JP2005278637A - Method for producing cytochrome p450 monooxygenase - Google Patents
Method for producing cytochrome p450 monooxygenase Download PDFInfo
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- JP2005278637A JP2005278637A JP2005038576A JP2005038576A JP2005278637A JP 2005278637 A JP2005278637 A JP 2005278637A JP 2005038576 A JP2005038576 A JP 2005038576A JP 2005038576 A JP2005038576 A JP 2005038576A JP 2005278637 A JP2005278637 A JP 2005278637A
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- Prior art keywords
- monooxygenase
- cytochrome
- ppiase
- producing
- derived
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Images
Abstract
Description
本発明は生触媒工学、すなわち生変換反応による有用物質合成領域に属するものである。より具体的には、本発明は、産業上有用な有機化合物の合成に使われている酵素であるシトクロムP450モノオキシゲナーゼ（以下、単に「P450モノオキシゲナーゼ」という場合がある。）を、機能を損なわない形で安定的に生産する方法を提供する。元来不安定な酵素であるP450モノオキシゲナーゼをコードする遺伝子を安定的に微生物内で機能発現することにより、種々の有用な生変換反応を効率的に行うことができる。 The present invention belongs to the field of biocatalyst engineering, that is, a useful substance synthesis region by bioconversion reaction. More specifically, the present invention impairs the function of cytochrome P450 monooxygenase (hereinafter sometimes simply referred to as “P450 monooxygenase”), which is an enzyme used in the synthesis of industrially useful organic compounds. Providing a stable production method without any problems. By stably expressing a gene encoding P450 monooxygenase, which is an originally unstable enzyme, in a microorganism, various useful bioconversion reactions can be efficiently performed.
P450モノオキシゲナーゼは、細菌から植物や哺乳動物に至る広範囲の生物種に分布している酸素添加酵素であり、酸素分子を還元して1分子の水を作ると共に、残りの酸素原子を様々な有機化合物(基質)に導入して、酸化生成物に変換する反応（一原子酸素添加反応）を触媒する鉄・イオウ酵素である。哺乳動物におけるP450モノオキシゲナーゼは非常によく研究されており、生体内に取り込まれた薬物や環境汚染毒物を酸化し水溶性を高め、体外への排出を促進する生体防御の役割を担う鍵酵素であり、また、生体内の二次代謝産物の生合成において水酸化反応等を担う鍵酵素でもある。P450モノオキシゲナーゼは、シトクロムP450本体タンパク質（CYP）以外に電子伝達タンパク質を必要とする複合酵素である。すなわち、P450が活性を発揮するには、NAD(P)Hから電子を伝達してくるタンパク質として、細菌や真核生物のミトコンドリアでは フェレドキシンレダクターゼとフェレドキシンの介在を必要とし、哺乳動物の肝臓ミクロソームでは NADPH-P450レダクターゼの介在を必要とする。P450モノオキシゲナーゼは部分的な構造の変化により多彩な化合物の代謝に多様化された酵素で、バイオテクノロジーの分野において既存の方法では難しいと思われている化合物の酸化処理が可能になると考えられている。したがって、本酵素は、新しい有機（低分子）化合物（たとえば，位置/立体特異的に導入された水酸化物など）の合成や、分解処理が難しい環境負荷物質の処理への応用に期待されている。 P450 monooxygenase is an oxygenated enzyme distributed in a wide range of biological species from bacteria to plants and mammals. It reduces oxygen molecules to make one molecule of water, and the remaining oxygen atoms are converted into various organic substances. It is an iron / sulfur enzyme that catalyzes a reaction (monoatomic oxygenation reaction) that is introduced into a compound (substrate) and converted into an oxidation product. P450 monooxygenase in mammals has been studied very well, and is a key enzyme that plays a role in the defense of the body that oxidizes drugs and environmental pollutants that are taken into the body to increase water solubility and promote their release outside the body. It is also a key enzyme responsible for hydroxylation and the like in the biosynthesis of secondary metabolites in vivo. P450 monooxygenase is a complex enzyme that requires an electron transfer protein in addition to cytochrome P450 body protein (CYP). In other words, in order for P450 to exert its activity, as a protein that transfers electrons from NAD (P) H, bacteria and eukaryotic mitochondria require the intermediation of ferredoxin reductase and ferredoxin, and in mammalian liver microsomes Requires the intervention of NADPH-P450 reductase. P450 monooxygenase is an enzyme that has been diversified into a variety of compound metabolisms due to partial structural changes, and is thought to be capable of oxidizing compounds that are considered difficult by existing methods in the field of biotechnology. Yes. Therefore, this enzyme is expected to be applied to the synthesis of new organic (low molecular weight) compounds (for example, hydroxide introduced in a position / stereospecific manner) and the treatment of environmentally hazardous substances that are difficult to decompose. Yes.
細菌をはじめとする微生物のP450研究は哺乳動物に比べると遅れていたが、細菌のゲノム解析の進展と相まって、細菌のP450研究が今日、盛んになってきている。細菌で最もよく研究されているP450モノオキシゲナーゼは、シュードモナス・プチダ由来のP450camで、本酵素は、樟脳（camphor）を基質として、5位に水酸基を導入する反応を触媒することが明らかにされており、P450cam遺伝子を導入・発現した組換え細菌を用いた解析も広く実施されている（Poulos, T.L., Finzel, B.C., and Howard, A,J., High-resolution crystal structure of cytochrome P450cam. J. Mol. Biol., 195, 687-700 (1987)）。また、高脂血症薬プラバスタチン（pravastatin;三共株式会社 商品名メバロチン）は、コンパクチン（compactin）カルボン酸の立体特異的な水酸化反応により合成されるが、この反応を担う酵素は、土壌細菌ストレプトマイセス・カルボフィラス（Streptmyces carbophilus）由来のP450モノオキシゲナーゼである（Matsuoka, T., Miyakoshi, S., Tanzawa, K., Nakahara, K., Hosobuchi, M., and Serizawa, N., Eur. J. Biochem., 184, 707-713, 1989）。 Research on P450s of microorganisms including bacteria has been delayed compared to mammals, but coupled with the progress of bacterial genome analysis, research on P450s of bacteria has become popular today. The most well-studied P450 monooxygenase in bacteria is P450cam derived from Pseudomonas putida, which has been shown to catalyze the reaction of introducing a hydroxyl group at the 5-position using camphor as a substrate. Analysis using recombinant bacteria into which P450cam gene has been introduced and expressed has also been widely carried out (Poulos, TL, Finzel, BC, and Howard, A, J., High-resolution crystal structure of cytochrome P450cam. Mol. Biol., 195, 687-700 (1987)). The hyperlipidemic drug pravastatin (trade name: mevalotin) is synthesized by stereospecific hydroxylation of compactin carboxylic acid, and the enzyme responsible for this reaction is streptococcal soil. P450 monooxygenase from Streptmyces carbophilus (Matsuoka, T., Miyakoshi, S., Tanzawa, K., Nakahara, K., Hosobuchi, M., and Serizawa, N., Eur. J) Biochem., 184, 707-713, 1989).
P450酵素は、肝ミクロソーム画分のものに関しては広く研究が行われ、試験用酵素も売り出されている。また、放線菌ストレプトマイセス・セリカラー（Streptomyces coelicolor）のゲノム配列がすでに決定され、本細菌が18個のP450配列を持っていることが明らかになったこともあり（D. C. Lamb et al, The cytochrome P450 complement (CYPome) of Streptomyces coelicolor A3 (2), J. Biol. Chem. 277, 24000-24005, 2002）、放線菌や他の細菌のP450を用いた研究も最近、盛んになりつつある。シトクロムP450モノオキシゲナーゼ遺伝子は、植物や動物由来のものと比べて、細菌（放線菌も細菌）由来のものは、大腸菌内で機能発現しやすいことがわかっていた。真核生物由来のもの（前者）が膜酵素であるのに対して、原核生物由来のもの（後者）が細胞質内の可溶性酵素ということが、その最大の理由であると考えられる。細菌バチルス・メガテリウム（Bacillus megaterium）やミクロテトラスポラ（Microtetraspora）属放線菌由来のP450モノオキシゲナーゼ遺伝子が大腸菌内で機能発現し、基質となる有機低分子化合物が水酸化されることが報告されている（小川 順ら, Bacillus megaterium由来のシトクロムP450 BM-3変異型酵素を用いるパラヒドロキノン類の酵素合成, 日本農芸化学会2003年大会講演要旨, p. 38, 2003、及び、有澤 章ら, 放線菌シトクロムP450および電子伝達タンパク質を共発現させた大腸菌による微生物変換系, 日本農芸化学会2003年大会講演要旨, p. 38, 2003）。しかし、細菌由来の同酵素でさえ、組換え大腸菌内で かなりの部分が正常に機能していないだけでなく、正常に機能していた酵素もしだいに失活していくことが観察された。P450モノオキシゲナーゼ酵素そのものが不安定酵素であると考えられている（H. Joo, Z. Lin, and F. H. Arnold, Laboratory evolution of peroxide-mediated cytochrome P450 hydroxylation. Nature 399, 670-673, 1999）。 P450 enzymes have been extensively studied for those in the liver microsomal fraction, and test enzymes are also on the market. In addition, the genome sequence of Streptomyces coelicolor has already been determined, and it has become clear that this bacterium has 18 P450 sequences (DC Lamb et al, The cytochrome). P450 complement (CYPome) of Streptomyces coelicolor A3 (2), J. Biol. Chem. 277, 24000-24005, 2002), studies on actinomycetes and other bacteria using P450 are also becoming popular recently. The cytochrome P450 monooxygenase gene was found to be more easily expressed in E. coli when it was derived from bacteria (actinomycetes were also bacteria) than from plants and animals. It is thought that the main reason is that the eukaryotic origin (the former) is a membrane enzyme, whereas the prokaryote origin (the latter) is a soluble enzyme in the cytoplasm. It has been reported that P450 monooxygenase genes derived from the bacteria Bacillus megaterium and Microtetraspora actinomycetes are functionally expressed in Escherichia coli and hydroxylate organic low-molecular compounds as substrates. (Ogawa Jun et al., Enzymatic synthesis of parahydroquinones using cytochrome P450 BM-3 mutant enzyme derived from Bacillus megaterium , Abstracts of the 2003 Annual Meeting of the Agricultural Chemical Society of Japan, p. 38, 2003, and Arisawa Akira et al., Actinomycetes Microbial transformation system by E. coli co-expressed with cytochrome P450 and electron transfer protein, Abstracts of the 2003 Annual Meeting of the Japanese Society for Agricultural Chemistry, p. 38, 2003). However, even the same enzyme derived from bacteria was observed not only to function a significant part in the recombinant E. coli, but also to gradually deactivate the functioning enzyme. P450 monooxygenase enzyme itself is considered to be an unstable enzyme (H. Joo, Z. Lin, and FH Arnold, Laboratory evolution of peroxide-mediated cytochrome P450 hydroxylation. Nature 399, 670-673, 1999).
