JP2004275013A - New enzyme - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an enzyme simply and accurately assaying hemoglobin Alc at a low cost therewith, and to provide a method for assaying, and to obtain a reagent. <P>SOLUTION: The enzyme has higher specificity for a glycated peptide at a glycated hemoglobin β chain N terminus than that for the glycated peptide at a glycated hemoglobin α chain N terminus. The method for assaying uses the enzyme. Hemoglobin A1c is assayed with the reagent. The hemoglobin A1c can specifically, simply and accurately be assayed at a low cost by using the enzyme and a protease having the higher specificity for the glycated peptide at the glycated hemoglobin β chain N terminus. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【００１５】
【表２】

【００１６】
【表３】

【００１７】
さらに本酵素を用いてヘモグロビンＡ１ｃを正確、簡便、安価に測定できることを見出し本発明を完成するに至った。
すなわち、本発明は
１）糖化ヘモグロビン瘢スＮ端の糖化ペプチドよりも、糖化ヘモグロビンβ鎖Ｎ端の糖化ペプチドに特異的に作用する酵素。
２）糖化ヘモグロビンβ鎖Ｎ端の糖化ペプチドに作用し、糖化ヘモグロビンα鎖Ｎ端の糖化ペプチドに実質的に作用しない酵素。
３）糖化ヘモグロビンβ鎖Ｎ端の糖化ペプチドが１−デオキシフルクトシル−Ｌ−バリル−Ｌ−ヒスチジンであり、ヘモグロビンα鎖Ｎ端の糖化ペプチドが、１−デオキシフルクトシル−Ｌ−バリル−Ｌ−ロイシンであることを特徴とする１）及び２）に記載の酵素。
４）酵素がフルクトシルアミンオキシダーゼである１）〜３）記載の酵素
５）酵素がネオコスモスポラ属、コニオケチジウム属、アルスリニウム属、ピレノケータ属、レプトスフェリア属、プレオスポラ属、オフィオボラス属、カーブラリア属、フォーマ属由来であることを特徴とする１）〜４）記載の酵素。
６）ネオコスモスポラ属、コニオケチジウム属、アルスリニウム属、ピレノケータ属、レプトスフェリア属、プレオスポラ属、オフィオボラス属、カーブラリア属、フォーマ属の培養物から糖化ヘモグロビンα鎖Ｎ端の糖化ペプチドよりも、糖化ヘモグロビンβ鎖Ｎ端の糖化ペプチドに特異的に作用する酵素を製造する方法。
７）糖化ヘモグロビンα鎖Ｎ端の糖化ペプチドよりも、糖化ヘモグロビンβ鎖Ｎ端の糖化ペプチドに特異性の高い酵素を用いることを特徴とする、アマドリ化合物の測定方法。
８）糖化ヘモグロビンβ鎖Ｎ端の糖化ペプチドに作用し、糖化ヘモグロビンα鎖Ｎ端の糖化ペプチドに実質的に作用しない酵素を用いることを特徴とするアマドリ化合物の測定方法。
９）アマドリ化合物がヘモグロビンＡ１ｃであることを特徴とする８）記載の方法。
１０）糖化ヘモグロビンα鎖Ｎ端の糖化ペプチドよりも、糖化ヘモグロビンβ鎖Ｎ端の糖化ペプチドに特異性の高い酵素を含有する試薬。
１１）プロテアーゼ、糖化ヘモグロビンα鎖Ｎ端の糖化ペプチドよりも、糖化ヘモグロビンβ鎖Ｎ端の糖化ペプチドに特異性の高い酵素を含有する試薬。に関する。
【００１８】
さらに詳しくは、ヘモグロビンＡ１ｃの本来の定義であるβ鎖のＮ末端バリンの糖化部位を特異的に認識し、測定するために必要な特異性を備えた酵素、該酵素を用いた測定方法及び試薬に関する。
【００１９】
【発明の実施の形態】
本発明について、以下具体的に説明する。
＜アマドリ化合物＞
本発明に於けるアマドリ化合物とは、タンパク質等のアミノ基をもつ化合物とグルコース等のアルデヒド基を持つ化合物がメイラード反応により、生じる下記の一般式（１）−（ＣＯ）−ＣＨＲ−ＮＨ−（Ｒは、水素原子か水酸基を示す）で表されるケトアミン構造を有する化合物のことを指す。アマドリ化合物には糖化ヘモグロビンや糖化アルブミンのような糖化タンパク質やペプチドが糖化された糖化ペプチド等が含まれる。
＜糖化ヘモグロビン＞
ヘモグロビンがメイラード反応により糖化されたアマドリ化合物のことを指し、α鎖及びβ鎖Ｎ末端のバリンや分子内のリジンが糖化されていると言われている。
＜ヘモグロビンＡ１ｃ＞
糖化ヘモグロビンのうちヘモグロビンβ鎖Ｎ末端のバリンが糖化された分子を指し、さらにアマドリ転移する前のシッフベースである不安定型ヘモグロビンとも区別される。
【００２０】
本発明に用いることが出来る酵素は、糖化ヘモグロビンα鎖Ｎ端の糖化ペプチドよりも、糖化ヘモグロビンβ鎖Ｎ端の糖化ペプチドに特異性の高い、若しくは、ＦＶＬよりもＦＶＨに特異性の高い酵素であればいかなる酵素を用いても良いが、糖化ヘモグロビンβ鎖Ｎ端の糖化ペプチドに作用し、糖化ヘモグロビンα鎖Ｎ端の糖化ペプチドに実質的に作用しないＦＡＯＤ、若しくは、ＦＶＨに作用し、ＦＶＬに実質的に作用しない酵素が好ましい。
【００２１】
特異性の高い酵素とは、通常、測定したい基質に対する活性を１００％とした時、測定対象でない基質に対する相対活性が例えば８０％以下であることをいい、好ましくは５０％以下、より好ましくは３０％以下である酵素をいう。また、実質的に作用しない酵素とは、通常、測定したい基質に対する活性を１００％とした時、測定対象でない基質に対する相対活性が例えば２０％以下であることをいい、好ましくは５％以下、より好ましくは１％以下である酵素をいう。
【００２２】
酵素の種類としてはでヒドロゲナーゼ、オキシダーゼ、キナーゼ等どのような酵素であっても糖化ヘモグロビンα鎖Ｎ端の糖化ペプチドよりも、糖化ヘモグロビンβ鎖Ｎ端の糖化ペプチドに特異性の高い酵素であれば用いることが出来るが、オキシダーゼが最も良く研究されており、使いやすさの点でオキシダーゼが好ましい。
【００２３】
以下に本発明に使用できる酵素の具体例を示すが、これによりなんら限定されるものではない。また、本発明の酵素を生産する能力を有するその変異株であっても使用できることは言うまでもない。表１にまとめたように、本発明に使用することが出来るオキシダーゼとしてはネオコスモスポラ属、コニオケチジウム属、アルスリニウム属、ピレノケータ属、レプトスフェリア属、プレオスポラ属、オフィオボラス属、カーブラリア属、フォーマ属に属する菌株が挙げられる。また、表１中のレプトスフェリア・ノドラム、レプトスフェリア・マクランス、プレオスポラ・ベタエはいずれも分生子世代がフォーマ属であり、本発明の酵素はフォーマ属にも広く分布していることがわかる。
【００２４】
また、より好ましくはネオコスモスポラ・バシンフェクタ ＮＢＲＣ７５９０菌株、コニオケチジウム・サボリＡＴＣＣ３６５４７菌株、アルスリニウム・エスピーＴＯ６（ＦＥＲＭ Ｐ−１９２１１）、アルスリニウム・ファエオスペルマムＮＢＲＣ３１９５０、アルスリニウム・ファエオスペルマムＮＢＲＣ６６２０、アルスリニウム・ジャポニカムＮＢＲＣ３１０９８、ピレノケータ・エスピーＹＨ８０７（ＦＥＲＭ Ｐ−１９２１０）、ピレノケータ・ゲンチアニコラＭＡＦＦ４２５５３１、ピレノケータ・テレストリスＮＢＲＣ３０９２９、レプトスフェリア・ノドラム（分生子世代名フォーマ・ヘンネレルギー）ＮＢＲＣ７４８０、レプトスフェリア・ドリオラムＪＣＭ２７４２、レプトスフェリア・マクランス（分生子世代名フォーマ・リンガム）ＭＡＦＦ７２６５２８、プレオスポラ・ハーブラムＮＢＲＣ３２０１２、プレオスポラ・ベタエ（分生子世代名フォーマ・ベタエ）ＮＢＲＣ５９１８、オフィオボラス・ヘルポトリカスＮＢＲＣ６１５８、カーブラリア・クラベータＹＨ９２３（ＦＥＲＭ Ｐ−１９２０９）などが挙げられる。
【００２５】
これらの菌株のうち、アルスリニウム・エスピーＴＯ６（ＦＥＲＭ Ｐ−１９２１１）、ピレノケータ・エスピーＹＨ８０７（ＦＥＲＭ Ｐ−１９２１０）、カーブラリア・クラベータＹＨ９２３（ＦＥＲＭ Ｐ−１９２０９）は本発明者等が新規に単離した菌株であり、独立行政法人産業技術総合研究所特許生物寄託センターに寄託した。
【００２６】
寄託したこれらの菌学的性質を示すと次の通りであり、以下のように同定した。
１．アルスリニウム・エスピー（Ａｒｔｈｒｉｎｉｕｍ ｓｐ．）ＴＯ６（ＦＥＲＭ Ｐ−１９２１１）について、宇田川俊一ら訳の「カビの分離・培養と同定」（医歯薬出版株式会社 １９８３）、宇田川俊一ら著の「菌類図鑑」（講談社サイエンティフィク １９８６）、Ｍ． Ｂ． Ｅｌｌｉｓ著「Ｄｍａｔａｃｅｏｕｓ Ｈｙｒｈｏｍｙｃｅｔｅｓ」（ｃｏｍｍｏｎｗｅａｌｔｈ ａｇｒｉｃｕｌｔｕｒａｌ ｂｕｒｅａｕｘ １９８８）を参考とし、その菌学的特性を検討した結果、
（１） 分生子は１細胞である。
（２） 分生子は外生する。
（３） 分生子は暗褐色である。
（４） 分生子は丸く、レンズ型で無色の帯がみられる。
（５） 分生子は分生子柄と１つの連結部で接する。
（６） 菌糸は有隔壁である。
（７） 分生子の大きさは６〜９μｍ×２〜４μｍである。
（８） 分生子柄は厚さ１．２〜２．５μｍである。
【００２７】
以上の形態的な特徴から、本菌株はアルスリニウム・サッカリコラ（Ａｒｔｈｒｉｎｉｕｍ Ｓａｃｃｈａｒｉｃｏｌａ）（分生子の大きさは９〜１０μｍ、分生子柄の厚さは２〜４μｍ）、アルスリニウム・ファエオスペルマム（Ａｒｔｈｒｉｎｉｕｍ ｐｈａｅｏｓｐｅｒｍｕｍ）（分生子の大きさは９〜１０μｍ、分生子柄の厚さは１〜１．５μｍ）、アルスリニウム・サッカリ（Ａｒｔｈｒｉｎｉｕｍ ｓａｃｃｈａｒｉ）（分生子の大きさは６〜８μｍ、分生子柄の厚さは１〜１．５μｍ）のいずれかに分類されると思われるが、これらの菌株とは分生子の大きさや分生子柄の厚みが若干異なることから、本菌株をアルスリニウム・エスピー（Ａｒｔｈｒｉｎｉｕｍ ｓｐ．）と同定した。
【００２８】
培地における生育状況
（１）オートミール寒天培地、２５℃、６日間培養後、直径約８．５ｃｍもしくはそれ以上に達し、コロニー裏面は無色で、白色で羊毛状のコロニーの上に暗褐色の胞子（塊）がみられる。
（２）ポテトデキストロース寒天培地上、２５℃、６日間培養後、直径約８．５ｃｍもしくはそれ以上に達し、コロニー裏面は無色で、白色で羊毛状のコロニーになる。
２．ピレノケータ・エスピー（Ｐｙｒｅｎｏｃｈａｅｔａ ｓｐ．）ＹＨ８０７（ＦＥＲＭ Ｐ−１９２１０）について、宇田川俊一ら訳の「カビの分離・培養と同定」（医歯薬出版株式会社 １９８３）、宇田川俊一ら著の「菌類図鑑」（講談社サイエンティフィク １９８６）、Ｂｒａｉａｎ Ｃ． Ｓｕｔｔｏｎ著「Ｔｈｅ Ｃｏｅｌｏｍｙｃｅｔｅｓ」（ｃｏｍｍｏｎｗｅａｌｔｈ ａｇｒｉｃｕｌｔｕｒａｌ ｂｕｒｅａｕｘ １９８０）を参考とし、その菌学的特性を検討した結果、
（１）分生子は楕円形で平滑な１細胞で、５〜７×１．５〜３．０μｍ。
（２）コロニーは暗緑褐色であり、色素は生成しない。
（３）胞子は連鎖せず、子実体（分生子殻）内部に形成される。
（４）菌糸は有隔壁である。
（５）分生子殻は暗褐色の球形〜亜球形もしくは倒洋ナシ形で１つの孔口をもつ。分生子殻は直径２００〜３００μｍで開口部周辺に剛毛を形成する。
【００２９】
以上の形態的な特徴から、本菌株はピレノケータ・ルビ−イダエイ（Ｐｙｒｅｎｏｃｈａｅｔａ ｒｕｂｉ−ｉｄａｅｉ）（分生子＞３．５μｍ×２．０〜２．５μｍ）に分類されると思われるが、これとは分生子のサイズが若干異なることから、本菌株をピレノケータ・エスピー（Ｐｙｒｅｎｏｃｈａｅｔａ ｓｐ．）と同定した。
【００３０】
培地における生育状況
（１）オートミール寒天培地上、２５℃、６日間培養後、直径約２．３ｃｍもしくはそれ以上に達し、コロニー裏面は暗緑褐色で暗緑褐色のコロニーになる。培地中に色素は生産しない。気生菌糸は疎である。
（２）ポテトデキストロース寒天培地上、２５℃、６日間培養後、直径約２．０ｃｍもしくはそれ以上に達し、コロニー裏面は暗緑褐色で暗緑褐色のコロニーになる。オートミール寒天培地上よりも気生菌糸が認められる。
（３）ＹＭ寒天培地上、２５℃、６日間培養すると褐色〜黒色の子実体（分生子殻）が生じる。
３．カーブラリア・クラベータ（Ｃｕｒｖｕｌａｒｉａ ｃｌａｖａｔａ）ＹＨ９２３（ＦＥＲＭ Ｐ−１９２０９）について、宇田川俊一ら訳の「カビの分離・培養と同定」（医歯薬出版株式会社 １９８３）、宇田川俊一ら著の「菌類図鑑」（講談社サイエンティフィク １９８６）、Ｍ． Ｂ． Ｅｌｌｉｓ著「Ｄｍａｔａｃｅｏｕｓ Ｈｙｒｈｏｍｙｃｅｔｅｓ」（ｃｏｍｍｏｎｗｅａｌｔｈ ａｇｒｉｃｕｌｔｕｒａｌ ｂｕｒｅａｕｘ １９８８）を参考とし、その菌学的特性を検討した結果、
（１）菌糸は有隔壁である。
（２）分生子はポロ型分生子で紡錘こん棒形、３つの隔壁を有し、ほとんど全てが下方より３番目の細胞がふくれている。基部にへそ（ｈｉｌｕｍ）があるが、顕著に突出していない。
（３）２番目の隔壁は正中にない。
（４）分生子の両端細胞はほとんど淡褐色、中間部細胞は褐色〜濃褐色、下方より３番目の細胞は最も濃色である。平坦でくびれなし、２６〜３４×１０〜１２μｍ。
（５）分生子柄は菌糸上に頂生または側生し、上方でしばしば湾曲し、ジグザグ状を呈する。
（６）分生子柄は無色〜淡褐色、平滑またはこぶ状、直径２．５〜４．０μｍ。
【００３１】
以上の形態的な特徴から、本菌株はカーブラリア・クラベータ（Ｃｕｒｖｕｌａｒｉａ ｃｌａｖａｔａ）と同定した。
培地における生育状況
（１）オートミール寒天培地、２５℃、７日間培養後、直径約５．５ｃｍもしくはそれ以上に達し、コロニー裏面及びコロニーは暗緑褐色で、ビロード状の気生菌糸が認められる。
（２）ポテトデキストロース寒天培地上、２５℃、７日間培養後、直径約４．０ｃｍもしくはそれ以上に達し、コロニー裏面は黒色で、灰白色で羊毛状の気生菌糸を形成する。
【００３２】
次に本発明に使用しうるＦＡＯＤ生産菌の培養方法について述べる。本発明ＦＡＯＤ生産菌の培養手段としては固体培養でも液体培養でもよいが、好ましくはフラスコまたはジャーファーメンター等による通気培養である。培地としては微生物の培養に通常用いられるものが広く使用される。炭素源としてはグルコース、グリセロール、ソルビトール、ラクトースまたはマンノースなど、窒素源としては酵母エキス、肉エキス、トリプトン、ペプトンなど、無機塩としては塩化ナトリウム、塩化マグネシウム、硫酸マグネシウム、塩化カルシウムなどを用いればよい。ｐＨはｐＨ５．０〜８．０、培養温度２５〜３７℃で目的とする酵素が最高力価となる培養時間、例えば２〜１０日間にて目的とする酵素を採取すればよい。
【００３３】
次いで酵素を採取するに当たっては培養液から菌体を遠心分離等によって分離し、菌体をリン酸緩衝液、トリス塩酸緩衝液などの緩衝液に懸濁した後、リゾチーム処理、超音波処理、ガラスビーズ破砕などの各種菌体破砕方法によって破砕して遠心分離し、可溶性画分を粗酵素液として回収する。
【００３４】
このようにして得られた粗製のＦＡＯＤ含有液を公知の蛋白質、酵素の単離、精製手段を用いて処理することにより、精製されたＦＡＯＤを得ることができる。例えばアセトン、エタノールなどの有機溶媒による分別沈殿法、硫安などによる塩析法、イオン交換クロマトグラフィー法、疎水クロマトグラフィー法、アフィニティークロマトグラフィー法、ゲルろ過法などの一般的な酵素精製法を適宜選択、組み合わせて精製ＦＡＯＤを得ることができ、適宜安定化剤、例えばショ糖、グリセロールまたはアミノ酸などを１〜５０％程度、補酵素などを０．０１〜０．１％程度として単独または２種以上適宜組み合わせて加えて凍結保存してもよい。
【００３５】
つぎに本発明で得られるＦＡＯＤの酵素作用および酵素活性測定法を述べる。酵素作用
酸素の存在化、ＦＶＨに作用し、過酸化水素、グルコソン、およびバリルヒスチジンを生成させる。
酵素活性測定方法
測定試薬
５０ｍＭ トリス塩酸緩衝液（ｐＨ７．５）
１ｍＭ フルクトシルバリルヒスチジン
０．０２％ ４−アミノアンチピリン
０． ０２％ ＴＯＯＳ
５Ｕ／ｍｌ ペルオキシダーゼ（メルク社製）
（ＴＯＯＳ：Ｎ−エチル−Ｎ−（２−ヒドロキシ−３−スルホプロピル）−３−メトキシアニリン）
測定試薬１ｍｌを光路長１ｃｍのセルにいれ、３７℃で５分間予備加温した後、０．０５ｍｌの酵素液を添加して５分間反応させる。反応後、０．５％ＳＤＳ（ソディウムドデシルスルフェイト）２ｍｌを添加して反応を停止させ、波長５５５ｎｍにおける吸光度（Ａａ）を測定する。また、ブランクとしてＦＶＨを含まない測定試薬を用いて同様の操作を行って吸光度を測定する（Ａｂ）。この吸光度（Ａａ）とブランクの吸光度（Ａｂ）の吸光度差（Ａａ−Ａｂ）より酵素活性を求める。酵素活性１単位は３７℃で１分間に１マイクロモルの過酸化水素を生成させる酵素量とした。
