JP2000023691A - Method for producing tetrapyrrole compound - Google Patents

Abstract

(57) Abstract: Production of a tetrapyrrole compound producing culture of a tetrapyrrole compound-producing bacterium in a medium to which 5-aminolevulinic acid or an ester, amide or salt of 5-aminolevulinic acid is added under non-light irradiation conditions Method. [Effect] A tetrapyrrole compound can be efficiently produced under non-light irradiation.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing a tetrapyrrole compound using a microorganism.
The present invention relates to a method for obtaining a tetrapyrrole compound in high yield without requiring light irradiation.

[0002]

2. Description of the Related Art Tetrapyrrole compounds such as porphyrins and chlorophylls play important roles in living organisms and accumulate in cancer cells. It is an important compound.

[0003] Some tetrapyrrole compounds are produced by chemical synthesis, but the resulting compounds are problematic in that they are mixtures, and it is difficult to obtain only the desired product in high yield.

Therefore, at present, tetrapyrrole compounds are synthesized using hemin extracted from animal blood as a mother nucleus. However, tetrapyrrole compounds from animal blood have problems in purity and hygiene, and in particular, when used as medicines, there is a concern that animal blood-derived viruses, pathogenic bacteria, prion which is a pathogen of mad cow disease, and the like may be mixed. In response to this, measures have been taken to utilize blood from healthy livestock, but this is not sufficient.

On the other hand, a method for culturing a tetrapyrrole compound-producing bacterium to obtain a tetrapyrrole compound from this culture has been studied, and the best method known so far is described by Kojima et al. Method using bacter (porphyrin 1996; 5 (4), 335-347)
Is mentioned. According to this method, the accumulated amount of the tetrapyrrole compound becomes a high concentration close to 500 mg / L. However, this method has not been put to practical use because it takes 18 days for culture.

It is known that photosynthetic microorganisms also have high biosynthesis ability of tetrapyrrole compounds, and there are many studies. However, photosynthetic microorganisms have low biosynthesis ability of tetrapyrrole compounds unless irradiated with light. In addition, there is no practical light irradiation culture tank, and many of the tetrapyrrole compounds are light-sensitive, and some of the produced tetrapyrrole compounds are decomposed under light irradiation. Was.

Accordingly, an object of the present invention is to find a method for industrially producing a tetrapyrrole compound without the need for light irradiation.

[0008]

Means for Solving the Problems In view of this situation, the present inventors have conducted intensive studies. As a result, if the photosynthetic microorganisms were cultured in a medium supplemented with 5-aminolevulinic acid or the like, it was unexpectedly irradiated with light. At least a considerable amount of the tetrapyrrole compound was produced, and it was found that the production capacity of the tetrapyrrole compound could be dramatically improved, and the present invention was completed.

That is, the present invention provides a method for producing 5-
Production of a tetrapyrrole compound characterized by culturing a tetrapyrrole compound producing bacterium in a medium to which an ester, amide or salt of aminolevulinic acid or 5-aminolevulinic acid has been added, and collecting the tetrapyrrole compound from the obtained culture It provides a method.

[0010]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In the present invention, 5-aminolevulinic acid, an ester, amide or salt of 5-aminolevulinic acid is used. Among them, specific examples of the ester or amide include methyl 5-aminolevulinate, ethyl 5-aminolevulinate, propyl 5-aminolevulinate, butyl 5-aminolevulinate, pentyl 5-aminolevulinate, Hexyl 5-aminolevulinate, heptyl 5-aminolevulinate, octyl 5-aminolevulinate, nonyl 5-aminolevulinate, dodecyl 5-aminolevulinate, hexadecyl 5-aminolevulinate, isopropyl 5-aminolevulinate, 5-aminolevulinic acid cyclohexyl ester, 5-aminolevulinic acid benzyl ester, 5-aminolevulinic acid phenethyl ester, 5
-Aminolevulinic acid-3-phenylpropyl ester,
5-aminolevulinic acid ethoxyethyl ester, 5-aminolevulinic acid-2- (hydroxymethyl) tetrafuranyl ester, 5-aminolevulinic acid-2- (hydroxymethyl) tetrahydropyranyl ester, 5-aminolevulinic acid acetamide, 5-aminolevulinic acid-n −
Hexanoic amide, 5-aminolevulinic acid-n-nonanoic amide and the like can be mentioned. Examples of the salt include inorganic salts such as hydrochloride, sulfate, hydrobromide, hydroiodide, nitrate, nitrite, and phosphate; succinate, oxalate, and fumarate Organic acids such as, maleate, malate, lactate, tartrate, citrate, acetate, propionate, butyrate, valerate, glycolate, methanesulfonate, toluenesulfonate, etc. Salts: alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt; and ammonium or alkyl ammonium salts. Furthermore, these 5-aminolevulinic acids, esters, amides or salts thereof may form hydrates or solvates. These compounds and methods for synthesizing them are described in JP-A-4-9360.

