JP2004242526A - Stabilized sarcosine oxidase - Google Patents
Stabilized sarcosine oxidase Download PDFInfo
- Publication number
- JP2004242526A JP2004242526A JP2003033641A JP2003033641A JP2004242526A JP 2004242526 A JP2004242526 A JP 2004242526A JP 2003033641 A JP2003033641 A JP 2003033641A JP 2003033641 A JP2003033641 A JP 2003033641A JP 2004242526 A JP2004242526 A JP 2004242526A
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- Prior art keywords
- sarcosine oxidase
- amino acid
- seq
- acid sequence
- modified
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
Description
【０００１】
【発明の属する技術分野】
本発明は、ザルコシンオキシダーゼ活性を有する蛋白質を蛋白工学的手法により改変することにより得られる、液状での安定性が向上したザルコシンオキシダーゼ、その製造法および用途に関する。
【０００２】
【従来の技術】
ザルコシンオキシダーゼ（ＥＣ １．５．３．１）は、臨床的に筋疾患，腎疾患の診断の指標となっている体液中のクレアチン，クレアチニンの測定用酵素として、他の酵素、例えばクレアチニナーゼ、クレアチナーゼ、ペルオキシダーゼと共に使用されている。ザルコシンオキシダーゼは基質であるザルコシンに水、酸素の存在下で作用して、グリシン、ホルムアルデヒドおよび過酸化水素を生成する。
【０００３】
このようなザルコシンオキシダーゼは、バチルス属、コリネバクテリウム属、シリンドロカルポン属、シュードモナス属、アースロバクター属等の細菌が生産することが知られている（例えば、特許文献１〜５および非特許文献１参照。）。また、これら生産菌のザルコシンオキシダーゼ遺伝子を、遺伝子工学的手法により、大腸菌等の宿主を用いた大量生産する技術についても報告されている（例えば、特許文献６〜８参照。）。
【０００４】
近年の臨床診断試薬の液状化に伴い、試薬成分の液状での安定化法が種々検討されているが、クレアチニンやクレアチン測定試薬に用いられるザルコシンオキシダーゼについても液状での安定性に優れたものが望まれている。我々のグループは、以前に野生型ザルコシンオキシダーゼを蛋白質工学的に改変し、金属イオンに対して安定性の向上した変異型ザルコシンオキシダーゼを報告した（例えば、特許文献９参照。）が、診断薬試薬中での長期保存安定性については更なる改良が期待されている。
本発明は、液状での安定性が向上した改変型ザルコシンオキシダーゼを提供することを目的とする。
【０００５】
【特許文献１】
特開昭５４−５２７８９号公報
【特許文献２】
特開昭６１−１６２１７４号公報
【特許文献３】
特開昭５６−９２７９０号公報
【特許文献４】
特開昭６０−４３３７９号公報
【特許文献５】
特開平２−２６５４７８号公報
【特許文献６】
特開平５−１１５２８１号公報
【特許文献７】
特開平６−１１３８４０号公報
【特許文献８】
特開平８−２３８０８７号公報
【特許文献９】
特開平７−１６３３４１号公報
【非特許文献１】
「Ｊ． Ｂｉｏｃｈｅｍ．」，１９８１年， ８９巻， ｐ．５９９
【０００６】
【課題を解決するための手段】
本発明者らは、上記目的を達成するために鋭意検討した結果、ザルコシンに対する作用性を損なわずに、液状での安定性が向上した改変型ザルコシンオキシダーゼを造成できることを見出し、本発明を完成させるに至った。
【０００７】
すなわち、本発明は以下のような構成から成る。
項１． ザルコシンオキシダーゼ活性を有する蛋白質を構成するアミノ酸配列の少なくとも１個のアミノ酸を付加、欠失、挿入あるいは置換により変換した蛋白質であって、ザルコシンオキシダーゼ活性を有し、液状での安定性が変換前に比べて向上していることを特徴とする改変型ザルコシンオキシダーゼ。
項２． ザルコシンオキシダーゼ活性を有する蛋白質を構成するアミノ酸配列の少なくとも１個のアミノ酸が他のアミノ酸に置換していることを特徴とする項１記載の改変型ザルコシンオキシダーゼ。
項３． ザルコシンオキシダーゼ活性を有する蛋白質が、配列表の配列番号１に記載されるアミノ酸配列と５０％以上の相同性を有する、項１〜２記載の改変型ザルコシンオキシダーゼ。
項４． ザルコシンオキシダーゼ活性を有する蛋白質が、配列表の配列番号１に記載されるアミノ酸配列と８０％以上の相同性を有する、 項１〜２記載の改変型ザルコシンオキシダーゼ。
項５． ザルコシンオキシダーゼ活性を有する蛋白質が、配列表の配列番号１に記載されるアミノ酸配列を有する、 項１〜２記載の改変型ザルコシンオキシダーゼ。
項６． 配列表の配列番号１に記載されるアミノ酸配列の１５５位〜２５０位間に対応する部位の少なくとも１つのアミノ酸が他のアミノ酸に置換されていることを特徴とする 項３〜５記載の改変型ザルコシンオキシダーゼ。
項７． 配列表の配列番号１に記載されるアミノ酸配列の８２位〜９２位、または、３５４位〜３６６位間に対応する部位の少なくとも１つのアミノ酸が他のアミノ酸に置換されていることを特徴とする 項３〜５記載の改変型ザルコシンオキシダーゼ。
項８． 配列表の配列番号１に記載されるアミノ酸配列の８９位、１５５位、１６６位、２０４位、２１３位、２３３位、２４０位、２５０位、３６４位に対応する部位からなる群より選ばれる少なくとも１つのアミノ酸が他のアミノ酸に置換されてされていることを特徴とする 項３〜５記載の改変型ザルコシンオキシダーゼ。
項９． 配列表の配列番号１に記載されるアミノ酸配列の８９位のリジンがアルギニンに置換されてされていることを特徴とする 項３〜５記載の改変型ザルコシンオキシダーゼ。
項１０． 配列表の配列番号１に記載されるアミノ酸配列の１５５位のシステインがイソロイシンに置換されてされていることを特徴とする 項３〜５記載の改変型ザルコシンオキシダーゼ。
項１１． 配列表の配列番号１に記載されるアミノ酸配列の１６６位のアスパラギンがリジンに置換されてされていることを特徴とする 項３〜５記載の改変型ザルコシンオキシダーゼ。
項１２． 配列表の配列番号１に記載されるアミノ酸配列の２０４位のメチオニンがアラニンに置換されてされていることを特徴とする 項３〜５記載の改変型ザルコシンオキシダーゼ。
項１３． 配列表の配列番号１に記載されるアミノ酸配列の２１３位のセリンがプロリンに置換されてされていることを特徴とする 項３〜５記載の改変型ザルコシンオキシダーゼ。
項１４． 配列表の配列番号１に記載されるアミノ酸配列の２３３位のシステインがセリンに置換されてされていることを特徴とする 項３〜５記載の改変型ザルコシンオキシダーゼ。
項１５． 配列表の配列番号１に記載されるアミノ酸配列の２４０位のアスパラギンがチロシンに置換されてされていることを特徴とする 項３〜５記載の改変型ザルコシンオキシダーゼ。
項１６． 配列表の配列番号１に記載されるアミノ酸配列の２５０位のグルタミン酸がグルタミンに置換されてされていることを特徴とする 項３〜５記載の改変型ザルコシンオキシダーゼ。
項１７． 配列表の配列番号１に記載されるアミノ酸配列の３６４位のアラニンがバリンに置換されてされていることを特徴とする 項３〜５記載の改変型ザルコシンオキシダーゼ。
項１８． 項１〜１７のいずれか１項に記載される改変型ザルコシンオキシダーゼをコードする遺伝子。
項１９． 項１８に記載の遺伝子を含むベクター。
項２０． 項１９に記載のベクターで形質転換された形質転換体。
項２１． 項２０に記載の形質転換体を培養し、該培養物からザルコシンオキシダーゼを採取することを特徴とする改変型ザルコシンオキシダーゼの製造法。
項２２． 項１〜１７項のいずれか１項に記載されるザルコシンオキシダーゼを含むクレアチン測定用試薬。
項２３． 項１〜１７項のいずれか１項に記載されるザルコシンオキシダーゼを含むクレアチニン測定用試薬。
【０００８】
【発明の実施の形態】
本発明の改変型ザルコシンオキシダーゼは、臨床検査分野におけるクレアチン、クレアチニンの分析に有用である。
【０００９】
本願発明の一実施態様は、ザルコシンオキシダーゼ活性を有する蛋白質を構成するアミノ酸配列の少なくとも１個のアミノ酸を付加、欠失挿入あるいは置換により変換した蛋白質であって、ザルコシンオキシダーゼ活性を有し、液状での安定性が変換前に比べて向上していることを特徴とする改変型ザルコシンオキシダーゼである。
【００１０】
本発明の改変型ザルコシンオキシダーゼは、液状での安定性が改変前に比べて向上していることを特徴とする。本発明における液状での安定性とは、例えば適当な緩衝液中に該改変型酵素を溶解して、適当な温度で一定期間保存した後の残存酵素活性の比率を意味する。
「適当な緩衝液」は、ザルコシンオキシダーゼの至適ｐＨであるｐＨ７〜８付近で十分な緩衝能を持つよう、その種類と濃度を選べば特に限定されないが、好ましくは５０ｍＭのリン酸カリウム緩衝液（ｐＨ７．５）、または、５０ｍＭのＰＩＰＥＳ−ＮａＯＨ緩衝液（ｐＨ７．５）が選択される。緩衝液には、さらに必要により界面活性剤、塩類、キレート剤、防腐剤などを含んでいてもよい。
「適当な温度で一定期間保存」の条件は特に限定されないが、好ましくは、液状診断薬試薬中での長期保存安定性を念頭に置いた加速（苛酷）試験の条件が選択される。具体的には、「４０℃、３日間保存」、または、「６０℃、３０分間保存」などが挙げられる。時間が許せば、液状診断薬中が実際に長期保存される温度として汎用される２℃〜１０℃の冷蔵条件下で６ヶ月以上の保存を選択してもよい。
保存における、ザルコシンオキシダーゼの濃度は、特に限定されないが、通常の診断試薬に使用される濃度を想定した１〜３０Ｕ／ｍｌが好ましく選択される。さらに好ましくは５〜２０Ｕ／ｍｌである。
「安定性が改変前に比べて向上している」とは、一定期間保存後の活性保持率が、同条件で測定した改変前酵素の活性保持率と比べて高いことをいう。
【００１１】
本発明の一実施態様としては、５０ｍＭのリン酸カリウム緩衝液（ｐＨ７．５）中で６０℃、３０分間保存した後の残存酵素活性率が、改変前に比べて向上した改変型ザルコシンオキシダーゼである。別な実施態様としては、２ｍＭのＥＤＴＡ、５０ｍＭのＮａＣｌ、０．１％（Ｗ／Ｖ）の２−メチルイソチアゾロン、０．１％（Ｗ／Ｖ）のトリトンＸ−１００を含む５０ｍＭのＰＩＰＥＳ−ＮａＯＨ緩衝液（ｐＨ７．５）中で４０℃、３日間保存した後の残存酵素活性率が、改変前に比べて向上した改変型ザルコシンオキシダーゼである。
【００１２】
本発明の改変に使用するザルコシンオキシダーゼは特に限定されるものではなく、例えば公知のバチルス属やシュードモナス属、コリネバクテリウム属等に由来するザルコシンオキシダーゼを用いることができる。
【００１３】
本発明では一例として、アースロバクター・エスピーＴＥ１８２６（微工研菌寄第１０６３７号）のザルコシンオキシダーゼを用いた（例えば、特許文献１０参照。）。本発明者らのグループは、既に、アースロバクター・エスピーＴＥ１８２６より抽出した染色体ＤＮＡよりザルコシンオキシダーゼ遺伝子の単離に成功し、そのＤＮＡの全構造を決定し（例えば、非特許文献２参照。）、本ザルコシンオキシダーゼを遺伝子工学的手法によって形質転換体に高生産させることに成功し、高純度なザルコシンオキシダーゼを安価に大量供給することを可能にしている（例えば、特許文献１１参照。）。アースロバクター・エスピーＴＥ１８２６のザルコシンオキシダーゼのアミノ酸配列を、配列表の配列番号１に示す。また、これらのアミノ配列をコードするＤＮＡ配列を、配列表の配列番号２に示す。