【００３６】
次に本発明の酵素を用いた測定法について述べる。
【００３７】
本発明に使用しうる測定方法については前記糖化ヘモグロビンα鎖Ｎ端の糖化ペプチドよりも、糖化ヘモグロビンβ鎖Ｎ端の糖化ペプチドに特異的に作用する酵素を用いた測定方法であればいかなる測定方法を用いても良いが、現在糖化ヘモグロビンの１デオキシフルクトシル基に直接作用する酵素は知られておらず、一度ヘモグロビンを断片化し、そのフラグメントに本発明の酵素を作用させれば良い。
【００３８】
ヘモグロビンの断片化方法としては化学法、酵素法が挙げられるが、その正確さ、簡便さから酵素法が好ましい。これまでヘモグロビンＡ１ｃのβ鎖Ｎ末端の糖化アミノ酸を切り出すプロテアーゼが報告されておりこれら公知のプロテアーゼを用いればよいが、ヘモグロビンＮ末端の糖化アミノ酸を切り出すとα鎖由来のものかβ鎖由来のものか見分けがつかなくなるのでヘモグロビンＡ１ｃのＮ末端の２アミノ酸以上からなる、糖化ペプチドを切り出すプロテアーゼが好ましく、Ｎ末端の２アミノ酸を切り出すプロテアーゼが最も好ましい。
【００３９】
好ましいプロテアーゼの例を具体的に以下に挙げるが、これは１例に過ぎない。スミチームＰ（パパイン；新日本化学社製）、ビオプラーゼＳＰ−１０、ビオプラーゼＡＬ１５ＦＧ（ナガセケムテックス社製）、オリエンターゼ１０Ｂ（阪急バイオインダストリー社製）、エンチロン（洛東化成工業）、プロテアーゼタイプＶＩＩＩ、プロテアーゼタイプＸＸＩＶ、プロテアーゼタイプＸＩＶ、 バチルスグロビギ（Ｂａｃｉｌｌｕｓ ｇｌｏｂｉｇｉｉ）由来プロテアーゼ、（以上シグマ社製）等。
【００４０】
本発明に於けるプロテアーゼの活性測定は、カゼイン−フォリン法で行った。活性の単位１Ｕは３７℃、１分間に１ｇのチロシンに相当するフォリン試薬の発色を示す酵素量を１Ｕと定義した。
【００４１】
さらにプロテアーゼを用いたヘモグロビンの分解方法、試薬において、前記プロテアーゼを単独で使用することはもちろんであるが、加えてその反応前後若しくは同時に他のエンドプロテアーゼ、または他のエキソプロテアーゼを作用させても良い。
【００４２】
本発明の糖化ヘモグロビンα鎖Ｎ端の糖化ペプチドよりも、糖化ヘモグロビンβ鎖Ｎ端の糖化ペプチドに特異的に作用する酵素を用いた測定方法に於ける酵素作用の検出は、例えばデヒドロゲナーゼを用いた場合には補酵素の変化量を、例えば補酵素としてＮＡＤを用いて生成される変化の量として還元型補酵素である還元型ＮＡＤをその極大吸収波長域である３４０ｎｍ付近の波長にて比色計で測定する等公知の技術を用い直接定量するか、若しくは生じた還元型補酵素を各種ジアフォラーゼ、またはフェナジンメトサルフェート等の電子キャリアー及びニトロテトラゾリウム、ＷＳＴ−１、ＷＳＴ−８（以上同人化学研究所社製）に代表される各種テトラゾリウム塩等の還元系発色試薬を用い間接的に定量してもよく、またこれ以外の公知の方法により直接、間接的に測定してもよい。
【００４３】
また例えばオキシダーゼを用いた場合には、酸素の消費量または反応生成物の量を測定することが好ましい。反応生成物として、例えばケトアミンオキシダーゼを用いた場合には反応により過酸化水素及びグルコソンが生成し、過酸化水素及びグルコソン共に公知の方法により直接、間接的に測定する事が出来る。
【００４４】
上記過酸化水素の量は、例えばパーオキシダーゼ等を用いて色素等を生成し、発色、発光、蛍光等により定量しても良く、また電気化学的手法によって定量しても良く、カタラーゼ等を用いてアルコールからアルデヒドを生成せしめて、生じたアルデヒドの量を定量しても良い。
【００４５】
過酸化水素の発色系は、パーオキシダーゼの存在下で４−ＡＡ若しくは３−メチル−２−ベンゾチアゾリノンヒドラゾン（ＭＢＴＨ）等のカップラーとフェノール等の色原体との酸化縮合により色素を生成するトリンダー試薬、パーオキシダーゼの存在下で直接酸化、呈色するロイコ型試薬（Ｎ−（カルボキシメチルアミノカルボニル）−４，４−ビス（ジメチルアミノ）ビフェニルアミン（ＤＡ６４）、１０−（カルボキシメチルアミノカルボニル）−３，７−ビス（ジメチルアミノ）フェノチアジン（ＤＡ６７）；以上和光純薬社製等）等を用いることが出来る。
【００４６】
また過酸化水素を電極を用いて測定する場合、電極には、過酸化水素との間で電子を授受する事の出来る材料である限り特に制限されないが、例えば白金、金若しくは銀等が挙げられ、電極測定方法としてはアンペロメトリー、ポテンショメトリー、クーロメトリー等の公知の方法を用いることが出来、さらにオキシダーゼまたは基質と電極との間の反応に電子伝達体を介在させ、得られる酸化、還元電流或いはその電気量を測定しても良い。電子伝達体としては電子伝達機能を有する任意の物質が使用可能であり、例えばフェロセン誘導体、キノン誘導体等の物質が挙げられる。またオキシダーゼ反応により生成する過酸化水素と電極の間に電子伝達体を介在させ得られる酸化、還元電流またはその電気量を測定しても良い。
【００４７】
本発明の酵素を用いたヘモグロビンＡ１ｃの測定用試薬の液組成については、使用するプロテアーゼ及び糖化ヘモグロビン瘢スＮ端の糖化ペプチドよりも、糖化ヘモグロビンβ鎖Ｎ端の糖化ペプチドに特異的に作用する酵素の至適ｐＨを考慮し、反応が効率よく進行するようにｐＨ及びプロテアーゼ濃度を決定すればよい。
【００４８】
例えばスミチームＰ（パパイヤ由来、新日本化学社製）をプロテアーゼとして用いる場合には、タンパク質分解活性がｐＨ５．５〜９．０付近で強いことから、反応のｐＨは５．５〜９．０を選択することができる。またプロテアーゼ濃度は実際に使用される反応時間中に被検液中のタンパク質を十分に分解し得る濃度で有れば良く０．０１〜１０００Ｕ／ｍｌが好ましく、０．１ 〜５００Ｕ／ｍｌ がさらに好ましい。
【００４９】
また、例えば糖化ヘモグロビンα鎖Ｎ端の糖化ペプチドよりも、糖化ヘモグロビンβ鎖Ｎ端の糖化ペプチドに特異的に作用する酵素としてフルクトシルアミンオキシダーゼ（Ａｒｔｈｒｉｎｉｕｍ ｓｐ． ＴＯ６由来）を用いる場合、至適ｐＨが７〜８であり、反応のｐＨは７〜８を選択できる。また酵素添加濃度は実際に使用される反応時間中に被検液から生成された糖化アミノ酸を十分に測定し得る濃度で有れば良く、０．０１Ｕ〜１０００Ｕ／ｍｌが好ましく、０．１Ｕ〜５００Ｕ／ｍｌがより好ましく、０．５Ｕ〜１００Ｕ／ｍｌが最も好ましい。
【００５０】
本発明に於ける糖化タンパク質定量用試薬としては、糖化ヘモグロビンα鎖Ｎ端の糖化ペプチドよりも、糖化ヘモグロビンβ鎖Ｎ端の糖化ペプチドに特異的に作用する酵素を含有するものとして調製すれば良く、好ましくはプロテアーゼ及び該糖化ヘモグロビンα鎖Ｎ端の糖化ペプチドよりも、糖化ヘモグロビンβ鎖Ｎ端の糖化ペプチドに特異的に作用する酵素を含有するものとして調整すれば良く、例えば液状品及び液状品の凍結物あるいは凍結乾燥品として提供できる。
【００５１】
さらに、本発明に基づく糖化蛋白質を定量する酵素反応組成には、例えば界面活性剤、塩類、緩衝剤、ｐＨ調製剤や防腐剤などを適宜選択して添加しても良い。
【００５２】
かくして調整された本発明のヘモグロビンＡ１ｃ測定用試薬を用いて、被検液中の糖化ヘモグロビンを測定するには、測定用試薬０．０１〜５．０ｍｌに溶血された被検液０．００１〜０．５ｍｌを加え、３７℃の温度にて反応させ、レートアッセイを行う場合には、反応開始後の一定時間後の２点間の数分ないし数十分間、例えば３分後と４分後の１分間、または３分ごと８分後の５分間における変化した補酵素、溶存酸素、過酸化水素若しくはその他生成物の量を直接または間接的に前記の方法で測定すれば良く、エンドポイントアッセイの場合には反応開始後一定時間後の変化した補酵素、溶存酸素、過酸化水素若しくはその他生成物の量を同様に測定すれば良い。この場合既知濃度のヘモグロビンＡ１ｃを含む試料を用いて測定した場合の吸光度等の変化と比較すれば被検液中のヘモグロビンＡ１ｃの量を求めることができる。
【００５３】
ここで述べた溶血された披検液とは、溶血操作を行った後のヘモグロビンＡ１ｃを含有する被検液であり、さらに詳しくは、全血遠心分離された赤血球、洗浄赤血球等の溶血液が挙げられる。
【００５４】
【実施例】
本発明を実施例に基づいて説明する。
【００５５】
【実施例１】
ネオコスモスポラ・バシンフェクタＮＢＲＣ７５９０菌株由来のＦＡＯＤの培養方法、精製方法および理化学的性質
＜培養方法および精製方法＞
５００ｍｌの三角フラスコ３本に１００ｍｌの２％のマンノース、３％の酵母エキス、０．１％のＫＨ_２ＰＯ_４、０．０５％ＭｇＳＯ_４・７Ｈ_２Ｏを含有する培地（ｐＨ６．０）をそれぞれ入れ、殺菌後、ネオコスモスポラ・バシンフェクタＮＢＲＣ７５９０菌株を植菌し、２８℃、４日間振とう培養した。
【００５６】
培養終了後、集菌し、３００ｍｌの１０ｍＭのトリス塩酸緩衝液（ｐＨ７．５）に懸濁させ、超音波破砕により菌体の可溶化を行った（１０Ｕ）。この可溶化液を１０ｍＭのトリス塩酸緩衝液（ｐＨ７．５）に１晩透析させた後、Ｑ−セファロース・ビッグビーズ（１００ｍｌ）樹脂（ファルマシア社製）イオン交換クロマトグラフィーを行った。溶出は０￣１Ｍのリニアグラジエントにより行い、０．２〜０．３ＭのＮａＣｌの溶出画分（９Ｕ）を回収した。
【００５７】
この酵素液に１５％硫酸アンモニウムの濃度となるように硫酸アンモニウムを溶解し、フェニルセファロース・ファーストフロー（５０ｍｌ）（ファルマシア社製）の疎水クロマトグラフィーを行った。溶出は１５〜０Ｍの硫酸アンモニウムのリニアグラジエントにより行い、５〜２％の硫酸アンモニウムの溶出画分（７Ｕ）を回収した。ついで、この酵素液を１０ｍＭのトリス塩酸緩衝液（ｐＨ７．０）にて１晩透析し、ハイドロキシアパタイト（３０ｍｌ）（ペンタックス社製）クロマトグラフィーを行った。溶出は０￣０．１Ｍのリン酸緩衝液（ｐＨ７．０）によるリニアグラジエントにより行い、０．０３￣０．０４Ｍのリン酸緩衝液の溶出画分（３Ｕ）を回収し、精製ＦＡＯＤを得た。
【００５８】
＜理化学的性質＞
（１）基質特異性
本発明ネオコスモスポラ・バシンフェクタ由来ＦＡＯＤの各種基質に対する特異性は表 の通りである。ＦＶに対して最も高い反応性を示し、ＦＶＨに対してＦＶの２９％反応し、ＦＶＬには全く作用しなかった。
（２）Ｋｍ値
本発明ネオコスモスポラ・バシンフェクタ由来ＦＡＯＤのＦＶＨに対するＫｍ値は３ｍＭであった。
（３）至適ｐＨ
前記酵素活性測定法に従って至適ｐＨを求めたもので、その結果を図１に示した。ｐＨ４．５〜５．５の範囲は１００ｍＭの酢酸緩衝液（図中、○）、ｐＨ５〜６の範囲は１００ｍＭのクエン酸緩衝液（図中、□）、ｐＨ６〜７．５の範囲は１００ｍＭのリン酸緩衝液（図中、△）７．５〜９の範囲は１００ｍＭのトリス塩酸緩衝液（図中、●）、ｐＨ９．５〜１０の範囲は１００ｍＭのグリシン緩衝液（図中、黒四角）を使用した場合の活性値を示すもので、至適ｐＨは６．３〜６．７にあった。
（４）ｐＨ安定性
０．５Ｕ／ｍｌの本発明ステフィリウム・エスピー由来ＦＡＯＤを１００ｍＭの各種緩衝液中で３７℃、１時間処理し、その残存活性を前記酵素活性測定法に従って測定した。その結果を図２に示した。ｐＨ４．５〜５．５の範囲は１００ｍＭの酢酸緩衝液（図中、○）、ｐＨ５〜６の範囲は１００ｍＭのクエン酸緩衝液（図中、□）、ｐＨ６〜７．５の範囲は１００ｍＭのリン酸緩衝液（図中、△）７．５〜９の範囲は１００ｍＭのトリス塩酸緩衝液（図中、●）、ｐＨ９．５〜１０の範囲は１００ｍＭのグリシン緩衝液（図中、黒四角）を使用した。ｐＨ５．７５〜７．３６の範囲で良好な安定性を示した。
（５）至適温度
前記酵素活性測定法に従って、温度３０〜６０℃の範囲で変化させて至適温度を求めた結果は図３に示す通りであり、至適温度は４５−５０℃であった。
（６）熱安定性
０．５Ｕ／ｍｌの本発明ステフィリウム・エスピー由来ＦＡＯＤを１００ｍＭのトリス塩酸緩衝液（ｐＨ７．５）中で各温度で１０分間加熱処理した後の残存活性を前記酵素活性測定法に従って測定した。その結果、図４に示す通り、少なくとも４５℃、１０分間加熱処理における残存活性が少なくとも８０％以上を示した。
（７）分子量
ＴＳＫ Ｇ−３０００ＳＷＸＬ（トーソー社製）を用いたＨＰＬＣにより分子量を測定した。その結果、分子量は３７，０００であった。
【００５９】
以上の結果を上記表２にまとめた。
【００６０】
【実施例２】
コニオケチジウム・サボリＡＴＣＣ菌株由来のＦＡＯＤの培養方法、精製方法および酵素学的性質
＜培養法および精製方法＞
実施例１で記載した培養方法により培養を行った。
【００６１】
培養終了後、集菌し、３００ｍｌの１０ｍＭのトリス塩酸緩衝液（ｐＨ７．５）に懸濁させ、超音波破砕により菌体の可溶化を行った（１５Ｕ）。この可溶化液を１０ｍＭのトリス塩酸緩衝液（ｐＨ７．５）に１晩透析させた後、Ｑ−セファロース・ビッグビーズ（１００ｍｌ）樹脂（ファルマシア社製）イオン交換クロマトグラフィーを行った。溶出は０￣１Ｍのリニアグラジエントにより行い、０．１５〜０．２５ＭのＮａＣｌの溶出画分（１３Ｕ）を回収した。
【００６２】
この酵素液に１５％硫酸アンモニウムの濃度となるように硫酸アンモニウムを溶解し、フェニルセファロース・ファーストフロー（５０ｍｌ）（ファルマシア社製）の疎水クロマトグラフィーを行った。溶出は１５〜０Ｍの硫酸アンモニウムのリニアグラジエントにより行い、４〜０％の硫酸アンモニウムの溶出画分（８Ｕ）を回収した。ついで、この酵素液を１０ｍＭのトリス塩酸緩衝液（ｐＨ７．０）にて１晩透析し、ハイドロキシアパタイト（３０ｍｌ）（ペンタックス社製）クロマトグラフィーを行った。溶出は０￣０．１Ｍのリン酸緩衝液（ｐＨ７．０）によるリニアグラジエントにより行い、０．０３￣０．０４Ｍのリン酸緩衝液の溶出画分（４Ｕ）を回収し、精製ＦＡＯＤを得た。
【００６３】
＜理化学的性質＞
（１）基質特異性
本発明コニオケチジウム・サボリ由来ＦＡＯＤの各種基質に対する特異性は表の通りである。ＦＶに対して最も高い反応性を示し、ＦＶＨに対してはＦＶの２６％反応し、ＦＶＬには全く作用しなかった。
（２）Ｋｍ値
本発明コニオケチジウム・サボリ由来ＦＡＯＤのＦＶＨに対するＫｍ値は１．６ｍＭであった。
（３）至適ｐＨ
実施例１と同様にして至適ｐＨを求めたもので、至適ｐＨは６．３〜６．７にあった。
（４）ｐＨ安定性
実施例１と同様にしてｐＨ安定性を求めたもので、ｐＨ５．８〜７の範囲で良好な安定性を示した。
（５）至適温度
実施例１と同様にして至適温度を求めたもので、至適温度は３７℃であった。（６）熱安定性
実施例１と同様にして至適温度を求めたもので、少なくとも３７℃、１０分間加熱処理における残存活性が少なくとも８０％以上を示した。
（７）分子量
ＴＳＫ Ｇ−３０００ＳＷＸＬ（トーソー社製）を用いたＨＰＬＣにより分子量を測定した。その結果、分子量は２７，０００であった。
【００６４】
以上の結果の結果は上記表２にまとめた。
【００６５】
【実施例３】
アルスレニウム・エスピーＴＯ６（ＦＥＲＭＰ−１９２１１）菌株由来のＦＡＯＤの培養方法、精製方法および酵素学的性質
＜培養方法＞
５００ｍｌ容坂口フラスコに１００ ｍｌのＹＭＧ培地（１．０％グルコース、１．０％ポリペプトン、０．３％酵母エキス、０．３％麦芽エキス、０．１％ ＫＨ_２ＰＯ_４、０．０５％ ＭｇＳＯ_４・７Ｈ_２Ｏ、ｐＨ ６．０）を入れ、殺菌後、アルスレニウム・エスピーＴＯ６を植菌し、３０℃、４日間振盪培養した。
＜精製方法＞
液体窒素で凍結した菌体を乳鉢で磨砕して菌体破砕を行い、４０ ｍｌの０．１Ｍトリス塩酸緩衝液（ｐＨ ７．３）を加えて、５℃で１晩放置した後、遠心分離して無細胞抽出液を得た。得られた無細胞抽出液に４０ ％ 飽和になるように硫酸アンモニウムを加え、遠心分離した上清を４０％ 飽和硫酸アンモニウムを加えた ０．１ Ｍトリス塩酸緩衝液（ｐＨ ７．３）で平衡化した Ｂｕｔｙｌ Ｔｏｙｏｐｅａｒｌ ６５０Ｍ （１８ｆ×１５０ ｍｍ， 東ソー製） のカラムに供与し、１０％飽和になるように硫酸アンモニウムを加えた ０．１ Ｍ トリス塩酸緩衝液（ｐＨ７．３）で洗浄を行い、０．１ Ｍ トリス塩酸 緩衝液（ｐＨ ７．３）で溶出した。その溶出液の活性画分に４０ ％ 飽和になるように硫酸アンモニウムを加え、４０％ 飽和硫酸アンモニウムを加えた０．１ Ｍトリス塩酸緩衝液（ｐＨ ７．３）で平衡化した Ｐｈｅｎｙｌ−５ＰＷ （８．０ｆ×７．５ ｍｍ， 東ソー製） のＨＰＬＣカラムに供与し、４０％ から ０％ までの飽和硫酸アンモニウムのリニアグラジエントで溶出した。その活性画分を ５０ ｍＭ トリス塩酸 緩衝液（ｐＨ ７．３）で透析した後、同緩衝液で平衡化した Ｐｏｒｏｓ ＨＱ／Ｈ （４．６ｆ×５０ ｍｍ， アプライドバイオシステムズ製） のカラムに供与し、０ から ０．５ Ｍ までの ＮａＣｌ のリニアグラジエントで溶出した。活性画分を回収し、蒸留水に対して透析し、精製ＦＡＯＤを得た。精製の過程を下記表にまとめた。
【００６６】
【表４】

