JP2004222569A - Corynebacterium, and cadaverine or salt thereof and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To isolate high-purity cadaverine from a fermentation liquid obtained by using a genetic engineering technique to enable corynebacteria to undergo cadaverine fermentation and subjecting the resultant recombinant corynebacteria to cadaverine fermentation. <P>SOLUTION: The cadaverine, cadaverine dihydrochloride or cadaverine dicarboxylate is produced by culturing corynebacteria having L-lysine decarboxylase activity and at least one of homoserine auxotrophy and S-(2-aminoethyl)-L-cysteine tolerance to obtain a culture fluid, which is subjected to a combination, as appropriate, of concentration, organic solvent addition, filtration, crystallization, extraction, distillation, etc. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【００６２】
【化４】

【００６３】
（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。
【００６４】
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。
【００６５】
コリネ型細菌が増殖し、発酵が十分進んでカダベリンが生成した後、菌体と培養上清を分離（分離方法としては、菌体を沈殿除去（遠心分離等）、濾別しても良いし、あるいは始めから菌体を保持材等で分離・保持あるいは固定化していても良い）して、カダベリン発酵液を得る。
【００６６】
このように得られたカダベリン発酵液からカダベリンまたはカダベリン二塩酸塩またはカダベリン・ジカルボン酸塩を採取する方法に制限はない。
【００６７】
例えば発酵液からカダベリンを回収する際には、カダベリン発酵の際の中和剤に制限はない。発酵液をアルカリでｐＨ１２から１４にし、極性有機溶媒で抽出すれば良い。
【００６８】
用いるアルカリに特に制限はない。好ましくは水酸化ナトリウム、水酸化カリウム、および／または水酸化カルシウムを用いることができる。
【００６９】
用いる極性有機溶媒に特に制限はない。好ましくはアニリン水溶液、シクロヘキサノン、１−オクタノール、イソブチルアルコール、シクロヘキサノール、および／またはクロロホルムを用いることができる。
【００７０】
抽出した極性有機溶媒を蒸留等の通常の方法で溶媒とカダベリンを分離すればよい。
【００７１】
それに対し、カダベリン二塩酸塩もしくはカダベリン・ジカルボン酸塩を回収する際には、カダベリン発酵の際の中和剤は好適な中和剤を選択することが好ましい。
【００７２】
例えばカダベリン二塩酸塩の場合では、中和剤は塩酸を用いることが好ましい。同様にカダベリン・ジカルボン酸塩の場合では、中和剤は先に述べたジカルボン酸を用いることが好ましい。
【００７３】
このように中和剤を選択することで望みのカダベリン塩を回収することができる。
【００７４】
例えば、カダベリン・ジカルボン酸塩を得る方法としては、中和剤を選択し得られた発酵液を濃縮し、有機溶媒を添加した晶析操作により採取できる。
【００７５】
晶析操作前に発酵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。
【００７６】
発酵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。
【００７７】
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、アセトニトリルおよび／またはアセトン等があげられる。
【００７８】
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あげられる。
【００７９】
用いる有機溶媒量は特に限定されないが、発酵液の重量に対して通常０．１〜５倍、より好ましくは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。発酵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。
【００８０】
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発酵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。発酵液中のカダベリン・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。
【００８１】
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。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分子とジカルボン酸２分子より形成された塩等が含まれていても良い。
【００８２】
次いで、濃縮工程で濃縮された発酵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。
【００８３】
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。
【００８４】
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。
【００８５】
このようにして得られたカダベリンもしくはカダベリン二塩酸塩もしくはカダベリン・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、０．００６重量％以下のカダベリンもしくはカダベリン二塩酸塩もしくはカタベリン・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１．４重量％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、０．００６重量％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１．４重量％以下のものが得られる。
【００８６】
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。
【００８７】
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もしくはカダベリン二塩酸塩もしくはカダベリン・ジカルボン酸塩は、ポリアミドの原料として有用である。
【００８８】
ポリアミドの製造方法としては、カダベリンとジカルボン酸の塩、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。
【００８９】
本発明で得られるカダベリンもしくはカダベリン二塩酸塩もしくはカダベリン・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。
【００９０】
なお、本発明においてＬＤＣ活性は、後述の実施例１の条件でＴＲ−ＣＡＤ１株の活性の、好ましくは０．０１倍（より好ましくは０．０５倍、更に好ましくは０．１倍）以上である。
【００９１】
【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。
【００９２】
［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。
【００９３】
［アジピン酸濃度の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。
【００９４】
実施例１（Ｌ−リジン脱炭酸活性を有し、かつホモセリンデヒドロゲナーゼ活性を欠損しているコリネバクテリウム・グルタミカムの作製）、比較例１
（１）ＨＯＭ遺伝子のクローニング
ＨＯＭ活性を欠損させるために、Ｎ末端から３００アミノ酸領域に該当する遺伝子をクローニングを行った。
【００９５】
データベース（ＧｅｎＢａｎｋ）に登録されているＨＯＭ遺伝子（Ａｃｃｅｓｓｉｏｎ Ｎｏ．ＢＡ００００３６）の塩基配列を参考にオリゴヌクレオチドプライマー５’−ｇａａｇａａｔｔｃｔａａａｃｃｔｃａｇｃａｔｃｔｇｃｃｃｃ−３’（配列番号：１）および５’−ｇａａｇｇａｔｃｃａａａｇｇａｃｔｔｇｔｔｔａｃｃｇａｃｇｃ−３’（配列番号：２）を合成した。コリネバクテリウム・グルタミカムＡＴＣＣ１３０３２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、トリス塩酸緩衝液ｐＨ８．０（２０ｍＭ）、塩化カリウム（２．５ｍＭ）、ゼラチン（１００μｇ／ｍｌ）、各ｄＮＴＰ（５０μＭ）、ＬＡＴａｑＤＮＡポリメラーゼ（２単位）（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サイクルポリメラーゼ連鎖反応反応させた（以下ＰＣＲ法と略す）。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、ＨＯＭ遺伝子を含む約０．９ｋｂのＤＮＡ断片をゲルから切り出しジーン・クリーン・キット（ＢＩＯ１０１社製）により精製した。この断片を、制限酵素のＥｃｏＲＩおよびＢａｍＨＩで消化し、得られた０．９ｋｂのＥｃｏＲＩ−ＢａｍＨＩ断片を、予めＥｃｏＲＩおよびＢａｍＨＩで消化しておいたｐＨＳＧ２９８（宝酒造製）のＥｃｏＲＩ／ＢａｍＨＩ間隙にライゲーションキットｖｅｒ．１（宝酒造社製）を用いて挿入し、得られたプラスミドをｐＨＯＭ１と命名した。
（２）ＬＤＣ発現カセットの作成
まず、ＬＤＣをコリネバクテリウム・グルタミカムで構成的に発現させるためのプロモーターとしてカナマイシン耐性遺伝子のプロモーターのクローニングを行った。
【００９６】
データベース（ＧｅｎＢａｎｋ）に登録されているｐＨＳＧ２９９（Ａｃｃｅｓｓｉｏｎ Ｎｏ．Ｍ１９４１５）の塩基配列を参考にオリゴヌクレオチドプライマー５’−ｇａａｃｃｇｃｇｇｃｃｔｇａａｔｃｇｃｃｃｃａｔｃａｔｃｃ−３’（配列番号：３）および５’−ｇａａｃｃａｔｇｇｃｃｃｃｔｔｇｔａｔｔａｃｔｇ−３’（配列番号：４）を合成した。プラスミドｐＨＳＧ２９９を増幅鋳型とし、オリゴヌクレオチド（配列番号：３）、（配列番号：４）をプライマーセットとしたＰＣＲ法により得られた産物を１％アガロースゲル電気泳動し、カナマイシン耐性遺伝子のプロモーター領域を含む０．３ｋｂのＤＮＡ断片をゲルから切り出しジーン・クリーン・キットにより精製した。この断片を、プラスミドベクターｐＴ７ｂｌｕｅ（Ｎｏｖａｇｅｎ社製）のＥｃｏＲＶ切断部位の３’−末端にＴ塩基が付加された間隙に、ライゲーションキットｖｅｒ．１を用いて挿入し、得られたプラスミドのうち制限酵素のＨｉｎｄＩＩＩおよびＳａｃＩＩで消化した際に３．２ｋｂの単一断片になるプラスミドをｐＫＭＰ１と命名した。
【００９７】
次に、ＬＤＣ遺伝子のクローニングを行った。
【００９８】
データベース（ＧｅｎＢａｎｋ）に登録されているＬＤＣ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’−ｇａａｃｃａｔｇｇａｃｇｔｔａｔｔｇｃａａ−３’（配列番号：５）、５’−ｇａａｃｃｇｃｇｇｔｔａｔｔｔｔｔｔｇｃｔｔｔｃｔｔｃｔｔｔ−３’（配列番号：６）を合成した。エシェリシア・コリ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オリゴヌクレオチド（配列番号：５）、（配列番号：６）をプライマーセットとしたＰＣＲ法により得られた産物を１％アガロースゲル電気泳動し、ＬＤＣ遺伝子を含む２．１ｋｂのＤＮＡ断片をゲルから切り出しジーン・クリーン・キットにより精製した。この断片を、プラスミドベクターｐＴ７ｂｌｕｅのＥｃｏＲＶ切断部位の３’−末端にＴ塩基が付加された間隙に、ライゲーションキットｖｅｒ．１を用いて挿入し、得られたプラスミドのうちＨｉｎｄＩＩＩおよびＮｃｏＩで消化した際に４．０ｋｂの単一断片になるプラスミドをｐＣＡＤＡと命名した。
【００９９】
最後に、ｐＫＭＰ１をＨｉｎｄＩＩＩおよびＮｃｏＩで消化し、この産物を１．２％アガロースゲル電気泳動し、カナマイシン耐性遺伝子のプロモーター領域を含む０．３ｋｂのＤＮＡ断片をゲルから切り出しジーン・クリーン・キットにより精製した。こうして得られたＨｉｎｄＩＩＩ−ＮｃｏＩ断片を、予めＨｉｎｄＩＩＩおよびＮｃｏＩで消化しておいたｐＣＡＤＡのＨｉｎｄＩＩＩ／ＮｃｏＩ間隙にライゲーションキットｖｅｒ．１を用い挿入し、得られたプラスミドをｐＴＭ１００と命名した。
（３）ＨＯＭ遺伝子破壊およびＬＤＣ遺伝子発現ベクターの作成
ｐＴＭ１００をＳａｃＩＩで消化し、この産物を１．０％アガロースゲル電気泳動し、ＬＤＣ発現カセットを含む２．４ｋｂのＤＮＡ断片をゲルから切り出しジーン・クリーン・キットにより精製した。こうして得られたＳａｃＩＩ断片を、予めＳａｃＩＩで消化しておいたｐＨＯＭ１のＳａｃＩＩ間隙にライゲーションキットｖｅｒ．１を用い挿入し、得られたプラスミドをｐＴＭ１０１と命名した（図１）。
（４）ｐＴＭ１０１の染色体への組み込み
コリネバクテリウム・グルタミカムＡＴＣＣ１３０３２（以下ＡＴＣＣ１３０３２株と略す）にプラスミドｐＴＭ１０１を、電気穿孔法［ＦＥＭＳ Ｍｉｃｒｏｂｉｏｌｏｇｙ Ｌｅｔｔｅｒｓ，６５，ｐ．２９９（１９８９）］により導入し、カナマイシン（２５μｇ／ｍｌ）が添加されているＬＢ（トリプトン（１０ｇ／ｌ）（Ｂａｃｔｏ社製）、酵母エキス（５ｇ／ｌ）（Ｂａｃｔｏ社製）、塩化ナトリウム（１０ｇ／ｌ））寒天培地上で選択した。
【０１００】
こうして選択された形質転換体から常法に従いゲノムＤＮＡ溶液を調整した。このゲノムＤＮＡを鋳型として、オリゴヌクレオチド（配列番号：５）（配列番号：６）をプライマーセットとして用いたＰＣＲ法を行い、得られた産物を１．０％アガロースゲルにて電気泳動したところ、２．１ｋｂの単一のバンドが観察された。このことから、選択された形質転換体が、ＨＯＭ遺伝子座に、ＬＤＣ遺伝子が挿入されていることが確認できた。この形質転換体を、コリネバクテリウム・グルタミカムＴＲ−ＣＡＤ１（以下ＴＲ−ＣＡＤ１株と略す）と命名した。
（５）ＴＲ−ＣＡＤ１株のＬＤＣ活性およびＨＯＭ活性の確認
まず、ＴＲ−ＣＡＤ１株（実施例１）がＬＤＣ活性を有しているかどうかを検査した。ＬＢ培地で１３０３２株（比較例１）を培養し、またカナマイシン（２５μｇ／ｍｌ）が添加されているＬＢ培地でＴＲ−ＣＡＤ１株を培養し、得られたそれぞれの菌体をリン酸緩衝液ｐＨ．６．０（５０ｍＭ）で洗浄し、超音波照射することでそれぞれの細胞破砕液を得た。この細胞破砕液を適量、Ｌ−リジン塩酸塩（５０ｍＭ）（シグマアルドリッチ社製）、ピリドキサルリン酸一水和物（０．０５ｍＭ）（シグマアルドリッチ社製）、リン酸緩衝液ｐＨ６．０（５０ｍＭ）よりなる反応液に溶解した。３０℃、１０分間反応させた後に、生成したカダベリンをＨＰＬＣにより分析した。また細胞破砕液中のタンパク質測定を、ローリー法（Ｌｏｕｒｙ ｅｔ．ａｌ．，Ｊｏｕｒｎａｌ ｏｆ Ｂｉｏｌｏｇｉｃａｌ Ｃｈｅｍｉｓｔｒｙ，１９３，ｐ．２６５−２７５，（１９５１））によって測定した。
【０１０１】
酵素活性を、Ｌ−リジンをカダベリンに１分間に１ｎｍｏｌの変換するとき１Ｕとし、結果をタンパク質重量当たりの比活性で表１に示した。
【０１０２】
【表１】

