JP3919918B2 - Process for producing optically active 2-halo-1- (substituted phenyl) ethanol - Google Patents
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Description
【０００１】
【発明の属する技術分野】
本発明は、光学活性２−ハロ−１−（置換フェニル）エタノールの製造法に関する。更に詳しくは光学活性２−ハロ−１−（置換フェニル）エタノールは、光学活性を必要とする医薬品、農薬等の合成原料として有用な光学活性置換スチレンオキサイドへ極めて容易に誘導することができる化合物であり、特に、光学活性３′−クロロスチレンオキサイドは、抗肥満薬の合成中間体として重要な化合物である。
【０００２】
【従来の技術】
光学活性２−ハロ−１−（置換フェニル）エタノールの製造方法として知られているものとしては、２−ハロ−１−（置換フェニル）エタノンに微生物を作用させることにより製造する方法が開示されている（特開平４−２１８３８４）が、この方法により製造できる化合物の立体配置は（−）体すなわち（Ｒ）体のみである。また、基質の仕込み濃度が低く実用的とは言い難い。一方、光学活性スチレンオキサイドの製造方法については、光学活性２−ハロ−１−（置換フェニル）エタノールをアルカリ条件下で閉環する方法が開示されている（特開平４−２１８３８４）。また、クロロ置換スチレンのノカルディア・コラリーナによるエポキシ化により７２〜８６％ｅｅを得たという報告（有機合成化学、４３、１６２（１９８７））がある。また、クロロ置換ベンズアルデヒドとジメチルスルフォニウムメテリドとの相間移動触媒反応によって合成する方法があるが、光学純度が極めて低い（特開昭５１−１０５０２４）。
【０００３】
【発明が解決しようとする課題】
本発明者らは、光学活性２−ハロ−１−（置換フェニル）エタノールの効率的な製造法を開発すべく検討を重ねた結果、２−ハロ−１−（置換フェニル）エタノンを立体選択的に還元し、光学活性２−ハロ−１−（置換フェニル）エタノールに変換する能力を有するいままでに報告例のない酵素源を発見し、本発明を完成するに至った。
【０００４】
【課題を解決するための手段】
本発明者らは上記課題を解決すべくを鋭意検討した結果、本発明を完成するに至った。
即ち、本発明の第１は、一般式［１］
【０００５】
【化９】
（式中、Ｘは塩素原子又は臭素原子を示し、置換基Ｒ１、Ｒ２、Ｒ３は水素原子、塩素原子、フッ素原子、メチル基、メトキシ基を示す。ただし、置換基すべてが水素原子の場合は除く。）で示される２−ハロ−１−（置換フェニル）エタノンを、
一般式［２］
【０００７】
【化１０】
（式中、Ｘ及び置換基Ｒ１、Ｒ２、Ｒ３は一般式［１］と同じ。）で示される（Ｒ）−２−ハロ−１−（置換フェニル）エタノールを生成する能力を有するオガタエア属、デバリオマイセス属に属する微生物群の中から選ばれた微生物の培養液、菌体または菌体処理物に作用させ、生成する一般式［２］で示される（Ｒ）−２−ハロ−１−（置換フェニル）エタノールを採取することを特徴とする（Ｒ）−２−ハロ−１−（置換フェニル）エタノールの製造法に関する。
【００１４】
本発明の第２は、一般式［１］
【００１５】
【化１３】
（式中、Ｘは塩素原子又は臭素原子を示し、置換基Ｒ１、Ｒ２、Ｒ３は水素原子、塩素原子、フッ素原子、メチル基、メトキシ基を示す。ただし、置換基すべてが水素原子の場合は除く。）で示される２−ハロ−１−（置換フェニル）エタノンを、
一般式［３］
【００１７】
【化１４】
（式中、Ｘ及び置換基Ｒ１、Ｒ２、Ｒ３は一般式［１］と同じ。）で示される（Ｓ）−２−ハロ−１−（置換フェニル）エタノールを生成する能力を有するヤマダジーマ属、シュードモナス属、ブレバンディモナス属、クリプトコッカス属に属する微生物群の中から選ばれた微生物の培養液、菌体または菌体処理物に作用させ、生成した一般式［３］で示される（Ｓ）−２−ハロ−１−（置換フェニル）エタノールを採取することを特徴とする（Ｓ）−２−ハロ−１−（置換フェニル）エタノールの製造法に関する。
【００２５】
【発明の実施の形態】
以下、本発明について詳述する。
本発明に用いる２−ハロ−１−（置換フェニル）エタノンを不斉還元し、２−ハロ−１−（置換フェニル）エタノールに変換する微生物は、以下に説明する方法によって見出すことができる。例えば、グルコース５％、ペプトン０．５％、リン酸二水素カリウム０．２％、リン酸水素二カリウム０．１％、硫酸マグネシウム０．０２％、酵母エキス０．１％の組成からなるＡ培地５０ｍｌ（ｐＨ６．５）を５００ｍｌ容坂口フラスコに入れ殺菌後、微生物を植え、３０℃で２〜３日間振とうする。その後、菌体を遠心分離により集め２−クロロ−１−（３′−クロロフェニル）エタノン０．１〜０．５％およびグルコース５％を含んだリン酸緩衝液２５ｍｌに懸濁し、５００ｍｌ容坂口フラスコ中で２〜３日間３０℃で振とうする。この際、遠心分離により得た菌体をデシケーター中またはアセトンにより乾燥したものを用いることもできる。さらに、これら微生物もしくはその処理物と２−クロロ−１−（３′−クロロフェニル）エタノンを反応させる際に、酸化型ニコチンアミドアデニンジヌクレオチド（ＮＡＤ）および／または酸化型ニコチンアミドアデニンジヌクレオチドりん酸（ＮＡＤＰ）とグルコース脱水素酵素を添加してもよい。変換反応ののち反応液と同体積の酢酸エチルを加え抽出を行ない生成する２−クロロ−１−（３′−クロロフェニル）エタノールを高速液体クロマトクロマトグラフィー（カラム：ダイセル化学工業株式会社製Chiralcel OJ、溶離液：ｎ−ヘキサン／イソプロパノール＝３９／１、流速：１ｍｌ／ｍｉｎ、検出：２１０ｎｍ、カラム温度：室温、溶出時間２−クロロ−１−（３′−クロロフェニル）エタノン：２８分、（Ｒ）−２−クロロ−１−（３′−クロロフェニル）エタノール：３７分、（Ｓ）−２−クロロ−１−（３′−クロロフェニル）エタノール：４５分）により分析する。
本発明に使用しうる微生物としては、２−ハロ−１−（置換フェニル）エタノンを不斉還元して光学活性な２−ハロ−１−（置換フェニル）エタノールに変換する能力を有する微生物としてオガタエア属、デバリオマイセス属、キャンディダ属、ヤマダジーマ属、シュードモナス属、ブレバンディモナス属、クリプトコッカス属に属する微生物が使用しうるが、具体的には例えば、オガタエア・ミニュータ・バー・ミニュータ(Ogataea minuta var.minuta) IFO 0975、オガタエア・ミニュータ・バー・ノンファーメンタンス(Ogataea minuta var.nonfermentans) IFO 1473、デバリオマイセス・カルソニー(Debaryomyces carsonii) IFO 0946、キャンディダ・パラプシロシス(Candida parapsilosis) IFO 0585、キャンディダ・ツェイラノイデス(Candida zeylanoides) IFO 0719、ヤマダジーマ・ファリノサ(Yamadazyma farinosa) IFO 0459、ヤマダジーマ・ファリノサ(Yamadazyma farinosa) IFO 0193、シュードモナス・プチダ(Pseudomonas putida) IFO 12966、ブレバンディモナス・ディミヌータ(Brevundimonas diminuta) IFO 12697、クリプトコッカス・フミコラス(Cryptococcus humicolus) IFO 0760、クリプトコッカス・フミコラス(Cryptococcus humicolus) JCM1460などを用いることができる。これら微生物は一般に、入手または購入が容易な保存株から得ることができる。また、自然界から分離することもできる。なお、これらの微生物に変異を生じさせてより本反応に有利な性質を有する菌株を得ることもできる。
これらの微生物の培養には、通常これらの微生物が資化しうる栄養源であれば何でも使用しうる。たとえば、グルコース、シュークロース、マルトース等の糖類、乳酸、酢酸、クエン酸、プロピオン酸等の有機酸類、エタノール、グリセリン等のアルコール類、パラフィン等の炭化水素類、大豆油、菜種油等の油脂類、またはこれらの混合物等の炭素源や、硫酸アンモニウム、リン酸アンモニウム、尿素、酵母エキス、肉エキス、ペプトン、コーンスチープリカー等の窒素源を混合することもできる。更に、その他の無機塩、ビタミン類等の栄養源を適宜混合することもできる。
微生物の培養は通常一般の条件により行なうことができ、例えば、ｐＨ４．０〜９．５、温度範囲２０℃〜４５℃の範囲で、好気的に１０〜９６時間培養する。２−ハロ−１−（置換フェニル）エタノンに微生物を反応させる場合においては、通常、上記微生物の培養液をそのまま反応に使用することもできるが、培養液の濃縮物も用いることができる。また、培養液中の成分が反応に悪影響を与える場合には、培養液を遠心分離等により処理して得られる菌体または菌体処理物を使用することが好ましい。