The above-mentioned 5-aminolevulinic acid and the like may be used alone or as a mixture of two or more. In addition, these 5-aminolevulinic acid and the like may be a fermented solution of a 5-aminolevulinic acid-producing bacterium containing 5-aminolevulinic acid. As the 5-aminolevulinic acid-producing microorganism,
For example, Rhodobacter Sp.
haeroides) CR-0072009 (FERM BP-6320), Rhodobacter Spheroises IFO1220
3. Rhodobacter Sphae
roides) CR-17 (FERM P-11752), Rhodobacter Sphaeroides CR-286 (FERM P-1254)
2), Rhodobacter Sphae
roides) CR-2S5 (FERM P-12543), Rhodobacter Sphaeroides CR-386 (FERM P-13)
159), Rhodobacter sp.
haeroides) CR-450 (FERM P-14085), Rhodobacter Sphaeroides CR-520 (FERM BP
-5255), Rhodobacter cephaloides
Sphaeroides) CR-649-B (FERM P-15974), Rhodobacter
Cephaloides (Rhodobacter Sphaeroides) CR-002 (FER
MP-155312) and the like. The fermentation broth can be used without particular limitation as long as it contains 5-aminolevulinic acid, as long as it does not contain a component that inhibits the production of a tetrapyrrole compound such as porphyrin.

The method of culturing photosynthetic bacteria in the presence of 5-aminolevulinic acid is disclosed in Japanese Patent Application Laid-Open No.
As already described in Japanese Patent No. 4937, in this method,
It is assumed that the photosynthetic bacteria are cultured under light irradiation as described in paragraph 0023 of the publication,
The method of the present invention in which a photosynthetic bacterium is cultured under non-light irradiation and a high yield of a tetrapyrrole compound can be obtained cannot be expected by those skilled in the art from this publication.

As the tetrapyrrole compound producing bacteria used in the present invention, the following microorganisms can be exemplified.

Bacteria belonging to the genus Rhodospirillum Bacteria belonging to the genus Rhodopseudomonas Bacteria belonging to the genus Rhodomicrobium Bacteria belonging to the genus Rhodocyclus Bacteria belonging to the genus Chromatium Thiobacteria genus Thiocistis (Thiocystis) Thiosartina bacterium Thiospirillum bacterium Amoebobacter bacterium Thiopedia bacterium Thiodicton bacterium Thiodictyon genus bacterium Thiocapsa bacterium Ectothiorhodospira bacterium Chloroflexus Chloronema bacterium Oscillochloris bacterium Chlorochromatium bacterium Pelochromatium bacterium Cylindroglia ogloea) Bacteria Pelodictyon Bacteria Clathrochloris Bacteria Ancalochloris Bacteria Chlorobium Bacteria Prosthecochloris Bacteria Rhodobacter Bacteria Various algae ( Phycomycetes)

More specifically, Rhodobactor Sphaeroides IFO 12203, CR-17
(FERM P-11752), CR-286 (FERM P-12542), CR-2S5 (FERM
P-12543), CR-386 (FERM P-13159), CR-450 (FERM P-1408)
5), CR-520 (FERM BP-5255), CR-649-B (FERM P-15974),
CR-002 (FERM P-15312), CR-0072009 (FERM BP-6320) and the like.

In the present invention, the method for culturing the tetrapyrrole compound-producing bacterium is not particularly limited, and may be set to respective optimum values such as the medium composition, the culturing temperature, and the culturing period depending on the type of microorganism and the target tetrapyrrole compound. I just need. In addition, 5-aminolevulinic acid or the like to be added to the culture medium of the microorganism can be appropriately set in an optimum amount and concentration depending on the target substance and the microorganism, but usually the concentration in the culture solution is 0.01 to 100 mM. It is particularly preferable to add so that the concentration becomes 0.03 to 30 mM. Also, when using a fermentation solution of a 5-aminolevulinic acid-producing bacterium, it is preferable to add the 5-aminolevulinic acid so that the 5-aminolevulinic acid concentration falls within the above range.

The time of addition of 5-aminolevulinic acid or the like can be appropriately selected depending on the target substance and the microorganism. It is desirable to administer it after the middle stage of cultivation since it reduces the production rate.

[0018] In addition, sequential or continuous addition of 5
It is preferable to add while keeping the concentration of -aminolevulinic acid or the like low (for example, 0.03 to 30 mM / medium) in order to improve the utilization of 5-aminolevulinic acid or the like. As a medium for growing microorganisms, a medium suitable for the growth of each microorganism is used. However, since most of 5-aminolevulinic acid and the like are decomposed by alkali, if the alkaline medium must be used, the aforementioned sequential and continuous addition method is used. Or an amide type 5-aminolevulinic acid having low alkali decomposability is preferably used.