但し、本発明は配列番号１に記載されるアミノ酸配列を有するザルコシンオキシダーゼを改変したものに限定されるものでなく、他のザルコシンオキシダーゼ活性を有する蛋白質を改変したものであってもよい。他のザルコシンオキシダーゼ活性を有する蛋白質の好適な例として、配列番号１に記載されるアミノ酸配列を有するザルコシンオキシダーゼと立体構造が類似したザルコシンオキシダーゼ、具体的にはアミノ酸配列の相同性が５０％以上である、更に好ましくはアミノ酸配列の相同性が８０％以上である、他のザルコシンオキシダーゼ活性を有する蛋白質が挙げられる。これはアミノ酸配列において５０％乃至は８０％以上の相同性を有し、同じ触媒活性を示す酵素蛋白質の場合、立体構造においても通常、類似性が高く、基質特異性に関与するアミノ酸残基や反応メカニズムが同じである場合が多いことを根拠とする。
また、本発明の改変型ザルコシンオキシダーゼは、本願発明の酵素特性の本質である安定性が損なわれない範囲で、更に１もしくは数個のアミノ酸が欠失、置換もしくは付加されたものであっても良い。具体例には、ザルコシンオキシダーゼの精製を簡素化するためにアミノ酸配列のＮ末端側、又はＣ末端側にヒスチジンタグを付加したものが例示される（例えば、非特許文献３参照）。
なお、本発明におけるアミノ酸配列の相同性は、公知の遺伝子解析ソフトなどを用いて求めることができる。ここで相同性とは比較対象とするアミノ酸配列と類似性を有する範囲において、一致するアミノ酸残基のパーセンテージをいう。
【００１４】
【特許文献１０】
特開平２−２６５４７８号公報
【特許文献１１】
特開平６−１１３８４０号公報
【非特許文献２】
「Ｊｏｕｒｎａｌ ｏｆ Ｆｅｒｍｅｎｔａｔｉｏｎ ａｎｄ Ｂｉｏｅｎｇｉｎｅｅｒｉｎｇ」，１９９３年， ７５巻４号 ，ｐ．２３９−２４４
【非特許文献３】
「実験医学」，２００２年，２０巻，ｐ４７９−４８２
【００１５】
本願発明の別の実施態様は、配列表の配列番号１に記載されるアミノ酸配列の少なくとも１個のアミノ酸が他のアミノ酸に置換していることを特徴とする、液状での安定性が変換前に比べて向上している改変型ザルコシンオキシダーゼである。
【００１６】
本願発明の別の実施態様は、配列表の配列番号１に記載されるアースロバクター・エスピーＴＥ１８２６のアミノ酸配列の１５５位〜２５０位、若しくは、アースロバクター・エスピーＴＥ１８２６以外のザルコシンオキシダーゼにおいて配列表の配列番号１に記載されるアミノ酸配列の１５５位〜２５０位に対応する部位の少なくとも１つのアミノ酸が他のアミノ酸に置換されていることを特徴とする、液状での安定性が変換前に比べて向上している改変型ザルコシンオキシダーゼである。
【００１７】
本願発明の別の実施態様は、配列表の配列番号１に記載されるアースロバクター・エスピーＴＥ１８２６のアミノ酸配列の８２位〜９２位、もしくは、アースロバクター・エスピーＴＥ１８２６以外のザルコシンオキシダーゼにおいて配列表の配列番号１に記載されるアミノ酸配列の８２位〜９２位に対応する部位の少なくとも１つのアミノ酸が他のアミノ酸に置換されていることを特徴とする、液状での安定性が変換前に比べて向上している改変型ザルコシンオキシダーゼである。
Ｘ線結晶解析により立体構造が明らかにされているザルコシンオキシダーゼの報告がある（例えば、非特許文献４参照）。その報告によれば、該ザルコシンオキシダーゼは配列表の配列番号１に記載されるアミノ酸配列と相同性を有し、アースロバクター・エスピーＴＥ１８２６のザルコシンオキシダーゼのアミノ酸配列である配列表の配列番号１に記載されるアミノ酸配列の８２位〜９２位、若しくは、アースロバクター・エスピーＴＥ１８２６以外のザルコシンオキシダーゼにおいて配列表の配列番号１に記載されるアミノ酸配列の８２位〜９２位に対応する部位が、ザルコシンオキシダーゼの触媒ドメインとＦＡＤ結合ドメインの連結部位を構成すると推察される。
【００１８】
本願発明の別の実施態様は、配列表の配列番号１に記載されるアースロバクター・エスピーＴＥ１８２６のアミノ酸配列の３６４位を含むαへリックスを構成すると推察される３５４位〜３６６位若しくはアースロバクター・エスピーＴＥ１８２６以外のザルコシンオキシダーゼにおいて配列表の配列番号１に記載されるアミノ酸配列の３５４位〜３６６位に対応する部位の間の少なくとも１つのアミノ酸が他のアミノ酸に置換されていることを特徴とする液状での安定性が変換前に比べて向上している改変型ザルコシンオキシダーゼである。
【非特許文献４】
「Ｓｔｒｕｃｔｕｒｅ」，１９９９年， ７巻３号，ｐ．３３１−３４５
【００１９】
好ましくは、配列表の配列番号１に記載されるアミノ酸配列の８９位、１５５位、１６６位、２０４位、２１３位、２３３位、２４０位、２５０位、３６４位、若しくは他のザルコシンオキシダーゼの対応する部位からなる群より選ばれる少なくとも１つのアミノ酸が他のアミノ酸に置換されている改変型ザルコシンオキシダーゼである。
【００２０】
更に好ましくは、次の群より選ばれる少なくとも１つのアミノ酸が他のアミノ酸に置換されている改変型ザルコシンオキシダーゼである。配列表の配列番号１に記載されるアミノ酸配列の８９位のリジンがアルギニンに置換、１５５位のシステインがイソロイシンに置換、１６６位のアスパラギンがリジンに置換、２０４位のメチオニンがアラニンに置換、２１３位のセリンがプロリンに置換、２３３位のシステインがセリンに置換、２４０位のアスパラギンがチロシンに置換、２５０位のグルタミン酸がグルタミンに置換、３６４位のアラニンがバリンに置換。
【００２１】
本願発明の別の実施態様は、上記の改変型ザルコシンオキシダーゼをコードする遺伝子、該遺伝子を含むベクター、該ベクターで形質転換された形質転換体、さらには、該形質転換体を培養し、該培養物からザルコシンオキシダーゼを採取することを特徴とする改変型ザルコシンオキシダーゼの製造法である。
【００２２】
本発明の改変型ザルコシンオキシダーゼの製造方法は、特に限定されないが、以下に示すような手順で製造することが可能である。ザルコシンオキシダーゼ活性を有するタンパク質を構成するアミノ酸配列を改変する方法としては、通常行われる遺伝情報を改変する手法が用いられる。すなわち、タンパク質の遺伝情報を有するＤＮＡの特定の塩基を変換することにより、或いは特定の塩基を挿入または欠失させることにより、改変蛋白質の遺伝情報を有するＤＮＡが作成される。ＤＮＡ中の塩基を変換する具体的な方法としては、例えば市販のキット（ＴｒａｎｓｆｏｒｍｅｒＭｕｔａｇｅｎｅｓｉｓ Ｋｉｔ；Ｃｌｏｎｅｔｅｃｈ製，ＥＸＯＩＩＩ／Ｍｕｎｇ Ｂｅａｎ Ｄｅｌｅｔｉｏｎ Ｋｉｔ；Ｓｔｒａｔａｇｅｎｅ製，ＱｕｉｃｋＣｈａｎｇｅ Ｓｉｔｅ Ｄｉｒｅｃｔｅｄ Ｍｕｔａｇｅｎｅｓｉｓ Ｋｉｔ；Ｓｔｒａｔａｇｅｎｅ製など）の使用、或いはポリメラーゼ連鎖反応法（ＰＣＲ）の利用が挙げられる。
【００２３】
作製された改変タンパク質の遺伝情報を有するＤＮＡは、プラスミドと連結された状態にて宿主微生物中に移入され、改変タンパク質を生産する形質転換体となる。この際のプラスミドとしては、例えば、エシェリヒア・コリー（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）を宿主微生物とする場合にはｐＢｌｕｅｓｃｒｉｐｔ，ｐＵＣ１８などが使用できる。宿主微生物としては、例えば、エシェリヒア・コリー Ｗ３１１０、エシェリヒア・コリーＣ６００、エシェリヒア・コリーＪＭ１０９、エシェリヒア・コリーＤＨ５αなどが利用できる。宿主微生物に組換えベクターを移入する方法としては、例えば宿主微生物がエシェリヒア属に属する微生物の場合には、カルシウムイオンの存在下で組換えＤＮＡの移入を行なう方法などを採用することができ、更にエレクトロポレーション法を用いても良い。更には、市販のコンピテントセル（例えば、コンピテントハイＪＭ１０９；東洋紡績製）を用いても良い。
【００２４】
こうして得られた形質転換体である微生物は、栄養培地で培養されることにより、多量の改変タンパク質を安定して生産し得る。形質転換体である宿主微生物の培養形態は宿主の栄養生理的性質を考慮して培養条件を選択すればよく、通常多くの場合は液体培養で行うが、工業的には通気撹拌培養を行うのが有利である。培地の栄養源としては微生物の培養に通常用いられるものが広く使用され得る。炭素源としては資化可能な炭素化合物であればよく、例えば、グルコ−ス，シュークロース、ラクトース、マルトース、フラクトース、糖蜜、ピルビン酸などが使用される。窒素源としては利用可能な窒素化合物であればよく、例えばペプトン、肉エキス、酵母エキス、カゼイン加水分解物、大豆粕アルカリ抽出物などが使用される。その他、リン酸塩、炭酸塩、硫酸塩、マグネシウム、カルシウム、カリウム、鉄、マンガン、亜鉛などの塩類、特定のアミノ酸、特定のビタミンなどが必要に応じて使用される。培養温度は菌が発育し、改変蛋白質を生産する範囲で適宜変更し得るが、エシェリヒア・コリーの場合、好ましくは２０〜４２℃程度である。培養時間は条件によって多少異なるが、改変タンパク質が最高収量に達する時期を見計らって適当時期に培養を終了すればよく、通常は６〜４８時間程度である。培地ｐＨは菌が発育し改変タンパク質を生産する範囲で適宜変更し得るが、特に好ましくはｐＨ６．０〜９．０程度である。
【００２５】
培養物中の改変タンパク質を生産する菌体を含む培養液をそのまま採取し利用することもできるが、一般には常法に従って改変タンパク質が培養液中に存在する場合は、濾過，遠心分離などにより、改変タンパク質含有溶液と微生物菌体とを分離した後に利用される。改変タンパク質が菌体内に存在する場合には、得られた培養物から濾過または遠心分離などの手段により菌体を採取し、次いでこの菌体を機械的方法またはリゾチームなどの酵素的方法で破壊し、また必要に応じてＥＤＴＡ等のキレート剤及びまたは界面活性剤を添加して改変タンパク質を可溶化し、水溶液として分離採取する。
【００２６】
この様にして得られた改変タンパク質含有溶液を、例えば、減圧濃縮、膜濃縮、更に硫酸アンモニウム、硫酸ナトリウムなどの塩析処理、或いは親水性有機溶媒、例えばメタノール、エタノール、アセトンなどによる分別沈澱法により沈澱せしめればよい。また、加熱処理や等電点処理も有効な精製手段である。吸着剤或いはゲル濾過剤などによるゲル濾過、吸着クロマトグラフィー、イオン交換クロマトグラフィー、アフィニティークロマトグラフィーにより、精製された改変タンパク質を得ることができる。
【００２７】
本願発明の別の実施態様は、上記の改変型ザルコシンオキシダーゼを含むクレアチン測定用試薬、および、クレアチニン測定試薬である。本願発明のクレアチン測定用試薬、および、クレアチニン測定試薬は、液状安定性を向上させた改変型ザルコシンオキシダーゼを用いることにより、
該試薬の有効期間の延長、或いは測定精度を向上させることができる。
【００２８】