上記微生物の菌体処理物としては特に限定されず、例えば、アセトンや五酸化二リンによる脱水処理またはデシケーターや扇風機を利用した乾燥によって得られる乾燥菌体、界面活性剤処理物、溶菌酵素処理物、固定化菌体または菌体を破砕した無細胞抽出標品などをあげることができる。更に、培養物より不斉還元反応を触媒する酵素を精製し、これを使用してもよい。
一般式［１］に示される２−ハロ−１−（置換フェニル）エタノンを一般式［２］、または［３］に示される光学活性な２−ハロ−１−（置換フェニル）エタノールに変換する能力を有する微生物の培養液、菌体あるいは菌体処理物、または当該能力を有する微生物から得られる当該活性を有する酵素を以下「酵素源」という。
また、微生物の培養液、菌体または菌体処理物としての当該能力の強弱に関係なく、その微生物より得られた酵素が当該活性を有していれば、その酵素をも酵素源とすることができる。こうした酵素としては、たとえば、４−クロロアセト酢酸エチルを還元して４−クロロ−３−ヒドロキシ酪酸エチルを与える酵素を用いることができる。この還元反応を触媒する酵素としては、キャンディダ属に属する微生物から得られる酵素であることが好ましい。
酵素源は多くの場合微生物から得ることができる。微生物の培養物からの酵素の抽出精製には、当業者に通常用いられている酵素の抽出精製法が用いられ得る。たとえば、培養液から遠心分離により菌体を集め、これを適当な緩衝液中に懸濁し、該菌体をグラスビーズ等の物理的手法、酵素等の生化学的手法などを用いて破砕または溶解したのち遠心分離により該溶液中の不溶物を除去して、粗酵素液を得ることができる。該粗酵素液を通常当業者が用いる方法、たとえば、硫酸アンモニウム沈殿、透析、クロマトグラフィーを単独または組み合わせて用いてさらに精製し得る。こうした粗酵素液を含む酵素精製操作の各段階の酵素画分が当該反応を触媒する場合、これらを酵素源として使用することができる。また、当該反応を触媒する酵素遺伝子を単離し、遺伝子工学的手法により得られる組み換え微生物の培養液、菌体または菌体処理物もまた酵素源として使用可能である。このようにして得られる当該反応を触媒する酵素としては、たとえば、ＰＣＴ／ＪＰ９７／０３０５１記載のキャンディダ属に属する微生物から得られる以下の（１）から（７）の理化学的性質を有する酵素Ｓ１を例示することができる。
（１）作用：
ＮＡＤＰＨを補酵素として、４−クロロアセト酢酸エチルに作用し、（Ｓ）−４−クロロ−３−ヒドロキシ酪酸エチルを生成する、
（２）基質特異性：
４−クロロアセト酢酸エチルに対して強い活性を示し、アセト酢酸エチルには実質的に活性を示さない、
（３）至適ｐＨ：ｐＨ５．５〜６．５、
（４）至適温度：５０〜５５℃、
（５）分子量：ゲル濾過分析において約７６０００、ＳＤＳポリアクリルアミド電気泳動分析において約３２０００、
（６）熱安定性：ｐＨ７．０で３０分間処理したときに約４０℃まで安定である、および
（７）阻害剤：水銀イオン、クエルセチンにより阻害される。
【００２６】
また、以下の（１）から（５）の理化学的性質を有するキャンディダ属微生物由来のカルボニル還元酵素Ｓ４も例示することができる。
（１）作用：
ＮＡＤＰＨを補酵素として、４−クロロアセト酢酸エチルに作用し、（Ｓ）−４−クロロ−３−ヒドロキシ酪酸エチルを生成する、
（２）基質特異性：
４−クロロアセト酢酸エチルに活性を示し、ケトパントイルラクトンに対して強い活性を示す、
（３）至適ｐＨ：ｐＨ６．０、
（４）至適温度：５０℃、および
（５）分子量：ゲル濾過分析において約８６０００、ＳＤＳポリアクリルアミド電気泳動分析において約２９０００。
還元反応の際には、基質である２−ハロ−１−（置換フェニル）エタノンを反応の初期に一括して添加してもよく、反応の進行にあわせて分割して添加してもよい。
また、反応時の温度は通常１０〜６０℃、好ましくは、２０〜４０℃であり。反応時のｐＨは２．５〜９、好ましくは、５〜９である。
反応液中の酵素源の量はこれらの基質を還元する能力に応じ適宜決定すればよい。また、反応液中の基質濃度は０．０１〜５０％（Ｗ／Ｖ）が好ましく、より好ましくは、０．１〜３０％である。
反応は通常、振とうまたは通気攪拌しながら行なう。反応時間は基質濃度、酵素源の量およびその他の反応条件により適宜決定される。通常、２〜１６８時間で反応が終了するように各条件を設定することが好ましい。
還元反応を促進させるために、反応液にグルコース、エタノールなどのエネルギー源を１〜３０％の割合で加えると優れた結果が得られるので好ましい。また、一般に生物学的方法による還元反応に必要とされている還元型ニコチンアミド・アデニンジヌクレオチド（ＮＡＤＨ）、還元型ニコチンアミド・アデニンジヌクレオチドりん酸（ＮＡＤＰＨ）等の補酵素を添加することにより、反応を促進させることもできる。具体的には、反応液に直接これらを添加してもよく、ＮＡＤＨ、ＮＡＤＰＨを生成する反応系を酸化型の補酵素とともに反応液に添加してもよい。例えば、ギ酸脱水素酵素がギ酸から二酸化炭素と水とを生成する際にＮＡＤをＮＡＤＨに還元する反応系や、グルコース脱水素酵素がグルコースからグルコノラクトンを生成する際にＮＡＤまたはＮＡＤＰをＮＡＤＨまたはＮＡＤＰＨにそれぞれ還元する反応系を利用することができる。また、トリトン（ナカライテスク株式会社製）、スパン（関東化学株式会社製）、ツイーン（ナカライテスク株式会社製）などの界面活性剤を反応液に添加することも効果的である。さらに、基質および／または還元反応の生成物であるアルコール体による反応の阻害を回避する目的で、酢酸エチル、酢酸ブチル、イソプロピルエーテル、トルエンなどの水に不溶な有機溶媒を反応液に添加してもよい。さらに、基質の溶解度を高める目的で、メタノール、エタノール、アセトン、テトラヒドロフラン、ジメチルスルホキシドなどの水に可溶な有機溶媒を添加することもできる。
還元反応により生成した光学活性な２−ハロ−１−（置換フェニル）エタノールの採取は、反応液から直接、あるいは菌体等を分離後、酢酸エチル、ｎ−ヘキサン等の溶剤で抽出し、脱水後、蒸留あるいはシリカゲルカラムクロマトグラフィー等により精製すれば高純度の光学活性２−ハロ−１−（置換フェニル）エタノールが容易に得られる。
【００２７】
上記の如くして得られた光学活性な２−ハロ−１−（置換フェニル）エタノールは、苛性ソーダ等のアルカリを等モル以上共存させ、加熱あるいは室温放置することにより容易に閉環し、光学活性な置換スチレンオキサイドに変換されうる。
【００２８】
【実施例】
以下、実施例により本発明をさらに詳しく説明するが、本発明はこれらの実施例により何ら限定されるものではない。なお、以下の記載において、「％」は特に断らない限り「重量％」を意味する。
実施例１
前記のＡ培地５０ｍｌを５００ｍｌ容坂口フラスコに入れ殺菌後、表１に示す微生物をそれぞれ植菌した。そして３０℃で２日間好気的に振とう培養を行なった。この培養液から遠心分離によって菌体を集め、２−クロロ−１−（３′−クロロフェニル）エタノン１％、酸化型ニコチンアミドアデニンジヌクレオチド（ＮＡＤ）０．０６％、酸化型ニコチンアミドアデニンジヌクレオチドりん酸（ＮＡＤＰ）０．０６％、グルコース５％、グルコース脱水素酵素（商品名：ＧＬＵＣＤＨ”Ａｍａｎｏ”II、天野製薬株式会社製）１４．３Ｕ／ｍｌを含有する５０ｍＭリン酸緩衝液（ｐＨ６．０）２０ｍｌに懸濁し、５００ｍｌ容坂口フラスコに入れて３０℃、２４時間振とう反応させた。反応後、反応液から同体積の酢酸エチルで光学活性２−クロロ−１−（３′−クロロフェニル）エタノールを２回抽出し、酢酸エチル層を高速液体クロマトグラフィーで分析して、反応率および光学純度を測定した。その結果を表１に示す。
【００２９】
【表１】
実施例２
表２に示す微生物について、グルコース１％、ペプトン１．５％、リン酸水素二カリウム０．３％、硫酸マグネシウム０．０２％、塩化ナトリウム０．２％、酵母エキス０．１％の組成からなるＢ培地（ｐＨ７．０）を用いたほかは実施例１と同様の操作を行ない、反応率および光学純度を測定した。その結果を表２に示す。
【００３１】
【表２】
実施例３
Ａ培地５０ｍｌを含む５００ｍｌ容坂口フラスコ２０本に、オガタエア・ミニュータ・バー・ノンファーメンタンス(Ogataea minuta var.nonfermentans) IFO 1473を植菌し、３０℃で４８時間振とう培養した。培養後菌体を遠心分離により集菌し、これにグルコース４０ｇ、ＮＡＤＰ２４０ｍｇ、ＮＡＤ２４０ｍｇ、グルコース脱水素酵素（商品名：ＧＬＵＣＤＨ”Ａｍａｎｏ”II、天野製薬株式会社製）８０ｍｇを含む５０ｍＭりん酸緩衝液（ｐＨ６．０）を加えて全量を８００ｍｌとした。さらに、基質である２−クロロ−１−（３′−クロロフェニル）エタノンを１．６ｇ加えて還元反応を開始した。反応は３０℃で、２Ｍ炭酸ナトリウム水溶液により反応液のｐＨを６．０に調整しながら攪拌しつつ行なった。反応液の一部を定期的にＨＰＬＣにより分析し、基質が枯渇している場合にはさらに基質１．６ｇを追加し反応を継続させた。この操作を繰り返しつつ約５０時間反応を行なった。反応終了時のＨＰＬＣ分析の結果によると、反応液の２−クロロ−１−（３′−クロロフェニル）エタノールの濃度は９．８ｍｇ／ｍｌであった。反応終了後、１０００ｍｌの酢酸エチルで２回抽出した。酢酸エチル層を無水硫酸ナトリウムで乾燥したのち、減圧下脱溶剤し、オイル状物質を得た。このものを減圧蒸留（１３０℃／３ｍｍＨｇ）し、無色オイル状の（Ｒ）−２−クロロ−１−（３′−クロロフェニル）エタノール８．８ｇを得た。その比旋光度は［α］（２０，Ｄ）＝−３３．６゜（ｃ＝１．０２）を示し、ＨＰＬＣ分析によれば光学純度９９．３％ｅｅであった。H-NMR（CDCl3）δppm 2.69(br,s,1H), 3.27〜3.90(m,2H),4.88(dd,1H),7.15〜7.54(m,4H)
