JP3850904B2 - Fructosyl amino acid oxidase and method for producing the same - Google Patents

Description

＊１：検出されず
＊２：フルクトシル牛血清アルブミン
＊３：フルクトシルヒト血清アルブミン
表１から、フルクトシルＮ^α−Ｚ−リジン及びフルクトシルバリンに対して高い特異性を有する。また、本発明のＦＡＯＤはフルクトシルポリリジンに対する活性を有し、さらに糖化タンパクのプロテアーゼ消化物に対する活性もある。
フサリウム属のＦＡＯＤ生産能力を有する菌株を下記表２に例示する。
表２ ＦＺＬ褐変化培地で培養したフザリウム属の菌から精製したＦＡＯＤの基質特異性
【表２】

１）：（フルクトシルN^α-Z-リジンに対する活性）／（フルクトシルバリンに対する活性）
【００１９】
表２に示されているように、本発明のＦＡＯＤは、フルクトシルリジン及びフルクトシルバリンの両者に対して活性を有しており、このことは該ＦＡＯＤが糖化ヘモグロビンの測定にも有用であることを示唆するものである。
【００２０】
３．ｐＨ及び温度条件
ｐＨ条件の検討
至適pＨは、通常のＦＡＯＤ活性測定法（後述の４．力価の測定法参照）で使用する緩衝液をｐＨ４〜１３の各種緩衝液（０.１Ｍリン酸カリウム緩衝液（ＫＰＢ）、トリス−塩酸緩衝液及びグリシン（Ｇly）−ＮaＯＨ緩衝液）に置き換えて酵素反応を行い検討した。
また、前記の各種緩衝液にＦＡＯＤを添加し、３０℃で１０分間インキュベートした後、 通常の条件（３０℃、ｐＨ８.０）で活性を測定する事によりpＨによる安定性を検討した。
温度条件の検討
至適温度を検討するために、反応温度を２０〜６０℃まで変化させて酵素活性を測定した。温度安定性については、０.１Ｍトリス-塩酸緩衝液（ｐＨ８.０）に溶解したＦＡＯＤを２０〜６０℃の各温度で１０分間インキュベートし、その酵素液を用いて通常の条件で残存活性を測定した。
上記方法で測定した結果、本発明のＦＡＯＤの至適ｐＨは、７.５〜９.０、より好ましくは８.５であり（図２）、安定なｐＨ域は、４.０〜１３.０であった。
また、ＦＡＯＤ酵素反応は２０〜５０℃、好ましくは２５〜４０℃、より好ましくは３５℃で効率よく進行した（図３）。安定な温度領域は２０〜５０℃であった。
【００２１】
４．力価の測定
酵素の力価測定は下記の方法で行った。
（１）生成する過酸化水素を比色法により測定する方法。
Ａ.速度法
１００ｍＭ ＦＺＬ溶液はあらかじめ得られたＦＺＬを蒸留水で溶解することによって調製した。４５mＭ ４−アミノアンチピリン、６０ユニット／ｍｌパーオキシダーゼ溶液、及び６０ｍＭ フェノール溶液それぞれ１００μｌと、０.１Ｍ トリス−塩酸緩衝液（ｐＨ８.０）１ｍｌ、及び酵素溶液５０μｌを混合し、全量を蒸留水で３.０ｍｌとする。３０℃で２分間インキュベートした後、１００ｍＭ ＦＺＬ溶液５０μｌを添加し、５０５ｎｍにおける吸光度を経時的に測定した。生成するキノン色素の分子吸光係数（５.１６×１０³Ｍ^-1cm^-1）から、１分間に生成する過酸化水素のマイクロモルを算出し、この数字を酵素活性単位（ユニット:Ｕ）とする。
【００２２】
Ｂ.終末法
上記Ａ法と同様に処理し、基質添加後、３０分間３０℃でインキュベートした後の５０５ｎｍにおける吸光度を測定し、あらかじめ標準過酸化水素溶液を用いて作成した検量線から生成した過酸化水素量を算出することにより、酵素活性を測定する。
（２）酵素反応による酸素吸収を測定する方法
０.１Ｍ トリス-塩酸緩衝液（pＨ８.０）1ｍｌと酵素溶液５０μｌを混合し、蒸留水で全量を３.０ｍｌとし、ランク ブラザーズ社の酸素電極のセルに入れる。３０℃で攪拌し、溶存酸素と温度を平衡化した後、５０ｍＭ ＦＺＬ １００μｌを添加し、酸素吸収を記録計で連続的に計測し、初速度を得る。標準曲線から１分間に吸収された酸素量を求め、これを酵素単位とする。
【００２３】
５．酵素の阻害、活性化及び安定化
（１）金属の影響
０.１Ｍ トリス-塩酸緩衝液（ｐＨ８.０）に、各種金属イオンを終濃度１mＭとなるように添加し、３０℃で５分間インキュベートした後、活性を測定した。結果を下記の表３に示す。
表３ 金属イオンのＦＡＯＤ−Ｌ活性への影響
【表３】

表３から明らかなように、本発明のＦＡＯＤの活性に対し、銅イオン、亜鉛イオンが阻害的であり、銀イオン及び水銀イオンは完全に阻害する。
【００２４】
（２）各種阻害物質の影響
上記（１）の金属イオンの影響に関する試験と同様の方法で試験した。ただし、パラクロロ安息香酸第二水銀は終濃度０.１mＭ、それ以外は１mＭとした。結果を表４に示す。また安定化の検討は、５０mＭ トリス-塩酸緩衝液(pＨ８.５)に２mＭジチオスレイトール（ＤＴＴ）を添加したものに対して精製酵素を一晩透析した後、活性を測定することにより行った。
表４ 各種物質のＦＡＯＤ活性への影響
【表４】

＊１：ＰＣＭＢ，パラクロロ安息香酸第二水銀
＊２：ＤＴＮＢ，５，５'−ジチオビス（２−ニトロ安息香酸）
＊３：検出できず
表４から明らかに、ＦＡＯＤ活性はＰＣＭＢ、ＤＴＮＢ、ヒドラジン、フェニルヒドラジンにより、強く阻害された。これより、酵素反応にはＳＨ基及びカルボニル基が重要な働きをしていることが予想される。
他方、ジチオスレイトールによって安定化され、保存に適した溶媒はジチオスレイトール２ｍＭを添加した５０mＭ トリス−塩酸緩衝液（pＨ８.５）である。
【００２５】
６．分子量
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）は、デービスの方法に従い、１０％ゲルを用いて、４０ｍＡで、３時間泳動し、タンパク染色は、クマシーブリリアントブルーＧ−２５０で行った。標準タンパクとしてホスホリラーゼＢ、牛血清アルブミン、オボアルブミン、カルボニックアンヒドラーゼ、大豆トリプシンインヒビターを同様に泳動し、検量線を作成して分子量を求めた。その結果、サブユニットの分子量は、約５１,０００（５１ｋＤａ）であった（図４)。
スーパーデックス２００ｐｇによるゲルろ過では、分子量は約１０６,０００（１０６ｋＤａ）と求められ（図５）、本発明のＦＡＯＤは二量体であることが示唆された。
【００２６】
７．等電点
ディスク焦点電気泳動法によって測定した結果、ＦＡＯＤはｐＩ＝６.８であった。
【００２７】
８．既知の酵素との比較
既存の菌由来フルクトシルアミノ酸オキシダーゼと、本発明のＦＡＯＤとを比較した。
表５ 種々の微生物由来のフルクトシルアミノ酸オキシダーゼの比較
【表５】

1): ホリウチら（T.Horiuchi et al.) Agric.Biol.Chem., 53(1), 103-110 (1989)
2): ホリウチら（T.Horiuchi et al.) Agric.Biol.Chem., 55(2), 333-338 (1991)
3): フルクトシルＮ^α−Ｚ−リジンに対する比活性
4): Ｎ^ε−Ｄ−フルクトシルＮ^α−ホルミルリジンに対する比活性
表５から、本発明のＦＡＯＤと他の２種の菌株由来のフルクトシルアミノ酸オキシダーゼとの間に、下記の相違点が認められる。
（１）分子量の相違：本発明のＦＡＯＤは他の２種の酵素よりも明らかに分子量が大きい。
（２）補酵素：本発明のＦＡＯＤは補酵素として共有結合的に結合したＦＡＤを有するのに対し、他の酵素はいずれも非共有結合的に結合したＦＡＤを有する。
（３）基質特異性：本発明のＦＡＯＤは、フルクトシルバリンよりもフルクトシルリジンに対する特異性が高いが、コリネバクテリウム属由来の酵素はフルクトシルリジンには作用せず、アスペルギルス属由来の酵素はフルクトシルリジンに作用するものの、フルクトシルバリンに対する活性に比べて、その活性が低い。
（４）ミハエリス定数：本発明のＦＡＯＤの基質フルクトシルリジンに対する親和性が、他の酵素のそれよりも高いことを示している。
（５）至適ｐＨ、至適温度、及びＳＨ試薬による阻害：本発明のＦＡＯＤと他の２酵素との相違を示している。
【００２８】
既述のごとく、本発明の酵素ＦＡＯＤは、アマドリ化合物の定量に有用である。従って、本発明は、アマドリ化合物を含有する試料と、本発明のＦＡＯＤとを接触させ、酸素の消費量又は過酸化水素の発生量を測定することを特徴とする、試料中のアマドリ化合物の分析法を提供するものである。本発明の分析法は、生体成分中の糖化タンパクの量及び／又は糖化率の測定、あるいはフルクトサミンの定量に基づいて行われる。
ＦＡＯＤの酵素活性は下記の反応に基づいて測定される。
Ｒ¹−ＣＯ−ＣＨ₂−ＮＨ−Ｒ² ＋ Ｏ₂ ＋ Ｈ₂Ｏ →
Ｒ¹−ＣＯ−ＣＨＯ ＋ Ｒ²−ＮＨ₂ ＋ Ｈ₂Ｏ₂
（式中、Ｒ¹はアルドース残基、Ｒ²はアミノ酸、タンパク質又はペプチド残基を表す）
被検液としては、アマドリ化合物を含有する任意の試料溶液を用いることができ、例えば、血液（全血、血漿又は血清）、尿等の生体由来の試料の外、醤油等の食品が挙げられる。
【００２９】
