JPS59144799A - Purification of coenzyme a - Google Patents

Abstract

PURPOSE:The purification of coenzyme A with industrial advantage is effected by adsorption of coenzyme A on a nonionic exchanging porous resin in an acidic aqueous solution of a specific pH followed by elution with water or a solvent of a specific pH. CONSTITUTION:Coenzyme A adsorbed on a nonionic exchanging porous resin in an aqueous solution of less than 2pH is eluted with water or an aqueous organic solvent of 3 to 7pH. EFFECT:The process of the present invention enables almost complete and rapid removal of impurities such as pigments or nucleic acids by use of a smaller amount of resin. Desalting process is unnecessary and no decomposition takes place during the concentration of coenzyme A.

Description

【発明の詳細な説明】 本発明はコエンザイムＡ（以下ＣｏＡと略記する）の精
製法に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for purifying coenzyme A (hereinafter abbreviated as CoA).

ＣｏＡは１９４５年Ｌｉｐｍａｒｕ１らが７　セｆ　／
ｌ／基転移因子の検索過程において、その研究の端を発
し。CoA was published in 1945 by Lipmaru1 et al.
The research began in the process of searching for l/transposition elements.

すでに四半世紀が過ぎた歴史的富貴な有機化合物である
。It is a historically rich organic compound that has already been around for a quarter of a century.

ＣｏＡは生体にとって重要な物質であり２例えば、糖お
よび脂質代謝、ステロイドおよびカロチノイドの生合成
などに重要な役割を果していることは周知の通りであり
、その多彩な生理作用により代謝障害改善剤等の医薬品
として期待される化合物である。CoA is an important substance for living organisms, and it is well known that it plays an important role in, for example, sugar and lipid metabolism, and the biosynthesis of steroids and carotenoids. Due to its various physiological actions, it is used as an agent for improving metabolic disorders, etc. It is a compound that is expected to be used as a drug.

ＣｏＡは微生物体内や動物組織内（二微量存在するが、
工業的には通常微生物の醗酵により製造される。CoA exists in microorganisms and animal tissues (although it exists in trace amounts,
Industrially, it is usually produced by microbial fermentation.

醗酵濾液又は菌体から純粋なＣｏＡを分離精製するため
には種々の不純物を除去する必要がある。In order to separate and purify pure CoA from fermentation filtrate or bacterial cells, it is necessary to remove various impurities.

従来、　　ＣｏＡの精製法としては金属イオン等による
沈殿法、有機溶媒による沈殿法、活性炭による吸脱着法
、アンバーライトＩＲＡ−４０１、ＤＥＡＥセファデッ
クス、ＱＡＥセファデックス等によるアニオン交換樹脂
法、アフィニティークロマトグラライによる方法、さら
には非イオン交換樹脂例えば、アンバーライ）　ＸＡＤ
−２、ダイヤイオン１（Ｐ−２０等による分配型クロマ
トグラフィーによる方法等が知られている。Conventionally, CoA purification methods include precipitation using metal ions, precipitation using organic solvents, adsorption/desorption using activated carbon, anion exchange resin using Amberlite IRA-401, DEAE Sephadex, QAE Sephadex, etc., and affinity chromatography. method, and even non-ion exchange resins such as Amberly) XAD
-2, a method using partition chromatography using Diaion 1 (P-20, etc.) is known.

しかしながら、これら従来の方法は一般に多量の有機溶
媒を必要としたり、多量のクロマト用担体を必要とした
り、また溶出液に塩類等を用いるので脱塩濃縮が必要と
なり、さらには。However, these conventional methods generally require a large amount of organic solvent, a large amount of chromatographic carrier, and use salts in the eluent, which requires desalting and concentration.

用いた樹脂量に対する溶出液量が厖大となり。The amount of eluate compared to the amount of resin used was enormous.

不安定なＣｏＡを取扱うには数々の問題を残している。Many problems remain in dealing with unstable CoA.

非イオン交換性の多孔性樹脂を利用するＣｏＡの精製法
に関しては１本発明者の一人小林時夫等の発明した方法
（特開昭５６−２５１．９９号）が知られているが、こ
の方法は明細書に［粗製ＣｏＡをｐＨ２〜７．好ましく
はｐＨ３〜５の水溶液とし。As for a CoA purification method using a non-ion exchange porous resin, a method invented by Tokio Kobayashi et al. The method is described in the specification [crude CoA at pH 2-7. Preferably, it is an aqueous solution with a pH of 3 to 5.

