JP3967438B2 - Kojibiose phosphorylase, its production method and use - Google Patents

Description

【００５７】
表１に示されるように、本発明のコージビオースホスホリラーゼは、コージビオースに極めて高い特異性で作用してＤ−グルコースとβ−Ｄ−グルコース−１リン酸とを生成することが判明した。一方、他の糖質には作用しないことが判明した。
【００５８】
【実験６】
〈コージビースホスホリラーゼのグルコシル転移糖生成反応における受容体特異性〉
受容体として表２に示す各種単糖類、オリゴ糖類および糖アルコール類のいずれかと、糖供与体としてβ−Ｄ−グルコース−１リン酸とを、固形物重量で等量溶解含有している水溶液に、実験２の方法で得た精製コージビオースホスホリラーゼをβ−Ｄ−グルコース−１リン酸１ｇ当たり１０単位ずつ加え、６０℃、ｐＨ５．５で２４時間反応させた。なお、受容体及び糖供与体の反応液中の濃度はいずれも１ｗ／ｖ％とした。酵素反応前後の反応液を、実験５と同様にＴＬＣに供した後発色させた。両反応液に由来するスポットを比較し、反応後に新たに生成したスポットより、転移糖を生成しているかどうかを判定した。また転移糖と判定されたスポットの発色強度を肉眼観察することにより、転移糖の生成率を相対的に評価した。結果を表２に示した。
【００５９】
【表２】
【００６０】
表２に示されるように、本発明のコージビオースホスホリラーゼは、β−Ｄ−グルコース−１リン酸を糖供与体として、Ｄ−グルコース、Ｌ−ソルボースなどの単糖類、マルトース、コージビオース、トレハロース、スクロースなどの二糖類、及びマルトトリオース、マルトテトラオース、マルトペンタオースなどの三糖以上のオリゴ糖類にグルコシル基を良く転移し、グルコシル転移糖を生成することが判った。なお、β−Ｄ−グルコース−１リン酸にかえてα−Ｄ−グルコース−１リン酸を糖供与体として用いた場合、グルコシル転移糖は得られなかった。
【００６１】
この実験６により、本発明のコージビオースホスホリラーゼによる糖転移反応で生成することが明らかとなったグルコシル転移糖のうちのいくつかの詳細な構造について、次の実験７乃至１２を示しながら説明する。
【００６２】
【実験７】
〈β−Ｄ−グルコース−１リン酸とＤ−グルコースとからのグルコシル転移糖〉
実験６で得たβ−Ｄ−グルコース−１リン酸とＤ−グルコースとを基質とした酵素反応液の糖成分をガスクロマトグラフィー（以下、「ＧＬＣ」と略称する。）で分析した。酵素反応液の一部を乾固し、ピリジンに溶解した後、トリメチルシリル化したものをＧＬＣの分析試料とした。ＧＬＣカラムは、２％シリコンＯＶ−１７／クロモゾルブＷ（ジー・エル・サイエンス株式会社製）を充填したステンレスカラム（３ｍｍφ×２ｍ）、キャリアーガスは、窒素ガスを流量４０ｍｌ／分で、カラムオーブン温度は、１６０℃から３２０℃まで７．５℃／分の昇温速度で分析した。検出は、水素炎イオン検出器を用いた。
【００６３】
その結果、本発明のコージビオースホスホリラーゼによるβ−Ｄ−グルコース−１リン酸とＤ−グルコースとからのグルコシル転移糖のピークの保持時間は、既知糖質コージビオースのそれと一致した。本発明のコージビオースホスホリラーゼによるβ−Ｄ−グルコース−１リン酸とＤ−グルコースとからのグルコシル転移糖は、コージビオースであると判断される。
【００６４】
【実験８】
〈グルコシルソルボース〉
本発明のコージビオースホスホリラーゼによるＬ−ソルボースへの糖転移反応で生成するグルコシル転移糖を確認するため、当該グルコシル転移糖を生成させ、単離し、構造を調べた。まず、β−Ｄ−グルコース−１リン酸を２．５％、Ｌ−ソルボースを２．５％含む水溶液をｐＨ５．０に調整し、これに実験２の方法で得た精製コージビオースホスホリラーゼをβ−Ｄ−グルコース−１リン酸１ｇ当たり１０単位加え、６０℃で４８時間反応させ、次いで１００℃で１０分間加熱して酵素を失活させた。実験７で示した方法に準じてこの反応終了液の一部をＧＬＣで分析した。その結果、反応終了液にはβ−Ｄ−グルコース−１リン酸及びＬ−ソルボースのいずれとも保持時間の異なる成分が多量生成していることが確認され、本成分が当該グルコシル転移糖と考えられた。また、ＧＬＣでの分析結果より求めた本反応における当該グルコシル転移糖の生成率は約２４％であった。本反応液の残り全量を活性炭で脱色、濾過し、Ｈ型及びＯＨ型イオン交換樹脂により脱塩して精製し、濃度約５０％まで濃縮して、以下に説明するカラムクロマトグラフィーを行い、当該グルコシル転移糖高含有画分を採取した。
【００６５】
分画用樹脂は、アルカリ金属型強酸性カチオン交換樹脂（東京有機化学工業株式会社製、商品名『ＸＴ−１０１６』、Ｎａ型、架橋度４％）を使用し、内径３ｃｍ、長さ１ｍのジャケット付きステンレス製カラム４本に水懸濁状で充填し、直列につなぎ、樹脂層全長を約４ｍになるようにした。カラム内温度を４０℃に維持しつつ、糖液を樹脂に対して５ｖ／ｖ％加え、これに４０℃の温水をＳＶ０．１５の流速で流して分画し、当該グルコシル転移糖高含有画分を採取した。
【００６６】
当該転移糖高含有画分を脱塩、精製し、濃度約４０％に濃縮してオクタデシルシリカゲルを充填したカラム（株式会社ワイエムシー、商品名『ＹＭＣ−ＰａｋＯＤＳ』）を用いたクロマトグラフィーを行い、当該グルコシル転移糖画分を採取した。採取された画分を濃度約４０％に濃縮し、再度オクタデシルシリカゲルを用いたカラムクロマトグラフィーに供する操作を繰り返し、採取された当該グルコシル転移糖高含有液を脱塩、精製、濃縮、真空乾燥して、当該グルコシル転移糖高含有粉末を、反応に供した糖質の固形物当たり収率約２０％で得た。実験７で示した方法に準じて本粉末標品をＧＬＣで分析したところ、本標品は固形物当たり約９８％当該グルコシル転移糖を含有していた。
【００６７】
次に、当該グルコシル転移糖高含有粉末を酸分解し、ＧＬＣ分析に供した。その結果、当該グルコシル転移糖は酸分解によりほぼ１対１のモル比でＤ−グルコースとＬ−ソルボースを生成することが分かった。また、当該グルコシル転移糖高含有粉末をメチル化した後、酸により加水分解し、続いて還元、アセチル化して得られた部分メチルヘキシトールアセテートをＧＬＣで分析すると２，３，４，６−テトラメチル−１，５−ジアセチルグルシトールが認められた。以上のことは、当該グルコシル転移糖が、Ｄ−グルコースとＬ−ソルボースが１対１のモル比で結合したものであり、更に、Ｄ−グルコースの１位のＯＨ基がその結合に関与していることを示唆している。なお、ソルボースのメチル化誘導体は認められなかったが、これは酸により分解されたためと思われる。
【００６８】
当該グルコシル転移糖の構造をさらに詳細に調べるため13Ｃ−核磁気共鳴スペクトルを測定した。その結果、13Ｃ−ＮＭＲスペクトル（１００ＭＨｚ,Ｄ2Ｏ）：σｐｐｍ ｆｒｏｍ ＴＳＰ１０１．２，１００．４，９９．５，９９．３，７７．１，７５．６，７４．９，７４．５，７４．０，７３．２，７２．２，６６．３，６３．２，６２．１の各値が得られた。この分析データから当該グルコシル転移糖はＤ−グルコースの１位がＬ−ソルボースの５位にα型で結合した２糖類であるグルコシルソルボース、すなわち、α−Ｄ−ｇｌｕｃｏｓｙｌ（１→５）−Ｌ−ｓｏｒｂｏｓｅであることが分かった。本物質は、従来知られていなかった新規な糖質である。
【００６９】
【実験９】
〈コージビオシルグルコース〉
本発明のコージビオースホスホリラーゼによるマルトースへの転移反応で生成するグルコシル転移糖を確認するため、当該グルコシル転移糖を生成させ、単離し、構造を調べた。まず、β−Ｄ−グルコース−１リン酸を５％、マルトースを１０％含む水溶液をｐＨ６．０に調整し、これに実験２の方法で得た精製コージビオースホスホリラーゼをβ−Ｄ−グルコース−１リン酸１ｇ当たり２０単位加え、６０℃で４８時間反応させ、次いで１００℃で１０分間加熱して酵素を失活させた。実験７で示した方法に準じてこの反応終了液の一部をＧＬＣで分析した。その結果、反応終了液にはβ−Ｄ−グルコース−１リン酸及びマルトースのいずれとも保持時間の異なる成分が多量生成していることが確認され、本成分が当該グルコシル転移糖と考えられた。また、ＧＬＣでの分析結果より求めた本反応における当該グルコシル転移糖の生成率は約４０％であった。本反応液の残り全量を実験７と同様に分画精製し、濃縮、真空乾燥して、当該グルコシル転移糖高含有粉末を、反応に供した糖質の固形物当たり収率約２０％で得た。本粉末標品は、当該グルコシル転移糖を固形物当たり約９８％含有していた。
【００７０】
次に、当該グルコシル転移糖高含有粉末をメチル化した後、酸により加水分解し、続いて還元、アセチル化して得られた部分メチルヘキシトールアセテートをＧＬＣ分析に供した。その結果、２，３，４，６−テトラメチル−１，５−ジアセチルグルシトールと３，４，６−トリメチル−１，２，５−トリアセチルグルシトールおよび２，３，６−トリメチル−１，４，５−トリアセチルグルシトールが約１対１対１の比率で認められた。このことは、当該グルコシル転移糖が、１位のＯＨ基のみが結合に関与しているグルコースと、１位と２位のＯＨ基がともに結合に関与しているか又は２位のＯＨ基のみが結合に関与しているグルコースと、１位と４位のグルコースがともに結合に関与しているか又は４位のＯＨ基のみが結合に関与しているグルコースとが、それぞれ１対１対１の割合で互いに結合しているオリゴ糖であることを示唆している。
【００７１】
当該グルコシル転移糖の構造をさらに詳細に調べるため13Ｃ−核磁気共鳴スペクトルを測定した。その結果、13Ｃ−ＮＭＲスペクトル（１００ＭＨｚ,Ｄ2Ｏ）：σｐｐｍ ｆｒｏｍ ＴＳＰ９９．５，９９．４，９９．１，９８．６，９４．６，７９．２，７８．８，７８．６，７８．０，７７．６，７７．３，７７．０，７６．１，７５．６，７５．３，７４．７，７４．２，７４．１，７３．８，７２．７，７２．２，７２．１，６３．６，６３．５，６３．２，６３．１の各値が得られた。この分析データから本グルコシル転移糖の構造はマルトースの非還元末端グルコース残基にＤ−グルコースがα型で１，２結合した３糖類であるコージビオシルグルコース、すなわち、α−Ｄ−ｇｌｕｃｏｓｙｌ（１→２）α−Ｄ−ｇｌｕｃｏｓｙｌ（１→４）−Ｄ−ｇｌｕｃｏｓｅであることが分かった。
【００７２】
【実験１０】
〈コージトリオース〉
本発明のコージビオースホスホリラーゼによるコージビオースへの糖転移反応で生成するグルコシル転移糖を確認するため、当該グルコシル転移糖を生成させ、単離し、構造を調べた。まず、２０％コージビオースを溶解含有する５ｍＭリン酸二水素ナトリウムをｐＨ５．５に調整し、これに実験２の方法で得た精製コージビオースホスホリラーゼをコージビオース１ｇ当たり１０単位加え、６０℃で４８時間反応させ、次いで１００℃で１０分間加熱して酵素を失活させた。実験７で示した方法に準じてこの反応終了液の一部をＧＬＣで分析した。その結果反応終了液にはコージビオースとも、また、Ｄ−グルコースやβ−Ｄ−グルコース−１リン酸とも保持時間の異なる成分が多量生成していることが確認され、本成分が当該グルコシル転移糖と考えられた。また、ＧＬＣでの分析結果より求めた本反応における当該グルコシル転移糖の生成率は約３０％であった。本反応液の残り全量を実験８と同様に分画精製し、濃縮、真空乾燥して、当該グルコシル転移糖高含有粉末を、反応に供したコージビオースの固形物当たり収率約１５％で得た。本粉末標品は、当該グルコシル転移糖を固形物当たり約９８％含有していた。
【００７３】
次に、当該グルコシル転移糖高含有粉末をメチル化した後、酸により加水分解し、続いて還元、アセチル化して得られた部分メチルヘキシトールアセテートをＧＬＣ分析に供した。その結果、２，３，４，６−テトラメチル−１，５−ジアセチルグルシトールと３，４，６−トリメチル−１，２，５−トリアセチルグルシトールが約１対２の比率で認められた。このことは、当該グルコシル転移糖が、１位のＯＨ基のみが結合に関与しているグルコースと、１位と２位のＯＨ基がともに結合に関与しているか又は２位のＯＨ基のみが結合に関与しているグルコースとが、１対２の割合で互いに結合しているオリゴ糖であることを示唆している。さらに、グルコースの結合様式の可能性を考慮に入れるとこのことは、当該グルコシル転移糖が、１位のＯＨ基のみが結合に関与しているグルコースと、１位と２位のＯＨ基がともに結合に関与しているグルコースと、２位のＯＨ基のみが結合に関与しているグルコースとが、それぞれ１対１対１の割合で互いに結合しているオリゴ糖であることを強く示唆している。
【００７４】
当該グルコシル転移糖の構造をさらに詳細に調べるため13Ｃ−核磁気共鳴スペクトルを測定した。その結果、13Ｃ−ＮＭＲスペクトル（１００ＭＨｚ,Ｄ2Ｏ）：σｐｐｍ ｆｒｏｍ ＴＳＰ９９．３，９９．０，９８．３，９７．６，９６．５，９２．２，８１．３，７８．８，７８．６，７８．５，７７．２，７７．１，７５．７，７５．６，７４．７，７４．６，７４．５，７４．３，７４．２，７４．１，７４．０，７３．９，７２．６，７２．３，７２．２，７２．１，７２．０，６３．６，６３．４，６３．２，６３．１，６３．１，６３．０の各値が得られた。この分析データから本グルコシルコージビオースの構造はコージビオースの非還元末端グルコース残基にＤ−グルコースがα型で１，２結合した３糖類であるコージトリオース、すなわち、α−Ｄ−ｇｌｕｃｏｓｙｌ（１→２）α−Ｄ−ｇｌｕｃｏｓｙｌ（１→２）−Ｄ−ｇｌｕｃｏｓｅであることが分かった。
【００７５】
【実験１１】
〈コージビオシルグルコシド〉
本発明のコージビオースホスホリラーゼによるトレハロースへの転移反応で生成するグルコシル転移糖を確認するため、当該グルコシル転移糖を生成させ、単離し、構造を調べた。まず、β−Ｄ−グルコース−１リン酸を５％、トレハロースを１０％含む水溶液をｐＨ５．５に調整し、これに実験２の方法で得た精製コージビオースホスホリラーゼをβ−Ｄ−グルコース−１リン酸１ｇ当たり１０単位加え、６０℃で４８時間反応させ、次いで１００℃で１０分間加熱して酵素を失活させた。実験７で示した方法に準じてこの反応終了液の一部をＧＬＣで分析した。その結果、反応終了液にはβ−Ｄ−グルコース及びトレハロースのいずれとも保持時間の異なる成分が多量生成していることが確認され、本成分が当該グルコシル転移糖と考えられた。また、ＧＬＣでの分析結果より求めた本反応における当該グルコシル転移糖の生成率は約７５％であった。本反応液の残り全量を実験８と同様に分画精製し、濃縮、真空乾燥して、当該グルコシル転移糖高含有粉末を、反応に供した糖質の固形物当たり収率約６５％で得た。本粉末標品は、当該グルコシル転移糖を固形物当たり約９８％含有していた。
【００７６】
次に、当該グルコシル転移糖高含有粉末をメチル化した後、酸により加水分解し、続いて還元、アセチル化して得られた部分メチルヘキシトールアセテートをＧＬＣ分析に供した。その結果、２，３，４，６−テトラメチル−１，５−ジアセチルグルシトールと３，４，６−トリメチル−１，２，５−トリアセチルグルシトールが２対１の比率で認められた。このことは、当該グルコシル転移糖が、１位のＯＨ基のみが結合に関与しているグルコースと、１位と２位のＯＨ基がともに結合に関与しているグルコースとが、２対１の割合で互いに結合しているオリゴ糖であることを示唆している。
【００７７】
当該グルコシル転移糖の構造をさらに詳細に調べるため13Ｃ−核磁気共鳴スペクトルを測定した。その結果、13Ｃ−ＮＭＲスペクトル（１００ＭＨｚ,Ｄ2Ｏ）：σｐｐｍ ｆｒｏｍ ＴＳＰ９８．６，９６．２，９３．３，７７．３，７５．８，７５．４，７５．１，７４．８，７４．６，７４．６，７４．０，７３．９，７２．５，７２．３，７２．２，６３．４，６３．４，６３．３の各値が得られた。この分析データから本グルコシルトレハロースの構造はトレハロースにＤ−グルコースが１，２結合した３糖類であるコージビオシルグルコシド（別名セラギノース）、すなわち、α−Ｄ−ｇｌｕｃｏｓｙｌ（１→２）α−Ｄ−ｇｌｕｃｏｓｙｌ（１，１）α−Ｄ−ｇｌｕｃｏｓｉｄｅであることが分かった。
【００７８】
【実験１２】
〈コージビオシルフラクトシド〉
本発明のコージビオースホスホリラーゼによるスクロースへの転移反応で生成するグルコシル転移糖を確認するため、当該グルコシル転移糖を生成させ、単離し、構造を調べた。まず、β−Ｄ−グルコース−１リン酸を５％、スクロースを１０％含む水溶液をｐＨ５．５に調整し、これに実験２の方法で得た精製コージビオースホスホリラーゼをβ−Ｄ−グルコース−１リン酸１ｇ当たり１０単位加え、６０℃で４８時間反応させ、次いで１００℃で１０分間加熱して酵素を失活させた。実験７で示した方法に準じてこの反応終了液の一部をＧＬＣで分析した。その結果、反応終了液にはβ−Ｄ−グルコース−１リン酸及びスクロースのいずれとも保持時間の異なる成分が多量生成していることが確認され、本成分が当該グルコシル転移糖と考えられた。また、ＧＬＣでの分析結果より求めた本反応における当該グルコシル転移糖の生成率は約７０％であった。本反応液の残り全量を実験８と同様に分画精製し、濃縮、真空乾燥して、当該グルコシル転移糖高含有粉末を、反応に供した糖質の固形物当たり収率約５０％で得た。本粉末標品は、当該グルコシル転移糖を固形物当たり約９８％含有していた。
