JP2001258583A - Method for purifying shikimic acid - Google Patents
Method for purifying shikimic acidInfo
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- JP2001258583A JP2001258583A JP2000073721A JP2000073721A JP2001258583A JP 2001258583 A JP2001258583 A JP 2001258583A JP 2000073721 A JP2000073721 A JP 2000073721A JP 2000073721 A JP2000073721 A JP 2000073721A JP 2001258583 A JP2001258583 A JP 2001258583A
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- shikimic acid
- solution
- acid
- exchange resin
- anion exchange
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/42—Hydroxy-carboxylic acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
- C07C51/47—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
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- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明はシキミ酸（shikimic
acid）のイオン交換樹脂による精製方法に関する。シ
キミ酸は、医薬品の合成原料等として、有用である。TECHNICAL FIELD The present invention relates to shikimic acid (shikimic acid).
acid) using an ion exchange resin. Shikimic acid is useful as a raw material for synthesis of pharmaceuticals and the like.
【０００２】[0002]
【従来の技術】従来、シキミ酸は合成法で製造されお
り、発酵法によるシキミ酸の製造は報告例が少なく、ま
た、純度の高い、精製されたシキミ酸の製造法に関する
例もない。また、従来のイオン交換樹脂では化学構造の
類似した物質同志の分離は困難な場合が多い。2. Description of the Related Art Conventionally, shikimic acid has been produced by a synthetic method, and there have been few reports on the production of shikimic acid by a fermentation method, and there is no example relating to a method of producing purified shikimic acid having high purity. Further, in the conventional ion exchange resin, it is often difficult to separate substances having similar chemical structures.
【０００３】シキミ酸の発酵液を強塩基性陰イオン交換
樹脂にて処理する報告が、１９６０年にSimonartとWiau
xにより報告されている(Nature, 186, 78-79, 1960)。
発酵液中のシキミ酸と他の類似化合物（発酵副生物）の
分析を目的として、著者らはシキミ酸発酵液を強陰イオ
ン交換樹脂に吸着させ、0.05N 酢酸・0.05N 酢酸ナトリ
ウム混合液で溶離しているが、発酵副生物であるデヒド
ロシキミ酸、5-ホスホ−シキミ酸との分離は達成されて
いない。[0003] In 1960, Simonart and Wiau reported that a fermentation broth of shikimic acid was treated with a strongly basic anion exchange resin.
x (Nature, 186, 78-79, 1960).
For the purpose of analyzing shikimic acid and other similar compounds (by-products of fermentation) in fermentation broth, the authors adsorb shikimic acid fermentation broth on strong anion exchange resin and use 0.05N acetic acid / 0.05N sodium acetate mixed solution. Although eluted, separation from the fermentation by-products dehydroshikimic acid and 5-phospho-shikimic acid has not been achieved.
【０００４】[0004]
【発明が解決しようとする課題】シキミ酸に化学構造の
類似したデヒドロシキミ酸及びその他の類縁化合物とシ
キミ酸との効率の良い分離を行うことができる、シキミ
酸の精製方法を提供する。SUMMARY OF THE INVENTION The present invention provides a method for purifying shikimic acid capable of efficiently separating shikimic acid from dehydroshikimic acid and other analogous compounds having a chemical structure similar to that of shikimic acid.
