JP5761548B2 - Inert hydrocarbon hydroxylation process and dummy molecules - Google Patents

Description

  The present invention relates to a method for hydroxylating an inert hydrocarbon using cytochrome P450 monooxygenase and a dummy molecule, and a dummy molecule used in this hydroxylation method.

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

  For example, cytochrome P450cam can catalyze camphor hydroxylation at a rate exceeding 1000 revolutions per minute. This catalytic activity is very high compared to the catalytic activity of mammalian P450 (several to several tens of rotations per minute). Cytochrome P450BM3 catalyzes the hydroxylation of the first to third (ω-1 to ω-3) methylene groups counted from the end of the fatty acid. The catalytic activity of cytochrome P450BM3 reaches several thousand revolutions per minute, and cytochrome P450BM3 has the maximum activity as cytochrome P450 monooxygenase.

  In addition, since eukaryote-derived P450 is a membrane protein, its affinity for water is extremely low. On the other hand, the above-mentioned cytochrome P450 monooxygenase has a high affinity for water and is easy to purify, crystallize and handle the enzyme protein.

  Cytochrome P450 uses an electron donor such as NADPH (hereinafter referred to as a P450 reduction system) and oxygen to catalyze hydroxylation of the substrate. In order to improve the catalytic activity of isolated cytochrome P450, it is necessary to efficiently advance electron transfer from the P450 reduction system to P450. The above-mentioned cytochrome P450BM3 is different from normal cytochrome P450, and a part of the P450 reduction system (FAD and FMN in cytochrome P450BM3) and P450 are fused and exist as one polypeptide. And it is one factor of the outstanding catalytic activity of cytochrome P450 monooxygenase that a part of P450 reduction system and P450 are fused and very close. In other words, since cytochrome P450BM3 is a fusion protein, it exhibits very good catalytic activity. P450cam is not a fusion protein. For this reason, putidaredoxin (Pdx) and putidaredoxin reductase (PdR) are essential for the catalytic reaction of P450cam. However, P450cam also exhibits excellent catalytic activity as described above.

  For these reasons, cytochrome P450 monooxygenase is considered suitable as a biocatalyst.

  By the way, in order to hydroxylate a substance that does not become a substrate of wild-type cytochrome P450 or a substrate that is hardly hydroxylated by wild-type cytochrome P450, a bacterium that produces wild-type cytochrome P450 is transformed, and these substances are transformed. Has been proposed (see, for example, Patent Documents 1 to 3).

  For example, wild-type cytochrome P450 monooxygenase has a very low catalytic activity against inert hydrocarbons such as cyclohexane, propane, benzene, ethane, and methane. However, the mutant cytochrome P450 monooxygenase may be able to catalyze the hydroxylation of these inert hydrocarbons.

  However, it is difficult to mass-produce mutant cytochrome P450 monooxygenase at low cost. Therefore, the mutant cytochrome P450 monooxygenase has a problem that it is not suitable for hydroxylating an inactive hydrocarbon in a large amount and continuously (ie, industrially).

JP 2000-508163 A Special table 2003-517815 gazette Japanese translation of PCT publication No. 2003-521889

  This invention is made | formed in view of the said situation, and it aims at providing the method which can hydroxylate an inactive hydrocarbon industrially using cytochrome P450 monooxygenase.

The method for hydroxylating an inert hydrocarbon of the present invention that solves the above problem includes a step of hydroxylating the inert hydrocarbon in the presence of cytochrome P450 monooxygenase, a dummy molecule, and the inert hydrocarbon. ,
The dummy molecule has a terminal structure capable of binding to the binding site of the cytochrome P450 monooxygenase, and an alkyl chain,
It is characterized in that at least a part of hydrogen contained in the alkyl chain is substituted with fluorine.

  The method for hydroxylating an inert hydrocarbon according to the present invention preferably includes any one of the following (1) to (5), and more preferably includes a plurality of (1) to (5).

  (1) The cytochrome P450 monooxygenase is a cytochrome P450 monooxygenase having a cytochrome P450 monooxygenase having 40% or more homology with the amino acid sequence of cytochrome P450BM3 and / or a cytochrome P450 monooxygenase having a homology of 40% or more with the amino acid sequence of cytochrome P450cam. is there.

  (2) Among the dummy molecules, at least hydrogen contained in the first to third alkyl groups counted from the end of the alkyl chain is substituted with fluorine.

  (3) The terminal structure is at least one selected from a hydrogen terminal, a hydroxyl group, an aldehyde group, and a carboxyl group.