【００６７】
＜理化学的性質＞
（１）基質特異性
本発明のアルスレニウム・エスピーＴＯ６由来ＦＡＯＤの各種基質に対する特異性は表３の通りである。反応時の各基質濃度を １ｍＭ とした。その他の反応条件は活性測定法に準じた。アルスレニウム・エスピーＴＯ６由来ＦＡＯＤはＦＶに対して最も高い反応性を示し、かつ、ＦＶＨにも高く反応するが、ＦＶＬへの作用はＦＶＨと比較して約４０分の１であり、本酵素はＦＶＬへ実質的に作用しないといえる。
（２）Ｋｍ値
本発明のアルスレニウム・エスピーＴＯ６由来ＦＡＯＤのＦＶＨに対するＫｍ値は０．４２ｍＭであった。
（３）至適ｐＨ
ＦＶＨを１ｍＭになるように溶解したｐＨ３．０〜１１．０の各種緩衝液（ｐＨ３〜５は１０ｍＭの酢酸緩衝液、ｐＨ５〜７は１０ｍＭのクエン酸緩衝液、ｐＨ７〜９は１０ｍＭのトリス塩酸緩衝液、ｐＨ９〜１１は１０ｍＭのホウ酸緩衝液）０．４５ｍｌを光路長１ｃｍのセルにいれ、３７℃で５分間予備加温した後、０．０５ｍｌの酵素液を添加して３０分間反応させた。反応後、測定試薬（０．０４％ ＴＯＯＳ、０．０４％ ４−アミノアンチピリン、パーオキシダーゼ５０Ｕを含む１００ｍＭトリス塩酸緩衝液（ｐＨ７．５））０．５ｍｌを加え、２分間発色させた後、０．５％ＳＤＳ２ｍｌを添加して反応を停止させ、波長５５５ｎｍにおける吸光度（Ａａ）を測定した。また、ブランクとして基質を含まない各種緩衝液を加え、同様の操作を行って吸光度を測定した（Ａｂ）。この吸光度（Ａａ）とブランクの吸光度（Ａｂ）の吸光度差（Ａａ−Ａｂ）より酵素活性を求めた。
【００６８】
その結果、アルスレニウム・エスピーＴＯ６由来ＦＡＯＤの至適ｐＨは７〜８にあった。
（４）ｐＨ安定性
酵素液を１０ｍＭの各種緩衝液中で４℃、２４時間処理し、その残存活性を前記酵素活性測定法に従って測定した。その結果、本発明のアルスレニウム・エスピーＴＯ６由来ＦＡＯＤはｐＨ７〜１１の範囲で良好な安定性を示した。
（５）至適温度
ＦＶＨを１ｍＭになるように溶解した１００ｍＭトリス塩酸緩衝液（ｐＨ７．５）０．４５ｍｌを光路長１ｃｍのセルにいれ、１５〜６０℃で５分間予備加温した後、０．０５ｍｌの酵素液を添加して１０分間反応させた。反応後氷中で冷却した後、測定試薬（０．０４％ ＴＯＯＳ、０．０４％ ４−アミノアンチピリン、パーオキシダーゼ５０Ｕを含む１００ｍＭトリス塩酸緩衝液（ｐＨ７．５））０．５ｍｌを加え、２分間発色させた後、０．５％ＳＤＳ２ｍｌを添加して反応を停止させ、波長５５５ｎｍにおける吸光度（Ａａ）を測定した。また、ブランクとしてＦＶＨを含まない各種緩衝液を加え、同様の操作を行って吸光度を測定した（Ａｂ）。この吸光度（Ａａ）とブランクの吸光度（Ａｂ）の吸光度差（Ａａ−Ａｂ）より酵素活性を求めた。
【００６９】
１５〜６０℃の範囲で変化させて至適温度を求めた結果、本発明のアルスレニウム・エスピーＴＯ６由来ＦＡＯＤの至適温度は３０〜４０℃であった。
（６）熱安定性
酵素液を１００ｍＭトリス塩酸緩衝液（ｐＨ７．５）中で各温度３０分間加熱処理した後の残存活性を前記酵素活性測定法に従って測定した。その結果、本発明のアルスレニウム・エスピーＴＯ６由来ＦＡＯＤは４０℃・３０分間の加熱処理までは８０％以上の残存活性を示した。
（７）分子量
ＹＭＣ−Ｐａｃｋ Ｄｉｏｌ−２００Ｇ（φ６．０×３００ ｍｍ、ワイエムシー製）によるゲル濾過から本酵素の分子量を求めた。標準蛋白質として牛血清アルブミン、オボアルブミン、大豆トリプシンインヒビター（全てシグマ製）を使用した。その結果、本酵素の分子量は約３４，０００であった。
【００７０】
Ｌａｅｍｍｌｉの方法での １０％ ｇｅｌ による ＳＤＳ−ＰＡＧＥ （ドデシル硫酸ナトリウム・ポリアクリルアミドゲル電気泳動）では、分子量は約 ５０，０００ であった。尚、標準蛋白質は ＳＤＳ−ＰＡＧＥ スタンダード Ｌｏｗ （バイオラッド社製） を使用した。
【００７１】
以上の結果から、本発明のアルスレニウム・エスピーＴＯ６由来ＦＡＯＤは単量体であることが明らかである。
【００７２】
【実施例４】
ピレノケータ・エスピーＹＨ８０７（ＦＥＲＭＰ−１９２１０菌株）菌株由来のＦＡＯＤの培養方法、精製方法および酵素学的性質
＜培養方法及び精製方法＞
実施例３で記載した製造及び精製法と同様の方法により活性画分を回収し、蒸留水に対して透析し、精製ＦＡＯＤを得た。精製の過程を下記表にまとめた。
【００７３】
【００７４】
【表５】

【００７５】
＜理化学的性質＞
（１）基質特異性
本発明のピレノケータ・エスピーＹＨ８０７由来ＦＡＯＤの各種基質に対する特異性は表３の通りである。反応時の各基質濃度を １ｍＭ とした。その他の反応条件は活性測定法に準じた。ピレノケータ・エスピーＹＨ８０７由来ＦＡＯＤはＦＶに対して最も高い反応性を示し、かつ、ＦＶＨにも高く反応するが、ＦＶＬへの作用はＦＶＨと比較して約１６０分の１であり、本酵素はＦＶＬへ実質的に作用しないといえる。
（２）Ｋｍ値
本発明のピレノケータ・エスピーＹＨ８０７由来ＦＡＯＤのＦＶＨに対するＫｍ値は０．９９ｍＭであった。
（３）至適ｐＨ
実施例３の（３）で記載した方法により本酵素の至適ｐＨを調べた結果、至適ｐＨは７〜８にあった。
（４）ｐＨ安定性
実施例３の（４）で記載した方法によりピレノケータ・エスピーＹＨ８０７由来ＦＡＯＤのｐＨ安定性を調べた結果、ｐＨ７〜１１の範囲で良好な安定性を示した。
（５）至適温度
実施例３の（５）で記載した方法に従って至適温度を求めた結果、本発明のピレノケータ・エスピーＹＨ８０７由来ＦＡＯＤの至適温度は３０〜４０℃であった。
（６）熱安定性
実施例３の（６）で記載した方法に従って熱安定性を求めた結果、ピレノケータ・エスピーＹＨ８０７由来ＦＡＯＤの熱安定性は、４０℃・３０分間の加熱処理までは８０％以上の残存活性を示した。
（７）分子量
実施例３の（７）で記載した方法に従ってゲル濾過ならびにＳＤＳ−ＰＡＧＥによる分子量を求めた結果、ピレノケータ・エスピーＹＨ８０７由来ＦＡＯＤの分子量はゲル濾過法で約３４，０００、ＳＤＳ−ＰＡＧＥで約５０，０００であった。この結果から、本発明のピレノケータ・エスピーＹＨ８０７由来ＦＡＯＤは単量体であることが明らかである。
【００７６】
【実施例５】
カーブラリア・クラベータＹＨ９２３（ＦＥＲＭＰ−１９２０９）菌株由来のＦＡＯＤの培養方法、精製方法および酵素学的性質について
＜培養方法および精製方法＞
実施例３で記載した製造及び精製法と同様の方法により活性画分を回収し、蒸留水に対して透析し、精製ＦＡＯＤを得た。精製の過程を下記表にまとめた。
【００７７】
【表６】

