JPS5853917B2 - Production method of coenzyme Q - Google Patents

Description

[Detailed description of the invention] The present invention relates to a method for producing coenzyme Q.

More specifically, the method for producing coenzyme Q is characterized in that 5sc-butyl alcohol or tert-amyl alcohol is used as an extraction solvent when coenzyme Q is extracted from living cells containing coenzyme Q.

Conventionally, aqueous suspensions of animal and plant tissues, microbial cells, etc. containing coenzyme Q have been treated with alkalinity, and the water has been removed and separated using organic solvents such as butyl alcohol, n-hexane, isooctane, and petroleum powder. It is known to obtain a hydrophobic solvent-extracted fraction containing coenzyme Q by extracting with dichloroethane, methyl imbutyl ketone or ethyl acetate (Japanese Unexamined Patent Publication No. 53-38690).

However, even if the living cells of bacteria containing coenzyme Q are extracted with n-butyl alcohol, hexane, methyl isobutyl ketone, ethyl acetate, etc. among the above organic solvents without alkaline treatment, coenzyme Q The extraction rate was low.

The present inventors have conducted various studies on organic solvents that can be separated from water when extracting coenzyme Q with a good extraction rate without alkali treatment of living bacteria containing coenzyme Q. In particular, 5ec-butyl alcohol or tert
- When amyl alcohol is used, viable bacteria can be grown at a pH of 10 or higher, at a heating temperature of 60 to 150'C, as in the conventional method.
The present invention was completed based on the discovery that coenzyme Q can be extracted in a high yield and in a short time by direct extraction without alkali treatment with a heating time of 10 minutes to 10 hours.

The present invention will be explained in detail below.

First, 5ec-butyl alcohol or tert-amyl alcohol can be separated from other waters in which coenzyme Q1o has a high solubility. An experimental example showing how high the extraction rate of the coenzyme Qzo from living bacterial cells is compared to organic solvents such as hexane is shown below.

Experimental example Baracoccus denitrificans ATCC19367
Table 1 shows the results of examining the extraction rate of coenzyme QIO after adding twice the amount of various organic solvents to an aqueous suspension of live bacterial cells (pH 11.0) and stirring at room temperature for 2 hours.

Generally, the extraction rate of a certain substance is considered to be related to the solubility of the substance, but as can be seen from Table 1, in the case of various butyl alcohols, the solubility of coenzyme QIO is 29 to 3.1.
? /l, but surprisingly,
The extraction rate of coenzyme QIO is much higher in 5ee-butyl alcohol than in other n-butyl alcohols or 1so-butyl alcohols.

Furthermore, the extraction rate is much higher than that of other organic solvents.

Also, with regard to ter-amyl alcohol, the extraction rate of coenzyme Q1o is much higher than with n-amyl alcohol, and also compared with organic solvents that can be separated from water other than 5ec-butyl alcohol.

The extraction rate is high.

No prior known literature suggests that such a high extraction rate is exhibited.

Examples of the viable bacterial cells containing coenzyme Q used in the present invention include a culture of bacteria containing coenzyme Q, bacterial cells collected after culturing, and an aqueous suspension of bacterial cells.

Further, the coenzyme Q in the coenzyme Q-containing material may be of any kind.

The stirring extraction with 5ec-butyl alcohol or tert-amyl alcohol is preferably carried out at room temperature for 1 to several hours.

The amount of solvent added and the extraction time can be changed as appropriate depending on the raw material, that is, the coenzyme Q-containing substance.

Pre-treatment for extraction (e.g. alkaline treatment, etc.) is not necessary and there are no particular restrictions on room temperature or pH during extraction, but it is best to perform extraction at room temperature and at pH 1.0 to 11 in order to further reduce the influence of phospholipids. It is.

The extract is purified by silica gel column chromatography and crystallized with ethanol, or the extract is concentrated, dissolved in a small amount of acetone, decolorized by activation treatment, cooled, and crystallized to obtain coenzyme Q crystals. is obtained.

Examples are shown below.

Example 1 Glucose 19/d11 Yeast extract 0.59/
dl.

Lease 1 with a pH of 7.2 and a composition of 1 peg 1 g/di, meat extract 0.5 g/dl, and salt 0.3 g/di.
After putting 001 into a fermenter and sterilizing it, inoculate it with culture solution 21 of Paracoccus denitrificans ATCC 19367, which had the same composition and had been cultured in a flask beforehand.
Aerobically cultured at ℃ for 96 hours with stirring number 15 rpm and aeration rate 40 min, live bacterial cells were collected from the culture solution and suspended in IOA water (
750g as dry bacterial cells, 315g containing coenzyme Q),
After adjusting the pH to 11, add 101 sec of butyl alcohol, stir and extract at room temperature for 1 hour, and centrifuge for 5 sec.
The ec-butyl alcohol layer was separated.

After repeating this operation once more, all extracts were collected.

The extract at this time contained 289 μ of coenzyme Q.

This extract was concentrated, passed through a column packed with silica gel 101, and then eluted with acetone.

The eluted fraction of coenzyme Q was concentrated, a small amount of activated carbon was added, and the furnace solution was cooled to obtain 237 cm of orange-yellow crystals.

The obtained substance was confirmed to be coenzyme QIO from the results of paper chromatography, thin layer chromatography, elemental analysis, nuclear magnetic resonance spectrum, and melting point.

Example 2 In the same manner as in Example 1, Pseudomonas aeruginosa ATCC 15246 was cultured, and viable cells were collected from the culture and suspended in 21 water (160 g as dry cells, coenzyme Q).
After adjusting the pH to 10, tert-
Added amyl alcohol 41, stirred and extracted at room temperature for 3 hours,
The tert-amyl alcohol layer was separated by centrifugation.

After repeating this operation once more, all extracts were collected.

The extract contained 99 μ of coenzyme Q.

This extract was concentrated, passed through a column packed with 50 g of silica gel, and then eluted with acetone.

The eluted fraction containing coenzyme Q was concentrated, a small amount of activated carbon was added, and the synovial fluid was cooled, yielding 71 cm of orange-yellow crystals.

This product was identified as Coenzyme Q by reverse phase thin layer chromatography.

Example 3 In the same manner as in Example 1, Agrobacterium tumefaciens ATCC4720 was cultured, and the culture was concentrated by centrifugation to obtain a 10A cell suspension. (
62 (Bi', coenzyme Q content 341■) as a dry bacterial cell
was extracted in the same manner as in Example 1, and coenzyme Q
An extract containing 320 μm was obtained.

This extract was concentrated to dryness under reduced pressure, the residue was dissolved in a small amount of acetone, a small amount of activated carbon was added for decolorization, and the p liquid was further concentrated and cooled to obtain crystals 273 of coenzyme Q1o.

Example 4 Proteus vulgaris ATCC was prepared in the same manner as in Example 1.
19181 was cultured and harvested, and a 1M aqueous suspension thereof (570 g as dry cells, coenzyme Q content 74 rnIl) was extracted and purified in the same manner as in Example 1 to obtain 61■ crystals of coenzyme Q.

This product was identified by reversed phase thin layer chromatography.
It was confirmed that it was coenzyme Q7.

Claims (1)

[Claims] 1. A method for producing coenzyme Q, which comprises using 5ec-butyl alcohol or tert-amyl alcohol as an extraction solvent when extracting coenzyme Q from living cells containing coenzyme Q.