JP3602162B2 - Method for producing 3-hydroxybutyrate dehydrogenase - Google Patents
Method for producing 3-hydroxybutyrate dehydrogenase Download PDFInfo
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- JP3602162B2 JP3602162B2 JP18104794A JP18104794A JP3602162B2 JP 3602162 B2 JP3602162 B2 JP 3602162B2 JP 18104794 A JP18104794 A JP 18104794A JP 18104794 A JP18104794 A JP 18104794A JP 3602162 B2 JP3602162 B2 JP 3602162B2
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- hydroxybutyrate dehydrogenase
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- hydroxybutyrate
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Description
【０００１】
【産業上の利用分野】
本発明はアルカリゲネス（Ａｌｃａｌｉｇｅｎｅｓ）属に属する３−ヒドロキシ酪酸脱水素酵素（３−ｈｙｄｒｏｘｙｂｕｔｙｒａｔｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ）生産菌を培養し、その培養物から３−ヒドロキシ酪酸脱水素酵素を採取してなる３−ヒドロキシ酪酸脱水素酵素の製造法に関する。
【０００２】
【従来技術及び課題】
３−ヒドロキシ酪酸脱水素酵素（ＥＣ．１．１．１．３０）は、ＮＡＤの存在下３ーヒドロキシ酪酸に作用して１モルのＮＡＤを消費し、１モルのアセト酢酸及び、１モルの還元型ＮＡＤに変換する触媒作用を示す酵素として知られている。
【０００３】
これまで３−ヒドロキシ酪酸脱水素酵素について、動物由来のものとしては例えばラット脳（Ｂｉｏｃｈｅｍ．Ｃｅｌｌ Ｂｉｏｌ．，６８，９８０−９８３（１９９０））、ラット肝臓（Ｂｉｏｃｈｅｍ．Ｃｅｌｌ Ｂｉｏｌ．，６８，１２２５−１２３０（１９９０））、ウシ心臓（Ａｒｃｈ．Ｂｉｏｃｈｅｍ．Ｂｉｏｐｈｙｓ．，２６２，８５−９８（１９８８））が報告されている。
【０００４】
また、微生物由来のものとしてはロードスピリラム・ルブラム（Ｒｈｏｄｏｓｐｉｒｉｌｌｕｍ ｒｕｂｒｕｍ）（Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．２３７，６０３−６０７（１９６２））、シュードモナス・レモイグネイ（Ｐｓｅｕｄｏｍｏｎａｓ ｌｅｍｏｉｇｎｅｉ）（Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．２４０，４０２３−４０２９（１９６５））、マイコバクテリウム・フレイ（Ｍｙｃｏｂａｃｔｅｒｉｕｍ ｐｈｌｅｉ）（Ｊ．Ｇｅｎ．Ｍｉｃｒｏｂｉｏｌ．１０４，１２３−１２６（１９７８））、パラコッカス・デニトリフィカンス（Ｐａｒａｃｏｃｃｕｓｄｅｎｉｔｒｉｆｉｃａｎｓ）（Ｂｉｏｃｈｉｍ．Ｂｉｏｐｈｙｓ．Ａｃｔａ８３９，３００−３０７（１９８５））、ズーグロエア・ラミゲラ（Ｚｏｏｇｌｏｅａ ｒａｍｉｇｅｒａ）（Ｊ．Ｂｉｏｃｈｅｍ．８９，６２５−６３５（１９８１））、ロードシュードモナス・スフェロイデス（Ｒｈｏｄｏｐｓｅｕｄｏｍｏｎａｓ ｓｐｈｅｒｏｉｄｅｓ）（Ｂｉｏｃｈｅｍ．Ｊ．２４１，２９７−３００（１９８７））、アゾスピリルム・ブラジレンズ（Ａｚｏｓｐｉｒｉｌｌｕｍ ｂｒａｓｉｌｅｎｓｅ）（Ｊ．Ｇｅｎ．Ｍｉｃｒｏｂｉｏｌ．１３６，６４５−６４９（１９９０））等が知られている。
【０００５】
【発明が解決しようとする課題】
しかしながら、これらの菌株以外の３−ヒドロキシ酪酸脱水素酵素生産能を有する菌株による該酵素の製造法の開発が望まれていた。
【０００６】
