JP4117823B2 - Method for producing thermostable hydrogenase - Google Patents

Description

＜２．耐熱性ヒドロゲナーゼ＞
本発明の耐熱性ヒドロゲナーゼは、上記方法によって作製されたものである。
＜３．DNA＞
本発明のDNAは、上記耐熱性ヒドロゲナーゼをコードするものである。本発明のDNAの一例としては、配列番号１記載の塩基配列で表されるDNAを挙げることができる。
＜４．微生物＞
本発明の微生物は、上記DNAを含み、耐熱性ヒドロゲナーゼを生産するものである。本発明の微生物としては、M2R株やM3R株を例示することができる。M2Rは、P181相当アミノ酸がイソロイシンに置換された変異型ヒドロゲナーゼをコードする遺伝子を持ち、M3R株は、L243相当アミノ酸がイソロイシンに置換された変異型ヒドロゲナーゼをコードする遺伝子を持つ。これらの菌株は、独立行政法人産業技術総合研究所特許微生物寄託センターに、それぞれ受託番号FERM P-18709、FERM P-18710として寄託されている（受託日：平成14年2月13日）。
＜５．耐熱性ヒドロゲナーゼの生産方法＞
本発明の耐熱性ヒロドゲナーゼの生産方法は、上記微生物を培養し、得られる培養物から耐熱性ヒドロゲナーゼを採取することを特徴とするものである。
【００１９】
微生物の培養方法は、使用する微生物の種類に応じて決めればよい。例えば、ラルストニア・ユトロファを使用する場合は、LB培地（1% 酵母エキス、0.5%トリプトン、1% NaCl）、FN培地(O. Lenz, E. Schwartz, M. Eitinger, B. Friedrich (1994) J. Bacteriol. 176, 4385-4393)などで、培養温度を30〜37℃に設定し、培養することができる。
【００２０】
培養物からの耐熱性ヒドロゲナーゼの採取は、微生物からタンパク質を採取する際に常用される方法に従って行うことができる。例えば、耐熱性ヒドロゲナーゼが菌体外に生産される場合には、培養液をそのまま使用するか、遠心分離等により菌体を除去し、その後、硫酸アンモニウム沈殿、ゲルクロマトグラフィー、イオン交換クロマトグラフィー、疎水クロマトグラフィー、アフィニティ−クロマトグラフィー等を単独又は適宜組み合わせて用いることにより、培養物中から耐熱性ヒドロゲナーゼを採取することができる。また、耐熱性ヒドロゲナーゼが菌体内に生産される場合には、超音波、フレンチプレス、ガラスビーズを使用するホモジナイザーなどを用いて菌体を破砕し、その破砕液について上記と同様の処理を行うことにより、培養物中から耐熱性ヒドロゲナーゼを採取することができる。
【００２１】
なお、ここでいう培養物とは、培養後の培地上清、培養により得られた細胞若しくは細胞の破砕物のいずれをも意味するものである。
＜６．微生物の作製方法＞
本発明の微生物の作製方法は、以下の（１）〜（６）の工程を含むものである。
【００２２】
工程（１）では、ヒドロゲナーゼの全部又は一部をコードするDNA断片であって、P181相当アミノ酸又はL243相当アミノをコードする配列がイソロイシンをコードする配列に置換されているDNA断片を作製する。
【００２３】
ヒドロゲナーゼは、上述の耐熱性ヒドロゲナーゼの作製方法と同様のものを使用することができる。DNA断片は、ヒドロゲナーゼの全長をコードするものであることが好ましいが、相同組換えを起こさせることができるのであれば、ヒドロゲナーゼの一部分をコードするものであってもよい。ヒドロゲナーゼのどのアミノ酸が、P181相当アミノ酸又はL243相当アミノに相当するかは、上述の耐熱性ヒドロゲナーゼの作製方法と同様に、アミノ酸配列の整列によりわかる。配列の置換は、PCRなどによって行うことができる。
【００２４】
工程（２）では、（１）で作製したDNA断片、抗生物質に対する耐性遺伝子、及び特定条件下で致死性を発揮する遺伝子を含むベクターを作製する。抗生物質耐性遺伝子としては、例えば、テトラサイクリン耐性遺伝子を使用できるが、他の遺伝子を使用してもよい。特定条件下で致死性を発揮する遺伝子としては、例えば、sacB遺伝子を使用することができるが、他の遺伝子を使用してもよい。sacB遺伝子は、高スクロース条件下で致死性を発揮する遺伝子であり、この遺伝子が組み込まれた菌株は、スクロースを高濃度で含む培地中では死滅してしまう。なお、sacB遺伝子の配列は、GenBankに登録されている（Accession No. X02730）。
【００２５】
工程（３）では、（２）で作製したベクターを微生物に導入する。ここで使用する微生物は特に限定されず、大腸菌などでよい。ベクターを導入する方法も特に限定されず、常法に従って行うことができる。
【００２６】
工程（４）では、（３）でベクターを導入した微生物とヒドロゲナーゼを生産する微生物を共存させる。このように２種類の微生物を共存させることにより、ベクターとヒドロゲナーゼ生産微生物のゲノムDNA間で相同組換えが起こる。なお、ここでいうヒドロゲナーゼ生産微生物とは、工程（１）におけるヒドロゲナーゼと同種のヒドロゲナーゼを生産する微生物である。
【００２７】
工程（５）では、ヒドロゲナーゼを生産する微生物を前述の抗生物質を含む培地中で培養し、（１）で作製したDNA断片、抗生物質に対する耐性遺伝子、及び特定条件下で致死性を発揮する遺伝子がゲノムDNA中に挿入された菌株を選抜する。
【００２８】
工程（６）では、（５）で選抜した菌株を前述の致死性遺伝子が致死性を発揮する条件下で培養し、抗生物質に対する耐性遺伝子及び特定条件下で致死性を発揮する遺伝子が脱落した菌株を選抜する。致死性を発揮する条件は、使用した致死性遺伝子に応じて決めればよく、例えば、致死性遺伝子としてsacB遺伝子を使用した場合には、高濃度のスクロースを含む培地での培養が、致死性を発揮する条件になる。
【００２９】
【実施例】
ヒドロゲノビブリオ・マリナス由来のヒドロゲナーゼは、高い酸素安定性と耐熱性を示す（特開2000-350585号公報）。この微生物由来のヒドロゲナーゼの大サブユニットに存在するイソロイシン残基の数を調べてみたところ、他の耐熱性ヒドロゲナーゼと同様にイソロイシン含量が高かった（表２）。
【００３０】
【表２】

