JPS6251598B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing coenzyme _Q10 . More specifically, the present invention involves culturing coenzyme Q ₁₀ -producing microorganisms belonging to the genus Pseudomonas in a medium supplemented with crushed tomatoes or carrots or their water-soluble fractions to produce coenzyme Q 10, and then collecting the coenzyme Q ₁₀ . The present invention relates to a method for producing coenzyme Q ₁₀ , which comprises the steps of: Coenzyme Q ₁₀ is a compound represented by the following structural formula, and plays an extremely important role as a component of the electron transport system in living organisms. It is already clear that this substance exhibits excellent pharmacological and physiological effects against various diseases. However, extracting it from animal heart muscles requires expensive raw materials and it is difficult to produce large-scale membranes, and even with synthetic methods, the yield is not always satisfactory due to the complicated operations involved. . In this respect, the production of coenzyme Q ₁₀ using microorganisms can be an economical method because of its relatively simple process. Regarding the fermentation method, coenzyme Q ₁₀ has been added to yeasts belonging to Rhodotorula, Sporobolomyces, Candida, Torulopsis, filamentous fungi belonging to Neurospora, Aspergillus, Pseudomonas, Agrobacterium, Rhodospirillum, and Rhodoseudomonas. Although it is known that coenzyme Q10 is contained in coenzyme Q10, the amount produced is extremely low, and it is far from being able to produce coenzyme _Q10 on an industrial scale. The present inventors have repeatedly investigated various compounds that can significantly increase the content of coenzyme _Q10 per unit bacterial cell in the case of culturing using bacteria belonging to the genus Pseudomonas. The present invention was completed based on the discovery that the content of coenzyme Q ₁₀ per unit cell can be significantly increased by adding the fraction to the growth medium. The microorganism used in the present invention is a bacterium belonging to the genus Pseudomonas that has the ability to produce coenzyme _Q10 .
50-122452) or the mutant strain Pseudomonas N842-M16 obtained therefrom (see
3155, Patent Application No. 122453) or Pseudomonas C1-36 (Feikoken Bacterial Serial No. 5209, Patent Application No. 1983)
No. 54-124727) and the like are preferable because they produce coenzyme Q ₁₀ in high yield. The culture medium in the present invention is not particularly limited; for example, carbon sources include hydrated carbon such as glucose,
Organic acids such as citric acid, methanol, alcohols such as glycerin can be used, nitrogen sources include ammonium sulfate, urea, ammonium sulfate, ammonium phosphate, ammonium chloride, ammonia gas, etc., and inorganic substances include phosphates, magnesium salts, calcium salts, Iron salts, manganese salts, and other trace metal salts can be used as necessary, and amino acids, vitamins, soybean protein hydrolysates, yeast extracts, peptones, casamino acids, and the like can be used as growth-promoting substances. For culturing, culture is carried out aerobically with shaking or stirring at pH 5 to 8 and 20 to 40°C for about 2 to 10 days. The crushed tomato or carrot or its water-soluble fraction used in the present invention may be crushed as is by a mixer or the like, or the supernatant obtained by removing solids from the crushed substance, or the supernatant can be fractionated using an ion exchange resin or the like. Any water-soluble fraction such as the fraction obtained by the above method may be used, and commercially available tomato juice may be used as it is or after being appropriately fractionated. The method and timing of addition of the additives are arbitrary; for example, they may be added all at once before the start of culture or during culture, or they may be added in portions depending on the fermentation state. The amount added is 2 to 80%, preferably 20 to 40% of the amount of the medium. After completion of the culture, the bacterial cells are separated from the culture solution by centrifugation or filtration using a conventional method. The cells obtained here contain abundant coenzyme Q ₁₀ , so after appropriately treating the cells, coenzyme Q ₁₀ can be used together with the cells in nutrients, medicines, or feeds. can. The mycological properties of representative strains suitable for use in the present invention are as follows.

【table】

[Table] The mycological properties of Pseudomonas N842-M16 strain are similar to Pseudomonas except that carotenoid production is lower than that of Pseudomonas N842.