発明者が所属する研究機関である(株)海洋バイオテクノロジー研究所では、積水化学工業(株)との共同研究により、免疫抑制剤FK506と結合するタンパク質であるPPIase（ペプチジルプロリルcis-transイソメラーゼ）遺伝子を好熱性古細菌ピロコッカス・ホリコシ（Pyrococcus horikoshii）等より単離した。本PPIaseは、古細菌の不安定な酵素であるロダネーゼ（rhodanese）や、不溶化しやすい鶏卵のリゾチーム抗体のFab断片等を、本来の立体構造に巻き戻し 可溶化させることができた（特開平10-91号公報）。しかも、ロダネーゼは本PPIase共存下で100℃まで安定であった。したがって、本PPIaseは高温でも安定な分子シャペロンであることがわかった。また、安定的に機能発現させたいタンパク質のN末に、前記PPIaseまたは大腸菌のシャペロニンGroELを融合させることにより、そのタンパク質が本来の立体構造に巻き戻されることがわかった。この融合タンパク質系を用いて、毒性の強いB型肝炎ウィルス表面抗原や不溶性のヒトIgGの重鎖等をコードする遺伝子が、安定的に組換え大腸菌内で機能発現されることがわかった（特許文献１、特許文献２）。最近、三井化学もPPIaseのシャペロン活性に着目し、大腸菌のPPIaseを利用することにより、大腸菌で生産が難しかったヒトのミッドカインや組織プラスミノーゲン活性化因子（TPA）の生産量の増強に成功したと発表している（日経バイオテク、2003年5月21日）。 In collaboration with Sekisui Chemical Co., Ltd., the research institution to which the inventor belongs, Marine Biotechnology Research Institute Co., Ltd., PPIase (peptidylprolyl cis - trans isomerase), a protein that binds to the immunosuppressant FK506 ) The gene was isolated from a thermophilic archaebacterium Pyrococcus horikoshii . This PPIase was able to unwind and solubilize Rhodanese, an unstable enzyme of archaebacteria, and the Fab fragment of lysozyme antibody of hen's egg, which is easily insolubilized, to the original three-dimensional structure (Japanese Patent Application Laid-Open No. 10-151). -91). Moreover, rhodanese was stable up to 100 ° C in the presence of this PPIase. Therefore, this PPIase was found to be a stable molecular chaperone even at high temperatures. It was also found that the PPIase or Escherichia coli chaperonin GroEL was fused to the N-terminal of the protein to be stably expressed in function, whereby the protein was unwound to its original three-dimensional structure. Using this fusion protein system, it was found that genes encoding highly toxic hepatitis B virus surface antigen, insoluble human IgG heavy chain, etc. were stably expressed in recombinant E. coli (patents) Literature 1, Patent Literature 2). Recently, Mitsui Chemicals also focused on the chaperone activity of PPIase and succeeded in enhancing the production of human midkine and tissue plasminogen activator (TPA), which were difficult to produce in E. coli, by using PPIase of E. coli. (Nikkei Biotech, May 21, 2003).
細菌バチルス・メガテリウム（Bacillus megaterium） BM-3株由来のP450モノオキシゲナーゼ（P450 BM-3）は、P450ドメインとNADPH-P450レダクターゼドメインが結合した、大変珍しい構造を有する酵素である。本酵素の塩基配列は、DDBJ Accession No. J04832に登録されている。本酵素は元々、C12〜C20の飽和脂肪酸のω末端（メチル基側）の1位、2位または3位を水酸化する酵素であったが、87番目のフェニルアラニンがバリンに変異したもの（以下、この変異酵素を「F87V」という場合がある。）は、ナフタレンや2-ブロモフェノール等の芳香族化合物を広く基質として認識し 水酸基を導入することができた（小川 順ら, Bacillus megaterium由来のシトクロムP450(BM-3)変異型酵素を用いるパラヒドロキノン類の酵素合成, 日本農芸化学会2003年大会講演要旨, p. 38, 2003、及び、Q. -W. Li, U. Schwaneberg, P. Fischer, and R. D. Schmid, Directed evolution of the fatty-acid hydoroxylase P450 BM-3 into an indole-hydroxylating catalyst. Chem. Eur. J. 6, 1531-1536, 2000）。 P450 monooxygenase (P450 BM-3) derived from the bacterium Bacillus megaterium BM-3 strain is an enzyme having a very unusual structure in which a P450 domain and a NADPH-P450 reductase domain are combined. The base sequence of this enzyme is registered in DDBJ Accession No. J04832. This enzyme was originally an enzyme that hydroxylates the 1st, 2nd, or 3rd position of the C12-C20 saturated fatty acid at the ω-terminal (methyl group side), but the 87th phenylalanine was mutated to valine This mutant enzyme is sometimes referred to as “F87V”.) Aromatic compounds such as naphthalene and 2-bromophenol were widely recognized as substrates and hydroxyl groups were introduced (from Ogawa Jun et al., Bacillus megaterium ) Enzymatic synthesis of parahydroquinones using cytochrome P450 (BM-3) mutant enzyme, Abstracts of the 2003 Annual Meeting of the Agricultural Chemical Society of Japan, p. 38, 2003, and Q. -W. Li, U. Schwaneberg, P. Fischer, and RD Schmid, Directed evolution of the fatty-acid hydoroxylase P450 BM-3 into an indole-hydroxylating catalyst. Chem. Eur. J. 6, 1531-1536, 2000).
P450モノオキシゲナーゼは、その構造だけでなく、基質特異性や反応特異性に関しても非常に多様であるので、生変換反応による有用物質合成領域への応用、すなわち、産業利用可能な様々な有機低分子化合物の合成に利用されることが期待されている酵素である。また実際に、高脂血症薬プラバスタチン（pravastatin；三共株式会社 商品名メバロチン）の製造に、放線菌が有するP450モノオキシゲナーゼの機能が利用されている。一方で、P450モノオキシゲナーゼは、失活しやすくターンオーバーが悪い不安定酵素として有名で、異種宿主中で安定的に生産する方法は知られていなかった。そのため、外来遺伝子としてP450モノオキシゲナーゼ遺伝子を導入・発現した組換え微生物を利用して、有用な有機化合物の実際の工業生産を行った例はほとんど存在しないと思われる。本発明では、実際の工業生産を可能にするため、外来のP450モノオキシゲナーゼ遺伝子を安定的に機能発現する方法を開発することを課題としている。 P450 monooxygenase is not only its structure but also very diverse in terms of substrate specificity and reaction specificity, so it can be applied to the synthesis of useful substances by biotransformation reaction, that is, various organic small molecules that can be used industrially. It is an enzyme that is expected to be used in the synthesis of compounds. Actually, the function of P450 monooxygenase possessed by actinomycetes is used for the production of the hyperlipidemic drug pravastatin (trade name: mevalotin, Sankyo Co., Ltd.). On the other hand, P450 monooxygenase is famous as an unstable enzyme that is easily inactivated and has poor turnover, and a method for stably producing it in a heterologous host has not been known. For this reason, there seems to be few examples of actual industrial production of useful organic compounds using recombinant microorganisms that have introduced and expressed the P450 monooxygenase gene as a foreign gene. In the present invention, in order to enable actual industrial production, an object is to develop a method for stably expressing a foreign P450 monooxygenase gene.
分子シャペロンとしては、GroEL等のシャペロニンやPPIase（ペプチジルプロリルcis-transイソメラーゼ）等多くのものが知られているが、発明者らは好熱性の古細菌由来のPPIaseに着目した。その理由は、古細菌由来のPPIaseは、失活しにくく、構造的にも真正細菌由来のPPIaseと異なっていること、さらには、ロダネーゼ遺伝子等の大腸菌での機能発現に実績があるからである。シャペロニンまたはPPIaseを利用したタンパク質の安定的生産の研究が盛んに行われてきたことは「背景技術」で述べたとおりである。一方、P450モノオキシゲナーゼに関する研究はきわめて盛んであることは言うまでもない。試しに、「PPIase」及び「P450」をキーワードとして、米国NCBI（National Center for Biotechnology Information）のEntrez-PubMedで検索すると、それぞれ、2,364件、及び、16,433件ヒットした。ところが、「PPIase & P450」で同データベースを検索すると、2件しかヒットがなく、しかもこの2件は、P450モノオキシゲナーゼの安定化には全く関係のない文献であった。P450モノオキシゲナーゼの安定化は、ニーズが多いはずにもかかわらず、今までPPIaseを利用した方法が存在しなかったのは、この方法の開発が困難であることを物語っている。本発明者らは、古細菌のPPIaseを利用するだけでなく、P450モノオキシゲナーゼの中では例外的に、１つのペプチドがP450モノオキシゲナーゼのホロ酵素である真正細菌バチルス・メガテリウム由来のP450モノオキシゲナーゼに着目してこれをP450モノオキシゲナーゼのモデルとして利用することにより、本発明を完成するに至った。 Many molecular chaperones are known, such as chaperonins such as GroEL and PPIases (peptidylprolyl cis - trans isomerase), but the inventors have focused on thermophilic archaeal PPIases. The reason is that PPIase derived from archaea is difficult to inactivate, is structurally different from PPIase derived from eubacteria, and has a proven record of functional expression in E. coli such as the rhodanese gene. . As described in “Background Art”, research on stable production of proteins using chaperonin or PPIase has been actively conducted. On the other hand, it goes without saying that research on P450 monooxygenase is very active. As a test, when searching with Entrez-PubMed of NCBI (National Center for Biotechnology Information) using “PPIase” and “P450” as keywords, 2,364 hits and 16,433 hits were found, respectively. However, when the database was searched with “PPIase & P450”, there were only two hits, and these two documents were completely unrelated to the stabilization of P450 monooxygenase. Despite the need for stabilization of P450 monooxygenase, there has been no method using PPIase until now, which means that it is difficult to develop this method. In addition to using archaeal PPIase, the present inventors, as an exception among P450 monooxygenases, are P450 monooxygenases derived from the authentic bacterium Bacillus megaterium, which is a holoenzyme of P450 monooxygenase. By paying attention and using this as a model of P450 monooxygenase, the present invention has been completed.
