JP2558524B2 - Process for producing pyrimine - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for efficiently producing pyrimine useful as an intermediate for ferropyrimine which is a natural pigment.

[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 belongs to the genus Pseudomonas in the past, produces pyrimine, and at the same time binds iron ions present in the culture 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] It is an object of the present invention to provide a method capable of producing pyrimine efficiently.

[Means for solving the problem]

The present invention was made based on the finding that the above problems can be efficiently achieved by culturing a pyrimine-producing bacterium with a plant and in a medium containing a specific element.

That is, the present invention, a pyrimine-producing bacterium belonging to the genus Pseudomonas is cultured in the presence of a plant in a medium containing a sugar, a nitrogen-containing compound, a magnesium compound, a phosphorus compound and an iron compound but not containing calcium ion and chloride ion. Then, there is provided a method for producing pyrimine, which comprises recovering the pyrimine produced.

Examples of the pyrimine-producing bacterium used here include those having the following mycological properties.

Mycological properties 1. Morphological properties Morphology: Single or not forming bacillus spores that form chains Reactivity: Yes, with polar flagella Large: 0.6-0.8 μx 1.0-3.5 μ Gram stain: Negative 2 .Growth condition in each medium (1) Broth culture Growth: Medium, Film: Ring formation Sediment: Slightly cloudy: Yes (2) Slope culture of broth Shape: Thread, Surface: Smooth and glossy Edge: All Edge, color: milky white Transparency: opaque (3) Broth agar plate culture Shape: round, peripheral: all edges, surface ridge shape: lenticular Surface: smooth and glossy, color: milky white (4) Bouillon agar puncture culture Grows on the surface and upper puncture 3. Physiological properties (1) Attitude toward oxygen: aerobic (2) Catalase: + (3) Oxidase: + (4) Arginine dehydrolase: − (5) Lipase (hydrolysis of Tween 80 Solution): + (6) Lecithinase (yolk decomposition) :-( 7) Hydrolysis of gelatin :-( 8) Hydrolysis of starch :-( 9) Hydrolysis of extracellular poly-β-hydroxybutyric acid (PHB): - (10) utilize to independently nutritionally growing H _2: - (11) generated in the dye: King a, B fluorescence medium, glutamate medium, Pseudomonas F agar, Pseudomonas P agar, TSI agar, potato dextrose agar address Does not produce pigment.

(12) Reducibility of nitrate :-( 13) Denitrification reaction :-( 14) Degradation position of protocatechurate: Ortho (15) Cellular accumulation of PHB: + (16) Levan formation from sucrose :-( 17) ) Growth temperature: 11-35 ℃ Optimum 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-Lysine: + L-Valine: ± β-Alanine: + Glycolate: + p-Hydroxybenzoate: + According to the above 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 positive for catalase and oxidase. It was

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
-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.

In addition, it has the above-mentioned mycological properties similar to Pseudomonas sp.K strain, but produces more than 400 μg / ml of pyrimine by culturing with a plant in LS basic medium, and the period from the start of culture to the production of pyrimine. Pseudomonas sp. K-1 strain (Microtechnology Research Institute
FERM BP-2932) can also be used.

Furthermore, Pseudomonas sp. K strain means that pyrimine is produced at 400 µg / ml or more by culturing with a plant in L-lysine- and LS basic medium, and the period from the culture to the start of pyrimine production is within 8 days. Pyrimine-producing bacteria of the genus Pseudomonas, for example, Pseudomonas sp. K-2 strain (FERM BP-2933, Institute for Microbiology Research) can be used.

In the present invention, when culturing a bacterium, it is characterized by culturing with a plant in a specific medium. Plants used here include lilies such as green onions, crucifers such as lettuce, cabbage, wasabi, radish and watercress, Lamiaceae such as perilla, basil and mint, and Polygonaceae such as sorel. Plants, legumes such as pods, plants of the Solanaceae such as tomatoes, eggplants and capsicums, and plants of the Tobacco 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 used for the mixed culture of the above-mentioned fungus and plant in the present invention does not contain calcium ion and chloride ion containing sugar, nitrogen-containing compound, magnesium compound, phosphorus compound and iron compound. Here, the sugar acts as a carbon source, and examples thereof include sugars such as glucose, fructose, and sucrose, and sugar alcohols such as glycerols. The nitrogen-containing compound functions as a nitrogen source, and examples thereof include inorganic nitrogen-containing compounds such as ammonium salts and nitrates. Magnesium compounds are compounds that supply magnesium ions, such as magnesium sulfate, phosphorus compounds are compounds that supply phosphorus ions, such as potassium phosphate and sodium phosphate, and iron compounds supply iron ions. Compounds such as iron sulfate. If divalent iron ions are allowed to exist in the medium, ferropyrimine can be directly obtained from pyrimine.