【０１０３】
１３０３２株はＬＤＣ活性を有していないが、ＴＲ−ＣＡＤ１株はＬＤＣ活性を有していた。
【０１０４】
次に、ＴＲ−ＣＡＤ１株のＨＯＭ活性を欠損しているかどうかを検査した。
【０１０５】
ＬＢ培地で１３０３２株を培養し、またカナマイシン（２５μｇ／ｍｌ）が添加されているＬＢ培地でＴＲ−ＣＡＤ１株を培養し、得られたそれぞれの菌体を生理食塩水で洗浄し、超音波照射することで得られるそれぞれの細胞破砕液を、トリス・ＨＣｌｐＨ７．０（１００ｍＭ）、硫酸アンモニウム（０．８Ｍ）、ＤＴＴ（１ｍＭ）、グリセリン（３０（ｖ／ｖ）％）より成る緩衝液に対して透析させた。こうして得られるそれぞれの酵素製剤を用いて、ＮＡＤＰＨの吸光度測定によりその消費量を定量し、表２のように、ＨＯＭ活性を測定した。タンパク質測定を、ローリー法によって測定した。
【０１０６】
【表２】

【０１０７】
酵素活性を、Ｌ−アスパルテイト−β−セミアルデヒドをＬ−ホモセリンに１分間に１ｎｍｏｌの変換するとき１Ｕとし、結果をタンパク質重量当たりの比活性で表３に示した。
【０１０８】
【表３】

【０１０９】
１３０３２株はＨＯＭ活性を有しているが、ＴＲ−ＣＡＤ１株はＨＯＭ活性を欠損していた。
【０１１０】
さらに、ＴＲ−ＣＡＤ１株がホモセリンを要求しているかどうかを検査した。このために、ＡＴＣＣ１３０３２株およびＴＲ−ＣＡＤ１株を、最少寒天培地（Ｋａｔｓｕｍａｔａ，Ｒ．ｅｔ ａｌ．Ｊｏｕｒｎａｌ ｏｆ Ｂａｃｔｅｒｉｏｌｏｇｙ，１５９，ｐ．３０６−３１１，（１９８４））、Ｄ，Ｌ−ホモセリン（Ｄ，Ｌ−Ｈｓｅ）（２００μｇ／ｍｌ）を有する最少寒天培地、Ｌ−メチオニン（Ｌ−Ｍｅｔ）（１００μｇ／ｍｌ）およびＬ−スレオニン（Ｌ−Ｔｈｒ）（１００μｇ／ｍｌ）を有する最少寒天培地上、およびＬ−メチオニン、Ｌ−スレオニン（各１００μｇ／ｍｌ）およびカナマイシン（Ｋｍ）（１０μｇ／ｍｌ）を有する最少寒天培地上で培養し、かつ増殖を３０℃で２４時間の培養後に判定した。結果を表４にまとめる。
【０１１１】
【表４】

【０１１２】
１３０３２株はホモセリンを要求しないが、ＴＲ−ＣＡＤ１株は、ホモセリンを要求した。
【０１１３】
こうしてＴＲ−ＣＡＤ１株はＬＤＣ活性を有し、かつＨＯＭ活性を欠損（ホモセリン栄養要求性）しているコリネバクテリウム・グルタミカムが作製できた。
【０１１４】
実施例２（Ｌ−リジン脱炭酸活性を有し、かつホモセリン要求性を有し、かつＳ−（２−アミノエチル）−Ｌ−システイン耐性を有するコリネバクテリウム・グルタミカムの作製）
（１）脱感作型ＡＫ遺伝子の作成
ＡＫ遺伝子に変異を導入し、脱感作型ＡＫ遺伝子を作成するためにＡＫ遺伝子をクローニングを行った。
【０１１５】
データベース（ＧｅｎＢａｎｋ）に登録されているＡＫ遺伝子（Ａｃｃｅｓｓｉｏｎ Ｎｏ．ＢＡ００００３６）の塩基配列を参考にオリゴヌクレオチドプライマー５’−ａｃａｇａａｔｔｃｇｔｇｇｃｃｃｔｇｇｔｃｇｔａｃａｇａａ−３’（配列番号：７）および５’−ｃａｔｃｔｃｇａｇｔｔａｇｃｇｔｃｃｇｇｔｇｃｃｔｇｃａｔ−３’（配列番号：８）を合成した。コリネバクテリウム・グルタミカムＡＴＣＣ１３０３２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、オリゴヌクレオチド（配列番号：７）、（配列番号：８）をプライマーセットとしたＰＣＲ法により得られた産物を１％アガロースゲル電気泳動し、ＡＫ遺伝子を含む約１．３ｋｂのＤＮＡ断片をゲルから切り出しジーン・クリーン・キットにより精製した。この断片を、ＥｃｏＲＩおよびＸｈｏＩで消化し、得られた１．３ｋｂのＥｃｏＲＩ−ＸｈｏＩ断片を、予めＥｃｏＲＩおよびＸｈｏＩで消化しておいたｐＨＳＧ３９６（宝酒造製）のＥｃｏＲＩ／ＸｈｏＩ間隙にライゲーションキットｖｅｒ．１を用いて挿入し、得られたプラスミドをｐＡＫ１と命名した。
【０１１６】
次に、クローニングしたＡＫ遺伝子の９３１番目から９３３番目のａｃｃ（Ｔｈｒ）をａｔｇ（Ｉｌｅ）に変異させるためにオリゴヌクレオチドプライマー５’−ｃｇａｃａｔｃａｔｃｔｔｃａｃｃｔｇｃｃ−３’（配列番号：９）および５’−ｇｇｃａｇｇｔｇａａｇａｔｇａｔｇｔｃｇ−３’（配列番号：１０）を合成した。ｐＡＫ１を増幅鋳型として、オリゴヌクレオチド（配列番号：９）、（配列番号：１０）をプライマーセットとしてＱｕｉｋＣｈａｎｇｅサイトダイレクテッド・ミュータジェネシス・キット（ストラタジーン社製）を用い得られたプラスミドをｐＴＭ１０２と命名した（図２）。このｐＴＭ１０２中のＡＫ遺伝子を常法によりシークエンスしたところ、目的通り９３１番目から９３３番目のａｃｃ（Ｔｈｒ）をａｔｇ（Ｉｌｅ）に変異されており、脱感作型ＡＫ遺伝子が作成できていることが確認できた。
（２）ｐＴＭ１０２の染色体への組み込み
データベース（ＧｅｎＢａｎｋ）に登録されているｐＦＫ３９８（Ａｃｃｅｓｓｉｏｎ Ｎｏ．Ｄ２９８２６）の塩基配列を参考にクロラムフェニコール耐性遺伝子のオリゴヌクレオチドプライマー５’−ａｃｇｇｔｃｇａｃｔｃｇｃａｇａａｔａａａｔａａａｔｃｃｔｇｇｔｇ−３’（配列番号：１１）および５’−ａｔｇａｇｇｃｃｔｇａｇａｇｇｃｇｇｔｔｔｇｃｇｔａｔｔｇｇａ−３’（配列番号：１２）を合成した。
【０１１７】
ＴＲ−ＣＡＤ１株にプラスミドｐＴＭ１０２を、電気穿孔法により導入し、カナマイシン（２５μｇ／ｍｌ）およびクロラムフェニコール（１０μｇ／ｍｌ）が添加されているＬＢ寒天培地上で選択した。
【０１１８】
こうして選択された形質転換体から常法に従いゲノムＤＮＡ溶液を調整した。このゲノムＤＮＡを鋳型として、オリゴヌクレオチド（配列番号：１１）（配列番号：１２）をプライマーセットとして用いたＰＣＲ法を行い、得られた産物を１．０％アガロースゲルにて電気泳動したところ、１．０ｋｂの単一のバンドが観察された。このことから、選択された形質転換体が、ＡＫ遺伝子座に、クロラムフェニコール耐性遺伝子が挿入されていることが確認できた。この形質転換体を、コリネバクテリウム・グルタミカムＴＲ−ＣＡＤ２（以下ＴＲ−ＣＡＤ２株と略す）と命名した。
（３）脱感作型ＡＫ遺伝子のＴＲ−ＣＡＤ２株の染色体上への導入の確認
データベース（ＧｅｎＢａｎｋ）に登録されているＡＫ遺伝子（Ａｃｃｅｓｓｉｏｎ Ｎｏ．ＢＡ００００３６）の塩基配列を参考に、ＡＫのＮ末端より上流０．１ｋｂのオリゴヌクレオチドプライマー５’−ｔｔｇｇａａｃｇｃｇｔｃｃｃａｇｔｇｇｃ−３’（配列番号：１３）を合成した。
【０１１９】
ＴＲ−ＣＡＤ２株から常法に従いゲノムＤＮＡ溶液を調整した。このゲノムＤＮＡを鋳型として、オリゴヌクレオチド（配列番号：１２）（配列番号：１３）をプライマーセットとして用いたＰＣＲ法を行い、得られた産物を１．０％アガロースゲルにて電気泳動し
、ＡＫ遺伝子およびクロラムフェニコール耐性遺伝子を含む３．１ｋｂのＤＮＡ断片をゲルから切り出しジーン・クリーン・キットにより精製した。この断片を、プラスミドベクターｐＴ７ｂｌｕｅのＥｃｏＲＶ切断部位の３’−末端にＴ塩基が付加された間隙に、ライゲーションキットｖｅｒ．１を用いて挿入し、得られたプラスミドをｐＡＫ２と命名した。このｐＡＫ２中のＡＫ遺伝子を常法によりシークエンスしたところ、目的通りの変異が含まれていることが確認できたため、ＴＲ−ＣＡＤ２株は染色体上で発現可能な脱感作型ＡＫ遺伝子が導入できたことが確認できた。
（４）ＴＲ−ＣＡＤ２株のＡＥＣ耐性の確認
ＴＲ−ＣＡＤ２株がＡＥＣ耐性であるかどうかを検査した。このために、ＡＴＣＣ１３０３２株およびＴＲ−ＣＡＤ２株を、最少寒天培地（Ｋａｔｓｕｍａｔａ，Ｒ．ｅｔ ａｌ．Ｊｏｕｒｎａｌ ｏｆ Ｂａｃｔｅｒｉｏｌｏｇｙ，１５９，ｐ．３０６−３１１，（１９８４））、Ｄ，Ｌ−ホモセリン（Ｄ，Ｌ−Ｈｓｅ）（２００μｇ／ｍｌ）を有する最小寒天培地上およびＤ，Ｌ−ホモセリン、ＡＥＣ（５０μｇ／ｍｌ）を有する最少寒天培地上で培養し、かつ増殖を３０℃で２４時間の培養後に判定した。結果を表５にまとめる。
【０１２０】
【表５】

