JPS6125490A - Preparation of coenzyme q10 - Google Patents

Abstract

PURPOSE:To obtain coenzyme Q10 in high yield, by culturing a microbial strain to Rhodepseudomonas genus. CONSTITUTION:A microbial strain belonging to Rhodopseudomonas genus, capable of producing coenzyme Q10 and resistance to quione compounds [e.g. Phodopseudomonas spheroides H-3161 (FERM-P No.7683)] is inoculated in a medium, and cultured aerobically at 5-9pH and 20-40 deg.C for 3-7 days. The objective coenzyme Q10 can be produced and accumulated in the culture product.

Description

[Detailed Description of the Invention] 4 minutes μ from 1! The present invention provides coenzyme Q1o (hereinafter referred to as COQ10) by fermentation method.
(referred to as ).

Conventional technology Conventionally, C.
The method for producing oQ10 is to use a mutant strain lacking or having a reduced ability to produce carotenoid pigments (Japanese Patent Publication No. 47-1999).
7954, JP-A-55-39730, JP-A-55-61797), a method using a mutant strain with improved ability to produce bacteriochlorophyll pigment (JP-A-56-7)
2694, Japanese Patent Application Laid-Open No. 57'-208992)
etc. are known.

Means for Solving the Problems According to the present invention, CoQ, which belongs to the genus Rhodopseudomonas,
+. Microorganisms that have production ability and are resistant to quinone compounds are cultured in a medium, and COQ10 is produced and accumulated in the culture, and Co Q + is produced from the culture. By collecting CoQ10, CoQ10 can be produced with good yield.

The microorganism used in the present invention belongs to the genus Rhodopseudomonas, and any microorganism can be used as long as it has the ability to produce COQ10 and is resistant to quinone compounds.

As a quinone compound, ■,4-benzoquinone.

Examples include 2-methyl-1,4-benzoquinone, dichlorodiazoquinone, 1,4-naphthoquinone, 2-methyl-104-naphthoquinone, anthraquinone, 2-methylanthraquinone, anthralphine, and quinizarin.

A microorganism having the above-mentioned properties can be obtained by using a microorganism originally capable of producing COQ10 as a parent strain and imparting properties of resistance to quinone compounds to this microorganism. It can also be obtained by the opposite method, that is, by imparting the ability to produce COQ10 to microorganisms that are resistant to quinone compounds.

For example, it can be obtained by treating microorganisms capable of producing C0QIO with various drugs or with X-rays, T-rays, Co irradiation, or the like.

The strains obtained in this way are cultured, and a strain whose C0QIO production ability is significantly improved over that of the parent strain is selected and used in the present invention.

Specific examples of bacterial strains used in the present invention include Rhodopseudomonas spheroides
onasspheroides) H-3161 (hereinafter referred to as H-3161), H-3751 (hereinafter referred to as H-3
751) and H-3752 (hereinafter referred to as H-3752)
) can be mentioned.

These H-3161, 8-3751 and H-3752
is the 76th Microbiological Research Institute at the Institute of Microbial Technology, Agency of Industrial Science and Technology.
No. 83, No. 7684, and No. 7685, respectively.

Next, an example of operation for obtaining the above-mentioned bacterial strain will be explained.

Rhodopseudomonas sphaeroides FERM-P600B (hereinafter referred to as FERM
- P6008) were placed in 0.05 M Tris-maleate buffer (pH 6.0) for approximately 10' cells.
N-methyl-N'-nitro N-nitrosoguanidine was suspended at a final concentration of 1ag/+n.s/ml.
After adding it to a total of 1 liter and leaving it to stand at room temperature for 30 minutes,
．． 4-benzoquinone (20 μg/ml), 1.4-naphthoquinone/7 (5 μg/ml), 2-methyl-1,4
- H-3161, H-375 from among the growing colonies were smeared onto a minimal medium agar plate medium having the following composition containing naphthoquinone (3 μg/ml).
1 and H-3752, respectively.

As is clear from Table 1, these mutant strains contain 104-benzoquinone, 1,4-naphthoquinone 12-methyl-1,
It can be clearly distinguished from the parent strain FERM-P6008 in that it has resistance to each of the 4-naphthoquinones.

Table 1: No additives ++ ++ ++ ++1.4
-Benzoquinone 20μg/ml-++1.4-Naphthoquinone 5μg/ml -++2-methyl-1.4
- 3 μg/ml -++ Naphthoquinone Note) ++; Sufficient growth m: No growth Composition of the above minimal medium agar plate medium Nigelcose 10g/
l, NH, CJ 4g/l, K1-1. PO41g/
β, 2HPO43g/J, Mg5O, 7HiOO,
4g/l, Fe5O-・7H200,01g/j! ,M
n5O,・4) (so 0.01g/l, biotin 5
0μg/1. Agar 20g/Il.

pH 7.2 The medium used in the present invention may be either a synthetic medium or a natural medium, as long as it contains a suitable amount of carbon sources, nitrogen sources, inorganic substances, and other nutrients that can be utilized by the strain used.

Carbon sources include glucose and sucrose.

Carbohydrates such as blackstrap molasses and starch, sugar alcohols such as glycerin and sorbitol, and alcohols such as methanol 7 to ethanol can be used.