すなわち、本発明は以下の（１）〜（１５）を提供するものである。 That is, the present invention provides the following (1) to (15).
（１）シトクロムP450モノオキシゲナーゼと古細菌由来のPPIaseの両者を生産可能な微生物を培養し、培養物又は菌体からシトクロムP450モノオキシゲナーゼを採取することを特徴とするシトクロームP450モノオキシゲナーゼの生産方法。 (1) A method for producing cytochrome P450 monooxygenase, comprising culturing a microorganism capable of producing both cytochrome P450 monooxygenase and archaeal PPIase, and collecting cytochrome P450 monooxygenase from the culture or the cells.
（２）シトクロムP450モノオキシゲナーゼが、細菌由来のシトクロムP450モノオキシゲナーゼであることを特徴とする（１）に記載のシトクロームP450モノオキシゲナーゼの生産方法。 (2) The method for producing cytochrome P450 monooxygenase according to (1), wherein the cytochrome P450 monooxygenase is a cytochrome P450 monooxygenase derived from bacteria.
（３）シトクロムP450モノオキシゲナーゼが、バチルス・メガテリウムBM-3株由来のシトクロムP450モノオキシゲナーゼ、又はその一部を改変したシトクロムP450モノオキシゲナーゼであることを特徴とする（１）に記載のシトクロームP450モノオキシゲナーゼの生産方法。 (3) The cytochrome P450 monooxygenase is a cytochrome P450 monooxygenase derived from a Bacillus megaterium BM-3 strain, or a cytochrome P450 monooxygenase obtained by modifying a part thereof. A method for producing oxygenase.
（４）バチルス・メガテリウムBM-3株由来のシトクロムP450モノオキシゲナーゼの一部を改変したシトクロムP450モノオキシゲナーゼが、バチルス・メガテリウムBM-3株由来のシトクロムP450モノオキシゲナーゼの87番目のフェニルアラニンをバリンに置換したシトクロムP450モノオキシゲナーゼであることを特徴とする（３）に記載のシトクロームP450モノオキシゲナーゼの生産方法。 (4) Cytochrome P450 monooxygenase modified from a part of cytochrome P450 monooxygenase derived from Bacillus megaterium BM-3 replaces 87th phenylalanine of cytochrome P450 monooxygenase derived from Bacillus megaterium BM-3 with valine. The method for producing cytochrome P450 monooxygenase according to (3), wherein the method is cytochrome P450 monooxygenase.
（５）微生物が、大腸菌である（１）乃至（４）のいずれか一項に記載のシトクロームP450モノオキシゲナーゼの生産方法。 (5) The method for producing cytochrome P450 monooxygenase according to any one of (1) to (4), wherein the microorganism is Escherichia coli.
（６）シトクロムP450モノオキシゲナーゼのN末端側に古細菌由来のPPIaseを付加した融合タンパク質を生産可能な微生物を培養し、培養物又は菌体から、活性のあるシトクロームP450モノオキシゲナーゼを含む融合タンパク質を採取することを特徴とするシトクロームP450モノオキシゲナーゼの生産方法。 (6) A microorganism capable of producing a fusion protein in which an archaebacterial PPIase is added to the N-terminal side of cytochrome P450 monooxygenase is cultured, and a fusion protein containing active cytochrome P450 monooxygenase is obtained from the culture or the cells. A method for producing cytochrome P450 monooxygenase, comprising collecting the cytochrome P450 monooxygenase.
（７）融合タンパク質が、シトクロムP450モノオキシゲナーゼと古細菌由来のPPIaseとの間に切断部位を含む融合タンパク質であることを特徴とする（６）に記載のシトクロームP450モノオキシゲナーゼの生産方法。 (7) The method for producing cytochrome P450 monooxygenase according to (6), wherein the fusion protein is a fusion protein containing a cleavage site between cytochrome P450 monooxygenase and archaeal PPIase.
（８）シトクロムP450モノオキシゲナーゼが、細菌由来のシトクロムP450モノオキシゲナーゼであることを特徴とする（６）又は（７）に記載のシトクロームP450モノオキシゲナーゼの生産方法。 (8) The method for producing cytochrome P450 monooxygenase according to (6) or (7), wherein the cytochrome P450 monooxygenase is a cytochrome P450 monooxygenase derived from bacteria.
（９）シトクロムP450モノオキシゲナーゼが、バチルス・メガテリウムBM-3株由来のシトクロムP450モノオキシゲナーゼ、又はその一部を改変したシトクロムP450モノオキシゲナーゼであることを特徴とする（６）又は（７）に記載のシトクロームP450モノオキシゲナーゼの生産方法。 (9) The cytochrome P450 monooxygenase is a cytochrome P450 monooxygenase derived from a Bacillus megaterium BM-3 strain, or a cytochrome P450 monooxygenase obtained by modifying a part thereof (6) or (7) Of production of cytochrome P450 monooxygenase.
（１０）バチルス・メガテリウムBM-3株由来のシトクロムP450モノオキシゲナーゼの一部を改変したシトクロムP450モノオキシゲナーゼが、バチルス・メガテリウムBM-3株由来のシトクロムP450モノオキシゲナーゼの87番目のフェニルアラニンをバリンに置換したシトクロムP450モノオキシゲナーゼであることを特徴とする（９）に記載のシトクロームP450モノオキシゲナーゼの生産方法。 (10) Cytochrome P450 monooxygenase modified from a part of cytochrome P450 monooxygenase derived from Bacillus megaterium BM-3 replaces 87th phenylalanine of cytochrome P450 monooxygenase derived from Bacillus megaterium BM-3 with valine. The method for producing cytochrome P450 monooxygenase according to (9), wherein the method is cytochrome P450 monooxygenase.
（１１）微生物が、大腸菌である（６）乃至（１０）のいずれかに記載のシトクロームP450モノオキシゲナーゼの生産方法。 (11) The method for producing cytochrome P450 monooxygenase according to any one of (6) to (10), wherein the microorganism is Escherichia coli.
（１２）シトクロムP450モノオキシゲナーゼと古細菌由来のPPIaseの両者を生産可能な微生物。 (12) A microorganism capable of producing both cytochrome P450 monooxygenase and archaeal PPIase.
（１３）シトクロムP450モノオキシゲナーゼのN末端側に古細菌由来のPPIaseを付加した融合タンパク質を生産可能な微生物。 (13) A microorganism capable of producing a fusion protein in which an archaebacterial PPIase is added to the N-terminal side of cytochrome P450 monooxygenase.
（１４）シトクロムP450モノオキシゲナーゼのN末端側に古細菌由来のPPIaseを付加した融合タンパク質。 (14) A fusion protein in which an archaeal PPIase is added to the N-terminal side of cytochrome P450 monooxygenase.
（１５）（１４）に記載の融合タンパク質をコードするDNA。 (15) A DNA encoding the fusion protein according to (14).
本発明は、古細菌由来のPPIaseを利用することによりP450モノオキシゲナーゼ遺伝子を大腸菌等の微生物宿主内で安定的に機能発現させる方法を提供する。そのような組換え微生物を用いて、生変換反応による産業上有用な有機（低分子）化合物の合成が可能になる。 The present invention provides a method for stably function-expressing a P450 monooxygenase gene in a microbial host such as Escherichia coli by using PPIase derived from archaea. Using such recombinant microorganisms, it is possible to synthesize industrially useful organic (low molecular weight) compounds by bioconversion reactions.
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
最初にP450モノオキシゲナーゼとPPIaseについて説明する。 First, P450 monooxygenase and PPIase will be described.