本発明のクレアチン測定試薬は、上記の液状での安定性が向上した改変型ザルコシンオキシダーゼ、クレアチンアミジノヒドロラーゼ、ペルオキシダーゼ、および過酸化水素検出試薬を含む。また、クレアチニン測定試薬は、上記の液状での安定性が向上した改変型ザルコシンオキシダーゼ、クレアチニンアミドヒドロラーゼ、クレアチンアミジノヒドロラーゼ、ペルオキシダーゼ、および過酸化水素検出試薬を含む。過酸化水素検出試薬とは、改変型ザルコシンオキシダーゼにより生成する過酸化水素をペルオキシダーゼの存在下で、生成色素として測定する試薬であり、酸化系発色試薬及び必要に応じて４−アミノアンチピリンや３−メチル−２−ベンゾチアゾリノンなどのカップラーである。本発明の過酸化水素測定試薬は、各種の市販のものなどを用いることができるが、特に限定されるものではない。更に上記のクレアチンまたはクレアチニン測定試薬は、金属塩、蛋白質、アミノ酸、糖類、有機酸などを安定化剤として使用することもできる。また通常、試薬性能に悪影響を及ぼさない範囲で防腐剤や界面活性剤を添加し、適当な緩衝液と共に使用される。緩衝液の種類、濃度およびｐＨは、各試薬成分の保存および酵素反応など目的に応じて一種もしくは複数が選択されるが、いずれの緩衝液を用いるに際しても、酵素反応時のｐＨとしては５．０〜１０．０の範囲で使用されることが好ましい。
【００２９】
本発明において、ザルコシンオキシダーゼ活性の測定は以下の条件で行う。
＜試薬＞
１００ｍＭトリス塩酸緩衝液（ｐＨ８．０）（２００ｍＭ ザルコシンおよび０．１％ トリトンＸ−１００を含む）
０．１％ ４−アミノアンチピリン
０．１％ フェノール
２５Ｕ／ｍｌ ペルオキシダーゼ
＜測定条件＞
上記トリス塩酸緩衝液、４−アミノアンチピリン溶液、フェノール溶液、ペルオキシダーゼ溶液を５：１：２：２の比率で混合し反応混液を調製する。反応混液１ｍｌを試験管に採り、３７℃で約５分間中予備加温した後、酵素溶液０．０５ｍｌを添加し、反応を開始させる。３７℃で正確に１０分間反応させた後、０．２５％ＳＤＳ水溶液２．０ｍｌを加えて反応を停止させ、この液の５００ｎｍの吸光度を測定する。盲検は酵素溶液の代わりに蒸留水を試薬混液に加えて、以下同様の操作で吸光度を測定する。上記条件下で１分間に１マイクロモルの過酸化水素を生成する酵素量を１単位とする。また、プロリンに対する反応性は、上記試薬中のザルコシンを同じ濃度のＬ−プロリンに置き換えた場合の活性の相対比として測定した。
【００３０】
【実施例】
以下、本発明を実施例により具体的に説明する。
しかし、本発明はこれらに限定されるものではない。たとえば、後述の実施例３で示された１３種類の改変型ザルコシンオキシダーゼのうち、ＳＡＯＭ１は、配列表・配列番号１に記載されたアミノ酸配列の８９位のリジンがアルギニンに置換された変異体であるが、該変異体の性能に実質的な影響を与えない範囲で、さらに、１もしくは数個のアミノ酸が欠失、置換もしくは付加されていてもさしつかえない。ＳＡＯＭ１以外の変異体についても同様である。
実施例１ ザルコシンオキシダーゼの発現プラスミドの構築
アースロバクター・エスピーＴＥ１８２６由来ザルコシンオキシダーゼの発現プラスミドｐＳＡＯＥＰ３は、特開平７−１６３３４１記載の方法に従って構築した。本発現プラスミドは、ＰＵＣ１８のマルチクローニングサイトに、ＴＥ１８２６のザルコシンオキシダーゼをコードする遺伝子を含む約１．７Ｋｂｐの挿入ＤＮＡ断片を含む。その塩基配列を配列表の配列番号２に、また該塩基配列から推定されるザルコシンオキシダーゼアミノ酸配列を配列表の配列番号１に示す。
【００３１】
実施例２ 改変型ザルコシンオキシダーゼ遺伝子の作製
ザルコシンオキシダーゼ遺伝子を含む発現プラスミドｐＳＡＯＥＰ３と、配列表の配列番号３記載の合成オリゴヌクレオチドおよびこれと相補的な合成オリゴヌクレオチドを用いて、ＱｕｉｃｋＣｈａｎｇｅＴＭ Ｓｉｔｅ−Ｄｉｒｅｃｔｅｄ Ｍｕｔａｇｅｎｅｓｉｓ Ｋｉｔ（ＳＴＲＡＴＡＧＥＮＥ製）を用いて、そのプロトコールに従って変異処理操作を行い、更に塩基配列を決定して、配列番号１記載のアミノ酸配列の８９番目のリジンがアルギニンに置換された変異型ザルコシンオキシダーゼをコードする組換えプラスミド（ｐＳＡＯＭ１）を取得した。
ｐＳＡＯＥＰ３と、配列表の配列番号４記載の合成オリゴヌクレオチドおよびこれと相補的な合成オリゴヌクレオチドを用いて、ＱｕｉｃｋＣｈａｎｇｅＴＭ Ｓｉｔｅ−Ｄｉｒｅｃｔｅｄ Ｍｕｔａｇｅｎｅｓｉｓ Ｋｉｔ（ＳＴＲＡＴＡＧＥＮＥ製）を用いて、上記と同様の操作により、配列番号１記載のアミノ酸配列の１５５番目のシステインがイソロイシンに置換された変異型ザルコシンオキシダーゼをコードする組換えプラスミド（ｐＳＡＯＭ２）を取得した。
ｐＳＡＯＥＰ３と、配列表の配列番号５記載の合成オリゴヌクレオチドおよびこれと相補的な合成オリゴヌクレオチドを用いて、上記と同様の操作により、配列番号１記載のアミノ酸配列の１６６番目のアスパラギンがリジンに置換された変異型ザルコシンオキシダーゼをコードする組換えプラスミド（ｐＳＡＯＭ３）を取得した。
ｐＳＡＯＥＰ３と、配列表の配列番号６記載の合成オリゴヌクレオチドおよびこれと相補的な合成オリゴヌクレオチドを用いて、上記と同様の操作により、配列番号１記載のアミノ酸配列の２０４番目のメチオニンがアラニンに置換された変異型ザルコシンオキシダーゼをコードする組換えプラスミド（ｐＳＡＯＭ４）を取得した。
ｐＳＡＯＥＰ３と、配列表の配列番号７記載の合成オリゴヌクレオチドおよびこれと相補的な合成オリゴヌクレオチドを用いて、上記と同様の操作により、配列番号１記載のアミノ酸配列の２１３番目のセリンがプロリンに置換された変異型ザルコシンオキシダーゼをコードする組換えプラスミド（ｐＳＡＯＭ５）を取得した。
ｐＳＡＯＥＰ３と、配列表の配列番号８記載の合成オリゴヌクレオチドおよびこれと相補的な合成オリゴヌクレオチドを用いて、上記と同様の操作により、配列番号１記載のアミノ酸配列の２３３番目のシステインがセリンに置換された変異型ザルコシンオキシダーゼをコードする組換えプラスミド（ｐＳＡＯＭ６）を取得した。
ｐＳＡＯＥＰ３と、配列表の配列番号９記載の合成オリゴヌクレオチドおよびこれと相補的な合成オリゴヌクレオチドを用いて、上記と同様の操作により、配列番号１記載のアミノ酸配列の２４０番目のアスパラギンがチロシンに置換された変異型ザルコシンオキシダーゼをコードする組換えプラスミド（ｐＳＡＯＭ７）を取得した。
ｐＳＡＯＥＰ３と、配列表の配列番号１０記載の合成オリゴヌクレオチドおよびこれと相補的な合成オリゴヌクレオチドを用いて、上記と同様の操作により、配列番号１記載のアミノ酸配列の２５０番目のグルタミン酸がグルタミンに置換された変異型ザルコシンオキシダーゼをコードする組換えプラスミド（ｐＳＡＯＭ８）を取得した。
ｐＳＡＯＥＰ３と、配列表の配列番号１１記載の合成オリゴヌクレオチドおよびこれと相補的な合成オリゴヌクレオチドを用いて、上記と同様の操作により、配列番号１記載のアミノ酸配列の３６４番目のアラニンがバリンに置換された変異型ザルコシンオキシダーゼをコードする組換えプラスミド（ｐＳＡＯＭ９）を取得した。
ｐＳＡＯＭ１と、配列表の配列番号７記載の合成オリゴヌクレオチドおよびこれと相補的な合成オリゴヌクレオチドを用いて、上記と同様の操作により、配列番号１記載のアミノ酸配列の８９番目のリジンがアルギニン、２１３番目のセリンがプロリンに置換された変異型ザルコシンオキシダーゼをコードする組換えプラスミド（ｐＳＡＯＭ１０）を取得した。
ｐＳＡＯＭ１０と、配列表の配列番号１０記載の合成オリゴヌクレオチドおよびこれと相補的な合成オリゴヌクレオチドを用いて、上記と同様の操作により、配列番号１記載のアミノ酸配列の８９番目のリジンがアルギニン、２１３番目のセリンがプロリン、２５０番目のグルタミン酸がグルタミンに置換された変異型ザルコシンオキシダーゼをコードする組換えプラスミド（ｐＳＡＯＭ１１）を取得した。
ｐＳＡＯＭ１１と、配列表の配列番号４、５、１１記載の各合成オリゴヌクレオチドおよびこれらと相補的な各合成オリゴヌクレオチドを用いて、上記と同様の操作の繰り返して、配列番号１記載のアミノ酸配列の８９番目のリジンがアルギニン、１５５番目のシステインがイソロイシン、１６６番目のアスパラギンがリジン、２１３番目のセリンがプロリン、２５０番目のグルタミン酸がグルタミン、３６４番目のアラニンがバリンに置換された変異型ザルコシンオキシダーゼをコードする組換えプラスミド（ｐＳＡＯＭ１２）を取得した。
ｐＳＡＯＭ１１と、配列表の配列番号６、８、９記載の各合成オリゴヌクレオチドおよびこれらと相補的な各合成オリゴヌクレオチドを用いて、上記と同様の操作の繰り返して、配列番号１記載のアミノ酸配列の８９番目のリジンがアルギニン、２０４番目のメチオニンがアラニン、２１３番目のセリンがプロリン、２３３番目のシステインがセリン、２４０番目のアスパラギンがチロシン、２５０番目のグルタミン酸がグルタミンに置換された変異型ザルコシンオキシダーゼをコードする組換えプラスミド（ｐＳＡＯＭ１３）を取得した。
【００３２】
実施例３ 改変型ザルコシンオキシダーゼの作製
ｐＳＡＯＭ１、ｐＳＡＯＭ２、ｐＳＡＯＭ３、ｐＳＡＯＭ４、ｐＳＡＯＭ５、ｐＳＡＯＭ６、ｐＳＡＯＭ７、ｐＳＡＯＭ８、ｐＳＡＯＭ９、ｐＳＡＯＭ１０、ｐＳＡＯＭ１１、ｐＳＡＯＭ１２、ｐＳＡＯＭ１３の各組み換えプラスミドでエシェリヒアコリーＪＭ１０９のコンピテントセルを形質転換し、該形質転換体をそれぞれ取得した。
４００ｍｌのＴｅｒｒｉｆｉｃ ｂｒｏｔｈを２Ｌ容坂口フラスコに分注し、１２１℃、２０分間オートクレーブを行い、放冷後別途無菌濾過したアンピシリンを１００μｌ／ｍｌになるように添加した。この培地に１００μｌ／ｍｌのアンピシリンを含むＬＢ培地で予め３０℃、１６時間培養したエシェリヒアコリーＪＭ１０９（ｐＳＡＯＭ１）の培養液を５ｍｌ接種し、３０℃で２０時間通気攪拌培養した。培養終了時のザルコシンオキシダーゼ活性は、前記活性測定において、培養液１ｍｌ当たり約９．５Ｕ／ｍｌであった。
上記菌体を遠心分離により集菌し、２０ｍＭリン酸緩衝液（ｐＨ７．５）に懸濁した後、超音波処理により破砕し、更に遠心分離を行い、上清液を粗酵素液として得た。得られた粗酵素液をポリエチレンイミンによる除核酸および硫安分画を行い、２０ｍＭリン酸緩衝液（ｐＨ７．５）で透析した後、ＤＥＡＥセファロースＣＬ−６Ｂ（アマシャムバイオサイエンス製）、更に１時間の熱処理により分離・精製し、精製酵素標品を得た。本方法により得られた標品は、ＳＤＳ−ＰＡＧＥ的にほぼ単一なバンドを示した。また、この変異体をＳＡＯＭ１と命名した。
ｐＳＡＯＭ２、ｐＳＡＯＭ３、ｐＳＡＯＭ４、ｐＳＡＯＭ５、ｐＳＡＯＭ６、ｐＳＡＯＭ７、ｐＳＡＯＭ８、ｐＳＡＯＭ９、ｐＳＡＯＭ１０、ｐＳＡＯＭ１１、ｐＳＡＯＭ１２、ｐＳＡＯＭ１３の各組み換えプラスミドによるエシェリヒアコリーＪＭ１０９形質転換体についても上記方法と同様にして精製酵素標品を取得した。得られた酵素標品をそれぞれＳＡＯＭ２、ＳＡＯＭ３、ＳＡＯＭ４、ＳＡＯＭ５、ＳＡＯＭ６、ＳＡＯＭ７、ＳＡＯＭ８、ＳＡＯＭ９、ＳＡＯＭ１０、ＳＡＯＭ１１、ＳＡＯＭ１２、ＳＡＯＭ１３と命名した。
【００３３】
比較例１ 野生型ザルコシンオキシダーゼの作製
比較例として、ｐＳＡＯＥＰ３によるエシェリヒアコリーＪＭ１０９形質転換体について、上記方法と同様にして、改変前の精製酵素標品を取得した。
【００３４】
実施例４ 改変型ザルコシンオキシダーゼの評価１
実施例３で取得した変異型ザルコシンオキシダーゼ（ＳＡＯＭ１、ＳＡＯＭ２、ＳＡＯＭ３、ＳＡＯＭ４、ＳＡＯＭ５、ＳＡＯＭ７、ＳＡＯＭ８、ＳＡＯＭ１０）および比較例１で取得した各種ザルコシンオキシダーゼをそれぞれ、５０ｍＭリン酸カリウム緩衝液（ｐＨ７．５）中に５Ｕ／ｍｌになるように加え、６０℃で３０分間保存した後の残存酵素活性率（％）を測定した。その結果を表１に示す。表１から判るように本発明の改変型ザルコシンオキシダーゼは改変前と比べて液状安定性が向上していることが確認された。
【００３５】
【表１】
実施例４ 改変型ザルコシンオキシダーゼの評価２