【００７９】
次に、当該グルコシル転移糖高含有粉末を酸分解し、ＧＬＣ分析に供した。その結果、当該グルコシル転移糖は酸分解によりほぼ２対１のモル比でＤ−グルコースとＤ−フラクトースを生成することが分かった。また、当該グルコシル転移糖高含有粉末をメチルした後、酸により加水分解し、続いて還元、アセチル化して得られた部分メチルヘキシトールアセテートをＧＬＣで分析すると２，３，４，６−テトラメチル−１，５−ジアセチルグルシトールと３，４，６−トリメチル−１，２，５−トリアセチルグルシトールが１対１の比率で認められた。以上のことは、当該グルコシル転移糖が、Ｄ−グルコースとＤ−フラクトースが２対１のモル比で結合したものであり、更に、当該グルコシル転移糖を構成するＤ−グルコースは、１位のＯＨ基のみが結合に関与するものと、１位と２位のＯＨ基がともに結合に関与するものとが、１対１のモル比で存在していることを示唆している。なお、Ｄ−フラクトースのメチル化誘導体は認められなかったが、これは酸により分解されたためと思われる。
【００８０】
当該グルコシル転移糖の構造をさらに詳細に調べるため13Ｃ−核磁気共鳴スペクトルを測定した。その結果、13Ｃ−ＮＭＲスペクトル（１００ＭＨｚ,Ｄ2Ｏ）：σｐｐｍ ｆｒｏｍ ＴＳＰ１０７．０，９９．３，９２．５，８４．０，７９．１，７８．５，７６．５，７５．７，７４．９，７４．７，７４．１，７４．０，７２．２，７２．１，６４．９，６４．６，６３．２，６３．１の各値が得られた。この分析データから本グルコシルスクロースの構造はスクロースのグルコース残基にＤ−グルコースがα型で１，２結合した３糖類であるコージビオシルフラクトシド、すなわち、α−Ｄ−ｇｌｕｃｏｓｙｌ（１→２）α−Ｄ−ｇｌｕｃｏｓｙｌ（１→２）β−Ｄ−ｆｒｕｃｔｏｓｉｄｅであることが分かった。本物質は、従来知られていなかった新規な糖質である。
【００８１】
【実験１３】
〈急性毒性〉
７週齢のｄｄ系マウスを使用して、実験８乃至１２で得たグルコシルソルボース高含有粉末、コージビオシルグルコース高含有粉末、コージトリオース高含有粉末、コージビオシルグルコシド高含有粉末及びコージビオシルフラクトシド高含有粉末を、それぞれ別個に経口投与して急性毒性テストをしたところ、いずれの場合にも投与可能な最大投与量すなわち、５０ｇ／ｋｇマウス体重においても死亡例は認められなかった。従って、本発明のこれら転移糖は、いずれも毒性の極めて低い物質である。
【００８２】
以下、本発明のコージビオースホスホリラーゼ及び当該コージビオースホスホリラーゼをコードする本発明のＤＮＡと、それを利用したグルコシル転移糖含有糖質の製造方法を実施例Ａで、このグルコシル転移糖含有糖質を含有せしめた組成物を実施例Ｂで示す。
【００８３】
【実施例Ａ−１】
〈酵素液〉
サーモアナエロビウム・ブロッキイ ＡＴＣＣ３５０４７を、実験１に示した方法で種培養した後、実験１に示した方法に準じて嫌気ファーメンターに調製した同じ組成の培地に、この培地体積当たり１ｖ／ｖ％の種培養液を接種し、培養温度６５℃で約３０時間培養した。培養液を遠心分離して得た菌体を超音波破砕し、破砕液上清のコージビオースホスホリラーゼ活性を測定した。この活性を培養液１ｍｌ当たりに換算すると、０．０８単位であった。破砕液上清を限外濾過膜にて濃縮し、その濃縮液を透析してｍｌ当たりコージビオースホスホリラーゼ活性約８単位を有する酵素液を、もとの培養液の総活性に対して約７０％の収率で得た。
【００８４】
【実施例Ａ−２】
〈ＤＮＡの調製〉
実験１に示した培地と同一組成の培地１１ｌで、サーモアナエロビウム・ブロッキイ（ＡＴＣＣ ３５０４７）を、実験１に示した方法に準じて６０℃で２４時間培養した。遠心分離により培養物から菌体を分離し、適量のトリス−ＥＤＴＡ−食塩緩衝液（以下、「ＴＥＳ緩衝液」と略記する。）（ｐＨ８．０）に浮遊させ、リゾチームを当該菌体浮遊液の体積に対し０．０５％（ｗ／ｖ）加えた後、３７℃で３０分間インキュベートした。その後この処理物を−８０℃で１時間保持して凍結させた後、ここに、予めＴＥＳ緩衝液（ｐＨ９．０）を加えて６０℃に加温しておいたＴＥＳ緩衝液／フェノール混液を加えて充分に攪拌し、さらに氷冷後遠心分離して形成された上層を採取した。この上層に、２倍容の冷エタノールを加えて生成した沈澱を採取し、ＳＳＣ緩衝液（ｐＨ７．１）の適量に溶解後、７．５μｇのリボヌクレアーゼ及び１２５μｇのプロテアーゼを加え、３７℃で１時間インキュベートした。ここに、クロロホルム／イソアミルアルコール混液を加えて攪拌後、静置して形成される上層を採取し、この上層に冷エタノールを加えて生成した沈澱を採取した。沈澱を冷７０％（ｖ／ｖ）エタノールで濯ぎ真空乾燥して、ＤＮＡを得た。得られたＤＮＡは、濃度約１ｍｇ／ｍｌとなるようにＳＳＣ緩衝液（ｐＨ７．１）に溶解し、−８０℃で凍結した。
【００８５】
【実施例Ａ−３】
〈形質転換体と組換えＤＮＡの調製〉
実施例Ａ−２の方法で得たＤＮＡ溶液を１ｍｌとり、ここに制限酵素ＨｉｎｄＩＩＩを約２０単位加え、３７℃で３０分間インキュベートしてＤＮＡを部分分解した後、蔗糖密度勾配超遠心法により、鎖長約２，０００乃至５，０００塩基対のＤＮＡ断片を採取した。別途、ストラタジーン・クローニング・システムズ製プラスミドベクター『ＢｌｕｅｓｃｒｉｐｔＩＩ ＳＫ（＋）』を常法により制限酵素Ｈｉｎｄ ＩＩＩを作用させて完全に切断した後、その切断されたプラスミドベクター０．３μｇと先に得たＤＮＡ断片約３μｇとを宝酒造製『ＤＮＡライゲーション・キット』を用いて、添付の説明書にしたがって操作して連結し、得られた組換えＤＮＡで、通常のコンピテントセル法によりストラタジーン・クローニング・システムズ製大腸菌『Ｅｐｉｃｕｒｉａｎ Ｃｏｌｉ ＸＬ１−Ｂｌｕｅ』１００μｌを形質転換して遺伝子ライブラリーを作製した。
【００８６】
このようにして得た遺伝子ライブラリーとしての形質転換体を、常法により調製した、１０ｇ／ｌ トリプトン、５ｇ／ｌ 酵母エキス、５ｇ／ｌ 塩化ナトリウム、７５ｍｇ／ｌ アンピシリンナトリウム塩及び５０ｍｇ／ｌ ５−ブロモ−４−クロロ−３−インドリル−β−ガラクトシドを含む寒天平板培地（ｐＨ７．０）に植菌し、３７℃で１８時間培養後、培地上に形成された白色のコロニー約４，０００個をアマシャム製ナイロン膜『Ｈｙｂｏｎｄ−Ｎ＋』上に固定した。別途、実験４の方法で明らかにした、配列表の配列番号７に示すアミノ酸配列における第１より７番目までのアミノ酸配列に基づき、５′−ＴＴＹＧＡＹＧＡＲＡＡＹＡＡＹＡＴＧＣＣ−３′で表される塩基配列のオリゴヌクレオチドを化学合成し、常法にしたがい［γ−32Ｐ］ＡＴＰ及びＴ４ポリヌクレオチドキナーゼを用いて同位体標識してプローブとしての合成ＤＮＡを得た。先に得たナイロン膜上に固定されたコロニーのうち、当該プローブと顕著な会合を示すコロニーを、通常のコロニーハイブリダイゼーション法を適用して選択し、当該形質転換体を『ＴＫＰ１』と命名した。
【００８７】
この形質転換体ＴＫＰ１を常法にしたがい、１００μｇ／ｍｌ アンピシリンナトリウム塩を含むＬ−ブロス培地（ｐＨ７．０）に植菌し、３７℃で２４時間回転振盪培養した。培養終了後、遠心分離により培養物から菌体を採取し、通常のアルカリ−ＳＤＳ法により組換えＤＮＡを抽出した。この組換えＤＮＡの塩基配列を、通常のジデオキシ法により分析したところ、当該組換えＤＮＡは、サーモアナエロビウム・ブロッキイ（ＡＴＣＣ ３５０４７）に由来する、鎖長３９５６塩基対の、配列表における配列番号９に示す塩基配列のＤＮＡを含んでいた。図５に示すように、この組換えＤＮＡにおいて、当該ＤＮＡは、制限酵素Ｈｉｎｄ ＩＩＩによる認識部位の下流に連結されていた。一方、この塩基配列から推定されるアミノ酸配列は、その配列番号９に併記したとおりであり、このアミノ酸配列と、実験４の方法で確認された本発明のコージビオースホスホリラーゼのＮ末端アミノ酸配列及び中間部部分アミノ酸配列である、配列表における配列番号１乃至３及び配列番号６乃至８に示すアミノ酸配列とを比較したところ、配列表における配列番号１、２及び３に示すアミノ酸配列は、それぞれ、配列番号９に併記したアミノ酸配列における第１乃至５番目、第５９０乃至５９４番目及び第３５７乃至３６１番目のアミノ酸配列と完全に一致した。また、配列表における配列番号６、７及び８に示すアミノ酸配列は、それぞれ、配列表における配列番号９に併記したアミノ酸配列における第１乃至１０番目、第５９０乃至５９９番目及び第３５７乃至３６６番目のアミノ酸配列と完全に一致した。以上のことは、本発明のコージビオースホスホリラーゼが配列表における配列番号４に示すアミノ酸配列を含むものであり、当該酵素はサーモアナエロビウム・ブロッキイ（ＡＴＣＣ ３５０４７）においては、配列表における配列番号５に示す塩基配列のＤＮＡによりコードされていることを示している。以上のようにして調製し、塩基配列を確認した組換えＤＮＡを『ｐＴＫＰ１』と命名した。
【００８８】
【実施例Ａ−４】
〈形質転換体によるコージビオースホスホリラーゼの産生〉
１６ｇ／ｌポリペプトン、１０ｇ／ｌ酵母エキス及び５ｇ／ｌ塩化ナトリウムを含む水溶液を５００ｍｌ容三角フラスコに１００ｍｌ入れ、オートクレーブで１２１℃で１５分間処理し、冷却し、無菌的にｐＨ７．０に調製した後、アンピシリンナトリウム塩１０ｍｇを無菌的に添加して液体培地を調製した。この液体培地に実施例Ａ−３の方法で得た形質転換体ＴＫＰ１を接種し、３７℃で約２０時間通気攪拌培養したものを種培養液とした。次に１０ｌ容ファーメンタに、種培養に用いたのと同一組成の培地を、種培養の場合に準じて７ｌ調製し、種培養液７０ｍｌ接種し、約２０時間通気攪拌培養した。この培養物を、常法にしたがい、遠心分離して菌体を回収し、１０ｍＭリン酸緩衝液（ｐＨ７．０）に懸濁し、超音波処理して菌体を破砕し、さらに遠心分離により不溶物を除去し、上清を採取した。その上清を１０ｍＭリン酸緩衝液に対して透析した後、透析液中のコージビオースホスホリラーゼ活性を測定したところ、培養物１ｌ当たり約５００単位の当該酵素が産生されていた。
【００８９】
第一の対照として、大腸菌ＸＬ１−Ｂｌｕｅ株を、培地にアンピシリンを添加していないこと以外はすべて上述の形質転換体の場合と同一条件で、培養し、培養物から菌体破砕物の上清を採取し、透析した。第二の対照として、サーモアナエロビウム・ブロッキイ（ＡＴＣＣ ３５０４７）を、アンピシリンを含有しないこと以外は上述の形質転換体の場合と同一組成の培地を用い、実験１に示した方法に準じて６０℃で静置培養し、さらに上述の形質転換体の場合と同じく培養物から菌体破砕物の上清を採取し、透析した。第一の対照の透析物には当該酵素活性は全く認められなかった。第二の対照の透析物には当該酵素活性が認められたが、この場合は、培養物１ｌ当たり約１００単位であり、形質転換体ＴＫＰ１の場合に比較して明らかに低い値であった。
【００９０】
この実施例Ａ−４の方法で得た透析物を、さらに実験２に示した方法に準じて、ＤＥＡＥ−トヨパール ６５０ゲル、ウルトロゲル ＡｃＡ４４を用いたカラムクロマトグラフィーに供して精製し、さらにこの精製酵素を実験３に示した方法に準じて分析した。ＳＤＳ−ポリアクリルアミドゲル電気泳動法による分子量８３，０００±５，０００ダルトン、ゲル濾過法による分子量５００，０００±３０，０００ダルトン、等電点ポリアクリルアミドゲル電気泳動法による等電点４．４±０．５、コージビオースホスホリラーゼ活性の至適温度６５℃付近、至適ｐＨ５．５付近、温度安定性は６５℃付近まで、ｐＨ安定性は約５．５乃至１０．０であり、実験１乃至２に示した方法で調製された当該酵素の理化学的性質と実質的に同一であった。以上の結果は、本発明のコージビオースホスホリラーゼは、組換えＤＮＡ技術によっても良好に製造でき、なお且つ酵素の生産性も有意に向上することを示している。
【００９１】
【実施例Ａ−５】
〈酵素液〉
実施例Ａ−３に示した方法で得た形質転換体ＴＫＰ１を実施例Ａ−４に示した方法で培養した。培養液を遠心分離して得た菌体を超音波破砕し、破砕液上清のコージビオースホスホリラーゼ活性を測定した。この活性を培養液１ｍｌ当たりに換算すると、約０．５単位であった。破砕液上清を限外濾過膜にて濃縮し、その濃縮液を透析してｍｌ当たりコージビオースホスホリラーゼ活性約１０単位を有する酵素液を、もとの培養液の総活性に対して約７０％の収率で得た。
【００９２】
【実施例Ａ−６】
〈コージビオース含有糖液〉
マルトースを５％含む２５ｍＭリン酸２カリウム−クエン酸緩衝液（ｐＨ６．０）に、市販の細菌由来マルトースホスホリラーゼをマルトース１ｇ当たり５単位、実施例Ａ−１の方法で調製したコージビオースホスホリラーゼをマルトース１ｇ当たり４０単位になるように加え、３０℃で７２時間反応させた。その反応液を１００℃で３０分間加熱し、酵素を失活させた後、冷却し、常法に従って活性炭で脱色、濾過し、Ｈ型及びＯＨ型イオン交換樹脂により脱塩して精製し、更に濃縮して濃度約７５％のシラップ状のコージビオース含有糖液を原料固形物当たり収率約９５％で得た。
【００９３】
本品は、コージビオースを固形物当たり約３０％含有するとともにコージビオシルグルコースをも含有しており、味質良好な甘味、適度の粘度、保湿性を有し、甘味剤、呈味改良剤、安定剤、ビフィズス菌増殖促進剤、ミネラル吸収促進剤などとして広く飲食物、化粧品、医薬品、成形物など各種組成物に有利に利用できる。
【００９４】
【実施例Ａ−７】
〈コージビオース高含有粉末〉
実施例Ａ−６の方法で反応、精製した固形物当たり約３０％のコージビオースを含有する糖液を原料として固形物濃度約２０％に調整し、これにグルコアミラーゼを固形物１ｇ当たり５単位加えてｐＨ４．５、４０℃で１６時間反応させ残存するマルトースを分解した。１００℃で３０分加熱して反応を停止した後、濃度約４０％まで濃縮した。本糖液中のコージビオース含有率を高めるため、アルカリ金属型強酸性カチオン交換樹脂（東京有機化学工業株式会社製、商品名『ＸＴ−１０１６』、Ｎａ型、架橋度４％）を使用し、内径３ｃｍ、長さ１ｍのジャケット付きステンレス製カラム４本に水懸濁状で充填し、直列につなぎ、樹脂層全長を約４ｍになるようにした。カラム内温度を４０℃に維持しつつ、糖液を樹脂に対して５ｖ／ｖ％加え、これに４０℃の温水をＳＶ０．１５の流速で流して分画し、コージビオース高含有画分を採取した。更に、精製、濃縮し、真空乾燥し、粉砕して、コージビオース高含有粉末を原料固形物当たり収率約２０％で得た。
【００９５】
本品は、固形物当たり約９０％のコージビオースを含有しており、味質良好な甘味、適度の保湿性を有し、甘味剤、呈味改良剤、安定剤、ビフィズス菌増殖促進剤、ミネラル吸収促進剤などとして広く飲食物、化粧品、医薬品、成形物など各種組成物に有利に利用できる。
【００９６】
【実施例Ａ−８】
〈グルコシルソルボース含有糖液〉
β−Ｄ−グルコース−１リン酸及びＬ−ソルボースをそれぞれ５％含む水溶液をｐＨ５．５に調整し、これに実施例Ａ−１の方法で調製したコージビオースホスホリラーゼをβ−Ｄ−グルコース−１リン酸１ｇ当たり１０単位になるように加え、６０℃で７２時間反応させた。その反応液を９０℃で３０分間加熱し、酵素を失活させた後、冷却し、常法に従って活性炭で脱色、濾過し、Ｈ型及びＯＨ型イオン交換樹脂により脱塩して精製し、更に濃縮して濃度約７５％のシラップ状のグルコシルソルボース含有糖液を原料固形物当たり収率約９５％で得た。
【００９７】
本品は、固形物当たりグルコシルソルボースを約３０％含有しており、味質良好な甘味、適度の粘度、保湿性を有し、甘味剤、呈味改良剤、安定剤、ビフィズス菌増殖促進剤、ミネラル吸収促進剤などとして広く飲食物、化粧品、医薬品、成形物など各種組成物に有利に利用できる。
【００９８】
【実施例Ａ−９】
〈グルコシルソルボース高含有糖液〉
β−Ｄ−グルコース−１リン酸を５％及びＬ−ソルボースを１０％含む水溶液をｐＨ６．０に調整し、これに実施例Ａ−１の方法で調製したコージビオースホスホリラーゼをβ−Ｄ−グルコース−１リン酸１ｇ当たり３０単位になるように加えて、６０℃で７２時間反応させた。反応後冷却し、市販パン酵母を固形物重量当たり湿重量で５％になるように加え、１規定の水酸化ナトリウム溶液を用いて該反応液をｐＨ５乃至６に制御しながら２７℃で６時間反応させた。その反応液を９０℃で３０分間加熱し、反応を停止させた後、冷却し、常法に従って活性炭で脱色、濾過し、Ｈ型及びＯＨ型イオン交換樹脂により脱塩して精製し、更に濃縮して濃度約７５％のシラップ状のグルコシルソルボース含有糖液を原料固形物当たり収率約６５％で得た。
【００９９】
本品は、固形物当たりグルコシルソルボースを約４０％含有しており、味質良好な甘味、適度の粘度、保湿性を有し、甘味剤、呈味改良剤、安定剤、ビフィズス菌増殖促進剤、ミネラル吸収促進剤などとして広く飲食物、化粧品、医薬品、成形物など各種組成物に有利に利用できる。