  (4) The inert hydrocarbon is at least one selected from cyclohexane, butane, propane, and benzene.

(5) The cytochrome P450 monooxygenase has a homology of 40% or more with the amino acid sequence of cytochrome P450BM3,
The dummy molecule is perfluorocarboxylic acid.

Composition comprising a dummy molecule of the present invention for solving the above-mentioned problems, coexist with cytochrome P450 monooxygenase and an inert hydrocarbon, a composition comprising a dummy molecule for hydroxylating said non hydrocarbon ,
A terminal structure capable of binding to the binding site of the cytochrome P450 monooxygenase, and an alkyl chain;
It is characterized in that at least a part of hydrogen contained in the alkyl chain is substituted with fluorine.

The composition containing the dummy molecule of the present invention preferably comprises any of the following (6) to (8), and more preferably comprises a plurality of (6) to (8).

  (6) At least hydrogen contained in the first to third alkyl groups counted from the end of the alkyl chain is substituted with fluorine.

  (7) The terminal structure is at least one selected from a hydrogen terminal, a hydroxyl group, an aldehyde group, and a carboxyl group.

(8) The cytochrome P450 monooxygenase has a homology of 40% or more with the amino acid sequence of cytochrome P450BM3,
The dummy molecule is perfluorocarboxylic acid.
Hereinafter, in the present specification, the “composition containing a dummy molecule” is simply abbreviated as a dummy molecule.

  Hereinafter, cytochrome P450 monooxygenase is abbreviated as P450. The catalytic reaction of P450 is considered to start as follows.

  In the active center of P450, there is a binding site to which the substrate binds. When the substrate binds to the binding site, water molecules present at the active center are pushed out of the active center. Hereinafter, this state is referred to as an activation switch being turned on. The active center then catalyzes the hydroxylation of the substrate after being subjected to the action of electrons, oxygen molecules, and the like. Therefore, it is considered that the catalytic reaction of P450 does not start unless the activation switch is turned on. Note that lauric acid, myristic acid, and palmitic acid are known as wild-type P450 substrates. These substrates have a terminal structure (carboxyl group) bonded to the binding site and an alkyl chain.

  The method for hydroxylating an inert hydrocarbon according to the present invention is a method for catalyzing the hydroxylation of an inert hydrocarbon by P450 in the presence of a dummy molecule. The dummy molecule has a portion (terminal structure) capable of binding to the binding site of P450 and an alkyl chain. For this reason, it is considered that when a dummy molecule and P450 coexist, the dummy molecule binds to the binding site of P450 instead of the substrate and an activation switch is turned on.

  As mentioned above, the catalytic activity of wild-type P450 against inert hydrocarbons is very low. However, when the activation switch is turned on, the catalytic activity of wild-type P450 against inert hydrocarbons is improved. Therefore, according to the method for hydroxylating an inert hydrocarbon of the present invention, the inert hydrocarbon can be hydroxylated using wild-type P450.

  In this dummy molecule, at least a part of hydrogen contained in the alkyl chain is substituted with fluorine. For this reason, it is considered that the dummy molecule increases the hydrophobicity of the active center and excludes water molecules from the active center. For this reason, according to the method for hydroxylating an inert hydrocarbon of the present invention, the activation switch in P450 is easily turned on, and the catalytic reaction is likely to proceed.

  By these actions, according to the method for hydroxylating an inert hydrocarbon of the present invention, the inert hydrocarbon can be hydroxylated even by wild-type P450. Therefore, according to the method for hydroxylating an inert hydrocarbon of the present invention, the inert hydrocarbon can be industrially hydroxylated using cytochrome P450 monooxygenase. In addition, according to the hydroxylation method of the inert hydrocarbon of this invention, it is also possible to hydroxylate an inert hydrocarbon using the mutant type P450.

It is explanatory drawing which represents typically the reactor used in the hydroxylation confirmation test of propane. It is a graph showing the relationship between the alkyl chain length of a dummy molecule, the propanol production | generation amount, and NADPH consumption. It is a graph showing the relationship between reaction time, propanol production | generation amount, and NADPH consumption. It is explanatory drawing which represents typically the active center of P450BM3 which the palmitic acid couple | bonded, and the active center of P450BM3 which PFC10 couple | bonded. It is a graph showing the relationship of the chain length of a dummy molecule, the presence or absence of hexane, and NADPH consumption. It is a graph showing the gas-chromatography analysis result obtained by the hydroxylation confirmation test of cyclohexane. It is a graph showing the relationship of the chain length of a dummy molecule, the amount of cyclohexanol production | generation, and NADPH consumption. It is a graph showing the gas-chromatography analysis result obtained by the hydroxylation confirmation test of benzene. It is a graph showing the relationship of the alkyl chain length of a dummy molecule, the amount of phenol production, and NADPH consumption. It is a graph showing the relationship of the alkyl chain length of a dummy molecule, 2-butanol production amount, and NADPH consumption.