本発明のＦＡＯＤをアマドリ化合物含有溶液に、適当な緩衝液中で作用させる。反応溶液のｐＨ、温度は、上記の条件を満たす範囲、即ち、ｐＨ４.０〜１３.０、好ましくは８.５、温度は２０〜５０℃、好ましくは３５℃である。緩衝液としてはトリス-塩酸等を用いる。ＦＡＯＤの使用量は、終点分析法においては通常、０.１ユニット／ml以上、好ましくは１〜１００ユニット／mlである。
【００３０】
本発明の分析法では、下記のいずれかのアマドリ化合物の定量法を用いる。
（１）過酸化水素発生量に基づく方法
当該技術分野で既知の過酸化水素の定量法、例えば、発色法、過酸化水素電極を用いる方法等で測定し、過酸化水素及びアマドリ化合物の量に関して作成した標準曲線と比較することにより、試料中のアマドリ化合物を定量する。具体的には、上記４の力価の測定に準じる。ただし、ＦＡＯＤ量は１ユニット／ｍｌとし適当に希釈した試料を添加し、生成する過酸化水素量を測定する。過酸化水素の発色系としては、パーオキシダーゼの存在下で４−アミノアンチピリン、３−メチル−２−ベンゾチアゾリノンヒドラゾン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発色する系を用いることができる。色原体として、フェノール誘導体、アニリン誘導体、トルイジン誘導体等があり、例えば、Ｎ−エチル−Ｎ−（２−ヒドロキシ−３−スルホプロピル）−ｍ−トルイジン、Ｎ,Ｎ−ジメチルアニリン、Ｎ,Ｎ−ジエチルアニリン、２,４−ジクロロフェノール、Ｎ−エチル−Ｎ−（２−ヒドロキシ−３−スルホプロピル）−３,５−ジメトキシアニリン、Ｎ−エチル−Ｎ−（３−スルホプロピル）−３,５−ジメチルアニリン、Ｎ−エチル−Ｎ−（２−ヒドロキシ−３−スルホプロピル）−３,５−ジメチルアニリン等が挙げられる。又パーオキシダーゼの存在下で酸化発色を示すロイコ型発色試薬も用いることができ、そのようなロイコ型発色試薬は、当業者に既知であり、ｏ−ジアニシジン、ｏ−トリジン、３,３−ジアミノベンジジン、３,３,５,５−テトラメチルベンジジン、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、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）フェノチアジン等が挙げられる。
（２）酸素の消費量に基づく方法
反応開始時の酸素量から反応終了時の酸素量を差し引いた値（酸素消費量）を測定し、酸素消費量とアマドリ化合物の量に関して作成した標準曲線と比較することにより、試料中のアマドリ化合物を定量する。具体的には、上記４の力価の測定に準じて行う。但し用いるＦＡＯＤ量は１ユニット／mlとし、適当に希釈した試料を添加し消費される酸素量を求める。
【００３１】
本発明方法は試料溶液をそのまま用いて行うこともできるが、対象となる糖化タンパクによっては、あらかじめ糖が結合したリジン及び／又はバリン残基を遊離させてから行うことが好ましい。
そのような目的には、タンパク質分解酵素を用いる場合（酵素法）と、塩酸等の化学物質を用いる場合（化学法）があるが、前者が好ましい。その場合、本発明方法には当業者に既知である、エンド型及びエキソ型のタンパク質分解酵素（プロテアーゼ）を用いることができる。エンド型のプロテアーゼには、例えばトリプシン、α−キモトリプシン、スブチリシン、プロティナーゼＫ、パパイン、カテプシンＢ、ペプシン、サーモリシン、プロテアーゼＸＩＶ、リジルエンドペプチダーゼ、プロレザー、ブロメラインＦ等がある。一方、エキソ型のプロテアーゼにはアミノペプチダーゼ、カルボキシペプチダーゼ等が挙げられる。酵素処理の方法も既知であり、例えば下記実施例に記載の方法で行うことができる。
【００３２】
上記のごとく、本発明のＦＡＯＤは、糖化タンパクに含まれるフルクトシルリジンに高い基質特異性を有するものであることから、血液試料中の糖化タンパクを測定することを含む、糖尿病の診断などに有用である。また、フルクトシルバリンにも特異性を有することから、糖化ヘモグロビンの測定にも有用である。
なお、検体として血液試料（全血、血漿又は血清）を用いる場合、採血した試料をそのまま、あるいは透折等の処理をした後用いる。
さらに、本発明方法に用いるＦＡＯＤ、パーオキシダーゼ等の酵素は、溶液状態で用いてもよいが、適当な固体支持体に固定化してもよい。例えば、ビーズに固定化した酵素をカラムに充填し、自動化装置に組み込むことにより、臨床検査など、多数の検体の日常的な分析を効率的に行うことができる。しかも、固定化酵素は再使用が可能であることから、経済効率の点でも好ましい。
さらには、酵素と発色色素とを適宜組み合わせ、臨床分析のみならず、食品分析にも有用なアマドリ化合物の分析のためのキットを得ることができる。
【００３３】
酵素の固定化は当該技術分野で既知の方法により行うことができる。例えば、担体結合法、架橋化法、包括法、複合法等によって行う。担体としては、高分子ゲル、マイクロカプセル、アガロース、アルギン酸、カラギーナン、などがある。結合は共有結合、イオン結合、物理吸着法、生化学的親和力を利用し、当業者既知の方法で行う。
固定化酵素を用いる場合、分析はフロー又はバッチ方式のいずれでもよい。上記のごとく、固定化酵素は、血液試料中の糖化タンパクの日常的な分析（臨床検査）に特に有用である。臨床検査が糖尿病診断を目的とする場合、診断の基準としては、結果を糖化タンパク濃度として表すか、試料中の全タンパク質濃度に対する糖化タンパク質の濃度の比率（糖化率）又はフルクトサミン値で表す。全タンパク質濃度は、通常の方法（２８０nmの吸光度、ブラッドフォード法、Lowry法、ビュレット法、アルブミンの自然蛍光、ヘモグロビンの吸光度など)で測定することができる。
【００３４】
本発明はまた、本発明のＦＡＯＤを含有するアマドリ化合物の分析試薬又はキットを提供するものである。
本発明のアマドリ化合物の定量のための試薬は、本発明のＦＡＯＤと好ましくはｐＨ７.５〜８.５、より好ましくはｐＨ８.０の緩衝液からなる。該ＦＡＯＤが固定化されている場合、固体支持体は高分子ゲルなどから選択され、好ましくはアルギン酸である。
試薬中のＦＡＯＤの量は、終点分析を行う場合、試料あたり、通常１〜１００ユニット／ml、緩衝液はトリス-塩酸（ｐＨ８.０）が好ましい。
過酸化水素の生成量に基づいてアマドリ化合物を定量する場合、発色系としては、先述の「（１）過酸化水素発生量に基づく方法」に記載の酸化縮合により発色する系、並びにロイコ型発色試薬等を用いることができる。
本発明のアマドリ化合物の分析試薬と、適当な発色剤ならびに比較のための色基準あるいは標準物質を組み合わせてキットとすることもできる。そのようなキットは、予備的な診断、検査に有用であると考えられる。
上記の分析試薬及びキットは生体成分中の糖化タンパク量及び／又は糖化率の測定、あるいは、フルクトサミンを定量するために、用いられるものである。
以下に実施例を挙げて本発明をさらに詳しく説明する。
【００３５】
【実施例】
実施例１ フサリウム・オキシスポルム・f.sp.・リニの培養とＦＡＯＤ−Ｌの精製
１）培養
フサリウム・オキシスポルム・f.sp.・リニ（IFO NO.5880；Fusarium oxysporum f.sp.lini）をＦＺＬ ０.５％、グルコース １.０％、リン酸二カリウム０.１％、リン酸一ナトリウム ０.１％、硫酸マグネシウム ０.０５％、塩化カルシウム ０.０１％、イーストエキス ０.２％を含有した培地（ｐＨ６.０)１０Ｌに植菌し、ジャーファーメンターを用いて通気量２Ｌ／分、攪拌速度４００ｒｐｍの条件で２８℃、８０時間攪拌培養した。培養物は瀘過して集めた。
２）粗酵素液の調製
菌糸体２７０ｇ（湿重量）を、２ｍＭのＤＴＴを含む、０.１Ｍトリス−塩酸緩衝液（ｐＨ８.５)８００mlに懸濁し、Ｄino−Ｍillにより菌糸体を破砕した。破砕液を９,５００ｒｐｍで２０分間遠心分離し、得られた上清（無細胞抽出液）を粗酵素液として、以下の方法で精製した。
３）精製
ステップ１：硫安分画
粗酵素液に４０％飽和になるように硫酸アンモニウム（以下、硫安と略す）を加え、遠心分離（４℃，１２,０００ｒｐｍ）して余分なタンパクを除去した。さらに、上清に硫安を７５％飽和になるように添加して沈殿を回収した。
ステップ２：疎水クロマトグラフィー（バッチ法）
ステップ１で得られた沈殿を、２ｍＭのＤＴＴを含有する５０ｍＭ トリス−塩酸緩衝液（ｐＨ８.５）（以下、緩衝液Ａと略す）に溶解し、等量の硫安４０％を含む緩衝液Ａを添加した。同粗酵素液にブチルトヨパール（butyl-ＴＯＹＯＰＥＡＲＬ）樹脂２００mlを加えて、バッチ法による吸着を行った。溶出も、緩衝液Ａを用いたバッチ法で行い、活性画分は硫安沈殿により濃縮した。
ステップ３：疎水クロマトグラフィー
２５％硫安を含む緩衝液Ａで平衡化したフェニルトヨパール（phenyl-ＴＯＹＯＰＥＡＲＬ）カラムに濃縮した活性画分を吸着させ、同緩衝液で洗浄後、２５〜０％硫安の直線勾配で溶出した。回収した活性画分は硫安沈殿により濃縮し、次のステップに用いた。
ステップ４：疎水クロマトグラフィー（カラム法）
回収した活性画分をブチルトヨパールカラム（４０％硫安を含む緩衝液Ａで平衡化）に用いた。濃縮液を吸着させ、同緩衝液で洗浄した。活性画分は４０〜０％硫安の直線勾配で得られた。
ステップ５：イオン交換クロマトグラフィー