【０１００】
【実施例Ａ−１０】
〈グルコシルソルボース高含有粉末〉
実施例Ａ−９の方法で反応、精製した固形物当たり約４０％のグルコシルソルボースを含有する糖液を原料として、これを固形物濃度約４５％に調整した。本糖液中のグルコシルソルボース含有率を高めるため、アルカリ金属型強酸性カチオン交換樹脂（東京有機化学工業株式会社製、商品名『ＸＴ−１０１６』、Ｎａ型、架橋度４％）を使用し、内径３ｃｍ、長さ１ｍのジャケット付きステンレス製カラム４本に水懸濁状で充填し、直列につなぎ、樹脂層全長を約４ｍになるようにした。カラム内温度を４０℃に維持しつつ、糖液を樹脂に対して５ｖ／ｖ％加え、これに４０℃の温水をＳＶ０．１５の流速で流して分画し、グルコシルソルボース高含有画分を採取した。更に、精製、濃縮し、真空乾燥し、粉砕して、グルコシルソルボース高含有粉末を原料固形物当たり収率約２５％で得た。
【０１０１】
本品は、固形物当たり約９０％のグルコシルソルボースを含有しており、味質良好な甘味、適度の保湿性を有し、甘味剤、呈味改良剤、安定剤、ビフィズス菌増殖促進剤、ミネラル吸収促進剤などとして広く飲食物、化粧品、医薬品、成形物など各種組成物に有利に利用できる。
【０１０２】
【実施例Ａ−１１】
〈コージビオシルフラクトシド含有糖液〉
コージビオースを１０％及びスクロースを１０％含む水溶液を調整し、これに実施例Ａ−１の方法で調製したコージビオースホスホリラーゼをコージビオース１ｇ当たり４０単位になるように加え、５ｍＭリン酸二水素ナトリウム塩存在下ｐＨ５．０、６０℃で７２時間反応させた。その反応液を９０℃で３０分間加熱し、酵素を失活させた後、冷却し、常法に従って活性炭で脱色、濾過し、Ｈ型及びＯＨ型イオン交換樹脂により脱塩して精製し、更に濃縮して濃度約７５％のシラップ状のコージビオシルフラクトシド含有糖液を原料固形物当たり収率約９５％で得た。
【０１０３】
本品は、固形物当たり約５５％のコージビオシルフラクトシドを含有しており、味質良好な甘味、適度の粘度、保湿性を有し、飲食物、化粧品、医薬品、成形物など各種組成物に有利に利用できる。
【０１０４】
【実施例Ａ−１２】
〈コージビオシルフラクトシド高含有粉末〉
実施例Ａ−１１の方法で反応、精製した、固形物当たり約５５％のコージビオシルフラクトシドを含有する糖液を原料として、これを固形物濃度約４５％に調整した。本糖液中のコージビオシルフラクトシド含有率を高めるため、アルカリ土類金属型強酸性カチオン交換樹脂（ダウケミカル社販売、商品名『ダウエックス５０Ｗ×４』、Ｃａ型）を用いた以外は、実施例Ａ−１０の方法に従ってカラムクロマトグラフィーを行い、コージビオシルフラクトシド高含有画分を採取した。更に、精製、濃縮し、真空乾燥し、粉砕して、コージビオシルフラクトシド高含有粉末を原料固形物当たり収率約４０％で得た。
【０１０５】
本品は、固形物当たり約９５％のコージビオシルフラクトシドを含有しており、味質良好な甘味、適度の保湿性を有し、飲食物、化粧品、医薬品、成形物など各種組成物に有利に利用できる。
【０１０６】
【実施例Ａ−１３】
〈コージビオシルグルコース含有糖液〉
β−Ｄ−グルコース−１リン酸を１０％及びマルトースを２０％含む水溶液をｐＨ５．０に調整し、これに実施例Ａ−１の方法で調製したコージビオースホスホリラーゼをマルトース１ｇ当たり４０単位になるように加え、６０℃で７２時間反応させた。その反応液を９０℃で３０分間加熱し、酵素を失活させた後、冷却し、常法に従って活性炭で脱色、濾過し、Ｈ型及びＯＨ型イオン交換樹脂により脱塩して精製し、更に濃縮して濃度約７５％のシラップ状のコージビオシルグルコース含有糖液を原料固形物当たり収率約９５％で得た。
【０１０７】
本品は、固形物当たり約４５％のコージビオシルグルコースを含有しており、味質良好な甘味、適度の粘度、保湿性を有し、飲食物、化粧品、医薬品、成形物など各種組成物に有利に利用できる。
【０１０８】
【実施例Ａ−１４】
〈コージビオシルグルコシド高含有糖液〉
β−Ｄ−グルコース−１リン酸を５％、トレハロースを１０％含む水溶液をｐＨ５．０に調整し、これに実施例Ａ−１の方法で調製したコージビオースホスホリラーゼをβ−Ｄ−グルコース−１リン酸１ｇ当たり２０単位になるように加え、６０℃で７２時間反応させた。その反応液に水酸化ナトリウムを加えてｐＨ１０以上のアルカリ性に保ちながら１００℃で加熱した後、冷却し、常法に従って活性炭で脱色、濾過し、Ｈ型及びＯＨ型イオン交換樹脂により脱塩して精製し、更に濃縮して濃度約７５％のシラップ状のコージビオシルグルコシド高含有糖液を原料固形物当たり収率約６０％で得た。
【０１０９】
本品は、固形物当たり約９５％のコージビオシルグルコシドを含有しており、味質良好な甘味、適度の粘度、保湿性を有し、飲食物、化粧品、医薬品、成形物など各種組成物に有利に利用できる。
【０１１０】
【実施例Ａ−１５】
〈コージビオース含有糖液〉
マルトースを５％含む２５ｍＭリン酸２カリウム−クエン酸緩衝液（ｐＨ６．０）に、市販の細菌由来マルトースホスホリラーゼをマルトース１ｇ当たり５単位、実施例Ａ−５の方法で調製したコージビオースホスホリラーゼをマルトース１ｇ当たり４０単位になるように加え、３０℃で７２時間反応させた。その反応液を１００℃で３０分間加熱し、酵素を失活させた後、冷却し、常法に従って活性炭で脱色、濾過し、Ｈ型及びＯＨ型イオン交換樹脂により脱塩して精製し、更に濃縮して濃度約７５％のシラップ状のコージビオース含有糖液を原料固形物当たり収率約９５％で得た。
【０１１１】
本品は、コージビオースを固形物当たり約３０％含有するとともにコージビオシルグルコースをも含有しており、味質良好な甘味、適度の粘度、保湿性を有し、甘味剤、呈味改良剤、安定剤、ビフィズス菌増殖促進剤、ミネラル吸収促進剤などとして広く飲食物、化粧品、医薬品、成形物など各種組成物に有利に利用できる。
【０１１２】
【実施例Ａ−１６】
〈コージビオシルグルコシド高含有糖液〉
β−Ｄ−グルコース−１リン酸を５％、トレハロースを１０％含む水溶液をｐＨ５．０に調整し、これに実施例Ａ−５の方法で調製したコージビオースホスホリラーゼをβ−Ｄ−グルコース−１リン酸１ｇ当たり２０単位になるように加え、６０℃で７２時間反応させた。その反応液に水酸化ナトリウムを加えてｐＨ１０以上のアルカリ性に保ちながら１００℃で加熱した後、冷却し、常法に従って活性炭で脱色、濾過し、Ｈ型及びＯＨ型イオン交換樹脂により脱塩して精製し、更に濃縮して濃度約７５％のシラップ状のコージビオシルグルコシド高含有糖液を原料固形物当たり収率約６０％で得た。
【０１１３】
本品は、固形物当たり約９５％のコージビオシルグルコシドを含有しており、味質良好な甘味、適度の粘度、保湿性を有し、飲食物、化粧品、医薬品、成形物など各種組成物に有利に利用できる。
【０１１４】
【実施例Ｂ−１】
〈甘味剤〉
実施例Ａ−１０の方法で得たグルコシルソルボース高含有粉末１重量部に対し、α−グリコシルステビオシド（東洋精糖株式会社製造、商品名α−Ｇスィート）０．０５重量部を加えて均一に混合して粉末甘味剤を製造した。本品は、上品な甘味で、砂糖の約２倍の甘味を有し、カロリーは甘味度当たり砂糖の約２分の１となる。したがって、この甘味剤は低カロリー甘味料として、カロリーの摂取を制限している人、例えば、肥満者、糖尿病者などのための低カロリー飲食物に対する味付けに好適である。また、この甘味剤は、虫歯誘発菌による酸の生成も少なく、不溶性グルカンの生成も少ないことにより、虫歯を抑制する飲食物などの味付けにも好適である。
【０１１５】
【実施例Ｂ−２】
〈ハードキャンディー〉
実施例Ａ−８の方法で得たグルコシルソルボース含有糖液３０重量部に対し、還元麦芽糖水飴（水分２５％）８０重量部を加えて混合溶解し、減圧下で水分が２％未満になるまで濃縮し、これにクエン酸１重量部及び適量のレモン香料と着色料とを混和し、次いで、常法に従って成形しハードキャンディーを製造した。本品は、上品な甘味を有し、吸湿性少なく、ダレを起こしにくい歯切れの良いハードキャンディーである。
【０１１６】
【実施例Ｂ−３】
〈チューインガム〉
実施例Ａ−１２の方法で得たコージビオシルフラクトシド高含有粉末４重量部に対し、グルコース３重量部および、柔らかくなる程度に加熱溶融したガムベース２重量部を加えて混合し、さらに適量のハッカ香料を混合した後、常法に従ってロールにより練り合わせ成形することによってチューインガムを製造した。本品は、テクスチャー、風味ともに優れた製品である。
【０１１７】
【実施例Ｂ−４】
〈チョコレート〉
実施例Ａ−７の方法で得たコージビオース高含有粉末１５重量部に対し、カカオペースト４０重量部、カカオバター１０重量部、蔗糖１０重量部及び全脂粉乳１５重量部を加えて混合し、レファイナーを通した。そして粒度を下げた後、コンチェに入れ、レシチン０．５重量部を加えて、５０℃で二昼夜練り上げた。次いで、常法に従い成型機に流し込み成型固化してチョコレートを製造した。本品は、ファットブルーム、シュガーブルームの恐れがなく、舌にのせた時の融け具合、風味ともに良好である。
【０１１８】
【実施例Ｂ−５】
〈カスタードクリーム〉
実施例Ａ−１０の方法で得たグルコシルソルボース高含有粉末４００重量部に対し、コーンスターチ５００重量部、マルトース５００重量部及び食塩５重量部を加え、篩いを通して充分に混合し、さらに鶏卵１，４００重量部を加えて攪拌し、これに沸騰した牛乳５，０００重量部を徐々に加え、さらにこれをとろ火にかけて攪拌を続け、コーンスターチが完全に糊化して全体が半透明になったとき火を止め、これを冷却して少量のバニラ香料を加えることによりカスタードクリームを製造した。本品は、なめらかで光沢を有し、甘味が強すぎず美味である。
【０１１９】
【実施例Ｂ−６】
〈ういろう〉
実施例Ａ−１１の方法で得たコージビオシルフラクトシド含有糖液９０重量部に対し、米粉９０重量部、コーンスターチ２０重量部、砂糖２０重量部、抹茶粉末１重量部及び水の適量を加えて混練した後、これを容器に入れて６０分間蒸して抹茶ういろうを製造した。本品は、照り、口当たり良好で、風味もよいものであった。また、澱粉の老化も抑制され、長時間安定であった。
【０１２０】
【実施例Ｂ−７】
〈べったら漬〉
実施例Ａ−１３の方法で得たコージビオシルグルコース含有糖液１重量部に対し、マルトース３重量部、甘草製剤０．０５重量部、リンゴ酸０．００８重量部、グルタミン酸ナトリウム０．０７重量部、ソルビン酸カリウム０．０３重量部及びプルラン０．２重量部を均一に混合してべったら漬の素を製造した。大根３０ｋｇを常法に従って食塩により下漬けし、次いで砂糖で中漬けしたものを、ここで得たべったら漬の素４ｋｇで調製した調味液に漬けて、べったら漬を製造した。本品は、色艶、香気ともに良好で、適度の甘味を有し歯切れも良かった。
【０１２１】
【実施例Ｂ−８】
〈乳酸菌飲料〉
実施例Ａ−６の方法で得たコージビオース含有糖液１３０重量部に対し、脱脂粉乳１７５重量部及びラクトスクロース高含有粉末（株式会社林原商事販売、登録商標「乳果オリゴ」）５０重量部を、水１，１５０重量部に溶解し、６５℃で３０分間殺菌し、４０℃に冷却後、これに、常法にしたがって、乳酸菌のスターターを３０重量部植菌し、３７℃で８時間培養して乳酸菌飲料を得た。本品は、風味良好な乳酸菌飲料である。また、本品は、オリゴ糖を含有し、乳酸菌を安定に保持するだけでなく、ビフィズス菌増殖促進作用をも有する。
【０１２２】
【実施例Ｂ−９】
〈合成酒〉
３０ｖ／ｖ％アルコール水溶液６５１８重量部（温度１５℃の場合）、７０％ぶどう糖水溶液６００重量部、実施例Ａ−６の方法で得たコージビオース含有糖液５０重量部、コハク酸１１．１重量部、７５％乳酸水溶液３．６６重量部、グルタミン酸ナトリウム２．３重量部、グリシン１．２重量部、アラニン１．２重量部、コハク酸ナトリウム２．２２重量部、食塩１．６重量部、天然塩１．４重量部及び水２５００重量部を混合して、アルコール含量約２０ｖ／ｖ％の原酒を得た。これを水で希釈して、アルコール含量１５乃至１６ｖ／ｖ％の製品を得た。本品は、まろやかな甘みと上品な風味を有し美味である。
【０１２３】
【実施例Ｂ−１０】
〈乳液〉
実施例Ａ−９の方法で得たグルコシルソルボース高含有糖液３．５重量部に対し、ポリオキシエチレンベヘニルエーテル０．５重量部、テトラオレイン酸ポリオキシエチレンソルビトール１重量部、親油型モノステアリン酸グリセリン１重量部、ベヘニルアルコール０．５重量部、アボガド油１重量部、α−グリコシルルチン１重量部、ビタミンＥ及び防腐剤の適量を加え、常法に従って加熱溶解し、これに１，３−ブチレングリコール５重量部、カルボキシビニルポリマー０．１重量部及び精製水８５．３重量部を加え、ホモゲナイザーにかけて乳化し、乳液を製造した。本品は、保湿性に優れ、日焼け止め、色白剤などとして有利に利用できる。
【０１２４】
【実施例Ｂ−１１】
〈スキンクリーム〉
実験１２の方法で得たコージビオシルフラクトシド高含有粉末４重量部に対し、モノステアリン酸ポリオキシエチレングリコール２重量部、自己乳化型モノステアリン酸グリセリン５重量部、α−グリコシルルチン２重量部、流動パラフィン１重量部、トリオクタン酸グリセリル１０重量部及び防腐剤の適量を加え、常法に従って加熱溶解し、これに１，３−ブチレングリコール５重量部、及び精製水６６重量部を更に加え、ホモゲナイザーにかけて乳化し、香料の適量を加えて攪拌混合し、スキンクリームを製造した。本品は、伸びのよいクリームで、日焼け止め、美肌剤、色白剤などとして有利に利用できる。
【０１２５】
【実施例Ｂ−１２】
〈練歯磨〉
実施例Ａ−１４の方法で得たコージビオシルグルコシド高含有糖液１５重量部に対し、第二リン酸カルシウム重量部、ラウリル硫酸ナトリウム１．５重量部、グリセリン２５重量部、ポリオキシエチレンソルビタンラウレート０．５重量部、サッカリン０．０２重量部、防腐剤０．０５重量部及び水１３重量部と混合して練歯磨を得た。本品は、光沢、洗浄力も良好で、練歯磨として好適である。
【０１２６】
【実施例Ｂ−１３】
〈経管栄養剤〉
実施例Ａ−７の方法で得たコージビオース高含有粉末８０重量部と、乾燥卵黄１９０重量部、脱脂粉乳２０９重量部、塩化ナトリウム４．４重量部、塩化カリウム１．８５重量部、硫酸マグネシウム４重量部、チアミン０．０１重量部、アスコルビン酸ナトリウム０．１重量部、ビタミンＥアセテート０．６重量部及びニコチン酸アミド０．０４重量部からなる配合物を調製した。この配合物２５ｇずつをラミネートアルミ製小包に充填し、ヒートシールして製品を得た。本品は、１袋分を約１５０乃３００ｍｌの水に溶解して栄養補給液とし、経管方法により、鼻腔、食道、胃などへ投与して使用する。
【０１２７】
【実施例Ｂ−１４】
〈錠剤〉
実験８の方法で得たグルコシルソルボース高含有粉末４重量部に対し、アスピリン５０重量部、マルトース１０重量部及びコーンスターチ４重量部を加え、均一に混合した後、直径１２ｍｍ、２０Ｒ杆を用いて１錠６８０ｍｇ、錠剤の厚さ５．２５ｍｍ、硬度８ｋｇ±１ｋｇで打錠した。本品は、適度の甘味を有する飲みやすい錠剤である。
【０１２８】
【実施例Ｂ−１５】
〈いちごジャム〉
生いちご１５０重量部、蔗糖６０重量部、マルトース２０重量部、実施例Ａ−１５の方法で得たコージビオース含有糖液４０重量部、ペクチン５重量部及びクエン酸１重量部を鍋で煮詰め、瓶詰めして製品を得た。本品は風味、色調とも良好なジャムである。
【０１２９】
【実施例Ｂ−１６】
〈加糖練乳〉
原乳１００重量部に実施例Ａ−１６の方法で得たコージビオシルグルコシド高含有糖液３重量部及び蔗糖１重量部を溶解し、プレートヒーターで加熱殺菌し、次いで濃度約７０％に濃縮し、無菌状態で缶詰して製品を得た。本品は温和な甘味で、風味もよく、乳幼児食品、フルーツ、コーヒー、ココア、紅茶などの調味用に有利に利用できる。
【０１３０】
【発明の効果】
叙上のように本発明は、従来知られていなかったコージビオースを生産し得るホスホリラーゼ、すなわち、コージビオースホスホリラーゼの発見に基づくものである。本発明のコージビオースホスホリラーゼは、至適温度、温度安定性における温度が高く、また、ｐＨ安定性におけるｐＨ域が広く、しかも至適ｐＨはそのｐＨ域内にあり、更に産生微生物からの酵素生産量も高い。したがって、β−Ｄ−グルコース−１リン酸を糖供与体として各種糖質の存在下で本発明の酵素を作用させれば、従来公知ではあるが入手の極めて困難であったコージビオース等や、新規のグルコシルソルボース等を始めとする各種グルコシル転移糖並びに当該転移糖含有糖質を大量且つ安価に製造することができる。
【０１３１】
この様にして製造されるグルコシル転移糖並びに当該転移糖含有糖質は、上品な甘味の甘味剤、呈味改良剤、品質改良剤、ボディー付与剤、粘度調節剤、保湿剤、照付与剤などとして、さらには、栄養補給剤などとして広く飲食物、化粧品、医薬品、成形物など各種組成物に供しうることが大きな特徴であることから、本発明は、食品、化粧品、医薬品分野のみならず、農水畜産業や、化学工業等の産業界に貢献すること誠に多大な意義のある発明といえる。
【０１３２】
【配列表】

