JPS5832877B2 - Production method of coenzyme Q↓1↓0 by fermentation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing coenzyme QIO by a fermentation method, and more specifically, to a method for producing coenzyme QIO by a fermentation method, and more specifically, to reduce or lack the carotenoid production ability of a microorganism that belongs to the genus Rhodotorula and has the ability to accumulate and produce coenzyme QIO within the microbial body. This invention relates to a method for producing coenzyme QIO using a deletion mutant.

Coenzyme QIO is widely distributed in living organisms and plays an important role as an electron carrier in the terminal electron respiratory system.In recent years, it has been used as a drug with excellent pharmacological effects on various diseases (such as congestive heart failure). , has been shown to exhibit physiological effects.

Conventionally, a method of extracting and purifying coenzyme QIO from animal or plant tissue is known as a method for obtaining coenzyme QIO.

However, this method has various problems such as resource constraints.

In addition, production methods using yeast (Rhodotorula sp.) are known (for example, Japanese Patent Publication No. 48-8836;
(See Publication No. 0-4758).

However, the productivity of coenzyme QIO is low in these methods, and the current situation is still unsatisfactory.

As a result of various studies in order to solve the drawbacks of these conventional techniques, the present inventors discovered that coenzyme QI belongs to the genus Rhodotorula.
The present invention has been completed by discovering that coenzyme Q1° can be obtained in higher yield and purity than the parent strain by using a mutant strain with reduced or deleted carotenoid pigment-producing ability of a microorganism that has the ability to accumulate and produce O.

That is, the gist of the present invention is to culture in a nutrient medium a mutant strain with reduced or deleted carotenoid production ability of a microorganism that belongs to the genus Rhodotorula and has the ability to accumulate and produce coenzyme QIO in the microbial body, and to The present invention relates to a method for producing coenzyme Qlo by a fermentation method characterized by collecting coenzyme QIO.

The microorganism used in the present invention may be any microorganism that belongs to the genus Rhodotorula and has the ability to accumulate and produce coenzyme Q1o, as long as it is a mutant strain that has a reduced or deleted ability to produce carotenoid pigments.

- For example, Rhodotorula graminis, DK-35
9-217-54 (FEI Microbiology Accession No. FEIKEN Microbiology 5)
No. 671).

This strain is Rhodotorula graminis wild strain DK-359.
(Feikoken Microorganism Accession No. 5670) by the following mutation treatment.

The wild strain DK-359 exhibits the following mycological properties.

1. Growth status on YM agar medium: Opaque reddish color.

The colony shape is circular, the ridge is convex at the center, the periphery is golden, the surface is granular and lack luster, and no pseudohyphae or hyphae are formed.

2. Ascospore formation: No formation 3. Physiological properties: (1) Optimal growth conditions: Temperature: 28-300C pH: 5-6 (2) Growth range temperature: 20-33°C pH: 2-8 3 Nitrate assimilation: Yes 4 Urea decomposition: Yes 5 Gelatin liquefaction: No 6 Carotenoid production: Yes 7 Significant organic acid production: No 8 Starch-like substance production: No 9 Vitamin requirement: No 4 , assimilation of carbon compounds: ■ D-arabinose: + 2 D-ribose: + 3 D-xylose: + 4 D-glucose: +, fermentability: 5 D-galactose: +, fermentability: 6 L-rhamnose :+ 7 L-sorbose: 8 Sucrose: +, fermentable: 9 Melibiose: (10) Raffinose: +, fermentable (II) Inosit: (12) D-mannitol: + α3) 2-keto D-glucone Acid acid: 04) Succinate: + (1) Citric acid: (16) Soluble starch: The strain used in the present invention can be obtained by ordinary mutation treatment, and specific examples are shown below.

A microorganism belonging to the genus Rhodotorula and capable of producing coenzyme QIO (parent strain, exhibiting reddish color) was grown in YM medium (malt extract 0.3 g/ct, Zs yeast extract 0.3 g/dll,
Peptone 0.5g/dA! , glucose 1 g/di, agar 2 g/dl, pH 7,0) at 30°C.
After growing for 8 hours, the bacterial cells were treated with 200 μg/rrtl of N-methyl-N'-nitroN-nitrosoguanidine. 0.05M Tris-maleate buffer (pH 6.0) containing
105-107 bacterial cells/suspended vertically, 30
Shake for 40 minutes at °C.

Then, centrifugation is performed at 5000 rpm for 5 minutes, and the suspension is suspended in physiological saline.

After further washing twice with physiological saline, approximately 103 to 104
Suspend in physiological saline at a concentration of bacterial cells/ml.

Transfer 0.1 ml of the resulting suspension to an agar plate (ethanol 2
TLl/dl, NH4Cl 0.2 g/dl, K
l(2PO40,21, p/ci 4 Na2HPO4
・] 2H201,2g/dl, MgSO4・2H
200.03g/dA, Na2SO40.05g/d
11 yeast extract 0.19/dA, agar 2ji/dl))
and cultured at 30° C. for 3 to 4 days.