【０１２１】
１３０３２株はＡＥＣ耐性がないが、ＴＲ−ＣＡＤ２株は、ＡＥＣ耐性であった。
【０１２２】
こうしてＴＲ−ＣＡＤ２株はＬＤＣ活性を有し、かつＨＯＭ活性を欠損（ホモセリン栄養要求性）し、かつＡＥＣ耐性であるコリネバクテリウム・グルタミカムが作製できた。
【０１２３】
実施例３および４、比較例２（カダベリン発酵）
ＡＴＣＣ１３０３２株（比較例２）、ＴＲ−ＣＡＤ１株（実施例３）およびＴＲ−ＣＡＤ２株（実施例４）を滅菌ＬＢ培地（ＡＴＣＣ１３０３２株は無添加、ＴＲ−ＣＡＤ１株はカナマイシン（２５μｇ／ｍｌ）添加、ＴＲ−ＣＡＤ２株はカナマイシン（２５μｇ／ｍｌ）およびクロラムフェニコール（１０μｇ／ｍｌ）添加）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た。
【０１２４】
この前々培養液を前々培養と同じ培地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た。
【０１２５】
次に表６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３０時間培養を行った。中和剤として塩酸水溶液（３Ｍ）およびアンモニア水（３Ｍ）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。この培養上清中のカダベリンをＨＰＬＣにより分析した。その結果を表７に示す。
【０１２６】
【表６】

【０１２７】
【表７】

【０１２８】
その結果、ＴＲ−ＣＡＤ１株およびＴＲ−ＣＡＤ２株でカダベリン発酵が行えた。
【０１２９】
実施例５（カダベリンの精製）
実施例４に示す方法でＴＲ−ＣＡＤ２株をカダベリン発酵した。この培養上清中に、総量として３９ｇのカダベリンを生成した。
【０１３０】
次にこの発酵液に水酸化ナトリウムを添加し、ｐＨを１３にした。次にクロロホルムを発酵液と等量加え、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１８．５ｇを単離した。
【０１３１】
実施例６（カダベリン二塩酸塩の精製）
実施例４に示す方法でＴＲ−ＣＡＤ２株をカダベリン発酵した。この培養上清中に、総量として３９ｇのカダベリンを生成した。
【０１３２】
次にその発酵液に塩酸を添加しｐＨ４．０とし、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（シグマアルドリッチ社製）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。カダベリン二塩酸塩の結晶４６．８ｇを得た。
【０１３３】
実施例７（カダベリン・アジピン酸塩の精製）
ＴＲ−ＣＡＤ２株を滅菌ＬＢ培地（カナマイシン（２５μｇ／ｍｌ）およびクロラムフェニコール（１０μｇ／ｍｌ）添加）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た。
【０１３４】
この前々培養液を前々培養と同じ培地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た。
【０１３５】
次に表６に示した発酵培地９５０ｍｌに前培養液全量を植菌し、滅菌した空気を１ｖｖｍ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３０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）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。この培養上清中のカダベリンをＨＰＬＣにより分析した。この培養上清中に、総量として４２ｇのカダベリンを生成した。
【０１３６】
次にその発酵液にアジピン酸を添加しｐＨ４．５とし、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（シグマアルドリッチ社製）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。こうしてカダベリン・アジピン酸塩の結晶７１．３ｇを得た。得られた結晶を水に溶解させカダベリン濃度およびアジピン酸濃度をＨＰＬＣで分析したところ、等モル塩であることがわかった。
【０１３７】
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、２−シクロヘキセン−１−オン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）。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。
【０１３８】
実施例８，９および１０ 比較例３（不純物の同定）
実施例５，６および７で得られたカダベリン、カダベリン二塩酸塩およびカダベリン・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８に示す。
【０１３９】
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）
【０１４０】
【表８】

【０１４１】
この結果、カダベリン発酵を行い生成したカダベリンを、カダベリン、カダベリン二塩酸塩およびカダベリン・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０．００６重量％以下かつ２，３，４，５−テトラヒドロピリジンの含有量が１．４重量％以下の高純度カダベリン、カダベリン二塩酸塩およびカダベリン・アジピン酸塩が得られた。
【０１４２】
実施例１１（ポリアミドの合成）
実施例７の方法で得られたカダベリン・アジピン酸塩を水に溶解させの５０重量％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。
【０１４３】
比較例４
比較例３の方法で得られたカダベリン・アジピン酸塩を水に溶解させ５０重量％水溶液５０．０ｇを調整し用いる以外は、実施例１１と全く同様の方法でポリアミドを得た。
【０１４４】
実施例１２（ポリアミドの評価）
実施例１１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９に示す。
【０１４５】
［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。
【０１４６】
［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。
【０１４７】
［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。
【０１４８】
［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。
【０１４９】
【表９】

【０１５０】
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。
【０１５１】
【発明の効果】
本発明によれば、カダベリン発酵可能なコリネ型細菌が提供され、該微生物を培養することにより、発酵液からカダベリン、カダベリン二塩酸塩もしくはカダベリン・ジカルボン酸塩としての単離方法が提供される。
【０１５２】
【配列表】