１．P450モノオキシゲナーゼ
P450モノオキシゲナーゼは、細菌から植物や哺乳動物に至る広範囲の生物種に分布している酸素添加酵素であり、酸素分子を還元して1分子の水を作ると共に、残りの酸素原子を様々な脂溶性の有機（低分子）化合物(基質)に導入して、酸化生成物に変換する反応（一原子酸素添加反応）を触媒する鉄・イオウ酵素である。哺乳動物のP450モノオキシゲナーゼは非常によく研究されており、本酵素は、生体内に取り込まれた薬物や環境汚染毒物を酸化し水溶性を高め、体外への排出を促進する生体防御の役割を担う鍵酵素（肝臓ミクロソーム型P450）であり、また、生体内の二次代謝産物の生合成において水酸化反応等を担う鍵酵素（ミトコンドリア型P450）でもある。ゲノム配列の解析からP450は、ヒトでは約57種類、細菌では、アグロバクテリウム・ツメファシエンス（Agrobacterium tumefaciens）が5種類、放線菌ストレプトマイセス・セリカラー（Streptomyces coelicolor）が18種類、ストレプトマイセス・エバーミチリス（S. avermitilis）が33種類、マイコバクテリウム・ツベルクロシスが20種類あることが知られている。なお、大腸菌はP450を有していない。
1. P450 monooxygenase
P450 monooxygenase is an oxygenated enzyme distributed in a wide range of biological species from bacteria to plants and mammals. It reduces oxygen molecules to make one molecule of water, and the remaining oxygen atoms are converted to various fats. It is an iron / sulfur enzyme that catalyzes a reaction (monoatomic oxygenation reaction) that is introduced into a soluble organic (low molecular weight) compound (substrate) and converted into an oxidation product. Mammalian P450 monooxygenase has been studied very well, and this enzyme plays a role in the defense of the body that oxidizes drugs and environmental pollutants that are taken into the body to increase water solubility and promote their release outside the body. It is a key enzyme (liver microsomal type P450) that bears, and also a key enzyme (mitochondrial type P450) that plays a role in hydroxylation in the biosynthesis of secondary metabolites in vivo. Genomic sequence analysis revealed that about 450 P450s were found in humans, 5 types in bacteria were Agrobacterium tumefaciens , 18 types in Streptomyces coelicolor , and Streptomyces evermitilis. It is known that there are 33 types of ( S. avermitilis ) and 20 types of Mycobacterium tuberculosis. E. coli does not have P450.
P450モノオキシゲナーゼは、シトクロムP450本体タンパク質（CYP）以外に電子伝達タンパク質を必要とする複合酵素である。すなわち、P450が活性を発揮するには、NAD(P)Hから電子を伝達してくるタンパク質として、細菌や真核生物のミトコンドリアでは フェレドキシンレダクターゼとフェレドキシンの介在を必要とし、哺乳動物の肝臓ミクロソームでは NADPH-P450レダクターゼの介在を必要とする。また、CYPそのものも きわめて多様である。すなわち、CYPファミリー間のアミノ酸配列の相同性は40％以下であり、共通部分は、ヘム鉄と結合するシステイン残基部分と両末端の共通配列部分である。P450モノオキシゲナーゼがdiversozymeと言われる所以である。P450モノオキシゲナーゼは部分的な構造の変化により多彩な化合物の代謝に多様化された酵素で、先端の生物工学（バイオテクノロジー）を活用することにより、既存の方法では難しいと思われている化合物の酸化処理が可能になると期待されている。したがって、本酵素は、新しい有機（低分子）化合物（たとえば，位置/立体特異的に導入された水酸化物など）の合成や、分解処理が難しい環境負荷物質の処理への応用に期待されている。また、作物植物に新たなP450モノオキシゲナーゼを導入して残留農薬の分解を促すことなども期待されている。 P450 monooxygenase is a complex enzyme that requires an electron transfer protein in addition to cytochrome P450 body protein (CYP). In other words, in order for P450 to exert its activity, as a protein that transfers electrons from NAD (P) H, bacteria and eukaryotic mitochondria require the intermediation of ferredoxin reductase and ferredoxin, and in mammalian liver microsomes Requires the intervention of NADPH-P450 reductase. The CYP itself is also very diverse. That is, the homology of amino acid sequences between CYP families is 40% or less, and the common part is a cysteine residue part that binds to heme iron and a common part part at both ends. This is why P450 monooxygenase is called diversozyme. P450 monooxygenase is an enzyme that has been diversified to metabolize various compounds due to partial structural changes. By utilizing advanced biotechnology, it is difficult for existing methods. It is expected that oxidation treatment will be possible. Therefore, this enzyme is expected to be applied to the synthesis of new organic (low molecular weight) compounds (for example, position / stereospecifically introduced hydroxides) and the treatment of environmentally hazardous substances that are difficult to decompose. Yes. It is also expected to introduce new P450 monooxygenase into crop plants to promote the degradation of residual pesticides.
細菌をはじめとする微生物のP450研究は哺乳動物に比べると遅れていたが、細菌のゲノム解析の進展と相まって、細菌のP450研究が今日、盛んになってきた。細菌で最もよく研究されているP450モノオキシゲナーゼは、シュードモナス・プチダ由来のP450camで、本酵素は、樟脳（camphor）を基質として、5位に水酸基を導入する反応を触媒することが明らかにされており、P450cam遺伝子を導入・発現した組換え細菌を用いた解析も広く実施されている（Poulos, T.L., Finzel, B.C., and Howard, A,J., High-resolution crystal structure of cytochrome P450cam. J. Mol. Biol., 195, 687-700 (1987)）。また、高脂血症薬プラバスタチン（pravastatin;三共株式会社 商品名メバロチン）は、コンパクチン（compactin）カルボン酸の立体特異的な水酸化反応により合成されるが、この反応を担う酵素は、土壌細菌ストレプトマイセス・カルボフィラス（Streptmyces carbophilus）由来のP450モノオキシゲナーゼである（Matsuoka, T., Miyakoshi, S., Tanzawa, K., Nakahara, K., Hosobuchi, M., and Serizawa, N., Purification and characterization of cytochrome P-450sca from Streptomyces carbophilus. ML-236B (compactin) induces a cytochrome P-450sca in Streptomyces carbophilus that hydroxylates ML-236B to pravastatin sodium (CS-514), a tissue-selective inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme-A reductase. Eur. J. Biochem., 184, 707-713, 1989）。最近、ロドコッカス属（Rhodococcus sp.）NCIMB 9784由来のP450モノオキシゲナーゼ（P450RhF）の構造が、P450本体タンパク質、及びフェレドキシンレダクターゼとフェレドキシン部分（電子伝達タンパク質）がすべて繋がった１つのポリペプチドであることが発見された。この酵素の遺伝子を発現した大腸菌は、7-エトキシクマリン（7-ethoxycoumarin）のO-脱アルキル化反応を触媒することが報告されている（Roberts, G.A., Grogan, G., Greter, A., Flitsch, S.L., and Turner, N.J., Identification of a new class of cytochrome P450 from a Rhodococcus sp. J. Bacteriol., 184, 3898-3908 (2002)）。 Research on P450 for microorganisms including bacteria has been delayed compared to mammals, but coupled with the progress of bacterial genome analysis, research on P450 for bacteria has become popular today. The most well-studied P450 monooxygenase in bacteria is P450cam derived from Pseudomonas putida, which has been shown to catalyze the reaction of introducing a hydroxyl group at the 5-position using camphor as a substrate. Analysis using recombinant bacteria into which P450cam gene has been introduced and expressed has also been widely carried out (Poulos, TL, Finzel, BC, and Howard, A, J., High-resolution crystal structure of cytochrome P450cam. Mol. Biol., 195, 687-700 (1987)). The hyperlipidemic drug pravastatin (trade name: mevalotin) is synthesized by stereospecific hydroxylation of compactin carboxylic acid, and the enzyme responsible for this reaction is streptococcal soil. P450 monooxygenase from Streptmyces carbophilus (Matsuoka, T., Miyakoshi, S., Tanzawa, K., Nakahara, K., Hosobuchi, M., and Serizawa, N., Purification and characterization of cytochrome P-450sca from Streptomyces carbophilus .ML-236B (compactin) induces a cytochrome P-450sca in Streptomyces carbophilus that hydroxylates ML-236B to pravastatin sodium (CS-514), a tissue-selective inhibitor of 3-hydroxy-3- methylglutaryl-coenzyme-A reductase. Eur. J. Biochem., 184, 707-713, 1989). Recently, the structure of P450 monooxygenase (P450RhF) derived from Rhodococcus sp. NCIMB 9784 is a single polypeptide in which the P450 body protein and the ferredoxin reductase and the ferredoxin moiety (electron transfer protein) are all connected. It's been found. E. coli expressing this enzyme gene has been reported to catalyze the O-dealkylation of 7-ethoxycoumarin (Roberts, GA, Grogan, G., Greter, A., Flitsch, SL, and Turner, NJ, Identification of a new class of cytochrome P450 from a Rhodococcus sp. J. Bacteriol., 184, 3898-3908 (2002)).
細菌バチルス・メガテリウムBM-3株由来のP450モノオキシゲナーゼは、P450ドメインとNADPH-P450レダクターゼドメインが結合した、大変珍しい構造を有する酵素である。本酵素の塩基配列は、DDBJ Accession No. J04832に登録されている。本酵素は元々、C12〜C20の飽和脂肪酸のω末端（メチル基側）の1位、2位または3位を水酸化する酵素であったが、87番目のフェニルアラニンがバリンに変異したもの（F87V）は、ナフタレンや2-ブロモフェノール等の芳香族化合物を広く基質として認識し 水酸基を導入することができた（小川 順ら, Bacillus megaterium由来のシトクロムP450(BM-3)変異型酵素を用いるパラヒドロキノン類の酵素合成, 日本農芸化学会2003年大会講演要旨, p. 38, 2003、及び、Q. -W. Li, U. Schwaneberg, P. Fischer, and R. D. Schmid, Directed evolution of the fatty-acid hydoroxylase P450 BM-3 into an indole-hydroxylating catalyst. Chem. Eur. J. 6, 1531-1536, 2000）。 P450 monooxygenase derived from the bacterial strain Bacillus megaterium BM-3 is an enzyme having a very unusual structure in which a P450 domain and a NADPH-P450 reductase domain are combined. The base sequence of this enzyme is registered in DDBJ Accession No. J04832. This enzyme was originally an enzyme that hydroxylates the 1st, 2nd, or 3rd position of ω-terminal (methyl group side) of saturated fatty acids of C12 to C20, but the 87th phenylalanine was mutated to valine (F87V ) Was able to introduce a hydroxyl group by widely recognizing aromatic compounds such as naphthalene and 2-bromophenol as a substrate (Jun Ogawa et al., A cytochrome P450 (BM-3) mutant enzyme derived from Bacillus megaterium ). Enzymatic synthesis of hydroquinones, Abstracts of the 2003 Annual Meeting of the Agricultural Chemical Society of Japan, p. 38, 2003, and Q. -W. Li, U. Schwaneberg, P. Fischer, and RD Schmid, Directed evolution of the fatty-acid hydoroxylase P450 BM-3 into an indole-hydroxylating catalyst. Chem. Eur. J. 6, 1531-1536, 2000).
