JPS6244915B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing coenzyme Q ₁₀ (hereinafter abbreviated as "CoQ ₁₀ ") using basidiomycetes.

CoQ ₁₀ is a 2,3-dimethoxy-
It is a 5-methyl-1,4-benzoquinone compound with 10 isoprene units.

CoQ ₁₀ mainly exists in the mitochondria of animals, plants, and microbial cells, and is known to be an important factor in the terminal electron transport chain within cells.In recent years, its properties have attracted attention as a medicine, and it has been used for treatments such as congestive heart failure. Many clinical effects are being reported, and its use as a medicine is expected to expand in the future.

Currently, attempts are being made to produce CoQ ₁₀ by synthetic methods, but due to its complex structure as described above,
When producing CoQ ₁₀ industrially, it is difficult to satisfy all aspects of manufacturing cost, manufacturing process, and yield.
Production methods using fermentation methods are being rediscovered.

Conventional methods for producing CoQ ₁₀ using fermentation methods include
Methods using the purple photosynthetic bacterium Rhodoseudomonas capsulatus (Japanese Patent Publication No. 48-21519), methods using yeasts belonging to Rhodotorula, Cryptococcus, and Sporobolomyces (Japanese Patent Publication No. 48-8836), etc. are known. Even when these bacteria and yeast are used, it is difficult to produce CoQ ₁₀ on an industrial scale due to the slow growth rate of the bacteria, the time it takes to cultivate the raw material, and the low amount of CoQ ₁₀ produced within the bacteria. has many problems.

Therefore, the present inventors investigated various methods for producing CoQ ₁₀ using an even better fermentation method, and found that
The present invention was achieved by discovering that Basidiomycetes belonging to the genus Ustilago have a remarkable ability to produce CoQ ₁₀ .

That is, the gist of the present invention belongs to the genus Ustilago,
By a fermentation method characterized by culturing a basidiomycete having the ability to produce CoQ ₁₀ in a nutrient medium to produce CoQ ₁₀ , and collecting it from the resulting bacterial cells.
This is a method for producing CoQ ₁₀ .

In addition, CoQ caused by basidiomycetes belonging to the genus Ustilago
Ustilago zeae is already known for its production (J. Biol.
Chem. Vol. 234, p. 2169, 1959), the amount of CoQ accumulated in the bacterial cells is only a trace, and the number of isoprene units in CoQ is not clear. Therefore, it has not been previously known that the basidiomycetes belonging to the genus Ustilago have the ability to produce CoQ ₁₀ , and that the basidiomycetes can be used for the fermentation production of CoQ ₁₀ , and the present invention is based on this new knowledge. It is based on

As the microorganisms used in the present invention, both wild strains and mutant strains of CoQ _10- producing bacteria belonging to the genus Ustilago can be used. Representative examples include Ustilago synodontheis IFO-9758, Ustilago esculenta IFO-9887, and Ustilago esculenta IFO-9887. My Days IFO−
6907, Ustilago cusanoi IFO-9070, and Ustilago labenhorstiana IFO-8995.

The carbon source added to the nutrient medium when culturing basidiomycetes in the present invention may be any carbon source as long as it can be assimilated, such as glucose,
Sugars such as sucrose, molasses, sulfite, pulp waste liquid, and wood saccharification liquid; Organic acids such as acetic acid and citric acid; Alcohols such as methanol and ethanol;
Liquid hydrocarbons, oils and fats, glycerin, etc. can be used. Inorganic or organic substances such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonia, urea, peptone, and casein are used as nitrogen sources, and other inorganic salts include potassium, magnesium, phosphoric acid, zinc, iron, manganese, copper, Various salts such as calcium are used. Further, as micronutrients, yeast extract, meat extract, corn steep liquor, vitamins, nucleic acids, amino acids, etc. may be added as necessary.

It is preferable to culture under aerobic conditions.
Generally, it is advantageous to carry out aerated agitation culture or shaking culture. Cultivation can be carried out at a temperature of 15 to 35°C, but especially
The temperature is preferably 25 to 33°C, and the pH of the culture solution may be 5 to 8.5, but 6 to 8 is particularly preferred. Moreover, the culture time requires about 24 to 72 hours. Furthermore, depending on the culture conditions, antifoaming agents such as silicone resins and fatty acid derivatives may be added as appropriate.

Extraction of CoQ ₁₀ from bacterial cells can be carried out in a conventional manner, for example by saponifying with methanol or ethanolic alkali in the presence of pyrogallol, and extracting n-hexane, n-heptane, petroleum ether from the saponified solution. In organic solvents that are immiscible with water, such as
Transfer CoQ ₁₀ . For further purification, this extract can be fractionated and purified by adsorption chromatography or thin layer chromatography using florisil, silica gel, alumina, or the like. CoQ ₁₀ isolated from bacterial cells was identified using various analytical methods such as thin layer chromatography, paper chromatography melting point measurement, elemental analysis, and infrared and ultraviolet absorption spectra. In addition, as a method for quantifying CoQ ₁₀ , the method of Rezdfan [Methods in
Methods in Enzymology
Volume 10, page 381, 1967] was used.

As described above, the method of the present invention utilizes microorganisms.
To produce CoQ ₁₀ , we used a basidiomycete that had not been previously recognized to have the ability to produce CoQ ₁₀ , but by using this basidiomycete, we were able to Good yield and large amount of CoQ ₁₀
This has the effect that it is possible to manufacture

In addition, when producing compounds using basidiomycetes, sugars such as glucose were conventionally used as carbon sources in the culture medium, but in the method of the present invention, the above-mentioned carbon sources are used to produce CoQ ₁₀ . A variety of materials can be used. Moreover, when a non-saccharide carbon source is used during culture, the yield of CoQ ₁₀ is better than when glucose is used as a carbon source.

