JPH067180A - Production of natural-type abscisic acid - Google Patents
Production of natural-type abscisic acidInfo
- Publication number
- JPH067180A JPH067180A JP16741792A JP16741792A JPH067180A JP H067180 A JPH067180 A JP H067180A JP 16741792 A JP16741792 A JP 16741792A JP 16741792 A JP16741792 A JP 16741792A JP H067180 A JPH067180 A JP H067180A
- Authority
- JP
- Japan
- Prior art keywords
- abscisic acid
- natural
- culture
- culture liquid
- natural abscisic
- 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.)
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【0001】[0001]
【産業上の利用分野】本発明は微生物の代謝機能を利用
した天然型アブシジン酸の製造方法に関する。FIELD OF THE INVENTION The present invention relates to a method for producing natural abscisic acid utilizing the metabolic function of microorganisms.
【0002】[0002]
【従来の技術】天然型アブシジン酸は1963年カリフ
ォルニア大学のアディコットなどにより、綿の幼果の落
下促進物質としてはじめて単離され、それ以降植物に対
する重要な生理作用が見出され、今日では植物ホルモン
の一種であることも確認されている。2. Description of the Related Art Natural abscisic acid was first isolated as a fall-promoting substance for cotton young fruit by Adikot of the University of California in 1963. Since then, an important physiological action on plants has been found. It is also confirmed to be a kind of.
【0003】天然型アブシジン酸の植物生理作用の特徴
は、他の植物ホルモンであるオーキシン、ジベレリン、
サイトカイニンなどと拮抗してそれらの作用を打消し、
植物の生育を抑制することにあるが、他に植物の気孔閉
塞作用などもある。将来、天然型アブシジン酸がこのよ
うな生理作用により、干ばつ、冷害などの気候変化に対
応できる物質として農業生産への応用が期待されてい
る。また、他には、果実の成熟促進、花芽形成のコント
ロールなどの生理作用があることが最近明らかにされつ
つあり、農業分野での利用が期待されている(バイオイ
ンダストリー 9巻2号5ページ(1992年))。さ
らには、ビール醸造における品質向上やコスト低下(特
開昭58−101677号公報)などへの利用など、食
品分野への利用などが進められている。The characteristic of the physiological action of natural abscisic acid is that other plant hormones such as auxin, gibberellin,
By competing with cytokinins, etc. to cancel their effects,
Although it is intended to suppress the growth of plants, it also has a stomata obstruction effect on plants. Due to such physiological effects, natural abscisic acid is expected to be applied to agricultural production in the future as a substance that can cope with climate change such as drought and cold damage. In addition, it has been recently revealed that there are other physiological actions such as promotion of fruit maturity and control of flower bud formation, and it is expected to be used in the agricultural field (Bioindustry Vol. 9, No. 2, page 5 ( 1992)). Furthermore, utilization in the food field, such as utilization for improving quality and reducing costs in beer brewing (Japanese Patent Laid-Open No. 58-101677), is being promoted.
【0004】従来、アブシジン酸の製造法としては、有
機合成法が検討されてきたが、この方法では光学活性の
天然型アブシジン酸を得ることはきわめて困難であり、
かつ、製造コストがきわめて高価であって、上記農業お
よび食品分野での利用は実質的に不可能であった。Conventionally, an organic synthesis method has been studied as a method for producing abscisic acid, but it is extremely difficult to obtain an optically active natural abscisic acid by this method.
In addition, the manufacturing cost is extremely high, and it is practically impossible to use it in the fields of agriculture and food.
【0005】近年では微生物の代謝機能を利用した発酵
培養による微生物法が有機合成にかわる有力な方法とし
て検討されるにいたり、セルコスポラ・ロシコラ(Cerco
spora ・rosicola) による製造法(特開昭58−363
93号公報、特開昭56−160996号公報)および
ボトリチス(Botrytis)菌による製造法(特公昭61−3
5838号公報)などが公知となっている。ボトリチス
菌については従来固体培養法での検討が行われていた
が、本発明者らの検討により、液体培養での生産も可能
となった(特開平2−60590号公報)。[0005] In recent years, the microbial method by fermentation culture utilizing the metabolic function of microorganisms has been studied as a powerful alternative to organic synthesis, and Cercospora rosicola (Cerco
spora rosicola) (JP-A-58-363)
93, JP-A-56-160996) and a production method using Botrytis bacteria (Japanese Patent Publication No. 61-3).
