JP2004041107A - Method for producing l-amino acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new method for producing a high-optical purity L-amino acid easily and efficiently, specifically using as a raw material a cinnamic acid derivative bearing substituent(s) on the phenyl group or an acrylic acid derivative bearing a heterocyclic ring. <P>SOLUTION: The method for producing the L-amino acid involves using a histidine ammonia lyase derived from microorganisms and animals and having the activity of optically selectively adding amino groups to various acrylic acid derivatives other than of making a reaction of giving histidine from urocanic acid as known conventionally. <P>COPYRIGHT: (C)2004,JPO

Description

（但し、式中Ｚはヘテロ原子を含んでいてもよい芳香環基を、Ｒは該芳香環上の置換基を表し、ｎは０以上の整数を表し、ｎが２以上の場合、Ｒは同一または相異なっていてもよい。但し、Ｚがイミダゾールであり、且つｎが０の場合を除く。）
で示されるアクリル酸誘導体に、アンモニアの存在下、ヒスチジンアンモニアリアーゼを作用させることを特徴とする、下記式（２）
【化７】

（但し、式中Ｚはヘテロ原子を含んでいてもよい芳香環基を、Ｒは該芳香環上の置換基を表し、ｎは０以上の整数を表し、ｎが２以上の場合、Ｒは同一または相異なっていてもよい。但し、Ｚがイミダゾールであり、且つｎが０を除く。）
で示されるＬ−アミノ酸の製造方法。
【００１５】
［２］Ｒ_ｎ−Ｚ−が下記式（３）
【化８】

（但し、Ｒは、ベンゼン環上の置換基であり、シアノ基、水酸基、カルボキシル基、アミド基、ハロゲン原子、アミノ基、ニトロ基、ヒドロキシメチル基、炭素数１〜６のアルキル基または炭素数１〜６のアルコキシ基を表す。ｎは０〜５の整数を表し、ｎが２以上の場合、Ｒは同一または相異なっていてもよい。）
で示される［１］に記載のＬ−アミノ酸の製造方法。
［３］Ｒの少なくとも一つが、炭素数１〜６のアルキル基、炭素数１〜６のアルコキシ基およびニトロ基であることを特徴とする［２］に記載のＬ−アミノ酸の製造方法。
［４］ｎが１であることを特徴とする［３］に記載のＬ−アミノ酸の製造方法。
【００１６】
［５］Ｒ_ｎ−Ｚ−が下記式（４）
【化９】

（但し、式中ＸはＳ、Ｏ、ＮＨまたはＮＲ^１を表し、Ｒ^１は炭素数１〜６のアルキル基を表し、式中ＹはＮ、ＣＨまたはＣＲ^２を表す。Ｒ、Ｒ^２は、シアノ基、水酸基、カルボキシル基、アミド基、ハロゲン原子、アミノ基、ニトロ基、ヒドロキシメチル基、炭素数１〜６のアルキル基または炭素数１〜６のアルコキシ基を表す。ｎは０〜３の整数を表し、ｎが２以上の場合、Ｒは同一または相異なっていてもよく、ＲとＲ^２は同一または相異なっていてもよく、隣り合うＲ、Ｒ^２は互いに結合し、炭素数１〜６であるヘテロ原子を含んでよい環を形成してもよい。但し、ＸがＮＨであり、且つＹがＮであり、且つｎが０の場合を除く。）
で示される［１］に記載のＬ−アミノ酸の製造方法。
［６］ＹがＣＨであることを特徴とする［５］に記載のＬ−アミノ酸の製造方法。
［７］ｎが０であり、且つＸがＳ、ＯまたはＮＨであることを特徴とする［６］に記載のＬ−アミノ酸の製造方法。
【００１７】
［８］Ｒ_ｎ−Ｚ−が下記式（５）
【化１０】