実施例３で取得した変異型ザルコシンオキシダーゼ（ＳＡＯＭ１、ＳＡＯＭ２、ＳＡＯＭ５、ＳＡＯＭ６、ＳＡＯＭ７、ＳＡＯＭ８、ＳＡＯＭ９）および比較例１で取得した各種ザルコシンオキシダーゼをそれぞれ、２ｍＭエチレンジアミン四酢酸ニ水素ニナトリウム、５０ｍＭＮａＣｌ、０．１％（Ｗ／Ｖ）２−メチルイソチアゾロン（ロッシュ・ダイアグノスティックス製）、０．１％（Ｗ／Ｖ）トリトンＸ−１００を含むＰＩＰＥＳ−ＮａＯＨ緩衝液（ｐＨ７．５）中に５Ｕ／ｍｌになるように加え、４０℃で３日間保存した後の残存酵素活性率（％）を測定した。その結果を表２に示す。表２から判るように本発明の改変型ザルコシンオキシダーゼは改変前と比べて液状安定性が向上していることが確認された。
【００３７】
【表２】
実施例５ 改変型ザルコシンオキシダーゼの評価３
実施例３で取得した変異型ザルコシンオキシダーゼ（ＳＡＯＭ１、ＳＡＯＭ１０、ＳＡＯＭ１１、ＳＡＯＭ１２、ＳＡＯＭ１３）および比較例１で取得した各種ザルコシンオキシダーゼの、クレアチニン測定試薬中の安定性を測定した。１ｍＭエチレンジアミン四酢酸ニ水素ニナトリウム、５０ｍＭ塩化ナトリウム、０．１％（Ｗ／Ｖ）２−メチルイソチアゾロン（ロッシュ・ダイアグノスティックス製）、０．１％（Ｗ／Ｖ）トリトンＸ−１００、０．０２％（Ｗ／Ｖ）４−アミノアンチピリン、０．０２％（Ｗ／Ｖ）ＴＯＯＳ（同仁化学研究所製）、１００Ｕ／ｍｌクレアチニンアミドヒドロラーゼ（東洋紡製；ＣＮＨ−２１１）、５０Ｕ／ｍｌクレアチンアミジノヒドロラーゼ（東洋紡製；ＣＲＨ−２２１）、１０Ｕ／ｍｌペルオキシダーゼ（東洋紡製；ＰＥＯ−３０１）を含む５０ｍＭＰＩＰＥＳ−ＮａＯＨ緩衝液（ｐＨ７．５）に、上記のザルコシンオキシダーゼを１０Ｕ／ｍｌになるよう加え、３５℃で２週間保存した後にザルコシンオキシダーゼ活性の残存率を測定した。その結果を表３に示す。表３から判るように本発明の改変型ザルコシンオキシダーゼは改変前と比べて、クレアチニン測定試薬中での液状安定性が向上していることが確認された。
【００３９】
【表３】
実施例６ 改変型ザルコシンオキシダーゼの評価４
実施例３で取得した変異型ザルコシンオキシダーゼ（ＳＡＯＭ１、ＳＡＯＭ１０、ＳＡＯＭ１１、ＳＡＯＭ１２、ＳＡＯＭ１３）および比較例１で取得した各種ザルコシンオキシダーゼの、クレアチン測定試薬中の安定性を測定した。１ｍＭエチレンジアミン四酢酸ニ水素ニナトリウム、５０ｍＭ塩化ナトリウム、０．１％（Ｗ／Ｖ）２−メチルイソチアゾロン（ロッシュ・ダイアグノスティックス製）、０．１％（Ｗ／Ｖ）トリトンＸ−１００、０．０２％（Ｗ／Ｖ）４−アミノアンチピリン、０．０２％（Ｗ／Ｖ）ＴＯＯＳ（同仁化学研究所製）、５０Ｕ／ｍｌクレアチンアミジノヒドロラーゼ（東洋紡製；ＣＲＨ−２２１）、１０Ｕ／ｍｌペルオキシダーゼ（東洋紡製；ＰＥＯ−３０１）を含む５０ｍＭＰＩＰＥＳ−ＮａＯＨ緩衝液（ｐＨ７．５）に、上記のザルコシンオキシダーゼを１０Ｕ／ｍｌになるよう加え、３５℃で２週間保存した後にザルコシンオキシダーゼ活性の残存率を測定した。その結果を表３に示す。表３から判るように本発明の改変型ザルコシンオキシダーゼは改変前と比べて、クレアチン測定試薬中での液状安定性が向上していることが確認された。
【００４１】
【表４】
【発明の効果】
本発明によって、ザルコシンオキシダーゼ活性を有する蛋白質を蛋白工学的手法により改変し、液状安定性が改良された改変型ザルコシンオキシダーゼを供給することが可能となった。本発明の改変型ザルコシンオキシダーゼを、臨床的に筋疾患，腎疾患の診断の指標となっている体液中のクレアチン，クレアチニンの測定用酵素として使用することで、該試薬の液状安定性を向上させることができる。
【００４３】
【配列表】
TECHNICAL FIELD OF THE INVENTION
The present invention relates to sarcosine oxidase having improved stability in a liquid state, which is obtained by modifying a protein having sarcosine oxidase activity by a protein engineering technique, a method for producing the same, and a use thereof.
[0002]
[Prior art]
Sarcosine oxidase (EC 1.5.3.1) is an enzyme for measuring creatine and creatinine in body fluids, which has been clinically used as an index for diagnosing muscular disease and renal disease. It has been used with enzymes such as Nase, Creatinase and Peroxidase. Sarcosine oxidase acts on the substrate sarcosine in the presence of water and oxygen to produce glycine, formaldehyde and hydrogen peroxide.
[0003]
Such sarcosine oxidase is known to be produced by bacteria such as Bacillus, Corynebacterium, Cylindrocarpon, Pseudomonas, and Arthrobacter (for example, Patent Documents 1 to 5 and See Non-Patent Document 1.) In addition, techniques for mass-producing the sarcosine oxidase gene of these producing bacteria using a host such as Escherichia coli by genetic engineering techniques have been reported (for example, see Patent Documents 6 to 8).
[0004]
With the recent liquefaction of clinical diagnostic reagents, various methods for stabilizing reagent components in liquid form have been studied, but sarcosine oxidase used in creatinine and creatine measurement reagents also has excellent liquid state stability. Is desired. Our group had previously reported a mutant sarcosine oxidase in which wild-type sarcosine oxidase was protein-engineered to improve the stability to metal ions (for example, see Patent Document 9). Further improvements in long-term storage stability in drug reagents are expected.
An object of the present invention is to provide a modified sarcosine oxidase having improved stability in a liquid state.
[0005]
[Patent Document 1]
JP-A-54-52789
[Patent Document 2]
JP-A-61-162174
[Patent Document 3]
JP-A-56-92790
[Patent Document 4]
JP-A-60-43379
[Patent Document 5]
JP-A-2-265478
[Patent Document 6]
JP-A-5-115281
[Patent Document 7]
JP-A-6-113840
[Patent Document 8]
JP-A-8-238087
[Patent Document 9]
JP-A-7-163341
[Non-patent document 1]
"J. Biochem.", 1981, vol. 89, p. 599
[0006]
[Means for Solving the Problems]
The present inventors have conducted intensive studies to achieve the above object, and as a result, have found that a modified sarcosine oxidase having improved stability in a liquid state can be formed without impairing the action on sarcosine, and completed the present invention. It led to.
[0007]
That is, the present invention has the following configuration.
Item 1. A protein in which at least one amino acid of an amino acid sequence constituting a protein having sarcosine oxidase activity has been converted by addition, deletion, insertion or substitution, has sarcosine oxidase activity, and has an improved stability in liquid form. A modified sarcosine oxidase characterized by being improved as compared to the previous case.
Item 2. Item 2. The modified sarcosine oxidase according to Item 1, wherein at least one amino acid of the amino acid sequence constituting the protein having sarcosine oxidase activity is substituted with another amino acid.
Item 3. Item 3. The modified sarcosine oxidase according to Items 1-2, wherein the protein having sarcosine oxidase activity has 50% or more homology with the amino acid sequence described in SEQ ID NO: 1 in the sequence listing.
Item 4. Item 3. The modified sarcosine oxidase according to Item 1 or 2, wherein the protein having sarcosine oxidase activity has 80% or more homology with the amino acid sequence described in SEQ ID NO: 1 in the sequence listing.