次に、ＤＥＡＥ−トヨパール（ＤＥＡＥ-ＴＯＹＯＰＥＡＲＬ）カラムクロマトグラフィーを行った（緩衝液Ａで平衡化）。洗浄画分にＦＡＯＤ活性が認められたため、これを回収して硫安で濃縮してから、次のステップに用いた。
ステップ６：ゲル濾過
最後にセファクリル−３００によるゲル瀘過をおこなった（０.１Ｍ ＮａＣl，２mＭ ＤＴＴを含む０.１Ｍトリス−塩酸緩衝液（ｐＨ８.５）で平衡化）。これにより、７０〜１００ユニットの酵素標品を得た。
【００３６】
精製酵素のＵＶ吸収スペクトルを図６に示す。図６は、本酵素がフラビン酵素であることを示している。
またＳＤＳ−ＰＡＧＥ（ドデシル硫酸ナトリウム・ポリアクリルアミド電気泳動）及びスーパーデックス２００pgを用いたゲル濾過により得られた精製酵素の標品の分子量の決定を行った。
ＳＤＳ−ＰＡＧＥは、デービスの方法に従い、１０％ゲルを用いて、４０ｍＡで３時間泳動し、タンパク染色はクマシーブリリアントブルーＧ−２５０でおこなった。標準タンパクとしてホスホリラーゼＢ、牛血清アルブミン、オボアルブミン、カルボニックアンヒドラーゼ、大豆トリプシンインヒビターを同様に泳動して検量線を作成した。その結果、精製酵素のサブユニット分子量は約５１,０００（５１ｋＤａ）であった。
一方、ゲルろ過は０.１Ｍ ＮａＣl含有０.１Ｍトリス−塩酸緩衝液（ｐＨ８.５）を用いて行った結果、図５に示すように、約１０６,０００（１０６ｋＤａ）であった。
さらに、本実施例で精製したＦＡＯＤ−Ｌの酵素活性、至適ｐＨ及び温度、pＨ及び温度安定性、金属及び阻害剤の影響等に関しては、前記に示した通りである。
【００３７】
実施例２ 糖化ヒト血清アルブミン濃度の測定
糖化ヒト血清アルブミン（シグマ社）を０.９％塩化ナトリウム水溶液で溶解させ、０〜１０％の範囲で濃度の異なる糖化ヒト血清アルブミン溶液を調製した。
これらの溶液を用いて以下の操作を行った。
１）プロテアーゼ処理
糖化アルブミン溶液 ６０μl
１２.５mg/ml プロテアーゼXIV（シグマ社）溶液 ６０μl
この混合液を３７℃で３０分間インキュベートし、その後、約９０℃で５分間、加熱して反応を停止させた。
２）活性測定
ＦＡＯＤ反応液は以下のようにして調製した。
４５mＭ ４−アミノアンチピリン溶液 ３０μｌ
６０mＭ Ｎ−エチル−Ｎ−（２−ヒドロキシ−
３−スルホプロピル）−m−トルイジン溶液 ３０μｌ
６０ユニット／ml パーオキシダーゼ溶液 ３０μｌ
０.１Ｍ トリス−塩酸緩衝液（pＨ８.０） ３００μｌ
６ユニット／ml ＦＡＯＤ−Ｌ溶液 ５０μｌ
蒸留水で全量を１mlとした。
６ユニット／ml ＦＡＯＤ−Ｌ溶液は、実施例１の方法で得たＦＡＯＤ−Ｌを６ユニット／mlになるよう、０.１Ｍトリス−塩酸緩衝液（pＨ８.０）で希釈して調製した。
ＦＡＯＤ反応液を３０℃で２分間インキュベートした後、上記の各プロテアーゼ処理溶液を１００μl加え、３０分後の５５５nmにおける吸光度を測定した。この方法で得られる糖化アルブミンの濃度と吸光度との関係を図７に示す。図中の縦軸は５５５nmの吸光度（過酸化水素の量に対応）、横軸は糖化アルブミンの濃度を表す。図は、糖化アルブミンの濃度と過酸化水素発生量が相関関係にあることを示している。
【００３８】
実施例３ ヒト血清アルブミンの糖化率の測定
０.９％塩化ナトリウム水溶液３mlに、糖化ヒト血清アルブミン（シグマ社）１５０mg、ヒト血清アルブミン（シグマ社）１５０mgをそれぞれ溶解した。これらの溶液を混合することにより、糖化率の異なる溶液を作製し、自動グリコアルブミン測定装置（京都第一科学）を用いて検定したところ、その糖化率は、２４.６％〜６１.１％であった。
これらの溶液を用いて以下の操作を行った。
１）プロテアーゼ処理
糖化アルブミン溶液 ６０μl
１２.５mg/ml プロテアーゼXIV（シグマ社）溶液 ６０μl
この溶液を３７℃で３０分間インキュベートし、その後、約９０℃で５分間加熱して反応を停止させた。
２）活性測定
ＦＡＯＤ反応液は以下のようにして調製した。
４５mＭ ４−アミノアンチピリン溶液 ３０μｌ
６０mＭ Ｎ−エチル−Ｎ−（２−ヒドロキシ−
３−スルホプロピル）−m−トルイジン溶液 ３０μｌ
６０ユニット／ml パーオキシダーゼ溶液 ３０μｌ
０.１Ｍ トリス−塩酸緩衝液（pＨ８.０） ３００μｌ
６ユニット／ml ＦＡＯＤ−Ｌ溶液 ５０μｌ
蒸留水で全量を１mlとした。
６ユニット／ml ＦＡＯＤ−Ｌ溶液は、実施例１の方法で得たＦＡＯＤ−Ｌを６ユニット／mlになるよう、０.１Ｍトリス−塩酸緩衝液（pＨ８.０）で希釈して調製した。
ＦＡＯＤ反応液を３０℃で２分間インキュベートした後、上記の各プロテアーゼ処理溶液を１００μl加え、３０分後の５５５nmにおける吸光度を測定した。この方法で得られるアルブミンの糖化率と吸光度との関係を図８に示す。図中の縦軸は５５５nmの吸光度（過酸化水素の量に対応）、横軸はアルブミンの糖化率を表す。図は、アルブミンの糖化率と過酸化水素発生量が相関関係にあることを示している。
【００３９】
実施例４ 糖化ヘモグロビン濃度の測定
グリコヘモグロビンコントロール（シグマ社）を蒸留水で溶解させ、０〜３０％の範囲で濃度の異なる糖化ヘモグロビン溶液を調製した。
これらの溶液を用いて以下の操作を行った。
１）プロテアーゼ処理
糖化ヘモグロビン溶液 ２５μl
５００ユニット／ml アミノペプチダーゼ溶液 ５μl
０.１Ｍ トリス−塩酸緩衝液（pＨ８.０） ２０μl
この混合液を３０℃で３０分間インキュベートした。その後、１０％トリクロロ酢酸を５０μl加えて撹拌し、０℃で３０分間静置した後１２０００rpmで１０分間遠心分離を行った。得られた上清に２Ｍ ＮaＯＨを約５０μl加え中性溶液にした。
２）活性測定
ＦＡＯＤ反応液は以下のようにして調製した。
３ｍＭ 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）ビフェニルアミン溶液 ３０μl
６０ユニット/ml パーオキシダーゼ溶液 ３０μl
０.１Ｍ トリス−塩酸緩衝液（pＨ８.０） ３００μl
４ユニット/ml ＦＡＯＤ−Ｌ溶液 １０μl
蒸留水で全量を１mlとした。
４ユニット／ml ＦＡＯＤ−Ｌ溶液は、実施例１の方法で得たＦＡＯＤ−Ｌを４ユニット／mlになるよう、０.１Ｍトリス−塩酸緩衝液（pＨ８.０）で希釈して調製した。
ＦＡＯＤ反応液を３０℃で２分間インキュベートした後、上記の各プロテアーゼ処理溶液を８０μl加え、３０分後の７２７nmにおける吸光度を測定した。この方法で得られる糖化ヘモグロビンの濃度と吸光度との関係を図９に示す。図中の縦軸は７２７nmの吸光度（過酸化水素の量に対応）、横軸は糖化ヘモグロビンの濃度を表す。図は、糖化ヘモグロビンの濃度と過酸化水素発生量が相関関係にあることを示している。
【００４０】
【発明の効果】
本発明のＦＡＯＤは、従来の同種の酵素フルクトシルアミノ酸オキシダーゼと異なり、フルクトシルリジン及びフルクトシルバリンのいずれにも特異的に作用し、また、前者に対する特異性がより高い。従って、新たな臨床分析及び食品分析法の開発に有用であり、糖尿病の診断や食品の品質管理の面で寄与するところが大きい。特に、血中の糖化タンパク量及び／又は糖化率あるいはフルクトサミン量を指標として、糖尿病の病状の診断に役立つと考えられる。また、本発明のＦＡＯＤを用いるアマドリ化合物の分析試薬及び分析方法によって、正確に糖化タンパクを定量することができ、糖尿病の診断、症状管理に貢献することができる。
【図面の簡単な説明】
【図１】 培養培地でのＦＡＯＤの生産量と培養時間の関係を示すグラフ。
【図２】 ＦＡＯＤの溶媒中での活性と至適pＨの関係を示すグラフ。
【図３】 ＦＡＯＤの溶媒中での活性と至適温度の関係を示すグラフ。
【図４】 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）におけるＦＡＯＤ−Ｌの泳動パターンを示す写真の模写図。
【図５】 スーパーデックス２００pgを用いたゲルろ過によるＦＡＯＤ−Ｌの分子量測定の結果を示すグラフ。
【図６】 ＦＡＯＤ−Ｌの吸収スペクトル。
【図７】 糖化ヒト血清アルブミンの濃度とＦＡＯＤ作用により生成された過酸化水素量との関係を示すグラフ。
【図８】 ヒト血清アルブミンの糖化率とＦＡＯＤ作用により生成された過酸化水素量との関係を示すグラフ。
【図９】 糖化ヘモグロビンの濃度とＦＡＯＤ作用により生成された過酸化水素量との関係を示すグラフ。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel fructosyl amino acid oxidase, and more particularly, to the genus Fusarium (Fusarium) -Derived fructosyl amino acid oxidase, a method for producing the enzyme, a method for analyzing an Amadori compound using the enzyme, and a reagent and a kit containing the enzyme.
