JPH06277081A - Production of 5-aminolevulinic acid by methane-producing bacterium - Google Patents

Abstract

PURPOSE:To continuously produce 5-aminolevulinic acid in high producing efficiency. CONSTITUTION:In producing 5-aminolevulinic acid by anaerobically culturing a culture medium containing a methane-producing bacterium, proliferation substrate of the methane producing bacterium and an inhibitor of 5-aminolevulinic acid dehydratase, glutamic acid is added to the culture medium. This method for continuously producing 5-aminolevulinic acid by using the methane-producing bacterium is to carry out anaerobic culture of the methane producing bacterium in a bioreactor retaining the methane-producing bacterium in high concentration while feeding a culture medium containing a proliferation substrate of the methane-producing bacterium, the inhibitor of 5-aminolevulinic acid dehydratase and glutamic acid into the bioreactor and take out the culture medium containing no bacterial cell from outlet of the bioreactor and recover 5- aminolevulinic acid in the culture medium.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing 5-aminolevulinic acid useful as a herbicide and an insecticide, and more particularly to a practical method for producing 5-aminolevulinic acid by methanogenic bacteria.

[0002]

As a method for producing 5-aminolevulinic acid, organic synthetic chemical methods and fermentation production methods using microorganisms are known, but the former method is not sufficient as a practical production method. In the latter, 5-aminolevulinic acid is produced by the latter fermentation production method. As this fermentation production method, a method using propionic acid bacteria, a method using photosynthetic bacteria, a method using methanogenic bacteria, and the like are known. Among these, the method using methanogenic bacteria focuses on the point of producing 5-aminolevulinic acid as an intermediate when synthesizing corinoid coenzyme in methanogenic bacteria. This corinoid coenzyme synthesis pathway is called C-5 pathway and is represented by the following metabolic pathway. Glutamic acid → ketoglutaric acid → 5-aminolevulinic acid → porphobilinogen → uroporphyrinogen → corinoid → corinoid coenzyme

As is clear from this C-5 pathway, 5-aminolevulinic acid as an intermediate does not accumulate by simply culturing methanogenic bacteria. Therefore, by using an inhibitor of 5-aminolevulinic acid dehydratase, It inhibits the biosynthetic pathway from the intermediate to porphobilinogen and produces 5-aminolevulinic acid.

[0004]

However, in the conventional fermentative production method using a propionic acid bacterium, the production of 5-aminolevulinic acid is easily accumulated in the cells because the produced organic acid is likely to inhibit the product. Since the cells must be crushed to recover the target product, continuous production is difficult and the production effect is poor. Further, the fermentation production method using photosynthetic bacteria has a drawback that the concentration of 5-aminolevulinic acid produced in the culture solution is low. further,
The culture substrate used in the fermentative production method using a propionic acid bacterium or a photosynthetic bacterium has a drawback that it is expensive as compared with the method using a methanogenic bacterium.

On the other hand, the fermentative production method using methanogenic bacteria has a low raw material cost required for culturing, and since the final products are methane gas and carbon dioxide gas, there is no product inhibition, and most of the produced 5-aminolevulinic acid. Has the advantage that the target substance can be easily recovered because it is metabolized to the outside of the cell and a continuous production process can be adopted. However, depending on the species of methanogenic bacteria, the ability to produce 5-aminolevulinic acid is Some were inferior to fungi and photosynthetic bacteria. The present invention has been made in view of the above circumstances, and provides a production method capable of achieving high production efficiency of 5-aminolevulinic acid and continuously producing it in the production of 5-aminolevulinic acid by methanogenic bacteria. The purpose is to do.

[0006]

Means for Solving the Problems As a result of various studies to achieve the above-mentioned object, the present inventors have found that by inhibiting the corinoid biosynthetic pathway of methanogenic bacteria with an inhibitor of 5-aminolevulinic acid dehydratase, 5- In producing aminolevulinic acid, it was found that the production of 5-aminolevulinic acid can be promoted by adding glutamic acid to the culture solution. Further, by continuously supplying a culture solution containing a growth substrate 5-aminolevulinic acid dehydratase inhibitor and glutamic acid of a methanogenic bacterium to a bioreactor holding a high concentration of methanogenic bacterium, continuous and high production efficiency can be obtained. It was found that 5-aminolevulinic acid can be produced by the above method, and the present invention has been completed.

