JPS6283895A - Production of coenzyme q10 - Google Patents

Abstract

PURPOSE:To readily, stably and efficiently produce the titled enzyme useful for medicines, etc., by cultivating a variant strain, capable of synthesizing carotenoid and derived from a microorganism of the genus Protomonas having the ability to produce coenzyme Q10. CONSTITUTION:A strain Protomonas extorquens BP-41, etc., is subjected to UV irradiation treatment, etc., to give a variant strain, capable of synthesizing carotenoid and having increased ability to produce coenzyme Q10, e.g. Protomonas extorquens DB-100, etc., which is then cultivated in a culture medium consisting of a carbon source L-arabinose, nitrogen source peptone, inorganic salt iron salt, growth promoting substance amino acid, etc., at 20-40 deg.C and 6-8pH by a batch culture method, etc., to produce and accumulate the coenzyme Q10 in microbial cells. The culture fluid is then subjected to centrifugal separation, etc., to separate and recover the microbial cells, which are extracted with methanol, etc., separated and purified to isolate and aimed coenzyme Q10 expressed by the formula.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing coenzyme Q10, and more particularly to a method for producing cofermented IQ,o using microorganisms.

Coenzyme Q10 has the structural formula shown below and plays an important role as an electron conductor in the terminal respiratory system in vivo, and is used as a cardiac function enhancer, pancreatic function enhancer, and myasthenia gravis treatment agent. It is a compound useful as a pharmaceutical agent such as a therapeutic agent for emphysema, a therapeutic agent for aplastic anemia, and a therapeutic agent for alopecia areata, and as a feed additive.

[Prior Art and Problems to be Solved by the Invention] Conventionally, coenzymes Q and O have been produced by extracting them from microbial, animal or plant tissues and further purifying them. Also,
In recent years, coenzymes Q and o have been obtained from bacterial cells obtained by culturing microorganisms.
There are also known methods for extracting.

However, coenzyme Q contained in microorganisms.

The content is relatively low, and for industrial production, it is desirable to obtain bacteria with a higher content of coenzyme Q.

The present inventors aimed to obtain a bacterial strain with a high content of coenzyme Q by the 11 mutation treatment.

[Means and actions for solving problems]

As a result of repeated research to find mutant strains that produce coenzyme Q in large quantities, the present inventors mutated a strain belonging to the genus Protomonas, resulting in a mutant strain capable of synthesizing p-tinoids, a strain with an increased ability to synthesize carotenoid, and a strain with increased carotenoid production. coenzyme Q by obtaining a reduced strain or a carotenoid-deficient strain. We succeeded in obtaining a strain containing a large amount of , and completed the present invention. That is, the present invention relates to coenzyme Q, which belongs to the genus Protomonas. It has a small production capacity.

The parent strain of the strain used in the present invention is a methanol-assimilating Durham-negative rod Pseudomonas that does not form spores, forms pink colonies, and has polar Ben hairs.
Pro taminobacter, Mycop
1ana.

This strain belongs to a group called the Th1obacillus genus. These strains were +9 by Urakami and Komagata.
In 1984, it was proposed as a bacterium of the genus Protomonas (International Journal
of Systematic Bacteriology
y, Vol. 34-pp. 188-201, 1984).

There are no particular restrictions on the mutation treatment method, but UV treatment, N-methyl-N'-nido-N-nido-N-nido-sogen (NTG) treatment, and ethylmethanosulfanalyte treatment, which are commonly used in practice, are not particularly limited. (EMS) treatment is preferred.

The carotenoid-synthesizing mutant strain is obtained by culturing the mutagenized bacterial solution on an agar medium and selecting based on changes in the pink color density of the resulting colonies.

For example, Protomonas extorfens BP-41
(Feikoken Bacteria Serial No. 4896), as a mutant strain with increased coenzyme Q production ability, for example, Protomonas extorfens DB-10, which is a carotenoid increase method.
0 (Koken Bokuyori No. 8590) and the carotenoid deletion strain DB-1115 (Koken Bokuyori No. 8391).
is induced. Also, from 1) 33-1115, assistant #
As a mutant strain with increased production ability of MotoQ, for example, HB-5 (Feikokenzonoyo No. 8392), which is a carotenoid increasing method, is used.
(No. 8395) and HB-8 (Feikoken Bibori No. 8395) are respectively induced.

The direct properties of these mutant strains are that the ability to synthesize carotenoids was changed and the ability to produce coenzymes Q and O was increased.
There is no substantial difference from -41.

In addition, although the strain was identified as Protomonas extorfens, in the paper by Uragami and Komagata, the medium for screening the same mutant strain was a medium in which both the parent strain and the mutant strain could grow and proliferate. There are no particular restrictions. The medium for growing the mutant strain, ie, the medium for producing coenzyme Q, may be any medium that allows the growth of the mutant strain, and is not particularly limited. i.e. carbon source,
Conventional media containing nitrogen sources, inorganic salts, and, if necessary, growth-promoting substances, are used.

Any carbon source that can be assimilated by these bacteria may be used, such as L-7 rabinose, D-xylose, D-mannose, D-fructose, galactose,
Glycerol, succinic acid, citric acid, acetic acid, methanol, and the like can be used alone or in combination. Among these, methanol is practically preferred.

As the nitrogen source, ammonium sulfate, urea, ammonium nitrate, ammonium phosphate, peptone, beef stock, etc. are used. As the inorganic salts, citrate salts, magnesium salts, iron salts, and other trace metal salts are used as necessary. Examples of growth-promoting substances used include amino acids, nucleic acids, vitamins, yeast extracts, and malt extracts. In addition, if the strain used is auxotrophic,
The auxotrophic substance is added to the medium.