（但し、式中Ｒはヘテロ環上の置換基であり、シアノ基、水酸基、カルボキシル基、アミド基、ハロゲン原子、アミノ基、ニトロ基、ヒドロキシメチル基、炭素数１〜６のアルキル基または炭素数１〜６のアルコキシ基を表す。ｎは０〜４の整数を表し、ｎが２以上の場合、Ｒは同一または相異なっていてもよく、隣り合うＲは互いに炭素数１〜６であるヘテロ原子を含んでよい環を形成してもよい。）
で示される［１］に記載のＬ−アミノ酸の製造方法。
［９］ｎが０であることを特徴とする［８］に記載のＬ−アミノ酸の製造方法。
【００１８】
［１０］ヒスチジンアンモニアリアーゼを、ヒスチジンアンモニアリアーゼを含む微生物の培養物、該培養物の処理組成物および／または該培養物より得られる酵素の形態で作用させることを特徴とする［１］ないし［９］のいずれかに記載のＬ−アミノ酸の製造方法。
［１１］ヒスチジンアンモニアリアーゼを含む微生物が、Ｐｓｅｕｄｏｍｏｎａｓ属、Ｂａｃｉｌｌｕｓ属、Ｋｌｅｂｓｉｅｌｌａ属、Ｒｈｉｚｏｂｉｕｍ属、Ａｌｃａｌｉｇｅｎｅｓ属、Ａｓｐｅｒｇｉｌｌｕｓ属、Ａｇｒｏｂａｃｔｅｒｉｕｍ属、Ｄｅｉｎｏｃｏｃｃｕｓ属、Ｅｓｃｈｅｒｉｃｈｉａ属、Ｓｔｒｅｐｔｏｍｙｃｅｓ属、Ｃａｕｌｏｂａｃｔｅｒ属、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ属およびＣｏｒｙｎｅｂａｃｔｅｒｉｕｍ属のいずれかから選ばれる少なくとも１種であることを特徴とする［１０］に記載のＬ−アミノ酸の製造方法。
［１２］ヒスチジンアンモニアリアーゼを含む微生物が、Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ　ＪＣＭ６１５６であることを特徴とする［１０］に記載のＬ−アミノ酸の製造方法。［１３］ヒスチジンアンモニアリアーゼを含む微生物が、Ｂａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ　ＭＩ１１３であることを特徴とする［１０］に記載のＬ−アミノ酸の製造方法。
［１４］ヒスチジンアンモニアリアーゼを含む微生物が、ヒスチジンアンモニアリアーゼ遺伝子により形質転換された微生物であることを特徴とする［１０］に記載のＬ−アミノ酸の製造方法。
【００１９】
［１５］ヒスチジンアンモニアリアーゼ遺伝子がＰｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ　ＪＣＭ６１５６由来のアミノ酸配列と９８％以上の相同性を有する配列をコードする遺伝子であることを特徴とする［１４］に記載のＬ−アミノ酸の製造方法。
［１６］ヒスチジンアンモニアリアーゼ遺伝子が微生物に由来するものであることを特徴とする［１４］に記載のＬ−アミノ酸の製造方法。
［１７］ヒスチジンアンモニアリアーゼ遺伝子の由来となる微生物が、Ｐｓｅｕｄｏｍｏｎａｓ属、Ｂａｃｉｌｌｕｓ属、Ｋｌｅｂｓｉｅｌｌａ属、Ｒｈｉｚｏｂｉｕｍ属、Ａｌｃａｌｉｇｅｎｅｓ属、Ａｓｐｅｒｇｉｌｌｕｓ属、Ａｇｒｏｂａｃｔｅｒｉｕｍ属、Ｄｅｉｎｏｃｏｃｃｕｓ属、Ｅｓｃｈｅｒｉｃｈｉａ属、Ｓｔｒｅｐｔｏｍｙｃｅｓ属、Ｃａｕｌｏｂａｃｔｅｒ属、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ属、Ｖｉｂｒｉｏ属およびＣｏｒｙｎｅｂａｃｔｅｒｉｕｍ属のいずれかから選ばれる少なくとも１種であることを特徴とする［１６］に記載のＬ−アミノ酸の製造方法。
【００２０】
［１８］ヒスチジンアンモニアリアーゼ遺伝子の由来となる微生物が、Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ　ＪＣＭ６１５６であることを特徴とする［１６］に記載のＬ−アミノ酸の製造方法。
［１９］ヒスチジンアンモニアリアーゼ遺伝子の由来となる微生物が、Ｂａｃｉｌｌｕｓｓｕｂｔｉｌｉｓ　ＭＩ１１３であることを特徴とする［１６］に記載のＬ−アミノ酸の製造方法。
［２０］ヒスチジンアンモニアリアーゼ遺伝子が動物に由来するものであることを特徴とする［１４］に記載のＬ−アミノ酸の製造方法。
［２１］動物が、マウス、ラット、ヒトのいずれかであることを特徴とする［２０］に記載のＬ−アミノ酸の製造方法。
【００２１】
［２２］Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ　ＪＣＭ６１５６由来のヒスチジンアンモニアリアーゼ遺伝子がコードするアミノ酸配列と８０％以上の相同性を有する配列をコードする遺伝子、該遺伝子を含むプラスミド、および該プラスミドで形質転換された形質転換微生物。
［２３］Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ　ＪＣＭ６１５６由来のヒスチジンアンモニアリアーゼ遺伝子、該遺伝子を含むプラスミド、および該プラスミドで形質転換された形質転換微生物。
【００２２】
【発明の実施の形態】
以下本発明について詳細に説明する。
【００２３】
本発明のＬ−アミノ酸の製造方法は、種々の置換基を有してもよく、またヘテロ原子を含んでもよい芳香環基を有するアクリル酸誘導体より、光学活性なアミノ酸を、１段階の酵素反応で簡便に得ることができるものである。
【００２４】
あらかじめ芳香環基に導入された置換基、例えばシアノ基、水酸基、カルボキシル基、アミド基、ハロゲン原子、アミノ基、ニトロ基、ヒドロキシメチル基、炭素数１〜６のアルキル基、炭素数１〜６のアルコキシ基は、本発明の温和な反応条件下では影響を受けることなく反応の終了まで保持され、対応する所望のＬ−フェニルアラニン誘導体をほぼ１００％の変換率で収率よく得ることができる。
【００２５】
また、本発明のＬ−アミノ酸の製造方法は、複素環構造を有するアクリル酸誘導体より、複素環構造を有する光学活性なアミノ酸を、一段の酵素反応で簡便に得ることができる。
【００２６】
当該アクリル酸誘導体中のフリル基、チエニル基、ピロリル基、イミダゾリル基等の複素環構造は、本発明における温和な反応条件下では影響を受けることなく反応の終了まで保持され、対応する所望のＬ−アミノ酸をほぼ１００％の変換率で収率よく得ることができる。
【００２７】
なお本発明の反応原料となる、種々の置換基を有するアクリル酸誘導体は、任意の置換基が導入されたアルデヒド誘導体のアルデヒド基と無水酢酸を作用させる、いわゆるパーキン反応による方法（Ａｒｃｈ．　Ｐｈａｒｍ．　（Ｗｅｉｎｈｅｉｍ）　（１９９４），　３２７（１０），　６１９−６２５等を参照）、もしくはピリジン溶媒中、ピペリジンの存在下マロン酸などを作用させる方法（Ｊ　Ｃｈｅｍ．　Ｓｏｃ．　（１９３９），　３５７−３６０等を参照）、ほか種々の改良された方法（Ｓｙｎｔｈ．　Ｃｏｍｍｕｎ．　（１９９９），　２９（４），　５７３−５８１等を参照）等を適用することにより調製される。
【００２８】
本発明の実施形態の一つとしては、培養されたヒスチジンアンモニアリアーゼ活性を持つ微生物の培養物を用いる。本発明における微生物の培養物とは微生物を培養した液および該培養液から微生物菌体のみを取り出した微生物菌体も示す。
【００２９】
本発明において適用される、ヒスチジンアンモニアリアーゼ活性を提供する微生物に特に制限はない。該酵素活性を有することが知られる微生物の具体例としては、Ｐｓｅｕｄｏｍｏｎａｓ属、Ｂａｃｉｌｌｕｓ属、Ｋｌｅｂｓｉｅｌｌａ属、Ｒｈｉｚｏｂｉｕｍ属、Ａｌｃａｌｉｇｅｎｅｓ属、Ａｓｐｅｒｇｉｌｌｕｓ属、Ａｇｒｏｂａｃｔｅｒｉｕｍ属、Ｄｅｉｎｏｃｏｃｃｕｓ属、Ｅｓｃｈｅｒｉｃｈｉａ属、Ｓｔｒｅｐｔｏｍｙｃｅｓ属、Ｃａｕｌｏｂａｃｔｅｒ属、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ属、Ｖｉｂｒｉｏ属、Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ属の各属の微生物が挙げられる。
【００３０】
さらに本発明の異なる実施形態の一つとしては、ｈａｌ遺伝子を微生物に導入して発現可能に存在させた、形質転換微生物を用いることもできる。
【００３１】
本発明で用いるｈａｌ遺伝子は、天然に存在するｈａｌ遺伝子の他、同じ機能を有する（同じ活性の変異体酵素をコードする）遺伝子であってもよい。該酵素遺伝子の由来生物からの単離、および該遺伝子を用いた形質転換微生物の作成方法、また該形質転換微生物より該酵素を分取する方法等については、例えばＰｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａを用いた事例としてＬａｎｇｅｒらの報告（Ｂｉｏｃｈｅｍｉｓｔｒｙ（１９９４），　３３，　６４６２−６４６７．）、またラットを用いた事例としてＴａｙｌｏｒらの報告（Ｊ．　Ｂｉｏｌ．　Ｃｈｅｍ．（１９９０），　２６５，　１８１９２−１８１９９．）が知られる。
【００３２】
ｈａｌ遺伝子の取得法、発現可能なプラスミドの作製法、及びこれらのプラスミドの各種生物への導入・発現法の例についてさらに詳しく説明する。
【００３３】
＜ｈａｌ遺伝子の取得法＞
動物由来のｈａｌ遺伝子は、公知の手法により作成したｃＤＮＡライブラリーから、ｈａｌ遺伝子の既知配列に相当する部分断片をプローブとして取得することができるが、対象によってはＡＴＣＣのごとき遺伝子バンクから当該ｃＤＮＡを入手することもできる。ｃＤＮＡライブラリーの作成には、まずｍＲＮＡを単離精製する必要があり、従来の方法によっても可能であるが、近年では組織から簡便にｔｏｔａｌＲＮＡを取得できるキットなども販売されている。ｃＤＮＡは、取得されたｔｏｔａｌＲＮＡまたはここからｏｌｉｇｏＴカラムなどによって単離したｍＲＮＡを鋳型にし、Ｒｅｖｅｒｓｅ　ｔｒａｎｓｃｒｉｐｔａｓｅを用いて得ることができる。こうして得たｃＤＮＡを適当なプラスミドベクター、例えばｐＢＲ３２２やｐＵＣ１８などに接続し、宿主とする大腸菌を形質転換する。
【００３４】
上記のようにして作成したｃＤＮＡライブラリーから目的のｈａｌ遺伝子を取得するには、ｈａｌ遺伝子の保存配列をコードするオリゴＤＮＡをプローブとして用いたコロニーハイブリダイゼーション法により得ることができる。
【００３５】
一方、微生物由来のｈａｌ遺伝子は、公知の手法により作成したｇｅｎｏｍｅＤＮＡライブラリーから、ｈａｌ遺伝子の保存配列に相当する部分断片をプローブとして取得することができる。ｇｅｎｏｍｅＤＮＡライブラリーの作成に必要なｇｅｎｏｍｅＤＮＡの取得には、従来の方法を用いて得ることもできるが、近年では微生物菌体から効率的にｇｅｎｏｍｅＤＮＡを取得できるキットなども販売されている。こうして得たｇｅｎｏｍｅＤＮＡを適当なプラスミドベクター、例えばｐＢＲ３２２やｐＵＣ１８などに接続し、宿主とする大腸菌を形質転換する。
【００３６】
上記のようにして作成したｇｅｎｏｍｅＤＮＡライブラリーから目的のｈａｌ遺伝子を取得するには、ｈａｌ遺伝子の保存配列をコードするオリゴＤＮＡをプローブとして用いたコロニーハイブリダイゼーション法により得ることができる。
【００３７】
さらに配列既知なものに関しては末端配列に相当する部分断片をプライマーとして、ｃＤＮＡあるいはｇｅｎｏｍｅＤＮＡを鋳型としたＰＣＲ法を行うことにより容易に十分な量のｈａｌ構造遺伝子断片を得ることができる。こうして得たｈａｌ遺伝子を適当なプラスミドベクター、例えばｐＢＲ３２２やｐＵＣ１８などに接続し、宿主とする大腸菌を形質転換する。
【００３８】
＜発現プラスミドの作製法および製造に用いる組換え微生物の作製法＞
ｈａｌ遺伝子は適当なベクター（具体例は後述）により、大腸菌等の細菌、Ｓａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｉｓｉａｅ等の酵母等の微生物、好ましくは大腸菌等の細菌に導入し、発現させることにより、目的のＬ−アミノ酸生成活性を有する組換え体を得ることができる。生成したＬ−アミノ酸は、後述するように微生物反応生成物を反応物から分離するための通常の方法により単離精製することができる。
外来遺伝子を含むプラスミドの作製法、大腸菌等の微生物へのプラスミドの導入および発現のための手順ないし方法は、遺伝子工学の分野で慣用されている方法（たとえば、”Ｖｅｃｔｏｒｓ　ｆｏｒ　ｃｌｏｎｉｎｇ　ｇｅｎｅｓ”，　Ｍｅｔｈｏｄｓ　ｉｎ　Ｅｎｚｙｍｏｌｏｇｙ，　２１６，　ｐ．　４６９−６３１，　１９９２，　Ａｃａｄｅｍｉｃ　Ｐｒｅｓｓ　、および、”Ｏｔｈｅｒ　ｂａｃｔｅｒｉａｌ　ｓｙｓｔｅｍｓ”，　Ｍｅｔｈｏｄｓ　ｉｎ　Ｅｎｚｙｍｏｌｏｇｙ，　２０４，　ｐ．３０５−６３６，　１９９　１，　Ａｃａｄｅｍｉｃ　Ｐｒｅｓｓ参照）に準じて実施すればよい。
【００３９】
大腸菌大腸菌への外来遺伝子（ｈａｌ遺伝子群）の導入法は、ハナハンの方法、ルビジウム法などすでに確立されたいくつかの効率的方法があり、それを用いて行えばよい（たとえば、Ｓａｍｂｒｏｏｋ，　Ｊ．，　Ｆｒｉｔｓｃｈ，Ｅ．　Ｆ．，　Ｍａｎｉａｔｉｓ，　Ｔ．，　”Ｍｏｌｅｃｕｌａｒ　ｃｌｏｎｉｎｇ　−Ａ　ｌａｂｏｒａｔｏｒｙ　ｍａｎｕａｌ．”　Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ，　１９８９参照）。大腸菌での外来遺伝子の発現は常法に従って行えばよいが（たとえば、前述の　”Ｍｏｌｅｃｕｌａｒ　ｃｌｏｎｉｎｇ　−　Ａ　ｌａｂｏｒａｔｏｒｙ　ｍａｎｕａｌ．”参照）、たとえば、ｐＵＣ　系やｐＢｌｕｅｓｃｒｉｐｔ　系等のｌａｃ　のプロモーター等を有する大腸菌用ベクターを用いて行うことができる。本発明者等は、ｔｒｃプロモーターを有する大腸菌用ベクターｐＴｒｃ９９Ａを用いて、プロモーターの下流にｈａｌ遺伝子を挿入し、大腸菌で発現させた。
【００４０】
酵母Ｓａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｉｓｉａｅへのｈａｌ遺伝子の導入法は、リチウム法などすでに確立された方法があり、それを用いて行えばよい（たとえば、秋山裕一監修バイオインダストリー協会編集、「酵母のニューバイオテクノロジー」医学出版センター刊参照）。酵母での外来遺伝子の発現は、ＰＧＫ　やＧ　ＰＤ　（ＧＡＰ）等のプロモーターおよびターミネーターを用いて、外来遺伝子をこのプロモーターとターミネーターの間に転写制御を受けるように挿入した発現カセットを構築し、この発現カセットを、Ｓａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｉｓｉａｅのベクター、たとえば、ＹＲｐ　系（酵母染色体のＡＲＳ　配列を複製起点とする酵母用マルチコピーベクター）、ＹＥｐ　系（酵母の２　　μｍ　ＤＮＡ　の複製起点を持つ酵母用マルチコピーベクター）、ＹＩｐ　系（酵母の複製起点を持たない酵母染色体組込み用ベクター）等のベクターに挿入することにより行うことができる（前述の「酵母のニューバイオテクノロジー」医学出版センター刊、日本農芸化学会ＡＢＣ　シリーズ「物質生産のための遺伝子工学」朝倉書店刊　参照）。
【００４１】
＜ヒスチジンアンモニアリアーゼ活性微生物の培養＞
本発明に関連する微生物（形質転換微生物を含む）の培養は、微生物の一般的な培養法に準じて行うことができる。
【００４２】
微生物を培養するための培地炭素源としては、微生物が炭素源とすることができるもの、例えばグルコースやシュークロース、フルクトース、廃糖蜜等の糖類、エタノールや酢酸、クエン酸、コハク酸、乳酸、安息香酸、脂肪酸などの有機物又はこれらのアルカリ金属塩、ｎ−パラフィンなどの脂肪族炭化水素類、また例えばペプトン、肉エキス、魚エキス、大豆粉、ふすま、麦芽抽出物、ジャガイモ抽出物等の天然有機物を、単独あるいはこれらの組み合わせにより、通常０．０１％〜３０％、好ましくは０．１％〜１０％程度の濃度で用いることができる。例えば大腸菌の形質転換微生物を用いる場合、グルコース、ペプトン、肉エキス、麦芽抽出物等が好適に用いられる。
【００４３】
微生物（形質転換微生物を含む）を培養するための培地窒素源としては、微生物が窒素源とすることができるもの、例えば硫酸アンモニウム、リン酸アンモニウム、硝酸ナトリウム、硝酸カリウムなどの無機窒素化合物、また尿素、尿酸などの含窒素有機物、ペプトン、肉エキス、魚エキス、大豆粉、麦芽抽出物、ジャガイモ抽出物等の天然有機物を、単独あるいはこれらの組み合わせにより、通常０．０１％〜３０％、好ましくは０．１％〜１０％程度の濃度で用いることができる。例えば大腸菌を形質転換微生物として用いる場合には、硫酸アンモニウム、ペプトン、肉エキス、麦芽抽出物等が好適に用いられる。
【００４４】
さらに必要に応じて、リン酸２水素カリウム等のリン酸塩、硫酸マグネシウム、硫酸第一鉄、酢酸カルシウム、塩化マンガン、硫酸銅、硫酸亜鉛、硫酸コバルト、硫酸ニッケルなどの金属塩が菌の生育改善のために添加される。添加濃度は培養条件により異なるものの、通常、リン酸塩に関しては０．０１％〜５％、マグネシウム塩においては１０ｐｐｍ〜１％、他の化合物では０．１ｐｐｍ〜１０００ｐｐｍ程度である。また選択する培地により、ビタミン類、アミノ酸、核酸などの供給源として例えば酵母エキス、カザミノ酸、酵母核酸を１ｐｐｍ〜１００ｐｐｍ程度添加することにより、菌の生育を改善することができる。
【００４５】
また培養中、必要に応じ、ヒスチジンアンモニアリアーゼ活性を高めるために、培養液中に酵素誘導源として、Ｌ−ヒスチジンを１０ｐｐｍ〜１％程度添加することができる。
【００４６】
形質転換微生物においては、必要に応じ、形質転換に供された該酵素遺伝子を宿主微生物で高度に発現させるため、ベクターの発現プロモーターに対応する誘導物質を培養中に添加することができる。例えばＬａｃプロモーターを用いる場合はＩＰＴＧ（イソプロピル−１−チオ−β−Ｄ−ガラクトシド）を０．０１ｍＭ〜１０ｍＭ程度、またアンピシリンやカナマイシン等の薬剤耐性プロモーターを用いる場合は対応する薬剤を０．１ｐｐｍ〜１０００ｐｐｍ程度添加する。
【００４７】
いずれの組成を用いた場合も培養のｐＨは、５〜９、好ましくは５．５〜８に調整される。培養液はそのまま反応に供することができるが、上記のごとき培地であらかじめ培養された微生物菌体を、遠心分離、膜ろ過などの方法により培養液から分取し、反応に供することは、培養液から持ち込まれる夾雑物を低減し、のちの生成物の分取を簡便にするために有用である。
【００４８】
培養して得た微生物は、そのまま遠心分離や膜分離など公知の方法により回収して、あるいは培養液そのものを目的反応に供することができる。
【００４９】
本発明の異なる実施形態の一つとしては、微生物（形質転換微生物を含む）より分離されたヒスチジンアンモニアリアーゼを用いる。すなわち、微生物より酵素を分離精製するための公知の方法、たとえば破砕した微生物菌体の画分からアセトン分画沈澱、硫安沈澱、各種分離カラムなどの常法により分離精製された該酵素を反応触媒として供することができる。種々の微生物からの具体的なヒスチジンアンモニアリアーゼの分離精製法としては、たとえばＡｃｈｒｏｍｏｂａｃｔｅｒ属微生物について特開昭５０−２４４８８号公報、またＰｓｅｕｄｏｍｏｎａｓ属微生物についてのＫｌｅｅらの報告、（Ｊｏｕｒｎａｌ　ｏｆ　Ｂｉｏｌｏｇｉｃａｌ　Ｃｈｅｍｉｓｔｒｙ，　２４５，　３１４３−３１５２（１９７０））等が知られる。
【００５０】
また本発明の異なる実施形態の一つとして、前記のごとき微生物（形質転換微生物を含む）の処理物を、目的反応に供する。微生物の処理物としては、例えば微生物の摩砕物・凍結乾燥物や微生物菌体からの無細胞抽出液、さらに抽出液から目的反応を触媒する成分を濃縮・抽出したもの、また、これら微生物体の処理物や抽出液、抽出成分を、難溶性の担体に固定化したもの、等を反応に供することができる。このような目的に供される固定化担体としては、ポリアクリル酸、ポリメタクリル酸、ポリアクリルアミド、ポリビニルアルコール、ポリ−Ｎ−ビニルホルムアミド、ポリアリルアミン、ポリエチレンイミン、メチルセルロース、グルコマンナン、アルギン酸塩、カラギーナン等、さらにこれらの（架橋）重合物など、微生物もしくはその抽出成分を包合した水難溶性の固形分を形成するような化合物を、単独もしくは混合して用いることができる。また、活性炭、多孔質セラミックス、グラスファイバー、多孔質ポリマー成型体、ニトロセルロース膜など、あらかじめ固形物として形成された物体上に形質転換微生物もしくはその抽出液、抽出成分を保持させたものを用いることもできる。
【００５１】
＜ヒスチジンアンモニアリアーゼ活性によるＬ−アミノ酸生成反応＞
本発明における、Ｌ−アミノ酸の生成反応は、以上のごとく種々の公知の方法で調製されるヒスチジンアンモニアリアーゼを作用させることによりなされる。反応原料としてアクリル酸誘導体濃度は、好ましくは１ｐｐｍ〜２０％、より好ましくは１０ｐｐｍ〜１０％であり、またアンモニアを終濃度として２Ｍ〜１２Ｍとなるよう添加することが好ましい。さらにｐＨを８〜１１に調整するのが好ましく、より好ましくは８．５〜１０．５である。反応時間は基質により異なるが、通常１時間〜２００時間程度反応することにより達せられる。ここにいうヒスチジンアンモニアリアーゼを作用させるとは、ヒスチジンアンモニアリアーゼを含む微生物の培養物、該培養物の処理組成物、該培養物より得られる酵素のいずれか、または、それらの混合物を作用させることをいい、微生物の培養物とは、培養液、培養物中の微生物菌体そのものも含み、菌体を分離して使用してもよい。更に、ヒスチジンアンモニアリアーゼを含む微生物としては、微生物、動物より単離された該酵素遺伝子で形質転換された微生物でもよい。
【００５２】
反応のｐＨを調整するために用いる酸としては、硫酸、塩酸、リン酸、ホウ酸、炭酸等の無機酸、また蟻酸、酢酸、プロピオン酸等の有機酸及びこれらの塩を用いることができる。この際、揮発性の酸を用いることは、反応液の除菌と留去により、脱塩の工程を省き簡便に生成物を分取することができ、有用である。このような酸としては炭酸が好適である。この場合の炭酸とは、二酸化炭素として水溶液中にバブリング等により溶解し、解離により生成する炭酸を含む。またこれらの酸とアンモニアの塩を、反応液へのアンモニア源として用いることもでき、上記のような理由からアンモニア源の一部または全部として炭酸アンモニウム、炭酸水素アンモニウムを用いることは好適である。
【００５３】
なお添加されるアクリル酸誘導体は必ずしも全量が溶解していなくてもよいが、反応液中での溶解性や分散性を改善するような溶媒、界面活性剤等を添加することもできる。また反応の進行による化合物の消費に応じて、連続的あるいは間欠的に化合物を添加してもよく、この場合の化合物の反応液中濃度は前記の限りではない。
【００５４】
反応液中に生成したＬ−アミノ酸は、その反応液中での性状により、遠心分離、膜ろ過、減圧乾燥、蒸留、溶媒抽出、塩析、イオン交換、各種クロマトグラフィーなど公知の方法で分取される。簡便には、以下のようにして達せられる。例えば、反応液からろ過、遠心、透析等により酵素、酵素処理物、形質転換微生物やその処理物を除去後、溶媒抽出、または反応液を酸性に調整し未反応の原料アクリル酸誘導体を析出させ、沈殿を除去する。
【００５５】
得られた上清のｐＨを再度、Ｌ−アミノ酸の等電点付近に調整し析出する誘導体を同様に回収する。このようにして反応液より生成物を収率・純度よく回収することができる。また、あらかじめ反応液から蒸留などにより余剰のアンモニアを除去すること、等電点での生成物の回収率を向上するために水を留去し濃度を高めておくこと等は有用である。
【００５６】
さらに簡便には、反応液のｐＨ調整を、揮発性の酸により実施する。変換率が十分高い場合は除菌後そのまま、また原料が多量に残存する場合は酸性として溶媒抽出し除去した後に水・酸塩基を留去することにより、生成物をＬ−アミノ酸のアンモニウム塩として単離することができる。このような方法に適した揮発性の酸としては炭酸及びそのアンモニウム塩が好適である。
【００５７】
反応生成物の性質によっては、反応液中に生成物が蓄積することにより、反応速度が低下する場合がある。このような場合は、生成物の濃度に応じて反応液中に、アンモニアを含む水、生理食塩水、反応緩衝液を追加し連続的に希釈してゆく方法は好適である。また反応速度が低下した時点で菌を分取し、上清を生産物溶液として回収し、分取した菌は再度反応原料を含む溶液あるいは懸濁液に戻すことにより、反応速度を回復することができる。これらの方法は、微生物のアンモニアリアーゼ活性が維持される範囲において、何回でも繰り返すことができる。
【００５８】
本発明の原料となる好適な化合物は、下記式（１）
【００５９】
【化１１】