実施例４
Ａ培地５０ｍｌを含む５００ｍｌ容坂口フラスコ２０本に、ヤマダジーマ・ファリノサ(Yamadazyma farinosa) IFO 0459を植菌し、３０℃で４８時間振とう培養した。培養後菌体を遠心分離により集菌し、これにグルコース４０ｇ、ＮＡＤＰ２４０ｍｇ、ＮＡＤ２４０ｍｇ、グルコース脱水素酵素（商品名：ＧＬＵＣＤＨ”Ａｍａｎｏ”II、天野製薬株式会社製）８０ｍｇを含む５０ｍＭりん酸緩衝液（ｐＨ６．０）を加えて全量を８００ｍｌとした。さらに、基質である２−クロロ−１−（３′−クロロフェニル）エタノンを１．６ｇ加えて還元反応を開始した。実施例３と同様の操作により還元反応を約５０時間行なった。反応終了時のＨＰＬＣ分析の結果によると、反応液の２−クロロ−１−（３′−クロロフェニル）エタノールの濃度は１１．８ｍｇ／ｍｌであった。反応終了後、１０００ｍｌの酢酸エチルで２回抽出した。酢酸エチル層を無水硫酸ナトリウムで乾燥したのち、減圧下脱溶剤し、オイル状物質を得た。このものを減圧蒸留（１３０℃／３ｍｍＨｇ）し、無色オイル状の（Ｓ）−２−クロロ−１−（３′−クロロフェニル）エタノール１０．６ｇを得た。その比旋光度は［α］（２０，Ｄ）＝３３．５゜（ｃ＝１．０２）を示し、ＨＰＬＣ分析によれば光学純度９９．１％ｅｅであった。H-NMR（CDCl3）δppm 2.69(br,s,1H), 3.27〜3.90(m,2H),4.88(dd,1H),7.15〜7.54(m,4H)
実施例５
Ｂ培地５０ｍｌを含む５００ｍｌ容坂口フラスコ２０本に、ブレバンディモナス・ディミヌータ(Brevundimonas diminuta) IFO 12697を植菌し、３０℃で２４時間振とう培養した。培養後菌体を遠心分離により集菌し、これにグルコース４０ｇ、ＮＡＤＰ２４０ｍｇ、ＮＡＤ２４０ｍｇ、グルコース脱水素酵素（商品名：ＧＬＵＣＤＨ”Ａｍａｎｏ”II、天野製薬株式会社製）８０ｍｇを含む５０ｍＭりん酸緩衝液（ｐＨ６．０）を加えて全量を８００ｍｌとした。さらに、基質である２−クロロ−１−（３′−クロロフェニル）エタノンを１．６ｇ加えて還元反応を開始した。実施例３と同様の操作により還元反応を約５０時間行なった。反応終了時のＨＰＬＣ分析の結果によると、反応液の（Ｓ）−２−クロロ−１−（３′−クロロフェニル）エタノールの濃度は１８．４ｍｇ／ｍｌ、光学純度は、８４．２％ｅｅであった。
実施例６
Ａ培地８０００ｍｌを調製し、２０００ｍｌ容坂口フラスコに４００ｍｌずつ分注して、１２０℃で２０分間蒸気殺菌した。これらの培地にあらかじめ同培地中で前培養しておいたオガタエア・ミニュータ・バー・ノンファーメンタンス(Ogataea minuta var.nonfermentans) IFO 1473の培養液を５ｍｌずつ接種し、３０℃で２日間振とう培養した。この培養液から遠心分離により菌体を集め、生理食塩水にて２回洗浄した。得られた湿菌体を４００ｍｌの５０ｍＭりん酸緩衝液（ｐＨ７．０）に懸濁し、次いで菌体をダイノミル（Ｄｙｎｏ−Ｍｉｌｌ社製）で破砕した。この菌体破砕物から遠心分離にて菌体残渣を除き、無細胞抽出液７００ｍｌを得た。大型試験菅に、２−クロロ−１−（３′−クロロフェニル）エタノン１００ｍｇ、ＮＡＤＰ０．６ｍｇ、グルコース５００ｍｇ、グルコース脱水素酵素（商品名：ＧＬＵＣＤＨ”Ａｍａｎｏ”II、天野製薬株式会社製）１４３Ｕを秤取り、これに調製した無細胞抽出液１０ｍｌを加えて、３０℃で２４時間振とう反応させた。反応後、反応液を１０ｍｌの酢酸エチルで２回抽出し、得られた有機層を実施例１に示した方法により分析したところ、２−クロロ−１−（３′−クロロフェニル）エタノールへの反応率は６０％、その光学純度は９９．３％、立体配置は（Ｒ）体であった。
参考例１ キャンディダ属微生物からの酵素Ｓ１の調製
酵素活性の測定は、２００ｍMりん酸緩衝液（ｐＨ７．０）に、基質４−クロロアセト酢酸エチル０．２ｍＭ、補酵素ＮＡＤＰＨ０．３２ｍＭおよび酵素を添加し、３０℃で波長３４０ｎｍの吸光度の減少を測定することにより行なう。この反応条件において、１分間に１μｍｏｌのＮＡＤＰＨをＮＡＤＰに酸化する酵素活性を１ｕｎｉｔと定義する。また、酵素反応により生成した４−クロロ−３−ヒドロキシ酪酸エチルの光学純度の測定は、光学分割カラムＣＨＩＲＡＬＣＥＬ ＯＢ（ダイセル化学工業株式会社製）を用いたＨＰＬＣ分析により行なう（移動相；ヘキサン／イソプロパノール＝９／１、流速；０．８ｍｌ／ｍｌ、カラム温度；０℃、検出；２１５ｎｍ）。
【００３３】
以下の組成からなる液体培地８０００ｍｌを調製し、２０００ｍｌ容坂口フラスコに４００ｍｌずつ分注して、１２０℃で２０分間蒸気殺菌した。
培地組成；
グルコース ５％
ポリペプトン ０．５％
ＫＨ2ＰＯ4 ０．２％
Ｋ2ＨＰＯ4 ０．１％
ＭｇＳＯ4／７Ｈ2Ｏ ０．０２％
水道水
ｐＨ ６．５
これらの培地に、あらかじめ同培地にて前培養しておいたキャンディダ・マグノリエ(Candida magnoliae IFO 0705)の培養液を５ｍｌずつ接種し、３０℃で３日間振とう培養した。遠心分離によりこの培養液から菌体を集め、生理食塩水にて２回洗浄を行ない、湿菌体２３０ｇを得た。このうち１８０ｇを３６０ｍｌの５０ｍＭりん酸緩衝液(ｐＨ７．０)に懸濁した後、菌体をダイノミル(Dyno-Mill社製)で破砕した。この菌体破砕物を遠心分離して残さを除き、無細胞抽出液７６０ｍｌを得た。この無細胞抽出液に４０％飽和となるように硫酸アンモニウムを添加し、生じた沈殿を遠心分離により除去し、上清を０．１％ＤＴＴを含む１０ｍＭりん酸緩衝液（ｐＨ７．０）に対して一晩透析した。これを同一緩衝液であらかじめ平衡化したDEAE sephacel(Pharmacia Biotech社製)カラムに供し、同緩衝液でカラムを洗浄した。素通りしてくる溶液から活性画分を回収し、終濃度４ＭとなるようにＮａＣlを添加した。この活性画分を４ＭのＮａＣｌと０．１ｍＭのＤＴＴを含む１０ｍＭりん酸緩衝液（ｐＨ７．０）であらかじめ平衡化しておいたPhenyl sepharose CL-4B(Pharmacia Biotech社製)カラムに供し、酵素を吸着させた。同一緩衝液でカラムを洗浄の後、１０ｍＭりん酸緩衝液（ｐＨ７．０）を用い、ＮａＣｌ（４Ｍから０Ｍまで）およびエチレングリコール（０％から５０％（Ｗ／Ｖ）まで）のリニアグラジエントにより、活性画分を溶出させた。これを１０ｍＭりん酸緩衝液（ｐＨ７．０）に対して一晩透析した。
この透析液を、０．１ｍＭＤＴＴを含む１０ｍＭりん酸緩衝液（ｐＨ７．０）であらかじめ平衡化しておいたMono Q HR5/5(Pharmacia Biotech社製)カラムに供し、同一緩衝液で洗浄した。洗浄液中の活性画分を集め、限外濾過にて濃縮し、０．２ＭＮａＣｌおよび０．１ｍＭＤＴＴを含む１０ｍＭりん酸緩衝液（ｐＨ７．０）にてあらかじめ平衡化しておいたSuperdex 200 HR10/30(Pharmacia Biotech社製)カラムに供し、同緩衝液で溶出した。活性画分を集め精製酵素標品とした。
参考例２ キャンディダ属微生物から得られた酵素Ｓ１の酵素化学的性質
酵素活性の測定は、基本的には、２００ｍMりん酸緩衝液（ｐＨ７．０）に、基質４−クロロアセト酢酸エチル０．２ｍＭ、補酵素ＮＡＤＰＨ０．３２ｍＭ、および酵素溶液０．１ｍｌを含む３．０ｍｌの反応液中、３０℃、１分間反応させ、波長３４０ｎｍの吸光度の減少を測定することにより行なった。
（１）作用：
ＮＡＤＰＨを補酵素として、４−クロロアセト酢酸エチルに作用し、光学純度９９％ｅｅ以上の（Ｓ）−４−クロロ−３−ヒドロキシ酪酸エチルを生成した。
（２）基質特異性：
表３に示す各種カルボニル化合物を基質として、４−クロロアセト酢酸エチルと同様の条件で反応を行なった結果、本酵素は表３に示すような基質特異性を示した。
【００３４】
【表３】
（３）至適ｐＨ：
緩衝液としてりん酸緩衝液、トリス−塩酸緩衝液を用いてｐＨ５．０〜８．５の範囲で、上記の方法により４−クロロアセト酢酸エチルを基質とした場合の酵素活性を測定した。その結果、至適ｐＨは５．５〜６．５であった。
（４）作用至適温度：
２０〜６０℃の温度での１分間の、４−クロロアセト酢酸エチルを基質とした場合の活性を測定した。その結果、至適温度は５０〜５５℃であった。