【００３１】
また、ヒドロゲノビブリオ・マリナス由来のヒドロゲナーゼ（大サブユニット）のアミノ酸配列と他の微生物の由来のヒドロゲナーゼのアミノ酸配列を整列させてみた（図１及び図２）。
【００３２】
デサルホビブリオ・ギガス由来のヒドロゲナーゼについては、その結晶構造が解析されており、タンパク質内部の疎水的コアの存在する位置（図中のInと記された部位）や大・小サブユニットの境界面の位置（図中のInterfaceと記された部位）が推定可能なので、ヒドロゲノビブリオ・マリナス由来のヒドロゲナーゼにおけるこれらの位置も推定することができる。これらの部位のアミノ酸について、ヒドロゲノビブリオ・マリナス由来のヒドロゲナーゼと他の微生物由来のヒドロゲナーゼを比較したところ、他の微生物では、バリン、ロイシン、アラニンなどであるのに対し、ヒドロゲノビブリオ・マリナスでは、より疎水的なイソロイシンになっていた。
【００３３】
以上のことから、非耐熱性ヒドロゲナーゼにおいて、疎水的コアや大−小サブユニット境界面に位置すると推定されるアミノ酸をイソロイシンに置換することにより、非耐熱性ヒドロゲナーゼを耐熱性ヒドロゲナーゼに変換できると予想される。
【００３４】
そこで、ラルストニア・ユウトロファ由来の非耐熱性ヒドロゲーナゼに耐熱性を付与するため、以下の実験を行った。
【００３５】
なお、図１及び図２において、アミノ酸配列上に示された矢印は、以下の実験で変異を導入したアミノ酸の位置を表している。また、アルファベットを伴う矢印はβ-シートを形成している部位を示し、数字を伴うボックスはα-ヘリックスを形成している部位を示す（いずれもデサルホビブリオ・ギガス由来のヒドロゲナーゼの結晶解析から推定されたものである。）。更に、図中のHma、Reu、Oca、Avi、Ach、Phy、Rca、Bja、Dgiは、それぞれヒドロゲノビブリオ・マリナス、ラルストニア・ユウトロファ、オリゴトロファ・カルボキシドボランス、アゾトバクタ・ビネランディ、アゾトバクタ・クロロコッカム、シュードモナス・ハイドロゲノボーラ、ロドバクタ・カプスラータ、ブラディリゾビウム・ジャポニカム、デサルホビブリオ・ギガスを表している。
【００３６】
〔実施例１〕 ラルストニア・ユウトロファの野生株の作製
Ralstonia eutropha H16株は細胞内に可溶性ヒドロゲナーゼと膜結合型ヒドロゲナーゼの２種類のヒドロゲナーゼを保有する（B. Friedrich, E. Schwartz (1993) Annu. Rev. Microbiol. 47, 351-383）。今回ヒドロゲナーゼに変異を導入するのは膜結合型のヒドロゲナーゼである。そのため、変異の効果をより明確に判断するために、Ralstonia eutropha H16株の可溶性ヒドロゲナーゼをマッサンツらの方法（C. Massanz, V.M. Fernandez, B. Friedrich (1997) Eur. J. Biochem. 245, 441-448）で欠落させたRalstonia eutropha HF387株を野生株として使用した。
【００３７】
〔実施例２〕 アミノ酸(１塩基)置換ヒドロゲナーゼの作製
Ralstonia eutropha H16株由来の膜結合型ヒドロゲナーゼ遺伝子がクローン化されたプラスミドpCH182（M. Bernhard, E. Schwartz, J. Rietdorf, B. Frisedrich (1996) J. Bacteriol. 178, 4522-4529）を保有する大腸菌をLB培地で培養し、QIAGENのPlasmid Midi Kitを用いてpCH182を抽出した。抽出したpCH182を鋳型DNAとして、２段階PCR法（O. P. Kuipers, H. J. Boot, W. M. de Vos (1991) Nucleic Acid Res. 19, 4558）を用いて、以下の５種類の変異を導入したDNA断片を調製した。
【００３８】
【表３】

【００３９】
２段階PCR法は、表６に記載したプライマーを変異導入用プライマーとして1回目のPCRを行い、得られたPCR産物をQIAGENのQIAquick Gel Extraction Kitを用いて精製した後、これをメガプライマーとして2回目のPCRを行った。PCRの条件は以下の通りである。
【００４０】
【表４】