[0002]
[Prior art]
In the case of an Amadori compound, when a substance having an amino group such as a protein, peptide or amino acid and a reducing sugar such as aldose coexist, the amino group and the aldehyde group are bound non-enzymatically and irreversibly. Generated by transition. The production rate of the Amadori compound is expressed by a function such as the concentration of the reactive substance, the contact time, and the temperature. Therefore, it is considered that various information related to substances containing these reactive substances can be obtained from the amount of production. Substances containing an Amadori compound include foods such as soy sauce and body fluids such as blood.
In the living body, a fructosylamine derivative, which is an Amadori compound in which glucose and amino acid are bonded, is generated. For example, a fructosylamine derivative in which hemoglobin in blood is glycated is called glycohemoglobin, a derivative in which albumin is glycated is called glycoalbumin, and a derivative in which protein in blood is glycated is called fructosamine. These blood concentrations reflect the average blood glucose level over a certain period in the past, and the measured values can be an important indicator for the diagnosis and management of symptoms of diabetes. It is extremely useful. In addition, by quantifying the Amadori compound in food, it is possible to know the storage status and period after the production of the food, which is useful for quality control.
Thus, quantitative analysis of Amadori compounds is useful in a wide range of fields including medicine and food.
[0003]
Conventionally, as a method for quantifying Amadori compounds, a method using high performance liquid chromatography [Chromatogr. Sci. 10: 659 (1979)], a method using a column packed with a solid bound with boric acid [Clin. Chem. 28: 2088-2094 (1982)], electrophoresis [Clin. Chem. 26: 1598-1602 (1980)], a method using an antigen-antibody reaction [JJCLA 18: 620 (1993), instruments / reagents 16: 33 -37 (1993)], determination of fructosamine [Clin.Chim.Acta 127: 87-95 (1982)], post-oxidation colorimetric determination using thiobarbituric acid [Clin.Chim.Acta 112: 197 -204 (1981)] is known, but expensive equipment is required, and it is not always an accurate and quick method.
[0004]
In recent years, due to the characteristics of enzymes (specificity of substrate, reaction, structure, position, etc.), the target substance can be selectively analyzed quickly and accurately. And in the field of food analysis.
There has already been proposed an analytical method for quantifying an Amadori compound by allowing an oxidoreductase to act on the Amadori compound and measuring the amount of oxygen consumed or the amount of hydrogen peroxide generated in the reaction (for example, Japanese Patent Publication No. 5). -33997, JP-B-6-65300, JP-A-2-195900, JP-A-3-155780, JP-A-4-4874, JP-A-5-192193, JP-A-6-46846. Issue gazette). Furthermore, glycated protein quantification methods for the diagnosis of diabetes are also disclosed (Japanese Patent Laid-Open Nos. 2-195899, 2-195900, 5-192193, and 6-46846). ).
[0005]
The decomposition reaction of an Amadori compound by an oxidoreductase can be represented by the following general formula.
R¹-CO-CH₂-NH-R²  + O₂  + H₂O →
R¹-CO-CHO + R²-NH₂  + H₂O₂
(Wherein R¹Is an aldose residue, R²Represents an amino acid, protein or peptide residue)
The following are known as enzymes that catalyze the above reaction.
1. Fructosyl amino acid oxidase: Corynebacterium (Corynebacterium) Genera (Japanese Patent Publication No. 5-33997, Japanese Patent Publication No. 6-65300), Aspergillus genus (Aspergillus(Japanese Patent Laid-Open No. 3-155780).
2. Fructosylamine deglycase: Candida (Candida(JP-A-6-46846).
3. Fructosyl amino acid degrading enzyme: Penicillium (Penicillium(Japanese Patent Laid-Open No. 4-4874).
4. Ketoamine oxidase: Corynebacterium genus, Fusarium genus, Acremonium genus or Debryomyces genus (Japanese Patent Laid-Open No. 5-192193)
5). Alkyl lysinase: Prepared by the method described in J. Biol. Chem. Vol. 239, pages 3790-3796 (1964).
[0006]
[Problems to be Solved by the Invention]
However, these enzyme methods have the following problems.
That is, glycated proteins in blood that serve as an index in the diagnosis of diabetes are glycated albumin, glycated hemoglobin, and fructosamine. Glycated albumin is produced by binding glucose to the ε position of a lysine residue in a protein molecule [J. Biol. Chem. 261: 13542-13545 (1986)]. Glycated hemoglobin [J. Biol. Chem. 254: 3892-3898 (1979)], glucose is also bound to the N-terminal valine of the hemoglobin β chain. Therefore, it was necessary to use enzymes with high specificity for fructosyl lysine and fructosyl valine for the measurement of glycated protein which is an indicator of diabetes. However, existing enzymes from the genus Corynebacterium do not act on fructosyl lysine, and enzymes derived from the genus Aspergillus have not been clarified for their action on glycated proteins or their hydrolysates. On the other hand, ketoamine oxidase described in JP-A-5-192193 can decompose fructosyl valine, but cannot accurately measure a glycated protein in which a sugar is bound to a lysine residue. Since fructosylamine degrigase has high activity in difructosyl lysine, it cannot specifically measure ε-glycosides of lysine residues, and it specifically measures glycated products of valine residues. I can't do that either. Furthermore, the method using alkyl lysinase also has an effect on substances other than saccharides bound to lysine, and has a problem of low specificity for saccharified products, and accurate measurement cannot be expected. An enzyme derived from the genus Penicillium described in JP-A-4-4874 is an enzyme that acts on fructosyl lysine and fructosyl alanine.
Thus, conventional enzymes are not suitable for accurate quantification of glycated proteins, and the development of enzymes with high specificity for fructosyl lysine and fructosyl valine has been awaited.
[0007]
Generally, in order for an analytical method using an enzyme to be accurate and useful, it is necessary to select an enzyme that is optimal for the purpose of the analysis. In other words, reproducible and accurate analysis may not be possible unless an appropriate enzyme is used in consideration of various conditions such as the type of test substance that is the enzyme substrate, the state of the measurement sample, and the measurement conditions. There is. In order to select such an enzyme, activity, substrate specificity, temperature stability, pH stability, etc. must be specified in advance for various enzymes. Therefore, it is desirable to produce more fructosyl amino acid oxidase and clarify their properties.
[0008]
[Means for Solving the Problems]
As a result of intensive research aimed at providing a novel fructosyl amino acid oxidase that specifically acts on amadori compounds, particularly glycated proteins, the present inventors have succeeded in the genus Fusarium (Fusarium) Bacterium of fructosyl lysine and / or fructosyl N^αIt was found that an enzyme having the desired activity was induced when cultured in the presence of -Z-lysine, and the present invention was completed.
That is, the present invention relates to the genus Fusarium (Fusarium), The fructosyl lysine and / or fructosyl N^αA fructosyl amino acid oxidase produced by culturing in a medium containing -Z-lysine is provided.
