JP4004081B2 - Fructosyl amino acid oxidase and method for producing the same - Google Patents

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＊１：検出されず
＊２：フルクトシルヒト血清アルブミン
表１から、本発明のＦＡＯＤはフルクトシル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。
ペニシリウム属のＦＡＯＤ産生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ペニシリウム属の菌から抽出したＦＡＯＤの基質特異性
【表２】

１）：検出されず
【００２０】
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と比較してフルクトシルバリンに対して高い活性を有しており、このことは該ＦＡＯＤが糖化ヘモグロビンの測定に有用であることを示唆するものである。
【００２１】
３．ｐＨおよび温度の条件
ｐＨ条件の測定
０.１Ｍ酢酸（Ａｃ）、リン酸カリウム（Ｋ−Ｐ）緩衝液、トリス-塩酸緩衝液およびグリシン（Ｇｌｙ）−ＮａＯＨ緩衝液（ｐＨ４.０〜１１.０）にＦＡＯＤを加え、２５℃、１０分間インキュベートした後、 通常の条件（２５℃、ｐＨ８.０）で活性を測定した。
上記方法で測定したとき、本発明のＦＡＯＤの安定なｐＨ域は、約ｐＨ４.０〜１１.０、好ましくはｐＨ６.０〜９.０であり、至適ｐＨは約７.５であった（図２参照）。
温度条件の測定
５０ｍＭ リン酸カリウム緩衝液（ｐＨ７.５）中で１５〜６０℃の温度条件にＦＡＯＤを加え、１０分インキュベートした後、通常の条件で活性を測定した。安定な温度領域は１５〜５０℃、好ましくは１５〜４５℃、より好ましくは１５℃であり、酵素反応は、１５〜４５℃、好ましくは１５〜４０℃、より好ましくは２５℃で効率良く進行する。（図３参照）
【００２２】
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 フルクトシルバリン（ＦＶ）溶液はあらかじめ得られたＦＶ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、および酵素溶液５０μｌを混合し、全量を蒸留水で２.９５ｍｌとする。２５℃で平衡化した後、１００ｍＭ ＦＶ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。
【００２３】
Ｂ.終末法
上記Ａ法と同様に処理し、基質添加後、３０分間２５℃でインキュベートした後の５０５ｎｍにおける吸光度を測定し、別に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。２５℃で攪拌し、溶存酸素と温度を平衡化した後、５０ｍＭ ＦＶ 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。
【００２４】
５．酵素の阻害、活性化および安定化
（１）金属の影響
０.１Ｍ トリス-塩酸緩衝液（ｐＨ８.０）の条件で、終濃度１ｍＭの各種金属イオンを添加し、５分間３０℃で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 金属イオンのペニシリウム・ヤンシネルムＳ−３４１３由来ＦＡＯＤ活性への影響
【表３】

表３から明らかに、本発明のＦＡＯＤの活性に対し、銅イオン、バリウムイオンが阻害的であり、コバルトイオン、亜鉛イオン、銀イオンおよび水銀イオンは強く阻害する。
【００２５】
（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（ＰＣＭＢ）は終濃度０.１ｍＭ、それ以外は１ｍＭとした。結果を表４に示す。安定化の検討は、５０mＭリン酸カリウム緩衝液(ｐＨ７.５)に０.１ｍＭのジチオスイトール（ＤＴＴ）を添加したものに対して一晩透析を行った後、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：ＤＮＴＢ，５，５'−ジチオビス（２−ニトロ安息香酸）
＊３：ＥＤＴＡ，エチレンジアミン四酢酸
表４から明らかに、ＦＡＯＤ活性はＰＣＭＢ、ＤＮＴＢ、ヒドラジン、フェニルヒドラジン、ヒドロキシルアミンにより、強く阻害され、酵素反応にはＳＨ基およ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０.１ｍＭを添加した５０mＭ リン酸カリウム緩衝液（pＨ７.５）である。
【００２６】
６．分子量
スーパーデックス２００ｐｇによるゲルろ過法で求めた結果、分子量は約３８,７００（３８.７ｋＤａ）であった（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）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。
また、ＳＤＳ−ＰＡＧＥにおいて、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、検量線を用いて分子量測定を行った結果、サブユニットの分子量は約４８,７００（４８.７ｋＤａ）であることが示された（図５）。従って、本発明のＦＡＯＤは単量体であることが明らかである。
【００２７】
７．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は単量体であるのに対し、他の２種の酵素は２量体であり、明らかに異なる。
（２）補酵素：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）至適ｐＨ、至適温度、およびＳＨ試薬による阻害等の差異はＦＡＯＤと他の２酵素との相違を示している。
【００２８】
さらに、特開平４−４８７４号公報記載のペニシリウム属由来フルクトシルアミノ酸分解酵素と、本発明のＦＡＯＤとを比較すると、下記の相違点が認められる。
（１）基質特異性：特開平４−４８７４号公報記載のフルクトシルアミノ酸分解酵素はフルクトシルリジンとフルクトシルアラニンに活性を有するのに対し、本発明のＦＡＯＤはフルクトシルリジンとフルクトシルバリンに活性を有し、活性はフルクトシルバリンに対する方が強い。
（２）誘導条件：特開平４−４８７４号公報記載のフルクトシルアミノ酸分解酵素はフルクトシルアミノ酸を含まない培地でも産生されるが、本発明のＦＡＯＤはフルクトシルリジンを含有する培地で培養することによって誘導される。
（３）製造方法：本発明のＦＡＯＤは褐変化培地を用いて培養し、製造する。
【００２９】
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、本発明はまた、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、酸素の消費量また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、あるいはフルクトシルア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、タンパク質また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、血漿または血清）、尿等の生体由来の試料の外、醤油等の食品が挙げられる。