  As P450 used in the method for hydroxylating an inert hydrocarbon of the present invention, cytochrome P450BM3 (CYP102A1) and / or cytochrome P450cam (camphor 5-monooxygenase, CYP101) can be particularly preferably selected. As described above, these P450s are excellent in catalytic activity.

Further, as P450, not only cytochrome P450BM3 and cytochrome P450cam, but also an enzyme partially different in amino acid sequence from these enzymes may be used. For example, P450 having a homology of 40% or more with the amino acid sequence of cytochrome P450BM3 (family enzyme of cytochrome P450BM3) or P450 having a homology of 55% or more with the amino acid sequence of cytochrome P450BM3 (subfamily enzyme of cytochrome P450BM3) It can be preferably used. Alternatively, P450 having a homology of 40% or more with the amino acid sequence of cytochrome P450cam (family enzyme of cytochrome P450cam) or P450 having a homology of 55% or more with the amino acid sequence of cytochrome P450cam (subfamily enzyme of cytochrome P450cam) It can be preferably used. The cytochrome P450BM3 family enzyme or subfamily enzyme is preferably capable of hydroxylating fatty acids, 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 cytochrome P450BM3 is described in, for example, J. Biol. chem. 264 (19), 10987-10998 (1989) and the NCBI beta base (National Center for Biotechnology Information http://www.ncbi.nlm.nih.gov/protein/P14779.2). Are known. The amino acid of cytochrome P450cam is also known as disclosed in the NCBI database (http://www.ncbi.nlm.nih.gov/protein/YP — 714029). The dummy molecule used in the method for hydroxylating an inert hydrocarbon of the present invention has a terminal structure and an alkyl chain. An example of a dummy molecule is shown in Formula 1 below.
(Formula 1)

  The terminal structure (R in Formula 1) only needs to be able to bind to the binding site of P450, and examples thereof include a hydrogen terminal, a hydroxyl group, an aldehyde group, and a carboxyl group. The terminal structure of the dummy molecule may be only 1 or plural. The plurality of terminal structures contained in one dummy molecule may be the same structure or different structures.

  The chain length of the alkyl chain in the dummy molecule is not particularly limited. Further, the dummy molecule may have only one alkyl chain, may be branched into a plurality, or may be cyclic. In the dummy molecule of the present invention, it is sufficient that part or all of the hydrogen contained in the alkyl chain is fluorine-substituted, but as will be described later, at least the first to third alkyl groups counted from the end of the alkyl chain. It is preferable that hydrogen contained in is substituted with fluorine. It is more preferable that at least hydrogen contained in the first to seventh alkyl groups counted from the end of the alkyl chain is substituted with fluorine.

As the dummy molecule, for example, the one shown in Formula 2 can be used.
(Formula 2)

  In the method for hydroxylating an inert hydrocarbon of the present invention, the reaction of P450, the dummy molecule and the inert hydrocarbon may be performed batchwise or continuously. When the reaction is carried out continuously, P450 may be immobilized on a known enzyme immobilization means (for example, a reaction vessel wall or a filter). Furthermore, the reaction system in the method for hydroxylating an inert hydrocarbon of the present invention preferably contains factors necessary for the reaction of P450 such as NADPH (NADP) and oxygen molecules.

  As P450, a wild type may be used or a mutant type may be used. However, in order to industrially hydroxylate an inert hydrocarbon as described above, it is preferable to use a wild type.

  Hereinafter, the method for hydroxylating an inert hydrocarbon and dummy molecules of the present invention will be described in detail.

1. Preparation of P450 [1] Preparation of cytochrome P450BM3 Cytochrome P450BM3 (hereinafter abbreviated as P450BM3) was partially purified by the following method.

  First, a pUC vector into which the full-length base sequence of P450BM3 was inserted into E. coli (DH5α strain) to express the P450BM3 gene.

  P450BM3 produced by Escherichia coli was extracted from Escherichia coli by culturing the Escherichia coli and sonicating the obtained Escherichia coli.

