JPS63317092A - Production of coenzyme q10 - Google Patents

Abstract

PURPOSE:To readily, efficiently and stably obtain a coenzyme Q10 useful in a medicine such as heart function-accentuating agent, feed additives, etc., by extracting a bacterium cell obtained by cultivating a coenzyme Q10 producing bacterium belonging to the genus Oligomonas. CONSTITUTION:A bacteria cell is separated and selected from a soil, etc., in a culture medium containing methanol to afford Oligonomonas methanolica strain exhibiting the following bacteriological and physiological properties: Shape and size of the cell: cocci or short rods, wide, 0.5-0.8mum, length, 0.5-1.5mum; Gram negative; capable of rearing and propagating in alkaline conditions; capable of reducing nitrate; capable of assimilating glucose and methanol, etc. Then the strain is aerobically cultivated in a culture medium containing about 6wt.% methanol, peptone, etc., at 20-42 deg.C, pH7-10 and 0.5-20ppm dissolved oxygen concentration. Then a bacterium cell is separated from the culture medium and dried and the resultant dried bacterium cell is heated with ethanol, etc., at 50-90 deg.C for about 1hr to provide an extract, which is fractionated with silica gel, etc., and purified to liberate the aimed coenzyme Q10.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing coenzyme Q +o, and more particularly to a method for producing coenzyme Q10 using microorganisms.

Coenzyme QIG plays an important role as an electron carrier in the terminal respiratory system in vivo, and is used as an agent for advanced cardiac function, a treatment for myasthenia gravis, a treatment for pulmonary emphysema, a treatment for aplastic anemia, and a treatment for alopecia areata. It is known as a compound that is useful as a pharmaceutical agent and feed additive.

[Prior art, problems to be solved by the invention] Conventionally,
Coenzyme Q10 is extracted from tissues of animals and plants, and is produced by further purification, which has problems with uniformity of quality and unstable supply of raw materials.

As one method for avoiding such drawbacks, a method has recently been known in which coenzyme Q10 is extracted from cells obtained by culturing microorganisms.

However, the productivity of coenzyme Q10 in microorganisms is still not sufficient for practical use, and the discovery of microorganisms with high productivity of coenzyme Q10 is expected.

The present invention uses microorganisms to efficiently produce coenzyme Q1.

The purpose is to provide a method for manufacturing.

[Means and actions for solving problems]

The present inventors conducted repeated research to find a strain that produces a large amount of coenzyme Q10, and as a result, a strain belonging to the genus Oligomonas produced a large amount of coenzyme Q + within its bacterial body.

The present invention was achieved by discovering that it is possible to produce and accumulate large quantities of.

That is, the present invention provides coenzyme Q belonging to the genus Oligomonas.
10 Coenzyme Q characterized by culturing producing bacteria to obtain bacterial cells, and separating and recovering coenzyme QIo from the obtained bacterial cells.
This is the manufacturing method of 1o.

The bacteria used in the present invention grow and multiply in alkaline conditions, can assimilate methanol as a sole carbon source, are Gram-negative, and produce coenzyme Q10. According to its mycological properties, this bacterium does not belong to any previously known genus or species;
It was determined that the bacteria belonged to a new genus.

The present inventors thought that this bacterium needed to establish a new home and named it the genus Oligomonas.

Among the alkaline methanol-assimilating bacteria of the present invention, a representative strain 0-3 (Feikoken Bacteria Collection No. 9280).

0-75 (Feikoken Bibori No. 9281) and 0-100
(Feikoken Bibori No. 9282) and their mycological properties are shown.

Mycological properties: (1) Microscopic form NazCO3 was cultured at 30°C for 3 days in broth liquid medium and broth agar medium adjusted to pH 9.0.

■ Cell shape and size Usually cocci or short bacilli, width 0.5-Q, 34° length 0.5-1.5- ■ Motility None.

■ Presence or absence of spores Not produced.

■ Gram stain Gram negative ■ Acid-fast negative (2) Growth status in various media ■ Juicy plate culture adjusted to pH 9.0 with Na2CO3 Cultured at 30°C for 3 days.

Shows medium growth.

Colony shape: external shape is circular, size is 2-3 nm, ridges are hemispherical, structure is homogeneous, surface is smooth, edges are smooth and gold-rimmed, color is yellowish white and glossy, transparency is opaque, hardness is butter quality.

■Methanol-containing agar plate culture Culture at 30'C for 3 days.

Same as ■.

■ Broth agar slant culture adjusted to pl+ 9.0 with Na2CO3. Cultured at 30"C for 3 days.

Grows vigorously and uniformly along the inoculation line.

The ridges are moderate, the surface is smooth, the edges are smooth and gold-rimmed, the color is yellowish-white and shiny, the transparency is opaque, and the hardness is putty.