【００３０】
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、ｐＨ６.０〜９.０、好ましくは７.５、温度は１５〜４５℃、好ましくは１５〜４０℃である。緩衝液としてはリン酸カリウム等を用いる。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。
【００３１】
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、過酸化水素およ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吸収される酸素量を求める。
【００３２】
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バリン及び／又はリジ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。
【００３３】
上記のごとく、本発明のＦＡＯＤは、フルクトシルバリンに高い特異性を有することから、糖化ヘモグロビンの測定に有用である。また、糖化タンパクに含まれるフルクトシルリジンにも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。
なお、検体として血液試料（全血、血漿または血清）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。
【００３４】
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）又はフルクトシルアミン量で表される。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、ビュレット法、Ｌowry法あるいは、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。
【００３５】
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、好ましくはｐＨ６.０〜９.０、より好ましくはｐＨ７.５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、緩衝液はリン酸カリウム（ｐＨ７.５）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の量及び／又は糖化率の測定、あるいはフルクトシルア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。
【００３６】
【実施例】
実施例１ ペニシリウム・ヤンシネルムＳ−３４１３由来のＦＡＯＤの製造および精製
ペニシリウム・ヤンシネルムＳ−３４１３（FERM BP-5475；Penicillium janthinellum S-3413）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５００ｒｐｍの条件で２８℃、３６時間攪拌培養した。培養物は瀘過して集めた。
菌糸体４１０ｇ（湿重量）を、０.１ｍＭのＤＴＴを含む、０.１Ｍリン酸カリウム緩衝液（ｐＨ７.５)８００mlに懸濁し、ダイノ・ミル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、得られた液を粗酵素液（無細胞抽出液）とし、以下の方法で精製した。
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、攪拌し、１２,０００ｒｐｍで１０分間遠心分離した。得られた上清に７５％飽和になるように硫安を加え、撹拌し、１２,０００ｒｐｍで１０分間遠心分離した。沈殿を０.１ｍＭのＤＴＴを含有する５０ｍＭ リン酸カリウム緩衝液（ｐＨ７.５)(以下、緩衝液Ａと略す）に溶解した。得られた酵素溶液を緩衝液Ａに対し一晩透析した。外液の交換は２回行った。透析後の酵素溶液は緩衝液Ａで平衡化したＤＥＡＥ−セファセルカラム（４.２×２６ｃｍ）にアプライした。活性画分は同緩衝液による洗浄画分に認められたので、これを集め、０−５５％飽和の硫安分画に供した。次に２５％飽和硫安を含む緩衝液Ａで平衡化したフェニル−セファロース６ＦＦ（Low Substitute）カラム（ＨＲ１０／１０）に吸着した。同緩衝液にて洗浄した後、硫安濃度２５−０％飽和の直線勾配で溶出した。活性画分を集め、硫安濃縮後、得られた酵素溶液を０.１mＭ ＤＴＴを含む０.２Ｍリン酸カリウム緩衝液（ｐＨ７.５）にて平衡化したスーパーデックス２００ｐｇカラムによりゲル瀘過を行い、７０〜１００ユニットの精製酵素を得た。
【００３７】
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。
得られた精製酵素標品は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）により分子量を決定した。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、クマシーブリリアントブルーＧ−２５０でタンパク染色を行った。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、検量線から分子量を求めた結果、サブユニットの分子量は約４８,７００（４８.７ｋＤａ）であることが示された（図５）。
また、スーパーデックス２００ｐｇによるゲルろ過による分子量測定では、図４の検量線図から明らかなように、約３８,７００（３８.７ｋＤａ）であった。
本実施例で調製したＦＡＯＤの酵素活性、ｐＨおよび温度安定性、金属および阻害物質による影響などに関しては、前記の値または性質を示した。
【００３８】
実施例２ 糖化ヘモグロビン量の測定
１) 試料の処理
０〜１５mgのグリコヘモグロビンコントロールＥ(シグマ社)を１００μlの蒸留水で溶解した。これらの試料に塩酸アセトン(１Ｎ塩酸／アセトン:１／１００)１mlを加え、１２０００回転で１０分間遠心分離した。沈澱物をジエチルエーテル５００μlで洗浄し、減圧乾固した。さらに８Ｍ尿素１００μlを加え、２０分間沸騰水中で加熱後冷却し、５．４ユニット／ml トリプシン３００μlと混合、３７℃で３時間インキュベートした。その後、沸騰水中で５分間加熱し、試料を調製した。
２) 活性測定
ＦＡＯＤ反応液は以下のようして調製した。
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
２５ユニット／ml ＦＡＯＤ溶液 ５μl
蒸留水で全量を１mlとした。
２５ユニット／ml ＦＡＯＤ溶液は、実施例１の方法で得たＦＡＯＤを２５ユニット／mlになるよう、０．１Ｍ リン酸カリウム緩衝液(pＨ７．５)で希釈して調製した。
このＦＡＯＤ反応液に上記の各処理基質を１５０μl加え、３０℃でインキュベートし、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量と吸光度との関係を図７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量を表す。図は、糖化ヘモグロビンの量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３ 糖化ヘモグロビン量の測定
