JP4356320B2 - Method for producing cadaverine dicarboxylate and polyamide - Google Patents

Description

【００３４】
【化４】

【００３５】
（但し、一般式（２）、（３）において、ｎ，ｏ，ｐ，ｑ＝０〜５）
更に好ましくは
一般式（１）において、
ｍ＝０〜１０
、および／または、該一般式（２）および／または（３）において、
ｎ，ｏ，ｐ，ｑ＝０〜１
である。
【００３６】
より更に好ましくは、アジピン酸、セバシン酸、１，１２−ドデカンジカルボン酸、コハク酸、イソフタル酸、テレフタル酸である。
【００３７】
水溶液中のＬ−リジンおよびジカルボン酸のモル比に制限はない。Ｌ−リジンは塩酸との塩を形成する際１：１のモル比でＬ−リジン・塩酸塩を形成する。そのためＬ−リジン・ジカルボン酸塩の状態では水溶液中にはＬ−リジンとジカルボン酸のモル比は１：０．５であるはずである。しかし、用いるＬ−リジン脱炭酸酵素の至適ｐＨ付近になるように調整するために、ジカルボン酸をさらに添加（Ｌ−リジン：ジカルボン酸＝１：０．５５〜０．７５［モル比］）することが好ましい。またはアルカリを添加する必要がある場合はアンモニアで調整することが好ましい。
【００３８】
Ｌ−リジン脱炭酸酵素は本来ビタミンＢ６と結合し存在するが、Ｌ−リジン・ジカルボン酸塩水溶液にビタミンＢ６を添加することにより、生産速度および反応収率を向上させることができる。ビタミンＢ６を添加する方法には特に制限はない。反応中に適宜添加しても良い。好ましくはＬ−リジン脱炭酸酵素が溶液状態であれば、酵素溶液中に添加することが好ましい。反応液中でのビタミンＢ６の濃度については特に制限はなく。好ましくは反応液に０．０５ｍＭのビタミンＢ６濃度となるように加えればよい。用いるビタミンＢ６に特に制限はない。好ましくは、ピリドキシン、ピリドキサミン、ピリドキサルおよびピリドキサルリン酸から選ばれる少なくとも１種である。さらに好ましくはピリドキサルリン酸である。
【００３９】
本発明において使用されるＬ−リジン脱炭酸酵素または、Ｌ−リジン脱炭酸酵素活性を有する細胞としては、特に制限はないが、例えば、Ｌ−リジン脱炭酸酵素が細胞内で高発現した組換え細胞（細胞内発現組み換え細胞）などを適当な培地で培養し、増殖した細胞を遠心分離、または、更に酵素の分離などの通常の方法により回収したものなどが挙げられる。
【００４０】
また、これとは別に、Ｌ−リジン脱炭酸酵素が細胞表面で局在化した組み換え細胞（細胞表面発現組み換え細胞）などを適当な培地で培養し、増殖した細胞を回収したものでも良い。
【００４１】
Ｌ−リジン脱炭酸酵素が細胞表面で局在化した組み換え細胞とは、少なくともＬ−リジン脱炭酸酵素が細胞表面に局在化している状態であり、細胞内に同時にＬ−リジン脱炭酸酵素が存在していても良い。
【００４２】
一方、細胞表面にＬ−リジン脱炭酸酵素を局在化させる具体的な方法としては、例えば、分泌シグナル配列の一部、細胞表面局在タンパク質の一部をコードする遺伝子配列、Ｌ−リジン脱炭酸酵素の構造遺伝子配列をこの順で有するＤＮＡを大腸菌に導入すればよい。
【００４３】
分泌シグナル配列の一部に制限はない。宿主においてタンパク質を分泌するために必要な配列であればよい。大腸菌においては、例えばリポプロテインの配列の一部であり、具体的には、アミノ酸配列としてＭＫＡＴＫＬＶＬＧＡＶＩＬＧＳＴＬＬＡＧＣＳＳＮＡＫＩＤＱ（アミノ酸の一文字表記）と翻訳される遺伝子配列であることが好ましい。
【００４４】
細胞表面局在タンパク質の一部をコードする遺伝子配列に特に制限はない。大腸菌においては、例えば外膜結合タンパク質の配列の一部であり、具体的にはＯｍｐＡ（外膜結合タンパク質）の４６番目のアミノ酸から１５９番目のアミノ酸までの配列の一部であることが好ましい。
【００４５】
Ｌ−リジン脱炭酸酵素遺伝子、リポプロテイン遺伝子およびＯｍｐＡ遺伝子をクローニングする方法に特に制限はない。既知の遺伝子情報に基づき、ＰＣＲ （ｐｏｌｙｍｅｒａｓｅ ｃｈａｉｎ ｒｅａｃｔｉｏｎ）法を用いて必要な遺伝領域を増幅取得する方法、既知の遺伝子情報に基づきゲノムライブラリーやｃＤＮＡライブラリーより相同性や酵素活性を指標としてクローニングする方法などが挙げられる。例えばエシェリシア・コリ Ｋ１２株の染色体ＤＮＡよりＰＣＲ法を用いてＬ−リジン脱炭酸酵素をコードする遺伝子であるｃａｄＡ遺伝子またはｌｄｃ遺伝子をクローニングする。この際使用する染色体ＤＮＡはエシェリシア・コリ由来であればどの菌株由来でもよい。なお、本発明においては、これらの遺伝子は、遺伝的多形性などによる変異型も含む。遺伝的多形性とは、遺伝子上の自然突然変異により遺伝子の塩基配列が一部変化しているものをいう。
【００４６】
Ｌ−リジン脱炭酸酵素が細胞表面に局在化していることの確認は、例えばＬ−リジン脱炭酸酵素を抗原として作製した抗体により、細胞表面発現組み換え細胞を免疫反応後、包埋、薄切りし、電子顕微鏡（免疫電顕法）により観察することで確認できる。
【００４７】
本発明では、Ｌ−リジン脱炭酸酵素は、Ｌ−リジン脱炭酸酵素の細胞内もしくは細胞表面での活性が上昇した組換え細胞から調製されたものが好ましく使用できる。細胞内もしくは細胞表面でＬ−リジン脱炭酸酵素の活性を上昇させる方法に特に制限はない。具体的には、例えば、Ｌ−リジン脱炭酸酵素の酵素量を増加させる方法、もしくは酵素の構造遺伝子自体に変異を導入して、酵素そのものの比活性を上昇させることなどが挙げられる。
【００４８】
細胞内もしくは細胞表面の酵素量を増加させる手段としては、遺伝子の転写調節領域の改良、遺伝子のコピー数の増加、蛋白への翻訳の効率化などが挙げられる。
【００４９】
転写調節領域の改良とは、遺伝子の転写量を増加させる改変を加えることをいう。例えば、プロモーターに変異を導入することによってプロモーター強化を行い、下流にある遺伝子の転写量を増加させることができる。プロモーターに変異を導入する以外にも、宿主内で強力に発現するプロモーターを導入しても良い。例えば大腸菌においては、ｌａｃ、ｔａｃ、ｔｒｐなどのプロモーターが挙げられる。また、エンハンサーを新たに導入することによって遺伝子の転写量を増加させることができる。染色体ＤＮＡのプロモーター等の遺伝子導入については、例えば特開平１−２１５２８０号公報に記載されている。
【００５０】
遺伝子のコピー数の上昇は、具体的には、遺伝子を多コピー型のベクターに接続して組換えＤＮＡを作製し、該組換えＤＮＡを宿主細胞に保持させることにより達成することができる。ここでベクターとは、プラスミドやファージ等広く用いられているものを含むが、これら以外にも、トランソポゾン（バーグ，Ｄ．Ｅ．（Ｂｅｒｇ，Ｄ．Ｅ）、外１名，「バイオ／テクノロジー（Ｂｉｏ／Ｔｅｃｈｎｏｌｏｇｙ）」，１９８３年，第１巻，ｐ．４１７）やＭｕファージ（特開平２−１０９９８５号公報）も含む。遺伝子を相同組換え用プラスミド等を用いた方法で染色体に組み込んでコピー数を上昇させることも可能である。
【００５１】
蛋白の翻訳効率を上昇させる方法としては、例えば原核生物においてはＳＤ配列、真核生物ではＫｏｚａｋのコンセンサス配列を導入、改変することや、使用コドンの最適化（特開昭５９−１２５８９５号公報）などが挙げられる。
【００５２】
Ｌ−リジン脱炭酸酵素の細胞内もしくは細胞表面での活性を上昇させる手段としては、Ｌ−リジン脱炭酸酵素の構造遺伝子自体に変異を導入して、Ｌ−リジン脱炭酸酵素そのものの活性を上昇させることも挙げられる。
【００５３】
遺伝子に変異を生じさせるには、部位特異的変異法（クラマー，Ｗ．（Ｋｒａｍｅｒ，Ｗ．）、外１名，「メゾットインエンザイモロジー（Ｍｅｔｈｏｄｓ ｉｎ Ｅｎｚｙｍｏｌｏｇｙ）」，１９８７年，第１５４巻，ｐ．３５０）、リコンビナントＰＣＲ法、特定の部分のＤＮＡを化学合成する方法、または当該遺伝子をヒドロキシアミン処理する方法や当該遺伝子を保有する菌株を紫外線照射処理、もしくはニトロソグアニジンや亜硝酸などの化学薬剤で処理する方法がある。
【００５４】
組換え細胞としては、微生物、動物、植物、または昆虫由来のものが好ましく使用できる。例えば動物を用いる場合、マウス、ラットやそれらの培養細胞などが用いられる。植物を用いる場合、例えばシロイヌナズナ、タバコやそれらの培養細胞が用いられる。また、昆虫を用いる場合、例えばカイコやその培養細胞などが用いられる。また、微生物を用いる場合、例えば、大腸菌などが用いられる。
【００５５】
また、Ｌ−リジン脱炭酸酵素を複数種組み合わせて使用しても良い。
【００５６】
Ｌ−リジン脱炭酸酵素を得るために、組換え細胞を培養する方法に特に制限はないが、例えば微生物を培養する場合、使用する培地は、炭素源、窒素源、無機イオンおよび必要に応じその他有機成分を含有する培地が用いられる。例えば、大腸菌の場合しばしばLB培地が用いられる。好ましい成分として、前記典型培地成分群があげられる。
【００５７】
更に好ましい培地は、Ｌ−リジン発酵を行った後の発酵液である。この発酵液にＬ−リジン脱炭酸酵素を得るために必要な栄養素を更に添加しても良い。
【００５８】
培養条件にも特に制限はなく、例えば大腸菌の場合、好気条件下で１６〜７２時間程度実施するのが良く、培養温度は３０℃〜４５℃に、特に好ましくは３７℃に、培養ｐＨは５〜８に、特に好ましくはｐＨ７に制御するのがよい。なおｐＨ調整には無機あるいは有機の酸性あるいはアルカリ性物質、さらにアンモニアガス等を使用することができる。
【００５９】
Ｌ−リジン脱炭酸酵素を高発現させた宿主がＬ−リジンまたはカダベリンまたはジカルボン酸を資化してしまう場合、Ｌ−リジン脱炭酸酵素を精製して用いる方が好ましい。
【００６０】
例えば、回収した細胞内発現組み換え細胞から細胞破砕液を調整するには、通常の方法が用いられる。すなわち、細胞内発現組み換え細胞を超音波処理、ダイノミル、フレンチプレス等の方法にて破砕し、遠心分離により細胞残渣を除去することにより細胞破砕液が得られる。
【００６１】
細胞破砕液からＬ−リジン脱炭酸酵素を精製するには、硫安分画、イオン交換クロマトグラフィー、疎水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、ゲル濾過クロマトグラフィー、等電点沈殿、熱処理、ｐＨ処理など酵素の精製に通常用いられる手法が適宜組み合わされて用いられる。
【００６２】
工業的には精製方法は簡便な方がよく、部分的に精製したＬ−リジン脱炭酸酵素を作用させることが好ましい。部分的な精製とは、工業的に容易な処理のみ行うことであり例えば熱処理、ｐＨ処理等が挙げられる。好ましくは熱処理である。さらに好ましくは単離精製したＬ−リジン脱炭酸酵素を作用させることである。Ｌ−リジン脱炭酸酵素を部分的に精製、または単離精製することで、Ｌ−リジンの分解やカダベリンの分解またはジカルボン酸の分解を引き起こす酵素を取り除くことができる。
【００６３】
本発明で使用する精製されたＬ−リジン脱炭酸酵素は、固定化することで繰り返しカダベリン合成反応に使用することができ、酵素の調製に必要なコストを低減させることができる。固定化方法としては、アクリルアミドなどの合成高分子に包括する方法、セファロースやスチレン樹脂を骨格とするイオン交換性担体や疎水性担体に吸着させる方法、または、ガラス担体に共有結合で結合させる方法などが挙げられる。
【００６４】
さらに、Ｌ−リジン脱炭酸酵素が細胞表面に局在化している細胞を作用させれば、Ｌ−リジンの分解およびカダベリンの分解を抑制することができ、より高生産速度でカダベリンが得られる。さらにはその細胞を回収することで繰り返し酵素反応に利用することができる。しかも酵素の調整が非常に簡便である。
【００６５】
これらＬ−リジン脱炭酸酵素活性を有する細胞をゲル状担体により包括固定化すると、細胞および酵素が安定化し、酵素の繰り返し利用が簡便にできるようになる。
【００６６】
ゲル状担体としては、特に制限はない。高分子多糖類でも合成高分子でもよく、好ましくは高分子多糖類である。より好ましくは、アルギン酸、カッパーカラギーナンまたはポリアクリルアミドのいずれかであり、更に好ましくはカッパーカラギーナンである。
【００６７】
包括固定する方法に特に制限はない。Ｌ−リジン脱炭酸酵素活性が低下しない程度の穏和な条件で行うことが好ましい。
【００６８】
包括固定するゲル状担体の量に特に制限はない。調整したＬ−リジン脱炭酸酵素活性を有する細胞のすべてを包括固定できればよい。
【００６９】
一般的に酵素というものは本来あるべきアミノ酸配列で自然界に存在するため、人工的にアミノ酸配列を付与もしくは欠如させると酵素の活性を失うことが多い。しかし本発明において細胞内にＬ−リジン脱炭酸酵素を発現させる場合、Ｌ−リジン脱炭酸酵素のＮ末端アミノ酸配列に１から３０個のアミノ酸配列を付与したＬ−リジン脱炭酸酵素が好ましく使用できる。ここで、Ｎ末端に付与するものは６から１０個のヒスチジン残基が好ましい。このように、Ｎ末端アミノ酸配列にアミノ酸を付与することで、Ｌ−リジン脱炭酸酵素の精製を簡便にできるようにすることができる。Ｎ末端アミノ酸配列に１から３０個のアミノ酸を付与する方法に特に制限はない。例えばＬ−リジン脱炭酸酵素遺伝子に付与したいアミノ酸配列に相当する塩基配列を遺伝子工学的手法を用い、組み換えればよい。用いる遺伝子工学的手法に特に制限はないが。好ましくはＰＣＲ法やＤＮＡ断片同士の連結による方法がある。
【００７０】
Ｌ−リジン脱炭酸酵素によるＬ−リジン・ジカルボン酸塩水溶液からカダベリンへの変換は、上記のようにして得られるＬ−リジン脱炭酸酵素を、Ｌ−リジン・ジカルボン酸塩水溶液に接触させることによって行うことができる。
【００７１】
用いるＬ−リジン脱炭酸酵素の量に特に制限はない。Ｌ−リジン・ジカルボン酸塩水溶液をカダベリン・ジカルボン酸塩水溶液に変換する反応を触媒するのに十分な量であればよい。好ましくは反応液中の酵素濃度が２５から７０ｍｇ／ｌである。好ましくは５０ｍｇ／ｌである。一方、休止細胞を使用する際は、反応液中の細胞濃度が５から１５ｇ／ｌである。好ましくは１０ｇ／ｌである。
【００７２】
なお、本発明においてＬ−リジン脱炭酸酵素活性を有する細胞又はＬ−リジン脱炭酸酵素の活性は、後述の実施例６又は参考例１（２）の条件で，ＪＭ１０９／ｐＴＶ１１８Ｎ株又はその細胞破砕液の活性の、好ましくは１０（より好ましくは５０）倍以上である。
【００７３】
反応温度は、通常、２８〜５５℃、好ましくは３５〜４８℃である。
【００７４】
反応液の状態に特に制限はない。好ましくは静置または攪拌状態である。攪拌状態にする方法には特に制限はない。好ましくは攪拌翼により攪拌する方法である。
【００７５】
反応時間は、使用する酵素活性、Ｌ−リジン・ジカルボン酸塩濃度などの条件によって異なるが、通常、１〜７２時間である。また、反応は、Ｌ−リジン・ジカルボン酸塩を供給しながら連続的に行ってもよい。
【００７６】
このように生成したカダベリン・ジカルボン酸塩を反応終了後、反応液から採取する方法としては、通常の単離または晶析を用いれば良く、例えば、好ましくは反応液を濃縮し、有機溶媒を添加した晶析操作により採取できる。
【００７７】
晶析操作前に水溶液中のカダベリンとジカルボン酸を等モルに調整することが好ましい。更に好ましくはジカルボン酸を過剰に加える方がよい。
【００７８】
本発明で、反応液に添加する有機溶媒は、アルコール類、ケトン類またはニトリル類が好ましい。更に好ましくは、炭素数６以下の脂肪族アルコール類、炭素数６以下の脂肪族ケトン類または炭素数６以下の脂肪族ニトリル類である。
【００７９】
例えば有機溶媒の具体的例として、メタノール、エタノール、プロパノール、ブタノール、ヘキサノール、イソプロパノール、アセトニトリルまたはアセトン等が挙げられる。
【００８０】
その他、有機溶媒は単独である必要はなく、混合溶媒で有っても良い。例えば、エタノール・アセトン混合液等が挙げられる。
【００８１】
本発明で用いる有機溶媒量は特に限定されないが、カダベリン・ジカルボン酸塩を含む水溶液の重量に対して通常０．１〜５倍、本発明を特に効果的に実施するためには０．３〜２倍が使用される。あるいは、カダベリン・ジカルボン酸塩重量に対して、好ましくは１〜１０（より好ましくは３〜７）倍である。
【００８２】
カダベリン・ジカルボン酸塩を含む水溶液と有機溶媒との接触方法は任意であり、バッチ法であっても連続法であってもよい。
【００８３】
晶析を効率的に行うために、および釜効率を良くするためにカダベリン・ジカルボン酸塩を含む水溶液は濃縮するのが好ましい。濃縮の程度は、無機塩およびカダベリン・ジカルボン酸塩が結晶として析出しない程度が好ましい。水溶液中のカダベリン・ジカルボン酸塩濃度、その他の塩濃度によって濃縮の程度は変わるが、重量基準で１／４〜１／３０まで濃縮するのが好ましく、１／１０〜１／２０まで濃縮するのがより好ましい。
【００８４】
濃縮方法は常圧濃縮、減圧濃縮のいずれでもよいが、濃縮時の液温は通常、水の沸点以下で行われる。かかる液温とするとカダベリン・ジカルボン酸塩が分解する恐れがない。
【００８５】
