JP6366051B2 - Hydrazine synthesis method - Google Patents

Description

  The present invention relates to a method for synthesizing hydrazine using an enzyme catalyst. More specifically, the enzyme catalyst relates to a method for synthesizing hydrazine that is derived from Anammox bacteria and can obtain hydrazine by enzyme-catalyzed reaction.

  Hydrazine is a useful chemical substance used in rocket fuel, fuel cells, etc., and has been conventionally produced by heating ammonia and sodium hypochlorite and chemically synthesizing them at high temperatures. Therefore, development of a method for synthesizing hydrazine under milder conditions is required, requiring a large amount of energy input.

  As a method related to the synthesis of hydrazine that is not based on chemical synthesis at high temperature, a method using a microorganism or an enzyme catalyst can be considered. In recent years, it has been noted that anaerobic ammonia oxidizing bacteria (Anamox bacteria) denitrify ammonia under anaerobic conditions. In general, the process of circulating organic nitrogen (Org-N) or ammonia into inorganic nitrogen by a microorganism or the like in vivo is a nitrification reaction that converts ammonia to nitrous acid or nitric acid under aerobic conditions. It was thought that the nitrous acid and nitric acid were converted to nitrogen gas under anaerobic conditions through a denitrification process. This nitrogen cycle required aerobic and anaerobic conditions. However, it is known that the above-mentioned Anammox bacteria perform an anaerobic ammonia oxidation reaction that converts ammonia into nitrogen even under anaerobic conditions. In this anaerobic ammonia oxidation reaction, hydrazine is considered to be synthesized as an intermediate (see FIG. 1).

For example, Non-Patent Document 1 includes an ammonium salt and nitrite ( ¹⁵ NO ²⁻ consisting of ¹⁵ N of a stable isotope), and hydrazine ( ²⁸ N ₂ H ₄ ) from cells of Anammox Kuenenia stuttgartiansis (Candidatus Kuenenia stuttgartiansis). ), The formation of ²⁹ N ₂ H ₄ was observed. That is, Anammox bacteria synthesize hydrazine in its denitrification step, and it has been confirmed that hydrazine is synthesized by coupling nitrogen atoms of ammonium salt and nitrite in vivo. And the protein which Anammox bacteria have is elucidated, and the specific enzyme is named hydrazine synthase (HZS). However, the name of HZS was named from the presumed function in the above-mentioned reaction, and it has not been proved that hydrazine synthesis can be catalyzed only by this enzyme.

  Furthermore, it is described that nitrogen gas was produced from ammonium salt and nitric oxide (NO) via hydrazine as an intermediate using the HZS of Anammox Kuennia stuttgartiensis and the electron acceptor Cytochrome c. However, in these studies, an increase in the concentration of hydrazine itself, which is presumed to be an intermediate, has not been confirmed, and a method for producing hydrazine using microorganisms or enzymes has not been known.

  Non-Patent Document 2 specifies the sequence of protein NaxLS found from Anammox bacteria, but does not describe its specific use.

Molecular mechanism of anaerobic ammonium oxidation, BoranKartal etal, Nature 479,127-130 (2011) A heterodimeric cytochrome c complex with a very low redoxpotential from ananaerobic ammonium-oxidizing enrichment culture, S. Ukita etal, FEMS Microbiol Lett313, 61-67 (2010).

  A conventional method for synthesizing hydrazine is based on a chemical synthesis method that requires a treatment such as heating at a high temperature. On the other hand, the possibility of synthesizing hydrazine by microorganisms has been studied as seen in Non-Patent Document 1, but none has been confirmed to synthesize hydrazine efficiently. Further, even when nitrogen gas is generated from the intermediate hydrazine in Non-Patent Document 1, the synthesis rate of hydrazine (molecular activity of HZS) determined from the generation of nitrogen gas was estimated to be a very small value. Under such circumstances, an object of the present invention is to provide a method for synthesizing hydrazine using a specific enzyme catalyst.

  As a result of intensive studies to solve the above problems, the present inventor has found that the following inventions meet the above object, and have reached the present invention.

