JPH01117793A - Production of ubiquinone coq9 - Google Patents

Abstract

PURPOSE:To obtain ubiquinone CoQ9 which is used as a remedy for cerebrovascular disorders, cardiac failure, hypertension or the like in high effi ciency by culturing a strain in Mortierella in an enriched medium. CONSTITUTION:A strain in Mortierella such as Mortierella isabellina IFO 7873 is cultured in a medium containing 300-10g/1,000ml of carbohydrate such as glucose, 50-10g/100ml of hydrocarbon, such as n-alkane, 100-10g/100ml of fatty acid of 8-22 carbon atoms such as caprylic acid, additionally nitrogen source such as urea or ammonium nitrate, inorganic salts such as potassium dihydrogen phosphate, growth factor such as yeast essence, and surface active agent such as polyoxyethylene (6) sorbitan fatty acid ester, at pH3.5-5.5 at 15-35 deg.C for 90-150hr by the shaking culture method to obtain the culture mixture. The cell bodies are separated from the mixture, crushed, extracted, then the lipid in the extract is saponified, the unsaponified fraction is extracted with a nonpolar organic solvent such as n-hexane to collect the subject ubiquinone CoQ9.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing ubiquinone-9, a type of coenzyme, using microorganisms.

Ubiquinone, known as coenzyme Q, is represented by the following formula (1) (where n is an integer from 1 to 12):
Co Qit C depending on the number of isoprenoid units in the side chain n
o Q*, “””Co Qxi.

Among these, CoQs with n=9 is called ubiquinone-9, and is known to be useful as a therapeutic agent for cerebrovascular disorders, heart failure, hypertension, and diabetes, and as a preventive of side effects of the anticancer drug Adriamycin.

Conventionally, methods for producing ubiquinone 9 include methods for obtaining ubiquinone 9 from cultures of microorganisms as shown in (1), (2), and (2) below, and from cultures of cultured plant cells as shown in (2) and (3). It is being

■ 7+W belonging to the genus Candida and the genus Shortmonas (Japanese Patent Publication No. 10752/1983).

■Bacteria belonging to the genus Achromobacter (Special Publication No. 44-269)
(Japanese Patent Application Laid-Open No. 08), ■Cultured cells of gramineous plants (Japanese Patent Application Laid-open No. 72895/1989), ■Cultured cells of safflower (Japanese Patent Laid-open No. 80491/1983), ■Bacteria belonging to the genus Graphiola (Japanese Patent Laid-Open No. 1983-80491) 56-14829
Publication No. 2).

The sun is shining - However, methods ① to ③ above produce a low amount of ubiquinone 9 from bacterial cells or cultured cells, and method ③ produces a large amount of ubiquinone 9 from dried bacterial cells, but Both methods had the disadvantage of low bacterial cell concentration during cultivation, and were industrially disadvantageous.

For this reason, it has been desired to develop an industrially advantageous manufacturing method that can efficiently produce ubiquinone 9.

Means and Action for Solving the Problems In view of the above circumstances, the inventors of the present invention, as a result of intensive studies, cultivated bacteria belonging to the genus Mortierella using a nutrient medium, and extracted It has been found that ubiquinone 9 can be efficiently produced by separating ubiquinone 9.

It has been known that when bacteria belonging to the genus Mortierella are cultured in the presence of a carbon source, lipids containing γ-lylunic acid can be collected from the culture (Japanese Unexamined Patent Application Publication No. 5-1171).
7-144986, Japanese Patent Application Laid-open No. 168391/1982), the present applicant has previously reported that lipids containing γ-lylunic acid can be produced more efficiently and at lower cost by using fatty acids or esters thereof as such carbon sources. I suggested what could be done (
(Japanese Patent Application No. 61-279147).

The present applicants further conducted research and cultivated the above-mentioned bacteria belonging to the genus Mortierella using a nutrient medium.

Surprisingly, such bacteria convert the carbon source into ubiquinone-9, a coenzyme, in the nutrient medium and grow while producing a large amount of ubiquinone-9, and what's more, this ubiquinone-9 is mixed in the lipid components of the culture. Therefore, we have discovered that ubiquinone 9 can be collected by extracting the lipid component from the grown bacterial cells and separating the unsaponifiable components in the lipid component using high performance liquid chromatography, etc., and have accomplished the present invention. reached.

Therefore, the present invention provides a method for producing ubiquinone 9, which comprises culturing a bacterium belonging to the genus Mortierella in a nutrient medium and separating ubiquinone 9 from the lipid components of the culture.

The present invention will be explained in more detail below.

In the present invention, there is no particular restriction on the type of fungus belonging to the genus Mortierella, and any species belonging to this genus can be used, but Mortierella Isabelina IF07873 and the like can be particularly preferably used.