このようにして、製造されたカダベリン・ジカルボン酸塩は、カダベリン１分子とジカルボン酸１分子よりなる塩であると考えて良いが、何等他の形態を排除するものでもなく、例えばカダベリン２分子とカルボン酸２分子より形成された塩等が含まれていても良い。
【００８６】
本発明の精製方法は、カダベリン・ジカルボン酸塩を含む水溶液に、不純物としてアミノ酸が含まれている場合の精製に特に有効である。含まれているアミノ酸に特に制限はない。好ましくは、非電荷・極性アミノ酸（グリシン、Ｌ−セリン、Ｌ−トレオニン、Ｌ−システイン、Ｌ−チロシン、Ｌ−アスパラギンおよびＬ−グルタミン）、酸性アミノ酸（Ｌ−アスパラギン酸およびＬ−グルタミン酸）および塩基性アミノ酸（Ｌ−リジン、Ｌ−アルギニンおよびＬ−ヒスチジン）のいずれかが含まれている場合の精製に特に有効である。更に好ましくは、Ｌ−アスパラギン酸、Ｌ−グルタミン酸およびＬ−リジンのいずれかが含まれている場合である。本発明において、水溶液中に含まれるアミノ酸濃度は、好適には０．０１〜１重量％である。前記数値範囲の下限とを下回るとカダベリンの生成が困難となる可能性があり、一方、上限値を上回ると単離・晶析が困難となる恐れがあり、いずれも好ましくない。
【００８７】
次いで、濃縮工程で濃縮されたカダベリン・ジカルボン酸塩水溶液に有機溶媒を添加し、析出した結晶を遠心分離などの通常の固液分離の方法によって取り除き、この有機溶媒からカダベリン・ジカルボン酸塩を通常の晶析分離操作によりカダベリン・ジカルボン酸塩を単離する。例えば、有機溶媒を添加した濃縮液をそのまま冷却してカダベリン・ジカルボン酸塩を晶析した後、結晶を遠心分離などの通常の固液分離の方法によって単離すると高純度のカダベリン・ジカルボン酸塩を得ることができる。
【００８８】
晶析行程でカダベリン・ジカルボン酸塩から分離された有機溶媒は晶析操作にリサイクルすることが好ましい。
【００８９】
かくして単離したカダベリン・ジカルボン酸塩は再度既知の晶析精製方法で、つまり、有機溶媒で均一溶液とした後、その均一液を冷却したり、熱時ろ過したろ液を冷却したりしてカダベリン・ジカルボン酸塩を結晶化させ、高純度品にできる。
【００９０】
このようにして得られたカダベリン・ジカルボン酸塩は、トリ−ｎ−ブチルアミンおよび２，３，４，５−テトラヒドロピリジンなどの不純物が少ない。本発明の方法で製造することで、以下の方法で分析した場合に、トリ−ｎ−ブチルアミンの含有量が、０．００６ｗｔ％以下のカタベリン・ジカルボン酸塩が得られる。さらに、以下の方法で分析した場合に、２，３，４，５−テトラヒドロピリジンの含有量が１．４ｗｔ％以下のものが得られる。さらに、トリ−ｎ−ブチルアミンの含有量が、０．００６ｗｔ％以下であり、かつ、２，３，４，５−テトラヒドロピリジンの含有量が１．４ｗｔ％以下のものが得られる。
【００９１】
トリ−ｎ−ブチルアミンおよび２，３，４，５−テトラヒドロピリジンの分析は、ＧＣ−ＭＳ法によって以下の条件で行う。
【００９２】
ＧＣ−ＭＳ：ＨＰ６９８０／ＨＰ５９７３Ａ
カラム：ＮＵＫＯＬ ３０ｍ×０．２４ｍｍＩ．Ｄ． ０．２μｍ Ｆｉｌｍ
オーブン：１２０℃（一定）
インジェクト：２００℃（Ｓｐｌｉｔ １０：１）
流速：Ｈｅ ２．４ｍｌ／ｍｉｎ（一定）
ＭＳ：２３０℃（ＳＣＡＮ ｍ／ｚ＝３０から４００）
このようにして得られたカダベリン・ジカルボン酸塩は、ポリアミドの原料として有用である。
【００９３】
ポリアミドの製造方法としては、こうして得られたカダベリン・ジカルボン酸塩および水の混合物を、加熱して脱水反応を進行させる加熱重縮合法が用いられる。また、加熱重縮合後、固相重合することによって、分子量を上昇させることも可能である。固相重合は、１００℃から融点の温度範囲で、真空中、あるいは不活性ガス中で加熱することにより進行し、加熱重縮合では分子量が不十分なポリアミドを高分子量化することができる。
【００９４】
本発明で得られるカダベリン・ジカルボン酸塩をポリアミドの原料とすることで、耐熱性の高いポリアミドが得られる。
【００９５】
【実施例】
以下に実施例を示し、本発明を更に具体的に説明するが、本発明はこれら実施例の記載に限定されるものではない。
【００９６】
[Ｌ−リジン濃度およびカダベリン濃度のＨＰＬＣによる分析方法]
使用カラム：ＣＡＰＣＥＬＬ ＰＡＫ Ｃ１８（資生堂）
移動相：０．１％（ｗ／ｗ）リン酸水溶液：アセトニトリル＝４．５：５．５
検出：ＵＶ３６０ｎｍ
サンプル前処理：分析サンプル２５μｌに内標として１，４−ジアミノブタン（０．０３Ｍ）を２５μｌ、炭酸水素ナトリウム（０．０７５Ｍ）を１５０μｌおよび２，４−ジニトロフルオロベンゼン（０．２Ｍ）のエタノール溶液を添加混合し３７℃で１時間保温する。上記反応溶液５０μｌを１ｍｌアセトニトリルに溶解後、１０，０００ｒｐｍで５分間遠心した後の上清１０μｌをＨＰＬＣ分析した。
【００９７】
[アジピン酸、コハク酸濃度のＨＰＬＣによる分析方法]
使用カラム：ＳＣＲ−１０１Ｈ（島津製作所）
移動相：０．０２５％（ｖ／ｖ）硫酸水溶液
検出：ＵＶ２１４ｎｍ
サンプル前処理：分析サンプルを、検量線が直線性を有することができる範囲内の濃度となるように、水で希釈し、１０μｌをＨＰＬＣ分析した。
【００９８】
参考例１（Ｌ−リジン脱炭酸酵素の調整）
（１）Ｌ−リジン脱炭酸酵素遺伝子のクローニングおよび細胞内発現ベクターの作製
Ｌ−リジン脱炭酸酵素を用いてＬ−リジン・ジカルボン酸塩からカダベリン・ジカルボン酸塩に変換させるために、エシェリシア・コリのＬ−リジン脱炭酸酵素遺伝子（ｃａｄＡ）のクローニングを行った。
【００９９】
データベース（ＧｅｎＢａｎｋ）に登録されているＬ−リジン脱炭酸酵素遺伝子（Ａｃｃｅｓｓｉｏｎ Ｎｏ．Ｍ７６４１１）の塩基配列を参考にオリゴヌクレオチドプライマー５’−ａｔｇａａｃｇｔｔａｔｔｇｃａａｔａｔｔｇ−３’（配列番号：１）、５’−ｇｃｔｇａｔｇｇｇｔｇａｇａｔａｇａａｔｇ −３’（配列番号：２）を合成した。エシェリシア・コリＫ１２株（ＡＴＣＣ１０７９８）から常法に従い調整したゲノムＤＮＡの溶液を増幅鋳型として０．２ｍｌのミクロ遠心チューブに０．２μｌづつ取り、各プライマーを２０ｐｍｏｌ、２０ｍＭトリス塩酸緩衝液（ｐＨ８．０）、２．５ｍＭ塩化カリウム、１００μｇ／ｍｌゼラチン、５０μＭ各ｄＮＴＰ、２単位 ＬＡＴａｑＤＮＡポリメラーゼ（宝酒造製）となるように各試薬を加え、全量を５０μｌとした。ＤＮＡの変性条件を９４℃、３０秒、プライマーのアニーリング条件を５５℃、３０秒、ＤＮＡプライマーの伸長反応条件を７２℃、３分の各条件でＢｉｏＲａｄ社のサーマルサイクラーを用い、３０サイクル反応させた（ポリメラーゼ連鎖反応：以後、ＰＣＲ法と記す）。尚、本実施例におけるＰＣＲ法は特に断らない限り、本条件にて行った。このＰＣＲ法により得られた産物を１％アガロースにて電気泳動し、ｃａｄＡ遺伝子を含む約２．１ｋｂのＤＮＡ断片を常法に従い調整した。この断片を、プラスミドベクターｐＴ７ｂｌｕｅ（Ｎｏｖａｇｅｎ社製）のＥｃｏＲＶ部位の３’−末端にＴ塩基が付加された間隙に、常法に従ったライゲーション反応により挿入し、得られたプラスミドをｐＴ７−ｃａｄＡと命名した。
【０１００】
このｐＴ７−ｃａｄＡを増幅鋳型として、オリゴヌクレオチド５’−ｃｇｃｃａｔｇｇｇｃｃａｔｃａｔｃａｔｃａｔｃａｔｃａｔｃａｔｃａｔａｔｇａａｃｇｔｔａｔｔｇｃａａｔａｔｔｇ−３’（配列番号：３）、５’−ｃｇｃｇｇａｔｃｃｇｃｔｇａｔｇｇｇｔｇａｇａｔａｇａａｔｇ−３’（配列番号：４）をプライマーセットとしたＰＣＲ法を行った。ここで得られる産物は、オリゴヌクレオチド（配列番号：３）由来の塩基配列と、オリゴヌクレオチド（配列番号：４）由来の塩基配列が、それぞれｃａｄＡ遺伝子を含むＤＮＡ断片の５’末端、および３’末端に付加されている。この産物を１％アガロースにて電気泳動し、約２．１ｋｂのＤＮＡ断片を常法に従い調整した。この断片を、プラスミドベクターｐＴ７ｂｌｕｅ（Ｎｏｖａｇｅｎ社製）のＥｃｏＲＶ部位の３’−末端にＴ塩基が付加された間隙に、常法に従ったライゲーション反応により挿入し、得られたプラスミドをｐＴ７−ｃａｄＡ１と命名した。
【０１０１】
このｐＴ７−ｃａｄＡ１をＮｃｏＩ、及びＢａｍＨＩで消化し、得られた２．１ｋｂのＮｃｏＩ−ＢａｍＨＩ断片を、予めＮｃｏＩ、及びＢａｍＨＩで消化しておいたｐＴＶ１１８Ｎ（宝酒造製）のＮｃｏＩ／ＢａｍＨＩ間隙に常法に従ったライゲーション反応により挿入し、得られたプラスミドをｐＬＤＣ１と命名した（図１）。このプラスミドを導入した大腸菌を培養することで、Ｌ−リジン脱炭酸酵素のＮ末端アミノ酸配列に６個のヒスジチン残基が付加された分子量約８０ｋＤａの組み換えＬ−リジン脱炭酸酵素を生産することができる。
【０１０２】
（２）組み換えＬ−リジン脱炭酸酵素の産生
ｐＬＤＣ１でエシェリシア・コリＪＭ１０９株をアンピシリン耐性に形質転換し、得られた形質転換体をＪＭ１０９／ｐＬＤＣ１株と命名した。
【０１０３】
次にＪＭ１０９／ｐＬＤＣ１株で組み換えＬ−リジン脱炭酸酵素を産生させた。まず、ＪＭ１０９／ｐＬＤＣ１株をそれぞれ５０μｇ／ｍｌのアンピシリンナトリウムを含んだ滅菌したＬＢ培地（ナカライテスク社製）（ＬＢ−ａｍｐ培地）５ｍｌに１白金耳植菌し、３７℃で２４時間振とうして前培養を行い、前培養液を得た。
【０１０４】
この前培養液をＬＢ−ａｍｐ培地５０ｍｌに全量植菌し、３７℃、振幅３０ｃｍで、１８０ｒｐｍの条件下で３時間培養した後にＩＰＴＧ（イソプロピル−１−チオ−β−Ｄ−ガラクトシド）（１ｍＭ）添加し、更に４時間培養した。対照実験として、ＪＭ１０９株をｐＴＶ１１８Ｎで形質転換した形質転換体（以後、ＪＭ１０９／ｐＴＶ１１８Ｎ株とする）を用い同様の培養を行った。こうして得られた菌体を集め、５ｍｌのＴＢＳ緩衝液（宝酒造製）に再懸濁後、超音波破砕および遠心分離により細胞破砕液を調製した。これらのL-リジン脱炭酸酵素活性の測定を単位時間当たりに生成するカダベリンの濃度を測定することにより行った。その結果、対照実験であるＪＭ１０９／ｐＴＶ１１８Ｎ株由来の細胞破砕液に対して、ＪＭ１０９／ｐＬＤＣ１株においてはＬ−リジン脱炭酸酵素活性は約１００倍に上昇していた。また、この細胞破砕液をＳＤＳ−ＰＡＧＥで分画し、Ｐｅｎｔａ−Ｈｉｓ Ａｎｔｉｂｏｄｙ抗体（ＱＩＡＧＥＮ社製）でウエスタンブロッティングを行った結果、ＪＭ１０９／ｐＬＤＣ１株由来の細胞破砕液のみから、分子量約８０ｋＤａの組み換えL-リジン脱炭酸酵素を検出した。
【０１０５】
（３）組み換えＬ−リジン脱炭酸酵素の精製
この組み換えＬ−リジン脱炭酸酵素は、Ｎ末端アミノ酸配列に６個のヒスチジン残基があることこから、ニッケルイオンとの相互作用を利用した精製を行った。
まず１０ｍｌのキレーティング セファロース ファースト フロー（Ｃｈｉｌａｔｉｎｇ ＳｅｐＨａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ）担体（アマシャム バイオサイエンス社製）を充填したカラムシステムを構築した。このカラムに５０ｍｌの５０ｍＭ硫酸ニッケル水溶液、５０ｍｌのＴＢＳ緩衝液の順で流した後、（２）と同様の方法で得られたＪＭ１０９／ｐＬＤＣ１株の５００ｍｌ培養液由来の５０ｍｌ細胞破砕液を流した。その後、１００ｍｌのイミダゾール（５ｍＭ）を含むＴＢＳ緩衝液、１００ｍｌのイミダゾール（５０ｍＭ）を含むＴＢＳ緩衝液をこの順序で流した。更に５０ｍｌのイミダゾール（６００ｍＭ）を含むＴＢＳ緩衝液を流した。カラムに流した各々の緩衝液のＬ−リジン脱炭酸酵素活性を（２）と同様の方法で測定したところ、イミダゾール（６００ｍＭ）を含むＴＢＳ緩衝液のみに活性があった。また、カラムに流した各々の緩衝液をＳＤＳ−ＰＡＧＥし、クマシーブリリアントブルーで染色たところ、イミダゾール（６００ｍＭ）を含むＴＢＳ緩衝液から、約８０ｋＤａの単一バンドを検出した。また、カラムに流した各々の緩衝液を（２）と同様の方法でウエスタンブロッティングを行ったところ、イミダゾール（６００ｍＭ）を含むＴＢＳ緩衝液のみに約８０ｋＤａタンパク質を検出し、この精製タンパク質はＬ−リジン脱炭酸酵素活性を有する組み換えＬ−リジン脱炭酸酵素であることを確認した。
【０１０６】
（４）Ｌ−リジン脱炭酸酵素の細胞表面発現ベクターの作製
まず、リポプロテインの配列の一部および外膜結合タンパク質（ＯｍｐＡ）の４６番目のアミノ酸から１５９番目のアミノ酸までの配列を一つのカセットとしてクローニングした。
【０１０７】
即ち、データベース（ＧｅｎＢａｎｋ）に登録されている外膜結合タンパク質（ｏｍｐＡ）（Ａｃｃｅｓｓｉｏｎ Ｎｏ．ＮＣ＿０００９１３）の塩基配列を参考にオリゴヌクレオチドプライマー５’−ａｔａａａｇｃｔｔａｔｇａａａｇｃｔａｃｔａａａｃｔｇｇ−３’（配列番号：５）５’−ａｔａｇｔｃｇａｃｇｔｔｇｔｃｃｇｇａｃｇａｇｔｇｃｃｇ−３’（配列番号：６）、５’−ａｔａａａｇｃｔｔａｔｇａａａｇｃｔａｃｔａａａｃｔｇｇｔａｃｔｇｇｇｃｇｃｇｇｔａａｔｃｃｔｇｇｇｔｔｃｔａｃｔｃｔｇｃｔｇｇｃａｇｇｔｔｇｃｔｃｃａｇｃａａｃｇｃｔａａａａｔｃｇａｔｃａｇａａｃｃｃｇｔａ−３’（配列番号：７）を合成した。エシェリシア・コリＫ１２株から常法に従い調整したゲノムＤＮＡの溶液を増幅鋳型として（配列番号：５）（配列番号：６）（配列番号：７）をプライマーセットとしたＰＣＲ法を行った。このＰＣＲ法により得られた産物を２％アガロースにて電気泳動し、ｏｍｐＡ遺伝子を含む約０．４ｋｂのＤＮＡ断片を常法に従い調整した。この断片を、プラスミドベクターｐＴ７ｂｌｕｅ（Ｎｏｖａｇｅｎ社製）のＥｃｏＲＶ部位の３’−末端にＴ塩基が付加された間隙に、常法に従ったライゲーション反応により挿入し、得られたプラスミドをｐＴＭ７と命名した。
【０１０８】
このｐＴＭ７をＨｉｎｄＩＩＩ、及びＳａｌＩで消化し、得られた約０．４ｋｂのＨｉｎｄＩＩＩ、ＳａｌＩ断片を、予めＨｉｎｄＩＩＩ、及びＳａｌＩで消化しておいたｐＵＣ１９（宝酒造製）のＨｉｎｄＩＩＩ／ＳａｌＩ間隙に常法に従ったライゲーション反応により挿入し、得られたプラスミドをｐＴＭ１４と命名した。
【０１０９】
次に、エシェリシア・コリＫ１２株のＬ−リジン脱炭酸酵素遺伝子（ｃａｄＡ）のクローニングを行った。
【０１１０】
データベース（ＧｅｎＢａｎｋ）に登録されているＬ−リジン脱炭酸酵素遺伝子の塩基配列を参考にオリゴヌクレオチドプライマー５’−ｔａｔｇｔｃｇａｃａｔｇａａｃｇｔｔａｔｔｇｃａａｔａｔ−３’（配列番号：８）、５’−ｇｇａｇａｇｃｔｃｔｔａｔｔｔｔｔｔｇｃｔｔｔｃｔｔｃｔｔｔ−３’（配列番号：９）を合成した。エシェリシア・コリＫ１２株から常法に従い調整したゲノムＤＮＡの溶液を増幅鋳型として（配列番号：８）（配列番号：９）をプライマーセットとしたＰＣＲ法を行った。
【０１１１】
このＰＣＲ法により得られた産物を１％アガロースにて電気泳動し、ｃａｄＡ遺伝子を含む約２．１ｋｂのＤＮＡ断片を常法に従い調整した。この断片を、プラスミドベクターｐＴ７ｂｌｕｅ（Ｎｏｖａｇｅｎ社製）のＥｃｏＲＶ部位の３’−末端にＴ塩基が付加された間隙に、常法に従ったライゲーション反応により挿入し、得られたプラスミドをｐＴＭ１５と命名した。
【０１１２】
これら調整したｐＴＭ１５をＳａｌＩ、及びＳａｃＩで消化し、得られた約２．１ｋｂのＳａｌＩ、ＳａｃＩ断片を、予めＳａｌＩ、及びＳａｃＩで消化しておいたｐＴＭ１４のＳａｌＩ／ＳａｃＩ間隙に常法に従ったライゲーション反応により挿入し、得られたプラスミドをｐＴＭ１６（図２）と命名した。
【０１１３】
このプラスミドを導入した大腸菌を培養することで、Ｌ−リジン脱炭酸酵素が細胞表面に局在化され、その大腸菌を回収するだけでＬ−リジン脱炭酸酵素を調整することができるようになる。
【０１１４】
（５）Ｌ−リジン脱炭酸酵素が細胞表面に局在化した細胞の調整
ｐＴＭ１６でエシェリシア・コリＪＭ１０９株をアンピシリン耐性に形質転換し、得られた形質転換体をＪＭ１０９／ｐＴＭ１６株と命名した。
【０１１５】
次にＪＭ１０９／ｐＴＭ１６株で組み換えＬ−リジン脱炭酸酵素を産生させた。まずＪＭ１０９／ｐＴＭ１６株をそれぞれ５０μｇ／ｍｌのアンピシリンナトリウムを含んだ滅菌ＬＢ培地（ＬＢ−ａｍｐ培地）５ｍｌに１白金耳植菌し、３７℃で２４時間振とうして前培養を行った。
【０１１６】
この前培養液をＬＢ−ａｍｐ培地５０ｍｌに全量植菌し、３７℃、振幅３０cmで、１８０ｒｐｍの条件下で３時間培養した後にＩＰＴＧ（１ｍＭ）を添加し、更に４時間培養した。こうして得られた菌体を遠心回収し、１０ｍｌのＴＢＳ緩衝液（宝酒造製）で３回洗浄後、遠心し菌体を回収した。
【０１１７】
実施例１，２、比較例１（Ｌ−リジン・ジカルボン酸水溶液を用いることの効果）
１，０００ｍｌのフラスコに、表１に示すようにＬ−リジンまたはＬ−リジンおよびジカルボン酸を水溶液５００ｍｌに加えた。その後、終濃度５０ｍｇ／ｌになるように参考例１（３）で得た精製した組み換えＬ−リジン脱炭酸酵素および終濃度０．０５ｍＭになるようにピリドキサルリン酸を加え作用させた。４５℃で４８時間反応した後の反応液中に含まれるカダベリンの濃度を測定した。その結果を表１に示す。
【０１１８】
【表１】