That is, the present invention relates to the following inventions.
<1> Using an enzyme catalyst, hydrazine is synthesized from a substrate (A) composed of an ammonium salt and at least one substrate (B) selected from the group consisting of hydroxylamine, nitrate, nitrite and nitric oxide. A hydrazine synthesis method comprising:
The subunit protein constituting the enzyme catalyst is at least one protein of the following (A), (B) or (C):
The enzyme catalyst is hydrazine synthase (I) which is a heterotrimeric heme protein of the following protein (1a), protein (1b) and protein (1c);
A hydrazine synthesis method using hydrazine synthase (II) which is a heterodimeric heme protein with the following protein (2a) and protein (2b).
(A) Protein (1a) is represented by SEQ ID NO: 1, protein (1b) is SEQ ID NO: 2, protein (1c) is SEQ ID NO: 3, protein (2a) is SEQ ID NO: 4, and protein (2b) is SEQ ID NO: 5, respectively. (B) In the above (a), one or several amino acids are deleted, substituted or added to the protein represented by the corresponding SEQ ID NO. A protein having a hydrazine synthesizing enzyme (I) or (II) and having hydrazine synthesizing activity (c) In the above (a), at least one protein is represented by a corresponding sequence number. A protein consisting of an amino acid sequence having 55% or more identity with the protein SEQ ID NO: <2> Recombinant hydrazine synthase (I) and recombinant hydrazine synthesis, wherein the elementary catalyst is a protein expressed from a gene encoding a subunit protein constituting at least one enzyme catalyst of the above (a), (b) or (c) The method for synthesizing hydrazine according to <1>, wherein the method is enzyme (II).
<3> Hydrazine from a substrate (A) comprising an ammonium salt and a substrate (B) which is at least one substrate selected from the group consisting of hydroxylamine, nitrate, nitrite and nitric oxide using an enzyme catalyst A hydrazine synthesis method for synthesizing
As the enzyme catalyst, hydrazine synthase (I ′) which is a heterotrimeric heme protein derived from Anammox,
A hydrazine synthesis method using hydrazine synthase (II ′), which is a heterodimeric heme protein derived from Anammox bacteria.
<4> The molecular mass of the enzyme (I ′) measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis is 91.5 to 96.5 kDa, 40.5 to 45.5 kDa, and 34.5 to 39.5 kDa. The method for synthesizing hydrazine according to <3>, wherein the enzyme (II ′) has a molecular mass of 11 to 17 kDa and 8 to 14 kDa as measured by tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis.
<5> Enzymes (I ′) and (II ′) crush the Anammox cells, centrifuge, salt the supernatant, centrifuge, and purify the resulting supernatant by hydrophobic column chromatography The method for synthesizing hydrazine according to <3> or <4>.
<6> When the enzymes (I ′) and (II ′) are purified by the hydrophobic column chromatography, the hemoproteins identified by monitoring the absorbance at a wavelength of 280 nm and the absorbance at a wavelength of 408 nm, The hydrazine synthesis method according to the above <5>, which is an enzyme obtained from a fraction around one peak and a fraction around a second peak.
<7> When the enzyme (II ′) is difficult to reduce and the enzyme (I ′) is in a reduced form, the absorption maximum is in the α band, the β band, and the Sole band characteristic of the c-type heme protein. The method for synthesizing hydrazine according to any one of the above <3> to <6>.
<8> The hydrazine is synthesized using a Tris-HCl buffer, a phosphate buffer, or a MOPS buffer (pH 7.0 to 8.0) as a buffer solution, according to any one of the above items <1> to <7>. Hydrazine synthesis method.
<9> The hydrazine synthesis method according to any one of <1> to <8>, wherein the substrate (B) is hydroxylamine.
<10> The hydrazine synthesis method according to any one of <1> to <9>, wherein the hydrazine synthesis method is performed in the presence of a reducing agent.
<11> The protein of the subunit is at least one protein of the following (A ′), (RO ′) or (C ′), and a heterodimeric hem with the following protein (2a) and the following protein (2b) Hydrazine synthase (II) for synthesizing protein hydrazine.
(A ′) Protein (2a) is a protein comprising the amino acid sequence represented by SEQ ID NO: 4 and protein (2b) is SEQ ID NO: 5 (B ′) In the above (A ′), at least one protein corresponds A protein having a hydrazine synthesizing activity according to the constitution of hydrazine synthase (II), consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added to the protein represented by SEQ ID NO. ′) In the above (A ′), at least one protein is a protein comprising an amino acid sequence that is 55% or more identical to the sequence number of the protein represented by the corresponding sequence number. <12> For hydrazine synthesis Hydrazine synthase (enzyme is a heterodimeric heme protein derived from Anammox bacteria) I').

  According to the present invention, by using a specific enzyme, hydrazine can be synthesized in vitro without requiring treatment such as high temperature heating.

It is a figure which shows the outline | summary of the biogeochemical nitrogen circulation of the natural world. It is an example of monitoring absorbance when fractionating the hydrazine synthase (I ′) and (II ′) of the present invention by gel filtration chromatography. It is an example of monitoring absorbance when fractionating the hydrazine synthase (I ′) and (II ′) of the present invention by gel filtration chromatography. It is a three-dimensional structural schematic diagram of hydrazine synthase (II ') (NaxLS) of the present invention. 2 is an absorption spectrum of a purified solution containing hydrazine synthase (I ′) of the present invention and its reduced heme protein. 2 is an absorption spectrum of a purified solution containing hydrazine synthase (II ′) of the present invention and its reduced heme protein. It is an example of a measurement result when molecular mass measurement of the hydrazine synthetase (I ') of this invention is carried out by SDS-PAGE. It is an example of a measurement result when molecular mass measurement of the hydrazine synthetase (II ') of this invention is carried out by SDS-PAGE. It is a measurement result when performing the TOF-MS test for confirming that the hydrazine is synthesize | combined with the hydrazine synthesis | combining method of this invention.

  DESCRIPTION OF EMBODIMENTS Embodiments of the present invention will be described in detail below. However, the description of the constituent elements described below is an example (representative example) of an embodiment of the present invention. It is not limited to the contents. In the present invention, “to” is used as an expression including numerical values or physical quantities before and after.

