JP2593819B2 - Pyrimine producing bacteria - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a pyrimine-producing bacterium useful as an intermediate of ferropyrimine, a natural pigment.

2. Description of the Related Art In recent years, natural dyes have attracted attention from the viewpoint of safety of synthetic dyes. Of these, ferropyrimin, a natural red pigment, is conventionally cultured with a GH strain belonging to the genus Pseudomonas, and at the same time as producing pyrimine, is combined with iron ions that have been present in the culture to form ferropyrimin, Ferropyrimin is collected from the culture (Biochemistry Vo
l.4, No. 10, 1965, pp. 2233-2236
page). Pyrimine has the following structural formula:

[0002]

Embedded image

The structural formula is described in the above biochemistry.

DISCLOSURE OF THE INVENTION The present invention provides a novel bacterium which is different from the bacterium used in the above-mentioned conventional method and which produces, for example, a pyrimine useful as an intermediate of ferropyrimine. The purpose is to do.

Means for Solving the Problems The present invention has been made based on the finding that pyrimines can be efficiently produced by culturing a novel Pseudomonas bacterium. That is, the present invention provides a pyrimine-producing bacterium having the following mycological properties.

Bacteriological properties 1. Morphological properties Form: singly or linked bacilli Bacteria that do not form spores Motility: Yes, with polar flagella Size: 0.6 × 0.8 μ × 1.0-3 0.5μ gram staining: negative 2. Growth condition in each medium (1) Broth culture Growth: Medium, film: formed a ring Sediment: slightly, turbidity: yes (2) Slope culture of bouillon Shape: filamentous, surface: smooth Perimeter: whole edge, color tone: milky white Transparency: opaque (3) Bouillon agar plate culture Shape: perfect circle, peripheral edge: whole edge Shape of surface protuberance: lens shape Surface: smooth and glossy, color tone: milky white ( 4) Broth agar culture grown on the surface and upper perforation

3. Physiological properties (1) Attitude to oxygen: aerobic (2) Catalase: + (3) Oxidase: + (4) Arginine dehydrolase:-(5) Lipase (hydrolysis of Tween 80): + (6) Lecithinase (yolk degradation):-(7) Hydrolysis of gelatin:-(8) Hydrolysis of starch:-(9) Hydrolysis of extracellular poly-β-hydroxybutyric acid (PHB):-(10) Growing autotrophically using H ₂ :-(11) Pigment generation: no fluorescent dye is produced on King A, B medium, glutamate medium, Pseudomonas F agar, Pseudomonas P agar, TSI agar, potato dextrose agar medium . (12) Reducing ability of nitrate:-(13) Denitrification reaction:-(14) Decomposition position of protocatechulate: ortho (15) Intracellular accumulation of PHB: + (16) Production of levan from sucrose:-(17 ) Growth temperature 11 ~ 35 ℃ Optimum temperature 18 ~
35 ° C (18) Growth pH 4.0-8.5 Optimum pH 4.5-8.0

(19) Utilization of carbon source D-glucose: + D-fructose: + D-arabinose: + trehalose: ± mannitol: + inositol: + glycerol: + L-valine: ± β-alanine: + glyco Rate: + p-hydroxybenzoate: +

In accordance with the above mycological properties, the taxonomic status of the strain of the present invention was determined by Bergey's Manual of Systematic Bact.
eriology) As determined by Volume 1 (1984), this strain is a gram-stained negative bacillus that exhibits polar motility and is aerobic and catalase-positive and oxidase-positive, and is considered to belong to the genus Pseudomonas. Was. Among the genus Pseudomonas, Pseudomonas solanacelam (according to the property of accumulating poly-β-hydroxybutyric acid as a carbon source storage substance in cells and not being able to grow at 40 ° C. and decomposing brotocatechuic acid at the ortho position) is considered. Ps
eudomonas solanacearum). However, as shown below, the strain is different from the present strain in that D-arabinose cannot be used as a carbon source.