【０００５】[0005]
【課題を解決するための手段】本研究者らは、シキミ酸
含有液からの効率の良いシキミ酸の精製法を開発すべく
鋭意検討した結果、アルカリ処理後、陰イオン交換樹脂
にシキミ酸含有液を吸着し、溶離剤で溶離することによ
り、効率的に不純物を除去できるシキミ酸精製方法を見
出し本発明に至った。すなわち、本願発明は、シキミ酸
含有液をアルカリ処理し、次いで陰イオン交換樹脂にシ
キミ酸を吸着後、溶離することを特徴とするシキミ酸の
精製方法に関する。陰イオン交換樹脂としては、強塩基
性樹脂が好ましく、溶離剤としては、強アルカリ性水溶
液が好ましく、特にＮａＯＨ水溶液が好ましい。また、
シキミ酸含有液のアルカリ処理工程と陰イオン交換樹脂
吸着工程をOH型陰イオン交換樹脂を用いることにより、
一段階で行うことも可能である。また、シキミ酸含有液
としては、シキミ酸を製造する能力を有する微生物を培
養して得られるシキミ酸発酵液または該発酵液から微生
物菌体を除いた水溶液が好ましい。The present inventors have conducted intensive studies to develop an efficient method for purifying shikimic acid from a liquid containing shikimic acid. As a result, after alkali treatment, the anion exchange resin contained shikimic acid. The present inventors have found a method for purifying shikimic acid that can efficiently remove impurities by adsorbing a liquid and eluting the liquid with an eluent. That is, the present invention relates to a method for purifying shikimic acid, comprising subjecting a shikimic acid-containing liquid to an alkali treatment, and then adsorbing and eluting shikimic acid to an anion exchange resin. As the anion exchange resin, a strongly basic resin is preferable, and as the eluent, a strongly alkaline aqueous solution is preferable, and particularly, an NaOH aqueous solution is preferable. Also,
By using an OH type anion exchange resin for the alkali treatment step and the anion exchange resin adsorption step of the shikimic acid-containing liquid,
It is also possible to do it in one step. Further, as the shikimic acid-containing liquid, a shikimic acid fermentation liquid obtained by culturing a microorganism capable of producing shikimic acid or an aqueous solution obtained by removing microbial cells from the fermentation liquid is preferable.
【０００６】[0006]
【発明の実施の形態】本発明において、使用されるシキ
ミ酸含有液とは、合成法（特開平１１？２１２６７参
照）及び発酵法により製造されるシキミ酸及び副生物を
含む液である。発酵液を遠心分離やMF・UF膜で処理し、
微生物を除いた除菌液、更に活性炭等で脱色した液や工
程中の処理液などシキミ酸を含有する液等でも使用可能
である。BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, the shikimic acid-containing liquid used is a liquid containing shikimic acid and by-products produced by a synthesis method (see JP-A-11-21267) and a fermentation method. The fermentation broth is processed by centrifugation or MF / UF membrane,
A solution containing shikimic acid, such as a bacteria-removing solution from which microorganisms have been removed, a solution decolorized with activated carbon, or a treatment solution during the process, can also be used.
【０００７】シキミ酸発酵液は、グルコースなどの炭素
源から、シキミ酸キナーゼ活性を欠損したバチルス属細
菌、具体的にはバチルス・ズブチリス１−１１８株（Yu
ki,S., Japan J. Genetics, 50 (2), 155-157 (197
5)）、及び同株から誘導されたバチルス・ズブチリスＩ
Ｄ３株（後述参考例１参照）を用いて、通常の培地、培
養方法で発酵することにより得られる。[0007] The shikimate fermentation liquor is prepared from a bacterium belonging to the genus Bacillus deficient in shikimate kinase activity from a carbon source such as glucose, specifically Bacillus subtilis strain 1-118 (Yu
ki, S., Japan J. Genetics, 50 (2), 155-157 (197
5)), and Bacillus subtilis I derived from the same strain
It is obtained by fermentation using a D3 strain (see Reference Example 1 below) in a usual medium and culture method.
【０００８】これらの合成反応液、発酵液、工程中の処
理液などのシキミ酸を含む液は、樹脂塔閉塞の原因とな
る固形物類を除去しておくのが好ましい。It is preferable to remove solids that cause blockage of the resin tower from a solution containing shikimic acid, such as a synthesis reaction solution, a fermentation solution, and a treatment solution during the process.
【０００９】アルカリ処理は、シキミ酸濃度1〜10 g/
L、pH11〜13好ましくは12〜13、温度10〜90℃好ましく
は20〜40℃で5〜30分の条件で行う。発酵副生物であ
り、類縁化合物であるデヒドロシキミ酸のアルカリ性条
件下での安定性を図１に示す。この図からもわかるよう
に、アルカリ性条件において、シキミ酸は安定であるの
に対し、デヒドロシキミ酸はすみやかに分解する。この
結果に基づき、シキミ酸とデヒドロシキミ酸の分離には
アルカリ処理が有効であることを見出した。In the alkali treatment, the concentration of shikimic acid is 1 to 10 g /
L, pH 11-13, preferably 12-13, temperature 10-90 ° C, preferably 20-40 ° C, for 5-30 minutes. FIG. 1 shows the stability of dehydroshikimic acid, a fermentation by-product and an analogous compound, under alkaline conditions. As can be seen from this figure, under alkaline conditions, shikimic acid is stable, whereas dehydroshikimic acid decomposes immediately. Based on this result, it was found that an alkali treatment was effective for separating shikimic acid and dehydroshikimic acid.