That is, according to the present invention, a culture solution containing methanogenic bacteria, a growth substrate of methanogenic bacteria and an inhibitor of 5-aminolevulinic acid dehydratase is anaerobically cultured to produce 5-aminolevulinic acid. 5 by methanogenic bacteria characterized by adding glutamic acid to the liquid
-A method for producing aminolevulinic acid. Further, the present invention provides a bioreactor for maintaining a high concentration of methanogenic bacteria, while supplying a culture solution containing a growth substrate of methanogenic bacteria, an inhibitor of 5-aminolevulinic acid dehydratase and glutamic acid, in the bioreactor. Anaerobic culturing of methanogenic bacteria is carried out, a culture solution containing no cells is taken out from the outlet of the bioreactor, and 5-aminolevulinic acid in the culture solution is recovered. It also provides a continuous production method of.

The present invention will be described in detail below. Examples of the methanogenic bacteria used in the present invention include Methanosarcina. Methane-assimilating species are advantageous for methanogenic bacteria in terms of the cost of the culture substrate, but if desirable methanogenic bacteria substrates such as acetic acid and hydrogen are available at a low cost, the methanogenic bacteria with the substrate utilization ability Bacteria may be used. Further, both high-temperature methanogenic bacteria and mesophilic methanogenic bacteria can be used. The culture solution used for culturing the methanogenic bacteria contains components such as a culture substrate (carbon source), nutrients (nitrogen source, vitamins, etc.), and infinite salts, as well as an inhibitor of 5-aminolevulinic acid dehydratase and glutamic acid. Included, and if necessary, a pH adjusting agent is added. Methanol-producing bacteria are preferably used as a culture substrate for methanogenic bacteria such as methanol, acetic acid, methylamine, carbon dioxide and hydrogen, and formic acid. One or more of these are selected depending on the type of methanogenic bacteria used. To use. Furthermore, it is also possible to use organic industrial wastewater containing the above-mentioned culture substrate as the culture solution. As nutrient components, nitrogen sources such as ammonium chloride, ammonium nitrate, ammonium sulfate, ammonium phosphate and urea, vitamins B ₁ and B ₂ ,
B _6, using biotin, folic acid, nicotinic acid, pantothenic acid, vitamins such as ribonucleic acid. Further, natural nutrition sources such as yeast extract, polypeptone, corn steve liquor and the like can also be used. As inorganic salts,
Calcium chloride, magnesium sulfate, potassium phosphate,
Salt, manganese chloride, ferrous sulfate, etc. are used.

As an inhibitor of 5-aminolevulinic acid dehydratase added to the culture medium, levulinic acid, 4,6-dioxoheptanoic acid or the like is used. These inhibitors are added depending on the cell concentration and the production amount of 5-aminolevulinic acid, but if the addition amount is too large or too small, it is not preferable for the production of 5-aminolevulinic acid. Usually, the addition amount of these is 1 to 50 m of 5-aminolevulinic acid based on the whole culture solution.
M, preferably 5-20 mM, 4,6-dioxoheptanoic acid is appropriately determined from the range of 1-5 mM, preferably 2-3 mM. Glutamic acid is produced by methanogenic bacteria 5
-Added for the purpose of promoting the production of aminolevulinic acid. The amount of glutamic acid added is determined by the species of methanogenic bacteria used for production, the bacterial concentration in the reactor, the glutamic acid utilization capacity of the methanogenic bacteria used, the tolerance to glutamic acid, etc. ~ 5
It is appropriately determined from the range of 0 mM, preferably 5 to 20 mM.

In the present invention, anaerobic culture is performed. To perform anaerobic culture, culture in an inert gas or carbon dioxide gas stream, sodium thiosulfate, titanium citrate,
A general method of adding a reducing agent such as L-cystine to the culture medium may be applied. Although a batch culture method can be used as the culture method of the present invention, a continuous culture method in which a bioreactor that holds methanogenic bacteria at a high concentration is used and a culture solution is continuously supplied to the bioreactor is adopted. It is preferable. As a method for maintaining a high concentration of methanogenic bacteria in the bioreactor, a bioreactor in which a carrier is used to retain the bacterial cells is used. An upflow anaerobic mudbed bioreactor that retains the cells by utilizing the self-granulation action of the cells is used. A bioreactor that retains the cells by entrapping the cells in a gel or the like is used. A separation membrane for concentrated bacterial cells is provided at the outlet of the bioreactor. By centrifugation. Install a natural (gravity) settling tank. Etc., and these methods are appropriately selected and used depending on the type of bacteria used and culture conditions. By using these methods, cultured bacteria (methanogenic bacteria) outside the culture system
Can be prevented from flowing out. Further, a culture solution containing no culture can be taken out from the bioreactor.