No. 5838) and the like are known. The Botrytis bacterium has been conventionally studied by a solid culture method, but the study by the present inventors has made it possible to produce it by liquid culture (JP-A-2-60590).
【0006】[0006]
【発明が解決しようとする課題】しかし、上記微生物法
の最大の欠点は、発酵生産による天然型アブシジン酸の
蓄積量がきわめて少量で、かつ発酵生産速度もきわめて
遅く、工業規模での生産手段としてはきわめて能率が悪
い点であった。However, the greatest drawback of the above-mentioned microbial method is that the amount of natural abscisic acid accumulated by fermentation is very small and the fermentation production rate is also very slow. Was a very inefficient point.
【0007】[0007]
【課題を解決するための手段】そこで本発明者らは、天
然型アブシジン酸を効率よく生産する方法に関し、鋭意
検討を進めた結果、不完全菌の一種であるボトリチス(B
otrytis)属に属し、天然型アブシジン酸生産能を有する
菌株を液体振とう培養するにあたり、培地中に発泡担体
を添加することにより、培地中に蓄積する天然型アブシ
ジン酸が著しく増加することを見出した。また、前記発
泡担体の大きさが1mm3 から150mm3の範囲であるこ
と、添加量が培養液1lあたり1.5から4gの範囲で
あること、または、前記発泡担体が主としてセルロース
からなることにより天然型アブシジン酸の培地中におけ
る生産量をさらに著しく増大させ得ることを見出した。[Means for Solving the Problems] Therefore, as a result of intensive studies on the method for efficiently producing natural abscisic acid, the present inventors have found that Botrytis (B
It was found that the addition of a foaming carrier to the medium significantly increases the amount of natural abscisic acid accumulated in the medium during liquid shaking culture of a strain belonging to the genus otrytis) and having a natural abscisic acid-producing ability. It was In addition, the size of the foam carrier is in the range of 1 mm 3 to 150 mm 3 , the addition amount is in the range of 1.5 to 4 g per liter of the culture solution, or the foam carrier is mainly composed of cellulose. It was found that the production amount of natural abscisic acid in the medium can be significantly increased.
【0008】すなわち、本発明は、ボトリチス(Botryti
s)属に属し、天然型アブシジン酸生産能を有する菌を、
発泡担体を含んだ培養液中で培養して天然型アブシジン
酸を生成蓄積せしめ、培養液から天然型アブシジン酸を
単離採取することを特徴とする天然型アブシジン酸の製
造方法であり、さらにあわせて前記発泡担体が主として
セルロースからなり、大きさが1mm3 から150mm3 の
範囲であり、添加量が培養液1lあたり1.5から4g
の範囲であることを特徴とする天然型アブシジン酸の製
造方法である。That is, the present invention relates to Botrytis.
s) a bacterium belonging to the genus and having a natural abscisic acid-producing ability,
A method for producing natural abscisic acid, which comprises culturing in a culture medium containing a foaming carrier to generate and accumulate natural abscisic acid, and isolating and collecting the natural abscisic acid from the culture medium. The foaming carrier is mainly composed of cellulose, and the size is in the range of 1 mm 3 to 150 mm 3 , and the addition amount is 1.5 to 4 g per liter of culture solution.
The method for producing natural abscisic acid is characterized in that
【0009】以下、本発明を詳述する。The present invention will be described in detail below.
【0010】本発明に使用される菌は、ボトリチス属に
属する天然型アブシジン酸生産菌であれば特に限定され
ず、通常の変異菌や変異処理によって生じた菌をも含む
ものである。The bacterium used in the present invention is not particularly limited as long as it is a natural abscisic acid-producing bacterium belonging to the genus Botrytis, and includes a normal mutated bacterium and a bacterium produced by a mutated treatment.
【0011】ボトリチス菌に属する天然型アブシジン酸
生産菌の菌株の具体例としてボトリチス・シネレア(Bot
rytis ・cinerea)FERM P−6156が挙げられ
る。As a specific example of a strain of a natural abscisic acid-producing bacterium belonging to Botrytis, Botrytis cinerea (Bot
rytis • cinerea) FERM P-6156.
