JP4969448B2 - Biological conversion reaction system of cytochrome P450 - Google Patents

Description

  The present invention relates to a biological conversion reaction system of a compound that can be a substrate of cytochrome P450 and a method for producing a biological conversion product of the compound using the conversion reaction system.

Cytochrome P450 enzymes encoded by the cytochrome P450 gene (hereinafter also simply referred to as P450) are a group of protoheme-containing proteins that bind to carbon monoxide in a reduced form and exhibit a Soret band near 450 nm. P450 is widely distributed across species in higher organisms including animals and plants, molds, yeasts, and bacteria, and is involved in the metabolism and degradation of various substances mainly including oxidation reactions. Numerous molecular species are known for P450, and it is known from systematic studies that it forms a superfamily.
Because of its functional role, P450 uses various substances such as in vivo and in vitro substances as substrates, hydroxylation reaction, epoxidation reaction, dealkylation reaction, denitrogenation reaction, or rarely NO reduction via monoatomic oxygenation reaction. It is known to catalyze various reactions such as a reduction reaction using electrons from NADH, such as an enzyme. These reactions are usually performed as strict reactions with respect to stereo-specificity and regio-specificity. Many of the drugs used for humans are metabolized and inactivated in the body by the action of specific P450s expressed in the liver, kidney, etc., such as hydroxylation, and conversely their pharmacological effects are improved, or Since side effects are enhanced, P450 is extremely important in the pharmaceutical industry from the viewpoint of drug metabolism research and prodrug development.
In recent years, the accumulation of genome information has revealed the existence of various P450 genes in animals and plants and microorganisms, and these P450 genes or functions of P450 are applied to substances or compound conversions for obtaining various useful compounds. It can be expected that it can be used industrially. The most specific industrial application of the P450 gene or P450 is substance conversion using a microorganism cell that expresses P450 that performs a desired conversion reaction, or a recombinant microorganism that expresses the P450 by genetic engineering techniques. Can be considered. For example, a method for producing active vitamin D ₃ by hydroxylating 1α-position and 25-position of vitamin D ₃ using actinomycete Pseudonocardia autotrophica has been put into practical use. In addition, yeast (see Non-Patent Document 1) in which mammalian P450 is expressed functionally, and specific literature names cited in this section are described below. The same applies hereinafter. (See Non-Patent Document 2) It is also possible to obtain drug metabolites. Furthermore, by incorporating heterogeneous ferredoxin genes and ferredoxin reductase genes as electron transfer means into a system for expressing various P450-encoding genes from various bacteria in the host E. coli, it is possible to effectively use various compounds that can serve as substrates. A conversion system has also been proposed (see Patent Document 1).
Such a biological or microbiological conversion reaction of a certain compound using cytochrome P450 gene or P450 has been carried out using a culture medium or cells of a microorganism expressing the enzyme. A method for producing a conversion product using such a conversion reaction usually takes the form of fermentation, in which a culture solution is prepared for each production lot, and culture of microorganisms having conversion ability and substrate conversion reaction are allowed to proceed. is necessary. In general, after the conversion reaction is completed, many processes are required to recover and purify the conversion product.
On the other hand, many of the substance conversion systems using hydrolases are modified by changing the enzyme or cells having the enzyme activity itself or a processed product to a conversion reaction system using a membrane reactor or an immobilized enzyme. Compared with the conversion method in the case of using a liquid or the like, considerable cost can be suppressed, and it has been established as an industrially advantageous conversion manufacturing method for specific substances.
The fact that a conversion reaction system using a membrane reactor or an immobilized enzyme similar to such a hydrolase cannot be diverted to P450 is due to the unique properties of the enzyme itself. Specifically, since a hydrolase reacts in a cofactor-independent manner, only that enzyme is necessary and sufficient in the reaction, whereas P450 reduces NADPH, ferredoxin, ferredoxin in the reaction involved. This is because a cofactor having an electron transfer function such as an enzyme is required. Therefore, the place of reaction using P450 is most desirably in a cell in which such a cofactor sufficiently functions and provides a reproducible environment.
Therefore, in order to realize a conversion production method of a target substance using a membrane reactor similar to a hydrolase or an immobilized enzyme using an enzyme having a characteristic requiring a cofactor such as P450, it is indispensable for an intracellular reaction system. How to replace the cofactor's functional role with readily available materials or means has been a technical challenge in the art. One means for solving this problem is, for example, the provision of a reaction system that can function P450 using an inexpensive chemical mediator having an electron transfer function. If such a reaction system can be provided, a biological conversion reaction using P450 may be allowed to proceed in a cell-free reaction tank. Furthermore, the combined use of techniques such as membrane separation and enzyme immobilization makes it possible to efficiently separate the target product converted from a compound that can serve as a substrate, and there is a possibility that an industrially useful continuous reaction system can be constructed.
Conventionally known substances that substitute for the cofactor function in the P450 reaction system under such physiological conditions include peroxides that can supply oxygen and electrons, such as hydrogen peroxide. Further, in another proposed system, a system that enables electron transfer from an electrode to P450 using a certain type of cobalt complex and enables hydroxylation of higher fatty acids is known (Non-patent Document 3). reference). As another system of further interest, recently, a reaction system has also been proposed in which the thermostable P450 enzymes CYP119 and P450BM3 are connected to an electrode with an appropriate mediator in between, and the enzyme is activated by transferring electrons from the electrode to P450. (See Non-Patent Document 4 and Non-Patent Document 5).
List of cited references
International Publication No. 03/088381 Pamphlet H. Murakami et al., J Biochem. vol. 108, 859-865, 1990 H. Iwata et al., Biochemical Pharmacology, vol. 55, 1315-1325, 1998 A. K. Udit et al., Journal of Inorganic Biochemistry, 98, 1547-1550, 2004. E. Blair et al., J. MoI. Am. Chem. Soc. 126, 8632-8633, 2004 A. K. Udit et al. Am. Chem. Soc. 126, 10218-10219, 2004