樹脂１２当り粗製ＣｏＡ約１０り以下の比率でカラムに
入れ、これをＦ■３〜５の水で比速度０３以下で展開溶
出する」旨記載されているように。The column is charged with a ratio of about 10 parts or less of crude CoA per 12 parts of resin, and the column is developed and eluted with F1 water of 3 to 5 at a specific velocity of 03 or less.'

分配型のクロマトグラフィ〜に関するものである。This relates to partition-type chromatography.

これに対し２本発明者等は、　　ＣｏＡが化学構造」二
、３個のリン酸エステル基、末端チオール基及びアデニ
ン部分の６位にアミノ基を有することに着目し、これら
解離基の解離を出来るだけおさえ、　　ＣｏＡ分子全体
の疎水性を高めることによって、樹脂への吸着量を増加
させる方法を検討した結果、ｐＨ２以下の酸性水溶液中
に於いて。In contrast, the present inventors focused on the fact that CoA has a chemical structure of two or three phosphate ester groups, a terminal thiol group, and an amino group at the 6-position of the adenine moiety, and investigated the dissociation of these dissociative groups. As a result of investigating a method of increasing the amount of CoA adsorbed to the resin by increasing the hydrophobicity of the entire CoA molecule as much as possible, we found that in an acidic aqueous solution with a pH of 2 or less.

ＣｏＡの樹脂に対する吸着量が著しく増加し、主要不純
物の核酸類や色素が殆んど吸着しないことを見出した。It was found that the amount of CoA adsorbed on the resin increased significantly, and that the main impurities, nucleic acids and pigments, were hardly adsorbed.

即ち本発明は、「コエンザイムＡをｐｆ（２以下の酸性
水溶液中で非イオン交換性の多孔性樹脂に吸着させ、吸
着したコエンザイムＡをメーＩ３乃至中性の水又は含水
有機溶媒で溶出することを特徴とするコエンザイムＡの
精製方法」に関するものである。That is, the present invention is directed to "adsorbing coenzyme A to a non-ion exchange porous resin in an acidic aqueous solution with a pf of 2 or less, and eluting the adsorbed coenzyme A with water or a water-containing organic solvent ranging from MeI3 to neutral water. A method for purifying coenzyme A characterized by the following.

本発明方法はブレビバクテリウム・アンモニアゲネスＩ
ＡＭ１６４１（微工研菌寄第１４７１号）等のＣｏＡ産
生微生物の醗酵濾液や粗製ＣｏＡを精製する場合に、従
来法（特開昭５６−２５１９９号）に比較して、樹脂使
用量が約吉以下２通液スピードが約１０倍の一回操作で
色素やアデノシンモノホスフェート（以下ＡＭＰという
）、アデノシンジホスフェート（以下ＡＤＰという）、
アデノシントリホスフェート（以下ＡＴＰという）等の
核酸類不純物をほぼ完全に除去し得るという格別優れた
特徴を有するが、一方で色素や核酸類以外の不純物例え
ば酸化型ＣｏＡ　、デポスホＣｏＡ等の分離性が劣るた
め一回の操作でただちに高純度品を得るための方法とし
ては適当でない。The method of the present invention uses Brevibacterium ammoniagenes I
When purifying the fermentation filtrate or crude CoA of CoA-producing microorganisms such as AM1641 (Feikoken Bacterial Serial No. 1471), the amount of resin used is approximately The following 2 liquid passage speeds are approximately 10 times faster in a single operation: dyes, adenosine monophosphate (hereinafter referred to as AMP), adenosine diphosphate (hereinafter referred to as ADP),
It has an exceptional feature of being able to almost completely remove nucleic acid impurities such as adenosine triphosphate (hereinafter referred to as ATP), but on the other hand, it is difficult to separate impurities other than dyes and nucleic acids such as oxidized CoA and depo-CoA. Because of its inferiority, it is not suitable as a method for immediately obtaining a high-purity product in a single operation.