[0009]
Fructosyl lysine and / or fructosyl N used for culture of the fructosyl amino acid oxidase producing bacterium of the present invention^α-Z-lysine-containing medium is glucose and lysine and / or N^α-Fructosyl lysine and / or fructosyl N obtained by autoclaving Z-lysine at a temperature of 100 to 150 ° C for 3 to 60 minutes^α-Z-lysine (hereinafter sometimes abbreviated as FZL). As will be described later, the fructosyl amino acid oxidase of the present invention is active on both fructosyl lysine and fructosyl valine, but the activity is characterized by the activity on the former being equal to or higher than the activity on the latter. In the present specification, the fructosyl amino acid oxidase of the present invention may be referred to as FAOD.
[0010]
The enzyme of the present invention can be used to treat Fusarium spp.^αIt can be produced by culturing in a medium containing -Z-lysine. As such bacteria, Fusarium oxysporum f.sp.Lini (IFO NO.5880) (Fusarium oxysporum f.sp.lini), Fusarium oxysporum f.sp.Batatus (IFO NO.4468) (Fusarium oxysporum f.sp.batatas), Fusarium oxysporum, f.sp.niveum (IFO NO.4471) (Fusarium oxysporum f.sp.niveum), Fusarium oxysporum, f.sp. kukumerinium (IFO NO.6384) (Fusarium oxysporum f.sp.cucumerinum), Fusarium oxysporum, f.sp. melon genae (IFO NO.7706) (Fusarium oxysporum f.sp.melongenae), Fusarium oxysporum f.sp.api (IFO NO.9964) (Fusarium oxysporum f.sp.apii), Fusarium oxysporum f.sp.Pini (IFO NO.9971) (Fusarium oxysporum f.sp.pini) And Fusarium oxysporum f.sp.Fragari (IFO NO.31180) (Fusarium oxysporum f.sp.fragariae) And the like.
[0011]
The FAODs of the present invention generally have the following physicochemical properties:
1) catalyzing the reaction of oxidizing an Amadori compound in the presence of oxygen to produce α-ketoaldehyde, an amine derivative and hydrogen peroxide;
2) The stable pH is 4.0-13.0, the optimum pH is 8.5;
3) The stable temperature is 20-50 ° C and the optimum temperature is 30-35 ° C;
4) When measured by a gel filtration method using 200 pg Superdex, the molecular weight is about 106,000 (106 kDa).
[0012]
The fructosyl lysine and / or FZL used for the production of the FAOD of the present invention comprises 0.01 to 50% by weight of glucose and lysine and / or N.^α-Z-lysine in an amount of 0.01 to 20% by weight is produced by autoclaving in a solution at 100 to 150 ° C for 3 to 60 minutes. Specifically, 200 g glucose, N in a 1000 ml total solution^αIt can be produced by dissolving 10 g of -Z-lysine and subjecting it to autoclaving usually at 120 ° C. for 20 minutes.
The fructosyl lysine and / or FZL-containing medium (hereinafter referred to as FZL medium) used for production of FAOD of the present invention is obtained by adding fructosyl lysine and / or FZL obtained by the above method to a normal medium. E.g. glucose 0.01-50% by weight, lysine and / or N^α-Z-lysine 0.01-20% by weight, K₂HPO_Four 0.1% by weight, NaH₂PO_Four 0.1% by weight, MgSO_Four・ 7H₂0.05% by weight of O, CaCl₂・ 2H₂It can be obtained by autoclaving a mixture (preferably pH 5.6-6.0) containing 0.01% by weight of O and 0.2% by weight of yeast extract at 100-150 ° C. for 3-60 minutes.
[0013]
The medium used for producing the FAOD of the present invention may be a normal synthetic or natural medium containing a carbon source, a nitrogen source, an inorganic substance, and other nutrient sources. Examples of the carbon source include glucose, xylose, and glycerin. As the nitrogen source, peptone, casein digest, yeast extract, and the like can be used. Furthermore, what is contained in a normal culture medium, such as sodium, potassium, calcium, manganese, magnesium, cobalt, can be used as an inorganic substance.
The FAOD of the present invention is best induced when cultured in a medium containing fructosyl lysine and / or FZL. As an example of a preferable medium, an FZL medium (1.0% glucose, 0.5% FZL, 1.0% K) using FZL obtained by the above method as a single nitrogen source and glucose as a carbon source.₂HPO_Four0.1% NaH₂PO_Four0.05% MgSO_Four・ 7H₂O, 0.01% CaCl₂・ 2H₂O and 0.01% vitamin mixture). A particularly preferred medium is 20 g (2%) glucose, 10 g FZL (1%), K in a total volume of 1,000 ml.₂HPO_Four 1.0 g (0.1%), NaH₂PO_Four1.0 g (0.1%), MgSO_Four・ 7H₂0.5 g (0.05%), CaCl₂・ 2H₂It is a medium (pH 5.6-6.0) containing 0.1 g (0.01%) of O and 2.0 g (0.2%) of yeast extract. FZL medium can be prepared by adding FZL to normal medium or adding glucose and N^αIt can be prepared by autoclaving a medium containing -Z-lysine. The medium obtained by any of the methods has a brown color due to the presence of fructosyl lysine and / or FZL, and FZL browning medium or GL (glycated lysine and / or glycated N^α-Z-lysine) called browning medium.
[0014]
The culture is usually performed at 25 to 37 ° C, preferably 28 ° C. The pH of the medium is in the range of 4.0 to 8.0, preferably 5.5 to 6.0. However, these conditions are appropriately prepared according to the state of each bacterium and are not limited to the above. For example, Fusarium oxysporum, f. sp. When Lini is cultured under these conditions for 20 to 100 hours, preferably 80 hours, FAOD is accumulated in the culture medium (FIG. 1).
The culture obtained in this manner can be used to remove nucleic acids, cell wall fragments and the like according to a conventional method to obtain an enzyme preparation.
Since the enzyme activity of the FAOD of the present invention is accumulated in the microbial cells, the microbial cells in the culture are crushed and used for enzyme production.
The disruption of the cells may be any of self-digestion using mechanical means or a solvent, freezing, sonication, pressurization and the like.
Methods for separating and purifying enzymes are also known, and they are purified by a combination of salting out using ammonium sulfate, precipitation with an organic solvent such as ethanol, ion exchange chromatography, hydrophobic chromatography, gel filtration, affinity chromatography, and the like.
For example, the culture is centrifuged or suction filtered to collect mycelia, washed, suspended in 0.1 M Tris-HCl (pH 8.5), and the mycelium is crushed with Dino-Mill. Subsequently, the supernatant obtained by centrifugation is purified as a cell-free extract by treatment with an ammonium sulfate fraction and phenyl-cephalose hydrophobic chromatography.
[0015]
However, for the purposes of the present invention, FAOD includes enzyme purification products and solutions in all purification stages, including culture solutions, so long as they can catalyze the oxidation reaction of Amadori compounds, regardless of their degree of purification. . Moreover, since the object of the present invention can be achieved only by a site involved in the catalytic activity in the enzyme molecule, any fragment having an Amadori compound oxidation activity is also included. The FAOD thus obtained is useful for the quantification of the Amadori compound, particularly the glycated protein for the diagnosis of diabetes.
Therefore, the present invention is characterized in that a fungus is cultured in a medium containing a glycated product of an amino acid having a free or protecting group and / or a glycated product of a protein to cause the fungus to produce fructosyl amino acid oxidase. A method for producing fructosyl amino acid oxidase is provided.
Furthermore, the present invention relates to a strain capable of producing a fructosyl amino acid oxidase belonging to the genus Fusarium, fructosyl lysine and / or fructosyl N.^αThe present invention provides a method for producing fructosyl amino acid oxidase, which comprises culturing in a medium containing Z-lysine and recovering fructosyl amino acid oxidase from the culture. Any FAOD produced by strains of these genera is useful for solving technical problems to be solved by the present invention. In the present specification, Fusarium oxysporum, f. sp. The LOD-derived FAOD is sometimes referred to as FAOD-L.
[0016]
The characteristics of the FAOD of the present invention will be described in detail below.
1. General induction characteristics
The FAOD of the present invention is an inducible enzyme induced by fructosyl lysine and / or FZL, and is a fructosyl lysine and / or FZL medium containing fructosyl lysine and / or FZL as a nitrogen source and glucose as a carbon source. Produced by culturing a genus fructosyl amino acid oxidase producing bacterium.
FAOD is glucose and lysine and / or N^αInduced in GL browning medium obtained by autoclaving together with -Z-lysine, but with glucose and lysine and / or N^αSince the enzyme is not induced in a medium prepared by separately autoclaving -Z-lysine, the enzyme acts specifically on the Amadori compound.
[0017]
2. Reaction specificity and substrate specificity
The FAOD of the present invention has the formula:
R¹-CO-CH₂-NH-R²  + O₂  + H₂O →
R¹-CO-CHO + R²-NH₂  + H₂O₂
(Wherein R¹Is an aldose residue, R²Represents an amino acid, protein or peptide residue)
It has a catalytic activity in the reaction shown by. In the above reaction formula, R¹Is —OH, — (CH₂)_n-Or-[CH (OH)]_n-CH₂OH (where n is an integer from 0-6) and R²Is -CHR^Three-[CONHR^Three]_mCOOH (wherein R^ThreeIs preferably an α-amino acid side chain residue, and m is an integer of 1 to 480). Above all, R^ThreeIs a side chain residue of an amino acid selected from lysine, polylysine, valine, asparagine and the like, and a compound in which n is 5 to 6 and m is 55 or less is preferable.
