JP2005041784A - Method for purifying sugar phosphate - Google Patents
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【０００１】
【発明の属する技術分野】
本発明は、グルコース−１−リン酸（以下「Ｇ−１−Ｐ」と略称する）等の糖リン酸エステル又はその塩の低コストな精製法に関する。
【０００２】
【従来の技術】
Ｇ−１−Ｐは、抗菌剤、抗腫瘍剤、心疾患治療薬等の医薬品や糖合成の基質として有用であり、更に近年では、体内への優れたカルシウム吸収作用に着目して、骨粗鬆症予防の新規食品素材としても精力的に研究が行われている。
【０００３】
Ｇ−１−Ｐの製造は、糖類とリン酸を基質として、これにイオン交換樹脂に固定化したマルトデキストリンフォスフォリラーゼ（ＭＤＰａｓｅ）を作用させる固定化酵素法（例えば特許文献１参照）など、主として酵素法によって行われている。また、Ｇ−１−Ｐは発酵法により製造することも可能であり、コリネバクテリウム属細菌を、糖及びリン酸又はその塩を含有する培地中で培養する発酵法によるＧ−１−Ｐの製造が提案されている（特許文献２参照）。
【０００４】
酵素法による反応終了液は、Ｇ−１−Ｐ以外に、未反応物である糖類及びリン酸（塩）を含んでいる。そのため、反応終了液を限外濾過後、透析法又は電気透析法によりリン酸又はその塩を除去し（特許文献３及び４参照）、続いてアセトン、エタノール等の水溶性の溶剤を用いた晶析により糖類を除去し、Ｇ−１−Ｐを精製している。しかしながら、Ｇ−１−Ｐの晶析には多量の溶剤が必要であり、高コストとなる。また水溶性の溶剤を再利用するためには共沸蒸留プロセスや浸透気化法（パーベーパレーション）等の特別な装置を用いた工程が必要となり、設備的な負荷が高くなるという問題がある。更には、発酵法による反応終了液には、上記糖類及びリン酸（塩）以外に、タンパク質、ペプチド、色素等が残存しており、これらも除去する必要があるが、溶剤晶析では除去することができない。また、仮に発酵法の反応液を、限外濾過、透析法又は電気透析法、活性炭処理等に付した後、更に溶剤晶析法で精製したとしても、発酵由来の香気成分等の低沸点成分が溶剤に濃縮されてくるため、溶剤を再利用することはできない。
【０００５】
【特許文献１】
特開平１−６３５８９号公報
【特許文献２】
特開２００２−３００８９９号公報
【特許文献３】
特開昭６１−１２９１８９号公報
【特許文献４】
特公平６−９６５８５号公報
【０００６】
【発明が解決しようとする課題】
そこで、本発明は、酵素法、発酵法の双方の反応終了液に適用でき、しかも低コストで、リン酸又はその塩、糖類等の不純物を除去し、Ｇ−１−Ｐ等の糖リン酸エステル又はその塩を精製できる方法を提供することを目的とする。
【０００７】
【課題を解決するための手段】
本発明者らは、発酵法又は酵素法による糖リン酸エステル製造の反応終了液に、１価イオン非選択性陰イオン交換膜を用いた電気透析を施せば、糖リン酸エステルイオンとリン酸のイオンは当該膜を通過するが、糖類、タンパク質等は通過しないため、糖類（及びタンパク質等）を低コストで分離することが可能であること、更に、これに１価イオン選択性陰イオン交換膜を用いた電気透析法による脱リン工程を組み合わせれば、糖類及びリン酸（塩）、更には発酵法による反応終了液に含まれるタンパク質（ペプチド）も除去することができ、低コストでＧ−１−Ｐの精製が可能となることを見出した。
【０００８】
すなわち本発明は、第一に、糖リン酸エステル又はその塩及び糖類を含有する混合水溶液を、１価イオン非選択性陰イオン交換膜を用いた電気透析に付すことにより、糖類を除去する糖リン酸エステル又はその塩の精製法を提供するものである。
【０００９】
更に本発明は、第二に、糖リン酸エステル又はその塩、糖類及びリン酸又はその塩を含有する混合水溶液を、１価イオン非選択性陰イオン交換膜を用いた電気透析、及び１価イオン選択性陰イオン交換膜を用いた電気透析に付すことにより、糖類及びリン酸又はその塩を除去する糖リン酸エステル又はその塩の精製法を提供するものである。