【００４１】
【表５】

【００４２】
【表６】

【００４３】
得られたDNA断片をpBluescript(Stratagene Cloning Systems)に挿入し、大腸菌XL-1 Blue(Stratagene Cloning Systems)に形質転換した。形質転換体をLB培地で培養し、QIAGENのQIAprep Spin Miniprep Kitを用いてプラスミドを調製してシークエンスを行った。アマシャムファルマシアバイオテクのDYEnamic Direct Cycle Sequencing Kit with 7-deaza dGTP (no primer)を用いてシークエンス反応を行い、DNA sequencer LIC-4200S (LI-COR, Inc.)により解析を行った。
【００４４】
〔実施例３〕 変異ヒドロゲナーゼのラルストニア・ユウトロファ野生株への形質転換
上記５種類の変異が導入されたヒドロゲナーゼ遺伝子を含む断片を制限酵素で切り出して、ラルストニア・ユウトロファへの相同組換え導入用スーサイド・ベクターpLO3に挿入した。スーサイド・ベクターpLO3は、レンツらの方法（O. Lenz, B. Friedrich (1998) Proc. Natl. Acad. Sci. USA 95, 12474-12479）に従って作製した。即ち、pNEB194(New England Biolabs, Inc.)のポリリンカー部位を含む0.08kbのHindIII-EcoRIをKlenow fragment(New England Biolabs, Inc.)で平滑末端化し、同じくKlenow fragmentで平滑末端化したpBR322(New England Biolabs, Inc.)のEcoRI-DraIサイトに連結した(pCH639)。pUD800（P. Gay, D. Le Cop, M. Steinmetz, T. Berkelman, C. I. Kado (1985) J. Bacteriol. 164, 918-921）のsacB遺伝子を含む1.96kb BamHI-NdeIを平滑末端化し、平滑末端化したpCH639のStyI-BsaIサイトに連結した(pCH640)。pCH640をNdeIで切断して平滑末端化した後、pJF145（J. P. Fuerste, W. Pansegrau, G. Ziegelin, M. Kroeger, E. Lanka (1979) Proc. Natl. Acad. Sci. USA 86, 1771-1775）由来のoriTを平滑末端化した0.47kb BamHI断片としてサブクローニングし、pLO3を作製した。
【００４５】
変異型ヒドロゲナーゼ遺伝子を挿入したスーサイド・ベクターpLO3をRalstonia eutropha HF387株に接合伝達し、相同組換えにより膜結合型ヒドロゲナーゼ遺伝子に変異が導入された組換え体を選抜した。接合伝達は、R. Simonらの方法（R. Simon, U. Priefer, U. Puehler (1983) Bio/Technology 1, 784-791）に従って行い、組換え体の選抜は、O. Lenzらの方法（O. Lenz, E. Schwartz, M. Eitinger, B. Friedrich (1994) J. Bacteriol. 176, 4385-4393）に従って行った。即ち、上述のプラスミドを保有する大腸菌をテトラサイクリン含有LB培地10mlで、 R.eutropha HF387株をLB培地10mlで37℃で一晩培養した。滅菌水で菌体を洗浄した後、菌体を1mlの滅菌水に懸濁し、0.2mlずつをLB平板培地上で混合して37℃で一晩培養することによってプラスミドの接合伝達を行わせた。次に、相同組換えにより野生型ヒドロゲナーゼ遺伝子に変異が導入された組換え体を選抜した。まず、pLO3に連結した変異型ヒドロゲナーゼ遺伝子とラルストニア・ユウトロファに存在する野生型ヒドロゲナーゼ遺伝子との間で相同組み換えを１ヶ所で起こした組換え体（pLO3がゲノム中にインテグレートされた状態）を、pLO3にコードされているテトラサイクリン耐性をマーカーとして選抜した。次に、この組換え体をLB培地10mlに植菌して37℃で一晩培養した後、15%のスクロースを含むLB平板培地に植菌して37℃で培養し、ゲノムにインテグレートされたpLO3上にある変異型ヒドロゲナーゼ遺伝子と野生型ヒドロゲナーゼ遺伝子との間の別の部位で、再度組み換えが生じることによってpLO3が脱落した組み換え体をsacBをマーカーとして選抜した。すなわち、この15%スクロース含有培地でコロニーを形成する高濃度スクロース耐性株を、外部から導入した変異型ヒドロゲナーゼ遺伝子と野生型ヒドロゲナーゼ遺伝子との間で遺伝子配列の交換が起こった相同組換え体として選抜した。
【００４６】
〔実施例４〕 ヒドロゲナーゼの活性中心の耐熱性評価
上記のようにして得られたヒドロゲナーゼ変異株をヒドロゲナーゼ発現用培地であるFGN培地（O. Lenz, E. Schwartz, M. Eitinger, B. Friedrich (1994) J. Bacteriol. 176, 4385-4393）で培養し、菌体から膜結合型ヒドロゲナーゼを含む膜画分をNishiharaらの方法（H. Nishihara, Y. Miyashita, K. Aoyama, T. Kodama, Y. Igarashi, Y. Takamura (1997) Biochem. Biophys. Res. Commun. 232, 766-770）により調製した。
【００４７】
この膜画分を70℃に加熱して経時的にサンプリングし、残存する重水素置換活性を測定し、これによりヒドロゲナーゼの活性中心の耐熱性を評価した。重水素置換反応の測定は、 Cammackらの方法（R. Cammack, V. M. Fernandez, E. C. Hatchikian (1994) Methods. Enzymol. 234, 43-68）に従って行った。即ち、あらかじめ重水素で飽和した10mlの50mM酢酸バッファーに、シリンジで酵素液を添加することにより反応を開始した。気相中に発生する水素の量を質量分析計で経時的に測定し、単位時間当たりの水素発生量で活性強度を表した。この結果を図３に示す。
【００４８】
図３に示すように、変異の導入されたヒドロゲナーゼは、いずれも野生型ヒドロゲナーゼよりも活性が高く、活性中心の耐熱性は向上した。
【００４９】
〔実施例５〕 ヒドロゲナーゼのサブユニット構造の耐熱性評価
実施例４で調製した膜画分を55℃に加熱して経時的にサンプリングし、残存するメチレンブルー還元活性を測定した。メチレンブルーを還元するためには、ヒドロゲナーゼの活性中心(大サブユニット)で水素から引き抜かれた電子が小サブユニットにうまく伝達されることが必要なので、メチレンブルー還元活性を測定することにより、サブユニット構造の耐熱性を評価することができる。メチレンブルー還元反応の測定は、SchinkとH. G. Schlegelの方法（B. Schink, H. G. Schlegel (1979) Biochem. Biophys. Acta 567, 315-324）に従って行った。この結果を図４に示す。
【００５０】
図４に示すように、サブユニット構造の安定性については、野生型ヒドロゲナーゼよりも安定性が低下したものもあり（V115I、V253I、N168I,L170I）、安定性が向上したのは、P181I及びL243Iの２つだけであった。
【００５１】
【発明の効果】
本発明は、ヒドロゲナーゼに耐熱性を付与する手段を提供する。これにより、非耐熱性のヒドロゲナーゼ生産菌を耐熱性ヒドロゲナーゼ生産菌に変えることができるようになる。従って、培養の容易な細菌を選択し、効率的に耐熱性ヒドロゲナーゼの生産が可能になる。
【配列表】