[0008]

[Table 1] Table 1 Properties Pseudomonas pseudomonas sp. K Solanaceram denitrification reaction-+ Utilization of carbon source +-D-arabinose

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 is provided by
Deposited as No. 8. Suitable pre-culturing conditions for the Pseudomonas sp. K strain used in the present invention include a method of inoculating bacteria grown on a broth agar medium at 25 ° C. for 2 to 3 days directly into an LS medium. It is also possible to culture in an LS medium. In the present invention, the cultivation of the fungus is characterized by culturing with the plant. Plants used here include plants of the lily family such as green onions, lettuce,
Brassicaceae plants such as cabbage, wasabi, radish and watercress, liana leaves, basil, sorel, mint and other Lamiaceae plants, legume plants such as pods, and pallidaceae plants such as Saintpaulia are preferably used. it can. Here, as the plant, it is preferable to use a plant obtained by aseptically planting a section of the leaf, stem, rhizome or the like of the plant on an LS agar medium supplemented with hormones such as α-naphthylacetic acid and kinetin and then inducing differentiation. Good.

[0010] It is also possible to sterilize the plant body, aseptically implant the explant into a medium (callus induction), and use the resulting callus or plant cells (tobacco, etc.). It is preferable that the bacteria be present in the medium in a number of 10 ⁵ to 10 ⁷ / ml. In the present invention, the medium composition used for the mixed cultivation of the above-mentioned bacteria and plants includes a carbon source: sugars such as glucose and fructose, or sugar alcohols such as glycerol; a nitrogen source: inorganic sources such as ammonium salts and nitrates. Nitrogen-containing compounds of inorganic salts: Components that are usually added to a medium, such as phosphates and sulfates of metals such as potassium and magnesium, are used. 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, iron citrate, or the like.
m should be present. The medium for producing pyrimine may be either solid or liquid, and can be cultured by various methods. Rotational shaking culture or aeration and stirring culture is preferred. Furthermore, it is preferable to 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 ° C, more preferably 20-25
C. Incubation time 14 to 20 days In order to obtain pyrimine produced in the medium by the above method, separation and purification means can be adopted by a conventional method.

According to the present invention, a novel pyrimine-producing bacterium is provided. Therefore, ferropyrimine as a red natural pigment 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.

[0012]

EXAMPLES Example 1 Linsmaier-Skoog basic medium (NH ₄ No ₃ 1650 mg, KNO
₃ 1900mg, CaCl ₂・ 2H ₂ O 440mg, 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 22.3mg, ZnSO 4} · 7H 2 O 8.6mg, KI 0.83
_{mg, Na 2 MoO 4 · 2H} 2 O 0.25mg, CoCl 2 · 6H 2 O 0.02
_{5mg, CuSO 4 · 5H 2 O} 0.025mg, Na 2 -EDTA 37.3m
_{g, FeSO 4 · 7H 2 O} 27.8mg, myo-inositol 0.4m
g, thiamine hydrochloride 0.1 mg, sucrose 3000 mg, distilled water 1
000 ml) with α-naphthylacetic acid and kinetin
0.1 mg was added, and 100 ml of a medium (hereinafter referred to as LS medium) adjusted to pH 5.8 was dispensed into a 300 ml Erlenmeyer flask. Ten cotton tubes were plugged and sterilized at 121 ° C for 15 minutes. did. After cooling, LS agar medium (0.8
% Agar) aseptically grown, inoculated with a horseradish seedling, and further inoculated with a Pseudomonas sp. K strain grown on a gravy agar medium, and rotated and shaken at 25 ° C under light irradiation (120 ° C).
rpm).

Twenty days later, the culture supernatant from which cells were removed by centrifugation was 1 liter in total, and contained 266 μg / ml of ferropyrimine as calculated from a calibration curve based on the absorbance at 558 nm. In order to separate ferropyrimine from the culture supernatant, first, twice the amount of methanol was added to the supernatant, and the resulting precipitate was filtered off and concentrated under reduced pressure. This is dissolved in a small amount of 50% methanol, and Kieselgel 6
0 (Merck 70-230 mesh) filled in a glass tube for chromatography with 50% methanol, 20 cm in diameter,
The sample was adsorbed on a column having a length of 30 cm. Then 50%
The red-purple section eluted and eluted with methanol was collected. Cellulose powder (Avicel = Funakoshi Chemical Co., Ltd.) was added to the concentrate, 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, the cellulose powder was charged into a glass tube for chromatography as it was, having a diameter of 2.0 cm and a length of 3 cm.
The powder was packed in a 0 cm column, methanol was added thereto, and column chromatography was carried out to collect a red-purple section flowing out. This fraction was concentrated under reduced pressure, dissolved in a small amount of methanol, and added to a 2.5 cm diameter, 45 cm column of Toyopearl HW-40 (F) (Tosoh Corporation) filled in a glass tube for chromatography with methanol. And eluted with methanol, and the eluted red-purple section was collected. This was concentrated under reduced pressure and dissolved in a small amount of water.

