JP2008148620A - Cmp−デアミノノイラミン酸の製造法 - Google Patents
Cmp−デアミノノイラミン酸の製造法 Download PDFInfo
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- JP2008148620A JP2008148620A JP2006339373A JP2006339373A JP2008148620A JP 2008148620 A JP2008148620 A JP 2008148620A JP 2006339373 A JP2006339373 A JP 2006339373A JP 2006339373 A JP2006339373 A JP 2006339373A JP 2008148620 A JP2008148620 A JP 2008148620A
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- Prior art keywords
- cmp
- kdn
- acid
- reaction
- enzyme
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Abstract
【解決手段】シチジン5’−トリリン酸(5’−CTP)とデアミノノイラミン酸(KDN)から酵素的にCMP−デアミノノイラミン酸(CMP−KDN)を製造する方法において、酵素としてCMP−N−アセチルノイラミン酸合成酵素(CMP−NeuAcシンセターゼ)を使用する、CMP−KDNの製造法を提供する。
また、工程1〜4よりなるCMP−KDNの精製法を提供する。
(工程1)CMP−KDN含有液に2価カチオンを添加し、共存するリン酸、ピロリン酸、ヌクレオチドを沈殿させる工程、(工程2)アルカリホスファターゼ反応によりヌクレオチドをヌクレオシドに変換する工程、(工程3)有機溶媒によりCMP−KDNを沈殿させる工程、及び(工程4)CMP−KDNを回収する工程。
【選択図】なし
Description
〔2〕KDNとして、N−アセチルノイラミン酸リアーゼを用いてマンノース及びピルビン酸から調製したもの、あるいはその反応液を使用する、〔1〕記載の方法。
〔3〕工程1〜4よりなるCMP−KDNの精製法。
工程1:CMP−KDN含有液に2価カチオンを添加し、共存するリン酸、ピロリン酸、ヌクレオチドを沈殿させる工程、
工程2:アルカリホスファターゼ反応によりヌクレオチドをヌクレオシドに変換する工程、
工程3:有機溶媒によりCMP−KDNを沈殿させる工程、及び
工程4:CMP−KDNを回収する工程
〔4〕工程4の前後に、イオン交換樹脂を用いて、CMP−KDNの塩の交換を行う、〔1〕記載の精製法。
本発明で使用するCMP−NeuAc合成酵素とは、5’−CTPとNeuAcを基質としてCMP−NeuAcを合成する反応を触媒する活性を有するものを意味し、微生物由来のCMP−NeuAc合成酵素は、上記反応以外にも5’−CTPとKDNを基質としてCMP−KDNを生成する触媒活性も併せ持つものである。
CMP−KDN合成反応に使用するKDNとしては、(1)ニジマス卵、精巣などの天然物より抽出、取得したもの(Anal.Biochem.,202,25-34(1992))、(2)ピルビン酸とマンノースからNeuAcリアーゼの触媒反応により可逆的に合成したもの(J.Am.Chem.Soc.,110,6481-6486(1988)、Tetrahedran,46,201-214(1990))、(3)ホスホエノールピルビン酸とマンノースからNeuAc−9リン酸シンセターゼの触媒反応によりKDN−9リン酸を不可逆的に合成した後、ホスファターゼによりKDNにしたもの(J.Biol.Chem.,275,17869-17877(2000))、あるいは微生物由来NeuAcシンセターゼの触媒反応により不可逆的に合成したものなどを利用することが可能であり、これらを各種クロマトグラフィーにより精製、単離したもの、あるいはKDN抽出液、あるいは合成反応液をそのまま使用することができる。
このようにして得られたCMP−KDN合成液は、その不安定さから高純度のものの取得が非常に困難であったが、2価カチオンを添加し、共存するリン酸、ピロリン酸、ヌクレオチドを沈殿させ(工程1)、アルカリホスファターゼ反応によりヌクレオチドをヌクレオシドに変換した後(工程2)、有機溶媒によりCMP−KDNを沈殿させ(工程3)、必要によりイオン交換カラムにより塩置換を行った後、CMP−KDNを回収する(工程4)ことで、高純度のCMP−KDNを簡単な操作により回収することができる。以下、各工程毎に説明する。