As for the culture conditions, a temperature suitable for growth and proliferation of each strain may be selected within a temperature range of 20 to 40°C. culture ip
H is usually selected from a pH range of 6 to 8, which is suitable for the growth and proliferation of each strain. The culture method may be either batch culture or continuous culture. When using ammonium salt as a nitrogen source, the pH of the culture medium decreases as the bacterial cells multiply, so in order to keep the pH of the medium constant during the culture period, ammonia, caustic potassium, or It is necessary to adjust the pH of the culture solution by adding caustic soda or the like. Among these, ammonia is particularly preferred.

After culturing m* in this manner, the bacterial cells are separated and recovered from the culture solution. For isolation and recovery of bacterial cells.

Conventional solid-liquid separation means are employed. For example, a method of centrifuging the culture solution as it is, a method of adding cells of other microorganisms larger than the present microorganism to the culture solution as a filter aid, or a method of filtering and separating bacterial cells from the culture solution by pre-coating. A method in which various flocculants are added to the culture solution to aggregate the bacterial cells, and the bacterial cells are separated from the culture solution by filtration or centrifugation. , further 50-10
A method of separating the microbial cells by heating at 0° C. can be applied. The isolated bacterial cells can be used as is or in 9t
The following coenzyme Q is dried using an M dryer or the like. Extraction and purification are performed.

Separation and extraction of coenzyme Q can be carried out according to conventional methods. For example, suspending bacterial cells in ethanol and
Heat extraction at 0 to 90°C for 1 to several hours. Alternatively, first use a mixture of methanol, sodium hydroxide, and Pigal-pol to saponify saponifiable substances such as paper lipids in the bacterial cells, and from this saponified liquid, extract a substance that does not mix with water, such as n-hexane. Add an organic solvent to extract coenzyme Q1o. This extract is then isolated by fractional purification using silica gel, futridyl, and the like.

Coenzyme Q obtained from bacterial cells. For identification, means such as high performance liquid chromatography, elemental analysis, melting point measurement, infrared and ultraviolet absorption spectra, nuclear magnetic resonance spectroscopy, and ff, 1 analysis are generally used.

〔Example〕

The present invention will be explained in more detail with reference to Examples.

Example pure water 11 Atasho, (NH4)2S043g, KH2PO
4164g, NazHt'042.1g, Mg5O<-
7HzOO, 29%CaC1w・2H203oq, Fe
(jHsoy-XH2030■, MnCl2-4H2
05m? , Zn5Oa・7H205■, CuSO4・
5H200, 5W, yeast extract 0.2g, calcium pantothenate 4■ and methanol 8p were dissolved in a bath, and p
Medium with H adjusted to 7.1 (hereinafter referred to as medium B)
Protomonas extorfens BP-4+ grown for 18 hours at 30°C was treated with 0.5 Da/-
10 in 0.1 M phosphate buffer (pH 7,0) containing
The mixture was suspended at a ratio of 50% to 50%, and shaken at 30°C for 30 minutes. After that, the bacterial cells collected by centrifugation were washed with 0.1M phosphate buffer (pH 7.0), and the bacterial cells were washed with 0.1M phosphate buffer (pH 7.0).
Cell suspension resuspended in phosphate buffer (108 cells/
ml) o, 1- was inoculated into medium B101dK.

Culture was performed for 2 days.

After diluting 0.1M of this culture solution with physiological saline, agar plate medium (agar was added to medium B to give a concentration of 2wt96)
After culturing for 4 days, carotenoid-synthesizing mutants were selected and isolated based on the intensity of pink color of the colonies.

Among these mutant strains, we selected Putomonas extorfens DB-100 (FeI-Ken Bacterial Collection No. 8
390), carotenoid deletion strain 1) B-11
15 (Choken Bacteria No. 8391) was obtained.

Furthermore, in order to increase the coenzyme Q content, mutation treatment was performed again using Protomonas extorfens DB-1115 as the parent strain in the same manner as above. Cultivation was carried out on an agar medium (medium B) for 4 days, and a ptinoid-synthesizing mutant strain with increased carotenoid content was isolated based on changes in the intensity of the pink color of Corny.

Among these mutant strains, the mutant strain with increased content of coenzymes Q and O is the same strain with increased carotenoid content.
B-5 (Feikoken Bibori No. 8392) and HB-8 (
8393) was obtained.

The culture solution of each strain precultured at 50°C for 3 days using medium B was inoculated at 1% by volume into medium B, and cultured with rotary shaking at 30°C. 48 hours after the start of culture, the methanol concentration in the culture solution became 0,001wt96 or less.

Extract this culture solution wt96 inpropanol aqueous solution 4,
After carrying out hexane transfer and further oxidizing the reduced coenzyme Q in the resulting hexan beef san solution with iron nitrate, the content of coenzyme Q in the hexan san solution was measured. Carotenoid content was also measured.

The results are shown in Table 1.

Table 1 〔Effect of the invention〕 According to the present invention, coenzyme Q is produced using bacteria.

can be obtained easily, stably and efficiently,
Furthermore, industrially produced chemical substances can also be used as raw materials.

Patent applicant: Mitsubishi Gas Chemical Co., Ltd. Representative Kazuyoshi Nagano

Claims (1)

[Claims] coenzyme Q_1_0, which is characterized in that coenzyme Q_1_0 is collected from the bacterial cells obtained by culturing a carotenoid synthesizing mutant strain derived from a microorganism belonging to the genus Protomonas and having coenzyme Q_1_0 producing ability. Manufacturing method.