Item 5. Item 3. The modified sarcosine oxidase according to Item 1 or 2, wherein the protein having sarcosine oxidase activity has the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing.
Item 6. Item 3. The modified form according to any one of Items 3 to 5, wherein at least one amino acid at a site corresponding to positions 155 to 250 in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing is substituted with another amino acid. Sarcosine oxidase.
Item 7. It is characterized in that at least one amino acid at the site corresponding to positions 82 to 92 or 354 to 366 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing has been substituted with another amino acid. Item 6. The modified sarcosine oxidase according to items 3 to 5.
Item 8. At least selected from the group consisting of sites corresponding to positions 89, 155, 166, 204, 213, 233, 240, 250, and 364 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing. Item 6. The modified sarcosine oxidase according to items 3 to 5, wherein one amino acid is substituted with another amino acid.
Item 9. Item 6. The modified sarcosine oxidase according to items 3 to 5, wherein the lysine at position 89 in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing has been substituted with arginine.
Item 10. Item 6. The modified sarcosine oxidase according to items 3 to 5, wherein cysteine at position 155 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing has been substituted with isoleucine.
Item 11. Item 6. The modified sarcosine oxidase according to items 3 to 5, wherein asparagine at position 166 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing has been substituted with lysine.
Item 12. Item 6. The modified sarcosine oxidase according to items 3 to 5, wherein methionine at position 204 in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing has been substituted with alanine.
Item 13. Item 6. The modified sarcosine oxidase according to Items 3 to 5, wherein the serine at position 213 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing has been replaced with proline.
Item 14. Item 3. The modified sarcosine oxidase according to items 3 to 5, wherein cysteine at position 233 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing is substituted with serine.
Item 15. Item 6. The modified sarcosine oxidase according to Items 3 to 5, wherein asparagine at position 240 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing is substituted with tyrosine.
Item 16. Item 6. The modified sarcosine oxidase according to items 3 to 5, wherein glutamic acid at position 250 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing is substituted with glutamine.
Item 17. Item 6. The modified sarcosine oxidase according to any one of Items 3 to 5, wherein alanine at position 364 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing is substituted with valine.
Item 18. Item 18. A gene encoding the modified sarcosine oxidase according to any one of Items 1 to 17.
Item 19. Item 18. A vector comprising the gene according to Item 18.
Item 20. Item 20. A transformant transformed with the vector according to item 19.
Item 21. Item 21. A method for producing a modified sarcosine oxidase, comprising culturing the transformant according to Item 20, and collecting sarcosine oxidase from the culture.
Item 22. Item 18. A creatine measurement reagent comprising the sarcosine oxidase according to any one of Items 1 to 17.
Item 23. Item 18. A creatinine measurement reagent containing the sarcosine oxidase according to any one of Items 1 to 17.
[0008]
BEST MODE FOR CARRYING OUT THE INVENTION
The modified sarcosine oxidase of the present invention is useful for analyzing creatine and creatinine in the field of clinical testing.
[0009]
One embodiment of the present invention is a protein in which at least one amino acid of an amino acid sequence constituting a protein having sarcosine oxidase activity is converted by addition, deletion insertion or substitution, and has sarcosine oxidase activity, A modified sarcosine oxidase characterized in that the stability in a liquid state is improved as compared with that before conversion.
[0010]
The modified sarcosine oxidase of the present invention is characterized in that the stability in a liquid state is improved as compared to before the modification. The stability in a liquid state in the present invention means, for example, the ratio of the residual enzyme activity after dissolving the modified enzyme in an appropriate buffer and storing it at an appropriate temperature for a certain period of time.
The “suitable buffer solution” is not particularly limited as long as its type and concentration are selected so as to have a sufficient buffering capacity around pH 7 to 8, which is the optimum pH of sarcosine oxidase, but is preferably 50 mM potassium phosphate buffer. Solution (pH 7.5) or 50 mM PIPES-NaOH buffer (pH 7.5) is selected. The buffer may further contain a surfactant, a salt, a chelating agent, a preservative, and the like, if necessary.