工程1は、CMP−KDN含有液に2価カチオンを添加し、共存するリン酸、ピロリン酸、ヌクレオチドを沈殿させる工程である。
工程2は、CMP−KDN含有液にホスファターゼを添加し、共存するヌクレオチドをヌクレオシドに変換する工程である。
工程3は、有機溶媒を添加し、CMP−KDNを沈殿させる工程である。
工程4は、沈殿したCMP−KDNを回収する工程である。
上記工程4終了前後、CMP−KDNはカルシウム塩、マンガン塩等の塩となっているため、必要に応じて、例えばナトリウム塩等に変換することも可能である。
このようにして得られたCMP−KDNはHPLCよる純度が95%以上で、5’−CMP等の夾雑物が極めて少ない高純度の製品である。
(1)KDN合成
400mMマンノース、400mMピルビン酸溶液(25ml)に2ユニット/ml反応液のNeuAcリアーゼを添加し、室温で反応を行った。63時間後、100℃、5分間の熱処理を行った後、HPPAE−PAD法でKDN生成量を測定したところ、270.3mMのKDNの生成を確認した。
20mM 5’−CTP、20mM KDN(上記(1)で合成した反応液13.8ml相当)、50mM塩化マグネシウムを含有する溶液(200ml)に1.75ユニット/ml反応液当たりのCMP−NeuAc合成酵素を添加し、40℃で反応を行った。反応中pHの低下を抑えるために水酸化ナトリウム溶液を適宜添加した。反応開始45分後、一部を分取し、HPLC分析により確認したところ、15.1mMのCMP−KDNの生成を確認した。対KDN及び対5’−CTPモル収率はそれぞれ75.5%と非常に高い反応率であった。
上記CMP−KDN合成反応、開始55分後、80mM相当量の塩化カルシウム溶液を添加することで反応を停止させ、続けて40ユニットのアルカリホスファターゼを添加し、40℃で反応を行った。反応途中、pHの低下を抑えるために水酸化ナトリウム溶液を適宜添加した。反応開始60分後に氷上に移すことで反応を終了させた。
Claims (4)
- シチジン5’−トリリン酸(5’−CTP)とデアミノノイラミン酸(KDN)から酵素的にCMP−デアミノノイラミン酸(CMP−KDN)を製造する方法において、酵素としてCMP−N−アセチルノイラミン酸合成酵素(CMP−NeuAcシンセターゼ)を使用する、CMP−KDNの製造法。
- KDNとして、N−アセチルノイラミン酸リアーゼを用いてマンノース及びピルビン酸から調製したもの、あるいはその反応液を使用する、請求項1記載の方法。
- 工程1〜4よりなるCMP−KDNの精製法。
工程1:CMP−KDN含有液に2価カチオンを添加し、共存するリン酸、ピロリン酸、ヌクレオチドを沈殿させる工程、
工程2:アルカリホスファターゼ反応によりヌクレオチドをヌクレオシドに変換する工程、
工程3:有機溶媒によりCMP−KDNを沈殿させる工程、及び
工程4:CMP−KDNを回収する工程 - 工程4の前後に、イオン交換樹脂を用いて、CMP−KDNの塩の交換を行う、請求項3記載の精製法。
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JPH06141880A (ja) * | 1992-11-05 | 1994-05-24 | Snow Brand Milk Prod Co Ltd | Cmp−kdnの合成方法 |
JP2003093091A (ja) * | 2001-07-19 | 2003-04-02 | Yamasa Shoyu Co Ltd | Cmp−n−アセチルノイラミン酸の製造法 |
WO2005030974A1 (ja) * | 2003-09-26 | 2005-04-07 | Yamasa Corporation | Cmp−n−アセチルノイラミン酸の製造法 |
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JPH06141880A (ja) * | 1992-11-05 | 1994-05-24 | Snow Brand Milk Prod Co Ltd | Cmp−kdnの合成方法 |
JP2003093091A (ja) * | 2001-07-19 | 2003-04-02 | Yamasa Shoyu Co Ltd | Cmp−n−アセチルノイラミン酸の製造法 |
WO2005030974A1 (ja) * | 2003-09-26 | 2005-04-07 | Yamasa Corporation | Cmp−n−アセチルノイラミン酸の製造法 |
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