【００１０】本発明方法において用いるイオン交換樹脂
としてはダイヤイオンR (Diaion PA-412)などの陰イオ
ン交換樹脂であればいずれでも良く、強塩基性樹脂が特
に好ましい。樹脂の使用量は、被処理液中のカチオン
量、即ちシキミ酸の他に液中に含まれている夾雑物アミ
ノ酸、有機酸、塩化物イオンなどの総モル当量が樹脂の
総交換容量以内となるようにすれば良い。The ion exchange resin used in the method of the present invention may be any anion exchange resin such as Diaion R (Diaion PA-412), and a strong basic resin is particularly preferred. The amount of resin used is such that the amount of cations in the liquid to be treated, that is, the total molar equivalents of contaminant amino acids, organic acids, chloride ions, etc. contained in the solution in addition to shikimic acid is within the total exchange capacity of the resin. What should be done.
【００１１】本発明における、アルカリ処理を行ったシ
キミ酸を含む発酵液、除菌液、脱色液、その他シキミ酸
含有液を樹脂処理する場合の好ましい態様は以下のとお
りである。In the present invention, preferred embodiments in the case of treating a fermented solution, a disinfecting solution, a decolorizing solution, and other solutions containing shikimic acid containing shikimic acid subjected to alkali treatment with a resin are as follows.
【００１２】アルカリ処理を行ったシキミ酸含有液を陰
イオン交換樹脂へ通液する。通液時の被処理液のpHは中
性からアルカリ性で温度90℃以下、SV＝1〜5好ましくは
SV=1〜3でシキミ酸を吸着する。吸着後、純水を1〜5RV
通液し、糖など樹脂吸着しない不純物を除去する。The shikimic acid-containing liquid having been subjected to the alkali treatment is passed through an anion exchange resin. The pH of the liquid to be treated during the passage is from neutral to alkaline at a temperature of 90 ° C. or lower, and SV = 1 to 5, preferably
Adsorb shikimic acid with SV = 1 ~ 3. After adsorption, 1-5 RV of pure water
The solution is passed through to remove impurities such as sugar that do not adsorb to the resin.
【００１３】次に、シキミ酸の溶離には、酸性水溶液あ
るいは強アルカリ性水溶液を溶離剤として用いることが
可能である。好ましい溶離剤としては、0.4〜1.0 Nの塩
酸水溶液、1〜2 Nの酢酸水溶液、0.2〜1.0 Nの苛性ソー
ダ溶液などがあげられる。これらの溶離剤をSV=1〜5好
ましくはSV=1〜3で通液し、シキミ酸を溶離することが
望ましい。しかし、デヒドロシキミ酸以外の分解物もし
くは類縁化合物との分離を行う場合は、溶離剤として酢
酸及び強アルカリ性水溶液を用いることが好ましく、と
くにNaOH、KOH水溶液が好ましい。図２に各溶離液での
シキミ酸画分のHPLC分析チャートを示す。（分析法は、
実施例１に後述する。）Next, for the elution of shikimic acid, an acidic aqueous solution or a strongly alkaline aqueous solution can be used as an eluent. Preferred eluents include a 0.4 to 1.0 N aqueous hydrochloric acid solution, a 1 to 2 N aqueous acetic acid solution, and a 0.2 to 1.0 N caustic soda solution. It is desirable to pass these eluents at SV = 1 to 5, preferably SV = 1 to 3, to elute shikimic acid. However, when separating from decomposed products or related compounds other than dehydroshikimic acid, it is preferable to use acetic acid and a strongly alkaline aqueous solution as an eluent, and particularly preferable to be an aqueous solution of NaOH or KOH. FIG. 2 shows an HPLC analysis chart of the shikimic acid fraction in each eluent. (The analysis method is
Example 1 will be described later. )
【００１４】NaOHやKOH等の強アルカリ性水溶液で溶離
した場合、陽イオンを除去するために、塩酸などでH型
に調整した強酸性カチオン交換樹脂などに通液し脱塩を
行う方が良い。シキミ酸含有貫流液を減圧濃縮後、冷却
晶析を行うことによりシキミ酸結晶を得ることができ
る。その他、通常の晶析方法が適用可能である。When eluting with a strong alkaline aqueous solution such as NaOH or KOH, desalting is preferably performed by passing the solution through a strongly acidic cation exchange resin adjusted to H-form with hydrochloric acid or the like in order to remove cations. The shikimic acid crystal can be obtained by concentrating the shikimic acid-containing flow-through liquid under reduced pressure and then performing cooling crystallization. In addition, a usual crystallization method can be applied.