（但し、式中Ｚはヘテロ原子を含んでいてもよい芳香環基を、Ｒは該芳香環上の置換基を表し、ｎは０以上の整数を表し、ｎが２以上の場合、Ｒは同一または相異なっていてもよい。但し、Ｚがイミダゾールであり、且つｎが０の場合を除く。）で示されるアクリル酸誘導体である。（１）式のアクリル酸部分の二重結合に対するＺとＣＯＯＨ基の立体配置はトランス、シスのいずれでも原料として使用し可能であるが、好ましくはトランス体である。
【００６０】
また、得られる化合物は、下記式（２）
【００６１】
【化１２】

（但し、式中Ｚはヘテロ原子を含んでいてもよい芳香環基を、Ｒは該芳香環上の置換基を表し、ｎは０以上の整数を表し、ｎが２以上の場合、Ｒは同一または相異なっていてもよい。但し、Ｚがイミダゾールであり、且つｎが０の場合を除く。）
で示されるＬ−アミノ酸である。
【００６２】
前記式中、一価以上の基であるＺとしては、たとえば下記式（６）〜（９）
【００６３】
【化１３】

で表される芳香環からなる基、下記式（１０）〜（１３）
【００６４】
【化１４】

で表される芳香環からなる基、下記式（１４）〜（１９）
【００６５】
【化１５】

で表される芳香環からなる基、下記式（２０）〜（２３）
【００６６】
【化１６】

で表される芳香環からなる基、下記式（２４）〜（２８）
【００６７】
【化１７】

で表される芳香環からなる基、下記式（２９）〜（３２）
【００６８】
【化１８】

で表される芳香環からなる基、下記式（３３）〜（３６）
【００６９】
【化１９】

で表される芳香環からなる基、下記式（３７）〜（４０）
【００７０】
【化２０】

で表される芳香環からなる基、下記式（４１）〜（４５）
【００７１】
【化２１】

で表される芳香環からなる基等が挙げられる。
【００７２】
Ｒとしては、シアノ基、水酸基、カルボキシル基、アミド基、ハロゲン原子、アミノ基、ニトロ基、ヒドロキシメチル基、炭素数１〜６のアルキル基または炭素数１〜６のアルコキシ基等が挙げられる。芳香環の種類にもよるが、好ましくは、シアノ基、水酸基、ハロゲン原子、ニトロ基、メチル基、エチル基、メトキシ基、エトキシ基が挙げられる。
【００７３】
ｎは、０以上で、芳香環の置換しうる位置の数から１を引いた数以下である整数とすることができる。ｎが２以上の場合には、Ｒは同一または相異なっていてもかまわない。
【００７４】
また本発明の原料となる好適な化合物は、前記式（１）において、Ｒ_ｎ−Ｚ−が下記式（３）
【００７５】
【化２２】

（但し、Ｒは、ベンゼン環上の置換基であり、シアノ基、水酸基、カルボキシル基、アミド基、ハロゲン原子、アミノ基、ニトロ基、ヒドロキシメチル基または炭素数１〜６のアルキル基もしくはアルコキシ基を表す。ｎは０〜５の整数を表し、ｎが２以上の場合、Ｒは同一または相異なっていてもよい。）で示される化合物が挙げられる。
【００７６】
また、好適に得られる化合物は、前記式（２）において、Ｒ_ｎ−Ｚ−が前記式（３）（但し、Ｒ、ｎは、前記に同じ意味を表す。）で示される化合物が挙げられる。
【００７７】
好ましくは、前記式（１）、（２）において、Ｒが、炭素数１〜６のアルキル基、炭素数１〜６のアルコキシ基およびニトロ基であり、更に好ましくは、Ｒが、炭素数１〜６のアルキル基、炭素数１〜６のアルコキシ基およびニトロ基であり、ｎが１である。
【００７８】
このような化合物の具体例としては２−シアノ桂皮酸、３−シアノ桂皮酸、４−シアノ桂皮酸、２−ヒドロキシ桂皮酸、３−ヒドロキシ桂皮酸、４−ヒドロキシ桂皮酸、３、４−ジヒドロキシ桂皮酸、２−ニトロ桂皮酸、３−ニトロ桂皮酸、４−ニトロ桂皮酸、２−カルボキシ桂皮酸、３−カルボキシ桂皮酸、４−カルボキシ桂皮酸、２−アミノ桂皮酸、３−アミノ桂皮酸、４−アミノ桂皮酸、２−クロロ桂皮酸、３−クロロ桂皮酸、４−クロロ桂皮酸、２−フルオロ桂皮酸、３−フルオロ桂皮酸、４−フルオロ桂皮酸、２−ブロモ桂皮酸、３−ブロモ桂皮酸、４−ブロモ桂皮酸、２−メチル桂皮酸、３−メチル桂皮酸、４−メチル桂皮酸、２−メトキシ桂皮酸、３−メトキシ桂皮酸、４−メトキシ桂皮酸、４−イソプロピル桂皮酸、４−ｔ−ブチル桂皮酸、等が挙げられる。
【００７９】
例えばＢａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ　ＭＩ１１３由来酵素を用いた場合、原料として２−シアノ桂皮酸、４−シアノ桂皮酸、２−メチル桂皮酸、３−メチル桂皮酸、４−メチル桂皮酸、２−メトキシ桂皮酸、３−メトキシ桂皮酸、４−メトキシ桂皮酸は好適であり、それぞれ２−シアノ−Ｌ−フェニルアラニン、４−シアノ−Ｌ−フェニルアラニン、２−メチル−Ｌ−フェニルアラニン、３−メチル−Ｌ−フェニルアラニン、４−メチル−Ｌ−フェニルアラニン、２−メトキシ−Ｌ−フェニルアラニン、３−メトキシ−Ｌ−フェニルアラニン、４−メトキシ−Ｌ−フェニルアラニン等が得られる。
【００８０】
また特に、本発明の原料となる好適な化合物は、前記式（１）において、Ｒ_ｎ−Ｚ−が下記式（４）
【００８１】
【化２３】

（但し、式中ＸはＳ、Ｏ、ＮＨまたはＮＲ^１を表し、Ｒ^１は炭素数１〜６のアルキル基を表し、式中ＹはＮ、ＣＨまたはＣＲ^２を表し、Ｒ、Ｒ^２は、シアノ基、水酸基、カルボキシル基、アミド基、ハロゲン原子、アミノ基、ニトロ基、ヒドロキシメチル基、炭素数１〜６のアルキル基または炭素数１〜６のアルコキシ基を表す。ｎは０〜３の整数を表し、ｎが２以上の場合、Ｒは同一または相異なっていてもよく、ＲとＲ^２は同一または相異なっていてもよく、隣り合うＲ、Ｒ^２は互いに結合し、炭素数１〜６であるヘテロ原子を含んでよい環を形成してもよい。但し、ＸがＮＨであり、且つＹがＮであり、且つｎが０の場合を除く。）が挙げられる。
【００８２】
また得られる化合物は、前記式（２）において、Ｒ_ｎ−Ｚ−が前記式（４）
（但し、式中Ｘ、Ｙ、Ｒ、Ｒ^１、Ｒ^２、ｎは、前記と同じ意味を表す。）で示されるＬ−アミノ酸である。
【００８３】
好ましくは前記式（１）、（２）でＹがＣＨであり、更に好ましくは、ＹがＣＨであり、且つｎが０であり、且つＸがＳ、ＯまたはＮＨである。
【００８４】
このような化合物の具体例としてはβ−（２−フリル）アクリル酸、β−（３−フリル）アクリル酸、β−（２−チエニル）アクリル酸、β（３−チエニル）アクリル酸、β−（２−ピロリル）アクリル酸、β−（３−ピロリル）アクリル酸等が挙げられる。
【００８５】
例えばＢａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ　ＭＩ１１３由来酵素を用いた場合、原料としてβ−（２−フリル）アクリル酸、β−（３−フリル）アクリル酸、β−（２−チエニル）アクリル酸、β−（３−チエニル）アクリル酸、β−（２−ピロリル）アクリル酸、β−（３−ピロリル）アクリル酸、イミダゾールアクリル酸（ウロカニン酸）は好適であり、対応して２−アミノ−３−（２−フリル）プロピオン酸、２−アミノ−３−（３−フリル）プロピオン酸、２−アミノ−３−（２−チエニル）プロピオン酸、２−アミノ−３−（３−チエニル）プロピオン酸、２−アミノ−３−（２−ピロリル）プロピオン酸、２−アミノ−３−（３−ピロリル）プロピオン酸が得られる。
【００８６】
また、本発明の原料となる好適な化合物は、前記式（１）において、Ｒ_ｎ−Ｚ−が下記式（５）
【００８７】
【化２４】

（但し、式中Ｒはヘテロ環上の置換基であり、シアノ基、水酸基、カルボキシル基、アミド基、ハロゲン原子、アミノ基、ニトロ基、ヒドロキシメチル基、炭素数１〜６のアルキル基または炭素数１〜６のアルコキシ基を表す。ｎは０〜４の整数を表し、ｎが２以上の場合、Ｒは同一または相異なっていてもよく、隣り合うＲは互いに炭素数１〜６であるヘテロ原子を含んでよい環を形成してもよい。）で示される化合物である。
【００８８】
また得られる化合物は、前記式（２）において、Ｒ_ｎ−Ｚ−が前記式（５）
（但し、式中ＸはＳ、Ｏ、ＮＨまたはＮＲ^１を表し、Ｒ^１は炭素数１〜６のアルキル基を表し、式中ＹはＮまたはＣを表し、式中Ｒはヘテロ環上の置換基であり、シアノ基、水酸基、カルボキシル基、アミド基、ハロゲン原子、アミノ基、ニトロ基、ヒドロキシメチル基または炭素数１〜６のアルキル基もしくはアルコキシ基を表す。ｎは０〜３の整数を表し、ｎが２以上の場合、Ｒは同一または相異なっていてもよく、隣り合うＲは互いに炭素数３〜５の、ヘテロ原子を含んでよい環を形成してもよい）で示される化合物である。
【００８９】
前記式（１）で示され、Ｒ_ｎ−Ｚ−が前記式（５）の例としては、β−（２−ピリジル）アクリル酸、β−（３−ピリジル）アクリル酸、β−（４−ピリジル）アクリル酸があげられ、生成するＬ−アミノ酸は、２−アミノ−３−（２−ピリジル）プロピオン酸、２−アミノ−３−（３−ピリジル）プロピオン酸、２−アミノ−３−（４−ピリジル）プロピオン酸である。
【００９０】
本発明の方法は、光学純度の高いＬ−アミノ酸を、有機合成的に簡便に得られるアクリル酸誘導体を基質として１段階の反応で効率よく得ることができる。このようにして得られるＬ−アミノ酸は、高度な光学純度が要求される医薬・農薬などファインケミカル分野の合成中間体として有用である。
【００９１】
【実施例】
以下実施例により本発明をさらに具体的に説明するが、本発明はこれら実施例によりなんら限定されるものではない。
【００９２】
なお実施例において、０．１Ｍ　ホウ酸ナトリウム緩衝液ｐＨ８．５、基質としてＬ−ヒスチジン１０ｍＭの存在下、３０℃において毎分１μｍｏｌｅのウロカニン酸を遊離する酵素量を１ｕｎｉｔと定義した。
【００９３】
また酵素タンパク質量の定量は、ビウレット法により、ウシ血清アルブミンを基準として定量し、酵素比活性はｕｎｉｔ／ｍｇタンパク質として表記した。
またアクリル酸誘導体、およびアミノ酸の同定は、^１Ｈ−ＮＭＲ、^１３Ｃ−ＮＭＲ、および反応液をＨＰＬＣに供し標品とＵＶ吸収強度を比較することにより実施した。
【００９４】
アミノ酸、アクリル酸誘導体の分離定量は、以下の分析条件で行った。
逆相ＨＰＬＣ分析条件
カラム：Ｓｈｏｄｅｘ（昭和電工株式会社登録商標）　ＲＳｐａｋ　ＮＮ−６１４（昭和電工株式会社製）
カラム温度：４０℃
溶離液：アセトニトリル／水／５０ｍＭ　Ｈ_３ＰＯ_４−ＫＨ_２ＰＯ_４水溶液（ｐＨ３）＝２０／７０／１０
流速：１．０ｍｌ／ｍｉｎ
検出：ＵＶ　２１０ｎｍの吸収による
【００９５】
また、得られたアミノ酸の光学純度の分析は、以下の条件による光学分割ＨＰＬＣで行った。
光学分割ＨＰＬＣ分析条件
カラム：Ｓｈｏｄｅｘ（昭和電工株式会社登録商標）　ＯＲｐａｋ　ＣＲＸ−８５３（昭和電工株式会社製）
カラム温度：室温（２２℃）
溶離液：アセトニトリル／水＝１５／８５　　ＣｕＳＯ_４　２．５ｍＭ
流速：１．０ｍｌ／ｍｉｎ．
検出方法：ＵＶ　２５６ｎｍの吸収による
【００９６】
［実施例１］　微生物の培養液から得た菌体による反応
Ｂａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ　ＭＩ１１３を、５００ｍｌフラスコ中、Ｌブロス１００ｍｌで３０℃、１８時間振盪培養した。培養液を遠心分離して得た菌体を、４Ｍアンモニア／炭酸アンモニウム（ｐＨ１０）２０ｍｌに再懸濁し、１％（ｗ／ｖ）相当の種々のアクリル酸誘導体を加え、３０℃で攪拌しながら反応を行った。反応８時間後、反応液中の生成物をＨＰＬＣにより定量した。それぞれの反応で得られた生成物と濃度、および光学純度について表１に示す。
【００９７】
【表１】

【００９８】
［実施例２］　微生物の培養物の処理組成物による反応
Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ　ＪＣＭ６１５６を、５００ｍｌフラスコ中、Ｌブロス１００ｍｌで３０℃、１８時間振盪培養した。全量を、ジャーファーメンター中、グルコース１％、尿素０．２％、酵母エキス０．２％、リン酸１カリウム０．２％、リン酸２ナトリウム０．０５％、硫酸マグネシウム０．０１％、硫酸第一鉄７水和物０．００１％、Ｌ−ヒスチジン０．１％　を含み、ｐＨ＝７に調整した培地２Ｌに移植し、３０℃、撹拌８００回転、通気１Ｌ／分　で３０時間培養した。菌体を全量、８０００回転１０分間の遠心により回収し、５０ｍＭ　リン酸緩衝液（ｐＨ７）２００ｍＬに懸濁し、氷冷しつつ超音波処理し菌体を破砕した。この懸濁液を１５０００回転３０分間の遠心に供し、得られた上清を、あらかじめ０．５Ｎ　ＮａＯＨで活性化したのち１０ｍＭリン酸緩衝液（ｐＨ＝７）で平衡化したＤＥＡＥセルロース充填カラムに供した。同ｐＨの１０ｍＭ？５００ｍＭリン酸緩衝液により吸着分を溶出させ、ヒスチジンアンモニアリアーゼ活性画分を分取した。この画分液全量を凍結乾燥に供した。得られた粗精製物の全タンパク量は５０．３ｍｇ、比活性は０．１７Ｕ／ｍｇであった。
【００９９】
実施例１の菌体に代えて、上記粗精製物１ｍｇを、４Ｍアンモニア／炭酸アンモニウム（ｐＨ１０）２０ｍｌに添加し、１％（ｗ／ｖ）相当の種々のアクリル酸誘導体を加え、３０℃で攪拌しながら反応を行った。反応８時間後、反応液中の生成物をＨＰＬＣにより定量した。それぞれの反応で得られた生成物と濃度、および光学純度について表２に示す。
【０１００】
【表２】