As nitrogen sources, inorganic and organic ammonium salts such as ammonium sulfate, ammonium chloride, ammonium acetate, etc.
Nitrogen-containing natural products such as yeast extract and cornstarch liquor can be used.

As the inorganic salt, potassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, etc. can be used.

Of course, when natural products are used, it may be possible to satisfy the requirements only with the inorganic salts contained therein.

Furthermore, if the microorganisms used in the present invention require specific nutrients for growth, appropriate amounts of those nutrients must be present in the culture medium, but these substances cannot be substituted with the natural products exemplified as nitrogen sources. May be included and added.

There are various substances in the culture solution, such as amino acids,
Nucleic acid related substances.

vitamins, organic acids, alcohols, sterols,
The amount of COQIO produced may increase by adding other CoQ10 biosynthesis precursors and related substances.

Cultivation is performed under aerobic conditions such as shaking culture 1 aerated stirring culture.

The appropriate culture temperature is 20 to 40°C, and the pH is 5 to 40°C.
A value of about 9 is appropriate.

The pH of the medium is adjusted using calcium carbonate, an acid or alkaline solution, a pH agent, or the like.

The culture period is usually 3 to 7 days, and COQ + O is mainly produced and accumulated in the bacterial cells. COQ+ from cultures.

Isolation is carried out by conventional methods such as solvent extraction, concentration, column chromatography, etc.

Examples are shown below.

Example 10 H-3161 strain was used as an inoculum. 10 This strain was added to glucose 2g/a2. Peptone 1g/a, yeast extract 1
g/d1. Salt 0.5g/dI! Seed medium (pH
7,2) A 21-volume Erlenmeyer flask containing 300 ml was inoculated and cultured with shaking at 30°C for 24 hours. This seed culture solution 30
Qml was inoculated into a 51-volume jar fermenter containing fermentation medium 3R with the following composition, and the temperature was 30°C and 1 ventilation II! /min, stirring number 3
The cells were cultured for 96 hours at 50 revolutions/min. For culturing, add 5 g of blackstrap molasses at the 24th and 48th hour, respectively.
(converted to sugar concentration) was fed. At this time, 150 r/ml of (:OQ10) was produced and accumulated in the culture solution.

As a control, the parent strain FE was cultured at the same time under the same conditions.
RM-P6008 was 131 r/ml.

Composition of fermentation medium: 5 g/# of blackstrap molasses (converted to sugar concentration).

Yeast extract 2g/a, ammonium sulfate 0.8g/d1
．． Potassium phosphate 0.05g/dl, dipotassium phosphate 0.05g/J, magnenium sulfate heptahydrate 0.
．． 025g/dll, calcium carbonate 2g/d, vitamin B, 018mg/d1. -:+tinic acid 08mg/
d1 (pH 7,2) After culturing, culture solution 2 of H-3161 strain! was centrifuged to obtain bacterial cells with a dry weight of 37 g. This was suspended in 2001711 water, and 400 ml of methanol was added.
Add 80g of sodium hydroxide and 15g of pyrogallol,
After refluxing at 85°C for 45 minutes, let it cool and 1! Two portions of n-hexane were added and extracted twice.

The n-hexane layer was separated, anhydrous sodium sulfate was added thereto for dehydration, and the mixture was concentrated under reduced pressure. The residue was dissolved in 4 Qml of acetone, insoluble matter was removed by filtration, and then concentrated again. The residue was dissolved in 1Qml of acetone, passed through a glue ram, and eluted with benzene.

Both fractions containing COQ10 were collected and concentrated, and the residue was dissolved in 5 ml of ethanol and cooled to obtain 155 mg of yellow crude crystals. Similarly, FERM-P4O10
133 mg was obtained from the culture solution.

Motojima confirmed that the COQ was 10 using reversed phase thin layer chromatography and high performance liquid chromatography.

Example 2゜ H-3751 and H-3752 were used as seed bacteria.

) l-3751 and H-37 in the seed medium shown in Example 1.
52 and cultured with shaking at 30°C for 24 hours. Fermentation medium 3 using these seed medium 3n+l as shown in Example 1
Inoculate a 25 Qml Erlenmeyer flask containing 0 ml of
Shaking culture was carried out at 0°C for 96 hours.

During the culture, 4 g/d of blackstrap molasses (in terms of sugar concentration) was fed at the 24th and 48th hours, respectively. At this time, the amount of CoQ10 produced and accumulated in the culture solution was
and 132 r/ml and 1 for H-3752, respectively.
33 r/m! Met. The parent strain FERM-P4O10, which was cultured under the same conditions as a control, was
/ml.

Effects of the invention Compared to the parent strain, the production amount of C0QIO is increased by 10% or more.

Claims (1)

[Claims] A microorganism belonging to the genus Rhodopseudomonas, capable of producing coenzyme Q_1_0, and resistant to quinone compounds is cultured in a medium, and coenzyme Q_1_0 is produced and accumulated in the culture, and coenzyme Q_1_0 is extracted from the culture. A method for producing coenzyme Q_1_0, which comprises collecting coenzyme Q_1_0.