  The obtained crushed liquid was fractionated (salted out) by the ammonium sulfate precipitation method, and the precipitate produced at an ammonium sulfate saturation of 40 to 60 (% saturation) was recovered. This precipitate was dispersed in Tris-HCl buffer solution pH 7.4 (hereinafter referred to as buffer A). The precipitate dispersion was dialyzed twice using 100 times (volume ratio) of buffer A as described above. By this operation, ammonium sulfate in the precipitate dispersion was desalted.

  The dialyzed precipitate dispersion (referred to as crude enzyme solution) was passed through an anion exchange column (DEAE650S or DE-52), and P450BM3 contained in the crude enzyme solution was adsorbed onto the column. P450BM3 adsorbed on the column was eluted using an aqueous KCl solution (concentration gradient 0 to 500 mmol / l), and a brown peak fraction which was the color of P450BM3 was collected.

  There is a possibility that the substrate is bound to P450BM3 contained in the recovered crude enzyme solution. In order to remove this substrate from P450BM3, NADPH in an amount of about 500 mmol / l was added to the recovered crude enzyme solution and reacted at 4 ° C. for 30 minutes. After the reaction, the crude enzyme solution was passed through a gel filtration column (Sephacryl S300) to separate the remaining NADPH and replace the buffer solution. The buffer solution used here is a buffer solution (hereinafter referred to as buffer solution B) in which KCl in an amount of 100 mmol / l is added to buffer solution A. The brown peak fraction obtained through the gel filtration column was collected, and the obtained fraction (referred to as P450BM3 solution) was used as P450 for the following tests.

2. Preparation of dummy molecule A C8-14 perfluoro fatty acid was prepared as a dummy molecule.

  In PFC8 to PFC14, all hydrogen contained in the alkyl chain is substituted with fluorine. A solution obtained by dissolving each dummy molecule in methanol was used as a dummy molecule solution in the following tests.

3. Inert hydrocarbon hydroxylation test using P450 and dummy molecules [1] Propane hydroxylation confirmation test Using the P450BM3 solution and PFC10 solution described above, propane, a kind of inert hydrocarbon, was hydroxylated.

  A mixed gas in which high purity propane gas and high purity oxygen gas were mixed at 80:20 (volume ratio) was prepared. This mixed gas was blown into the buffer solution B described above to prepare a mixed gas saturated buffer solution B. P450BM3 solution, PFC10 solution, and NADPH were added to this mixed gas saturated buffer B to initiate the propane hydroxylation reaction. The mixed gas saturated buffer B was contained in 90% by volume or more in the reaction solution at the start of the reaction. In the reaction solution at the start of the reaction, the concentration of P450BM3 was 500 nmol / l, the concentration of PFC10 was 100 μmol / l, and the concentration of NADPH was 5 mmol / l. Further, this reaction liquid contained 0.5% by volume of methanol.

  As shown in FIG. 1, the reaction was performed in a sealed vial. A balloon filled with the above-mentioned mixed gas was connected to this vial. The reaction was allowed to proceed while stirring the reaction solution under the pressure of the balloon. 30 minutes after the start of the reaction, alcohol (propanol) produced by the reaction was detected and quantified by gas chromatography. As a control experiment, a system in which only 0.5% by volume of methanol was added instead of PFC10 and a system in which propane gas was not added were reacted in the same manner, and propanol in the reaction solution was analyzed by gas chromatography. .

  Gas chromatography analysis was performed as follows.

  First, the alcohol in the reaction solution was derivatized to nitrite. The derivatization of the alcohol is carried out according to the literature (Nguyen et al., Anal. Sci. 2001, 17, 639-643 and Peters et al., J. AM. Chem. Soc. 2003, 125, 13442-13450). It was. Specifically, 0.15 g of sodium nitrite was added to 1 ml of the reaction solution, and then 0.5 ml of hexane was added. Then, 150 ml of 20% sulfuric acid was added dropwise to the reaction solution on ice, and the reaction solution was allowed to react for 15 minutes. The alcohol in it was derivatized.

  After derivatization, the hexane phase was recovered, and the recovered hexane phase was washed with pure water. The hexane phase after washing was used as an analytical sample for gas chromatography. The gas chromatograph used was GC-2014 having an FID detector manufactured by Shimadzu Corporation, and the column used was an Rtx-1 column manufactured by Restek (inner diameter 0.53 mm, liquid layer thickness 3 mm, column length 60 m). The sample inlet temperature in the gas chromatograph was 250 ° C. The temperature program of the column oven was set to be maintained at 50 ° C. for 5 minutes, then heated to 200 ° C. over 6 minutes and maintained for 3 minutes. The detector was set at 250 ° C.

