JPH08168391A - Production of 5-aminolevulinic acid - Google Patents

Abstract

PURPOSE: To provide a method for producing a large amount of 5-aminolevulinic acid without irradiation with light rays. CONSTITUTION: This method for producing 5-aminolevulinic acid comprises culturing a photosynthetic bacterium capable of producing 5-aminolevulinic acid under at least one of the following conditions (a), (b) and (c). (a) The concentration of dissolved oxygen in a culture solution is <1ppm, (b) the oxidation- reduction potential in the culture solution is -180 to 50mV and (c) a bacterium respiration rate is 5×10<-9> to KrM-2×10<-8> [mol of O2 /ml.min.cell]. (KrM shows the maximum of the bacterium respiration rate when oxygen is excessively supplied).

Description

Detailed Description of the Invention

[0001]

INDUSTRIAL APPLICABILITY The present invention is capable of culturing photosynthetic bacteria without irradiating light, and by controlling oxygen supply
It relates to a method capable of producing aminolevulinic acid in high yield.

[0002]

BACKGROUND OF THE INVENTION 5-Aminolevulinic acid is widely present in biological systems as a metabolic intermediate in the pigment biosynthetic pathway that biosynthesizes tetrapyrrole compounds (vitamin B ₁₂ , heme, chlorophyll, etc.), and plays an important role in the body. It is a compound that plays. That is, 5-aminolevulinic acid is biosynthesized from glycine and succinyl CoA by 5-aminolevulinic acid synthase or by glutamic acid in a living system and metabolized by 5-aminolevulinic acid dehydratase.

Further, 5-aminolevulinic acid is a herbicide,
It has excellent effects as an insecticide, plant growth regulator, photosynthesis enhancer for plants, and is not toxic to humans and animals. It is highly degradable and has no residual in the environment. A compound (Japanese Patent Application Laid-Open No. 61-502814)
No. 2-138201).

However, 5-aminolevulinic acid has a high production cost and is not practical for use as a herbicide, a plant growth regulator, or a plant photosynthesis enhancer (CHEMIC).
ALWEEK / October, 29, 1984). Under such circumstances, many chemical synthesis methods have been studied (for example, JP-A-2-76841 and JP-A-2-2613).
No. 89) has not yet been developed as a sufficiently satisfactory method.

On the other hand, a method for producing 5-aminolevulinic acid using a microorganism has also been investigated. For example, the genus Propionibacterium,
Methanobacterium
m) genus or methanosarcina
Although a method using na) or the like (Japanese Patent Laid-Open No. 5-184376, etc.) has been proposed, the production amount is very small, and it is not industrially satisfactory.

[0006] In addition, Rhodobacter
The method using the genus er) (JP-A-6-141875) has a higher production amount than the method using the above-mentioned microorganism, but light irradiation is required for synthesizing a considerable amount of photosynthetic bacteria including Rhodobacter. However, even in the production of 5-aminolevulinic acid, which is a precursor of the dye, sufficient light must be irradiated, and many problems remain for practical use such as high cost.

[0007] In order to solve this problem, a method has been proposed in which a bacterium of the genus Rhodobacter is mutated to prepare a mutant strain and 5-aminolevulinic acid is produced under heterotrophic conditions that do not require light irradiation. (Kaihei 4-333521), the production amount was smaller than the method using light irradiation.

On the other hand, oxygen is indispensable for energy production in culturing microorganisms under heterotrophic conditions that do not require light irradiation.

However, it is said that oxygen inhibits pigment synthesis of photosynthetic bacteria, especially purple non-sulfur bacteria, and 5-aminolevulinic acid synthase is also inactivated by oxygen [protein, nucleic acid, enzyme, Vol. 15, No.
3, 195 (1970)].

[0010]

Therefore, the object of the present invention is to culture bacteria under aerobic conditions, and to obtain 5-aminolevulinic acid in high yield regardless of whether light is irradiated or not. It is to provide a method that can be produced in.

[0011]

[Means for Solving the Problems] In view of the above situation, the present inventors have conducted diligent research, and as a result, if photosynthetic bacteria producing 5-aminolevulinic acid were used and oxygen supply was limited under the following conditions, bacterial cells were The present invention has been completed by finding that a large amount of 5-aminolevulinic acid can be produced without impairing the amount of oxygen required for the energy generation of.

That is, the present invention provides a photosynthetic bacterium which produces 5-aminolevulinic acid, wherein the dissolved oxygen concentration in the following culture medium (a), (b) and (c) (a) is less than 1 ppm (b) Redox potential of -180 to 50 mV (c) Bacterial respiration rate of 5 x 10 ^-9 to KrM-2 x 10 ^-8
[Mol of O ₂ / ml · min · cell] (where KrM represents the maximum value of the bacterial respiration rate when oxygen is excessively supplied) is cultivated under at least one condition 5 -A method for producing aminolevulinic acid is provided.