（５）熱安定性：
本酵素をｐＨ７．０において４０℃で３０分間加熱したのちの４−クロロアセト酢酸エチルを基質とした場合の活性を測定した。その結果加熱処理前の９０％の活性が残存していた。
（６）阻害剤：
反応液に、表４に示す濃度の各種の化合物を添加して、４−クロロアセト酢酸エチルを基質とした場合の活性を測定した。表４に示すように、本酵素はクエルセチンおよび水銀イオンにより阻害をうけた。
【００３６】
【表４】
（７）分子量：
酵素の分子量の測定は、TSK-G3000Wカラムを用い、溶離液として０．１ＭＮａ2ＳＯ4および０．０５％ＮａＮ3を含む１００ｍＭりん酸緩衝液（ｐＨ７．０）を用いた。この場合の分子量は約７６０００であった。酵素のサブユニットの分子量は、２％の２−メルカプトエタノール存在下、１０％ＳＤＳ−ポリアクリルアミドゲル電気泳働分析により標準蛋白の相対移動度から算出した。その結果、本酵素のサブユニットの分子量は約３２０００であった。
（８）有機溶媒耐性：
本酵素が溶解しているｐＨ７．０のりん酸緩衝液に同体積の酢酸エチルまたは酢酸ブチルを添加し、２８℃で３０分間振とうののち、遠心分離により水相と有機相を分離した。水相中の残存する酵素活性を４−クロロアセト酢酸エチルを基質として測定した。その結果、酢酸エチルを用いた場合では７２％、酢酸ブチルを用いた場合には８５％の酵素活性の残存が認められた。
実施例７ キャンディダ属微生物からの酵素Ｓ４の調製
参考例１と同様の操作により得られた無細胞抽出液１１００ｍｌに６０ｇのポリエチレングリコールを攪拌しながら加え、添加後約２時間放置した。その後遠心分離により得られた上清を０．１ｍＭＤＴＴを含む１０ｍＭりん酸緩衝液（ｐＨ７．０）に対し透析し、同緩衝液で平衡化したDEAE sephacel(Pharmacia Biotech社製)カラムに供した。つぎに同緩衝液でカラムを洗浄ののち、ＮａＣｌの０Ｍから０．２Ｍのリニアグラジエントにより活性画分を溶出させた。この画分を同緩衝液に対して透析ののち、２Ｍ硫酸アンモニウムを含む同緩衝液で平衡化したButyl toyopearl 650（東ソー社製）に供した。硫酸アンモニウムを含まない同緩衝液により活性画分を溶出し、限外濾過により脱塩および濃縮した。この酵素液を同緩衝液で平衡化したMono Q HR 10/10（Pharmacia Biotech社製)カラムに供した。０．２５ＭのＮａＣｌにより活性画分を溶出し、同緩衝液に対して透析した。これを、２Ｍ硫酸アンモニウムを含む同緩衝液で平衡化したAlkyl Superose HR 10/10（Pharmacia Biotech社製)に供した。エチレングリコールおよび硫酸アンモニウムによるリニアグラジエントにより溶出すると、目的の活性は前者の０％から１０％および後者の２Ｍから０Ｍの画分に見い出され、これを精製酵素標品とした。
実施例８ キャンディダ属微生物から得られた酵素Ｓ４の酵素化学的性質
参考例２と同様の操作により酵素Ｓ４の諸性質を調べた。
（１）作用：
ＮＡＤＰＨを補酵素として、４−クロロアセト酢酸エチルに作用し、光学純度５１％ｅｅの（Ｓ）−４−クロロ−３−ヒドロキシ酪酸エチルを生成する。
（２）基質特異性：
表５に示す各種カルボニル化合物を基質として、４−クロロアセト酢酸エチルと同様の条件で反応を行なった結果、表５に示すような基質特異性を示した。
【００３８】
【表５】
（３）至適ｐＨ：
緩衝液としてりん酸緩衝液、トリス塩酸緩衝液を用いてｐＨ５．０〜ｐＨ８．５の範囲で、参考例２の方法により酵素活性を測定した。その結果、４−クロロアセト酢酸エチルに作用する至適ｐＨは６．０であった。
（４）至適温度：
２０℃〜６０℃の温度で１分間の、４−クロロアセト酢酸エチルを基質とした場合の酵素の活性を測定して至適温度を求めたところ、５０℃であった。
（５）分子量：
酵素の分子量の測定は、参考例２の方法により行ない、ゲル濾過分析により約８６０００、ＳＤＳポリアクリルアミドゲル電気泳動分析により、約２９０００と算出された。
実施例９
参考例１で精製した酵素Ｓ１を５ｕｎｉｔｓ、２−クロロ−１−（３′−クロロフェニル）エタノン２５ｍｇ、ＮＡＤＰ０．１６ｍｇ、グルコース２８ｍｇ、グルコース脱水素酵素（商品名：ＧＬＵＣＤＨ”Ａｍａｎｏ”II、天野製薬株式会社製）６ｕｎｉｔｓを含む１００ｍＭりん酸緩衝液（ｐＨ６．５）２．５ｍｌを、３０℃で２４時間攪拌した。反応液を酢酸エチルで抽出し、脱溶剤したのち抽出物の分析を行なうと、反応率８０％、光学純度９９％ｅｅ以上の（Ｓ）−２−クロロ−１−（３′−クロロフェニル）エタノールが生成していた。
実施例１０
実施例７で精製した酵素Ｓ４を用いて、実施例９と同様の操作を行ない、反応物を分析したところ、反応率５０％、光学純度９１％ｅｅの（Ｒ）−２−クロロ−１−（３′−クロロフェニル）エタノールが生成していた。
実施例１１
Ａ培地５０ｍｌを５００ｍｌ容坂口フラスコに入れ殺菌後、表６に示す微生物をそれぞれ植菌した。３０℃で２日間好気的に振とう培養を行なった。この培養液から菌体を遠心分離によって集め、２−ブロモ−１−（３′−クロロフェニル）エタノン０．５％を０．３％グルコース含有１００ｍＭりん酸緩衝液（ｐＨ７．０）２５ｍｌに懸濁し、５００ｍｌ容坂口フラスコに入れて３０℃で４８時間振とう反応させた。反応後、反応液から等量の酢酸エチルで２−ブロモ−１−（３′−クロロフェニル）エタノールを抽出し、酢酸エチル層をガスクロマトグラフィーで分析し、反応率を調べた。また、実施例１と同じ方法により光学純度を測定した。これらの結果を表６に示した。
【００４０】
【表６】
実施例１２
Ａ培地３Ｌを含む５Ｌ容ミニジャーファーメンターにクリプトコッカス・フミコラス(Cryptococcus humicolus)IFO0760を植菌し、３０℃、通気１ｖｖｍ、攪拌５００ｒｐｍにて２４時間培養した。終了後、遠心分離により菌体を集め、７５０ｍｌの水に懸濁し、２−ブロモ−１−（３′−クロロフェニル）エタノン７．５ｇ、グルコース３８ｇを添加し、ｐＨを１Ｎ苛性ソーダ水溶液で７．０に保ちながら３０℃、攪拌１５０ｒｐｍで２４時間反応させた。反応後、７５０ｍｌの酢酸エチルで２回抽出し、これを脱水し、減圧下脱溶剤し、油状物質を得た。このものを蒸留し（１３０℃／３ｍｍＨｇ）、無色オイル状の（Ｓ）−２−ブロモ−１−（３′−クロロフェニル）エタノール２．５ｇを得た。その比旋光度は［α］（２０、Ｄ）２４．５（ｃ＝１，メタノール）を示し、ＨＰＬＣによる光学純度は９７％ｅｅであった。
1H-NMR δppm 2.88(br,1H),3.35〜3.90(m,4H),4.90(dd,2H),6.98〜7.51(m,4H)
参考例３
実施例３で得られた（Ｒ）−２−クロロ−１−（３′−クロロフェニル）エタノール２ｇを等モル相当の４０％苛性ソーダ水溶液、塩化メチレン１０ｍｌと混合し、５０℃で６時間反応した。冷却後、塩化メチレン１０ｍｌを加え、有機層を飽和食塩水で洗浄し脱水濾過した後、塩化メチレンを脱溶剤し、粗エポキシド油状物を得た。このものを減圧蒸留（８０℃、４ｍｍＨｇ）により精製し、無色オイル状の（Ｒ）―３′−クロロスチレンオキサイド１ｇを得た。その比旋光度は［α］（２０，Ｄ）＝−６７．５（ｃ＝１）であった。
1H-NMR(CDCl3) δppm 2.63(dd,1H),3.15(dd,1H),4.17(dd,1H),7.01〜7.51(m,4H)
【００４２】
【発明の効果】
本発明によれば、光学活性２−ハロ−１―（置換フェニル）エタノールを効率的にかつ工業的規模で生産することが可能となる。得られる光学活性２−ハロ−１―（置換フェニル）エタノールは医薬品等の合成原料として有用な光学活性置換スチレンオキサイドへ容易に導くことができる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a process for producing optically active 2-halo-1- (substituted phenyl) ethanol. More specifically, optically active 2-halo-1- (substituted phenyl) ethanol is a compound that can be very easily derived into optically active substituted styrene oxide useful as a synthetic raw material for pharmaceuticals, agricultural chemicals and the like that require optical activity. In particular, optically active 3'-chlorostyrene oxide is an important compound as a synthetic intermediate for anti-obesity drugs.