【０１０１】
［実施例３］動物ヒスチジンアンモニアリアーゼ遺伝子（ｈａｌ）断片の取得
（マウスｈａｌ遺伝子断片の取得）
ｈａｌ遺伝子の取得には、マウスの肝臓の組織からＲＮｅａｓｙ　Ｍｉｎｉ　Ｋｉｔ（ＱＩＡＧＥＮ社製）を用いてｔｏｔａｌＲＮＡを取得した。方法は全て同キットの標準的な使用法に従った。即ち、３０ｍｇのマウス肝臓をホモジナイザーで破砕し、添付のＲＬＴ緩衝液中０．６ｍｌに懸濁した。懸濁液を遠心機（１５，０００ｒｐｍ）で３分間遠心し、上清０．５ｍｌを新しい容器に取り、等量の７０％エタノールを加えて混合した。混合液の０．７ｍｌをＲＮｅａｓｙカラムに供し、遠心機（１０，０００ｒｐｍ）で１５秒遠心して通過液を捨て、添付のＲＷ１緩衝液をカラムに加え、同じように遠心して液を通過させた。さらに添付のＲＰＮ緩衝液０．５ｍｌをカラムに供し、ｔｏｔａｌＲＮＡをカラムより溶出させた。
【０１０２】
通常は得られたｔｏｔａｌＲＮＡよりｍＲＮＡ精製しｃＤＮＡライブラリーを作製するが、マウスｈａｌ遺伝子の配列が既知であったためｔｏｔａｌＲＮＡを鋳型としたＲＴ−ＰＣＲ法により直接目的遺伝子を増幅した。ＲＴ−ＰＣＲに用いるプライマーは、マウスｈａｌ遺伝子の両末端配列とした。
配列番号１：マウスｈａｌ遺伝子フォワードプライマー：５’−ａｔｇｃｃｔａｇｇｔａｃａｃａｇｔｇ３’
配列番号２：マウスｈａｌ遺伝子リバースプライマー：５’−ｔｔａａａｇａｔｃｇｔｃａｇａｃｔｃ３’
ＲＴ−ＰＣＲにはＯｎｅｓｔｅｐ　ＲＴ−ＰＣＲ　Ｋｉｔ（ＱＩＡＧＥＮ社製）を用い、同キットの標準的な使用法に従って目的遺伝子断片の増幅を行った。鋳型ｔｏｔａｌＲＮＡ溶液の量、アニーリング温度、伸張反応時間の長さ等を最適化した。反応液のうち、ＲＮＡテンプレート（ｔｏｔａｌＲＮＡ）を除く全てを混合し、ＲＮＡテンプレート（ｔｏｔａｌＲＮＡ）を最後に混合した。ミネラルオイルを重層し、あらかじめ５０℃に加温したサーマルサイクラーに反応液をセットし、ＲＴ−ＰＣＲを行った結果、目的遺伝子断片長２〜２．２ｋｂの断片がほぼ特異的に増幅された。この断片をアガロースゲル電気泳動に供して分離精製し、制限酵素によるカットパターンを調べたところ、マウスｈａｌ遺伝子と一致した。
【０１０３】

（ＲＴ−ＰＣＲ条件）
逆転写反応　　　　　　　　　　　　　５０℃、３０分
ＰＣＲ初期活性化　　　　　　　　　　９５℃、１５分
続いて、
変性　　　　　　　　　　　　　　　９４℃、３０秒
アニーリング　　　　　　　　　　　４８℃、３０秒
伸張　　　　　　　　　　　　　　　７２℃、６０秒
（４０サイクル）
さらに、
最終伸張　　　　　　　　　　　　　７２℃、１０分
（ラットｈａｌ遺伝子断片の取得）
ラットｈａｌ遺伝子はＡＴＣＣ（Ａｍｅｒｉｃａｎ　Ｔｙｐｅ　Ｃｕｌｔｕｒｅ　Ｃｏｌｌｅｃｔｉｏｎ）　Ｎｏ．６３１４９としてｐＢｌｕｅｓｃｒｉｐｔ　ＫＳ−上にクローニングされ、Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　ＨＢ１０１株を形質転換したものが販売されているため、これを購入して用いた。
【０１０４】
［実施例４］動物ｈａｌ遺伝子発現系の構築
（マウスｈａｌ遺伝子）
実施例３で得られたｈａｌ遺伝子断片の両末端を平滑化し、発現ベクターｐＴｒｃ９９ＡのＳｍａＩサイトに接続した。このプラスミドｐＴｒｍｈで宿主Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　ＪＭ１０９株を形質転換し、実施例１に従って培養しｈａｌ活性を測定したところ、殆ど活性が認められなかった。
【０１０５】
発現を改善するため、ベクター上のＲｉｂｏｓｏｍｅ　Ｂｉｎｄｉｎｇ　Ｓｉｔｅ（ＲＢＳ）と開始コドンの距離を調節した。即ち、ベクター上のＲＢＳから８塩基下流から、上流に向かって２３塩基の相補鎖をリバースプライマーとし、ｈａｌ遺伝子の増幅に用いたフォワードプライマーと組み合わせて、ベクターとｈａｌ構造遺伝子を含む約６．２ｋｂのＤＮＡ断片を増幅した。
配列番号３：ＲＢＳ〜開始コドン間短縮用のリバースプライマー：
５’−ｇｇｔｃｔｇｔｔｔｃｃｔｇｔｇｔｇａａａｔｔｇ−３’
【０１０６】
生じた断片を０．７％アガロースゲル電気泳動に供し目的の断片を抽出精製し、得られた断片を平滑化した後セルフライゲーションして得られたプラスミド、ｐＴｒｍｈｓで宿主Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　ＪＭ１０９株を形質転換した。得られた形質転換微生物を実施例１に従って培養し、ｈａｌ活性を測定した（表３参照）。
（ラットｈａｌ遺伝子）
購入した形質転換微生物から当該プラスミドを抽出精製し、発現用プラスミドを再構築した。即ち、配列番号４及び５で示されるラットのｈａｌ遺伝子ｃＤＮＡの両末端配列をプライマーとしてｈａｌ構造遺伝子部分のみを増幅し、この断片を平滑化した後、発現ベクターｐＴｒｃ９９ＡのＳｍａＩサイトに接続した。得られたプラスミドをｐＴｒｒｈと命名した。
配列番号４：ラットｈａｌ遺伝子フォワードプライマー：５’−ａｔｇｃｃｔａｇｇｔａｃａｃｇｇｔｇ３’
配列番号５：ラットｈａｌ遺伝子リバースプライマー：５’−ｔｔａａａｇａｔｃｇｔｃａｇａｃｔｃ３’

（反応条件）
変性　　　　　　　　　　　　　　　　９６℃、３０秒
アニーリング　　　　　　　　　　　　５０℃、３０秒
伸張　　　　　　　　　　　　　　　　７２℃、３００秒
（３０サイクル）
さらに、
最終伸張　　　　　　　　　　　　　　７２℃、５分
【０１０７】
マウスと同様に、このプラスミドを鋳型としてベクター上のＲｉｂｏｓｏｍｅ　ＢｉｎｄｉｎｇＳｉｔｅ（ＲＢＳ）と開始コドンの距離を短縮するＰＣＲ反応を行った。即ち、配列番号３で示されるリバースプライマーとｈａｌ遺伝子の増幅に用いたフォワードプライマーと組み合わせてベクターとｈａｌ構造遺伝子を含む約６．２ｋｂのＤＮＡ断片を増幅した。
【０１０８】
生じた断片を０．７％アガロースゲル電気泳動に供し目的の断片を抽出精製し、得られた断片を平滑化した後セルフライゲーションして得られたプラスミド、ｐＴｒｒｈｓで宿主Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　ＪＭ１０９株を形質転換した。得られた形質転換微生物を実施例１と同様に培養し、ｈａｌ活性を測定した（表３参照）。なおＬ−ヒスチジンの生成反応は、実施例１に準じて実施した。またウロカニン酸の生成反応は、１ｍｌの培養液から菌体を遠心分離し、０．１％　Ｌ−ヒスチジンを含む０．１Ｍ　ピロリン酸緩衝液（ｐＨ９．３）１ｍｌに再懸濁し、３０℃、１８時間反応後に反応液中の生成物をＨＰＬＣで確認した。
【０１０９】
【表３】

【０１１０】
［実施例５］動物ｈａｌ遺伝子による形質転換微生物を用いたＬ−アミノ酸の生産実施例４で得られた形質転換微生物ｐＴｒｒｈｓ／ＪＭ１０９株を用いて、各種のＬ−アミノ酸の生産試験を行った。即ち、形質転換微生物ｐＴｒｒｈｓ／ＪＭ１０９株を栄養培地（Ｌブロス）２Ｌで１６時間培養し、０．２ｍＭのＩＰＴＧを添加してさらに４時間培養した培養液から、遠心（８，０００ｒｐｍ）して菌体を分離した。培養液１００ｍＬ分の菌体を各種のアクリル酸誘導体０．１％を含む４Ｍアンモニア／炭酸アンモニウム（ｐＨ１０）反応液に２０ｍＬに再懸濁し、３０℃で８時間反応させて生成物の蓄積をＨＰＬＣで定量した。その結果表４に示すように、各種のＬ−アミノ酸の蓄積が観察された。
【０１１１】
【表４】

【０１１２】
［実施例６］微生物ｈａｌ遺伝子断片の取得
微生物ｈａｌ遺伝子は、各微生物細胞を直接鋳型としたＰＣＲにより取得した。増幅用のプライマーには、各株と同じ属で知られているｈａｌ遺伝子の末端配列を用いた。
配列番号６：Ｐｓｅｕｄｏｍｏｎａｓ用フォワードプライマー：５’−　ａｔｇａｃｃｇａａｃｔｃａｃｃ　−３’
配列番号７：Ｐｓｅｕｄｏｍｏｎａｓ用リバースプライマー：５’−　ｔｔａｃａｇｇｃｔｔｇｇｃａｇ　−３’
配列番号８：Ｂａｃｉｌｌｕｓ用フォワードプライマー：５’−　ａｔｇｇｔｇａｃｔｔｔａｇａｃ　−３’
配列番号９：Ｂａｃｉｌｌｕｓ用リバースプライマー：５’−　ｔｃａｔａｔｇｔｔｃａｔｃｃｃ　−３’

（反応条件）
変性　　　　　　　　　　　　　９６℃、４５秒
アニーリング　　　　　　　　　５０℃、３０秒
伸張　　　　　　　　　　　　　７２℃、１２０秒
（３０サイクル）
さらに、
最終伸張　　　　　　　　　　　７２℃、５分
増幅反応終了後、反応液中の生成物をアガロースゲル電気泳動で確認したところ、幾つかの株で約１．５ｋｂの断片が特異的に生成した（表２参照）。
【０１１３】
【表５】

【０１１４】
［実施例７］断片のＴ／Ａクローニング及び配列決定
実施例６で得られた断片をアガロースゲル電気泳動して抽出精製し、ベクターｐＴ７Ｂｌｕｅ（Ｎｏｖａｇｅｎ社製）のＥｃｏＲＩサイトにライゲーションした（Ｔ／Ａクローニング）。挿入した断片の塩基配列をＴ７　ｐｒｏｍｏｔｅｒ　ｐｒｉｍｅｒとＭ１３　Ｍ４　ｐｒｉｍｅｒを用いて解読した。その結果、Ｂａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ　ＭＩ１１３より得たｈａｌ遺伝子は既知のＢａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ　１６８株のｈａｌ遺伝子配列と完全に一致した。一方、Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ　ＪＣＭ６１５６より得たｈａｌ遺伝子は、Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ　ＡＴＣＣ　１２６３３と相同性を示すが、異なる配列を有する、配列番号１０で示されるアミノ酸配列をコードするｈａｌ遺伝子であることが判明した。
【０１１５】
［実施例８］微生物ｈａｌ遺伝子発現系の構築
ｐＴ７Ｂｌｕｅ上の挿入ｈａｌ断片を制限酵素ＢａｍＨＩとＨｉｎｄＩＩＩで切り出し、アガロースゲル電気泳動で分離精製後、同じ組み合わせの制限酵素で切断した発現ベクターｐＴｒｃ９９Ａとライゲーションした。
【０１１６】
このプラスミドを鋳型としてベクター上のＲｉｂｏｓｏｍｅ　Ｂｉｎｄｉｎｇ　Ｓｉｔｅ（ＲＢＳ）と開始コドンの距離を短縮するＰＣＲ反応を行った。即ち、配列番号３で示されるリバースプライマーと、各微生物のｈａｌ遺伝子の増幅に用いたフォワードプライマーと組み合わせてベクターとｈａｌ構造遺伝子を含む約５．７ｋｂのＤＮＡ断片を増幅した。
【０１１７】
生じた断片を０．７％アガロースゲル電気泳動に供し目的の断片を抽出精製し、断片を平滑化した後セルフライゲーションしたもので宿主Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　ＪＭ１０９株を形質転換した。それぞれに由来するｈａｌ遺伝子を有するプラスミドの名称は、Ｂａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ　ＭＩ１１３株由来をｐＴｒｂｃｈ３、Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ　ＪＣＭ６１５６由来をｐＴｒｐｓｈ１、Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ　ＡＴＣＣ１２６３３由来をｐＴｒｐｓｈ２と命名した。得られた形質転換微生物を実施例１と同様に培養し、ｈａｌ活性を測定した（表６参照）。なお活性測定は実施例１および４に準じて実施した。この結果、得られた微生物由来のｈａｌ遺伝子のうち、特にＰｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ　ＪＣＭ６１５６について、アミノ脱離方向の活性に対しアミノ付加反応活性が高いことが判った。
【０１１８】
【表６】

【０１１９】
［実施例９］微生物ｈａｌ遺伝子による形質転換微生物を用いたＬ−アミノ酸の生成
実施例８で得られた形質転換微生物ｐＴｒｐｓｈ１／ＪＭ１０９株を用いて、各種のヒスチジン誘導体の生産試験を行った。即ち、形質転換微生物ｐＴｒｐｓｈ１／ＪＭ１０９株を栄養培地（Ｌブロス）２Ｌで１６時間培養し、０．２ｍＭのＩＰＴＧを添加してさらに４時間培養した培養液から、遠心（８，０００ｒｐｍ）して菌体を分離した。培養液１００ｍＬ分の菌体を各種のアクリル酸誘導体１％を含む４Ｍアンモニア／炭酸アンモニウム（ｐＨ１０）反応液２０ｍＬに再懸濁し、３０℃で８時間反応させて生成物の蓄積をＨＰＬＣで定量した。その結果表４に示すように、各種のＬ−アミノ酸の蓄積が観察された。
【０１２０】
【表７】

【０１２１】
【発明の効果】
本発明によれば、微生物あるいは動物のヒスチジンアンモニア−リアーゼを用いることによって、アクリル酸誘導体を原料として、簡便で効率よく、対応するＬ−アミノ酸を得ることができる。
【０１２２】
本発明のＬ−アミノ酸の製造法により得られるＬ−アミノ酸は、医薬中間体、農薬中間体をはじめとする生理活性有機化合物のキラルビルディングブロックとして幅広い分野に有用であり、主として医薬中間体として有用である。
【０１２３】
【図面の簡単な説明】
【図１】マウスｈａｌ遺伝子発現系の構築
【図２】ラットｈａｌ遺伝子発現系の構築
【図３】Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ　ＪＣＭ６１５６　ｈａｌ遺伝子発現系の構築
【図４】Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ　ＡＴＣＣ１２６３３　ｈａｌ遺伝子発現系の構築
【図５】Ｂａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ　ＭＩ１１３　ｈａｌ遺伝子発現系の構築
【０１２４】
【配列表】