Methanogenic bacteria produce 5-aminolevulinic acid by being anaerobically cultured in a bioreactor, and most of the produced 5-aminolevulinic acid is metabolized outside the cells. Then, the culture solution containing 5-aminolevulinic acid produced by the culture is taken out from the outlet of the bioreactor. In the present invention, the culture of methanogenic bacteria is 25 to 45 ° C., preferably 36 ° C. for mesophilic bacteria.
~ 38 ° C, 45 ~ 60 ° C in the case of thermophilic bacterium, preferably 5
Carried out at a temperature of 0-55 ° C. The pH of the culture solution is 6.0.
~ 8.0, preferably 6.8-7.2. The culturing is usually completed in 2 to 8 days, but it can be appropriately changed depending on other culturing conditions.

As a method for recovering 5-aminolevulinic acid in the culture broth taken out from the outlet of the bioreactor, a method of adsorbing using an ion exchange resin, a method of solvent extraction, etc. are adopted. The method using an ion exchange resin is preferable because it can recover an inhibitor of 5-aminolevulinic acid dehydratase and glutamic acid that promotes production of 5-aminolevulinic acid. On the other hand, methane gas generated by culturing in the bioreactor can be stored in a gas holder and used as fuel gas or the like.

Next, the continuous production method of aminolevulinic acid according to the present invention will be described with reference to FIG. FIG. 1 is a schematic view of a continuous culture apparatus which is preferably used for continuous culture using methanogenic bacteria as an example of the production apparatus according to the present invention. This continuous culture device is connected to a bioreactor 1 that holds methanogenic bacteria at a high concentration, a culture solution supply device 2 that supplies a culture solution into the bioreactor 1, and an outlet of the bioreactor 1, and from the bioreactor 1 A solid-liquid separator 3 for separating solids from the culture solution to be taken out, and 5-aminolevulinic acid in the culture solution containing no bacterial cells separated by the solid-liquid separator 3 and 5-aminolevulinic acid A 5-aminolevulinic acid recovery device 1 is provided as a main component, which sends the recovery residual liquid as a culture stock solution to the culture liquid supply device 2. The culture solution supply device 2 includes a growth substrate storage tank 6 for methanogenic bacteria, a nutrition factor storage tank 7 such as a nitrogen source, inorganic salts, and vitamins, an inhibitor storage tank 8 for 5-aminolevulinic acid dehydratase 8, and a glutamic acid storage tank 9. , Ph that stores the pH regulator of the culture broth
It comprises a regulator storage tank 10 and a mixture regulator 11 which mixes the stock culture solution sent from the 5-aminolevulinic acid recovery device 4 and the additives in the respective storage tanks 6 to 10 to prepare the culture solution and supplies it to the bioreactor 1. ing. The 5-aminolevulinic acid recovery device 4 is configured by connecting a plurality of ion exchange resin columns, and is configured to elute 5-aminolevulinic acid with the solvent sent from the recovery solvent storage tank 12 and send it to the 5-aminolevulinic acid generator 13. ing.

In the present invention, 5-aminolevulinic acid can be produced with high efficiency as described below using the above continuous culture apparatus. That is, bioreactor 1
While maintaining methanogenic bacteria at a high concentration inside, supply a culture solution suitable for culturing this bacterium to the bioreactor,
Anaerobic culture is performed in the bioreactor 1. The culture solution taken out from the outlet of the bioreactor is passed through the solid-liquid separation device 3, the culture solution containing no bacteria is taken out, and sent to the 5-aminolevulinic acid recovery device 4, where 5-aminolevulinic acid in the culture solution is recovered and the culture residue is left. The solution is sent to the culture solution supply device 2 as a culture stock solution. Various additives are mixed in the culture solution supply device 2 to regenerate the culture solution, and the methanogenic bacteria are continuously cultured by supplying the culture solution to the bioreactor 1. 5
The 5-aminolevulinic acid recovered by the aminolevulinic acid recovery device 4 is eluted at a suitable time with a recovery solvent such as methanol and sent to the purification device 13, where it is subjected to electrophoresis, ion exchange, reverse phase chromatography, etc. After purification, the product is stored in the product storage tank 14. The methane gas generated by the culture in the bioreactor 1 is stored in the gas holder 5. The present invention will be described below with reference to examples, but the present invention is not limited thereto.