The present invention synthesizes hydrazine from an ammonium salt substrate (A) and at least one substrate (B) selected from the group consisting of hydroxylamine, nitrate, nitrite and nitric oxide using an enzyme catalyst. A method for synthesizing hydrazine,
The subunit protein constituting the enzyme catalyst is at least one protein of the following (A), (B) or (C):
The enzyme catalyst is hydrazine synthase (I) which is a heterotrimeric heme protein of the following protein (1a), the following protein (1b) and the following protein (1c);
A hydrazine synthesizing method comprising using hydrazine synthase (II) which is a heterodimeric heme protein with the following protein (2a) and the following protein (2b);
(A) Protein (1a) is represented by SEQ ID NO: 1, protein (1b) is SEQ ID NO: 2, protein (1c) is SEQ ID NO: 3, protein (2a) is SEQ ID NO: 4, and protein (2b) is SEQ ID NO: 5, respectively. (B) In (a) above, at least one protein is deleted, substituted or added to the protein represented by the corresponding SEQ ID NO. A protein having a hydrazine synthesizing activity according to the structure of hydrazine synthases (I) and (II); (c) In (i) above, at least one protein is represented by a corresponding sequence number. Related to a protein consisting of an amino acid sequence having an identity with the SEQ ID NO of 55% or more It is. By this synthesis method, hydrazine can be biosynthesized by enzyme catalysis.

  The present invention also provides a hydrazine from a substrate (A) comprising an ammonium salt and at least one substrate (B) selected from the group consisting of hydroxylamine, nitrate, nitrite and nitric oxide using an enzyme catalyst. Hydrazine synthesis method for synthesizing hydrazine synthase (I ') which is a heterotrimeric heme protein derived from Anammox bacteria and hydrazine synthesis which is a heterodimeric heme protein derived from Anammox bacteria The present invention relates to a hydrazine synthesis method characterized by using an enzyme (II ′).

  That is, the present invention is a method for synthesizing hydrazine from a substrate (A) and a substrate (B). Hereinafter, each substrate will be described. An ammonium salt is used as the substrate (A). Here, the ammonium salt is a salt generated by the combination of ammonia and an acid, and specifically includes ammonium chloride, ammonium nitrate, ammonium sulfate and the like. Since the synthesis method of the present invention is generally carried out in an aqueous solution, those that are easily dissociated as ammonium ions in the aqueous solution and that are extremely difficult to change pH when dissolved are preferably used. Further, ammonium ions in which organic nitrogen is decomposed by microorganisms or the like in an aqueous solution or the like and exist as ammonium ions may be used as a substrate, and the ammonium salt including the ammonium ions is referred to as an ammonium salt which is the substrate (A) of the present invention.

  As the substrate (B) of the present invention, at least one substrate selected from the group consisting of hydroxylamine, nitrate, nitrite and nitric oxide can be used. These substances selected as the substrate (B) are substances produced by reducing nitrate ions with an enzyme produced by Anammox bacteria, and are particularly relevant to the process of Denitrification by Anammox bacteria. is there. Among these, by using hydroxylamine, hydrazine can be efficiently synthesized by the hydrazine synthase (I) or (I ′) of the present invention and the hydrazine synthase (II) or (II ′). This is preferable because it is possible. When other nitrates, nitrites, and nitric oxide are used, it is preferable to provide a step of converting to hydroxylamine by other chemical methods or enzyme catalysis. The step of converting to hydroxylamine may be performed in the same field (generally in an aqueous solution) as the reaction for synthesizing hydrazine. Examples of the enzyme that converts to hydroxylamine by enzyme catalysis include hydroxylamine oxidoreductase (HAO), nitrite reductase (NIR), and the like.

  As described above, in the present invention, it is preferable to use hydroxylamine as the substrate (B). The present inventors have found that hydroxylamine can be used to efficiently produce hydrazine synthase (I) or (I ′) and hydrazine synthase (II) or (II ′).

  In the present invention, hydrazine is preferably synthesized in the presence of a reducing agent. That is, it is preferable to carry out the reaction in the presence of a reducing agent at the reaction site for carrying out the hydrazine synthesis reaction of the present invention. More specifically, the present invention is performed mainly in a liquid in which a substrate, an enzyme, and the like are mixed. By mixing a reducing agent with this liquid, the state in the presence of the reducing agent can be achieved. By synthesizing hydrazine in a state where this reducing agent is mixed in the reaction field, hydrazine can be synthesized with higher efficiency.