The condition of “storage at an appropriate temperature for a fixed period” is not particularly limited, but preferably, conditions of an accelerated (severe) test are selected, taking into account long-term storage stability in a liquid diagnostic reagent. Specific examples include “store at 40 ° C. for 3 days” or “store at 60 ° C. for 30 minutes”. If time permits, storage for 6 months or more may be selected under refrigerated conditions of 2 ° C. to 10 ° C., which is generally used as a temperature at which the liquid diagnostic agent is actually stored for a long period of time.
The concentration of sarcosine oxidase in storage is not particularly limited, but is preferably selected from 1 to 30 U / ml assuming the concentration used for a usual diagnostic reagent. More preferably, it is 5 to 20 U / ml.
The phrase "stability is improved as compared with that before modification" means that the activity retention after storage for a certain period of time is higher than the activity retention of the enzyme before modification measured under the same conditions.
[0011]
As one embodiment of the present invention, a modified sarcosine oxidase in which the residual enzyme activity after storage in a 50 mM potassium phosphate buffer (pH 7.5) at 60 ° C. for 30 minutes is improved as compared to before modification. It is. In another embodiment, 50 mM PIPES- containing 2 mM EDTA, 50 mM NaCl, 0.1% (W / V) 2-methylisothiazolone, 0.1% (W / V) Triton X-100. It is a modified sarcosine oxidase in which the residual enzyme activity after storage in an NaOH buffer (pH 7.5) at 40 ° C. for 3 days is improved as compared to before the modification.
[0012]
The sarcosine oxidase used in the modification of the present invention is not particularly limited. For example, sarcosine oxidase derived from a known genus Bacillus, Pseudomonas, Corynebacterium or the like can be used.
[0013]
In the present invention, as an example, sarcosine oxidase of Arthrobacter sp. TE1826 (No. 10637 of B.I.K.) is used (for example, see Patent Document 10). The group of the present inventors has already succeeded in isolating the sarcosine oxidase gene from chromosomal DNA extracted from Arthrobacter sp. TE1826 and determined the entire structure of the DNA (for example, see Non-Patent Document 2). ), The present sarcosine oxidase was successfully produced in a transformant by a genetic engineering technique at a high level, and high-purity sarcosine oxidase could be supplied in large quantities at low cost (for example, see Patent Document 11). ). The amino acid sequence of sarcosine oxidase of Arthrobacter sp. TE1826 is shown in SEQ ID NO: 1 in the sequence listing. A DNA sequence encoding these amino sequences is shown in SEQ ID NO: 2 in the sequence listing.
However, the present invention is not limited to a modified sarcosine oxidase having the amino acid sequence shown in SEQ ID NO: 1, but may be a modified sarcosine oxidase protein having another sarcosine oxidase activity. Preferable examples of other proteins having sarcosine oxidase activity include sarcosine oxidase having a three-dimensional structure similar to that of sarcosine oxidase having the amino acid sequence of SEQ ID NO: 1; % Or more, more preferably 80% or more homologous amino acid sequence and having another sarcosine oxidase activity. This is because, in the case of an enzyme protein having 50% to 80% or more homology in the amino acid sequence and exhibiting the same catalytic activity, the three-dimensional structure usually has high similarity and amino acid residues involved in substrate specificity. It is based on the fact that the reaction mechanism is often the same.
Further, the modified sarcosine oxidase of the present invention is one in which one or several amino acids are further deleted, substituted or added within a range that does not impair the stability which is the essential characteristic of the enzyme of the present invention. Is also good. Specific examples include those in which a histidine tag is added to the N-terminal side or C-terminal side of the amino acid sequence to simplify purification of sarcosine oxidase (for example, see Non-Patent Document 3).
In addition, the homology of the amino acid sequence in the present invention can be determined using known gene analysis software or the like. Here, homology refers to the percentage of identical amino acid residues within a range having similarity with the amino acid sequence to be compared.
[0014]
[Patent Document 10]
JP-A-2-265478
[Patent Document 11]
JP-A-6-113840
[Non-patent document 2]
"Journal of Fermentation and Bioengineering", 1993, Vol. 75, No. 4, p. 239-244
[Non-Patent Document 3]
"Experimental Medicine", 2002, Volume 20, p. 479-482
[0015]
Another embodiment of the present invention is characterized in that at least one amino acid of the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing has been substituted with another amino acid, and the stability in liquid state has been changed before the conversion. It is a modified sarcosine oxidase that has been improved as compared to.
[0016]
Another embodiment of the present invention relates to a sarcosine oxidase other than positions 155 to 250 of the amino acid sequence of Arthrobacter sp. TE1826 described in SEQ ID NO: 1 of the sequence listing, or sarcosine oxidase other than Arthrobacter sp. TE1826. Wherein at least one amino acid at a site corresponding to positions 155 to 250 of the amino acid sequence described in SEQ ID NO: 1 in the column list has been substituted with another amino acid, and the stability in liquid state has been improved before conversion. It is a modified sarcosine oxidase that is improved as compared with the present invention.
[0017]
Another embodiment of the present invention relates to sarcosine oxidase other than positions 82 to 92 of the amino acid sequence of Arthrobacter sp. TE1826 described in SEQ ID NO: 1 in the sequence listing, or sarcosine oxidase other than Arthrobacter sp. Wherein at least one amino acid at a site corresponding to positions 82 to 92 of the amino acid sequence described in SEQ ID NO: 1 in the column list has been substituted with another amino acid, and the stability in liquid form has been improved before conversion. It is a modified sarcosine oxidase that is improved as compared with the present invention.
There is a report of sarcosine oxidase whose tertiary structure is clarified by X-ray crystallography (for example, see Non-Patent Document 4). According to the report, the sarcosine oxidase has homology to the amino acid sequence described in SEQ ID NO: 1 of the sequence listing, and has the amino acid sequence of sarcosine oxidase of Arthrobacter sp. No. 82 to No. 92 of the amino acid sequence described in No. 1 or a site corresponding to No. 82 to No. 92 of the amino acid sequence described in SEQ ID NO: 1 in sarcosine oxidase other than Arthrobacter sp. TE1826 Is presumed to constitute the linking site between the catalytic domain of sarcosine oxidase and the FAD binding domain.
[0018]
Another embodiment of the present invention relates to an amino acid sequence of Arthrobacter sp. TE1826 set forth in SEQ ID NO: 1 in the sequence listing, which is presumed to constitute an α helix containing position 364, or positions 354 to 366 or Arthro helix. In sarcosine oxidase other than Bacter sp. TE1826, at least one amino acid between the sites corresponding to positions 354 to 366 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing is replaced with another amino acid. It is a modified sarcosine oxidase characterized by improved stability in a liquid state as compared to before conversion.
[Non-patent document 4]
"Structure", 1999, Vol. 7, No. 3, p. 331-345
[0019]
Preferably, positions 89, 155, 166, 204, 213, 233, 240, 250, 364, or other sarcosine oxidases of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing. A modified sarcosine oxidase in which at least one amino acid selected from the group consisting of corresponding sites is substituted with another amino acid.
[0020]
More preferably, it is a modified sarcosine oxidase in which at least one amino acid selected from the following group is substituted with another amino acid. Lysine at position 89 in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing is substituted with arginine; cysteine at position 155 is substituted with isoleucine; asparagine at position 166 is substituted with lysine; methionine at position 204 is substituted with alanine; Serine at the position is substituted with proline, cysteine at the position 233 is substituted with serine, asparagine at the position 240 is substituted with tyrosine, glutamic acid at the position 250 is substituted with glutamine, and alanine at the position 364 is substituted with valine.
[0021]
Another embodiment of the present invention is a gene encoding the above-mentioned modified sarcosine oxidase, a vector containing the gene, a transformant transformed with the vector, and further, culturing the transformant, A method for producing a modified sarcosine oxidase, which comprises collecting sarcosine oxidase from a culture.
[0022]
The method for producing the modified sarcosine oxidase of the present invention is not particularly limited, but it can be produced by the following procedure. As a method for modifying an amino acid sequence constituting a protein having sarcosine oxidase activity, a commonly used technique for modifying genetic information is used. That is, a DNA having the genetic information of the modified protein is prepared by converting a specific base of the DNA having the genetic information of the protein or by inserting or deleting a specific base. As a specific method for converting bases in DNA, for example, commercially available kits (Transformer Mutagenesis Kit; manufactured by Clonetech, EXOIII / Mung Bean Deletion Kit; manufactured by Stratagene, used by QuickChange Site Digest, a company of QuickChange Site, and the like) The use of the chain reaction method (PCR) is mentioned.
[0023]
The prepared DNA having the genetic information of the modified protein is transferred into a host microorganism in a state of being linked to a plasmid, and becomes a transformant that produces the modified protein. As a plasmid at this time, for example, when Escherichia coli is used as a host microorganism, pBluescript, pUC18 and the like can be used. Examples of the host microorganism include Escherichia coli W3110, Escherichia coli C600, Escherichia coli JM109, and Escherichia coli DH5α. As a method for transferring the recombinant vector to the host microorganism, for example, when the host microorganism is a microorganism belonging to the genus Escherichia, a method for transferring the recombinant DNA in the presence of calcium ions can be used. Electroporation may be used. Further, a commercially available competent cell (for example, competent high JM109; manufactured by Toyobo) may be used.
[0024]
The microorganism, which is a transformant thus obtained, can stably produce a large amount of the modified protein by being cultured in a nutrient medium. The culture form of the host microorganism, which is a transformant, may be selected in consideration of the nutritional and physiological properties of the host, and is usually liquid culture in most cases. Is advantageous. As the nutrient source of the medium, those commonly used for culturing microorganisms can be widely used. The carbon source may be any carbon compound that can be assimilated, and for example, glucose, sucrose, lactose, maltose, fructose, molasses, pyruvic acid and the like are used. Any available nitrogen compound may be used as the nitrogen source, and examples thereof include peptone, meat extract, yeast extract, casein hydrolyzate, and soybean meal alkaline extract. In addition, phosphates, carbonates, sulfates, salts such as magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, and specific vitamins are used as needed. The culture temperature can be appropriately changed within a range in which the bacteria grow and produce the modified protein. In the case of Escherichia coli, the culture temperature is preferably about 20 to 42 ° C. The cultivation time varies somewhat depending on the conditions, but the cultivation may be terminated at an appropriate time in anticipation of the time when the modified protein reaches the maximum yield, and is usually about 6 to 48 hours. The pH of the medium can be appropriately changed within a range in which the bacteria grow and produce the modified protein, and particularly preferably, the pH is about 6.0 to 9.0.