[0018]
The activity of each FAOD substrate of the present invention is shown in Table 1 below.
Table 1  Substrate specificity of purified FAOD-L
[Table 1]

* 1: Not detected
* 2: Fructosyl bovine serum albumin
* 3: Fructosyl human serum albumin
From Table 1, fructosyl N^αHas high specificity for -Z-lysine and fructosyl valine. The FAOD of the present invention has activity against fructosylpolylysine, and further has activity against protease digests of glycated proteins.
The strains having the ability to produce Fusarium FAOD are exemplified in Table 2 below.
Table 2  Substrate specificity of FAOD purified from Fusarium spp. Cultured in FZL browning medium
[Table 2]

1): (Fructosyl N^α-Activity against Z-lysine) / (Activity against fructosyl valine)
[0019]
As shown in Table 2, the FAOD of the present invention has activity against both fructosyl lysine and fructosyl valine, which is useful for measuring glycated hemoglobin. It suggests that there is.
[0020]
3. pH and temperature conditions
Examination of pH conditions
The optimum pH is determined by using various buffer solutions (0.1 M potassium phosphate buffer (KPB), Tris, pH 4 to 13 as buffers used in the usual FAOD activity measurement method (see 4. Method for Measuring Titer described later). -Hydrochloric acid buffer solution and glycine (Gly) -NaOH buffer solution) were replaced by an enzyme reaction and examined.
Moreover, after adding FAOD to the above-mentioned various buffers and incubating at 30 ° C. for 10 minutes, the stability by pH was examined by measuring the activity under normal conditions (30 ° C., pH 8.0).
Examination of temperature conditions
In order to examine the optimum temperature, the enzyme activity was measured by changing the reaction temperature from 20 to 60 ° C. Regarding temperature stability, FAOD dissolved in 0.1 M Tris-HCl buffer (pH 8.0) was incubated at each temperature of 20-60 ° C. for 10 minutes, and the residual activity was obtained under normal conditions using the enzyme solution. It was measured.
As a result of the measurement by the above method, the optimum pH of the FAOD of the present invention is 7.5 to 9.0, more preferably 8.5 (FIG. 2), and the stable pH range is 4.0 to 13. 0.
The FAOD enzyme reaction proceeded efficiently at 20 to 50 ° C., preferably 25 to 40 ° C., more preferably 35 ° C. (FIG. 3). The stable temperature range was 20-50 ° C.
[0021]
4). Titer measurement
The enzyme titer was measured by the following method.
(1) A method of measuring generated hydrogen peroxide by a colorimetric method.
A. Velocity method
The 100 mM FZL solution was prepared by dissolving FZL obtained in advance with distilled water. 100 μl each of 45 mM 4-aminoantipyrine, 60 unit / ml peroxidase solution, and 60 mM phenol solution, 1 ml of 0.1 M Tris-HCl buffer (pH 8.0), and 50 μl of enzyme solution were mixed, and the whole volume was made up with distilled water. Make up to 3.0 ml. After incubating at 30 ° C. for 2 minutes, 50 μl of 100 mM FZL solution was added, and the absorbance at 505 nm was measured over time. Molecular absorption coefficient of the quinone dye produced (5.16 × 10^ThreeM^-1cm^-1) To calculate the micromoles of hydrogen peroxide generated per minute, and this number is used as the enzyme activity unit (unit: U).
[0022]
B. Terminal law
The treatment was performed in the same manner as in the above method A. After adding the substrate, the absorbance at 505 nm was measured after 30 minutes of incubation at 30 ° C., and the amount of hydrogen peroxide generated from a calibration curve prepared in advance using a standard hydrogen peroxide solution was determined. By calculating, the enzyme activity is measured.
(2) Method for measuring oxygen absorption by enzyme reaction
Mix 1 ml of 0.1 M Tris-HCl buffer (pH 8.0) and 50 μl of the enzyme solution to make 3.0 ml with distilled water, and put it into a cell of Rank Brothers oxygen electrode. After stirring at 30 ° C. to equilibrate the dissolved oxygen and temperature, 100 μl of 50 mM FZL is added, and oxygen absorption is continuously measured with a recorder to obtain the initial velocity. The amount of oxygen absorbed per minute is determined from a standard curve, and this is used as an enzyme unit.
[0023]
5). Enzyme inhibition, activation and stabilization
(1) Influence of metal
Various metal ions were added to 0.1 M Tris-HCl buffer (pH 8.0) to a final concentration of 1 mM, incubated at 30 ° C. for 5 minutes, and the activity was measured. The results are shown in Table 3 below.
Table 3  Effect of metal ions on FAOD-L activity
[Table 3]

As is apparent from Table 3, copper ions and zinc ions are inhibitory to the activity of the FAOD of the present invention, and silver ions and mercury ions are completely inhibited.
[0024]
(2) Effects of various inhibitors
It tested by the method similar to the test regarding the influence of the metal ion of said (1). However, mercuric parachlorobenzoate had a final concentration of 0.1 mM, and other concentrations were 1 mM. The results are shown in Table 4. The stabilization was examined by dialyzing the purified enzyme overnight against 50 mM Tris-HCl buffer (pH 8.5) with 2 mM dithiothreitol (DTT) and measuring the activity. .
Table 4  Effects of various substances on FAOD activity
[Table 4]

* 1: PCMB, mercuric parachlorobenzoate
* 2: DTNB, 5,5′-dithiobis (2-nitrobenzoic acid)
* 3: Not detected
As apparent from Table 4, FAOD activity was strongly inhibited by PCMB, DTNB, hydrazine, and phenylhydrazine. From this, it is expected that the SH group and the carbonyl group play an important role in the enzyme reaction.
On the other hand, a solvent stabilized by dithiothreitol and suitable for storage is 50 mM Tris-HCl buffer (pH 8.5) supplemented with 2 mM dithiothreitol.
[0025]
6). Molecular weight
SDS-PAGE (sodium dodecyl sulfate / polyacrylamide gel electrophoresis) was run for 3 hours at 40 mA using 10% gel according to the method of Davis, and protein staining was performed with Coomassie Brilliant Blue G-250. Phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase, and soybean trypsin inhibitor were similarly run as standard proteins, and a calibration curve was prepared to determine the molecular weight. As a result, the molecular weight of the subunit was about 51,000 (51 kDa) (FIG. 4).
In gel filtration with Superdex 200 pg, the molecular weight was determined to be about 106,000 (106 kDa) (FIG. 5), suggesting that the FAOD of the present invention is a dimer.
[0026]
7). Isoelectric point
As a result of measurement by disc focus electrophoresis, FAOD was pI = 6.8.
[0027]
8). Comparison with known enzymes
The existing fungus-derived fructosyl amino acid oxidase was compared with the FAOD of the present invention.
Table 5  Comparison of fructosyl amino acid oxidases from various microorganisms
[Table 5]

1): T. Horiuchi et al. Agric. Biol. Chem., 53 (1), 103-110 (1989)
2): T. Horiuchi et al. Agric. Biol. Chem., 55 (2), 333-338 (1991)
3): Fructosyl N^αSpecific activity for -Z-lysine
4): N^ε-D-fructosyl N^α-Specific activity against formyllysine
From Table 5, the following differences are observed between the FAOD of the present invention and the fructosyl amino acid oxidase derived from the other two strains.
(1) Difference in molecular weight: The FAOD of the present invention clearly has a higher molecular weight than the other two enzymes.
(2) Coenzyme: The FAOD of the present invention has FAD covalently bound as a coenzyme, while all other enzymes have non-covalently bound FAD.
(3) Substrate specificity: Although the FAOD of the present invention has higher specificity for fructosyl lysine than fructosyl valine, the enzyme derived from Corynebacterium does not act on fructosyl lysine and is derived from Aspergillus. Although the enzyme acts on fructosyl lysine, its activity is low compared to its activity on fructosyl valine.
(4) Michaelis constant: This shows that the affinity of the FAOD of the present invention for the substrate fructosyl lysine is higher than that of other enzymes.
(5) Optimal pH, optimal temperature, and inhibition by SH reagent: This shows the difference between the FAOD of the present invention and the other two enzymes.
[0028]
As described above, the enzyme FAOD of the present invention is useful for quantifying Amadori compounds. Accordingly, the present invention provides an analysis of an Amadori compound in a sample, which comprises contacting a sample containing an Amadori compound with the FAOD of the present invention and measuring the amount of oxygen consumed or the amount of hydrogen peroxide generated. It provides the law. The analysis method of the present invention is performed based on the measurement of the amount and / or saccharification rate of glycated protein in biological components, or the determination of fructosamine.
The enzyme activity of FAOD is measured based on the following reaction.
R¹-CO-CH₂-NH-R²  + O₂  + H₂O →
R¹-CO-CHO + R²-NH₂  + H₂O₂
(Wherein R¹Is an aldose residue, R²Represents an amino acid, protein or peptide residue)
As the test solution, any sample solution containing an Amadori compound can be used, and examples include foods such as soy sauce in addition to biological samples such as blood (whole blood, plasma or serum) and urine. .
[0029]
The FAOD of the present invention is allowed to act on an Amadori compound-containing solution in a suitable buffer. The pH and temperature of the reaction solution are in a range satisfying the above conditions, that is, pH 4.0 to 13.0, preferably 8.5, and the temperature is 20 to 50 ° C., preferably 35 ° C. Tris-hydrochloric acid or the like is used as the buffer. The amount of FAOD used is usually 0.1 units / ml or more, preferably 1 to 100 units / ml in the end point analysis method.