【0012】次に、本発明において天然型アブシジン酸
生産に使用される培地としては液体培地が用いられる。
この液体培地には、フスマ、小麦、米、カンショ、バレ
イショ、麦芽糖、麦芽エキス、蔗糖、デキストリン、廃
糖密、澱粉などの炭素源、脱脂大豆粉、大豆粉、グルテ
ン、酵母エキス、ペプトン、肉エキス、コーンスティー
プリカー、硫酸アンモニウム、尿素、硝酸ナトリウムな
どの窒素源がそれぞれ単独で、または二種以上混合して
含有される。この他、たとえば、マグネシウム塩、カリ
ウム塩、ナトリウム塩、リン酸塩などの無機物質、さら
にはビタミン類、油脂類、その他を添加することができ
る。さらには、培養培地すなわち液体培地に柑橘類の熱
水抽出液を添加することが天然型アブシジン酸の生成量
を高めることに有効である。Next, a liquid medium is used as the medium used for the production of natural abscisic acid in the present invention.
This liquid medium includes bran, wheat, rice, sweet potato, potato, maltose, malt extract, sucrose, dextrin, waste sugar concentrate, carbon sources such as starch, defatted soybean powder, soybean powder, gluten, yeast extract, peptone, meat. Nitrogen sources such as extract, corn steep liquor, ammonium sulfate, urea and sodium nitrate are contained singly or as a mixture of two or more kinds. In addition to these, for example, inorganic substances such as magnesium salts, potassium salts, sodium salts, and phosphates, as well as vitamins, oils and fats, and the like can be added. Furthermore, adding a hot water extract of citrus to a culture medium, that is, a liquid medium is effective in increasing the production amount of natural abscisic acid.
【0013】本発明においては発泡担体を液体培地中に
添加し、培養することが重要である。In the present invention, it is important to add the foaming carrier to the liquid medium and culture it.
【0014】ここで発泡担体は、特に限定されずウレタ
ンフォームなどの他いかなるものも使用可能であるが、
特に主としてセルロースからなるものが好ましい。ま
た、発泡担体の大きさは1mm3 から150mm3 の範囲の
粒状物であることが好ましく、さらに好ましくは1〜5
mm角程度の立方体形状のものが用いられるが、球状など
他の形状でもよい。また、発泡担体の添加量は培養液1
lあたり1.5から4gの範囲であることが好ましく、
さらに好ましくは2.5〜3.5g程度が添加される。Here, the foam carrier is not particularly limited, and any other material such as urethane foam can be used.
In particular, those mainly composed of cellulose are preferable. The size of the foam carrier is preferably a granular material in the range of 1 mm 3 to 150 mm 3 , and more preferably 1 to 5
A cubic shape with a size of about mm square is used, but other shapes such as spherical shape may be used. The amount of the foaming carrier added is 1
It is preferably in the range of 1.5 to 4 g per liter,
More preferably, about 2.5 to 3.5 g is added.
【0015】液体培地に発泡担体を添加し、常法により
滅菌処理を実施し、実質的な無菌培地とした後、菌を接
種する。A foaming carrier is added to the liquid medium and sterilized by a conventional method to make a substantially sterile medium, and then inoculated with bacteria.
【0016】滅菌処理を行った培地にはボトリチス属に
属する天然型アブシジン酸生産菌株の菌糸または胞子ま
たは両者のいずれを植菌してもよい。The sterilized medium may be inoculated with either the hyphae or spores of a natural abscisic acid-producing strain belonging to the genus Botrytis or both.
【0017】培養条件は、培養温度が通常10から35
℃、好ましくは20から30℃、培地のpHが3から1
2、好ましくは4から8、培養期間が通常1から30
日、好ましくは5から15日程度であり、雑菌の混入を
排除した培養系を用い培養する。 さらに、本発明では
通気撹拌培養することが好ましい。静置培養においても
天然型アブシジン酸は生成するが通気撹拌培養によりそ
の生成は顕著に促進される。The culturing condition is that the culturing temperature is usually 10 to 35.
℃, preferably 20 to 30 ℃, pH of the medium is 3 to 1
2, preferably 4 to 8, culture period is usually 1 to 30
The culture is carried out for a day, preferably about 5 to 15 days, using a culture system free from contamination by various bacteria. Further, in the present invention, it is preferable to perform aeration stirring culture. Natural type abscisic acid is produced even in static culture, but its production is remarkably promoted by aeration and agitation culture.
【0018】次に培養終了後、培養液より天然型アブシ
ジン酸を単離採取するには通常の方法を採用することが
でき、たとえば、以下に述べる方法が用いられる。After completion of the culture, a conventional method can be employed for isolating and collecting natural abscisic acid from the culture medium, and for example, the method described below is used.