【図面の簡単な説明】
【図１】ＨＯＭ遺伝子座へのＬＤＣ遺伝子挿入破壊ベクターｐＴＭ１０１のフィジカルマップを示す図である。
【図２】脱感作型ＡＫ遺伝子ベクターｐＴＭ１０２のフィジカルマップを示す図である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to cadaverine or cadaverine dihydrochloride or cadaverine dicarboxylate, a method for producing them, and a bacterium suitably used in the production method. Cadaverine or cadaverine dihydrochloride or cadaverine dicarboxylate is expected as a raw material for polyamide, and its demand is increasing.
[0002]
[Prior art]
Conventionally, it is known that cadaverine can be obtained by boiling L-lysine in cyclohexanol containing a small amount of tetralin peroxide (see Non-Patent Document 1). However, the high temperature reaction requires large amounts of energy and organic solvents, and the reaction yield is very low (36%).
[0003]
In addition, a method of synthesizing 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 can be obtained by heating and stirring L-lysine at 155 ° C. for 3 hours in cyclohexanol containing sodium methoxide using 3-methyl-cyclohexenone as a catalyst (see Patent Document 1). ).
[0005]
Moreover, cadaverine obtained by this method contains a basic compound such as tri-n-butylamine or 2,3,4,5-tetrahydropyridine as an impurity.
[0006]
In addition, cadaverine is a biogenic amine that is ubiquitous in living organisms, and its biosynthetic system is being elucidated (see Non-Patent Document 3). As the biosynthetic pathway, L-lysine decarboxylase (hereinafter abbreviated as LDC), which catalyzes the decarboxylation of cadaverine from L-lysine, is known. For example, an LDC gene derived from Escherichia coli is known as the gene (see Non-Patent Document 4).
[0007]
Also, a method for producing cadaverine by fermentation with recombinant Escherichia coli (see Patent Document 2) and a method for producing cadaverine by enzymatic method using recombinant Escherichia coli (see Patent Document 3) are known.
[0008]
Further, a polyamide has been produced by purifying a free diamine and a free dicarboxylic acid as raw materials and then mixing them in equimolar amounts 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 particularly using a solvent (see Non-Patent Document 5). Therefore, there was no need to study the crystallization of diamine / dicarboxylic acid from the aqueous solution.
[0009]
However, in order to produce a polyamide raw material by fermentation different from the chemical synthesis method, it is common to use water as a solvent, and to perform fermentation, it is generally necessary to adjust the pH of the solution. . 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 a production method by fermentation of succinic acid, which is a dicarboxylic acid, crystallization and purification are performed as calcium succinate (see Patent Document 4).
[0010]
On the other hand, the following method is known as a method for producing L-lysine by fermentation using a microorganism belonging to the genus Corynebacterium.
(1) As a method using an L-lysine-producing mutant, for example, S- (2-aminoethyl) -L-cysteine (hereinafter abbreviated as AEC) resistant mutant (see Non-Patent Document 6), L-lysine Homoserine-requiring mutant (see Non-patent Document 6), respiratory system inhibitor-resistant mutant (see Patent Document 5), pyrimidine analog-resistant mutant (see Patent Document 6), and purine analog-resistant mutant (see Patent Document 7) See, for example).
(2) As a method using a recombinant bacterium using a gene recombination technique, a vector plasmid having a marker gene that replicates autonomously in a microorganism belonging to the genus Corynebacterium and gives a selectable phenotype, and the vector plasmid can be efficiently used. A method for introducing the bacterium into the bacterium is disclosed, and a method for efficiently producing L-lysine by increasing the intracellular copy number of a gene involved in the synthesis of L-lysine using the method is disclosed (Patent Documents). 8).
[0011]
The control of L-lysine biosynthesis includes concerted feedback inhibition of aspartokinase (hereinafter abbreviated as AK), which catalyzes the biosynthesis of asparatyl phosphate from L-aspartic acid, by L-lysine and L-threonine. Well known. A microorganism belonging to the genus Corynebacterium having a gene encoding a mutant AK (hereinafter abbreviated as desensitized AK) from which this feedback inhibition has been released (hereinafter abbreviated as a desensitized AK gene) has AEC resistance. Is known to secrete L-lysine out of the cells (see Non-Patent Document 6). Furthermore, desensitized AK released from concerted feedback inhibition by L-lysine and L-threonine and feedback inhibition by L-lysine alone is also known (see Patent Document 9).
[0012]
It is known that homoserine auxotrophy can be obtained by deficient homoserine dehydrogenase activity (see Non-Patent Document 6).
[0013]
However, no practical production technology has been established for the production of cadaverine or cadaverine dihydrochloride or cadaverine dicarboxylate, and the development of a method for producing cadaverine or cadaverine dihydrochloride or cadaverine dicarboxylate of high purity has not been established. Is desired.
[0014]
[Patent Document 1]
JP-A-60-23328, pp. 11-12
[0015]
[Patent Document 2]
JP-A-2002-223770, page 5
[0016]
[Patent Document 3]
JP-A-2002-223771, pages 4 to 5
[0017]
[Patent Document 4]
JP-A-62-294090, page 6
[0018]
[Patent Document 5]
Japanese Patent Publication No. 5-55114, pages 1 to 4
[0019]
[Patent Document 6]
JP-A-59-88094, pages 1 to 3
[0020]
[Patent Document 7]
JP-B-63-062199, pages 1 to 5
[0021]
[Patent Document 8]
JP-B-5-11959, pp. 7-19
[0022]
[Patent Document 9]
WO 00/63388, pages 10 to 29
[0023]
[Non-patent document 1]
Tadashi Suyama and Kiyozo Kanao, "Decarboxylation of Amino Acids (Report 4)", Pharmaceutical Magazine, 1965, Vol. 85, No. 6, p. 531-533
[0024]
[Non-patent document 2]
Mitsunori Hashimoto, 4 others, "Chemistry Letters", 1986, p. 893-896
[0025]
[Non-Patent Document 3]
Celia white tabor, et al., “Microbiological Reviews”, 1985, vol. 49, p. 81-99
[0026]
[Non-patent document 4]
Shi-yuanengeng, et al., “Journal of Bacteriology”, 1992, Vol. 174, p. 2659-2669
[0027]
[Non-Patent Document 5]
Mitsuaki Mukoyama, "Industrial Organic Chemistry", 4th edition, Tokyo Kagaku Dojin, p. 270-273, 275-281
[0028]
[Non-patent Document 6] Hiroshi Aida, "Amino Acid Fermentation", Gakkai Shuppan Center, 1986, p. 273-305
[0029]
[Problems to be solved by the invention]
When cadaverine or cadaverine dihydrochloride or cadaverine dicarboxylate containing a large amount of tri-n-butylamine and 2,3,4,5-tetrahydropyridine is used as a raw material of the polymer, for example, cadaverine or cadaverine dihydrochloride Or, when synthesizing a polyamide from a raw material containing cadaverine dicarboxylate by heating polycondensation, the reaction temperature is high, so cadaverine or cadaverine dihydrochloride or tri-n-butylamine contained in cadaverine dicarboxylate And a basic compound such as 2,3,4,5-tetrahydropyridine causes a decomposition reaction of the polyamide, resulting in a problem that the resulting polyamide has insufficient heat resistance.
[0030]
Furthermore, tri-n-butylamine is toxic to aquatic organisms and has a problem that the lethal dose to fish is 20 to 40 mg / l.
[0031]
An object of the present invention is to provide a method for producing cadaverine or cadaverine dihydrochloride or cadaverine dicarboxylate by fermentation of cadaverine or cadaverine dihydrochloride or cadaverine dicarboxylate having a small amount of such impurities. .
[0032]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the present inventors have conducted intensive studies on a method for producing cadaverine or cadaverine dihydrochloride or cadaverine dicarboxylate by fermentation, and as a result, it has L-lysine decarboxylase activity. And growing coryneform bacteria having at least one characteristic of homoserine auxotrophy or S- (2-aminoethyl) -L-cysteine resistance, and cadaverine or cadaverine from the culture supernatant of the cells. It has been found that dihydrochloride or cadaverine dicarboxylate can be obtained.
[0033]
That is, the present invention provides "(1) having L-lysine decarboxylase activity and having at least one characteristic of homoserine auxotrophy or S- (2-aminoethyl) -L-cysteine resistance. Coryneform bacteria.
(2) A method for producing cadaverine or a salt thereof, comprising a step of growing the microorganism and a step of collecting and purifying cadaverine from a culture supernatant of the cells.
(3) Cadaverine or cadaverine dihydrochloride or cadaverine dicarboxylate having a content of 2,3,4,5-tetrahydropyridine of 1.4% by weight or less.
(4) Cadaverine or cadaverine dihydrochloride or cadaverine dicarboxylate having a tri-n-butylamine content of 0.006% by weight or less.
(5) A polyamide containing cadaverine or a salt thereof obtained by the method (2) or cadaverine or cadaverine dihydrochloride or cadaverine dicarboxylate according to the above (3) or (4) as a raw material. ".
[0034]
BEST MODE FOR CARRYING OUT THE INVENTION
Cadaverine is 1,5-pentanediamine and is a useful compound as a polymer raw material.
[0035]
Examples of the coryneform bacterium in the present invention include Agrococcus, Agromyces, Arthrobacter, Aureobacterium, Brevibacterium, and Cellulomonas. Bacteria belonging to the genera, genus Clavibacter, genus Corynebacterium, genus Microbacterium, genus Rathaibacter, genus Terrabactor, genus Turicella. Preferable examples include bacteria belonging to the genus Corynebacterium and the genus Brevibacterium. As specific examples, for example, the following strains are used. Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869, Corynebacterium glutamicum ATCC 13870, Corynebacterium carnae ATCC 15991, Corynebacterium acetoglutamicum ATCC 15806.
[0036]