で表される。１，４−ナフトキノンは次式

で表される。１，４−ジヒドロキシ−２−ナフトエ酸は次式

で表される。ビタミンＫ_３は次式

で表される。１，４−ベンゾキノンは次式

で表される。
このようなキノン類縁化合物を含んで成る本発明に従うシトクロムＰ４５０の反応系は、従来反応に不可欠とされ、制限要因とされてきた電子伝達コファクターを排除することができ、安価で有用な物質変換に利用することができる。
本発明で使用する、初発電子供給手段とは、Ｐ４５０とキノン類縁化合物と，場合により、Ｐ４５０の基質となり得る化合物の存在する系において、電子を供給することのできる物質もしくは構造物を意味する。かような物質の代表的なものとしては、還元型ニコチンアミドアデニンジヌクレオチド（ＮＡＤＨ）及び還元型ニコチンアミドアデニンジヌクレオチドリン酸（ＮＡＤＰＨ）などを挙げることができる。また、例えば，非特許文献５に記載されるように、例えば、グラファイト電極に一定の固定媒体またはリガンドを介して、必要により改変したＰ４５０またはその活性ドメインを固定すると、電極からＰ４５０に電子を伝達でき、例えば、一方では、共存する二原子酸素（ｄｉｏｘｙｇｅｎ）を水までに変換できることが知られている。限定されるものでないが、このような電子を伝達できる電極であって、本発明の目的に沿うものであれば、本発明にいう初発電子供給手段に包含される。
以上の本発明の反応系は、例えば、通常の酵素変換を想定して組まれた反応形態のように、ＮＡＰＤＨまたはＮＡＤＨ、キノン類縁体、Ｐ４５０を含む適当な緩衝液を入れた反応槽に基質を添加する方法で、バッチまたは連続的な反応を行うことで実用に適する。
一方、初発電子供給手段に電極を選択する場合は電極を設置した反応槽内にキノン類縁体、およびＰ４５０を含む適当な緩衝液を入れ、さらに基質を添加しながら通電してバッチまたは連続的な反応を行う。この場合は電極からキノンへの電子伝達を円滑に行うためにフェリシアニド（ｆｅｒｒｉｃｉａｎｉｄｅ，化学式 Ｆｅ（ＣＮ）_６ ^３−）等の添加物を加えることもできる。このように反応系を構築することで、所望の基質をその酸化型化合物へ変換できるバイオリアクターを構築することもできる。
本発明の反応系では、初発電子供給手段、Ｐ４５０、キノン類縁化合物、存在させる場合の基質となり得る化合物は、バッチ式に、または連続様式でＰ４５０以外の要素を供給でき、それらの使用割合は、通常、
ＮＡＤＨまたはＮＡＤＰＨ ０．１〜１ｍＭ
Ｐ４５０ ０．１〜１０μＭ
キノン類縁化合物 ０．０１〜１μＭ
基質有機化合物 １ｍＭ〜１０００ｍＭ
とすることができる。
こうして、基質となり得る化合物からそれらの酸化型化合物に変換する工程を含んで成る該酸化型化合物の製造方法もさらなる別の態様の本発明として提供できる。反応系を稼動する温度は、使用するＰ４５０の温度感受性に応じて決定することができる。例えば、上述した耐熱性のＰ４５０酵素ＣＹＰ１１９の場合には、３０〜８０℃で本発明の系を使用できる。かような反応系は通常、その系で働くＰ４５０酵素の至適ｐＨにて十分な緩衝効果をもつような緩衝剤を使用して、至適ｐＨに調整した緩衝液、例えばｐＨ７．３付近が至適ｐＨであれば１００ｍＭリン酸カリウム緩衝液（ｐＨ７．３）などを使用することができる。また、この緩衝液には必要に応じてＰ４５０酵素を安定化するのに寄与する物質、例えばグリセロールやジチオスレイトールなどを補助的に添加してもよい。Although the above-mentioned peroxide is relatively inexpensive and provides a system that can supply oxygen and electrons simultaneously, inactivation of the enzyme by the peroxide is a major problem. A system using a cobalt complex or the like as a chemical mediator is not necessarily an effective method because it has disadvantages such as costly preparation of the chemical mediator, deterioration due to natural oxidation, and low reaction efficiency.
Therefore, the object of the present invention does not require the following (i) complicated pretreatment to compensate for the above-mentioned drawbacks.
(Ii) It is inexpensive and easy to obtain, replacing the cofactor function by simply adding to the reaction system.
(Iii) To provide a reaction system involving P450 that uses a chemical mediator that satisfies conditions that do not easily oxidize relatively naturally and (iv) does not impair enzyme function, and that uses them.
The present inventors have conducted intensive research in order to realize a combination of P450 with a high applied value suitable for searching for a substance that can be a new chemical mediator. As a result, the present inventors have found that quinone analogs that have not been reported as chemical mediators of cytochrome P450 activate P450, particularly P450 derived from actinomycetes, in an electron transfer protein-independent manner, thereby converting the substrate. It has been found that the reaction proceeds effectively. The present invention is based on such knowledge. Therefore, the present invention provides a reaction system for performing a microbiological conversion reaction of a compound that can be a substrate of cytochrome P450 in the presence of cytochrome P450 as a means to solve the above-described problems,
(A) cytochrome P450,
There is provided a reaction system comprising (b) an initial electron supply means, and (c) a quinone analog.
Also provided is a method for producing a biological conversion product comprising a step of performing a biological conversion of a compound that can be a substrate of cytochrome P450, particularly an oxidized form, in the reaction system.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION The reaction system referred to in the present invention uses a cytochrome P450 appropriately selected according to the substance to be converted (or a compound that can serve as a substrate), so that a desired biological substance can be obtained. It means an entity applicable to a composition, an apparatus or a bioreactor for obtaining a conversion product, a kit used for assaying a specific substrate, or the like. As used herein, the term biological transformation is a concept for chemical transformation.