  As a result of gas chromatographic analysis of the reaction solution, in the reaction system containing P450BM3, dummy molecules, and propane gas, only one peak not found in the control test was obtained. This peak is considered to be the peak of the reaction product. When propane is hydroxylated, 1-propanol or 2-propanol may be produced. Therefore, when 2-propanol was analyzed by gas chromatography, the peak of 2-propanol (retention time) coincided with the peak of the reaction product. From this result, it was found that the reaction product was 2-propanol.

  In the gas chromatography analysis, no peak corresponding to derivatized propanol was detected. From this result, it was found that propanol derivatized to nitrite is unstable under this condition and cannot be detected as nitrite, and decomposed and detected as propanol itself.

  In addition, as a result of quantifying 2-propanol in the reaction solution using 1 mmol / l ethanol as an internal standard, 2-propanol exceeding 600 μmol / l was produced when reacted for 30 minutes under the above conditions.

[2] Evaluation of dummy molecule when propane is used as substrate A dummy molecule having an alkyl chain length of 10 to 13 (PFC10 to PFC13) was used to examine the correlation between the alkyl chain length of the dummy molecule and the hydroxylation of propane. P450BM3 solution was used as P450. The reaction conditions are the same as those described above. When 1 mol of propanol is produced, 1 mol of NADPH is consumed. For this reason, the coupling rate can be calculated as propanol production (μmol / min) ÷ NADPH consumption (μmol / min) × 100. The amount of NADPH was calculated using the decrease in absorbance in the UV spectrum (340 nm) and the molar extinction coefficient ε340 nm = 6200 M ⁻¹ cm ^{−1 of} NADPH. A graph showing the relationship between the alkyl chain length of the dummy molecule, the amount of propanol produced, and the amount of NADPH consumed is shown in FIG. Table 1 shows the relationship between the alkyl chain length of the dummy molecule, the amount of propanol produced, the amount of NADPH consumed, and the coupling rate.

  As shown in Table 1 and FIG. 2, NADPH was consumed when any of PFC9 to PFC13 was used as the dummy molecule. For this reason, it is considered that the activation switch of P450BM3 was turned on by dummy molecules (PFC9 to PFC13).

  In addition, propanol was produced from propane when any of PFC9 to PFC13 was used as the dummy molecule. From these results, it can be seen that by using these dummy molecules, the propane hydroxylation activity of P450BM3 can be increased, and propane can be hydroxylated. Moreover, the amount of propanol produced by P450BM3 showed a high value in the order of PFC10> PFC11> PFC12 ≧ PFC9> PFC13. From this result, in order to increase the propane hydroxylation activity of P450BM3, the alkyl chain length of the dummy molecule is preferably 9-12, more preferably 10-11, and the alkyl chain length is It can be seen that 10 is particularly preferred.

[3] Time course of propane hydroxylation by P450BM3 and PFC10 Using P450BM3 and PFC10, propanol production and NADPH consumption were measured at 10 minutes, 30 minutes, 60 minutes and 180 minutes after the start of the reaction. The time course of propane hydroxylation was investigated. The reaction conditions are the same as those described above. A graph showing the relationship between reaction time, propanol production and NADPH consumption is shown in FIG. Table 2 shows the relationship between the reaction time, propanol production, NADPH consumption, and coupling rate.

  As shown in Table 2 and FIG. 3, the propane hydroxylation reaction by P450BM3 and PFC10 proceeded until NADPH was consumed.

[4] Coordination of substrate and dummy molecule at active center of P450BM3 FIG. 4 (a) schematically shows the active center of P450BM3 to which palmitic acid, which is a substrate of wild-type P450BM3, is bound, and PFC10 binds to it. An explanatory view schematically showing the active center of P450BM3 is shown in FIG. FIGS. 4A and 4B are obtained by replacing the substrate of the substrate-bound P450BM3 crystal structure with PFC10. This crystal structure is structural data stored in a database of a PDB (Protein Structure Data Bank Protein Data Bank), and its entry ID (PDB ID) is 1FAG.

  P450 has a heme in the active site. Heme is a reaction field for hydroxylation reaction, and as shown in FIGS. 4A and 4B, a binding site to which a substrate or a dummy molecule is bonded is present above the heme.