【０１１９】
この結果、原料としてＬ−リジン・ジカルボン酸塩水溶液を用いると、ｐＨの調整をしなくてもカダベリン・ジカルボン酸塩が得られた。
【０１２０】
実施例３、比較例２（Ｌ−リジン発酵をジカルボン酸で中和することの効果）コリネバクテリウム・グルタミカ ＡＴＣＣ１３２８６株を滅菌ＬＢ培地５ｍｌに１白金耳植菌し、３０℃で２４時間振とうして前々培養を行って、前々培養液を得た。
【０１２１】
この前々培養液を滅菌ＬＢ培地５０ｍｌに全量植菌し、３０℃、振幅３０ｃｍで、１８０ｒｐｍの条件下で２４時間培養して前培養を行って、前培養液を得た。
【０１２２】
次に表２に示す発酵培地９５０ｍｌに前培養液全量を植菌し、滅菌した空気を１ｖｖｍ（１分間に培養液体積と同等の体積の空気を通気する）で通気しながら、３０℃、攪拌翼回転数８００ｒｐｍ、ｐＨを７に調整しながら７５時間培養を行った。中和剤として、滅菌したアジピン酸水溶液（１０ｇ／ｌ）およびアンモニア水（３Ｍ）（実施例３の場合）、もしくは、塩酸水溶液（３Ｍ）およびアンモニア水（３Ｍ）（比較例２の場合）で行った。 培養終了後、４℃、８，０００ｒｐｍで１０分間遠心分離することで菌体を除去し、培養上清を回収した。この培養上清中に、実施例３および比較例２共に総量として１０ｇのＬ−リジンを生成した。
【０１２３】
【表２】