２．PPIase
PPIase（ペプチジルプロリルcis-transイソメラーゼ）は、ペプチド中のプロリンイミド結合の回転を速めてタンパク質の折り畳みを促進する酵素で、免疫抑制剤のシクロスポリンAと結合するシクロフィリン、FK506と結合するFKBP、これらと結合しないパーブリンの3種類がある。超好熱古細菌のゲノムはPPIaseとしてFKBP遺伝子しか有していない。超好熱古細菌のPPIaseは他の生物種のPPIaseにはない、変性タンパク質の凝集を阻止して折りたたむシャペロン活性を有する（A. Ideno, T. Yoshida, T IIDA, M. Furutani, and T. Maruyama. FK506-binding protein of the hyperthermophilic archaeum, Thermococcus sp. KS-1, a cold-shock-inducible peptidyl-prolyl cis-trans isomerase with activities to trap and refold denatured proteins. Biochem J. 357(Pt 2):465-71, 2001）。またタンパク質が熱凝集することを抑制する凝集抑制活性も有している（A. Ideno, M. Furutani, Y. Iba, Y. Kurosawa, and T. Maruyama. FK506 binding protein from the hyperthermophilic archaeon Pyrococcus horikoshii suppresses the aggregation of proteins in Escherichia coli. Appl Environ Microbiol. Feb;68(2):464-469, 2002.）。これらの性質はその高い熱安定性と併せて、変性しやすいタンパク質の安定化やタンパク質生産の際生じる不活性型タンパク質の再活性化など産業分野への応用が期待されているものである。
2. PPIase
PPIase (peptidylprolyl cis - trans isomerase) is an enzyme that accelerates protein folding by accelerating the rotation of the proline imide bond in peptides. Cyclophilin that binds to the immunosuppressant cyclosporin A, FKBP that binds FK506, and these There are three types of perbrin that do not bind to. The genome of hyperthermophilic archaea has only FKBP gene as PPIase. The hyperthermophilic archaeal PPIase has a chaperone activity that prevents the aggregation of denatured protein and folds, unlike other PPIases (A. Ideno, T. Yoshida, T IIDA, M. Furutani, and T. Maruyama.FK506-binding protein of the hyperthermophilic archaeum, Thermococcus sp.KS-1, a cold-shock-inducible peptidyl-prolyl cis-trans isomerase with activities to trap and refold denatured proteins.Biochem J.357 (Pt 2): 465 -71, 2001). It also has an aggregation inhibitory activity that suppresses thermal aggregation of proteins (A. Ideno, M. Furutani, Y. Iba, Y. Kurosawa, and T. Maruyama. FK506 binding protein from the hyperthermophilic archaeon Pyrococcus horikoshii suppresses The aggregation of proteins in Escherichia coli . Appl Environ Microbiol. Feb; 68 (2): 464-469, 2002.). These properties, together with their high thermal stability, are expected to be applied to industrial fields such as stabilization of proteins that are easily denatured and reactivation of inactive proteins that occur during protein production.
発明者が所属する研究機関である(株)海洋バイオテクノロジー研究所では、積水化学工業(株)との共同研究により、免疫抑制剤FK506と結合するタンパク質であるPPIase遺伝子を好熱性古細菌ピロコッカス・ホリコシ（Pyrococcus horikoshii）等より単離した。本PPIaseは、古細菌の不安定な酵素であるロダネーゼ（rhodanese）や、不溶化しやすい鶏卵のリゾチーム抗体のFab断片等を、本来の立体構造に巻き戻し 可溶化させることができた（特開平10-91号公報）。しかも、ロダネーゼは本PPIase共存下で100℃まで安定であった。したがって、本PPIaseは高温でも安定な分子シャペロンであることがわかった。また、安定的に機能発現させたいタンパク質のN末に、前記PPIaseまたは大腸菌のシャペロニンGroELを融合させることにより、そのタンパク質が本来の立体構造に巻き戻されることがわかった。この融合タンパク質系を用いて、毒性の強いB型肝炎ウィルス表面抗原や不溶性のヒトIgGの重鎖等をコードする遺伝子が、安定的に組換え大腸菌内で機能発現されることがわかった（特許文献１、特許文献２）。最近、三井化学もPPIaseのシャペロン活性に着目し、大腸菌のPPIaseを利用することにより、大腸菌で生産が難しかったヒトのミッドカインや組織プラスミノーゲン活性化因子（TPA）の生産量の増強に成功したと発表している（日経バイオテク、2003年5月21日）。 In collaboration with Sekisui Chemical Co., Ltd., the research institution to which the inventor belongs is the Marine Biotechnology Research Institute Co., Ltd., and the PPIase gene, a protein that binds to the immunosuppressant FK506, Isolated from Pyrococcus horikoshii and the like. This PPIase was able to unwind and solubilize Rhodanese, an unstable enzyme of archaebacteria, and the Fab fragment of lysozyme antibody of hen's egg, which is easily insolubilized, to the original three-dimensional structure (Japanese Patent Application Laid-Open No. 10-151). -91). Moreover, rhodanese was stable up to 100 ° C in the presence of this PPIase. Therefore, this PPIase was found to be a stable molecular chaperone even at high temperatures. It was also found that the PPIase or Escherichia coli chaperonin GroEL was fused to the N-terminal of the protein to be stably expressed in function, whereby the protein was unwound to its original three-dimensional structure. Using this fusion protein system, it was found that genes encoding highly toxic hepatitis B virus surface antigen, insoluble human IgG heavy chain, etc. were stably expressed in recombinant E. coli (patents) Literature 1, Patent Literature 2). Recently, Mitsui Chemicals also focused on the chaperone activity of PPIase and succeeded in enhancing the production of human midkine and tissue plasminogen activator (TPA), which were difficult to produce in E. coli, by using PPIase of E. coli. (Nikkei Biotech, May 21, 2003).
次に、本発明のP450モノオキシゲナーゼの生産方法について説明する。 Next, a method for producing the P450 monooxygenase of the present invention will be described.
本発明のP450モノオキシゲナーゼの生産方法は、古細菌由来のPPIaseのシャペロン活性を利用することにより、機能のあるP450モノオキシゲナーゼを安定的に生産することができる。古細菌由来のPPIaseのシャペロン活性を利用する方法としては、以下の二通りの方法がある。 The method for producing P450 monooxygenase of the present invention can stably produce functional P450 monooxygenase by utilizing the chaperone activity of archaeal PPIase. There are the following two methods for utilizing the chaperone activity of PPIase derived from archaea.
第一の方法は、P450モノオキシゲナーゼと古細菌由来のPPIaseの両者を生産可能な微生物を培養し、培養物又は菌体からP450モノオキシゲナーゼを採取することを特徴とするものである。 The first method is characterized in that a microorganism capable of producing both P450 monooxygenase and archaeal PPIase is cultured, and P450 monooxygenase is collected from the culture or the cells.
P450モノオキシゲナーゼと古細菌由来のPPIaseの両者を生産可能な微生物は、P450モノオキシゲナーゼ遺伝子を発現するベクターと古細菌由来のPPIase遺伝子を発現するベクターを作製し、これらを同一の宿主に導入することにより作製できる。 For microorganisms capable of producing both P450 monooxygenase and archaeal PPIase, create a vector that expresses the P450 monooxygenase gene and a vector that expresses the archaeal PPIase gene, and introduce them into the same host. Can be produced.
P450モノオキシゲナーゼが酵素活性を発揮するためには、酵素本体の他に電子伝達タンパク質も必要であり、また、酵素本体と電子伝達タンパク質は通常別々のタンパク質として生産される。従って、ベクター作製の際には、通常、P450モノオキシゲナーゼ遺伝子だけではなく、電子伝達タンパク質をコードする遺伝子も挿入しておく必要がある。 In order for P450 monooxygenase to exhibit enzyme activity, an electron transfer protein is required in addition to the enzyme main body, and the enzyme main body and the electron transfer protein are usually produced as separate proteins. Therefore, when preparing a vector, it is usually necessary to insert not only the P450 monooxygenase gene but also a gene encoding an electron transfer protein.
使用するP450モノオキシゲナーゼ遺伝子は、植物や動物などの真核生物由来のものであってもよいが、細菌由来のものであることが好ましい。細菌由来のP450モノオキシゲナーゼ遺伝子は特に限定されないが、バチルス・メガテリウムBM-3株由来の遺伝子（DDBJ Accession No. J04832）、ロドコッカス属 NCIMB9784由来の遺伝子（GenBank Accession No. AF459424）、シュードモナス・プチダ由来の遺伝子（P450cam、DDBJ Accession No. M12546）などを使用するのが好ましい。また、天然のP450モノオキシゲナーゼ遺伝子の配列を一部改変した遺伝子を使用してもよい。このような配列の一部改変した遺伝子としては、バチルス・メガテリウムBM-3株由来のP450モノオキシゲナーゼの87番目のフェニルアラニンをバリンに置換した酵素をコードする遺伝子（DDBJ Accession No. J04832の塩基配列において、261番目のアデニンがグアニンに（アミノ酸には影響しない）、262番目のチミンがグアニンに置き換わり、その結果、P450モノオキシゲナーゼの87番目のフェニルアラニンがバリンに置き換わる。）を挙げることができる。 The P450 monooxygenase gene to be used may be derived from eukaryotes such as plants and animals, but is preferably derived from bacteria. The P450 monooxygenase gene derived from bacteria is not particularly limited, but is derived from a gene derived from Bacillus megaterium BM-3 (DDBJ Accession No. J04832), a gene derived from Rhodococcus genus NCIMB9784 (GenBank Accession No. AF459424), Pseudomonas putida It is preferable to use a gene (P450cam, DDBJ Accession No. M12546) or the like. Alternatively, a gene obtained by partially modifying the sequence of the natural P450 monooxygenase gene may be used. Such a partially modified gene includes a gene encoding an enzyme in which 87th phenylalanine of P450 monooxygenase derived from Bacillus megaterium BM-3 strain is replaced with valine (in the base sequence of DDBJ Accession No. J04832). , The 261st adenine is replaced by guanine (which does not affect the amino acid), the 262th thymine is replaced by guanine, and as a result, the 87th phenylalanine of the P450 monooxygenase is replaced by valine.