These wild-type strains can be subjected to feedback inhibition by L-lysine or L-lysine and L-threonine. A method for obtaining a strain in which the feedback inhibition has been released includes, for example, a mutation operation to a wild-type strain [N- A method using methyl-N'-nitro-N-nitrosoguanidine (NTG); Microorganism Experiment Manual, 1986, page 131, Kodansha Scientific Co., Ltd.], and selected using AEC resistance as an index. A method of obtaining from a mutant strain that has become productive can be given. As preferred mutants, L-lysine-producing bacteria or wild-type AK genes obtained by mutation treatment from Corynebacterium glutamicum wild-type strain ATCC13032 are obtained, and then the NTG method or the in vitro mutation method (“Molecular”). and General Genetics ", 1978, Vol. 145, p. 101), in which the gene is mutated to have AEC resistance and desensitized using L-lysine productivity as an index. And the like.
[0037]
A more preferable method for imparting AEC resistance to a wild-type strain is a method using a genetic engineering technique. There is no particular limitation on the method using genetic engineering techniques. For example, a method using a recombinant DNA that can be replicated in coryneform bacteria or a method in which a target gene in a chromosome is recombined by homologous recombination. S- (2-aminoethyl) -L-cysteine resistance can be obtained by having a mutant aspartokinase gene in which the amino acid residue at position 311 in the amino acid sequence of SEQ ID NO: 15 is substituted with an amino acid other than Thr. it can.
[0038]
As a vector used for producing a recombinant DNA replicable in a coryneform bacterium, any vector can be used as long as it can replicate autonomously in a coryneform bacterium. Specifically, pCG1 (JP-A-57-134500), pCG2 (JP-A-58-35197), pCG4 (JP-A-57-183799), and pCG11 (JP-A-57-134500). Gazettes), pCG116, pCE54, pCB101 (JP-A-58-105999), pCE51, pCE52, and pCE53 ("Molecular and General Genetics", 1984, Vol. 196, p. 175). Preferably, one having a relatively small size such as pCG116, having many cloning sites, and having a selectable marker gene such as a drug resistance gene is used.
[0039]
As a method for introducing the above-mentioned recombinant DNA into coryneform bacteria, a protoplast method (for example, JP-A-57-186492 and JP-A-57-18649), an electroporation method ("Journal of Bacteriology"). , 1993, Vol. 175, p. 4096).
[0040]
A more preferred genetic engineering technique is a method in which the AK gene on the chromosome of the coryneform bacterium is recombined with the desensitized AK gene by homologous recombination. Further, the desensitized AK gene is preferably obtained by a technique for introducing a desired mutation into the AK gene (for example, a method using a QuikChange site-directed mutagenesis kit manufactured by Stratagene).
[0041]
Also, the wild type strain has no homoserine auxotrophy. As a method for obtaining a homoserine auxotroph, as in the previous example, for example, after performing a mutation procedure from a wild-type strain, a mutation that is selected using homoserine auxotrophy as an index and becomes L-lysine-producing is obtained. The method of acquiring from a stock can be given.
[0042]
As a more preferable method for imparting homoserine auxotrophy to a wild-type strain, homoserine dehydrogenase activity is deleted by inserting another gene into a homoserine dehydrogenase gene (hereinafter abbreviated as HOM gene) in a chromosome by homologous recombination. There is a method. There are no particular restrictions on other genes. Preferably, the LDC gene is used, and more preferably, the LDC gene is a promoter and a cassette which can be constitutively expressed in a wild-type strain.
[0043]
The AK gene, the HOM gene, and the LDC gene can be obtained from a strain containing these genes by, for example, isolating them by the method of Saito et al. (“Biochimica et Biophysica Acta”, 1963, Vol. 72, p. 619). Can be. That is, chromosomal DNA is prepared and cut with an appropriate restriction enzyme. After cleavage, the obtained fragment is ligated to a vector (for example, a plasmid) capable of autonomous replication in the microorganism, and this is introduced into a host microorganism deficient in these enzyme activities. It can be obtained by isolating a transformant expressing these activities from the microorganism and isolating the gene from the transformant.
[0044]
As the host microorganism, any microorganism that can express these genes can be used. Preferable is Escherichia coli. The autonomously replicable vector may be any vector that can autonomously replicate in a microorganism into which the vector has been introduced. For example, vectors that can replicate autonomously in Escherichia coli, such as pUC18, pUC19, pHSG298, pHSG299, pHSG396, and pHSG398, can be mentioned. Ligation of a vector and a DNA fragment containing these genes can be performed by an ordinary method using T4 DNA ligase or the like.
[0045]
For example, in the case of Escherichia coli, introduction into a host can be carried out by the method of Hanahan et al. (“Journal of Molecular Biology”, 1983, Vol. 166, p. 557) or the like.
[0046]
Preferably, the AK gene, HOM gene and LDC gene can be isolated and obtained by the following method.
[0047]
Oligomeric DNA is synthesized based on the known nucleotide sequence information of the AK gene derived from Corynebacterium glutamicum, the nucleotide sequence information of the HOM gene, and the known nucleotide sequence information of the LDC gene derived from Escherichia coli. The DNA fragment containing the gene is amplified and isolated by PCR. The DNA fragment is ligated to a vector having a selectable marker gene and introduced into an appropriate host microorganism such as Escherichia coli or coryneform bacterium. By isolating the vector introduced from the microorganism, the AK gene, HOM gene and LDC gene can be obtained. It is not necessary to use a strain deficient in these enzymes as a host microorganism, and it can be obtained.
[0048]
LDC is an enzyme that converts L-lysine into cadaverine, and is known to be present not only in microorganisms of the genus Escherichia such as Escherichia coli K12 but also in many other organisms.
[0049]
There is no particular limitation on the LDC gene used in the present invention, but for example, Bacillus halodurans, Bacillus subtilis, Escherichia coli, Selenomonas lumennium, Selenium, Vibrio cholerae, Vibrio parahaemolyticus, Streptomyces coelicolor, Streptomyces epiroseus, Streptomyces corosicles ), Eubacterium acididaminophilum, Salmonella typhimurium, Hafnia alveii, Neisseria nigeris, and Neisseria meningitias (Neisseria thiemia piercina). -A material derived from Pyrococcus abyssi is preferably used. More preferably, it is derived from Escherichia coli whose safety is recognized.
[0050]
The coryneform bacterium used in the present invention has LDC activity and at least one property of homoserine auxotrophy or AEC resistance, and preferably has LDC activity and homoserine auxotrophy. It is a coryneform bacterium having sex and AEC resistance.
[0051]
When these coryneform bacteria are cultured, cadaverine can be produced and accumulated in the culture solution.
[0052]
As a culture medium, an ordinary nutrient medium containing a carbon source, a nitrogen source, inorganic salts, and the like can be used.
[0053]
As the carbon source, for example, sugars such as glucose, fructose, sucrose, maltose, and starch hydrolysate, alcohols such as ethanol, and organic acids such as acetic acid, lactic acid, and succinic acid can be used.
[0054]
Nitrogen sources include various inorganic and organic ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium carbonate, and ammonium acetate, urea and other nitrogen-containing compounds, meat extract, yeast extract, corn steep liquor, soybean hydrolyzate And other nitrogen-containing organic substances.
[0055]
As the inorganic salt, potassium hydrogen phosphate, potassium hydrogen phosphate, ammonium sulfate, sodium chloride, magnesium sulfate, calcium carbonate and the like can be used.
[0056]
In addition, trace nutrients such as biotin, thiamine, and vitamin B6 can be added as needed. These trace nutrients can be replaced with a medium additive such as meat extract, yeast extract, corn steep liquor, casamino acid and the like.
[0057]
The culturing conditions are not particularly limited, and the culturing is performed under aerobic conditions such as shaking culture and deep-aeration stirring culture. The cultivation temperature is generally between 25C and 42C, particularly preferably between 28C and 38C. The culturing time is not particularly limited, and is usually 1 to 6 days.
[0058]
In the cadaverine fermentation, it is preferable to use ammonia and hydrochloric acid or dicarboxylic acid for adjusting the culture pH. It is preferable to control the culture pH to 5 to 8, particularly preferably to 6.5 to 7.5, by using these neutralizing agents. The state of the neutralizing agent is not limited, and it is used as a gas, liquid, solid or aqueous solution. Particularly preferred is an aqueous solution.
[0059]
There is no particular limitation on the dicarboxylic acid used as the neutralizing agent, preferably,
The functional group is an aliphatic and / or aromatic dicarboxylic acid having only two carboxyl 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 herein means a polyamide polymerization reaction (reaction conditions include, for example, a reaction temperature of 250 to 270 ° C., ² Reacts with an amino group or a carboxyl group during a reaction time of 1 to 5 hours to cause a branch of the polymer or reduce the crystallinity of the polymer (crystallinity of 80% or less). . For example, an amino group or a carboxyl group corresponds to this, but other than the above, an acidic group (such as a sulfonic acid group, a phosphoric acid group, or a phenolic hydroxyl group), a basic group (such as a hydrazino group), or a protonic polar group (such as A hydroxyl group, a cleavable group (epoxy group, peroxide group, etc.) and other highly reactive groups (isocyanate group, etc.) are often applicable. On the other hand, halogen substituents and aromatic substituents are relatively stable, and ethers, esters, amides and the like are also stable and often have low functionality.
[0060]
The dicarboxylic acid is more preferably a dicarboxylic acid represented by the following general formula (1), (2) or (3).
HOOC- (CH ₂ ) _m -COOH (1)
(However, in the general formula (1), m = 0 to 16)
[0061]
Embedded image