[0002]
[Prior art]
As a known method for producing optically active 2-halo-1- (substituted phenyl) ethanol, a method for producing 2-halo-1- (substituted phenyl) ethanone by causing a microorganism to act is disclosed. However, the configuration of the compound that can be produced by this method is only the (−) isomer, ie, the (R) isomer. Moreover, it is difficult to say that the substrate concentration is low and practical. On the other hand, as a method for producing optically active styrene oxide, a method of ring-closing optically active 2-halo-1- (substituted phenyl) ethanol under alkaline conditions is disclosed (Japanese Patent Laid-Open No. 4-218384). There is also a report that 72-86% ee was obtained by epoxidation of chloro-substituted styrene with Nocardia Coralina (Organic Synthetic Chemistry, 43, 162 (1987)). Also, there is a method of synthesizing by a phase transfer catalyzed reaction of chloro-substituted benzaldehyde and dimethylsulfonium meteride, but the optical purity is extremely low (Japanese Patent Laid-Open No. 51-105024).
[0003]
[Problems to be solved by the invention]
As a result of repeated studies to develop an efficient production method of optically active 2-halo-1- (substituted phenyl) ethanol, the present inventors have stereoselectively selected 2-halo-1- (substituted phenyl) ethanone. Thus, the present invention has been completed by discovering an unprecedented source of enzyme having the ability to be reduced to optically active 2-halo-1- (substituted phenyl) ethanol.
[0004]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventors have completed the present invention.
That is, the first of the present invention is the general formula [1]
[0005]
[Chemical 9]
(In the formula, X represents a chlorine atom or a bromine atom, and the substituents R1, R2, and R3 represent a hydrogen atom, a chlorine atom, a fluorine atom, a methyl group, and a methoxy group. However, when all the substituents are hydrogen atoms, 2-halo-1- (substituted phenyl) ethanone represented by
General formula [2]
[0007]
[Chemical Formula 10]
(Wherein X and substituents R 1, R 2 and R 3 are the same as in general formula [1].) Ogataae having the ability to produce (R) -2-halo-1- (substituted phenyl) ethanol, (R) -2-halo-1- (substituted) represented by the general formula [2] produced by reacting with a culture solution, fungus body or treated cell of a microorganism selected from the group of microorganisms belonging to the genus Devariomyces The present invention relates to a method for producing (R) -2-halo-1- (substituted phenyl) ethanol, characterized by collecting phenyl) ethanol.
[0014]
First of the present invention 2 Is the general formula [1]
[0015]
Embedded image
(In the formula, X represents a chlorine atom or a bromine atom, and the substituents R1, R2, and R3 represent a hydrogen atom, a chlorine atom, a fluorine atom, a methyl group, and a methoxy group. However, when all the substituents are hydrogen atoms, 2-halo-1- (substituted phenyl) ethanone represented by
General formula [3]
[0017]
Embedded image
(Wherein X and substituents R 1, R 2 and R 3 are the same as those in the general formula [1].) Having the ability to produce (S) -2-halo-1- (substituted phenyl) ethanol, (S) − represented by the general formula [3] generated by acting on a culture solution, fungus body or treated product of a microorganism selected from the group of microorganisms belonging to the genus Pseudomonas, Brebandimonas, and Cryptococcus The present invention relates to a method for producing (S) -2-halo-1- (substituted phenyl) ethanol, which comprises collecting 2-halo-1- (substituted phenyl) ethanol.
[0025]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
A microorganism that asymmetrically reduces 2-halo-1- (substituted phenyl) ethanone used in the present invention and converts it into 2-halo-1- (substituted phenyl) ethanol can be found by the method described below. For example, A composed of 5% glucose, 0.5% peptone, 0.2% potassium dihydrogen phosphate, 0.1% dipotassium hydrogen phosphate, 0.02% magnesium sulfate, and 0.1% yeast extract. 50 ml of medium (pH 6.5) is placed in a 500 ml Sakaguchi flask, sterilized, and then microorganisms are planted and shaken at 30 ° C. for 2 to 3 days. Thereafter, the cells were collected by centrifugation and suspended in 25 ml of a phosphate buffer containing 0.1-0.5% 2-chloro-1- (3′-chlorophenyl) ethanone and 5% glucose, and a 500 ml Sakaguchi flask. Shake at 30 ° C for 2-3 days. Under the present circumstances, what dried the microbial cell obtained by centrifugation in the desiccator or acetone can also be used. Furthermore, oxidized nicotinamide adenine dinucleotide (NAD) and / or oxidized nicotinamide adenine dinucleotide phosphate when reacting these microorganisms or treated products thereof with 2-chloro-1- (3′-chlorophenyl) ethanone (NADP) and glucose dehydrogenase may be added. After the conversion reaction, 2-chloro-1- (3′-chlorophenyl) ethanol, which is extracted by adding the same volume of ethyl acetate as the reaction solution, is extracted with high performance liquid chromatography (column: Chiralcel OJ, manufactured by Daicel Chemical Industries, Ltd. Eluent: n-hexane / isopropanol = 39/1, flow rate: 1 ml / min, detection: 210 nm, column temperature: room temperature, elution time 2-chloro-1- (3′-chlorophenyl) ethanone: 28 minutes, (R) 2-chloro-1- (3′-chlorophenyl) ethanol: 37 minutes, (S) -2-chloro-1- (3′-chlorophenyl) ethanol: 45 minutes).
As a microorganism that can be used in the present invention, Ogata Air is a microorganism having the ability to asymmetrically reduce 2-halo-1- (substituted phenyl) ethanone and convert it into optically active 2-halo-1- (substituted phenyl) ethanol. Microorganisms belonging to the genus, Debariomyces, Candida, Yamadazima, Pseudomonas, Brevandimonas, Cryptococcus can be used.Specifically, for example, Ogataea minuta var.minuta (Ogataea minuta var.minuta ) IFO 0975, Ogataea minuta var.nonfermentans IFO 1473, Debaryomyces carsonii IFO 0946, Candida parapsilosis IFO 0585, Candida ceiranoides Candida zeylanoides) IFO 0719, Yamadazima Farinosa (Yamadazyma farinosa) IFO 0459, Yamadazyma farinosa IFO 0193, Pseudomonas putida IFO 12966, Brevundimonas diminuta IFO 12697, Cryptococcus fumicocus 760 -Cumiptococcus humicolus JCM1460 etc. can be used. These microorganisms can generally be obtained from stocks that are readily available or purchased. It can also be separated from nature. It is also possible to obtain a strain having properties more advantageous for this reaction by causing mutation in these microorganisms.
For culturing these microorganisms, any nutrient source that can be assimilated by these microorganisms can be used. For example, sugars such as glucose, sucrose and maltose, organic acids such as lactic acid, acetic acid, citric acid and propionic acid, alcohols such as ethanol and glycerin, hydrocarbons such as paraffin, fats and oils such as soybean oil and rapeseed oil, Alternatively, a carbon source such as a mixture thereof, or a nitrogen source such as ammonium sulfate, ammonium phosphate, urea, yeast extract, meat extract, peptone, corn steep liquor or the like can be mixed. Furthermore, other nutrients such as inorganic salts and vitamins can be appropriately mixed.
The culture of microorganisms can be usually performed under general conditions. For example, the microorganism is aerobically cultured for 10 to 96 hours at a pH of 4.0 to 9.5 and a temperature range of 20 ° C to 45 ° C. In the case of reacting a microorganism with 2-halo-1- (substituted phenyl) ethanone, the culture solution of the microorganism can be used for the reaction as it is, but a concentrate of the culture solution can also be used. In addition, when the components in the culture solution adversely affect the reaction, it is preferable to use microbial cells or processed microbial cells obtained by treating the culture solution by centrifugation or the like.
