JPS58149688A - Preparation of coenzyme q10 - Google Patents

Abstract

PURPOSE:To prepare coenzyme Q10 in high yield and purity, by culturing a novel bacterial strain belonging to Alcaligenes genus. CONSTITUTION:A novel bacterial strain belonging to Alcaligenes genus, i.e. Alcaligenes sp.DK-605 (FERM No.6342) or Alcaligenes sp. DK-515 (FERM No.6341), is inoculated in a nutrient medium and cultured at 5-8pH and 20- 40 deg.C for 2-6 days under aerobic condition, and the coenzyme Q10 is separated from the cultured bacterial cells.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing coenzyme Q1o, and more particularly to a method for producing coenzyme QIO using Alcaligenes sp, DK-605 or Alcaligenes sp, DK-515.

Coenzyme Q1o is widely intercalated 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 microorganisms are also known. For example, microorganisms that produce coenzyme QIO include Ω bacteria such as Pseudomonas denitrificans, Agrobacterium toumefaciens, and Gluconobacter saboxidans, Rhodospirillium rubra, and Rhodopseudomonas capsulitus. Photosynthetic bacteria such as Rhodotorula spp., Cryptococcus spp., Loboromyces sp. The genera Aureobasidium, Theileteiopsis, and Ephsobasibium are known.

Furthermore, microorganisms belonging to alkaline earth metals, such as Alcaligenes faecalis AJ2560
No. 49) describes a method for producing coenzyme QIO (Japanese Patent Publication No. 19034/1983). However, in these methods, the productivity of coenzyme Q1o is low, and the current situation is not yet satisfactory.

As a result of various studies to solve the drawbacks of these conventional techniques, the present inventors discovered that a certain new strain belonging to alkaline earth metals produces coenzyme Q1o in high yield and purity. Completed the invention.

That is, the gist of the present invention is Alcaligenes SP.

DK-605 or Alcaligenes sp, DK-515
The present invention relates to a method for producing coenzyme QIO, which comprises culturing coenzyme QIO and collecting coenzyme QIO from the produced bacterial cells.

Alcaligenes used in the present invention
s) ip, DK-605 and Alcaligenes sp, D
Both K-515 are new strains isolated from soil bacteria by screening, and have been deposited with the Institute of Microbiology, Agency of Industrial Science and Technology under accession numbers 6342 and 6341, respectively.

These new strains have the following mycological properties: Alcaligenes 8P, DK-605 (1) Morphological properties They were statically cultured for 1 to 2 days at 30 DEG C. in a broth medium and observed.

11) Cell size: 0°9-1.2μ x 1.2-2.3μ coccoid bacilli (co
ccobacilli) or short rods (2) Populations alone or often in duplicate (3) Without motile 4) With or without spores 5) Negative for Durham stain ■ Growth status in each medium Cultivation in each medium at 30°C I went there for 3 days.

ll) Meat juice liquid culture Growth: Moderate Film: Forms a ring Sediment: Yes Turbidity: Yes (2) Meat juice agar plate culture Shape: Perfect circular rim: Full surface Shape of ridges: Lenticular surface: Smooth and glossy Color tone: Milky white (3) Meat juice agar slant culture Shape: Thread-like Surface: Smooth and glossy Margin: All bound Color tone: Milky white Transparency: Opaque (4) Meat juice agar slant culture Grows along the surface and upper puncture■, physiological properties Il+ Nitrate reduction knee (2) Denitrification reaction knee 131 MR test knee + 41 VP test -1 (5) Indole formation knee (6) Hydrogen sulfide production knee (7) Starch hydrolysis knee (8) Utilization of inorganic nitrogen sources : Use only ammonium salts and nitrates as nitrogen sources (9) Formation of pigments: For example, NH4Cl Q, 1%,
MgSO4-7H200.03%, Na2S04
0.05%, KH2PO40.21%, Na2HPO4.12 H2O1.2%, yeast extract 0.1%, glucose 2% and distilled water (remainder) medium may produce a reddish-brown pigment.

αω urease Neat tm oxidase: + 021 Catalase: + +131 Attitude towards oxygen: Aerobic - liquefaction of gelatin (15) Lidomus milk: unchanged 16) Growth temperature = 25-35℃ Optimum growth temperature
:30℃ (1η Growth pH: 5.Q~7.3 Optimum growth pH: 6.Q~7.0 Production of acid and gas from DI sugar nigelcose, galactose, maltose, sucrose,
Formation of acid and gas from lactose, raffinose, glycerol is not observed III Carbon source assimilation glucose +, mannit +, galactose +, sorbit +, mannose
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so-erythrite-, sorbose-shi, glucono-
δ-lactone +, arabinose +, 2-keto-logluconate-, xylose +, 5-keto-logluconate-, ribose +, sodium citrate-, maltose-, sodium succinate +,
Lactose +, Aspartic acid +, Sucrose +, Asparagine +, Trehalose +
, histidine +, melibiose +, propionic acid -, melezitose -, butyric acid
-, raffinose +, vinegar dredged sodium
+, inulin -, phenol -, soluble starch, ethanol +, glycerin
+, methanol +.