【図面の簡単な説明】
【図１】ヒドロゲナーゼ大サブユニットのアライメントの結果を示す図である（前半）。
【図２】ヒドロゲナーゼ大サブユニットのアライメントの結果を示す図である（後半）。
【図３】ヒドロゲナーゼ画分の重水素置換活性の経時的変化を示す図である。
【図４】ヒドロゲナーゼ画分のメチレンブルー還元活性の経時的変化を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing a thermostable hydrogenase, a thermostable hydrogenase produced by the method, a DNA encoding the thermostable hydrogenase, a thermostable hydrogenase-producing microorganism containing the DNA, a method for producing the microorganism, and the microorganism The present invention relates to a method for producing a thermostable hydrogenase used.
[0002]
[Prior art]
Hydrogenase is an enzyme that catalyzes the reduction of an electron carrier by hydrogen and the reverse generation of hydrogen, and is expected to be used in various industrial fields such as industrial production of hydrogen and catalyst for fuel cells. The hydrogenase utilized for such industrial use must be highly stable and cannot be easily deactivated by heat.
[0003]
Several hydrogenases that are resistant to heat are known. For example, hydrogenases derived from Hydrogenogenibibrio marinus (Japanese Patent Laid-Open No. 2000-350585) and hydrogenases derived from the thermophilic bacterium Pyrococcus furious (R. Sapra, MFJMVerhagen, and MWWAdams, J. Bacteriol. 182: 3423-3428 (2000)). If these enzymes can be produced by microorganisms such as Escherichia coli using gene recombination technology, the productivity of thermostable hydrogenase will be dramatically improved. However, in order for a class I hydrogenase to exert its function, a large number of cofactors are required. Even if a hydrogenase gene is simply introduced into Escherichia coli or the like, an active hydrogenase cannot be obtained. In addition, if a cofactor gene is introduced into a host together with a hydrogenase gene, a theoretically active hydrogenase should be obtained. However, there have been no successful cases so far.
[0004]
From the above, active thermostable hydrogenase can only be obtained from microorganisms that have the ability to produce this enzyme. However, thermostable hydrogenase-producing bacteria are mostly thermophilic, except for the above-mentioned hydrogenovibrio marinas. It is. Furthermore, there is no case where an active hydrogenase is obtained by introducing a thermophilic hydrogenase gene into Escherichia coli. In addition, thermophilic bacteria must be cultured at a high temperature close to 100 ° C., and there is a big problem in culture costs.
[0005]
[Problems to be solved by the invention]
If there is a technique for imparting heat resistance to a non-thermostable hydrogenase, there is no need to cultivate thermophilic bacteria or the like that are difficult to culture in order to obtain a thermostable hydrogenase.
[0006]
The present invention has been made under the above technical background, and an object thereof is to provide a means for improving the heat resistance of hydrogenase.
[0007]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventor has replaced the 181st proline or the 243rd leucine of the large subunit of hydrogenase derived from Ralstonia eutropha with isoleucine, whereby the heat resistance of hydrogenase is increased. Has been found to be significantly improved, and the present invention has been completed.
[0008]
That is, the first of the present invention is a method for producing a thermostable hydrogenase, characterized in that in the hydrogenase, an amino acid corresponding to the 181st proline or the 243rd leucine in the amino acid sequence shown in SEQ ID NO: 2 is substituted with isoleucine. is there.
[0009]
The second of the present invention is a thermostable hydrogenase produced by the above method.
[0010]
The third of the present invention is a DNA encoding the thermostable hydrogenase.
[0011]
A fourth aspect of the present invention is a microorganism that contains the above DNA and produces a thermostable hydrogenase.
[0012]
A fifth aspect of the present invention is a method for producing a thermostable hydrogenase, which comprises culturing the microorganism and collecting thermostable hydrogenase from the obtained culture.
[0013]
A sixth aspect of the present invention is a method for producing a microorganism that produces a thermostable hydrogenase including the following steps.
(1) A DNA fragment encoding all or part of a hydrogenase, wherein a sequence encoding an amino acid corresponding to the 181st proline or 243rd leucine of the amino acid sequence shown in SEQ ID NO: 2 is a sequence encoding isoleucine Step (2) for producing a substituted DNA fragment (3) Step for producing a vector comprising the DNA fragment produced in (1), a gene resistant to antibiotics, and a gene that exhibits lethality under specific conditions (3) ( Steps for introducing the vector prepared in 2) into the microorganism (4) Steps for allowing the microorganism introduced with the vector in (3) and the microorganism producing hydrogenase to coexist (5) The microorganism producing the hydrogenase containing the aforementioned antibiotics The DNA fragment prepared in (1), the antibiotic resistance gene, and the gene that exhibits lethality under specific conditions Steps (6) and (5) for selecting a strain inserted in the DNA of the strain are cultured under conditions where the aforementioned lethal gene exhibits lethality, and the antibiotic resistance gene and under specific conditions A process of selecting a strain from which a gene exhibiting lethality has been lost.
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
<1. Method for producing thermostable hydrogenase>
The method for producing a thermostable hydrogenase of the present invention is characterized in that a specific amino acid is substituted with isoleucine in the hydrogenase.
[0015]
As the hydrogenase, class I hydrogenase can be used, but other hydrogenase may be used. Class I hydrogenase refers to a type of hydrogenase that is membrane-bound and has a nickel / iron cluster inside the molecule. Usually, a gene encoding this hydrogenase is a small subunit gene, a large subunit on the genomic DNA. It is known that the subunit genes are adjacent to each other in the order (L.-F. Wu and MAMadrand, FEMS Microbiology Reviews 104: 243-270 (1993)).
[0016]
Examples of the hydrogenase used in the present invention include Ralstonia eutropha, Oligotropha carboxidovorans, Azotobacter vinelandii, Azotobacter chroococcum, Examples include hydrogenase derived from Pseudomonas hydrogenovora), Rhodobacter capsulatus, Bradyrhizobium japonicum, but are not limited thereto. In addition, since all these hydrogenases are well-known and the amino acid sequence is registered into GenBank etc. (Table 1), those skilled in the art can use these hydrogenases suitably as needed.
[0017]
The above-mentioned specific amino acid is an amino acid corresponding to the 181st proline of the large subunit of hydrogenase derived from Ralstonia jutropha (the amino acid sequence of this subunit is as shown in SEQ ID NO: 2) (hereinafter referred to as “P181 equivalent”). Amino acid)), or an amino acid corresponding to the 243rd leucine (hereinafter referred to as “L243 equivalent amino acid”). Which amino acid of hydrogenase corresponds to the amino acid corresponding to P181 or L243 is determined by comparing the amino acid sequence of the large subunit of hydrogenase derived from Ralstonia / eutropha with the amino acid sequence of the large subunit of the hydrogenase, etc. It can be seen by trying to align as an index. Table 1 shows amino acids corresponding to P181 or L243 of the hydrogenases exemplified above.
[0018]
[Table 1]