【００１０】
【発明の実施の形態】
本発明の第一の精製法において使用する、「糖リン酸エステル又はその塩及び糖類を含有する混合水溶液」としては、発酵法による反応終了液、酵素法による反応終了液のいずれでもよく、更にはこれらを電気透析等に付すことによりリン酸又はその塩を除去したものでもよい。また、本発明の第二の精製法において使用する、「糖リン酸エステル又はその塩、糖類及びリン酸又はその塩を含有する混合水溶液」としては、発酵法による反応終了液、酵素法による反応終了液のいずれでもよい。
【００１１】
本発明で精製の対象とする糖リン酸エステル又はその塩としては、ヘキソース、ペントース等の単糖類若しくは二糖類等の多糖類のリン酸エステル、又はそれらのナトリウム塩、カリウム塩等のアルカリ金属塩が挙げられる。より具体的には、Ｇ−１−Ｐ、グルコース−６−リン酸、マンノース−６−リン酸、ガラクトース−６−リン酸、フルクトース−６−リン酸、グルコース−１，６−二リン酸、フルクトース−１，６−二リン酸、又はそれらのナトリウム塩、カリウム塩等のアルカリ金属塩が挙げられ、本発明は特にＧ−１−Ｐの精製に好適である。
【００１２】
また、前記混合水溶液に含まれる糖類としては、未反応物としてのヘキソース、ペントース等の単糖類及び二糖類等の多糖類、例えばグルコース、マルトース、マルトトリオース、マルトテトラオース、マルトペンタオース、マンノース、ガラクトース、フルクトース等が挙げられる。更に前記混合水溶液に含まれるリン酸又はその塩としては、オルトリン酸のほか、ピロリン酸等の縮合リン酸、又はそれらのナトリウム塩、カリウム塩等のアルカリ金属塩が挙げられる。
【００１３】
本発明では、前記混合水溶液を一段階又は相異なる二段階の電気透析に付すことにより、糖リン酸エステルを精製するものであるが、電気透析に先立ち、限外ろ過等のろ過を施すことが好ましく、また、混合水溶液として発酵法による反応終了液を用いる場合は、発酵菌体分離のため、精密濾過処理を施すことが好ましい。また発酵液の種類によっては、電気透析脱糖工程での糖リン酸エステルの透過性を悪くする夾雑物質が存在する場合がある。そのような発酵液の場合には、上記ろ過に加え、更に、活性炭による処理を施すことが好ましい。
【００１４】
糖類除去のための電気透析（脱糖工程）では、１価イオン非選択性陰イオン交換膜を使用する。１価イオン非選択性陰イオン交換膜とは、１価陰イオンの選択性のない陰イオン交換膜をいい、例えばセレミオンＡＭＶ（旭硝子社製）、Ａ２０１（旭化成社製）、ＡＣＭ（トクヤマ社製）等の市販品を使用することができる。一方、陽イオン交換膜は１価イオン非選択性である必要はないが、１価イオン非選択性であることが好ましい。これらは、電気透析装置において交互に配列され、糖リン酸エステル及び不純物を含む混合水溶液（反応終了液）を多室型透析槽の隔室（脱塩室）に、また隣り合う隔室（濃縮液室）には低濃度の塩（電解質）の水溶液を供給する。
【００１５】
脱糖工程における混合水溶液（反応終了液）の濃度、電解質溶液の濃度、電気透析時間、通電電流量、膜面積等は、目的に応じて条件を設定すればよいが、通電電流量は限界電流密度を超えない範囲で操作するのが好ましい。この工程により、１価イオン非選択性陰イオン交換膜を、アニオンである糖リン酸エステルイオンとリン酸イオンだけが透過して隣の隔室へ移るため、糖類、更には発酵法の反応終了液に含まれるタンパク質、ペプチド、色素等を分離することができる。
【００１６】
リン酸（塩）除去のための電気透析（脱リン工程）では、１価イオン選択性陰イオン交換膜を使用する。１価イオン選択性陰イオン交換膜とは、１価陰イオンのみを透過させる選択性を付与された陰イオン交換膜をいい、例えばセレミオンＡＳＶ（旭硝子社製）、Ａ１９２（旭化成社製）、ＡＣＳ（トクヤマ社製）等の市販品を使用することができる。一方、陽イオン交換膜は１価イオン選択性である必要はないが、１価イオン選択性であることが好ましい。これらは、電気透析装置において交互に配列され、糖リン酸エステル及び不純物を含む混合水溶液（反応終了液）を多室型透析槽の隔室（脱塩室）に、また隣り合う隔室（濃縮液室）には水又は低濃度の塩（電解質）の水溶液を供給する。