１) 試料の処理
３０mgのグリコヘモグロビンコントロールＥ(シグマ社)を蒸留水２００μlで溶解し、８Ｍ尿素、０．２％ＥＤＴＡ・２ナトリウムを含む５７０mＭトリス−塩酸緩衝液(pＨ８．８)１mlと、２−メルカプトエタノール４０μlを添加し、窒素封入下で２時間静置した。その後、１Ｍのヨード酢酸ナトリウム４００μlを添加し、３０分間静置後、２−メルカプトエタノール４０μlを添加した。０．１Ｍ重炭酸アンモニウムに対して透析した後、１０mg／ml ＴＰＣＫ−トリプシン１０μlと混合し、３７℃で３時間インキュベートした。その後、沸騰水中で５分間加熱し試料を調製した。
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
２５ユニット／ml ＦＡＯＤ溶液 １０μl
処理試料 ０〜１３．２mg
蒸留水で全量を９００μlとした。
２５ユニット／ml ＦＡＯＤ溶液は、実施例１の方法で得たＦＡＯＤを２５ユニット／mlになるよう、０．１Ｍ リン酸カリウム緩衝液(pＨ７．５)で希釈して調製した。
このＦＡＯＤ反応液を３０℃でインキュベートし、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量と吸光度との関係を図８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量を表す。図は、糖化ヘモグロビンの量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】
実施例４ ヘモグロビンＡ１c値の測定
ヘモグロビンＡ０試薬(シグマ社)を蒸留水で２．３mＭになるように溶解した。この溶液を自動グリコヘモグロビン測定装置(京都第一科学)を用いて分画し、ヘモグロビンＡ１c画分とヘモグロビンＡ０画分を分取、精製した。両画分を比率混合することにより、ヘモグロビンＡ１c値０％〜５２．０％の基質試料を得た。
１) 試料の処理
基質試料 ２５０μg
５００ユニット／ml アミノペプチダーゼ溶液 ５μl
１．０Ｍ トリス−塩酸緩衝液(pＨ８．０) １５μl
これらを混合し、蒸留水で全量を２００μlとした。
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２００μl加えて撹拌し、０℃で２０分間静置した後１２０００回転で１０分間遠心分離を行った。得られた上清に５Ｎ ＮaＯＨを約４０μl加え、中性溶液にした。
２) 活性測定
ＦＡＯＤ反応液は以下のようして調製した。
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 １００μl
６０ユニット／ml パーオキシダーゼ溶液 １００μl
０．１Ｍ トリス−塩酸緩衝液(pＨ８．０) １０００μl
１６ユニット／ml ＦＡＯＤ溶液 １５μl
蒸留水で全量を２．６mlとした。
１６ユニット／ml ＦＡＯＤ溶液は、実施例１の方法で得たＦＡＯＤを１６ユニット／mlになるよう、０．１Ｍ リン酸カリウム緩衝液(pＨ７．５)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、上記の各処理基質を４００μl加え、さらに３０分インキュベートした後の７２７nmにおける吸光度を測定した。この方法で得られる基質のヘモグロビンＡ１c値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)、横軸はヘモグロビンＡ１c値を表す。図は、ヘモグロビンＡ１c値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。
【００４１】
【発明の効果】
本発明のＦＡＯＤは、フルクトシルバリンおよびフルクトシルリジンのいずれにも特異的に作用する。従って、新たな臨床分析およ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／又は糖化率又はフルクトシルア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およ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】 ＦＡＯＤの培養培地での産生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】 スーパーデックス２００pgを用いたゲルろ過による分子量測定の結果を示すグラフ。
【図５】 ペニシリウム・ヤンシネルムＳ−３４１３由来の精製ＦＡＯＤを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）にかけて得た移動パターンを示す写真。
【図６】 精製Ｓ−３４１３由来ＦＡＯＤの吸収スペクトル。
【図７】 糖化ヘモグロビン量と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】 糖化ヘモグロビン量と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】 ヘモグロビンＡ１c値と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]
[Industrial application fields]
The present invention relates to a novel fructosyl amino acid oxidase, and more particularly, to the genus Penicillium (Penicillium) -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 kit containing the enzyme.
[0002]
[Prior art]
When a substance having an amino group such as a protein, peptide or amino acid and a reducing sugar such as aldose coexist, an Amadori compound binds an amino group and an aldehyde group 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.
For example, 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 the reducing ability of the fructosylamine derivative in an alkaline solution 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. No. 7, JP-A-7-289253). 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). JP-A-7-289253).