<2. Thermostable hydrogenase>
The thermostable hydrogenase of the present invention is produced by the above method.
<3. DNA>
The DNA of the present invention encodes the above thermostable hydrogenase. As an example of the DNA of the present invention, DNA represented by the base sequence described in SEQ ID NO: 1 can be mentioned.
<4. Microorganism>
The microorganism of the present invention contains the above DNA and produces a thermostable hydrogenase. Examples of the microorganism of the present invention include M2R strains and M3R strains. M2R has a gene encoding a mutant hydrogenase in which the P181 equivalent amino acid is substituted with isoleucine, and the M3R strain has a gene encoding a mutant hydrogenase in which the L243 equivalent amino acid is substituted with isoleucine. These strains are deposited under the accession numbers FERM P-18709 and FERM P-18710, respectively, at the National Institute of Advanced Industrial Science and Technology Patent Microorganism Depositary (Trust Date: February 13, 2002).
<5. Method for producing thermostable hydrogenase>
The method for producing a thermostable hydrogenase of the present invention is characterized by culturing the microorganism and collecting the thermostable hydrogenase from the obtained culture.
[0019]
What is necessary is just to determine the cultivation method of microorganisms according to the kind of microorganisms to be used. For example, when using Ralstonia eutropha, LB medium (1% yeast extract, 0.5% tryptone, 1% NaCl), FN medium (O. Lenz, E. Schwartz, M. Eitinger, B. Friedrich (1994) J Bacteriol. 176, 4385-4393) etc., the culture temperature can be set at 30-37 ° C.
[0020]
Collection of the thermostable hydrogenase from the culture can be performed according to a method commonly used when collecting proteins from microorganisms. For example, when thermostable hydrogenase is produced outside the cells, use the culture solution as it is or remove the cells by centrifugation or the like, and then perform ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, hydrophobicity The thermostable hydrogenase can be collected from the culture by using chromatography, affinity-chromatography, etc. alone or in appropriate combination. In addition, when thermostable hydrogenase is produced in the microbial cells, the microbial cells should be crushed using an ultrasonic wave, a French press, a homogenizer using glass beads, etc., and the crushed solution should be treated in the same manner as described above. Thus, thermostable hydrogenase can be collected from the culture.
[0021]
In addition, the culture here means both the culture medium supernatant after the culture, the cells obtained by the culture, or the disrupted cells.
<6. Preparation method of microorganism>
The method for producing a microorganism of the present invention includes the following steps (1) to (6).
[0022]
In step (1), a DNA fragment that encodes all or a part of the hydrogenase, wherein a sequence encoding an amino acid corresponding to P181 or an amino acid corresponding to L243 is substituted with a sequence encoding isoleucine, is prepared.
[0023]
As the hydrogenase, the same method as the method for producing the thermostable hydrogenase described above can be used. The DNA fragment preferably encodes the full length of hydrogenase, but may encode a part of hydrogenase as long as it can cause homologous recombination. Which amino acid of the hydrogenase corresponds to the amino acid corresponding to P181 or amino acid corresponding to L243 can be determined by alignment of the amino acid sequences, as in the method for producing the thermostable hydrogenase described above. Sequence replacement can be performed by PCR or the like.
[0024]
In the step (2), a vector containing the DNA fragment prepared in (1), a resistance gene for antibiotics, and a gene that exhibits lethality under specific conditions is prepared. As the antibiotic resistance gene, for example, a tetracycline resistance gene can be used, but other genes may be used. As a gene that exhibits lethality under specific conditions, for example, the sacB gene can be used, but other genes may be used. The sacB gene is a gene that exhibits lethality under high sucrose conditions, and a strain into which this gene has been incorporated is killed in a medium containing sucrose at a high concentration. The sequence of the sacB gene is registered in GenBank (Accession No. X02730).
[0025]
In step (3), the vector prepared in (2) is introduced into a microorganism. The microorganism used here is not particularly limited and may be Escherichia coli or the like. The method for introducing the vector is not particularly limited, and can be performed according to a conventional method.
[0026]
In step (4), the microorganism introduced with the vector in (3) and the microorganism producing hydrogenase are allowed to coexist. By coexisting two types of microorganisms in this way, homologous recombination occurs between the vector and the genomic DNA of the hydrogenase-producing microorganism. The hydrogenase-producing microorganism here is a microorganism that produces the same kind of hydrogenase as the hydrogenase in step (1).
[0027]
In step (5), a microorganism that produces hydrogenase is cultured in a medium containing the aforementioned antibiotic, the DNA fragment prepared in (1), a resistance gene for the antibiotic, and a gene that exhibits lethality under specific conditions Select strains inserted into the genomic DNA.
[0028]
In step (6), the strain selected in (5) was cultured under the condition that the lethal gene exhibits lethality, and the resistance gene for antibiotics and the gene that exhibited lethality under specific conditions were lost. Select strains. The condition for exerting lethality may be determined according to the lethality gene used.For example, when the sacB gene is used as the lethality gene, culturing in a medium containing a high concentration of sucrose is not lethal. It becomes a condition to exert.
[0029]
【Example】
A hydrogenase derived from Hydrogenobibrio marinas exhibits high oxygen stability and heat resistance (Japanese Patent Laid-Open No. 2000-350585). When the number of isoleucine residues present in the large subunit of the hydrogenase derived from this microorganism was examined, the content of isoleucine was high as in other thermostable hydrogenases (Table 2).
[0030]
[Table 2]

