JP6679084B2 - Materials for the hydroxylation of hydrocarbon-based substrates and their use - Google Patents

Description

  The present specification relates to materials and their use for hydroxylating hydrocarbon-based substrates.

  Cytochrome P450, which is an enzyme derived from bacteria, is known to have higher catalytic activity than cytochrome P450 derived from eukaryote. As cytochrome P450 derived from bacteria, cytochrome P450 monooxygenase such as cytochrome P450cam derived from Pseudomonas aeruginosa and cytochrome P450BM3 derived from Bacillus megaterium, which is a bacterium belonging to the genus Bacillus, is known.

The cytochrome P450 derived from these bacteria has high catalytic activity for hydroxylating substrates such as fatty acids. Moreover, since it has a relatively high affinity for water, it is easy to obtain and handle.
For these reasons, cytochrome P450 monooxygenase is considered suitable as a biocatalyst.

  Such wild-type cytochromes do not necessarily have high catalytic activity for hydroxylating industrially useful hydrocarbon substrates. Therefore, various mutant cytochrome P450s have been proposed (see, for example, Patent Documents 1 to 3).

  On the other hand, the present inventors incorporate a pseudo substrate (dummy molecule or decoy molecule) into a hydrocarbon that does not exhibit high catalytic activity so as to cause the cytochrome P450 to malfunction, thereby catalyzing the hydroxylation reaction with high efficiency. It has already been reported (Patent Document 4). The decoy molecule has a terminal structure capable of binding to the binding site of cytochrome P450 monooxygenase, and an alkyl chain, and is characterized in that at least one molecule contained in the alkyl chain is substituted with fluorine.

  Here, the hydrogen molecule of the alkyl chain substituted with fluorine is used as a decoy molecule because the fluorine atom radius is close to hydrogen, so that the fluorinated product obtained by substituting the hydrogen of the substrate with fluorine is similar to the original substrate, This is because cytochrome P450 cannot hydroxylate the C—H bond and thus does not itself serve as a substrate.

Tokuyo 2000-508163 Japanese Patent Publication No. 2003-517815 Japanese Patent Publication No. 2003-521889 JP, 2012-24009, A

  Since the decoy molecule proposed by the present inventors contains fluorine, the manufacturing cost is high, and problems such as persistent decomposition, environmental persistence and bioaccumulation may occur.

  However, when the present inventors utilized a non-fluorinated fatty acid as a decoy molecule, they were unable to hydroxylate a hydrocarbon-based substrate.

  In addition, since the mutant cytochrome P450 monooxygenase has the modified active site and the like, it was not possible for a person skilled in the art to predict that it would function by applying the decoy molecule applied to the wild-type cytochrome P450 monooxygenase.

  Thus, the present specification provides a more practical decoy molecule and its use as a material for the hydroxylation of hydrocarbon-based substrates.

  The inventors of the present invention firstly diligently studied to make it function as a decoy molecule without containing fluorine. As a result, the terminal structure capable of binding to the substrate binding site of cytochrome P450 monooxygenase and the non-fluorinated alkyl chain were mediated by a linker containing a peptide bond without fluorinating the alkyl chain in the decoy molecule. It was found that the hydrocarbon-based substrate can be hydroxylated by using it as a decoy molecule.

  Further, the present inventors have also found that a decoy molecule having a fluoroalkyl chain through a peptide linker functions as a decoy molecule for mutant cytochrome P450 monooxygenase.

  The present specification provides the following means based on these findings.

（上記式（１）中、Ａは、第１の部位を表し、Ｂは第２の部位を表し、Ｒは、水素原子、アルキル基、チオアルキル基、アルキケニル基、シクロアルキル基、シクロアルケニル基、アリール基及びアラルキル基から選択される１又は２以上の基を含む有機官能基を表し、ｎは１以上の整数を表す。）
（４）前記Ｒは、ロイシン、メチオニン、フェニルアラニン及びトリプトファンからなる群から選択されるアミノ酸の側鎖を表す、（３）に記載のデコイ分子。
（５）前記シトクロムＰ４５０モノオキシゲナーゼは、以下の（ａ）及び（ｂ）のシトクロムＰ４５０モノオキシゲナーゼから選択される１種又は２種以上である、（１）〜（４）のいずれかに記載のデコイ分子。
（ａ）シトクロムＰ４５０ＢＭ３のアミノ酸配列と４０％以上の同一性を有するシトクロムＰ４５０モノオキシゲナーゼ
（ｂ）シトクロムＰ４５０ｃａｍのアミノ酸配列と４０％以上の同一性を有するシトクロムＰ４５０モノオキシゲナーゼ
（６）前記不活性化炭化水素は、シクロヘキサン、ブタン、プロパン及びベンゼンからなる群から選択される１種又は２種以上である、（１）〜（５）のいずれかに記載のデコイ分子。
（７）炭化水素系基質の水酸化方法であって、
（１）〜（６）のいずれかに記載のデコイ分子、シトクロムＰ４５０モノオキシゲナーゼ及び炭化水素系基質の存在下で、前記シトクロムＰ４５０モノオキシゲナーゼにより前記炭化水素系基質を水酸化する工程、
を備える、方法。
（８）炭化水素系基質の水酸化物の生産方法であって、
（１）〜（６）のいずれかに記載のデコイ分子、シトクロムＰ４５０モノオキシゲナーゼ及び炭化水素系基質の存在下で、前記シトクロムＰ４５０モノオキシゲナーゼにより前記炭化水素系基質を水酸化する工程、
を備える、方法。
（９）シトクロムＰ４５０モノオキシゲナーゼで炭化水素を水酸化するためのデコイ分子のスクリーニング方法であって、
シトクロムＰ４５０モノオキシゲナーゼ、前記シトクロムＰ４５０モノオキシゲナーゼの基質結合部位に結合可能な末端構造である第１の部位と、フッ素化されていないアルキル鎖である第２の部位と、前記第１の部位と前記第２の部位とを連結する、ペプチド結合を含むリンカーと、を備える、１又は２以上のデコイ分子候補及び炭化水素系基質の存在下で、前記シトクロムＰ４５０モノオキシゲナーゼを前記炭化水素系基質に作用させる工程、
を備え、
前記シトクロムＰ４５０モノオキシゲナーゼによる前記炭化水素系基質の水酸化を評価する、スクリーニング方法。 (1) A decoy molecule for hydroxylating a hydrocarbon substrate with cytochrome P450 monooxygenase,
A first site which is a terminal structure capable of binding to the substrate binding site of the cytochrome P450 monooxygenase;
A second moiety which is a non-fluorinated alkyl chain,
A linker containing a peptide bond, which connects the terminal structure and the alkyl chain,
A decoy molecule comprising.
(2) The decoy molecule according to (1), wherein the first site is selected from the group consisting of a hydrogen atom, a hydroxyl group, an aldehyde group, a carboxyl group and a carboxyl derivative group.
(3) The decoy molecule according to (1) or (2), which is represented by the following formula (1).