【０１２４】
次にそれぞれの培養上清に終濃度５０ｍｇ／ｌになるように参考例１（３）で得た精製した組み換えＬ−リジン脱炭酸酵素および終濃度０．０５ｍＭになるようにピリドキサルリン酸を加え、４５℃で４８時間作用させた。この反応液中に、実施例３および比較例２共に総量として６．６ｇのカダベリンを生成した。
【０１２５】
次にそれぞれの反応液を２Ｌ３つ口フラスコ内で減圧下で水を留去した。スラリーの粘度が上昇し攪拌操作性が悪化し始めたところで濃縮を終了した。素早く８０ｇのエタノールを加え、６５℃まで加熱し溶解させた。溶解せずに析出した結晶をろ過により固液分離した。その後均一に溶解したエタノールを徐々に冷却し、晶析、固液分離、乾燥した。実施例３では、カダベリン・アジピン酸の等モル塩である結晶１２．２ｇを得た。対して比較例２では、カダベリン二塩酸塩の結晶６．８ｇを得た。
【０１２６】
比較例２ではこの後さらに、得られたカダベリン二塩酸塩の結晶を水に溶解し、水酸化ナトリウムを添加し、この水溶液のｐＨを１３以上にした。次にクロロホルムを水溶液と等量加え、クロロホルム相にカダベリンを抽出した。最後にこのクロロホルム相を、減圧蒸留（３０ｍｍＨｇ、８０℃）することによりフリーのカダベリン３．６ｇを単離した。 この単離したカダベリンと等モルのアジピン酸５．１ｇを水に一旦溶解させ減圧下で水を留去し、乾燥することでカダベリン・アジピン酸の等モル塩である結晶８．７ｇを得た。
【０１２７】
この結果、Ｌ−リジン発酵を塩酸で中和せずに、アジピン酸で中和することでカダベリン・アジピン酸塩を得る工程が簡略化される。そのため当然ながら対原料収率が向上する。
【０１２８】
実施例４〜６（Ｌ−リジン脱炭酸酵素の状態の効果）
実施例３の方法と同様にＬ−リジン発酵を行い、総量として１０ｇのＬ−リジンを含む培養上清を得た。
【０１２９】
この培養上清に終濃度０．０５ｍＭになるようにピリドキサルリン酸を加え、最後に参考例１（２）および（５）に記載の方法により調整したＪＭ１０９／ｐＴＶ１１８Ｎ菌体（実施例６）、ＪＭ１０９／ｐＬＤＣ１菌体（実施例４）およびＪＭ１０９／ｐＴＭ１６菌体（実施例５）を加え、４５℃で２４時間作用させた。経時的にカダベリンの濃度を測定した結果を図３に示す。
【０１３０】
この結果、Ｌ−リジン脱炭酸酵素を細胞表面に局在化させることで高生産速度でカダベリンが得られた。
【０１３１】
実施例７（Ｌ−リジン発酵培地でＬ−リジン脱炭酸酵素を調整する効果）
実施例３の方法と同様にＬ−リジン発酵を行い、総量として１０ｇのＬ−リジンを含む培養上清を得た。
【０１３２】
この培養上清に終濃度１０ｇ／ｌになるように滅菌したグルコースを添加し、ＪＭ１０９／ｐＴＭ１６菌体を１白金耳植菌し、滅菌した空気を１ｖｖｍで通気しながら、３７℃、攪拌翼回転数４００ｒｐｍ、ｐＨを７に調整しながら２４時間培養を行った。中和剤として滅菌したアジピン酸水溶液（１０ｇ／ｌ）およびアンモニア水（３Ｍ）を使用した。培養終了後、４℃、８，０００ｒｐｍで１０分間遠心分離することで菌体を除去し、培養上清を回収した。この培養上清中に、全量として６．８ｇのカダベリンを生成した。
【０１３３】
この培養上清を実施例３に記載の方法でカダベリン・アジピン酸塩を精製し、カダベリン・アジピン酸の等モル塩である結晶１２．５ｇを得た。
【０１３４】
この結果一つの発酵装置を用いるだけでカダベリン・アジピン酸を得ることができた。
【０１３５】
比較例３（既存の化学合成法によるカダベリンの調製）
Ｌ−リジン一塩酸塩２０ｇ（フルカ社製）シクロヘキサノール１００ｍｌ（シグマアルドリッチジャパン製）に懸濁し、次いで２８％ナトリウムメトキシド／メタノール溶液（シグマアルドリッチジャパン製）２１．２ｍｌ、３−メチル−シクロヘキセノン１ｍｌ（シグマアルドリッチジャパン製）を加え、１５５℃で３時間加熱撹拌した。反応終了後、反応混合物に塩化水素４ｇ（シグマアルドリッチジャパン製）を含むイソプロパノール溶液２０ｍｌ（シグマアルドリッチジャパン製）を加え、析出した生成物を回収し、乾燥することによりカダベリン二塩酸塩を得た（特開昭６０−２３３２８号公報の実施例３記載の方法）。
【０１３６】
得られたカダベリン二塩酸塩を水に溶解させ、この水溶液に、水酸化ナトリウム水溶液を添加することによってカダベリン二塩酸塩をカダベリンに変換し、クロロホルムで抽出して、減圧蒸留（３０ｍｍＨｇ、８０℃）することにより、カダベリンを得た。この単離したカダベリンと等モルのアジピン酸を水に一旦溶解させ減圧下で乾燥することでカダベリン・アジピン酸の等モル塩である結晶を得た。
【０１３７】
実施例３、６比較例３の不純物の同定
実施例３および６で得られたカダベリン・アジピン酸塩の不純物の分析を、下記に示す条件でＧＣ−ＭＳ法により行い、比較例３で調製したカダベリン・アジピン酸塩の不純物分析と比較した結果を表３に示す。
【０１３８】
ＧＣ−ＭＳ：ＨＰ６９８０／ＨＰ５９７３Ａ
カラム：ＮＵＫＯＬ ３０ｍ×０．２４ｍｍＩ．Ｄ． ０．２μｍ Ｆｉｌｍ
オーブン：１２０℃（一定）
インジェクト：２００℃（Ｓｐｌｉｔ １０：１）
流速：Ｈｅ ２．４ｍｌ／ｍｉｎ（一定）
ＭＳ：２３０℃（ＳＣＡＮ ｍ／ｚ＝３０から４００）
【０１３９】
【表３】

【０１４０】
この結果、Ｌ−リジン脱炭酸酵素を用い生成したカダベリンをカダベリン・アジピン酸塩に精製することにより、水性生物に有毒で、また酸と接触すると反応してしまうトリ−ｎ−ブチルアミンの含有量が０．００６ｗｔ％以下かつ２，３，４，５−テトラヒドロピリジンの含有量が１．４ｗｔ％以下の高純度カダベリン・アジピン酸塩が得られた。
【０１４１】
実施例７（ポリアミドの合成）
実施例３の方法で得られたカダベリン・アジピン酸の等モル塩を水に溶解させの５０ｗｔ％水溶液５０．０ｇを調整した。この溶液を試験管に仕込み、オートクレーブに入れて、密閉し、窒素置換した。ジャケット温度を２６５℃に設定し、加熱を開始した。缶内圧力が１７．５ｋｇ／ｃｍ²に到達した後、缶内圧力を１７．５ｋｇ／ｃｍ²で３時間保持した。その後、ジャケット温度を２７５℃に設定し、２時間かけて缶内圧力を常圧に放圧した。その後、缶内温度が２４５℃に到達した時点で、加熱を停止した。室温に放冷後、試験管をオートクレーブから取り出し、ポリアミドを得た。
【０１４２】
比較例４
比較例３の方法で得られたカダベリン・アジピン酸の等モル塩を水に溶解させ５０ｗｔ％水溶液５０．０ｇを調整し用いる以外は、実施例７と全く同様の方法でポリアミドを得た。
【０１４３】
実施例７，比較例４のポリアミドの評価
実施例７で得られたポリアミドおよび比較例４で得られたポリアミドを下記に示す方法で比較評価した。その結果を表４に示す。
［ポリアミド中の２，３，４，５−テトラヒドロピリジン、およびトリ−ｎ−ブチルアミンの定量（ＧＣ−ＭＳ）］
ポリアミド約１５ｇを精秤して、メタノールでソックスレー抽出し、その抽出液を、下記条件でＧＣ−ＭＳ分析して、ポリアミド中に含まれる２，３，４，５−テトラヒドロピリジン、およびトリ−ｎ−ブチルアミンを定量した。
装置：ヒューレットパッカード製 ＨＰ５８９０質量検出器
カラム：５％−ジフェニル−９５％−ジメチルポリシロキサン
カラム温度：Ｉｎｉｔｉａｌ １００℃
Ｆｉｎａｌ ２５０℃
昇温速度：１０℃／ｍｉｎ
注入口温度：２３０℃
検出器温度：２８０℃
キャリアガス：ヘリウム
注入口圧力：５０ｋｇ／ｃｍ²
試料注入量：１μｌ。
【０１４４】
［ＤＳＣ（示差走査熱量測定）］
セイコー電子工業製 ロボットＤＳＣ ＲＤＣ２２０を用い、窒素雰囲気下、ポリアミドを約５ｍｇを採取し、次の条件で測定した。
融点＋２５℃に昇温して３分間保持し、ポリアミドを完全に融解させた後、２０℃／分の降温速度で、３０℃まで降温し、３分間保持した後、３０℃から融点＋２５℃まで２０℃／分の昇温速度で昇温したときに観測される吸熱ピークの温度、および熱量を求めた。
【０１４５】
［相対粘度（ηｒ）］
９８％硫酸中、０．０１ｇ／ｍｌ濃度、２５℃でオストワルド式粘度計を用いて測定を行った。
［溶融滞留試験］
試験管にポリアミド約５ｇを仕込み、窒素雰囲気下、融点＋２０℃の温度のシリコンバスに浸漬し、ポリアミドが完全に溶融してから３０分間放置した後、ポリアミドを回収して相対粘度測定を行った。
【０１４６】
【表４】

【０１４７】
その結果、本発明の方法によって得られた高純度カダベリン・ジカルボン酸塩を使用することにより、相対粘度（ηｒ）の保持率の高い（耐熱性の高い）ポリアミドが得られた。
【０１４８】
【発明の効果】
本発明によれば、一つの発酵装置で高反応収率、高生産速度および高純度でのカダベリン・ジカルボン酸塩の工業的製造方法およびカダベリン・ジカルボン酸塩の単離方法が提供される。
【０１４９】
【配列表】