The microbial cell processed product is not particularly limited. For example, a dried microbial cell obtained by dehydration using acetone or diphosphorus pentoxide or drying using a desiccator or a fan, a surfactant-treated product, or a lysed enzyme-treated product. Examples thereof include immobilized cells or cell-free extract preparations obtained by disrupting cells. Furthermore, an enzyme that catalyzes the asymmetric reduction reaction may be purified from the culture and used.
The 2-halo-1- (substituted phenyl) ethanone represented by the general formula [1] is converted into the optically active 2-halo-1- (substituted phenyl) ethanol represented by the general formula [2] or [3]. An enzyme having the activity obtained from a culture solution of a microorganism having ability, a microbial cell or a treated product of the microorganism, or a microorganism having the ability is hereinafter referred to as “enzyme source”.
In addition, if the enzyme obtained from the microorganism has the activity regardless of the strength of the ability as a culture solution, microbial cell or treated microbial product, the enzyme should also be used as the enzyme source. Can do. As such an enzyme, for example, an enzyme that reduces ethyl 4-chloroacetoacetate to give ethyl 4-chloro-3-hydroxybutyrate can be used. The enzyme that catalyzes this reduction reaction is preferably an enzyme obtained from a microorganism belonging to the genus Candida.
The enzyme source can often be obtained from a microorganism. For extraction and purification of enzymes from microbial cultures, enzyme extraction and purification methods commonly used by those skilled in the art can be used. For example, cells are collected from the culture solution by centrifugation, suspended in an appropriate buffer solution, and disrupted or dissolved using physical methods such as glass beads or biochemical methods such as enzymes. Thereafter, the insoluble matter in the solution is removed by centrifugation to obtain a crude enzyme solution. The crude enzyme solution can be further purified by methods usually used by those skilled in the art, for example, ammonium sulfate precipitation, dialysis, or chromatography alone or in combination. When the enzyme fraction in each stage of the enzyme purification operation including such a crude enzyme solution catalyzes the reaction, these can be used as an enzyme source. In addition, an enzyme gene that catalyzes the reaction is isolated, and a recombinant microorganism culture solution, fungus body, or treated cell product obtained by genetic engineering techniques can also be used as an enzyme source. As an enzyme that catalyzes the reaction thus obtained, for example, an enzyme S1 having the following physicochemical properties (1) to (7) obtained from a microorganism belonging to the genus Candida described in PCT / JP97 / 03051 Can be illustrated.
(1) Action:
Using NADPH as a coenzyme, it acts on ethyl 4-chloroacetoacetate to produce ethyl (S) -4-chloro-3-hydroxybutyrate,
(2) Substrate specificity:
Show strong activity against ethyl 4-chloroacetoacetate and substantially no activity against ethyl acetoacetate,
(3) Optimal pH: pH 5.5-6.5,
(4) Optimal temperature: 50-55 ° C.
(5) Molecular weight: about 76000 in gel filtration analysis, about 32000 in SDS polyacrylamide electrophoresis analysis
(6) Thermal stability: stable to about 40 ° C. when treated at pH 7.0 for 30 minutes, and
(7) Inhibitor: Inhibited by mercury ion and quercetin.
[0026]
Further, carbonyl reductase S4 derived from Candida microorganisms having the following physicochemical properties (1) to (5) can also be exemplified.
(1) Action:
Using NADPH as a coenzyme, it acts on ethyl 4-chloroacetoacetate to produce ethyl (S) -4-chloro-3-hydroxybutyrate,
(2) Substrate specificity:
Active against ethyl 4-chloroacetoacetate and strong against ketopantoyl lactone,
(3) Optimal pH: pH 6.0,
(4) Optimal temperature: 50 ° C., and
(5) Molecular weight: about 86000 in gel filtration analysis, about 29000 in SDS polyacrylamide electrophoresis analysis.
In the reduction reaction, the substrate 2-halo-1- (substituted phenyl) ethanone may be added all at once at the beginning of the reaction, or may be added in portions as the reaction proceeds.
Moreover, the temperature at the time of reaction is 10-60 degreeC normally, Preferably it is 20-40 degreeC. The pH during the reaction is 2.5-9, preferably 5-9.
The amount of the enzyme source in the reaction solution may be appropriately determined according to the ability to reduce these substrates. In addition, the substrate concentration in the reaction solution is preferably 0.01 to 50% (W / V), more preferably 0.1 to 30%.
The reaction is usually carried out with shaking or stirring with aeration. The reaction time is appropriately determined depending on the substrate concentration, the amount of enzyme source, and other reaction conditions. Usually, it is preferable to set each condition so that the reaction is completed in 2 to 168 hours.
In order to promote the reduction reaction, it is preferable to add an energy source such as glucose or ethanol to the reaction solution at a ratio of 1 to 30% because excellent results can be obtained. In addition, by adding coenzymes such as reduced nicotinamide / adenine dinucleotide (NADH) and reduced nicotinamide / adenine dinucleotide phosphate (NADPH), which are generally required for reduction reactions by biological methods. The reaction can also be promoted. Specifically, these may be added directly to the reaction solution, or a reaction system that generates NADH and NADPH may be added to the reaction solution together with the oxidized coenzyme. For example, a reaction system that reduces NAD to NADH when formate dehydrogenase generates carbon dioxide and water from formic acid, or NAD or NADP is converted to NADH or glucose when dehydrogenase generates gluconolactone from glucose. A reaction system that reduces each to NADPH can be used. It is also effective to add a surfactant such as Triton (manufactured by Nacalai Tesque), Span (manufactured by Kanto Chemical Co., Ltd.), Tween (manufactured by Nacalai Tesque) to the reaction solution. In addition, an organic solvent insoluble in water such as ethyl acetate, butyl acetate, isopropyl ether, toluene or the like is added to the reaction solution for the purpose of avoiding the inhibition of the reaction by the substrate and / or the alcohol which is the product of the reduction reaction. Also good. Furthermore, for the purpose of increasing the solubility of the substrate, an organic solvent soluble in water such as methanol, ethanol, acetone, tetrahydrofuran, dimethyl sulfoxide and the like can be added.
Collection of optically active 2-halo-1- (substituted phenyl) ethanol produced by the reduction reaction can be carried out directly from the reaction solution or after separation of the cells, etc., followed by extraction with a solvent such as ethyl acetate or n-hexane, and dehydration Thereafter, if it is purified by distillation or silica gel column chromatography, highly pure optically active 2-halo-1- (substituted phenyl) ethanol can be easily obtained.
[0027]
The optically active 2-halo-1- (substituted phenyl) ethanol obtained as described above easily closes the ring by coexisting an equimolar amount of alkali such as caustic soda and left standing at room temperature. Can be converted to substituted styrene oxide.
[0028]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited at all by these Examples. In the following description, “%” means “% by weight” unless otherwise specified.
Example 1
50 ml of the A medium was put into a 500 ml Sakaguchi flask and sterilized, and then the microorganisms shown in Table 1 were inoculated. Then, shaking culture was performed aerobically at 30 ° C. for 2 days. Bacteria were collected from the culture by centrifugation, 2-chloro-1- (3′-chlorophenyl) ethanone 1%, oxidized nicotinamide adenine dinucleotide (NAD) 0.06%, oxidized nicotinamide adenine dinucleotide. 50 mM phosphate buffer (pH 6.) containing 0.06% phosphate (NADP), glucose 5%, glucose dehydrogenase (trade name: GLUCDH “Amano” II, Amano Pharmaceutical Co., Ltd.) 14.3 U / ml 0) Suspended in 20 ml, placed in a 500 ml Sakaguchi flask and allowed to react with shaking at 30 ° C. for 24 hours. After the reaction, optically active 2-chloro-1- (3′-chlorophenyl) ethanol was extracted twice from the reaction solution with the same volume of ethyl acetate, and the ethyl acetate layer was analyzed by high performance liquid chromatography. Purity was measured. The results are shown in Table 1.
[0029]
[Table 1]
Example 2
About microorganisms shown in Table 2, glucose 1%, peptone 1.5%, dipotassium hydrogen phosphate 0.3%, magnesium sulfate 0.02%, sodium chloride 0.2%, yeast extract 0.1% The reaction rate and optical purity were measured in the same manner as in Example 1 except that B medium (pH 7.0) was used. The results are shown in Table 2.