Cytochrome P450 is derived from higher organisms such as animals and plants, and is distributed in a wide range of organisms from molds, mushrooms, yeasts, and bacteria. When it functions in cells, ferredoxin and ferredoxin reductase are usually used. Alternatively, it is a general term for active enzyme-like substances that require a protein having an electron transfer function such as cytochrome P450 reductase as a cofactor of the conversion reaction by P450. Such cytochrome P450 can be used without regard to its origin as long as it is a P450 that expresses a predetermined catalytic function by substituting the quinone analog for the cofactor in accordance with the object of the present invention. However, for industrial use, preferably, soluble P450 derived from bacteria that is relatively easy to express in microorganisms, such as MoxA or Actinomyces nocardia falci (actinomycete Nocardia falci) which is a P450 derived from the actinomycete Nonomura Rectica naina (formerly Microtetraspora lecticanatena) IFO14525. And CYP154A, which is P450 derived from Nocardia farcinica) IFM 10152. Specific examples of P450 derived from other microorganisms include those listed in Patent Document 1.
Such P450 is also described in part in Patent Document 1, but it can be obtained using various public information today. In recent years, in addition to gene databases such as GenBank and EMBL, a very large number of P450 gene sequences and information on their origins can be easily obtained from sites that publish P450 gene information and genome information on the Internet. Among such information sources are D.I. The P450 gene database http: // drnelson. utmem. edu / bacteria. A public database of actinomycetes genomes having a relatively wide variety of P450 genes among html and bacteria, such as the genome database of Nocardia Farcinica IFM 10152 (Nocardia farcinica IFM 10152) containing 26 species in the P450 gene, http: // nocardia. nih. go. jp / (this database is supplemented by the content of a paper by Ishikawa et al., Proc Natl Acad Sci US A. 2004, 101 (41): 14925-30) and Streptomyces evermitilis MA- containing 33 P450 genes Genomic database of 4680 (Streptomyces avermitilis MA-4680), http: // avermitilis. ls. kitasato-u. ac. jp / (this database is supplemented by the article by Lamb et al., Biochem Biophys Res Commun. 2003, 307 (3): 610-9). Those skilled in the art can obtain P450 by cloning the target P450 gene or its homologous gene from the derived organism or its related species using these gene information, and expressing those genes in an appropriate host. it can. Therefore, P450 obtained in this way can also be used in the present invention.
Among the specific acquisition methods, the easiest one is based on the available gene sequence information, and using primers from DNA sequences corresponding to the N-terminal sequence and C-terminal sequence portion of the P450 coding region of a specific gene sequence. Prepare and amplify the P450 gene coding region by PCR. Next, this amplified fragment is inserted into an expression vector and transformed into a host in which the expression system functions.
For example, a method for obtaining a gene encoding MoxA (moxA) can be carried out by a method well known in the art. Details of these operations as a whole are described in the above-mentioned Patent Document 1 by the present applicant (the description is incorporated herein by reference). The nucleotide sequence (amino acid sequence in the coding sequence) including the peripheral region of the P450 gene thus obtained is shown in SEQ ID NO: 1 in the sequence listing. The continuous nucleotide sequence from base 313 to base 1533 of this sequence corresponds to the P450 gene (moxA). Further, it is P450 having homology of 40% or more, preferably 60% or more, more preferably 80% or more, particularly preferably 90% or more with MoxA at the amino acid sequence level, and its functional properties are similar to those of MoxA. In particular, many P450 groups belonging to the CYP105 family based on the classification definition of Nevert et al. (DR Nelson et.al., Pharmacogenetics, 6, 1-42, 1996) It can be used in the reaction system of the invention. As used herein, the homology as described above refers to commercially available software that can evaluate the homology of sequences, such as GENETYX Version 6.0 (Genetics Corporation (English: GENETYX CORPORATION)). It can be evaluated using the amino acid sequence homology search function of Shibuya-ku, Tokyo). The CYP154A gene is a P450 gene identified by the gene code nfa22930 in the genome database of the aforementioned Nocardia Farcinica IFM 10152. The base sequence and coding sequence of this gene are shown in SEQ ID NO: 2. Based on the same classification definition as MoxA, many P450 groups belonging to the CYP154 family can also be used in the reaction system of the present invention. The “P450 group” refers to P450 having a similar structure and function having 40% or more homology with CYP154A at the amino acid sequence level.