高純度品は通常予備精製された粗製ＣｏＡを出発原料と
してイオン交換クロマト法又はゲル濾過法により精製す
ることにより製造されている。High-purity products are usually produced by using prepurified crude CoA as a starting material and purifying it by ion exchange chromatography or gel filtration.

本発明は、醗酵濾液より粗製ＣｏＡを得るための予備精
製法又は粗製ＣｏＡより色素及び核酸類を除去するため
の精製法として工業的に極めて優れた方法を提供するも
のである。The present invention provides an industrially excellent method as a preliminary purification method for obtaining crude CoA from a fermentation filtrate or as a purification method for removing pigments and nucleic acids from crude CoA.

本発明を好適に実施するためには、非イオン交換性の多
孔性樹脂をカラムに充填し、ｐＨ２以下好ましくは１．
８の酸性水（希塩酸などで調整）を通塔して充分平衡化
し、これに同−ｐＨの醗酵濾液又は粗製ＣｏＡ水溶液を
比速度（ＳＶ）３．５程度の一定速度で通液する。通液
後間−声の酸性水で洗浄した後、ｐＨ３乃至中性の水又
は含水有機溶媒で吸着したＣｏＡを溶出する。In order to suitably carry out the present invention, a column is filled with a non-ion exchange porous resin, and the pH is 2 or less, preferably 1.
8 acidic water (adjusted with dilute hydrochloric acid, etc.) is passed through the column to achieve sufficient equilibration, and the fermentation filtrate or crude CoA aqueous solution having the same pH is passed through the column at a constant specific velocity (SV) of about 3.5. After passing through the solution, it is washed with acidic water, and then the adsorbed CoA is eluted with water having a pH of 3 to neutral or a water-containing organic solvent.

本発明によれば、１２の樹脂で約５０ｍｇまでのＣｏＡ
を処理することが出来る。According to the present invention, up to about 50 mg of CoA in 12 resins
can be processed.

醗酵濾液を処理する場合は、共存する酸化型ＣｏＡは粗
製ＣｏＡ中に混合物として採取される。When processing the fermentation filtrate, the coexisting oxidized CoA is collected as a mixture in the crude CoA.

粗製ＣｏＡを処理する場合は、あらかじめ酸化型ＣｏＡ
を還元しておくのが望ましい。When processing crude CoA, oxidized CoA must be prepared in advance.
It is desirable to return the

本発明によれば、溶出液として微酸性の水又は含水有機
溶媒を用いるため、脱塩工程など繁雑な操作が不要であ
り、溶出液を低温濃縮もしくは凍結乾燥で効率良く濃縮
できるため、　　ＣＯＡ濃縮工程での分解は殆んど起こ
らない。According to the present invention, since slightly acidic water or a water-containing organic solvent is used as the eluent, there is no need for complicated operations such as a desalting step, and the eluate can be efficiently concentrated by low temperature concentration or freeze drying. Almost no decomposition occurs during the process.

使用後の非イオン交換性の多孔性樹脂はアセトン水、メ
タノール水などの含水有機溶媒で簡単に再生出来、繰返
し再利用出来る。After use, the non-ion exchange porous resin can be easily regenerated with a water-containing organic solvent such as acetone water or methanol water, and can be reused repeatedly.

次に１本発明の詳細な説明するため、試験例を示す。Next, in order to explain the present invention in detail, test examples will be shown.

試験例　　ＣｏＡ及び核酸類のＨＰ−２０樹脂に対する
吸着性に及ぼすｐＨの影響 （１）　　試験方法 ＦＯＰ−２０樹脂７００〜（２，５ｍ／）を蒸留水に分
散させ、これを希塩酸で所定ｐｉ（（１，５，２，０，
３，０，５，０及び６０）に平衡化されるよう調整し、
併わせて液量を５−に調整した。Test Example Effect of pH on Adsorption of CoA and Nucleic Acids to HP-20 Resin (1) Test Method FOP-20 resin 700 ~ (2.5 m/) is dispersed in distilled water, and diluted with dilute hydrochloric acid to a specified pi ( (1, 5, 2, 0,
3,0,5,0 and 60),
At the same time, the liquid volume was adjusted to 5-.