[0031]
[Table 2]
Example 3
Ogataea minuta var. Nonfermentans IFO 1473 was inoculated into 20 500 ml Sakaguchi flasks containing 50 ml of medium A, and cultured with shaking at 30 ° C. for 48 hours. After incubation, the cells were collected by centrifugation, and this was collected with 50 mM phosphate buffer (containing 40 g glucose, 240 mg NADP, 240 mg NAD, 80 mg glucose dehydrogenase (trade name: GLUCDH “Amano” II, Amano Pharmaceutical Co., Ltd.)) pH 6.0) was added to make the total volume 800 ml. Furthermore, 1.6 g of the substrate 2-chloro-1- (3′-chlorophenyl) ethanone was added to initiate the reduction reaction. The reaction was carried out at 30 ° C. with stirring while adjusting the pH of the reaction solution to 6.0 with a 2M aqueous sodium carbonate solution. A part of the reaction solution was periodically analyzed by HPLC. When the substrate was depleted, 1.6 g of the substrate was further added to continue the reaction. The reaction was carried out for about 50 hours while repeating this operation. According to the result of HPLC analysis at the end of the reaction, the concentration of 2-chloro-1- (3′-chlorophenyl) ethanol in the reaction solution was 9.8 mg / ml. After completion of the reaction, extraction was performed twice with 1000 ml of ethyl acetate. The ethyl acetate layer was dried over anhydrous sodium sulfate and then desolvated under reduced pressure to obtain an oily substance. This was distilled under reduced pressure (130 ° C./3 mmHg) to obtain 8.8 g of colorless oily (R) -2-chloro-1- (3′-chlorophenyl) ethanol. The specific rotation was [α] (20, D) = − 33.6 ° (c = 1.02), and the optical purity was 99.3% ee according to HPLC analysis. H-NMR (CDCl Three ) Δppm 2.69 (br, s, 1H), 3.27 to 3.90 (m, 2H), 4.88 (dd, 1H), 7.15 to 7.54 (m, 4H)
Example 4
Yamadazyma farinosa IFO 0459 was inoculated into 20 500 ml Sakaguchi flasks containing 50 ml of medium A, and cultured with shaking at 30 ° C. for 48 hours. After incubation, the cells were collected by centrifugation, and this was collected with 50 mM phosphate buffer (containing 40 g glucose, 240 mg NADP, 240 mg NAD, 80 mg glucose dehydrogenase (trade name: GLUCDH “Amano” II, Amano Pharmaceutical Co., Ltd.)) pH 6.0) was added to make the total volume 800 ml. Furthermore, 1.6 g of the substrate 2-chloro-1- (3′-chlorophenyl) ethanone was added to initiate the reduction reaction. The reduction reaction was carried out for about 50 hours in the same manner as in Example 3. According to the result of HPLC analysis at the end of the reaction, the concentration of 2-chloro-1- (3′-chlorophenyl) ethanol in the reaction solution was 11.8 mg / ml. After completion of the reaction, extraction was performed twice with 1000 ml of ethyl acetate. The ethyl acetate layer was dried over anhydrous sodium sulfate and then desolvated under reduced pressure to obtain an oily substance. This was distilled under reduced pressure (130 ° C./3 mmHg) to obtain 10.6 g of colorless oily (S) -2-chloro-1- (3′-chlorophenyl) ethanol. The specific rotation was [α] (20, D) = 33.5 ° (c = 1.02), and the optical purity was 99.1% ee according to HPLC analysis. H-NMR (CDCl Three ) Δppm 2.69 (br, s, 1H), 3.27 to 3.90 (m, 2H), 4.88 (dd, 1H), 7.15 to 7.54 (m, 4H)
Example 5
Brevundimonas diminuta IFO 12697 was inoculated into 20 500 ml Sakaguchi flasks containing 50 ml of B medium and cultured with shaking at 30 ° C. for 24 hours. After incubation, the cells were collected by centrifugation, and this was collected with 50 mM phosphate buffer (containing 40 g glucose, 240 mg NADP, 240 mg NAD, 80 mg glucose dehydrogenase (trade name: GLUCDH “Amano” II, Amano Pharmaceutical Co., Ltd.)) pH 6.0) was added to make the total volume 800 ml. Furthermore, 1.6 g of the substrate 2-chloro-1- (3′-chlorophenyl) ethanone was added to initiate the reduction reaction. The reduction reaction was carried out for about 50 hours in the same manner as in Example 3. According to the results of HPLC analysis at the end of the reaction, the concentration of (S) -2-chloro-1- (3′-chlorophenyl) ethanol in the reaction solution was 18.4 mg / ml, and the optical purity was 84.2% ee. there were.
Example 6
8000 ml of A medium was prepared, 400 ml was dispensed into a 2000 ml Sakaguchi flask, and steam sterilized at 120 ° C. for 20 minutes. Inoculate 5 ml each of Ogataea minuta var. Nonfermentans IFO 1473 culture medium pre-cultured in the same medium and shake at 30 ° C. for 2 days. Cultured. Bacteria were collected from this culture solution by centrifugation and washed twice with physiological saline. The obtained wet cells were suspended in 400 ml of 50 mM phosphate buffer (pH 7.0), and then the cells were crushed with Dynomill (Dyno-Mill). The cell residue was removed from the crushed cell by centrifugation to obtain 700 ml of a cell-free extract. In a large test bowl, 2-chloro-1- (3′-chlorophenyl) ethanone 100 mg, NADP 0.6 mg, glucose 500 mg, glucose dehydrogenase (trade name: GLUCDH “Amano” II, Amano Pharmaceutical Co., Ltd.) 143U are weighed. 10 ml of the prepared cell-free extract was added thereto, and the mixture was shaken at 30 ° C. for 24 hours. After the reaction, the reaction solution was extracted twice with 10 ml of ethyl acetate, and the obtained organic layer was analyzed by the method shown in Example 1. As a result, the reaction to 2-chloro-1- (3′-chlorophenyl) ethanol was performed. The ratio was 60%, the optical purity was 99.3%, and the configuration was (R).
Reference Example 1 Preparation of enzyme S1 from Candida microorganisms
The enzyme activity was measured by adding the substrate 4-chloroacetoacetate 0.2 mM, coenzyme NADPH 0.32 mM and enzyme to 200 mM phosphate buffer (pH 7.0), and measuring the decrease in absorbance at a wavelength of 340 nm at 30 ° C. To do so. Under this reaction condition, the enzyme activity that oxidizes 1 μmol of NADPH to NADP per minute is defined as 1 unit. The optical purity of ethyl 4-chloro-3-hydroxybutyrate produced by the enzyme reaction is measured by HPLC analysis using an optical resolution column CHIRALCEL OB (manufactured by Daicel Chemical Industries, Ltd.) (mobile phase; hexane / isopropanol). = 9/1, flow rate; 0.8 ml / ml, column temperature; 0 ° C., detection; 215 nm).
[0033]
8000 ml of a liquid medium having the following composition was prepared, dispensed 400 ml each into a 2000 ml Sakaguchi flask, and steam sterilized at 120 ° C. for 20 minutes.
Medium composition;
Glucose 5%
Polypeptone 0.5%
KH 2 PO Four 0.2%
K 2 HPO Four 0.1%
MgSO Four / 7H 2 O 0.02%
Tap water
pH 6.5
Each of these media was inoculated with 5 ml of a Candida magnoliae IFO 0705 culture solution pre-cultured in the same medium, and cultured with shaking at 30 ° C. for 3 days. Bacteria were collected from this culture solution by centrifugation and washed twice with physiological saline to obtain 230 g of wet cells. Of these, 180 g was suspended in 360 ml of 50 mM phosphate buffer (pH 7.0), and the cells were disrupted with Dynomill (Dyno-Mill). This cell disruption was centrifuged to remove the residue, and 760 ml of cell-free extract was obtained. Ammonium sulfate was added to this cell-free extract so as to be 40% saturation, the resulting precipitate was removed by centrifugation, and the supernatant was added to 10 mM phosphate buffer (pH 7.0) containing 0.1% DTT. And dialyzed overnight. This was applied to a DEAE sephacel (Pharmacia Biotech) column pre-equilibrated with the same buffer, and the column was washed with the same buffer. The active fraction was recovered from the passing solution, and NaCl was added to a final concentration of 4M. This active fraction was applied to a Phenyl sepharose CL-4B (Pharmacia Biotech) column pre-equilibrated with 10 mM phosphate buffer (pH 7.0) containing 4 M NaCl and 0.1 mM DTT. Adsorbed. After washing the column with the same buffer, a linear gradient of NaCl (4M to 0M) and ethylene glycol (0% to 50% (W / V)) using 10 mM phosphate buffer (pH 7.0) The active fraction was eluted. This was dialyzed overnight against 10 mM phosphate buffer (pH 7.0).
This dialysate was applied to a Mono Q HR5 / 5 (Pharmacia Biotech) column that had been equilibrated in advance with 10 mM phosphate buffer (pH 7.0) containing 0.1 mM DTT, and washed with the same buffer. The active fractions in the washing solution were collected, concentrated by ultrafiltration, and Superdex 200 HR10 / 30 (equilibrated in advance with 10 mM phosphate buffer (pH 7.0) containing 0.2 M NaCl and 0.1 mM DTT). (The column was manufactured by Pharmacia Biotech) and eluted with the same buffer. The active fraction was collected and used as a purified enzyme preparation.
Reference Example 2 Enzymatic properties of enzyme S1 obtained from Candida microorganisms
The enzyme activity was measured basically by 3.0 ml containing 200 mM phosphate buffer (pH 7.0), substrate 4-chloroacetoacetate 0.2 mM, coenzyme NADPH 0.32 mM, and enzyme solution 0.1 ml. In the reaction solution at 30 ° C. for 1 minute, and the decrease in absorbance at a wavelength of 340 nm was measured.
(1) Action:
NADPH was used as a coenzyme to act on ethyl 4-chloroacetoacetate to produce ethyl (S) -4-chloro-3-hydroxybutyrate having an optical purity of 99% ee or higher.
(2) Substrate specificity:
As a result of carrying out the reaction under the same conditions as in ethyl 4-chloroacetoacetate using various carbonyl compounds shown in Table 3 as substrates, the enzyme showed substrate specificity as shown in Table 3.
[0034]
[Table 3]
(3) Optimum pH:
The enzyme activity was measured using 4-chloroacetoacetate as a substrate in the pH range of 5.0 to 8.5 using a phosphate buffer and Tris-HCl buffer as the buffer. As a result, the optimum pH was 5.5 to 6.5.
(4) Optimum temperature of action:
The activity was measured using ethyl 4-chloroacetoacetate as a substrate for 1 minute at a temperature of 20 to 60 ° C. As a result, the optimum temperature was 50 to 55 ° C.
(5) Thermal stability:
After the enzyme was heated at 40 ° C. for 30 minutes at pH 7.0, the activity was measured when ethyl 4-chloroacetoacetate was used as a substrate. As a result, 90% of the activity before the heat treatment remained.