【課題を解決するための手段】
本発明者らは、３−ヒドロキシ酪酸脱水素酵素の工業的生産の可能な製造法を求めて鋭意研究を重ねた結果、奈良県五条市のジャガイモ畑の土壌から単離したアルカリゲネス属菌ＮＯ．９８１菌株が３−ヒドロキシ酪酸脱水素酵素生産能を有することを初めて見いだし、本発明を完成した。
【０００７】
本発明は上記の知見に基づいて完成されたもので、アルカリゲネス属に属する３−ヒドロキシ酪酸脱水素酵素生産菌を培養し、その培養物から３−ヒドロキシ酪酸脱水素酵素を採取することを特徴とする３−ヒドロキシ酪酸脱水素酵素の製造法である。
以下に本発明について詳細に説明する。
【０００８】
まず、本発明の３−ヒドロキシ酪酸脱水素酵素生産菌について、アルカリゲネス属に属する３−ヒドロキシ酪酸脱水素酵素を生産する能力を有する微生物であれば何ら限定されるものではなく、３−ヒドロキシ酪酸脱水素酵素生産能を有する変種や変異株であってもよく、好ましくはアルカリゲネス属に属するＮＯ．９８１株が挙げられ、本菌株は工業技術院技術研究所（現；工業技術院生命工学技術研究所）に寄託番号微工研条寄第２５７０号（ＦＥＲＭ ＢＰ−２５７０）として寄託したものである。なお本菌株の菌学的性質、同定及び命名については特開平３−１２７９８５号公報、米国特許第５１７３４１６明細書に詳細に記載されている。
【０００９】
本発明を実施するにあたり、その培養形態としては液体培養、個体培養いずれも可能であるが工業的には通気撹拌培養を行うのが有利である。また使用する培養源としては一般に微生物培養に用いられる炭素源、窒素源、無機塩及びその他の微量栄養源の他、アルカリゲネス属に属する微生物の利用できる栄養源であればすべて使用できる。
【００１０】
炭素源としてはグルコース、フルクトース、サッカロース、キシロース、マルトース、グリセロール、デキストリン、でんぷん、アミノ酸等の他、脂肪酸、油脂、有機酸などが単独でまたは組み合わせて用いられる。窒素源としては無機窒素源、有機窒素源のいずれも使用可能であり、無機栄養源としては硫酸アンモニウム、硝酸アンモニウム、尿素、硝酸ソーダ、塩化アンモニウム等が挙げられる。また有機窒素源としては大豆、米、トウモロコシ、小麦等の粉、コーンスティープリカー、ペプトン、肉エキス、カゼイン、アミノ酸、酵母エキス等が挙げられる。無機塩及び微量栄養素としてはリン酸、マグネシュウム、カリウム、鉄、カルシウム、亜鉛等の塩類の他ビタミン、非イオン性界面活性剤、消泡剤等の菌の生育や３−ヒドロキシ酪酸脱水素酵素の生産を促進するものであれば必要に応じて使用できる。
【００１１】
培養は好気的条件で、培養温度は菌が発育し、３−ヒドロキシ酪酸脱水素酵素が産生する範囲であればよく、通常１５℃〜３７℃、好ましくは２５゜Ｃ〜３５゜Ｃである。培養時間は条件により異なるが３−ヒドロキシ酪酸脱水素酵素が最も産生される時間まで培養すればよく、通常２４〜１００時間程度である。
３−ヒドロキシ酪酸脱水素酵素は主としてその菌体内に含有、蓄積されており、その菌体内から抽出すればよい。３−ヒドロキシ酪酸脱水素酵素の抽出法を例示すればまず培養物を固液分離し、得られた湿潤菌体をリン酸緩衝液やトリス−塩酸緩衝液などの溶液に分散し、リゾチーム処理、超音波処理、フレンチプレス処理、ダイノミル処理などの種種の菌体処理手段を適宜選択組み合わせて、粗製の３−ヒドロキシ酪酸脱水素酵素含有液を得る。
【００１２】
粗製の３−ヒドロキシ酪酸脱水素酵素含有液から公知のタンパク質や酵素などの単離、精製手段を用いて精製３−ヒドロキシ酪酸脱水素酵素を得る。例えば、粗製の３−ヒドロキシ酪酸脱水素酵素含有液にアセトン、メタノール、エタノールなどの有機溶媒による分別沈澱法、硫安、食塩などによる塩析法などを適用して３−ヒドロキシ酪酸脱水素酵素を沈澱させ、回収する。さらに、この沈澱物を必要に応じ透析、等電点沈澱を行った後、電気泳動法などで単一の帯を示すまで、イオン交換体、ゲル濾過剤、吸着体などを用いるカラムクロマトグラフィーなどにより精製する。また、これらの方法を適当に組み合わせることにより３−ヒドロキシ酪酸脱水素酵素の精製度が上がる場合は適宜組み合わせて行うことができる。
【００１３】