[0062]
Embedded image

[0063]
(However, in general formulas (2) and (3), n, o, p, and q = 0 to 5)
More preferably
In the general formula (1),
m = 0 to 10
And / or in the general formulas (2) and / or (3),
n, o, p, q = 0 to 1
It is.
[0064]
Still more preferred are adipic acid, sebacic acid, 1,12-dodecanedicarboxylic acid, succinic acid, isophthalic acid and terephthalic acid.
[0065]
After the coryneform bacterium grows and fermentation proceeds sufficiently to produce cadaverine, the cells and the culture supernatant are separated (as a separation method, the cells may be removed by sedimentation (such as centrifugation), or may be separated by filtration, or From the beginning, the cells may be separated / held / fixed with a holding material or the like) to obtain a cadaverine fermented solution.
[0066]
There is no limitation on the method for collecting cadaverine or cadaverine dihydrochloride or cadaverine dicarboxylate from the cadaverine fermented liquor thus obtained.
[0067]
For example, when recovering cadaverine from a fermentation liquor, there is no limitation on the neutralizing agent for cadaverine fermentation. The fermentation liquor may be adjusted to pH 12 to 14 with alkali and extracted with a polar organic solvent.
[0068]
There is no particular limitation on the alkali used. Preferably, sodium hydroxide, potassium hydroxide, and / or calcium hydroxide can be used.
[0069]
There is no particular limitation on the polar organic solvent used. Preferably, an aqueous aniline solution, cyclohexanone, 1-octanol, isobutyl alcohol, cyclohexanol, and / or chloroform can be used.
[0070]
The extracted polar organic solvent may be separated from cadaverine by a usual method such as distillation.
[0071]
On the other hand, when recovering cadaverine dihydrochloride or cadaverine dicarboxylate, it is preferable to select a suitable neutralizing agent as the neutralizing agent during cadaverine fermentation.
[0072]
For example, in the case of cadaverine dihydrochloride, it is preferable to use hydrochloric acid as the neutralizing agent. Similarly, in the case of cadaverine dicarboxylate, it is preferable to use the above-mentioned dicarboxylic acid as the neutralizing agent.
[0073]
By selecting a neutralizing agent in this manner, a desired cadaverine salt can be recovered.
[0074]
For example, as a method for obtaining cadaverine dicarboxylate, a fermentation solution obtained by selecting a neutralizing agent can be concentrated and collected by a crystallization operation in which an organic solvent is added.
[0075]
It is preferable to adjust cadaverine and dicarboxylic acid in the fermentation solution to be equimolar before the crystallization operation. It is more preferable to add a dicarboxylic acid in excess.
[0076]
As the organic solvent added to the fermentation solution, alcohols, ketones or nitriles are preferable. More preferred are aliphatic alcohols having 6 or less carbon atoms, aliphatic ketones having 6 or less carbon atoms, and aliphatic nitriles having 6 or less carbon atoms.
[0077]
For example, specific examples of the organic solvent include methanol, ethanol, propanol, butanol, hexanol, isopropanol, acetonitrile, and / or acetone.
[0078]
In addition, the organic solvent does not need to be used alone, and may be a mixed solvent. For example, a mixed solution of ethanol and acetone may be used.
[0079]
The amount of the organic solvent used is not particularly limited, but is usually 0.1 to 5 times, more preferably 0.3 to 2 times, the weight of the fermentation liquor. Alternatively, it is preferably 1 to 10 (more preferably 3 to 7) times the weight of cadaverine dicarboxylate. The method of contacting the fermentation liquid with the organic solvent is optional, and may be a batch method or a continuous method.
[0080]
The fermentation broth is preferably concentrated for efficient crystallization and for improving pot efficiency. The concentration is preferably such that the inorganic salt and cadaverine dicarboxylate do not precipitate as crystals. The concentration varies depending on the concentration of cadaverine dicarboxylate and other salts in the fermentation liquor, but it is preferable that the concentration be reduced to 1/4 to 1/30, more preferably 1/10 to 1/20 by weight. Is more preferred.
[0081]
The concentration method may be either normal pressure concentration or reduced pressure concentration, but the liquid temperature during concentration is usually lower than the boiling point of water. At such a liquid temperature, there is no fear that the cadaverine dicarboxylate is decomposed. The cadaverine dicarboxylate thus prepared may be considered to be a salt composed of one molecule of cadaverine and one molecule of dicarboxylic acid, but it does not exclude any other form, for example, two molecules of cadaverine. A salt or the like formed from two molecules of dicarboxylic acid may be contained.
[0082]
Next, an organic solvent is added to the fermentation liquor concentrated in the concentration step, and the precipitated crystals are removed by a usual solid-liquid separation method such as centrifugation, and cadaverine dicarboxylate is separated from the organic solvent by a usual crystallization separation. Cadaverine dicarboxylate is isolated by operation. For example, after cooling the concentrated solution to which the organic solvent is added as it is to crystallize cadaverine dicarboxylate, the crystals are isolated by a usual solid-liquid separation method such as centrifugation to obtain high-purity cadaverine dicarboxylate. Can be obtained.
[0083]
The organic solvent separated from cadaverine dicarboxylate in the crystallization step is preferably recycled to the crystallization operation.
[0084]
The cadaverine dicarboxylate thus isolated is again purified by a known crystallization purification method, that is, after forming a homogeneous solution with an organic solvent, cooling the homogeneous solution, or cooling the filtrate filtered while hot. Cadaverine dicarboxylate can be crystallized to produce high purity products.
[0085]
The cadaverine or cadaverine dihydrochloride or cadaverine dicarboxylate obtained in this manner has few impurities such as tri-n-butylamine and 2,3,4,5-tetrahydropyridine. By the production according to the method of the present invention, cadaverine or cadaverine dihydrochloride or cataverine dicarboxylate having a tri-n-butylamine content of 0.006% by weight or less is obtained when analyzed by the following method. Can be Further, when analyzed by the following method, a product having a content of 2,3,4,5-tetrahydropyridine of 1.4% by weight or less is obtained. Furthermore, a product having a tri-n-butylamine content of 0.006% by weight or less and a 2,3,4,5-tetrahydropyridine content of 1.4% by weight or less is obtained.
[0086]
Analysis of tri-n-butylamine and 2,3,4,5-tetrahydropyridine is performed by GC-MS under the following conditions.
[0087]
GC-MS: HP6980 / HP5973A
Column: NUKOL 30m × 0.24mmI. D. 0.2μm Film
Oven: 120 ° C (constant)
Injection: 200 ° C (Split 10: 1)
Flow rate: He 2.4 ml / min (constant)
MS: 230 ° C (SCAN m / z = 30 to 400)
The cadaverine or cadaverine dihydrochloride or cadaverine dicarboxylate thus obtained is useful as a raw material for polyamide.
[0088]
As a method for producing a polyamide, a heating polycondensation method in which a mixture of cadaverine and a dicarboxylic acid and water is heated to cause a dehydration reaction to proceed is used. In addition, it is also possible to increase the molecular weight by performing solid phase polymerization after heat polycondensation. Solid-state polymerization proceeds by heating in a vacuum or an inert gas at a temperature in the range of 100 ° C. to the melting point, and heating polycondensation can increase the molecular weight of a polyamide having an insufficient molecular weight.
[0089]
By using cadaverine or cadaverine dihydrochloride or cadaverine dicarboxylate obtained in the present invention as a raw material for polyamide, a polyamide having high heat resistance can be obtained.
[0090]
In the present invention, the LDC activity is preferably 0.01 times (more preferably 0.05 times, more preferably 0.1 times) or more of the activity of the TR-CAD1 strain under the conditions of Example 1 described below. is there.
[0091]
【Example】
Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.
[0092]
[Analysis method of 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: UV 360 nm
Sample pretreatment: 25 μl of 1,4-diaminobutane (0.03 M), 150 μl of sodium bicarbonate (0.075 M) and ethanol of 2,4-dinitrofluorobenzene (0.2 M) as an internal standard in 25 μl of an analysis sample The solution is added and mixed and incubated at 37 ° C. for 1 hour. After dissolving 50 µl of the above reaction solution in 1 ml of acetonitrile, 10 µl of the supernatant after centrifugation at 10,000 rpm for 5 minutes was analyzed by HPLC.
[0093]
[Analysis method of adipic acid concentration by HPLC]
Column used: SCR-101H (Shimadzu Corporation)
Mobile phase: 0.025% (v / v) sulfuric acid aqueous solution
Detection: UV 214 nm
Sample pretreatment: The analytical sample was diluted with water so that the calibration curve had a concentration within a range capable of having linearity, and 10 μl was analyzed by HPLC.
[0094]
Example 1 (Production of Corynebacterium glutamicum having L-lysine decarboxylation activity and lacking homoserine dehydrogenase activity), Comparative Example 1
(1) Cloning of HOM gene
To delete the HOM activity, a gene corresponding to the 300 amino acid region from the N-terminus was cloned.
[0095]
Oligonucleotide primers 5'-gaagaattcttaaacctcagcatctgcccc-3 '(SEQ ID NO: 1) and 5'-gaaggccccaaaggacttgtctagcttagtaccc sequence with reference to the nucleotide sequence of the HOM gene (Accession No. BA0000036) registered in the database (GenBank). : 2) was synthesized. Take 0.2 μl of a genomic DNA solution prepared from Corynebacterium glutamicum ATCC13032 according to a conventional method into a 0.2 ml microcentrifuge tube as an amplification template, 20 pmol of each primer, Tris-HCl buffer pH 8.0 (20 mM), Each reagent was added so that potassium chloride (2.5 mM), gelatin (100 μg / ml), each dNTP (50 μM), and LATaq DNA polymerase (2 units) (manufactured by Takara Shuzo) were used to make the total volume 50 μl. DNA denaturation conditions were 94 ° C for 30 seconds, primer annealing conditions were 55 ° C for 30 seconds, DNA primer extension reaction conditions were 72 ° C for 3 minutes, and each cycle was performed using a BioRad thermal cycler for 30 cycles. The reaction was performed (hereinafter abbreviated as PCR method). The PCR method in this example was performed under the same conditions unless otherwise specified. The product obtained by this PCR method was electrophoresed on 1% agarose, a DNA fragment of about 0.9 kb containing the HOM gene was cut out from the gel, and purified by Gene Clean Kit (manufactured by BIO101). This fragment was digested with restriction enzymes EcoRI and BamHI, and the resulting 0.9 kb EcoRI-BamHI fragment was ligated into the EcoRI / BamHI gap of pHSG298 (Takara Shuzo) previously digested with EcoRI and BamHI. ver. 1 (manufactured by Takara Shuzo) and the resulting plasmid was named pHOM1.
(2) Creation of LDC expression cassette
First, a kanamycin resistance gene promoter was cloned as a promoter for constitutively expressing LDC in Corynebacterium glutamicum.
[0096]
The oligonucleotide primers 5′-gaaccgcgggcctgaatcgccccatcatcc-3 ′ (SEQ ID NO: 3) and 5′-gaaccatgggcccctgtattgt-3 ′ (sequence number: 3 ′) with reference to the nucleotide sequence of pHSG299 (Accession No. M19415) registered in the database (GenBank). 4) was synthesized. The product obtained by the PCR method using the plasmid pHSG299 as an amplification template and the oligonucleotides (SEQ ID NO: 3) and (SEQ ID NO: 4) as primer sets was subjected to 1% agarose gel electrophoresis to remove the promoter region of the kanamycin resistance gene. A 0.3 kb DNA fragment was cut out of the gel and purified using a Gene Clean Kit. This fragment was ligated with the ligation kit ver. In the space where a T base was added to the 3′-end of the EcoRV cleavage site of the plasmid vector pT7blue (manufactured by Novagen). The resulting plasmid was designated pKMP1 by inserting a plasmid into a single 3.2 kb fragment upon digestion with the restriction enzymes HindIII and SacII.
[0097]
Next, the LDC gene was cloned.
[0098]
Oligonucleotide primers 5'-gaaccatgggacgttatttgcaa-3 '(SEQ ID NO: 5) and 5'-gaaccgcgtttttttctttttt sequence, referring to the nucleotide sequence of the LDC gene (Accession No. M76411) registered in the database (GenBank). : 6) was synthesized. Using a solution of genomic DNA prepared from Escherichia coli ATCC 10798 according to a conventional method as an amplification template, a product obtained by a PCR method using oligonucleotides (SEQ ID NO: 5) and (SEQ ID NO: 6) as a primer set was 1% agarose gel. After electrophoresis, a 2.1 kb DNA fragment containing the LDC gene was excised from the gel and purified using a Gene Clean kit. This fragment was ligated with the ligation kit ver. In the space where a T base was added to the 3′-end of the EcoRV cleavage site of the plasmid vector pT7blue. The plasmid obtained by inserting the plasmid into a single fragment of 4.0 kb when digested with HindIII and NcoI was named pCADA.
[0099]
Finally, pKMP1 was digested with HindIII and NcoI, the product was subjected to 1.2% agarose gel electrophoresis, a 0.3 kb DNA fragment containing the promoter region of the kanamycin resistance gene was cut out from the gel, and purified using a Gene Clean kit. did. The HindIII-NcoI fragment thus obtained was ligated to the ligation kit ver. In the HindIII / NcoI gap of pCADA which had been digested with HindIII and NcoI in advance. 1 and the resulting plasmid was named pTM100.
(3) HOM gene disruption and creation of LDC gene expression vector
pTM100 was digested with SacII, the product was subjected to 1.0% agarose gel electrophoresis, and a 2.4 kb DNA fragment containing the LDC expression cassette was excised from the gel and purified using a Gene Clean Kit. The SacII fragment thus obtained was ligated to the SacII gap of pHOM1 which had been digested with SacII beforehand by ligation kit ver. The resulting plasmid was named pTM101 (FIG. 1).
(4) Integration of pTM101 into chromosome
Plasmid pTM101 was introduced into Corynebacterium glutamicum ATCC13032 (hereinafter abbreviated as ATCC13032 strain) by electroporation [FEMS Microbiology Letters, 65, p. 299 (1989)] and kanamycin (25 μg / ml) added to LB (tryptone (10 g / l) (Bacto), yeast extract (5 g / l) (Bacto), sodium chloride ( 10 g / l)) Selection on agar medium.
[0100]
A genomic DNA solution was prepared from the thus selected transformants according to a conventional method. Using this genomic DNA as a template, a PCR method was performed using an oligonucleotide (SEQ ID NO: 5) (SEQ ID NO: 6) as a primer set, and the resulting product was electrophoresed on a 1.0% agarose gel. A single band of 2.1 kb was observed. From this, it was confirmed that the LDC gene was inserted into the HOM locus in the selected transformant. This transformant was named Corynebacterium glutamicum TR-CAD1 (hereinafter abbreviated as TR-CAD1 strain).
(5) Confirmation of LDC activity and HOM activity of TR-CAD1 strain
First, it was examined whether the TR-CAD1 strain (Example 1) had LDC activity. The 13032 strain (Comparative Example 1) was cultured in the LB medium, and the TR-CAD1 strain was cultured in the LB medium to which kanamycin (25 μg / ml) was added. . After washing with 6.0 (50 mM) and irradiating with ultrasonic waves, each cell lysate was obtained. An appropriate amount of the cell lysate is L-lysine hydrochloride (50 mM) (Sigma Aldrich), pyridoxal phosphate monohydrate (0.05 mM) (Sigma Aldrich), phosphate buffer pH 6.0 (50 mM). Was dissolved in the reaction solution. After reacting at 30 ° C. for 10 minutes, the produced cadaverine was analyzed by HPLC. The protein in the cell lysate was measured by the Lowry method (Lowry et. Al., Journal of Biological Chemistry, 193, p. 265-275, (1951)).
[0101]
The enzyme activity was 1 U when converting L-lysine to cadaverine at 1 nmol per minute, and the results are shown in Table 1 in terms of specific activity per protein weight.
[0102]
[Table 1]

[0103]
The 13032 strain did not have LDC activity, whereas the TR-CAD1 strain had LDC activity.
[0104]
Next, it was examined whether the HOM activity of the TR-CAD1 strain was deficient.
[0105]
The 13032 strain was cultured in the LB medium, and the TR-CAD1 strain was cultured in the LB medium to which kanamycin (25 μg / ml) was added. The obtained cells were washed with physiological saline and irradiated with ultrasonic waves. The cell lysate obtained by the above-mentioned process was subjected to a buffer solution composed of Tris-HCl pH 7.0 (100 mM), ammonium sulfate (0.8 M), DTT (1 mM), and glycerin (30 (v / v)%). Dialysis was performed. Using each of the enzyme preparations thus obtained, the amount of consumption was quantified by measuring the absorbance of NADPH, and the HOM activity was measured as shown in Table 2. Protein measurements were measured by the Lowry method.
[0106]
[Table 2]

[0107]
The enzymatic activity was defined as 1 U when converting 1 nmol of L-aspartate-β-semialdehyde to L-homoserine per minute, and the results are shown in Table 3 in terms of specific activity per protein weight.
[0108]
[Table 3]