【００７８】
理化学的性質
（１）基質特異性
本発明のカーブラリア・クラベータＹＨ９２３由来ＦＡＯＤの各種基質に対する特異性は表３の通りである。反応時の各基質濃度を １ｍＭ とした。その他の反応条件は活性測定法に準じた。カーブラリア・クラベータＹＨ９２３由来ＦＡＯＤはＦＶに対して最も高い反応性を示し、かつ、ＦＶＨにも高く反応するが、ＦＶＬへの作用はＦＶＨと比較して約３０分の１であり、本酵素はＦＶＬへ実質的に作用しないといえる。
（２）Ｋｍ値
本発明のカーブラリア・クラベータＹＨ９２３由来ＦＡＯＤのＦＶＨに対するＫｍ値は０．９５ｍＭであった。
（３）至適ｐＨ
実施例３の（３）で記載した方法により本酵素の至適ｐＨを調べた結果、至適ｐＨは７〜８にあった。
（４）ｐＨ安定性
実施例３の（４）で記載した方法によりカーブラリア・クラベータＹＨ９２３由来ＦＡＯＤのｐＨ安定性を調べた結果、ｐＨ７〜１１の範囲で良好な安定性を示した。
（５）至適温度
実施例３の（５）で記載した方法に従って至適温度を求めた結果、本発明のカーブラリア・クラベータＹＨ９２３由来ＦＡＯＤの至適温度は５０〜５５℃であった。
（６）熱安定性
実施例３の（６）で記載した方法に従って熱安定性を求めた結果、カーブラリア・クラベータＹＨ９２３由来ＦＡＯＤの熱安定性は、５０℃・３０分間の加熱処理までは８０％以上の残存活性を示した。
（７）分子量
実施例３の（７）で記載した方法に従ってゲル濾過ならびにＳＤＳ−ＰＡＧＥによる分子量を求めた結果、カーブラリア・クラベータＹＨ９２３由来ＦＡＯＤの分子量はゲル濾過法で約３４，０００、ＳＤＳ−ＰＡＧＥで約５０，０００であった。この結果から、本発明のカーブラリア・クラベータＹＨ９２３由来ＦＡＯＤは単量体であることが明らかである。
【００７９】
【実施例６】
レプトスフェリア・ノドラムＮＢＲＣ７４８０菌株由来ＦＡＯＤの製造及び精製
＜培養方法及び精製方法＞
実施例３で記載した製造及び精製法と同様の方法により活性画分を回収し、蒸留水に対して透析し、精製ＦＡＯＤを得た。精製の過程を下記表６にまとめた。
【００８０】
【表７】

【００８１】
理化学的性質
（１）基質特異性
本発明のレプトスフェリア・ノドラムＮＢＲＣ７４８０由来ＦＡＯＤの各種基質に対する特異性は表３の通りである。反応時の各基質濃度を １ｍＭ とした。その他の反応条件は活性測定法に準じた。レプトスフェリア・ノドラムＮＢＲＣ７４８０由来ＦＡＯＤはＦＶに対して最も高い反応性を示し、かつ、ＦＶＨにも高く反応するが、ＦＶＬへの作用はＦＶＨと比較して約５分の１であり、本酵素はＦＶＬへ実質的に作用しないといえる。
（２）Ｋｍ値
本発明のレプトスフェリア・ノドラムＮＢＲＣ７４８０由来ＦＡＯＤのＦＶＨに対するＫｍ値は０．８５ｍＭであった。
（３）至適ｐＨ
実施例３の（３）で記載した方法によりレプトスフェリア・ノドラムＮＢＲＣ７４８０由来ＦＡＯＤの至適ｐＨを調べた結果、至適ｐＨは７〜８にあった。
（４）ｐＨ安定性
実施例３の（４）で記載した方法によりレプトスフェリア・ノドラムＮＢＲＣ７４８０由来ＦＡＯＤのｐＨ安定性を調べた結果、ｐＨ７〜１１の範囲で良好な安定性を示した。
（５）至適温度
実施例３の（５）で記載した方法に従って至適温度を求めた結果、本発明のレプトスフェリア・ノドラムＮＢＲＣ７４８０由来ＦＡＯＤの至適温度は３０〜４０℃であった。
（６）熱安定性
実施例３の（６）で記載した方法に従って熱安定性を求めた結果、レプトスフェリア・ノドラムＮＢＲＣ７４８０由来ＦＡＯＤの熱安定性は、４０℃・３０分間の加熱処理までは８０％以上の残存活性を示した。
（７）分子量
実施例３の（７）で記載した方法に従ってゲル濾過ならびにＳＤＳ−ＰＡＧＥによる分子量を求めた結果、レプトスフェリア・ノドラムＮＢＲＣ７４８０由来ＦＡＯＤの分子量はゲル濾過法で約３４，０００、ＳＤＳ−ＰＡＧＥで約５０，０００であった。この結果から、本発明のレプトスフェリア・ノドラムＮＢＲＣ７４８０由来ＦＡＯＤは単量体であることが明らかである。
【００８２】
【実施例７】
プレオスポラ・ハーブラムＮＢＲＣ３２０１２菌株由来のＦＡＯＤの培養方法、精製方法および酵素学的性質
＜培養方法及び精製方法＞
実施例３で記載した製造及び精製法と同様の方法により活性画分を回収し、蒸留水に対して透析し、精製ＦＡＯＤを得た。精製の過程を下記表７にまとめた。
【００８３】
【表８】

【００８４】
理化学的性質
（１）基質特異性
本発明のプレオスポラ・ハーブラムＮＢＲＣ３２０１２由来ＦＡＯＤの各種基質に対する特異性は表３の通りである。反応時の各基質濃度を１ｍＭ とした。その他の反応条件は活性測定法に準じた。プレオスポラ・ハーブラムＮＢＲＣ３２０１２由来ＦＡＯＤはＦＶに対して最も高い反応性を示し、かつ、ＦＶＨにも高く反応するが、ＦＶＬへの作用はＦＶＨと比較して約１５６分の１であり、本酵素はＦＶＬへ実質的に作用しないといえる。
（２）Ｋｍ値
本発明のプレオスポラ・ハーブラムＮＢＲＣ３２０１２由来ＦＡＯＤのＦＶＨに対するＫｍ値は０．８ｍＭであった。
（３）至適ｐＨ
実施例３の（３）で記載した方法により本酵素の至適ｐＨを調べた結果、至適ｐＨは７〜８にあった。
（４）ｐＨ安定性
実施例３の（４）で記載した方法によりプレオスポラ・ハーブラムＮＢＲＣ３２０１２由来ＦＡＯＤのｐＨ安定性を調べた結果、ｐＨ７〜１１の範囲で良好な安定性を示した。
（５）至適温度
実施例３の（５）で記載した方法に従って至適温度を求めた結果、プレオスポラ・ハーブラムＮＢＲＣ３２０１２由来ＦＡＯＤの至適温度は３０〜４０℃であった。
（６）熱安定性
実施例３の（６）で記載した方法に従って熱安定性を求めた結果、プレオスポラ・ハーブラムＮＢＲＣ３２０１２由来ＦＡＯＤの熱安定性は、４０℃・３０分間の加熱処理までは８０％以上の残存活性を示した。
（７）分子量
実施例３の（７）で記載した方法に従ってゲル濾過ならびにＳＤＳ−ＰＡＧＥによる分子量を求めた結果、プレオスポラ・ハーブラムＮＢＲＣ３２０１２由来ＦＡＯＤの分子量はゲル濾過法で約３４，０００、ＳＤＳ−ＰＡＧＥで約５０，０００であった。この結果から、本発明のプレオスポラ・ハーブラムＮＢＲＣ３２０１２由来ＦＡＯＤは単量体であることが明らかである。
【００８５】
【実施例８】
オフィオボラス・ヘルポトリカスＮＢＲＣ６１５８菌株由来のＦＡＯＤの培養方法、精製方法および酵素学的性質
＜培養方法＞
５００ｍｌ容坂口フラスコに１００ ｍｌのＹＭＧ培地（１．０％グルコース、１．０％ポリペプトン、０．３％酵母エキス、０．３％麦芽エキス、０．１％ ＫＨ_２ＰＯ_４、０．０５％ ＭｇＳＯ_４・７Ｈ_２Ｏ、ｐＨ ６．０）を入れ、殺菌後、ＦＡＯＤ生産菌株を植菌し、３０℃、１０日間振盪培養した。
＜精製方法＞
実施例で記載した精製法と同様の方法により活性画分を回収し、蒸留水に対して透析し、精製ＦＡＯＤを得た。精製の過程を下記表にまとめた。
【００８６】
【表９】

【００８７】
理化学的性質
（１）基質特異性
本発明のオフィオボラス・ヘルポトリカスＮＢＲＣ６１５８由来ＦＡＯＤの各種基質に対する特異性は表３の通りである。反応時の各基質濃度を １ｍＭ とした。その他の反応条件は活性測定法に準じた。オフィオボラス・ヘルポトリカスＮＢＲＣ６１５８由来ＦＡＯＤはＦＶに対して最も高い反応性を示し、かつ、ＦＶＨにも高く反応するが、ＦＶＬへの作用はＦＶＨと比較して約１５６分の１であり、本酵素はＦＶＬへ実質的に作用しないといえる。
（２）Ｋｍ値
本発明のオフィオボラス・ヘルポトリカスＮＢＲＣ６１５８由来ＦＡＯＤのＦＶＨに対するＫｍ値は０．８ｍＭであった。
（３）至適ｐＨ
実施例３の（３）で記載した方法によりオフィオボラス・ヘルポトリカスＮＢＲＣ６１５８由来ＦＡＯＤの至適ｐＨを調べた結果、至適ｐＨは７〜８にあった。
（４）ｐＨ安定性
実施例３の（４）で記載した方法によりオフィオボラス・ヘルポトリカスＮＢＲＣ６１５８由来ＦＡＯＤのｐＨ安定性を調べた結果、ｐＨ７〜１１の範囲で良好な安定性を示した。
（５）至適温度
実施例３の（５）で記載した方法に従って至適温度を求めた結果、本発明のオフィオボラス・ヘルポトリカスＮＢＲＣ６１５８由来ＦＡＯＤの至適温度は３０〜４０℃であった。
（６）熱安定性
実施例３の（６）で記載した方法に従って熱安定性を求めた結果、オフィオボラス・ヘルポトリカスＮＢＲＣ６１５８由来ＦＡＯＤの熱安定性は、４０℃・３０分間の加熱処理までは８０％以上の残存活性を示した。
（７）分子量
実施例３の（７）で記載した方法に従ってゲル濾過ならびにＳＤＳ−ＰＡＧＥによる分子量を求めた結果、オフィオボラス・ヘルポトリカスＮＢＲＣ６１５８由来ＦＡＯＤの分子量はゲル濾過法で約３４，０００、ＳＤＳ−ＰＡＧＥで約５０，０００であった。この結果から、本発明のオフィオボラス・ヘルポトリカスＮＢＲＣ６１５８由来ＦＡＯＤは単量体であることが明らかである。
【００８８】
【実施例９】
ヘモグロビンＡ１ｃの測定
＜本発明のプロテアーゼを用いた糖化ヘモグロビンの測定＞
＜溶血試薬＞
１５０ｍＭ トリス緩衝液（和光純薬社製）ｐＨ７．５
１％ Ｐｏｌｙｏｘｙｅｔｈｙｌｅｎｅ ｌａｕｒｙｌ ｅｔｈｅｒ（和光純薬社製）
＜Ｒ１；プロテアーゼ試薬＞
１５０ｍＭ トリス緩衝液（和光純薬社製）ｐＨ７．５
４０００Ｕ／ｍｌ ストレプトマイセス属由来プロテアーゼ（プロテアーゼタイプＸＩＶ；シグマ社製）
＜Ｒ２；発色試薬＞
１５０ｍＭ トリス緩衝液（和光純薬社製）ｐＨ８．０
５ｍＭ ＤＡ６４（和光純薬社製）
２４Ｕ／ｍｌ フルクトシルアミンオキシダーゼ（Ａｒｔｈｒｉｎｉｕｍ ｓｐ． ＴＯ６由来）
２０Ｕ／ｍｌ ＰＯＤ（シグマ社製）
＜反応手順＞
上記溶血試薬０．９ｍｌに全血より分離した赤血球若しくは標準糖化ペプチド溶液０．１ｍｌを添加し、３７℃−１０分間インキュベーションし溶血試料とした。溶血試料はヘモグロビン濃度を求める目的でＡ５７０ｎｍを測定した。続いてＲ１試薬２４０μｌおよび溶血試料６μｌを混合し、３７℃５分反応を行い７５０ｎｍを測光した（Ａ０）。さらにＲ２試薬６０μｌを添加し３７℃−５分間インキュベーションし７５０ｎｍを測光した（Ａ１）。ブランクの測定は、試料に蒸留水を用いてブランクの吸光度変化（ブランクΔＡ＝Ａ１ブランク−Ａ０ブランク）を測定した。また試料に検体及び糖化ヘモグロビン値既知の試料を用いて感度（感度ΔＡ＝（Ａ１−Ａ０）−ブランクΔＡ）を求め、糖化ヘモグロビン濃度を算出した。さらに糖化ヘモグロビン濃度をヘモグロビン濃度で除し、糖化ヘモグロビン値を算出した。
＜試料＞
健常者全血５検体、糖尿病患者全血５検体
１００μＭ、８０μＭ、６０μＭ、４０μＭ、２０μＭのＦＶＨ若しくはＦＶＬ
＜ヘモグロビンＡ１ｃのＨＰＬＣを用いた測定＞
ＨＰＬＣ法の測定はＨｂＡ１ｃ測定装置（アークレイ社製）にて測定した。
標準糖化ペプチド（ＦＶＨ，ＦＶＬ）の測定結果を図５に、検体の測定結果を表９に示す。
【００８９】
【表１０】