これらの方法によって得られる酵素は安定化剤として、各種の塩類、糖類、タンパク質、脂質、界面活性剤などを加えあるいは加える事なく、限外濾過濃縮、凍結乾燥の方法により、液状または固形の３−ヒドロキシ酪酸脱水素酵素を得ることができ、また、適宜凍結乾燥を行ってもよく、この場合安定化剤としてサッカロース、マンニトール、食塩、アルブミンなどを０．５〜１０％程度添加してもよい。
【００１４】
つぎに本発明で得られる３−ヒドロキシ酪酸脱水素酵素の理化学的性質及び酵素活性測定法を述べる。
３−ヒドロキシ酪酸脱水素酵素の活性測定法
０．２Ｍのトリス−塩酸緩衝液（ｐＨ８．５）０．２ｍｌ、５０ｍＭの３−ヒドロキシ酪酸０．１ｍｌ、１０ｍＭのＮＡＤ０．１ｍｌ、１００Ｕ／ｍｌのジアホラーゼ０．０５ｍｌ、０．２５％のＮＴＢ（ニトロテトラゾニウムブルー）０．０２ｍｌ、１０％のトリトンＸ−１００を０．０１ｍｌ、及び蒸留水０．５２ｍｌよりなる反応液１ｍｌを３７゜Ｃで１分間予備加温した後、２０μｌの酵素液を添加して１０分間反応させる。反応後、０．１Ｎの塩酸を添加して反応を停止させ、５分以内に層長１．０ｃｍセルを用いて波長５５０ｎｍにおける吸光度を測定する（Ａｓ）。また盲検として酵素液のかわりに蒸留水２０μｌを用いて同一の操作を行って吸光度を測定する（Ａｂ）、この酵素使用の吸光度（Ａｓ）と盲検の吸光度（Ａｂ）の吸光度差（Ａｓ−Ａｂ）より酵素活性を求める。酵素活性１単位は３７゜Ｃで１分間に１μモルの還元型ＮＡＤを生成させる酵素量とし、計算式は下記の通りである。
【００１５】
【数１】
理化学的性質
（１）酵素作用：基質として３−ヒドロキシ酪酸を用いた酵素作用を以下に示す。
【００１７】
【化１】
（２）基質特異性：３−ヒドロキシ酪酸に基質特異性を示す。各種基質に対する特異性は表１の通りである。
【００１９】
【表１】
（３）Ｋｍ 値：１．６±０．５（ｍＭ）（３−ヒドロキシ酪酸に対して）
０．１２±０．０２（ｍＭ）（ＮＡＤに対して）
４）等電点：５．０±０．２（キャリアー−アンホラインを用いた電気泳動法にて）
（５）分子量：６００００±５０００（ＴＳＫ Ｇ−３０００ＳＷによるゲル濾過法にて）、３００００±５０００（ＳＤＳポリアクリルアミドゲル電気泳動法にて）
（６）至適ｐＨ
前記酵素活性測定法にしたがって至適ｐＨを求めたもので、その結果を図１に示した。ｐＨ５．０〜６．０の範囲は１００ｍＭ酢酸緩衝液、ｐＨ６．０〜７．５の範囲は１００ｍＭリン酸緩衝液、ｐＨ７．５〜９．０の範囲は１００ｍＭトリス−塩酸緩衝液、ｐＨ９．０〜１１．０の範囲は１００ｍＭグリシン−水酸化ナトリウム緩衝液を使用した場合の活性値を示すもので、至適ｐＨは８〜９にあった。
（７）ｐＨ安定性
１００ｍＭ各種緩衝液（３−ヒドロキシ酪酸脱水素酵素１Ｕ／ｍｌ）を３７゜Ｃ、６０分間処理し、その残存活性を前記酵素活性測定法に従って測定した。その結果を図２に示す。ｐＨ４．０〜５．０は１００ｍＭクエン酸緩衝液、ｐＨ５．０〜６．０は１００ｍＭ酢酸緩衝液、ｐＨ６．０〜７．５は１００ｍＭリン酸緩衝液、ｐＨ７．５〜９．０は１００ｍＭトリス−塩酸緩衝液、ｐＨ９．０〜１１．０は１００ｍＭグリシン−水酸化ナトリウム緩衝液を使用した。ｐＨ７．５〜１１の範囲で最も良好な安定性を示した。
（８）至適温度
前記酵素活性測定法に従って、温度を２５゜Ｃ〜６０゜Ｃの範囲で変化させて至適温度を求めた結果は図３に示すとおりであり、本酵素の至適温度は４５〜５０゜Ｃであった。
（９）熱安定性
１００ｍＭトリス−塩酸緩衝液（ｐＨ ８．５）（３−ヒドロキシ酪酸脱水素酵素１Ｕ／ｍｌ）を各温度で１０分間加熱処理した後の残存活性を前記酵素活性測定法に従って測定した。その結果、図４に示すとおり少なくとも３７゜Ｃまで安定であった。
（１０）金属イオンの影響
各種金属イオン（１ｍＭ）の本酵素活性への影響について調べた結果は表２に示すとおりで、銅イオンによる強い阻害がみられた。
【００２１】
【表２】
【実施例】
ついで、本発明の実施例を詳しく述べるが、本発明はなんらこれにより限定されるものではない。
【００２３】
【実施例１】
アルカリゲネス・エスピーＮＯ．９８１株をグルタミン酸２％、酵母エキス５．０％からなる培地（ｐＨ７．０）１００ｍｌに接種し、２８゜Ｃ、４８時間培養し、得られた種培養液を上記と同一組成培地に消泡剤を加えた培地（ｐＨ７．０）２０ｌに添加し、２８゜Ｃで４８時間培養した。培養終了後、培養物を４５００ｒｐｍで３０分間、遠心し、菌体５６０ｇを回収した。