【００１７】
１価イオン選択性陰イオン交換膜は、糖リン酸エステルイオンやリン酸多価イオンは通過できず、リン酸１価イオン（Ｈ２ＰＯ４ −）のみが通過する。リン酸は溶液のｐＨによって荷電状態が異なり、ｐＨを下げると１価イオンになる。従って、リン酸除去の効率の点及び糖リン酸エステルの安定性の点から、混合水溶液のｐＨは、２〜１０、特に４〜７に調整することが好ましい。脱リン工程におけるその他の設定条件は、前記同様、目的に応じて条件を設定すればよく、通電電流量は限界電流密度を超えない範囲で操作するのが好ましい。
【００１８】
以上の２種の電気透析を行う順番は、反応終了液が発酵法、酵素法のいずれによるものであるかを問わず、どちらを先に行うこともできるが、発酵法による反応終了液を精製する場合は、先に脱リン工程を行った場合、続く脱糖工程での糖リン酸エステルの透過性が低下する傾向にあるため、まず脱糖工程、次いで脱リン工程の順番で行うのが好ましい。
【００１９】
電気透析終了液の溶液は、必要に応じて、蒸発等による濃縮し、水酸化ナトリウム、水酸化カリウム等の添加により中和塩とし、更に噴霧乾燥等することにより、糖リン酸エステルを粉末状で得ることができる。この場合の蒸発濃縮と中和の順番は、高ｐＨでの加熱による着色等を回避するため、蒸発濃縮を先に行うのが好ましい。
【００２０】
【実施例】
実施例１
コリネバクテリウム・カルナエ（Ｃ． ｃａｌｌｕｎａｅ）ＩＦＯ１５３５９を、酵母エキス０．３％、アミノ酸混合液１．０％、水あめ１０％、塩化カルシウム０．０１％、硫酸マグネシウム７水和物０．０２％、塩化鉄（ＩＩＩ）６水和物０．００２５％、硫安０．５％、尿素１．０％、リン酸二水素カリウム・リン酸水素二カリウム緩衝液４００ｍＭ（ｐＨ７．０）の条件下で、３０℃、５日間培養し、７．１ｇ／ＬのＧ−１−Ｐ含有発酵液を得た。発酵液を孔径０．２５μｍの精密濾過膜（旭化成社製ＰＭＰ−１０２）にて菌体分離を行った。
得られた精密濾過液を、旭化成社製電気透析装置アシライザーＧ３を用いた電気透析処理に付した。１価陰イオン非選択透過膜としてＡ２０１（旭化成社製）、１価陽イオン非選択透過膜としてＫ５０１ＳＢ（旭化成社製）を使用したイオン交換膜カートリッジＡＣ−２２０−４００型を使用した。電気透析処理を２時間行ったところ、濃縮液室でのＧ−１−Ｐの回収率は８５％、リン酸イオンの回収率は９８％、糖の除去率は９４％（フェノール硫酸法により測定。以下同じ）、タンパク質・ペプチドの除去率は９３％（Ｌｏｗｒｙ法により測定。以下同じ）であった。また、得られた濃縮液の色相ΔＥ（Ｌａｂ）を色差計にて測定した。ΔＥ（Ｌａｂ）は色差計を用いて試料溶液の水に対する明度（Ｌ）、色度−黄（ａ）、色度−青（ｂ）の差を測定し、それらを２乗平均して得られる値であり、値が大きいほど色が濃いことを意味する。精密濾過液の色相がΔＥ（Ｌａｂ）＝３４であったのに対し、濃縮液の色相はΔＥ（Ｌａｂ）＝３でありほぼ透明であった。
【００２１】
実施例２
実施例１にて１価イオン非選択膜を用いた電気透析処理を行った液を、１価イオン選択膜を用いた電気透析処理に付した。１価陰イオン選択透過膜としてＡ１９２（旭化成社製）、１価陽イオン選択透過膜としてＫ１９２（旭化成社製）を使用したイオン交換膜カートリッジＡＣ−１１０−４００型を使用した。電気透析処理を２時間行ったところ、脱塩室でのＧ−１−Ｐの回収率は９０％であり、リン酸イオンの除去率は９９．９％であった。
その後、水酸化ナトリウムにて中和処理を行った後、噴霧乾燥して、Ｇ−１−Ｐ含有粉末を得た。得られたＧ−１−Ｐ含有粉末のＧ−１−Ｐ純度は、Ｇ−１−Ｐ・２Ｎａ・４Ｈ２Ｏとして６８％であった。
【００２２】
実施例３