【０１３３】

【０１３４】

【０１３５】

【０１３６】

【０１３７】

【０１３８】

【０１３９】

【０１４０】

【図面の簡単な説明】
【図１】本発明のコージビオースホスホリラーゼの酵素活性に及ぼす温度の影響を示す図である。
【図２】本発明のコージビオースホスホリラーゼの酵素活性に及ぼすｐＨの影響を示す図である。
【図３】本発明のコージビオースホスホリラーゼの安定性に及ぼす温度の影響を示す図である。
【図４】本発明のコージビオースホスホリラーゼの安定性に及ぼすｐＨの影響を示す図である。
【図５】本発明の組換えＤＮＡの制限酵素地図を示す図である。図中矢印は、本発明のコージビオースホスホリラーゼをコードするＤＮＡを示す。
【表２】

【表２】

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel enzyme cordobiose phosphorylase, a method for producing the same, and uses thereof. More specifically, inorganic phosphate and / or a salt thereof (hereinafter referred to as “inorganic phosphate” throughout the present specification unless inconvenience occurs). In the presence of D-glucose and β-D-glucose-1-phosphate and / or a salt thereof (hereinafter referred to as “β-D-glucose unless otherwise noted”). -1 phosphate ")), and conversely, a novel enzyme cordobiose phosphorylase which has the action of producing cordobiose and inorganic phosphate from β-D-glucose-1 phosphate and D-glucose. And its production method, glucosyltransferase-containing saccharides produced using this cordobiose phosphorylase, and these saccharides are contained. It relates to a composition.
[0002]
[Prior art]
In recent years, oligosaccharides such as maltose and trehalose and their functions have attracted attention, and various production methods of these oligosaccharides have been widely studied from various fields. As methods for producing these oligosaccharides, utilization of various phosphorylases such as maltose phosphorylase, trehalose phosphorylase, sucrose phosphorylase, cellobiose phosphorylase, laminaribiose phosphorylase is known. Various microorganisms are known as a source of these phosphorylases.
[0003]
These phosphorylases, their actions and source microorganisms are summarized in “Enzyme Handbook” Asakura Shoten (1982). However, phosphorylase capable of producing cordobiose is not yet known, and its provision is desired.
[0004]
[Problems to be solved by the invention]
The present invention provides a novel enzyme, cordobiose phosphorylase, as well as a method for producing the enzyme, a glucosyltransferase-containing saccharide using the enzyme, and a use thereof.
[0005]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the present inventors searched for an unknown cordierbiose phosphorylase and searched for microorganisms that produce the enzyme. As a result, the microorganism Thermoanaerobium brokii (ATCC 35047) belonging to the genus Thermoanaerobium has been found to produce a novel enzyme, cordobiose phosphorylase, and a method for producing this enzyme Established. Furthermore, it contains a novel and / or known glucosyl transfer saccharide-containing saccharide obtained by allowing this enzyme to act in the presence of various saccharides using β-D-glucose-1-phosphate as a sugar donor, and contains the saccharide A method for producing the caulked composition was established to complete the present invention.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
The cordobiose phosphorylase of the present invention includes all enzymes having the action of degrading cordobiose in the presence of inorganic phosphate to produce D-glucose and β-D-glucose-1 phosphate, The origin and origin are not questioned. Examples of such cordobiose phosphorylase include enzymes that exhibit the following actions and have physicochemical properties.
(1) Action
(A) Cozybiose is decomposed in the presence of inorganic phosphate to produce D-glucose and β-D-glucose-1 phosphate.
(B) Cozybiose and inorganic phosphoric acid are produced from β-D-glucose-1-phosphate and D-glucose, and β-D-glucose-1 phosphate is used as a sugar donor, and other carbohydrates have glucosyl groups. Catalyzes the transfer of
(2) Molecular weight
83,000 ± 5,000 daltons by SDS-gel electrophoresis.
(3) Isoelectric point
PI 4.4 ± 0.5 by an ampholine-containing electrophoresis.
(4) Optimal temperature
pH 5.5, around 65 ° C. for 30 minutes reaction.
(5) Optimum pH
The pH was around 5.5 after reaction at 60 ° C for 30 minutes.
(6) Temperature stability
Stable up to around 65 ° C under pH 5.5 and 1 hour holding conditions.
(7) pH stability
PH of about 5.5 to 10.0 at 4 ° C. for 24 hours.
(8) Inhibition
This enzyme activity is inhibited by 1 mM Hg ++.
[0007]
More specifically, such a cordobiose phosphorylase includes, for example, the amino acid sequence shown in SEQ ID NO: 1, 2, or 3 in the sequence listing as a partial amino acid sequence, and the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing as a whole Examples thereof include proteins and functional derivatives thereof. As used herein, the functional derivative means a substance obtained by substituting one or more of the amino acids in the amino acid sequence with another amino acid within a range that does not substantially lose the action as the above-described cordobiose phosphorylase, the amino acid sequence In which one or more amino acids are added to the N-terminal and / or C-terminal, in which one or more amino acids are inserted in the middle of the amino acid sequence, and in the N-terminal and / or C-terminal It means one in which one or more amino acids have been deleted and one in which one or more amino acids in the middle of the amino acid sequence have been deleted. The above-described cordobiose phosphorylase of the present invention is a mutant obtained by treating the microorganism with a mutagen or the like, even if it is isolated from a natural source such as a culture of the microorganism producing the enzyme. It may be separated from the culture or may be artificially synthesized by applying a recombinant DNA technique or a peptide synthesis method. As will be described in detail later, according to the production method of the present invention, the cordobiose phosphorylase can be obtained in a favorable manner both from a natural source and when recombinant DNA technology is applied. .
[0008]
The DNA of the present invention includes all of the DNAs encoding the above-described cordobiose phosphorylase of the present invention. Examples of such DNA include DNA containing the base sequence shown in SEQ ID NO: 5 in the sequence listing, which encodes the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing, and functional derivatives thereof. As used herein, the functional derivative means that one or more of the bases in the base sequence are replaced with other bases as long as the protein encoded by the DNA does not substantially lose its action as the above-mentioned cordobiose phosphorylase. In which 1 or more bases are added to the 5 ′ end and / or 3 ′ end of the base sequence, in which 1 or more bases are inserted in the middle of the base sequence, in the base sequence Those with one or more bases at the 5 'and / or 3' ends deleted, those with one or more bases in the base sequence deleted, and those with a base sequence complementary to these base sequences Means. Such functional derivatives include, for example, a base sequence in which one or more of the bases are substituted with other bases in the base sequence shown in SEQ ID NO: 5 in the sequence listing without changing the amino acid sequence encoded by the base sequence. The DNA consisting of The DNA of the present invention includes not only the DNA as described above, but also DNA other than the base sequence encoding the cordobiose phosphorylase at the 5 ′ end and / or 3 ′ end of the DNA, such as a start codon. In addition, DNA comprising one or more selected from a termination codon, a Shine-Dalgarno sequence, a base sequence encoding a signal peptide, a recognition sequence by an appropriate restriction enzyme, a promoter, an enhancer, a terminator, and the like is also included.
[0009]
In the present invention, the source and preparation method of such DNA are not limited. For example, natural sources of such DNA include microorganisms of the genus Thermoanaerobium, including Thermoanaerobium brokkii (ATCC 35047), and bacteria obtained by culturing the microorganisms by a conventional method If the DNA fraction is collected from the crushed material, DNA containing the DNA of the present invention can be obtained. The DNA thus obtained can be used in the present invention by itself, but when the DNA of the present invention is obtained as a recombinant DNA obtained by inserting a fragment containing the DNA into a vector capable of autonomous replication. This is extremely advantageous in practicing the present invention. The recombinant DNA is prepared by, for example, applying a general recombinant DNA technique to the DNA obtained as described above to produce a gene library, and the base sequence of the DNA encoding the cordobiose phosphorylase of the present invention, For example, it can be obtained by applying a hybridization method or the like that selects a target recombinant DNA from the gene library based on the base sequence shown in SEQ ID NO: 5 in the sequence listing. The recombinant DNA thus obtained is amplified when a transformant obtained by introduction into an appropriate host such as Escherichia coli is cultured, and if a general alkaline-SDS method or the like is applied to the culture, the present invention The desired amount of DNA can be obtained very easily. On the other hand, using the crushed cell of the microorganism or the DNA recovered therefrom as a template, using the DNA chemically synthesized based on the nucleotide sequence shown in SEQ ID NO: 5 in the sequence listing as a primer, and applying the PCR method according to a conventional method Alternatively, the DNA of the present invention can also be easily obtained by chemically synthesizing a DNA containing the base sequence shown in SEQ ID NO: 5 in the sequence listing. In order to obtain the DNA of the present invention as the above-mentioned functional derivative, for example, a site-directed mutagenesis method is applied to the recombinant DNA, or the recombinant DNA is used as a template and converted into a desired sequence. It can be obtained by applying a PCR method using a chemically synthesized DNA containing the nucleotide sequence as a primer.
[0010]
Further, the DNA of the present invention includes a recombinant DNA inserted into an autonomously replicable vector. As described above, such a recombinant DNA is not only extremely useful for obtaining the DNA of the present invention, but also very useful for producing the cordobiose phosphorylase of the present invention as described in detail later. . Such a recombinant DNA is relatively easy to obtain by inserting the DNA into a desired vector once the target DNA is obtained as described above, and usually by applying a general recombinant DNA technique. It is. The vector into which the DNA of the present invention can be inserted may be any vector as long as it has the property of autonomous replication in an appropriate host. For example, pUC18, Bluescript II SK (+), pKK223 using Escherichia coli as a host. PHY300PLK, pHV14, TRp7, YEp7, pBS7, etc. using two or more types of hosts such as p-3, λgt, λC, etc., such as pUB110, pTZ4, pC194, ρ11, φ1, φ105, etc. using Bacillus subtilis as the host are all advantageously used. be able to. An example of a method for inserting the DNA of the present invention into such a vector will be described. First, the DNA of the present invention obtained as described above or a DNA containing the DNA and an appropriate vector are cleaved with a restriction enzyme, Next, the generated DNA fragment and the vector fragment are ligated. Examples of restriction enzymes used here include Acc I, Bam HI, Bgl II, Bst XI, Eco RI, Hind III, Not I, Pst I, Sac I, Sal I, Sma I, Spe I, Xba I, Any of Xho I and the like can be advantageously used. Further, when ligating the DNA fragment and the vector fragment, it is optional to intervene chemically synthesized DNA having an appropriate restriction enzyme recognition sequence or the like, if necessary. For DNA ligation, DNA ligase may be allowed to act inside or outside the cell after annealing both.
[0011]
Furthermore, the DNA of the present invention includes a form of a transformant introduced into an appropriate host microorganism. Such a transformant is extremely useful for obtaining the cordobiose phosphorylase of the present invention and the DNA of the present invention. As the host microorganism of such a transformant, for example, any of Escherichia coli, Bacillus subtilis, actinomycetes, yeast and the like can be advantageously used. Such a transformant is usually obtained by introducing the recombinant DNA obtained as described above into an appropriate host. Specifically, when the host is Escherichia coli, the host is treated with the recombinant DNA and calcium. The culture may be performed in the presence of ions. On the other hand, when the host is Bacillus subtilis, a competent cell method or a protoplast method may be applied. Each of the individual methods for obtaining the DNA of the present invention described above is conventional in the art. For example, J. Sambrook et al., “Molecular Cloning a Laboratory Manual”, 2nd edition, 1989. It is described in detail in the issue of Cold Spring Harbor Laboratory.
[0012]
The method for producing Kojibiose phosphorylase according to the present invention is characterized by culturing a microorganism capable of producing the enzyme and collecting the enzyme from the culture. It doesn't matter. Examples of such microorganisms include, for example, microorganisms belonging to the genus Thermoanaerobium, more preferably Thermoanaerobium brokki (ATCC 35047), and transformants obtained by introducing the DNA of the present invention into an appropriate host microorganism. Can be mentioned.
[0013]
In the production method of the present invention, the medium used for culturing the microorganism may be any medium that can grow the microorganism and produces the enzyme of the present invention, and may be either a synthetic medium or a natural medium. As the carbon source, any substance that can be assimilated by microorganisms may be used. For example, sugars such as maltose, trehalose, dextrin, starch, molasses, and sugar-containing substances such as yeast extract may be used. it can. The concentration of these carbon sources in the medium is appropriately selected depending on the type of carbon source. For example, the sugar concentration in the culture solution is desirably 20 w / v% or less, and usually 5 w / v% or less is preferable from the growth and proliferation of bacteria. Examples of the nitrogen source include inorganic nitrogen compounds such as ammonium salts and nitrates, and organic nitrogen-containing materials such as urea, corn steep liquor, casein, peptone, yeast extract and meat extract. In addition, as the inorganic component, for example, calcium salt, magnesium salt, potassium salt, sodium salt, phosphate, manganese salt, zinc salt, iron salt, copper salt, molybdenum salt, cobalt salt, etc. are used as necessary. It is done.
[0014]
For the culture, the conditions under which the microorganisms used for production grow well can be appropriately selected and applied. For example, when using the aforementioned microorganism belonging to the genus Thermoanaerobium, the temperature is usually 50 to 80 ° C., preferably 60 to 70 ° C., pH 5 to 8, preferably pH 6.5 to 7.5, Done anaerobically. The culture time may be a time during which the microorganism can grow, and is preferably 10 to 50 hours. On the other hand, when the above-described transformant microorganism is used, although it depends on the microorganism species, the culture time is usually maintained under aerobic conditions such as aeration stirring while maintaining the temperature at 20 to 65 ° C. and pH 2 to 9. It may be about 1 to 6 days.
[0015]
In this way, after culturing the microorganism, the enzyme of the present invention is recovered from the obtained culture. This enzyme activity is usually observed in the microbial cells of the culture, and the microbial cells or the treated microbial cells may be recovered as a crude enzyme, or the entire culture can be used as a crude enzyme. A normal solid-liquid separation means is employed for separation of the extracellular culture solution and the bacterial cells. For example, a means for centrifuging the culture itself as it is, a means for adding a filter aid to the culture or pre-coating for filtration separation, a means for membrane filtration separation using a flat membrane, a hollow fiber membrane, etc. are adopted. Is done. The bacterial cells or the processed bacterial cells can be used as a crude enzyme solution as they are. Further, if necessary, it can be used as a partially purified enzyme. For example, in the case of a transformant using Bacillus subtilis or the like as a host, the enzyme may be produced in the medium used for the culture depending on the type of recombinant DNA used for the transformation. In such a case, the culture supernatant can be used as a crude enzyme.
[0016]
Examples of the treated cells include dried cells, freeze-dried products, surfactant-treated products, enzyme-treated products, ultrasonic crushed materials, mechanically ground products, mechanical pressure-treated products, and bacterial cell protein fractions. Minute, microbial cells or immobilized microbial cells. As the enzyme, either a crude enzyme or a purified enzyme is used, and the processed bacterial cells are usually used for the purification of the enzyme, for example, ammonium sulfate salting out method, acetone and alcohol precipitation method, flat membrane, hollow fiber membrane, etc. Membrane concentration and dialysis methods are used.
[0017]
Furthermore, the bacterial cells and the treated bacterial cells can be immobilized by ordinary means. For example, a binding method with an ion exchanger, a covalent bond / adsorption method with a resin and a membrane, a comprehensive method using a polymer substance, and the like are employed.
[0018]
The crude enzyme may be used as it is, but can also be purified by ordinary means. As an example, after dialysis of a crude enzyme preparation obtained by subjecting a cell-treated product to ammonium sulfate salting out and concentration, anion exchange column chromatography using DEAE-Toyopearl resin (manufactured by Tosoh Corporation), followed by CM-Toyopearl resin Cation exchange column chromatography using Tosoh Corp., hydrophobic column chromatography using butyl Toyopearl resin (Tosoh Corp.), gel filtration using Ultrogel AcA44 resin (Sepracol, France) A single enzyme can be obtained electrophoretically by column chromatography.
[0019]
The activity of the cordobiose phosphorylase of the present invention is measured as follows. After adding 0.2 ml of the enzyme solution to 2 ml of 20 mM McKilvein buffer (pH 5.5) containing 1.0 w / v% cordobiose as a substrate and reacting at 60 ° C. for 30 minutes, 0.5 ml of the reaction solution was added at 100 ° C., 10 ° C. Heat for minutes to stop the reaction. To this reaction stop solution, 0.5 ml of D-glucose oxidase / peroxidase reagent is added and stirred, and left at 40 ° C. for 30 minutes, then 2.5 ml of 5N hydrochloric acid is added and stirred, and the absorbance at 525 nm is measured. One unit of enzyme activity is defined as the amount of enzyme that produces 1 μmol of D-glucose per minute under the above reaction conditions. In addition, the activities of maltose phosphorylase and trehalose phosphorylase can be measured by the same procedure, except that Kojibiose as a substrate is used in place of maltose and trehalose, respectively.
[0020]
The enzyme reaction for producing a glucosyl transfer sugar-containing saccharide using the cordobiose phosphorylase of the present invention is usually performed by using β-D-glucose-1-phosphate as a sugar donor as a substrate and other suitable saccharides. For example, monosaccharides such as D-glucose and L-sorbose, disaccharides such as maltose, cordobiose, trehalose and sucrose, and oligosaccharides composed of trisaccharides such as maltotriose, maltotetraose and maltopentaose. The enzyme is allowed to act as a receptor under conditions as described below. As a result, depending on the receptor used, for example, Codybiose, glucosyl sorbose, Codybiosyl glucose, Cody triose, Codybiosyl glucoside, Codybiosyl fructoside, Codybiosyl maltose, Codybiosyl maltotriose and Various glucosyl transfer sugars including Kojibiosyl maltotetraose can be produced. Of these transfer sugars, glucosyl sorbose and kojibiosyl fructoside are novel carbohydrates first discovered in the present invention.
[0021]
For β-D-glucose-1-phosphate as a sugar donor, a commercially available reagent may be used as it is, or an appropriate phosphorylase is used as a sugar in the presence of inorganic phosphate and / or a salt thereof. It may be prepared by acting on quality. Specifically, for example, in the presence of inorganic phosphoric acid and / or a salt thereof, Codybiose phosphorylase is allowed to act on Codybiose, Maltose phosphorylase is allowed to act on maltose, or Trehalose phosphorylase is allowed to act on trehalose. It may be prepared. Furthermore, when any of the β-D-glucose-1-phosphate production reaction by the action of these phosphorylases is carried out in the same reaction system as the glucosyl transfer sugar production reaction using the cordobiose phosphorylase, Since β-D-glucose-1-phosphate can be directly supplied to the transfer sugar production reaction system, the production cost can be reduced and the production process can be simplified, which is extremely advantageous. As a receptor, it is also possible to advantageously use two or more of the above saccharides simultaneously. In addition, although inorganic phosphoric acid here includes not only orthophosphoric acid but condensed phosphoric acid, it is usually desirable to use orthophosphoric acid. In addition, the salt herein includes all phosphate ion compounds derived from the above-mentioned inorganic phosphoric acid. Of these, sodium salts and potassium salts with high water solubility are usually used. Is desirable.
[0022]