(In the formula (1), A represents a first site, B represents a second site, R represents a hydrogen atom, an alkyl group, a thioalkyl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group, (Represents an organic functional group containing one or more groups selected from an aryl group and an aralkyl group, and n represents an integer of 1 or more.)
(4) The decoy molecule according to (3), wherein R represents a side chain of an amino acid selected from the group consisting of leucine, methionine, phenylalanine and tryptophan.
(5) The cytochrome P450 monooxygenase is one kind or two or more kinds selected from the following cytochrome P450 monooxygenases (a) and (b), and (1) to (4) Decoy molecule.
(A) Cytochrome P450 monooxygenase having 40% or more identity with the amino acid sequence of cytochrome P450BM3 (b) Cytochrome P450 monooxygenase having 40% or more identity with the amino acid sequence of cytochrome P450cam (6) The inactivated carbonization Hydrogen is 1 type (s) or 2 or more types selected from the group which consists of cyclohexane, butane, propane, and benzene, The decoy molecule in any one of (1)-(5).
(7) A method for hydroxylating a hydrocarbon-based substrate,
Hydroxylating the hydrocarbon-based substrate with the cytochrome P450 monooxygenase in the presence of the decoy molecule according to any one of (1) to (6), the cytochrome P450 monooxygenase, and the hydrocarbon-based substrate,
Comprising a method.
(8) A method for producing a hydrocarbon-based substrate hydroxide, comprising:
Hydroxylating the hydrocarbon-based substrate with the cytochrome P450 monooxygenase in the presence of the decoy molecule according to any one of (1) to (6), the cytochrome P450 monooxygenase, and the hydrocarbon-based substrate,
Comprising a method.
(9) A method for screening a decoy molecule for hydroxylating a hydrocarbon with a cytochrome P450 monooxygenase, which comprises:
Cytochrome P450 monooxygenase, a first site that is a terminal structure capable of binding to the substrate binding site of the cytochrome P450 monooxygenase, a second site that is a non-fluorinated alkyl chain, the first site and the above A peptide-containing linker connecting the second site, and the cytochrome P450 monooxygenase acting on the hydrocarbon-based substrate in the presence of one or more decoy molecule candidates and the hydrocarbon-based substrate. The process of
Equipped with
A screening method for evaluating hydroxylation of the hydrocarbon-based substrate by the cytochrome P450 monooxygenase.

(10) A decoy molecule for hydroxylating a hydrocarbon substrate with a mutant cytochrome P450 monooxygenase, comprising:
A first site which is a terminal structure capable of binding to the substrate binding site of the mutant cytochrome P450 monooxygenase;
A second moiety that is at least partially fluorinated alkyl chain;
A linker containing a peptide bond, which connects the terminal structure and the alkyl chain,
A decoy molecule comprising.
(11) A method for producing a hydrocarbon-based substrate hydroxide, comprising:
Hydroxylating the hydrocarbon-based substrate with the mutant cytochrome P450 monooxygenase in the presence of the decoy molecule according to (10), the mutant cytochrome P450 monooxygenase, and the hydrocarbon-based substrate,
Comprising a method.
(12) The method according to (11), wherein the hydrocarbon-based substrate is methane or ethane.
(13) A method for screening a decoy molecule for hydroxylating a hydrocarbon substrate with a mutant cytochrome P450 monooxygenase, which comprises:
Mutant cytochrome P450 monooxygenase, a first site that is a terminal structure capable of binding to the substrate binding site of the mutant cytochrome P450 monooxygenase, and a second site that is at least a part of a fluorinated alkyl chain, In the presence of one or more decoy molecule candidates, the mutant cytochrome P450 monooxygenase, and a hydrocarbon-based substrate, which comprises a linker containing a peptide bond that connects the first site and the second site. And a step of allowing the mutant cytochrome P450 monooxygenase to act on the hydrocarbon-based substrate,
Equipped with
A screening method for evaluating hydroxylation of the hydrocarbon-based substrate by the mutant cytochrome P450 monooxygenase.

  The present specification relates to a decoy molecule and its use for hydroxylating a hydrocarbon-based substrate with a cytochrome P450 monooxygenase.

  At the active center of cytochrome P450 monooxygenase (hereinafter simply referred to as P450), there is a binding site to which a substrate binds. When the substrate binds to the binding site, water molecules existing in the active center are pushed out from the active center. Hereinafter, this state is referred to as the activation switch being turned on. The active center then catalyzes the hydroxylation of the substrate after being subjected to the action of electrons or oxygen molecules. Therefore, it is considered that the catalytic reaction of P450 does not start unless the activation switch is turned on. As a substrate of wild-type P450, for example, lauric acid, myristic acid, palmitic acid are known. These substrates generally have an end structure attached to the attachment site and an alkyl chain.

(Decoy molecule of the first aspect)
The decoy molecule disclosed in the present specification includes a first site (terminal structure) capable of binding to the binding site of P450, and a second site that is an alkyl chain. Further, it has a linker containing a peptide bond connecting them. Since the decoy molecule has the first site and the second site, it is considered that when P450 coexists, the dummy molecule binds to the P450 binding site instead of the substrate, and the activation switch is turned on.

  In this decoy molecule, the first site and the second site are linked by a linker. Although not speculative and binding to the disclosure herein, it is believed that such decoy molecules are more likely to reach the active center and rapidly displace water molecules from the active center. Further, by providing such a linker, even if it is not fluorinated, hydroxylation of the decoy molecule can be suppressed or avoided, and the hydrocarbon-based substrate can be hydroxylated.

  By nature, the catalytic activity of wild-type P450 on hydrocarbon-based substrates is very low. However, the activation switch of the decoy molecule improves the catalytic activity of wild-type P450 on hydrocarbon substrates. Thus, wild-type P450 can be used to hydroxylate hydrocarbon-based substrates.

  As described above, by using the decoy molecule disclosed in the present specification, the use of fluorine can be eliminated, and the hydrocarbon-based substrate can be hydroxylated by the wild-type P450. Therefore, by using the decoy molecule disclosed in the present specification, a useful hydroxide can be obtained by industrially hydroxylating a hydrocarbon-based substrate.

(Decoy molecule of the second aspect)
Other decoy molecules disclosed herein are suitable decoy molecules for hydroxylating a hydrocarbon-based substrate with a mutant P450. According to this decoy molecule, it is possible to favorably function as a decoy molecule even for mutant P450. Therefore, by using this decoy molecule, the function of mutant P450 can be further enhanced.

  Although not binding to the disclosure of the present specification, the decoy molecule has a terminal structure through a peptide linker and a fluorinated alkyl chain, so that even if it is a mutated P450, its substrate binding site and It is considered that it can interact well with the active center, and as a result, the catalytic ability of the mutant P450 can be further enhanced.