コリネバクテリウム・グルタミカム（Ｃ． ｇｌｕｔａｍｉｃｕｍ）ＪＣＭ１３２１を酵母エキス０．５％、アミノ酸混合液１．０％、水あめ１０％、塩化カルシウム０．０１％、硫酸マグネシウム７水和物０．０２％、塩化鉄（ＩＩＩ）６水和物０．００２５％、硫安０．５％、リン酸
二水素カリウム・リン酸水素二カリウム緩衝液４００ｍＭ（ｐＨ７．０）の条件下で、３０℃、５日間培養し、８．０ｇ／ＬのＧ−１−Ｐ含有発酵液を得た。得られた発酵液を実施例１と同様に精密濾過膜処理により菌体分離を行い、実施例１と同様に１価イオン非選択透過膜にて電気透析処理を２時間行った。濃縮液室でのＧ−１−Ｐの回収率は６３％、リン酸イオンの回収率は９８％、糖の除去率は９４％、タンパク質・ペプチドの除去率は８８％であった。
【００２３】
実施例４
実施例３にて１価イオン非選択膜を用いた電気透析処理を行った液を、実施例２と同様に１価イオン選択膜を用いた電気透析処理に２時間付したところ、脱塩室でのＧ−１−Ｐの回収率は９２％であり、リン酸イオンの除去率は９９．９％であった。
その後、水酸化ナトリウムにて中和処理を行った後、噴霧乾燥して、Ｇ−１−Ｐ含有粉末を得た。得られたＧ−１−Ｐ含有粉末のＧ−１−Ｐ純度は、Ｇ−１−Ｐ・２Ｎａ・４Ｈ２Ｏとして６２％であった。
【００２４】
実施例５
実施例３にて発酵液を精密濾過膜処理により菌体分離を行った後、粉末活性炭２％を添加し、濾過した。その後実施例１と同様に１価イオン非選択透過膜にて電気透析処理を２時間行った。濃縮液室でのＧ−１−Ｐの回収率は９３％、リン酸イオンの回収率は９９％、糖の除去率は９４％、タンパク質・ペプチドの除去率は９６％であった。
【００２５】
実施例６
実施例５にて１価イオン非選択膜を用いた電気透析処理を行った液を、実施例２と同様に１価イオン選択膜を用いた電気透析処理に２時間付したところ、脱塩室でのＧ−１−Ｐの回収率は９２％であり、リン酸イオンの除去率は９９．９％であった。その後、水酸化ナトリウムにて中和処理を行った後、噴霧乾燥して、Ｇ−１−Ｐ含有粉末を得た。得られたＧ−１−Ｐ含有粉末のＧ−１−Ｐ純度は、Ｇ−１−Ｐ・２Ｎａ・４Ｈ２Ｏとして７４％であった。
【００２６】
比較例１
実施例３で得られた精密濾過した液を限外濾過（旭化成社製ＡＩＰ−１０１０）により除タンパクを行った。その後、実施例２、４及び６と同様に１価イオン選択透過膜にて電気透析処理を２時間行った。脱塩室でのＧ−１−Ｐの回収率は９２％であり、リン酸イオンの除去率は９９．９％であった。更に逆浸透膜（日東電工社製ＮＴＲ−７４５０）にて約１０倍に液を濃縮し、活性炭１０％を添加・濾過し脱色を行った。逆浸透濃縮液の色相がΔＥ（Ｌａｂ）＝１６６であったのに対し、活性炭処理によりΔＥ（Ｌａｂ）＝２となった。得られた活性炭処理液を水酸化ナトリウムで中和し、エタノールを元液に対して２３０％添加し、沈殿・ろ別し、減圧乾燥機にて４０℃一昼夜乾燥を行った。エタノール晶析でのＧ−１−Ｐ回収率は９６％、全工程における糖及びタンパク質・ペプチドの除去率はそれぞれ９２％、８９％であった。得られたＧ−１−Ｐ含有粉末のＧ−１−Ｐ純度は、Ｇ−１−Ｐ・２Ｎａ・４Ｈ２Ｏとして６４％であった。
【００２７】
以上の結果を表１に示した。
【００２８】
【表１】
以上の結果、１価イオン非選択透過膜と１価イオン選択透過膜を組み合わせることにより効率的にＧ−１−Ｐを精製することができた。
【００３０】
【発明の効果】
本発明の精製法は、酵素法、発酵法の双方の反応終了液に適用でき、しかも低コストで、リン酸又はその塩、糖類等の不純物を除去し、Ｇ−１−Ｐ等の糖リン酸エステル又はその塩を精製することができる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a low-cost method for purifying a sugar phosphate ester such as glucose-1-phosphate (hereinafter abbreviated as “G-1-P”) or a salt thereof.