As the medium used in the present invention, specifically,
Sugar 20 to 80 g, preferably 30 to 60 g, nitrogen-containing compound 1.0 to 5.0
g, preferably 1.5 to 3.5 g, magnesium compound 0.1 to 0.5
g, preferably 0.3-0.4 g, phosphorus compound 0.1-0.8 g, preferably 0.15-0.5 g and iron compound 0.01-0.2 g, preferably
An example of the medium is 0.02-0.15 g, and the balance is water.
In addition, components other than calcium ion and chloride ion can be added to this medium. For example, 0 to 0.2 g of EDTA alkali metal salt, 0 to 20 g of agar, and the like.

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 pH 3.8 to 6.0 of medium in culture, preferably pH 4 .0-4.5 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〕

INDUSTRIAL APPLICABILITY According to the present invention, there is provided a novel method for producing pyrimine which is industrially investigated and has high production efficiency. Therefore, the red natural pigment ferropyrimine can be easily obtained only 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 S modified medium (sucrose 30000 mg, NH ₄ NO ₃ 1650 mg, M
gSO ₄ / 7H ₂ O 370mg, KH ₂ PO ₄ 500mg, FeSO ₄ / 7H ₂ O 27.
8mg, Na ₂ -EDTA 37.3mg, distilled water 1000 ml, pH 7.0) 50 ml
Two pieces were placed in an Erlenmeyer flask with a capacity of 300 ml, and a cotton plug was put on them 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 then grown on GS modified medium (preparation method is shown below).
Inoculate 1 platinum loop of sp.K strain and rotate at 25 ° C under light irradiation.
0 rpm) was 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 160 μg / ml.

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 · 7
Composition consisting of H ₂ O 27.8 mg, Mn ₂ -EDTA 37.3 mg, agar 20000 mg, and distilled water 1000 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.

LS agar medium Linsmaier-Skoog basal medium (NH ₄ NO ₃ 1650 mg, KNO _{3
1900mg, 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 · SH ₂ O 22.3 mg, Zn
SO ₄・ 7H ₂ O 8.6mg, KI 0.83mg, Na ₂ MoO ₄・ 2H ₂ O 0.25m
_{g, CoCl 2 · 6H 2 O} 0.025mg, CuSO 4 · 5H 2 O 0.025mg, Na 2
_{-EDTA 37.3mg, FeSO 4 · 7H 2} O 27.8mg, myoinositol 100mg, thiamine hydrochloride 0.4 mg, sucrose 3000 mg, distilled water 1000ml) to α- naphthyl acetate and kinetin was 0.1mg each addition, and addition of agar 0.8% A medium whose pH has been adjusted to 5.8.

Example 2 Pyrimine was prepared in the same manner as in Example 1 except that Pseudomonas sp. K-1 strain was used as a pyrimine-producing bacterium and the cells were removed by centrifugation after 12 days instead of after 17 days. Was produced, the content of ferropyrimine in the culture was 1360 μg / ml.

Example 3 Pyrimine was prepared in the same manner as in Example 1 except that Pseudomonas sp. K-2 strain was used as a pyrimine-producing bacterium and the cells were removed by centrifugation after 13 days instead of after 17 days. Was produced, the content of ferropyrimine in the culture was 940 μg / ml.

Claims (1)

(57) [Claims] 1. A pyrimine-producing bacterium belonging to the genus Pseudomonas is cultivated in the presence of a plant in a medium containing a sugar, a nitrogen-containing compound, a magnesium compound, a phosphorus compound and an iron compound but not calcium ion and chloride ion, Next, a method for producing pyrimine, which comprises recovering the produced pyrimine.