It is almost the same as N842. The bacterial cells that are the raw material for the treatment for the isolation of coenzyme Q ₁₀ can be either live bacterial cells, dried bacterial cells, or processed bacterial cells. An example of a method for isolating coenzyme _Q10 is to first add a mixture of methanol, caustic soda and pyrogallol to a solution containing microbial cells for saponification, and then extract under reflux at 60 to 90°C for 1 to 2 hours. Next, the extract is extracted with a solvent such as n-hexane, and the solvent layer is washed with water and dehydrated, then concentrated, and this is added to a column such as silica gel, and when developed with benzene etc., coenzyme Q ₁₀ is eluted. The eluted fraction is concentrated to dryness and the ethanol-soluble portion is left to cool, yielding red-yellow crude crystals of coenzyme Q ₁₀ . If recrystallization is repeated further, the coenzyme
Although pure crystals of Q ₁₀ can be obtained, separation using clathrate compounds, molecular distillation, etc. are also effective. Identification of coenzyme Q ₁₀ is done by UV spectrum, melting point measurement,
The results were compared with standard products using reverse phase thin layer chromatography using acetone/water (95:5) as a developing solvent, IR, NMR, mass spectra, etc. EXAMPLES Next, the present invention will be specifically explained with reference to Examples, but the present invention is not limited thereto. Example 1 Glucose, 300 g, potassium dihydrogen phosphate (KH ₂ PO ₄ ) 15 g, dipotassium hydrogen phosphate (K ₂ HPO ₄ ) 15 g, ammonium chloride (NH ₄ Cl)
75g, magnesium sulfate heptahydrate ( _MgSO4 .
7H ₂ O) 1.5g, sodium sulfate (Na ₂ SO ₄ ) 7.5
Pseudomonas N842 was added to a medium containing 75 g of corn staple liquor (PH6.5), and 3.75 g of commercially available tomatoes crushed with a juicer to remove the solids, and tap water added to make a total volume of 15.
(Feikoken Bibori No. 3154) was cultured for 48 hours in 300 ml of an aqueous medium containing 2% glucose, 1% peptone, and 1% yeast extract (PH6.5), and the culture was inoculated as a seed. A 30-volume jar fermentor was used for agitation culture at 30° C. with air aerated at a rate of 15 per minute. If the pH drops, use ammonia water.
The pH was adjusted to 6.5. After completion of the culture, a wet bacterial cell paste was obtained by centrifugation. This is as a dried bacterial cell.
68g, and the bacterial cells contained 2.2mg of coenzyme _Q10 per 1g of dry matter. That is, culture solution 1
The amount of coenzyme Q ₁₀ produced per serving was 9.97 mg. The obtained wet bacterial cells were mixed with 300 ml of water, 2 methanol, 40 g of pyrogallol, and 300 g of sodium hydroxide, and heated under reflux at 85° C. for 1 hour. After cooling 2
The extract was extracted twice with n-hexane, and the n-hexane layer was taken, washed with water, dried over Glauber's salt, and concentrated to dryness. The acetone-soluble portion of the dried solid product is taken, the acetone is distilled off, and the residue is dissolved in n-hexane. This solution was mixed with ether/n using a silica gel column.