【００９０】
図５から分かるように、本発明の酵素を用いた測定試薬はＦＶＬには作用せず、ＦＶＨにのみ作用することから、正確にヘモグロビンβ鎖Ｎ末端の糖化ペプチドのみを検出できることが明らかであり、糖化ヘモグロビンではなくヘモグロビンＡ１ｃを正確に測定していることが明らかであった。
また表１０よりＨＰＬＣ法で測定したヘモグロビンＡ１ｃの測定値と酵素法の値が極めて良く一致することから、正確にヘモグロビンＡ１ｃを測定していることが明白であった。
【００９１】
【発明の効果】
本発明の糖化ヘモグロビンα鎖Ｎ端の糖化ペプチドよりも、糖化ヘモグロビンβ鎖Ｎ端の糖化ペプチドに特異性の高い酵素、該酵素を用いた測定方法、試薬を用いろことにより、ヘモグロビンＡ１ｃをより簡便、正確、安価に測定することが可能になる。
【図面の簡単な説明】
【図１】図１はネオコスモスポラ・バシンフェクタ由来のＦＡＯＤの至適ｐＨ曲線を示す。
【図２】図２はネオコスモスポラ・バシンフェクタ由来のＦＡＯＤのｐＨ安定性曲線を示す。
【図３】図３はネオコスモスポラ・バシンフェクタ由来のＦＡＯＤの至適温度曲線を示す。
【図４】図４はネオコスモスポラ・バシンフェクタ由来のＦＡＯＤの熱安定性曲線を示す。
【図５】図５は標準糖化ペプチド（ＦＶ、ＦＶＨ）の測定曲線を示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an enzyme having higher specificity for a glycated peptide at the N-terminal of glycated hemoglobin β-chain than a glycated peptide at the N-terminal of glycated hemoglobin α-chain, a measuring method and a reagent using the enzyme. More specifically, the present invention relates to an enzyme for simply, accurately and inexpensively measuring hemoglobin A1c in a clinical setting, a measuring method and a reagent using the enzyme.
[0002]
[Prior art]
When a reducing sugar such as glucose coexists, a glycated protein is produced by the non-enzymatic and irreversible binding of an amino group and an aldehyde group of a protein and Amadori transfer. In vivo, glycohemoglobin in which hemoglobin in blood is glycated, glycoalbumin in which albumin is glycated, fructosamine in which blood protein is glycated, and the like are known. These blood concentrations reflect the average blood glucose level for a certain period in the past, and the measured value can be an important indicator for the diagnosis and management of symptoms of diabetes pathology. Extremely useful.
[0003]
Among them, the most general and clinical data that can be used as an index for diagnosing diabetes is the blood hemoglobin A1c concentration, and it is particularly required to establish a simple and accurate measurement method. Also, by quantifying the Amadori compound in foods such as soy sauce and miso, it is possible to know the preservation state and the period of the food after production, which is considered to be useful for quality control.
[0004]
Conventionally, as a method for measuring an Amadori compound, a method using high performance liquid chromatography (Non-patent Document 1), a method using a column packed with a solid to which boric acid is bound (Non-patent Document 2), an antigen-antibody reaction, Utilization method (Non-patent document 3) A method for colorimetric determination of reducing ability using a tetrazolium salt (Non-patent document 4), a method for colorimetric determination using thiobarbituric acid (Non-patent document 5), and the like are known. I have.
[0005]
However, all of these methods are complicated in operation, require expensive equipment, and are not always accurate and quick methods. At present, an enzymatic method has been proposed as a method for measuring glycoproteins which is simpler, cheaper, and more accurate than the above method, in a short time. Patent Literatures 1) to 7) are known as methods for quantifying Amadori compounds, and Patent Literatures 8) to 11) are known as methods for measuring glycated proteins for diagnosing diabetes.
[0006]
Enzymes that act on glycated amino acids or glycated peptides used in the above methods have so far been of the genus Corynebacterium (Patent Document 12), the genus Aspergillus (Patent Document 13), the genus Nissilium (Patent Document 14), and the genus Fusarium. (Patent Documents 15 to 17), Fructosylamine oxidases derived from the genus Gibberella (Patent Documents 18 and 19), the genus Candida (Patent Document 20), and the genus Aspergillus (Patent Documents 21 and 22) have been reported.
[0007]
Hemoglobin is a tetramer having a molecular weight of 64,500 consisting of two polypeptides, an α chain and a β chain. The N-terminal amino acid sequence of the α-chain of hemoglobin is valine-leucine-serine-..., And the N-terminal amino acid sequence of the β-chain is valine-histidine-leucine-. Hemoglobin A1c is defined as glycated β-chain N-terminal valine, and is distinguished from glycated hemoglobin in which it is not known which amino acid in hemoglobin is glycated. At present, hemoglobin A1c is frequently used in clinical practice due to its accuracy, and measurement of glycated hemoglobin has almost disappeared.
[0008]
As a method for measuring glycated hemoglobin using an enzyme, a method has been devised in which a protease capable of cleaving the C-terminal side of leucine and dipeptidyl carboxypeptidase are allowed to act to release only the N-terminal glycated valine of the β-chain (Patent Document 23). However, in this method, a large amount of peptide is released by the protease, so that a large amount of expensive dipeptidyl carboxypeptidase is required to completely decompose the intended fructosyl valyl histidyl leucine. is there.
[0009]
In addition, a measurement method using FAOD acting on glycated valylhistidine at the N-terminus was reported (Patent Document 24), but there was no mention of the reactivity of glycated hemoglobin to a peptide released from the α chain. Was. In other words, rather than specifically recognizing and measuring the glycation site of the N-terminal valine of the β-chain of hemoglobin, N-terminal glycated valine of both the α-chain and the β-chain is measured, and the original hemoglobin A1c, That is, it did not measure the N-terminal glycated valine of the β chain.
Until now, there has been no simple, inexpensive, and rapid method for measuring HbA1c using an enzyme.
[0010]
[Non-patent document 1]
Chromatogr. Sci. , 10: 659 (1979).
[Non-patent document 2]
Clin. Chem. 26: 1598 (1982)
[Non-Patent Document 3]
JJCLA 18: 620 (1993)
[Non-patent document 4]
Clin. Chim. Acta 127: 87 (1982).
[Non-Patent Document 5]
Clin. Chim. Acta 112: 197 (1981)
[Patent Document 1]
Japanese Patent Publication No. 05-33997
[Patent Document 2]
Japanese Patent Publication No. 6-65300
[Patent Document 3]
JP-A-02-195900
[Patent Document 4]
JP-A-03-155780
[Patent Document 5]
JP-A-04-4874
[Patent Document 6]
JP 05-192193 A
[Patent Document 7]
JP 06-46846 A
[Patent Document 8]
JP-A-02-195899
[Patent Document 9]
JP-A-02-19590
[Patent Document 10]
JP 05-192193 A
[Patent Document 11]
JP 06-46846 A
[Patent Document 12]
JP-A-61-268178
[Patent Document 13]
JP-A-3-155780
[Patent Document 14]
JP-A-4-4874
[Patent Document 15]
JP-A-5-192193
[Patent Document 16]
JP-A-7-289253
[Patent Document 17]
JP-A-8-154672
[Patent Document 18]
JP-A-5-192193
[Patent Document 19]
JP-A-7-289253
[Patent Document 20]
JP-A-6-46846
[Patent Document 21]
JP-A-10-33177
[Patent Document 22]
JP-A-10-33180
[Patent Document 23]
JP 2000-300294 A
[Patent Document 24]
JP 2001-95598 A
[0011]
[Problems to be solved by the invention]
An object of the present invention is to provide an enzyme having higher specificity for a glycated peptide at the N-terminal of glycated hemoglobin β-chain than a glycated peptide at the N-terminal of glycated hemoglobin α-chain, a measuring method and a reagent using the enzyme, and More specifically, an object of the present invention is to provide an enzyme, a measuring method, and a reagent for easily, accurately, and inexpensively measuring hemoglobin A1c using an enzyme.
[0012]
[Means for Solving the Problems]
The present inventors have searched for an enzyme that acts only on the N-terminal of the hemoglobin β chain in order to solve the above problems. In the search, the sequence of the hemoglobin α-chain is VLS from the N-terminus, and the sequence of the β-chain is VHL from the N-terminus. Therefore, the N-terminus is the same for the α-chain and β-chain, and the glycated amino acid at the N-terminus is released. Thus, it is impossible to search for an enzyme that acts only on the N-terminus of hemoglobin β chain. Therefore, the present inventors used 1 deoxyfructosyl valylleucine (hereinafter abbreviated as FVL) and 1 deoxyfructosyl valyl histidine (hereinafter abbreviated as FVH) which are model substrates of N-terminal glycated products of hemoglobin α-chain and β-chain. It was synthesized and purified using the method described above (Hashiba H, J. Agric. Food Chem. 24:70, 1976).
[0013]
The present inventors cultured natural enzymes (fungi isolated from soils and the like in various places) and purchased preserved bacteria, and screened a strain producing FAOD which acts on FVH and does not substantially act on FVL. As a result of diligent studies, Neocosmospora vasinfecta NBRC 7590, Coniochaetidium savoryi ATCC 36547, Arthrinium Sp. (Arthrinium phaeosperum) NBRC 31950, Arthrinium phaeospermum NB C6620, Arthrinium japonicum NBRC 31098, Pyrenoceta sp. YH807 (FERM P-19210), Pyrenoketa Gentiana retino Nentiana NFarentiana Pentre Nancy Renaissance, Ptynochaeta Nentiana FP Nodrum (Leptosphaeria nodorum) (Phona hennebergii) NBRC7480, Leptosphaeria doliorum JCM2742, Leptosphere A-Makuransu (Leptosphaeria maculans) (conidia generation name forma Lingam (Phoma lingum)) MAFF7
26528, Pleospora herbarum NBRC 32012, Pleospora betae (conidia generation name Phoma betae) NBRC 5918, Ophioboras herpotricus Bolphia vulsa Bercula (Rhob) It has been discovered that a microorganism such as (FERM P-19209) produces an enzyme having higher specificity for a glycated hemoglobin β-chain N-terminal glycated peptide than a glycated hemoglobin α-chain N-terminal glycated peptide. The productivity is shown in Table 1 below, and the properties of the purified enzymes are shown in Tables 2 and 3 below.
[0014]
[Table 1]

[0015]
[Table 2]

[0016]
[Table 3]