[0025]
The culture solution containing the cells producing the modified protein in the culture can be directly collected and used. However, in general, when the modified protein is present in the culture solution according to a conventional method, filtration, centrifugation, etc. It is used after separating the modified protein-containing solution from the microbial cells. When the modified protein is present in the cells, the cells are collected from the obtained culture by means such as filtration or centrifugation, and then the cells are disrupted by a mechanical method or an enzymatic method such as lysozyme. The modified protein is solubilized by adding a chelating agent such as EDTA and / or a surfactant, if necessary, and separated and collected as an aqueous solution.
[0026]
The modified protein-containing solution thus obtained is subjected to, for example, concentration under reduced pressure, membrane concentration, salting-out treatment with ammonium sulfate, sodium sulfate, or the like, or a fractional precipitation method using a hydrophilic organic solvent such as methanol, ethanol, acetone, or the like. It may be allowed to settle. Heat treatment and isoelectric point treatment are also effective purification means. A purified modified protein can be obtained by gel filtration using an adsorbent or a gel filtration agent, adsorption chromatography, ion exchange chromatography, or affinity chromatography.
[0027]
Another embodiment of the present invention is a creatine measurement reagent containing the above-mentioned modified sarcosine oxidase and a creatinine measurement reagent. The creatine measurement reagent of the present invention, and a creatinine measurement reagent, by using a modified sarcosine oxidase with improved liquid stability,
The validity of the reagent can be extended or the measurement accuracy can be improved.
[0028]
The creatine measurement reagent of the present invention includes the above-mentioned modified sarcosine oxidase, creatine amidinohydrolase, peroxidase, and a hydrogen peroxide detection reagent having improved stability in a liquid state. Further, the creatinine measurement reagent includes the above-mentioned modified sarcosine oxidase, creatinine amide hydrolase, creatine amidinohydrolase, peroxidase, and hydrogen peroxide detection reagent having improved stability in a liquid state. The hydrogen peroxide detection reagent is a reagent for measuring hydrogen peroxide generated by the modified sarcosine oxidase in the presence of peroxidase as a dye, and includes an oxidizing color developing reagent and 4-aminoantipyrine or 3 A coupler such as -methyl-2-benzothiazolinone. Various commercially available reagents and the like can be used as the reagent for measuring hydrogen peroxide of the present invention, but are not particularly limited. Furthermore, the above-mentioned creatine or creatinine measurement reagent can also use metal salts, proteins, amino acids, saccharides, organic acids, and the like as stabilizers. Usually, preservatives and surfactants are added within a range that does not adversely affect the reagent performance, and used together with an appropriate buffer. The type, concentration, and pH of the buffer solution may be selected from one or more depending on the purpose such as storage of each reagent component and enzyme reaction. When any buffer solution is used, the pH at the time of the enzyme reaction is 5. It is preferably used in the range of 0 to 10.0.
[0029]
In the present invention, sarcosine oxidase activity is measured under the following conditions.
<Reagent>
100 mM Tris-HCl buffer (pH 8.0) (containing 200 mM sarcosine and 0.1% Triton X-100)
0.1% 4-aminoantipyrine
0.1% phenol
25U / ml peroxidase
<Measurement conditions>
The above Tris-HCl buffer, 4-aminoantipyrine solution, phenol solution, and peroxidase solution are mixed at a ratio of 5: 1: 2: 2 to prepare a reaction mixture. 1 ml of the reaction mixture is placed in a test tube, preliminarily heated at 37 ° C. for about 5 minutes, and 0.05 ml of the enzyme solution is added to start the reaction. After the reaction at 37 ° C. for exactly 10 minutes, the reaction is stopped by adding 2.0 ml of a 0.25% SDS aqueous solution, and the absorbance at 500 nm of this solution is measured. In the blind test, distilled water is added to the reagent mixture instead of the enzyme solution, and the absorbance is measured in the same manner as described below. The amount of the enzyme that produces 1 micromol of hydrogen peroxide per minute under the above conditions is defined as one unit. The reactivity with proline was measured as a relative ratio of the activity when sarcosine in the above reagent was replaced with the same concentration of L-proline.
[0030]
【Example】
Hereinafter, the present invention will be described specifically with reference to examples.
However, the present invention is not limited to these. For example, among the 13 types of modified sarcosine oxidases shown in Example 3 described below, SAOM1 is a mutant in which lysine at position 89 in the amino acid sequence described in SEQ ID NO: 1 has been substituted with arginine. However, as long as the performance of the mutant is not substantially affected, one or several amino acids may be further deleted, substituted or added. The same applies to mutants other than SAOM1.
Example 1Construction of sarcosine oxidase expression plasmid
The expression plasmid pSAOEP3 for sarcosine oxidase derived from Arthrobacter sp. TE1826 was constructed according to the method described in JP-A-7-163341. This expression plasmid contains an inserted DNA fragment of about 1.7 Kbp containing a gene encoding sarcosine oxidase of TE1826 at the multiple cloning site of PUC18. The nucleotide sequence is shown in SEQ ID NO: 2 in the sequence listing, and the amino acid sequence of sarcosine oxidase deduced from the nucleotide sequence is shown in SEQ ID NO: 1 in the sequence listing.
[0031]
Example 2Construction of modified sarcosine oxidase gene
Using an expression plasmid pSAOEP3 containing a sarcosine oxidase gene, a synthetic oligonucleotide represented by SEQ ID NO: 3 in the sequence listing and a synthetic oligonucleotide complementary thereto, QuickChangeTM Using Site-Directed Mutagenesis Kit (manufactured by STRATAGENE), a mutagenesis operation was performed according to the protocol, the nucleotide sequence was determined, and the lysine at position 89 in the amino acid sequence described in SEQ ID NO: 1 was substituted with arginine. A recombinant plasmid (pSAOM1) encoding sarcosine oxidase was obtained.
Using pSAOEP3, a synthetic oligonucleotide represented by SEQ ID NO: 4 in the sequence listing, and a synthetic oligonucleotide complementary thereto, QuickChangeTM Using a Site-Directed Mutagenesis Kit (manufactured by STRATAGENE), a recombinant plasmid encoding a mutated sarcosine oxidase in which the cysteine at position 155 of the amino acid sequence described in SEQ ID NO: 1 has been replaced with isoleucine by the same operation as described above ( pSAOM2) was obtained.
Using pSAOEP3, a synthetic oligonucleotide represented by SEQ ID NO: 5 in the sequence listing, and a synthetic oligonucleotide complementary thereto, the 166th asparagine in the amino acid sequence represented by SEQ ID NO: 1 was replaced with lysine by the same operation as described above. The obtained recombinant plasmid (pSAOM3) encoding the mutant sarcosine oxidase was obtained.
Using pSAOEP3, a synthetic oligonucleotide represented by SEQ ID NO: 6 in the sequence listing, and a synthetic oligonucleotide complementary thereto, the methionine at position 204 in the amino acid sequence represented by SEQ ID NO: 1 was replaced with alanine by the same operation as described above. The obtained recombinant plasmid (pSAOM4) encoding the mutant sarcosine oxidase was obtained.
Using pSAOEP3, a synthetic oligonucleotide represented by SEQ ID NO: 7 in the sequence listing, and a synthetic oligonucleotide complementary thereto, the serine at position 213 of the amino acid sequence represented by SEQ ID NO: 1 was replaced with proline by the same operation as described above. The obtained recombinant plasmid (pSAOM5) encoding the mutant sarcosine oxidase was obtained.
Using pSAOEP3, a synthetic oligonucleotide represented by SEQ ID NO: 8 in the sequence listing, and a synthetic oligonucleotide complementary thereto, the cysteine at position 233 of the amino acid sequence represented by SEQ ID NO: 1 was replaced with serine by the same operation as described above. The obtained recombinant plasmid (pSAOM6) encoding the mutant sarcosine oxidase was obtained.
Using pSAOEP3, a synthetic oligonucleotide represented by SEQ ID NO: 9 in the sequence listing, and a synthetic oligonucleotide complementary thereto, asparagine at position 240 in the amino acid sequence represented by SEQ ID NO: 1 was replaced with tyrosine by the same operation as described above. The obtained recombinant plasmid (pSAOM7) encoding the mutant sarcosine oxidase was obtained.
Using pSAOEP3, a synthetic oligonucleotide represented by SEQ ID NO: 10 in the sequence listing, and a synthetic oligonucleotide complementary thereto, the glutamic acid at position 250 of the amino acid sequence represented by SEQ ID NO: 1 was replaced with glutamine by the same operation as described above. A recombinant plasmid (pSAOM8) encoding the mutated sarcosine oxidase thus obtained was obtained.
Using pSAOEP3, a synthetic oligonucleotide represented by SEQ ID NO: 11 in the Sequence Listing and a synthetic oligonucleotide complementary thereto, the alanine at position 364 of the amino acid sequence represented by SEQ ID NO: 1 was replaced with valine by the same operation as described above. The obtained recombinant plasmid (pSAOM9) encoding the mutant sarcosine oxidase was obtained.
Using pSAOM1, a synthetic oligonucleotide represented by SEQ ID NO: 7 in the sequence listing, and a synthetic oligonucleotide complementary thereto, the lysine at position 89 in the amino acid sequence represented by SEQ ID NO: 1 was replaced with arginine, 213 A recombinant plasmid (pSAOM10) encoding a mutant sarcosine oxidase in which the second serine was replaced with proline was obtained.
Using pSAOM10, a synthetic oligonucleotide represented by SEQ ID NO: 10 in the sequence listing, and a synthetic oligonucleotide complementary thereto, the lysine at position 89 in the amino acid sequence represented by SEQ ID NO: 1 was replaced with arginine, 213 A recombinant plasmid (pSAOM11) encoding a mutant sarcosine oxidase in which the second serine was replaced with proline and the 250th glutamate was replaced with glutamine was obtained.
Using pSAOM11, the synthetic oligonucleotides described in SEQ ID NOs: 4, 5, and 11 in the sequence listing and the synthetic oligonucleotides complementary thereto, the same operation as described above was repeated to obtain the amino acid sequence described in SEQ ID NO: 1. A mutated sarcosine oxidase in which the lysine at position 89 is arginine, the cysteine at position 155 is isoleucine, the asparagine at position 166 is lysine, the serine at position 213 is proline, the glutamic acid at position 250 is glutamine, and the alanine at position 364 is valine. Was obtained (pSAOM12).