  As described above, when propane is hydroxylated, the hydroxylation activity of P450BM3 becomes very high when PFC10 having an alkyl chain length of 10 is used as a dummy molecule. This is because the alkyl chain length of PFC10 (alkyl chain length 10) is 6 shorter than palmitic acid (alkyl chain length 16), which is a substrate of wild-type P450BM3, so that the space in which propane is taken up above the reaction field hem. This is considered to be ensured.

  Moreover, it is considered that the hydrophobicity of the reaction field is enhanced by binding of PFC10, that is, a fluorine-substituted dummy molecule, to the binding site of the active center. For this reason, it is also considered that hydrophobic propane easily enters the active center reaction field, and water molecules are excluded from the reaction field. For this reason, it is considered that the activation switch is turned on and the hydroxylation reaction of propane proceeds.

  Further, when PFC9 (alkyl chain length 9) is used as a dummy molecule, it is considered that the activation switch does not sufficiently enter because the alkyl chain length of the dummy molecule is short. For this reason, when PFC9 is used as the dummy molecule, it can be interpreted that both the propanol production amount (that is, the hydroxylation activity of P450) and the NADPH consumption amount are lower than when PFC10 is used as the dummy molecule.

  In addition, when PFC13 (alkyl chain length 13) is used as a dummy molecule, the amount of propanol produced (hydroxylation activity of P450) decreases due to the long alkyl chain length of the dummy molecule. This is considered to be because if the alkyl chain length of the dummy molecule is excessively long, the space above the reaction field (the space into which the substrate is taken in) becomes narrow and propane is hardly taken up. In this case as well, since the activation switch is turned on, the consumption of NADPH proceeds sufficiently quickly.

  In consideration of turning on the activation switch and sufficiently increasing the hydroxylation activity of P450BM3, it is particularly preferable to use PFC10 (perfluorodecanoic acid) as a dummy molecule for hydroxylating propane with P450BM3. Conceivable.

[5] Cyclohexane hydroxylation confirmation test Cyclohexane, which is a kind of inert hydrocarbon, was hydroxylated using the above-mentioned P450BM3 solution and dummy molecule (PFC 8-14) solution.

  The P450BM3 solution, the dummy molecule solution, NADPH and cyclohexane were added to the buffer B described above to initiate the hydroxylation reaction of cyclohexane. Cyclohexane used was dissolved in ethanol. In the reaction solution at the start of the reaction, the concentration of P450BM3 is 100 nmol / l, the concentration of the dummy molecule is 0.1 mmol / l, the concentration of NADPH is 1 mmol / l, and the concentration of cyclohexane is 10 mmol / l. there were. Further, this reaction solution contained 1% by volume of ethanol.

  The reaction solution was allowed to stand overnight at room temperature with stirring to allow the cyclohexane hydroxylation reaction to proceed. Thereafter, the alcohol (cyclohexanol) produced by the reaction was detected and quantified by gas chromatography. As a control experiment, a system in which only 0.5% by volume of ethanol was added instead of the dummy molecule solution (no dummy molecule) was reacted in the same manner, and cyclohexanol in the reaction solution was analyzed by gas chromatography.

  As a result of gas chromatographic analysis of the reaction solution, cyclohexanol was detected from the reaction solution containing dummy molecules, P450BM3, and cyclohexane, as shown in FIG. No significant amount of cyclohexanol was detected from the reaction solution containing no dummy molecule. This result shows that cyclohexane hydroxylation activity of P450BM3 can be improved by coexisting dummy molecules (PFCs 8 to 14).

[6] Evaluation of dummy molecules when cyclohexane is used as a substrate Using dummy molecules having an alkyl chain length of 8 to 14 (PFC8 to PFC14), the correlation between the alkyl chain length of the dummy molecule and the hydroxylation of cyclohexane was investigated. P450BM3 solution was used as P450. The reaction conditions were the same as those described above except that the reaction time was 10 minutes. The amount of cyclohexanol produced by the reaction (μmol / min) and the amount of NADPH consumed by the reaction (μmol / min) were compared for each dummy molecule used. Moreover, the ratio (coupling rate (%)) with respect to the NADPH consumption (total amount) of the amount of NADPH utilized for cyclohexanol production was calculated.

  A graph showing the relationship between the chain length of the dummy molecule, the amount of cyclohexanol produced and the amount of NADPH consumed is shown in FIG. Table 3 shows the relationship between the alkyl chain length of the dummy molecule, the amount of cyclohexanol produced, the amount of NADPH consumed, and the coupling rate.