  As described above, the decoy molecule of the second aspect disclosed in the present specification can be suitably applied to mutant P450, and further enhances its catalytic ability to obtain a useful hydroxide. be able to.

  In the present specification, the hydrocarbon-based substrate may include aromatic compounds, alkanes, alkenes, cycloalkanes, cycloalkenes and the like. The aromatic compound may be mono-substituted or poly-substituted. Examples of the substituent include alkyl having 1 to 4 carbons, alkenyl having 2 to 4 carbons, hydroxyl group, halogen atom and the like. The alkyl or alkenyl substituent may have a keto group or an aldehyde group. The aromatic compound may be mononuclear or polynuclear. It is preferably mononuclear or dinuclear. Further, the aromatic compound may be condensed with two or more nuclei or may be condensed with a non-aromatic ring. Examples of such aromatic compounds include mononuclear or binuclear aromatic compounds, and more specifically, optionally substituted benzene, naphthalene, indene, and the like.

  Examples of the alkane include linear or branched alkanes having about 1 to 15 carbon atoms. For example, methane, ethane, n-propane, n-butane, n-pentane, n-hexane, n-heptane, n-octane, n-nonane, n-decane, n-undecane and n-dodecane and two or more of these. A branched chain having a branch of

  Examples of the alkene include the above-mentioned alkanes having one or more unsaturated bonds. Examples include alkenes with 1 unsaturation.

  Examples of the cycloalkane and cycloalkene include optionally substituted cycloalkane and cycloalkene having 4 to 8 carbon atoms. For example, cyclopentane, cyclopentene, cyclohexane, cyclohexene, cycloheptane, cycloheptene and the like can be mentioned. The cycloalkane and cycloalkene may have one or more substituents, for example, may have 1 to 5 or less substituents. As the substituent, the above-mentioned substitution can be applied. Examples of cycloalkanes and cycloalkenes include cyclohexane and cyclohexene, which are unsubstituted or have about 1 to 3 substituents.

  In the present specification, hydroxylation of a hydrocarbon-based substrate means that the C—H bond of the hydrocarbon-based substrate is oxidized by an oxidation reaction, and as a result, a hydroxyl group is introduced into a carbon atom.

  Hereinafter, various embodiments disclosed in the present specification will be described in detail.

(Decoy molecule of the first aspect)
The decoy molecule of the first aspect disclosed in the present specification (hereinafter referred to as the first decoy molecule) can introduce a hydroxyl group into a carbon atom of a hydrocarbon-based substrate with P450.

(P450: cytochrome P450 monooxygenase)
P450 can include a known cytochrome P450 monooxygenase. As P450, P450 derived from bacteria can be preferably used, and above all, P450 derived from Pseudomonas aeruginosa and P450 derived from Bacillus can be used. Examples of such P450s include cytochrome P450BM3 (CYP102A1) and cytochrome P450cam (camphor 5-monooxygenase, CYP101).

As P450, P450 having an amino acid sequence identity of 40% or more with P450 whose amino acid sequence is known can also be used. For example, P450 (family enzyme of cytochrome P450BM3) having 40% or more identity with the amino acid sequence of cytochrome P450BM3, or P450 having 55% or more identity with the amino acid sequence of cytochrome P450BM3 (subfamily of cytochrome P450BM3 (CYP102A etc. The enzyme))) can be preferably used. Alternatively, P450 (family enzyme of cytochrome P450cam) having 40% or more identity with the amino acid sequence of cytochrome P450cam or P450 (subfamily enzyme of cytochrome P450cam) having 55% or more identity with amino acid sequence of cytochrome P450cam It can be preferably used. The cytochrome P450BM3 family enzyme or subfamily enzyme is preferably capable of hydroxylating a fatty acid, and the cytochrome P450cam family enzyme or subfamily enzyme is preferably capable of hydroxylating camphor (C ₁₀ H ₁₆ O). .

  The amino acid sequence of such cytochrome P450 BM3 is described in, for example, J. Biol. chem. 264 (19), 10987-10998 (1989) and NCBI beta base (National Center for Biotechnology Information http://www.ncbi.nlm.nih.gov/protein/P147799.2). The amino acid of cytochrome P450cam is also disclosed in the NCBI database (http://www.ncbi.nlm.nih.gov/protein/YP_714029).

  P450 may be a wild type or a mutant type. As the mutant type, for example, a mutant type of P450 derived from bacteria can be preferably used. For example, a mutant form of cytochrome P450BM3 or cytochrome P450cam can be used. Regarding the details and acquisition method of these mutants, for example, in addition to Patent Documents 1 to 3 described above, CJC Whitehouse, SG Bell, LL Wong, Chem. Soc. Rev. 2012, 41, 1218-1260 (above, All variants of cytochrome P450BM3), F. Xu, SG Bell, J. Lednik, A. Insley, ZH Rao, LL Wong, Angew. Chem. Int. Ed. 2005, 44, 4029-4032, H. Joo, ZL Lin, FH Arnold, Nature 1999, 399, 670-673 (above, mutant form of cytochrome P450cam) and the like.

  For the first decoy molecule, it may be preferable to use wild-type P450.

(Constitution of decoy molecule)
The first decoy molecule comprises a first site which is a terminal structure capable of binding to the substrate binding site of the cytochrome P450 monooxygenase, a second site which is a non-fluorinated alkyl chain, the terminal structure and the And a linker containing a peptide bond that connects the alkyl chain.

(First site: terminal structure capable of binding to P450 substrate binding site)
The first site is a terminal structure capable of binding to the substrate binding site of P450 used. More specifically, it is a terminal structure capable of binding to the opening of the substrate binding site of P450. In general, the substrate-binding site of P450 reaches the heme molecule from the surface of the molecule and is in the form of a tunnel mainly composed of hydrophobic amino acid residues. Then, for example, hydrogen bonding between positively charged amino acid sequence residues of arginine residue and tyrosine residue on the surface of the molecule where the substrate binding site such as cytochrome P450BM-3 opens and the substrate is considered to be essential for the oxidation reaction. There is.

  The first part preferably has a structure capable of forming a hydrogen bond so that it can interact with the opening of the substrate binding part. The first site can have, for example, one or more atoms or groups selected from the group consisting of hydrogen atom, hydroxyl group, aldehyde group, carboxyl group and carboxyl derivative group. Examples of the carboxyl derivative group include alkyl esters, amides and anhydrides having about 1 to 4 carbon atoms. Further, examples of the carboxyl derivative group include an aminoacyl group in which one or more amino acids are introduced into the carboxyl group. One or two or more of these atoms and groups may be combined. The first part preferably contains at least a carboxyl group.

