JPH06261776A - Production of indigo by enzymic method - Google Patents
Production of indigo by enzymic methodInfo
- Publication number
- JPH06261776A JPH06261776A JP5688293A JP5688293A JPH06261776A JP H06261776 A JPH06261776 A JP H06261776A JP 5688293 A JP5688293 A JP 5688293A JP 5688293 A JP5688293 A JP 5688293A JP H06261776 A JPH06261776 A JP H06261776A
- Authority
- JP
- Japan
- Prior art keywords
- indigo
- microbial cell
- aqueous solution
- solution containing
- indole
- 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.)
- Pending
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【0001】[0001]
【産業上の利用分野】本発明は、インドールから酵素法
により効率よくインジゴを製造する方法に関する。TECHNICAL FIELD The present invention relates to a method for efficiently producing indigo from indole by an enzymatic method.
【0002】[0002]
【従来の技術】従来、インジゴは化学合成法により製造
され、工業用染料として広く利用されている。しかしな
がら、化学合成法は反応が多段階になるので収率が悪
く、また化学的分解による副産物が多い等の欠点があっ
た。また、インジゴを製造する種々の方法の中で有望視
されている方法として、キシレンオキシゲナーゼまたは
ナフタレンオキシゲナーゼを含有する微生物を酵素触媒
として用い、インドールから製造する方法が知られてい
る{Burt D. Ensley, Barry J. Ratzkin, Thimthy D. O
sslund and Mary J. Simon; Science, vol.222, p167-1
69(1983)}。しかしながら、実際的な製造技術を確立す
るには至っておらず、効率よくインジゴを製造する方法
の開発が望まれている。2. Description of the Related Art Conventionally, indigo is produced by a chemical synthesis method and is widely used as an industrial dye. However, the chemical synthesis method has drawbacks in that the reaction is multistage and the yield is poor, and there are many by-products due to chemical decomposition. In addition, as a promising method among various methods for producing indigo, a method of producing from indole using a microorganism containing xylene oxygenase or naphthalene oxygenase as an enzyme catalyst is known {Burt D. Ensley , Barry J. Ratzkin, Thimthy D. O
sslund and Mary J. Simon; Science, vol.222, p167-1
69 (1983)}. However, a practical manufacturing technique has not been established yet, and development of a method for efficiently manufacturing indigo is desired.
【0003】[0003]
【発明が解決しようとする課題】本発明は、酵素法によ
り、効率よく、かつ高収率でインジゴを製造することを
目的とする。SUMMARY OF THE INVENTION It is an object of the present invention to produce indigo efficiently and in high yield by an enzymatic method.
【0004】[0004]
【課題を解決するための手段】本発明者らは、インジゴ
生成能を有する菌体またはその処理物を利用した酵素法
によるインジゴの製造方法を確立すべく、反応条件等に
ついて鋭意検討した結果、該菌体を予め塩化ナトリウム
含有水溶液で洗浄することにより、菌体の透過性を向上
させ、効率よくインジゴを製造可能なことを見いだし、
本発明を完成するに至った。Means for Solving the Problems As a result of extensive studies on reaction conditions, etc., in order to establish a method for producing indigo by an enzymatic method using a bacterium having indigo-forming ability or a treated product thereof, the present inventors have By previously washing the cells with a sodium chloride-containing aqueous solution, the permeability of the cells was improved, and it was found that indigo can be efficiently produced,
The present invention has been completed.
【0005】本発明においては、シュードモナス属に属
するインジゴ生成能を有する菌体が用いられるが、特に
シュードモナス(Pseudomonas) sp.MY-6菌株が好適に用
いられる。本菌株の菌学的性質とその分類学的性質は特
開平4−287690号公報に示した。なお、本菌株は
工業技術院生命工学工業技術研究所に生命研菌寄第11
963号(FERM P−11963)として寄託され
ている。In the present invention, a bacterium belonging to the genus Pseudomonas and having the ability to generate indigo is used, and a Pseudomonas sp. MY-6 strain is particularly preferably used. The mycological properties and taxonomic properties of this strain are shown in JP-A-4-287690. This strain was sent to the Institute of Biotechnology, Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology.
Deposited as 963 (FERM P-11963).
【0006】微生物の培養に用いる培地の炭素源として
は、グルコース等の炭水化物、キシレン、トルエン等の
芳香族化合物、フマル酸等の有機酸が利用できるが、そ
れらの中でもm−キシレン、p−キシレン、フマル酸、
コハク酸、クエン酸が好適に用いられる。As a carbon source of a medium used for culturing microorganisms, carbohydrates such as glucose, aromatic compounds such as xylene and toluene, and organic acids such as fumaric acid can be used. Among them, m-xylene and p-xylene are available. , Fumaric acid,
Succinic acid and citric acid are preferably used.
【0007】窒素源としては、塩化アンモニウム、硫酸
アンモニウム、リン酸アンモニウム等のアンモニウム
塩;硝酸ナトリウム、硝酸カリウム、硝酸アンモニウム
等の硝酸塩;アンモニア等を用いることができる。As the nitrogen source, ammonium salts such as ammonium chloride, ammonium sulfate and ammonium phosphate; nitrates such as sodium nitrate, potassium nitrate and ammonium nitrate; ammonia and the like can be used.
