JPS6336789A - Production of coenzyme q10 - Google Patents

Abstract

PURPOSE:To make it possible to produce coenzyme Q10 readily and stably, by cultivating a bacterium belonging to the genus Hyphomonas, capable of producing coenzyme Q10 to form a mold and separating and recovering the coenzyme Q10 from the formed mold. CONSTITUTION:A bacterium belonging to the genus Hyphomonas, capable of producing coenzyme Q10 to form a mold. A strain belonging to the genus Hyphomonas janashiana is preferable as the bacterium belonging to the genus Hyphomonas. Either a natural medium or a synthetic medium may be used as the medium. The coenzyme Q10 is separated and recovered by a method to centrifuge the culture solution directly, etc. The coenzyme Q10 is a compound which has a structural formula shown by formula I, plays an important role as an electron carrier for peripheral respiratory system and is useful as a sthenia agent for cardiac function, remedy for myasthenia gravis, remedy for pulmonary emphysema, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention provides coenzyme Q. More specifically, the present invention relates to a method for producing coenzyme QIo using microorganisms.

Coenzyme Q. has the structural formula shown below, plays an important role as an electron carrier in the terminal respiratory system in vivo, and is a cardiac function enhancer and a therapeutic agent for type 5 myasthenia.

It is a compound useful as a pharmaceutical agent such as a treatment for emphysema, a treatment for aplastic anemia, and a treatment for alopecia areata, as well as a feed additive.

[Prior art 1 Problems to be solved by the invention] Conventionally,
Coenzyme Q10 is produced by extracting from animal or plant tissue and further purifying it. In addition, in recent years, coenzyme Q has been produced from bacterial cells obtained by culturing microorganisms. There are also known methods for extracting.

However, the productivity of coenzyme Q10 by microorganisms is still not sufficient; The discovery of microorganisms with high productivity is desired.

The present inventors aimed to obtain a microorganism with high productivity of coenzyme QI6.

[Means for Solving the Problem 9 Effects] As a result of repeated research to find a strain that produces coenzyme Q+o in multiple ways, the present inventors found that a strain belonging to the genus Hyhomonas produces multiple coenzyme QI0. They discovered this and arrived at the present invention.

That is, the present invention provides coenzyme Q belonging to the genus Hyhomonas.
10. Culture the producing bacteria to obtain bacterial cells, and from the obtained bacterial cells, coenzyme Q. Coenzyme Q1 characterized by separating and recovering
0 manufacturing method.

The bacteria used in the present invention may be any strain as long as it belongs to the genus Hyhomonas, but Ilyhomonas jana-s
Bacterial strains belonging to P. hiana) are preferred. A representative strain of this strain is, for example, Hyhomonas diana siana ATCC33883. Hyhomonas zianasiana is known as a marine bacterium, and its mycological properties include In5t, J, 5yst, Bacteriol,
vol.

35, NO, 3, P, 237-243 (1985)
It is described in.

As the medium used for culture, these coenzyme Q.

There is no particular restriction on the medium as long as it allows production bacteria to grow, and it may be either a natural medium or a synthetic medium.

In addition, Hyhomonas diana siana is grown in a seawater medium containing polypeptone, yeast extract, etc., or in this medium, the concentration of sodium chloride and cum is 2 to 6 w instead of seawater.
A medium using a t% aqueous sodium chloride solution is preferred.

Regarding culture conditions, the culture temperature is usually 20 to 40°C.
In addition, the culture pl+ may be in the range of 6 to 8, and the temperature and pl+ suitable for growth and proliferation may be selected for each strain.

The culture method may be either batch culture or continuous culture.

The bacterial cells are separated and collected from the culture solution obtained by culturing the bacteria. A known solid-liquid separation means is used to separate and recover the bacterial cells. For example, the culture solution may be centrifuged as is, cells of other microorganisms larger than the target bacteria in the culture solution may be added as a filter aid, or cells may be filtered and separated from the culture solution by pre-coating. A method is to add a flocculant to the culture solution to agglutinate the bacterial cells and separate them by filtration or centrifugation, or to reduce the phi of the culture solution to 5 or less and heat the bacterial cells at 50 to 100°C. A method of agglutinating bacteria and separating the aggregated bacterial cells can be applied. Bacterial cells that remain isolated,
Or, for example, coenzyme Q from dried bacterial cells using a spray dryer or the like. This coenzyme Q10 is further purified.

Coenzyme Q. The separation, extraction and purification of coenzyme QIo
It can be carried out according to the usual separation extraction method and purification method applied to. That is, for example, the bacterial cells are made cloudy in ethanol, methanol, or acetone, and extracted by heating at 50 to 90°C for one to several hours to obtain an extract. Alternatively, first, saponifiable substances such as phospholipids in the bacterial cells are saponified using a mixture of methanol, sodium hydroxide, and pyrogallol to obtain a saponified substance.

From these extractions or saponification, for example, n
-Is it like hexane? Coenzyme QIO is extracted with three medium to obtain an extract. Next, coenzyme Q is extracted from this extract using, for example, a porous synthetic resin such as Ha.f porous polymer, silica gel, and florisil.
The IO is separated, separated and purified.

High performance liquid chromatography and elemental analysis are generally used to identify and quantify coenzyme QIO 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 per pure water 11: 5 g of peptone, 1 g of yeast extract, 0.1 g of ferric citrate, 19.45 g of NaCl
4MgC1z・611z08.8g, NazSO
n4011zO3,24g, CaCIz・611z
Dissolve 1.8 g of O and 0.55 g of KCl, p)l
The medium was placed in a 20 On + 7 volume Erlenmeyer flask adjusted to 7.6 and autoclaved at 120°C for 20 minutes.

Hyhomonas diananiana ATCC obtained by pre-cultivating this medium for 2 days with a medium having the same composition as this medium.
33883 preculture solution to 1 volume ■%, seedlings were planted,
Rotary shaking culture was performed at 30°C. The culture was terminated 48 hours after the start of the culture, and the resulting culture solution was centrifuged to obtain bacterial cells.

Coenzyme QIG is extracted from this bacterial cell at a concentration of 90% isopropanol.
Extract with isopropanol aqueous solution of νolχ, then
After oxidizing the pentagonal coenzyme Q10 in the hexane solution with iron nitrate, coenzyme QIO was identified in the hexane solution by high performance liquid chromatography, and its content was measured.

As a result, 1.50 μ of coenzyme QI per 1 g of dry bacterial cells
Q was obtained.

〔Effect of the invention〕

According to the present invention, coenzyme Ok+ is produced using bacteria.

can be manufactured easily and stably.

In particular, when Hyhomonas diana siana is used as the bacterium, there is an advantage that seawater can be used as the water for the culture medium.

Claims (1)

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