- Develop with a hexane mixture and collect the fraction containing coenzyme _Q10 . The solvent was distilled off from this eluate, and the residue was dissolved in a small amount of ethanol and left in a cool place to precipitate coenzyme Q ₁₀ crystals. Recrystallization from ethanol was repeated three times to obtain 57 mg of coenzyme Q ₁₀ crystals. On the other hand, the above medium 15 without the addition of tomato youth
The same culture as above was carried out, and a wet bacterial cell paste was obtained by the same method as above. 60g as dried bacterial cells
It contained 1.6 mg of coenzyme _Q10 per gram of bacterial cells. That is, the production amount of coenzyme Q ₁₀ per culture solution was 6.40 mg. Coenzyme Q ₁₀ was extracted and purified by the same method as above to obtain 38 mg of coenzyme Q ₁₀ crystals. Considering the effect of tomato youth on the production and accumulation of coenzyme Q ₁₀ based on the above results, we found that the addition of tomato youth to the culture medium
_Q10 increases by approximately 56% per 1 culture solution, and 1 dry bacterial cell
Per gram, it increased by about 38%. Example 2 In the same manner as in Example 1, Pseudomonas N842 was cultured in the same manner as in Example 1 except that instead of tomato juice, a water-soluble fraction obtained by crushing 1.5 kg of carrots and removing solid matter was added. The cells were separated to obtain a cell paste. It weighs 56g as dry bacterial cells,
This bacterial cell contained 2.7 mg of coenzyme Q ₁₀ per gram of dry matter. The bacterial cells were extracted and purified in the same manner as in Example 1 to obtain 59 mg of pure coenzyme Q ₁₀ crystals. When using the method of the present invention, compared to using crushed carrots or their water-soluble fractions without additives,
Coenzyme Q ₁₀ increased by about 57% per 1 culture solution, and by about 69% per 1 g of dry bacterial cells. Example 3 In the medium of Example 1, the same amount of tomato youth as in Example 1 was dialyzed instead of tomato youth,
Obtain the external dialysis fluid and use this as a cation exchange resin.
Pseudomonas N842− as in Example 1 using a medium supplemented with a solution passed through Amberlite IRC 50H ⁺ .
M16 (Feikoken Bacteria No. 3155) was cultured and the bacterial cells were isolated to obtain a bacterial paste. 77g as dried bacterial cells
This bacterial cell contained 3.8 mg of coenzyme per gram of dry matter. The bacterial cells were extracted and purified in the same manner as in Example 1 to obtain 110 mg of coenzyme Q ₁₀ crystals. On the other hand, Pseudomonas was grown in the same manner as in Example 1 using the above medium without adding the tomato youth fraction.
N842-M16 was cultured and the bacterial cells were separated to obtain a bacterial cell paste. The dry bacterial mass was 78 g, and this bacterial cell contained 3.0 mg of coenzyme Q ₁₀ per gram of dry matter. The bacterial cells were extracted and purified in the same manner as in Example 1 to obtain 80 mg of coenzyme Q ₁₀ crystals. Considering the effect of tomato youth fractions on the production and accumulation of coenzyme Q ₁₀ based on the above results, the addition of tomato youth to the culture medium increased coenzyme Q ₁₀ by about 30% per culture solution. The amount increased by about 28% per bacterial cell. Example 4 20 ml of the medium from Example 1 except ammonium chloride was placed in a test tube, and Pseudomonas N842
-M16 was planted and cultured with shaking at 30°C for 4 days.
The quantitative value of coenzyme _Q10 obtained by this culture was 20 mg per 1 medium, and 3.4 mg per 1 g of dry bacterial cells.
It was hot. On the other hand, Pseudomonas N842-M16 was cultured in the same manner as above in the above medium without tomato fraction added. The obtained quantitative value of coenzyme Q ₁₀ is per 1 medium.
The amount was 14.5 mg, and 2.4 mg per gram of dry bacterial cells. Example 5 Pseudomonas C1-36 (Feikoken Bacteria Serial No. 5209) was cultured in the same manner as in Example 4 using the medium of Example 2 except that ammonium chloride was removed. The quantitative value of coenzyme _Q10 obtained by this culture was 95 mg per 1 medium, and per 1 g of dry bacterial cells.
It was 15.5 mg. Culture was performed in the same manner as above without adding the carrot fraction. The quantitative value of coenzyme Q ₁₀ obtained by this culture was 72 mg per 1 medium and 13 mg per 1 g of dry bacterial cells.

Claims (1)

[Claims] 1. A microorganism of the genus Pseudomonas that has the ability to accumulate and produce coenzyme Q ₁₀ within the bacterial body is cultured in a medium supplemented with crushed tomato or carrot or their water-soluble fraction to produce and accumulate coenzyme Q ₁₀ , and A method for producing coenzyme Q 10, which comprises then collecting the coenzyme Q ₁₀ .