[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(Japanese Patent Laid-Open No. 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, pp. 3790-3796 (1964).
[0006]
[Problems to be Solved by the Invention]
However, these enzyme methods have the following problems.
That is, the indicators 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)]. In glycated hemoglobin, glucose is also bound to the N-terminal valine of the hemoglobin β chain [J. Biol. Chem. 254: 3892-3898 (1979)]. Therefore, it has been 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 is an enzyme capable of degrading fructosyl valine and cannot accurately measure a glycated protein in which a sugar is bound to a lysine residue. Since fructosylamine degrigase has a high activity in difructosyl lysine, it cannot specifically measure the glycated product at the ε position of the lysine residue, and it can specifically measure the glycated product of the valine residue. It cannot be measured. 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 an Amadori compound, particularly a glycated protein, the present inventors have found that Penicillium (Penicillium) Fungus of fructosyllysine and / or fructosyl^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 Penicillium (Penicillium) Of the bacteria^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 fructosyl amino acid oxidase producing bacteria of the present invention^α-Z-lysine-containing medium contains glucose and lysine and / or N^α-Fructosyllysine 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 valine and fructosyl lysine, but the activity is characterized by higher activity on the former than on the latter. In the present specification, the fructosyl amino acid oxidase of the present invention is sometimes referred to as FAOD.
[0010]
The enzyme of the present invention can be used to treat bacteria of the genus Penicillium having fructosyl amino acid oxidase producing ability with fructosyl lysine and / or fructosyl N.^αIt can be produced by culturing in a medium containing -Z-lysine. As such fungus, Penicillium Jansinerum S-3413 (Penicillium janthinellum S-3413) (FERM BP-5475), Penicillium Yansinerum (IFO NO.4651,6581,7905) (Penicillium janthinellum), Penicillium oxalicum (IFO NO.5748) (Penicillium oxalicum), Penicillium yabanikum (IFO NO.7994) (Penicillium javanicum), Penicillium chrysogenum (IFO NO.4897) (Penicillium chrysogenum), Penicillium cyanium (IFO NO.5337) (Penicillium cyaneum) 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 form α-ketoaldehyde, an amine derivative and hydrogen peroxide;
2) The stable pH is 4.0 to 11.0 and the optimum pH is 7.5;
3) The stable temperature is 15-50 ° C and the optimum temperature is 25 ° C;
4) When measured by a gel filtration method using 200 pg Superdex, the molecular weight is about 38,700 (38.7 kDa).
[0012]
Fructosyl lysine and / or FZL used in the production of the FAOD of the present invention comprises glucose in an amount of 0.01 to 50% by weight, 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 the production of the 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 preferred medium, 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 mixtures). 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・ 2H₂A 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]
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 adjusted according to the state of each bacterium and are not limited to the above. For example, when Penicillium yansinerum S-3413 strain is cultured under these conditions for 20 to 48 hours, preferably 36 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 bacteria in the culture are disrupted and the enzyme is extracted.
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 mycelium is collected by centrifugation or suction filtration of the culture, washed, suspended in 50 mM potassium phosphate buffer (pH 7.5), and the mycelium is crushed by dynomill. Subsequently, the centrifuged supernatant is purified as a cell-free extract by treatment with an ammonium sulfate fraction and DEAE-Sephacel ion exchange 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 quantifying Amadori compounds, particularly glycated proteins for the diagnosis of diabetes.
Therefore, the present invention relates to a strain belonging to the genus Penicillium and capable of producing fructosyl amino acid oxidase as fructosyl lysine and / or fructosyl N.^αThe present invention provides a method for producing fructosyl amino acid oxidase, comprising culturing in a medium containing -Z-lysine and recovering fructosyl amino acid oxidase from the culture.
[0016]
Among the bacteria producing FAOD of the present invention, Penicillium yancinerum S-3413 (Penicillium janthinellum S-3413) (hereinafter referred to as S-3413 strain) is a strain newly isolated from soil by the present inventors. As a result of examining the mycological characteristics of this strain with reference to “Mycological Encyclopedia” by Koichi Udagawa et al. (Kodansha Scientific 1993), Penicillium Yansinerum (Penicillium janthinellum).
This strain has been deposited with the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology under the accession number FERM BP-5475.
(1) Do not form a child generation.
(2) Penicillus formation was observed.
(3) The phialide is quite a shape, suddenly narrows and becomes a long tip.
(4) Penicillary branches irregularly and is an open type.
(5) No sclerotia is formed.
(6) The conidia chain is extremely open.
further,
(7) Growth status in medium
Grow quickly on Czapek agar. When cultured in a thermostat at 25 ° C. for 10 days, it spreads in the form of wool and exhibits a pale yellow or grayish green color. In addition, deep wrinkles are formed radially.
[0017]
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 fructosyl lysine and / or fructosyl N.^αA fructosyl amino acid belonging to the genus Penicillium in a fructosyl lysine and / or FZL medium with fructosyl lysine and / or FZL as a nitrogen source and glucose as a carbon source, induced by Z-lysine (FZL) Produced by culturing an oxidase-producing strain.