The microorganism used in the present invention is 5-
Photosynthetic bacteria capable of producing aminolevulinic acid, particularly purple non-sulfur bacteria, are preferred. Specifically, Rhodobacter (R
Rhodospirillum (Rhod)
genus ospirillum, Rhodopi
la), Rhodopseud
homonas genus, Rhodomicrobium (Rhodomi)
genus crobium, Rhodocy
and microorganisms belonging to the genus clas) or mutants thereof. These microorganisms are commercially available, for example, from the Vergise's Manual of Detergent Bacteriology [Bergey's Manual of Determin
inatiate Bacteriology, Vol.
3, (1989)], page 1658. In the present invention, it is preferable to use a strain having a high production ability of 5-aminolevulinic acid as much as possible even under aerobic conditions or in a complex medium to which natural components are added. Rhodobacter sphaeroides C deposited as FERM P-14672 at the National Institute of Advanced Industrial Science and Technology
The R-520 strain can be exemplified.

As the medium for culturing the photosynthetic bacterium producing 5-aminolevulinic acid, any medium can be used as long as the microorganism can grow sufficiently, but it can be assimilated in the medium. It is preferable to appropriately contain a carbon source and a nitrogen source. As the carbon source, sugars such as glucose, acids such as acetic acid, malic acid, lactic acid, and succinic acid can be used. As the nitrogen source, ammonium nitrogen compounds such as ammonium sulfate and ammonium chloride, inorganic nitrogen sources such as nitrate nitrogen compounds such as sodium nitrate, organic nitrogen compounds such as urea, polypeptone and yeast extract can be used.

Furthermore, trace components such as inorganic salts, alanine,
Amino acids such as valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, glutamine, asparagine, tyrosine, lysine, arginine, histidine, aspartic acid, glutamic acid; yeast extract, dry yeast, peptone, Natural ingredients such as meat extract, malt extract, corn steep liquor, casamino acid and the like can be appropriately added. Further, when producing 5-aminolevulinic acid, it is preferable to add glycine and levulinic acid to the medium. The amount of glycine added is 5 to 100 mM,
In particular, the amount is preferably 10 to 60 mM, and the amount of levulinic acid added is preferably 1 to 60 mM, particularly preferably 5 to 30 mM. Since the addition of glycine and levulinic acid may reduce the growth rate of the strain, it is advisable to add them at the time when they have grown to some extent.

The culturing temperature and pH for culturing may be the conditions under which the above-mentioned strains grow, for example, a temperature of 20 to 40 ° C.,
It is preferable to adjust the pH to 6-8. When the pH changes during the production of 5-aminolevulinic acid, it is preferable to adjust the pH using an alkaline solution such as sodium hydroxide, ammonia, potassium hydroxide or the like, or an acid such as hydrochloric acid, sulfuric acid, phosphoric acid or the like. In addition, it is not necessary to specifically irradiate with light upon culturing.

In the present invention, the oxygen supply is limited as described above in order to efficiently produce 5-aminolevulinic acid. Specifically, the culture is performed under one or more of the following conditions.

(A) The dissolved oxygen concentration in the culture broth is less than 1 ppm, preferably less than 0.5 ppm, more preferably less than 0.1 ppm (below the detection limit of the measuring instrument at this stage). This concentration may be measured using a dissolved oxygen meter. (B) The redox potential in the culture medium is −180 to 50 mV, but especially −100 to 20 mV, and further −50 to 0 mV.
It is preferable that The redox potential may be measured using a redox potential meter.

(C) Bacterial respiration rate is 5 × 10 ^{-9 to} KrM-
2 × 10 ⁻⁸ [mol of O ₂ / ml · min · cell], but especially 1 × 10 ^{−8 to} KrM-4 × 10 ⁻⁸ [mol of O ₂ / ml · mi]
[n.cell] is preferable. Here, KrM refers to the maximum value of the respiration rate of the bacterium when oxygen is excessively supplied, but this varies depending on the bacterium, for example, Rhodobacter cephaloides (Rhodobacter).
Sphaeroides) or Rhodobacter capsulatus (Rhodobacter capsula)
in the case of tus), about 8 × 10 ^-8 mol ofO ₂ / ml ・ min ・ cel
is l. Therefore, the bacterial respiration rate when using this bacterium is 5
× 10 ^{-9 to} 6 × 10 ^-8 [mol of O ₂ / ml · min · cell] 1 × 10 ⁻⁸ to 4 × 10 ⁻⁸ [mol of O ₂ / ml · min]
・ Cell].

