JPS6125490A - Preparation of coenzyme q10 - Google Patents
Preparation of coenzyme q10Info
- Publication number
- JPS6125490A JPS6125490A JP14532984A JP14532984A JPS6125490A JP S6125490 A JPS6125490 A JP S6125490A JP 14532984 A JP14532984 A JP 14532984A JP 14532984 A JP14532984 A JP 14532984A JP S6125490 A JPS6125490 A JP S6125490A
- Authority
- JP
- Japan
- Prior art keywords
- coenzyme
- medium
- culture
- produced
- coq10
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
羞1よりμ四分!
本発明は発酵法による補酵素Q1o(以下、COQ10
と称す)の製造法に関する。[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 ).
従来の技術
従来、ロドシュードモナス属に属する微生物を用いるC
oQ10の製造方法としては、カロチノイド色素生成能
欠失あるいは低下変異株を使用する方法(特公昭47−
7954号公報、特開昭55−39730号公報、特開
昭55−61797号公報)、バクテリオクロロフィル
色素生成能向上変異株を使用する方法(特開昭56−7
2694号公報、特開昭57’−208992号公報)
等が知られている。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.
問題点を解°するための手段
本発明によれば、ロドシュードモナス属に属し、CoQ
+。生産能を有し、かつキノン化合物に抵抗性を有する
微生物を培地に培養し、培養物中にC0Q10を生成蓄
積させ、培養物からCo Q +。を採取することによ
り、好収率でCoQ10を生産できる。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.
本発明に使用する微生物はロドシュードモナス属に属し
、COQ10生産能を有し、かつキノン化合物に抵抗性
を有する微生物であればいずれも用いられる。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.
キノン化合物としては、■、4−ベンゾキノン。As a quinone compound, ■,4-benzoquinone.
2−メチル−1,4−ベンゾキノン、ジクロロジアゾキ
ノン、1.4−ナフトキノン、2−メチル−104−ナ
フトキノン、アントラキノン、2−メチルアントラキノ
ン、アントラルフィン、キニザリン等があげられる。Examples include 2-methyl-1,4-benzoquinone, dichlorodiazoquinone, 1,4-naphthoquinone, 2-methyl-104-naphthoquinone, anthraquinone, 2-methylanthraquinone, anthralphine, and quinizarin.
上述の性質を有する微生物は、元来COQ10生産能を
有する微生物を親株としてこれにキノン化合物に抵抗性
の性質を付与することによって得ることができる。また
、その逆の方法、すなわち、キノン化合物に抵抗性の微
生物にCOQ10生産能の性質を付与することによって
得ることもできる。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.
例えば、C0QIO生産能を有する微生物を種々の薬剤
処理又はX線、T線、Co照射等の処理をすることによ
って得られる。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.
このようにして得られる菌株を培養して、C0QIO生
産能が親株より顕著に向上した菌株を選択して本発明に
使用する。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.
本発明に使用する菌株の具体例としては、ロドシュード
モナス争スフェロイデス(Rhodopse、udom
onasspheroides) H−3161(以下
、H−3161と称す)、H−3751(以下、H−3
751と称す)及びH−3752(以下、H−3752
と称す)があげられる。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.
これらのH−3161,8−3751及びH−3752
は工業技術院微生物工業技術研究所に微工研菌寄第76
83号、第7684号及び第7685号として、それぞ
れ寄託されている。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.
次に、上記菌株を得る操作例を説明する。Next, an example of operation for obtaining the above-mentioned bacterial strain will be explained.
C,OQ Io生産能を有するロドシュードモナス・ス
フェロイデスFERM−P600B (以下、FERM
−P6008と称す)の細胞を0.05 Mトリス・マ
レイン酸緩衝液(pH6,0)中に約10’ cell
s/mlの濃度に懸濁し、ここにN−メチル−N′−二
トローN−ニトロソグアニジンヲ最終濃度1ag/+n
lになるように添加し、室温で30分間静置した後、1
.4−ベンゾキノン(20μg/ml)、1.4−ナフ
トキ/7 (5μg/ml) 、 2−メチル−1,4
−ナフトキノン(3μg /ml )を含む下記のごと
き組成を有する最少培地寒天平板培地にそれぞれ塗抹し
、生育するコロニーの中からH−3161,H−375
1及びH−3752をそれぞれ選択した。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.
これらの変異株は第1表から明らかな如く、104−ベ
ンゾキノン、1.4−ナフトキノン12−メチル−1,
4−ナフトキノンの各々の抵抗性を有する点で親株であ
るFERM−P6008と明らかに区別できる。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.