(Second site: non-fluorinated alkyl chain)
The chain length of the alkyl chain in the second portion is not particularly limited, and can be appropriately set as necessary, for example, according to the P450 used or the hydrocarbon substrate to be hydroxylated. The alkyl chain may be linear or branched. When the branched chain is used, the branched chain may be an alkyl group having about 1 to 4 carbon atoms. The form of the alkyl chain can be appropriately set depending on the P450 used, the hydrocarbon-based substrate to be hydroxylated, and the like. The alkyl chain may be cyclic or may have a cyclic portion.

  Typically, the number of carbon atoms in the alkyl chain is preferably 5 or more, more preferably 6 or more, still more preferably 7 or more, still more preferably 8 or more, and The number is preferably 9 or more, and more preferably 10 or more. The carbon number of the alkyl chain is preferably 15 or less, more preferably 14 or less, still more preferably 13 or less, still more preferably 12 or less. The alkyl chain is preferably linear.

(Linker)
The first decoy molecule can include a linker containing a peptide bond connecting the first site and the second site connecting them. By providing such a linker, although not theoretically clear, even a decoy molecule having a non-fluorinated alkyl chain can be made to function.

  The linker may have a peptide bond, and may further have, for example, one or several or less (preferably one or two) alkylene groups between the linker and the first site. Examples of the alkylene chain include a methylene group, an ethylene group and a propylene group. The first decoy molecule having such a linker is represented by, for example, the following formula (1). The linker is a unit represented by [] in the formula (1).

  In the above formula (1), A represents a first site, B represents a second site, and R represents a hydrogen atom, an alkyl group, a thioalkyl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group, an aryl group. And an organic functional group containing one or more groups selected from aralkyl groups, and n can be an integer of 1 or more.

  The definition of the alkane described above can be applied to the alkyl group of R and the alkyl of the thioalkyl group in the formula (1). Further, the thioalkyl group can have a form in which about 1 or 2 carbon atoms in the alkyl group are substituted with sulfur atoms.

  As the alkenyl in the alkenyl group in R, the above-mentioned definition of the alkene can be applied. Further, the definitions of cycloalkane and cycloalkene described above can be applied to cycloalkyl and cycloalkenyl in the cycloalkyl group and cycloalkenyl group.

  As for the aryl in the aryl group, the above-mentioned definition of aromatic can be applied. Further, the definition of an alkyl group and the definition of an aryl group can be applied to the aralkyl group.

  R is preferably the side chain of an amino acid. The amino acid may be L-form or D-form, but the L-form can be preferably used. As the amino acids, in addition to the 20 types of natural amino acids that are the main components of proteins, various types of natural and unnatural amino acids can be used.

  R is more preferably the side chain of an amino acid residue having a hydrophobic side chain. Such amino acid residues include alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine and proline. From the viewpoint of hydrophobicity, leucine, methionine, phenylalanine and tryptophan are more preferable. Further, preferably, methionine, phenylalanine and tryptophan are mentioned.

  Although n in formula (1) is not particularly limited, it is preferably several or less, for example, one or two.

  The first decoy molecule represented by the formula (1) can be produced, for example, by introducing one amino acid into a carboxylic acid having an alkyl chain at the second site. The carboxyl group (first site) of the “N-acylamino acid” thus obtained corresponds to A of Formula (1), the alkyl group of the acyl group (second site) corresponds to B thereof, and the amino acid The side chain of is corresponding to the same R. Furthermore, two or more amino acids can be introduced into the alkylcarboxylic acid.

  This reaction is represented by the formula (1) by, for example, protecting the carboxyl group of an amino acid by esterification, then condensing the carboxylic acid with the carboxyl-protected amino acid, and then deprotecting. "N-acyl amino acid" can be obtained. Below, as a typical scheme, a synthetic scheme of N-acyl amino acid via methyl ester of amino acid is illustrated.

(Amino acid methyl esterification)
For example, put amino acid (19 mmol) in a 50 mL eggplant flask, dissolve in methanol (19 mL), cool to -20 ° C, and slowly add thionyl chloride (20 mmol, 1.45 mL) to this solution. After stirring for 1 hour at ° C, stir for 16 hours at room temperature. The reaction solution is evaporated under reduced pressure, the obtained white solid is washed with diethyl ether, and dried under reduced pressure.

(Condensation reaction of carboxylic acid and amino acid)
Then, in a 50 mL two-necked flask, for example, as methyl ester of amino acid, L-phenylalanine methyl ester hydrochloride (4 mmol, 863 mg), nonanoic acid (4 mmol, 643 mg), 1-hydroxybenzotriazole (HOBt ・ H ₂ O) (5 mmol, 676 mg) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC-HCl) (5 mmol, 978 mg) were added, and dimethylformamide (5 (mL) and dissolve. Subsequently, N, N'-diisopropylethylamine (DIPEA) (10 mmol, 1.29 g) was added, and the mixture was stirred at room temperature for 6 hours, then ion-exchanged water in the same volume as the reaction solution was added, and extracted 3 times with ethyl acetate. I do. The extracted organic phases are combined, washed with a saturated sodium hydrogen carbonate aqueous solution and a saturated saline solution, magnesium sulfate is added and the mixture is dried for 30 minutes, filtered, and then the solvent is distilled off by an evaporator. It was purified with a silica gel column using ethyl acetate: hexane = 3: 7 as a developing solvent. The corresponding fractions are collected, the solvent is evaporated and dried under vacuum to give a solid.

(Deprotection of methyl ester)
Add 1.0 M LiOH / THF = 4: 1 solution to the synthesized N-acyl amino acid methyl ester and stir at room temperature for 24 hours. The pH is adjusted to 1 by adding 2M HCl on an ice bath, extraction is performed 3 times with Et ₂ O, magnesium sulfate is added and the mixture is dried for 30 minutes, filtered, and the solvent is distilled off by an evaporator. The obtained solid was dissolved in a small amount of ethyl acetate, hexane was added on a water bath at 70 ° C until just before precipitation of the solid, and the mixture was allowed to stand at room temperature for recrystallization. The precipitated solid is suction filtered and vacuum dried to obtain a solid.

  Further, in the "N-acyl amino acid", the first site is a carboxylic acid, but by using an unnatural amino acid in place of an amino acid that condenses with an alkylcarboxylic acid, the first site is a functional group other than carboxylic acid. Can be the basis. For example, in order to obtain a carboxy group derivative such as an alkyl ester or amide, the carboxyl group of an amino acid is changed to a methyl ester group as a protecting group, and esterified with ethanol or a corresponding alcohol is condensed with an alkylcarboxylic acid Can be obtained by Further, for example, in order to make the first site an amide group, it can be obtained by condensing methylamine, ethylamine or the like with the carboxyl of an amino acid and then condensing with an alkylcarboxylic acid. Further, for example, to make the first site an aldehyde group, the carboxyl group of the amino acid is reduced to an aldehyde group, followed by acetal protection, and after condensation reaction with an alkylcarboxylic acid, deprotection with an acid is performed. Obtainable.

