JP2558526B2 - Pyrimine-producing bacterium and method 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, It is carried out by the method of collecting ferropyrimine from the culture (Biochemistry Vol.4 No.10, 1965).
Year 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 spores of rod-shaped bacilli alone or in motility: Yes, with polar flagella Size: 0.6-0.8μ × 1.0-3.5μ Gram stain: Negative 2. Each medium (1) Broth culture Growth: Medium Capsule: Ring formation Sediment: Slight turbidity: Yes (2) Bouillon slope culture Shape: Thread surface: Smooth and glossy Edge: Full edge Color: Milky white Transparency : Opaque (3) Broth agar plate culture Shape: Round Edge: Full edge Surface ridge shape: Lenticular Surface: Smooth and glossy, color tone: Milky white 3. Physiological properties (1) Attitude toward oxygen: aerobic (2) Catalase: + (3) Oxidase: + (4) Arginine dehydrolase:-(5) Lipase: + (hydrolysis of Tween 80) ( ) Lecithinase: - (yolk decomposition) (7) Hydrolysis of gelatin: - (8) Hydrolysis of starch: - Hydrolysis of (9) extracellular poly -β- hydroxybutyrate (PHB): - (10) H ₂ Grow autotrophically by using :-( 11) Pigment formation: Pseudomonas F agar and Pseudomonas P agar do not produce a fluorescent pigment.

(12) Reducibility of nitrate:-(13) Denitrification reaction:-(14) Decomposition position of protocatechurate: Ortho (15) Cellular accumulation of PHB: + (16) Levan production from sucrose:-(17) ) Growth temperature 11-35 ℃ Optimum temperature 18-35 ℃ (18) Growth pH: 3.8-8.5 Optimum pH 4.5-8.0 (19) Carbon source utilization: D-glucose: + D-fructose: + D- Arabinose: + Trehalose: ± Mannitol: + Inositol: + Glycerol: + L-lysine: + L-valine: ± β-alanine: + Glycolate: + p-Hydroxybenzoate: + According to the above mycological properties, The categorical status of the strains of the invention "The Manual of Bacteriology" by Vergie
(Bergey's Manual of Systematic Bacteriology) Volu
As a result of determination by me 1 (1984), this strain was considered to belong to the genus Pseudomonas because it was a Gram-staining negative bacillus, which showed motility due to polar hairs, and was aerobic and 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 carbon source
-This strain (Pseudomonas sp. K-1) differs from Pseudomonas solanacerum in that it cannot utilize arabinose.

Therefore, it was judged appropriate that this strain be a new strain belonging to the genus Pseudomonas, and Pseudomonas sp.
It was named a strain. This strain has been deposited as FERM BP-2932, a microbiological research institute.

The Pseudomonas sp. K strain deposited as Microindustrial Research Institute No. 9628 means that K-1 strain produces pyrimine at 400 μg / ml or more by culturing with a plant in LS basic medium, and The difference is that it takes less than 8 days to start producing pyrimine.

Suitable pre-culture conditions for the Pseudomonas sp. K-1 strain used in the present invention include broth agar medium and GS modified medium (described later).
For example, a method of directly inoculating the LS medium with the cells that have been cultured at 25 ° C for 2 to 3 days can be mentioned, but the LS medium can also be cultured from the beginning.

In the present invention, the cultivation of the fungus is characterized by culturing with the plant. Plants used here include lilies such as green onions, crucifers such as lettuce, cabbage, wasabi, radish, and watercress, perilla, basil,
Mint and other Lamiaceae plants, Soler and other Polygonaceae plants, Peas and other legume plants, tomatoes, eggplants,
Plants of the Solanaceae family such as capsicum and plants of the Tobacco family Nicotiana family such as Saintpaulia 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. Alternatively, sterilized seeds may be planted in the LS agar medium and germinated and grown.