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for producing ammonia by adding ammonia to an acrylic acid derivative by the action of histidine ammonia lyase to obtain a corresponding optically active amino acid. Optically active amino acids are useful as raw materials for synthesizing pharmaceuticals, agricultural chemicals and other fine chemicals.
[0002]
[Prior art]
As methods for obtaining optically active amino acids, many organic synthetic reactions using asymmetric catalysts and fermentative production methods utilizing the specificity of microorganisms have been studied.
[0003]
For example, a method is known in which an ammonia lyase is allowed to act on an acrylic acid derivative in the presence of ammonia to add an amino group to the α-position carbon of the acrylic acid group to obtain an L-amino acid. This utilizes the reverse reaction of an enzyme that originally catalyzes the ammonia elimination reaction from amino acids. For example, British Patent No. 1489468 and JP-A-53-96388 disclose a method for obtaining optically active optically active phenylalanine by reacting cinnamic acid with a microorganism-derived phenylalanine ammonia-lyase or a microorganism having the enzyme activity. Many have been reported. These utilize a reaction of adding an amino group to the α-carbon of cinnamic acid by the action of the enzyme under a high ammonia concentration, and can produce L-phenylalanine with high optical purity.
[0004]
The method for obtaining an L-amino acid by ammonia lyase has excellent characteristics such as avoidance of side reactions under mild reaction conditions, high conversion, and high optical selectivity, as compared with an organic synthesis method. In particular, in the field of medicine, the analysis of material dynamics and information transmission in living organisms based on genetic information has been advanced, and drug design methods have been diversified in recent years. Examples of applied research using amino acids are increasing. As described above, the reaction with ammonia lyase is suitable for obtaining high-purity unnatural L-amino acids required for such uses. For example, in U.S. Pat. No. 5,981,239, Rhodotorula (Rhodotorula) A method for producing L-amino acids from acrylic acid derivatives using phenylalanine ammonia lyase of the genus microorganism is disclosed. JP-A-63-148992 discloses a method in which a cinnamic acid derivative having a fluorinated phenyl group is used as a raw material to obtain optically active fluorinated phenylalanine by the action of the same enzyme. Poppe et al. Also discuss the substrate specificity of phenylalanine ammonia lyase in Current Opinion in Chemical Biology, 5: 512-524 (2001). However, there are problems in practical use, such as the fact that the target compound is limited to some phenyl-substituted products, and that sufficient studies have not been made industrially.
[0005]
Histidine ammonia lyase is known as an enzyme similar to phenylalanine ammonia lyase, which catalyzes asymmetric amino addition. Histidine ammonia lyase is known to be widely distributed in animals and bacteria.
[0006]
In bacteriaPseudomonasGenus,BacillusGenus,KlebsiellaGenus,RhizobiumGenus,AlcaligenesGenus,AspergillusGenus,AgrobacteriumGenus,DeinococcusGenus,EscherichiaGenus,StreptomycesGenus,CaulobacterGenus,StreptococcusGenus,VibrioGenus,CorynebacteriumThe reaction has been confirmed in genera and the like, and many studies have been made on its structure and reaction mechanism, including its relation to various related ammonia lyases.
[0007]
On the other hand, in animals, histidine ammonia lyase has been studied in detail in humans, mice, rats and the like, but is considered to be ubiquitous. It is an enzyme essential for histidine metabolism in the body. Particularly in humans, detailed medical and physiological studies have been made on the mechanism and treatment of inborn errors of metabolism based on genetic defects, so-called histidineemia.
[0008]
Furthermore, many histidine ammonia lyase genes (hereinafter, referred to as “hal genes”) have already been reported. In other words, from microorganisms,Agrobacterium tumefaciens(Science @ (2001), $ 294, $ 2317-2323.),Bacillus subtilis(J. @Bacteriol. (1988), $ 170, $ 3199-3205.),Bacillus halodurans(Nucleic Acids Res. (2000), 28, 4317-4331.),Caulobacter crescentus(Proc.Natl.Acad.Sci. U. S. A. (2001), $ 98, $ 4136-4141. ),Deinococcus radiodurans(Science (1999), $ 286, $ 1571-1577.),Pseudomonas putida(J. @Bacteriol. (1990), @ 172, @ 2224-2229.),Pseudomonas aeruginosa(Nature (2000), $ 406, $ 959-964.),Streptomyces griseus(J. @Bacteriol. (1992), @ 174, @ 1647-1655.),Rhizobium meliloti(EMBL / GenBank / DDBJ \ databases \ as \ O31197),Streptococcus pyogenes(Proc. Natl. Acad. Sci. USA (2001), 98, 4658-4663.),Streptomyces coelicolor(EMBL / GenBank / DDBJ \ databases \ as \ Q9EWW1),Corynebacterium(WO2001079469) and the like are known. In animal origin,Homo sapiens(Human, Genomics (1995), $ 29, $ 98-104.),Mus musculus(Mouse, Genomics (1993), $ 16, $ 231-240.),Rattus norvegicus(Rat, J. Biol. Chem. (1990), 265, 18192-18199.) And the like. Attempts have been made to express some of these genes using appropriate expression vectors, and studies on the catalytic reaction mechanism of enzymes by methods such as point mutation have already been reported (WO200107469, @Biochemistry (1994). ), 33, ６２6462-6467., J. Biol. Chem. (1990), 265, 18192-18199.).
[0009]
As described above, although there are many research reports on histidine ammonia lyase, an attempt from the viewpoint of substance production using the enzyme has been made by a method of producing L-histidine from urocanic acid disclosed in JP-A-63-148892. Other than that, there are few known examples such as Japanese Patent Publication, Journal of Bacteriology, 162, 1:98 (1985). Regarding the substrate specificity of the enzyme for compounds other than histidine or urocanic acid, biochemical and biophysical research communication, 87, {1: 343 (1979)}, nitrohistidine, and The Journal of Biochemical 250 Cry. In (2), there is only an example that discusses the ammonia elimination activity for fluorohistidine and the production of fluorohistidine by an ammonia addition reaction to fluorourocanic acid. As described above, reports on the reaction of unnatural L-amino acid production by addition of an amino group using the enzyme are very limited, and there are no known practical studies from the viewpoint of industrial production.
[0010]
[Problems to be solved by the invention]
One object of the present invention is to provide a novel method for obtaining L-amino acids having high optical purity, in a simple manner, efficiently and in a variety of ways. An object is to provide a novel method for efficiently obtaining various L-amino acids having high optical purity.
[0011]
[Means for Solving the Problems]
The present inventors have searched ammonia lyase capable of catalyzing a stereoselective amino group addition reaction for acrylic acid derivatives having various substituents, targeting the whole animal kingdom of animals, plants and microorganisms.
[0012]
As a result, the present inventors have found that, besides the conventionally known reaction of giving histidine from urocanic acid to histidine ammonia lyase derived from microorganisms and animals, the activity of stereoselectively adding an amino group to various acrylic acid derivatives is also known. I found that there is. As described above, the metabolic reaction of histidine in vivo involving histidine ammonia lyase has been studied in detail, and the reaction of producing histidine by the reverse reaction was also known, but other than histidine, The potential for producing various amino acids, including unnatural amino acids, has been little studied.
[0013]
The present inventors have further studied diligently, and found that amino acids can be produced with extremely high efficiency by using microorganisms that variously express the histidine ammonia lyase gene derived from various microorganisms and animals. It was completed.
[0014]
That is, the present invention includes, for example, the following items.
[1] The following formula (1)
Embedded image

(Wherein, Z represents an aromatic ring group which may contain a hetero atom, R represents a substituent on the aromatic ring, n represents an integer of 0 or more, and when n is 2 or more, R represents They may be the same or different, provided that Z is imidazole and n is 0.)
Wherein histidine ammonia lyase is allowed to act on an acrylic acid derivative represented by the following formula (2):
Embedded image

(Wherein, Z represents an aromatic ring group which may contain a hetero atom, R represents a substituent on the aromatic ring, n represents an integer of 0 or more, and when n is 2 or more, R represents They may be the same or different, provided that Z is imidazole and n excludes 0.
A method for producing an L-amino acid represented by the formula:
[0015]
[2] R_n-Z- is the following formula (3)
Embedded image

(However, R is a substituent on the benzene ring, and is a cyano group, a hydroxyl group, a carboxyl group, an amide group, a halogen atom, an amino group, a nitro group, a hydroxymethyl group, an alkyl group having 1 to 6 carbon atoms or a carbon number. Represents an alkoxy group of 1 to 6. n represents an integer of 0 to 5, and when n is 2 or more, R may be the same or different.)
The method for producing an L-amino acid according to [1], which is represented by [1].
[3] The method for producing an L-amino acid according to [2], wherein at least one of R is an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, and a nitro group.
[4] The method for producing an L-amino acid according to [3], wherein n is 1.
[0016]
[5] R_n-Z- is the following formula (4)
Embedded image

(Where X is S, O, NH or NR¹And R¹Represents an alkyl group having 1 to 6 carbon atoms, wherein Y is N, CH or CR²Represents R, R²Represents a cyano group, a hydroxyl group, a carboxyl group, an amide group, a halogen atom, an amino group, a nitro group, a hydroxymethyl group, an alkyl group having 1 to 6 carbon atoms or an alkoxy group having 1 to 6 carbon atoms. n represents an integer of 0 to 3, and when n is 2 or more, Rs may be the same or different;²May be the same or different, and adjacent R, R²May combine with each other to form a ring that may contain a heteroatom having 1 to 6 carbon atoms. However, this excludes the case where X is NH, Y is N, and n is 0. )
The method for producing an L-amino acid according to [1], which is represented by [1].
[6] The method for producing an L-amino acid according to [5], wherein Y is CH.
[7] The method for producing an L-amino acid according to [6], wherein n is 0 and X is S, O or NH.
[0017]
[8] R_n-Z- is the following formula (5)
Embedded image