■ Methanol-containing agar slant culture Culture at 30°C for 3 days.

Same as ■.

■ Meat juice culture 3 adjusted to pH 9.0 with NazCO:+
Culture at 0°C for 3 days.

Grows all over. There is precipitation.

■ Peptone water 3 adjusted to pH 9.0 with NazCO=
Culture at 0°C for 5 days.

Grows all over. However, growth is not vigorous.

■ Methanol-containing liquid medium Culture at 30°C for 3 days.

Grows all over. There is precipitation.

■ Broth agar puncture culture adjusted to pu 9.0 with NazCOl. Cultured at 30'C for 3 days.

Grows uniformly in the form of small papillae. On the surface of the medium, the diameter is 2-4
It grows in a circular shape about the size of a bell.

■ Puncture culture containing methanol Culture at 30°C for 3 days.

Grows uniformly in the form of small papillae. On the surface of the medium, the diameter is 2-4
[Grows in a circular shape about 1ll1.

[Phase] Broth gelatin multilayer culture adjusted to pH 9.0 with Na2CO3. Cultured at 20°C for 10 days.

Although bacterial growth is observed, the gelatin is not liquefied.

■ Cultured for 4 weeks at 30°C in lidmus milk adjusted to pH 9.0 with NazCO=.

Although bacterial growth is observed, lidmus milk does not coagulate.

(3) Physiological properties ■ Nitrate reduction Reduces nitrate to nitrite.

However, the reducing ability of the 0-3 strain is weak.

■ VR' test negative ■ Formation of indole negative ■ Formation of hydrogen sulfide negative ■ Starch hydrolysis negative ■ Utilization of citric acid (Christen)
sen medium) Negative ■ Use of nitrogen sources Ammonium salts, nitrates, urea and peptone are used as nitrogen sources.

■ Pigment production: No pigment production.

■ Urease positive [Phase] Catalase Positive ■Generation of ammonia　Generate.

@ Denitrification reaction negative ■ Oxidase positive Q O-F test negative ■ Growth range Grows in the pH range of 7 to 10. pl+ 7.5-9.
5 is preferred. Particularly preferred are p118-9.

It does not grow at 5°C937°C.

@) Assimilation of IJi species + (W): Grows weakly ■　tJl! # Assimilation of carbon sources other than N■ Salt tolerance It grows weakly in a medium containing 3% by weight NaCl.

■ Vitamin requirement Requires biotin.

@CC (guanine cytosine) content 0-3 63.9molχ0-75
64.7molχ0-100
64.4 mol'''t■ Main bacterial cell fatty acid composition Monounsaturated fatty acid CIl+□ @ Main hydroxy acid 3-hydroxy acid C108゜3-hydroxy acid C14.

0 Quinone type Ubiquinone Q-10 [Phase] Separation source soil Bergey's Manual of Systematic Bacteriology
fSystematic Bacteriology
) Volume 1 (editors Krieg and Holt): Williams & Wilkins
.

(1984), these strains are Durham-negative, cocci or short bacilli, and aerobic;
Gram-Negative of 5ection 4
e Aerobic Rods and Cocc
It is considered to be included in iJ. This 5ectio
37 genera are described in n. Comparing this bacterium with these genera, it can be said that it is a coccus or a short bacillus, is non-motile, and is close to the genus Paracoccus in terms of GC content. However, this bacterium does not match the description regarding the genus Paracoccus in that it does not have denitrifying ability. In addition, bacterial species belonging to the genus r'aracoccus include P. denitrificans and P. halo.
denitrificans, but they differ from these species in terms of denitrification ability and growth pl+.

Therefore, the present inventors assigned this bacterium to a new family and named it the genus Oligomonas, and also named the bacterium Oligomonas methanolica (Oligomonas).
Igomonas methanolica).

In the present invention, the experimental method for investigating mycological properties is described in the above-mentioned Harsey's Manual, edited by the Institute of Medical Science Alumni Association [Bacteriology Practice Summary J (1958) and Hase's
Based on "Classification and Identification of Microorganisms" (1975), edited by Sokoi.

Furthermore, a medium having the following composition was used as a methanol-containing agar plate medium and a methanol-containing agar slant medium (the same applies to Examples). That is, (NHs)zsOa 3g, 1l
zPO41.4g, NaJPOs 2.1g 5M
g50448200.2 g, CaC1z・211z
030mg, FeCJsO7・XIIzo 30+ng
.

MnCIz'411205mg+ ZnS044Hz0
5mg, Cu5Oa・5t1200.5mg and Dirco agar (Bacto agar)
-agar) 15 g in pure water 11, 1 kg
/ After sterilizing with crA G for 20 minutes, 10% N by weight
a2CO.