【0008】無機物としては、リン酸カリウム、硫酸マ
グネシウム、鉄、マンガン、亜鉛、銅等を用いることが
できる。また、必要に応じて、ビタミン、アミノ酸また
は酵母エキス、ペプトン等の栄養源を添加することがで
きる。As the inorganic substance, potassium phosphate, magnesium sulfate, iron, manganese, zinc, copper or the like can be used. If necessary, nutritional sources such as vitamins, amino acids or yeast extract and peptone can be added.
【0009】培養温度は20〜50℃、好ましくは30
〜40℃であり、培養中の培地のpHは6〜9、好ましく
は7〜8である。培養は好気的に行う。The culture temperature is 20 to 50 ° C., preferably 30.
-40 ° C, and the pH of the medium in culture is 6-9, preferably 7-8. Culture is performed aerobically.
【0010】培養した菌体を本酵素反応に利用する場
合、該菌体は予め塩化ナトリウム含有水溶液で洗浄した
後用いる。洗浄用の塩化ナトリウム濃度は0.2〜20
%、好ましくは0.5〜10%である。洗浄後の菌体は
そのまま使用することができるし、該菌体を必要により
固定化して用いることもできる。When the cultured bacterial cells are used in the present enzymatic reaction, the bacterial cells are washed with an aqueous solution containing sodium chloride before use. Sodium chloride concentration for cleaning is 0.2 to 20
%, Preferably 0.5 to 10%. The washed bacterial cells can be used as they are, or the bacterial cells can be immobilized if necessary before use.
【0011】該菌体またはその処理物をインドールと反
応させるには、通常の酵素反応と同様に、例えば0.1
Mリン酸緩衝液(pH6〜9)あるいは水溶液(pH6〜
9)中で、20〜50℃、好ましくは30〜40℃の温
度で、通常10〜72時間反応させる。反応は撹拌しな
がら行うのが好ましい。To react the microbial cell or a treated product thereof with indole, for example, in the same manner as in a usual enzymatic reaction, for example, 0.1
M phosphate buffer (pH 6-9) or aqueous solution (pH 6-
In 9), the reaction is carried out at a temperature of 20 to 50 ° C., preferably 30 to 40 ° C. for usually 10 to 72 hours. The reaction is preferably carried out with stirring.
【0012】反応液に添加する菌体またはその処理物の
添加量は、特に制限されるものではないが、一般に0.
5〜10%(wt/vol) が用いられる。反応後、反応液か
らのインジゴの分離・精製は、それ自体既知の方法、例
えば溶媒(クロロホルム)抽出等の方法で行うことがで
きる。The amount of the bacterial cells or the treated product thereof added to the reaction solution is not particularly limited, but is generally 0.
5-10% (wt / vol) is used. After the reaction, indigo can be separated and purified from the reaction solution by a method known per se, for example, a method such as solvent (chloroform) extraction.
【0013】[0013]
参考例 (NH4)2 SO4 :3g、KH2 PO4 :0.5g、K
2 HPO4 :0.5g、MgSO4 ・7H2 O:0.5
g、NaCl:0.5g、FeSO4 ・7H2O:10m
g、CaCl2 ・2H2 O:10mg、酵母エキス1g及
び蒸留水:1000ml(pH7.0)の培地100mlを5
00ml容の三角フラスコに分注し、120℃、15分間
滅菌処理したものにm−キシレン0.3mlを添加後、シ
ュードモナス・sp.MY-6 菌株を植菌し、30℃にて24
時間振とう培養した。Reference Example (NH 4) 2 SO 4: 3g, KH 2 PO 4: 0.5g, K
2 HPO 4: 0.5g, MgSO 4 · 7H 2 O: 0.5
g, NaCl: 0.5g, FeSO 4 · 7H 2 O: 10m
g, CaCl 2 .2H 2 O: 10 mg, yeast extract 1 g, and distilled water: 1000 ml (pH 7.0)
Aliquots were dispensed into a 100 ml Erlenmeyer flask and sterilized at 120 ° C. for 15 minutes, 0.3 ml of m-xylene was added, and Pseudomonas sp. MY-6 strain was inoculated at 24 ° C. at 30 ° C.
The culture was shaken for an hour.
【0014】また、上記と同様の培地1000mlを5リ
ットル容の三角フラスコに入れ、120℃、15分間滅
菌処理したものにm−キシレン3mlを添加後、上記振と
う培養液20mlを接種し、これを30℃にて24時間振
とうした。得られた培養液の1000mlを遠心分離(8
000rpm、15分、4℃)して集菌した該集菌体を、次
のように前処理に供試した。1000 ml of the same medium as described above was placed in a 5 liter Erlenmeyer flask and sterilized at 120 ° C. for 15 minutes, 3 ml of m-xylene was added, and 20 ml of the shaking culture solution was inoculated. Was shaken at 30 ° C. for 24 hours. 1000 ml of the obtained culture solution was centrifuged (8
The collected cells were collected at 000 rpm, 15 minutes, 4 ° C.) and subjected to pretreatment as follows.