  As the reducing agent, any reducing agent that does not inhibit hydrazine synthesis may be used, and any known inorganic or organic reducing agent can be used. Specific reducing agents include sodium dithionite (dithionite), thioglycolic acid or a salt thereof, glycerin monothioglycolate, 2-mercaptopropionic acid, 2-mercaptoethylamine, thiolactic acid, thioglycerin, sodium sulfite, Sodium bisulfite, sodium metasulfite, hydroquinone, benzyl viologen, methyl viologen and the like can be used, and any of them may be used alone or in appropriate combination. As the reducing agent, it is preferable to use a water-soluble reducing agent so as to disperse in an aqueous solution used for hydrazine synthesis. A reducing agent having a relatively low standard oxidation-reduction potential is preferable, and a reducing agent having a standard oxidation-reduction potential of −300 mV or less and −750 mV or more is particularly preferable. The reducing agent preferably used is organic and water-soluble and has a standard oxidation-reduction potential in the above-mentioned range, and specifically, dithionite is a preferable reducing agent. The amount of the reducing agent added is appropriately set according to the standard oxidation-reduction potential and the substrate or enzyme used in the hydrazine synthesis reaction. For example, in the solution (concentration at the time of use) used as the reaction site for hydrazine synthesis, It can be set to about 0.1 to 100 mmol / L. When the concentration of the reducing agent is low, the hydrazine synthesis activity may be difficult to improve. When dithionite is used as the reducing agent, the lower limit of the concentration during use is preferably 1 mmol / L or more, more preferably 3 mmol / L or more. Further, the upper limit of the concentration during use is preferably 50 mmol / L or less, and more preferably 15 mmol / L or less.

  As described above, the substrate (B) is a substance produced by reducing nitrite ions with an enzyme produced by Anammox bacteria, and is particularly relevant to the process of denitrifying Anammox bacteria. . Here, this substrate (B) becomes hydrazine in the presence of the ammonium salt which is the substrate (A) and the hydrazine synthase of the present invention, but against the oxidation number −2 of the nitrogen atom of hydrazine. Thus, since the oxidation number of the nitrogen atom of nitric oxide is +2, it is considered that assisting the reduction functions effectively. In addition, hydrazine synthases (II) and (II ′), which are proteins used in the synthesis of hydrazine found in the present invention, are difficult to reduce, but are slightly reduced by the reducing agent described above, and are reduced. Enzymes (II) and (II ′) can deliver electrons to heme of the prosthetic group having a low standard redox potential of hydrazine synthases (I) and (I ′), and (I) and (I ′) Are considered to function more effectively. Therefore, it is considered that the synthesis efficiency of hydrazine is improved even when hydroxylamine whose oxidation number is close to the oxidation number of the nitrogen atom of hydrazine is used as a substrate.

The synthesis method of the present invention is characterized in that hydrazine synthase (I) or (I ′) and hydrazine synthase (II) or (II ′) are used in combination as an enzyme catalyst.
Hydrazine synthase (I ′) and hydrazine synthase (II ′) used in the present invention can be obtained from Anammox bacteria, and hydrazine synthase (I ′) is an enzyme of heterotrimeric heme protein, hydrazine Synthase (II ′) is a heterodimeric heme protein enzyme.
On the other hand, hydrazine synthase (I) and hydrazine synthase (II) can be obtained from the same base sequence as that obtained from Anammox bacteria, or from a protein produced so as to satisfy certain requirements such as identity. It can be an enzyme.

[Hydrazine synthase (I) or (I ′) (HZS)]
Hydrazine synthase (I) or (I ′) is known as an enzyme (HZS) presumed to be a hydrazine synthase, and is also disclosed in the above-mentioned Non-Patent Document 1 and the like. In the present invention, this hydrazine synthase (I) or (I ′) is used as one of the enzyme catalysts.

[Hydrazine synthase (II) or (II ′) (NaxLS)]
Hydrazine synthase (II) or (II ′) was also disclosed in the above-mentioned Non-Patent Document 2 as a protein of Anammox bacteria, but its function and use were unknown. In the present invention, the hydrazine synthase (II) or (II ′) is used as one of enzyme catalysts.
The hydrazine synthase (II) or (II ′) is an enzyme of a heterodimeric heme protein, and each protein unit is known as NaxL or NaxS. The entire heterodimeric heme protein may be referred to as NaxLS.

  In the present invention, in particular, hydrazine synthase (I) (or (I ′)) and hydrazine synthase (II) (or (II ′)) are combined and subjected to an enzyme-catalyzed reaction using a specific substrate. Hydrazine was synthesized by the above method, and the synthesized hydrazine not only exists as an intermediate, but also increases in concentration in an aqueous solution, so that it can be used as a production method.

  Here, in the present invention, the subunit protein constituting the enzyme catalyst is at least one protein of the following (A), (B) or (C), and the enzyme catalyst is the following protein (1a): A hydrazine synthase (I) that is a heterotrimeric heme protein of the following protein (1b) and the following protein (1c), and a heterodimeric heme protein of the following protein (2a) and the following protein (2b) A hydrazine synthesis method characterized by using hydrazine synthase (II). Here, (a) protein (1a) is SEQ ID NO: 1, protein (1b) is SEQ ID NO: 2, protein (1c) is SEQ ID NO: 3, protein (2a) is SEQ ID NO: 4, and protein (2b) is SEQ ID NO: 5. (B) In (i) above, at least one protein is deleted from the protein represented by the corresponding SEQ ID NO., One or several amino acids are deleted, A protein comprising a substituted or added amino acid sequence and having hydrazine synthesizing activity by the constitution of hydrazine synthases (I) and (II); (c) a sequence corresponding to at least one protein in (a) above; A protein consisting of an amino acid sequence whose identity with the sequence number of the protein represented by the number is 55% or more A.