【００１５】一方、酢酸水溶液で溶離した場合、そのま
ま溶離液を減圧濃縮し、冷却晶析を行うことにより、シ
キミ酸結晶を得ることができる。On the other hand, when elution is carried out with an aqueous acetic acid solution, the eluate is directly concentrated under reduced pressure, and then cooled and crystallized, whereby shikimic acid crystals can be obtained.
【００１６】また、上記のアルカリ処理と陰イオン交換
樹脂吸着は、苛性ソーダなどでOH型に再生した樹脂へ吸
着することにより同時に行うことができる。The alkali treatment and the adsorption of the anion exchange resin can be performed simultaneously by adsorbing the resin regenerated to the OH type with caustic soda.
【００１７】即ち、NaOH等の強アルカリ性水溶液を用い
てOH型に調整した陰イオン交換樹脂に、シキミ酸濃度1
〜10 g/Lになるように希釈したシキミ酸含有液を通液す
る。この通液により、アルカリ処理と樹脂吸着を同時に
行うことが可能である。通液後、純水を1〜3 RV通液し
た後、前述と同じく、酸性水溶液あるいは強アルカリ性
水溶液を溶離剤として用いて、シキミ酸を溶離する。溶
離後の処理は、前述の通りである。That is, a shikimic acid concentration of 1 is added to an anion exchange resin adjusted to OH type using a strong alkaline aqueous solution such as NaOH.
Pass the shikimic acid-containing solution diluted to 1010 g / L. By this liquid passing, alkali treatment and resin adsorption can be performed simultaneously. After passing through the solution, pure water is passed through 1-3 RV, and shikimic acid is eluted using an acidic aqueous solution or a strongly alkaline aqueous solution as an eluent as described above. Processing after elution is as described above.
【００１８】以下、参考例により本発明にて使用できる
発酵液の調製法を具体的に説明する。 ＜参考例＞ （１） 菌株 シキミ酸生産菌として、バチルス・ズブチリスＩＤ３株
（ａｒｏＩ１１６，ａｍｙ４，ａｍｙ：：Ｐｒ
（ｒｐｍＡ）−ａｒｏＤ（＋）−ｐｈｅＡ）を用いた。
バチルス・ズブチリスＩＤ３株は、1999年3月1日に、ル
シアン・ナショナル・コレクション・オブ・インダスト
リアル・マイクロオーガニズムス（VKPM）・デポジタリ
ー GNIIgenetika（Russian National Collection of In
dustrial Microorganisms（VKPM）Depositary, GNIIgen
etika）（住所：1, Dorozhny Proezd., 1,113545, Mosc
ow, Russia）に、登録番号VKPM B-7755のもとに寄託さ
れている。Hereinafter, a method for preparing a fermentation broth usable in the present invention will be described in detail with reference to Reference Examples. Reference Example (1) Bacterial strain As a shikimic acid-producing bacterium, Bacillus subtilis ID3 strain (aroI116, amy4, amy :: Pr) was used.
(RpmA) -aroD (+)- pheA) was used.
Three Bacillus subtilis ID shares were acquired on March 1, 1999 by the Lucian National Collection of Industrial Microorganisms (VKPM) Depositary GNIIgenetika (Russian National Collection of In
dustrial Microorganisms (VKPM) Depositary, GNIIgen
etika) (Address: 1, Dorozhny Proezd., 1,113545, Mosc
ow, Russia) under the registration number VKPM B-7755.
【００１９】（２） 種培養 上記菌株は、１０ｍｇ／Ｌのエリスロマイシンを含むＬ
Ｂ培地寒天のプレートまたは斜面上で３７℃２４時間生
育させた。次に、種培地として使用した７００ｍｌ容量
のフラスコ中のＬＢ培地３０ｍｌ中で菌体をインキュベ
ートした。種培養は３７℃、６〜７時間ロータリーシェ
ーカーでインキュベートした。(2) Seed culture The above strain is L-type containing 10 mg / L erythromycin.