  R in the formula (1) can be further modified into various forms by utilizing the side chain of the amino acid.

  As described above, the first decoy molecule includes the first site, the second site, and the linker that connects them. It is believed that the linker enhances the interaction between the first site of the first decoy molecule and the substrate binding site of P450 or its opening.

  When the first decoy molecule is introduced with a hydroxyl group into the hydrocarbon-based substrate by P450, the hydrophilicity of the hydrocarbon-based substrate is improved and the first decoy molecule is rapidly discharged from the hydrophobic substrate-binding site. For this reason, introduction of a hydroxyl group into only one or a specific carbon atom tends to occur easily. Therefore, the first decoy molecule can be preferably used for such hydroxylation.

  In addition, the first decoy molecule tends to have a higher coupling efficiency (%), that is, a higher ratio (%) of the number of moles of NADPH consumed with respect to the amount of hydroxylation (number of moles) of the produced alcohol or the like (for example, , A second decoy molecule and the like described later), there are few side reactions, and the life of the enzyme can be extended.

(Hydrocarbon-based substrate hydroxylation method and hydrocarbon-based substrate hydroxide production method)
These methods disclosed herein can include the step of hydroxylating a first decoy molecule, P450 and a hydrocarbon-based substrate with the P450 in the presence of the substrate. According to these methods, it is possible to practically avoid various disadvantages caused by fluorine and to oxidize various substrates to obtain hydroxide.

  For the first decoy molecule, the hydrocarbon-based substrate, and P450, the various aspects already described can be applied. For hydroxylation of a hydrocarbon-based substrate, those skilled in the art can select an appropriate P450 from known P450s, design and obtain the first decoy molecule, and carry out the hydroxylation reaction.

  In this method, NADPH (NADP), oxygen molecules, and the like are preferably present in a state of being appropriately present for the purpose of maintaining the enzymatic activity of P450. Further, in the state of the hydrocarbon-based substrate (gas, gas, etc.), the substrate supply mode and the like can be changed as appropriate.

  This method may be carried out batchwise or continuously. P450 may be immobilized by using a known immobilizing means, and other known methods applied to industrial methods using enzymes may be appropriately applied.

  Although these methods do not limit the hydrocarbon-based substrate, they can be suitably used for the hydroxylation of ethane, propane, butane, heptane, cyclohexane, benzene and the like.

(Screening method for decoy molecules for hydroxylating a hydrocarbon substrate with P450)
The screening method disclosed in the present specification includes P450, a first site that is a terminal structure capable of binding to a substrate binding site of P450, a second site that is a non-fluorinated alkyl chain, and The P450 in the presence of one or more decoy molecule candidates and a hydrocarbon-based substrate, which comprises a linker containing a peptide bond that connects the one site and the second site. And a step of acting on. According to the present screening method, the hydroxylation of the hydrocarbon substrate of P450 by the decoy molecule candidate can be evaluated, and the decoy molecule can be screened for hydroxylating the desired hydrocarbon substrate. In this screening method, a suitable P450 can be screened by combining 1 or 2 or more candidates for P450.

  In this screening method, the above-mentioned action steps can be carried out in the same manner as the above-mentioned hydroxylation method.

  In this screening method, a first decoy molecule candidate in which the alkyl chain length, the alkyl chain form, the type of the second site, and the linker form in the first decoy molecule are appropriately combined can be prepared. Further, as described above, various known P450 candidates can be prepared for P450. When using the first decoy molecule, it may be preferable to use wild type P450.

(Decoy molecule of the second aspect)
The decoy molecule of the second aspect disclosed in the present specification (hereinafter referred to as the second decoy molecule) can introduce a hydroxyl group into a carbon atom of a hydrocarbon-based substrate by using a mutant P450.

  The second decoy molecule has a first site that is a terminal structure capable of binding to the substrate binding site of the mutant cytochrome P450 monooxygenase, and a second site that is an alkyl chain in the already-described first decoy molecule. The same configuration can be adopted except that at least a part of the above is fluorinated.

  Since such a second decoy molecule can suitably function against the mutant P450, it becomes possible to modify or enhance the catalytic activity of the mutant P450 by the second decoy molecule.

  The first site in the second decoy molecule is a substrate binding site of mutant P450, more specifically, a terminal structure capable of binding in the vicinity of its opening. Since the site of the mutant P450 is basically equivalent to the wild type P450, the first site of the second decoy molecule may be substantially the same as the first decoy molecule.

  The second site of the second decoy molecule may be fluorinated by substituting at least a part of hydrogen atoms bonded to carbon atoms with fluorine atoms. All hydrogen atoms may be replaced by fluorine atoms. Preferably, at least the hydrogen atom bonded to the 1st to 3rd carbon atoms counted from the end of the alkyl chain is substituted with fluorine. Further, preferably, at least the hydrogen atom bonded to the 1st to 7th carbon atoms counted from the end of the alkyl chain is substituted with fluorine.

  The second decoy molecule is preferably used for the mutant P450. Examples of the mutant P450 include various P450 mutants described above. For example, a mutant form of A328F of P450BM-3 can be mentioned. The A328F variant is A. Seifert, S. Vomund, K. Grohmann, S. Kriening, VB Urlacher, S. Laschat and J. Pleiss, ChemBioChem, 2009, 10, 853-861, A. Rentmeister, TR Brown, CD. Snow, MN Carbone and FH Arnold, ChemCatChem, 2011, 3, 1065-1071, E. Weber, A. Seifert, M. Antonovici, C. Geinitz, J. Pleiss and VB Urlacher, Chem. Commun., 2011, 47, 944-946 etc. Further, similar to P328BM of P450BM-3, preferred mutant P450s include P450BM-3A264V, A264I, A82W, A328V and A328I. Double mutants such as A264V / A328F, A264V / A328V, A264V / A328I are also included. These are located near the heme of the active center.

  The second decoy molecule can also be applied to the mutant P450, so that it becomes possible to efficiently hydroxylate a hydrocarbon-based substrate that was conventionally difficult in the wild type. For example, it can be suitably applied to alkanes having about 1 to 3 carbon atoms such as methane, ethane, and propane. More preferred are methane and ethane. It is significant that the second decoy molecule can be applied to mutant P450BM3 and the like to oxidize methane and ethane, especially methane using oxygen enzymatically. Previously, it was not reported that methane was oxidized by an enzyme having P450-containing heme iron as an active center to introduce a hydroxyl group.

  In addition, with respect to the second decoy molecule, various aspects of the first decoy molecule can be applied as they are, except for the aspect in which at least a part of the second site is fluorinated.