第1表
無添加 ++ ++ ++ ++1.4
−ベンゾキノン 20μg/ml−++1.4−ナフト
キノン 5μg/ml −++2−メチルー1.4
− 3μg/ml −++ナフトキノン
注)++;充分な生育
m:生育せず
上記最少培地寒天平板培地の組成ニゲルコース10g/
l、NH,CJ 4g/l、K1−1.PO41g/
β、に2HPO43g/J、Mg5O,・7HiOO,
4g/l、Fe5O−・7H200,01g/j!、M
n5O,・4)(so 0.01g/l、ビオチン5
0μg/1.寒天20g/Il。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.
pH7,2
本発明に使用する培地としては、使用菌株の利用しうる
炭素源、窒素源、無機物その他の栄養物を程よく含有す
る培地ならば、合成培地、天然培地のいずれも使用でき
る。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.
炭素源としては、グルコース、サッカロース。Carbon sources include glucose and sucrose.
廃糖蜜、殿粉等の炭水化物、グリセリン、ソルビトール
等の糖アルコール類、メタノール7〜エタノール等のア
ルコール類等が使用できる。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.
窒素源としては、硫酸アンモニウム、塩化アンモニウム
、酢酸アンモニウム等の無機及び有機アンモニウム塩、
酵母エキス、コーンスチーフリカー等の窒素含有天然物
が使用できる。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.
無機塩としては、リン酸第−カリウム、リン酸第二カリ
ウム、硫酸マグネシウム、塩化ナトリウム、硫酸第一鉄
、硫酸マンガン等が使用できる。As the inorganic salt, potassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, etc. can be used.
もちろん、天然物を用いたときなどにはそれに含まれる
無機塩のみで満足させることが可能なこともある。Of course, when natural products are used, it may be possible to satisfy the requirements only with the inorganic salts contained therein.
また、本発明に使用する微生物が生育のために特定の栄
養素を必要とする場合には、その栄養素を適当量培地に
存在させなければならないが、これらの物質は窒素源と
して例示した天然物に含まれて添加される場合がある。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.
そして培養液中に各種の物質、たとえば、アミノ酸類、
核酸関連物質。There are various substances in the culture solution, such as amino acids,
Nucleic acid related substances.
ビタミン類、有機酸類、アルコール類、ステロール類、
その他CoQ10生合成前駆物質およびその関連物質な
どを添加することによりC0QIO生成量が増加する場
合がある。vitamins, organic acids, alcohols, sterols,
The amount of COQIO produced may increase by adding other CoQ10 biosynthesis precursors and related substances.
培養は、振盪培養1通気攪拌培養などの好気的条件下で
行う。Cultivation is performed under aerobic conditions such as shaking culture 1 aerated stirring culture.
培養温度は20〜40℃の範囲が適当であり、pH5〜
9程度が適当である。The appropriate culture temperature is 20 to 40°C, and the pH is 5 to 40°C.
A value of about 9 is appropriate.
培地のpH調節は炭酸カルシウム、酸あるいはアルカリ
溶液、pH1lll剤などによって行う。The pH of the medium is adjusted using calcium carbonate, an acid or alkaline solution, a pH agent, or the like.
培養期間は通常3〜7日間でCOQ + oは主として
菌体中に生成蓄積する。培養物からのCOQ+。The culture period is usually 3 to 7 days, and COQ + O is mainly produced and accumulated in the bacterial cells. COQ+ from cultures.
の単離は常法、例えば溶媒抽出、濃縮、カラムクロマト
グラフィー等によって行う。Isolation is carried out by conventional methods such as solvent extraction, concentration, column chromatography, etc.
以下に、実施例を示す。Examples are shown below.
実施例10
種菌としてH−3161株を用いた。10この菌株をグ
ルコース2g/a2.ペプトン1g/a、酵母エキス1
g/d1.食塩0.5g/dI!からなる種培地(pH
7,2)300mlを含む21容三角フラスコに接種し
、30℃で24時間振盪培養した。この種培養液30
Qmlを下記組成の発酵培地3Rを含む51容ジヤ一発
酵槽に接種し、温度30℃1通気II!/分、攪拌数3
50回転/分の条件下で96時間培養した。培養にあた
っては24時間目と48時間目にそれぞれ廃糖蜜5g/
〃(糖濃度換算)をフィードした。このとき培養液中に
(:OQ10は150 r /ml生成蓄積していた。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.