The medium was grown at 37 ° C. for 24 hours on a plate or a slope of medium B agar. Next, cells were incubated in 30 ml of LB medium in a 700 ml flask used as a seed medium. Seed cultures were incubated on a rotary shaker at 37 ° C. for 6-7 hours.
【００２０】（３）ジャーファーメンターでの培養 上記菌株の培養は、「マルビシ」研究用ファーメンター
（Ｖ〜１−１．２Ｌ）中で実施された。酵母エキスに含
まれているため、下記培地には芳香族アミノ酸（フェニ
ルアラニン、トリプトファン、およびチロシン）を加え
なかった。(3) Cultivation in a jar fermenter The culture of the above strain was carried out in a fermenter (V-1-1. 2 L) for "Malvisi" research. Aromatic amino acids (phenylalanine, tryptophan, and tyrosine) were not added to the following medium because they were contained in the yeast extract.
【００２１】初期培養培地の構成（ｉｎ ｇ／Ｌ）： グルコース ：１００（１Ｌにつき〜１５０ｇが
フィード溶液として加えられる） （ＮＨ４）２ＳＯ４ ：２ ＮＨ４Ｃｌ ：３ ＫＨ２ＰＯ４ ：３ ＭｇＳＯ４ ：０．４ ＦｅＳＯ４ ：０．０２ 酵母エキス ：１５ （「シグマ」Ｙ４０００） エリスロマイシン ：１０ｍｇ／Ｌ ｐＨ７．０ ファーメンター中の培地の初期容量：５００ｍｌ 接種サイズ：６％（Ｅｍ １０ｍｇ／Ｌを含むＬＢ培地
３０ｍｌ中の６時間培養） フィード溶液（２００〜２５０ｍｌ）は濃度７００ｇ／
Ｌのグルコースを含有する。Composition of initial culture medium (in g / L): Glucose: 100 (-150 g per liter is added as a feed solution) (NH 4 ) 2 SO 4 : 2 NH 4 Cl: 3 KH 2 PO 4 : 3 MgSO 4 : 0.4 FeSO 4 : 0.02 Yeast extract: 15 (“Sigma” Y4000) Erythromycin: 10 mg / L pH 7.0 Initial volume of medium in fermenter: 500 ml Inoculation size: 6% (Em 10 mg / L) For 6 hours in 30 ml of LB medium containing). The feed solution (200 to 250 ml) has a concentration of 700 g /
Contains L glucose.
【００２２】供給率： 培養初期から１９時間まで：３〜３．２ｍｌ／Ｌ・時間 １９時間後：４．５ｍｌ／Ｌ・時間 温度：３７℃、 アジテーション：１０００〜１１００r.p.m.、 エアー：０．６Ｌ／分Feed rate: From the initial stage of culture to 19 hours: 3 to 3.2 ml / L · hour After 19 hours: 4.5 ml / L · hour Temperature: 37 ° C., Agitation: 1000 to 1100 rpm, Air: 0. 6L / min
【００２３】[0023]
【実施例】以下、実施例により本発明を具体的に説明す
る。 ＜実施例１＞参考例に従って調整されたシキミ酸発酵液
(シキミ酸13 g/L)を遠心分離(5000G)により菌体を分離
した除菌液100mlに活性炭(和光純薬）5gを加え、インペ
ラーで撹拌(200 rpm)しながら、60℃で１時間脱色を行
った。ろ紙(5C, Advantech Toyo)により活性炭を除去
し、脱色液を得た。強塩基性陰イオン交換樹脂(Diaion
PA-412) 50 mlをガラスカラムに詰め、2N NaOHを用い
てOH型に再生した。脱色液を純水で希釈した溶液(5.1 g
/L, 100 ml)をOH型に再生した強塩基性陰イオン交換樹
脂に通液し、シキミ酸を吸着した。純水100 mlを通液し
たあと、0.2N NaOH100 ml、0.4N NaOH 200 ml で順次溶
離し、シキミ酸を含む画分を取得した(シキミ酸0.50 m
g)。この画分からH型に調整した強酸性陽イオン交換樹
脂(Diaion SK1B)を用いて、ナトリウムイオンを除去し
た後、濃縮し、シキミ酸濃度270g/Lの濃縮液 (1.7ml)
を得た。得られた濃縮液を４℃で静置晶析を行い、シキ
ミ酸結晶(0.12 g、純度99%)を取得した。The present invention will be described below in detail with reference to examples. <Example 1> Shikimic acid fermented liquid prepared according to Reference Example
5 g of activated carbon (Wako Pure Chemical Industries, Ltd.) was added to 100 ml of the sterilized solution obtained by separating cells by centrifugation (5000 g) of (shikimic acid 13 g / L), and the mixture was stirred at 60 ° C. for 1 hour with an impeller (200 rpm). Decolorization was performed. The activated carbon was removed with a filter paper (5C, Advantech Toyo) to obtain a decolorized solution. Strongly basic anion exchange resin (Diaion