The substrate concentration in the glucosyl transferase production reaction using the cordobiose phosphorylase of the present invention is not particularly limited. Generally, β-D-glucose-1-phosphate as a sugar donor is 1 to 20 w / w% (hereinafter, unless otherwise specified, w / w% is abbreviated as%). It is preferable to use the receptor as an aqueous solution of about 1 to 20%. On the other hand, as described above, when the β-D-glucose-1-phosphate production reaction by the action of an appropriate phosphorylase is carried out in the same reaction system as the glucosyl transfer sugar production reaction, the following is desirable. That is, in the case of the action of cordobiose phosphorylase, instead of β-D-glucose-1-phosphate which is a substrate for the glucosyl transfer reaction, an aqueous solution of about 1 to 20% cordierbiose is used, and further 0.5 to 20 mM. A phosphate such as sodium dihydrogen phosphate to the extent is allowed to coexist. In the case of other phosphorylase action, instead of β-D-glucose-1-phosphate as a substrate of the glucosyl transfer reaction, for example, about 1 to 20% maltose or trehalose and about 0.5 to 20 mM are used. It is desirable that a phosphate such as sodium dihydrogen phosphate coexists with each other, and that maltose phosphorylase or trehalose phosphorylase be coexistent with each other in an amount of about 0.1 to 50 units per gram of the sugar based on the sugar used. .
[0023]
The reaction temperature may be a temperature at which the enzyme is not inactivated in the presence of the substrate, that is, up to about 70 ° C., preferably in the range of about 15 to 65 ° C. The reaction pH is usually adjusted to a range of about 4.0 to 9.0, preferably a pH of about 5.0 to 7.5. The reaction time may be appropriately selected depending on the progress of the enzyme reaction, and is usually about 0.1 to 100 hours at a use amount of about 0.1 to 50 units per gram of the substrate solid. In addition, as described above, when the β-D-glucose-1-phosphate production reaction by the action of other phosphorylases is carried out in the same reaction system as the glucosyl transfer sugar production reaction using the cordobiose phosphorylase. Is preferably adjusted to a reaction temperature or reaction pH at which any phosphorylase used is not inactivated, depending on the stability of the phosphorylase to be coexisted.
[0024]
Thus, glucosyl transfer sugars are produced in the reaction product depending on the carbohydrate used as the substrate. The production rate varies depending on the substrate concentration of the enzyme reaction, the type of substrate used, the reaction conditions, and the like. For example, when β-D-glucose-1-phosphate with a concentration of 5% and sorbose with a concentration of 25% are used as substrates, the production rate of glucosyl sorbose is about 30%. Throughout the present specification, the production rate means the percentage of the weight of the produced transfer sugar with respect to the total sugar weight in the reaction solution.
[0025]
In addition, in order to increase the glucosyl transfer sugar content in the reaction product as much as possible, an enzyme source having an activity of decomposing and removing D-glucose produced in the reaction solution is coexistent to promote the transfer reaction. it can. In this method, when the β-D-glucose-1-phosphate production reaction by the appropriate phosphorylase described above is carried out in the same system as the glucosyl transfer reaction and a sugar donor is directly supplied, D-by-product generated in the reaction process is produced. It can be particularly advantageously applied to decompose and remove glucose and promote the transfer reaction.
[0026]
Examples of the enzyme source having D-glucose degrading activity include microorganisms having D-glucose degrading activity, cultures of microorganisms, microbial cells or treated cells, or enzymes having D-glucose degrading activity. As the microorganism having D-glucose degrading activity, any microorganism can be used as long as it has a high D-glucose degrading activity and has little or no transfer sugar degrading activity. Preferably, yeast is used. As the enzyme having D-glucose degrading activity, for example, glucose oxidase, catalase, pyranose oxidase, glucose dehydrogenase, glucokinase, hexokinase and the like can be used. Glucose oxidase or catalase is preferably used.
[0027]
The reaction solution in which the glucosyl transfer sugar is produced as described above is subjected to conventional methods such as filtration and centrifugation to remove insoluble matters, followed by decolorization with activated carbon and desalting with H-type and OH-type ion exchange resins. After the purification process, etc., it is concentrated to make a syrup-like product. If necessary, it is optional to further dry it by a method such as spray drying into a powdered product.
[0028]
In addition, the glucosyl transfer saccharide-containing carbohydrate of the present invention can be separated from the enzyme reaction solution and purified to obtain a high content of the glucosyl transfer saccharide. As the method, for example, a method for removing monosaccharides by fermentation using yeast (yeast fermentation method), a method for separating and removing contaminating carbohydrates by membrane filtration, column chromatography and the like can be appropriately employed. In particular, the objective glucosyl is obtained by removing contaminating carbohydrates by column chromatography using a salt-type strongly acidic cation exchange resin disclosed in JP-A-58-23799 and JP-A-58-72598. The method of collecting a fraction having a high transfer sugar content can be advantageously carried out on an industrial scale. At this time, it is optional to employ any of the known fixed floor method, moving floor method, and simulated moving floor method.
[0029]
Thus, the liquid from which the contaminating saccharide has been separated is subjected to a purification process such as decolorization with activated carbon, desalting with H-type or OH-type ion exchange resin, after removing insoluble matter by filtration, centrifugation, etc., by a conventional method. After that, it is concentrated to a syrup product. If necessary, it is optional to further dry it by a method such as spray drying into a powdered product.
[0030]
The glucosyl transfer sugar-containing carbohydrate of the present invention thus obtained usually contains 5% or more, preferably 10% or more, of glucosyl transfer sugar per solid.
[0031]
The glucosyl transfer sugar-containing saccharide produced by the cordobiose phosphorylase of the present invention thus obtained has a good taste and sweetness, and also has osmotic pressure control, moisture retention, illumination, and crystallization prevention. It has properties such as anti-cariogenic properties, bifidobacteria growth promoting properties, and mineral absorption promoting properties, and is widely used in food, beverages, foods, feeds, feeds, cosmetics, and pharmaceuticals. It is advantageously used for various compositions such as articles, molded articles, and other daily necessities, agricultural, forestry and fishery products, reagents, chemical industrial products and the like.
[0032]
Glucosyl transfer sugar-containing saccharide can be used as a seasoning for sweetening as it is, but if necessary, for example, flour, glucose, maltose, trehalose, sucrose, isomerized sugar, honey, maple sugar, One or more other sweeteners such as sorbitol, maltitol, lactitol, dihydrochalcone, stevioside, α-glycosyl stevioside, rebaudioside, glycyrrhizin, L-aspartyl-L-phenylalanine methyl ester, saccharin, glycine, alanine, etc. It may be used by mixing with an appropriate amount of, and if necessary, it can also be used by mixing with a bulking agent such as dextrin, starch, lactose and the like.
[0033]
In addition, the taste of saccharides containing glucosyl transfer sugar is well harmonized with various substances having other tastes such as acidity, salt to taste, astringency, umami, bitterness, etc. It can be advantageously used for sweetening foods, improving taste and improving quality.
[0034]
For example, soy sauce, powdered soy sauce, miso, powdered miso, moromi, hashio, sprinkle, mayonnaise, dressing, vinegar, three cups of vinegar, powdered sushi vinegar, Chinese element, tentsuyu, noodle soup, sauce, ketchup, takuanzuke, Chinese cabbage It can be advantageously used for various seasonings such as nori, yakiniku sauce, curry roux, stew, soup, dashi, compound seasonings, mirin, new mirin, table sugar, coffee sugar.
[0035]
In addition, for example, Japanese rice cakes such as rice crackers, hail, rice cakes, rice cakes, manju, oirou, chanterelles, sheep mushrooms, water sheep rice cakes, brocade balls, jelly, castella, rice cakes, bread, biscuits, crackers, cookies, pie , Pudding, butter cream, custard cream, cream puff, waffle, sponge cake, donut, chocolate, chewing gum, caramel, candy and other Western confectionery, ice cream, sherbet and other ice confectionery, fruit syrup pickled, syrup such as ice honey, flower Pastes such as paste, peanut paste, fruit paste, spread, fruit such as jam, marmalade, syrup pickles, sugar cane, processed foods of vegetables, pickles such as Fukujin pickled, bedara pickled, Senzuke pickled, ryaky pickled, ham, sausage, etc. Livestock meat products, fish Manufactured with fish products such as ham, fish sausage, kamaboko, chikuwa, tempura, various delicacies such as sea urchin, salted squid, vinegared konbu, suki-shime, fugumi-rin, dried fish, shellfish, small fish, shellfish, etc. Stewed food such as rutsuda stew, boiled beans, potato salad, konbu roll, dairy products, fish meat, livestock meat, fruits, bottled vegetables, canned foods, sake, synthetic liquor, liqueur, Western liquor, tea, coffee, cocoa , Juices, carbonated drinks, lactic acid drinks, soft drinks such as lactic acid bacteria drinks, pudding mixes, hot cake mixes, instant foods such as instant soups, instant soups, baby food, therapeutic foods, drinks, cooked rice, noodles, frozen foods It can be advantageously used for sweetening various foods such as food, improving taste, and improving physical properties.
[0036]
Moreover, it can also be used in order to improve the palatability of feed, feed, etc. for domestic animals such as livestock, poultry and fish. In addition, tobacco, toothpaste, lipstick, lip balm, oral solution, tablets, troches, liver oil drop, mouth freshener, mouth fragrance, mouthwash, etc. It can be advantageously used as a sweetener for various compositions, as a taste improver, a taste corrector, and as a quality improver.
[0037]
As a quality improver and stabilizer, it can be advantageously applied to various physiologically active substances that tend to lose the activity of active ingredients or health foods and pharmaceuticals containing them. For example, interferon-α, interferon-β, interferon-γ, tsumore necrosis factor-α, tsumore necrosis factor-β, macrophage migration inhibitory factor, colony stimulating factor, transfer factor, interleukin 1, interleukin 2, Cytokin-containing fluids such as interleukin 6, interleukin 12, interleukin 15 and interleukin 18; hormone-containing fluids such as insulin, growth hormone, prolactin, erythropoietin, tissue plasminogen activator, egg cell stimulating hormone, placental hormone, BCG Vaccine, Japanese encephalitis vaccine, measles vaccine, polio live vaccine, seedling, tetanus toxoid, hub antitoxin, liquid containing biologics such as human immunoglobulin, penicill , Erythromycin, chloramphenicol, tetracycline, streptomycin, kanamycin sulfate and other antibiotic-containing liquids, thiamine, riboflavin, L-ascorbic acid, liver oil, carotenoids, ergosterol, tocopherol, vitamin-containing liquids, lipase, elastase, Enzyme-containing liquids such as urokinase, protease, β-amylase, isoamylase, glucanase, and lactase, ginseng extract, suppon extract, chlorella extract, aloe extract, propolis extract and other extracts, live bacteria such as viruses, lactic acid bacteria, and yeast, Various physiologically active substances such as royal jelly can easily produce stable and high-quality health foods and pharmaceuticals without losing the activity of the active ingredients.
[0038]
As described above, the composition referred to in the present invention includes not only foods and beverages, cosmetics, and pharmaceuticals used orally or parenterally, but also other items such as daily necessities, agricultural, forestry and fishery products, reagents, chemicals, and the like. It has a wide range of uses such as industrial products.
[0039]
In addition, the method of incorporating the glucosyl transfer sugar-containing carbohydrate of the present invention into these compositions may be included in the process until the product is completed, for example, mixing, dissolution, dipping, infiltration, spraying, application Well-known methods such as spraying, pouring, and solidification are appropriately selected. The amount to be contained varies depending on the composition, but generally, an amount of 0.1% or more, desirably 0.5% or more is suitable as the glucosyl transfer sugar.
[0040]
Next, the present invention will be described more specifically by experiments.
[0041]
[Experiment 1]
<Production of Kojibiose phosphorylase>
“ATCC Catalog of Bacteria and Bacteriophages, 18th Edition” (American Type) except that 0.5 w / v% trehalose was used instead of 0.5 w / v% glucose as the carbon source In accordance with the preparation method of Thermoanaerobium brokki medium described in Culture Collection (1992), pages 452 to 456, 100 ml each of the medium was prepared in a 100 ml pressure-resistant bottle and then inoculated with Thermoanaerobium brokki ATCC35047. The seed culture was obtained by static culture at 60 ° C. for 48 hours.
[0042]
About 10 l of medium with the same composition as in the case of seed culture is placed in 4 stainless steel bottles with a volume of 11 l, heat-sterilized and cooled to a temperature of 60 ° C., then seeded with 1 v / v% of the seed culture solution per volume of the medium Then, static culture was performed at a temperature of 60 ° C. for about 40 hours.
[0043]
About 40 l of the culture solution was centrifuged to obtain 92 g of cultured cells. The bacterial cells were suspended in 10 mM phosphate buffer, sonicated, and then centrifuged to obtain a microbial disrupted supernatant. The activity of cordobiose phosphorylase was 0.1 unit / ml in terms of per culture broth.
[0044]
[Experiment 2]
<Purification of Kojibiose phosphorylase>
The cell disruption supernatant obtained in Experiment 1 was concentrated in a UF membrane, and about 360 ml of a concentrated enzyme solution having about 10 units of cordobiose phosphorylase per ml was recovered.
[0045]
Of the resulting concentrated enzyme solution, 300 ml was dialyzed against 10 mM phosphate buffer (pH 7.0) for 24 hours and centrifuged to remove insolubles. The dialysate supernatant (380 ml) DEAE-Toyopearl 650 gel The sample was subjected to ion exchange column chromatography (gel amount: 380 ml) using Tosoh Corporation.
[0046]
Cozybiose phosphorylase of the present invention DEAE-Toyopearl 650 gel And was eluted from the column with a linear gradient from 0 M saline to 0.5 M saline. The enzyme activity fraction eluted with about 0.2M sodium chloride was collected and further purified by the following method. Dialyze the same buffer containing 1.5M ammonium sulfate, centrifuge the dialyzed solution to remove insolubles, and then use hydrophobic column chromatography using Butyl Toyopearl 650 Gel (manufactured by Tosoh Corporation) ( Gel amount 100 ml). The adsorbed cordobiose phosphorylase was eluted from the column with a linear gradient from 1.5 M ammonium sulfate to 0 M ammonium sulfate, and the enzyme activity fraction was recovered.
[0047]
Subsequently, gel filtration chromatography (gel amount: 300 ml) using Ultrogel AcA44 (Sepracol, France) was performed, and the eluted enzyme activity fraction was collected.
[0048]
The recovery rate of the purified enzyme preparation obtained by the above purification means was about 20% based on the cell disruption supernatant in terms of activity. The specific activity of the purified enzyme preparation was 71.4 units per mg of protein. The protein was quantified using the bovine serum albumin as a standard according to the Raleigh method.
[0049]
When the purity of the enzyme preparation was assayed by gel electrophoresis containing 7.5 w / v% polyacrylamide in the purified enzyme preparation, the protein band was a single and high purity preparation.
[0050]
[Experiment 3]
<Properties of cordobiose phosphorylase>
The Kojibiose phosphorylase preparation obtained in Experiment 2 was subjected to SDS-polyacrylamide gel electrophoresis (gel concentration: 10 w / v%) and compared with a molecular weight marker (manufactured by Nippon Bio-Rad Laboratories Co., Ltd.). The molecular weight of the enzyme was measured and found to be 83,000 ± 5,000 daltons. The molecular weight was measured by gel filtration using a TSKgel G4000SW column (φ7.5 mm × 600 mm, manufactured by Tosoh Corporation). As a result, the molecular weight was 500,000 ± 30,000 Dalton.
[0051]
This enzyme is subjected to isoelectric focusing polyacrylamide gel electrophoresis containing 2 w / v% ampholine (manufactured by Pharmacia El Kavy, Sweden) after purification, and the pH of the protein band and gel is measured after electrophoresis. The isoelectric point was found to be 4.4 ± 0.5.
[0052]
The effects of temperature and pH on the cordobiose phosphorylase activity of the present invention were examined according to the activity measurement method. That is, when investigating the influence of temperature, the reaction is carried out at any temperature of about 40 ° C. to about 80 ° C. instead of the reaction temperature of 60 ° C. in the activity measuring method, and when examining the influence of pH, the activity measurement is performed. In place of the buffer used in the method to a pH of about 4 to about 8 Has buffer capacity Reaction was performed using a buffer solution, and then the reaction was stopped in the same manner as in the activity measurement method, and the amount of glucose produced was measured. The results of the measurement results expressed as relative values with respect to the maximum value are shown in FIG. 1 (effect of temperature) and FIG. 2 (effect of pH). The optimum temperature of the enzyme was pH 5.5 at a reaction of 30 minutes and around 65 ° C., and the optimum pH was around 5.5 at a reaction of 60 ° C. and 30 minutes. The temperature stability of this enzyme is determined by maintaining the enzyme activity remaining after 10 mM McKilbain buffer solution (pH 5.5) in which the enzyme is dissolved is kept at a temperature of about 40 ° C. to 80 ° C. for 1 hour and cooled with water. It was determined according to the activity measurement method. Further, the pH stability is adjusted to pH 5.5 after dissolving the enzyme in a buffer solution having a buffer capacity at any pH of about 3.5 to about 10 and holding at 4 ° C. for 24 hours, The remaining enzyme activity was determined according to the activity measurement method. The results of the measurement results expressed as relative values with respect to the maximum values are shown in FIG. 3 (temperature stability) and FIG. 4 (pH stability). The temperature stability of this enzyme was up to around 65 ° C., and the pH stability was about 5.5 to 10.0. The enzyme activity was inhibited by 1 mM Hg ++.
[0053]
[Experiment 4]
<Partial amino acid sequence of cordobiose phosphorylase>
(1) N-terminal amino acid sequence
A portion of the purified enzyme preparation obtained by the method of Experiment 2 was dialyzed against distilled water, and about 40 μg of protein was used as a sample for N-terminal sequence analysis. The N-terminal sequence was analyzed from the N-terminal to 5 residues using a protein sequencer model 473A (Applied Biosystems, USA). The sequence obtained from the N-terminus was the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. Furthermore, when the same sample was used and analyzed in more detail by the same method, the enzyme was found to contain the amino acid sequence shown in SEQ ID NO: 6 in the sequence listing at the N-terminus.
[0054]
(2) Intermediate partial amino acid sequence
A part of the purified enzyme preparation obtained by the method of Experiment 2 was dialyzed against 10 mM Tris / HCl buffer (pH 9.0), and then diluted to a concentration of 1 mg / ml with the same buffer. 10 μg of lysyl endopeptidase (sold by Wako Pure Chemical Industries, Ltd.) was added to this sample solution (1 ml), and the mixture was reacted at 30 ° C. for 22 hours for peptide formation. Reverse phase HPLC was performed to isolate the resulting peptide. A microbonder sphere C18 column (diameter 2.1 mm × length 150 mm, manufactured by Waters, USA) was used, and a flow rate of 0.9 ml / min from a 0.1 v / v% trifluoroacetic acid-0% acetonitrile solution at room temperature was reduced to 0. 120% up to 1v / v% trifluoroacetic acid-48v / v% acetonitrile solution Linear gradient It went on condition of. The peptide eluted from the column was detected by measuring the absorbance at a wavelength of 210 nm. Two peptides, KP15 (retention time of 66 minutes) and KP23 (retention time of 96 minutes), which were well separated from other peptides, were collected and dried in vacuo, and then 200 μl of 0.1 v / v% trifluoroacetic acid-50 v Dissolved in / v% acetonitrile solution. These peptide samples were subjected to a protein sequencer, and amino acid sequences from the N-terminal to 5 residues were analyzed. From peptide KP15, the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing was obtained, and from peptide KP23, the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing was obtained. Furthermore, when analyzed in more detail by the same method using the same sample, peptide KP15 and peptide KP23 contain the amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8 in the N-terminal part thereof, respectively. There was found.
[0055]
[Experiment 5]
<Substrate specificity in the glycolysis reaction of Kojibies phosphorylase>
D-glucose, maltose, sucrose, lactose, trehalose, neotrehalose, cellobiose, melibiose, cordierbiose, isomaltose, sophorose, gentiobiose, nigerose and laminaribiose Codybiose phosphorylase was added in an amount of 10 units per gram of saccharide solid, and allowed to act at 60 ° C. and pH 5.5 for 24 hours in the presence of 5 mM sodium dihydrogen phosphate. The sugar concentration in each reaction solution was 2 w / v%. The reaction solution before and after the enzyme reaction was subjected to thin layer chromatography (hereinafter abbreviated as “TLC”) using Kieselgel 60 (Merck, Inc .; aluminum plate, 20 × 20 cm). TLC was developed once at room temperature using 1-butanol: pyridine: water = 7: 3: 1 (volume ratio) as a developing solvent, and then sprayed with a 20 v / v% sulfuric acid-methanol solution, and about 10 at 110 ° C. Color was developed by heating for minutes. The spots derived from both reaction solutions were compared to determine the presence or absence of enzyme action on each carbohydrate. The results are shown in Table 1.
[0056]
[Table 1]