[0030]
In the analysis method of the present invention, any of the following Amadori compound quantification methods is used.
(1) Method based on hydrogen peroxide generation
Samples were measured by hydrogen peroxide quantification methods known in the art, for example, a color development method, a method using a hydrogen peroxide electrode, etc., and compared with a standard curve prepared for the amounts of hydrogen peroxide and Amadori compounds. Quantify the Amadori compound in it. Specifically, it conforms to the measurement of titer 4 above. However, the amount of FAOD is 1 unit / ml, an appropriately diluted sample is added, and the amount of hydrogen peroxide produced is measured. As a coloring system of hydrogen peroxide, a system that develops color by oxidative condensation of a coupler such as 4-aminoantipyrine or 3-methyl-2-benzothiazolinone hydrazone with a chromogen such as phenol in the presence of peroxidase. Can be used. Examples of chromogens include phenol derivatives, aniline derivatives, toluidine derivatives and the like, for example, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine, N, N-dimethylaniline, N, N -Diethylaniline, 2,4-dichlorophenol, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (3-sulfopropyl) -3, Examples include 5-dimethylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline. A leuco-type color reagent that exhibits oxidative coloration in the presence of peroxidase can also be used, and such leuco-type color reagents are known to those skilled in the art and include o-dianisidine, o-tolidine, and 3,3-diamino. Benzidine, 3,3,5,5-tetramethylbenzidine, N- (carboxymethylaminocarbonyl) -4,4-bis (dimethylamino) biphenylamine, 10- (carboxymethylaminocarbonyl) -3,7-bis ( And dimethylamino) phenothiazine.
(2) Method based on oxygen consumption
The value obtained by subtracting the amount of oxygen at the end of the reaction (oxygen consumption) from the amount of oxygen at the start of the reaction (oxygen consumption) is compared with the standard curve prepared for the oxygen consumption and the amount of the Amadori compound. Quantify. Specifically, the measurement is performed according to the measurement of the titer 4 described above. However, the amount of FAOD used is 1 unit / ml, and an appropriately diluted sample is added to determine the amount of oxygen consumed.
[0031]
The method of the present invention can be carried out using the sample solution as it is, but depending on the target glycated protein, it is preferable to carry out after releasing lysine and / or valine residues to which sugar is bound in advance.
For such purposes, there are a case where a proteolytic enzyme is used (enzymatic method) and a case where a chemical substance such as hydrochloric acid is used (chemical method), but the former is preferable. In that case, endo-type and exo-type proteolytic enzymes (proteases) known to those skilled in the art can be used in the method of the present invention. Examples of endo-type proteases include trypsin, α-chymotrypsin, subtilisin, proteinase K, papain, cathepsin B, pepsin, thermolysin, protease XIV, lysyl endopeptidase, proleather, and bromelain F. On the other hand, examples of exo-type proteases include aminopeptidases and carboxypeptidases. The method of enzyme treatment is also known, and can be performed, for example, by the method described in the following examples.
[0032]
As described above, since the FAOD of the present invention has high substrate specificity for fructosyl lysine contained in glycated protein, it is useful for diagnosis of diabetes including measurement of glycated protein in a blood sample. It is. Moreover, since it has specificity also to fructosyl valine, it is useful also in the measurement of glycated hemoglobin.
In addition, when using a blood sample (whole blood, plasma, or serum) as a specimen, the collected blood sample is used as it is or after it has been subjected to a treatment such as folding.
Furthermore, enzymes such as FAOD and peroxidase used in the method of the present invention may be used in a solution state, but may be immobilized on a suitable solid support. For example, a daily analysis of a large number of specimens such as clinical examinations can be efficiently performed by packing an enzyme immobilized on beads into a column and incorporating the enzyme into an automated apparatus. Moreover, since the immobilized enzyme can be reused, it is preferable from the viewpoint of economic efficiency.
Furthermore, a kit for analyzing an Amadori compound useful not only for clinical analysis but also for food analysis can be obtained by appropriately combining an enzyme and a coloring dye.
[0033]
Enzyme immobilization can be performed by methods known in the art. For example, it is carried out by a carrier binding method, a crosslinking method, a comprehensive method, a composite method or the like. Examples of the carrier include polymer gel, microcapsule, agarose, alginic acid, carrageenan and the like. The binding is performed by a method known to those skilled in the art using a covalent bond, an ionic bond, a physical adsorption method, and biochemical affinity.
When using an immobilized enzyme, the analysis may be either flow or batch. As described above, the immobilized enzyme is particularly useful for routine analysis (clinical examination) of glycated proteins in blood samples. When the clinical test is aimed at diagnosing diabetes, the diagnosis is expressed as a glycated protein concentration, or the ratio of the glycated protein concentration to the total protein concentration in the sample (glycation rate) or the fructosamine value. The total protein concentration can be measured by a usual method (absorbance at 280 nm, Bradford method, Lowry method, Burette method, natural fluorescence of albumin, absorbance of hemoglobin, etc.).
[0034]
The present invention also provides an analytical reagent or kit for an Amadori compound containing the FAOD of the present invention.
The reagent for quantifying the Amadori compound of the present invention comprises the FAOD of the present invention and preferably a buffer solution having a pH of 7.5 to 8.5, more preferably pH 8.0. When the FAOD is immobilized, the solid support is selected from a polymer gel or the like, and is preferably alginic acid.
When the end point analysis is performed, the amount of FAOD in the reagent is usually 1 to 100 units / ml per sample, and the buffer is preferably tris-hydrochloric acid (pH 8.0).
In the case of quantifying the Amadori compound based on the amount of hydrogen peroxide produced, the color developing system includes a system that develops color by oxidative condensation as described in the above-mentioned “(1) Method based on the amount of hydrogen peroxide generated”, and leuco-type color development. A reagent etc. can be used.
The analysis reagent of the Amadori compound of the present invention can be combined with an appropriate color former and a color standard or standard substance for comparison to make a kit. Such a kit is considered useful for preliminary diagnosis and testing.
The analytical reagents and kits described above are used for measuring the amount of glycated protein and / or the saccharification rate in biological components, or for quantifying fructosamine.
Hereinafter, the present invention will be described in more detail with reference to examples.
[0035]
【Example】
Example 1  Culture of Fusarium oxysporum, f.sp., Lini and purification of FAOD-L
1) Culture
Fusarium oxysporum f.sp. Rini (IFO NO.5880;Fusarium oxysporum f.sp.lini) FZL 0.5%, glucose 1.0%, dipotassium phosphate 0.1%, monosodium phosphate 0.1%, magnesium sulfate 0.05%, calcium chloride 0.01%, yeast extract 0.0 The cells were inoculated into 10 L of a medium (pH 6.0) containing 2%, and cultured with a jar fermenter at 28 ° C. for 80 hours under an aeration rate of 2 L / min and a stirring speed of 400 rpm. The culture was collected by filtration.
2) Preparation of crude enzyme solution
270 g (wet weight) of mycelium was suspended in 800 ml of 0.1 M Tris-HCl buffer (pH 8.5) containing 2 mM DTT, and the mycelium was crushed with Dino-Mill. The disrupted solution was centrifuged at 9,500 rpm for 20 minutes, and the resulting supernatant (cell-free extract) was purified as a crude enzyme solution by the following method.
3) Purification
Step 1: Ammonium sulfate fraction
Ammonium sulfate (hereinafter abbreviated as ammonium sulfate) was added to the crude enzyme solution so as to be 40% saturation, and excess protein was removed by centrifugation (4 ° C., 12,000 rpm). Furthermore, ammonium sulfate was added to the supernatant to 75% saturation, and the precipitate was recovered.
Step 2: Hydrophobic chromatography (batch method)
The precipitate obtained in Step 1 is dissolved in 50 mM Tris-HCl buffer (pH 8.5) (hereinafter abbreviated as Buffer A) containing 2 mM DTT, and Buffer A containing an equal amount of 40% ammonium sulfate. Was added. 200 ml of butyl-TOYOPARL resin was added to the crude enzyme solution, and adsorption by a batch method was performed. Elution was also performed by a batch method using buffer A, and the active fraction was concentrated by ammonium sulfate precipitation.
Step 3: Hydrophobic chromatography
The concentrated active fraction was adsorbed onto a phenyl-TOYOPEARL column equilibrated with buffer A containing 25% ammonium sulfate, washed with the same buffer solution, and eluted with a linear gradient of 25 to 0% ammonium sulfate. The collected active fraction was concentrated by ammonium sulfate precipitation and used in the next step.
Step 4: Hydrophobic chromatography (column method)
The collected active fraction was used in a butyl Toyopearl column (equilibrated with buffer A containing 40% ammonium sulfate). The concentrated solution was adsorbed and washed with the same buffer. The active fraction was obtained with a linear gradient of 40-0% ammonium sulfate.
Step 5: Ion exchange chromatography
Next, DEAE-TOYOPARL column chromatography was performed (equilibrated with buffer A). Since the washed fraction showed FAOD activity, it was collected and concentrated with ammonium sulfate, and then used in the next step.
Step 6: Gel filtration
Finally, gel filtration with Sephacryl-300 was performed (equilibrated with 0.1 M Tris-HCl buffer (pH 8.5) containing 0.1 M NaCl and 2 mM DTT). This gave 70-100 units of enzyme preparation.
[0036]
The UV absorption spectrum of the purified enzyme is shown in FIG. FIG. 6 shows that this enzyme is a flavin enzyme.