対照として同一の条件で同時に培養した親株であるFE
RM−P6008は131 r /mlであった。As a control, the parent strain FE was cultured at the same time under the same conditions.
RM-P6008 was 131 r/ml.
発酵培地の組成:廃糖蜜5g/#(糖濃度換算)。Composition of fermentation medium: 5 g/# of blackstrap molasses (converted to sugar concentration).
酵母エキス2g/a、硫酸アンモニウム0.8g/d1
.リン酸−カリウム0.05g/dl、 リン酸二カ
リウム0.05g/J、硫酸マグネンウム・7水塩0.
.025g/dll、炭酸カルシウム2g/d、ビタミ
ンB、 018mg/d1.−:+チン酸08mg/
d1(pH7,2)
培養終了後、H−3161株の培養液2!を遠心分離し
、乾燥重量として37gに相当する菌体を得た。これを
2001711の水に懸濁し、メタノール400m1.
水酸化ナトリウム80g、ピロガロール15gを加え、
85℃で45分間還流後放冷し、1!ずつのn−ヘキサ
ンを加え、2回抽出した。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.
n−へキサン層を鴬めてこれに無水芒硝を加えて脱水後
、減圧下で濃縮した。残渣を4 Qmlのアセトンに溶
解して不溶物を濾別除去した後、再び濃縮した。残渣を
lQmlのアセトンに溶解後、ノリ力ゲル力ラムに流し
て、ベンゼンにて溶出した。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.
COQ10を含む両分を集めて濃縮し、残渣を5mlの
エタノールに溶解後冷却することにより、黄色の粗結晶
155mgを得た。また、同様にFERM−P4O10
の培養液からは133mgを得た。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.
本島は、逆相薄層クロマトグラフィー、高速液体クロマ
トグラフィーなどによりCOQ10であることを確言忍
した。Motojima confirmed that the COQ was 10 using reversed phase thin layer chromatography and high performance liquid chromatography.
実施例2゜ 種菌としてH−3751及びH−3752を用いた。Example 2゜ H-3751 and H-3752 were used as seed bacteria.
実施例1に示した種培地に)l−3751及びH−37
52をそれぞれ接種し、30℃で24時間振盪培養した
。これら種培地3n+lを実施例1に示した発酵培地3
0m1を含む25 Qml容三角フラスコに接種し、3
0℃で96時間振盪培養した。) 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.
培養にあたっては、24時間目及び48時間目にそれぞ
れ廃糖蜜4g/d(糖濃度換算)をフィードした。この
とき培養液中のCoQ10生成蓄積量は、H−3751
及びH−3752でそれぞれ132 r /ml及び1
33 r /m!であった。対照として同一条件で培養
した親株であるFERM−P4O10はl 20 r
/mlであった。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.
発明の効果
親株に比べて、C0QIOの生成量は10%以上向上す
る。Effects of the invention Compared to the parent strain, the production amount of C0QIO is increased by 10% or more.
Claims (1)
を有し、かつキノン化合物に抵抗性を有する微生物を培
地に培養し、培養物中に補酵素Q_1_0を生成蓄積さ
せ、該培養物から補酵素Q_1_0を採取することを特
徴とする補酵素Q_1_0の製造法。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.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP14532984A JPS6125490A (en) | 1984-07-13 | 1984-07-13 | Preparation of coenzyme q10 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP14532984A JPS6125490A (en) | 1984-07-13 | 1984-07-13 | Preparation of coenzyme q10 |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS6125490A true JPS6125490A (en) | 1986-02-04 |
JPH0365952B2 JPH0365952B2 (en) | 1991-10-15 |
Family
ID=15382646
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP14532984A Granted JPS6125490A (en) | 1984-07-13 | 1984-07-13 | Preparation of coenzyme q10 |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS6125490A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1125596A2 (en) | 2000-02-16 | 2001-08-22 | Terumo Kabushiki Kaisha | Intravascular catheter |
-
1984
- 1984-07-13 JP JP14532984A patent/JPS6125490A/en active Granted
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1125596A2 (en) | 2000-02-16 | 2001-08-22 | Terumo Kabushiki Kaisha | Intravascular catheter |
US6482180B2 (en) | 2000-02-16 | 2002-11-19 | Terumo Kabushiki Kaisha | Intravascular catheter |
Also Published As
Publication number | Publication date |
---|---|
JPH0365952B2 (en) | 1991-10-15 |
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