When the cloned P450 gene is expressed in a suitable host, various expression systems can be used. However, it is desirable to use a microorganism host that is convenient for industrial conversion using P450. For example, Escherichia coli can be used as a host. In this case, various vectors such as pUC18, pTrcHis and pET11a can be used for expression of the P450 gene. When using actinomycetes as a host, vectors such as pIJ702 and pIJ943 can be used. When yeast is used as a host, vectors such as pPICZ, pPIC9K, pMET, and pTEF1 / Bsd can be used.
In addition, P450 protein can be purified from cells expressing P450 according to biochemical methods that are already generally known. In particular, in the case of expressing in an E. coli recombinant, a method in which a histidine-tag is connected to the C-terminus of the target P450 and expressed, and is easily purified using a nickel-NTA agarose column or the like. Is established.
Based on such a purification method, P450 prepared as an enzyme source can be used as a catalyst in the reaction system of the present invention. The P450 enzyme source may be a cell itself expressing P450 that catalyzes a desired reaction, or a recombinant of the P450 gene itself.
Furthermore, the P450 enzyme source is not limited to the P450 protein extracted and purified from these cells, but to reduce the purification cost, the P450 protein-containing cell itself or a processed product thereof, that is, a cell-free extract or a partial purification treatment is applied. You can also use it. Except for the case where a purified protein is used as the P450 enzyme source, strictly speaking, there is a possibility that a protein having an electron transfer function is mixed as a contaminating protein. Regardless of the presence or absence of a nuisance electron transfer protein, the added quinone analog effectively utilizes the phenomenon of substituting the role of the electron transfer protein. Good.
The compound that can be a substrate of cytochrome P450 referred to in the present invention means any P450 listed above or a compound that can be a substrate of the functionally equivalent active enzyme-like substance or a substrate-like substance. The compound that can be a substrate is not only a substrate that can be confirmed from the above-mentioned various information sources, but with reference to the substrate, it is structurally similar or not similar to them, but in fact, it can react with any cytochrome P450 as a substrate. Compounds selected as being able to participate are included.
Quinone is a general term for compounds formed by moving two CH atom groups of an aromatic compound into CO atom groups and moving them as much as necessary to make double bonds into a quinoid structure. The quinone analogs used in the reaction system of the present invention are all compounds corresponding to the definition of quinone that can replace the cofactor function in the P450 reaction system under physiological conditions in accordance with the object of the present invention. Include. Although not limited, quinone analogs include aromatic quinones such as benzoquinone and naphthoquinone, various derivatives obtained by modifying these compounds with amino groups, carboxyl groups, and methyl groups, and naphthoic acid. Mention may be made of reduced quinone compounds. Specific examples of the compound include 2-amino-3-carboxy-1,4-naphthoquinone (hereinafter also referred to as ACNQ), 1,4-naphthoquinone, 1,4-dihydroxy-2-naphthoic acid, vitamin K _3. 1,4-benzoquinone and the like. The structural formulas of ACNQ, 1,4-naphthoquinone, 1,4-dihydroxy-2-naphthoic acid, vitamin K ₃ and 1,4-benzoquinone are shown below.
ACNQ is the following formula