【００２４】
この菌体に１０ｍＭＥＤＴＡ、５０ｍＭトリス−塩酸緩衝液（ｐＨ７．５）を含む０．１％リゾチーム溶液２ｌを加え、３７゜Ｃ、３０分間反応させて、可溶化を行った。その後、これを４５００ｒｐｍで３０分間遠心分離して不溶物を除去して、その上清１８７０ｍｌ（４１１００Ｕ）を得た。
ついでこの上清に５％硫酸プロタミン１８．７ｍｌを加えて沈でん物を遠心除去し、その上清を２０ｍＭトリス−塩酸緩衝液（ｐＨ８．５）にて一夜透析し、ＤＥＡＥ−セファロース（２．７×３０ｃｍ）（ファルマシア社製）イオン交換クロマトグラフィーを行った。溶出は塩化カリウムの０〜１Ｍのリニアグラジエントにより行い、０．２０．３ＭＫＣｌの溶出画分（３１７００Ｕ）を回収した。この酵素溶液に４Ｍ濃度となるようにＮａＣｌを溶解し、フェニルセファロース（２．６×１９．５ｃｍ）（ファルマシア社製）の疎水クロマトグラフィーを行った。溶出は４Ｍ〜０ＭのＮａＣｌ直線濃度勾配により行い、１Ｍ〜０．５ＭのＮａＣｌの溶出画分（２０８０Ｕ）を回収した。
【００２５】
ついで、この酵素液を１０ｍＭのトリス−塩酸緩衝液（ｐＨ８．５）にて一夜透析し、Ｑ−セファロース（２．６×１９．５ｃｍ）（ファルマシア社製）のイオン交換クロマトグラフィーを行った。溶出は０〜０．４ＭのＮａＣｌのリニアグラジエントにより行い、０．２〜０．２５ＭのＮａＣｌの溶出画分（２００００Ｕ）を回収した。更に、この酵素液を１０ｍＭトリス−塩酸緩衝液（ｐＨ７．５）にて一夜透析し、ハイドロキシアパタイト（１．６×２６．５ｃｍ）（ペンタックス社製）クロマトグラフィーを行った。溶出は０〜０．３Ｍリン酸緩衝液による直線グラジエントにより行い、０．０６〜０．１Ｍのリン酸緩衝液の溶出画分（１６０００Ｕ）を回収し、凍結乾燥して精製３−ヒドロキシ酪酸脱水素酵素（２１３Ｕ／ｍｇ、７５ｍｇ）を得た。
【００２６】
【発明の効果】
本発明により、アルカリゲネス属に属する微生物による新規な製造法を提供できるものであり、本酵素を用いるケトン体の１つの３−ヒドロキシ酪酸の測定用酵素をして提供できた。
【図面の簡単な説明】
【図１】図１は本発明の３−ヒドロキシ酪酸脱水素酵素の至適ｐＨ曲線を示す。
【図２】図２は本発明の３−ヒドロキシ酪酸脱水素酵素のｐＨ安定曲線を示す。
【図３】図３は本発明の３−ヒドロキシ酪酸脱水素酵素の至適温度曲線を示す。
【図４】図４は本発明の３−ヒドロキシ酪酸脱水素酵素の熱安定曲線を示す。[0001]
[Industrial applications]
The present invention provides 3-hydroxybutyrate dehydrogenase dehydration by culturing 3-hydroxybutyrate dehydrogenase-producing bacteria belonging to the genus Alcaligenes and collecting 3-hydroxybutyrate dehydrogenase from the culture. The present invention relates to a method for producing an enzyme.
[0002]
[Prior art and problems]
3-Hydroxybutyrate dehydrogenase (EC 1.1.1.130) acts on 3-hydroxybutyrate in the presence of NAD to consume 1 mole of NAD, 1 mole of acetoacetic acid and 1 mole of reduction It is known as an enzyme having a catalytic action of converting to type NAD.
[0003]
So far, 3-hydroxybutyrate dehydrogenase has been derived from animals such as rat brain (Biochem. Cell Biol., 68, 980-983 (1990)) and rat liver (Biochem. Cell Biol., 68, 1225). 1230 (1990)) and bovine heart (Arch. Biochem. Biophys., 262, 85-98 (1988)).