[0015]

【Example】

Example 1 As a methanogenic bacterium, methanol-utilizing methanogenic bacterium methanosarcina.sp.CHTI55
Was cultured in a DSM318 medium containing 10 mM of levulinic acid, which is an inhibitor of 5-aminolevulinic acid dehydratase, and 10 mM of glutamic acid, which is a promoter of 5-aminolevulinic acid production. This culture was performed by static culture in a 100 ml anaerobic vial for 1 week at a culture temperature of 53 ° C., after which the concentration of 5-aminolevulinic acid in the culture solution was measured. For comparison, culture was performed under the same conditions except that glutamic acid was not added, and Table 1 shows the concentration of 5-aminolevulinic acid and the amount of 5-aminolevulinic acid produced per dry cell in each case.

[0016]

[Table 1]

[0017]

As described above, the following effects can be obtained in the present invention. A methanogenic bacterium is used for the production of 5-aminolevulinic acid, and 5 in the C-5 metabolic pathway is used.
-The biosynthetic pathway from aminolevulinic acid to porphobilinogen is inhibited by an inhibitor of 5-aminolevulinic acid dehydratase, and glutamic acid, which promotes the production of 5-aminolevulinic acid, is added and cultured. The production efficiency of 5-aminolevulinic acid per body weight can be increased. In addition, using propionic acid bacteria and photosynthetic bacteria 5
-Compared to the aminolevulinic acid production method, the culture substrate and culture solution are cheaper, and the final products of fermentation are methane gas and carbon dioxide gas, so that the product inhibition that often occurs in other fermentation methods does not easily occur. Furthermore, since most of the 5-aminolevulinic acid produced by the method of the present invention is metabolized to the outside of the cell, it is easy to recover the target substance, and a bioreactor holding a high concentration of methanogenic bacteria is used. Then, a continuous production process of 5-aminolevulinic acid can be adopted. When this continuous production process is adopted, there is almost no liquid flowing out of the system, heat loss in culturing methanogenic bacteria that is cultivated at high temperatures is reduced, and the methane gas produced by fermentation can be used as an energy source so that thermal efficiency is improved. It is also advantageous in respect of. In addition, since the recovery residual liquid, levulinic acid, glutamic acid, etc. after recovering 5-aminolevulinic acid from the culture solution flowing out from the bioreactor is readjusted by the culture solution regenerator and supplied to the bioreactor, the use of the culture solution Can achieve excellent effects such as increased efficiency

[Brief description of drawings]

FIG. 1 is a schematic view of a continuous culture device used in the method for producing 5-aminolevulinic acid using methanogenic bacteria of the example of the present invention.

[Explanation of symbols]

 1 Bioreactor 2 Culture liquid supply device 3 Solid-liquid separation device 4 5-Aminolevulinic acid recovery device 5 Gas holder 6 Growth substrate storage tank 7 Nutrient factor storage tank 8 Inhibitor storage tank 9 Glutamic acid storage tank 10 Ph regulator storage tank 11 Mixing adjustment tank 12 Recovery solvent Storage tank 13 5-Aminolevulinic acid purifier 14 Product storage tank

Claims (2)

[Claims] 1. A method for producing 5-aminolevulinic acid by anaerobically culturing a culture solution containing a methanogenic bacterium, a growth substrate of a methanogenic bacterium and an inhibitor of 5-aminolevulinic acid dehydratase, and producing glutamic acid in the culture solution. A method for producing 5-aminolevulinic acid by a methanogenic bacterium, which comprises adding. 2. A bioreactor for maintaining a high concentration of methanogenic bacteria, while supplying a culture medium containing a growth substrate of methanogenic bacteria, an inhibitor of 5-aminolevulinic acid dehydratase and glutamic acid, in the bioreactor. Anaerobic culture of methanogenic bacteria is performed, a culture solution containing no cells is taken out from the bioreactor outlet, and 5-aminolevulinic acid in the culture solution is recovered. Continuous production method.