It is represented by 1,4-naphthoquinone has the following formula

It is represented by 1,4-dihydroxy-2-naphthoic acid has the formula

It is represented by Vitamin K ₃ is the following formula

It is represented by 1,4-benzoquinone has the following formula

It is represented by
The reaction system of cytochrome P450 according to the present invention comprising such a quinone analog can eliminate the electron transfer cofactor which has been indispensable for the conventional reaction and has been regarded as a limiting factor, and is an inexpensive and useful substance conversion. Can be used.
The initial electron supply means used in the present invention means a substance or a structure capable of supplying electrons in a system in which P450, a quinone-related compound, and optionally a compound that can be a substrate of P450 are present. Representative examples of such substances include reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH). For example, as described in Non-Patent Document 5, for example, when P450 or its active domain modified as necessary is fixed to a graphite electrode via a fixed medium or ligand, electrons are transferred from the electrode to P450. For example, it is known that, on the one hand, coexisting dioxygen can be converted to water. Although it is not limited, if it is an electrode which can transmit such an electron and is in accordance with the objective of this invention, it will be included by the initial electron supply means said to this invention.
The above reaction system of the present invention is, for example, a substrate in a reaction tank containing an appropriate buffer containing NAPDH or NADH, a quinone analog, and P450, as in a reaction form constructed assuming normal enzyme conversion. It is suitable for practical use by performing a batch or continuous reaction.
On the other hand, when an electrode is selected as the initial electron supply means, an appropriate buffer solution containing quinone analog and P450 is placed in a reaction vessel in which the electrode is installed, and a batch or continuous process is performed by adding current while adding a substrate. Perform the reaction. In this case, an additive such as ferricyanide (chemical formula Fe (CN) ₆ ³⁻ ) can be added to smoothly transfer electrons from the electrode to the quinone. By constructing the reaction system in this way, it is possible to construct a bioreactor that can convert a desired substrate into its oxidized compound.
In the reaction system of the present invention, the initial electron supply means, P450, a quinone analog, and a compound that can be a substrate when present can supply elements other than P450 in a batch manner or in a continuous manner, and their use ratio is as follows: Normal,
NADH or NADPH 0.1-1 mM
P450 0.1-10 μM
Quinone analogs 0.01-1 μM
Substrate organic compound 1 mM to 1000 mM
It can be.
Thus, a method for producing the oxidized compound comprising the step of converting a compound that can serve as a substrate into the oxidized compound can also be provided as another aspect of the present invention. The temperature at which the reaction system is operated can be determined according to the temperature sensitivity of P450 used. For example, in the case of the above-described thermostable P450 enzyme CYP119, the system of the present invention can be used at 30 to 80 ° C. Such a reaction system usually uses a buffer that has a sufficient buffering effect at the optimum pH of the P450 enzyme working in the system, and a buffer solution adjusted to the optimum pH, for example, around pH 7.3. If the pH is optimum, 100 mM potassium phosphate buffer (pH 7.3) or the like can be used. In addition, a substance that contributes to stabilizing the P450 enzyme, such as glycerol or dithiothreitol, may be supplementarily added to the buffer as necessary.

FIG. 1 is a graph showing the relationship between the addition of ACNQ or the like and the amount of 4′-hydroxylation conversion of diclofenac by MoxA. In the figure, a is a system to which MoxA and diclofenac are added, but ACNQ, NADH + NADPH and glucose dehydrogenase + glucose are not added, b is a system to which MoxA, diclofenac and ACNQ are added, but NADH + NADPH and glucose dehydrogenase + glucose are not added , C represents a system to which MoxA, diclofenac, ACNQ and NADH + NADPH are added, but glucose dehydrogenase + glucose is not added, and d represents a system to which MoxA, diclofenac, ACNQ, NADH + NADPH and glucose dehydrogenase + glucose are added.
FIG. 2 is a graph showing the effect of various quinone compounds on the 4′-hydroxylation of diclofenac. In the figure, ■ indicates an ACNQ addition system, ▲ indicates a 1,4-dihydroxy-2-naphthoic acid addition system, □ indicates a vitamin K ₃ addition system, * indicates a 1,4-benzoquinone addition system, And ● represent a 1,4-naphthoquinone-added system, and ◆ represents a quinone-free system.
FIG. 3 is a graph showing the relationship between the presence or absence of addition of 1,4-naphthoquinone and the amount of 7-hydroxycoumarin produced by CYP154A. In the figure, a is a system in which a purified CYP154A solution is not added, but 1,4-naphthoquinone, NADPH and 7-ethoxycoumarin are added, and b is a system in which 1,4-naphthoquinone is not added, but a purified CYP154A solution, NADPH and 7- The system to which ethoxycoumarin has been added, c represents the system to which purified CYP154A solution, 1,4-naphthoquinone, NADPH and 7-ethoxycoumarin have been added.

  Hereinafter, reaction examples using MoxA, which is P450 derived from Nonomura Air Recticaena IFO 14525, and CYP154A, which is P450 derived from Nocardia farcinica IFM 10152, will be described in detail, but the present invention is not limited to these examples. It is not limited.