(Where R is a substituent on the heterocycle, and is a cyano group, a hydroxyl group, a carboxyl group, an amide group, a halogen atom, an amino group, a nitro group, a hydroxymethyl group, an alkyl group having 1 to 6 carbon atoms, Represents an alkoxy group having a number of 1 to 6. n represents an integer of 0 to 4, and when n is 2 or more, Rs may be the same or different, and adjacent Rs have 1 to 6 carbon atoms. (It may form a ring that may contain a heteroatom.)
The method for producing an L-amino acid according to [1], which is represented by [1].
[9] The method for producing an L-amino acid according to [8], wherein n is 0.
[0018]
[10] The histidine ammonia lyase is acted on in the form of a culture of a microorganism containing histidine ammonia lyase, a treatment composition of the culture and / or an enzyme obtained from the culture [1] to [1]. [9] The method for producing an L-amino acid according to any of [9].
[11] A microorganism containing histidine ammonia lyase,PseudomonasGenus,BacillusGenus,KlebsiellaGenus,RhizobiumGenus,AlcaligenesGenus,AspergillusGenus,AgrobacteriumGenus,DeinococcusGenus,EscherichiaGenus,StreptomycesGenus,CaulobacterGenus,StreptococcusGenus andCorynebacteriumThe method for producing an L-amino acid according to [10], which is at least one member selected from the genus.
[12] The microorganism containing histidine ammonia lyase isPseudomonas putida方法 The method for producing an L-amino acid according to [10], which is JCM6156. [13] A microorganism containing histidine ammonia lyase isBacillus subtilis方法 The method for producing an L-amino acid according to [10], which is MI113.
[14] The method for producing an L-amino acid according to [10], wherein the microorganism containing histidine ammonia lyase is a microorganism transformed with the histidine ammonia lyase gene.
[0019]
[15] the histidine ammonia lyase genePseudomonas putida[4] The method for producing an L-amino acid according to [14], which is a gene encoding a sequence having 98% or more homology with the amino acid sequence derived from JCM6156.
[16] The method for producing an L-amino acid according to [14], wherein the histidine ammonia lyase gene is derived from a microorganism.
[17] The microorganism from which the histidine ammonia lyase gene is derived,PseudomonasGenus,BacillusGenus,KlebsiellaGenus,RhizobiumGenus,AlcaligenesGenus,AspergillusGenus,AgrobacteriumGenus,DeinococcusGenus,EscherichiaGenus,StreptomycesGenus,CaulobacterGenus,StreptococcusGenus,VibrioGenus andCorynebacteriumThe method for producing an L-amino acid according to [16], wherein the method is at least one member selected from the genus.
[0020]
[18] The microorganism from which the histidine ammonia lyase gene is derived,Pseudomonas putida方法 The method for producing an L-amino acid according to [16], wherein the method is JCM6156.
[19] The microorganism from which the histidine ammonia lyase gene is derived,Bacillus subtilis方法 The method for producing an L-amino acid according to [16], which is MI113.
[20] The method for producing an L-amino acid according to [14], wherein the histidine ammonia lyase gene is derived from an animal.
[21] The method for producing an L-amino acid according to [20], wherein the animal is any one of a mouse, a rat, and a human.
[0021]
[22]Pseudomonas putida(4) A gene encoding a sequence having at least 80% homology with the amino acid sequence encoded by the histidine ammonia lyase gene derived from JCM6156, a plasmid containing the gene, and a transformed microorganism transformed with the plasmid.
[23]Pseudomonas putidaヒ Histidine ammonia lyase gene derived from JCM6156, a plasmid containing the gene, and a transformed microorganism transformed with the plasmid.
[0022]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
[0023]
The method for producing an L-amino acid according to the present invention comprises the steps of converting an optically active amino acid from an acrylic acid derivative having an aromatic ring group which may have various substituents and containing a hetero atom by a one-step enzymatic reaction. And can be easily obtained.
[0024]
Substituents previously introduced into the aromatic ring group, for example, a cyano group, a hydroxyl group, a carboxyl group, an amide group, a halogen atom, an amino group, a nitro group, a hydroxymethyl group, an alkyl group having 1 to 6 carbon atoms, and 1 to 6 carbon atoms Under the mild reaction conditions of the present invention, the alkoxy group of the formula (1) is maintained without being affected until the end of the reaction, and the corresponding desired L-phenylalanine derivative can be obtained in good yield with almost 100% conversion.
[0025]
In addition, according to the method for producing an L-amino acid of the present invention, an optically active amino acid having a heterocyclic structure can be easily obtained from an acrylic acid derivative having a heterocyclic structure by a one-step enzymatic reaction.
[0026]
The heterocyclic structure such as a furyl group, a thienyl group, a pyrrolyl group, an imidazolyl group in the acrylic acid derivative is retained under the mild reaction conditions of the present invention without being affected until the end of the reaction, and the corresponding desired L Amino acids can be obtained in good yields with a conversion of almost 100%.
[0027]
The acrylic acid derivative having various substituents, which is a reaction raw material of the present invention, is obtained by a method based on a so-called Perkin reaction in which an aldehyde group of an aldehyde derivative having an arbitrary substituent introduced therein is reacted with acetic anhydride (Arch. @Pharm. (See Weinheim) (1994), (327) (10), and (619-625) or a method of reacting malonic acid or the like in the presence of piperidine in a pyridine solvent (J Chem. Soc. (1939), 357-360, etc.). ), And various improved methods (see Synth. {Commun.} (1999), $ 29 (4), $ 573-581, etc.) and the like.
[0028]
In one embodiment of the present invention, a cultured culture of a microorganism having histidine ammonia lyase activity is used. The culture of a microorganism in the present invention also refers to a liquid in which the microorganism is cultured and a microorganism in which only the microorganism is removed from the culture.
[0029]
The microorganism that provides histidine ammonia lyase activity applied in the present invention is not particularly limited. Specific examples of microorganisms known to have the enzyme activity include:PseudomonasGenus,BacillusGenus,KlebsiellaGenus,RhizobiumGenus,AlcaligenesGenus,AspergillusGenus,AgrobacteriumGenus,DeinococcusGenus,EscherichiaGenus,StreptomycesGenus,CaulobacterGenus,StreptococcusGenus,VibrioGenus,CorynebacteriumMicroorganisms of each genus of the genus are included.
[0030]
Further, as another embodiment of the present invention, a transformed microorganism in which the hal gene has been introduced into a microorganism so that the hal gene can be expressed can also be used.
[0031]
The hal gene used in the present invention may be a naturally-occurring hal gene or a gene having the same function (encoding a mutant enzyme having the same activity). Isolation of the enzyme gene from the origin organism, and a method for preparing a transformed microorganism using the gene, and a method for separating the enzyme from the transformed microorganism, etc.Pseudomonas putida(Biochemistry (1994), 33, ６２6462-6467.), And Taylor et al. (J. Biol. Chem. (1990), 265, 18192-18199) using rats. .) Are known.
[0032]
Examples of a method for obtaining the hal gene, a method for preparing an expressible plasmid, and a method for introducing and expressing these plasmids in various organisms will be described in further detail.
[0033]
<Method for obtaining hal gene>
The hal gene derived from an animal can be obtained from a cDNA library prepared by a known method using a partial fragment corresponding to the known sequence of the hal gene as a probe, but depending on the target, the cDNA can be obtained from a gene bank such as ATCC. You can also get it. In order to prepare a cDNA library, it is necessary to first isolate and purify mRNA, which can be performed by a conventional method. In recent years, kits and the like that can easily obtain total RNA from tissues have been sold. The cDNA can be obtained by using Reverse RNA transcriptase using the obtained total RNA or mRNA isolated therefrom by an oligoT column or the like as a template. The cDNA thus obtained is connected to an appropriate plasmid vector, for example, pBR322 or pUC18, and Escherichia coli as a host is transformed.
[0034]
The target hal gene can be obtained from the cDNA library prepared as described above by a colony hybridization method using an oligo DNA encoding a conserved sequence of the hal gene as a probe.
[0035]
On the other hand, a hal gene derived from a microorganism can be obtained as a probe from a genome DNA library prepared by a known method, using a partial fragment corresponding to the conserved sequence of the hal gene. Genome DNA required for preparing a genome DNA library can be obtained by a conventional method. In recent years, kits and the like that can efficiently obtain genome DNA from microbial cells have been sold. The genome DNA thus obtained is connected to a suitable plasmid vector, for example, pBR322 or pUC18, and Escherichia coli as a host is transformed.
[0036]
The target hal gene can be obtained from the genome DNA library prepared as described above by a colony hybridization method using an oligo DNA encoding a conserved sequence of the hal gene as a probe.
[0037]
Further, with respect to those having a known sequence, a sufficient amount of the hal structural gene fragment can be easily obtained by performing a PCR method using a partial fragment corresponding to the terminal sequence as a primer and cDNA or genome DNA as a template. The hal gene thus obtained is ligated to a suitable plasmid vector, for example, pBR322 or pUC18, to transform Escherichia coli as a host.
[0038]
<Method for preparing expression plasmid and method for preparing recombinant microorganism used for production>
The hal gene can be obtained by using a suitable vector (specific examples will be described later),Saccharomyces cerevisiaeBy introducing the gene into a microorganism such as yeast such as Escherichia coli, preferably a bacterium such as Escherichia coli and expressing the same, a recombinant having the desired L-amino acid producing activity can be obtained. The produced L-amino acid can be isolated and purified by a usual method for separating a microbial reaction product from a reaction product as described later.
Procedures and methods for preparing a plasmid containing a foreign gene and for introducing and expressing the plasmid into a microorganism such as Escherichia coli are methods commonly used in the field of genetic engineering (for example, "Vectors for cloning genes", "Methods in"). Enzymology, 216, p. 469-631, 1992, ｃAcademic Press Systems ’, and ＯOther Bacterial Systems’, Methods in Enzymology, 204, p.305-636, 199de.
[0039]
Escherichia coli There are several well-established methods for introducing a foreign gene (hal gene group) into Escherichia coli, such as the Hanahan's method and the rubidium method, which may be performed using such methods (for example, Sambrook, J. Fritsch, E. F., Maniatis, T., "Molecular Cloning-A Laboratory Manual." Cold Spring Harbor Laboratory Press, 1989). Expression of a foreign gene in Escherichia coli may be carried out according to a conventional method (for example, see the above-mentioned "Molecular cloning"-"A laboratory"). For example, an Escherichia coli vector having a lac promoter such as a pUC or pBluescript or the like. Can be performed. The present inventors inserted the hal gene downstream of the promoter using the E. coli vector pTrc99A having a trc promoter, and expressed it in E. coli.
[0040]
yeastSaccharomyces cerevisiaeThere are already established methods such as the lithium method for introducing the hal gene into E. coli, and may be performed using such methods (see, for example, the Bioindustry Association, edited by Yuichi Akiyama, “New Yeast Biotechnology for Yeast”, published by Medical Publishing Center). ). Expression of a foreign gene in yeast is performed by using a promoter and terminator such as PGK or G PD (GAP) to construct an expression cassette in which a foreign gene is inserted between the promoter and the terminator so as to be under transcriptional control. Expression cassetteSaccharomyces cerevisiaeFor example, YRp system (multicopy vector for yeast having an ARS sequence of yeast chromosome as an origin of replication), YEp system (multicopy vector for yeast having a replication origin of 2 μm DNA of yeast), YIp system (multicopy vector for yeast) It can be performed by inserting into a vector such as a yeast chromosome integration vector having no origin of replication (the above-mentioned “New Yeast Biotechnology” published by Medical Press Center, ABC 農 series of Japanese Society for Agricultural Chemistry “ Genetic Engineering, Asakura Publishing Co., Ltd.).
[0041]
<Culture of histidine ammonia lyase active microorganism>
Culturing of microorganisms (including transformed microorganisms) related to the present invention can be performed according to a general culture method of microorganisms.
[0042]
As a medium carbon source for culturing microorganisms, those which can be used as carbon sources by microorganisms, for example, glucose, sucrose, fructose, sugars such as molasses, ethanol, acetic acid, citric acid, succinic acid, lactic acid, benzoic acid Organic substances such as acids and fatty acids or alkali metal salts thereof, aliphatic hydrocarbons such as n-paraffin, and natural organic substances such as peptone, meat extract, fish extract, soybean flour, bran, malt extract, potato extract, etc. Can be used alone or in combination at a concentration of usually about 0.01% to 30%, preferably about 0.1% to 10%. For example, when a transformed microorganism of Escherichia coli is used, glucose, peptone, meat extract, malt extract and the like are preferably used.
[0043]
As a medium nitrogen source for culturing microorganisms (including transformed microorganisms), those which can be used as a nitrogen source by the microorganism, for example, inorganic nitrogen compounds such as ammonium sulfate, ammonium phosphate, sodium nitrate and potassium nitrate, urea, Natural organic substances such as nitrogen-containing organic substances such as uric acid, peptone, meat extract, fish extract, soybean flour, malt extract, potato extract, etc., alone or in combination, are usually 0.01% to 30%, preferably 0%. It can be used at a concentration of about 1% to 10%. For example, when Escherichia coli is used as a transforming microorganism, ammonium sulfate, peptone, meat extract, malt extract and the like are preferably used.
[0044]
If necessary, phosphates such as potassium dihydrogen phosphate, and metal salts such as magnesium sulfate, ferrous sulfate, calcium acetate, manganese chloride, copper sulfate, zinc sulfate, cobalt sulfate, and nickel sulfate may be used to grow the bacteria. Added for improvement. The addition concentration varies depending on the culture conditions, but is usually 0.01% to 5% for phosphate, 10 ppm to 1% for magnesium salt, and about 0.1 ppm to 1000 ppm for other compounds. Depending on the selected medium, the growth of the bacterium can be improved by adding, for example, yeast extract, casamino acid, or yeast nucleic acid at a concentration of about 1 ppm to 100 ppm as a source of vitamins, amino acids, nucleic acids, and the like.
[0045]
During the culture, if necessary, L-histidine can be added to the culture solution in an amount of about 10 ppm to 1% as an enzyme induction source in order to increase histidine ammonia lyase activity.
[0046]
In the transformed microorganism, if necessary, an inducer corresponding to the expression promoter of the vector can be added during the culture in order to highly express the transformed enzyme gene in the host microorganism. For example, when the Lac promoter is used, IPTG (isopropyl-1-thio-β-D-galactoside) is used at about 0.01 mM to 10 mM. When a drug-resistant promoter such as ampicillin or kanamycin is used, the corresponding drug is used at 0.1 ppm to 0.1 ppm. Add about 1000 ppm.
[0047]
In any case, the pH of the culture is adjusted to 5 to 9, preferably 5.5 to 8. The culture solution can be subjected to the reaction as it is, but the microbial cells pre-cultured in the medium as described above are separated from the culture solution by a method such as centrifugation or membrane filtration, and subjected to the reaction. This is useful for reducing foreign substances brought in from the reactor and simplifying the subsequent product separation.
[0048]
The microorganism obtained by culturing can be directly recovered by a known method such as centrifugation or membrane separation, or the culture solution itself can be subjected to a target reaction.
[0049]
In one of the different embodiments of the present invention, histidine ammonia lyase isolated from microorganisms (including transformed microorganisms) is used. That is, a known method for separating and purifying an enzyme from a microorganism, for example, the enzyme separated and purified by a conventional method such as acetone fractionation precipitation, ammonium sulfate precipitation, and various separation columns from a fraction of crushed microbial cells is used as a reaction catalyst. Can be offered. Specific methods for separating and purifying histidine ammonia lyase from various microorganisms include, for example,AchromobacterJapanese Patent Application Laid-Open No. 50-24488 for genus microorganisms,PseudomonasKlee et al. Report on genus microorganisms (Journal of Biological Chemistry, 245, 3143-3152 (1970)) and the like are known.
[0050]
In another embodiment of the present invention, a processed product of the microorganism (including a transformed microorganism) as described above is subjected to a target reaction. Examples of processed microorganisms include, for example, ground and freeze-dried microorganisms and cell-free extracts from microbial cells, and those obtained by concentrating and extracting components that catalyze the target reaction from the extract, and A processed product, an extract, or an extract obtained by immobilizing an extract component on a poorly soluble carrier can be used for the reaction. Examples of the immobilization carrier for such a purpose include polyacrylic acid, polymethacrylic acid, polyacrylamide, polyvinyl alcohol, poly-N-vinylformamide, polyallylamine, polyethyleneimine, methylcellulose, glucomannan, alginate, and carrageenan. Further, a compound which forms a hardly water-soluble solid content containing a microorganism or an extractable component thereof, such as a (cross-linked) polymer, can be used alone or in combination. In addition, use of activated carbon, porous ceramics, glass fibers, porous polymer moldings, nitrocellulose membranes, etc., in which transforming microorganisms or their extracts or components are retained on objects previously formed as solids You can also.
[0051]
<L-amino acid production reaction by histidine ammonia lyase activity>
In the present invention, the reaction for producing an L-amino acid is carried out by reacting histidine ammonia lyase prepared by various known methods as described above. The concentration of the acrylic acid derivative as a reaction raw material is preferably 1 ppm to 20%, more preferably 10 ppm to 10%, and it is preferable to add ammonia so that the final concentration becomes 2 M to 12 M. Further, the pH is preferably adjusted to 8 to 11, more preferably 8.5 to 10.5. The reaction time varies depending on the substrate, but is usually achieved by reacting for about 1 to 200 hours. The term "acting histidine ammonia lyase" as used herein means that a culture of a microorganism containing histidine ammonia lyase, a treatment composition of the culture, any of the enzymes obtained from the culture, or a mixture thereof is acted on. The culture of a microorganism includes a culture solution and the microbial cells themselves in the culture, and the cells may be separated and used. Furthermore, the microorganism containing histidine ammonia lyase may be a microorganism or a microorganism transformed with the enzyme gene isolated from an animal.
[0052]
Examples of the acid used for adjusting the pH of the reaction include inorganic acids such as sulfuric acid, hydrochloric acid, phosphoric acid, boric acid, and carbonic acid, and organic acids such as formic acid, acetic acid, and propionic acid, and salts thereof. At this time, using a volatile acid is useful because the desalting step can be omitted and the product can be easily collected by removing bacteria and distilling off the reaction solution. Carbonic acid is preferred as such an acid. The carbonic acid in this case includes carbonic acid which is dissolved as a carbon dioxide in an aqueous solution by bubbling or the like and is generated by dissociation. Further, a salt of these acids and ammonia can be used as an ammonia source for the reaction solution, and it is preferable to use ammonium carbonate or ammonium hydrogen carbonate as part or all of the ammonia source for the above-described reason.
[0053]
Note that the acrylic acid derivative to be added does not necessarily have to be completely dissolved, but a solvent, a surfactant, or the like that improves the solubility or dispersibility in the reaction solution can also be added. The compound may be added continuously or intermittently depending on the consumption of the compound as the reaction proceeds. In this case, the concentration of the compound in the reaction solution is not limited to the above.
[0054]
The L-amino acid produced in the reaction solution is separated by a known method such as centrifugation, membrane filtration, drying under reduced pressure, distillation, solvent extraction, salting out, ion exchange, or various types of chromatography, depending on the properties of the reaction solution. Is done. Conveniently, this can be achieved as follows. For example, filtration, centrifugation, dialysis, etc. remove the enzyme, the enzyme-treated product, the transformed microorganism and the processed product from the reaction solution, and then extract the solvent or adjust the reaction solution to acidic to precipitate the unreacted raw material acrylic acid derivative. , Remove the precipitate.
[0055]
The pH of the obtained supernatant is adjusted again to around the isoelectric point of the L-amino acid, and the precipitated derivative is recovered in the same manner. Thus, the product can be recovered from the reaction solution with high yield and purity. It is also useful to remove excess ammonia from the reaction solution by distillation or the like in advance, or to evaporate water to increase the concentration in order to improve the product recovery rate at the isoelectric point.
[0056]
More simply, the pH of the reaction solution is adjusted with a volatile acid. When the conversion rate is sufficiently high, the product is converted into an ammonium salt of L-amino acid by removing the water and acid base after removing the bacteria as they are, and removing the water and acid base after acid-extracting the solvent if the raw material remains in a large amount. Can be isolated. As volatile acids suitable for such a method, carbonic acid and its ammonium salt are preferred.
[0057]
Depending on the nature of the reaction product, the accumulation of the product in the reaction solution may decrease the reaction rate. In such a case, a method in which water containing ammonia, physiological saline, and a reaction buffer are added to the reaction solution to dilute it continuously according to the concentration of the product is preferable. When the reaction rate decreases, the bacteria are collected, the supernatant is collected as a product solution, and the collected bacteria are returned to the solution or suspension containing the reaction raw materials to recover the reaction rate. Can be. These methods can be repeated any number of times as long as the ammonia lyase activity of the microorganism is maintained.
[0058]
A preferred compound as a raw material of the present invention is represented by the following formula (1)
[0059]
Embedded image