  That is, for example, the protein (1a) is a protein comprising the amino acid sequence represented by SEQ ID NO: 1, or one or several amino acids are deleted, substituted or substituted with respect to the amino acid sequence represented by SEQ ID NO: 1. Identical to a protein consisting of an added amino acid sequence and having hydroxylamine hydrazine synthesizing activity by the constitution of hydrazine synthase (I) or (II), or a protein consisting of the amino acid sequence represented by SEQ ID NO: 1. It is at least one of proteins consisting of an amino acid sequence having a sex of 55% or more, and (b) and (c) can be satisfied at the same time, and when the identity is 100% in (c) Exactly (a) is also satisfied at the same time. The protein (1a) has a predetermined relationship with the protein comprising the amino acid sequence represented by SEQ ID NO: 1 and the protein comprising the amino acid sequence represented by SEQ ID NO: 1 such as (b) and (c). It is a concept that comprehensively expresses proteins, and other proteins (1b), (1c), (2a), and (2b) also include proteins each having a corresponding sequence number and proteins having a predetermined relationship therewith. Each concept is expressed as a concept.

  The enzyme catalyst of the present invention comprises a plurality of subunit proteins. That is, hydrazine synthase (I) is a trimeric heme protein and consists of three subunit proteins, and hydrazine synthase (II) is a dimeric heme protein and consists of two subunit proteins.

  The hydrazine synthase (I) and hydrazine synthase (II) used in the present invention can be an enzyme composed of subunit proteins that satisfy the following requirement (a). That is, (a) protein (1a) is SEQ ID NO: 1, protein (1b) is SEQ ID NO: 2, protein (1c) is SEQ ID NO: 3, protein (2a) is SEQ ID NO: 4, and protein (2b) is SEQ ID NO: 5. Each protein consists of an amino acid sequence represented.

  The protein represented by SEQ ID NO: 1, the protein represented by SEQ ID NO: 2, and the protein represented by SEQ ID NO: 3 will be described later as Anammox battery strain KSU-1 (hereinafter abbreviated as “Anamox KSU-1”). The amino acid sequence is specified from the derived hydrazine synthase (I ′). The protein represented by SEQ ID NO: 4 and the protein represented by SEQ ID NO: 5 are those in which the amino acid sequence is specified from Anammox KSU-1 derived hydrazine synthase (II ′) described later.

  The proteins having the amino acid sequences represented by SEQ ID NOs: 1, 2, 4, and 5 each have a c-type heme-binding motif, and hydrazine synthase (I) formed by heme-binding to the proteins having these amino acid sequences. (II) is an enzyme composed of a heme protein when all function as an enzyme.

  Furthermore, the hydrazine synthase (I), (II) of the present invention is as follows: “(b) In the above (b), at least one protein is one or a number relative to the protein represented by the corresponding SEQ ID NO. A protein having an amino acid sequence in which one amino acid is deleted, substituted or added, and having hydrazine synthesizing activity according to the constitution of hydrazine synthase (I), (II) "or" (c) in (i) above, Each of the trimeric heme proteins and the dimers such as “a protein comprising an amino acid sequence having 55% or more identity with the sequence number of the protein represented by each corresponding sequence number” This can also be achieved by having a hydrazine synthesizing enzyme activity when it is made into body hemoprotein. As described above, the proteins represented by SEQ ID NOs: 1, 2, 3, 4, and 5 are hydrazine synthase sequences identified from the Anammox KSU-1 strain. It has hydrazine synthase and can also be achieved as a protein having the same amino acid sequence without being limited to living organisms. In particular, in the case of deletion, substitution or addition, the sequence that affects the binding property of the protein to heme is preferably the same, and deletion, substitution and addition are allowed in other portions. More specifically, known protein sequences that have been estimated to be hydrazine synthases derived from other strains, and protein sequences corresponding to hydrazine synthases (II) and (II ′) disclosed by the present invention. In comparison, deletion, substitution, and addition tend to be allowed where the commonality is low.

  Further, in the above (c), the identity (Identity) with the protein represented by SEQ ID NO: 1, 2, 3, 4 and 5 is 55% or more and 60% or more, respectively, with the protein of the corresponding SEQ ID NO: It is preferable that it is 80% or more. The proteins represented by SEQ ID NOs: 1 to 5 are amino acid sequences identified from Anammox KSU-1 as described above, and have the same identity as the amino acid sequences identified from the proteins of Anammox Kuenenia stuttgartiansis, which is another strain. 84-59% (depending on subunit). In addition, these amino acid sequences are relatively long as 126 to 810 amino acids, and only proteins that have been estimated to be hydrazine synthase derived from Anammox bacteria have been found to be 30% or more in identity. There is no characteristic amino acid sequence. In general, in the case of a protein having a sequence length of this degree, it is expected that the same function is achieved if the identity is 40% or more. In the present invention, BLAST (Basic Local Alignment Search Tool), which is one of algorithms widely used in this field, was used as an algorithm for obtaining amino acid sequence identity.