[0017]
Further, they have found that hemoglobin A1c can be measured accurately, simply and inexpensively using the present enzyme, and have completed the present invention.
That is, the present invention
1) An enzyme that specifically acts on the glycated peptide at the N-terminal of glycated hemoglobin β chain rather than the glycated peptide at the N-terminal of glycated hemoglobin scar.
2) An enzyme that acts on the glycated peptide at the N-terminal of the glycated hemoglobin β chain and does not substantially act on the glycated peptide at the N-terminal of the glycated hemoglobin α chain.
3) The glycated peptide at the N-terminal of the glycated hemoglobin β chain is 1-deoxyfructosyl-L-valyl-L-histidine, and the glycated peptide at the N-terminal of the hemoglobin α chain is 1-deoxyfructosyl-L-valyl-L-. The enzyme according to 1) or 2), which is leucine.
4) The enzyme according to 1) to 3), wherein the enzyme is fructosylamine oxidase.
5) The enzyme according to 1) to 4), wherein the enzyme is derived from the genus Neocosmospora, the genus Coniochetium, the genus Arsulinium, the genus Pyrenoceta, the genus Leptosperia, the genus Pleospora, the genus Ophioboras, the genus Carbralia, or the genus Former.
6) Glycated hemoglobin β rather than glycated hemoglobin α-chain N-terminal glycated peptide from cultures of Neocosmospora, Coniochetium, Arsulinium, Pyrenoceta, Leptosperia, Pleospora, Ophioboras, Carbralia, Forma A method for producing an enzyme that specifically acts on a glycated peptide at the N-terminal of a chain.
7) A method for measuring an Amadori compound, comprising using an enzyme having higher specificity for a glycated peptide at the N-terminal of glycated hemoglobin β-chain than at a glycated peptide at the N-terminal of glycated hemoglobin α-chain.
8) A method for measuring an Amadori compound, comprising using an enzyme that acts on a glycated peptide at the N-terminal of glycated hemoglobin β chain and does not substantially act on a glycated peptide at the N-terminal of glycated hemoglobin α chain.
9) The method according to 8), wherein the Amadori compound is hemoglobin A1c.
10) A reagent containing an enzyme having higher specificity for the glycated peptide at the N-terminal of glycated hemoglobin β-chain than at the glycated peptide at the N-terminal of glycated hemoglobin α-chain.
11) A reagent containing a protease or an enzyme having higher specificity for a glycated peptide at the N-terminal of glycated hemoglobin β-chain than at a glycated peptide at the N-terminal of glycated hemoglobin α-chain. About.
[0018]
More specifically, an enzyme having the specificity necessary for specifically recognizing and measuring the glycation site of the N-terminal valine of the β chain, which is the original definition of hemoglobin A1c, a measuring method and a reagent using the enzyme About.
[0019]
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention will be specifically described below.
<Amadori compound>
The Amadori compound in the present invention is a compound represented by the following general formula (1)-(CO) -CHR-NH- () generated by a Maillard reaction between a compound having an amino group such as a protein and a compound having an aldehyde group such as glucose. R represents a hydrogen atom or a hydroxyl group) and represents a compound having a ketoamine structure. Amadori compounds include glycated proteins such as glycated hemoglobin and glycated albumin, and glycated peptides obtained by glycating peptides.
<Saccharified hemoglobin>
It refers to an Amadori compound in which hemoglobin is saccharified by the Maillard reaction, and it is said that valine at the N-terminal of α-chain and β-chain and lysine in the molecule are saccharified.
<Hemoglobin A1c>
Among glycated hemoglobins, valine at the N-terminal of the hemoglobin β chain refers to a saccharified molecule, and is further distinguished from unstable hemoglobin, which is a Schiff base before Amadori transfer.
[0020]
The enzyme that can be used in the present invention is an enzyme having higher specificity for glycated hemoglobin β-chain N-terminal glycated peptide than glycated hemoglobin α-chain N-terminal glycated peptide, or higher specificity for FVH than FVL. Any enzyme may be used as long as it acts on glycated hemoglobin β-chain N-terminal glycated peptide and acts substantially on glycated hemoglobin α-chain N-terminal glycated peptide on FAOD or FVH, and acts on FVL. Enzymes that do not substantially act are preferred.
[0021]
An enzyme having high specificity generally means that the relative activity to a substrate not to be measured is, for example, 80% or less, preferably 50% or less, more preferably 30%, when the activity for the substrate to be measured is 100%. % Or less. In addition, an enzyme that does not substantially act means that the relative activity to a substrate that is not a measurement target is, for example, 20% or less, preferably 5% or less, when the activity for a substrate to be measured is 100%. It refers to an enzyme that is preferably 1% or less.
[0022]
Regarding the type of enzyme, any enzyme such as hydrogenase, oxidase, and kinase can be used as long as it has a higher specificity for the glycated peptide at the N-terminal of glycated hemoglobin β-chain than the glycated peptide at the N-terminal of glycated hemoglobin α-chain. Although oxidases can be used, oxidases have been best studied and oxidases are preferred for ease of use.
[0023]
Specific examples of the enzyme that can be used in the present invention are shown below, but the present invention is not limited thereto. Needless to say, mutants having the ability to produce the enzyme of the present invention can also be used. As summarized in Table 1, the oxidases that can be used in the present invention belong to the genus Neocosmospora, the genus Conioquethidium, the genus Arsulinium, the genus Pyrenocatora, the genus Leptosperia, the genus Pleospora, the genus Ophioboras, the genus Carburaria, and the genus Forma Strains. In addition, all of the conidia generations of Leptosperia nodrum, Leptosperia macrans, and Pleospora betta in Table 1 belong to the Forma genus, indicating that the enzyme of the present invention is widely distributed in the Forma genus.
[0024]
More preferably, Neocosmospora bacinfecta NBRC 7590 strain, Coniocetium savory ATCC 36547 strain, Arsulinium sp. NBRC 31098, Pyrenocator SP YH807 (FERM P-19210), Pyrenocator Gentianikola MAFF4255531, Pyrenocator telestris NBRC 30929, Leptospheria nodrum (conidia generation name Forma Hennerelgie) NBRC7480, Leptospheria magellan J. Ikuko generation name Ma Lingam) MAFF726528, Pureosupora-Haburamu NBRC32012, Pureosupora-Betae (conidia generation name forma Betae) NBRC5918, Ofioborasu-Herupotorikasu NBRC6158, Curvularia-Kurabeta YH923 (FERM P-19209) and the like.
[0025]
Among these strains, Arthrinium sp. TO6 (FERM P-19211), Pyrenocator sp. YH807 (FERM P-19210), and Carbraria clavator YH923 (FERM P-19209) are newly isolated strains by the present inventors. And deposited it at the Patent Organism Depositary, the National Institute of Advanced Industrial Science and Technology.
[0026]
The deposited mycological properties are as follows, and were identified as follows.
1. About Arthrinium sp. TO6 (FERM P-19211), "Separation, cultivation and identification of mold" translated by Shunichi Udagawa et al. (Medical Publishing Co., Ltd. 1983), "Science book of fungi" written by Shunichi Udagawa et al. (Kodansha Scientific 1986); B. As a result of examining the bacteriological properties of the substance, with reference to "Dmateaceous Hyrhomyocetes" (Commonwealth agical cultural bureauux 1988) by Ellis,
(1) The conidium is one cell.
(2) Conidia exogenously.
(3) Conidia are dark brown.
(4) Conidia are round, lens-shaped and colorless.
(5) The conidium touches the conidium at one junction.
(6) The hypha is a septum.
(7) The size of the conidia is 6 to 9 μm × 2 to 4 μm.
(8) The conidiophore has a thickness of 1.2 to 2.5 μm.
[0027]
From the above morphological characteristics, this strain is Arthrinium saccharicola (Arthrinium saccharicola) (conidium size is 9 to 10 μm, conidiophore thickness is 2 to 4 μm), Arthrinium phaeospermum ) (The size of conidia is 9-10 μm, the thickness of conidia is 1-1.5 μm), Arthrinium sacchari (The size of conidia is 6-8 μm, the thickness of conidia Is considered to be classified into any one of 1 to 1.5 μm), but the strain is slightly different from these strains in the size of conidia and the thickness of the conidia. Therefore, this strain was named Arthrinium sp. ).
[0028]
Growth status in medium
(1) Oatmeal agar medium, after culturing at 25 ° C. for 6 days, reached a diameter of about 8.5 cm or more, the back of the colonies was colorless, and dark brown spores (mass) were observed on white wool-like colonies. Can be
(2) After culturing on a potato dextrose agar medium at 25 ° C. for 6 days, the diameter of the colony reaches about 8.5 cm or more, and the back of the colony is colorless, white and wool-like colony.
2. About Pyrenochaeta sp. YH807 (FERM P-19210), "Separation, culture and identification of mold" translated by Shunichi Udagawa et al. (Ichiyaku Shuppan Co., Ltd. 1983), "Science book of fungi" written by Shunichi Udagawa et al. (Kodansha Scientific 1986); As a result of examining its mycological properties with reference to "The Coelomycetes" (Commonwealth agical cultural bureaux 1980) by Sutton,
(1) Conidia are single cells that are elliptical and smooth, 5 to 7 × 1.5 to 3.0 μm.
(2) The colonies are dark green-brown and do not produce pigment.
(3) The spores are not linked and are formed inside the fruit body (conidia).
(4) The hypha is a septum.
(5) Conidial shells are dark brown spherical to subspherical or pear-shaped and have one hole. The conidial shell is 200-300 μm in diameter and forms bristles around the opening.
[0029]
From the above morphological characteristics, it is considered that this strain is classified into Pyrenoceta rubi-idaei (conidia> 3.5 μm × 2.0-2.5 μm). This strain was identified as Pyrenochaeta sp. Because the conidia differed slightly in size.
[0030]
Growth status in medium
(1) After culturing on an oatmeal agar medium at 25 ° C. for 6 days, the diameter reaches about 2.3 cm or more, and the back of the colony becomes dark green-brown and dark green-brown colony. No dye is produced in the medium. Aerial hyphae are sparse.
(2) After culturing on a potato dextrose agar medium at 25 ° C. for 6 days, the diameter reaches about 2.0 cm or more, and the back of the colonies becomes dark green-brown and dark green-brown colonies. Aerial mycelia are observed more than on oatmeal agar medium.
(3) When cultured on a YM agar medium at 25 ° C. for 6 days, brown to black fruiting bodies (conidia) are formed.
3. Regarding the Curvularia clavata YH923 (FERM P-19209), "Separation, culture and identification of mold" translated by Shunichi Udagawa et al. Kodansha Scientific 1986); B. As a result of examining the bacteriological properties of the compound by referring to "Dmateaceous Hyrhomyocetes" by Ellis (commonwealth agical cultural bureaux 1988),
(1) The hypha is a septum.
(2) The conidia are polo-type conidia and have a spindle-rod shape and three partition walls, and almost all of them are swollen by the third cell from the bottom. There is a navel (hilum) at the base, but it is not significantly protruding.
(3) The second partition is not in the middle.
(4) The cells at both ends of the conidium are almost light brown, the middle cells are brown to dark brown, and the third cell from the bottom is darkest. Flat and constricted, 26-34 × 10-12 μm.
(5) The conidiophores are apical or lateral on the mycelium, often curved upward, and have a zigzag shape.
(6) Conidiophores are colorless to pale brown, smooth or bumpy, 2.5-4.0 μm in diameter.
[0031]
Based on the above morphological features, this strain was identified as Curvularia clavata.
Growth status in medium
(1) After culturing at 25 ° C. for 7 days in an oatmeal agar medium, the diameter reaches about 5.5 cm or more, the colony back and the colony are dark greenish brown, and velvety aerial hyphae are observed.
(2) After culturing on a potato dextrose agar medium at 25 ° C. for 7 days, the diameter of the colony reaches about 4.0 cm or more, and the back of the colony is black, grayish white and forms a wool-like aerial mycelium.
[0032]
Next, a method of culturing FAOD-producing bacteria that can be used in the present invention will be described. The culture means of the FAOD-producing bacteria of the present invention may be solid culture or liquid culture, but is preferably aeration culture using a flask or a jar fermenter. As the medium, those commonly used for culturing microorganisms are widely used. As a carbon source, glucose, glycerol, sorbitol, lactose or mannose, etc., as a nitrogen source, yeast extract, meat extract, tryptone, peptone, etc., and as inorganic salts, sodium chloride, magnesium chloride, magnesium sulfate, calcium chloride, etc. may be used. . The target enzyme may be collected at a pH of 5.0 to 8.0 and a culture temperature of 25 to 37 ° C., and a culture time at which the target enzyme has the highest titer, for example, 2 to 10 days.
[0033]
Next, when collecting the enzyme, the cells were separated from the culture solution by centrifugation or the like, and the cells were suspended in a buffer solution such as a phosphate buffer solution and a Tris-HCl buffer solution, followed by lysozyme treatment, ultrasonic treatment, and glass treatment. The cells are disrupted by various cell disruption methods such as bead disruption and centrifuged, and the soluble fraction is recovered as a crude enzyme solution.
[0034]
The thus-obtained crude FAOD-containing solution is treated using known protein and enzyme isolation and purification means, whereby purified FAOD can be obtained. For example, a general enzyme purification method such as a fractional precipitation method using an organic solvent such as acetone or ethanol, a salting-out method using ammonium sulfate, an ion exchange chromatography method, a hydrophobic chromatography method, an affinity chromatography method, and a gel filtration method is appropriately selected. And a combination thereof to obtain a purified FAOD. If necessary, a stabilizer such as sucrose, glycerol or amino acid may be used in an amount of about 1 to 50%, and a coenzyme or the like may be used in an amount of about 0.01 to 0.1%. It may be added in appropriate combination and stored frozen.
[0035]
Next, a method for measuring the enzyme activity and enzyme activity of FAOD obtained by the present invention will be described. Enzymatic action
Acts on the presence of oxygen, FVH, producing hydrogen peroxide, glucosone, and valylhistidine.
Enzyme activity measurement method
Measurement reagent
50 mM Tris-HCl buffer (pH 7.5)
1 mM fructosyl valyl histidine
0.02% 4-aminoantipyrine
0. 02% TOOS
5U / ml peroxidase (Merck)
(TOOS: N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline)
1 ml of the measuring reagent is placed in a cell having an optical path length of 1 cm, preliminarily heated at 37 ° C. for 5 minutes, and then 0.05 ml of an enzyme solution is added and reacted for 5 minutes. After the reaction, 2 ml of 0.5% SDS (sodium dodecyl sulfate) is added to stop the reaction, and the absorbance (Aa) at a wavelength of 555 nm is measured. The same operation is performed using a measurement reagent containing no FVH as a blank, and the absorbance is measured (Ab). The enzyme activity is determined from the difference (Aa-Ab) between the absorbance (Aa) and the absorbance (Ab) of the blank. One unit of the enzyme activity was defined as the amount of the enzyme capable of producing 1 micromol of hydrogen peroxide per minute at 37 ° C.
[0036]
Next, a measuring method using the enzyme of the present invention will be described.
[0037]
Regarding the measuring method that can be used in the present invention, any measuring method using an enzyme that specifically acts on the glycated peptide of the glycated hemoglobin β-chain N-terminal rather than the glycated hemoglobin α-chain glycated peptide can be used. However, an enzyme that directly acts on the 1-deoxyfructosyl group of glycated hemoglobin is not known at present, and it is only necessary to once fragment hemoglobin and then react the fragment with the enzyme of the present invention.
[0038]
The method of fragmenting hemoglobin includes a chemical method and an enzymatic method, and the enzymatic method is preferred because of its accuracy and simplicity. Proteases that cleave the glycated amino acid at the N-terminus of the β-chain of hemoglobin A1c have been reported, and these known proteases may be used. When glycated amino acids at the N-terminus of hemoglobin are excised, those derived from the α-chain or β-chain can be used. Since it is difficult to distinguish the protease, a protease which cuts out a glycated peptide consisting of two or more N-terminal amino acids of hemoglobin A1c is preferable, and a protease which cuts out two N-terminal amino acids is most preferable.
[0039]
Examples of preferred proteases are specifically shown below, but this is only one example. Sumiteam P (papain; manufactured by Shin Nippon Chemical Co., Ltd.), bioprase SP-10, bioprase AL15FG (manufactured by Nagase ChemteX Corporation), orientase 10B (manufactured by Hankyu Bio-Industry Co., Ltd.), Entilon (Rakuto Kasei Kogyo), protease type VIII, Protease type XXIV, protease type XIV, Bacillus globigi-derived protease (all manufactured by Sigma) and the like.
[0040]
The activity of the protease in the present invention was measured by the casein-forin method. The unit of activity, 1 U, was defined as 1 U, which is the amount of enzyme that shows color of the Folin reagent corresponding to 1 g of tyrosine per minute at 37 ° C.
[0041]
Further, in the method and reagents for decomposing hemoglobin using a protease, the protease may be used alone, or in addition, another endoprotease or another exoprotease may be allowed to act before or after the reaction or simultaneously. .
[0042]
In the measurement method using an enzyme that specifically acts on the glycated peptide at the N-terminal of glycated hemoglobin β-chain rather than the glycated peptide at the N-terminal of glycated hemoglobin α-chain of the present invention, for example, dehydrogenase was used to detect the enzymatic action. In this case, the amount of change in the coenzyme, for example, the amount of change produced using NAD as a coenzyme, is reduced colorimetrically as a reduced coenzyme at a wavelength around 340 nm, which is its maximum absorption wavelength range. Either direct quantification using a known technique such as measurement with a meter, or the resulting reduced coenzyme, or an electron carrier such as various diaphorases or phenazine methosulfate, and nitrotetrazolium, WST-1, WST-8 (the above-mentioned coterie chemistry research) Indirect determination using a reducing color reagent such as various tetrazolium salts typified by Toshosha Co., Ltd. The measurement may be directly or indirectly performed by the method described above.
[0043]
For example, when oxidase is used, it is preferable to measure the amount of consumed oxygen or the amount of reaction products. When, for example, ketoamine oxidase is used as a reaction product, hydrogen peroxide and glucosone are generated by the reaction, and both hydrogen peroxide and glucosone can be measured directly and indirectly by a known method.
[0044]
The amount of the hydrogen peroxide is, for example, a dye or the like is generated using peroxidase or the like, and color development, luminescence, may be quantified by fluorescence, or may be quantified by an electrochemical method, using catalase or the like. Alternatively, an aldehyde may be generated from the alcohol, and the amount of the generated aldehyde may be determined.
[0045]
The coloring system of hydrogen peroxide generates a dye by oxidative condensation of a coupler such as 4-AA or 3-methyl-2-benzothiazolinone hydrazone (MBTH) with a chromogen such as phenol in the presence of peroxidase. Leuco-type reagent (N- (carboxymethylaminocarbonyl) -4,4-bis (dimethylamino) biphenylamine (DA64), 10- (carboxymethylamino) Carbonyl) -3,7-bis (dimethylamino) phenothiazine (DA67); such as those manufactured by Wako Pure Chemical Industries, Ltd.).
[0046]
When measuring hydrogen peroxide using an electrode, the electrode is not particularly limited as long as it is a material that can exchange electrons with hydrogen peroxide, and examples thereof include platinum, gold, and silver. As the electrode measurement method, known methods such as amperometry, potentiometry, and coulometry can be used, and furthermore, an oxidase or reduction current obtained by interposing an electron carrier in the reaction between the oxidase or the substrate and the electrode can be used. Alternatively, the quantity of electricity may be measured. Any substance having an electron transfer function can be used as the electron carrier, and examples thereof include substances such as ferrocene derivatives and quinone derivatives. Alternatively, oxidation, reduction current or the amount of electricity obtained by interposing an electron carrier between hydrogen peroxide generated by the oxidase reaction and the electrode may be measured.
[0047]
Regarding the liquid composition of the reagent for measuring hemoglobin A1c using the enzyme of the present invention, the reagent specifically acts on the glycated peptide at the N-terminal of glycated hemoglobin β chain rather than the protease used and the glycated peptide of the glycated hemoglobin scar N-terminal. Considering the optimum pH of the enzyme, the pH and the protease concentration may be determined so that the reaction proceeds efficiently.
[0048]
For example, when Sumiteam P (derived from papaya, manufactured by Shin Nippon Chemical Co., Ltd.) is used as a protease, since the proteolytic activity is strong at around pH 5.5 to 9.0, the reaction pH should be 5.5 to 9.0. You can choose. The protease concentration may be a concentration that can sufficiently decompose the protein in the test solution during the reaction time actually used, and is preferably 0.01 to 1000 U / ml, more preferably 0.1 to 500 U / ml. preferable.
[0049]
Further, for example, when fructosylamine oxidase (derived from Arthrinium sp. TO6) is used as an enzyme that specifically acts on the glycated peptide at the N-terminal of glycated hemoglobin β-chain rather than the glycated peptide at the N-terminal of glycated hemoglobin α-chain, the optimum pH is obtained. Is 7 to 8, and the pH of the reaction can be selected from 7 to 8. The concentration of the enzyme to be added may be a concentration capable of sufficiently measuring glycated amino acids generated from the test solution during the reaction time actually used, and is preferably 0.01 U to 1000 U / ml, more preferably 0.1 U to 1000 U / ml. 500 U / ml is more preferred, and 0.5 U to 100 U / ml is most preferred.
[0050]
The reagent for quantifying glycated protein in the present invention may be prepared as a reagent containing an enzyme that specifically acts on the glycated peptide at the N-terminal of glycated hemoglobin β-chain rather than the glycated peptide at the N-terminal of glycated hemoglobin α-chain. Preferably, the preparation may be carried out so as to contain an enzyme that specifically acts on the glycated peptide at the N-terminal of glycated hemoglobin β-chain rather than the protease and the glycated peptide at the N-terminal of glycated hemoglobin α-chain. Can be provided as a frozen product or a lyophilized product.
[0051]
Furthermore, a surfactant, salts, a buffer, a pH adjuster, a preservative, and the like may be appropriately selected and added to the enzyme reaction composition for quantifying a glycated protein according to the present invention.
[0052]
In order to measure glycated hemoglobin in a test solution using the thus-prepared reagent for measuring hemoglobin A1c of the present invention, a test solution prepared by lysing hemolysin in 0.01 to 5.0 ml of a test solution having a concentration of 0.001 to 0.001 was used. When performing a rate assay by adding 0.5 ml and reacting at a temperature of 37 ° C., several minutes to several tens of minutes between two points after a certain time after the start of the reaction, for example, three minutes and four minutes The amount of altered coenzyme, dissolved oxygen, hydrogen peroxide or other products in the next 1 minute or every 5 minutes after 8 minutes can be measured directly or indirectly by the above method, and the endpoint In the case of an assay, the amounts of coenzyme, dissolved oxygen, hydrogen peroxide or other products that have changed a certain time after the start of the reaction may be measured in the same manner. In this case, the amount of hemoglobin A1c in the test solution can be determined by comparing the change in absorbance or the like when measured using a sample containing a known concentration of hemoglobin A1c.
[0053]
The hemolyzed test solution described herein is a test solution containing hemoglobin A1c after performing a hemolysis operation, and more specifically, hemolyzed blood such as red blood cells and washed red blood cells that have been subjected to whole blood centrifugation. No.
[0054]
【Example】
The present invention will be described based on examples.
[0055]
Embodiment 1
Culture method, purification method and physicochemical properties of FAOD derived from Neocosmospora basinfecta NBRC 7590 strain
<Culture method and purification method>
100 ml of 2% mannose, 3% yeast extract, 0.1% KH in three 500 ml Erlenmeyer flasks ₂ PO ₄ , 0.05% MgSO ₄ ・ 7H ₂ O-containing medium (pH 6.0) was added to each, and after sterilization, Neocosmospora bacinfecta NBRC7590 strain was inoculated and cultured with shaking at 28 ° C for 4 days.
[0056]
After completion of the culture, the cells were collected, suspended in 300 ml of 10 mM Tris-HCl buffer (pH 7.5), and the cells were solubilized by sonication (10 U). This lysate was dialyzed overnight against 10 mM Tris-HCl buffer (pH 7.5), and then subjected to ion-exchange chromatography on Q-Sepharose Big Beads (100 ml) resin (Pharmacia). Elution was performed with a linear gradient of 0 to 1 M, and an eluted fraction (9 U) of 0.2 to 0.3 M NaCl was collected.
[0057]
Ammonium sulfate was dissolved in the enzyme solution to a concentration of 15% ammonium sulfate, and subjected to hydrophobic chromatography on phenyl sepharose first flow (50 ml) (Pharmacia). Elution was performed with a linear gradient of 15 to 0 M ammonium sulfate, and an eluted fraction (7 U) of 5 to 2% ammonium sulfate was collected. Then, the enzyme solution was dialyzed overnight against a 10 mM Tris-HCl buffer (pH 7.0), and subjected to hydroxyapatite (30 ml) (Pentax) chromatography. Elution was performed by a linear gradient using a phosphate buffer of 0￣0.1 M (pH 7.0), and an eluted fraction (3 U) of a phosphate buffer of 0.03￣0.04 M was collected to obtain a purified FAOD. Was.
[0058]
<Physicochemical properties>
(1) Substrate specificity
The specificity of the FAOD derived from Neocosmospora bacinfector of the present invention for various substrates is as shown in the table. It showed the highest reactivity to FV, reacted 29% of FV to FVH, and had no effect on FVL.
(2) Km value
The Km value for FVH of FAOD derived from Neocosmospora bacinfecta of the present invention was 3 mM.
(3) Optimum pH
The optimum pH was determined according to the enzyme activity measurement method, and the results are shown in FIG. The range of pH 4.5 to 5.5 is 100 mM acetate buffer (○ in the figure), the range of pH 5 to 6 is 100 mM citrate buffer (□ in the figure), and the range of pH 6 to 7.5 is 100 mM. Phosphate buffer solution (△ in the figure), the range of 7.5-9 is 100 mM Tris-HCl buffer (●, figure), and the range of pH 9.5-10 is 100 mM glycine buffer (black, figure). (Square) shows the activity value when used, and the optimum pH was 6.3 to 6.7.
(4) pH stability
0.5 U / ml of the inventive FAOD derived from Stefilium sp. Was treated in 100 mM of various buffer solutions at 37 ° C. for 1 hour, and the residual activity was measured according to the enzyme activity measurement method described above. The result is shown in FIG. The range of pH 4.5 to 5.5 is 100 mM acetate buffer (○ in the figure), the range of pH 5 to 6 is 100 mM citrate buffer (□ in the figure), and the range of pH 6 to 7.5 is 100 mM. Phosphate buffer solution (△ in the figure), the range of 7.5-9 is 100 mM Tris-HCl buffer (●, figure), and the range of pH 9.5-10 is 100 mM glycine buffer (black, figure). Square) was used. Good stability was exhibited in the pH range of 5.75 to 7.36.
(5) Optimal temperature
According to the enzyme activity measurement method, the temperature was changed in the range of 30 to 60 ° C. to determine the optimum temperature. The result is as shown in FIG. 3, and the optimum temperature was 45 to 50 ° C.
(6) Thermal stability
The residual activity of 0.5 U / ml FAOD derived from Stefylium sp. Of the present invention after heat treatment in 100 mM Tris-HCl buffer (pH 7.5) at each temperature for 10 minutes was measured according to the enzyme activity measurement method described above. As a result, as shown in FIG. 4, the residual activity in the heat treatment at 45 ° C. for 10 minutes was at least 80% or more.
(7) Molecular weight
The molecular weight was measured by HPLC using TSK G-3000SWXL (manufactured by Tosoh Corporation). As a result, the molecular weight was 37,000.
[0059]
The above results are summarized in Table 2 above.
[0060]
Embodiment 2
Culture method, purification method and enzymatic properties of FAOD derived from Coniocetium savory ATCC strain
<Culture method and purification method>
Culturing was performed by the culturing method described in Example 1.
[0061]
After completion of the culture, the cells were collected, suspended in 300 ml of 10 mM Tris-HCl buffer (pH 7.5), and the cells were solubilized by sonication (15 U). This lysate was dialyzed overnight against 10 mM Tris-HCl buffer (pH 7.5), and then subjected to ion-exchange chromatography on Q-Sepharose Big Beads (100 ml) resin (Pharmacia). Elution was performed with a linear gradient of 0-1M, and an eluted fraction (13U) of 0.15-0.25M NaCl was collected.
[0062]
Ammonium sulfate was dissolved in the enzyme solution to a concentration of 15% ammonium sulfate, and subjected to hydrophobic chromatography on phenyl sepharose first flow (50 ml) (Pharmacia). Elution was performed with a linear gradient of 15 to 0 M ammonium sulfate, and an eluted fraction (8 U) of 4 to 0% ammonium sulfate was collected. Then, the enzyme solution was dialyzed overnight against a 10 mM Tris-HCl buffer (pH 7.0), and subjected to hydroxyapatite (30 ml) (Pentax) chromatography. Elution was performed with a linear gradient using a phosphate buffer of 0￣0.1 M (pH 7.0), and an eluted fraction (4 U) of a phosphate buffer of 0.03￣0.04 M was collected to obtain a purified FAOD. Was.
[0063]
<Physicochemical properties>
(1) Substrate specificity
The specificity of FAOD derived from Coniocetium savory of the present invention for various substrates is as shown in the table. It showed the highest reactivity to FV, reacted to FVH at 26% of FV, and had no effect on FVL.
(2) Km value
The Km value for FVH of FAOD derived from Coniocetium savory of the present invention was 1.6 mM.
(3) Optimum pH
The optimum pH was determined in the same manner as in Example 1, and the optimum pH was 6.3 to 6.7.
(4) pH stability
The pH stability was determined in the same manner as in Example 1, and showed good stability in the pH range of 5.8 to 7.
(5) Optimal temperature
The optimum temperature was determined in the same manner as in Example 1, and the optimum temperature was 37 ° C. (6) Thermal stability
The optimum temperature was determined in the same manner as in Example 1. The residual activity in the heat treatment at 37 ° C. for 10 minutes was at least 80% or more.
(7) Molecular weight
The molecular weight was measured by HPLC using TSK G-3000SWXL (manufactured by Tosoh Corporation). As a result, the molecular weight was 27,000.
[0064]
The results of the above results are summarized in Table 2 above.
[0065]
Embodiment 3
Culture method, purification method and enzymatic properties of FAOD derived from Arslenium sp. TO6 (FERMP-19211) strain
<Culture method>
In a 500 ml Sakaguchi flask, 100 ml of YMG medium (1.0% glucose, 1.0% polypeptone, 0.3% yeast extract, 0.3% malt extract, 0.1% KH ₂ PO ₄ , 0.05% MgSO ₄ ・ 7H ₂ O, pH 6.0), sterilized, and inoculated with Arslenium sp. TO6, followed by shaking culture at 30 ° C. for 4 days.
<Purification method>
The cells frozen in liquid nitrogen are ground in a mortar to disrupt the cells, 40 ml of 0.1 M Tris-HCl buffer (pH 7.3) is added, the mixture is left at 5 ° C. overnight, and then centrifuged. Separation gave a cell-free extract. Ammonium sulfate was added to the obtained cell-free extract so as to be 40% saturated, and the supernatant obtained by centrifugation was equilibrated with 0.1 M Tris-HCl buffer (pH 7.3) to which 40% saturated ammonium sulfate was added. Donated to a column of Butyl Toyopearl 650M (18f × 150 mm, manufactured by Tosoh Corporation), washed with 0.1 M Tris-HCl buffer (pH 7.3) to which ammonium sulfate was added so as to be 10% saturated, and washed with 0.1%. Elution was performed with M Tris-HCl buffer (pH 7.3). Ammonium sulfate was added to the active fraction of the eluate so as to be 40% saturated, and Phenyl-5PW (8.10) equilibrated with 0.1 M Tris-HCl buffer (pH 7.3) to which 40% saturated ammonium sulfate was added. (0f × 7.5 mm, manufactured by Tosoh Corporation) and eluted with a linear gradient of saturated ammonium sulfate from 40% to 0%. The active fraction was dialyzed against 50 mM Tris-HCl buffer (pH 7.3), and then applied to a column of Poros HQ / H (4.6 f × 50 mm, manufactured by Applied Biosystems) equilibrated with the same buffer. And eluted with a linear gradient of NaCl from 0 to 0.5M. The active fraction was collected and dialyzed against distilled water to obtain a purified FAOD. The purification process is summarized in the table below.
[0066]
[Table 4]