(Wherein, Z represents an aromatic ring group which may contain a hetero atom, R represents a substituent on the aromatic ring, n represents an integer of 0 or more, and when n is 2 or more, R represents And the same or different, except that Z is imidazole and n is 0). Regarding the configuration of Z and the COOH group with respect to the double bond of the acrylic acid moiety in the formula (1), any of trans and cis can be used as a raw material, but a trans form is preferred.
[0060]
The compound obtained has the following formula (2)
[0061]
Embedded image

(Wherein, Z represents an aromatic ring group which may contain a hetero atom, R represents a substituent on the aromatic ring, n represents an integer of 0 or more, and when n is 2 or more, R represents They may be the same or different, provided that Z is imidazole and n is 0.)
L-amino acid represented by
[0062]
In the above formula, Z which is a monovalent or higher valent group is, for example, the following formulas (6) to (9)
[0063]
Embedded image

A group consisting of an aromatic ring represented by the following formulas (10) to (13)
[0064]
Embedded image

A group comprising an aromatic ring represented by the following formulas (14) to (19)
[0065]
Embedded image

A group consisting of an aromatic ring represented by the following formulas (20) to (23)
[0066]
Embedded image

A group consisting of an aromatic ring represented by the following formulas (24) to (28)
[0067]
Embedded image

A group consisting of an aromatic ring represented by the following formulas (29) to (32)
[0068]
Embedded image

A group consisting of an aromatic ring represented by the following formulas (33) to (36)
[0069]
Embedded image

A group consisting of an aromatic ring represented by the following formulas (37) to (40)
[0070]
Embedded image

A group consisting of an aromatic ring represented by the following formulas (41) to (45)
[0071]
Embedded image

And the like consisting of an aromatic ring represented by
[0072]
Examples of R include a cyano group, a hydroxyl group, a carboxyl group, an amide group, a halogen atom, an amino group, a nitro group, a hydroxymethyl group, an alkyl group having 1 to 6 carbon atoms, and an alkoxy group having 1 to 6 carbon atoms. Although it depends on the type of the aromatic ring, preferred are a cyano group, a hydroxyl group, a halogen atom, a nitro group, a methyl group, an ethyl group, a methoxy group, and an ethoxy group.
[0073]
n can be an integer greater than or equal to 0 and less than or equal to the number obtained by subtracting 1 from the number of substitutable positions on the aromatic ring. When n is 2 or more, R may be the same or different.
[0074]
Further, a preferred compound as a raw material of the present invention is a compound represented by the formula (1)_n-Z- is the following formula (3)
[0075]
Embedded image

(Where R is a substituent on the benzene ring, and is a cyano group, a hydroxyl group, a carboxyl group, an amide group, a halogen atom, an amino group, a nitro group, a hydroxymethyl group, an alkyl group having 1 to 6 carbon atoms or an alkoxy group) And n represents an integer of 0 to 5, and when n is 2 or more, Rs may be the same or different.).
[0076]
Further, a compound which can be suitably obtained is a compound represented by the formula (2)_nAnd -Z- is a compound represented by the formula (3) (wherein R and n have the same meanings as described above).
[0077]
Preferably, in the above formulas (1) and (2), R is an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, and a nitro group. An alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms and a nitro group, and n is 1.
[0078]
Specific examples of such compounds include 2-cyanocinnamic acid, 3-cyanocinnamic acid, 4-cyanocinnamic acid, 2-hydroxycinnamic acid, 3-hydroxycinnamic acid, 4-hydroxycinnamic acid, and 3,4-dihydroxy Cinnamic acid, 2-nitrocinnamic acid, 3-nitrocinnamic acid, 4-nitrocinnamic acid, 2-carboxycinnamic acid, 3-carboxycinnamic acid, 4-carboxycinnamic acid, 2-aminocinnamic acid, 3-aminocinnamic acid 4-aminocinnamic acid, 2-chlorocinnamic acid, 3-chlorocinnamic acid, 4-chlorocinnamic acid, 2-fluorocinnamic acid, 3-fluorocinnamic acid, 4-fluorocinnamic acid, 2-bromocinnamic acid, 3 -Bromocinnamic acid, 4-bromocinnamic acid, 2-methylcinnamic acid, 3-methylcinnamic acid, 4-methylcinnamic acid, 2-methoxycinnamic acid, 3-methoxycinnamic acid, 4-methoxycinnamic acid, 4-isopropyl Cinnamic acid, 4-t Butyl cinnamic acid, and the like.
[0079]
For exampleBacillus subtilisWhen an enzyme derived from MI113 is used, 2-cyanocinnamic acid, 4-cyanocinnamic acid, 2-methylcinnamic acid, 3-methylcinnamic acid, 4-methylcinnamic acid, 2-methoxycinnamic acid, and 3-methoxycinnamic acid are used as raw materials. Acids and 4-methoxycinnamic acid are preferred, and 2-cyano-L-phenylalanine, 4-cyano-L-phenylalanine, 2-methyl-L-phenylalanine, 3-methyl-L-phenylalanine, 4-methyl-L, respectively. -Phenylalanine, 2-methoxy-L-phenylalanine, 3-methoxy-L-phenylalanine, 4-methoxy-L-phenylalanine and the like are obtained.
[0080]
Particularly, a preferred compound as a raw material of the present invention is a compound represented by the formula (1)_n-Z- is the following formula (4)
[0081]
Embedded image

(Where X is S, O, NH or NR¹And R¹Represents an alkyl group having 1 to 6 carbon atoms, wherein Y is N, CH or CR²R, R²Represents a cyano group, a hydroxyl group, a carboxyl group, an amide group, a halogen atom, an amino group, a nitro group, a hydroxymethyl group, an alkyl group having 1 to 6 carbon atoms or an alkoxy group having 1 to 6 carbon atoms. n represents an integer of 0 to 3, and when n is 2 or more, Rs may be the same or different;²May be the same or different, and adjacent R, R²May combine with each other to form a ring that may contain a heteroatom having 1 to 6 carbon atoms. However, this excludes the case where X is NH, Y is N, and n is 0. ).
[0082]
Further, the obtained compound is represented by the formula (2)_n-Z- is the above formula (4)
(Where X, Y, R, R¹, R², N represent the same meaning as described above. )).
[0083]
Preferably, in the formulas (1) and (2), Y is CH, more preferably, Y is CH, n is 0, and X is S, O or NH.
[0084]
Specific examples of such compounds include β- (2-furyl) acrylic acid, β- (3-furyl) acrylic acid, β- (2-thienyl) acrylic acid, β (3-thienyl) acrylic acid, β- (2-pyrrolyl) acrylic acid, β- (3-pyrrolyl) acrylic acid, and the like.
[0085]
For exampleBacillus subtilisWhen an enzyme derived from MI113 is used, β- (2-furyl) acrylic acid, β- (3-furyl) acrylic acid, β- (2-thienyl) acrylic acid, β- (3-thienyl) acrylic acid, β- (2-pyrrolyl) acrylic acid, β- (3-pyrrolyl) acrylic acid, imidazoleacrylic acid (urocanic acid) are preferred and correspondingly 2-amino-3- (2-furyl) propionic acid, -Amino-3- (3-furyl) propionic acid, 2-amino-3- (2-thienyl) propionic acid, 2-amino-3- (3-thienyl) propionic acid, 2-amino-3- (2- Pyrrolyl) propionic acid and 2-amino-3- (3-pyrrolyl) propionic acid are obtained.
[0086]
Further, a preferred compound as a raw material of the present invention is a compound represented by the formula (1)_n-Z- is the following formula (5)
[0087]
Embedded image

(Where R is a substituent on the heterocycle, and is a cyano group, hydroxyl group, carboxyl group, amide group, halogen atom, amino group, nitro group, hydroxymethyl group, alkyl group having 1 to 6 carbon atoms or carbon atom Represents an alkoxy group having a number of 1 to 6. n represents an integer of 0 to 4, and when n is 2 or more, Rs may be the same or different, and adjacent Rs have 1 to 6 carbon atoms. Which may form a ring which may contain a heteroatom).
[0088]
Further, the obtained compound is represented by the formula (2)_n-Z- is the above formula (5)
(Where X is S, O, NH or NR¹And R¹Represents an alkyl group having 1 to 6 carbon atoms, wherein Y represents N or C, wherein R is a substituent on a heterocyclic ring, a cyano group, a hydroxyl group, a carboxyl group, an amide group, a halogen atom, an amino Represents a group, a nitro group, a hydroxymethyl group or an alkyl or alkoxy group having 1 to 6 carbon atoms. n represents an integer of 0 to 3, and when n is 2 or more, Rs may be the same or different, and adjacent Rs mutually form a ring having 3 to 5 carbon atoms and containing a hetero atom. May be used).
[0089]
In the formula (1), R_nExamples of -Z- in the formula (5) include β- (2-pyridyl) acrylic acid, β- (3-pyridyl) acrylic acid, and β- (4-pyridyl) acrylic acid, and the L formed is -Amino acids are 2-amino-3- (2-pyridyl) propionic acid, 2-amino-3- (3-pyridyl) propionic acid, 2-amino-3- (4-pyridyl) propionic acid.
[0090]
According to the method of the present invention, an L-amino acid having a high optical purity can be efficiently obtained by a one-step reaction using an acrylic acid derivative which can be easily obtained organically as a substrate. The L-amino acid thus obtained is useful as a synthetic intermediate in the field of fine chemicals such as pharmaceuticals and agricultural chemicals requiring high optical purity.
[0091]
【Example】
Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to these Examples.
[0092]
In the examples, the amount of enzyme that releases 1 μmole urocanic acid per minute at 30 ° C. in the presence of 0.1 M sodium borate buffer pH 8.5 and L-histidine 10 mM as a substrate was defined as 1 unit.
[0093]
The amount of the enzyme protein was determined by the biuret method based on bovine serum albumin, and the specific activity of the enzyme was expressed as unit / mg protein.
The identification of acrylic acid derivatives and amino acids¹H-NMR,¹³C-NMR and the reaction were performed by subjecting the reaction solution to HPLC and comparing the sample with the UV absorption intensity.
[0094]
Separation and quantification of amino acids and acrylic acid derivatives were performed under the following analysis conditions.
Reverse phase HPLC analysis conditions
Column: Shodex (registered trademark of Showa Denko KK) @RSpak NN-614 (manufactured by Showa Denko KK)
Column temperature: 40 ° C
Eluent: acetonitrile / water / 50 mM @ H₃PO₄-KH₂PO₄Aqueous solution (pH 3) = 20/70/10
Flow rate: 1.0 ml / min
Detection: by absorption at UV @ 210 nm
[0095]
In addition, the analysis of the optical purity of the obtained amino acid was performed by optical resolution HPLC under the following conditions.
Optical resolution HPLC analysis conditions
Column: Shodex (registered trademark of Showa Denko Corporation) ORpak CRX-853 (manufactured by Showa Denko Corporation)
Column temperature: room temperature (22 ° C)
Eluent: acetonitrile / water = 15/85 CuSO₄2.5 mM
Flow rate: 1.0 ml / min.
Detection method: UV @ 256 nm absorption
[0096]
[Example 1] Reaction with cells obtained from a culture solution of a microorganism
Bacillus subtilis@ MI113 was shake-cultured in a 500 ml flask with 100 ml of L broth at 30 ° C. for 18 hours. The cells obtained by centrifuging the culture were resuspended in 20 ml of 4M ammonia / ammonium carbonate (pH 10), and various acrylic acid derivatives equivalent to 1% (w / v) were added thereto. The reaction was performed. After 8 hours of the reaction, the product in the reaction solution was quantified by HPLC. Table 1 shows the products obtained in each reaction, the concentration, and the optical purity.
[0097]
[Table 1]

[0098]
Example 2 Reaction of a culture of a microorganism with a treatment composition
Pseudomonas putida@ JCM6156 was shake-cultured in a 500 ml flask with 100 ml of L broth at 30 ° C. for 18 hours. In a jar fermenter, the whole amount is glucose 1%, urea 0.2%, yeast extract 0.2%, monopotassium phosphate 0.2%, disodium phosphate 0.05%, magnesium sulfate 0.01%, Transfer to 2 L of a medium containing 0.001% of ferrous sulfate heptahydrate and 0.1% of L-histidine and adjusted to pH = 7, culturing for 30 hours at 30 ° C., 800 rpm of stirring, and aeration of 1 L / min. did. The whole cells were collected by centrifugation at 8000 rpm for 10 minutes, suspended in 200 mL of 50 mM phosphate buffer (pH 7), and sonicated while cooling on ice to break the cells. This suspension was subjected to centrifugation at 15,000 rpm for 30 minutes, and the obtained supernatant was loaded on a DEAE cellulose packed column which had been previously activated with 0.5N NaOH and then equilibrated with 10 mM phosphate buffer (pH = 7). Provided. 10 mM at the same pH? The adsorbed portion was eluted with a 500 mM phosphate buffer, and a histidine ammonia lyase active fraction was collected. The entire amount of this fraction was subjected to lyophilization. The total amount of protein in the obtained crude product was 50.3 mg, and the specific activity was 0.17 U / mg.
[0099]
Instead of the cells of Example 1, 1 mg of the above crude product was added to 20 ml of 4 M ammonia / ammonium carbonate (pH 10), and various acrylic acid derivatives equivalent to 1% (w / v) were added. The reaction was performed with stirring. After 8 hours of the reaction, the product in the reaction solution was quantified by HPLC. Table 2 shows the products and concentrations obtained in each reaction, and the optical purity.
[0100]
[Table 2]

[0101]
Example 3 Obtaining Animal Histidine Ammonia Lyase Gene (hal) Fragment
(Acquisition of mouse hal gene fragment)
To obtain the hal gene, total RNA was obtained from the liver tissue of a mouse using RNeasy Mini Kit (manufactured by QIAGEN). All methods followed standard use of the kit. That is, 30 mg of mouse liver was crushed with a homogenizer and suspended in 0.6 ml of the attached RLT buffer. The suspension was centrifuged in a centrifuge (15,000 rpm) for 3 minutes, 0.5 ml of the supernatant was taken in a new container, and an equal volume of 70% ethanol was added and mixed. 0.7 ml of the mixed solution was applied to an RNeasy column, centrifuged with a centrifuge (10,000 rpm) for 15 seconds to discard the passing solution, and the attached RW1 buffer was added to the column, followed by similar centrifugation to allow the solution to pass. Further, 0.5 ml of the attached RPN buffer was applied to the column, and total RNA was eluted from the column.
[0102]
Usually, mRNA is purified from the obtained totalRNA to prepare a cDNA library. However, since the sequence of the mouse hal gene was known, the target gene was directly amplified by RT-PCR using totalRNA as a template. Primers used for RT-PCR were both terminal sequences of the mouse hal gene.
SEQ ID NO: 1: mouse hal gene forward primer: 5'-atgcctagtagtacacagtg3 '
SEQ ID NO: 2: Mouse hal gene reverse primer: 5'-ttaagacatcgtcagactc3 '
RT-PCR was performed using Onestep RT-PCR Kit (manufactured by QIAGEN), and the target gene fragment was amplified according to the standard usage of the kit. The amount of the template total RNA solution, the annealing temperature, the length of the extension reaction time, and the like were optimized. All of the reaction solutions except the RNA template (totalRNA) were mixed, and the RNA template (totalRNA) was finally mixed. A mineral oil was overlaid, the reaction solution was set in a thermal cycler preheated to 50 ° C., and RT-PCR was performed. As a result, a fragment having a target gene fragment length of 2 to 2.2 kb was almost specifically amplified. This fragment was subjected to agarose gel electrophoresis to separate and purify, and the cut pattern by the restriction enzyme was examined, which was consistent with the mouse hal gene.
[0103]