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 autoclaving Z-lysine separately, the enzyme acts specifically on the Amadori compound.
[0018]
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.
[0019]
The activity of each FAOD substrate of the present invention is shown in Table 1 below.
Table 1  Substrate specificity of purified FAOD from Penicillium yancinerum S-3413
[Table 1]

* 1: Not detected
* 2: Fructosyl human serum albumin
From Table 1, the FAOD of the present invention is fructosyl N^αHas high specificity for -Z-lysine and fructosyl valine.
The strains of Penicillium having the ability to produce FAOD are exemplified in Table 2 below.
Table 2  Substrate specificity of FAOD extracted from Penicillium spp. Cultured in FZL browning medium
[Table 2]

1): Not detected
[0020]
As shown in Table 2, the FAOD of the present invention has higher activity against fructosyl valine compared to fructosyl lysine, which indicates that the FAOD is useful for measuring glycated hemoglobin. It is suggested that.
[0021]
3. pH and temperature conditions
Measurement of pH conditions
FAOD was added to 0.1 M acetic acid (Ac), potassium phosphate (K-P) buffer, Tris-hydrochloric acid buffer, and glycine (Gly) -NaOH buffer (pH 4.0 to 11.0). After incubating for 10 minutes, the activity was measured under normal conditions (25 ° C., pH 8.0).
When measured by the above method, the stable pH range of the FAOD of the present invention was about pH 4.0 to 11.0, preferably pH 6.0 to 9.0, and the optimum pH was about 7.5. (See FIG. 2).
Measurement of temperature conditions
FAOD was added to a temperature condition of 15 to 60 ° C. in 50 mM potassium phosphate buffer (pH 7.5), and the mixture was incubated for 10 minutes, and then the activity was measured under normal conditions. The stable temperature range is 15 to 50 ° C., preferably 15 to 45 ° C., more preferably 15 ° C., and the enzymatic reaction proceeds efficiently at 15 to 45 ° C., preferably 15 to 40 ° C., more preferably 25 ° C. To do. (See Figure 3)
[0022]
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
A 100 mM fructosyl valine (FV) solution was prepared by dissolving FV 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 entire volume was made up with distilled water. 2.95 ml. After equilibration at 25 ° C., 50 μl of 100 mM FV 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 defined as the enzyme activity unit (unit: U).
[0023]
B. Terminal law
The amount of hydrogen peroxide generated from a calibration curve prepared in advance using a standard hydrogen peroxide solution after measuring the absorbance at 505 nm after treatment at 25 ° C. for 30 minutes after adding the substrate. The enzyme activity is measured by calculating
(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 a total volume of 3.0 ml with distilled water, and put it into a cell of Rank Brothers oxygen electrode. After stirring at 25 ° C. to equilibrate the dissolved oxygen and temperature, 100 μl of 50 mM FV 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.
[0024]
5. Enzyme inhibition, activation and stabilization
(1) Influence of metal
Various metal ions with a final concentration of 1 mM were added under conditions of 0.1 M Tris-HCl buffer (pH 8.0), and incubated at 30 ° C. for 5 minutes, and then the activity was measured. The results are shown in Table 3 below.
Table 3  Effect of metal ions on FAOD activity derived from Penicillium yancinerum S-3413
[Table 3]

As apparent from Table 3, copper ions and barium ions are inhibitory to the activity of the FAOD of the present invention, and cobalt ions, zinc ions, silver ions and mercury ions are strongly inhibited.
[0025]
(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 (PCMB) was set to a final concentration of 0.1 mM, and other concentrations were set to 1 mM. The results are shown in Table 4. Stabilization was performed by dialysis overnight against 50 mM potassium phosphate buffer (pH 7.5) supplemented with 0.1 mM dithiositol (DTT) and then measuring the activity. It was.
Table 4  Effects of various substances on FAOD activity
[Table 4]

* 1: PCMB, mercuric parachlorobenzoate
* 2: DNTB, 5,5′-dithiobis (2-nitrobenzoic acid)
* 3: EDTA, ethylenediaminetetraacetic acid
As apparent from Table 4, the FAOD activity is strongly inhibited by PCMB, DNTB, hydrazine, phenylhydrazine, and hydroxylamine, and it is expected that the SH group and the carbonyl group play an important role in the enzymatic reaction.
On the other hand, a solvent stabilized by dithiothreitol and suitable for storage is 50 mM potassium phosphate buffer (pH 7.5) supplemented with 0.1 mM dithiothreitol.
[0026]
6). Molecular weight
As a result of gel filtration using Superdex 200 pg, the molecular weight was about 38,700 (38.7 kDa) (FIG. 4).
SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) 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.
In SDS-PAGE, phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase, and soybean trypsin inhibitor were migrated in the same manner as standard proteins, and the molecular weight was measured using a calibration curve. Was shown to be about 48,700 (48.7 kDa) (FIG. 5). Therefore, it is clear that the FAOD of the present invention is a monomer.
[0027]
7. 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: While the FAOD of the present invention is a monomer, the other two kinds of enzymes are dimers and are clearly different.