Construction of plasmid (1) Construction of pMoxAhis Primer MoxA-1F (5′-GCCCCCCATATGACGGAAGAACGTCCGCCGACGGAACTG-3 ′) (see SEQ ID NO: 3) and MoxA-Rhis (5′-GGCCAGGCTCCCAGGTGACCGGGAGTTCGAGA-3 ′) The polymerase chain reaction (PCR) was carried out under the following reaction conditions using the whole DNA of Nonuraea lecticcatena IFO 14525 as a template to amplify the moxA gene.
(Reaction solution composition)
Sterile purified water 30 μl
2-fold concentrated GC buffer I (Takara Shuzo) 50 μl
dNTP mixed solution (dATP, dGTP, dTTP,
dCTP 2.5 mM each) 16 μl
MoxA-1F primer (100 pmol / μl) 1 μl
MoxA-Rhis primer (100 pmol / μl) 1 μl
Nonomuraea lecticatena
IFO 14525 total DNA (100 ng / μl) 1 μl
LA Taq polymerase (5 units / μl,
(Takara Shuzo) 1 μl
Dispense 50 μl into two microtubes.
(Temperature conditions)
94 ° C. 3 minutes (98 ° C. 20 seconds, 63 ° C. 30 seconds, 68 ° C. 2 minutes) 25 cycles, 72 ° C. 5 minutes The DNA fragment amplified by this reaction was purified using QIAquick PCR Purification Kit (Qiagen) did. This DNA solution was treated with restriction enzymes Nde I and Hind III in a reaction volume of 50 μl, and then electrophoresed on a 0.8% agarose gel. After the electrophoresis, the fragment was recovered from the gel slice containing the 1.2 kb moxA gene fragment cut out from this gel using QIAquick Gel Extraction Kit (manufactured by Qiagen) and purified. This fragment was ligated to the Nde I site and Hind III site of E. coli plasmid vector pET29b (+) (Novagen) with T4 DNA ligase, and transformed into E. coli DH5α to construct plasmid pMoxAhis.
(2) Construction of pNfa2293His Primers Nfa2293-1F (5′-GCCCCCCATATGGAGTCAACCCAGATCGCCG-3 ′) (see SEQ ID NO: 5) and Nfa2293-3R (5′-GCAAGCTCTCCCGCGTCAGGTACACCGG-3 ′) (see SEQ ID No. c in ac) PCR was carried out using the total DNA of IFM 10152 as a template under the following reaction conditions to amplify the CYP154A gene.
(Reaction solution composition)
Sterile purified water 71μl
10-fold concentrated KOD-Plus-buffer (manufactured by Toyobo) 10 μl
25 mM MgSO _{4 4} μl
dNTP mixed solution (dATP, dGTP, dTTP,
dCTP 2 mM each) 10 μl
Nfa 2293-1F primer (50 pmol / μl) 1 μl
Nfa 2293-3R primer (50 pmol / μl) 1 μl
Nocardia farcinica IFM 10152
Total DNA (100 ng / μl) 1 μl
KOD-Plus-polymerase
(1 unit / μl, manufactured by Toyobo Co., Ltd.) 2 μl
Dispense 50 μl into two microtubes.
(Temperature conditions)
94 ° C for 3 minutes, (98 ° C for 20 seconds, 63 ° C for 30 seconds, 68 ° C for 2 minutes) 25 cycles, 72 ° C for 5 minutes The DNA fragment amplified by this reaction was purified using QIAquick PCR Purification Kit (Qiagen) did. This DNA solution was treated with restriction enzymes Nde I and Hind III in a reaction volume of 50 μl, and then electrophoresed on a 0.8% agarose gel. After the electrophoresis, the fragment was recovered from the gel slice containing the 1.2 kb CYP154A gene fragment cut out from the gel using QIAquick Gel Extraction Kit (manufactured by Qiagen) and purified. This fragment was ligated to the Nde I site and Hind III site of E. coli plasmid vector pET29 (+) (manufactured by Novagen) with T4 DNA ligase and transformed into E. coli DH5α to construct plasmid pNfa2293His.