An exhaust oxygen / carbon dioxide analyzer may be used to measure the bacterial respiration rate.

The bacillus respiration rate is calculated by the general formula of Hirose et al. (Agric. Blo).
1 Chem., 29, 931, 1965), in order to further correct the variation in the amount of bacterial cells per unit volume, a method of determining by interrupting with the amount of bacterial cells was adopted. That is, it is as shown in the following equation.

[0022]

[Equation 1]

Rab / a: Bacterial respiration rate (mol of O ₂ / ml.
min · cell) Q: Aeration rate (ml / min) V: Infused solution volume (ml) T: Culture temperature (° C) x ₀ , x ₁ : Oxygen concentration (%) y ₀ , y ₁ at the air outlet and inlet : Carbon dioxide concentration (%) at air outlet and inlet a: Amount of cells, dry cell weight / L (c
ell)

The bacterial respiration rate represents the oxygen absorption rate of the bacterial cells in the culture tank, and the larger the value, the more active the oxygen utilization.

Since all of the indices (a) to (c) often show the same tendency, at least one may be used, but it is preferable to analyze two or more values.

As a method for satisfying these conditions,
Various means may be employed. For example, the amount of aeration to the fermenter, the stirring rate, the increase / decrease in the amount of culture medium fed, the adjustment of the oxygen partial pressure in the aeration gas, the addition of a reducing substance, the adjustment of the medium supply amount, etc. Further, since the present invention limits the oxygen supply amount, it may decrease the growth rate of the bacterium, so in that case, sufficient oxygen supply is performed for the growth of the microbial cells, and control is performed when the microbial cells have grown to some extent. Just start.

The 5-aminolevulinic acid in the culture broth obtained as described above can be purified by a conventional method. For example, 5-aminolevulinic acid is secreted outside the cells, so it may be separated from the culture solution by a means such as using an ion exchange resin.

[0028]

EFFECTS OF THE INVENTION According to the method of the present invention, a large amount of 5-aminolevulinic acid can be produced from a producing bacterium without light irradiation.

[0029]

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

Example 1 1 L of a medium (medium 1) having the composition shown in Table 1 was placed in a 2 L fermentor, sterilized at 121 ° C. for 15 minutes, and cooled to room temperature.

[0031]

[Table 1]

20% of the medium 1 was previously added to the above fermenter.
The CR-520 strain (FERM P-1) was grown by shaking culture under aerobic conditions in a 1 L Sakaguchi flask containing 0 ml.
4672) (KrM = 8.2 × 10 ⁻⁸ ) and inoculated with 30
The culture was carried out with aeration and agitation at ℃, aeration rate of 0.1 v / v / m and agitation rate of 200 rpm. All cultures were performed under non-light irradiation conditions. 48 hours after the start of the culture, the bacterial cell concentration in the culture medium was 0.64 g per 1 L of the culture medium. Next, glycine, levulinic acid, glucose, and yeast extract are each 60 m
M, 5mM, 50mM, 1% added, pH
The adjustment with 1N sodium hydroxide and 1N sulfuric acid was started so that the value became 6.5 to 7.0. Air volume 0.0
14 v / v / m, N ₂ gas 0.086 v
/ V / m. The stirring number was 200 rpm. Culture was performed under these conditions until after 84 hours. Table 2 shows the maximum amount of 5-aminolevulinic acid accumulated in this culture, the dissolved oxygen concentration in the culture medium after 48 hours, the average redox potential from 48 hours to 84 hours, and the average bacterial respiration rate. The dissolved oxygen concentration is the dissolved oxygen indicator M-1 manufactured by Able
032 and the oxygen membrane electrode for fermentation, the redox potential was measured using a digital ORP controller manufactured by Mitsuwa Biosystems and the ORP electrode manufactured by Ingold, and the bacterial respiration rate was measured using an exhaust gas analyzer WSMR-1400LB manufactured by Westlon. .

Example 2 The same treatment as in Example 1 was carried out except that the stirring number after 48 hours was 300 rpm. Table 2 shows the maximum amount of 5-aminolevulinic acid accumulated in this culture, the dissolved oxygen concentration in the culture medium after 48 hours, the average redox potential from 48 hours to 84 hours, and the average bacterial respiration rate.

Example 3 The same treatment as in Example 1 was carried out except that the stirring number after 48 hours was 400 rpm. Table 2 shows the maximum amount of 5-aminolevulinic acid accumulated in this culture, the dissolved oxygen concentration in the culture medium after 48 hours, the average redox potential from 48 hours to 84 hours, and the average bacterial respiration rate.