[0057]
As shown in Table 1, it was found that the cordobiose phosphorylase of the present invention acts on cordobiose with extremely high specificity to produce D-glucose and β-D-glucose-1-phosphate. On the other hand, it was found that it does not act on other carbohydrates.
[0058]
[Experiment 6]
<Receptor specificity in glucosyl transfer saccharide-producing reaction of Cozybee phosphorylase>
An aqueous solution containing any of the various monosaccharides, oligosaccharides and sugar alcohols shown in Table 2 as the acceptor and β-D-glucose-1-phosphate as the sugar donor dissolved in an equal amount by weight of the solid. Purified cordobiose phosphorylase obtained by the method of Experiment 2 was added in units of 10 units per gram of β-D-glucose-1-phosphate, and reacted at 60 ° C. and pH 5.5 for 24 hours. The concentrations of the acceptor and sugar donor in the reaction solution were both 1 w / v%. The reaction solution before and after the enzyme reaction was subjected to TLC in the same manner as in Experiment 5 and then developed. The spots derived from both reaction solutions were compared, and it was determined from the newly generated spots after the reaction whether or not transfer sugars were generated. Further, by visually observing the color intensity of the spot determined to be a transfer sugar, the generation rate of the transfer sugar was relatively evaluated. The results are shown in Table 2.
[0059]
[Table 2]
[0060]
As shown in Table 2, the cordobiose phosphorylase of the present invention comprises β-D-glucose-1 phosphate as a sugar donor, monosaccharides such as D-glucose and L-sorbose, maltose, cordobiose, trehalose, It has been found that glucosyl groups are well transferred to disaccharides such as sucrose and oligosaccharides such as maltotriose, maltotetraose, and maltopentaose to produce trisaccharose. When α-D-glucose-1-phosphate was used as a sugar donor instead of β-D-glucose-1-phosphate, no glucosyl transfer sugar was obtained.
[0061]
The detailed structures of some of the glucosyltransferases that have been clarified to be produced by the transglycosylation reaction using the cordobiose phosphorylase of the present invention by Experiment 6 will be described with reference to Experiments 7 to 12 below. .
[0062]
[Experiment 7]
<Glucosyl transfer sugar from β-D-glucose-1-phosphate and D-glucose>
The sugar component of the enzyme reaction solution using β-D-glucose-1-phosphate and D-glucose as substrates as obtained in Experiment 6 was analyzed by gas chromatography (hereinafter abbreviated as “GLC”). A part of the enzyme reaction solution was dried, dissolved in pyridine, and then trimethylsilylated as an analytical sample for GLC. The GLC column is a stainless steel column (3 mmφ × 2 m) packed with 2% silicon OV-17 / chromosolve W (manufactured by GL Sciences Inc.), the carrier gas is nitrogen gas at a flow rate of 40 ml / min, and the column oven temperature. Was analyzed from 160 ° C. to 320 ° C. at a heating rate of 7.5 ° C./min. For detection, a flame ion detector was used.
[0063]
As a result, the retention time of the peak of glucosyl transfer sugar from β-D-glucose-1-phosphate and D-glucose by the cordobiose phosphorylase of the present invention was consistent with that of known carbohydrate cordobiose. The glucosyl transfer saccharide from β-D-glucose-1-phosphate and D-glucose by the cordobiose phosphorylase of the present invention is judged to be cordobiose.
[0064]
[Experiment 8]
<Glucosyl sorbose>
In order to confirm the glucosyltransferase produced by the transglycosylation reaction to L-sorbose by the cordobiose phosphorylase of the present invention, the glucosyltransferase was produced, isolated, and the structure was examined. First, an aqueous solution containing 2.5% of β-D-glucose-1-phosphate and 2.5% of L-sorbose was adjusted to pH 5.0, and the purified cordobiose phosphorylase obtained by the method of Experiment 2 was added thereto. 10 units per gram of β-D-glucose-1-phosphate were added, reacted at 60 ° C. for 48 hours, and then heated at 100 ° C. for 10 minutes to deactivate the enzyme. In accordance with the method shown in Experiment 7, a part of this reaction completed solution was analyzed by GLC. As a result, it was confirmed that a component having a different retention time was produced in both the β-D-glucose-1-phosphate and L-sorbose in the reaction completion solution, and this component is considered to be the glucosyl transferase. It was. Moreover, the production rate of the glucosyl transferase in this reaction obtained from the analysis result by GLC was about 24%. The remaining amount of the reaction solution is decolorized with activated carbon, filtered, purified by desalting with H-type and OH-type ion exchange resins, concentrated to about 50%, and subjected to column chromatography described below. A fraction containing a high amount of glucosyl transfer sugar was collected.
[0065]
As the fractionation resin, an alkali metal type strongly acidic cation exchange resin (manufactured by Tokyo Organic Chemical Industry Co., Ltd., trade name “XT-1016”, Na type, cross-linking degree 4%) is used, and the inner diameter is 3 cm and the length is 1 m. Four jacketed stainless steel columns were filled with water suspension and connected in series so that the total length of the resin layer was about 4 m. While maintaining the temperature in the column at 40 ° C., the sugar solution is added to the resin at 5 v / v%, and the water is fractionated by flowing warm water at 40 ° C. at a flow rate of SV0.15. Minutes were collected.
[0066]
The fraction with high transfer sugar content was desalted and purified, and chromatographed using a column (YMC Corporation, trade name “YMC-PakODS”) packed with octadecyl silica gel after concentration to about 40%, The glucosyl transfer sugar fraction was collected. Concentrate the collected fraction to a concentration of about 40% and repeat the operation for column chromatography using octadecyl silica gel again. The collected glycosyl transfer sugar-rich solution is desalted, purified, concentrated and vacuum dried. Thus, the high glucosyl transfer sugar-containing powder was obtained at a yield of about 20% per saccharide solid substance subjected to the reaction. When this powder sample was analyzed by GLC according to the method shown in Experiment 7, this sample contained about 98% of the glucosyl transferase per solid.
[0067]
Next, the high glucosyl transfer sugar-containing powder was acid-decomposed and subjected to GLC analysis. As a result, it was found that the glucosyl transferase produced D-glucose and L-sorbose at a molar ratio of about 1: 1 by acid decomposition. Further, the methyl glycosyl transfer sugar-rich powder was methylated and then hydrolyzed with acid, followed by reduction and acetylation. Tetramethyl-1,5-diacetylglucitol was observed. As for the above, the said glucosyl transfer sugar is what D-glucose and L-sorbose couple | bonded by the molar ratio of 1: 1, Furthermore, OH group of 1-position of D-glucose is concerned in the coupling | bonding. Suggests that No methylated derivative of sorbose was observed, but this was probably due to decomposition by acid.
[0068]
In order to investigate the structure of the glucosyl transfer sugar in more detail, 13C-nuclear magnetic resonance spectrum was measured. As a result, 13 C-NMR spectrum (100 MHz, D 2 O): σ ppm from TSP 101.2, 100.4, 99.5, 99.3, 77.1, 75.6, 74.9, 74.5, 74.0 , 73.2, 72.2, 66.3, 63.2, 62.1 were obtained. From this analysis data, the glucosyl transferase is glucosyl sorbose, which is a disaccharide in which the 1-position of D-glucose is linked to the 5-position of L-sorbose in the α-form, that is, α-D-glucosyl (1 → 5) -L- It turned out to be sorbose. This substance is a novel carbohydrate that has not been known so far.
[0069]
[Experiment 9]
<Kojibiosyl glucose>
In order to confirm the glucosyltransferase produced by the transfer reaction to maltose by the cordobiose phosphorylase of the present invention, the glucosyltransferase was produced, isolated, and the structure was examined. First, an aqueous solution containing 5% β-D-glucose-1-phosphate and 10% maltose was adjusted to pH 6.0, and the purified cordierbiose phosphorylase obtained by the method of Experiment 2 was added to β-D-glucose- 20 units were added per gram of phosphoric acid, reacted at 60 ° C. for 48 hours, and then heated at 100 ° C. for 10 minutes to deactivate the enzyme. In accordance with the method shown in Experiment 7, a part of this reaction completed solution was analyzed by GLC. As a result, it was confirmed that a component having a different retention time was produced in both the β-D-glucose-1-phosphate and maltose in the reaction end solution, and this component was considered to be the glucosyl transferase. Moreover, the production rate of the glucosyl transferase in this reaction determined from the analysis result by GLC was about 40%. The remaining total amount of this reaction solution was fractionated and purified in the same manner as in Experiment 7, concentrated, and vacuum dried to obtain the glucosyl transfer sugar-rich powder with a yield of about 20% per saccharide solid substance subjected to the reaction. It was. This powder preparation contained about 98% of the glucosyl transfer sugar per solid.
[0070]
Next, after methylating the said glucosyl transfer sugar high content powder, it hydrolyzed with an acid, and the partial methyl hexitol acetate obtained by reducing and acetylating subsequently was used for the GLC analysis. As a result, 2,3,4,6-tetramethyl-1,5-diacetylglucitol and 3,4,6-trimethyl-1,2,5-triacetylglucitol and 2,3,6-trimethyl were obtained. -1,4,5-triacetylglucitol was found in a ratio of about 1: 1. This is because the glucosyl transferase is composed of glucose in which only the 1-position OH group is involved in binding, and both the 1-position and 2-position OH groups are involved in binding, or only the 2-position OH group is involved. Glucose involved in binding and glucose in which both 1-position and 4-position glucose are involved in binding, or glucose in which only the 4-position OH group is involved in binding are in a ratio of 1: 1 to 1, respectively. This suggests that they are oligosaccharides bonded to each other.
[0071]
In order to investigate the structure of the glucosyl transfer sugar in more detail, a 13 C-nuclear magnetic resonance spectrum was measured. As a result, 13 C-NMR spectrum (100 MHz, D 2 O): σ ppm from TSP 99.5, 99.4, 99.1, 98.6, 94.6, 79.2, 78.8, 78.6, 78.0 77.6, 77.3, 77.0, 76.1, 75.6, 75.3, 74.7, 74.2, 74.1, 73.8, 72.7, 72.2, 72. Each value of .1, 63.6, 63.5, 63.2, 63.1 was obtained. From this analysis data, the structure of the present glucosyl transferase is a cordybiosyl glucose, which is a trisaccharide in which D-glucose is α-type and 1,2-linked to the non-reducing terminal glucose residue of maltose, that is, α-D-glucosyl (1 → 2) It was found to be α-D-glucosyl (1 → 4) -D-glucose.
[0072]
[Experiment 10]
<Koji Triose>
In order to confirm the glucosyl transferase produced by the transglycosylation reaction to cordobiose by the cordobiose phosphorylase of the present invention, the glucosyl transferase was produced, isolated, and the structure was examined. First, 5 mM sodium dihydrogen phosphate dissolved in 20% cordobiose was adjusted to pH 5.5, and 10 units of purified cordierbiose phosphorylase obtained by the method of Experiment 2 was added per 1 g of cordierbiose, and the mixture was heated at 60 ° C. for 48 hours. Then, the enzyme was inactivated by heating at 100 ° C. for 10 minutes. In accordance with the method shown in Experiment 7, a part of this reaction completed solution was analyzed by GLC. As a result, With Kojibiose In addition, it was confirmed that D-glucose and β-D-glucose-1-phosphate produced a large amount of components having different retention times, and this component was considered to be the glucosyl transferase. Moreover, the production rate of the glucosyltransferase in this reaction determined from the analysis result by GLC was about 30%. The entire remaining amount of this reaction solution was fractionated and purified in the same manner as in Experiment 8, concentrated, and vacuum-dried to obtain the glucosyl transfer sugar-rich powder with a yield of about 15% per solid of cordobiose subjected to the reaction. . This powder preparation contained about 98% of the glucosyl transfer sugar per solid.
[0073]
Next, after methylating the said glucosyl transfer sugar high content powder, it hydrolyzed with an acid, and the partial methyl hexitol acetate obtained by reducing and acetylating subsequently was used for the GLC analysis. As a result, 2,3,4,6-tetramethyl-1,5-diacetylglucitol and 3,4,6-trimethyl-1,2,5-triacetylglucitol were in a ratio of about 1: 2. Admitted. This is because the glucosyl transferase is composed of glucose in which only the 1-position OH group is involved in binding, and both the 1-position and 2-position OH groups are involved in binding, or only the 2-position OH group is involved. This suggests that the glucose involved in the binding is an oligosaccharide bound to each other at a ratio of 1: 2. Furthermore, taking into account the possibility of glucose binding mode, this indicates that the glucosyl transfer saccharide has both glucose in which only the 1-position OH group is involved in binding and the 1- and 2-position OH groups. Strongly suggesting that the glucose involved in the binding and the glucose in which only the 2-position OH group is involved in the binding are oligosaccharides bound to each other at a ratio of 1: 1 to 1: 1, respectively. Yes.
[0074]
In order to investigate the structure of the glucosyl transfer sugar in more detail, 13C-nuclear magnetic resonance spectrum was measured. As a result, 13 C-NMR spectrum (100 MHz, D 2 O): σ ppm from TSP 99.3, 99.0, 98.3, 97.6, 96.5, 92.2, 81.3, 78.8, 78.6 78.5, 77.2, 77.1, 75.7, 75.6, 74.7, 74.6, 74.5, 74.3, 74.2, 74.1, 74.0, 73 .9, 72.6, 72.3, 72.2, 72.1, 72.0, 63.6, 63.4, 63.2, 63.1, 63.1, 63.0 Obtained. From this analytical data, the structure of the present glucosyl cordobiose is cordierose, which is a trisaccharide in which D-glucose is α-linked in the form of 1,2-linked to the non-reducing terminal glucose residue of cordobiose, that is, α-D-glucosyl (1 → 2) It was found to be α-D-glucosyl (1 → 2) -D-glucose.
[0075]
[Experiment 11]
<Cody biosyl glucoside>
In order to confirm the glucosyl transferase produced by the transfer reaction to trehalose by the cordobiose phosphorylase of the present invention, the glucosyl transferase was produced, isolated, and the structure was examined. First, an aqueous solution containing 5% β-D-glucose-1-phosphate and 10% trehalose was adjusted to pH 5.5, and the purified cordierbiose phosphorylase obtained by the method of Experiment 2 was added to β-D-glucose- 10 units were added per 1 g of phosphoric acid, reacted at 60 ° C. for 48 hours, and then heated at 100 ° C. for 10 minutes to deactivate the enzyme. In accordance with the method shown in Experiment 7, a part of this reaction completed solution was analyzed by GLC. As a result, it was confirmed that a component having a different retention time was produced in both the β-D-glucose and trehalose in the reaction completion solution, and this component was considered to be the glucosyl transferase. Moreover, the production rate of the glucosyl transferase in this reaction obtained from the analysis result by GLC was about 75%. The remaining total amount of this reaction solution was fractionated and purified in the same manner as in Experiment 8, concentrated, and vacuum dried to obtain the glucosyl transfer sugar-rich powder with a yield of about 65% per saccharide solid substance subjected to the reaction. It was. This powder preparation contained about 98% of the glucosyl transfer sugar per solid.
[0076]
Next, after methylating the said glucosyl transfer sugar high content powder, it hydrolyzed with an acid, and the partial methyl hexitol acetate obtained by reducing and acetylating subsequently was used for the GLC analysis. As a result, 2,3,4,6-tetramethyl-1,5-diacetylglucitol and 3,4,6-trimethyl-1,2,5-triacetylglucitol were found in a ratio of 2 to 1. It was. This means that the glucosyltransferase has a two-to-one relationship between glucose in which only the 1-position OH group is involved in binding and glucose in which both the 1- and 2-position OH groups are involved in binding. This suggests that the oligosaccharides are bound to each other in proportion.
[0077]
In order to investigate the structure of the glucosyl transfer sugar in more detail, 13C-nuclear magnetic resonance spectrum was measured. As a result, 13 C-NMR spectrum (100 MHz, D 2 O): σ ppm from TSP 98.6, 96.2, 93.3, 77.3, 75.8, 75.4, 75.1, 74.8, 74.6 , 74.6, 74.0, 73.9, 72.5, 72.3, 72.2, 63.4, 63.4, 63.3. From this analytical data, the structure of the present glucosyl trehalose is cordierbiosyl glucoside (also known as ceraginose), which is a trisaccharide having D-glucose 1 or 2 linked to trehalose, that is, α-D-glucosyl (1 → 2) α-D- It was found to be glucosyl (1,1) α-D-glucoside.
[0078]
[Experiment 12]
<Kojibiosyl fructoside>
In order to confirm the glucosyl transferase produced by the transfer reaction to sucrose by the cordobiose phosphorylase of the present invention, the glucosyl transferase was produced, isolated, and the structure was examined. First, an aqueous solution containing 5% β-D-glucose-1-phosphate and 10% sucrose was adjusted to pH 5.5, and the purified cordierbiose phosphorylase obtained by the method of Experiment 2 was added to β-D-glucose- 10 units were added per 1 g of phosphoric acid, reacted at 60 ° C. for 48 hours, and then heated at 100 ° C. for 10 minutes to deactivate the enzyme. In accordance with the method shown in Experiment 7, a part of this reaction completed solution was analyzed by GLC. As a result, it was confirmed that a component having a different retention time was produced in both the β-D-glucose-1-phosphate and sucrose in the reaction end solution, and this component was considered to be the glucosyltransferase. Moreover, the production rate of the glucosyl transferase in this reaction determined from the analysis result by GLC was about 70%. The remaining total amount of this reaction solution was fractionated and purified in the same manner as in Experiment 8, concentrated, and vacuum dried to obtain the glucosyl transfer sugar-rich powder with a yield of about 50% per saccharide solid substance subjected to the reaction. It was. This powder preparation contained about 98% of the glucosyl transfer sugar per solid.
[0079]
Next, the high glucosyl transfer sugar-containing powder was subjected to acid decomposition and subjected to GLC analysis. As a result, it was found that the glucosyl transfer sugar produced D-glucose and D-fructose at a molar ratio of about 2 to 1 by acid decomposition. Further, when methyl of the glucosyl transfer sugar-rich powder was hydrolyzed with acid, and subsequently reduced and acetylated, the partial methyl hexitol acetate obtained by GLC was analyzed by GLC, 2,3,4,6-tetra Methyl-1,5-diacetylglucitol and 3,4,6-trimethyl-1,2,5-triacetylglucitol were found in a 1: 1 ratio. From the above, the glucosyl transferase is a combination of D-glucose and D-fructose in a molar ratio of 2 to 1, and the D-glucose constituting the glucosyl transferase is OH at the 1-position. This suggests that the group in which only the group is involved in bonding and the group in which both the 1-position and 2-position OH groups are involved in bonding exist in a molar ratio of 1: 1. In addition, although the methylated derivative of D-fructose was not recognized, it seems that this was decomposed | disassembled by the acid.
[0080]
In order to investigate the structure of the glucosyl transfer sugar in more detail, a 13 C-nuclear magnetic resonance spectrum was measured. As a result, 13 C-NMR spectrum (100 MHz, D 2 O): σ ppm from TSP107.0, 99.3, 92.5, 84.0, 79.1, 78.5, 76.5, 75.7, 74.9 , 74.7, 74.1, 74.0, 72.2, 72.1, 64.9, 64.6, 63.2, 63.1 were obtained. From this analysis data, the structure of the present glucosyl sucrose is cordierbiosyl fructoside, which is a trisaccharide in which D-glucose is α-linked and 1,2-linked to the glucose residue of sucrose, that is, α-D-glucosyl (1 → 2). It was found to be α-D-glucosyl (1 → 2) β-D-fructoside. This substance is a novel carbohydrate that has not been known so far.
[0081]
[Experiment 13]
<Acute toxicity>
Using 7-week-old dd mice, the glucosyl sorbose-rich powder, the cordierbiosyl-glucose-rich powder, the cordierioose-rich powder, the cordierbiosylglucoside-rich powder and the cordiobio obtained in Experiments 8 to 12 When acute toxicity tests were carried out by orally administering each of the powders containing a high content of silfrucside, no deaths were observed at the maximum dose that could be administered, that is, the body weight of the mouse of 50 g / kg. Accordingly, all of these transfer sugars of the present invention are extremely toxic substances.
[0082]
Hereinafter, the cordierbiose phosphorylase of the present invention, the DNA of the present invention encoding the cordobiose phosphorylase, and a method for producing a glucosyltransferase-containing saccharide using the same will be described in Example A. Example B shows a composition containing.
[0083]
Example A-1
<Enzyme solution>
Thermoanaerobium broccii ATCC 35047 was seed-cultured by the method shown in Experiment 1 and then added to a medium having the same composition prepared in an anaerobic fermenter according to the method shown in Experiment 1, with 1 v / v% per volume of the medium. The seed culture solution was inoculated and cultured at a culture temperature of 65 ° C. for about 30 hours. The bacterial cells obtained by centrifuging the culture broth were subjected to ultrasonic disruption, and the Codybiose phosphorylase activity of the supernatant of the disrupted solution was measured. When this activity was converted per 1 ml of the culture solution, it was 0.08 unit. The lysate supernatant is concentrated with an ultrafiltration membrane, and the concentrated solution is dialyzed to obtain an enzyme solution having about 8 units of cordobiose phosphorylase activity per ml with respect to the total activity of the original culture solution. % Yield.
[0084]
Example A-2
<Preparation of DNA>
Thermoanaerobium brokkii (ATCC 35047) was cultured at 11 ° C. in the same composition as that shown in Experiment 1 for 24 hours at 60 ° C. according to the method shown in Experiment 1. The cells are separated from the culture by centrifugation, suspended in an appropriate amount of Tris-EDTA-saline buffer (hereinafter abbreviated as “TES buffer”) (pH 8.0), and lysozyme is suspended in the cell suspension. After adding 0.05% (w / v) to the volume of the solution, it was incubated at 37 ° C. for 30 minutes. Thereafter, the treated product was kept at −80 ° C. for 1 hour to freeze, and then a TES buffer / phenol mixed solution in which a TES buffer solution (pH 9.0) was previously added and heated to 60 ° C. was then added. In addition, the mixture was sufficiently stirred, and the upper layer formed by centrifugation after cooling with ice was collected. A precipitate formed by adding 2 volumes of cold ethanol to this upper layer was collected, dissolved in an appropriate amount of SSC buffer (pH 7.1), 7.5 μg ribonuclease and 125 μg protease were added, and 1 ° C. at 37 ° C. Incubated for hours. A mixed solution of chloroform / isoamyl alcohol was added thereto, stirred, and then allowed to stand to collect an upper layer. A precipitate formed by adding cold ethanol to the upper layer was collected. The precipitate was rinsed with cold 70% (v / v) ethanol and vacuum dried to obtain DNA. The obtained DNA was dissolved in SSC buffer (pH 7.1) so as to have a concentration of about 1 mg / ml, and frozen at −80 ° C.
[0085]
Example A-3
<Preparation of transformant and recombinant DNA>
Take 1 ml of the DNA solution obtained by the method of Example A-2, add about 20 units of restriction enzyme HindIII here, incubate at 37 ° C. for 30 minutes to partially decompose the DNA, and then by sucrose density gradient ultracentrifugation, A DNA fragment having a chain length of about 2,000 to 5,000 base pairs was collected. Separately, the plasmid vector “Bluescript II SK (+)” manufactured by Stratagene Cloning Systems was completely cleaved by the action of restriction enzyme Hind III by a conventional method, and 0.3 μg of the cleaved plasmid vector was obtained in advance. About 3 μg of the DNA fragment was ligated using the “DNA ligation kit” manufactured by Takara Shuzo according to the attached instructions, and the resulting recombinant DNA was subjected to the Stratagene cloning clone by the ordinary competent cell method. A gene library was prepared by transforming 100 μl of E. coli “Epicurian Coli XL1-Blue” manufactured by Systems.
[0086]
The thus obtained transformant as a gene library was prepared by a conventional method, and 10 g / l tryptone, 5 g / l yeast extract, 5 g / l sodium chloride, 75 mg / l ampicillin sodium salt and 50 mg / l 5 -Inoculated on an agar plate medium (pH 7.0) containing bromo-4-chloro-3-indolyl-β-galactoside, cultured at 37 ° C for 18 hours, and then about 4,000 white colonies formed on the medium. The pieces were fixed on an Amersham nylon membrane “Hybond-N +”. Separately, the oligonucleotide of the base sequence represented by 5′-TTYGAYGAARAAYAYATGCC-3 ′ based on the first to seventh amino acid sequences in the amino acid sequence shown in SEQ ID NO: 7 of the Sequence Listing, which was clarified by the method of Experiment 4 Was synthesized by isotope labeling using [γ- 32 P] ATP and T4 polynucleotide kinase according to a conventional method to obtain a synthetic DNA as a probe. Among the colonies fixed on the nylon membrane obtained earlier, a colony showing a remarkable association with the probe was selected by applying a normal colony hybridization method, and the transformant was named “TKP1”. .
[0087]
This transformant TKP1 was inoculated in an L-broth medium (pH 7.0) containing 100 μg / ml ampicillin sodium salt according to a conventional method, and cultured with shaking at 37 ° C. for 24 hours. After completion of the culture, the cells were collected from the culture by centrifugation, and the recombinant DNA was extracted by the usual alkali-SDS method. When the base sequence of this recombinant DNA was analyzed by a normal dideoxy method, the recombinant DNA was derived from Thermoanaerobium brokki (ATCC 35047) and has a chain length of 3956 base pairs in SEQ ID NO: 9 of the base sequence shown in FIG. As shown in FIG. 5, in this recombinant DNA, the DNA was ligated downstream of the recognition site by the restriction enzyme Hind III. On the other hand, the amino acid sequence deduced from this base sequence is as shown in SEQ ID NO: 9, and this amino acid sequence and the N-terminal amino acid sequence of the present invention Combibiose phosphorylase confirmed by the method of Experiment 4 and When comparing the amino acid sequences shown in SEQ ID NOs: 1 to 3 and SEQ ID NOs: 6 to 8 in the Sequence Listing, which are the middle part amino acid sequences, the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3 in the Sequence Listing are respectively It completely matched with the first to fifth, 590th to 594th and 357th to 361th amino acid sequences in the amino acid sequence shown in SEQ ID NO: 9. In addition, the amino acid sequences shown in SEQ ID NOs: 6, 7, and 8 in the sequence listing are the 1st to 10th, 590th to 599th, and 357th to 366th amino acids in the amino acid sequence shown in SEQ ID NO: 9 in the sequence listing, respectively. Completely matched the amino acid sequence. The above is the fact that the cordobiose phosphorylase of the present invention contains the amino acid sequence shown in SEQ ID NO: 4 in the Sequence Listing. It shows that it is encoded by DNA of the base sequence shown in FIG. The recombinant DNA prepared as described above and confirmed in base sequence was named “pTKP1”.
[0088]
Example A-4
<Production of Kojibiose phosphorylase by transformants>
100 ml of an aqueous solution containing 16 g / l polypeptone, 10 g / l yeast extract and 5 g / l sodium chloride was placed in a 500 ml Erlenmeyer flask, treated in an autoclave at 121 ° C. for 15 minutes, cooled and aseptically adjusted to pH 7.0. Thereafter, 10 mg of ampicillin sodium salt was aseptically added to prepare a liquid medium. This liquid medium was inoculated with the transformant TKP1 obtained by the method of Example A-3 and cultured at 37 ° C. for about 20 hours with aeration and stirring as a seed culture solution. Next, 7 l of a medium having the same composition as that used for seed culture was prepared in a 10 l fermenter according to the seed culture, inoculated with 70 ml of the seed culture, and cultured with aeration and stirring for about 20 hours. According to a conventional method, the culture is centrifuged to recover the cells, suspended in 10 mM phosphate buffer (pH 7.0), sonicated to disrupt the cells, and further centrifuged to insoluble. Things were removed and the supernatant was collected. The supernatant was dialyzed against 10 mM phosphate buffer, and then the Codybiose phosphorylase activity in the dialysate was measured. As a result, about 500 units of the enzyme were produced per liter of culture.