【0019】まず、培養液から遠心分離により菌体を除
去し、その上清から酢酸エチル、クロロホルムなどの有
機溶媒で天然型アブシジン酸を抽出し、活性炭、シリカ
ゲル、合成吸着樹脂などに吸着させた後、適当な有機溶
媒で脱着し、濃縮結晶化するなどの方法で単離精製で
き、通常の有機化合物の精製法を応用することで単離精
製が可能である。First, bacterial cells were removed from the culture solution by centrifugation, and natural abscisic acid was extracted from the supernatant with an organic solvent such as ethyl acetate or chloroform and adsorbed on activated carbon, silica gel, synthetic adsorption resin or the like. After that, it can be isolated and purified by a method such as desorption with an appropriate organic solvent and concentrated crystallization, and it can be isolated and purified by applying a general method for purifying an organic compound.
【0020】[0020]
【実施例】以下、実施例により本発明を具体的に示す。EXAMPLES The present invention will be specifically described below with reference to examples.
【0021】実施例1 (培地の調整)表1に示した液体培地100mlを500
ml容エーレンマイヤーフラスコに入れ、発泡担体として
表2に示したセルロース発泡体(東レファインケミカル
(株)製)を各3.5gずつ添加し、120℃15分蒸
気滅菌した。Example 1 (Preparation of medium) 100 ml of the liquid medium shown in Table 1 was added to 500
The mixture was placed in a ml-volume Erlenmeyer flask, 3.5 g of each of the cellulose foams (manufactured by Toray Fine Chemical Co., Ltd.) shown in Table 2 was added as a foaming carrier, and steam sterilization was performed at 120 ° C. for 15 minutes.
【0022】[0022]
【表1】 [Table 1]
【0023】(ボトリチス・シネレア種胞子の調製)表
2に示した培地にボトリシス・シネレア(FERM P
−6156)の保存用種菌を接種し、25℃で静置培養
した。この場合、接種後4日間は暗所におき、これに続
く4日間は360nmに極大値を有する波長光に照射させ
ながら培養し、さらにこれに続く1日間を再び暗所で静
置培養した。この培養菌体を滅菌した0.1%“Twe
en”80溶液に懸濁させ、濾過を行い、実質的にボト
リチス菌の胞子だけを含む水溶液を得た。(Preparation of Botrytis cinerea spores) Botrytis cinerea (FERM P) was added to the medium shown in Table 2.
-6156) was inoculated with the inoculum for storage, and statically cultured at 25 ° C. In this case, the cells were left in the dark for 4 days after the inoculation, cultivated while irradiating with the wavelength light having the maximum value at 360 nm for the following 4 days, and further cultivated in the dark for the following 1 day. The cultured cells were sterilized with 0.1% “Twe
It was suspended in an en "80 solution and filtered to obtain an aqueous solution containing substantially only Botrytis spores.
【0024】[0024]
【表2】 [Table 2]
【0025】(天然型アブシジン酸の生産)上記液体培
地に上記で得られたボトリチス・シネレア(FERM
P−6156)の胞子溶液を植菌し、25℃、振幅3c
m、172rpm で10日間通気撹拌培養した。(Production of Natural Type Abscisic Acid) Botrytis cinerea (FERM) obtained above in the above liquid medium
P-6156) inoculated with spore solution at 25 ° C, amplitude 3c
The cells were cultured with aeration and stirring at m, 172 rpm for 10 days.
【0026】培養終了後、遠心分離により菌体を除き、
上清液を高速液体クロマトグラフィー(カラム:資生堂
CAPCELL PAK C18 4.6x250mm、
展開溶媒:0.1Mリン酸/メタノール=1/1)で分
析し、天然型アブシジン酸を定量した。After the completion of the culture, the cells are removed by centrifugation,
The supernatant was subjected to high performance liquid chromatography (column: Shiseido CAPCELL PAK C18 4.6 × 250 mm,
The developing solvent: 0.1 M phosphoric acid / methanol = 1/1) was used for analysis to quantify natural abscisic acid.
【0027】結果を表3に示す。The results are shown in Table 3.
【0028】[0028]
【表3】 [Table 3]
【0029】実施例2 培地の調製・培養方法・種胞子の調製はすべて実施例1
と同様に行った。セルロース発泡体は3mm角立方体のも
のを、表4に示した量添加した。Example 2 Preparation of medium, culture method, and preparation of seed spores were all performed in Example 1.