In addition, there are no restrictions on the types of ingredients in the nutrient medium, and a variety of ingredients can be used, but in particular, suitable carbon sources, nitrogen sources, inorganic salts, growth factors, surfactants, etc. may be used as necessary for culturing. It is preferable to include supplements, other nutritional sources, etc. Here, specific examples of the components of the nutrient medium include carbohydrates such as glucose (medium concentration: 300
~Log/100100O, hydrocarbons such as n-alkanes (50~Log/100d), carbon atoms 8 to 22 such as capric acid, caprylic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, etc. fatty acids or their alkyl esters, palm oil mainly composed of palmitic acid and oleic acid, soybean oil, corn oil, peanut oil, rice bran oil, mainly composed of oleic acid and linoleic acid, mainly oleic acid and linoleic acid. Nitrogen sources include urea, peptone, soybean protein, etc. (100-10 g/1000 m11), etc., such as rapeseed oil, which is composed of acid and erucic acid, and castor oil, which is mainly composed of oleic acid and lysyl acid. .

Organic nitrogen sources such as corn, steep liquor, etc. (NH4)
2So4. Inorganic salts such as inorganic nitrogen sources such as NH, No, etc. K Hz P O4e M g SO4 7H
,O.

FeSO4・7H,O, Zn5O,・7H,○.

Examples of growth factors include yeast extract and malt extract, and examples of surfactants include polyoxyethylene (60) sorbitan fatty acid ester.

In the present invention, there are no limitations on the method for culturing the bacterial cells, but
It is preferable to perform shaking culture, aerated agitation culture, or dry culture in a liquid medium, or to perform solid culture by impregnating a sponge or the like with the liquid medium. In addition, the pH of the medium is 3.
5-5.5, culture temperature 15-35℃, culture time 90℃
It is preferable to set the time to about 150 hours.

Furthermore, after the completion of the culture, known means can be used to collect lipid components from the culture, for example, after collecting bacteria from the culture, the cells are crushed with a homogenizer using a crushing aid. In addition, a method of extracting lipids using an extraction solvent can also be suitably employed. Here, as the extraction solvent,
It is preferable to use a mixed solvent of a non-polar solvent and a polar solvent.

Specifically, a mixed solvent of n-hexane and ethanol, a mixed solvent of chloroform and methanol, etc. are exemplified, and among them, a mixed solvent of n-hexane and ethanol can be preferably used. In addition, the mixing ratio of the non-polar solvent and the polar solvent in the above mixed solvent is 3:1 (volume/volume, the same applies hereinafter) ~
The ratio is preferably 1:3, especially 2:1.

In the present invention, ubiquinone 9 is extracted from the lipid components of the culture.
There are no limitations on the method for separating ubiquinone, but since ubiquinone 9 exists in the unsaponifiable components of lipid components, the lipid components are extracted with the above mixed solvent, salted out with saline, etc., and then the nonpolar solvent layer is separated. However, a method may be adopted in which the lipid obtained from this non-polar solvent layer is saponified with a methanolic alkali, the unsaponifiable matter is extracted with a non-polar solvent, and ubiquinone 9 is separated from this. In addition, after saponifying the lipids, extraction is performed with a non-polar solvent such as n-hexane to obtain fatty acids and unsaponifiable substances,
A method of separating ubiquinone 9 from unsaponifiables may also be adopted.

Note that the above-mentioned lipid component contains γ-lylunic acid and the like along with ubiquinone 9, but since these are present in the saponified component, they can be efficiently separated from ubiquinone 9.

Furthermore, methods for collecting ubiquinone 9 from unsaponifiable components include methods such as dissolving it in a nonpolar solvent and performing thin layer chromatography or column chromatography, among which methods include column chromatography using ODS as a packing material. By performing this, ubiquinone 9 can be collected with good yield.

As explained above, according to the method for producing ubiquinone 9 of the present invention, the bacterial cells of the genus Mortierella proliferate at a high concentration while producing a large amount of ubiquinone 9.
can be obtained efficiently and is industrially advantageous.

EXAMPLES The present invention will be specifically described below with reference to Examples, but the present invention is not limited to the Examples below.

[Example 1] i 80g of Shengsangluglucose, (NH*)tSO43g, K
H2PO45g, Mg5O*7HzO2,5g.

KCQ 0.5g, Fe50.・7H,050, Yeast extract 5g, Soybean protein 5g, Ca CQ, ・2H
,01,2■,CuSO4・5H200,2ff1g,
znSo4.7 Hz OL-Omg was dissolved in distilled water IQ and adjusted to pH 4.5. Add 1.8Q of this medium to 3
I put it in a Q-Long jar fermenter. After inoculating this with Morteierella Isabelina IF07873 strain,
Temperature: 30°C, ventilation amount: 0.5VVM, stirring speed: 500rp
The cells were cultured for 96 hours at m. After culturing, the bacteria were collected using two pieces of glass fiber paper (bacterial yield: dry bacterial weight: 37 g/Q-
broth).