(Hydrocarbon-based substrate hydroxylation method and hydrocarbon-based substrate hydroxide production method)
In these methods disclosed in the present specification, in addition to using the second decoy molecule, the hydroxylation method and the hydroxide production method for the first decoy molecule are applied except that the mutant P450 is used. You can

(Method for screening decoy molecule for hydroxylation of hydrocarbon substrate with mutant cytochrome P450 monooxygenase)
The screening method disclosed herein includes a mutant cytochrome P450 monooxygenase, a first site that is a terminal structure capable of binding to a substrate binding site of the mutant P450, and an alkyl chain at least partially fluorinated. One or more second decoy molecule candidates and a hydrocarbon-based substrate, each of which has a second site that is and a linker containing a peptide bond that connects the first site and the second site. In the presence of the enzyme, the step of allowing the mutant cytochrome P450 monooxygenase to act on the hydrocarbon-based substrate can be provided. According to the present screening method, the hydroxylation of the mutant P450 hydrocarbon substrate by the second candidate decoy molecule can be evaluated, and the second decoy molecule for hydroxylating a desired hydrocarbon substrate can be identified. Can be screened. In the present screening method, a suitable mutant P450 can also be screened by combining one or more candidates for the mutant P450.

  In this screening method, the above-mentioned action steps can be carried out in the same manner as the above-mentioned hydroxylation method.

  In this screening method, as in the case of the first decoy molecule, the first decoy molecule candidate is prepared by appropriately combining the alkyl chain length, the alkyl chain form, the second site type, and the linker form. You can Further, as described above, various known P450 candidates can be prepared for the mutant P450.

(Kit for hydroxylation containing decoy molecule and decoy molecule composition)
The present specification can further provide a kit for hydroxylation, which comprises the decoy molecule of the first aspect and / or the decoy molecule of the second aspect already described. Such a kit can also include one or two or more decoy molecules having different structures for each decoy molecule of each of the first and second aspects. In addition, such kits can also include one or more P450s and mutant P450s.

  The decoy molecule may be provided as a single reagent, or may be provided as a composition in which P450 and / or mutant P450 to be applied are combined in advance. By being provided as a composition, the hydroxylation reaction can be easily carried out.

  Hereinafter, the disclosure of the present specification will be described with reference to specific examples, but the disclosure of the present specification is not limited to the following examples.

(P450 expression purification)
DNA (amino acid sequence, SEQ ID NO: 1) encoding the entire length of cytochrome P450BM3 (CYP102A1) in Escherichia coli BL21 (DE3) strain (CJC Whitehouse, SG Bell, LL Wong, Chem. Soc. Rev. 2012, 41, 1218-1260. 2) of the CYP102A subfamily disclosed in Fig. 2) was inserted into the pUC vector into which the DNA encoding the PUCBM3 gene was expressed. In order to extract and purify the produced P450BM3 from E. coli, the E. coli was disrupted by sonication, the disrupted solution was centrifuged and the supernatant was collected, and this supernatant was once bound to the anion exchange column (DE-52). Elution was performed with a gradient of 0 to 250 mM KCl, and a brown peak fraction showing P450BM3 was collected.

  Check the purity of the eluted peak fractions by SDS-PAGE, mix the highly purified fractions, concentrate with an ultrafiltration kit with a molecular weight cut-off (MWCO) of 30,000, and then use buffer A (20 mM Tris -HCl (pH 7.4)) and the KCl concentration was adjusted to 5 mM or less for desalting. Then, using an anion exchange column (DEAE650S), KCl was subjected to a concentration gradient in the range of 0 to 1000 mM to elute BM3, and the same purification was performed. Finally, Buffer B (20 mM Tris-HCl (pH7.4), 100 mM KCl) was passed through a gel filtration column (Sephacryl S-300HR) to elute P450BM3, and the obtained brown peak fraction was collected, This was used as a wild-type P450BM3 sample in the following reaction.

(Benzene hydroxylation reaction 1)
In this example, N-acyl amino acids obtained by modifying C9 alkylcarboxylic acid (nonanoic acid) with various amino acids were synthesized, and these N-acyl amino acids were used as decoy molecules and added to wild-type P450BM3 to obtain wild-type P450BM3. It was confirmed that P450BM3 can be provided with benzene hydroxylation activity. Hereinafter, all concentrations are shown as final concentrations.

  Nonanoic acid was modified with glycine (Gly), alanine (Ala), leucine (Leu), methionine (Met), phenylalanine (Phe) and tryptophan (Trp) to synthesize various N-acyl amino acids. Regarding the introduction of the amino acid into the nonanoic acid, as already described, the amino acid was methyl-esterified, condensed with nonanoic acid, and then deprotected to obtain various N-acyl amino acids. The scheme is shown below. Various reagents were used at the ratios shown below with respect to 1 equivalent of amino acid.

  Fill 90% or more of the total volume of the reaction solution with buffer B saturated with high-purity oxygen gas, add 500 nM wild-type P450BM3, 10 mM benzene, 100 μM each N-acyl amino acid, and finally add 5 mM NADPH. The reaction started. Since it was necessary to use a DMSO solution to dissolve the N-acyl amino acid, the reaction solution consequently contained 0.5 v / v% DMSO.

  The reaction was performed in a closed vial, and the reaction solution was allowed to proceed at 25 ° C or lower while stirring. After the reaction for 10 minutes, the reaction was stopped by adding 0.15 mL of 1M hydrochloric acid, and the mixture was stirred for 10 minutes. The reaction solution was neutralized with 0.15 mL of a 1 M aqueous sodium hydroxide solution, 1.3 mL of acetonitrile was added, and then filtered through a filter and analyzed by HPLC. LC-10ADVP manufactured by Shimadzu was used for HPLC, and a reverse phase column Inertsil ODS-3 (internal diameter 4.6 mm, column length 250 mm, particle diameter 5 μm) manufactured by GL Sciences was used as the column. Buffer B / acetonitrile = 1: 1 was used as an eluent, and measurement was performed at a flow rate of 0.5 mL / min for 35 minutes.

  In addition, the same operation was performed except that nonanoic acid was used instead of the N-nonanoyl amino acid, and a comparative example was obtained. Further, the same operation was performed as a system in which N-acyl amino acid was not added and only 0.5 v / v% DMSO was added instead.

  On the HPLC chromatogram of each reaction solution, a new peak which was not found in Comparative Example and Control Example was observed. Since it was confirmed that the retention time of the new peak coincided with the retention time of phenol, it was found that phenol was produced as a reaction product in this reaction solution. The amount of phenol and the amount of NADPH consumed in each reaction solution were also measured. Table 1 shows the amount of phenol produced per minute with respect to the added N-nonanoyl amino acid, the amount of NADPH consumed, and the ratio of the amount of NADPH used to produce phenol (coupling ratio).