Using pSAOM11, the synthetic oligonucleotides described in SEQ ID NOs: 6, 8, and 9 in the sequence listing and the synthetic oligonucleotides complementary thereto, the same operation as described above was repeated to obtain the amino acid sequence described in SEQ ID NO: 1. A mutant sarcosine oxidase in which the lysine at position 89 is arginine, the methionine at position 204 is alanine, the serine at position 213 is proline, the cysteine at position 233 is serine, the asparagine at position 240 is tyrosine, and the glutamic acid at position 250 is glutamine. Was obtained (pSAOM13).
[0032]
Example 3Preparation of modified sarcosine oxidase
pSAOM1, pSAOM2, pSAOM3, pSAOM4, pSAOM5, pSAOM6, pSAOM7, pSAOM8, pSAOM9, pSAOM10, pSAOM11, pSAOM12, and pSAOM13 were transformed with each recombinant plasmid. .
400 ml of Terrific broth was dispensed into a 2 L Sakaguchi flask, autoclaved at 121 ° C. for 20 minutes, allowed to cool, and then separately added with sterile-filtered ampicillin to 100 μl / ml. To this medium was inoculated 5 ml of a culture solution of Escherichia coli JM109 (pSAOM1) previously cultured at 30 ° C. for 16 hours in an LB medium containing 100 μl / ml ampicillin, followed by aeration and stirring at 30 ° C. for 20 hours. The sarcosine oxidase activity at the end of the culture was about 9.5 U / ml per 1 ml of the culture in the above-mentioned activity measurement.
The cells were collected by centrifugation, suspended in 20 mM phosphate buffer (pH 7.5), disrupted by sonication, and further centrifuged to obtain a supernatant as a crude enzyme solution. . The obtained crude enzyme solution was subjected to nucleic acid removal and ammonium sulfate fractionation using polyethyleneimine, dialyzed against 20 mM phosphate buffer (pH 7.5), and then DEAE Sepharose CL-6B (manufactured by Amersham Bioscience) for another 1 hour. Separation and purification were performed by heat treatment to obtain a purified enzyme preparation. The sample obtained by this method showed an almost single band on SDS-PAGE. This mutant was named SAOM1.
An Escherichia coli JM109 transformant obtained by purifying an Escherichia coli JM109 transformant using the recombinant plasmids of pSAOM2, pSAOM3, pSAOM4, pSAOM5, pSAOM6, pSAOM7, pSAOM8, pSAOM9, pSAOM10, pSAOM11, pSAOM12 and pSAOM13 was also obtained using the recombinant plasmid. The obtained enzyme preparations were named SAOM2, SAOM3, SAOM4, SAOM5, SAOM6, SAOM7, SAOM8, SAOM9, SAOM10, SAOM11, SAOM12, and SAOM13, respectively.
[0033]
Comparative Example 1Production of wild-type sarcosine oxidase
As a comparative example, a purified enzyme preparation before modification was obtained for Escherichia coli JM109 transformant using pSAOEP3 in the same manner as described above.
[0034]
Example 4Evaluation of modified sarcosine oxidase 1
The mutant sarcosine oxidase (SAOM1, SAOM2, SAOM3, SAOM4, SAOM5, SAOM7, SAOM8, SAOM10) obtained in Example 3 and the various sarcosine oxidases obtained in Comparative Example 1 were each used in a 50 mM potassium phosphate buffer (pH 7). .5) at 5 U / ml, and after storage at 60 ° C. for 30 minutes, the residual enzyme activity ratio (%) was measured. Table 1 shows the results. As can be seen from Table 1, it was confirmed that the modified sarcosine oxidase of the present invention had improved liquid stability as compared with before modification.
[0035]
[Table 1]
Example 4Evaluation of modified sarcosine oxidase 2
The mutated sarcosine oxidase (SAOM1, SAOM2, SAOM5, SAOM6, SAOM7, SAOM8, SAOM9) obtained in Example 3 and the various sarcosine oxidases obtained in Comparative Example 1 were respectively treated with 2 mM disodium dihydrogen ethylenediaminetetraacetate and 50 mM NaCl. In a PIPES-NaOH buffer (pH 7.5) containing 0.1% (W / V) 2-methylisothiazolone (manufactured by Roche Diagnostics) and 0.1% (W / V) Triton X-100. And stored at 40 ° C. for 3 days, and the residual enzyme activity ratio (%) was measured. Table 2 shows the results. As can be seen from Table 2, it was confirmed that the modified sarcosine oxidase of the present invention had improved liquid stability as compared with before modification.
[0037]
[Table 2]
Example 5Evaluation of modified sarcosine oxidase 3
The stability of the mutant sarcosine oxidase (SAOM1, SAOM10, SAOM11, SAOM12, SAOM13) obtained in Example 3 and the various sarcosine oxidases obtained in Comparative Example 1 in the creatinine measurement reagent was measured. 1 mM ethylenediaminetetraacetic acid disodium dihydrogen, 50 mM sodium chloride, 0.1% (W / V) 2-methylisothiazolone (manufactured by Roche Diagnostics), 0.1% (W / V) Triton X-100, 0.02% (W / V) 4-aminoantipyrine, 0.02% (W / V) TOOS (manufactured by Dojindo Laboratories), 100 U / ml creatinine amidohydrolase (manufactured by Toyobo; CNH-211), 50 U / ml The above-mentioned sarcosine oxidase was added to 10 U / ml in 50 mM PIPES-NaOH buffer (pH 7.5) containing creatine amidinohydrolase (manufactured by Toyobo; CRH-221) and 10 U / ml peroxidase (manufactured by Toyobo; PEO-301). In addition, after storage at 35 ° C for 2 weeks, the residual ratio of sarcosine oxidase activity was It was boss. Table 3 shows the results. As can be seen from Table 3, it was confirmed that the modified sarcosine oxidase of the present invention had improved liquid stability in the creatinine measurement reagent as compared with before modification.
[0039]
[Table 3]
Example 6Evaluation of modified sarcosine oxidase 4
The stability of the mutant sarcosine oxidase (SAOM1, SAOM10, SAOM11, SAOM12, SAOM13) obtained in Example 3 and the various sarcosine oxidases obtained in Comparative Example 1 in the creatine measurement reagent was measured. 1 mM ethylenediaminetetraacetic acid disodium dihydrogen, 50 mM sodium chloride, 0.1% (W / V) 2-methylisothiazolone (manufactured by Roche Diagnostics), 0.1% (W / V) Triton X-100, 0.02% (W / V) 4-aminoantipyrine, 0.02% (W / V) TOOS (manufactured by Dojindo Laboratories), 50 U / ml creatine amidinohydrolase (manufactured by Toyobo; CRH-221), 10 U / ml The above-mentioned sarcosine oxidase was added to a 50 mM PIPES-NaOH buffer solution (pH 7.5) containing peroxidase (manufactured by Toyobo; PEO-301) to a concentration of 10 U / ml, and after storage at 35 ° C. for 2 weeks, the activity of sarcosine oxidase activity was measured. The residual ratio was measured. Table 3 shows the results. As can be seen from Table 3, it was confirmed that the modified sarcosine oxidase of the present invention had improved liquid stability in the creatine measurement reagent as compared with before modification.
[0041]
[Table 4]
【The invention's effect】
According to the present invention, it has become possible to supply a modified sarcosine oxidase having improved liquid stability by modifying a protein having sarcosine oxidase activity by a protein engineering technique. By using the modified sarcosine oxidase of the present invention as an enzyme for measuring creatine and creatinine in body fluids, which is clinically used as an indicator for diagnosis of muscular disease and renal disease, the liquid stability of the reagent is improved. Can be done.
[0043]
[Sequence list]
Claims (23)
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| JP2003033641A JP2004242526A (en) | 2003-02-12 | 2003-02-12 | Stabilized sarcosine oxidase |
| PCT/JP2003/014423 WO2004044193A1 (en) | 2002-11-13 | 2003-11-13 | Modified sarcosine oxidase, process for producing the same and reagent composition using the same |
| US10/534,583 US7229812B2 (en) | 2002-11-13 | 2003-11-13 | Modified sarcosine oxidase, process for producing the same and reagent composition using the same |
| CN200380103232.XA CN1711353B (en) | 2002-11-13 | 2003-11-13 | Modified sarcosine oxidase, preparation method and reagent composition thereof |
| AU2003284548A AU2003284548A1 (en) | 2002-11-13 | 2003-11-13 | Modified sarcosine oxidase, process for producing the same and reagent composition using the same |
| EP03774008.1A EP1561812B1 (en) | 2002-11-13 | 2003-11-13 | Modified sarcosine oxidase, process for producing the same and reagent composition using the same |
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| JP2003033641A JP2004242526A (en) | 2003-02-12 | 2003-02-12 | Stabilized sarcosine oxidase |
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| JP2008262875A Division JP2009077720A (en) | 2008-10-09 | 2008-10-09 | Stabilized sarcosine oxidase |
| JP2008262876A Division JP2009050272A (en) | 2008-10-09 | 2008-10-09 | Stabilized sarcosine oxidase |
| JP2008262877A Division JP2009050273A (en) | 2008-10-09 | 2008-10-09 | Stabilized sarcosine oxidase |
| JP2008262880A Division JP2009072195A (en) | 2008-10-09 | 2008-10-09 | Stabilized sarcosine oxidase |
| JP2008262879A Division JP2009055914A (en) | 2008-10-09 | 2008-10-09 | Stabilized sarcosine oxidase |
| JP2008262878A Division JP2009050274A (en) | 2008-10-09 | 2008-10-09 | Stabilized sarcosine oxidase |
| JP2008263716A Division JP2009022302A (en) | 2008-10-10 | 2008-10-10 | Stabilized sarcosine oxidase |
| JP2008263717A Division JP2009022303A (en) | 2008-10-10 | 2008-10-10 | Stabilized sarcosine oxidase |
| JP2008263719A Division JP2009055915A (en) | 2008-10-10 | 2008-10-10 | Stabilized sarcosine oxidase |
| JP2008263720A Division JP2009082132A (en) | 2008-10-10 | 2008-10-10 | Stabilized sarcosine oxidase |
| JP2008263718A Division JP2009022304A (en) | 2008-10-10 | 2008-10-10 | Stabilized sarcosine oxidase |
| JP2008263715A Division JP2009077721A (en) | 2008-10-10 | 2008-10-10 | Stabilized sarcosine oxidase |
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