The molecular weight of the purified enzyme preparation obtained by gel filtration using SDS-PAGE (sodium dodecyl sulfate / polyacrylamide electrophoresis) and 200 pg Superdex was determined.
SDS-PAGE was performed for 3 hours at 40 mA using a 10% gel according to the method of Davis, and protein staining was performed with Coomassie Brilliant Blue G-250. As a standard protein, phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase, and soybean trypsin inhibitor were similarly migrated to prepare a calibration curve. As a result, the subunit molecular weight of the purified enzyme was about 51,000 (51 kDa).
On the other hand, as a result of performing gel filtration using 0.1 M Tris-HCl buffer (pH 8.5) containing 0.1 M NaCl, it was about 106,000 (106 kDa) as shown in FIG.
Furthermore, the enzyme activity, optimum pH and temperature, pH and temperature stability of FAOD-L purified in this example, the influence of metals and inhibitors, etc. are as described above.
[0037]
Example 2  Measurement of glycated human serum albumin concentration
Glycated human serum albumin (Sigma) was dissolved in 0.9% aqueous sodium chloride solution to prepare glycated human serum albumin solutions having different concentrations in the range of 0 to 10%.
The following operations were performed using these solutions.
1) Protease treatment
60 μl of glycated albumin solution
12.5 mg / ml Protease XIV (Sigma) 60 μl
This mixture was incubated at 37 ° C. for 30 minutes, and then heated at about 90 ° C. for 5 minutes to stop the reaction.
2) Activity measurement
The FAOD reaction solution was prepared as follows.
30 μl of 45 mM 4-aminoantipyrine solution
60 mM N-ethyl-N- (2-hydroxy-
3-sulfopropyl) -m-toluidine solution 30 μl
60 units / ml peroxidase solution 30 μl
0.1 M Tris-HCl buffer (pH 8.0) 300 μl
6 units / ml FAOD-L solution 50 μl
The total volume was made up to 1 ml with distilled water.
The 6 unit / ml FAOD-L solution was prepared by diluting the FAOD-L obtained by the method of Example 1 with 0.1 M Tris-HCl buffer (pH 8.0) to 6 units / ml.
After incubating the FAOD reaction solution at 30 ° C. for 2 minutes, 100 μl of each protease treatment solution was added, and the absorbance at 555 nm after 30 minutes was measured. The relationship between the concentration of glycated albumin obtained by this method and the absorbance is shown in FIG. In the figure, the vertical axis represents the absorbance at 555 nm (corresponding to the amount of hydrogen peroxide), and the horizontal axis represents the concentration of glycated albumin. The figure shows that there is a correlation between the concentration of glycated albumin and the amount of hydrogen peroxide generated.
[0038]
Example 3  Measurement of glycation rate of human serum albumin
150 mg of glycated human serum albumin (Sigma) and 150 mg of human serum albumin (Sigma) were dissolved in 3 ml of 0.9% sodium chloride aqueous solution. By mixing these solutions, solutions having different saccharification rates were prepared and tested using an automated glycoalbumin measuring apparatus (Kyoto Daiichi Kagaku). The saccharification rate was 24.6% to 61.1%. Met.
The following operations were performed using these solutions.
1) Protease treatment
60 μl of glycated albumin solution
12.5 mg / ml Protease XIV (Sigma) 60 μl
This solution was incubated at 37 ° C. for 30 minutes, and then heated at about 90 ° C. for 5 minutes to stop the reaction.
2) Activity measurement
The FAOD reaction solution was prepared as follows.
30 μl of 45 mM 4-aminoantipyrine solution
60 mM N-ethyl-N- (2-hydroxy-
3-sulfopropyl) -m-toluidine solution 30 μl
60 units / ml peroxidase solution 30 μl
0.1 M Tris-HCl buffer (pH 8.0) 300 μl
6 units / ml FAOD-L solution 50 μl
The total volume was made up to 1 ml with distilled water.
The 6 unit / ml FAOD-L solution was prepared by diluting the FAOD-L obtained by the method of Example 1 with 0.1 M Tris-HCl buffer (pH 8.0) to 6 units / ml.
After incubating the FAOD reaction solution at 30 ° C. for 2 minutes, 100 μl of each protease treatment solution was added, and the absorbance at 555 nm after 30 minutes was measured. FIG. 8 shows the relationship between the saccharification rate and absorbance of albumin obtained by this method. In the figure, the vertical axis represents absorbance at 555 nm (corresponding to the amount of hydrogen peroxide), and the horizontal axis represents albumin saccharification rate. The figure shows that there is a correlation between the saccharification rate of albumin and the amount of hydrogen peroxide generated.
[0039]
Example 4  Measurement of glycated hemoglobin concentration
Glycohemoglobin control (Sigma) was dissolved in distilled water to prepare glycated hemoglobin solutions having different concentrations in the range of 0 to 30%.
The following operations were performed using these solutions.
1) Protease treatment
Glycated hemoglobin solution 25 μl
500 units / ml aminopeptidase solution 5 μl
0.1 M Tris-HCl buffer (pH 8.0) 20 μl
This mixture was incubated at 30 ° C. for 30 minutes. Thereafter, 50 μl of 10% trichloroacetic acid was added and stirred, allowed to stand at 0 ° C. for 30 minutes, and then centrifuged at 12000 rpm for 10 minutes. About 50 μl of 2M NaOH was added to the resulting supernatant to make a neutral solution.
2) Activity measurement
The FAOD reaction solution was prepared as follows.
3 mM N- (carboxymethylaminocarbonyl) -4,4-
Bis (dimethylamino) biphenylamine solution 30 μl
60 units / ml peroxidase solution 30 μl
0.1 M Tris-HCl buffer (pH 8.0) 300 μl
4 units / ml FAOD-L solution 10 μl
The total volume was made up to 1 ml with distilled water.
The 4 unit / ml FAOD-L solution was prepared by diluting the FAOD-L obtained by the method of Example 1 with 0.1 M Tris-HCl buffer (pH 8.0) so as to be 4 units / ml.
After incubating the FAOD reaction solution at 30 ° C. for 2 minutes, 80 μl of each protease treatment solution was added, and the absorbance at 727 nm after 30 minutes was measured. FIG. 9 shows the relationship between the concentration of glycated hemoglobin and the absorbance obtained by this method. The vertical axis in the figure represents the absorbance at 727 nm (corresponding to the amount of hydrogen peroxide), and the horizontal axis represents the concentration of glycated hemoglobin. The figure shows that the concentration of glycated hemoglobin is correlated with the amount of hydrogen peroxide generated.
[0040]
【The invention's effect】
Unlike the conventional enzyme fructosyl amino acid oxidase, FAOD of the present invention specifically acts on both fructosyl lysine and fructosyl valine, and has higher specificity for the former. Therefore, it is useful for the development of new clinical analysis and food analysis methods, and greatly contributes to the diagnosis of diabetes and the quality control of food. In particular, it is considered useful for the diagnosis of the pathology of diabetes using the amount of glycated protein and / or glycation rate in blood or fructosamine amount as an index. In addition, the glycated protein can be accurately quantified by the reagent and method for analyzing an Amadori compound using the FAOD of the present invention, which can contribute to diabetes diagnosis and symptom management.
[Brief description of the drawings]
FIG. 1 is a graph showing the relationship between the production amount of FAOD in a culture medium and the culture time.
FIG. 2 is a graph showing the relationship between the activity of FAOD in a solvent and the optimum pH.
FIG. 3 is a graph showing the relationship between the activity of FAOD in a solvent and the optimum temperature.
FIG. 4 is a copy of a photograph showing the migration pattern of FAOD-L in SDS-PAGE (sodium dodecyl sulfate / polyacrylamide gel electrophoresis).
FIG. 5 is a graph showing the results of molecular weight measurement of FAOD-L by gel filtration using 200 pg Superdex.
FIG. 6 shows an absorption spectrum of FAOD-L.
FIG. 7 is a graph showing the relationship between the concentration of glycated human serum albumin and the amount of hydrogen peroxide produced by the FAOD action.
FIG. 8 is a graph showing the relationship between the saccharification rate of human serum albumin and the amount of hydrogen peroxide produced by the FAOD action.
FIG. 9 is a graph showing the relationship between the concentration of glycated hemoglobin and the amount of hydrogen peroxide produced by the FAOD action.

Claims (3)

Fusarium oxysporum & f.sp. linear (IFO NO.5880) a fructosyl amino acid oxidase produced by culturing (Fusarium oxysporum f.sp.lini), having physicochemical properties described below, fructosyl Amino acid oxidase:
1) catalyzing the reaction of oxidizing an Amadori compound in the presence of oxygen to produce α-ketoaldehyde, an amine derivative and hydrogen peroxide;
2) The stable pH is 4.0-13.0, the optimum pH is 8.5;
3) The stable temperature is 20-50 ° C and the optimum temperature is 30-35 ° C;
4) When measured by gel filtration, the molecular weight is approximately 106,000 (106 kDa). In fructosyl lysine and / or fructosyl N ^alpha -Z- lysine-containing medium, culturing Fusarium oxysporum & f.sp. linear (IFO NO.5880) (Fusarium oxysporum f.sp.lini ), fructosyl from the culture The method for producing fructosyl amino acid oxidase according to claim 1, wherein the amino acid oxidase is recovered. An analytical reagent or kit for an Amadori compound containing the fructosyl amino acid oxidase according to claim 1 .

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