The aqueous solution was added aseptically and the pH was adjusted to 9.0. Furthermore, methanol 8- was added aseptically to prepare a plate culture medium or a slant culture medium. Furthermore, as the methanol-containing liquid medium, the above-mentioned medium to which agar was not added was used.

Isolation of this bacterium from soil was carried out in a conventional manner using the methanol-containing medium described above.

The medium used for culturing this bacterium must contain a carbon source that can be assimilated by this bacterium, and may be either a synthetic medium or a natural medium as long as it contains an appropriate amount of nitrogen source and inorganic substances. good.

There are no particular restrictions on the carbon source as long as it is a carbon source that can be assimilated by this bacterium. In addition to methanol, synthetic carbon sources such as ethanol can be suitably used, but other carbon sources such as molasses and peptone can also be used. and natural products such as meat extracts,
arabinose, xylose, glucose, mannose,
Alcohols such as fructose and galactose, sorbitol, nitol and inositol, and organic acids such as succinic acid can also be used. Among these, methanol is the most preferred as an industrial fermentation raw material. The concentration of these carbon sources in the medium is appropriately selected depending on the type of carbon source.

For example, when the carbon source is methanol, the methanol concentration in the medium or culture solution is preferably 6% by weight or less, and particularly preferably 3% by weight or less from the viewpoint of good bacterial growth and proliferation.

Nitrogen sources include, for example, inorganic nitrogen compounds such as ammonium salts, nitrates and/or e.g. urea,
Organic nitrogen-containing substances such as corn stave liquor, casein, peptone, yeast extract, and meat extract are used.

In addition, examples of inorganic components include calcium salts, magnesium salts, potassium salts, sodium salts, phosphates,
Manganese salts, zinc salts, iron salts, copper salts, molybdenum salts, cobalt salts, boron compounds and iodine compounds are used.

In addition, growth promoting substances such as amino acids, nucleic acids, vitamins, yeast extract and malt extract are also used.

In addition, if the bacteria used are auxotrophic,
It is necessary to make the required substance exist.

The culture conditions include a temperature of 20-42°C, preferably 25-42°C.
0"C, pf 17 to 10, preferably 7.5 to 9.5. Cultivation is carried out aerobically under these conditions. If culture is performed outside these conditions, the growth of this bacterium will not occur. Although it will be relatively worse, it does not prevent culturing outside these conditions.

Further, there is no particular restriction on the dissolved oxygen concentration of the culture solution, but it is usually preferably 0.5 to 20 ppm. For this purpose, measures such as adjusting the amount of aeration, stirring, using oxygen or a mixed gas of oxygen and air as the aeration gas, and increasing the pressure inside the culture tank are adopted. Also,
The culture method may be either batch culture or continuous culture.

When ammonium salt is used as a nitrogen source, ammonia is consumed for bacterial cell production during the culture period, and the pH of the culture solution decreases. In this case, in order to maintain the pH of the culture solution at a predetermined value, an alkali such as ammonia, caustic potash, and caustic soda is added, and it is preferable to add ammonia.

After culturing the bacteria in this manner, the bacterial bodies are separated from the culture solution. For separation, ordinary solid-liquid separation means are employed. In other words, solid-liquid separation means include, for example, centrifuging the culture solution itself, adding other microorganisms larger than the present bacteria to the culture solution as a filter aid, or pre-coating the culture solution. Means for separating bacterial cells by filtration, means for flocculating bacterial cells by adding various flocculants to the culture solution, and separating the flocculated cells from the culture solution by filtration or centrifugation, and the pH of the culture solution.
5 or less, or by adjusting the pH to 5 or less and heating at 50 to 50°C to aggregate the bacterial cells, and then separating the aggregated bacterial cells from the culture solution by filtration or centrifugation. etc. can be applied.

Coenzyme Q10 is extracted from isolated bacterial cells or dried bacterial cells, for example, by spray drying, and this coenzyme Q10 is further purified if necessary.

Separation, extraction and purification of coenzyme Q10
This can be carried out by conventional separation/extraction methods and purification methods that are applied to. That is, for example, by suspending bacterial cells in ethanol, methanol, or acetone,
Extract by heating at 50-90°C for 1 hour to obtain an extract. Alternatively, a saponification method is obtained by first saponifying saponifiable substances such as phospholipids in the bacterial cells using a mixture of methanol, sodium hydroxide, and pyrogallol. Coenzyme Q10 is extracted from these extracts or saponification using an organic solvent such as n-hexane to obtain an extract. Then, coenzyme QIO is fractionated, isolated, and purified from this extract using, for example, a porous synthetic resin such as a high-porous polymer, silica gel, and Florisil.

High performance liquid chromatography and elemental analysis are generally used to identify and quantify coenzyme Q lo obtained from bacterial cells.