(2) Coenzyme: FAOD has FAD covalently bound as a coenzyme, while all other enzymes have non-covalently bound FAD.
(3) Differences such as optimum pH, optimum temperature, and inhibition by SH reagent indicate differences between FAOD and the other two enzymes.
[0028]
Further, when the Penicillium genus fructosyl amino acid degrading enzyme described in JP-A-4-4874 is compared with the FAOD of the present invention, the following differences are observed.
(1) Substrate specificity: The fructosyl amino acid-degrading enzyme described in JP-A-4-4874 has activity on fructosyl lysine and fructosyl alanine, whereas the FAOD of the present invention is fructosyl lysine and fructosyl valine. The activity is stronger against fructosyl valine.
(2) Induction conditions: The fructosyl amino acid degrading enzyme described in JP-A-4-4874 is produced even in a medium not containing fructosyl amino acid, but the FAOD of the present invention is cultured in a medium containing fructosyl lysine. Induced by.
(3) Production method: The FAOD of the present invention is produced by culturing using a browning medium.
[0029]
As described above, the enzyme FAOD of the present invention is useful for quantifying Amadori compounds. Accordingly, the present invention also provides a sample of an Amadori compound in a sample, characterized in that a sample containing the Amadori compound is brought into contact with the FAOD of the present invention to measure oxygen consumption or hydrogen peroxide generation. Analytical methods are provided. 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 fructosylamine.
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. .
[0030]
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 6.0 to 9.0, preferably 7.5, and the temperature is 15 to 45 ° C, preferably 15 to 40 ° C. As the buffer, potassium phosphate or the like is used. 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.
[0031]
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, color development methods, methods using hydrogen peroxide electrodes, etc., and compared with standard curves prepared for the amount 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 the hydrogen peroxide coloring system, a system that develops color by oxidative condensation between a coupler such as 4-aminoantipyrine and 3-methyl-2-benzothiazolinone hydrazone and a chromogen such as phenol in the presence of peroxidase is used. be able to. 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 absorbed.
[0032]
The method of the present invention can be carried out using the sample solution as it is, but depending on the glycated protein of interest, it is preferable to carry out after releasing the valine and / or lysine residue to which the 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.
[0033]
As described above, since the FAOD of the present invention has high specificity for fructosyl valine, it is useful for measurement of glycated hemoglobin. Moreover, since fructosyl lysine contained in glycated protein also has high substrate specificity, it is useful for diagnosis of diabetes including measurement of glycated protein in a blood sample.
When a blood sample (whole blood, plasma or serum) is used as a specimen, the collected sample is used as it is or after being subjected to a treatment such as through 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.
[0034]
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 intended to diagnose diabetes, the standard for diagnosis is expressed as the glycated protein concentration, or the ratio of the glycated protein concentration to the total protein concentration in the sample (glycation rate) or the amount of fructosylamine. Is done. The total protein concentration can be measured by a usual method (absorbance at 280 nm, Bradford method, Burette method, Lowry method, natural fluorescence of albumin, absorbance of hemoglobin, etc.).
[0035]
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, preferably a buffer solution having a pH of 6.0 to 9.0, more preferably pH 7.5. When the FAOD is immobilized, the solid support is selected from a polymer gel or the like, and is preferably alginic acid.
When performing end point analysis, the amount of FAOD in the reagent is usually 1 to 100 units / ml per sample, and the buffer is preferably potassium phosphate (pH 7.5).
When the Amadori compound is quantified 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 hydrogen peroxide generation amount”, and a leuco-type color developing. 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 and / or saccharification rate of glycated protein in biological components, or for quantifying fructosylamine.
Hereinafter, the present invention will be described in more detail with reference to examples.
[0036]
【Example】
Example 1  Production and purification of FAOD from Penicillium yancinerum S-3413
Penicillium yansinerum S-3413 (FERM BP-5475;Penicillium janthinellum S-3413) FZL 0.5%, glucose 1.0%, dipotassium phosphate 0.1%, monosodium phosphate 0.1%, magnesium sulfate 0.05%, calcium chloride 0.01%, yeast The cells were inoculated into 10 L of a medium (pH 6.0) containing 0.2% of the extract, and cultured with stirring at 28 ° C. for 36 hours using a jar fermenter under the conditions of an aeration rate of 2 L / min and a stirring speed of 500 rpm. The culture was collected by filtration.
410 g (wet weight) of mycelium was suspended in 800 ml of 0.1 M potassium phosphate buffer (pH 7.5) containing 0.1 mM DTT, and the mycelium was crushed by a Dino mill. The disrupted solution was centrifuged at 9,500 rpm for 20 minutes, and the resulting solution was used as a crude enzyme solution (cell-free extract) and purified by the following method.