[0014] Then Dowex 50 (H ^-) resin to create a column packed with a diameter 1.0cm 15cm long glass tube for chromatography with water, adsorbed by the addition of the sample, then 1N-NH ₃ OH Was added and the reddish purple section eluted was collected. 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 dye. 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 measured. As a result, it was confirmed that the dye was dextrorotatory D ⁺ -type ferropyrimine. . Further, when the absorption spectrum of the ultraviolet visible region was measured in an aqueous solution, it was found that Biochemistry Vol. 4 No. 10 (1965) No. 2235
It coincided with the spectrum pattern of FIG. 1 in the upper left column of the page.
Incidentally, the above experiment, except excluding the FeSO ₄ · 7H ₂ O from the medium, carried in the same manner to obtain a Pirimin. Then
An aqueous solution of Pirimin was added FeSO ₄ · 7H ₂ O, red Feropirimin occurs, the spectrum was the same as described above.

[0015] Example 2 LS medium (except excluding FeSO ₄ · 7H ₂ O) Erlenmeyer flask 300ml those dispensed 100ml addressed fraction prepared ten, were sterilized by a cotton plug 121 ° C. 15 min. After cooling, each of them was inoculated with a horseradish seedling that had been aseptically grown on an LS agar medium, and the Pseudomonas putida K strain shown in Example 1 and the horseradish seedling at 25 ° C. 20
When 10 ml of the mixed culture solution was inoculated for 25 days, the culture was rotated and shaken (120 rpm) at 25 ° C under light irradiation, and after 14 days, the accumulated amount of pyrimine was 298 mcg / ml on average.

──────────────────────────────────────────────────の Continuation of the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location C12R 1:38) (72) Inventor Osamu Fujii 1-5-7 Miguisakaemachi, Higashi Osaka City, Osaka Prefecture House Foods Co., Ltd. (72) Inventor Shinsuke Imai 1-5-7 Mikitei Sakaecho, Higashi Osaka-shi, Osaka House Foods Co., Ltd. (56) References BIOCHEMSTRY 4-10! (1965) p. 2233−2236

Claims (1)

(57) [Claims] A pyrimine-producing bacterium Pseudomonas sp. K belonging to the genus Pseudomonas having the following mycological properties. Bacteriological properties 1. Morphological properties Form: singly or linked bacilli Not forming spores Motility: Yes, with polar flagella Size: 0.6 × 0.8μ × 1.0-3.5μ g Staining: Negative 2. Growth condition in each medium (1) Broth culture Growth: Medium, film: formed a ring Sediment: Slightly, turbidity: Yes (2) Slope culture of bouillon Shape: Filament, Surface: Smooth and glossy Margin: whole edge, color: milky white Transparency: opaque (3) Bouillon agar plate shape: round, peripheral, whole edge Surface bulge: lens-like Surface: smooth and glossy, color: milky white (4) bouillon Agar puncture culture Growth along surface and upper perforation 3. Physiological properties (1) Attitude to oxygen: aerobic (2) Catalase: + (3) Oxidase: + (4) Arginine dehydrolase:-(5) Lipase (Hydrolysis of Tween 80): + (6) Lecithinase ( Yellow decomposition): - (7) Hydrolysis of gelatin: - (8) Hydrolysis of starch: - (9) Hydrolysis of extracellular poly -β- hydroxybutyrate (PHB): - (10) utilize of H ₂ And autotrophic growth:-(11) Pigment production: No fluorescent pigment is produced on King A, B medium, glutamate medium, Pseudomonas F agar, Pseudomonas P agar, TSI agar, potato dextrose agar. (12) Reducing ability of nitrate:-(13) Denitrification reaction:-(14) Decomposition position of protocatechulate: ortho (15) Intracellular accumulation of PHB: + (16) Production of levan from sucrose:-(17 ) Growth temperature 11 ~ 35 ℃ Optimum temperature 18 ~
35 ° C. (18) Growth pH 4.0 to 8.5 Optimum pH 4.5 to 8.0 (19) Utilization of carbon source D-glucose: + D-fructose: + D-arabinose: + trehalose: ± mannitol : + Inositol: + glycerol: + L-valine: ± β-alanine: + glycolate: + p-hydroxybenzoate: +