[0002]
[Prior art]
G-1-P is useful as an antibacterial agent, antitumor agent, therapeutic agent for heart disease and the like, and as a substrate for sugar synthesis. In recent years, G-1-P has been used to prevent osteoporosis by focusing on excellent calcium absorption action in the body. As a new food material, research is being conducted energetically.
[0003]
The production of G-1-P uses saccharides and phosphoric acid as substrates, and an immobilized enzyme method (for example, see Patent Document 1) in which maltodextrin phosphorylase (MDPase) immobilized on an ion exchange resin is allowed to act. It is mainly performed by enzymatic methods. G-1-P can also be produced by fermentation, and G-1-P produced by fermentation in which a bacterium belonging to the genus Corynebacterium is cultured in a medium containing sugar and phosphoric acid or a salt thereof. Manufacturing has been proposed (see Patent Document 2).
[0004]
The reaction end solution by the enzyme method contains saccharides and phosphoric acid (salt) which are unreacted substances in addition to G-1-P. Therefore, after ultrafiltration of the reaction completion solution, phosphoric acid or a salt thereof is removed by dialysis or electrodialysis (see Patent Documents 3 and 4), followed by crystals using a water-soluble solvent such as acetone or ethanol. Sugars are removed by analysis to purify G-1-P. However, a large amount of solvent is required for crystallization of G-1-P, resulting in high cost. Further, in order to reuse the water-soluble solvent, a process using a special apparatus such as an azeotropic distillation process or an osmotic vaporization method (pervaporation) is required. Furthermore, in addition to the above saccharides and phosphoric acid (salt), proteins, peptides, pigments, and the like remain in the reaction completion solution by the fermentation method, and these also need to be removed, but they are removed by solvent crystallization. I can't. Moreover, even if the reaction solution of the fermentation method is subjected to ultrafiltration, dialysis method or electrodialysis method, activated carbon treatment, etc., and further purified by a solvent crystallization method, low boiling point components such as fermentation-derived aroma components Is concentrated in the solvent, the solvent cannot be reused.
[0005]
[Patent Document 1]
JP-A-1-63589 [Patent Document 2]
JP 2002-300899 A [Patent Document 3]
JP 61-129189 A [Patent Document 4]
Japanese Examined Patent Publication No. 6-96585 [0006]
[Problems to be solved by the invention]
Therefore, the present invention can be applied to reaction completion solutions of both enzymatic methods and fermentation methods, and at low cost, removes impurities such as phosphoric acid or a salt thereof, saccharides, and sugar phosphates such as G-1-P. It aims at providing the method which can refine | purify ester or its salt.
[0007]
[Means for Solving the Problems]
When the present inventors performed electrodialysis using a monovalent ion non-selective anion exchange membrane on a reaction completion solution for sugar phosphate production by a fermentation method or an enzyme method, sugar phosphate ester ions and phosphate Ions pass through the membrane, but saccharides, proteins, etc. do not pass through, so it is possible to separate saccharides (and proteins, etc.) at low cost, and monovalent ion selective anion exchange By combining the dephosphorylation step by electrodialysis using a membrane, saccharides and phosphoric acid (salts), as well as proteins (peptides) contained in the reaction completion solution by the fermentation method can be removed. It was found that -1-P could be purified.
[0008]
That is, the present invention firstly provides a saccharide from which a saccharide is removed by subjecting a mixed aqueous solution containing a sugar phosphate ester or a salt thereof and a saccharide to electrodialysis using a monovalent ion-nonselective anion exchange membrane. A method for purifying a phosphate ester or a salt thereof is provided.
[0009]
Furthermore, the present invention secondly, a mixed aqueous solution containing a sugar phosphate ester or a salt thereof, a saccharide and phosphoric acid or a salt thereof is electrodialyzed using a monovalent ion-nonselective anion exchange membrane, and monovalent The present invention provides a method for purifying a sugar phosphate ester or a salt thereof, which removes sugar and phosphoric acid or a salt thereof by subjecting to electrodialysis using an ion selective anion exchange membrane.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
As the “mixed aqueous solution containing a sugar phosphate ester or a salt thereof and a saccharide” used in the first purification method of the present invention, either a reaction completion solution by a fermentation method or a reaction completion solution by an enzyme method may be used. May be obtained by removing phosphoric acid or a salt thereof by subjecting them to electrodialysis or the like. In addition, the “mixed aqueous solution containing a sugar phosphate ester or a salt thereof, a saccharide and phosphoric acid or a salt thereof” used in the second purification method of the present invention includes a reaction completion solution by a fermentation method and a reaction by an enzyme method. Any of the end liquids may be used.