■Vitamin requirement: None 211 cc content: 65.8 mol%■ Morphological properties Observations were made on the specimens that were kept in a broth medium at 30°C for 1 to 2 days.

11) Cell size, shape 09-12μ x 1.2-2.3μ coccobacillus (cocc)
obacil li) or short stem growth (2) Eastern times alone or often in duplicate (3) No motility 4) No spores 5) Negative Durham stain ■ Growth status on each medium Culture on each medium is 30 It was carried out for 3 days at ℃.

Ill Meat juice liquid culture Growth: Moderate Film: Forms a ring Sediment: Yes Turbidity: Yes (2) Meat juice agar plate culture Shape: Perfect circle Periphery: Gold rim Surface ridge shape: Lens-shaped Surface: Smooth and glossy Color tone: Milky white ( 3) Meat juice agar slant culture Shape: filamentous Surface: smooth and glossy Border: golden edge Color tone: milky Transparency: opaque (4) Growth along the meat juice agar puncture culture surface and upper puncture ■, physiological properties 11) Nitrate Reducing knee (2) Denitrification reaction knee +31 MR test knee +41 VP test knee (5) Indole formation knee (6) Hydrogen sulfide production knee (7) Starch hydrolysis knee (8) Utilization of inorganic nitrogen source: ammonium salt , using only nitrate as nitrogen source 19) Formation of pigments: e.g. N) (4Cl O, 1%,
MgSO4-7H200.03%, Na2S04
0.05%, KH2PO40.21%, Na2HPO4-12H2O1.2%, yeast extract 01%, glucose 2% and distilled water (remainder) The medium may produce a reddish-brown pigment.

(101 Urease 20 (11) Oxidase: + (12 Catalase: 10 (13) Attitude towards oxygen: Aerobic (141 Liquefaction of gelatin 5) Lidomus milk: Slightly alkaline 06) Growth temperature = 25-37℃ Optimum growth temperature 1 : 30-33℃ Till growth pH: 5.0-7.3 Optimum growth pH: 6.0-65 (Acid and gas generation from around 181 sugars,
galactose, maltose, sucrose, lactose,
No acid or gas production was observed from raffinose and glyceptol (191 carbon sources assimilable glucose +, mannit +, galactose +, sorbit +, mannose).
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, meso-engineering IJ)lJtsu)-, sorbose
glucono-δ-lactone +, arabinose +,
2-keto-logluconate −, xylose +
, 5-Geto D-gluconate-, ribose
+, Sodium citrate +, Maltose -, Kono Risu) IJum +, Lactose +, Aspartic acid +, Sucrose +, Asparagine
+, trehalose +, histidine +,
Melibiose, propionic acid -, melezitose -, butyric acid -, raffinose +
, sodium acetate, inulin, phenol, soluble starch, ethanol
+, glycerin +, methanol +
.

■Vitamin requirement: None Regarding the creation method of these two strains, patent Ia was filed on the same day.
n+ and (2).

When culturing these strains, the culture medium includes a carbon source that can be assimilated by the microorganisms used (e.g., glucose, molasses, acetic acid, succinic acid, ethanol, methanol, etc.) and an assimilable nitrogen source (e.g., yeast extract, peptone, corn steep liquor, etc.). , ammonia, ammonium sulfate, ammonium nitrate, etc.) and various inorganic salts necessary for growth.

The culture is carried out at a temperature of 20 to 40°C, preferably 25 to 35°C, with the pH of the medium adjusted to 5 to 8, preferably 6 to 7.
This is done with aerobic shaking or stirring for at least about 2 days, preferably 2 to 6 days. After culturing, use the conventional method (
For example, the bacterial cells are separated by centrifugation, filtration, etc.).

Since coenzyme Q1o is abundantly accumulated in the obtained bacterial cells, it is possible to treat the bacterial cells appropriately and provide the coenzyme still contained in the bacterial cells to nutrients, medicines, feed, etc. can.

In addition, in order to separate coenzyme QIO from bacterial cells, the bacterial cells may be either live cells, dried bacterial cells, or processed bacterial cells.