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

Example 1 20g glucose, 1g ammonium sulfate, 1g potassium phosphate, 0.5g magnesium sulfate heptahydrate,
A culture medium was prepared by dissolving 5 mg of ferrous sulfate heptahydrate, 3 g of polypeptone, and 1 g of yeast extract in 1 part of tap water. After adjusting this medium 5 and setting the pH to 7.0, it was poured into a 10-volume jar fermenter.
Sterilized at 121°C for 12 minutes. Ustilago synodonteis IFO-9758, which had been precultured in a Sakaguchi flask using a medium with the same composition as above, was inoculated into the above jar fermenter, and cultured at a culture temperature of 30°C, an aeration rate of 1 VVM, and a stirring speed of 500 rpm. Ta. After culturing for 35 hours, the bacteria were collected using a centrifuge, and 510 viable bacteria were collected.
g (46.4 g in terms of dry matter) was obtained.

Next, suspend 100 g of the above-mentioned live bacterial cells in 20 ml of water, add 200 ml of methanol and 10 ml of pyrogallol.
g, 10 ml of 60% caustic soda was added, heated under reflux at 85°C for 1 hour, cooled by adding 400 ml of water, and then
- Extraction was carried out twice by adding 200 ml of hexane.
The n-hexane layer was washed three times with 100 ml of water, and after dehydration by adding anhydrous sodium sulfate to the organic solvent layer, the n-hexane was distilled off using an evaporator.

20 ml of acetone was added to the residue, and then the acetone was distilled off and dried. The dried product was redissolved in n-hexane, applied to an alumina column, and the column was first washed with 100 ml each of n-hexane solutions containing 2% and then 4% ethyl ether. - Elution was carried out using a hexane solution. After elution, the CoQ _10- containing fractions were combined and concentrated under reduced pressure, and the dried product was dissolved in 5 ml of ethanol.

Leave this melt in a cold room to produce yellow coarse crystals23
I got mg.

The crude crystals were subjected to two reconsolidation operations from ethanol to obtain 12 mg of yellow plate-like crystals. The identity of CoQ ₁₀ was confirmed by thin layer chromatography, elemental analysis, melting point, ultraviolet absorption spectrum, and mass spectrometry.

Example 2 Ustilago esculenta IFO-9887 was cultured in a medium having the same composition as in Example 1, and viable cells were grown.
407g (37g in terms of dry bacterial cells) was obtained. This was extracted and purified in the same manner as in Example 1, and 8 mg of CoQ ₁₀
I got it.

Example 3 Ustilago mayidas IFO-6907 was cultured in a medium having the same composition as in Example 1, and 330 viable cells were grown.
g (30 g in terms of dry bacterial cells) was obtained. Example 1
15 mg of CoQ ₁₀ was obtained by extraction and purification in the same manner as above.

Example 4 Ustilago cusanoi IFO-9070 was cultured in a medium having the same composition as in Example 1, and 264 g of viable bacterial cells were grown.
(24 g in terms of dry bacterial cells) was obtained. This was extracted and purified in the same manner as in Example 1 to obtain 6 mg of CoQ ₁₀ .

Example 5 Ustilago labenhorstiana IFO-8995 was cultured in a medium having the same composition as in Example 1 to obtain 374 g of viable cells (34 g in terms of dry cells). This was extracted and purified in the same manner as in Example 1 to obtain 8 mg of CoQ ₁₀
I got it.

Example 6 Ustilago mayidas IFO-6907 in Example 1
Medium composition used in glucose and hexadecane 20
The cells were cultured for 72 hours under the culture conditions shown in Example 1 using the medium replaced with ml/ml, and 297 g of viable cells (27 g in terms of dry cells) were obtained. This was extracted and purified in the same manner as in Example 1 to obtain 20 mg of CoQ ₁₀ .

Example 7 Ustilago labenhorstiana IFO-8995 was cultured under the culture conditions shown in Example 1 using a medium in which the glucose in the medium composition used in Example 1 was replaced with ethanol, while maintaining the ethanol concentration at 0.5% or less. Culture to 3%, live bacteria
220 g (20 g in terms of dry bacterial cells) was obtained. This was extracted and purified in the same manner as in Example 1 to obtain 12 mg of CoQ ₁₀ .

Claims (1)

[Claims] 1. Cultivating a basidiomycete belonging to the genus Ustilago and having the ability to produce coenzyme Q ₁₀ in a nutrient medium to produce coenzyme Q ₁₀ , and collecting it from the resulting bacterial cells. Coenzyme Q ₁₀ produced by fermentation method characterized by
manufacturing method. 2 Basidiomycetes that belong to the genus Ustilago and have the ability to produce coenzyme Q ₁₀ are Ustilago cynodontis IFO-9758 and Ustilago esculenta IFO-
9887, Ustilago Maydeis
mydis) IFO-6907, Ustilago kusanoi IFO-9070 and Ustilago labenhorstiana (Ustilago kusanoi)
rabenhorstiana) IFO-8995, the method for producing coenzyme Q ₁₀ according to claim 1. 3. The method for producing coenzyme Q ₁₀ according to claim 1 or 2, wherein the carbon source of the nutrient medium is saccharides, organic acids, alcohols, hydrocarbons, fatty acids, fats and oils, or glycerin.