It is also possible to use the callus or plant cells (tobacco etc.) obtained by sterilizing the plant body and aseptically implanting the explant into the medium (callus induction). The amount of plant added is arbitrary, but 0.01 to 5 g, preferably 0.1 to 0.5 g, per 100 parts by weight of the medium.
It is good to do. In addition, it is preferable to inoculate about 10 ⁵ to 10 ⁷ / ml of the bacteria in the medium.

The medium composition used for the mixed culture of the above-mentioned fungus and plant in the present invention includes carbon sources such as sugars such as glucose, fructose and sucrose, or sugar alcohols of glycerol such as nitrogen sources such as ammonium salts and nitrates. Inorganic Nitrogen-Containing Compound Inorganic salt: a component such as a metal phosphate such as potassium or magnesium added to a normal medium, such as a sulfate. In order to directly obtain ferropyrimine from pyrimine, it is preferable that divalent iron ions be present in the medium. Such iron ions can be supplied in the form of a salt such as iron sulfate, and it is preferable that 10 ppm or more, preferably 20 to 100 ppm, be present as a salt in the medium.

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.

Initial medium pH 3.8 to 7.0 More preferably pH 5.0 to 6.0 Culture temperature 15 to 30 ° C, more preferably 20 to 25 ° C Culture time 8 to 20 days Medium pH during culture pH 3.8 to 6.0, preferably 4.0 to 4.5 In order to obtain the pyrimine produced in the medium by the above method, separation and purification means can be adopted by a conventional method.

〔The invention's effect〕

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 19
_{00mg, CaCl 2 · 2H 2 O} 440mg, MgSO 4 · 7H 2 O 370mg, 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・
_{6H 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 100 mg, thiamine hydrochloride 0.4 mg, sucrose 3000 mg, distilled water 1000 ml) α-naphthyl acetic acid and kinetin 0.1 mg l / each were added, and the medium was adjusted to pH 5.8 ( Hereinafter referred to as LS medium) 50m
Ten pieces of l were dispensed into a 300 ml Erlenmeyer flask, which were capped with cotton plugs to sterilize at 121 ° C for 15 minutes. After cooling, this was seeded with horseradish seedlings that had been aseptically grown on LS agar medium (containing 0.8% agar) in advance, and further grown on GS improved medium (conditioning method is shown below) sp. Pseudomonas sp. K-
1 strain of 1 platinum loop was inoculated and shaken under light at 25 ° C (150r).
pm) Cultured.

After 17 days, the culture supernatant obtained by centrifugation to remove bacterial cells was filtered with a 0.45 μm membrane filter. After the FeSO ₄ .7H ₂ O solution (278 mg /) was added to this solution, the absorbance at 558 nm was measured and the content of ferropyrimine in the culture solution was determined from the calibration curve to be 1750 μg / ml. Pyrimine was produced from the 5th day of culture.

GS modified medium (a) Glucose 30,000 mg, meat extract (manufactured by Difco) 7
_{70mg, NH 4 NO 3 1650mg,} MgSO 4 · 7H 2 O 370mg, FeSO 4 · 7H 2
O 27.8 mg, Na ₂ -EDTA 37.3 mg, agar 20000 mg, distilled water 100
Composition consisting of 0 ml (adjusted with pH 6.0: 1 N-KON).

(B) 1M-potassium phosphate buffer (pH 5.0) The above (a) and (b) were autoclaved at 121 ° C. for 15 minutes, and then 2 ml of (b) was added to 11 ml of (a) to prepare GS. The medium was adjusted.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location C12R 1:38)

Claims (2)

(57) [Claims] 1. A pyrimine-producing bacterium Pseudomonas sp. K-1 belonging to the genus Pseudomonas, which has the following mycological properties. Denitrification reaction-Use as carbon source D-arabinose + L-lysine + LS By culturing with a plant in a basic medium, pyrimine is produced at 400 µg / ml or more, and the period from the culture to the start of pyrimine production is 8 days. Within. 2. A method for producing pyrimine, which comprises culturing the pyrimine-producing bacterium according to claim 1 together with a plant and collecting the pyrimine from the culture.