[0004]
In addition, as those derived from microorganisms, Rhodospirillum rubrum (J. Biol. Chem. 237, 603-607 (1962)), Pseudomonas lemoignei (J. Biol. C. 240, J. Biol. 4023-4029 (1965)), Mycobacterium Frey (Mycobacterium phlei) (J.Gen.Microbiol.104,123-126 (1978 )), Paracoccus denitrificans (Paracoccusdenitrificans) (Biochim.Biophys.Acta839, 300-307 (1985)), Zoogloea ramigera ( Zoogloea ramiger) a) (J.Biochem.89,625-635 (1981)) , load Pseudomonas sphaeroides (Rhodopseudomonas spheroides) (Biochem.J.241,297-300 (1987 )), Azospirillum, Brazil lens (Azospirillum brasilense) (J Gen. Microbiol. 136, 645-649 (1990)).
[0005]
[Problems to be solved by the invention]
However, it has been desired to develop a method for producing a 3-hydroxybutyrate dehydrogenase using a strain other than these strains having the ability to produce 3-hydroxybutyrate dehydrogenase.
[0006]
[Means for Solving the Problems]
The present inventors have conducted intensive studies in search of a production method capable of industrial production of 3-hydroxybutyrate dehydrogenase, and as a result, the genus Alcaligenes genus NO. Isolated from the soil of a potato field in Gojo City, Nara Prefecture, Japan. It has been found for the first time that 981 strain has the ability to produce 3-hydroxybutyrate dehydrogenase, and the present invention has been completed.
[0007]
The present invention has been completed on the basis of the above findings, and is characterized by culturing a 3-hydroxybutyrate dehydrogenase producing bacterium belonging to the genus Alcaligenes, and collecting 3-hydroxybutyrate dehydrogenase from the culture. This is a method for producing 3-hydroxybutyrate dehydrogenase.
Hereinafter, the present invention will be described in detail.