【図面の簡単な説明】
【図１】 Ｌ−リジン脱炭酸酵素細胞内発現ベクターｐＬＤＣ１のフィジカルマップを示す図である。
【図２】 Ｌ−リジン脱炭酸酵素細胞表面発現ベクターｐＴＭ１６のフィジカルマップを示す図である。
【図３】 実施例４〜６における時間とカダベリン生産量の関係を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing cadaverine dicarboxylate. Cadaverine dicarboxylate is expected as a raw material for polyamide, and its demand is increasing.
[0002]
[Prior art]
Conventionally, it is known that cadaverine is obtained by boiling L-lysine in cyclohexanol containing a trace amount of tetralin peroxide (see Non-Patent Document 1). However, the high temperature reaction requires a large amount of energy and organic solvent, and the reaction yield is very low (36%).
[0003]
Further, a method of synthesizing from lysine using vinyl ketones such as 2-cyclohexen-1-one as a catalyst is known (see Non-Patent Document 2).
[0004]
Furthermore, it is known that cadaverine is obtained by heating and stirring L-lysine in cyclohexanol containing sodium methoxide with 3-methyl-cyclohexenone as a catalyst at 155 ° C. for 3 hours (see Patent Document 1).
[0005]
Further, cadaverine obtained by this method contains basic compounds such as tri-n-butylamine and 2,3,4,5-tetrahydropyridine as impurities.
[0006]
Cadaverine is a biogenic amine that exists universally in the living body, and its biosynthetic system is being elucidated (see Non-Patent Document 3). Further, an L-lysine decarboxylase gene derived from Escherichia coli is known (see Non-Patent Document 4).
[0007]
Further, a production method of cadaverine by fermentation in recombinant E. coli (see Patent Document 2) and a production method by enzymatic method in recombinant E. coli (see Patent Document 3) are known.
[0008]
On the other hand, studies for localizing proteins on the cell surface have been conducted in many bacteria. Research on localizing proteins on the cell surface of E. coli has been actively conducted (see Non-Patent Document 5).
[0009]
Polyamide was produced by purifying free diamine and free dicarboxylic acid as raw materials and then mixing them in an equimolar amount to carry out a polymerization reaction. Since conventional diamines and dicarboxylic acids use petroleum-derived raw materials, free diamines and dicarboxylic acids were obtained without using any solvent (see Non-Patent Document 6). Therefore, it was not necessary to examine the crystallization of diamine / dicarboxylic acid from an aqueous solution.
[0010]
However, in order to produce a polyamide raw material by a fermentation or enzymatic method different from the chemical synthesis method, it is common to use water as a solvent, and to perform the fermentation or enzymatic method, the pH of the solution must be adjusted. Generally necessary. As these neutralizing agents, alkali metal hydroxides or inorganic acids are generally used, and diamines or dicarboxylic acids are crystallized as salts thereof. For example, in the manufacturing method by fermentation of succinic acid which is a dicarboxylic acid, it crystallizes and refine | purifies as calcium succinate (refer patent document 4).
[0011]
However, no practical production technique has been established for the production of cadaverine dicarboxylate, and development of a method for producing high-purity cadaverine dicarboxylate with little by-product waste is desired.
[0012]
[Patent Document 1]
JP-A-60-23328, pages 11-12
[Patent Document 2]
JP 2002-223770 A, page 5
[Patent Document 3]
JP-A-2002-223771 pages 4-5
[Patent Document 4]
Japanese Patent Application Laid-Open No. 62-294090, page 6
[Non-Patent Document 1]
Masaru Suyama, Kiyozo Kaneo, “Decarboxylation of Amino Acids (Part 4)”, Pharmaceutical Journal, 1965, Vol. 85, No. 6, p. 531-533
[Non-Patent Document 2]
Mitsunori Hashimoto, 4 others, “Chemistry letters”, 1986, p. 893-896
[Non-Patent Document 3]
Celia White Taber, Herbert Taber, “Microbiological Reviews”, 1985, Vol. 49, p. 81-99
[Non-Patent Document 4]
Shi-Yuannmeng, 1 other, “Journal of Bacteriology”, 1992, 174, p. 2659-2669
[Non-Patent Document 5]
George Georgeou, five others, “Protein Engineering”, 1996, Vol. 9, No. 2, p. 239-247
[Non-Patent Document 6]
Mitsuaki Mukaiyama, “Industrial Organic Chemistry”, 4th edition, Tokyo Chemical Doujin, p. 270-273, 275-281
[0013]
[Problems to be solved by the invention]
When cadaverine dicarboxylate containing a large amount of tri-n-butylamine and 2,3,4,5-tetrahydropyridine is used as the raw material of the polymer, for example, the polyamide is heated from the raw material containing cadaverine dicarboxylate. When synthesizing by condensation, since the reaction temperature is high, basic compounds such as tri-n-butylamine and 2,3,4,5-tetrahydropyridine contained in cadaverine dicarboxylate decompose the polyamide. There is a problem that the heat resistance is insufficient in the polyamide obtained.
[0014]
In addition, tri-n-butylamine is toxic to aquatic organisms and has a problem that the lethal dose to fish is 20-40 mg / l.
[0015]
When hydrochloric acid is used as a neutralizing agent when L-lysine is produced by fermentation, and hydrochloric acid is used as a neutralizing agent when producing cadaverine from the fermentation broth by an enzymatic method, cadaverine is crystallized as dihydrochloride. Will do. Then, cadaverine dihydrochloride needs to be freed by an ion exchange method, an extraction method, or the like, which complicates the process, necessitates a solvent or a disposal process for by-products.
[0016]
In addition, the fermentation apparatus and the enzyme preparation apparatus are generally performed in separate reactors, and the maintenance and management of the apparatus become complicated.
[0017]
The fermentation broth contains amino acids, but the solubility of amino acids in water is high, and the solubility of cadaverine dicarboxylate in water is also high. Therefore, cadaverine dicarboxylate cannot be crystallized directly from the aqueous solution.
[0018]
If cadaverine dicarboxylate contains an amino acid when polymerized to polyamide, the amino acid is polymerized to cause polymer branching or to lower the crystallinity of the polymer.
[0019]
The object of the present invention is to use such a low-impurity cadaverine dicarboxylate in a simpler production process without using an alkali metal hydroxide or an inorganic acid as a neutralizing agent during fermentation and enzymatic reaction. It is to provide a method for producing cadaverine dicarboxylate from an aqueous solution.
[0020]
[Means for Solving the Problems]
In order to solve the above problems, the present inventors conducted extensive research on a method for producing cadaverine dicarboxylate, and as a result, caused L-lysine decarboxylase to act on an L-lysine dicarboxylate aqueous solution. It was found that cadaverine dicarboxylate can be obtained efficiently.
[0021]
  That is, the present invention
“(1) A method for producing cadaverine dicarboxylate characterized in that L-lysine decarboxylase is allowed to act on an aqueous solution of L-lysine dicarboxylate to isolate cadaverine dicarboxylate from the reaction solution.
(2) Method (1) aboveFor polyamide raw materials obtained byCadaverine dicarboxylateHeat polycondensationpolyamideManufacturing method. It is.
[0022]
DETAILED DESCRIPTION OF THE INVENTION
Cadaverine is 1,5-pentanediamine and is a useful compound as a polymer raw material.
[0023]
In the present invention, the L-lysine decarboxylase is not limited as long as it is at least an enzyme that converts L-lysine into cadaverine, and also has an enzyme action other than L-lysine decarboxylizing ability. May be. However, in that case, in the present invention, it is not preferable to cause a side reaction or the like that greatly impedes the production of cadaverine dicarboxylate. It is known that the L-lysine decarboxylase is present not only in microorganisms belonging to the genus Escherichia, including Escherichia coli K12, but also in many organisms.
[0024]
The L-lysine decarboxylase used in the present invention is not particularly limited. For example, Bacillus halodurans, Bacillus subtilis, Escherichia coli, Selenomonas luminantium ( Selenomonas ruminantium, Vibrio cholerae, Vibrio parahemolyticus, Streptomyces coelikara, Streptomyces coelicolor, Streptomyces coelicolor rodens), Eubacterium acidaminophilum, Salmonella typhimurium, Hafnia alvide, Neisseria menisumide, Neisseria plasma. Those derived from Pyrococcus abyssi are preferably used. More preferably, it is derived from Escherichia coli that has been confirmed to be safe.
[0025]
Since the product in the present invention is cadaverine, which is an alkaline compound, the pH of the reaction solution changes to the alkali side as the reaction proceeds. However, if L-lysine decarboxylase is allowed to act on an L-lysine / dicarboxylate aqueous solution, cadaverine can be efficiently produced without the need to control the pH of the reaction solution.
[0026]
In the lysine fermentation, it is preferable to use ammonia and dicarboxylic acid for adjusting the culture pH, and it is preferable to control the culture pH to 5-8, particularly preferably pH 7, using these neutralizing agents. In addition, there is no restriction | limiting in the state of a neutralizing agent, It uses by gas, a liquid, solid, or aqueous solution. Particularly preferred is an aqueous solution.
[0027]
In the present invention, the L-lysine / dicarboxylate aqueous solution is preferably a fermentation broth obtained by culturing L-lysine fermenting microorganisms, and more preferably, when the L-lysine fermenting microorganisms are cultured, It is a fermentation broth performed while adjusting the pH. At that time, the microorganism used as the cell having L-lysine decarboxylase activity is not particularly limited, and is preferably a microorganism belonging to the genus Corynebacterium or the genus Brevibacterium, more preferably Corynebacterium glutamicum.
[0028]
As the medium to be used, a medium containing a carbon source, a nitrogen source, inorganic ions, and other organic components as required is used. For example, carbon sources include glucose, lactose, galactose, fructose, arabinose, maltose, xylose, trehalose, sugars such as ribose and starch hydrolysates, alcohols such as glycerol, mannitol and sorbitol, gluconic acid, fumaric acid, Organic acids such as citric acid and succinic acid can be used. As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen such as soybean hydrolysate, urea, ammonia gas, aqueous ammonia and the like can be used. As organic micronutrients, it is desirable to contain appropriate amounts of various substances, required substances such as vitamins such as vitamin B1, nucleic acids such as RNA, or yeast extract. In addition to these, small amounts of calcium phosphate, calcium sulfate, iron ions, manganese ions, etc. are added as inorganic ions as necessary. Examples of the carbon source, nitrogen source, organic micronutrients, and inorganic ions listed above are hereinafter referred to as typical medium component groups.
[0029]
More preferably, in the case of Corynebacterium glutamicum, magnesium sulfate, citric acid, iron sulfate, sodium chloride, calcium chloride, potassium phosphate, potassium hydrogen phosphate, urea, L-threonine, L-methionine, L-leucine, biotin And a medium containing thiamine.
[0030]
The culture conditions are not particularly limited. For example, in the case of Corynebacterium glutamicum, the culture is preferably performed for about 16 to 150 hours under aerobic conditions, and the culture temperature is 25 ° C to 42 ° C, particularly preferably 30 ° C. is there.
[0031]
The dicarboxylic acid used as the neutralizing agent is not particularly limited, and is preferably an aliphatic and / or aromatic dicarboxylic acid having only two carboxyl groups as functional groups. An aliphatic or aromatic dicarboxylic acid having only two carboxyl groups as a functional group is one having substantially no functional group other than the two carboxyl groups. The functional group here is a polyamide polymerization reaction (reaction conditions are, for example, a reaction temperature of 250 to 270 ° C., a pressure of 10 to 20 kg / cm²The reaction time is 1 to 5 hours), and the group reacts with an amino group or a carboxyl group to cause branching of the polymer or to lower the crystallinity of the polymer (the crystallinity is 80% or less). . For example, an amino group or a carboxyl group corresponds to this, but other than that, an acidic group (sulfonic acid group, phosphoric acid group, phenolic hydroxyl group, etc.), a basic group (hydrazino group, etc.) or a protonic polar group ( A hydroxyl group or the like), a cleavable group (such as an epoxy group or a peroxide group), or other highly reactive groups (such as an isocyanate group). On the other hand, halogen substituents and aromatic substituents are relatively stable, ethers, esters, amides, and the like are also stable and often have low functionality.
[0032]
The dicarboxylic acid is more preferably a dicarboxylic acid represented by the following general formula (1), (2) or (3).
HOOC- (CH₂)_m-COOH (1)
(However, in general formula (1), m = 0 to 16)
[0033]
[Chemical 3]

[0034]
[Formula 4]