  As shown in Table 1, decoy molecules obtained by modifying nonanoic acid with amino acids having hydrophobic side chains of glycine, alanine, leucine, methionine, phenylalanine, and tryptophan are all capable of oxidizing benzene to phenol. all right. On the other hand, no phenol was produced in the nonanoic acid which was not modified with an amino acid (comparative example) and the control example.

  It was also found that the phenol hydroxylation activity and NADPH consumption were changed by changing the amino acid site of N-acyl amino acid. In the hydroxylation reaction of benzene, C9-Phe modified with phenylalanine showed the highest oxidative activity when a nonoic acid having an alkyl chain having 9 carbon atoms was modified with an amino acid.

(Benzyl hydroxylation reaction 2)
In this example, the chain length of the alkyl chain of N-acyl amino acid and the benzene oxidation activity were evaluated. In this example, according to Example 2, octanoic acid, (C8), nonanoic acid (C9), decanoic acid (C10) and undecanoic acid (C11) were modified with phenylalanine to synthesize various N-acylphenylalanines. . The various decoy molecules were subjected to a benzene hydroxylation reaction in the same manner as in Example 2. Table 2 shows the results.

  As shown in Table 2, phenol hydroxylation activity and NADPH consumption varied depending on the type of amino acid. Among phenylalanine-modified N-acylphenylalanines, N-decanoylphenylalanine (C10-Phe) having an alkyl chain with 10 carbon atoms showed the highest oxidative activity in benzene hydroxylation. Furthermore, among N-acyl leucines modified with leucine instead of phenylalanine, the highest oxidative activity was obtained when N-decanoyl leucine (C10-Leu) having an alkyl chain having 10 carbon atoms was added.

  The cytochrome P450BM3 (CYP102A5) (CJC Whitehouse, SG Bell, LL Wong, Chem. Soc. Rev. 2012, 41, 1218-1260. Fig. 2 is A5 in the alignment result of the CYP102A subfamily disclosed in Fig. 2. ) Is also operated in the same manner as in Example 1 to obtain A5 wild-type P450BM3, and the benzene oxidation activity of N-decanoylleucine (C10-Leu) is determined in the same manner as described above. Upon evaluation, oxidation to phenol was confirmed as in the case of A1 wild type.

(Propane hydroxylation reaction)
In this example, it was evaluated whether N-nonanoylleucine (C9-Leu) obtained by modifying nonanoic acid with leucine has a propane hydroxylation activity with respect to wild-type P450BM3.

  A mixed gas of high-purity propane gas and high-purity oxygen gas was blown into the buffer solution B to be saturated to obtain a mixed gas saturated buffer solution B. 90% or more of the total volume of the reaction solution was filled with this mixed gas saturation buffer B, 500 nM P450BM3 and 100 μM N-nonanoylleucine were added, and finally 5 mM NADPH was added to start the reaction.

  The reaction was carried out in a closed vial, the vial was connected to a balloon filled with a mixed gas of propane gas and oxygen gas, and the reaction solution was allowed to proceed while stirring under the pressure of the balloon. After the reaction for 10 minutes, the alcohol was derivatized to a nitrite in order to detect and quantify the produced alcohol. Derivatization to nitrites follows the literature Nguyen et al., Anal. Sci. 2001, 17, 639-643 and Peters et al., J. Am. Chem. Soc. 2003, 125, 13442-13450. After adding 0.20 g of sodium nitrite and 1.0 mL of hexane to 1 ml of the reaction sample, 150 μl of 20% sulfuric acid was added dropwise on ice and the reaction was performed for 15 minutes. After derivatization, the hexane phase was recovered and washed with pure water. The hexane phase after washing was analyzed by gas chromatography. As the gas chromatograph, GC-2014 having a FID detector manufactured by Shimadzu was used, and as the column, an Rtx-1 column (internal diameter 0.53 mm, film thickness 3 μm, column length 60 m) manufactured by Restek was used. The separation conditions were set such that the sample inlet temperature was 250 ° C., the temperature program of the column oven was maintained at 50 ° C. for 5 minutes, the temperature was raised to 200 ° C. over 6 minutes, and then maintained for 3 minutes. The detector was set at 250 ° C. The control was a system in which N-nonanoylleucine was not added, but only 0.5 v / v% DMSO was added instead, and the same procedure was used.

  When the sample obtained in this reaction was examined by gas chromatography, only one new peak, which was not found in the control, was obtained. The propane oxidation reaction may produce 1-propanol or 2-propanol. Then, when 2-propanol was directly injected and the retention time was determined, it was detected at a position just overlapping with the reaction product. From this, it was found that the reaction product was 2-propanol. In addition, it was revealed that propanol derivatized to nitrite cannot be detected as nitrite under this condition due to the instability of the derivative, but is decomposed and detected as propanol itself.

  When 2-propanol was quantified using 1 mM 3-pentanol as an internal standard, more than 220 μM of 2-propanol was produced under the above conditions, and the coupling rate (%) was 44%.

(Methane hydroxylation reaction)
In this example, it was evaluated whether N-decanoylphenylalanine (C10-Phe) obtained by modifying decanoic acid with phenylalanine has a methane hydroxylation activity against a P450BM3 mutant P450BM3A328F mutant. P450BM3 A328F was prepared by introducing a point substitution mutation of A328F into the previously described amino acid sequence of P450BM3 using the Quik Change Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA).

90% of the total volume of the reaction solution with a mixed gas saturated buffer solution (20 mM Tris-HCl (pH 7.4), 100 mM KCl) which was saturated by blowing a mixed gas of high purity ¹³ C methane gas and high purity oxygen gas into buffer solution B. Satisfy the above, add 2μM P450BM3 A328F mutant, 100μM N-decanoylphenylalanine, catalase 5 × 10 ⁷ unit, SOD (superoxide dismutase) 2 × 10 ³ unit, and finally add 5 mM NADPH to react. Started. Catalase and SOD were added to suppress the inactivation of P450. Since the DMSO solution was used to dissolve N-decanoylphenylalanine, the reaction solution contained 0.5 v / v% DMSO.

  The reaction was carried out in a closed vial, and after 10 minutes of reaction, it was analyzed by GC-MS.

When the sample obtained in this reaction was examined by GC-MS, a peak derived from ¹³ C methanol was obtained. When ¹³ C methanol was quantified using 100 μM tert-butyl alcohol as an internal standard, 6.8 μM ¹³ C methanol was produced when the reaction was performed for 10 minutes under the above conditions, and the catalyst rotation speed of 3.4 rotations was obtained in 10 minutes. showed that.

  Further, except that N-perfluorononanoyltryptophan (fluorine-substituted decoy molecule) was used in place of N-decanoylphenylalanine, the methane hydroxylation reaction was carried out in the same manner as above, and methanol was produced. The catalyst rotation number was 20 times.