  As shown in Table 3 and FIG. 7, NADPH was consumed when any of PFC8 to PFC14 was used as the dummy molecule. As described above, since NADPH is consumed when the activation switch is inserted, it is considered that the activation switch is turned on due to the presence of the dummy molecule. Note that cyclohexanol was not detected when the reaction was similarly carried out for 10 minutes without adding the dummy molecule.

  In addition, cyclohexanol was produced from cyclohexane when any of PFC8 to PFC14 was used as the dummy molecule. From this result, it is understood that cyclohexane hydroxylation activity of P450BM3 can be increased by using a dummy molecule having an alkyl chain length (here, carbon number) of about 8 to 14, and cyclohexane can be hydroxylated with high reactivity. Further, the amount of cyclohexanol produced and the amount of NADPH consumed were different depending on the alkyl chain length of the dummy molecule. The amount of cyclohexanol produced by P450BM3 showed a higher value in the order of PFC9> PFC10> PFC8> PFC11> PFC12> PFC13> PFC14. From these results, in order to increase the cyclohexane hydroxylation activity of P450BM3, the dummy molecule preferably has an alkyl chain length of 8 to 12, more preferably an alkyl chain length of 8 to 11, and an alkyl chain length of It can be seen that 9 is particularly preferred.

[7] Benzene Hydroxylation Confirmation Test Benzene, which is a kind of inert hydrocarbon, was hydroxylated using the P450BM3 solution and the dummy molecule (PFC8 to PFC14) solution described above. Benzene dissolved in ethanol was used. Other than that, it is the same as the above-mentioned hydroxylation of cyclohexane. Detection was performed by HPLC.

  In the reaction solution at the start of the reaction, the concentration of P450BM3 was 500 nmol / l, the concentration of dummy molecules was 100 μmol / l, and the concentration of NADPH was 5 mmol / l. Further, this reaction solution contained 1% by volume of ethanol. Ten minutes after the start of the reaction, the alcohol (phenol) produced by the reaction was detected and quantified by HPLC. As a control experiment, a system in which buffer B was added instead of dummy molecules was reacted in the same manner, and phenol in the reaction solution was analyzed.

  As a result of gas chromatographic analysis of the reaction solution, as shown in FIG. 8, in the reaction system containing P450BM3, dummy molecules and benzene, a benzene peak and a phenol peak were confirmed. Although not shown, in the reaction system of the control experiment, only the benzene peak was confirmed, and the phenol peak was not confirmed. From this result, it was confirmed that benzene in the reaction solution was hydroxylated by P450BM3 and a dummy molecule to produce phenol. From this result, it is understood that the benzene hydroxylation activity of P450BM3 can be improved by using dummy molecules (PFC8 to PFC14).

[8] Evaluation of dummy molecules in the case of using benzene as a substrate Using dummy molecules having an alkyl chain length of 8 to 14 (PFC8 to PFC14), the correlation between the alkyl chain length of the dummy molecule and hydroxylation of benzene was examined. P450BM3 solution was used as P450. The reaction conditions are the same as those described above. A graph showing the relationship between the alkyl chain length of the dummy molecule, the amount of phenol produced and the NADPH consumption is shown in FIG. Table 4 shows the relationship between the alkyl chain length of the dummy molecule, the amount of phenol produced, the NADPH consumption, and the coupling rate.

  As shown in Table 4 and FIG. 9, NADPH was consumed when any of PFC8 to PFC14 was used as the dummy molecule. For this reason, it is considered that the activation switch of P450BM3 was turned on by PFC8 to PFC14.

  In addition, phenol was produced from benzene when any of PFC8 to PFC14 was used as the dummy molecule. From this result, it can be seen that by using these dummy molecules, the benzene hydroxylation activity of P450BM3 can be increased, and benzene can be hydroxylated. Moreover, the amount of phenol produced by P450BM3 showed a high value in the order of PFC9> PFC10> PFC11> PFC12> PFC8> PFC13> PFC14. From this result, in order to increase the benzene hydroxylation activity of P450BM3, the alkyl chain length of the dummy molecule is preferably 9-12, more preferably 9-11, and the alkyl chain length is It can be seen that 9 is particularly preferred.