Ammonium sulfate (hereinafter abbreviated as ammonium sulfate) was added to the crude enzyme solution so as to be 40% saturated, stirred, and centrifuged at 12,000 rpm for 10 minutes. Ammonium sulfate was added to the obtained supernatant to 75% saturation, stirred, and centrifuged at 12,000 rpm for 10 minutes. The precipitate was dissolved in 50 mM potassium phosphate buffer (pH 7.5) (hereinafter abbreviated as buffer A) containing 0.1 mM DTT. The resulting enzyme solution was dialyzed overnight against buffer A. The external liquid was exchanged twice. The enzyme solution after dialysis was applied to a DEAE-Sephacel column (4.2 × 26 cm) equilibrated with buffer A. The active fraction was found in the washing fraction with the same buffer, and was collected and subjected to 0-55% saturated ammonium sulfate fraction. Next, it was adsorbed on a phenyl-Sepharose 6FF (Low Substitute) column (HR 10/10) equilibrated with buffer A containing 25% saturated ammonium sulfate. After washing with the same buffer, elution was performed with a linear gradient of ammonium sulfate concentration 25-0% saturation. The active fractions were collected and concentrated with ammonium sulfate, and the resulting enzyme solution was subjected to gel filtration with a Superdex 200 pg column equilibrated with 0.2 M potassium phosphate buffer (pH 7.5) containing 0.1 mM DTT. 70-100 units of purified enzyme were obtained.
[0037]
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 was determined by SDS-PAGE (sodium dodecyl sulfate / polyacrylamide gel electrophoresis). SDS-PAGE was run 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, soybean trypsin inhibitor were migrated in the same manner, and the molecular weight was determined from the calibration curve. As a result, the subunit molecular weight was about 48,700 (48.7 kDa). (FIG. 5).
Further, in the molecular weight measurement by gel filtration using Superdex 200 pg, it was about 38,700 (38.7 kDa) as is apparent from the calibration curve of FIG.
Regarding the enzyme activity, pH and temperature stability, effects of metals and inhibitors, etc. of FAOD prepared in this example, the above values or properties are shown.
[0038]
Example 2  Measurement of glycated hemoglobin
1) Sample processing
0 to 15 mg of glycohemoglobin control E (Sigma) was dissolved in 100 μl of distilled water. To these samples, 1 ml of acetone hydrochloride (1N hydrochloric acid / acetone: 1/100) was added and centrifuged at 12,000 rpm for 10 minutes. The precipitate was washed with 500 μl of diethyl ether and evaporated to dryness. Further, 100 μl of 8M urea was added, heated in boiling water for 20 minutes, cooled, mixed with 300 μl of 5.4 units / ml trypsin, and incubated at 37 ° C. for 3 hours. Then, it heated for 5 minutes in boiling water and prepared the sample.
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
25 units / ml FAOD solution 5 μl
The total volume was made up to 1 ml with distilled water.
The 25 unit / ml FAOD solution was prepared by diluting the FAOD obtained by the method of Example 1 with 0.1 M potassium phosphate buffer (pH 7.5) to 25 units / ml.
150 μl of each of the above treated substrates was added to this FAOD reaction solution, incubated at 30 ° C., and the absorbance at 727 nm was measured after 30 minutes. FIG. 7 shows the relationship between the amount of glycated hemoglobin obtained by this method and the absorbance. In the figure, the vertical axis represents the absorbance at 727 nm (corresponding to the amount of hydrogen peroxide), and the horizontal axis represents the amount of glycated hemoglobin. The figure shows that there is a correlation between the amount of glycated hemoglobin and the amount of hydrogen peroxide generated.
[0039]
Example 3  Measurement of glycated hemoglobin
1) Sample processing
30 mg of glycohemoglobin control E (Sigma) was dissolved in 200 μl of distilled water, 1 ml of 570 mM Tris-HCl buffer (pH 8.8) containing 8 M urea and 0.2% EDTA · sodium, and 40 μl of 2-mercaptoethanol. Was added and allowed to stand under nitrogen sealing for 2 hours. Thereafter, 400 μl of 1M sodium iodoacetate was added, and after standing for 30 minutes, 40 μl of 2-mercaptoethanol was added. After dialyzing against 0.1 M ammonium bicarbonate, it was mixed with 10 μl of 10 mg / ml TPCK-trypsin and incubated at 37 ° C. for 3 hours. Then, it heated for 5 minutes in boiling water and prepared the sample.
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
25 units / ml FAOD solution 10 μl
Treated sample 0 to 13.2 mg
The total volume was made up to 900 μl with distilled water.
The 25 unit / ml FAOD solution was prepared by diluting the FAOD obtained by the method of Example 1 with 0.1 M potassium phosphate buffer (pH 7.5) to 25 units / ml.
This FAOD reaction solution was incubated at 30 ° C., and the absorbance at 727 nm after 30 minutes was measured. FIG. 8 shows the relationship between the amount of glycated hemoglobin obtained by this method and the absorbance. In the figure, the vertical axis represents the absorbance at 727 nm (corresponding to the amount of hydrogen peroxide), and the horizontal axis represents the amount of glycated hemoglobin. The figure shows that there is a correlation between the amount of glycated hemoglobin and the amount of hydrogen peroxide generated.
[0040]
Example 4  Measurement of hemoglobin A1c value
Hemoglobin A0 reagent (Sigma) was dissolved with distilled water to 2.3 mM. This solution was fractionated using an automatic glycohemoglobin measuring apparatus (Kyoto Daiichi Kagaku), and the hemoglobin A1c fraction and the hemoglobin A0 fraction were fractionated and purified. By mixing both fractions in proportion, a substrate sample having a hemoglobin A1c value of 0% to 52.0% was obtained.