各ｐＨに平衡化された樹脂分散液に、それぞれ同−田の
蒸留水で濃度１０％に溶解したＡＭＰ。AMP was dissolved at a concentration of 10% in each pH-equilibrated resin dispersion with distilled water from the same field.

ＡＤＰ、ＡＴＰ及びＣｏＡの溶液０．５−を添加し、１
０分間激しく振盪し、１時間静置後上澄みの未吸着成分
を定量し、各成分のそれぞれのｐＨに於ける吸着率を求
めた。Add 0.5- solution of ADP, ATP and CoA, 1
After shaking vigorously for 0 minutes and standing still for 1 hour, the amount of unadsorbed components in the supernatant was determined, and the adsorption rate of each component at each pH was determined.

（２）試験結果 本試験の結果は第１表に示す通りであり、ＨＰ−２０樹
脂に対し、　ｐＨ２以下の水溶液中でＣｏＡは非常に高
い吸着性を示すが、ｐＨが中性に近付くにつれて溶出が
起ることが認められた。(2) Test results The results of this test are shown in Table 1. CoA shows very high adsorption to HP-20 resin in an aqueous solution with a pH of 2 or less, but as the pH approaches neutrality, It was observed that elution occurred.

これに対し、核酸類のＡＴＰとＡＤＰは全く吸着せず、
Ａ、ＭＰは若干吸着するが　ＣｏＡに比較しく７） 次に本発明の実施態様を具体的に説明するたてその吸着
率はｐ）１１．５の場合２０／９４．　　ｐＨ２，０の
場合１１／７６と著しく低い値を示した。On the other hand, ATP and ADP of nucleic acids do not adsorb at all,
A, MP is slightly adsorbed, but compared to CoA7) Next, the embodiment of the present invention will be specifically explained.The adsorption rate is 20/94 for p)11.5. In the case of pH 2.0, it showed a significantly low value of 11/76.

第１表 備考 ■　ＣｏＡは高速液体クロマトグラフィーにより定量し
た。Table 1 Notes ■ CoA was determined by high performance liquid chromatography.

カラム：ウォーターズ社製マイクロボンダパック０１８ 移動相：０．００５Ｍ硫酸テトラ−ｎ−ブチルアンモニ
ウムを含む０．０７５Ｍ！Ｊン酸緩衝液とアセトニトリ
ルの混液（４：１） ■　核酸類は２６０ｎｍの吸光度により定量した。Column: Microbondapak 018 manufactured by Waters Mobile phase: 0.075M containing 0.005M tetra-n-butylammonium sulfate! Mixture of J acid buffer and acetonitrile (4:1) (4) Nucleic acids were quantified by absorbance at 260 nm.

（８） 氷晶は、還元型ＣｏＡとして純度３０チ、工程間実施例
１゜ ブレビバクテリウム・アンモニャゲネスＴ、Ａ、Ｍ−１
６４１株の醗酵濾液２（１０ｍｌ　（ＣｏＡ　１．３ｍ
９／−を含む）を３規定塩酸水でｐＨ１，８に調整して
おく。(8) The ice crystals had a purity of 30% as reduced CoA, and interprocess example 1° Brevibacterium ammoniagenes T, A, M-1.
Fermentation filtrate 2 of 641 strains (10ml (CoA 1.3m
9/-) to pH 1.8 with 3N hydrochloric acid.

一方、あらかじめ準備したダイヤイオント（Ｐ−２０樹
脂５０−をガラス製カラム（直径１．８　ｃｍ、高さ４
９ｍ）へ蒸留水で充填し、その後ｐＨ１，８の塩酸水で
充分平衡化しておく。先の酸性に調整した醗酵濾液を５
Ｖ＝３．５で通液後、　ｐＨ２，０の塩酸水１５０−で
充分洗滌して、３００ｆｎ１．の５０ｑ６メタノール水
で溶出した。On the other hand, the previously prepared diamond iont (P-20 resin 50-) was placed in a glass column (diameter 1.8 cm, height 4 cm).
9m) with distilled water, and then sufficiently equilibrated with hydrochloric acid water of pH 1.8. The fermentation filtrate adjusted to acidity was
After passing through the solution at V=3.5, it was thoroughly washed with 150-hydrochloric acid solution having a pH of 2.0, and was heated to 300 fn1. It was eluted with 50q6 methanol water.