[0031]
In addition, the amino acid sequence of hydrogenase (large subunit) derived from Hydrogenobibrio marinas and the amino acid sequence of hydrogenase derived from other microorganisms were aligned (FIGS. 1 and 2).
[0032]
The crystal structure of the hydrogenase derived from desulphobibrio gigas has been analyzed, and the position of the hydrophobic core in the protein (site marked with In) and the interface between the large and small subunits Since the position (site marked as Interface in the figure) can be estimated, it is also possible to estimate these positions in the hydrogenase derived from hydrogen Vibrio marinas. Regarding the amino acids at these sites, hydrogenase derived from hydrogenobiblio marinas and hydrogenase derived from other microorganisms were compared with other microorganisms such as valine, leucine, alanine, etc. , Became more hydrophobic isoleucine.
[0033]
Based on the above, in non-thermostable hydrogenases, it is expected that non-thermostable hydrogenases can be converted to thermostable hydrogenases by substituting amino acids presumed to be located at the hydrophobic core and large / small subunit interface with isoleucine. Is done.
[0034]
Therefore, in order to impart heat resistance to the non-heat-resistant hydrogenase derived from Ralstonia / Yutropa, the following experiment was conducted.
[0035]
1 and 2, the arrows shown on the amino acid sequences represent the positions of amino acids into which mutations have been introduced in the following experiments. Also, the arrow with the alphabet indicates the site forming the β-sheet, and the box with the number indicates the site forming the α-helix (both from the crystallographic analysis of the hydrogenase derived from desulphobibrio gigas) Estimated.) In addition, Hma, Reu, Oca, Avi, Ach, Phy, Rca, Bja, Dgi in the figure are hydrogenovibrio marinas, ralstonia eutropha, oligotropha carboxydoborans, azotobacter vinerandi, azotobacter chlorococcam, respectively. It represents Pseudomonas hydrogenobola, Rhodobacta capslatata, Bradyrizobium japonicam, and Desarhovibrio gigas.
[0036]
[Example 1] Production of wild strain of Ralstonia eutropha
Ralstonia eutropha strain H16 has two types of hydrogenase, a soluble hydrogenase and a membrane-bound hydrogenase (B. Friedrich, E. Schwartz (1993) Annu. Rev. Microbiol. 47, 351-383). It is membrane-bound hydrogenase that introduces mutations in hydrogenase this time. Therefore, in order to judge the effect of mutation more clearly, soluble hydrogenase of Ralstonia eutropha H16 strain was analyzed by the method of Massantz et al. (C. Massanz, VM Fernandez, B. Friedrich (1997) Eur. J. Biochem. 245, 441- The Ralstonia eutropha HF387 strain deleted in 448) was used as a wild strain.
[0037]
[Example 2] Preparation of amino acid (1-base) substituted hydrogenase
Contains plasmid pCH182 (M. Bernhard, E. Schwartz, J. Rietdorf, B. Frisedrich (1996) J. Bacteriol. 178, 4522-4529), a clone of the membrane-bound hydrogenase gene from Ralstonia eutropha strain H16 E. coli was cultured in LB medium, and pCH182 was extracted using QIAGEN's Plasmid Midi Kit. Using the extracted pCH182 as a template DNA, a DNA fragment with the following five mutations is prepared using a two-step PCR method (OP Kuipers, HJ Boot, WM de Vos (1991) Nucleic Acid Res. 19, 4558) did.
[0038]
[Table 3]