使用するPPIaseをコードする遺伝子は、古細菌由来のものであれば特に限定されず、例えば、ピロコッカス・ホリコシ（Pyrococcus horikoshii）由来の遺伝子[遺伝子番号 PH1399; http://www.ncbi.nlm.nih.gov/]、メタノバクテリウム・サーモオートトロフィカム（Methanobacterium thermoautotrophicum）由来の遺伝子[遺伝子番号 MTH1125; http://www.ncbi.nlm.nih.gov/]などを使用することができる。 The gene encoding PPIase to be used is not particularly limited as long as it is derived from archaea. For example, a gene derived from Pyrococcus horikoshii [gene number PH1399; http: //www.ncbi.nlm.nih .gov /], a gene derived from Methanobacterium thermoautotrophicum [gene number MTH1125; http://www.ncbi.nlm.nih.gov/] and the like can be used.
宿主とする微生物は、両方の遺伝子を発現させることのできる微生物であれば特に限定されないが、大腸菌であることが好ましい。 The microorganism used as the host is not particularly limited as long as it can express both genes, but Escherichia coli is preferable.
大腸菌等の微生物内における上記の二つの遺伝子の発現は、遺伝子工学実験の常法に基づいて行うことができる。大腸菌や放線菌等の種々の微生物のベクターの情報や外来遺伝子の導入・発現法は、多くの実験書に記載されているので（たとえば、Sambrook, J., Russel,D. W., Molecular Cloning A Laboratory Manual, 3rd Edition, CSHL Press, 2001; Hopwood, D. A., Bibb, M. J., Chater, K. F., Bruton, C. J., Kieser, H. M., Lydiate, D. J., Smith, C. P., Ward, J. M., Schrempf, H. Genetic manipulation of Streptomyces: A laboratory manual, The John Innes Institute, Norwich, UK, 1985）、それらに従ってベクターの選択、遺伝子の導入、発現を行うことができる。 Expression of the above two genes in a microorganism such as E. coli can be performed based on a conventional method of genetic engineering experiment. Information on vectors of various microorganisms such as E. coli and actinomycetes and methods for introducing and expressing foreign genes are described in many experimental documents (for example, Sambrook, J., Russel, DW, Molecular Cloning A Laboratory Manual). , 3 rd Edition, CSHL Press, 2001; Hopwood, DA, Bibb, MJ, Chater, KF, Bruton, CJ, Kieser, HM, Lydiate, DJ, Smith, CP, Ward, JM, Schrempf, H. Genetic manipulation of Streptomyces : A laboratory manual, The John Innes Institute, Norwich, UK, 1985), vector selection, gene introduction, and expression can be performed accordingly.
微生物の培養及び菌体等からの酵素の採取は、常法に従って行うことができる。 The cultivation of microorganisms and the collection of enzymes from cells etc. can be performed according to conventional methods.
第二の方法は、P450モノオキシゲナーゼのN末端側に古細菌由来のPPIaseを付加した融合タンパク質を生産可能な微生物を培養し、培養物又は菌体から、活性のあるP450モノオキシゲナーゼを含む融合タンパク質を採取することを特徴とするものである。必要に応じて、この融合タンパク質からP450モノオキシゲナーゼを遊離させ、採取することもできる。 The second method is to culture a microorganism capable of producing a fusion protein in which an archaebacterial PPIase is added to the N-terminal side of P450 monooxygenase, and from the culture or cell body, a fusion protein containing active P450 monooxygenase It is characterized by collecting. If necessary, P450 monooxygenase can be released from the fusion protein and collected.
P450モノオキシゲナーゼのN末端側に古細菌由来のPPIaseを付加した融合タンパク質を生産可能な微生物は、P450モノオキシゲナーゼ遺伝子の5'末端側に古細菌由来のPPIase遺伝子を付加した融合遺伝子を発現するベクターを作製し、それを宿主微生物に導入することにより作製できる。 Microorganisms capable of producing fusion proteins with an archaeal PPIase added to the N-terminal side of P450 monooxygenase express a fusion gene with an archaeal PPIase gene added to the 5 'end of the P450 monooxygenase gene Can be prepared and introduced into a host microorganism.
使用するP450モノオキシゲナーゼ遺伝子及び古細菌由来のPPIase遺伝子は、第一の方法と同様のものでよい。 The P450 monooxygenase gene and the archaeal PPIase gene used may be the same as in the first method.
上述したように、P450モノオキシゲナーゼ遺伝子を発現するベタクーには通常電子伝達タンパク質遺伝子も挿入しておく必要があるので、融合遺伝子を発現するベクターは、P450モノオキシゲナーゼ遺伝子、電子伝達タンパク質遺伝子、及び古細菌由来のPPIase遺伝子の三者を含むことになる。このようなベクターの作製には、図１に示すプラスミドを使用するのが好ましい。 As described above, a vector expressing a P450 monooxygenase gene, an electron transfer protein gene, an old protein transfer gene, and an archaeal vector must be inserted into a betaku expressing a P450 monooxygenase gene. It will include the three PPIase genes derived from bacteria. For the preparation of such a vector, it is preferable to use the plasmid shown in FIG.
具体的には、古細菌由来のPPIase遺伝子の後に適当な長さのリンカーを介して、電子伝達タンパク質遺伝子を繋ぐ。リンカー部分にλファージのatt配列を加えておけば、λファージの部位特異的組換え系（attB x attP または attL x attR）を利用して、対応するatt配列に囲まれたP450モノオキシゲナーゼ遺伝子を容易に挿入することができる。この部位特異的組換え実験には、インビトロジェン社のGateway Technologyキットを用いることができる。電子伝達タンパク質は種々のものを用いることができるが、たとえば、ロドコッカス属NCIMB 9784由来のP450モノオキシゲナーゼ（P450−フェレドキシンレダクターゼ−フェレドキシンの融合タンパク質のような構造をしている）におけるフェレドキシンレダクターゼ−フェレドキシン部分を用いればよい。 Specifically, an electron transfer protein gene is connected to a PPIase gene derived from archaea via a linker having an appropriate length. If the att sequence of λ phage is added to the linker part, the P450 monooxygenase gene surrounded by the corresponding att sequence can be obtained using the site-specific recombination system of λ phage ( attB x attP or attL x attR ). Can be inserted easily. For this site-specific recombination experiment, Invitrogen's Gateway Technology kit can be used. Various electron transfer proteins can be used. For example, a ferredoxin reductase-ferredoxin moiety in P450 monooxygenase derived from Rhodococcus NCIMB 9784 (structured like a fusion protein of P450-ferredoxin reductase-ferredoxin) May be used.
また、融合タンパク質中の古細菌由来のPPIaseとP450モノオキシゲナーゼの間には、酵素などによって切断される部位が挿入されていることが好ましい。このような切断部位としては、実施例で構築したトロンビン切断部位を例示できるが、これに限定されるわけではない。融合タンパク質からP450モノオキシゲナーゼを遊離させる方法は特に限定されないが、切断部位を挿入した場合には、その切断部位を切断する酵素等を作用させればよい。 Moreover, it is preferable that a site to be cleaved by an enzyme or the like is inserted between archaeal PPIase and P450 monooxygenase in the fusion protein. Examples of such cleavage sites include, but are not limited to, thrombin cleavage sites constructed in the examples. The method for releasing P450 monooxygenase from the fusion protein is not particularly limited, but when a cleavage site is inserted, an enzyme or the like that cleaves the cleavage site may be allowed to act.
以下、本発明を実施例に基づき具体的に説明する。ただし、本発明はこれらの実施例に限定されるものではない。 Hereinafter, the present invention will be specifically described based on examples. However, the present invention is not limited to these examples.
〔実施例１〕 共発現系用プラスミドの構築
バチルス・メガテリウムBM-3株由来のP450モノオキシゲナーゼ（monooxygenase）（DDBJ Accession No. J04832）の変異酵素であるF87V（87番目のフェニルアラニンをバリンに置換したもの）は、その構造遺伝子をPCRにより増幅し、ベクタープラスミドpUC18のEcoRI−BamHI部位にクローニングした。以後、このプラスミドをpUC18-F87Vと呼ぶ。 PCRによる増幅の条件は以下の通りである。
[Example 1] Construction of plasmid for co-expression system F87V (87th phenylalanine was substituted with valine), a mutant enzyme of P450 monooxygenase (DDBJ Accession No. J04832) derived from Bacillus megaterium BM-3 strain The product was amplified by PCR and cloned into the Eco RI- Bam HI site of the vector plasmid pUC18. Hereinafter, this plasmid is referred to as pUC18-F87V. The conditions for PCR amplification are as follows.
PCR増幅に用いたプライマー
Primer1 (36mer)
5'- gTgAAAgAggAATTCCATgACAATTAAAgAAATgCC -3'（配列番号１）
Primer2 (42mer)
5'- CgggATTCTTAATgATgATgATgATgATgCCCAgCCCACACg -3'（配列番号２）
下線部はEcoRIサイト（Primer1）、BamHIサイト（Primer2）をそれぞれ示している。
Primers used for PCR amplification
Primer1 (36mer)
5'- gTgAAAgAg gAATTC CATgACAATTAAAgAAATgCC-3 '(SEQ ID NO: 1)
Primer2 (42mer)
5'- Cg ggATTC TTAATgATgATgATgATgATgCCCAgCCCACACg -3 '(SEQ ID NO: 2)
Underlined parts indicate Eco RI site (Primer1) and Bam HI site (Primer2), respectively.