PA-412) 50 ml was packed in a glass column, and regenerated to the OH form using 2N NaOH. Decolorized solution diluted with pure water (5.1 g
/ L, 100 ml) was passed through a strongly basic anion exchange resin regenerated to the OH form to adsorb shikimic acid. After passing through 100 ml of pure water, elution was carried out sequentially with 100 ml of 0.2N NaOH and 200 ml of 0.4N NaOH to obtain a fraction containing shikimic acid (0.55 m
g). Using a strongly acidic cation exchange resin (Diaion SK1B) adjusted to H-type from this fraction, after removing sodium ions, concentrated, and concentrated with a shikimic acid concentration of 270 g / L (1.7 ml)
I got The resulting concentrated solution was subjected to static crystallization at 4 ° C. to obtain shikimic acid crystals (0.12 g, purity 99%).
【００２４】尚、シキミ酸の分析は、ODSカラム(Inerts
il ODS-3 4x250 mm, GLサイエンス製)をカラムオーブン
にて15℃に温度を維持し、移動層(0.05 % 酢酸、0.11 %
トリエチルアミン)を流速0.5 ml/minで通液して、UV224
nmで検出した。The analysis of shikimic acid was carried out using an ODS column (Inerts
il ODS-3 4x250 mm, manufactured by GL Science) in a column oven at 15 ° C, and the moving bed (0.05% acetic acid, 0.11%
Triethylamine) at a flow rate of 0.5 ml / min.
Detected in nm.
【００２５】＜実施例２＞上記実施例１にて、調整した
脱色液に苛性ソーダを加え、シキミ酸5g/L及び0.2N NaO
Hとなるように純水で希釈し、30℃、30分間震盪しアル
カリ処理を行った。その溶液100 mlを強塩基性陰イオン
交換樹脂(Diaion PA-412)に通液し、シキミ酸を吸着さ
せ、実施例１の方法に従い、溶離・脱塩・濃縮・晶析を
実施した(純度99%)。<Example 2> In Example 1, caustic soda was added to the prepared decolorizing solution, and 5 g / L of shikimic acid and 0.2 N NaO
The mixture was diluted with pure water so as to obtain H, and shaken at 30 ° C. for 30 minutes to perform an alkali treatment. 100 ml of the solution was passed through a strongly basic anion exchange resin (Diaion PA-412) to adsorb shikimic acid, and elution, desalting, concentration and crystallization were carried out according to the method of Example 1. 99%).
【００２６】＜実施例３＞上記実施例１にて、調整した
脱色液をシキミ酸の濃度が5 g/Lになるように希釈し、
その溶液50 mlをOH型に再生した強塩基性陰イオン交換
樹脂(Diaion PA-412)に通液し、シキミ酸を吸着させ
た。100 mlの純水で洗浄し、0.5 M 酢酸 100 ml、1.0 N
酢酸 100 ml で順次溶離し、シキミ酸を含む画分を取
得した。得られた画分を実施例１の方法に従い、濃縮・
晶析を実施したが、脱塩は行わなかった（純度98%）。<Example 3> The decolorized solution prepared in Example 1 was diluted so that the concentration of shikimic acid was 5 g / L.
50 ml of the solution was passed through a strongly basic anion exchange resin (Diaion PA-412) regenerated to the OH form to adsorb shikimic acid. Wash with 100 ml of pure water, 100 ml of 0.5 M acetic acid, 1.0 N
Elution was performed sequentially with 100 ml of acetic acid to obtain a fraction containing shikimic acid. According to the method of Example 1, the obtained fraction was concentrated and concentrated.
