JPH01148193A - Production of 5-aminolevulic acid - Google Patents

Abstract

PURPOSE:To obtain 5-aminolevulic acid (5-ALA) which is useful in agrochemicals economically, by culturing a microorganism in Methanosarcina in a medium. CONSTITUTION:A microorganism in Methanosarcina, capable of producing 5-ALA is obtained by mutagenic treatment of a wild strain such as Methanosarcina barkeri DSM804 strain (A). Then, a culture medium (B) is prepared by adding less than 2V/V% of carbon source such as methanol, nitrogen source such as ammonium chloride, inorganic salts such as NaCl, when needed, 1-5mM of succinic acid as a precursor of 5-ALA, and 4,6- diketoheptanoic acid. Then, the strain A is immobilized on a support such as perforated stone, the culture medium is supplied to the immobilized support continuously and cultivation is conducted anaerobically at a pH of 5.8-7, 25-50 deg.C for 3-8days to obtain the culture mixture. Then, the mixture is centrifuged, the supernatant is adjusted in its pH to about 2.5, subjected to ion- exchange treatment, elution and concentration whereby 5-aminolevulic acid is collected.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing 5-aminolevulinic acid (hereinafter abbreviated as 5-ALA) by culturing a microorganism belonging to the genus Methanosarcina.

5-ALA is a type of plant hormone and a compound that is attracting attention as an agricultural chemical.It is also a compound that is attracting attention as a pesticide. It is also known as a precursor of tetravirol compounds such as phycobilin and Factor' 430.

(Prior Art) Conventionally, as a method for producing 5-ALA using microorganisms, Methanobacterium thermoautod074cam (M
ethanobacterium thermoout
otr.

phtcum) is known (Archives of Microbiology (Arch, M.
icr.

biol, ), 135, 237-240. (198
3)) Various studies have also been conducted on the metabolic pathways related to the production of 5-ALA, including animals and yeast.

Succinyl-C in bacteria including photosynthetic bacteria.

Shemin pathway synthesized from A and glycine, higher plants,
In green algae, the C-5 pathway from succinic acid to α-ketoglutaric acid is known. Glutamic acid or 4.5-dioxyvaleric acid is assumed to be an intermediate in the C-5 pathway, but enzymatic proof has not yet been provided.

(Problems to be Solved by the Invention) The method for producing 5-ALA by culturing Methanobacterium thermoautotrophicum requires the use of carbon dioxide gas and hydrogen as a carbon source and an energy source.
In order to further accumulate 5-ALA, it is necessary to add a high concentration of levulinic acid, which inhibits 5-ALA dehydratase, which is a 5-ALA dehydratase, and this is not an economically advantageous method.

(Means for Solving the Problem) The present inventors conducted intensive research on natural and known preserved bacterial strains with the aim of obtaining microorganisms capable of producing 5-ALA. - It was discovered that ALA accumulates in large amounts in the culture medium, leading to the present invention.

That is, the present invention provides a method for producing 5-aminolevulinic acid, which comprises culturing a microorganism belonging to the genus Methanosarcina and having the ability to produce 5-aminolevulinic acid, producing 5-aminolevulinic acid, and collecting the same. It is.

The microorganism used in the present invention belongs to the genus Methanosarcina and has the ability to produce 5-ALA.
The strain may be a wild strain, a mutant strain, a strain obtained by IMIIIM hybridization, genetic recombination, or other genetic methods; for example, DSM (D
eutsche S amalung van M i
Methanosarcina barkeri available from
Berqe'l'S
1 lantlal of determinative
e baCt4)riol.

gV 8th edition.

The culture medium used in the present invention contains a carbon source and a nitrogen source.

Sulfur sources, inorganic salts, and other nutrients as needed.

A medium containing trace elements and the like to create an anaerobic atmosphere is used.

Carbon sources include methanol, carbon dioxide, and acetic acid.

Methylamine and the like are used, but methanol is particularly preferred, and its concentration is preferably 2% or less.

As methanol concentration increases, it inhibits the growth of microorganisms, so maintain the methanol concentration during culture at 0.05 to 1%.
Cultivation can also be carried out by sequentially adding methanol.

Examples of the nitrogen source include ammonium sulfate, ammonium chloride, ammonium phosphate, urea, ammonium nitrate, and sodium nitrate, which are commonly used for culturing microorganisms.

Inorganic salts include potassium phosphate, magnesium sulfate,
In addition to sodium chloride, calcium chloride, ferrous sulfate, sodium selenate, manganese chloride, etc., additions of zinc, boric acid, copper, nickel, molybdenum, etc. are also effective.

Trace elements include biotin, folic acid, vitamin B,
BSB6. Nicotinic acid, pantothenic acid.

In addition to vitamins such as p-aminobenzoic acid and ribic acid, corn staple liquor, yeast extract, peptone, etc. can also be added as natural nutritional sources.

Furthermore, succinic acid, which is considered as a precursor of 5-ALA,
The amount of 5-ALA produced can be increased by adding α-ketoglutaric acid, glutamic acid, 4°5-dioxyvaleric acid, or levulinic acid, which is a 5-ALA dehydratase inhibitor.