[0008]
First, the 3-hydroxybutyrate dehydrogenase-producing bacterium of the present invention is not particularly limited as long as it is a microorganism capable of producing 3-hydroxybutyrate dehydrogenase belonging to the genus Alcaligenes. It may be a variant or a mutant having an enzyme-producing ability, and is preferably a NO. 981 strain, and this strain was deposited at the National Institute of Advanced Industrial Science and Technology (currently, the National Institute of Bioscience and Biotechnology) under the deposit number No. 2570 (FERM BP-2570). . The mycological properties, identification and nomenclature of this strain are described in detail in JP-A-3-127895 and US Pat. No. 5,173,416.
[0009]
In practicing the present invention, the culture form may be either liquid culture or solid culture, but industrially, it is advantageous to carry out aeration-agitation culture. As a culture source to be used, in addition to carbon sources, nitrogen sources, inorganic salts and other trace nutrients generally used for microorganism culture, any nutrient sources that can be used by microorganisms belonging to the genus Alcaligenes can be used.
[0010]
As a carbon source, glucose, fructose, saccharose, xylose, maltose, glycerol, dextrin, starch, amino acids and the like, as well as fatty acids, oils and fats, organic acids and the like are used alone or in combination. Either an inorganic nitrogen source or an organic nitrogen source can be used as the nitrogen source, and examples of the inorganic nutrient sources include ammonium sulfate, ammonium nitrate, urea, sodium nitrate, and ammonium chloride. Examples of the organic nitrogen source include flours such as soybean, rice, corn, and wheat, corn steep liquor, peptone, meat extract, casein, amino acids, and yeast extract. Inorganic salts and micronutrients include phosphoric acid, magnesium, potassium, iron, calcium, zinc and other salts, as well as the growth of bacteria such as vitamins, nonionic surfactants, and antifoaming agents, and the use of 3-hydroxybutyrate dehydrogenase. Anything that promotes production can be used as needed.
[0011]
The cultivation is performed under aerobic conditions, and the culturing temperature may be within a range in which the bacteria grow and 3-hydroxybutyrate dehydrogenase is produced, and is usually 15 ° C to 37 ° C, preferably 25 ° C to 35 ° C. . The cultivation time varies depending on the conditions, but the cultivation may be performed up to the time when 3-hydroxybutyrate dehydrogenase is most produced, and is usually about 24 to 100 hours.
3-Hydroxybutyrate dehydrogenase is mainly contained and accumulated in the cells, and may be extracted from the cells. To illustrate the method for extracting 3-hydroxybutyrate dehydrogenase, first, a culture is separated into a solid and a liquid, and the obtained wet cells are dispersed in a solution such as a phosphate buffer or a tris-hydrochloride buffer, and treated with lysozyme. Various kinds of cell treatment means such as ultrasonic treatment, French press treatment, and dynomill treatment are appropriately selected and combined to obtain a crude 3-hydroxybutyrate dehydrogenase-containing solution.
[0012]
A purified 3-hydroxybutyrate dehydrogenase is obtained from the crude 3-hydroxybutyrate dehydrogenase-containing solution by means of isolation and purification of known proteins and enzymes. For example, a 3-hydroxybutyrate dehydrogenase is precipitated on a crude 3-hydroxybutyrate dehydrogenase-containing liquid by applying a fractional precipitation method using an organic solvent such as acetone, methanol, ethanol, or the like, or a salting-out method using ammonium sulfate, sodium chloride, or the like. Allow to collect. Further, after dialysis and isoelectric focusing of the precipitate as necessary, column chromatography using an ion exchanger, a gel filtration agent, an adsorbent, etc. until a single band is shown by electrophoresis or the like. To purify. In addition, when the purification degree of 3-hydroxybutyrate dehydrogenase is increased by appropriately combining these methods, they can be appropriately combined.
[0013]
The enzyme obtained by these methods can be used as a stabilizer by adding or removing various salts, saccharides, proteins, lipids, surfactants, etc., by ultrafiltration and lyophilization to obtain a liquid or solid solution. -Hydroxybutyrate dehydrogenase can be obtained, and lyophilization may be appropriately performed. In this case, about 0.5 to 10% of saccharose, mannitol, salt, albumin, etc. may be added as a stabilizer. .
[0014]
Next, the physicochemical properties of 3-hydroxybutyrate dehydrogenase obtained by the present invention and a method for measuring enzyme activity will be described.