In the case of a microorganism of a type in which biosynthesis of a tetrapyrrole compound is inhibited by the produced heme, iron is excluded from the medium, dipyridyl, phenanthroline,
It is desirable to add a chelating agent such as EDTA to reduce the amount of heme produced or to obtain and use a mutant strain with low sensitivity to heme. Examples of obtaining such mutants include CR-286 (FERM P-12542) and CR-286.
520 (FERM BP-5255) and the like.

Examples of the tetrapyrrole compound produced according to the present invention include porphyrin, chlorophyll, chlorin, choline, cobalamin, heme, hemin and the like. The type of the tetrapyrrole compound depends on the selection of the microorganism to be used. Will be

The produced tetrapyrrole compound accumulates in the medium, accumulates in the cells, or both depending on the type of microorganism and the type of tetrapyrrole compound to be produced. Separated and extracted.

[0022]

According to the present invention, the production of a tetrapyrrole compound by a microorganism can be greatly increased even under non-light irradiation, and the compound can be produced efficiently.

[0023]

EXAMPLES Next, the present invention will be further described with reference to examples, but the present invention is not limited to these examples.

Example 1 A 1.5 L medium shown in Table 1 was placed in a Maruishi 3L jar fermenter (MDL300 type 3L) and autoclaved. After cooling to room temperature, the photosynthetic bacterium Rhodobacter sphaeroides IFO 122
03 was inoculated and cultured for 24 hours under the conditions of an aeration rate of 0.2 vvm (0.36 L / min), a stirring rotation speed of 400 rpm, and a pH of 7.0. The cell mass after 24 hours was OD ₆₆₀ = 3.5.
After 24 hours of culture, the medium components shown in Table 2 were added, 5-aminolevulinic acid hydrochloride was continuously added at a rate of 0.8 mM / hr for 20 hours, and the aeration rate was 0.2 vvm.
m and pH 6.3 for 24 hours. The culture solution was sampled at the start of addition of 5-aminolevulinic acid hydrochloride and at 8, 20, and 28 hours, and the porphyrin concentration of the centrifuged supernatant was determined by the method of Sato (J. Nut).
r. Sei. vitaminol, 27, 439-44
7, 1981) was 8, 25, respectively.
It was 0, 900, and 1050 mg / L (FIG. 1). No light irradiation was performed throughout the culture period. When porphyrin obtained at 8 hours was analyzed by HPLC, it was almost coproporphyrin III (FIG. 2).

[0025]

[Table 1]

[0026]

[Table 2]

Comparative Example 1 The procedure of Example 1 was repeated except that 5-aminolevulinic acid hydrochloride was not added, and the final porphyrin accumulation was 9 mg / L.

Comparative Example 2 Glycine 6 which is known to promote the production of tetrapyrrole compounds instead of adding 5-aminolevulinic acid hydrochloride
When 0 mM was added twice in culture for 24 hours and 48 hours, the final porphyrin accumulation was 48 mg / L (FIG. 1).

Example 2 The same procedure as in Example 1 was carried out except that the strain used was changed to IFO 12203 and to Rhodobacter cephaloides CR-286.
320 and 1630 mg / L (FIG. 3).

From these Examples and Comparative Examples, it is clear that the addition of 5-aminolevulinic acid or the like dramatically improves the productivity of the tetrapyrrole compound, and its performance is similar to that of glycine which is a known tetrapyrrole productivity improving substance. It turns out that it surpasses much.

[Brief description of the drawings]

FIG. 1 is a graph showing the amount of porphyrin produced.

FIG. 2 is a view showing the results of HPLC analysis of produced porphyrins.

FIG. 3 is a graph showing the amount of porphyrin produced.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor: Satoshi Tanaka 1134-2, Gondogendo, Satte City, Saitama Prefecture Inside the R & D Center, Cosmo Research Institute, Inc. (72) Seiji Nishikawa 1134-2, Gongendo, Satte City, Saitama Prefecture Kosmo Research Institute R & D Center (72) Inventor Shinya Miyachi 1134-2 Gongendo, Satte City, Saitama Prefecture Kosmo Research Institute R & D Center (72) Koji Hotta Gongen Satte City, Saitama Prefecture 1134-2 F-term (reference) 4S064 AE57 CA02 CB29 CD13 CE10 DA05 DA14

Claims (4)

[Claims] 1. A tetrapyrrole compound-producing bacterium is cultured in a medium to which 5-aminolevulinic acid or an ester, amide or salt of 5-aminolevulinic acid is added under non-light irradiation conditions. A method for producing a tetrapyrrole compound. 2. The method according to claim 1, wherein the tetrapyrrole compound is porphyrin. 3. The method for producing a tetrapyrrole compound according to claim 1, wherein the tetrapyrrole compound producing bacterium is a photosynthetic microorganism. 4. The method for producing a tetrapyrrole compound according to claim 1, wherein 5-aminolevulinic acid or an ester, amide or salt of 5-aminolevulinic acid is added to the medium sequentially or continuously.