[0039]
In the two-step PCR method, PCR is performed for the first time using the primers shown in Table 6 as primers for mutagenesis, and the resulting PCR product is purified using QIAGEN's QIAquick Gel Extraction Kit. A second PCR was performed. PCR conditions are as follows.
[0040]
[Table 4]

[0041]
[Table 5]

[0042]
[Table 6]

[0043]
The obtained DNA fragment was inserted into pBluescript (Stratagene Cloning Systems) and transformed into E. coli XL-1 Blue (Stratagene Cloning Systems). Transformants were cultured in LB medium, and plasmids were prepared and sequenced using QIAGEN's QIAprep Spin Miniprep Kit. A sequence reaction was performed using DYEnamic Direct Cycle Sequencing Kit with 7-deaza dGTP (no primer) of Amersham Pharmacia Biotech, and analysis was performed with DNA sequencer LIC-4200S (LI-COR, Inc.).
[0044]
[Example 3] Transformation of mutant hydrogenase into wild-type Ralstonia eutropha strain A fragment containing the hydrogenase gene into which the above five types of mutations were introduced was excised with a restriction enzyme, and suicide for introducing homologous recombination into Ralstonia eutropha Inserted into vector pLO3. Suicide vector pLO3 was prepared according to the method of Lenz et al. (O. Lenz, B. Friedrich (1998) Proc. Natl. Acad. Sci. USA 95, 12474-12479). That is, 0.08 kb HindIII-EcoRI containing the polylinker site of pNEB194 (New England Biolabs, Inc.) was blunt-ended with Klenow fragment (New England Biolabs, Inc.), and also blunt-ended with pBR322 (New England Biolabs, Inc.) (PCH639). 1.96kb BamHI-NdeI containing the sacB gene of pUD800 (P. Gay, D. Le Cop, M. Steinmetz, T. Berkelman, CI Kado (1985) J. Bacteriol. 164, 918-921) It was ligated to the StyI-BsaI site of pCH639 terminated (pCH640). pCH640 was digested with NdeI to make blunt ends, and then pJF145 (JP Fuerste, W. Pansegrau, G. Ziegelin, M. Kroeger, E. Lanka (1979) Proc. Natl. Acad. Sci. USA 86, 1771-1775 ) OriT was subcloned as a blunt-ended 0.47 kb BamHI fragment to prepare pLO3.
[0045]
The suicide vector pLO3 into which the mutant hydrogenase gene was inserted was transferred to the Ralstonia eutropha HF387 strain, and a recombinant in which the mutation was introduced into the membrane-bound hydrogenase gene was selected by homologous recombination. Conjugate transfer is performed according to the method of R. Simon et al. (R. Simon, U. Priefer, U. Puehler (1983) Bio / Technology 1, 784-791), and selection of recombinants is performed by the method of O. Lenz et al. (O. Lenz, E. Schwartz, M. Eitinger, B. Friedrich (1994) J. Bacteriol. 176, 4385-4393). That is, Escherichia coli carrying the above-mentioned plasmid was cultured overnight at 37 ° C. in 10 ml of tetracycline-containing LB medium and R. eutropha HF387 strain in 10 ml of LB medium. After washing the cells with sterilized water, the cells were suspended in 1 ml of sterilized water, 0.2 ml each was mixed on the LB plate medium, and cultured overnight at 37 ° C. to allow plasmid conjugation transfer. . Next, recombinants in which a mutation was introduced into the wild type hydrogenase gene by homologous recombination were selected. First, a recombinant in which homologous recombination occurred between the mutant hydrogenase gene linked to pLO3 and the wild-type hydrogenase gene present in Ralstonia eutropha in one place (pLO3 integrated in the genome) The tetracycline resistance encoded by is selected as a marker. Next, this recombinant was inoculated into 10 ml of LB medium and cultured overnight at 37 ° C., then inoculated into LB plate medium containing 15% sucrose, cultured at 37 ° C., and integrated into the genome. Recombinants in which pLO3 was lost due to recombination at another site between the mutant hydrogenase gene and the wild type hydrogenase gene on pLO3 were selected using sacB as a marker. That is, a high-concentration sucrose-resistant strain that forms colonies in this medium containing 15% sucrose was selected as a homologous recombinant in which a gene sequence was exchanged between an externally introduced mutant hydrogenase gene and a wild-type hydrogenase gene. did.
[0046]
[Example 4] Evaluation of thermostability of the active center of hydrogenase The hydrogenase mutant obtained as described above was prepared as an FGN medium (O. Lenz, E. Schwartz, M. Eitinger, B. Friedrich ( 1994) J. Bacteriol. 176, 4385-4393), and the membrane fraction containing membrane-bound hydrogenase from the cells was obtained by the method of Nishihara et al. (H. Nishihara, Y. Miyashita, K. Aoyama, T. Kodama, Y. Igarashi, Y. Takamura (1997) Biochem. Biophys. Res. Commun. 232, 766-770).
[0047]
This membrane fraction was heated to 70 ° C. and sampled over time, and the remaining deuterium displacement activity was measured, thereby evaluating the heat resistance of the active center of hydrogenase. The deuterium substitution reaction was measured according to the method of Cammack et al. (R. Cammack, VM Fernandez, EC Hatchikian (1994) Methods. Enzymol. 234, 43-68). That is, the reaction was started by adding the enzyme solution with a syringe to 10 ml of 50 mM acetate buffer previously saturated with deuterium. The amount of hydrogen generated in the gas phase was measured over time with a mass spectrometer, and the activity intensity was expressed as the amount of hydrogen generated per unit time. The result is shown in FIG.
[0048]
As shown in FIG. 3, all of the hydrogenases introduced with mutations had higher activity than the wild type hydrogenase, and the heat resistance of the active center was improved.
[0049]
[Example 5] Evaluation of heat resistance of subunit structure of hydrogenase The membrane fraction prepared in Example 4 was heated to 55 ° C and sampled over time, and the remaining methylene blue reducing activity was measured. In order to reduce methylene blue, it is necessary that the electrons extracted from hydrogen at the active center (large subunit) of hydrogenase are successfully transferred to the small subunit. The heat resistance of can be evaluated. The methylene blue reduction reaction was measured according to the method of Schink and HG Schlegel (B. Schink, HG Schlegel (1979) Biochem. Biophys. Acta 567, 315-324). The result is shown in FIG.
[0050]
As shown in FIG. 4, the stability of the subunit structure is lower than that of the wild type hydrogenase (V115I, V253I, N168I, L170I), and the stability is improved by P181I and L243I. There were only two.
[0051]
【The invention's effect】
The present invention provides a means for imparting heat resistance to hydrogenases. Thereby, a non-thermostable hydrogenase producing bacterium can be changed to a thermostable hydrogenase producing bacterium. Therefore, it is possible to select bacteria that can be easily cultured and efficiently produce thermostable hydrogenase.
[Sequence Listing]