[0035]
(However, in the general formulas (2) and (3), n, o, p, q = 0 to 5)
More preferably
In general formula (1),
m = 0-10
And / or in the general formula (2) and / or (3),
n, o, p, q = 0 to 1
It is.
[0036]
Still more preferred are adipic acid, sebacic acid, 1,12-dodecanedicarboxylic acid, succinic acid, isophthalic acid and terephthalic acid.
[0037]
There is no limitation on the molar ratio of L-lysine and dicarboxylic acid in the aqueous solution. When L-lysine forms a salt with hydrochloric acid, L-lysine hydrochloride is formed at a molar ratio of 1: 1. Therefore, in the state of L-lysine dicarboxylate, the molar ratio of L-lysine and dicarboxylic acid should be 1: 0.5 in the aqueous solution. However, in order to adjust the L-lysine decarboxylase to be used so that it is close to the optimum pH, dicarboxylic acid is further added (L-lysine: dicarboxylic acid = 1: 0.55-0.75 [molar ratio]). It is preferable to do. Or when it is necessary to add an alkali, it is preferable to adjust with ammonia.
[0038]
Although L-lysine decarboxylase is inherently bound to vitamin B6, the production rate and reaction yield can be improved by adding vitamin B6 to the L-lysine / dicarboxylate aqueous solution. There is no restriction | limiting in particular in the method of adding vitamin B6. You may add suitably during reaction. If L-lysine decarboxylase is in a solution state, it is preferably added to the enzyme solution. There is no restriction | limiting in particular about the density | concentration of vitamin B6 in a reaction liquid. Preferably, the vitamin B6 concentration of 0.05 mM may be added to the reaction solution. There is no restriction | limiting in particular in the vitamin B6 to be used. Preferably, it is at least one selected from pyridoxine, pyridoxamine, pyridoxal and pyridoxal phosphate. More preferred is pyridoxal phosphate.
[0039]
The L-lysine decarboxylase or the cell having L-lysine decarboxylase activity used in the present invention is not particularly limited. For example, recombination in which L-lysine decarboxylase is highly expressed in the cell. Examples include cells obtained by culturing cells (intracellularly expressed recombinant cells) and the like in an appropriate medium, and recovering the proliferated cells by a usual method such as centrifugation or further enzyme separation.
[0040]
Apart from this, recombinant cells in which L-lysine decarboxylase is localized on the cell surface (cell surface-expressed recombinant cells) and the like are cultured in an appropriate medium, and the proliferated cells may be recovered.
[0041]
A recombinant cell in which L-lysine decarboxylase is localized on the cell surface is a state in which at least L-lysine decarboxylase is localized on the cell surface. May exist.
[0042]
On the other hand, specific methods for localizing L-lysine decarboxylase on the cell surface include, for example, a part of a secretory signal sequence, a gene sequence encoding a part of a cell surface localized protein, and L-lysine desorption. What is necessary is just to introduce | transduce into the colon_bacillus | E._coli DNA which has the structural gene sequence of a carbonic acid enzyme in this order.
[0043]
There is no restriction on part of the secretory signal sequence. Any sequence may be used as long as it is necessary for secreting the protein in the host. In Escherichia coli, for example, it is a part of a lipoprotein sequence. Specifically, it is preferably a gene sequence translated as MKATKLVLGAVIGLSTLLAGCSSSNAKIDQ (amino acid one letter code) as an amino acid sequence.
[0044]
There is no particular limitation on the gene sequence encoding a part of the cell surface localized protein. In E. coli, for example, it is a part of the sequence of the outer membrane binding protein, and specifically, it is preferably a part of the sequence from the 46th amino acid to the 159th amino acid of OmpA (outer membrane binding protein).
[0045]
There is no particular limitation on the method for cloning the L-lysine decarboxylase gene, lipoprotein gene and OmpA gene. Based on known gene information, PCR (polymerase chain reaction) method is used to amplify and acquire necessary genetic regions, cloning based on known gene information from genomic and cDNA libraries using homology and enzyme activity as indicators The method of doing is mentioned. For example, the cadA gene or the ldc gene, which is a gene encoding L-lysine decarboxylase, is cloned from the chromosomal DNA of Escherichia coli K12 strain by PCR. The chromosomal DNA used here may be derived from any strain as long as it is derived from Escherichia coli. In the present invention, these genes include mutants due to genetic polymorphism and the like. Genetic polymorphism means that the base sequence of a gene is partially changed due to a natural mutation on the gene.
[0046]
Confirmation that L-lysine decarboxylase is localized on the cell surface is performed by, for example, embedding and slicing cell surface-expressed recombinant cells after immunoreaction with an antibody prepared using L-lysine decarboxylase as an antigen. This can be confirmed by observation with an electron microscope (immunoelectron microscopy).
[0047]
In the present invention, as L-lysine decarboxylase, those prepared from recombinant cells in which the activity of L-lysine decarboxylase in the cell or on the cell surface is preferably used. There is no particular limitation on the method for increasing the activity of L-lysine decarboxylase in the cell or on the cell surface. Specifically, for example, a method for increasing the enzyme amount of L-lysine decarboxylase, or introducing a mutation into the enzyme structural gene itself to increase the specific activity of the enzyme itself can be mentioned.
[0048]
Examples of means for increasing the amount of enzyme in the cell or on the cell surface include improving the transcriptional regulatory region of the gene, increasing the copy number of the gene, and increasing the efficiency of translation into a protein.
[0049]
Improving the transcriptional regulatory region means adding a modification that increases the amount of gene transcription. For example, the promoter can be strengthened by introducing a mutation into the promoter, and the transcription amount of the downstream gene can be increased. In addition to introducing mutations into the promoter, a promoter that is strongly expressed in the host may be introduced. For example, in E. coli, promoters such as lac, tac, and trp can be mentioned. In addition, the amount of gene transcription can be increased by newly introducing an enhancer. The introduction of a gene such as a chromosomal DNA promoter is described in, for example, JP-A-1-215280.
[0050]
Specifically, the increase in the copy number of a gene can be achieved by connecting the gene to a multi-copy type vector to produce a recombinant DNA and allowing the host cell to hold the recombinant DNA. Here, the vector includes those widely used such as plasmids and phages, but besides these, transposon (Burg, DE (Berg, DE), one other person, “Bio / Technology ( Bio / Technology) ", 1983, Vol. 1, p. 417) and Mu phage (Japanese Patent Laid-Open No. 2-109985). It is also possible to increase the copy number by incorporating a gene into a chromosome by a method using a plasmid for homologous recombination.
[0051]
Methods for increasing the translation efficiency of proteins include, for example, introduction and modification of SD sequences in prokaryotes and Kozak consensus sequences in eukaryotes, and optimization of codons used (Japanese Patent Laid-Open No. 59-125895). Etc.
[0052]
As a means for increasing the activity of L-lysine decarboxylase in the cell or on the cell surface, a mutation is introduced into the structural gene itself of L-lysine decarboxylase to increase the activity of L-lysine decarboxylase itself. Can also be mentioned.
[0053]
In order to cause mutations in genes, site-specific mutagenesis (Kramer, W.), 1 other, “Methods in Enzymology”, 1987, Vol. 154, p. 350), recombinant PCR method, method of chemically synthesizing a specific portion of DNA, or a method of treating the gene with hydroxyamine or a strain carrying the gene with ultraviolet irradiation, or chemistry such as nitrosoguanidine or nitrous acid There is a method of treating with drugs.
[0054]
As the recombinant cell, those derived from microorganisms, animals, plants, or insects can be preferably used. For example, when animals are used, mice, rats and cultured cells thereof are used. When plants are used, for example, Arabidopsis thaliana, tobacco and cultured cells thereof are used. In addition, when insects are used, for example, silkworms and cultured cells thereof are used. In addition, when microorganisms are used, for example, E. coli is used.
[0055]
Moreover, you may use L-lysine decarboxylase in combination of multiple types.
[0056]
In order to obtain L-lysine decarboxylase, there is no particular limitation on the method for culturing recombinant cells. For example, when culturing microorganisms, the medium to be used is a carbon source, a nitrogen source, inorganic ions, and other as required. A medium containing organic components is used. For example, in the case of E. coli, LB medium is often used. As a preferable component, the typical medium component group can be mentioned.
[0057]
A more preferable medium is a fermentation broth after performing L-lysine fermentation. Nutrients necessary for obtaining L-lysine decarboxylase may be further added to this fermentation broth.
[0058]
The culture conditions are not particularly limited. For example, in the case of Escherichia coli, the culture is preferably performed for about 16 to 72 hours under aerobic conditions, the culture temperature is 30 ° C. to 45 ° C., particularly preferably 37 ° C. It is good to control to 5-8, Most preferably, pH7. In addition, an inorganic or organic acidic or alkaline substance, ammonia gas or the like can be used for pH adjustment.
[0059]
When a host that highly expresses L-lysine decarboxylase assimilates L-lysine, cadaverine, or dicarboxylic acid, it is preferable to purify and use L-lysine decarboxylase.
[0060]
For example, a normal method is used to prepare a cell disruption solution from the collected intracellular-expressed recombinant cells. That is, a cell disruption solution is obtained by disrupting intracellularly expressed recombinant cells by a method such as ultrasonic treatment, dynomill, French press, etc., and removing cell residues by centrifugation.
[0061]
In order to purify L-lysine decarboxylase from the cell lysate, enzyme such as ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration chromatography, isoelectric precipitation, heat treatment, pH treatment, etc. Techniques usually used for purification are used in appropriate combination.
[0062]
Industrially, the purification method should be simple, and it is preferable to act on partially purified L-lysine decarboxylase. Partial purification means performing only industrially easy treatment, and examples thereof include heat treatment and pH treatment. Heat treatment is preferred. More preferably, the isolated and purified L-lysine decarboxylase is allowed to act. By partially purifying or isolating and purifying L-lysine decarboxylase, an enzyme that causes degradation of L-lysine, cadaverine, or dicarboxylic acid can be removed.
[0063]
The purified L-lysine decarboxylase used in the present invention can be repeatedly used for cadaverine synthesis reaction by immobilization, and the cost required for enzyme preparation can be reduced. Examples of the immobilization method include a method encompassing synthetic polymers such as acrylamide, a method of adsorbing to an ion-exchange carrier or a hydrophobic carrier having a skeleton of Sepharose or styrene resin, or a method of covalently bonding to a glass carrier. Is mentioned.
[0064]
Furthermore, if cells in which L-lysine decarboxylase is localized on the cell surface are allowed to act, degradation of L-lysine and cadaverine can be suppressed, and cadaverine can be obtained at a higher production rate. Furthermore, the cells can be collected and used repeatedly for enzyme reactions. Moreover, the enzyme preparation is very simple.
[0065]
When these cells having L-lysine decarboxylase activity are entrapped and immobilized with a gel carrier, the cells and the enzyme are stabilized, and the enzyme can be used repeatedly easily.
[0066]
There is no restriction | limiting in particular as a gel-like support | carrier. High molecular polysaccharides or synthetic polymers may be used, and high molecular polysaccharides are preferred. More preferably, it is any one of alginic acid, copper carrageenan or polyacrylamide, and more preferably copper carrageenan.
[0067]
There is no particular limitation on the method of fixing inclusive. It is preferable to carry out under mild conditions such that L-lysine decarboxylase activity does not decrease.
[0068]
There is no restriction | limiting in particular in the quantity of the gel-like support | carrier fixed inclusive. It suffices if all the cells having adjusted L-lysine decarboxylase activity can be comprehensively fixed.
[0069]
In general, an enzyme is an amino acid sequence that should originally exist and exists in nature. Therefore, when an amino acid sequence is artificially added or lost, the activity of the enzyme is often lost. However, in the present invention, when L-lysine decarboxylase is expressed in cells, L-lysine decarboxylase in which 1 to 30 amino acid sequences are added to the N-terminal amino acid sequence of L-lysine decarboxylase can be preferably used. . Here, 6 to 10 histidine residues are preferable for the N-terminal. Thus, L-lysine decarboxylase can be easily purified by adding an amino acid to the N-terminal amino acid sequence. There is no particular limitation on the method of adding 1 to 30 amino acids to the N-terminal amino acid sequence. For example, the base sequence corresponding to the amino acid sequence to be added to the L-lysine decarboxylase gene may be recombined using a genetic engineering technique. There are no particular restrictions on the genetic engineering technique used. A PCR method and a method by linking DNA fragments are preferable.
[0070]
The conversion of the L-lysine dicarboxylate aqueous solution into cadaverine by L-lysine decarboxylase is performed by bringing the L-lysine decarboxylase obtained as described above into contact with the L-lysine dicarboxylate aqueous solution. It can be carried out.
[0071]
There is no particular limitation on the amount of L-lysine decarboxylase used. The amount may be sufficient to catalyze the reaction for converting the L-lysine / dicarboxylate aqueous solution into the cadaverine / dicarboxylate aqueous solution. Preferably, the enzyme concentration in the reaction solution is 25 to 70 mg / l. Preferably it is 50 mg / l. On the other hand, when resting cells are used, the cell concentration in the reaction solution is 5 to 15 g / l. Preferably it is 10 g / l.
[0072]
In the present invention, the cell having L-lysine decarboxylase activity or the activity of L-lysine decarboxylase is determined under the conditions of Example 6 or Reference Example 1 (2) described later, JM109 / pTV118N strain or cell disruption thereof. The activity of the liquid is preferably 10 (more preferably 50) times or more.
[0073]
The reaction temperature is usually 28 to 55 ° C, preferably 35 to 48 ° C.
[0074]
There is no restriction | limiting in particular in the state of a reaction liquid. Preferably, it is left standing or stirred. There is no particular limitation on the method of stirring. A method of stirring with a stirring blade is preferred.
[0075]
The reaction time varies depending on conditions such as enzyme activity and L-lysine / dicarboxylate concentration used, but is usually 1 to 72 hours. The reaction may be carried out continuously while supplying L-lysine dicarboxylate.
[0076]
As a method for collecting the cadaverine dicarboxylate thus produced from the reaction solution after completion of the reaction, normal isolation or crystallization may be used. For example, the reaction solution is preferably concentrated and an organic solvent is added. Can be collected by the crystallization operation.
[0077]
It is preferable to adjust cadaverine and dicarboxylic acid in the aqueous solution to equimolarity before the crystallization operation. More preferably, the dicarboxylic acid is added in excess.
[0078]
In the present invention, the organic solvent added to the reaction solution is preferably an alcohol, a ketone or a nitrile. More preferred are aliphatic alcohols having 6 or less carbon atoms, aliphatic ketones having 6 or less carbon atoms, or aliphatic nitriles having 6 or less carbon atoms.
[0079]
For example, specific examples of the organic solvent include methanol, ethanol, propanol, butanol, hexanol, isopropanol, acetonitrile or acetone.
[0080]
In addition, the organic solvent does not need to be single, and may be a mixed solvent. For example, an ethanol / acetone mixed solution may be used.
[0081]
The amount of the organic solvent used in the present invention is not particularly limited, but is usually 0.1 to 5 times the weight of the aqueous solution containing cadaverine dicarboxylate, and 0.3 to 0.3 to effectively carry out the present invention. Twice is used. Alternatively, it is preferably 1 to 10 (more preferably 3 to 7) times the weight of cadaverine dicarboxylate.
[0082]
The contact method of the aqueous solution containing cadaverine dicarboxylate and the organic solvent is arbitrary, and may be a batch method or a continuous method.
[0083]
In order to perform crystallization efficiently and to improve the pot efficiency, it is preferable to concentrate the aqueous solution containing cadaverine dicarboxylate. The degree of concentration is preferably such that the inorganic salt and cadaverine dicarboxylate do not precipitate as crystals. Although the degree of concentration varies depending on the concentration of cadaverine dicarboxylate in the aqueous solution and other salt concentrations, it is preferable to concentrate to 1/4 to 1/30 on a weight basis, and to 1/10 to 1/20. Is more preferable.
[0084]
The concentration method may be either normal pressure concentration or reduced pressure concentration, but the liquid temperature at the time of concentration is usually performed below the boiling point of water. With such a liquid temperature, there is no possibility that cadaverine dicarboxylate is decomposed.
[0085]
The cadaverine dicarboxylate thus produced may be considered as a salt composed of one molecule of cadaverine and one molecule of dicarboxylic acid, but does not exclude any other form. A salt or the like formed from two molecules of carboxylic acid may be contained.
[0086]
The purification method of the present invention is particularly effective for purification when an aqueous solution containing cadaverine dicarboxylate contains amino acids as impurities. There is no restriction | limiting in particular in the amino acid contained. Preferably, uncharged / polar amino acids (glycine, L-serine, L-threonine, L-cysteine, L-tyrosine, L-asparagine and L-glutamine), acidic amino acids (L-aspartic acid and L-glutamic acid) and bases It is particularly effective for purification in the case where any of the amino acids (L-lysine, L-arginine and L-histidine) is contained. More preferably, any of L-aspartic acid, L-glutamic acid and L-lysine is contained. In the present invention, the concentration of the amino acid contained in the aqueous solution is preferably 0.01 to 1% by weight. If the value falls below the lower limit of the numerical range, cadaverine may be difficult to produce. On the other hand, if the value exceeds the upper limit, isolation and crystallization may become difficult.
[0087]
Next, an organic solvent is added to the cadaverine dicarboxylate aqueous solution concentrated in the concentration step, and the precipitated crystals are removed by an ordinary solid-liquid separation method such as centrifugation, and cadaverine dicarboxylate is usually removed from the organic solvent. The cadaverine dicarboxylate is isolated by the following crystallization separation operation. For example, after cooling the concentrated liquid added with the organic solvent as it is to crystallize cadaverine dicarboxylate, the crystal is isolated by a usual solid-liquid separation method such as centrifugation, so that high purity cadaverine dicarboxylate is obtained. Can be obtained.
[0088]
The organic solvent separated from cadaverine dicarboxylate in the crystallization process is preferably recycled to the crystallization operation.
[0089]
The cadaverine dicarboxylate thus isolated is again obtained by a known crystallization purification method, that is, after making a homogeneous solution with an organic solvent, the homogeneous solution is cooled, or the filtrate that has been filtered hot is cooled. Cadaverine dicarboxylate can be crystallized to produce a high purity product.
[0090]
The cadaverine dicarboxylate thus obtained is low in impurities such as tri-n-butylamine and 2,3,4,5-tetrahydropyridine. When produced by the method of the present invention, when analyzed by the following method, a catavelin dicarboxylate having a tri-n-butylamine content of 0.006 wt% or less is obtained. Furthermore, when the following method is used for analysis, a 2,3,4,5-tetrahydropyridine content of 1.4 wt% or less is obtained. Furthermore, the tri-n-butylamine content is 0.006 wt% or less and the 2,3,4,5-tetrahydropyridine content is 1.4 wt% or less.
[0091]
Analysis of tri-n-butylamine and 2,3,4,5-tetrahydropyridine is carried out under the following conditions by the GC-MS method.
[0092]
GC-MS: HP6980 / HP5973A
Column: NUKOL 30m x 0.24mmI. D. 0.2μm Film
Oven: 120 ° C (constant)
Inject: 200 ° C. (Split 10: 1)
Flow rate: He 2.4 ml / min (constant)
MS: 230 ° C. (SCAN m / z = 30 to 400)
The cadaverine dicarboxylate thus obtained is useful as a raw material for polyamide.
[0093]
As a method for producing polyamide, a heating polycondensation method is used in which a mixture of cadaverine dicarboxylate and water thus obtained is heated to advance a dehydration reaction. It is also possible to increase the molecular weight by solid phase polymerization after heat polycondensation. Solid-phase polymerization proceeds by heating in a temperature range from 100 ° C. to the melting point in a vacuum or in an inert gas, and polyamide having an insufficient molecular weight by heating polycondensation can be increased in molecular weight.
[0094]
By using the cadaverine dicarboxylate obtained in the present invention as a raw material for polyamide, a polyamide having high heat resistance can be obtained.
[0095]
【Example】
Examples Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the description of these examples.
[0096]
[Analysis method of L-lysine concentration and cadaverine concentration by HPLC]
Column used: CAPCELL PAK C18 (Shiseido)
Mobile phase: 0.1% (w / w) phosphoric acid aqueous solution: acetonitrile = 4.5: 5.5
Detection: UV360nm
Sample pretreatment: 25 μl of analytical sample, 25 μl of 1,4-diaminobutane (0.03 M), 150 μl of sodium bicarbonate (0.075 M) as an internal standard, ethanol of 2,4-dinitrofluorobenzene (0.2 M) The solution is added and mixed and incubated at 37 ° C. for 1 hour. After dissolving 50 μl of the reaction solution in 1 ml acetonitrile, 10 μl of the supernatant after centrifugation at 10,000 rpm for 5 minutes was analyzed by HPLC.
[0097]
[Analytical HPLC analysis of adipic acid and succinic acid concentrations]
Column used: SCR-101H (Shimadzu Corporation)
Mobile phase: 0.025% (v / v) sulfuric acid aqueous solution
Detection: UV214nm
Sample pretreatment: The analysis sample was diluted with water so that the calibration curve had a concentration within a range where linearity was possible, and 10 μl was subjected to HPLC analysis.
[0098]
Reference Example 1 (adjustment of L-lysine decarboxylase)
(1) Cloning of L-lysine decarboxylase gene and preparation of intracellular expression vector
In order to convert L-lysine dicarboxylate to cadaverine dicarboxylate using L-lysine decarboxylase, the L-lysine decarboxylase gene (cadA) of Escherichia coli was cloned.
[0099]
Reference is made to the nucleotide sequence of the L-lysine decarboxylase gene (Accession No. M76411) registered in the database (GenBank), and the oligonucleotide primer 5'-atgaacgtttgcaatattg-3 '(SEQ ID NO: 1), 5'-gctgatgggtgagatagatg- 3 ′ (SEQ ID NO: 2) was synthesized. 0.2 μl of a genomic DNA solution prepared from Escherichia coli K12 strain (ATCC10798) according to a conventional method is used as an amplification template in a 0.2 ml microcentrifuge tube. ), 2.5 mM potassium chloride, 100 μg / ml gelatin, 50 μM each dNTP, 2 units LATaq DNA polymerase (manufactured by Takara Shuzo) was added to each reagent to make a total volume of 50 μl. The DNA denaturation conditions were 94 ° C. for 30 seconds, the primer annealing conditions were 55 ° C. for 30 seconds, the DNA primer extension reaction conditions were 72 ° C. for 3 minutes, and each cycle was carried out for 30 cycles using a BioRad thermal cycler. (Polymerase chain reaction: hereinafter referred to as PCR method). The PCR method in this example was performed under these conditions unless otherwise specified. The product obtained by this PCR method was electrophoresed with 1% agarose, and a DNA fragment of about 2.1 kb containing the cadA gene was prepared according to a conventional method. This fragment was inserted into a gap in which T base was added to the 3′-end of the EcoRV site of plasmid vector pT7blue (manufactured by Novagen) by a ligation reaction according to a conventional method, and the resulting plasmid was designated as pT7-cadA. Named.
[0100]
Using this pT7-cadA as an amplification template, oligonucleotide 5'-cgccatgggcccatcatcatcatcatcatcatatagaacgtttattgcaatattg-3 '(SEQ ID NO: 3) and 5'-cgcgatccgctgagtgggtgagatag4) (primer set: sequence number: PCR) The product obtained here has a nucleotide sequence derived from an oligonucleotide (SEQ ID NO: 3) and a nucleotide sequence derived from an oligonucleotide (SEQ ID NO: 4), each having a 5 ′ end and a 3 ′ It is added to the end. This product was electrophoresed with 1% agarose, and a DNA fragment of about 2.1 kb was prepared according to a conventional method. This fragment was inserted into a gap in which T base was added to the 3′-end of the EcoRV site of plasmid vector pT7blue (manufactured by Novagen) by a ligation reaction according to a conventional method, and the resulting plasmid was designated as pT7-cadA1. Named.
[0101]
This pT7-cadA1 was digested with NcoI and BamHI, and the obtained 2.1 kb NcoI-BamHI fragment was digested with NcoI / BamHI of pTV118N (Takara Shuzo) previously digested with NcoI and BamHI. The resulting plasmid was named pLDC1 (FIG. 1). By culturing Escherichia coli into which this plasmid has been introduced, a recombinant L-lysine decarboxylase having a molecular weight of about 80 kDa in which 6 histidine residues are added to the N-terminal amino acid sequence of L-lysine decarboxylase can be produced it can.
[0102]
(2) Production of recombinant L-lysine decarboxylase
Escherichia coli JM109 strain was transformed to ampicillin resistance with pLDC1, and the resulting transformant was named JM109 / pLDC1 strain.
[0103]
Next, recombinant L-lysine decarboxylase was produced in the JM109 / pLDC1 strain. First, 1 platinum ear of the JM109 / pLDC1 strain was inoculated into 5 ml of sterilized LB medium (Nacalai Tesque) (LB-amp medium) containing 50 μg / ml ampicillin sodium and shaken at 37 ° C. for 24 hours. Then, pre-culture was performed to obtain a pre-culture solution.
[0104]
This pre-cultured solution was inoculated in a total volume of 50 ml of LB-amp medium, cultured at 37 ° C., amplitude of 30 cm, and 180 rpm for 3 hours, and then IPTG (isopropyl-1-thio-β-D-galactoside) (1 mM) The mixture was added and further cultured for 4 hours. As a control experiment, the same culture was performed using a transformant obtained by transforming the JM109 strain with pTV118N (hereinafter referred to as JM109 / pTV118N strain). The bacterial cells thus obtained were collected, resuspended in 5 ml of TBS buffer (Takara Shuzo), and a cell disruption solution was prepared by ultrasonic disruption and centrifugation. These L-lysine decarboxylase activities were measured by measuring the concentration of cadaverine produced per unit time. As a result, the L-lysine decarboxylase activity increased about 100 times in the JM109 / pLDC1 strain compared to the cell lysate derived from the control experiment JM109 / pTV118N strain. Further, this cell lysate was fractionated by SDS-PAGE and subjected to Western blotting with a Penta-His Antibody antibody (QIAGEN). As a result, recombination with a molecular weight of about 80 kDa was obtained only from the cell lysate derived from the JM109 / pLDC1 strain. L-lysine decarboxylase was detected.
[0105]
(3) Purification of recombinant L-lysine decarboxylase
This recombinant L-lysine decarboxylase was purified using interaction with nickel ions because of the 6 histidine residues in the N-terminal amino acid sequence.
First, a column system packed with 10 ml of chelating Sepharose Fast Flow carrier (manufactured by Amersham Bioscience) was constructed. After flowing 50 ml of 50 mM nickel sulfate aqueous solution and 50 ml of TBS buffer in this order, 50 ml cell lysate derived from 500 ml culture of JM109 / pLDC1 obtained in the same manner as in (2) was flowed. . Thereafter, a TBS buffer containing 100 ml of imidazole (5 mM) and a TBS buffer containing 100 ml of imidazole (50 mM) were run in this order. Further, a TBS buffer containing 50 ml of imidazole (600 mM) was flowed. When the L-lysine decarboxylase activity of each buffer passed through the column was measured by the same method as in (2), only the TBS buffer containing imidazole (600 mM) was active. In addition, each buffer solution passed through the column was subjected to SDS-PAGE and stained with Coomassie Brilliant Blue, and a single band of about 80 kDa was detected from the TBS buffer containing imidazole (600 mM). Moreover, when each buffer solution passed through the column was subjected to Western blotting in the same manner as in (2), an approximately 80 kDa protein was detected only in a TBS buffer solution containing imidazole (600 mM). The recombinant L-lysine decarboxylase having lysine decarboxylase activity was confirmed.
[0106]
(4) Preparation of cell surface expression vector of L-lysine decarboxylase
First, a part of the lipoprotein sequence and the sequence from the 46th amino acid to the 159th amino acid of the outer membrane associated protein (OmpA) were cloned as one cassette.
[0107]
That is, with reference to the base sequence of outer membrane binding protein (ompA) (Accession No. NC_000913) registered in the database (GenBank), the oligonucleotide primer 5'-ataaagctttagaatactactaactggg-3 '(SEQ ID NO: 5) 5'-atagcgacgtgtgtgtgt -3 ′ (SEQ ID NO: 6), 5′-ataaagctttagagaagctactaaaactggtactggggcgcggtaatcctggtttctactctctctggcaggttgctccagcaacctctaaaatccatacatc PCR was carried out using a genomic DNA solution prepared from Escherichia coli K12 strain according to a conventional method as an amplification template and using (SEQ ID NO: 5) (SEQ ID NO: 6) (SEQ ID NO: 7) as a primer set. The product obtained by this PCR method was electrophoresed with 2% agarose, and a DNA fragment of about 0.4 kb containing the ompA gene was prepared according to a conventional method. This fragment was inserted into a gap in which T base was added to the 3′-end of the EcoRV site of plasmid vector pT7blue (manufactured by Novagen) by a ligation reaction according to a conventional method, and the resulting plasmid was designated as pTM7. .
[0108]
This pTM7 was digested with HindIII and SalI, and the resulting 0.4 kb HindIII and SalI fragment was routinely inserted into the HindIII / SalI gap of pUC19 (Takara Shuzo) previously digested with HindIII and SalI. The resulting plasmid was inserted by a ligation reaction, and the resulting plasmid was named pTM14.
[0109]
Next, the L-lysine decarboxylase gene (cadA) of Escherichia coli K12 strain was cloned.
[0110]
With reference to the nucleotide sequence of the L-lysine decarboxylase gene registered in the database (GenBank), the oligonucleotide primer 5′-tatgtcgacatgaacgtttattgcaatat-3 ′ (SEQ ID NO: 8), 5′-ggagagtctttttttttttcttttttttttt3 ′ (SEQ ID NO: 8) 9) was synthesized. PCR was carried out using a solution of genomic DNA prepared from Escherichia coli K12 strain according to a conventional method as an amplification template and using (SEQ ID NO: 8) (SEQ ID NO: 9) as a primer set.
[0111]
The product obtained by this PCR method was electrophoresed with 1% agarose, and a DNA fragment of about 2.1 kb containing the cadA gene was prepared according to a conventional method. This fragment was inserted into a gap in which T base was added to the 3′-end of the EcoRV site of plasmid vector pT7blue (manufactured by Novagen) by a ligation reaction according to a conventional method, and the resulting plasmid was designated as pTM15. .
[0112]
The prepared pTM15 was digested with SalI and SacI, and the resulting approximately 2.1 kb SalI and SacI fragment was applied to the SalI / SacI gap of pTM14 previously digested with SalI and Sacl. The plasmid obtained by insertion by ligation reaction was named pTM16 (FIG. 2).
[0113]
By culturing E. coli into which this plasmid has been introduced, L-lysine decarboxylase is localized on the cell surface, and it becomes possible to adjust L-lysine decarboxylase simply by recovering the E. coli.
[0114]
(5) Preparation of cells in which L-lysine decarboxylase is localized on the cell surface
Escherichia coli JM109 strain was transformed to ampicillin resistance with pTM16, and the resulting transformant was named JM109 / pTM16 strain.
[0115]
Next, recombinant L-lysine decarboxylase was produced in the JM109 / pTM16 strain. First, 1 platinum ear of the JM109 / pTM16 strain was inoculated into 5 ml of sterilized LB medium (LB-amp medium) containing 50 μg / ml ampicillin sodium, and precultured by shaking at 37 ° C. for 24 hours.
[0116]
The whole amount of this preculture was inoculated into 50 ml of LB-amp medium, cultured at 37 ° C. and amplitude of 30 cm for 3 hours under the condition of 180 rpm, added with IPTG (1 mM), and further cultured for 4 hours. The bacterial cells thus obtained were collected by centrifugation, washed 3 times with 10 ml of TBS buffer (Takara Shuzo), and then centrifuged to collect the bacterial cells.
[0117]
Examples 1, 2 and Comparative Example 1 (Effect of using L-lysine / dicarboxylic acid aqueous solution)
As shown in Table 1, L-lysine or L-lysine and dicarboxylic acid were added to 500 ml of an aqueous solution in a 1,000 ml flask. Thereafter, the purified recombinant L-lysine decarboxylase obtained in Reference Example 1 (3) to a final concentration of 50 mg / l and pyridoxal phosphate were added to a final concentration of 0.05 mM. The concentration of cadaverine contained in the reaction solution after 48 hours of reaction at 45 ° C. was measured. The results are shown in Table 1.
[0118]
[Table 1]