[0011]
Examples of sugar phosphates or salts thereof to be purified in the present invention include phosphate esters of polysaccharides such as monosaccharides or disaccharides such as hexose and pentose, or alkali metal salts such as sodium salts and potassium salts thereof. Is mentioned. More specifically, G-1-P, glucose-6-phosphate, mannose-6-phosphate, galactose-6-phosphate, fructose-6-phosphate, glucose-1,6-diphosphate, Examples thereof include alkali metal salts such as fructose-1,6-diphosphate or sodium salts and potassium salts thereof, and the present invention is particularly suitable for purification of G-1-P.
[0012]
Examples of the saccharide contained in the mixed aqueous solution include saccharides such as hexose and pentose as unreacted substances and polysaccharides such as disaccharides, such as glucose, maltose, maltotriose, maltotetraose, maltopentaose, mannose. , Galactose, fructose and the like. Furthermore, examples of phosphoric acid or a salt thereof contained in the mixed aqueous solution include orthophosphoric acid, condensed phosphoric acid such as pyrophosphoric acid, and alkali metal salts such as sodium salt and potassium salt thereof.
[0013]
In the present invention, the sugar phosphate ester is purified by subjecting the mixed aqueous solution to one-stage or two-stage electrodialysis, but prior to electrodialysis, filtration such as ultrafiltration may be performed. Moreover, when using the reaction completion liquid by a fermentation method as mixed aqueous solution, it is preferable to perform a microfiltration process for fermentation microbial cell separation. Further, depending on the type of the fermentation broth, there may be a contaminant that deteriorates the permeability of the sugar phosphate ester in the electrodialysis desugaring step. In the case of such a fermentation broth, it is preferable to perform a treatment with activated carbon in addition to the above filtration.
[0014]
In electrodialysis (desugaring step) for saccharide removal, a monovalent ion non-selective anion exchange membrane is used. The monovalent ion non-selective anion exchange membrane means an anion exchange membrane having no monovalent anion selectivity. For example, Selemion AMV (Asahi Glass Co., Ltd.), A201 (Asahi Kasei Co., Ltd.), ACM (Tokuyama Corp.) ) Etc. can be used. On the other hand, the cation exchange membrane does not need to be monovalent ion non-selective, but is preferably monovalent ion non-selective. These are alternately arranged in the electrodialyzer, and a mixed aqueous solution (reaction completed solution) containing sugar phosphates and impurities is placed in a compartment (desalting chamber) of a multi-chamber dialysis tank and adjacent compartments (concentration). A low-concentration salt (electrolyte) aqueous solution is supplied to the liquid chamber.
[0015]
The concentration of the mixed aqueous solution (reaction completed solution), the concentration of the electrolyte solution, the electrodialysis time, the energization current amount, the membrane area, etc. in the desugaring step may be set according to the purpose. It is preferable to operate within a range not exceeding the density. This step allows the monovalent ion non-selective anion exchange membrane to pass only through the sugar phosphate ester ions and phosphate ions, which are anions, and move to the adjacent compartment. Proteins, peptides, pigments, etc. contained in the liquid can be separated.
[0016]
In electrodialysis (dephosphorization step) for removing phosphoric acid (salt), a monovalent ion selective anion exchange membrane is used. The monovalent ion-selective anion exchange membrane refers to an anion exchange membrane imparted with a selectivity that allows only monovalent anions to pass through. For example, Selemion ASV (Asahi Glass Co., Ltd.), A192 (Asahi Kasei Co., Ltd.), ACS Commercial products such as (manufactured by Tokuyama Corporation) can be used. On the other hand, the cation exchange membrane need not be monovalent ion-selective, but is preferably monovalent ion-selective. These are alternately arranged in the electrodialyzer, and a mixed aqueous solution (reaction completed solution) containing sugar phosphates and impurities is placed in a compartment (desalting chamber) of a multi-chamber dialysis tank and adjacent compartments (concentration). Water or an aqueous solution of salt (electrolyte) having a low concentration is supplied to the liquid chamber.