Preparation of P450 enzyme Plasmid pMoxAhis DNA or pNfa2293His was transformed into E. coli BL21 (DE3). A 20% glycerol preservation culture of the obtained strain was prepared, 10 μl thereof was added to 2 ml of LB medium supplemented with 25 μg / ml (final concentration) kanamycin, and cultured with shaking at 30 ° C. for 16 hours at 220 rpm. 500 μl of this culture solution was added to M9mix medium (M9 salt, 0.4% glucose, 0.5% casamino acid, 20 μl / ml thymine, 0.1 mM CaCl ₂ , 1 mM MgCl ₂ ) supplemented with kanamycin 25 μg / ml (final concentration). , 0.1 mM FeSO ₄ ) and shaking culture at 37 ° C. for 2.5 hours, 50 μl of 100 mM IPTG and 50 μl of 80 mg / ml 5-aminolevulinic acid were added successively, and shaking culture at 22 ° C. and 120 rpm for 16 hours. did. By centrifuging, the cells recovered from this culture solution were washed once with 10 ml of Tris buffer (50 mM Tris-HCl (pH 7.4), 10% glycerol), and then the cells were suspended in 10 ml of the same buffer. It became cloudy. 900 μl of BugBaster (manufactured by Novagen), 3 μl of Benzonase (manufactured by Merck, 25 units / μl) and 900 μl of lysozyme solution (40 mg / ml) were sequentially added to 9 ml of this cell suspension, and incubated at 30 ° C. for 20 minutes. . The supernatant obtained after centrifuging this lysate (3000 × g, 4 ° C., 10 minutes) is passed through a ProBond column (manufactured by Invitrogen) that has been pre-washed with ion-exchanged water and equilibrated with Tris buffer. I let you. The column was then washed with 10 ml of Tris buffer. Next, after washing the column with 5 ml of Tris buffer containing 50 mM imidazole, MoxA or CYP154A was eluted with 5 ml of Tris buffer containing 200 mM imidazole. The eluate was dialyzed in CV-1 buffer (50 mM sodium phosphate buffer (pH 7.4), 10% glycerol, 2 mM dithiothreitol, 1 mM EDTA, 1 mM glucose) to obtain a purified P450 solution. It was. This solution showed an almost single band at about 45 kD, which was consistent with the estimated molecular weight of P450 protein by SDS-PAGE. Further, according to the method of Omura and Sato et al. (J. Biol. Chem. 1964239: 2370-378), the P450 concentration contained in the solution is measured by measuring the difference spectrum before and after the carbon monoxide bond under reducing conditions. As a result, 6.1 μM of the MoxA solution and 11.2 μM of P450 were contained in the CYP154A solution. These purified P450 solutions were used in the reactions described in the examples below.

Diclofenac hydroxylation using ACNQ A diclofenac hydroxylation test was conducted under the following conditions.
(Reaction solution composition)
6.1 μM Purified MoxA solution 234 μl
10 mM ACNQ (DMSQ solution) 5 μl
50 mM NADH 25 μl
50 mM NADPH 25 μl
1 unit / μl glucose dehydrogenase (manufactured by Toyobo) 10 μl
2M glucose 10 μl
100 mM diclofenac sodium (DMSO solution) 5 μl
In addition to this, the final volume was adjusted to 500 μl with CV-1 buffer. In addition, following the above reaction solution composition, (1) ACNQ, NADH, NADPH, glucose dehydrogenase, glucose not added, (2) NADH, NADPH In order to compare glucose dehydrogenase, glucose not added, and (3) glucose dehydrogenase, glucose not added, a reaction solution was prepared and reacted at 30 ° C. under 200 rpm for 20 hours.
After the reaction, 500 μl of methanol was added, vortexed, and the supernatant obtained by centrifugation was analyzed by HPLC under the following conditions.
(HPLC analysis conditions)
Column: J'sphere ODS-H80 (75 × 4.6 mm id manufactured by YMC Corporation)
Mobile phase: 0.85% phosphoric acid aqueous solution-methanol (40:60)
Flow rate: 1.0 ml / min
Column temperature: 40 ° C
Detection wavelength: 267 nm
Retention time: Diclofenac: 11.5 min, 4′-hydroxydiclofenac: 4.0 min
As a result, as shown in FIG. 1, hydroxylation of diclofenac was observed only when ACNQ was added to the reaction system. At this time, the amount of hydroxide conversion increased by 7 times or more in a system in which NADPH and NADH were added from ACNQ alone, and when glucose dehydrogenase and glucose were added to regenerate NADH, the conversion amount was further doubled. Increased.

Effect of various quinones on hydroxylation of diclofenac Under the conditions shown below,
(Reaction solution composition)
6.1 μM purified MoxA solution 40 μl
50 mM NADPH 10 μl
10 mM various quinone-DMSO solutions * 1 μl
100 mM diclofenac sodium 1 μl
48 μl of CV-1 buffer
* ACNQ, 1,4-naphthoquinone, 1,4-dihydroxy-2-naphthoic acid, vitamin K ₃ and 1,4-benzoquinone were tested for various quinones in the above reaction solution composition. Only DMSO was used as a control (no quinone added).
After the reaction, 5 μl of 1N hydrochloric acid was added and extracted with 100 μl of ethyl acetate. 50 μl of this ethyl acetate extract was transferred to a sample tube and dried under reduced pressure using an evaporator. This was dissolved in 200 μl of methanol and analyzed by HPLC under the same conditions as described in Example 3 as a sample solution.
As a result of this test, as shown in FIG. 2, hydroxylation activity of diclofenac was observed in the reaction system to which any of the five quinones was added. The quinones relatively suitable for this reaction were ACNQ and 1,4-naphthoquinone due to the amount of hydroxide converted. The conversion amount of 4′-hydroxy diclofenac obtained in the presence of various quinone analogs is the amount of 4′-hydroxy diclofenac obtained after 18 hours when 1,4-naphthoquinone is added under the present reaction conditions. Is expressed as a relative value where 100 is 100.