[9] Evaluation of dummy molecules when butane is used as a substrate Using dummy molecules having an alkyl chain length of 8 to 14 (PFC8 to PFC14) and dummy molecules consisting of partially fluorinated fatty acids (referred to as PartialFC10) The correlation between the alkyl chain length of the dummy molecule and the hydroxylation of butane was investigated. P450BM3 solution was used as P450. The reaction conditions are the same as those described above. A graph showing the relationship between the alkyl chain length of the dummy molecule, the amount of 2-butanol produced, and the NADPH consumption is shown in FIG. Table 5 shows the relationship between the alkyl chain length of the dummy molecule, the amount of 2-butanol produced, the NADPH consumption, and the coupling rate. The Partial FC 10 is a dummy molecule in which the 2, 2, 3, and 3 positions of the PFC 10 are not fluorinated but remain hydrogen. For Partial FC10, only 2-butanol production was measured.

  As shown in Table 5 and FIG. 10, NADPH was consumed when any of PFC8 to PFC14 was used as the dummy molecule. For this reason, it is considered that the activation switch of P450BM3 was turned on by PFC8 to PFC14.

  In addition, 2-butanol was produced from butane when any of PFC8 to PFC14 was used as the dummy molecule. From this result, it can be seen that the use of these dummy molecules can increase the butane hydroxylation activity of P450BM3 and hydroxylate butane. Moreover, the 2-butanol production amount by P450BM3 showed the high value in order of PFC9> PFC10> PFC11> PFC12> PFC13> PFC14> PFC18. From this result, in order to increase the butane hydroxylation activity of P450BM3, the alkyl chain length of the dummy molecule is preferably 9-12, more preferably 9-10, and the alkyl chain length is It can be seen that 9 is particularly preferred. Even when Partial FC10 was used as a dummy molecule, the amount of 2-butanol produced was very high. From this result, it can be seen that the dummy molecule does not have to substitute all the hydrogen contained in the alkyl chain with fluorine, and only needs to substitute a part of hydrogen with fluorine.

  The method for hydroxylating an inert hydrocarbon and the dummy molecule of the present invention can be used as a method for producing an alcohol such as cyclohexanol, butanol, propanol, or phenol by hydroxylating an inert hydrocarbon.

Claims (10)

Including the step of coexisting cytochrome P450 monooxygenase, a dummy molecule, and an inert hydrocarbon to hydroxylate the inert hydrocarbon,
The dummy molecule has a terminal structure capable of binding to the binding site of the cytochrome P450 monooxygenase, and an alkyl chain,
A method for hydroxylating an inert hydrocarbon, wherein at least a part of hydrogen contained in the alkyl chain is substituted with fluorine.   The cytochrome P450 monooxygenase is a cytochrome P450 monooxygenase having 40% or more homology with a cytochrome P450 monooxygenase having a homology of 40% or more with the amino acid sequence of cytochrome P450BM3 and / or a cytochrome P450 monooxygenase having a homology of 40% or more with the amino acid sequence of cytochrome P450cam. 2. The method for hydroxylating an inert hydrocarbon according to 1.   3. The inert hydrocarbon water according to claim 1, wherein hydrogen contained in at least the first to third alkyl groups counted from the end of the alkyl chain in the dummy molecule is substituted with fluorine. 4. Oxidation method.   The method for hydroxylating an inert hydrocarbon according to any one of claims 1 to 3, wherein the terminal structure is at least one selected from a hydrogen terminal, a hydroxyl group, an aldehyde group, and a carboxyl group.   The method for hydroxylating an inert hydrocarbon according to any one of claims 1 to 4, wherein the inert hydrocarbon is at least one selected from cyclohexane, butane, propane, and benzene. The cytochrome P450 monooxygenase has at least 40% homology with the amino acid sequence of cytochrome P450BM3 ,
The method for hydroxylating an inert hydrocarbon according to any one of claims 1 to 5, wherein the dummy molecule is perfluorocarboxylic acid. A composition comprising a dummy molecule for hydroxylating the inert hydrocarbon in the presence of cytochrome P450 monooxygenase and the inert hydrocarbon,
A terminal structure capable of binding to the binding site of the cytochrome P450 monooxygenase, and an alkyl chain;
A composition comprising a dummy molecule, wherein at least a part of hydrogen contained in the alkyl chain is substituted with fluorine. The composition containing a dummy molecule according to claim 7, wherein hydrogen contained in at least the first to third alkyl groups counted from the end of the alkyl chain is substituted with fluorine. The composition containing a dummy molecule according to claim 7 or 8, wherein the terminal structure is at least one selected from a hydrogen terminal, a hydroxyl group, an aldehyde group, and a carboxyl group. The cytochrome P450 monooxygenase has at least 40% homology with the amino acid sequence of cytochrome P450BM3,
The said dummy molecule is a perfluoro carboxylic acid, The composition containing the dummy molecule as described in any one of Claims 7-9.