1) Sample processing
Substrate sample 250μg
500 units / ml aminopeptidase solution 5 μl
1.0 M Tris-HCl buffer (pH 8.0) 15 μl
These were mixed and made up to 200 μl with distilled water.
This mixture was incubated at 30 ° C. for 30 minutes. Thereafter, 200 μl of 10% trichloroacetic acid was added and stirred, allowed to stand at 0 ° C. for 20 minutes, and then centrifuged at 12,000 rpm for 10 minutes. About 40 μl of 5N 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 100 μl
60 units / ml peroxidase solution 100 μl
0.1 M Tris-HCl buffer (pH 8.0) 1000 μl
16 units / ml FAOD solution 15 μl
The total volume was adjusted to 2.6 ml with distilled water.
The 16 unit / ml FAOD solution was prepared by diluting the FAOD obtained by the method of Example 1 with 0.1 M potassium phosphate buffer (pH 7.5) so as to be 16 units / ml.
After incubating the FAOD reaction solution at 30 ° C. for 2 minutes, 400 μl of each of the above treated substrates was added, and the absorbance at 727 nm was measured after further incubation for 30 minutes. FIG. 9 shows the relationship between the hemoglobin A1c value of the substrate obtained by this method and the absorbance. In the figure, the vertical axis represents the absorbance at 727 nm (corresponding to the amount of hydrogen peroxide), and the horizontal axis represents the hemoglobin A1c value. The figure shows that there is a correlation between the hemoglobin A1c value and the amount of hydrogen peroxide generated.
[0041]
【The invention's effect】
The FAOD of the present invention specifically acts on both fructosyl valine and fructosyl lysine. 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 to be useful for diagnosis of the pathology of diabetes using the amount of glycated protein and / or glycation rate or fructosylamine amount in blood as an index. In addition, the glycated protein can be accurately quantified by the analysis reagent and analysis method for the Amadori compound using the FAOD of the present invention, which can contribute to the diagnosis and symptom management of diabetes.
[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 graph showing the result of molecular weight measurement by gel filtration using Superdex 200 pg.
FIG. 5 is a photograph showing a migration pattern obtained by subjecting purified FAOD derived from Penicillium yancinerum S-3413 to SDS-PAGE (sodium dodecyl sulfate / polyacrylamide gel electrophoresis).
FIG. 6 shows an absorption spectrum of purified S-3413-derived FAOD.
FIG. 7 is a graph showing the relationship between the amount of glycated hemoglobin and the amount of hydrogen peroxide generated by the FAOD action.
FIG. 8 is a graph showing the relationship between the amount of glycated hemoglobin and the amount of hydrogen peroxide generated by the FAOD action.
FIG. 9 is a graph showing the relationship between the hemoglobin A1c value and the amount of hydrogen peroxide generated by the FAOD action.

Claims (9)

A fructosyl amino acid oxidase produced by culturing Penicillium janthinellum S-3413 (FERM BP-5475) in a fructosyl lysine-containing medium, having the following physicochemical properties: Tosyl amino acid oxidase:
1) catalyzing the reaction of oxidizing an Amadori compound in the presence of oxygen to form α-ketoaldehyde, an amine derivative and hydrogen peroxide;
2) The stable pH is 4.0 to 11.0 and the optimum pH is 7.5;
3) The stable temperature is 15-50 ° C and the optimum temperature is 25 ° C;
4) When measured by gel filtration using 200 pg Superdex, the molecular weight is about 38,700 (38.7 kDa),
5) Has activity against fructosyl lysine and fructosyl valine.   The fructosyl amino acid oxidase according to claim 1, wherein the fructosyl amino acid oxidase has higher activity against fructosyl valine compared to fructosyl lysine.   Penicillium janthinellum S-3413 (FERM BP-5475).   By culturing Penicillium janthinellum S-3413 (FERM BP-5475) 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. The method for producing fructosyl amino acid oxidase according to claim 1 or 2, wherein fructosyl amino acid oxidase is produced. Culturing Penicillium janthinellum S-3413 (FERM BP-5475) in a fructosyl lysine and / or fructosyl N ^α -Z-lysine-containing medium and recovering fructosyl amino acid oxidase from the culture 5. The method of claim 4, wherein:   A sample containing an Amadori compound is brought into contact with the fructosyl amino acid oxidase according to claim 1 or 2, and the amount of oxygen consumed or the amount of hydrogen peroxide generated is measured. Analytical method.   The method according to claim 6, wherein the sample is a biological component, and the analysis of the Amadori compound is performed by measuring the amount and / or glycation rate of glycated protein in the biological component, or quantifying fructosylamine. .   An analytical reagent or kit for an Amadori compound comprising the fructosyl amino acid oxidase according to claim 1 or 2.   9. The analytical reagent or kit according to claim 8, which is used for measuring the amount and / or saccharification rate of a glycated protein in a biological component, or for quantifying fructosylamine.

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