PCR増幅条件
９４℃ ２分処理後、９４℃ １５秒、６１℃ ３０秒、６８℃ ４分を２５サイクル行った。KOD Plus DNA polymerase (TOYOBO)を用いた。
PCR amplification conditions After treatment at 94 ° C. for 2 minutes, 25 cycles of 94 ° C. for 15 seconds, 61 ° C. for 30 seconds, and 68 ° C. for 4 minutes were performed. KOD Plus DNA polymerase (TOYOBO) was used.
ピロコッカス・ホリコシ（Pyrococcus horikoshii）由来のPPIase遺伝子はPCRにより増幅し、ベクタープラスミドpET21aベクターのNdeI-HindIII部位にクローニングした後、PPIase遺伝子とこれに隣接するT7プロモーターをSphI−SalIで切り出し、pACYC184の同部位にクローニングし、PPIase発現プラスミドを作製した。以後、このプラスミドをpACPhFK-１と呼ぶ（Ideno, A., Furutani, M., Iba, Y., Kurosawa, Y., and Maruyama, T., FK506 binding protein from the hyperthermophilic archaeon Pyrococcus horikoshii suppresses the aggregation of proteins in Escherichia coli. Appl Environ Microbiol. 68(2):464-469 (2002)）。 Pyrococcus horikoshii (Pyrococcus horikoshii) from PPIase gene was amplified by PCR, was cloned into Nde I- Hin dIII sites of vector plasmid pET21a vector, excised T7 promoter adjacent thereto and PPIase gene Sph I- Sal I And cloned into the same site of pACYC184 to prepare a PPIase expression plasmid. Hereinafter, this plasmid is called pACPhFK-1 (Ideno, A., Furutani, M., Iba, Y., Kurosawa, Y., and Maruyama, T., FK506 binding protein from the hyperthermophilic archaeon Pyrococcus horikoshii suppresses the aggregation of proteins in Escherichia coli . Appl Environ Microbiol. 68 (2): 464-469 (2002)).
大腸菌JM109(DE3)株をはじめにpACPhFK-１で形質転換した。得られた大腸菌を用いてコンピテントセルを作製し、更にpUC18-F87Vで形質転換した。この操作により、pACPhFK-１とpUC18-F87Vの２つを有した大腸菌JM109(DE3)/ pACPhFK-１/ pUC18-F87Vが得られた。 E. coli JM109 (DE3) was first transformed with pACPhFK-1. Competent cells were prepared using the obtained Escherichia coli, and further transformed with pUC18-F87V. By this operation, Escherichia coli JM109 (DE3) / pACPhFK-1 / pUC18-F87V having two of pACPhFK-1 and pUC18-F87V was obtained.
また比較の対象としてPPIase遺伝子を持たない大腸菌も作製した。これはPPIase遺伝子が入っていないベクターであるpACYC184とpUC18-F87Vの２プラスミドを有するJM109(DE3)/ pACYC184/ pUC18-F87Vである。 E. coli without PPIase gene was also prepared for comparison. This is JM109 (DE3) / pACYC184 / pUC18-F87V having two plasmids, pACYC184 and pUC18-F87V, which are vectors not containing the PPIase gene.
〔実施例２〕 共発現系による化合物の生変換能力の解析
構築した共発現系JM109(DE3)/ pACPhFK-１/ pUC18-F87V及びP450単独発現系JM109(DE3)/ pACYC184/ pUC18-F87Vは各々LB培地（アンピシリン100 μg/ml及びクロラムフェニコール25 μg/mlを含む）4 mLで16時間、30℃で培養した。この前培養液全量をM9＋グルコース培地（アンピシリン100 μg/ml及びクロラムフェニコール25 μg/mlを含む）100 mLに全量植菌し、4時間培養した。この培養液に誘導物質としてIPTGを1 mMになるよう加え、更に30℃で16時間培養した。その後集菌し、10 mMトリスバッファー（ｐH＝7.5）40 mLに懸濁、基質としてインドールを1 mM、または5 mMになるよう添加した。30℃で16時間振盪培養し基質との反応を行わせた後、細胞懸濁液と等量、すなわち40 mLのメタノールを入れ、30分間攪拌した。次に8,000回転で5分間遠心を行い、上清80 μlをHPLCでの解析に用いた。
[Example 2] Analysis of compound biotransformation ability by co-expression system The constructed co-expression system JM109 (DE3) / pACPhFK-1 / pUC18-F87V and P450 single expression system JM109 (DE3) / pACYC184 / pUC18-F87V are each The cells were cultured at 30 ° C. for 16 hours in 4 mL of LB medium (containing 100 μg / ml of ampicillin and 25 μg / ml of chloramphenicol). The total amount of this preculture was inoculated into 100 mL of M9 + glucose medium (containing 100 μg / ml ampicillin and 25 μg / ml chloramphenicol), and cultured for 4 hours. To this culture broth, IPTG was added as an inducer to 1 mM, and further cultured at 30 ° C. for 16 hours. Thereafter, the cells were collected, suspended in 40 mL of 10 mM Tris buffer (pH = 7.5), and indole as a substrate was added to 1 mM or 5 mM. After shaking culture at 30 ° C. for 16 hours for reaction with the substrate, an amount equivalent to the cell suspension, that is, 40 mL of methanol was added and stirred for 30 minutes. Next, centrifugation was performed at 8,000 rpm for 5 minutes, and 80 μl of the supernatant was used for analysis by HPLC.
インドールはP450モノオキシゲナーゼ(F87V)により水酸化反応を触媒され、インディゴに変換される。共発現系、及びP450単独発現系でのインディゴ生成をHPLCで検出、比較した結果を表１に示す。実験は３回行い、その平均値が示してある。なお、インドール及びインディゴにおけるHPLCでの保持時間はそれぞれ、９.５分、及び、４.８分であった。 Indole is catalyzed by hydroxylation by P450 monooxygenase (F87V) and converted to indigo. Table 1 shows the results of detection and comparison of indigo production in the co-expression system and the P450 single expression system by HPLC. The experiment was performed three times and the average value is shown. The retention times by HPLC in indole and indigo were 9.5 minutes and 4.8 minutes, respectively.
表１に示すように、PPIaseとの共発現系はP450単独発現系と比較し、１ｍMインドールを基質とした場合で約１．７倍、５ｍMインドールで約３．３倍の変換効率を示した。 As shown in Table 1, the co-expression system with PPIase showed a conversion efficiency of about 1.7 times when using 1 mM indole as a substrate and about 3.3 times when using 5 mM indole as compared with the P450 single expression system. .
〔実施例３〕 融合タンパク質作製用発現プラスミドの構築
バチルス・メガテリウムBM-3株由来のP450モノオキシゲナーゼの変異酵素であるF87V は、その構造遺伝子1.3 kbをPCRにより増幅した。用いたプライマーの配列を以下に示す。
[Example 3] Construction of expression plasmid for production of fusion protein F87V, which is a mutant enzyme of P450 monooxygenase derived from Bacillus megaterium BM-3 strain, amplified its structural gene 1.3 kb by PCR. The primer sequences used are shown below.
Primer1 (30mer)
5'- GGAGCTCACAATTAAAGAAATGCCTCAGCC-3'（配列番号３）
Primer2 (36mer)
5'- CGCCTCGAGTTAATGATGATGATGATGATGCCCAGC -3'（配列番号４）
下線部はSacIサイト（Primer1）、XhoIサイト（Primer2）をそれぞれ示している。
Primer1 (30mer)
5'-G GAGCTC ACAATTAAAGAAATGCCTCAGCC-3 '(SEQ ID NO: 3)
Primer2 (36mer)
5'- CGC CTCGAG TTAATGATGATGATGATGATGCCCAGC -3 '(SEQ ID NO: 4)
The underlined portions indicate the Sac I site (Primer 1) and the Xho I site (Primer 2), respectively.
PCR増幅条件
９４℃ ２分処理後、９４℃ １５秒、６１℃ ３０秒、６８℃ ４分を２５サイクル行った。KOD Plus DNA polymerase (TOYOBO)を用いた。
PCR amplification conditions After treatment at 94 ° C. for 2 minutes, 25 cycles of 94 ° C. for 15 seconds, 61 ° C. for 30 seconds, and 68 ° C. for 4 minutes were performed. KOD Plus DNA polymerase (TOYOBO) was used.
サーモコッカス属（Thermococcus sp.） KS-1由来のPPIase遺伝子18 kbはPCRにより増幅した。用いたプライマーの配列を以下に示す。 Thermococcus sp. An 18 kb PPIase gene derived from KS-1 was amplified by PCR. The primer sequences used are shown below.
Primer3 (28mer)
5'- GGCCATGGGAAAAGTTGAAGCTGGTGAT -3'（配列番号５）
Primer4 (26mer)
5'- CCACTAGTAGCTTCTGAGTCCTCCTC -3'（配列番号６）
下線部はNcoIサイト（Primer3）、SpeIサイト（Primer4）をそれぞれ示している。
Primer3 (28mer)
5'-GG CCATGG GAAAAGTTGAAGCTGGTGAT-3 '(SEQ ID NO: 5)
Primer4 (26mer)
5'- CC ACTAGT AGCTTCTGAGTCCTCCTC -3 '(SEQ ID NO: 6)
Underlined indicates Nco I site (Primer3), Spe I site (Primer4), respectively.
PPIase遺伝子とP450モノオキシゲナーゼ遺伝子の間にトロンビン切断部位を構築するため、相互に相補的な次の配列を持つ2本のオリゴヌクレオチド(Throm-F、Throm-R)を合成しハイブリダイズさせ2本鎖DNA断片を作製した。以下この2本鎖DNA断片をThrom断片と呼ぶ。 In order to construct a thrombin cleavage site between the PPIase gene and the P450 monooxygenase gene, two oligonucleotides (Throm-F, Throm-R) having the following sequences complementary to each other were synthesized and hybridized. Strand DNA fragments were prepared. Hereinafter, this double-stranded DNA fragment is referred to as a Throm fragment.