(RT-PCR conditions)
Reverse transcription reaction @ 50 ° C, 30 minutes
PCR initial activation @ 95 ° C, 15 minutes
continue,
Denaturation @ 94 ° C, 30 seconds
Annealing @ 48 ° C, 30 seconds
Extension @ 72 ° C, 60 seconds
(40 cycles)
further,
Final extension @ 72 ° C, 10 minutes
(Acquisition of rat hal gene fragment)
The rat hal gene is ATCC (American Type Culture Collection) No. 63149 as cloned on pBluescript @ KS-Escherichia coliBecause a transformant of the HB101 strain was sold, it was purchased and used.
[0104]
[Example 4] Construction of animal hal gene expression system
(Mouse hal gene)
Both ends of the hal gene fragment obtained in Example 3 were blunt-ended and connected to the SmaI site of the expression vector pTrc99A. This plasmid pTrmhEscherichia coliThe JM109 strain was transformed and cultured according to Example 1, and the hal activity was measured. As a result, almost no activity was observed.
[0105]
To improve expression, the distance between Ribosome \ Binding \ Site (RBS) on the vector and the start codon was adjusted. That is, a complementary strand of 23 bases from 8 bases downstream to the upstream from the RBS on the vector is used as a reverse primer, and combined with the forward primer used for amplification of the hal gene, about 6.2 kb containing the vector and the hal structural gene. Was amplified.
SEQ ID NO: 3: Reverse primer for shortening between RBS and start codon:
5'-ggtctgtttttcctgtgtgaaattg-3 '
[0106]
The resulting fragment was subjected to 0.7% agarose gel electrophoresis to extract and purify the target fragment. The resulting fragment was blunted, and then self-ligated to obtain a plasmid, pTrmhs.Escherichia coliThe JM109 strain was transformed. The obtained transformed microorganism was cultured according to Example 1, and the hal activity was measured (see Table 3).
(Rat hal gene)
The plasmid was extracted and purified from the purchased transformed microorganism, and the expression plasmid was reconstructed. That is, only the hal structural gene portion was amplified using both terminal sequences of the rat hal gene cDNA represented by SEQ ID NOs: 4 and 5 as primers, and this fragment was blunted and then connected to the SmaI site of the expression vector pTrc99A. The resulting plasmid was named pTrrrh.
Sequence number 4: Rat hal gene forward primer: 5'-atgcctagggtacacgggtg3 '
SEQ ID NO: 5: rat hal gene reverse primer: 5'-ttaagacatcgtcagactc3 '

(Reaction conditions)
Denaturation @ 96 ° C, 30 seconds
Annealing @ 50 ° C, 30 seconds
Extension @ 72 ° C, 300 seconds
(30 cycles)
further,
Final extension @ 72 ° C, 5 minutes
[0107]
Similarly to the mouse, a PCR reaction was performed using this plasmid as a template to shorten the distance between Ribosome @ BindingSite (RBS) on the vector and the start codon. That is, in combination with the reverse primer represented by SEQ ID NO: 3 and the forward primer used for amplifying the hal gene, a DNA fragment of about 6.2 kb containing the vector and the hal structural gene was amplified.
[0108]
The resulting fragment was subjected to 0.7% agarose gel electrophoresis to extract and purify the target fragment. The resulting fragment was blunted, and then self-ligated to obtain a plasmid, pTrrhs.Escherichia coliThe JM109 strain was transformed. The obtained transformed microorganism was cultured in the same manner as in Example 1, and the hal activity was measured (see Table 3). The reaction for producing L-histidine was carried out according to Example 1. The urocanic acid production reaction was carried out by centrifuging the cells from 1 ml of the culture solution, resuspending the cells in 1 ml of 0.1 M {pyrophosphate buffer (pH 9.3) containing 0.1% L-histidine, and heating at 30 ° C. After the reaction for 18 hours, the product in the reaction solution was confirmed by HPLC.
[0109]
[Table 3]

[0110]
[Example 5] Production of L-amino acid using transformed microorganism by animal hal gene Using the transformed microorganism pTrrhs / JM109 strain obtained in Example 4, production tests of various L-amino acids were performed. That is, the transformed microorganism pTrhrs / JM109 strain was cultured in 2 L of nutrient medium (L broth) for 16 hours, 0.2 mM IPTG was added thereto, and the culture was further cultured for 4 hours. The body was separated. The cells of 100 mL of the culture solution were resuspended in 20 mL of a 4 M ammonia / ammonium carbonate (pH 10) reaction solution containing 0.1% of various acrylic acid derivatives, reacted at 30 ° C. for 8 hours, and the accumulation of the product was analyzed by HPLC. Quantified. As a result, as shown in Table 4, accumulation of various L-amino acids was observed.
[0111]
[Table 4]

[0112]
[Example 6] Obtaining microbial hal gene fragment
The microorganism hal gene was obtained by PCR using each microorganism cell directly as a template. For the amplification primer, the terminal sequence of the hal gene known in the same genus as each strain was used.
SEQ ID NO: 6:PseudomonasForward primer for use: 5 '-{atgaccgaactcacc} -3'
SEQ ID NO: 7:PseudomonasReverse primer for use: 5 '-{ttaagggctttggcag -3'
SEQ ID NO: 8:BacillusForward primer: 5 '-{atgggtgactttagac} -3'
SEQ ID NO: 9:BacillusReverse primer for use: 5 '-{tcatatgttcatccc} -3'

(Reaction conditions)
Denaturation @ 96 ° C, 45 seconds
Annealing @ 50 ° C, 30 seconds
Extension @ 72 ° C, 120 seconds
(30 cycles)
further,
Final extension @ 72 ° C, 5 minutes
After completion of the amplification reaction, the products in the reaction solution were confirmed by agarose gel electrophoresis. As a result, fragments of about 1.5 kb were specifically generated in some strains (see Table 2).
[0113]
[Table 5]

[0114]
Example 7 T / A cloning and sequencing of fragments
The fragment obtained in Example 6 was extracted and purified by agarose gel electrophoresis, and ligated to the EcoRI site of the vector pT7Blue (Novagen) (T / A cloning). The base sequence of the inserted fragment was read using T7 promoter / primer and M13 / M4 primer. as a result,Bacillus subtilisＨHal gene obtained from MI113 is knownBacillus subtilisIt completely matched the hal gene sequence of the # 168 strain. on the other hand,Pseudomonas putidaＨ The hal gene obtained from JCM6156 isPseudomonas putidaThe hal gene, which shows homology to 相同 ATCC 12633 but has a different sequence, was found to encode the amino acid sequence shown in SEQ ID NO: 10.
[0115]
[Example 8] Construction of microorganism hal gene expression system
The inserted hal fragment on pT7Blue was cut out with restriction enzymes BamHI and HindIII, separated and purified by agarose gel electrophoresis, and ligated with the expression vector pTrc99A cut with the same combination of restriction enzymes.
[0116]
Using this plasmid as a template, a PCR reaction was performed to shorten the distance between Ribosome \ Binding \ Site (RBS) on the vector and the start codon. That is, a DNA fragment of about 5.7 kb containing the vector and the hal structural gene was amplified by combining the reverse primer represented by SEQ ID NO: 3 with the forward primer used for amplifying the hal gene of each microorganism.
[0117]
The resulting fragment was subjected to 0.7% agarose gel electrophoresis to extract and purify the target fragment, blunt the fragment, and then self-ligated.Escherichia coliThe JM109 strain was transformed. The name of the plasmid having the hal gene derived from each is:Bacillus subtilisPTrbch3 derived from MI113 strain,Pseudomonas putidaＰ pTrpsh1 derived from JCM6156,Pseudomonas putida由来 ATCC12633-derived was named pTrpsh2. The obtained transformed microorganism was cultured in the same manner as in Example 1, and the hal activity was measured (see Table 6). The activity was measured according to Examples 1 and 4. As a result, among the obtained hal genes derived from microorganisms,Pseudomonas putidaIt was found that JCM6156 has a higher amino addition reaction activity than the activity in the direction of amino desorption.
[0118]
[Table 6]

[0119]
[Example 9] Production of L-amino acid using microorganism transformed by microorganism hal gene
Using the transformed microorganism pTrpsh1 / JM109 strain obtained in Example 8, production tests of various histidine derivatives were performed. That is, the transformed microorganism pTrpsh1 / JM109 strain was cultured in 2 L of a nutrient medium (L broth) for 16 hours, 0.2 mM IPTG was added thereto, and the culture was further cultured for 4 hours, followed by centrifugation (8,000 rpm). The body was separated. The cells of 100 mL of the culture solution were resuspended in 20 mL of a 4 M ammonia / ammonium carbonate (pH 10) reaction solution containing 1% of various acrylic acid derivatives, reacted at 30 ° C. for 8 hours, and the accumulation of the product was quantified by HPLC. . As a result, as shown in Table 4, accumulation of various L-amino acids was observed.
[0120]
[Table 7]

[0121]
【The invention's effect】
According to the present invention, a corresponding L-amino acid can be obtained simply and efficiently from an acrylic acid derivative as a raw material by using histidine ammonia-lyase of a microorganism or an animal.
[0122]
The L-amino acids obtained by the method for producing L-amino acids of the present invention are useful in a wide range of fields as chiral building blocks for bioactive organic compounds including pharmaceutical intermediates and agricultural chemical intermediates, and are mainly useful as pharmaceutical intermediates It is.
[0123]
[Brief description of the drawings]
FIG. 1. Construction of mouse hal gene expression system
FIG. 2 Construction of rat hal gene expression system
FIG. 3Pseudomonas putidaConstruction of ６１JCM6156 hal gene expression system
FIG. 4Pseudomonas putidaConstruction of {ATCC12633} hal gene expression system
FIG. 5Bacillus subtilisConstruction of {MI113} hal gene expression system
[0124]
[Sequence list]

Claims (23)

The following equation (1)

(Wherein, Z represents an aromatic ring group which may contain a hetero atom, R represents a substituent on the aromatic ring, n represents an integer of 0 or more, and when n is 2 or more, R represents They may be the same or different, provided that Z is imidazole and n is 0.)
Wherein histidine ammonia lyase is allowed to act on an acrylic acid derivative represented by the following formula (2):

(Wherein, Z represents an aromatic ring group which may contain a hetero atom, R represents a substituent on the aromatic ring, n represents an integer of 0 or more, and when n is 2 or more, R represents They may be the same or different, provided that Z is imidazole and n is 0.)
A method for producing an L-amino acid represented by the formula: R _n -Z- is the following formula (3)

(However, R is a substituent on the benzene ring, and is a cyano group, a hydroxyl group, a carboxyl group, an amide group, a halogen atom, an amino group, a nitro group, a hydroxymethyl group, an alkyl group having 1 to 6 carbon atoms or a carbon number. Represents an alkoxy group of 1 to 6. n represents an integer of 0 to 5, and when n is 2 or more, R may be the same or different.)
The method for producing an L-amino acid according to claim 1, which is represented by: The method for producing an L-amino acid according to claim 2, wherein at least one of R is an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, and a nitro group. The method for producing an L-amino acid according to claim 3, wherein n is 1. R _n -Z- is the following formula (4)

(Where X represents S, O, NH or NR ¹ , R ¹ represents an alkyl group having 1 to 6 carbon atoms, and Y represents N, CH or CR ^2. R and R ² represent , A cyano group, a hydroxyl group, a carboxyl group, an amide group, a halogen atom, an amino group, a nitro group, a hydroxymethyl group, an alkyl group having 1 to 6 carbon atoms or an alkoxy group having 1 to 6 carbon atoms, where n is 0 to 3 When n is 2 or more, R may be the same or different, R and R ² may be the same or different, adjacent R and R ² are bonded to each other, and the number of carbon atoms is A ring may be formed which may contain 1 to 6 hetero atoms, provided that X is NH, Y is N, and n is 0.
The method for producing an L-amino acid according to claim 1, which is represented by: The method for producing an L-amino acid according to claim 5, wherein Y is CH. The method for producing an L-amino acid according to claim 6, wherein n is 0 and X is S, O or NH. R _n -Z- is the following formula (5)

(Where R is a substituent on the heterocycle, and is a cyano group, hydroxyl group, carboxyl group, amide group, halogen atom, amino group, nitro group, hydroxymethyl group, alkyl group having 1 to 6 carbon atoms or carbon atom Represents an alkoxy group having a number of 1 to 6. n represents an integer of 0 to 4, and when n is 2 or more, Rs may be the same or different, and adjacent Rs have 1 to 6 carbon atoms. (It may form a ring that may contain a heteroatom.)
The method for producing an L-amino acid according to claim 1, which is represented by: The method for producing an L-amino acid according to claim 8, wherein n is 0. The histidine ammonia lyase is acted on in the form of a culture of a microorganism containing histidine ammonia lyase, a treatment composition of the culture and / or an enzyme obtained from the culture. 3. The method for producing an L-amino acid according to item 1. Microorganisms, including histidine ammonia lyase, Pseudomonas spp, Bacillus spp, Klebsiella spp, Rhizobium genus Alcaligenes sp, Aspergillus sp, Agrobacterium genus Deinococcus spp, Escherichia spp, Streptomyces spp., Caulobacter spp, from any of the genus Streptococcus and Corynebacterium spp The method for producing an L-amino acid according to claim 10, wherein the method is at least one selected from the group consisting of: The method for producing an L-amino acid according to claim 10, wherein the microorganism containing histidine ammonia lyase is Pseudomonas putida JCM6156. The method for producing an L-amino acid according to claim 10, wherein the microorganism containing histidine ammonia lyase is Bacillus subtilis MI113. The method for producing an L-amino acid according to claim 10, wherein the microorganism containing histidine ammonia lyase is a microorganism transformed by a histidine ammonia lyase gene. The method for producing an L-amino acid according to claim 14, wherein the histidine ammonia lyase gene is a gene encoding a sequence having 98% or more homology with the amino acid sequence derived from Pseudomonas putida JCM6156. The method for producing an L-amino acid according to claim 14, wherein the histidine ammonia lyase gene is derived from a microorganism. Microorganism from which it is derived histidine ammonia-lyase gene, Pseudomonas spp, Bacillus spp, Klebsiella spp, Rhizobium spp, Alcaligenes sp, Aspergillus sp, Agrobacterium genus, Deinococcus spp, Escherichia spp, Streptomyces spp, Caulobacter spp, Streptococcus spp, Vibrio spp and 17. The method for producing an L-amino acid according to claim 16, wherein the method is at least one selected from the genus Corynebacterium . The method for producing an L-amino acid according to claim 16, wherein the microorganism from which the histidine ammonia lyase gene is derived is Pseudomonas putida JCM6156. The method for producing an L-amino acid according to claim 16, wherein the microorganism from which the histidine ammonia lyase gene is derived is Bacillus subtilis MI113. The method for producing an L-amino acid according to claim 14, wherein the histidine ammonia lyase gene is derived from an animal. The method for producing an L-amino acid according to claim 20, wherein the animal is any one of a mouse, a rat, and a human. A gene encoding a sequence having at least 80% homology with the amino acid sequence encoded by the histidine ammonia lyase gene derived from Pseudomonas putida JCM6156, a plasmid containing the gene, and a transformed microorganism transformed with the plasmid. A histidine ammonia lyase gene derived from Pseudomonas putida JCM6156, a plasmid containing the gene, and a transformed microorganism transformed with the plasmid.

Images