  In the same manner, P450BM-3A 264V, the same 264I, the same A82W, the same A328V, the same A328I, A264V / A328F, A264V / A328V, and the double mutants such as A264V / A328I were also confirmed to oxidize methane. However, it was confirmed that methanol was produced.

  The P450BM3 A328F mutant was replaced with its wild type, and N-decanoylphenylalanine and N-perfluorononanoyltryptophan were used as decoy molecules to perform a methane hydroxylation reaction in the same manner as above. It was confirmed that no methanol was produced at all.

  From the above, it was found that the decoy molecule obtained by introducing an amino acid into an alkylcarboxylic acid is applicable to both wild-type and mutant P450s. It was also found that the decoy molecule obtained by introducing an amino acid into a fluorocarboxylic acid having a fluorine atom in place of a hydrogen atom has a higher hydroxylation activity-enhancing activity for mutant P450. That is, it was found that the fluoro N-acyl amino acid obtained by introducing an amino acid into fluorocarboxylic acid had higher compatibility with mutant P450. It was also found that such decoy molecules can enzymatically convert methane to methanol.

(Ethanol hydroxylation reaction)
In this example, it was evaluated whether N-decanoylphenylalanine (C10-Phe) obtained by modifying decanoic acid with phenylalanine has a methane hydroxylation activity against a P450BM3 mutant P450BM3A328F mutant.

A mixed gas saturated buffer solution (20 mM Tris-HCl (pH 7.4), 100 mM KCl) obtained by blowing a saturated mixture gas of high-purity ¹³ C ethane gas and high-purity oxygen gas into buffer solution B to be saturated was used, except that A hydroxylation reaction was performed in the same manner as in 5, and after 10 minutes of reaction, analysis was performed by GC-MS. As a result, it was found that ethanol was produced.

Claims (15)

An agent for hydroxylating a hydrocarbon substrate with cytochrome P450 monooxygenase, comprising:
A first site which is a terminal structure capable of binding to the substrate binding site of the cytochrome P450 monooxygenase;
A second moiety which is a non-fluorinated alkyl chain,
A linker containing a peptide bond, which connects the terminal structure and the alkyl chain,
An agent containing a decoy molecule for hydroxylating a hydrocarbon-based substrate with a cytochrome P450 monooxygenase, comprising:   The agent according to claim 1, wherein the first site is selected from the group consisting of a hydrogen atom, a hydroxyl group, an aldehyde group, a carboxyl group and a carboxyl derivative group. The agent according to claim 1, which is represented by the following formula (1).

(In the formula (1), A represents a first site, B represents a second site, R represents a hydrogen atom, an alkyl group, a thioalkyl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group, (Represents an organic functional group containing one or more groups selected from an aryl group and an aralkyl group, and n represents an integer of 1 or more.)   The agent according to claim 3, wherein R represents a side chain of an amino acid selected from the group consisting of leucine, methionine, phenylalanine and tryptophan. The agent according to any one of claims 1 to 4, wherein the cytochrome P450 monooxygenase is one or more selected from the following cytochrome P450 monooxygenases (a) and (b).
(A) Cytochrome P450 monooxygenase having 40% or more identity with the amino acid sequence of cytochrome P450BM3 (b) Cytochrome P450 monooxygenase having 40% or more identity with the amino acid sequence of cytochrome P450cam   The agent according to claim 5, wherein the cytochrome P450 monooxygenase is a mutant having one or more amino acid substitution mutations in cytochrome P450BM3 or cytochrome P450cam, and is a mutant having cytochrome P450 monooxygenase activity. .   The agent according to claim 1, wherein the hydrocarbon-based substrate is one or more selected from the group consisting of cyclohexane, butane, propane, benzene and ethane. A method for hydroxylating a hydrocarbon-based substrate, comprising:
Hydroxylating the hydrocarbon-based substrate with the cytochrome P450 monooxygenase in the presence of the agent according to any one of claims 1 to 7, the cytochrome P450 monooxygenase and the hydrocarbon-based substrate,
Comprising a method. A method for producing a hydroxide of a hydrocarbon-based substrate, comprising:
Hydroxylating the hydrocarbon-based substrate with the cytochrome P450 monooxygenase in the presence of the agent according to any one of claims 1 to 7, the cytochrome P450 monooxygenase and the hydrocarbon-based substrate,
Comprising a method. A method for screening decoy molecules for hydroxylating hydrocarbons with cytochrome P450 monooxygenase, comprising:
Cytochrome P450 monooxygenase, a first site which is a terminal structure capable of binding to the substrate binding site of the cytochrome P450 monooxygenase, a second site which is a non-fluorinated alkyl chain, the first site and the above A peptide-bonded linker connecting to the second site, and the cytochrome P450 monooxygenase acting on the hydrocarbon-based substrate in the presence of one or more decoy molecule candidates and the hydrocarbon-based substrate. The process of
Equipped with
A screening method for evaluating hydroxylation of the hydrocarbon-based substrate by the cytochrome P450 monooxygenase. An agent for hydroxylating methane with a mutant cytochrome P450 monooxygenase,
A first site which is a terminal structure capable of binding to the substrate binding site of the mutant cytochrome P450 monooxygenase;
A second moiety that is at least partially fluorinated alkyl chain;
A linker containing a peptide bond, which connects the terminal structure and the alkyl chain,
An agent comprising a decoy molecule for hydroxylating methane with a mutant cytochrome P450 monooxygenase, comprising:   The agent according to claim 11, wherein the mutant cytochrome P450 monooxygenase is one or more amino acid substitution mutants in the amino acid sequence represented by SEQ ID NO: 1, and has cytochrome P450 monooxygenase activity. A method for producing methane hydroxide,
Hydroxylating methane with the mutant cytochrome P450 monooxygenase in the presence of the agent according to claim 11, the mutant cytochrome P450 monooxygenase and methane,
Comprising a method.   The mutant cytochrome P450 monooxygenase is one or two or more amino acid substitution mutations in the amino acid sequence represented by SEQ ID NO: 1 and shows 90% or more identity with the amino acid sequence represented by SEQ ID NO: 1. 14. The method of claim 13, which has and comprises cytochrome P450 monooxygenase activity. A method for screening a decoy molecule for hydroxylating methane with a mutant cytochrome P450 monooxygenase, comprising:
Mutant cytochrome P450 monooxygenase, a first site that is a terminal structure capable of binding to the substrate binding site of the mutant cytochrome P450 monooxygenase, and a second site that is at least a fluorinated alkyl chain, In the presence of one or more candidate decoy molecules, the mutant cytochrome P450 monooxygenase and methane, which comprises a linker containing a peptide bond that connects the first site and the second site, A step of acting a mutant cytochrome P450 monooxygenase on methane,
Equipped with
A screening method for evaluating the hydroxylation of methane by the mutant cytochrome P450 monooxygenase.