[0119]
As a result, when L-lysine dicarboxylate aqueous solution was used as a raw material, cadaverine dicarboxylate was obtained without adjusting the pH.
[0120]
Example 3 and Comparative Example 2 (Effect of Neutralizing L-Lysine Fermentation with Dicarboxylic Acid) Corynebacterium glutamica ATCC 13286 strain was inoculated in 1 platinum ear in 5 ml of sterilized LB medium and shaken at 30 ° C. for 24 hours. Then, pre-culture was performed to obtain a pre-culture solution.
[0121]
The pre-culture medium was inoculated in a total volume of 50 ml of sterilized LB medium, pre-cultured at 30 ° C. with an amplitude of 30 cm and 180 rpm for 24 hours to obtain a pre-culture liquid.
[0122]
Next, inoculate 950 ml of the fermentation medium shown in Table 2 with the entire pre-culture solution and stir at 30 ° C. while aerating sterilized air at 1 vvm (venting air of a volume equivalent to the culture solution volume per minute). Culturing was carried out for 75 hours while adjusting the blade rotation speed to 800 rpm and pH to 7. As a neutralizing agent, sterilized adipic acid aqueous solution (10 g / l) and aqueous ammonia (3M) (in the case of Example 3), or aqueous hydrochloric acid solution (3M) and aqueous ammonia (3M) (in the case of Comparative Example 2) went. After completion of the culture, the cells were removed by centrifugation at 8,000 rpm for 10 minutes at 4 ° C., and the culture supernatant was recovered. In this culture supernatant, 10 g of L-lysine was produced as a total amount in both Example 3 and Comparative Example 2.
[0123]
[Table 2]