[0109]
The 13032 strain had HOM activity, whereas the TR-CAD1 strain lacked HOM activity.
[0110]
Furthermore, it was examined whether the TR-CAD1 strain required homoserine. For this purpose, the ATCC13032 strain and the TR-CAD1 strain were transformed with a minimal agar medium (Katsumasata, R. et al. Journal of Bacteriology, 159, p.306-311, (1984)), D, L-homoserine (D, L). -Hse) (200 μg / ml) on a minimal agar medium with L-methionine (L-Met) (100 μg / ml) and L-threonine (L-Thr) (100 μg / ml), and L -Cultivation on minimal agar medium with methionine, L-threonine (100 μg / ml each) and kanamycin (Km) (10 μg / ml), and proliferation was determined after incubation at 30 ° C. for 24 hours. The results are summarized in Table 4.
[0111]
[Table 4]

[0112]
The 13032 strain did not require homoserine, whereas the TR-CAD1 strain required homoserine.
[0113]
Thus, Corynebacterium glutamicum having the TRCAD1 strain having LDC activity and lacking HOM activity (homoserine auxotrophy) could be produced.
[0114]
Example 2 (Preparation of Corynebacterium glutamicum having L-lysine decarboxylation activity, having homoserine requirement, and having S- (2-aminoethyl) -L-cysteine resistance)
(1) Creation of desensitized AK gene
A mutation was introduced into the AK gene, and the AK gene was cloned to prepare a desensitized AK gene.
[0115]
Oligonucleotide primers 5′-acagaattcgtggccctggtcgtacagaa-3 ′ (SEQ ID NO: 7) and 5′-cattcgagtagtagggtcgtggtgccgccgccgccgccggtccggt : 8) was synthesized. Using a solution of genomic DNA prepared from Corynebacterium glutamicum ATCC13032 according to a conventional method as an amplification template, a product obtained by PCR using oligonucleotides (SEQ ID NO: 7) and (SEQ ID NO: 8) as primer sets was used to obtain 1 % Agarose gel electrophoresis, a DNA fragment of about 1.3 kb containing the AK gene was excised from the gel and purified using a Gene Clean kit. This fragment was digested with EcoRI and XhoI, and the resulting 1.3 kb EcoRI-XhoI fragment was ligated into the EcoRI / XhoI gap of pHSG396 (Takara Shuzo), which had been digested with EcoRI and XhoI in advance. 1 and the resulting plasmid was named pAK1.
[0116]
Next, oligonucleotide primers 5′-cgacatcatcttcacctgcc-3 ′ (SEQ ID NO: 9) and 5′-ggcaggtgaagatgatcgg-to mutate the 931st to 933th acc (Thr) of the cloned AK gene to atg (Ile). 3 ′ (SEQ ID NO: 10) was synthesized. The plasmid obtained using a QuikChange site-directed mutagenesis kit (manufactured by Stratagene) using pAK1 as an amplification template and oligonucleotides (SEQ ID NO: 9) and (SEQ ID NO: 10) as primer sets was named pTM102. (Fig. 2). When the AK gene in this pTM102 was sequenced by a conventional method, it was found that the 931st to 933th acc (Thr) was mutated to atg (Ile) as intended, and a desensitized AK gene could be produced. It could be confirmed.
(2) Integration of pTM102 into chromosome
With reference to the base sequence of pFK398 (Accession No. D29826) registered in the database (GenBank), the oligonucleotide primer 5′-acgggtcgactcgcagaataaataaattcgtgggtgggg-3g (SEQ ID NO: 11) and 5′-atggggtggggtggtgggggcgg of the chloramphenicol resistance gene. -3 '(SEQ ID NO: 12) was synthesized.
[0117]
Plasmid pTM102 was introduced into the TR-CAD1 strain by electroporation and selected on LB agar medium supplemented with kanamycin (25 μg / ml) and chloramphenicol (10 μg / ml).
[0118]
A genomic DNA solution was prepared from the thus selected transformants according to a conventional method. Using this genomic DNA as a template, a PCR method using an oligonucleotide (SEQ ID NO: 11) (SEQ ID NO: 12) as a primer set was performed, and the obtained product was electrophoresed on a 1.0% agarose gel. A single band of 1.0 kb was observed. From this, it was confirmed that the selected transformant had the chloramphenicol resistance gene inserted at the AK locus. This transformant was named Corynebacterium glutamicum TR-CAD2 (hereinafter abbreviated as TR-CAD2 strain).
(3) Confirmation of introduction of desensitized AK gene into chromosome of TR-CAD2 strain
With reference to the base sequence of the AK gene (Accession No. BA0000036) registered in the database (GenBank), an oligonucleotide primer 5′-ttggaacgcgtcccaggtggc-3 ′ (0.1 kb) upstream from the N-terminal of AK (SEQ ID NO: 13) Was synthesized.
[0119]
A genomic DNA solution was prepared from the TR-CAD2 strain according to a conventional method. Using this genomic DNA as a template, PCR was performed using oligonucleotides (SEQ ID NO: 12) (SEQ ID NO: 13) as a primer set, and the resulting product was electrophoresed on a 1.0% agarose gel.
A 3.1 kb DNA fragment containing the AK gene and the chloramphenicol resistance gene was excised from the gel and purified using a Gene Clean Kit. This fragment was ligated with the ligation kit ver. In the space where a T base was added to the 3′-end of the EcoRV cleavage site of the plasmid vector pT7blue. 1 and the resulting plasmid was named pAK2. When the AK gene in pAK2 was sequenced by a conventional method, it was confirmed that the desired mutation was contained. Therefore, the TR-CAD2 strain was able to introduce a desensitized AK gene that can be expressed on the chromosome. That was confirmed.
(4) Confirmation of AEC resistance of TR-CAD2 strain
It was examined whether the TR-CAD2 strain was AEC resistant. For this purpose, the ATCC13032 strain and the TR-CAD2 strain were transformed with a minimal agar medium (Katsumasata, R. et al. Journal of Bacteriology, 159, p.306-311, (1984)), D, L-homoserine (D, L). -Hse) (200 μg / ml) and on a minimal agar medium with D, L-homoserine, AEC (50 μg / ml) and growth was determined after 24 hours of culture at 30 ° C. . The results are summarized in Table 5.
[0120]
[Table 5]

[0121]
The 13032 strain was not AEC resistant, whereas the TR-CAD2 strain was AEC resistant.
[0122]
Thus, TR-CAD2 strain was able to produce Corynebacterium glutamicum having LDC activity, deficient HOM activity (homoserine auxotrophy), and AEC resistance.
[0123]
Examples 3 and 4, Comparative Example 2 (cadaverine fermentation)
ATCC13032 strain (Comparative Example 2), TR-CAD1 strain (Example 3) and TR-CAD2 strain (Example 4) were added to a sterilized LB medium (ATCC13032 strain was not added, and TR-CAD1 strain was added kanamycin (25 μg / ml). The TR-CAD2 strain was inoculated with one platinum loop into 5 ml of kanamycin (25 μg / ml) and chloramphenicol (10 μg / ml), and shaken at 30 ° C. for 24 hours to perform pre-preculture.
[0124]
This pre-pre-culture solution was inoculated into 50 ml of the same medium as in the pre-pre-culture, and cultured at 30 ° C., an amplitude of 30 cm and 180 rpm for 24 hours to perform pre-culture.
[0125]
Next, 950 ml of the fermentation medium shown in Table 6 was inoculated with the entire amount of the preculture liquid, and cultured for 30 hours while adjusting the pH to 7 at 30 ° C., the number of rotations of stirring blades at 800 rpm while passing sterilized air at 1 vvm. Was. As a neutralizing agent, an aqueous solution of hydrochloric acid (3M) and aqueous ammonia (3M) were used. 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. Cadaverine in the culture supernatant was analyzed by HPLC. Table 7 shows the results.
[0126]
[Table 6]

[0127]
[Table 7]

[0128]
As a result, cadaverine fermentation was performed with the TR-CAD1 strain and the TR-CAD2 strain.
[0129]
Example 5 (Purification of cadaverine)
The cadaverine fermentation of the TR-CAD2 strain was performed by the method shown in Example 4. In this culture supernatant, 39 g of cadaverine was produced in total.
[0130]
Next, sodium hydroxide was added to the fermented liquor to adjust the pH to 13. Next, chloroform was added in the same amount as the fermentation liquid, and cadaverine was extracted into the chloroform phase. Finally, 18.5 g of free cadaverine was isolated by subjecting the chloroform phase to distillation under reduced pressure (30 mmHg, 80 ° C.).
[0131]
Example 6 (Purification of cadaverine dihydrochloride)
The cadaverine fermentation of the TR-CAD2 strain was performed by the method shown in Example 4. In this culture supernatant, 39 g of cadaverine was produced in total.
[0132]
Next, hydrochloric acid was added to the fermented liquid to adjust the pH to 4.0, and water was distilled off under reduced pressure in a 2 L three-necked flask. The concentration was terminated when the viscosity of the slurry increased and the stirring operability began to deteriorate. 80 g of ethanol (manufactured by Sigma-Aldrich) was quickly added, and the mixture was heated to 65 ° C. and dissolved. Crystals precipitated without dissolution were separated into solid and liquid by filtration. Thereafter, the uniformly dissolved ethanol was gradually cooled, crystallized, solid-liquid separated, and dried. 46.8 g of crystals of cadaverine dihydrochloride were obtained.
[0133]
Example 7 (Purification of cadaverine adipate)
One loopful of the TR-CAD2 strain was inoculated into 5 ml of a sterilized LB medium (added with kanamycin (25 μg / ml) and chloramphenicol (10 μg / ml)) and shaken at 30 ° C. for 24 hours to perform pre-preculture. Was.
[0134]
This pre-pre-culture solution was inoculated into 50 ml of the same medium as in the pre-pre-culture, and cultured at 30 ° C., an amplitude of 30 cm and 180 rpm for 24 hours to perform pre-culture.
[0135]
Next, the entire amount of the preculture was inoculated into 950 ml of the fermentation medium shown in Table 6, and cultivated for 30 hours while adjusting the pH to 7 at 30 ° C., the stirring blade rotation speed at 800 rpm while passing sterilized air at 1 vvm. went. As a neutralizing agent, sterilization was carried out with an aqueous solution of adipic acid (10 g / l) and aqueous ammonia (3 M). 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. Cadaverine in the culture supernatant was analyzed by HPLC. A total of 42 g of cadaverine was produced in the culture supernatant.
[0136]
Next, adipic acid was added to the fermented liquid to adjust the pH to 4.5, and water was distilled off under reduced pressure in a 2 L three-necked flask. The concentration was terminated when the viscosity of the slurry increased and the stirring operability began to deteriorate. 80 g of ethanol (manufactured by Sigma-Aldrich) was quickly added, and the mixture was heated to 65 ° C. and dissolved. Crystals precipitated without dissolution were separated into solid and liquid by filtration. Thereafter, the uniformly dissolved ethanol was gradually cooled, crystallized, solid-liquid separated, and dried. Thus, 71.3 g of crystals of cadaverine adipate were obtained. The obtained crystals were dissolved in water, and cadaverine concentration and adipic acid concentration were analyzed by HPLC. As a result, it was found that the crystals were equimolar salts.
[0137]
Comparative Example 3 (Preparation of cadaverine by existing chemical synthesis method)
20 g of L-lysine monohydrochloride (Fluka) and 100 ml of cyclohexanol (Sigma-Aldrich Japan) were suspended, and then 21.2 ml of a 28% sodium methoxide / methanol solution (Sigma-Aldrich Japan) and 2-cyclohexene-1- 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 (manufactured by Sigma-Aldrich Japan) containing 4 g of hydrogen chloride (manufactured by Sigma-Aldrich Japan) was added to the reaction mixture, and the precipitated product was collected and dried to obtain cadaverine dihydrochloride ( Method described in Example 3 of JP-A-60-23328). The obtained cadaverine dihydrochloride is dissolved in water, and cadaverine dihydrochloride is converted to cadaverine by adding an aqueous sodium hydroxide solution to the aqueous solution, extracted with chloroform, and distilled under reduced pressure (30 mmHg, 80 ° C.). As a result, cadaverine was obtained. The isolated cadaverine and an equimolar amount of adipic acid were once dissolved in water and dried under reduced pressure to obtain a crystal as an equimolar salt of cadaverine-adipic acid.
[0138]
Examples 8, 9 and 10 Comparative Example 3 (Identification of impurities)
The cadaverine, cadaverine dihydrochloride and cadaverine adipate obtained in Examples 5, 6 and 7 were analyzed for impurities by GC-MS under the following conditions, and the cadaverine adipin prepared in Comparative Example 3 was analyzed. Table 8 shows the results of comparison with the impurity analysis of the acid salt.
[0139]
GC-MS: HP6980 / HP5973A
Column: NUKOL 30m × 0.24mmI. D. 0.2μm Film
Oven: 120 ° C (constant)
Injection: 200 ° C (Split 10: 1)
Flow rate: He 2.4 ml / min (constant)
MS: 230 ° C (SCAN m / z = 30 to 400)
[0140]
[Table 8]