  More specifically, the identity between SEQ ID NOs: 1, 2, and 3, and the proteins represented by SEQ ID NOs: 6, 7, and 8, which are proteins derived from Anammox Kuennia stuttgartiensis, are 82%, 84%, and 79%, respectively. It is. In addition, the proteins represented by SEQ ID NOs: 6, 7, and 8 are also hydrazine synthases that synthesize hydrazine as an intermediate in the denitrification reaction in Anammox Kuennia stuttgartiansis, and are used as the enzymes of the heterotrimeric heme protein of the present invention. It can be used as hydrazine synthase (I).

  Furthermore, SEQ ID NOs: 9 and 10 are given as proteins derived from Anammox Kuenenia stuttgartiensis, and the identity of these proteins with SEQ ID NOs: 4 and 5 is 75% and 59%, respectively. The proteins represented by SEQ ID NOs: 9 and 10 can also be used as hydrazine synthase (II) as an enzyme of heterodimeric heme protein.

  In addition, the subunit protein constituting the enzyme catalyst may satisfy at least one of the following conditions (a), (b), or (c), but simultaneously satisfies the requirements of (b) and (c). In the above (a), hydrazine synthesis consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and an amino acid sequence having 55% or more identity with the sequence number of each protein It is preferable that the protein has a hydrazine synthesis activity depending on the structure of the enzymes (I) and (II).

  There are no particular limitations on the method for producing the proteins shown in hydrazine synthases (I) and (II). Any of those produced by a microorganism such as Coli can be used.

  By the protein expressed from the gene encoding the protein (1a), (1b), (1c), (2a), (2b) of the above (a), (b) or (c) of the hydrazine synthase (I) Recombinant hydrazine synthase (I) and hydrazine synthase (II) can be used. Examples of genes encoding each protein include SEQ ID NOs: 11 to 15. These SEQ ID NOs: 11 to 15 were identified as genes related to the synthesis of hydrazine synthase extracted from the aforementioned Anammox KSU-1 strain. The base sequences of the DNAs represented by SEQ ID NOs: 11 to 15 are the complement sequences of the genes to be encoded. SEQ ID NO: 11 is one of the proteins (1a), SEQ ID NO: 12 is SEQ ID NO: 2 which is one of the proteins (1b), and SEQ ID NO: 13 is one of the proteins (1c). SEQ ID NO: 14 corresponds to SEQ ID NO: 4 that is one of the proteins (2a), and SEQ ID NO: 15 corresponds to SEQ ID NO: 5 that is one of the proteins (2b).

  The amino acid and gene sequences of the Anammox KSU-1 strain represented by SEQ ID NOs: 1 to 5 and 11 to 15 of the present invention are registered in the nucleic acid database under the number BAFH01000004. SEQ ID NOs: 1 and 11 are “GAB63748 KSU1D — 439 (ZP 10100787)”, SEQ ID NOs: 2 and 12 are “GAB63748 KSU1d — 440 (ZP 10100908)”, SEQ ID NOs: 3 and 13 are “GAB63750 KSU1D — 441 (ZP 10100809)”, Is registered as “GAB63547 KSU1d — 238 (ZP 10100756)”, and SEQ ID NOs: 5 and 15 are registered as “GAB63548 KSU1d — 239 (ZP 10100657)”.

  The present invention also provides hydrazine synthase (I ′), which is a heterotrimeric heme protein derived from Anammox bacteria, and hydrazine synthase (II ′), which is a heterodimeric heme protein derived from Anammox bacteria, as enzyme catalysts. It can also be achieved in an embodiment using

  The hydrazine synthases (I ′) and (II ′) of the present invention are enzymes derived from Anammox bacteria that can be obtained from Anammox bacteria. Here, the Anammox bacterium is a general term for bacteria that perform anaerobic ammonia oxidation reaction (anaerob ammonia oxidation). Anammox bacteria are not isolated and cultured. Generally, Anammox bacteria are preferentially cultivated from pre-cultured Anammox bacteria, which is obtained by attaching cultured Anammox sludge to a nonwoven fabric and continuously cultivating it. Can do. Examples of the Anammox bacteria include Candidatus Kuenenia stuttgartiensis (in the present specification, it is sometimes referred to as “Ammamox Kuenenia stuttgartidians and Candidacetidiaidiaidia and Candidas etidiumidiaidiaid.

  Examples of the Anammox bacteria present in seawater include Candidatus Scalindua brodae, Candidatus Scalindua sorokinii, and Candidatus Scalindua wagneri.

  Furthermore, Anammox bacteria strain KSU-1 (sometimes abbreviated as “Ammamox KSU-1”), which is a strain of Anammox bacteria possessed by the present inventors, Anammox Bacterium strain BRW-1 (“Ammoxx ASah-1”) May be abbreviated.) And the like.