[0124]
Next, the purified recombinant L-lysine decarboxylase obtained in Reference Example 1 (3) to a final concentration of 50 mg / l and pyridoxal phosphate to a final concentration of 0.05 mM were added to each culture supernatant, It was allowed to act for 48 hours at 45 ° C. In this reaction solution, a total amount of 6.6 g of cadaverine was produced in both Example 3 and Comparative Example 2.
[0125]
Next, each reaction liquid was distilled off under reduced pressure in a 2 L three-necked flask. Concentration was terminated when the viscosity of the slurry increased and stirring operability began to deteriorate. 80 g of ethanol was quickly added and heated to 65 ° C. to dissolve. Crystals precipitated without dissolving were separated into solid and liquid by filtration. Thereafter, the uniformly dissolved ethanol was gradually cooled, followed by crystallization, solid-liquid separation, and drying. In Example 3, 12.2 g of crystals which are equimolar salts of cadaverine and adipic acid were obtained. On the other hand, in Comparative Example 2, 6.8 g of cadaverine dihydrochloride crystals were obtained.
[0126]
In Comparative Example 2, the obtained cadaverine dihydrochloride crystals were further dissolved in water, and sodium hydroxide was added to adjust the pH of the aqueous solution to 13 or more. Next, chloroform was added in an amount equivalent to the aqueous solution, and cadaverine was extracted from the chloroform phase. Finally, 3.6 g of free cadaverine was isolated from the chloroform phase by distillation under reduced pressure (30 mmHg, 80 ° C.). This isolated cadaverine and equimolar 5.1 g of adipic acid were once dissolved in water, water was distilled off under reduced pressure, and dried to obtain 8.7 g of crystals which are equimolar salts of cadaverine and adipic acid. .
[0127]
As a result, the process of obtaining cadaverine adipate is simplified by neutralizing L-lysine fermentation with adipic acid without neutralizing with hydrochloric acid. Therefore, naturally, the yield for raw materials is improved.
[0128]
Examples 4-6 (Effect of the state of L-lysine decarboxylase)
L-lysine fermentation was performed in the same manner as in Example 3 to obtain a culture supernatant containing 10 g of L-lysine as a total amount.
[0129]
To this culture supernatant, pyridoxal phosphate was added to a final concentration of 0.05 mM, and finally JM109 / pTV118N cells (Example 6) and JM109 prepared by the method described in Reference Examples 1 (2) and (5) were used. / PLDC1 cells (Example 4) and JM109 / pTM16 cells (Example 5) were added and allowed to act at 45 ° C. for 24 hours. The results of measuring the concentration of cadaverine over time are shown in FIG.
[0130]
As a result, cadaverine was obtained at a high production rate by localizing L-lysine decarboxylase on the cell surface.
[0131]
Example 7 (Effect of adjusting L-lysine decarboxylase in L-lysine fermentation medium)
L-lysine fermentation was performed in the same manner as in Example 3 to obtain a culture supernatant containing 10 g of L-lysine as a total amount.
[0132]
Glucose sterilized to a final concentration of 10 g / l was added to the culture supernatant, 1 platinum ear of JM109 / pTM16 cells was inoculated, and the agitated blade was rotated at 37 ° C. while aerating sterilized air at 1 vvm. The culture was performed for 24 hours while adjusting the pH to several 400 rpm and 7. Sterilized aqueous solution of adipic acid (10 g / l) and aqueous ammonia (3M) were used as neutralizing agents. After completion of the culture, the cells were removed by centrifugation at 8,000 rpm for 10 minutes at 4 ° C., and the culture supernatant was recovered. In this culture supernatant, 6.8 g of cadaverine was produced in total.
[0133]
Cadaverine adipate was purified from the culture supernatant by the method described in Example 3 to obtain 12.5 g of crystals that are equimolar salts of cadaverine adipic acid.
[0134]
As a result, cadaverine and adipic acid could be obtained by using only one fermentation apparatus.
[0135]
Comparative Example 3 (Preparation of cadaverine by existing chemical synthesis method)
Suspended in 20 g of L-lysine monohydrochloride (Fluka) cyclohexanol 100 ml (manufactured by Sigma-Aldrich Japan), then 21.2 ml of 28% sodium methoxide / methanol solution (manufactured by Sigma-Aldrich Japan), 3-methyl-cyclohexenone 1 ml (manufactured by Sigma Aldrich Japan) was added, and the mixture was heated and stirred at 155 ° C for 3 hours. After completion of the reaction, 20 ml of an isopropanol solution containing 4 g of hydrogen chloride (manufactured by Sigma-Aldrich Japan) (manufactured by Sigma-Aldrich Japan) was added to the reaction mixture, and the precipitated product was recovered and dried to obtain cadaverine dihydrochloride ( (Method described in Example 3 of JP-A-60-23328).
[0136]
The obtained cadaverine dihydrochloride is dissolved in water, and to this aqueous solution, cadaverine dihydrochloride is converted to cadaverine by adding an aqueous sodium hydroxide solution, extracted with chloroform, and distilled under reduced pressure (30 mmHg, 80 ° C.). As a result, cadaverine was obtained. The isolated cadaverine and equimolar adipic acid were once dissolved in water and dried under reduced pressure to obtain crystals that are equimolar salts of cadaverine and adipic acid.
[0137]
Examples 3 and 6 Identification of Impurities of Comparative Example 3
The analysis of the impurities of cadaverine adipate obtained in Examples 3 and 6 was performed by the GC-MS method under the conditions shown below, and the results compared with the impurity analysis of cadaverine adipate prepared in Comparative Example 3 Is shown in Table 3.
[0138]
GC-MS: HP6980 / HP5973A
Column: NUKOL 30m x 0.24mmI. D. 0.2μm Film
Oven: 120 ° C (constant)
Inject: 200 ° C. (Split 10: 1)
Flow rate: He 2.4 ml / min (constant)
MS: 230 ° C. (SCAN m / z = 30 to 400)
[0139]
[Table 3]

[0140]
As a result, the cadaverine produced using L-lysine decarboxylase is purified to cadaverine adipate, so that the content of tri-n-butylamine which is toxic to aquatic organisms and reacts when contacted with acid is increased. A high-purity cadaverine adipate having 0.006 wt% or less and 2,3,4,5-tetrahydropyridine content of 1.4 wt% or less was obtained.
[0141]
Example 7 (Synthesis of polyamide)
An equimolar salt of cadaverine and adipic acid obtained by the method of Example 3 was dissolved in water to prepare 50.0 g of a 50 wt% aqueous solution. This solution was placed in a test tube, placed in an autoclave, sealed, and purged with nitrogen. The jacket temperature was set to 265 ° C. and heating was started. The pressure inside the can is 17.5 kg / cm²The pressure inside the can is 17.5 kg / cm²For 3 hours. Thereafter, the jacket temperature was set to 275 ° C., and the internal pressure of the can was released to normal pressure over 2 hours. Thereafter, the heating was stopped when the internal temperature reached 245 ° C. After cooling to room temperature, the test tube was removed from the autoclave to obtain polyamide.
[0142]
Comparative Example 4
A polyamide was obtained in the same manner as in Example 7, except that an equimolar salt of cadaverine and adipic acid obtained by the method of Comparative Example 3 was dissolved in water to prepare and use 50.0 g of a 50 wt% aqueous solution.
[0143]
Evaluation of polyamides of Example 7 and Comparative Example 4
The polyamide obtained in Example 7 and the polyamide obtained in Comparative Example 4 were comparatively evaluated by the methods shown below. The results are shown in Table 4.
[Quantification of 2,3,4,5-tetrahydropyridine and tri-n-butylamine in polyamide (GC-MS)]
About 15 g of polyamide is precisely weighed and subjected to Soxhlet extraction with methanol. The extract is subjected to GC-MS analysis under the following conditions to obtain 2,3,4,5-tetrahydropyridine contained in polyamide and tri-n. -Butylamine was quantified.
Apparatus: HP5890 mass detector manufactured by Hewlett-Packard
Column: 5% -diphenyl-95% -dimethylpolysiloxane
Column temperature: Initial 100 ° C
Final 250 ° C
Temperature increase rate: 10 ° C / min
Inlet temperature: 230 ° C
Detector temperature: 280 ° C
Carrier gas: helium
Inlet pressure: 50 kg / cm²
Sample injection volume: 1 μl.
[0144]
[DSC (Differential Scanning Calorimetry)]
Using a DSC RDC220 robot manufactured by Seiko Denshi Kogyo, about 5 mg of polyamide was collected under a nitrogen atmosphere and measured under the following conditions.
The temperature was raised to the melting point + 25 ° C. and held for 3 minutes to completely melt the polyamide. Then, the temperature was lowered to 30 ° C. at a rate of 20 ° C./min. The temperature of the endothermic peak and the amount of heat observed when the temperature was raised at a rate of temperature increase of 20 ° C./min were determined.
[0145]
[Relative viscosity (ηr)]
Measurement was performed using an Ostwald viscometer at a concentration of 0.01 g / ml in 25% sulfuric acid in 98% sulfuric acid.
[Melt retention test]
About 5 g of polyamide was placed in a test tube and immersed in a silicon bath having a melting point of + 20 ° C. in a nitrogen atmosphere. After the polyamide was completely melted, it was allowed to stand for 30 minutes, and then the polyamide was recovered and the relative viscosity was measured. .
[0146]
[Table 4]

[0147]
As a result, by using the high purity cadaverine dicarboxylate obtained by the method of the present invention, a polyamide having a high relative viscosity (ηr) retention (high heat resistance) was obtained.
[0148]
【The invention's effect】
According to the present invention, an industrial production method of cadaverine dicarboxylate and a method of isolating cadaverine dicarboxylate with high reaction yield, high production rate and high purity in one fermentation apparatus are provided.
[0149]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a diagram showing a physical map of L-lysine decarboxylase intracellular expression vector pLDC1.
FIG. 2 is a diagram showing a physical map of L-lysine decarboxylase cell surface expression vector pTM16.
FIG. 3 is a graph showing the relationship between time and cadaverine production in Examples 4-6.

Claims (13)

  A method for producing cadaverine dicarboxylate characterized by allowing L-lysine decarboxylase to act on an L-lysine dicarboxylate aqueous solution and isolating cadaverine dicarboxylate from the reaction solution.   A method for producing cadaverine dicarboxylate, which comprises reacting L-lysine dicarboxylate with an aqueous solution of L-lysine dicarboxylate and recovering cadaverine dicarboxylate from the reaction solution by a crystallization step.   3. The fermentation solution according to claim 1, wherein the L-lysine / dicarboxylate aqueous solution is a fermentation solution obtained by adjusting the pH of the culture solution with dicarboxylic acid when the L-lysine fermentation microorganism is cultured. The manufacturing method of cadaverine dicarboxylate of description.   The method for producing cadaverine dicarboxylate according to claim 3, wherein the L-lysine fermenting microorganism is a genus Corynebacterium and / or a genus Brevibacterium.   The cadaverine dicarboxylate according to any one of claims 1 to 4, wherein the dicarboxylic acid is an aliphatic and / or aromatic dicarboxylic acid having only two carboxyl groups as functional groups. Production method. 6. The method for producing cadaverine dicarboxylate according to claim 5, wherein the dicarboxylic acid is any one of the dicarboxylic acids represented by the following general formulas (1), (2), and (3).
_{HOOC- (CH 2) m -COOH (} 1)
(However, in general formula (1), m = 0 to 16)

(However, in the general formulas (2) and (3), n, o, p, q = 0 to 5)   The method for producing cadaverine dicarboxylate according to any one of claims 1 to 6, wherein L-lysine decarboxylase is prepared using the fermentation broth according to any one of claims 3 to 6.   The cadaverine dicarboxylate according to any one of claims 1 to 7, wherein the L-lysine decarboxylase is L-lysine decarboxylase of a cell having L-lysine decarboxylase activity. Production method.   The method for producing cadaverine dicarboxylate according to any one of claims 1 to 8, wherein the L-lysine decarboxylase is L-lysine decarboxylase localized on the cell surface.   The cadaverine dicarboxylate according to any one of claims 1 to 9, wherein an organic solvent is added when the cadaverine dicarboxylate is isolated or crystallized from the reaction solution after the reaction. Production method.   The method for producing cadaverine dicarboxylate according to claim 10, wherein the organic solvent is an alcohol, a ketone, or a nitrile.   The organic solvent is any one of aliphatic alcohols having 6 or less carbon atoms, aliphatic ketones having 6 or less carbon atoms, or aliphatic nitriles having 6 or less carbon atoms. A method for producing cadaverine dicarboxylate. A method for producing a polyamide, comprising subjecting a cadaverine dicarboxylate for a polyamide raw material obtained by the method for producing a cadaverine dicarboxylate according to any one of claims 1 to 12 to heat polycondensation.

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