JPS61293391A - Production of coenzyme q - Google Patents

Abstract

PURPOSE:To extract coenzyme Q efficiently at a low cost, by extracting the coenzyme Q from dried microbial cells with hydrophilic dimethyl sulfoxide which is a hydrophilic solvent alone or in a mixture with another solvent in producing the coenzyme Q from the microbial cells. CONSTITUTION:Microbial cells are separated from a culture fluid of coenzyme Q by centrifugation and dried. Coenzyme Q is then extracted from the dried microbial cells using dimethyl sulfoxide alone or in combination with another solvent, e.g. methyl alcohol or isopropyl alcohol, as an extracting solvent. After the coenzyme Q ia extracted into the hydrophilic solvent, the coenzyme Q is transferred and dissolved in n-hexane, etc., in which the coenzyme is readily soluble, separated, dehydrated and concentrated to dryness. The resultant residue is purified by column chromatography, etc. and crystallized.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing coenzyme Q, and particularly to a method for producing coenzyme Q in high yield from microbial cells.

Coenzyme Q can be obtained by synthesis or by extraction from animal and plant tissues, mitochondria of microorganisms, etc. where it exists. Coenzyme Q is a quinone derivative having the general formula below.

(However, n in the formula represents an integer of 12 or less) Coenzyme Q
In vivo, it is involved in the terminal electron transport chain of the respiratory enzyme system,
It is a substance that shows excellent pharmacological effects on various diseases such as heart disease, high blood pressure, and malignant tumors.

The coenzyme Q of the present invention particularly includes coenzyme QIO, and specifically, is contained in microbial cells produced by fermentation.

(Prior art) Conventionally, the extraction method for coenzyme Q from natural products involves direct oxidation with an alcoholic alkaline hydroxide solution in the presence of an antioxidant such as pyrogallol, and then transfer to a hydrophobic solvent such as hexane. and then concentrated to dryness, followed by purification using column chromatography, etc., extraction with hydrocarbons, acetone, ethyl alcohol/ether (3:1), etc., or hot methyl alcohol → hot ether → ether/methyl. A method has been adopted in which extraction is repeated in sequence with alcohol (3:1) or hot acetone → ethyl alcohol → ether.

(Problems to be Solved by the Invention) Conventionally, this method has disadvantages in that the chemicals are expensive, the extraction equipment is large, and it requires a complicated process. There was a need for an industrial extraction method without drawbacks.

(Means for solving problems) The present invention provides an industrial extraction method that does not have the aforementioned drawbacks.

In extracting coenzyme Q from microbial cells containing coenzyme Q, the present invention involves drying the cells that have been centrifuged from a coenzyme Q culture solution, and dimethyl sulfoxide alone as an extraction solvent from the dried cells. This is a method for producing coenzyme Q, which is characterized in that coenzyme Q is extracted using a solvent or in combination with another solvent.

As the mixed solvent with dimethyl sulfoxide, a hydrophilic solvent selected from the group consisting of methyl alcohol, ethyl alcohol, Improvil alcohol n-propyl alcohol, and acetone is used. In particular, when isopropyl alcohol, in which coenzyme Q has a relatively high solubility, is mixed, the amount used is small and the extraction rate is excellent. Dimethyl sulfoxide has good permeability to bacteria, and by using it as a mixed solvent in this way, an excellent extraction rate can be obtained. The mixing ratio of dimethyl sulfoxide and other solvents is
The amount of dimethyl sulfoxide in the mixed solvent is about 10
~90% by volume, preferably about 40-60% by volume. The extraction temperature is about 20-100°C, preferably about 50-100°C.
The temperature is 80°C. Extraction takes 30 minutes to 5 hours, preferably about 2
Extract with stirring for ~3 hours.

In one preferred embodiment of the invention, the mixed solvent ratio is 1:1.
Extraction is carried out under conditions of stirring at a temperature of 70° C. for 2 hours.

After extracting coenzyme Q into a hydrophilic solvent, it is dissolved by stirring into a hydrophobic solvent such as n-hexane in which coenzyme Q is easily soluble, and separated into two layers. The solution layer is collected, washed with water, dehydrated with anhydrous sodium sulfate, etc., and concentrated to dryness. The obtained concentrated residue was dissolved in acetone, and after cooling, insoluble matter was removed by filtration, and purified by column chromatography using silica gel as a packing material and n-hexane as a developing agent. Concentrate the image to dryness,
The residue is dissolved in ethyl alcohol and crystallized under cooling. The crystallized coenzyme Q is filtered off and dried under reduced pressure.

In the extraction of coenzyme Q of the present invention, since chemicals such as acids and alkalis are not used, the material of the equipment can be easily selected and wastewater treatment equipment is not required.

The hydrophilic solvent separated by two-layer separation can be separated and purified by filtration, distillation, etc. and recovered. So you can reuse this.

The dried bacterial cells used in the present invention can be prepared, for example, as follows. If the amount is small, centrifuge the cultured cells, wash them with water, and filter the washed cells.
Dry at a low temperature (20 to 40°C) while crushing the material in the process of drying in a mortar. To obtain a large amount of dry powder, the cultured cells are centrifuged, washed and separated, and the moisture content is adjusted to about 85%. Using a spray dryer, the inlet temperature is 170-2.
00℃, outlet temperature (cyclone inlet temperature) 70-90℃
Dry with. No decomposition of coenzyme Q occurs even when operating under these conditions. Further, the moisture content in the dried bacterial cells is about 3 to 10% by weight, for example, 5 to 7% by weight.

Hereinafter, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to this.