Means such as melting point measurement, infrared absorption spectroscopy, ultraviolet absorption spectroscopy, nuclear magnetic resonance spectroscopy, and mass spectrometry are used.

〔Example〕

The present invention will be explained in more detail with reference to Examples. Note that the present invention is not limited to the examples.

Example 1 Approximately 0.5 to 1 g of a soil sample of ``Oligo ``Eggplant methanolica fungus'' was aseptically placed in sterile water 10-, and this suspension 1- was placed on a methanol-containing agar plate medium so that the liquid depth was almost uniform. After allowing water to be absorbed into the agar medium, static culture was performed at 28"C for about 7 days. A portion of the colony that had formed on this agar medium was further cultured on an agar plate medium containing methanol. The single colony obtained was inoculated onto a methanol-containing agar slant medium and cultured to obtain Oligomonas methanolica 0-3, Oligomonas methanolica 0-
75 and 0-100, respectively.

Production of coenzyme Q10 per 11 of pure water, (NH4) zsO, 3g, 1 (
zPOn 1.8 g.

NazHPO42,1g, MgSO4'7HzO0,
2g, CaC1g・2Hz0301g, FeCJsO
t'XHz0 30mg+ MnCIz・4H205m
g.

ZnSO4・7Hz0 5 mg, CuSO4°5L
A medium containing 5 mg of OQ, 2.5 g of NazCO, 2 ml of biotin and 8 ml of methanol (hereinafter referred to as medium A) 30II1. was dispensed into a 100-capacity Erlenmeyer flask and sterilized at 120°C for 20 minutes. Methanol-containing agar medium supplemented with 20 g/l of agar (na2CO3-free medium at 120°C)
sterilized for 20 minutes, then separately sterilized 10 wt% Na
Oligomonas methanolica 0-100 (made by adding 0.25 CO3 aqueous solution per 10 d of culture medium) at 30''C for 3 days (Feikoken Bacterial Serial No. 9282)
Inoculate medium A in a 100-capacity Erlenmeyer flask and shake at 30°C with a rotary shaker at 220 rpm.
Rotary shaking culture was performed once per minute. This culture solution was used as a seed mother solution.

Industrial water 11af,:,su, (NH4) zsOa
1 g 1Mg5O, a.

7tlz0 1.5 g, KII2PO44.5g
, FeCJsOy・XHz030mg, CaC]
z'2Hz0 90mg, ZnSO4'411z0
15mg.

MnCl2°411z0 15111g, CLIS
O4°51hO1.5mg.

(Nlla)aMO7(h4'4Hzo 1.0mg
, CoCIz・211z0 1.0ffllLFI3
PO4t, omg, NaCl 50mg, Kl
A medium (medium B) containing 1.0 mg of biotin and 80 II g of biotin was placed in a 30 ffi culture tank, and after sterilization, the pi was adjusted to pi-19.0 with aqueous ammonia, and 40 m of methanol was added.
1 and 200 mj of seed mother liquor were added respectively. As the bacteria proliferated, the methanol concentration in the culture solution decreased, but this methanol concentration was analyzed by gas chromatography, and methanol was replenished so that the methanol concentration in the culture solution was 0.1 to 0.62% by weight. . Culture temperature 30
°C1 culture solution pH 9.0, stirrer rotation speed (stirring blade radius 71 mm) 800 times/min and aeration rate 1vvm
When aeration (using air) agitation culture was carried out, bacteria proliferated in about 4 hours. After 48 hours from the start of the culture, the culture solution was centrifuged, and the resulting bacterial cells were freeze-dried to obtain 90 g of dried bacterial cells.

Coenzyme Q Io was extracted from the dried bacterial cells with an aqueous solution of isopropanol with an inpropanol concentration of 90 volumes χ,
Next, hexane transfer was performed, and after oxidizing the reduced coenzyme Q10 in the obtained hexane solution with iron nitrate, coenzyme Q10 was identified in this hexane solution by high performance liquid chromatography, and its content was measured. . The results show that 1.60 μ of coenzyme Q10 was obtained per 1 g of dry bacterial cells.

〔Effect of the invention〕

According to the present invention, substances that are stable and easily available through industrial production can also be used as raw materials, and further,
Coenzyme Q10 can be easily, efficiently (and stably produced) using bacteria.

Moreover, in particular, since the bacteria in the present invention are alkaliphilic bacteria, contamination by undesirable bacteria during culture is significantly reduced.

Patent applicant: Mitsubishi Gas Chemical Co., Ltd. Representative Nagano Japanese book

Claims (1)

[Claims] A method for producing coenzyme Q_1_0, which comprises culturing coenzyme Q_1_0-producing bacteria belonging to the genus Oligomonas to obtain bacterial cells, and separating and recovering coenzyme Q_1_0 from the obtained bacterial cells.