Crystallization was performed but no desalting was performed (purity 98%).
【００２７】＜実施例４＞上記実施例１をスケールアッ
プして調整した脱色液をシキミ酸の濃度が5.0 g/Lにな
るように希釈し、その溶液10 LをOH型に再生した強塩基
性陰イオン交換樹脂(Diaion PA-412) 5Lに通液し、シキ
ミ酸を吸着させた。純水10 Lを通液したあと、0.2N NaO
H 10L、0.4N NaOH 20Lで順次溶離し、シキミ酸を含む画
分を取得した(シキミ酸49 g)。上記実施例1に従い、H型
に調整した強酸性陽イオン交換樹脂(Diaion SK1B) 5Lを
用いて、ナトリウムイオンを除去した後、シキミ酸濃度
37%まで濃縮した。濃縮液を300 mlの晶析槽に移し、イ
ンペラ−により撹拌しながら(200 rpm) 、濃縮液を40℃
から10℃まで１時間当たり5℃づつ徐々に冷却し、10℃
で50時間晶析を行った（4.3g, 98%）。Example 4 A decolorizing solution prepared by scaling up the above Example 1 was diluted so that the concentration of shikimic acid was 5.0 g / L, and 10 L of the solution was regenerated into an OH type strong base. The solution was passed through 5 L of an anion exchange resin (Diaion PA-412) to adsorb shikimic acid. After passing 10 L of pure water, 0.2N NaO
Elution was carried out sequentially with 10 L of H and 20 L of 0.4N NaOH to obtain a fraction containing shikimic acid (49 g of shikimic acid). According to Example 1 above, using a strongly acidic cation exchange resin (Diaion SK1B) 5L adjusted to H-type, after removing sodium ions, shikimic acid concentration
Concentrated to 37%. The concentrate was transferred to a 300 ml crystallization tank, and the concentrate was stirred at 200 rpm with an impeller (200 rpm).
To 10 ° C, gradually cool at 5 ° C per hour to 10 ° C
For 50 hours (4.3 g, 98%).
【００２８】[0028]
【発明の効果】本発明の精製法により、シキミ酸含有液
から構造類似のデヒドロシキミ酸及びその他副生物、類
縁化合物を除去することが可能となり、純度の高いシキ
ミ酸を製造できる。According to the purification method of the present invention, it is possible to remove dehydroshikimic acid and other by-products and analogous compounds having a similar structure from a shikimic acid-containing solution, and it is possible to produce shikimic acid with high purity.
【図１】 シキミ酸及びデヒドロシキミ酸のアルカリ条
件における安定性を示す図である。FIG. 1 is a diagram showing the stability of shikimic acid and dehydroshikimic acid under alkaline conditions.
【図２】 各種溶離剤で得られたシキミ酸溶離液のHPLC
分析結果を示す図である。[Figure 2] HPLC of shikimic acid eluate obtained with various eluents
It is a figure showing an analysis result.
フロントページの続き (51)Int.Cl.7 識別記号 ＦＩ テーマコート゛(参考） //(Ｃ１２Ｐ 7/42 (Ｃ１２Ｐ 7/42 Ｃ１２Ｒ 1:125） Ｃ１２Ｒ 1:125） Continued on the front page (51) Int.Cl. 7 Identification symbol FI Theme coat ゛ (Reference) // (C12P 7/42 (C12P 7/42 C12R 1: 125) C12R 1: 125)
Claims (4)
で陰イオン交換樹脂にシキミ酸を吸着後、溶離すること
を特徴とするシキミ酸の精製方法。1. A method for purifying shikimic acid, comprising treating a shikimic acid-containing liquid with an alkali, and then adsorbing and eluting shikimic acid on an anion exchange resin.
イオン交換樹脂吸着工程をOH型陰イオン交換樹脂を用い
ることにより、一段階で行うことを特徴とする特許請求
の範囲第1項記載の方法。2. The method according to claim 1, wherein the alkali treatment step of the shikimic acid-containing liquid and the anion exchange resin adsorption step are carried out in one step by using an OH type anion exchange resin. Method.