Among these, Candidatus Kuenenia stuttgartiensis, Candidatus Jettenia asiatica, Candidatus Anammoxoglobus propionicus, Candidatus Brocadia anammoxidans, Anammox bacterium strain KSU-1, Anammox bacterium strain BRW-1 from hydrazine synthase is present in seawater Candidatus Scalindua brodae, etc. Unlike that, it is preferably used because it is considered to have a hydrazine synthase that easily reacts in an aqueous solution having a low sodium chloride concentration. More preferably, hydrazine synthase derived from Anammox KSU-1 or Anammox Asahi BRW-1. In addition, Anammox KSU-1 and Anammox Asahi BRW-1 are accustomed in the Faculty of Biological Life Sciences of the school corporation Kimigasu Gakuen Sojo University, as will be described later.
Moreover, you may use as an enzyme derived from these single bacteria, and may be used in combination. Since hydrazine synthase (I ′) and hydrazine synthase (II ′) have a high complementary relationship between the enzymes, it is better to use enzymes derived from the same fungus.

  The hydrazine synthase of the present invention is a protein derived from Anammox bacteria and has hydrazine synthesis activity.

  The hydrazine synthase (I ′) of the present invention has a c-type heme binding motif and is a c-type heme protein. Also, hydrazine synthase (II ′) has a c-type heme binding motif and is a c-type heme protein.

  The molecular mass of the enzyme (I ′) measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis (“SDS-PAGE”) is 91.5-96.5 kDa (representative value is 94 kDa) and 40.5-45. It is a heterotrimer of a protein of .5 kDa (representative value is 43 kDa) and a protein of 34.5 to 39.5 kDa (representative value is 37 kDa). The molecular mass of the enzyme (II ′) measured by tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis (Tricine SDS-PAGE) is 11 to 17 kDa (representative value is 14 kDa) and 8 to 14 kDa (representative value is 11 kDa). ) Protein heterodimer. The molecular mass of the enzyme (I ′) can be measured by SDS-PAGE usually performed by those skilled in the art. On the other hand, the molecular mass of the enzyme (II ′) having a small apparent molecular mass can be measured by Tricine SDS-PAGE (Tricine SDS-PAGE) using tricine as the cathode side electrode solution, which is usually performed by those skilled in the art. The molecular mass of each protein of the enzymes (I ′) and (II ′) varies depending on the species, genus, strain, and amino acid sequence of the protein from which the enzyme is derived. The molecular mass of each is determined as a trimer or heterodimer. The molecular mass of the protein constituting the subunit of the enzyme (II ′) may vary depending on the positional relationship with the molecular weight marker when measured by Tricine SDS-PAGE. The mass difference is about 3 kDa and can be confirmed as a heterodimer.

  In addition, Table 1 shows values obtained from amino acid sequences, in which the molecular mass of each protein constituting the hydrazine synthase of the present invention is known from the respective gene sequences. Thus, the molecular mass varies depending on the species, genus and strain from which the enzyme is derived, and also varies depending on amino acid deletion, substitution, addition, and degree of identity. As described above, since the protein constituting the hydrazine synthase of the present invention is a heme protein, the molecular mass obtained by adding the molecular mass of heme to the molecular mass determined from the amino acid sequence is the molecular mass as the heme protein. Become. In the present invention, the molecular mass consisting of this amino acid sequence is preferably 89.6 to 93.6 kDa, including the molecular mass of heme in the protein subunit constituting the enzyme (I) and the enzyme (I ′). 37.5 to 41.5 kDa, 39.2 to 43.2 kDa, and the subunits of the proteins constituting the enzyme (II) and the enzyme (II ′) are 14.8 to 16.8 kDa, 13.3 to 15 .3 kDa.

* In Table 1, Candidus Kuennia stuttgartiansis is abbreviated as “Kuenenia”.

The hydrazine synthase (I ′) and (II ′) of the present invention are proteins that can be obtained from Anammox bacteria, but when obtained by a purification method as described later, when hydrophobic column chromatography is used in the purification process, It is important to confirm which fraction contains the protein. As shown in FIG. 2, as a method for confirming whether a protein is contained in each fraction, when the enzymes (I ′) and (II ′) are purified by the hydrophobic column chromatography, the wavelength is 280 nm. There is a method of confirming that the enzyme is obtained from the fraction around the first peak and the fraction around the second peak of the heme protein confirmed by monitoring the absorbance at 408 nm and the wavelength of 408 nm. . Observe the presence or absence of heme because the absorbance (Abs ₂₈₀ ) at a wavelength of 280 nm, which is often used when confirming that a protein is contained, and the enzymes (I ′) and (II ′) of the present invention are heme proteins. This is confirmed by monitoring the absorbance (Abs ₄₀₈ ) at a wavelength of ₄₀₈ nm suitable for the above. At this time, when purifying using hydrophobic column chromatography as described later, the fraction around the first peak near the mutation point where Abs ₂₈₀ and Abs ₄₀₈ start to increase together (indicated by ii in FIG. 2). ) Contains a large amount of enzyme (II ′), and the fraction near the second peak (abbreviated as i in FIG. 2), which has a characteristic peak in combination with Abs ₂₈₀ and Abs ₄₀₈ , is enzyme (I ′). Many are included. Therefore, when a protein derived from Anammox is purified using hydrophobic column chromatography, a clear peak is observed as indicated by ii in FIG. 3 where Abs ₂₈₀ and Abs ₄₀₈ begin to increase. ) Enzyme (II ′) and Enzyme (I ′) can be obtained with the vicinity of the first peak and the vicinity of the first peak followed by Abs 280 and Abs 408 having both characteristic peaks. it can.