[0089]
As a first control, Escherichia coli XL1-Blue strain was cultured under the same conditions as in the case of the above-described transformant except that no ampicillin was added to the medium. Was collected and dialyzed. As a second control, Thermoanaerobium brokkii (ATCC 35047) was used in the same manner as in the case of the transformant except that it did not contain ampicillin. The culture was allowed to stand at 0 ° C., and the supernatant of the crushed cell was collected from the culture and dialyzed as in the case of the transformant described above. No enzyme activity was observed in the first control dialysate. The enzyme activity was observed in the second control dialysate, which in this case was about 100 units per liter of culture, which was clearly lower than that of transformant TKP1.
[0090]
According to the method shown in Experiment 2, the dialyzate obtained by the method of Example A-4 was further DEAE-Toyopearl 650 gel The purified enzyme was purified by column chromatography using Ultrogel AcA44, and the purified enzyme was analyzed according to the method shown in Experiment 3. Molecular weight 83,000 ± 5,000 dalton by SDS-polyacrylamide gel electrophoresis, molecular weight 500,000 ± 30,000 dalton by gel filtration, isoelectric point 4.4 ± by polyacrylamide gel electrophoresis 0.5, optimum temperature of cordobiose phosphorylase activity around 65 ° C., optimum pH around 5.5, temperature stability up to around 65 ° C., pH stability about 5.5 to 10.0, Experiment 1 It was substantially the same as the physicochemical properties of the enzyme prepared by the method shown in FIGS. The above results indicate that the cordobiose phosphorylase of the present invention can be produced satisfactorily by recombinant DNA technology, and the productivity of the enzyme is also significantly improved.
[0091]
Example A-5
<Enzyme solution>
The transformant TKP1 obtained by the method shown in Example A-3 was cultured by the method shown in Example A-4. The bacterial cells obtained by centrifuging the culture broth were subjected to ultrasonic disruption, and the Codybiose phosphorylase activity of the supernatant of the disrupted solution was measured. When this activity was converted per 1 ml of the culture solution, it was about 0.5 units. The supernatant of the crushed liquid is concentrated with an ultrafiltration membrane, and the concentrated liquid is dialyzed to obtain an enzyme solution having about 10 units of cordobiose phosphorylase activity per ml with respect to the total activity of the original culture solution. % Yield.
[0092]
Example A-6
<Corgebiose-containing sugar solution>
Combibiose phosphorylase prepared by the method of Example A-1 in a 25 mM dipotassium phosphate-citrate buffer solution (pH 6.0) containing 5% maltose and 5 units of commercially available maltose phosphorylase derived from bacteria. It added so that it might become 40 units per gram of maltose, and it was made to react at 30 degreeC for 72 hours. The reaction solution was heated at 100 ° C. for 30 minutes to inactivate the enzyme, then cooled, decolorized and filtered with activated carbon according to a conventional method, purified by desalting with H-type and OH-type ion exchange resins, By concentrating, a syrup-like cordierbiose-containing sugar solution having a concentration of about 75% was obtained at a yield of about 95% per raw material solid.
[0093]
This product contains about 30% of Codybiose per solid and also contains Codybiosyl glucose, has good taste sweetness, moderate viscosity, moisturizing, sweetener, taste improver, Widely used as stabilizers, bifidobacteria growth promoters, mineral absorption promoters, etc., can be advantageously used in various compositions such as foods, drinks, cosmetics, pharmaceuticals, and molded products.
[0094]
Example A-7
<Cojibiose high content powder>
A sugar solution containing about 30% cordierbiose per solid obtained by reaction and purification by the method of Example A-6 was used as a raw material to adjust the solid concentration to about 20%, and 5 units of glucoamylase was added to 1 g of the solid. The reaction was carried out at pH 4.5 and 40 ° C. for 16 hours to decompose the remaining maltose. The reaction was stopped by heating at 100 ° C. for 30 minutes, and then concentrated to a concentration of about 40%. In order to increase the content of cordobiose in the sugar solution, an alkali metal type strongly acidic cation exchange resin (manufactured by Tokyo Organic Chemical Industry Co., Ltd., trade name “XT-1016”, Na type, cross-linking degree 4%) is used. Four stainless steel columns with a jacket of 3 cm and a length of 1 m were filled with water suspension and connected in series so that the total length of the resin layer was about 4 m. While maintaining the temperature in the column at 40 ° C, the sugar solution is added to the resin at 5 v / v%, and 40 ° C warm water is flowed at a flow rate of SV0.15 for fractionation, and a fraction containing high Cozybiose is collected. did. Further, purification, concentration, vacuum drying, and pulverization gave a powder having a high content of cordobiose with a yield of about 20% per raw material solid.
[0095]
This product contains approximately 90% cordierbiose per solid, has good sweetness and moderate moisture retention, sweetener, taste improver, stabilizer, bifidobacterial growth promoter, mineral Widely used as an absorption accelerator and the like, it can be advantageously used in various compositions such as foods and drinks, cosmetics, pharmaceuticals, and molded products.
[0096]
[Example A-8]
<Glucosyl sorbose-containing sugar solution>
An aqueous solution containing 5% each of β-D-glucose-1-phosphate and L-sorbose was adjusted to pH 5.5, and cordierbiose phosphorylase prepared by the method of Example A-1 was added to β-D-glucose- It added so that it might become 10 units per 1g of phosphoric acid, and was made to react at 60 degreeC for 72 hours. The reaction solution was heated at 90 ° C. for 30 minutes to inactivate the enzyme, then cooled, decolorized and filtered with activated carbon according to a conventional method, purified by desalting with H-type and OH-type ion exchange resins, By concentrating, a syrup-like glucosyl sorbose-containing sugar solution having a concentration of about 75% was obtained with a yield of about 95% per raw material solid.
[0097]
This product contains about 30% glucosyl sorbose per solid, has good taste sweetness, moderate viscosity, and moisture retention, sweetener, taste improver, stabilizer, bifidobacteria growth promoter As a mineral absorption promoter, it can be used advantageously in various compositions such as foods, drinks, cosmetics, pharmaceuticals, and molded products.
[0098]
Example A-9
<Glucosyl sorbose-rich sugar solution>
An aqueous solution containing 5% of β-D-glucose-1-phosphate and 10% of L-sorbose was adjusted to pH 6.0, and cordierbiose phosphorylase prepared by the method of Example A-1 was added to β-D- It added so that it might become 30 units per g of glucose-1 phosphate, and it was made to react at 60 degreeC for 72 hours. After the reaction, the reaction mixture was cooled, commercial baker's yeast was added to a wet weight of 5% based on the solid weight, and the reaction solution was controlled at pH 5 to 6 using 1N sodium hydroxide solution for 6 hours at 27 ° C. Reacted. The reaction solution was heated at 90 ° C. for 30 minutes to stop the reaction, then cooled, decolorized and filtered with activated carbon according to a conventional method, purified by desalting with H-type and OH-type ion exchange resins, and further concentrated. As a result, a syrup-like glucosyl sorbose-containing sugar solution having a concentration of about 75% was obtained at a yield of about 65% per solid material.
[0099]
This product contains about 40% glucosyl sorbose per solid, has good taste sweetness, moderate viscosity, and moisture retention, sweetener, taste improver, stabilizer, bifidobacteria growth promoter As a mineral absorption promoter, it can be used advantageously in various compositions such as foods, drinks, cosmetics, pharmaceuticals, and molded products.
[0100]
Example A-10
<Glucosyl sorbose high content powder>
Using a sugar solution containing about 40% glucosyl sorbose as a raw material by the method of Example A-9, the solid concentration was adjusted to about 45%. In order to increase the glucosyl sorbose content in the sugar solution, an alkali metal type strongly acidic cation exchange resin (manufactured by Tokyo Organic Chemical Industry Co., Ltd., trade name “XT-1016”, Na type, crosslinking degree 4%) is used. Four jacketed stainless steel columns having an inner diameter of 3 cm and a length of 1 m were filled with water suspension and connected in series so that the total length of the resin layer was about 4 m. While maintaining the temperature in the column at 40 ° C., the sugar solution is added to the resin at 5 v / v%, and 40 ° C. warm water is flowed at a flow rate of SV0.15 to perform fractionation, and a fraction containing high glucosyl sorbose is obtained. Collected. Furthermore, purification, concentration, vacuum drying, and pulverization gave a powder containing high glucosyl sorbose in a yield of about 25% per raw material solid.
[0101]
This product contains about 90% glucosyl sorbose per solid, has good taste sweetness, moderate moisture retention, sweetener, taste improver, stabilizer, bifidobacterial growth promoter, As a mineral absorption accelerator, it can be widely used in various compositions such as foods, cosmetics, pharmaceuticals and molded products.
[0102]
Example A-11
<Corgebiosyl fructoside-containing sugar solution>
An aqueous solution containing 10% cordobiose and 10% sucrose was prepared, and to this was added cordierbiose phosphorylase prepared by the method of Example A-1 to 40 units per gram cordierbiose, 5 mM sodium dihydrogen phosphate The reaction was carried out in the presence at pH 5.0 and 60 ° C. for 72 hours. The reaction solution was heated at 90 ° C. for 30 minutes to inactivate the enzyme, then cooled, decolorized and filtered with activated carbon according to a conventional method, purified by desalting with H-type and OH-type ion exchange resins, By concentrating, a syrup-like cordobiosyl fructoside-containing sugar solution having a concentration of about 75% was obtained with a yield of about 95% per raw material solid.
[0103]
This product contains about 55% Kojibiosyl fructoside per solid, has good sweetness, moderate viscosity, and moisturizing properties, and various compositions such as food and drink, cosmetics, pharmaceuticals, and molded products It can be used to advantage.
[0104]
Example A-12
<Cody biosyl fructoside-rich powder>
The sugar solution containing about 55% of cordobiosyl fructoside per solid, which was reacted and purified by the method of Example A-11, was adjusted to a solid concentration of about 45%. In order to increase the content of Kojibiosyl fructoside in this molasses, an alkaline earth metal type strongly acidic cation exchange resin (Dow Chemical Co., trade name “Dowex 50W × 4”, Ca type) was used. Then, column chromatography was performed according to the method of Example A-10, and a fraction containing a high content of cordobisyl fructoside was collected. Furthermore, purification, concentration, vacuum drying, and pulverization yielded a powder having a high content of cordobiosyl fructoside in a yield of about 40% per raw material solid.
[0105]
This product contains about 95% Kojibiosyl fructoside per solid, has good sweetness and moderate moisturizing properties, and is used in various compositions such as food and drink, cosmetics, pharmaceuticals, and molded products. It can be used advantageously.
[0106]
Example A-13
<Kojibiosyl glucose-containing sugar solution>
An aqueous solution containing 10% β-D-glucose-1 phosphate and 20% maltose was adjusted to pH 5.0, and cordierbiose phosphorylase prepared by the method of Example A-1 was adjusted to 40 units per gram of maltose. And reacted at 60 ° C. for 72 hours. The reaction solution was heated at 90 ° C. for 30 minutes to inactivate the enzyme, then cooled, decolorized and filtered with activated carbon according to a conventional method, purified by desalting with H-type and OH-type ion exchange resins, By concentrating, a syrup-like cordierbiosylglucose-containing sugar solution having a concentration of about 75% was obtained with a yield of about 95% per raw material solid.
[0107]
This product contains about 45% kojibiosylglucose per solid, has good sweetness, moderate viscosity, and moisturizing properties, and various compositions such as foods, cosmetics, pharmaceuticals, and molded products Can be advantageously used.
[0108]
Example A-14
<Corgebiosyl glucoside-rich sugar solution>
An aqueous solution containing 5% β-D-glucose-1 phosphate and 10% trehalose was adjusted to pH 5.0, and then Codybiose phosphorylase prepared by the method of Example A-1 was added to β-D-glucose- It added so that it might become 20 units per 1g of phosphoric acid, and it was made to react at 60 degreeC for 72 hours. Sodium hydroxide was added to the reaction solution and heated at 100 ° C. while maintaining an alkalinity of pH 10 or higher, then cooled, decolorized with activated carbon and filtered according to conventional methods, and desalted with H-type and OH-type ion exchange resins. Purification and further concentration yielded a syrup-like cordierbiosylglucoside-rich sugar solution having a concentration of about 75% in a yield of about 60% per raw material solid.
[0109]
This product contains about 95% kojibiosyl glucoside per solid, has good sweetness, moderate viscosity, and moisturizing properties, and various compositions such as foods, cosmetics, pharmaceuticals, and molded products. Can be advantageously used.
[0110]
Example A-15
<Corgebiose-containing sugar solution>
Combibiose phosphorylase prepared by the method of Example A-5, 5 units of commercially available maltose phosphorylase derived from bacteria in 25 mM dipotassium phosphate-citrate buffer (pH 6.0) containing 5% maltose It added so that it might become 40 units per gram of maltose, and it was made to react at 30 degreeC for 72 hours. The reaction solution was heated at 100 ° C. for 30 minutes to inactivate the enzyme, then cooled, decolorized and filtered with activated carbon according to a conventional method, purified by desalting with H-type and OH-type ion exchange resins, By concentrating, a syrup-like cordierbiose-containing sugar solution having a concentration of about 75% was obtained at a yield of about 95% per raw material solid.
[0111]
This product contains about 30% of cordobiose per solid and also contains cordobiosyl glucose, has a good taste sweetness, moderate viscosity, and moisture retention, sweetener, taste improver, Widely used as stabilizers, bifidobacteria growth promoters, mineral absorption promoters, etc., can be advantageously used in various compositions such as foods, drinks, cosmetics, pharmaceuticals, and molded products.
[0112]
Example A-16
<Corgebiosyl glucoside-rich sugar solution>
An aqueous solution containing 5% β-D-glucose-1-phosphate and 10% trehalose was adjusted to pH 5.0, and then Combibiose phosphorylase prepared by the method of Example A-5 was added to β-D-glucose- It added so that it might become 20 units per 1g of phosphoric acid, and it was made to react at 60 degreeC for 72 hours. Sodium hydroxide was added to the reaction solution and heated at 100 ° C. while maintaining an alkalinity of pH 10 or higher, then cooled, decolorized with activated carbon and filtered according to conventional methods, and desalted with H-type and OH-type ion exchange resins. Purification and further concentration yielded a syrup-like cordierbiosylglucoside-rich sugar solution having a concentration of about 75% in a yield of about 60% per raw material solid.
[0113]
This product contains about 95% kojibiosyl glucoside per solid, has good sweetness, moderate viscosity, and moisturizing properties, and various compositions such as foods, cosmetics, pharmaceuticals, and molded products. Can be advantageously used.
[0114]
Example B-1
<Sweetener>
To 1 part by weight of the glucosyl sorbose-rich powder obtained by the method of Example A-10, 0.05 part by weight of α-glycosyl stevioside (manufactured by Toyo Seika Co., Ltd., trade name α-G sweet) is added and mixed uniformly. Thus, a powder sweetener was produced. The product is refined and has about twice the sweetness of sugar, and calories are about half that of sugar per degree of sweetness. Therefore, this sweetener is suitable as a low-calorie sweetener for seasoning low-calorie foods and drinks for people who restrict intake of calories, such as obese and diabetics. In addition, this sweetener is suitable for seasoning foods and the like that suppress dental caries because it produces less acid by caries-inducing bacteria and produces less insoluble glucan.
[0115]
Example B-2
<Hard Candy>
To 30 parts by weight of the glucosyl sorbose-containing sugar solution obtained by the method of Example A-8, 80 parts by weight of reduced maltose starch syrup (water content 25%) is added and mixed and dissolved until the water content is less than 2% under reduced pressure. Concentrated and mixed with 1 part by weight of citric acid and appropriate amounts of lemon flavor and colorant, and then molded according to a conventional method to produce a hard candy. This product is a crisp hard candy that has an elegant sweetness, low hygroscopicity, and does not cause dripping.
[0116]
Example B-3
<Chewing gum>
3 parts by weight of glucose and 2 parts by weight of a gum base heated and melted to a degree of softening were added to and mixed with 4 parts by weight of the cordieroyl fructoside-rich powder obtained by the method of Example A-12. After the mint flavor was mixed, the chewing gum was manufactured by kneading with a roll according to a conventional method. This product is excellent in both texture and flavor.
[0117]
Example B-4
<chocolate>
A refiner containing 15 parts by weight of Cozybiose-rich powder obtained by the method of Example A-7 was mixed with 40 parts by weight of cocoa paste, 10 parts by weight of cocoa butter, 10 parts by weight of sucrose and 15 parts by weight of whole milk powder. I passed through. After reducing the particle size, the mixture was placed in a conche, 0.5 parts by weight of lecithin was added, and the mixture was kneaded at 50 ° C. for two days and nights. Subsequently, it poured into the molding machine according to the conventional method, and it solidified by shaping | molding and manufactured chocolate. This product has no fear of fat bloom or sugar bloom and has good melting and flavor when placed on the tongue.
[0118]
Example B-5
<custard cream>
500 parts by weight of corn starch, 500 parts by weight of maltose and 5 parts by weight of salt are added to 400 parts by weight of the high glucosyl sorbose powder obtained by the method of Example A-10, and the mixture is thoroughly mixed through a sieve. Add 5,000 parts by weight and stir, and gradually add 5,000 parts by weight of boiled milk to it, and continue to stir it over the heat until the corn starch is completely gelatinized and the whole becomes translucent. The custard cream was prepared by cooling and adding a small amount of vanilla flavor. This product is smooth and glossy and is not too sweet and delicious.
[0119]
Example B-6
<Uiro>
Add 90 parts by weight of rice flour, 20 parts by weight of corn starch, 20 parts by weight of sugar, 1 part by weight of powdered green tea powder and appropriate amounts of water to 90 parts by weight of the sugar solution containing Kojibiosyl fructoside obtained by the method of Example A-11. After kneading, this was put in a container and steamed for 60 minutes to produce a matcha uiro. This product had good shine, good mouthfeel and good flavor. Moreover, the aging of starch was suppressed and it was stable for a long time.
[0120]
Example B-7
<Betara pickles>
3 parts by weight of maltose, 0.05 parts by weight of licorice preparation, 0.008 parts by weight of malic acid, 0.07 parts by weight of sodium glutamate based on 1 part by weight of the sugar solution containing kojibiosyl glucose obtained by the method of Example A-13 Parts, 0.03 parts by weight of potassium sorbate, and 0.2 parts by weight of pullulan were uniformly mixed to prepare a pickle. In accordance with a conventional method, 30 kg of radish was submerged with salt and then soaked with sugar. The soup was then soaked in a seasoning solution prepared with 4 kg of the base of the pickles. This product had good color and aroma, moderate sweetness, and crispness.
[0121]
[Example B-8]
<Lactic acid bacteria beverage>
With respect to 130 parts by weight of the Kojibiose-containing sugar solution obtained by the method of Example A-6, 175 parts by weight of skim milk powder and 50 parts by weight of lactosucrose-containing powder (Hayashibara Shoji Co., Ltd., registered trademark “milk oligo”) Dissolve in 1,150 parts by weight of water, sterilize at 65 ° C. for 30 minutes, cool to 40 ° C., inoculate 30 parts by weight of a starter of lactic acid bacteria, and culture at 37 ° C. for 8 hours. Thus, a lactic acid bacteria beverage was obtained. This product is a lactic acid bacteria beverage with good flavor. In addition, this product contains oligosaccharides and not only stably retains lactic acid bacteria, but also has a bifidobacteria growth promoting action.
[0122]
Example B-9
<Synthetic sake>
6518 parts by weight of a 30 v / v% alcohol aqueous solution (at a temperature of 15 ° C.), 600 parts by weight of a 70% aqueous glucose solution, 50 parts by weight of a sugar solution containing cordobiose obtained by the method of Example A-6, and 11.1 parts by weight of succinic acid 3.66 parts by weight of 75% lactic acid aqueous solution, 2.3 parts by weight of sodium glutamate, 1.2 parts by weight of glycine, 1.2 parts by weight of alanine, 2.22 parts by weight of sodium succinate, 1.6 parts by weight of sodium chloride, natural 1.4 parts by weight of salt and 2500 parts by weight of water were mixed to obtain a raw liquor having an alcohol content of about 20 v / v%. This was diluted with water to obtain a product having an alcohol content of 15 to 16 v / v%. This product has a mellow sweetness and refined flavor and is delicious.
[0123]
Example B-10
<Latex>
With respect to 3.5 parts by weight of the glucosyl sorbose-rich sugar solution obtained by the method of Example A-9, 0.5 parts by weight of polyoxyethylene behenyl ether, 1 part by weight of polyoxyethylene sorbitol tetraoleate, lipophilic mono Add 1 part by weight of glyceryl stearate, 0.5 part by weight of behenyl alcohol, 1 part by weight of avocado oil, 1 part by weight of α-glycosyl rutin, vitamin E and preservatives, and dissolve by heating according to a conventional method. -5 parts by weight of butylene glycol, 0.1 part by weight of carboxyvinyl polymer and 85.3 parts by weight of purified water were added and emulsified with a homogenizer to produce an emulsion. This product is excellent in moisture retention and can be advantageously used as a sunscreen, whitening agent, and the like.
[0124]
Example B-11
<Skin cream>
2 parts by weight of polyoxyethylene glycol monostearate, 5 parts by weight of glyceryl monostearate, 2 parts by weight of α-glycosyl rutin with respect to 4 parts by weight of the powder containing high-quality cordobiosyl fructoside obtained by the method of Experiment 12 In addition, 1 part by weight of liquid paraffin, 10 parts by weight of glyceryl trioctanoate and an appropriate amount of preservative were added and dissolved by heating according to a conventional method. To this, 5 parts by weight of 1,3-butylene glycol and 66 parts by weight of purified water were further added The mixture was emulsified with a homogenizer, and an appropriate amount of a fragrance was added and stirred and mixed to produce a skin cream. This product is a cream with good elongation and can be advantageously used as a sunscreen, skin cleansing agent, fair skin agent and the like.
[0125]
Example B-12
<Toothpaste>
With respect to 15 parts by weight of the high-concentration sugar solution obtained by the method of Example A-14, dicalcium phosphate parts by weight, sodium lauryl sulfate 1.5 parts by weight, glycerin 25 parts by weight, polyoxyethylene sorbitan laurate A toothpaste was obtained by mixing 0.5 part by weight, 0.02 part by weight saccharin, 0.05 part by weight preservative and 13 parts by weight water. This product has good gloss and detergency and is suitable as a toothpaste.
[0126]
Example B-13
<Tube nutritional supplement>
80 parts by weight of Kojibiose-rich powder obtained by the method of Example A-7, 190 parts by weight of dried egg yolk, 209 parts by weight of skim milk powder, 4.4 parts by weight of sodium chloride, 1.85 parts by weight of potassium chloride, magnesium sulfate 4 A formulation consisting of parts by weight, 0.01 parts by weight thiamine, 0.1 parts by weight sodium ascorbate, 0.6 parts by weight vitamin E acetate and 0.04 parts by weight nicotinamide was prepared. Each 25 g of this blend was filled into a laminated aluminum parcel and heat sealed to obtain a product. This product is used by dissolving one bag in about 150 to 300 ml of water to give a nutritional supplement and administering it to the nasal cavity, esophagus, stomach, etc. by the tube method.
[0127]
Example B-14
<tablet>
After adding 50 parts by weight of aspirin, 10 parts by weight of maltose and 4 parts by weight of corn starch to 4 parts by weight of the powder containing high glucosyl sorbose obtained by the method of Experiment 8, the mixture was uniformly mixed, and then 1 using a 12 mm diameter, 20R soot. Tableting was performed with a tablet of 680 mg, a tablet thickness of 5.25 mm, and a hardness of 8 kg ± 1 kg. This product is an easy-to-drink tablet with moderate sweetness.
[0128]
Example B-15
<Strawberry jam>
150 parts by weight of raw strawberries, 60 parts by weight of sucrose, 20 parts by weight of maltose, 40 parts by weight of a sugar solution containing Kojibiose obtained by the method of Example A-15, 5 parts by weight of pectin and 1 part by weight of citric acid are boiled in a pan and bottled. And got the product. This product is a jam with good flavor and color.
[0129]
Example B-16
<Sweetened condensed milk>
In 100 parts by weight of raw milk, 3 parts by weight of sugar solution containing Kojibiosylglucoside and 1 part by weight of sucrose obtained by the method of Example A-16 are dissolved, sterilized by heating with a plate heater, and then concentrated to a concentration of about 70%. And canned under aseptic conditions to obtain a product. This product has a mild sweet taste and good flavor and can be advantageously used for seasoning infant foods, fruits, coffee, cocoa, tea and the like.
[0130]
【The invention's effect】
As described above, the present invention is based on the discovery of a phosphorylase capable of producing cordobiose, which has not been known so far, that is, cordobiose phosphorylase. The cordobiose phosphorylase of the present invention has a high temperature in the optimum temperature and temperature stability, a wide pH range in the pH stability, and the optimum pH is within the pH range, and further enzyme production from the producing microorganism The amount is also high. Therefore, if β-D-glucose-1-phosphate is used as a sugar donor and the enzyme of the present invention is allowed to act in the presence of various sugars, Cojibiose, which has been conventionally known but extremely difficult to obtain, Various glucosyl transfer sugars including glucosyl sorbose and the transfer sugar-containing saccharides can be produced in large quantities and at low cost.
[0131]
The glucosyl transfer sugar and the transfer sugar-containing sugar produced in this way are refined sweeteners, taste improvers, quality improvers, body imparting agents, viscosity modifiers, moisturizers, illumination imparting agents, etc. Furthermore, since it is a great feature that it can be widely applied to various compositions such as foods and drinks, cosmetics, pharmaceuticals, and molded products as nutritional supplements, the present invention is not limited to the food, cosmetics, and pharmaceutical fields, Contributing to the agricultural, aquatic and livestock industries and the chemical industry is an invention that has great significance.
[0132]
[Sequence Listing]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a graph showing the influence of temperature on the enzyme activity of the cordobiose phosphorylase of the present invention.
FIG. 2 is a graph showing the influence of pH on the enzyme activity of the cordobiose phosphorylase of the present invention.
FIG. 3 is a graph showing the influence of temperature on the stability of the cordobiose phosphorylase of the present invention.
FIG. 4 is a diagram showing the influence of pH on the stability of the cordobiose phosphorylase of the present invention.
FIG. 5 is a diagram showing a restriction enzyme map of the recombinant DNA of the present invention. The arrow in the figure indicates the DNA encoding the cordobiose phosphorylase of the present invention.
[Table 2]