Deethylation of 7-ethoxycoumarin using 1,4-naphthoquinone A 7-ethoxycoumarin deethylation test was carried out under the following conditions.
(Reaction solution composition)
11.2 μM purified CYP154A solution 80 μl
10 mM 1,4-naphthoquinone (DMSO solution) 4 μl
100 mM NADPH 10 μl
100 mM 7-ethoxycoumarin (DMSO solution) 0.8 μl
Adjust the final volume to 400 μl with CV-1 buffer.
(1) No addition of purified CYP154A solution,
(2) A reaction solution was prepared in order to compare those without addition of 1,4-naphthoquinone, and the reaction was carried out at 30 ° C. under a condition of 125 rpm for 4 hours.
After the reaction, extraction was performed with 400 μl of ethyl acetate. 200 ul of this ethyl acetate extract was transferred to a test tube and dried under reduced pressure using an evaporator. The dried product was dissolved in 4 ml of 25 mM potassium phosphate buffer (pH 7.4) to obtain a sample solution. In order to detect 7-hydroxycoumarin produced as a result of deethylation of 7-ethoxycoumarin, fluorescence at 450 nm at an excitation wavelength of 380 nm was measured with a fluorescence detector.
As a result, as shown in FIG. 3, deethylation of 7-ethoxycoumarin by CYP154A was observed in the presence of 1,4-naphthoquinone.

According to the present invention, since an oxidative reaction system capable of effectively converting a certain organic compound which is a cell-free system can be provided, the present invention can be applied to, for example, the manufacture of pharmaceuticals with modified biological activity, research on drug metabolism. It can be used in industries that provide tools and the like.
[Sequence Listing]

Claims (13)

A reaction system for conducting a biological conversion reaction of a compound that can be a substrate of cytochrome P450 in the presence of cytochrome P450,
(A) cytochrome P450 derived from actinomycetes ,
(B) NADH and NADPH, and an initial electron supply means selected from the group consisting of electrodes capable of supplying electrons;
(C) (1) 1,4-naphthoquinone, 1,4-benzoquinone, 2-amino-3-carboxy-1,4-naphthoquinone, 1,4-dihydroxy-2-naphthoic acid, and vitamin K ₃ , and ( 2) A compound other than the compound described in (1), which is selected from compounds in which 1,4-benzoquinone or 1,4-naphthoquinone is modified with an amino group, a carboxyl group or a methyl group, and under physiological conditions And a quinone analog selected from the group consisting of compounds capable of substituting the cofactor function in the cytochrome P450 reaction system. The reaction system according to claim 1, wherein cytochrome P450 has 90 % or more identity with MoxA or CYP154A derived from actinomycetes or MoxA at the amino acid sequence level and has a function of P450. The reaction system according to claim 1 or 2, wherein the cytochrome P450 is MoxA or CYP154A derived from actinomycetes . The quinone analog is selected from the group consisting of 1,4-naphthoquinone, 1,4-benzoquinone, 2-amino-3-carboxy-1,4-naphthoquinone, 1,4-dihydroxy-2-naphthoic acid, and vitamin K ₃ The reaction system according to any one of claims 1 to 3. The reaction system according to any one of claims 1 to 4, wherein the initial electron supply means is one or two selected from NADH and NADPH. The reaction system according to any one of claims 1 to 4, wherein the initial electron supply means is an electrode capable of supplying electrons. A reaction system for performing a biological conversion reaction of a compound that can be a substrate of cytochrome P450 in the presence of cytochrome P450,
(A) cytochrome P450 derived from actinomycetes ,
(B) NADH and NADPH, and an initial electron supply means selected from the group consisting of electrodes capable of supplying electrons;
(C) (1) 1,4-naphthoquinone, 1,4-benzoquinone, 2-amino-3-carboxy-1,4-naphthoquinone, 1,4-dihydroxy-2-naphthoic acid, and vitamin K ₃ , and ( 2) A compound other than the compound described in (1), which is selected from compounds in which 1,4-benzoquinone or 1,4-naphthoquinone is modified with an amino group, a carboxyl group or a methyl group, and under physiological conditions A quinone analog selected from the group consisting of compounds capable of substituting a cofactor function in a cytochrome P450 reaction system, wherein a biological conversion of a compound that can be a substrate of cytochrome P450 in the reaction system is included. A method for producing a biological transformation product characterized by the above. The method according to claim 7, wherein the biological transformation is an oxidized form. The production method according to claim 7 or 8, wherein cytochrome P450 has 90 % or more identity with MoxA or CYP154A derived from actinomycetes or MoxA at the amino acid sequence level and has a function of P450. The quinone analog is selected from the group consisting of 1,4-naphthoquinone, 1,4-benzoquinone, 2-amino-3-carboxy-1,4-naphthoquinone, 1,4-dihydroxy-2-naphthoic acid, and vitamin K ₃ The manufacturing method in any one of Claims 7-9. The method according to any one of claims 7 to 10, wherein the cytochrome P450 is MoxA or CYP154A derived from actinomycetes . The manufacturing method according to any one of claims 7 to 11, wherein the initial electron supply means is one or two selected from NADH and NADPH. The manufacturing method according to claim 7, wherein the initial electron supply means is an electrode capable of supplying electrons.

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