【0015】実施例 (洗浄操作)集菌した菌体を表−1の塩化ナトリウム含
有水各50mlに懸濁し、同上の条件で遠心分離し、再
び、表1の濃度の塩化ナトリウム含有水各50mlに懸濁
して同様に遠心分離した。該菌体を、反応液(インドー
ル2mM、100mMリン酸緩衝液、pH7.0)1000ml
に懸濁後、3リットル容の通気撹拌装置に入れ、、30
℃で48時間反応させた。途中、24時間後にインドー
ルを2mM添加した。Example (Washing operation) The collected bacterial cells were suspended in 50 ml each of sodium chloride-containing water shown in Table 1, centrifuged under the same conditions as above, and again 50 ml each of sodium chloride-containing water having the concentration shown in Table 1. And suspended in a similar manner and centrifuged. 1000 ml of the reaction solution (indole 2 mM, 100 mM phosphate buffer, pH 7.0)
After suspending in, place in a 3-liter aeration stirrer,
The reaction was carried out at 48 ° C for 48 hours. On the way, 2 mM of indole was added 24 hours later.
【0016】生成したインジゴ量を、常法 [H. Keil,
C. M. Saint and P. A. Williams, Journal of Bacteri
ology, 169, No.2, p764-770(1987)]に従い定量した。
なお、対照として、培養集菌後菌体を洗浄せずにそのま
ま反応に供した系および、蒸留水で同様に洗浄を行った
系での結果を比較した。結果を表1に、洗浄を行わなか
った実験系を100とする相対値で示した。The amount of indigo produced was measured by the conventional method [H. Keil,
CM Saint and PA Williams, Journal of Bacteri
ology, 169, No. 2, p764-770 (1987)].
As a control, the results were compared between a system in which the cells were directly subjected to the reaction without being washed after the culture was collected and a system in which the cells were similarly washed with distilled water. The results are shown in Table 1 as relative values with the experimental system not washed as 100.
【0017】[0017]
【表1】 [Table 1]
【0018】[0018]
【発明の効果】本発明の方法によれば、酵素法により、
効率よく、かつ高収率でインドールよりインジゴを製造
することができる。According to the method of the present invention, the enzymatic method is used.
Indigo can be produced from indole efficiently and in high yield.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 山懸 恒 茨城県牛久市田宮町駅区画整理48街区2番 地フジハイム2−102 (72)発明者 寺沢 真人 茨城県稲敷郡阿見町中央1−11−5−401 (72)発明者 湯川 英明 茨城県稲敷郡阿見町中央6−23−9 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hisashi Yamagake Tamiya-cho, Ushiku-shi, Ibaraki Land readjustment 48 Block 2-Fujiheim 2-102 (72) Inventor Masato Terazawa 1-11 Chuo, Ami-machi, Inashiki-gun, Ibaraki 5-401 (72) Inventor Hideaki Yukawa 6-23-9 Chuo, Ami Town, Inashiki District, Ibaraki Prefecture
Claims (1)
能を有する菌体またはその処理物を、インドールを含有
する水溶液に作用させて酵素的にインジゴを生成させる
方法に於いて、予め該菌体を塩化ナトリウム含有水溶液
で洗浄することを特徴とする酵素法によるインジゴの製
造方法。1. A method for enzymatically producing indigo by allowing a bacterial cell belonging to the genus Pseudomonas having the ability to produce indigo or a treated product thereof to act on an aqueous solution containing indole to produce indigo beforehand. A method for producing indigo by an enzymatic method, which comprises washing with an aqueous solution containing water.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP5688293A JPH06261776A (en) | 1993-03-17 | 1993-03-17 | Production of indigo by enzymic method |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP5688293A JPH06261776A (en) | 1993-03-17 | 1993-03-17 | Production of indigo by enzymic method |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH06261776A true JPH06261776A (en) | 1994-09-20 |
Family
ID=13039794
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP5688293A Pending JPH06261776A (en) | 1993-03-17 | 1993-03-17 | Production of indigo by enzymic method |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH06261776A (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6004772A (en) * | 1995-02-28 | 1999-12-21 | Canon Kabushiki Kaisha | Oxygenase expressing microorganism strain JM1 (FERM BP-5352) for degrading organic compounds without an inducer |
CN103060218A (en) * | 2011-10-18 | 2013-04-24 | 大连理工大学 | Phenol-degrading bacteria and method for preparing indigo by conversing indole |
-
1993
- 1993-03-17 JP JP5688293A patent/JPH06261776A/en active Pending
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6004772A (en) * | 1995-02-28 | 1999-12-21 | Canon Kabushiki Kaisha | Oxygenase expressing microorganism strain JM1 (FERM BP-5352) for degrading organic compounds without an inducer |
CN103060218A (en) * | 2011-10-18 | 2013-04-24 | 大连理工大学 | Phenol-degrading bacteria and method for preparing indigo by conversing indole |
CN103060218B (en) * | 2011-10-18 | 2014-04-23 | 大连理工大学 | Phenol-degrading bacteria and method for preparing indigo by conversing indole |
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