[0067]
<Physicochemical properties>
(1) Substrate specificity
Table 3 shows the specificity of the FAOD derived from Arslenium sp. TO6 of the present invention for various substrates. The concentration of each substrate during the reaction was 1 mM. Other reaction conditions were in accordance with the activity measurement method. FAOD derived from Arslenium sp. TO6 shows the highest reactivity to FV and also reacts highly to FVH, but its action on FVL is about 1/40 compared to FVH. It can be said that it does not substantially affect FVL.
(2) Km value
The Km value for FVH of the FAOD derived from Arslenium sp. TO6 of the present invention was 0.42 mM.
(3) Optimum pH
Various buffers of pH 3.0 to 11.0 in which FVH is dissolved to 1 mM (pH 3 to 5 is 10 mM acetate buffer, pH 5 to 7 is 10 mM citrate buffer, pH 7 to 9 is 10 mM Tris-HCl) 0.45 ml of a buffer solution (pH 9-11, 10 mM borate buffer solution) was placed in a cell having an optical path length of 1 cm, preliminarily heated at 37 ° C. for 5 minutes, and 0.05 ml of an enzyme solution was added thereto and reacted for 30 minutes. I let it. After the reaction, 0.5 ml of a measuring reagent (100 mM Tris-HCl buffer (pH 7.5) containing 0.04% TOOS, 0.04% 4-aminoantipyrine and 50 U of peroxidase) was added, and the color was developed for 2 minutes. The reaction was stopped by adding 2 ml of 0.5% SDS, and the absorbance (Aa) at a wavelength of 555 nm was measured. In addition, various buffers containing no substrate were added as blanks, and the same operation was performed to measure the absorbance (Ab). The enzyme activity was determined from the difference (Aa-Ab) between the absorbance (Aa) and the absorbance of the blank (Ab).
[0068]
As a result, the optimum pH of the FAOD derived from Arslenium sp. TO6 was 7 to 8.
(4) pH stability
The enzyme solution was treated in various buffer solutions of 10 mM at 4 ° C. for 24 hours, and the residual activity was measured according to the enzyme activity measurement method. As a result, the FAOD derived from Arslenium sp. TO6 of the present invention showed good stability in the pH range of 7 to 11.
(5) Optimal temperature
0.45 ml of 100 mM Tris-HCl buffer (pH 7.5) in which FVH is dissolved to 1 mM is placed in a cell having an optical path length of 1 cm, preliminarily heated at 15 to 60 ° C. for 5 minutes, and then 0.05 ml of the enzyme solution is added. Was added and reacted for 10 minutes. After the reaction, the mixture was cooled in ice, and 0.5 ml of a measuring reagent (100 mM Tris-HCl buffer (pH 7.5) containing 0.04% TOOS, 0.04% 4-aminoantipyrine and 50 U of peroxidase) was added. After the color was developed for 2 minutes, 2 ml of 0.5% SDS was added to stop the reaction, and the absorbance (Aa) at a wavelength of 555 nm was measured. In addition, various buffers not containing FVH were added as blanks, and the absorbance was measured by performing the same operation (Ab). The enzyme activity was determined from the difference (Aa-Ab) between the absorbance (Aa) and the absorbance of the blank (Ab).
[0069]
As a result of determining the optimum temperature by changing the temperature in the range of 15 to 60 ° C., the optimum temperature of the FAOD derived from Arslenium sp. TO6 of the present invention was 30 to 40 ° C.
(6) Thermal stability
The remaining activity after heating the enzyme solution in 100 mM Tris-HCl buffer (pH 7.5) for 30 minutes at each temperature was measured according to the enzyme activity measurement method described above. As a result, the FAOD derived from Arslenium sp. TO6 of the present invention showed a residual activity of 80% or more until heat treatment at 40 ° C. for 30 minutes.
(7) Molecular weight
The molecular weight of the enzyme was determined by gel filtration using YMC-Pack Diol-200G (φ6.0 × 300 mm, manufactured by YMC). Bovine serum albumin, ovalbumin, and soybean trypsin inhibitor (all manufactured by Sigma) were used as standard proteins. As a result, the molecular weight of the present enzyme was about 34,000.
[0070]
The molecular weight was about 50,000 by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) using 10% gel by the method of Laemmli. The standard protein used was SDS-PAGE Standard Low (manufactured by Bio-Rad).
[0071]
From the above results, it is clear that the FAOD derived from Arslenium sp. TO6 of the present invention is a monomer.
[0072]
Embodiment 4
Culture method, purification method and enzymatic properties of FAOD derived from Pyrenocator sp. YH807 (FERMP-19210 strain)
<Culture method and purification method>
The active fraction was recovered by the same method as the production and purification method described in Example 3, and dialyzed against distilled water to obtain a purified FAOD. The purification process is summarized in the table below.
[0073]
[0074]
[Table 5]