をＮａＯＨ水溶液で溶離することを特徴とする特許請求
の範囲第１項記載の方法。3. The method according to claim 1, wherein the shikimic acid adsorbed on the anion exchange resin is eluted with an aqueous NaOH solution.
力を有する微生物を培養して得られるシキミ酸発酵液ま
たは該発酵液から微生物菌体を除いた水溶液であること
を特徴とする特許請求の範囲第１項記載の方法。4. The shikimic acid-containing solution is a shikimic acid fermentation solution obtained by culturing a microorganism capable of producing shikimic acid or an aqueous solution obtained by removing microbial cells from the fermentation solution. 2. The method according to claim 1, wherein
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2000073721A JP2001258583A (en) | 2000-03-16 | 2000-03-16 | Method for purifying shikimic acid |
| PCT/JP2001/002086 WO2001068891A1 (en) | 2000-03-16 | 2001-03-16 | Method of purifying shikimic acid |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2000073721A JP2001258583A (en) | 2000-03-16 | 2000-03-16 | Method for purifying shikimic acid |
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| WO (1) | WO2001068891A1 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2012188374A (en) * | 2011-03-09 | 2012-10-04 | Sophia School Corp | Method for acquiring shikimic acid, and process for producing shikimic acid |
| JP2015096076A (en) * | 2009-12-29 | 2015-05-21 | ローム アンド ハース カンパニーＲｏｈｍ Ａｎｄ Ｈａａｓ Ｃｏｍｐａｎｙ | Process for separation of organic acids and amino acids from fermentation broths |
| CN111072468A (en) * | 2019-12-25 | 2020-04-28 | 东莞市东阳光生物合成药有限公司 | Method for extracting shikimic acid and shikimic acid extract |
| CN111087296A (en) * | 2019-12-20 | 2020-05-01 | 东莞市东阳光生物合成药有限公司 | Method for extracting shikimic acid and shikimic acid extract |
| CN111675611A (en) * | 2020-06-28 | 2020-09-18 | 湖南杰萃生物技术有限公司 | Method for extracting shikimic acid from ginkgo leaves |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011110927A1 (en) * | 2010-03-12 | 2011-09-15 | Council Of Scientific & Industrial Research | A method for the extraction of shikimic acid |
| MY160083A (en) | 2012-11-26 | 2017-02-15 | Sime Darby Malaysia Berhad | A method for isolating shikimic acid from oil palm waste |
| CN109721487B (en) * | 2019-01-15 | 2021-11-30 | 浙江海正药业股份有限公司 | Process for efficiently purifying shikimic acid by using continuous ion exchange technology |
| WO2021193565A1 (en) * | 2020-03-27 | 2021-09-30 | 住友ベークライト株式会社 | Production method for cyclic compound or derivative thereof |
| CN114213241B (en) * | 2021-12-28 | 2023-09-01 | 浙江来益生物技术有限公司 | Method for extracting shikimic acid from shikimic acid fermentation liquor |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001026567A (en) * | 1999-05-07 | 2001-01-30 | Toray Ind Inc | Purification of shikimic acid |
-
2000
- 2000-03-16 JP JP2000073721A patent/JP2001258583A/en active Pending
-
2001
- 2001-03-16 WO PCT/JP2001/002086 patent/WO2001068891A1/en active Application Filing
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2015096076A (en) * | 2009-12-29 | 2015-05-21 | ローム アンド ハース カンパニーＲｏｈｍ Ａｎｄ Ｈａａｓ Ｃｏｍｐａｎｙ | Process for separation of organic acids and amino acids from fermentation broths |
| JP2012188374A (en) * | 2011-03-09 | 2012-10-04 | Sophia School Corp | Method for acquiring shikimic acid, and process for producing shikimic acid |
| CN111087296A (en) * | 2019-12-20 | 2020-05-01 | 东莞市东阳光生物合成药有限公司 | Method for extracting shikimic acid and shikimic acid extract |
| CN111072468A (en) * | 2019-12-25 | 2020-04-28 | 东莞市东阳光生物合成药有限公司 | Method for extracting shikimic acid and shikimic acid extract |
| CN111675611A (en) * | 2020-06-28 | 2020-09-18 | 湖南杰萃生物技术有限公司 | Method for extracting shikimic acid from ginkgo leaves |
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| Publication number | Publication date |
|---|---|
| WO2001068891A1 (en) | 2001-09-20 |
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