In addition, 5-A
It is possible to significantly increase the amount of LA produced. This 4
,6-siketohebutanoic acid is effective at concentrations as low as 1-5mM.

Cultivation in the present invention must be performed anaerobically. To perform the culture anaerobically, in addition to adding a reducing agent such as L-cysteine, sodium thiosulfate, or titanium citrate to the medium, nitrogen gas from which oxygen has been removed, carbon dioxide gas, or a mixture of these gases is added to the medium. It is possible to provide aeration and also to prevent oxygen from being mixed into the gas phase of the culture container.

Batch culture may be used as the culture method, but in order to improve production efficiency, it is preferable to immobilize the microorganisms on a carrier and continuously supply the culture medium. Any type of porous carrier that can be attached can be used, but specifically,
Examples include foamed brick and porous silica.

The culture temperature is 25 to 50°C, preferably 30 to 37°C. IpH is 5.8-7. O1 preferably 6.
Adjust to 3-6.8. Culture usually ends in 3 to 8 days, but this can be changed as appropriate depending on other culture conditions.

5-ALA can be collected from a culture using a conventional method. For example, the culture is first centrifuged to remove bacterial cells,
After adjusting the pH of the supernatant to 2.5, it was adsorbed onto a Na+ type ion exchange resin, eluted with pl (5,0 sodium citrate/citric acid v11i solution, and the 5-ALA fraction was concentrated. ``A further purification step may be added if necessary.

The present invention will be explained below with reference to Examples, but the present invention is not limited thereto.

(Example) Example 1 Methanosarcina Berkeli DSM804 was cultured at 37°C for 60 hours in the basal medium shown in Table 1 in which the gas phase was replaced with nitrogen. After culturing, the bacteria were removed by centrifugation, and the amount of 5-ALA produced in the resulting supernatant was measured, and the result was 0.
．． 5-ALA of 8.1/1 was obtained.

Example 2 Methanosarcina barkeri DSM804 was added to Example 1
6 ml of the culture was inoculated into a basal medium containing each concentration of 4.6-siketohebutanoic acid shown in Table 2, and the mixture was incubated at 37°C under anaerobic conditions.

Cultured for 80 hours. The production amount of 5-ALA and the bacterial cell yield are shown in Table 2.

Example 3 Methanosarcina barkeri DSM804 was added to Example 1
The cells were cultured in the same manner as above, and 6 ml of the culture was inoculated into a basal medium containing each concentration of levulinic acid shown in Table 3, and cultured under anaerobic conditions at 37°C for 80 hours. The amount of 5-ALA produced and the amount of bacterial biomass are shown in Table 3.

Example 4 Methanosarcina barkeri DSM804 was added to Example 1
Culture in the same manner as above, add 6 ml of levulinic acid to 20 mH of levulinic acid,
L-alanine 15 nN and α of each concentration shown in Table 4
- They were each inoculated into a basal medium containing ketoglutaric acid and cultured at 37°C for 80 hours under anaerobic conditions. 5-AL
The production amount of A and the amount of bacterial cell culture are shown in Table 4.

Example 5 Methanosarcina barkeri DSM804 was added to Example 1
8.5 ml of the culture was inoculated into a base reactor filled with porous silica (Nagao Porcel, manufactured by Nagao Soda) as a carrier and basic medium, and incubated at 37°C and 48°C.
Cultured for hours. Thereafter, basal medium containing levulinic acid at each concentration shown in Table 5 was continuously supplied.

The feed rate was finally fixed at a dilution rate of 13.1/daV. 5-ALA productivity at steady-state values is shown in Table-5.

Table-1 Basic medium composition Imidazole 5.44 gK2)
IPO40,6961r KM2PO40,454g NH4C11,OOt MgS04・7 H201-005r NaC14,5g CaC1・2H200,25g Fe50 ・7H204,0■ Na25ea3 o,04 MMn
Cl ・4 H200,125+w vitamin solution 1
) 20ml mineral solution 2
)6ml titanium citrate 0,075+eno
1L-cysteine/hydrochloric acid 0.3 g methanol 8 gj pH6.3-6.8 1) Vitamin solution biotin 2N folic acid 2゜pyridoxine/salt a 10 ■ Thiamine/hydrochloric acid 5 ■ Riboflavin 5 ■ Nicotinic acid 5 ■ Calcium pantothenate 5 , P-aminobenzoic acid 5mg lipoic acid
5N 112) Mineral solution Z n 5040, 1 g MnCl ・4H200,04g H3BO40,3g Coal ・6HO0,2g Cue 12 ・2H200-01t NiCl2・6 H2・0 0 ,02 ,tNi
Cl ・6 H200,02t NaMoO4-2H200,03g 11Table-2 Table-3 Table-4 Table-5 (Effect of the invention) The present invention makes it possible to produce 5-ALA in an economically advantageous method. became.

Claims (1)

[Claims] A method for producing 5-aminolevulinic acid, which comprises culturing a microorganism belonging to the genus Methanosarcina and having the ability to produce 5-aminolevulinic acid, producing 5-aminolevulinic acid, and collecting the same.