I went the same way. Cellulose foam having a cubic shape of 3 mm square was added in the amount shown in Table 4.
【0030】結果を表4に示す。The results are shown in Table 4.
【0031】[0031]
【表4】 [Table 4]
【0032】実施例3 実施例1で示した培地に、3mm角立方体セルロース発泡
体を1g/l添加し、実施例1に示した方法で培養を行
い、得られた培養液から遠心分離により菌体を除き、そ
の上清液をアンバーライトXAD7樹脂を充填したカラ
ムに吸着させ、水洗後、50%メタノールで溶出した。
溶出液を濃縮し、pHを3に調節した後、酢酸エチルで
抽出し、無水硫酸ナトリウムで脱水し、濃縮液をシリカ
ゲルクロマトグラフィーにかけ、n−ヘキサン/酢酸エ
チル=3/1で留出した。精製された天然型アブシジン
酸区分を分別し、濃縮後純粋な天然型アブシジン酸結晶
80mgを得た。Example 3 To the medium shown in Example 1, 1 g / l of 3 mm square cubic cellulose foam was added, cultivated by the method shown in Example 1, and the obtained culture broth was centrifuged. The body was removed, and the supernatant was adsorbed on a column filled with Amberlite XAD7 resin, washed with water, and then eluted with 50% methanol.
The eluate was concentrated and the pH was adjusted to 3, then extracted with ethyl acetate, dehydrated with anhydrous sodium sulfate, and the concentrate was subjected to silica gel chromatography to distill off with n-hexane / ethyl acetate = 3/1. The purified natural abscisic acid fraction was separated and concentrated to obtain 80 mg of pure natural abscisic acid crystals.
【0033】得られた天然型アブシジン酸の結晶の旋光
度はThe optical rotation of the obtained crystals of natural abscisic acid is
【数1】 であった。[Equation 1] Met.
【0034】[0034]
【発明の効果】本発明によれば、微生物を利用した発酵
培養により、天然型アブシジン酸を高い蓄積量で得るこ
とができ、工業的規模での生産を可能にすることができ
る。EFFECTS OF THE INVENTION According to the present invention, natural abscisic acid can be obtained in a high accumulation amount by fermentation culture using microorganisms, and production on an industrial scale can be realized.
Claims (4)
型アブシジン酸生産能を有する菌を、発泡担体を含んだ
培養液中で培養して天然型アブシジン酸を生成蓄積せし
め、培養液から天然型アブシジン酸を単離採取すること
を特徴とする天然型アブシジン酸の製造方法。1. A bacterium belonging to the genus Botrytis and having a natural abscisic acid-producing ability is cultivated in a culture medium containing a foaming carrier to produce and accumulate natural abscisic acid. A method for producing natural abscisic acid, which comprises isolating and collecting abscisic acid.
3 の範囲である請求項1記載の天然型アブシジン酸の製
造方法。2. The size of the foam carrier is from 1 mm 3 to 150 mm
The method for producing natural abscisic acid according to claim 1, which is in the range of 3 .
1.5から4gの範囲である請求項1記載の天然型アブ
シジン酸の製造方法。3. The method for producing natural abscisic acid according to claim 1, wherein the amount of the foamed carrier added is in the range of 1.5 to 4 g per liter of the culture solution.
請求項1記載の天然型アブシジン酸の製造方法。4. The method for producing natural abscisic acid according to claim 1, wherein the foam carrier is mainly composed of cellulose.
Priority Applications (1)
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JP16741792A JPH067180A (en) | 1992-06-25 | 1992-06-25 | Production of natural-type abscisic acid |
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JP16741792A JPH067180A (en) | 1992-06-25 | 1992-06-25 | Production of natural-type abscisic acid |
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JPH067180A true JPH067180A (en) | 1994-01-18 |
Family
ID=15849316
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN100427606C (en) * | 2005-10-08 | 2008-10-22 | 中国科学院成都生物研究所 | Method for preparing 14C or 3H marked natural active abscisic acid |
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- 1992-06-25 JP JP16741792A patent/JPH067180A/en active Pending
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN100427606C (en) * | 2005-10-08 | 2008-10-22 | 中国科学院成都生物研究所 | Method for preparing 14C or 3H marked natural active abscisic acid |
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