Isolation of coenzyme Q1o can be carried out by conventional methods, for example as follows: First, a mixture of methanol, sodium hydroxide and pyrogallol is added to an image-containing solution for saponification. 1-2 at 60-90℃
Extract by heating under reflux for an hour. Next, the extract is extracted with a solvent such as n-hexane, and the solvent phase is washed with water, dehydrated, and then concentrated. When the concentrate is added to a column such as silica gel and developed with a solvent such as benzene, coenzyme Q1o is eluted. Both parts containing coenzyme Qlo are concentrated to dryness, and the ethanol-soluble part is left to cool to obtain red-yellow crude crystals of coenzyme Qlo. Furthermore, pure crystals of coenzyme Q1o can be obtained by repeating recrystallization from ζethanol.In addition, separation using clathrate compounds, molecular distillation, etc. are also effective.

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

Example 1 A medium (pH 6.5) consisting of 2% by weight of glucose, 1% by weight of peptone, 1% by weight of yeast extract, and the remainder of water was added to a jar fermentor with a capacity of 7 tons, and to this was added 150 tons of a medium with the same composition. A culture of Alcaligenes sp. I got it. This is 22.59 as a dry bacterial cell, and this bacterial cell has coenzyme Q102. Contains Ogg.

The obtained wet bacterial cells were mixed with 100 ml of water and 20 ml of methanol.
409 and heated under reflux at 85° C. for 1 hour.

After cooling, the mixture was extracted twice with 1 t of n-hexane. The n-hexane extracts were combined, dehydrated by adding anhydrous Glauber's salt, then concentrated under reduced pressure, and the residue was dissolved in 20 ml of acetone.
Insoluble matter was removed. The acetone solution was concentrated, and the residue was redissolved in acetone smt, applied to a silica gel column, and eluted with benzene.

Both fractions containing coenzyme Q1o were combined and concentrated. When the residue was dissolved in ethanol and allowed to stand, 40 mg of yellow crude crystals of coenzyme Q1o were precipitated. Rf value of reverse phase thin phase chromatography of pure product obtained by recrystallizing crude crystals from ethanol, retention time of high performance liquid chromatography, melting point, N
The MR spectrum, elemental analysis values, infrared absorption spectrum, and ultraviolet absorption spectrum were consistent with those of the standard coenzyme QIO.

Example 2 As a medium, glucose 2% by weight, KH2PO40,2
1% by weight, Na2HPO4-12H20 1.2% by weight, N04C1 0.5% by weight, M g NO 4 ・
The same procedure as in Example 1 was repeated except for using a medium consisting of 0.03% by weight of 7H20, 0.05% by weight of 2S04, 0.1% of enzyme extract, and the remaining water to obtain crude crystals of coenzyme Q1o 39w9. Ta.

Example 3 Alcaligenes sp. Alcaligenes sp. instead of DK-605. Culture was performed in the same manner as in Example 1 except for using DK-515. A bacterial cell with a dry weight of 21 tons was obtained.

This bacterial cell contains 1.4 μ of coenzyme Q1o per 1f of dry bacterial cell.
was included. Crude crystals of coenzyme QIO from this bacterial cell 2
61q was obtained.

Example 4 The same procedure as in Example 2 was repeated except that Alcaligenes sp, DK-605 was replaced with Alcaligenes 5P-DK-515 to obtain crude crystals of coenzyme QIO 2.
I got 4 ■.

Patent Applicant: Daikin Industries, Ltd. Agent Masu Aoyama, Physician (and 2 others) Procedures: Amendment (spontaneous) 1. Indication of the case: Patent Application No. 26592, filed in 1982. 2. Name of the invention: Process for producing coenzyme Q 0 3. Relationship with the case of the person making the amendment Patent applicant address New Hankyu Building, 1-12-39 Umeda, Kita-ku, Osaka, Osaka Prefecture Name (285) Representative of Daikin Industries, Ltd. Yama 1) Minoru 5, Amendment order Date: (voluntary) 6. Subject of amendment 2: “Detailed description of the invention” in the specification
In Column 7, the following parts of the detailed statement of amendments will be amended.

10 Detailed Description of the Invention Column (1) At the end of page 3, line 8, "flucalidenel" was corrected to "alcaligenes."

(2) In line 3 at the end of page 7 and line 9 on page 12, [Frucose] was corrected to [Fructose].

that's all

Claims (1)

[Scope of Claims] 1. Coenzyme Q1, which is characterized by culturing Alcaligenes Sap, DK-605 or Alcaligenes sp, 0K-515, and collecting coenzyme Q1o from the produced box.
Method of manufacturing o. 2. The production method according to claim 1, using Alcaligenes sp, DK-605. 3. The manufacturing method according to claim 1, using Alcaligenes 8P and DK-515.