[0075]
<Physicochemical properties>
(1) Substrate specificity
Table 3 shows the specificity of the Pyrenocator sp. YH807-derived FAOD of the present invention for various substrates. The concentration of each substrate during the reaction was 1 mM. Other reaction conditions were in accordance with the activity measurement method. FAOD derived from Pyrenocator sp. YH807 shows the highest reactivity to FV and also reacts to FVH at a high level, but its action on FVL is about 160 times lower than that of FVH. Can be said to have no substantial effect.
(2) Km value
The Km value for FVH of FAOD derived from Pyrenocator sp. YH807 of the present invention was 0.99 mM.
(3) Optimum pH
As a result of examining the optimum pH of the present enzyme by the method described in (3) of Example 3, the optimum pH was 7 to 8.
(4) pH stability
As a result of examining the pH stability of FAOD derived from Pyrenocator sp. YH807 by the method described in (4) of Example 3, good stability was shown in the pH range of 7 to 11.
(5) Optimal temperature
As a result of determining the optimum temperature according to the method described in (5) of Example 3, the optimum temperature of FAOD derived from Pyrenocator SP YH807 of the present invention was 30 to 40 ° C.
(6) Thermal stability
As a result of determining the thermal stability according to the method described in (6) of Example 3, the thermal stability of FAOD derived from Pyrenocator SP YH807 shows a residual activity of 80% or more until heat treatment at 40 ° C. for 30 minutes. Was.
(7) Molecular weight
As a result of determining the molecular weight by gel filtration and SDS-PAGE according to the method described in (7) of Example 3, the molecular weight of FAOD derived from Pyrenocator sp. YH807 was about 34,000 by gel filtration, and about 50,000 by SDS-PAGE. 000. From these results, it is clear that the FAOD derived from Pyrenocator SP YH807 of the present invention is a monomer.
[0076]
Embodiment 5
Culture method, purification method, and enzymatic properties of FAOD derived from Carbralia clabetata YH923 (FERMP-19209) strain
<Culture method and purification method>
The active fraction was recovered by the same method as the production and purification method described in Example 3, and dialyzed against distilled water to obtain a purified FAOD. The purification process is summarized in the table below.
[0077]
[Table 6]

[0078]
Physicochemical properties
(1) Substrate specificity
The specificity of the FAOD derived from the Carbalia clabetar YH923 of the present invention for various substrates is shown in Table 3. The concentration of each substrate during the reaction was 1 mM. Other reaction conditions were in accordance with the activity measurement method. Although the FAOD derived from Carbraria cravata YH923 shows the highest reactivity to FV and also reacts highly to FVH, the effect on FVL is about 30 times lower than that of FVH. Can be said to have no substantial effect.
(2) Km value
The Km value for the FVH of the FAOD derived from the Carbraria cravata YH923 of the present invention was 0.95 mM.
(3) Optimum pH
As a result of examining the optimum pH of the present enzyme by the method described in (3) of Example 3, the optimum pH was 7 to 8.
(4) pH stability
As a result of examining the pH stability of the FAOD derived from Carburaria cratabeta YH923 by the method described in (4) of Example 3, good stability was shown in the pH range of 7 to 11.
(5) Optimal temperature
As a result of determining the optimum temperature in accordance with the method described in (3) of Example 3, the optimum temperature of the FAOD derived from the Carbralia clabetar YH923 of the present invention was 50 to 55 ° C.
(6) Thermal stability
As a result of determining the thermal stability according to the method described in (6) of Example 3, the thermal stability of the FAOD derived from the Carbralia Clavator YH923 shows a residual activity of 80% or more until the heat treatment at 50 ° C. for 30 minutes. Was.
(7) Molecular weight
The molecular weight of gel-filtration and SDS-PAGE was determined in accordance with the method described in (7) of Example 3, and as a result, the molecular weight of FAOD derived from Carburaria clavator YH923 was about 34,000 by gel filtration, and about 50,000 by SDS-PAGE. 000. From this result, it is clear that the FAOD derived from Carbraria clavator YH923 of the present invention is a monomer.
[0079]
Embodiment 6
Production and purification of FAOD derived from Leptosperia nodrum NBRC 7480 strain
<Culture method and purification method>
The active fraction was recovered by the same method as the production and purification method described in Example 3, and dialyzed against distilled water to obtain a purified FAOD. The purification process is summarized in Table 6 below.
[0080]
[Table 7]

[0081]
Physicochemical properties
(1) Substrate specificity
Table 3 shows the specificity of the FAOD derived from Leptosperia nodrum NBRC7480 of the present invention for various substrates. The concentration of each substrate during the reaction was 1 mM. Other reaction conditions were in accordance with the activity measurement method. FAOD derived from Leptosperia nodrum NBRC7480 shows the highest reactivity to FV and also reacts to FVH at a high level, but its action on FVL is about one-fifth compared to FVH. It can be said that it does not substantially affect FVL.
(2) Km value
The Km value of the FAOD derived from Leptosperia nodrum NBRC7480 of the present invention with respect to FVH was 0.85 mM.
(3) Optimum pH
As a result of examining the optimal pH of FAOD derived from Leptosperia nodrum NBRC7480 by the method described in (3) of Example 3, the optimal pH was 7 to 8.
(4) pH stability
As a result of examining the pH stability of FAOD derived from Leptosperia nodrum NBRC7480 by the method described in (4) of Example 3, good stability was shown in the pH range of 7 to 11.
(5) Optimal temperature
As a result of determining the optimum temperature according to the method described in (5) of Example 3, the optimum temperature of FAOD derived from Leptosperia nodrum NBRC7480 of the present invention was 30 to 40 ° C.
(6) Thermal stability
As a result of determining the thermal stability according to the method described in (6) of Example 3, the thermal stability of the FAOD derived from Leptosperia nodrum NBRC7480 showed a residual activity of 80% or more until heat treatment at 40 ° C. for 30 minutes. Indicated.
(7) Molecular weight
The molecular weight of the FAOD derived from Leptosperia nodrum NBRC7480 was determined to be about 34,000 by gel filtration and about 50 by SDS-PAGE, as determined by gel filtration and SDS-PAGE according to the method described in (7) of Example 3. 2,000. From these results, it is clear that FAOD derived from Leptosperia nodrum NBRC7480 of the present invention is a monomer.
[0082]
Embodiment 7
Culture method, purification method and enzymatic properties of FAOD derived from Preospora herb ram NBRC32012 strain
<Culture method and purification method>
The active fraction was recovered by the same method as the production and purification method described in Example 3, and dialyzed against distilled water to obtain a purified FAOD. The purification process is summarized in Table 7 below.
[0083]
[Table 8]

[0084]
Physicochemical properties
(1) Substrate specificity
Table 3 shows the specificity of the FAOD derived from Pleospora herb NBRC32012 of the present invention for various substrates. The concentration of each substrate during the reaction was 1 mM. Other reaction conditions were in accordance with the activity measurement method. FAOD derived from Preospora herbrum NBRC32012 shows the highest reactivity to FV and also responds to FVH at a high level, but its action on FVL is about 156 times lower than that of FVH. Can be said to have no substantial effect.
(2) Km value
The Km value of the FAOD derived from Pleospora herb NBRC32012 of the present invention with respect to FVH was 0.8 mM.
(3) Optimum pH
As a result of examining the optimum pH of the present enzyme by the method described in (3) of Example 3, the optimum pH was 7 to 8.
(4) pH stability
As a result of examining the pH stability of FAOD derived from Preospora herb NBRC32012 by the method described in (4) of Example 3, good stability was shown in the pH range of 7 to 11.
(5) Optimal temperature
As a result of determining the optimal temperature according to the method described in (5) of Example 3, the optimal temperature of FAOD derived from Preospora herb NBRC32012 was 30 to 40 ° C.
(6) Thermal stability
As a result of determining the thermal stability according to the method described in (6) of Example 3, the thermal stability of FAOD derived from Pleospora herb NBRC32012 shows a residual activity of 80% or more until heat treatment at 40 ° C. for 30 minutes. Was.
(7) Molecular weight
According to the method described in Example 3, (7), the molecular weight was determined by gel filtration and SDS-PAGE. As a result, the molecular weight of FAOD derived from Preospora herbrum NBRC32012 was about 34,000 by gel filtration, and about 50,000 by SDS-PAGE. 000. From these results, it is clear that FAOD derived from Pleospora herb NBRC32012 of the present invention is a monomer.
[0085]
Embodiment 8
Culture method, purification method and enzymatic properties of FAOD derived from Ophiobolas herpotrichus NBRC6158 strain
<Culture method>
In a 500 ml Sakaguchi flask, 100 ml of YMG medium (1.0% glucose, 1.0% polypeptone, 0.3% yeast extract, 0.3% malt extract, 0.1% KH ₂ PO ₄ , 0.05% MgSO ₄ ・ 7H ₂ O, pH 6.0), sterilized, and inoculated with a FAOD-producing strain, followed by shaking at 30 ° C. for 10 days.
<Purification method>
The active fraction was collected by the same method as the purification method described in the examples, and dialyzed against distilled water to obtain purified FAOD. The purification process is summarized in the table below.
[0086]
[Table 9]

[0087]
Physicochemical properties
(1) Substrate specificity
Table 3 shows the specificity of FAOD of the present invention derived from Ophiobolas herpotrichus NBRC6158 for various substrates. The concentration of each substrate during the reaction was 1 mM. Other reaction conditions were in accordance with the activity measurement method. Although FAOD derived from Ophiobolus herpotrichus NBRC6158 shows the highest reactivity to FV and also reacts to FVH at a high level, its effect on FVL is about 156 times lower than that of FVH. Can be said to have no substantial effect.
(2) Km value
The Km value of the FAOD of the present invention derived from Ophiobolas herpotrichus NBRC6158 with respect to FVH was 0.8 mM.
(3) Optimum pH
As a result of examining the optimum pH of FAOD derived from Ophiobolus herpotrichus NBRC6158 by the method described in (3) of Example 3, the optimum pH was found to be 7 to 8.
(4) pH stability
As a result of examining the pH stability of FAOD derived from Ophiobolas herpotrichus NBRC6158 by the method described in (4) of Example 3, good stability was shown in the pH range of 7 to 11.
(5) Optimal temperature
As a result of determining the optimum temperature according to the method described in (5) of Example 3, the optimum temperature of the FAOD derived from Ophiobolas herpotrichus NBRC6158 of the present invention was 30 to 40 ° C.
(6) Thermal stability
As a result of determining thermal stability according to the method described in (6) of Example 3, the thermal stability of FAOD derived from Ophiobolus herpotrichus NBRC6158 shows a residual activity of 80% or more until heat treatment at 40 ° C. for 30 minutes. Was.
(7) Molecular weight
As a result of determining the molecular weight by gel filtration and SDS-PAGE according to the method described in (7) of Example 3, the molecular weight of FAOD derived from Ophiobolus herpotrichus NBRC6158 was about 34,000 by gel filtration, and about 50,000 by SDS-PAGE. 000. From these results, it is clear that the FAOD derived from Ophiobolus herpotrichus NBRC6158 of the present invention is a monomer.
[0088]
Embodiment 9
Measurement of hemoglobin A1c
<Measurement of glycated hemoglobin using the protease of the present invention>
<Hemolysis reagent>
150 mM Tris buffer (manufactured by Wako Pure Chemical Industries) pH 7.5
1% Polyoxyethylene lauryl ether (Wako Pure Chemical Industries, Ltd.)
<R1: Protease reagent>
150 mM Tris buffer (manufactured by Wako Pure Chemical Industries) pH 7.5
4000 U / ml Streptomyces genus protease (protease type XIV; manufactured by Sigma)
<R2: coloring reagent>
150 mM Tris buffer (manufactured by Wako Pure Chemical Industries) pH 8.0
5 mM DA64 (manufactured by Wako Pure Chemical Industries, Ltd.)
24 U / ml fructosylamine oxidase (from Arthrinium sp. TO6)
20U / ml POD (Sigma)
<Reaction procedure>
Red blood cells separated from whole blood or a standard glycated peptide solution (0.1 ml) were added to the above hemolytic reagent (0.9 ml) and incubated at 37 ° C. for 10 minutes to obtain a hemolyzed sample. The hemolyzed sample was measured at A570 nm for the purpose of determining hemoglobin concentration. Subsequently, 240 μl of the R1 reagent and 6 μl of the hemolyzed sample were mixed, reacted at 37 ° C. for 5 minutes, and measured at 750 nm (A0). Further, 60 μl of R2 reagent was added, and incubated at 37 ° C. for 5 minutes, and the light was measured at 750 nm (A1). The blank was measured by measuring the absorbance change of the blank (blank ΔA = A1 blank−A0 blank) using distilled water as a sample. The sensitivity (sensitivity ΔA = (A1−A0) −blank ΔA) was determined using a sample and a sample whose glycated hemoglobin value was known, and the glycated hemoglobin concentration was calculated. Furthermore, the glycated hemoglobin concentration was divided by the hemoglobin concentration to calculate a glycated hemoglobin value.
<Sample>
5 healthy blood samples, 5 diabetic whole blood samples
100 μM, 80 μM, 60 μM, 40 μM, 20 μM FVH or FVL
<Measurement of hemoglobin A1c using HPLC>
The HPLC method was measured with an HbA1c measuring device (Arkray).
FIG. 5 shows the measurement results of the standard glycated peptides (FVH, FVL), and Table 9 shows the measurement results of the samples.
[0089]
[Table 10]

[0090]
As can be seen from FIG. 5, since the measurement reagent using the enzyme of the present invention does not act on FVL but acts only on FVH, it is clear that only the glycosylated peptide at the N-terminus of the hemoglobin β chain can be accurately detected. It was clear that hemoglobin A1c was accurately measured instead of glycated hemoglobin.
In addition, Table 10 shows that the measured value of hemoglobin A1c measured by the HPLC method and the value of the enzymatic method are in very good agreement, so that it was clear that hemoglobin A1c was accurately measured.
[0091]
【The invention's effect】
By using an enzyme having a higher specificity for the glycated peptide of the glycated hemoglobin β-chain N-terminal than the glycated peptide of the glycated hemoglobin α-chain N-terminal of the present invention, a measuring method using the enzyme, and using the reagent, the hemoglobin A1c can be further purified Measurement can be performed simply, accurately, and inexpensively.
[Brief description of the drawings]
FIG. 1 shows the optimal pH curve of FAOD derived from Neocosmospora basinfecta.
FIG. 2 shows the pH stability curve of FAOD from Neocosmospora basinfecta.
FIG. 3 shows an optimum temperature curve of FAOD derived from Neocosmospora basinfecta.
FIG. 4 shows the thermal stability curve of FAOD from Neocosmospora basinfecta.
FIG. 5 shows a measurement curve of a standard glycated peptide (FV, FVH).

Claims (11)

An enzyme having higher specificity for a glycated hemoglobin β-chain N-terminal glycated peptide than a glycated hemoglobin α-chain N-terminal glycated peptide. An enzyme that acts on a glycated peptide at the N-terminal of glycated hemoglobin β-chain and does not substantially act on a glycated peptide at the N-terminal of glycated hemoglobin α-chain. The glycated peptide at the N-terminal of glycated hemoglobin β chain is 1-deoxyfructosyl-L-valyl-L-histidine, and the glycated peptide at the N-terminal of hemoglobin α chain is 1-deoxyfructosyl-L-valyl-L-leucine. The enzyme according to claim 1, wherein the enzyme is present. 4. The enzyme according to claim 1, wherein the enzyme is fructosylamine oxidase. The enzyme according to any one of claims 1 to 4, wherein the enzyme is derived from the genus Neocosmospora, the genus Coniochetium, the genus Arsulinium, the genus Pyrenocatora, the genus Leptosperia, the genus Pleospora, the genus Ophioboras, the genus Carburaria, or the genus Forma. Glycated hemoglobin β-chain N rather than glycated hemoglobin α-chain N-terminal glycated peptide from cultures of Neocosmospora, Coniochetium, Arsurinium, Pyrenocator, Leptosperia, Pleospora, Ophioboras, Carbralia, Forma A method for producing an enzyme that specifically acts on a glycated peptide at the end. A method for measuring an Amadori compound, comprising using an enzyme having higher specificity for a glycated peptide at the N-terminal of glycated hemoglobin β-chain than at a glycated peptide at the N-terminal of glycated hemoglobin α-chain. A method for measuring an Amadori compound, comprising using an enzyme that acts on a glycated peptide at the N-terminal of glycated hemoglobin β-chain and that does not substantially act on a glycated peptide at the N-terminal of glycated hemoglobin α-chain. The method according to claim 8, wherein the Amadori compound is hemoglobin A1c. A reagent containing an enzyme having a higher specificity for a glycated hemoglobin β-chain N-terminal glycated peptide than for a glycated hemoglobin α-chain N-terminal glycated peptide. A reagent containing an enzyme having higher specificity for a glycated hemoglobin β-chain N-terminal glycated peptide than for a protease or glycated hemoglobin α-chain N-terminal glycated peptide.

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