[0017]
The monovalent ion-selective anion exchange membrane cannot pass sugar phosphate ester ions or phosphate polyvalent ions, and only phosphate monovalent ions (H 2 PO 4 − ) pass through. Phosphoric acid has a different charge state depending on the pH of the solution, and becomes monovalent ions when the pH is lowered. Therefore, the pH of the mixed aqueous solution is preferably adjusted to 2 to 10, particularly 4 to 7, from the viewpoint of the efficiency of removing phosphoric acid and the stability of the sugar phosphate ester. Other setting conditions in the dephosphorization process may be set according to the purpose as described above, and it is preferable to operate the energization current amount in a range not exceeding the limit current density.
[0018]
The order of performing the above two types of electrodialysis can be performed first regardless of whether the reaction end solution is the fermentation method or the enzyme method, but the reaction end solution by the fermentation method is purified. In this case, when the dephosphorization step is performed first, the permeability of the sugar phosphate ester in the subsequent desugaring step tends to decrease. Therefore, the dephosphorization step and then the dephosphorization step are performed first. preferable.
[0019]
The solution of the electrodialysis finish solution is concentrated by evaporation, etc., if necessary, neutralized by adding sodium hydroxide, potassium hydroxide, etc., and further spray-dried, etc. Can be obtained at In this case, the order of evaporation concentration and neutralization is preferably performed first in order to avoid coloring and the like due to heating at high pH.
[0020]
【Example】
Example 1
Corynebacterium carnae IFO15359, yeast extract 0.3%, amino acid mixture 1.0%, starch syrup 10%, calcium chloride 0.01%, magnesium sulfate heptahydrate 0.02%, Under conditions of iron (III) chloride hexahydrate 0.0025%, ammonium sulfate 0.5%, urea 1.0%, potassium dihydrogen phosphate / dipotassium hydrogen phosphate buffer 400 mM (pH 7.0), By culturing at 30 ° C. for 5 days, a fermented liquid containing 7.1 g / L of G-1-P was obtained. The fermentation broth was subjected to cell separation using a microfiltration membrane (PMP-102 manufactured by Asahi Kasei Co., Ltd.) having a pore size of 0.25 μm.
The obtained microfiltrate was subjected to an electrodialysis treatment using an electrodialyzer acylator G3 manufactured by Asahi Kasei Corporation. An ion exchange membrane cartridge AC-220-400 type using A201 (Asahi Kasei Co., Ltd.) as the monovalent anion non-selective permeable membrane and K501SB (Asahi Kasei Co., Ltd.) as the monovalent cation non-selective permeable membrane was used. When electrodialysis was performed for 2 hours, the G-1-P recovery rate in the concentrate chamber was 85%, the phosphate ion recovery rate was 98%, and the sugar removal rate was 94% (measured by the phenol-sulfuric acid method). The same applies hereinafter), and the protein / peptide removal rate was 93% (measured by the Lowry method; the same applies hereinafter). Further, the hue ΔE (Lab) of the obtained concentrated liquid was measured with a color difference meter. ΔE (Lab) is obtained by measuring the lightness (L), chromaticity-yellow (a), and chromaticity-blue (b) difference of the sample solution with respect to water using a color difference meter, and averaging these values. Value, meaning that the larger the value, the darker the color. The hue of the microfiltrate was ΔE (Lab) = 34, whereas the hue of the concentrate was ΔE (Lab) = 3 and was almost transparent.
[0021]
Example 2
The liquid subjected to electrodialysis treatment using a monovalent ion non-selective membrane in Example 1 was subjected to electrodialysis treatment using a monovalent ion selective membrane. An ion exchange membrane cartridge AC-110-400 type using A192 (manufactured by Asahi Kasei) as the monovalent anion selective permeable membrane and K192 (manufactured by Asahi Kasei) as the monovalent cation selective permeable membrane was used. When electrodialysis was performed for 2 hours, the G-1-P recovery rate in the desalting chamber was 90%, and the phosphate ion removal rate was 99.9%.
Then, after neutralizing with sodium hydroxide, it was spray-dried to obtain a G-1-P-containing powder. The G-1-P purity of the obtained G-1-P-containing powder was 68% as G-1-P · 2Na · 4H 2 O.
[0022]
Example 3
Corynebacterium glutamicum ( C. glutamicum ) JCM1321 with yeast extract 0.5%, amino acid mixture 1.0%, starch syrup 10%, calcium chloride 0.01%, magnesium sulfate heptahydrate 0.02%, chloride Cultured at 30 ° C. for 5 days under conditions of iron (III) hexahydrate 0.0025%, ammonium sulfate 0.5%, potassium dihydrogen phosphate / potassium hydrogen phosphate buffer 400 mM (pH 7.0) 8.0 g / L of G-1-P-containing fermentation broth was obtained. The obtained fermentation broth was subjected to cell separation by microfiltration membrane treatment in the same manner as in Example 1, and electrodialysis treatment was carried out for 2 hours using a monovalent ion non-selective permeable membrane in the same manner as in Example 1. The G-1-P recovery rate in the concentrate chamber was 63%, the phosphate ion recovery rate was 98%, the sugar removal rate was 94%, and the protein / peptide removal rate was 88%.