Among the colonies that appeared, those that were white or yellow in color and had a low or absent ability to produce reddish pigments like the parent strain were selected as strains with reduced or deleted ability to produce carotenoid pigments.

The carbon source of the nutrient medium used in the production method of the present invention may be any carbon source that can be used by the microorganisms used. Organic acids, alcohols such as ethanol, propatool, and butanol, fats and oils such as soybean oil, and higher fatty acids are used, and mixtures thereof can also be used.

As a nitrogen source, ammonium salts such as ammonium sulfate and ammonium chloride,
Urea, amino acids or mixtures thereof can be used.

As inorganic salts, potassium salts, magnesium salts, phosphates, zinc salts, manganese salts such as potassium chloride, magnesium sulfate, potassium phosphate, zinc sulfate, etc. can be used.

As trace elements, vitamins, yeast extract, molasses, corn steep liquor, etc. may be added as necessary.

Furthermore, the amount of coenzyme QIO produced can be increased by adding various substances such as sterols, amino acids, benzoic acid, and other coenzyme QIO biosynthetic precursors and related substances to the medium or culture solution. It is.

Cultivation is carried out by aerated agitation culture, but either batch or continuous culture may be used.

The pH of the culture solution is preferably 3 to 7.

As the neutralizing agent, aqueous ammonia, sodium hydroxide, calcium hydroxide, etc. are used.

The culture period is 2-7 days.

Coenzyme Q1o can be isolated from microbial organisms by extracting with organic solvents or by using the quartz method to obtain an extract containing the coenzyme and fractionating it by column chromatography.
Alternatively, it can be carried out by performing partition extraction with an organic solvent, concentrating both components containing coenzyme Q1o, and then crystallizing from ethanol.

Next, Examples will be shown to specifically explain the manufacturing method of the present invention.

Example 1 2 ml of ethanol and NH4Cl for 100 TL of water
1g, KH2PO40, 1g, MgSO4・7H200
,03g, Mg5040.05g and yeast extract 0.03g.
300 yd of seed medium at pH 7.0 added at the rate of 1 g
was added to a 21 Erlenmeyer flask, sterilized under 1200CX for 5 minutes, and Rhodotorula gramis DK-
359-21754 (Feikoken Microbiology Accession No. 5671) (mutant strain lacking carotenoid pigment production ability)
was inoculated and cultured with shaking at 30°C for 24 hours.

300 TL of the culture solution was inoculated into a jar fermenter (volume 71) containing medium 31 with the same composition as above, rotation speed 600 rpm, aeration volume IV, V, M, temperature 30°C.
The cells were cultured under aeration and agitation for 96 hours under the following culture conditions.

811r19 of coenzyme Q1o per 11 culture solutions were found.

Further, in this case, no co-production of reddish carotenoid pigments was observed.

In addition, during culturing, an appropriate amount of ethanol as a carbon source was added as necessary.

After culturing for 96 hours, the cells were collected by centrifugation to obtain 6007 nl of manga (106 g of dried cells).

Suspend the bacterial cells in 480ml of water and add 150ml of methanol to this.
0TL11 150 g of sodium hydroxide and pyrogazol 61 are added for saponification, and n-hexane is added to the extract for extraction three times.

The n-hexane extracts were combined, washed with water, concentrated, and cooled to remove the precipitate.

Glauber's salt was added to the obtained tear fluid, and after dehydration, it was developed with hexane:ether (8:2) using a silica gel column to obtain a coenzyme QIO fraction.

This fraction was concentrated to dryness, dissolved in ethanol, cooled and recrystallized.

Repeat this operation to obtain 99 yellow crystals of coenzyme Q1o.
61n9 was obtained.

Retention time, melting point, NM of high performance liquid chromatography of crystals obtained by recrystallizing this from ethanol
The R spectrum, elemental analysis values, infrared absorption spectrum, and ultraviolet absorption spectrum curves all matched those of the standard coenzyme QIO.

On the other hand, under the same conditions, Rhodotorula gramis wild strain DK-359 (Feikoken Microorganism Accession No. 5670) used as a control was cultured, but the production amount of coenzyme QIO was 42.7 ■ /l, carotenoid co-production was observed.

Claims (1)

[Claims] 1 Belongs to the genus Rhodotorula and contains coenzyme Q1 in the microorganism.
Coenzyme QIO produced by a fermentation method characterized in that a mutant strain with reduced or deleted carotenoid production ability of a microorganism that has the ability to accumulate and produce o is cultured in a nutrient medium, and coenzymes Q and 0 are collected from the produced cells. manufacturing method. 2. The carotenoid-producing ability reduced or deleted mutant strain is Rhodotorula graminis, DK-359-217-54 (Feikoken Microorganisms Accession No. 5671) Manufacturing method.