Supervise the above bacterial cells in a blood dish with n-hexane/ethanol = 2/1 (volume/
The cells were homogenized using a homogenizer using a volume of the extract as an extraction solvent and glass beads as a crushing aid to crush the bacterial cells and extract the lipids. This extract was collected, salted out and washed with brine, and the n-hexane layer was collected and dehydrated with anhydrous sodium sulfate.
- Hexane was distilled off to obtain lipids. Lipids are dried bacterial cells 1
0.33 g/g was obtained.

For ubiquinone 9, 1 g of the lipid obtained by the above method was suspended in 10 mQ of water,
80 dl of methanol, 8 g of NaOH, 1 g of pyrogallol
was added and heated under reflux for 1 hour. After cooling, n-hexane 1
00- and 50 all of water were added to perform extraction twice. Furthermore, this n-hexane layer was washed three times with water, dehydrated by adding anhydrous sodium sulfate, and then n-hexane was distilled off to obtain a dry product.

The obtained dried product was dissolved in 10ml of n-hexane, and ubiquinone 9 was separated by high performance liquid chromatography (column: oDS, 4.5m$X3Qam, developing solvent: ethanol/water = 99.510.5 (volume/ capacity), flow rate:
0, 8 mQ/min, detector: ultraviolet absorption 275n
m).

Using pure ubiquinone 9 (manufactured by Sigma), quantification was performed using R and T absolute calibration methods, and qualitative analysis was performed using thin layer chromatography and ultraviolet absorption spectroscopy. There was a 1000pp seal.

(Example 2) 50g of Sumio Ritsukayo crude palm oil, 25g of soybean protein, 10g of yeast extract
, KHzPO* 15g, Mg5O, -7HzOL, 5
g, Fe3O3・7H,050■, CaCQ,・2H,
01,2mg, CuSO4”5H,00,2ag.

Zn5O, -7H,01, OI+g, 2.5 g of sucrose fatty acid ester, and 2.5 g of fatty acid monoglyceride were dissolved in distilled water IQ, and the pH was adjusted to 4.5.

1.8Q of this culture medium was charged into a 3Q capacity jar fermenter, and Mortierella Isabelina IF0787 was added to it.
When the three bacterial strains were inoculated and cultured for 150 hours under the same conditions as in Example 1, the bacterial cell yield was 46 g as a dry bacterial weight.
/Q-broth.

When the extraction process was carried out in the same manner as in Example 1, 0.25 g of lipid was obtained per 1 g of dried bacterial cells.

Amount of ubiquinone 9 Ubiquinone 9 was separated from lipids in the same manner as in Example 1.
When quantified, ubiquinone 9 was found to be 2900pp in lipids.
m existed.

[Example 3] KOHl was added to lipid 40H obtained in the same manner as in Example 2.
After adding 800 mQ of methanol and refluxing on a water bath for 1 hour, methanol was distilled off.

This was dissolved in water, excess sulfuric acid was added, and acid decomposition was performed on a water bath for 1 hour. After cooling, the mixture was extracted with n-hexane and the n-hexane was distilled off to obtain fatty acids and other saponified products. This was dissolved in 50 d of methanol and subjected to column chromatography using ODS as a packing material (column: 50 φ
X 50 (!@ w developing solvent niacetonitrile, flow rate 40 ml/l 1 in, detector RI), after dissolving the fatty acid, the developing solvent was changed to ethanol to elute unsaponifiables adsorbed on the column. This was collected, ethanol was distilled off, and after concentration, ubiquinone 9 was separated using column chromatography (column = 50φ x 50a11, packing material ODS, developing solvent: ethanol/water = 99.510,
5 (V/V), flow rate 40 d/ml (detector: ultraviolet absorption 275 nm), fractions of ubiquinone 9 were collected and the solvent was distilled off, resulting in crude crystals 11 of ubiquinone 9.
0■ was obtained.

Applicant: Laion Co., Ltd. Agent: Patent Attorney Takashi Kojima

Claims (1)

[Scope of Claims] 1. A method for producing ubiquinone 9, which comprises culturing a bacterium belonging to the genus Mortierella in a nutrient medium, and separating ubiquinone 9 from the lipid components of the culture. 2. The production method according to claim 1, wherein after saponifying the lipid components of the culture, ubiquinone 9 is collected by extracting the unsaponifiable matter with a non-polar organic solvent.