Method for measuring the activity of 3-hydroxybutyrate dehydrogenase 0.2 ml of 0.2 M Tris-HCl buffer (pH 8.5), 0.1 ml of 50 mM 3-hydroxybutyric acid, 0.1 ml of 10 mM NAD, 100 U / ml of diaphorase 1 ml of a reaction solution containing 0.05 ml, 0.02 ml of 0.25% NTB (nitrotetrazonium blue), 0.01 ml of 10% triton X-100 and 0.52 ml of distilled water at 37 ° C. After preheating for 20 minutes, 20 μl of the enzyme solution is added and reacted for 10 minutes. After the reaction, the reaction is stopped by adding 0.1N hydrochloric acid, and the absorbance at a wavelength of 550 nm is measured within 5 minutes using a 1.0 cm layer cell (As). As a blind test, the same operation is performed using 20 μl of distilled water instead of the enzyme solution, and the absorbance is measured (Ab). The absorbance difference (As) between the absorbance of the enzyme (As) and the absorbance of the blind test (Ab) is measured. -Determine the enzyme activity from Ab). One unit of the enzyme activity is the amount of the enzyme that produces 1 μmol of reduced NAD per minute at 37 ° C., and the calculation formula is as follows.
[0015]
(Equation 1)
Physicochemical properties (1) Enzymatic action: Enzymatic action using 3-hydroxybutyric acid as a substrate is shown below.
[0017]
Embedded image
(2) Substrate specificity: 3-hydroxybutyric acid shows substrate specificity. Table 1 shows the specificity for various substrates.
[0019]
[Table 1]
(3) Km value: 1.6 ± 0.5 (mM) (based on 3-hydroxybutyric acid)
0.12 ± 0.02 (mM) (relative to NAD)
4) Isoelectric point: 5.0 ± 0.2 (by electrophoresis using carrier-ampholine)
(5) Molecular weight: 60000 ± 5000 (by gel filtration method using TSK G-3000SW), 30,000 ± 5000 (by SDS polyacrylamide gel electrophoresis method)
(6) Optimum pH
The optimum pH was determined according to the enzyme activity measurement method, and the results are shown in FIG. The range of pH 5.0 to 6.0 is 100 mM acetate buffer, the range of pH 6.0 to 7.5 is 100 mM phosphate buffer, the range of pH 7.5 to 9.0 is 100 mM Tris-HCl buffer, pH 9.0. The range of 0 to 11.0 indicates the activity value when a 100 mM glycine-sodium hydroxide buffer was used, and the optimum pH was 8 to 9.
(7)
(8) Optimal temperature According to the enzyme activity measurement method, the temperature was changed in the range of 25 ° C. to 60 ° C. to determine the optimal temperature. The result is shown in FIG. Was 45-50 ° C.
(9)
(10) Influence of metal ions The results of investigating the effects of various metal ions (1 mM) on the activity of this enzyme are shown in Table 2, and strong inhibition by copper ions was observed.
[0021]
[Table 2]
【Example】
Next, examples of the present invention will be described in detail, but the present invention is not limited thereto.
[0023]
Embodiment 1
Alkaligenes SP NO. The 981 strain was inoculated into 100 ml of a medium (pH 7.0) containing 2% of glutamic acid and 5.0% of yeast extract, and cultured at 28 ° C. for 48 hours. The obtained seed culture was defoamed to a medium having the same composition as above. The solution was added to 20 l of a medium (pH 7.0) to which the agent was added, and cultured at 28 ° C for 48 hours. After the completion of the culture, the culture was centrifuged at 4500 rpm for 30 minutes to recover 560 g of cells.
[0024]
Two liters of a 0.1% lysozyme solution containing 10 mM EDTA and 50 mM Tris-HCl buffer (pH 7.5) was added to the cells, and reacted at 37 ° C. for 30 minutes to solubilize the cells. Thereafter, this was centrifuged at 4500 rpm for 30 minutes to remove insolubles, and 1870 ml (41100 U) of the supernatant was obtained.
