces were dispensed into an Erlenmeyer flask with a volume of l, and the pieces were capped with a cotton plug and sterilized at 121 ° C. for 15 minutes. After cooling, these were inoculated with horseradish seedlings that had been aseptically grown on LS agar medium (containing 0.8% agar) in advance, and then inoculated with Pseudomonas sp. K strains grown on broth agar medium, and under light irradiation. The cells were cultivated at 25 ° C with rotary shaking (120 rpm).

After 20 days, the total number of culture supernatants obtained by centrifugation to remove bacterial cells was 1 and it contained 266 μg / ml ferropyrimine calculated from the calibration curve by the absorbance at 558 nm.
In order to separate ferropyrimine from this culture supernatant,
First, twice the amount of methanol was added to the supernatant, the resulting precipitate was separated by filtration and then concentrated under reduced pressure. Dissolve this in a small amount of 50% methanol and add Kieselgel 60 (Merck 70-230mesh) to 5
A column having a diameter of 20 cm and a length of 30 cm packed in a glass tube for chromatography with 0% methanol was added and adsorbed.
Then, a reddish purple section which was eluted with 50% methanol and which was flowing out was collected.

Cellulose powder (Avicel = Funakoshi Chemical Co., Ltd.) was added to this concentrated solution, mixed well, dried under reduced pressure at an external temperature of 40 ° C. or less, and then ground well in a mortar to obtain a uniform powder. Next, column chromatography was carried out by adding methanol to a column having a diameter of 2.0 cm and a length of 30 cm, which was packed with cellulose powder as it was in a glass tube for chromatography. Collected. After concentrating this section under reduced pressure, it was dissolved in a small amount of methanol and Toyopearl HW-40 (F) (Tosoh Corp.) was added to a column of 2.5 cm in diameter and 45 cm in which a glass tube for chromatography was packed with methanol. It was eluted with methanol, and the reddish-purple sections that eluted were collected. This was concentrated under reduced pressure and dissolved in a small amount of water.

Then, Dowex 50 (H ⁺ ) resin was filled with water into a glass tube for chromatography to prepare a column having a diameter of 1.0 cm and a length of 15 cm, and the above sample was added to the column for adsorption and then 1N-NH 2
₃ OH was added to collect the reddish purple fraction that was eluting. After concentrating to dryness under reduced pressure, the residue was dissolved in a small amount of water, and acetone was added thereto.
This was collected, washed with acetone-hexane, and dried under reduced pressure to obtain 3 mg of a dark purple pigment.

The ¹³ C-NMR spectrum of this dye was measured in methanol, the ¹ H-NMR spectrum of the dye component was measured, and the optical rotation was also measured, and it was confirmed that the dye was dextrorotatory D ⁺ -type ferropyrimine. .

In addition, when the UV-visible absorption spectrum was measured in an aqueous solution, Biochemistry Vol.4 No.10 (1965)
It coincided with the spectrum pattern of FIG. 1 in the upper left column of page 2235.

The above experiment was performed in the same manner except that FeSO ₄ .7H ₂ O was removed from the medium to obtain pyrimine. Then
When FeSO ₄ .7H ₂ O was added to the aqueous solution of pyrimine, a red color of ferropyrimine was produced, and its spectrum was the same as that described above.

Example 2 Ten LS media (excluding FeSO ₄ .7H ₂ O) dispensed into a 300 ml Erlenmeyer flask for 100 ml were prepared, and a cotton plug was sterilized at 121 ° C. for 15 minutes. After cooling, each of them was inoculated with horseradish seedlings that had been aseptically grown on LS agar medium in advance, and a mixed culture solution of Pseudomonas putida K strain and horseradish seedlings shown in Example 1 at 25 ° C. for 20 days was added. When 10 ml was inoculated and cultured under light irradiation at 25 ° C. under rotary shaking (120 rpm), the accumulated amount of pyrimine after 14 days was on average 298 mcg / ml.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Osamu Fujii 1-5-7 Mikitei-cho, Higashi-Osaka City, Osaka Prefecture House Food Industry Co., Ltd. (72) Shinsuke Imai 1-5 Mikitei-cho, Higashi-Osaka City, Osaka Prefecture No. 7 House Food Industry Co., Ltd. (56) References Biochemistry 4 [10] (1965) (US) P. 2233-2236

Claims (3)

(57) [Claims] 1. A method for producing pyrimine, which comprises cultivating a pyrimine-producing bacterium of the genus Pseudomonas with a plant and collecting the pyrimine from the culture. 2. A Pyrimine-producing bacterium is a Pseudomonas sp. K.
The method according to claim 1, which is Microorganisms Research Institute, Ltd. 3. The plant according to claim 1, wherein the plant is a plant, plant tissue, callus of plant, or plant cell selected from the group consisting of Liliaceae, Brassicaceae, Lamiaceae, Leguminaceae and Saccharidae. Manufacturing method.