JP2558542B2 - 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 as compared with synthetic pigments in terms of safety. 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 bacillus spores alone or in chains Motility: Yes, with flagella Size: 0.6-0.8μ × 1.0-3.5μ Gram stain: Negative 2. Each medium Growth condition (1) Broth culture Growth: Moderate, film: Ring formed Sediment: Slightly turbid, Yes (2) Slope culture broth Shape: Filamentous, surface: smooth and glossy Edge: All edges, color tone : Milky white Transparency: Opaque (3) Broth agar plate culture Shape: Round, Peripheral: Full edge, Surface ridge shape: Lenticular Surface: Smooth and glossy, Color: Milky white (4) Broth agar stab culture surface and top Growth along the buccal area 3. Physiological properties (1) Attitude toward oxygen: aerobic (2) Catalase: + (3) Oxidase: + (4) Arginine dehydrolase:-(5) Lipase: + (Tween 80 Water Solution) (6) Lecithinase:-(Egg yolk decomposition) (7) Gelatin hydrolysis:-(8) Starch hydrolysis:-(9) Extracellular poly-β-hydroxybutyric acid (PHB) hydrolysis:-( 10) Grow autotrophically using H ₂ : − (11) Production of pigment: Pseudomonas F agar and Pseudomonas P agar do not produce fluorescent pigment.

(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 11-40 ℃ Optimum temperature 30-32 ℃ (18) Growth pH: 3.8-8.5 Optimum pH 5.5-6.5 (19) Carbon source assimilation: D-glucose: + D-fructose: + D- Arabinose: + trehalose: ± mannitol: + inositol: + glycerol: + L-lysine: -L-valine: ± β-alanine: + glycolate: + p-hydroxybenzoede: + According to the above-mentioned mycological properties, The categorical status of the strains of the present invention is described in "Bergey's Manual of Systematic Bacteriolog" by Vergie.
y) As a result of Volume 1 (1984), this strain is a Gram-staining bacillus that exhibits motility due to polar hairs and is aerobic and catalase-positive and oxidase-positive, so it is considered to belong to the genus Pseudomonas. It was

Of the genus Pseudomonas, poly-β-hydroxybutyrate accumulates intracellularly as a carbon source storage substance, grows at 40 ° C, and shows the property of decomposing protocatechurate at the ortho position, and it has no arginine decomposing property and denitrification reaction. Pseudomonas cepacia (Pseudomona)
s cepacia) and Pseudomonas gladioli (Pseudomon)
as gladioli) exists.

However, as shown below, the characteristics are clearly different from Pseudomonas cepacia in that sucrose cannot be used as a carbon source and that glycine can be used. Further, it differs from Pseudomonas gladioli in that it does not produce levan from sucrose, has no gelatin hydrolyzability, and has no lecithinase.

Therefore, it was judged appropriate that this strain is a new strain belonging to the genus Pseudomonas, and Pseudomonas sp.
It was named a strain. This strain has been deposited as FERM BP-2933, a microbiology strain.

Incidentally, the Pseudomonas sp. K strain deposited as the Japan Institute of Microbiology No. 9628 refers to the K-2 strain as L-lysine- and LS.
It is different in that pyrimine is produced in an amount of 400 μg / ml or more by culturing with a plant in a basic medium, and the period from the culture to the start of pyrimine production is within 8 days.

Suitable pre-culture conditions for the Pseudomonas sp. K-2 strain used in the present invention include broth agar medium and GS modified medium (described later).
Examples of the method include culturing at 25 ° C. for 2 to 3 days in the same manner and directly inoculating the LS medium with the bacteria, but it is also possible to culture in the LS medium from our laboratory.

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 3.8 to 6.0 during culture, pH of medium 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 ₃ 190
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・
_{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 30000 mg, distilled water 1000 ml) to α-naphthyl acetic acid and kinetin 0.1 mg / respectively, a medium adjusted to pH 5.8 ( Hereinafter referred to as LS medium) 50 ml
10 pieces of agarose were dispensed into a 300 ml Erlenmeyer flask were prepared, and a cotton plug was placed on them to sterilize at 121 ° C. for 15 minutes. After cooling, this was seeded with horseradish seedlings that had been aseptically grown in LS agar medium (containing 0.8 agar) in advance, and further grown in GS modified medium (preparation method is shown below) sp.K sp.K. -2
1 platinum loop of the strain was inoculated and shaken under light at 25 ° C (150 rp)
m) Cultured.

After 17 days, the culture supernatant obtained by centrifugation to remove bacterial cells was filtered with a 0.45 μm membrane filter. After adding a FeSO ₄ .7H ₂ O solution (278 mg /) 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 1650 μg / ml. Pyrimine was produced from the 6th 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 6.0) After the above (a) and (b) were autoclaved at 121 ° C. for 15 minutes, 2 ml of (b) was added to 11 ml of (a) to prepare GS. The medium was adjusted.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location C12R 1:38) (72) Inventor Rie Okabe 1-5-7 Mikitei-cho, Higashiosaka-shi, Osaka House Food Industry Co., Ltd. (56) References Biochemistry 4 [10] (1965) (US) P. 2233−2236

Claims (2)

(57) [Claims] 1. A pyrimine-producing bacterium, Pseudomonas sp. K-2, belonging to the genus Pseudomonas, which comprises the following mycological substance. Denitrification-Use as carbon source D-arabinose + L-lysine-LS Pyrimine is produced at 400 µg / ml or more by culturing with a plant in a basic medium, and the period from 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.