Example 4 The same treatment as in Example 1 was carried out except that the stirring number after 48 hours was 500 rpm. Table 2 shows the maximum amount of 5-aminolevulinic acid accumulated in this culture, the dissolved oxygen concentration in the culture medium after 48 hours, the average redox potential from 48 hours to 84 hours, and the average bacterial respiration rate.

Example 5 The same treatment as in Example 1 was carried out except that the stirring number after 48 hours was 600 rpm. Table 2 shows the maximum amount of 5-aminolevulinic acid accumulated in this culture, the dissolved oxygen concentration in the culture medium after 48 hours, the average redox potential from 48 hours to 84 hours, and the average bacterial respiration rate.

Comparative Example 1 After 48 hours, the stirring number was 400 rpm and the aeration rate was air 0.
The same process as in Example 1 was performed except that the amount was 5 v / v / m. Maximum 5-aminolevulinic acid accumulation in this culture, 48
Table 2 shows the dissolved oxygen concentration in the culture solution after the lapse of time, the average redox potential from 48 hours to 84 hours, and the average bacterial respiration rate from 48 hours to 84 hours.

[0038]

[Table 2]

Example 6 The strain used was Rhodobacter capsulatus (Rh
odobactercapsulatus) ATCC1
1166 (KrM = 8.5 × 10 ⁻⁸ ) was treated in the same manner as in Example 1 except that the stirring number after 48 hours was 400 rpm. The maximum amount of 5-aminolevulinic acid accumulated in this culture was 0.086 mM, the dissolved oxygen concentration in the culture medium after 48 hours was not detected (less than 0.1 ppm), and the average redox from 48 hours to 84 hours was detected. The potential is -31m
The average bacterial respiration rate from 48 hours to 84 hours after V was 2.7 × 10 ⁻⁸ [mol of O ₂ / ml · min · cell].

Comparative Example 2 The strain used was Rhodobacter capsulatus (Rh
odobactercapsulatus) ATCC1
The same process as in Comparative Example 1 was performed except that 1166 was used.
The maximum amount of 5-aminolevulinic acid accumulated in this culture was not detected (less than 0.01 mM), the dissolved oxygen concentration in the culture solution after 18 hours was 18 ppm, and the average redox potential from 48 hours to 84 hours was , 131 mV, the average bacterial respiration rate from 48 hours to 84 hours was 8.5 × 10 ^-8 [mo
l of O ₂ / ml · min · cell].

Example 7 A medium (medium 1) having the composition shown in Table 1 was placed in a 30 L fermentor and sterilized at 121 ° C. for 30 minutes. CR- was grown by shaking culture under aerobic conditions in a 1 L Sakaguchi flask containing 200 ml of the culture medium 1 in the above fermentor.
520 strain (FERM P-14672) was inoculated, and
The culture was carried out with aeration and agitation at ℃, aeration rate of 0.1 v / v / m and agitation rate of 200 rpm. All cultures were performed under dark conditions. 48 hours after the start of culture, the bacterial cell concentration in the culture solution was 1 L of the culture solution.
It was 0.68 g per unit. Next, glycine, levulinic acid, glucose, and yeast extract are 60 mM and 5 m, respectively.
M, 50 mM, 1% added, and pH 6.5
To 7.0, 1N sodium hydroxide and 1N
Adjustment with sulfuric acid was started. Air flow rate is 0.028v /
It was reduced to v / m, and N ₂ gas was supplied at 0.172 v / v / m. The stirring number was 300 rpm. Culture under these conditions 8
It went until 4 hours later. The maximum amount of 5-aminolevulinic acid accumulated in this culture was 12.8 mM, and the dissolved oxygen concentration in the culture medium after 48 hours was not detected (less than 0.1 ppm).
The average redox potential from the time after 84 hours is -2.
The average bacterial respiration rate from 2 hours after 48 hours to 84 hours was 2.2 × 10 ⁻⁸ [mol of O ₂ / ml · min · cell].

Front page continuation (72) Inventor Toru Tanaka 1134-2 Gongendo, Satte City, Saitama Prefecture Cosmo Research Institute Co., Ltd. Research and Development Center (72) Inventor, Koji Horita 1134-2 Gongendo, Satte City, Saitama Prefecture Co., Ltd. Sumo Research Institute R & D Center

Claims (1)

[Claims] 1. A photosynthetic bacterium that produces 5-aminolevulinic acid is oxidized by the following (a), (b) and (c) (a) the dissolved oxygen concentration in the culture broth is less than 1 ppm (b) Reduction potential of -180 to 50 mV (c) Bacterial respiration rate of 5 x 10 ^-9 to KrM-2 x 10 ^-8
[Mol of O ₂ / ml · min · cell] (where KrM represents the maximum value of the bacterial respiration rate when oxygen is excessively supplied) is cultivated under at least one condition 5 -A method for producing aminolevulinic acid.