[0141]
As a result, by purifying cadaverine produced by cadaverine fermentation into cadaverine, cadaverine dihydrochloride and cadaverine adipate, tri-n-butylamine is toxic to aqueous organisms and reacts upon contact with acid. Cadaverine, cadaverine dihydrochloride and cadaverine adipate having a content of 0.006% by weight or less and a content of 2,3,4,5-tetrahydropyridine of 1.4% by weight or less were obtained. .
[0142]
Example 11 (Synthesis of polyamide)
Cadaverine adipate obtained by the method of Example 7 was dissolved in water to prepare 50.0 g of a 50% by weight aqueous solution. This solution was charged into a test tube, placed in an autoclave, sealed, and replaced with nitrogen. The jacket temperature was set at 265 ° C. and heating was started. After the pressure in the can reached 17.5 kg / cm 2, the pressure in the can was maintained at 17.5 kg / cm 2 for 3 hours. Thereafter, the jacket temperature was set at 275 ° C., and the pressure in the can was released to normal pressure over 2 hours. Thereafter, when the temperature in the can reached 245 ° C, the heating was stopped. After cooling to room temperature, the test tube was taken out of the autoclave to obtain a polyamide.
[0143]
Comparative Example 4
A polyamide was obtained in exactly the same manner as in Example 11, except that cadaverine adipate obtained by the method of Comparative Example 3 was dissolved in water, and 50.0 g of a 50% by weight aqueous solution was prepared and used.
[0144]
Example 12 (Evaluation of polyamide)
The polyamide obtained in Example 11 and the polyamide obtained in Comparative Example 4 were comparatively evaluated by the following method. Table 9 shows the results.
[0145]
[Quantification of 2,3,4,5-tetrahydropyridine and tri-n-butylamine in polyamide (GC-MS)]
Approximately 15 g of polyamide was precisely weighed and subjected to Soxhlet extraction with methanol, and the extract was subjected to GC-MS analysis under the following conditions, and the 2,3,4,5-tetrahydropyridine and tri-n contained in the polyamide were analyzed. -Butylamine was quantified.
Equipment: Hewlett-Packard HP5890 mass detector
Column: 5% -diphenyl-95% -dimethylpolysiloxane
Column temperature: Initial 100 ° C
Final 250 ° C
Heating 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.
[0146]
[DSC (Differential Scanning Calorimetry)]
Using a robot DSC RDC220 manufactured by Seiko Denshi Kogyo, about 5 mg of polyamide was sampled under a nitrogen atmosphere and measured under the following conditions.
After the temperature was raised to the melting point + 25 ° C. and maintained for 3 minutes to completely melt the polyamide, the temperature was lowered to 30 ° C. at a temperature lowering rate of 20 ° C./min. The temperature of the endothermic peak and the calorific value observed when the temperature was raised at a rate of 20 ° C./min were determined.
[0147]
[Relative viscosity (ηr)]
The measurement was carried out in 98% sulfuric acid at 0.01 g / ml at 25 ° C. using an Ostwald viscometer.
[0148]
[Melting retention test]
About 5 g of polyamide was charged into a test tube, immersed in a silicon bath having a temperature of melting point + 20 ° C. in a nitrogen atmosphere, and allowed to stand for 30 minutes after the polyamide was completely melted. Then, the polyamide was recovered and the relative viscosity was measured. .
[0149]
[Table 9]

[0150]
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 rate (high heat resistance) was obtained.
[0151]
【The invention's effect】
According to the present invention, there is provided a coryneform bacterium capable of cadaverine fermentation, and a method for isolating cadaverine, cadaverine dihydrochloride or cadaverine dicarboxylate from a fermentation broth by culturing the microorganism.
[0152]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a diagram showing a physical map of an LDC gene insertion disruption vector pTM101 at the HOM locus.
FIG. 2 is a diagram showing a physical map of a desensitized AK gene vector pTM102.

Claims (24)

A coryneform bacterium having L-lysine decarboxylase activity and having at least one characteristic of homoserine auxotrophy or S- (2-aminoethyl) -L-cysteine resistance. The coryneform bacterium according to claim 1, wherein the coryneform bacterium lacks homoserine dehydrogenase activity. The coryneform bacterium according to claim 1 or 2, wherein the coryneform bacterium lacks homoserine dehydrogenase activity due to gene insertion mutagenesis. The coryneform bacterium according to claim 3, wherein the gene insertion is an insertion of an L-lysine decarboxylase gene expression cassette (promoter and L-lysine decarboxylase gene). The coryneform bacterium according to any one of claims 1 to 4, wherein the genus of the coryneform bacterium is at least one genus selected from the group consisting of Corynebacterium and Brevibacterium. The coryneform bacterium according to any one of claims 1 to 5, wherein the coryneform bacterium has a mutant aspartokinase gene in which the amino acid residue at position 311 in the amino acid sequence of SEQ ID NO: 15 is substituted with an amino acid other than Thr. A method for producing cadaverine or a salt thereof, comprising the steps of: growing the coryneform bacterium according to any one of claims 1 to 6, and recovering and purifying cadaverine or a salt thereof from a culture supernatant of the cells. Production method. 7. A step of growing the coryneform bacterium according to any one of claims 1 to 6, wherein the culture supernatant of the cells is adjusted to pH 12 to 14 with alkali, and cadaverine is extracted and recovered with a polar organic solvent. Manufacturing method. The method for producing cadaverine according to claim 8, wherein the alkali is at least one selected from the group consisting of sodium hydroxide, potassium hydroxide and calcium hydroxide. The cadaverine according to claim 8, wherein the polar organic solvent is at least one selected from the group consisting of an aqueous aniline solution, cyclohexanone, 1-octanol, isobutyl alcohol, cyclohexanol, and chloroform. Manufacturing method. A step of growing the coryneform bacterium according to any one of claims 1 to 6 while maintaining the pH of the culture solution with hydrochloric acid, and recovering cadaverine dihydrochloride from a culture supernatant of the cells by a crystallization step. A method for producing cadaverine dihydrochloride, which is characterized in that: A step of growing the coryneform bacterium according to any one of claims 1 to 6 while maintaining the pH of the culture solution with dicarboxylic acid, and recovering cadaverine dicarboxylate from a culture supernatant of the cells by a crystallization step. A method for producing cadaverine dicarboxylate, comprising: The method for producing cadaverine dicarboxylate according to claim 12, wherein the dicarboxylic acid is an aliphatic and / or aromatic dicarboxylic acid having only two carboxyl groups as a functional group. The dicarboxylic acid is
The method for producing cadaverine dicarboxylate according to claim 12, which is any one of dicarboxylic acids represented by the following general formulas (1), (2) and (3).
_{HOOC- (CH 2) m -COOH (} 1)
(However, in the general formula (1), m = 0 to 16)

(However, in general formulas (2) and (3), n, o, p, and q = 0 to 5) In the general formula (1),
m = 0 to 10
And / or in the general formulas (2) and / or (3),
n, o, p, q = 0 to 1
13. The method for producing cadaverine dicarboxylate according to claim 12, wherein: The cadaverine dicarboxylate according to claim 12, wherein the dicarboxylic acid is any of adipic acid, sebacic acid, 1,12-dodecanedicarboxylic acid, succinic acid, isophthalic acid and terephthalic acid. Method. The method for producing cadaverine dicarboxylate according to any one of claims 12 to 16, wherein an organic solvent is added when crystallizing cadaverine dicarboxylate from the culture solution. The method for producing cadaverine dicarboxylate according to claim 17, wherein the organic solvent is at least one selected from the group consisting of alcohols, ketones, and nitriles. The organic solvent is at least one selected from the group consisting of aliphatic alcohols having 6 or less carbon atoms, aliphatic ketones having 6 or less carbon atoms, and aliphatic nitriles having 6 or less carbon atoms. A method for producing the cadaverine dicarboxylate according to claim 17. The method for producing cadaverine dicarboxylate according to claim 17, wherein the organic solvent is at least one selected from the group consisting of methanol, ethanol, propanol, butanol, hexanol, isopropanol, acetonitrile, and acetone. . Cadaverine or cadaverine dihydrochloride or cadaverine dicarboxylic acid having a content of 2,3,4,5-tetrahydropyridine of 1.4% by weight or less, produced by the method according to any one of claims 7 to 20. salt. Cadaverine or cadaverine dihydrochloride or cadaverine dicarboxylate having a tri-n-butylamine content of 0.006% by weight or less, produced by the method according to any one of claims 7 to 20. The content of 2,3,4,5-tetrahydropyridine produced by the method according to any one of claims 7 to 20 is 1.4% by weight or less and the content of tri-n-butylamine is 0.1%. Not more than 006% cadaverine or cadaverine dihydrochloride or cadaverine dicarboxylate A cadaverine, cadaverine dihydrochloride or cadaverine dicarboxylate obtained by the method according to any one of claims 1 to 20, or a cadaverine, cadaverine dihydrochloride or cadaverine according to any one of claims 21 to 23. -A polyamide containing a dicarboxylate as a raw material.

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