Throm-F
5'- GGACTAGTCTGGTTCGGCGTGGATCC CATATG GAATTCGG -3'（配列番号７）
Throm-R
5'- CCGAATTC CATATG GGATCCACGCCGAACCAGACTAGTCC -3'（配列番号８）
この断片はSpeI（ACTAGT）、BamHI(GGATCC)、 NdeI(CATATG)、 EcoRI(GAATTC)の制限酵素サイトを含んでいる。上記Throm-F及び Throm-Rの配列においてこれらのサイトは下線で示している。
Throm-F
5'-GG ACTAGT CTGGTTCGGCGT GGATCC CATATG GAATTC GG-3 '(SEQ ID NO: 7)
Throm-R
5'- CC GAATTC CATATG GGATCC ACGCCGAACCAG ACTAGT CC -3 '(SEQ ID NO: 8)
This fragment contains restriction sites for Spe I (ACTAGT), Bam HI (GGATCC), Nde I (CATATG), and Eco RI (GAATTC). These sites are underlined in the Throm-F and Throm-R sequences.
上述の方法で増幅したPPIase遺伝子はNcoI及びSpeI、 Throm断片はSpeIとEcoRIで各々処理し、ベクタープラスミドpET21dのNcoI-EcoRIサイトへタンデムに挿入した。得られた発現プラスミドのSacI−XhoIサイトへ更にP450遺伝子をクローニングし、PPIaseとP450の融合タンパク質遺伝子発現系を構築した。以下、このプラスミドをpFusionP450と呼ぶ。pFusionP450のマップは図２に示されている。なお、プラスミドpFusionP450により作られるFKBPとP450の分子間にはリンカーペプチドがあり（アミノ酸配列 TSLVPRGSHMEFEL）トロンビン切断部位（下線部）を有している。 PPIase gene amplified in the manner described above Nco I and Spe I, Throm fragment respectively treated with Spe I and Eco RI, and inserted in tandem into Nco I- Eco RI site of vector plasmid pET21d. The P450 gene was further cloned into the Sac I- Xho I site of the obtained expression plasmid, and a PPIase-P450 fusion protein gene expression system was constructed. Hereinafter, this plasmid is referred to as pFusionP450. A map of pFusionP450 is shown in FIG. There is a linker peptide (amino acid sequence TS LVPRG SHMEFEL) between the FKBP and P450 molecules produced by the plasmid pFusionP450 and a thrombin cleavage site (underlined).
比較対象として、P450遺伝子のみをpET21dのSacI−XhoI部位へクローニングしたプラスミドpETP450も作製した。 For comparison, a plasmid pETP450 was also prepared in which only the P450 gene was cloned into the Sac I- Xho I site of pET21d.
〔実施例４〕 融合タンパク質法によるF87Vの安定発現
大腸菌BL21 (DE3)株をpFusionP450またはpETP450で形質転換し、各々LB培地（アンピシリン100 μg/mlを含む）3 mLで4時間、30℃で培養した。この前培養液全量をLB培地（アンピシリン100 μg/ml含む）40 mLに全量植菌し、3時間培養した。この培養液に誘導物質としてIPTGを0.5 ｍMになるよう加え、更に30℃で16時間培養した。その後集菌し、10 ｍMリン酸ナトリウムバッファー（ｐH＝8.0）2 ｍLに懸濁、氷上で超音波破砕を行い32,000回転で30分間超遠心を行い、得られた上清を無細胞抽出液とした。BL21(DE3)/pFusionP450または/pETP450より調製した無細胞抽出液のタンパク質濃度を測定し、約6 μgをSDS/PAGEゲル（7.5-15％）で泳動した。得られたバンドを解析したところ、pFusionP450を用いた系はpETP450の系と比較して約2.7倍量のP450が発現していることが明らかとなった。
[Example 4] Stable expression of F87V by fusion protein method Escherichia coli BL21 (DE3) strain was transformed with pFusionP450 or pETP450, and each was cultured in 3 mL of LB medium (containing ampicillin 100 µg / ml) for 4 hours at 30 ° C. did. The entire amount of this preculture was inoculated into 40 mL of LB medium (containing 100 μg / ml of ampicillin) and cultured for 3 hours. To this culture broth, IPTG was added as an inducer to a concentration of 0.5 mM, and further cultured at 30 ° C. for 16 hours. Thereafter, the cells are collected, suspended in 2 mL of 10 mM sodium phosphate buffer (pH = 8.0), sonicated on ice and ultracentrifuged at 32,000 rpm for 30 minutes, and the resulting supernatant is used as a cell-free extract. did. The protein concentration of the cell-free extract prepared from BL21 (DE3) / pFusionP450 or / pETP450 was measured, and about 6 μg was run on an SDS / PAGE gel (7.5-15%). Analysis of the obtained band revealed that the system using pFusionP450 expressed approximately 2.7 times the amount of P450 compared to the system using pETP450.
次に、P450単独発現系及びPPIase-P450融合タンパク質の無細胞抽出液について、P450の熱安定性を検討した。実際には、前もって、各無細胞抽出液のP450モノオキシゲナーゼ活性を測定し、60℃、20分の処理を行った後に、残存活性を測定した。活性の測定は、0.1 Mリン酸ナトリウムバッファー(pH8.0)に無細胞抽出液（タンパク量200〜400μg）及び基質としてラウリン酸ナトリウムを最終濃度0.5 ｍMになるよう加え、NADPH（終濃度0.2 μM）を添加後素早く混合し、分光光度計で340 nmに示されるNADPHの吸光度の減少を追うことによって行った。各々の無細胞抽出液の活性を熱処理前と後で比較した結果、表２に示すように、PPIaseとの融合タンパク質化したP450BM-3モノオキシゲナーゼF87V[大腸菌(pFusionP450)の無細胞抽出液]の方が、単独のP450BM-3モノオキシゲナーゼF87V[大腸菌(pETP450)の無細胞抽出液]よりも熱安定性に優れていることが判明した。 Next, P450 single expression system and PPIase-P450 fusion protein cell-free extract were examined for P450 thermal stability. Actually, the P450 monooxygenase activity of each cell-free extract was measured in advance, and after treatment at 60 ° C. for 20 minutes, the residual activity was measured. The activity was measured by adding a cell-free extract (protein amount 200-400 μg) and sodium laurate as a substrate to a final concentration of 0.5 mM in 0.1 M sodium phosphate buffer (pH 8.0) and adding NADPH (final concentration 0.2 μM). ) Was added quickly after addition and followed by a spectrophotometer following the decrease in NADPH absorbance as shown at 340 nm. As a result of comparing the activity of each cell-free extract before and after heat treatment, as shown in Table 2, P450BM-3 monooxygenase F87V [cell-free extract of Escherichia coli (pFusionP450)] fused with PPIase It was found that this was superior in thermal stability to single P450BM-3 monooxygenase F87V [cell-free extract of E. coli (pETP450)].
〔実施例５〕 融合タンパク質法によるP450camの安定発現
シュードモナス・プチダ由来のP450cam（DDBJ Accession No. M12546）は、その構造遺伝子1.3 kbをPCRにより増幅した。用いたプライマーの配列を以下に示す。
[Example 5] Stable expression of P450cam by fusion protein method P450cam derived from Pseudomonas putida (DDBJ Accession No. M12546) was amplified by PCR with a structural gene of 1.3 kb. The primer sequences used are shown below.
Primer1 (30mer)
5'- GGACTAGTATGACGACTGAAACCATACAAA-3'（配列番号９）
Primer2 (30mer)
5'- CATATGTTATACCGCTTTGGTAGTCGCCGG -3'（配列番号１０）
下線部はSpeIサイト（Primer1）、NdeIサイト（Primer2）をそれぞれ示している。
Primer1 (30mer)
5'-GG ACTAGT ATGACGACTGAAACCATACAAA-3 '(SEQ ID NO: 9)
Primer2 (30mer)
5′- CATATG TTATACCGCTTTGGTAGTCGCCGG-3 ′ (SEQ ID NO: 10)
The underlined portions indicate the SpeI site (Primer1) and the NdeI site (Primer2), respectively.
PCR増幅条件
９４℃ ２分処理後、９４℃ １５秒、６１℃ ３０秒、６８℃ ４分を２５サイクル行った。KOD Plus DNA polymerase (TOYOBO)を用いた。〔実施例３〕で構築した発現プラスミドのSpeI−NdeIサイトにこの断片をクローニングし、融合プラスミドpFusionP450camを作製した。また比較対象としてP450cam遺伝子をpET21aにクローニングしたpETP450camプラスミドも作製した。大腸菌BL21 (DE3)株をpFusionP450camまたはpETP450camで形質転換し、実施例４と同様の手法で培養、無細胞抽出液を調製した。各無細胞抽出液のタンパク質濃度を測定し、約6 μgをSDS/PAGEゲル（7.5-15％）で泳動し得られたバンドを解析したところ、pFusionP450camを用いた系はpETP450の系と比較して約2.１倍量のP450camが発現していることが明らかとなった。
PCR amplification conditions After treatment at 94 ° C. for 2 minutes, 25 cycles of 94 ° C. for 15 seconds, 61 ° C. for 30 seconds, and 68 ° C. for 4 minutes were performed. KOD Plus DNA polymerase (TOYOBO) was used. This fragment was cloned into the SpeI-NdeI site of the expression plasmid constructed in [Example 3] to prepare a fusion plasmid pFusionP450cam. For comparison, a pETP450cam plasmid was also prepared by cloning the P450cam gene into pET21a. Escherichia coli BL21 (DE3) strain was transformed with pFusionP450cam or pETP450cam, cultured in the same manner as in Example 4, and a cell-free extract was prepared. When the protein concentration of each cell-free extract was measured and the band obtained by running about 6 μg on an SDS / PAGE gel (7.5-15%) was analyzed, the system using pFusionP450cam was compared with the system of pETP450. It was revealed that about 2.1 times as much P450cam was expressed.
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