[Table 2]

Claims (16)

Cozybiose is decomposed in the presence of inorganic phosphoric acid and / or its salt to produce D-glucose and β-D-glucose-1 phosphate and / or its salt, and β-D-glucose-1 phosphate and / or its salt Alternatively, it has the action of producing cordobiose and inorganic phosphoric acid and / or a salt thereof from a salt thereof and D-glucose, and β-D-glucose-1 phosphate and / or a salt thereof as a sugar donor. Which has the action of catalyzing the transfer of a glucosyl group to the saccharide of the above, contains one or more of the amino acid sequences shown in SEQ ID NOs: 1 to 3 in the sequence listing as partial amino acid sequences, and has the following physicochemical properties Aus phosphorylase.
(1) Molecular weight
83,000 ± 5,000 daltons by SDS-gel electrophoresis.
(2) Optimal temperature
pH 5.5, around 65 ° C. for 30 minutes reaction.
(3) Optimum pH
The pH was around 5.5 after reaction at 60 ° C for 30 minutes.
(4) Temperature stability
Stable up to around 65 ° C under pH 5.5 and 1 hour holding conditions.
(5) pH stability
PH of about 5.5 to 10.0 at 4 ° C. for 24 hours.   In the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing, or in the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing, one or more of the amino acids are substituted within the range that does not substantially lose the action as a cordobiose phosphorylase The cordobiose phosphorylase according to claim 1, comprising an added or deleted amino acid sequence.   A DNA encoding the cordobiose phosphorylase according to claim 2.   The DNA according to claim 3, comprising the base sequence shown in SEQ ID NO: 5 in the sequence listing or a base sequence complementary to the base sequence.   The DNA according to claim 3 or 4, wherein one or a plurality of bases is substituted with another base without changing the encoded amino acid sequence based on the degeneracy of the gene.   A recombinant DNA comprising the DNA according to any one of claims 3 to 5 and a vector capable of autonomous replication.   A transformant obtained by introducing the recombinant DNA according to claim 6 into an appropriate host.   Cultivating the thermoanaerobium brokkii having the ability to produce cordobiose phosphorylase according to claim 1 or 2 or the transformant according to claim 7 in a nutrient medium, and collecting cordobiose phosphorylase produced from the culture. A method for producing Kojibiose phosphorylase, characterized in that A method for producing a glucosyltransferase-containing saccharide, wherein the cordobiose phosphorylase according to claim 1 is allowed to act on β-D-glucose-1-phosphate and / or a salt thereof and another saccharide.   β-D-glucose-1 phosphate and / or salt thereof causes cordierbiose to act on cordobiose in the presence of inorganic phosphate and / or salt thereof, or presence of inorganic phosphate and / or salt thereof The glucosyl transfer sugar-containing saccharide according to claim 9, which is produced by allowing maltose phosphorylase to act on maltose or trehalose phosphorylase to act on trehalose in the presence of inorganic phosphate and / or a salt thereof. Production method.   The method for producing a glucosyltransferase-containing saccharide according to claim 9 or 10, wherein the other saccharide is a saccharide selected from D-glucose, L-sorbose, maltose, cordobiose, trehalose and sucrose.   The glucosyl transfer according to any one of claims 9 to 11, which includes a method selected from yeast fermentation and column chromatography as means for removing contaminating saccharides other than glucosyl transfer saccharide produced and / or remaining in the reaction solution. A method for producing a sugar-containing carbohydrate. The cordobiose phosphorylase according to claim 1 is allowed to act on maltose together with maltose phosphorylase in the presence of inorganic phosphate and / or a salt thereof, or acts on trehalose together with trehalose phosphorylase in the presence of inorganic phosphate and / or a salt thereof. A method for producing Kojibiose, characterized in that:   The α-D-glucosyl (1 → 5) -L-sorbose, cordobiosylglucose or cordierbiosyl fructoside produced by the method for producing a glucosyltransferase-containing saccharide according to any one of claims 9 to 12. Contains carbohydrates.   Glucosyl sorbose represented by α-D-glucosyl (1 → 5) -L-sorbose.   Codybiosyl fructoside represented by α-D-glucosyl (1 → 2) α-D-glucosyl (1 → 2) β-D-fructoside.

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