ＣｏＡを含む各溶出フラクションを薄層クロマトグラフ
ィーおよび高速液体クロマトグラフィーで検出し、主分
画１６０コを集め、１規定の苛性ソーダでｐｉ（３，０
に調整後、４０℃以下でメタノールを回収し、凍結乾燥
すると８２３りの粗物質が得られた。Each eluted fraction containing CoA was detected by thin layer chromatography and high performance liquid chromatography, and 160 main fractions were collected and diluted with 1N caustic soda at pi (3,0
After adjusting the methanol to 40° C. or below, methanol was recovered and freeze-dried to obtain 823 crude materials.

実施例２゜ 粗物質１．０３　Ｆ　（還元型ＣｏＡ　３９．３　％を
含有）を蒸留水２−に溶解後、１規定の苛性ソーダでβ
（７，０に調整後、チオグリセロール０３−を加えて、
窒素ガスを封入して室温で１夜放置して還元を行った。Example 2 After dissolving 1.03 F of crude material (containing 39.3% reduced CoA) in distilled water, β was dissolved with 1N caustic soda.
(After adjusting to 7.0, add thioglycerol 03-,
The mixture was sealed with nitrogen gas and left overnight at room temperature to carry out reduction.

還元終了後５℃に冷却したアセトン２０−を加え生じた
黄白色沈殿物を冷却遠心分離して１？（純度３９３％）
の還元型ＣｏＡを得た。上記粗物質を蒸留水５０ｔｎｔ
に溶解後、１規定塩酸水でｐＨ１，８に調整しておく。After the reduction was completed, 20°C of acetone was cooled to 5°C, and the resulting yellow-white precipitate was cooled and centrifuged. (purity 393%)
Reduced CoA was obtained. The above crude substance was mixed with 50 tons of distilled water.
After dissolving in water, adjust the pH to 1.8 with 1N hydrochloric acid.

一方、あらかじめ準備したダイヤイオンＨＰ−２０樹脂
５０−をガラス製カラム（直径１．８ｃｒｎ＋　高さ３
８０）へ水で充填後、　ｐＨ１，８の塩酸水で充分平衡
化しておく。On the other hand, a glass column (diameter 1.8 crn + height 3
After filling the tank (80) with water, equilibrate it thoroughly with hydrochloric acid water with a pH of 1.8.

先のＣｏＡを含む酸性溶液を流速５Ｖ＝３．５で通液後
、　ｐＨ２，０の塩酸水１００−で充分洗滌して蒸留水
４００−で溶出した。After passing the above-mentioned acidic solution containing CoA at a flow rate of 5V=3.5, it was thoroughly washed with 100% hydrochloric acid solution having a pH of 2.0, and eluted with 400% distilled water.

ＣｏＡを含む各溶出フラクションを薄層クロマトグラフ
ィーおよび高速液体クロマトグラフィーで検出し、主分
画３６０−を集め、１規定の苛性ソーダでｐＨ３，０に
調整後、凍結乾燥すると４００〜の淡黄白色の粉末が得
られた。Each eluted fraction containing CoA was detected by thin layer chromatography and high performance liquid chromatography, and the main fraction 360- was collected, adjusted to pH 3.0 with 1N caustic soda, and then lyophilized to give a pale yellowish white fraction with a pH of 400~. A powder was obtained.

水晶は、還元型ＣｏＡとして純度６５．０％、工程間収
率６６．２チであった。The crystal had a purity of 65.0% as reduced CoA and an inter-process yield of 66.2%.

特許出願人 わかもと製薬株式会社 ９４４−patent applicant Wakamoto Pharmaceutical Co., Ltd. 944-

Claims (1)

【特許請求の範囲】[Claims] コエンザイムＡをｐＨ２以下の酸性水溶液中で非イオン
交換性の多孔性樹脂に吸着させ、吸着したコエンザイム
ＡをｐＨ３乃至中性の水又は含水有機溶媒で溶出するこ
とを特徴とするコエンザイムＡの精製方法。A method for purifying coenzyme A, which comprises adsorbing coenzyme A on a non-ion exchange porous resin in an acidic aqueous solution with a pH of 2 or less, and eluting the adsorbed coenzyme A with water or a water-containing organic solvent with a pH of 3 or neutral. .