Example 1 Aureobasidium (Aur) producing coenzyme Q1o
eobasidium) sp, 14 (Microorganism Deposit Number: Fiber Science and Technology Research Institute No. 5912), 16.9 g of urea,
KH2PO460g.

Mg5044H2[]6gXFeCL ・68zO0
, 18g, dibenzoylthiamine hydrochloride 12.4mg
A medium containing 2250 ppm of p-hydroxybenzoic acid, a trace amount of inorganic salt, and 12β of tap water was heated with aeration stirring type 30I! while controlling the ethyl alcohol concentration to 5000 ppm. Cultured in jar fermenters. During the culture, KH2PO, MgSL7H20, FeCl2
・Supplying 6H20, dibenzoylthiamine hydrochloride, and trace amounts of inorganic salts at 30°C and pH 5.5 for 6 days (14
After culturing (4 hours) and centrifuging the obtained culture solution (containing 3.42 kg of dry bacterial cells) and washing with water,
A portion was adjusted to 85% moisture and dried with a spray dryer. Dry powder 43.6g (absolutely dry amount 40g, Coenzyme Q
10 content (12.7 mg) was taken out, poured into a mixed solvent of 75 ml of dimethyl sulfoxide and 75 ml of isopropyl alcohol, and extracted with stirring at 70 to 75°C for 2 hours. Then add 300ml of n-hexane and add 15ml of n-hexane.
Shake for a minute and add coenzyme Q. After dissolving, leave it still, 2
Layer separation was performed to separate the n-hexane layer. After performing this operation three times, all the n-hexane extracted layers were collected, washed three times with an equal volume of 900 ml of water, and dehydrated with anhydrous sodium sulfate. Since the extract at this stage contains substances that interfere with quantitative determination using high-performance liquid chromatography, a portion of the extract
The extraction rate was determined by saponifying the % amount and found to be 93.1%. Next, the mixture was concentrated to dryness under reduced pressure, the residue was dissolved in a small amount of acetone, cooled, the precipitate was removed, and the mixture was concentrated to dryness under reduced pressure. Thereafter, it was subjected to column chromatography packed with 25 g of silica gel and eluted with n-hexane. The eluted fraction of coenzyme QIO was concentrated to dryness under reduced pressure, dissolved in a small amount of ethyl alcohol, and cooled to yield an orange-yellow coenzyme Ql.
Crystals of o were precipitated. 2 more with ethyl alcohol
Recrystallization was repeated several times, separated from the ethanol mother liquor, and dried under reduced pressure to obtain 9.965 mg of coenzyme Q1o crystals (yield: 8665%).

Example 2 The cells obtained by culturing the same bacteria as in Example 1 using the same culture method were centrifuged, washed with water, and dried at 40° C. while being crushed in a mortar. Using these dried bacterial cells, we extracted coenzyme Q by changing the mixing ratio of dimethyl sulfoxide and isopropyl alcohol as follows.

was extracted.

The dried bacterial cells were added to a mixed solution of dimethyl sulfoxide and isopropyl alcohol, and stirred for 2 hours under heating.

After cooling, 300 ml of n-hexane was added, shaken for 15 minutes, and allowed to stand to separate the n-hexane layer. This operation is
After repeating the extraction, the total n-beef sun extract was collected, washed three times with an equal amount of water, dehydrated with anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. The extract at this stage contained substances that would interfere with quantitative analysis using high performance liquid chromatography, so it was quantified after saponification. For saponification, add 1 g of pyrogallol to the concentrate.
This was carried out by adding 4 g of sodium hydroxide, 30 ml of methyl alcohol, and 10 ml of water, and refluxing at 90° C. for 1 hour. After quenching, 100 ml of n-hexane was added, shaken for 15 minutes, and allowed to stand to separate the n-hexane layer.

This operation was performed three times. Collect all n-hexane layers and
The mixture was washed three times with 00+nl of water, dehydrated with anhydrous sodium sulfate, and then concentrated to dryness under reduced pressure. The concentrate was dissolved in a small amount of acetone and quantified by high performance liquid chromatography. These results are shown in Table 1.

Example 3 The same bacteria as in Example 1 were used and cultured in the same manner. However, coenzyme Q1o was extracted using a mixed solvent of dimethyl sulfoxide and ethyl alcohol, and was stirred for 2 hours. The following steps were carried out in the same manner as in Example 2. These results are shown in Table 1.

Example 4 The same bacteria as in Example 1 were used and cultured in the same manner. However, coenzyme Q1° was extracted using a mixed solvent of dimethyl sulfoxide and methyl alcohol, and stirring was performed for 2 hours. The following procedure was carried out in the same manner as in Example 2. These results are shown in Table 1.

Example 5 The same bacteria as in Example 1 were used and cultured. However, dried bacterial cells 20
．． 6 g (coenzyme Q, content: 14.1 mg) was added to a mixed solvent of 45 ml of dimethyl sulfoxide and 45 ml of acetone, and extraction was performed with stirring at 50° C. for 2 hours.

The following steps were carried out in the same manner as in Example 2. The extraction rate at this time was 87.4%.

(Effects of the Invention) As is clear from Example 1 and Table 1, the present invention can extract coenzyme Q with extremely high efficiency through a simple process using inexpensive chemicals and small-sized extraction equipment. Therefore, the present invention is extremely useful industrially.

Claims (1)

[Claims] 1. When extracting coenzyme Q from microbial cells containing coenzyme Q, the cells that have been centrifuged from the culture solution of coenzyme Q are dried, and the hydrophilic solvent dimethyl is used as an extraction solvent from the dried cells. A method for producing coenzyme Q, which comprises extracting coenzyme Q by using sulfoxide alone or by mixing it with another solvent.