Then, 18.7 ml of 5% protamine sulfate was added to the supernatant, and the precipitate was removed by centrifugation. The supernatant was dialyzed overnight against a 20 mM Tris-HCl buffer (pH 8.5) to give DEAE-Sepharose (2.7). × 30 cm) (manufactured by Pharmacia) was subjected to ion exchange chromatography. Elution was performed with a linear gradient of 0 to 1 M of potassium chloride, and an eluted fraction (31700 U) of 0.20.3 M KCl was collected. NaCl was dissolved in the enzyme solution to a concentration of 4 M, and phenyl sepharose (2.6 × 19.5 cm) (manufactured by Pharmacia) was subjected to hydrophobic chromatography. Elution was performed by a linear concentration gradient of 4M to 0M NaCl, and an eluted fraction (2080U) of 1M to 0.5M NaCl was collected.
[0025]
Then, the enzyme solution was dialyzed overnight against a 10 mM Tris-HCl buffer (pH 8.5), and subjected to ion-exchange chromatography on Q-Sepharose (2.6 × 19.5 cm) (Pharmacia). Elution was performed with a linear gradient of 0 to 0.4 M NaCl, and an eluted fraction (20,000 U) of 0.2 to 0.25 M NaCl was collected. Further, the enzyme solution was dialyzed overnight against a 10 mM Tris-HCl buffer (pH 7.5), and subjected to hydroxyapatite (1.6 × 26.5 cm) (Pentax) chromatography. Elution was performed with a linear gradient using a 0 to 0.3 M phosphate buffer, and an eluted fraction (16000 U) of a 0.06 to 0.1 M phosphate buffer was collected, freeze-dried, and purified 3-hydroxybutyric acid dehydrated. An enzymatic enzyme (213 U / mg, 75 mg) was obtained.
[0026]
【The invention's effect】
According to the present invention, a novel production method using a microorganism belonging to the genus Alcaligenes can be provided, and an enzyme for measurement of one 3-hydroxybutyric acid in a ketone body using the present enzyme can be provided.
[Brief description of the drawings]
FIG. 1 shows an optimal pH curve of the 3-hydroxybutyrate dehydrogenase of the present invention.
FIG. 2 shows a pH stability curve of the 3-hydroxybutyrate dehydrogenase of the present invention.
FIG. 3 shows an optimal temperature curve of the 3-hydroxybutyrate dehydrogenase of the present invention.
FIG. 4 shows a thermostability curve of the 3-hydroxybutyrate dehydrogenase of the present invention.
Claims (2)
（１）酵素作用
ＮＡＤ＋の存在下に３−ヒドロキシ酪酸に作用して、アセト酢酸、ＮＡＤＨ及びＨ ＋を生成する作用を有する。
（２）基質特異性
３−ヒドロキシ酪酸に基質特異性を示す。
（３）分子量
６００００±５０００（ゲル濾過法にて）
（４）至適ｐＨ
８〜９
（５）至適温度
４５〜５０℃
（６）ｐＨ安定性
ｐＨ７．５〜１１の範囲で最も良好な安定性を示す。A method for producing 3-hydroxybutyrate dehydrogenase, comprising culturing 3-hydroxybutyrate dehydrogenase-producing bacteria belonging to the genus Alcaligenes in a medium, and collecting 3-hydroxybutyrate dehydrogenase from the culture. However, the physicochemical properties of the 3-hydroxybutyrate dehydrogenase are as follows.
(1) acting on the 3-hydroxybutyric acid in the presence of enzymatic NAD +, it has the effect of producing acetoacetic acid, NADH and H +.
(2) Substrate specificity 3-hydroxybutyric acid shows substrate specificity.
(3) Molecular weight 60000 ± 5000 (by gel filtration method)
(4) Optimum pH
8-9
(5) Optimal temperature 45-50 ° C
(6) pH stability The best stability is exhibited in the pH range of 7.5 to 11.
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|---|---|---|---|
| JP18104794A JP3602162B2 (en) | 1994-08-02 | 1994-08-02 | Method for producing 3-hydroxybutyrate dehydrogenase |
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| JP18104794A JP3602162B2 (en) | 1994-08-02 | 1994-08-02 | Method for producing 3-hydroxybutyrate dehydrogenase |
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| JP3602162B2 true JP3602162B2 (en) | 2004-12-15 |
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| JP6945469B2 (en) * | 2018-02-20 | 2021-10-06 | 大阪瓦斯株式会社 | Method for producing acetoacetic acid |
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