[0023]
Example 4
The liquid that had been subjected to electrodialysis treatment using a monovalent ion non-selective membrane in Example 3 was subjected to electrodialysis treatment using a monovalent ion selective membrane in the same manner as in Example 2 for 2 hours. The recovery rate of G-1-P was 92%, and the phosphate ion removal rate was 99.9%.
Then, after neutralizing with sodium hydroxide, it was spray-dried to obtain a G-1-P-containing powder. The G-1-P purity of the obtained G-1-P-containing powder was 62% as G-1-P · 2Na · 4H 2 O.
[0024]
Example 5
In Example 3, the fermentation broth was subjected to cell separation by microfiltration membrane treatment, and then 2% powdered activated carbon was added and filtered. Thereafter, as in Example 1, electrodialysis treatment was performed for 2 hours using a monovalent ion non-selective permeable membrane. The G-1-P recovery rate in the concentrate chamber was 93%, the phosphate ion recovery rate was 99%, the sugar removal rate was 94%, and the protein / peptide removal rate was 96%.
[0025]
Example 6
When the liquid subjected to electrodialysis treatment using a monovalent ion non-selective membrane in Example 5 was subjected to electrodialysis treatment using a monovalent ion selective membrane in the same manner as in Example 2 for 2 hours, a desalting chamber was obtained. The recovery rate of G-1-P was 92%, and the phosphate ion removal rate was 99.9%. Then, after neutralizing with sodium hydroxide, it was spray-dried to obtain a G-1-P-containing powder. The G-1-P purity of the obtained G-1-P-containing powder was 74% as G-1-P · 2Na · 4H 2 O.
[0026]
Comparative Example 1
The microfiltered liquid obtained in Example 3 was deproteinized by ultrafiltration (AIP-1010 manufactured by Asahi Kasei Corporation). Thereafter, electrodialysis treatment was performed for 2 hours using a monovalent ion selective permeable membrane in the same manner as in Examples 2, 4 and 6. The recovery rate of G-1-P in the desalting chamber was 92%, and the phosphate ion removal rate was 99.9%. Further, the solution was concentrated about 10 times with a reverse osmosis membrane (NTR-7450 manufactured by Nitto Denko Corporation), and 10% activated carbon was added and filtered to perform decolorization. The hue of the reverse osmosis concentrate was ΔE (Lab) = 166, whereas ΔE (Lab) = 2 was obtained by the activated carbon treatment. The obtained activated carbon treatment liquid was neutralized with sodium hydroxide, 230% of ethanol was added to the original liquid, precipitated and filtered, and dried at 40 ° C. overnight in a vacuum dryer. The G-1-P recovery rate in ethanol crystallization was 96%, and the sugar and protein / peptide removal rates in all steps were 92% and 89%, respectively. The G-1-P purity of the obtained G-1-P-containing powder was 64% as G-1-P · 2Na · 4H 2 O.
[0027]
The above results are shown in Table 1.
[0028]
[Table 1]
As a result, G-1-P could be efficiently purified by combining a monovalent ion non-selective permeable membrane and a monovalent ion selective permeable membrane.
[0030]
【The invention's effect】
The purification method of the present invention can be applied to reaction completion solutions of both enzymatic methods and fermentation methods, and at low cost, removes impurities such as phosphoric acid or its salts, saccharides, and sugar phosphorus such as G-1-P. Acid esters or their salts can be purified.
Claims (2)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011115302A1 (en) * | 2010-03-19 | 2011-09-22 | 味の素株式会社 | Method for producing phosphoric acid from bone, and method and device for employing phosphoric acid produced from bone in the fermentation of sugar |
| WO2016038142A1 (en) | 2014-09-10 | 2016-03-17 | Pfeifer & Langen GmbH & Co. KG | Process for the enzymatic preparation of a product glucoside and of a co-product from an educt glucoside |
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| WO2016038142A1 (en) | 2014-09-10 | 2016-03-17 | Pfeifer & Langen GmbH & Co. KG | Process for the enzymatic preparation of a product glucoside and of a co-product from an educt glucoside |
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