(6) Inhibitor:
Various compounds having concentrations shown in Table 4 were added to the reaction solution, and the activity when ethyl 4-chloroacetoacetate was used as a substrate was measured. As shown in Table 4, this enzyme was inhibited by quercetin and mercury ions.
[0036]
[Table 4]
(7) Molecular weight:
The molecular weight of the enzyme was measured using a TSK-G3000W column with 0.1M Na as the eluent. 2 SO Four And 0.05% NaN Three 100 mM phosphate buffer solution (pH 7.0) was used. The molecular weight in this case was about 76000. The molecular weight of the subunit of the enzyme was calculated from the relative mobility of the standard protein by 10% SDS-polyacrylamide gel electrophoresis analysis in the presence of 2% 2-mercaptoethanol. As a result, the molecular weight of the subunit of this enzyme was about 32000.
(8) Organic solvent resistance:
The same volume of ethyl acetate or butyl acetate was added to a pH 7.0 phosphate buffer in which this enzyme was dissolved, and the mixture was shaken at 28 ° C. for 30 minutes, and then the aqueous phase and the organic phase were separated by centrifugation. The remaining enzyme activity in the aqueous phase was measured using ethyl 4-chloroacetoacetate as a substrate. As a result, 72% of the remaining enzyme activity was observed when ethyl acetate was used, and 85% when butyl acetate was used.
Example 7 Preparation of enzyme S4 from Candida microorganisms
60 g of polyethylene glycol was added to 1100 ml of the cell-free extract obtained by the same operation as in Reference Example 1 with stirring, and the mixture was allowed to stand for about 2 hours after the addition. Thereafter, the supernatant obtained by centrifugation was dialyzed against 10 mM phosphate buffer (pH 7.0) containing 0.1 mM DTT and applied to a DEAE sephacel (Pharmacia Biotech) column equilibrated with the same buffer. Next, the column was washed with the same buffer, and the active fraction was eluted with a linear gradient of NaCl from 0 M to 0.2 M. This fraction was dialyzed against the same buffer and then subjected to Butyl toyopearl 650 (manufactured by Tosoh Corporation) equilibrated with the same buffer containing 2M ammonium sulfate. The active fraction was eluted with the same buffer containing no ammonium sulfate, desalted and concentrated by ultrafiltration. This enzyme solution was applied to a Mono Q HR 10/10 (Pharmacia Biotech) column equilibrated with the same buffer. The active fraction was eluted with 0.25 M NaCl and dialyzed against the same buffer. This was subjected to Alkyl Superose HR 10/10 (Pharmacia Biotech) equilibrated with the same buffer containing 2M ammonium sulfate. When eluted with a linear gradient with ethylene glycol and ammonium sulfate, the desired activity was found in the former fractions of 0% to 10% and the latter 2M to 0M, and this was used as a purified enzyme preparation.
Example 8 Enzymatic chemistry of enzyme S4 obtained from Candida microorganisms
Various properties of enzyme S4 were examined by the same operation as in Reference Example 2.
(1) Action:
Using NADPH as a coenzyme, it acts on ethyl 4-chloroacetoacetate to produce ethyl (S) -4-chloro-3-hydroxybutyrate having an optical purity of 51% ee.
(2) Substrate specificity:
As a result of performing the reaction under the same conditions as for ethyl 4-chloroacetoacetate using various carbonyl compounds shown in Table 5 as substrates, the substrate specificity as shown in Table 5 was shown.
[0038]
[Table 5]
(3) Optimum pH:
The enzyme activity was measured by the method of Reference Example 2 in the range of pH 5.0 to pH 8.5 using phosphate buffer and Tris-HCl buffer as buffer. As a result, the optimum pH acting on ethyl 4-chloroacetoacetate was 6.0.
(4) Optimal temperature:
It was 50 degreeC when the activity of the enzyme when using 4-chloroacetoacetic acid ethyl as a substrate for 1 minute at the temperature of 20 to 60 degreeC was calculated | required, and the optimal temperature was calculated | required.
(5) Molecular weight:
The molecular weight of the enzyme was measured by the method of Reference Example 2, and calculated to be about 86000 by gel filtration analysis and about 29000 by SDS polyacrylamide gel electrophoresis analysis.
Example 9
5 units of enzyme S1 purified in Reference Example 1, 25 mg of 2-chloro-1- (3′-chlorophenyl) ethanone, 0.16 mg of NADP, 28 mg of glucose, glucose dehydrogenase (trade name: GLUCDDH “Amano” II, Amano Pharmaceutical Co., Ltd.) 2.5 ml of 100 mM phosphate buffer solution (pH 6.5) containing 6 units was stirred at 30 ° C. for 24 hours. The reaction solution was extracted with ethyl acetate, the solvent was removed, and the extract was analyzed. (S) -2-chloro-1- (3'-chlorophenyl) ethanol having a reaction rate of 80% and an optical purity of 99% ee or higher. Was generated.
Example 10
Using the enzyme S4 purified in Example 7, the same operation as in Example 9 was performed, and the reaction product was analyzed. As a result, (R) -2-chloro-1- with a reaction rate of 50% and an optical purity of 91% ee was obtained. (3'-Chlorophenyl) ethanol was produced.
Example 11
50 ml of medium A was placed in a 500 ml Sakaguchi flask and sterilized, and then the microorganisms shown in Table 6 were inoculated. The culture was aerobically shaken at 30 ° C. for 2 days. Bacteria were collected from this culture by centrifugation, and 0.5% 2-bromo-1- (3′-chlorophenyl) ethanone was suspended in 25 ml of 100 mM phosphate buffer (pH 7.0) containing 0.3% glucose. In a 500 ml Sakaguchi flask, the reaction was shaken at 30 ° C. for 48 hours. After the reaction, 2-bromo-1- (3′-chlorophenyl) ethanol was extracted from the reaction solution with an equal amount of ethyl acetate, and the ethyl acetate layer was analyzed by gas chromatography to examine the reaction rate. Further, the optical purity was measured by the same method as in Example 1. These results are shown in Table 6.
[0040]
[Table 6]
Example 12
Cryptococcus humicolus IFO0760 was inoculated into a 5 L minijar fermenter containing 3 L of A medium, and cultured at 30 ° C., aeration 1 vvm, and stirring at 500 rpm for 24 hours. After completion, the cells were collected by centrifugation, suspended in 750 ml of water, 7.5 g of 2-bromo-1- (3′-chlorophenyl) ethanone and 38 g of glucose were added, and the pH was adjusted to 7.0 with 1N aqueous sodium hydroxide solution. The mixture was allowed to react at 30 ° C. with stirring at 150 rpm for 24 hours. After the reaction, the mixture was extracted twice with 750 ml of ethyl acetate, dehydrated, and desolvated under reduced pressure to obtain an oily substance. This was distilled (130 ° C./3 mmHg) to obtain 2.5 g of colorless oily (S) -2-bromo-1- (3′-chlorophenyl) ethanol. The specific rotation was [α] (20, D) 24.5 (c = 1, methanol), and the optical purity by HPLC was 97% ee.
1H-NMR δppm 2.88 (br, 1H), 3.35 to 3.90 (m, 4H), 4.90 (dd, 2H), 6.98 to 7.51 (m, 4H)
Reference example 3
2 g of (R) -2-chloro-1- (3′-chlorophenyl) ethanol obtained in Example 3 was mixed with an equimolar equivalent of 40% aqueous sodium hydroxide solution and 10 ml of methylene chloride, and reacted at 50 ° C. for 6 hours. After cooling, 10 ml of methylene chloride was added, and the organic layer was washed with saturated brine, dehydrated and filtered, and then methylene chloride was removed from the solvent to obtain a crude epoxide oil. This was purified by distillation under reduced pressure (80 ° C., 4 mmHg) to obtain 1 g of colorless oily (R) -3′-chlorostyrene oxide. The specific rotation was [α] (20, D) = − 67.5 (c = 1).
1H-NMR (CDCl Three ) δppm 2.63 (dd, 1H), 3.15 (dd, 1H), 4.17 (dd, 1H), 7.01 to 7.51 (m, 4H)
[0042]
【The invention's effect】
According to the present invention, optically active 2-halo-1- (substituted phenyl) ethanol can be efficiently produced on an industrial scale. The obtained optically active 2-halo-1- (substituted phenyl) ethanol can be easily led to an optically active substituted styrene oxide useful as a synthetic raw material for pharmaceuticals and the like.
Claims (4)
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| ATE416255T1 (en) * | 2002-04-30 | 2008-12-15 | Kaneka Corp | CARBONYL REDUCTASE, ITS GENE AND USE |
| AU2003264502A1 (en) | 2002-09-19 | 2004-04-08 | Kaneka Corporation | Novel carbonyl reductase, gene thereof and method of using the same |
| DE10248277A1 (en) * | 2002-10-16 | 2004-05-06 | X-Zyme Gmbh | Preparation of optically active 1-hydroxy-2-alkylaminoethane compounds, useful as bronchodilators for treating e.g. asthma, by reacting an amine with reactive 2-substituted ethanol, also new intermediates |
| JP4736654B2 (en) * | 2005-09-09 | 2011-07-27 | 住友化学株式会社 | Reductase gene and its use |
| JP4772434B2 (en) * | 2005-09-09 | 2011-09-14 | 住友化学株式会社 | Process for producing optically active 2-hydroxy-5- (4-methoxyphenyl) -pentanoic acid ester |
| WO2007099764A1 (en) * | 2006-02-28 | 2007-09-07 | Kaneka Corporation | Novel carbonyl reductase, gene for the reductase, and method for production of optically active alcohol using the reductase or the gene |
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