[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram showing alignment results of a large hydrogenase subunit (first half).
FIG. 2 is a diagram showing the result of alignment of the hydrogenase large subunit (second half).
FIG. 3 is a graph showing the change over time in the deuterium displacement activity of the hydrogenase fraction.
FIG. 4 is a graph showing the change over time of the methylene blue reducing activity of the hydrogenase fraction.

Claims (8)

  In the large subunit of class I hydrogenase, when aligned with the amino acid sequence of SEQ ID NO: 2 as an index, the amino acid corresponding to the 181st proline or the 243rd leucine in the amino acid sequence is replaced with isoleucine A method for producing a thermostable hydrogenase, comprising:   The method for producing a thermostable hydrogenase according to claim 1, wherein the large subunit of class I hydrogenase is a large subunit of class I hydrogenase derived from Ralstonia jutropha. A thermostable hydrogenase produced by the method according to claim 2 .   DNA encoding the thermostable hydrogenase according to claim 3.   A microorganism comprising the DNA according to claim 4 and producing a thermostable hydrogenase.   A method for producing a thermostable hydrogenase, comprising culturing the microorganism according to claim 5 and collecting thermostable hydrogenase from the obtained culture. A method for producing a microorganism by producing a thermostable hydrogenase comprising the following steps , which is a method for producing a microorganism by a homologous recombination method.
(1) A DNA fragment encoding all or part of the large subunit of class I hydrogenase, which is aligned with the amino acid sequence described in SEQ ID NO: 2 using the amino acid identity as an index, and the 181st amino acid sequence A DNA fragment in which the sequence encoding the amino acid corresponding to proline or the 243rd leucine is substituted with a sequence encoding isoleucine (2), the DNA fragment prepared in (1), an antibiotic resistance gene, And a step (3) for producing a vector containing a gene that exhibits lethality under a specific condition, a step (4) for introducing the vector produced in (2) into the microorganism, and a microorganism and a class I hydrogenase introduced with the vector in (3) (5) A microorganism producing a class I hydrogenase in a medium containing the aforementioned antibiotics Select in the steps (6) and (5) of selecting the strains in which the DNA fragments prepared in (1), the antibiotic resistance genes, and the genes that exhibit lethality under specific conditions are inserted into the genomic DNA A step of culturing the obtained strain under the condition that the lethality gene exhibits the lethality, and selecting a strain in which the resistance gene to the antibiotic and the gene exhibiting the lethality under the specific condition have been dropped   The method for producing a microorganism producing a thermostable hydrogenase according to claim 7, wherein the large subunit of class I hydrogenase is a large subunit of class I hydrogenase derived from Ralstonia jutropha.

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