  It can be confirmed that the hydrazine synthase (II ′) of the present invention is a hardly-reducing heme protein. As a method for confirming that hydrazine synthase (II ′) is difficult to reduce, it can be confirmed from an absorption spectrum when dithionite is added to a purified solution of hydrazine synthase (II ′). There is a method of confirming that the characteristic peak seen at a wavelength of about 419 nm hardly changes even after addition of dithionite when the absorption spectrum of the purified solution of oxidized hydrazine synthase (II ′) is observed (FIG. 6). See spectrum). This is also described in the above-mentioned Non-Patent Document 2, in which the heme of a protein that is a subunit of hydrazine synthase (II ′) (NaxLS in the non-patent document) is a heterodimeric heme protein. This is described in detail together with the fact that it has a three-dimensional structure coordinated by histidine and cysteine side chains in the protein (see FIG. 4).

  It can be confirmed that the hydrazine synthase (I ′) of the present invention is a reduced type and has absorption maxima (peaks appear) in the α band, β band and Sole band. That is, as shown in the spectrum diagram of FIG. 5, the peak of the oxidized enzyme (I ′) at 408 nm is shifted to 419 nm in the reduced form reduced by dithionite, and the wavelength is near 523 nm (α band). It can be confirmed from the fact that a peak appears at a wavelength of 553 nm (β band).

[Method of obtaining enzyme]
When the hydrazine synthase (I ′) or (II ′) of the present invention is obtained by a method derived from a living body, it can be obtained from an anaerobic ammonia oxidizing bacterium (Anammox bacterium). For example, anaerobic ammonia-oxidizing bacteria purified from sludge obtained by culturing (acclimating) anaerobic ammonia-oxidizing bacteria and using cell membrane disruption, centrifugation, salting-out, fractionation by liquid chromatography, etc. From these proteins, hydrazine synthase (I ′) and hydrazine synthase (II ′) can be obtained.

A specific method for purifying the hydrazine synthase of the present invention will be described in detail below.
Acclimatization of sludge containing Anammox bacteria (hereinafter abbreviated as “Anamox sludge”) is acclimatized by a method of continuously culturing Anammox sludge that has been cultivated by attaching it to a nonwoven fabric or the like. Specifically, it can be selectively cultured in a reactor that continuously supplies a degassed inorganic medium mainly composed of nitrous acid and ammonia. For example, in the case of the Anammox KSU-1 strain, the inside of the reactor turns red as the acclimatization progresses. As described above, the Anammox bacterium is a bacterium that cannot be cultivated alone. However, the anammox-dominant sludge can be obtained by acclimatization by such a method.

  The destruction of Anammox cells in the Anammox sludge can be performed, for example, by grinding with an ultrasonic treatment or a mortar. Specifically, cell-free extract preparation buffer was added to Anammox sludge, and sonicated to make the sludge almost uniform paste, then ground in a mortar and further sonicated to destroy the cells. Can be.

  After disrupting the cells, by centrifuging the cells, the supernatant of the cell extract containing the protein derived from Anammox bacteria (cell-free extract) can be used more efficiently for the protein and cell debris used in the present invention. Can be separated.

  Further, the cell-free extract is subjected to salting-out with ammonium sulfate or the like, stirred, and then centrifuged to remove impurities by precipitation. Further, the target hydrazine synthase (I ′) or hydrazine synthesis A supernatant (high protein extract) having a high protein concentration such as enzyme (II ′) can be obtained.

  By purifying this high protein extract by hydrophobic column chromatography or the like, purified hydrazine synthase (I ′) and hydrazine synthase (II ′) can be obtained. In order to confirm the fractions containing each protein, it can be confirmed by monitoring the absorbance of the heme (Abs 408) and the absorbance of the protein (Abs 280). Can be managed simply by fraction number.

  Furthermore, the hydrazine synthase (I ') and hydrazine synthase purified to a higher concentration by further concentrating the fraction fractionated by hydrophobic column chromatography with a centrifugal filter or purifying the fraction by gel filtration chromatography. (II ′).

  Depending on the purification conditions, whether or not hydrazine synthase (I ′) and hydrazine synthase (II ′) are contained in each protein fraction purified by hydrophobic column chromatography, gel filtration chromatography, etc. For simplicity, it can be confirmed by measuring the absorbance at a wavelength of 419 nm as described above, or by measuring Abs280 or Abs408 because it is a heme protein.

  As a more accurate confirmation method, when observed by SDS-PAGE, hydrazine synthase (II ′) is Tricine SDS-PAGE as described above, and bands are observed at positions of 14 kDa and 11 kDa as representative values. This also confirms that the enzyme is a heterodimeric heme protein. On the other hand, hydrazine synthase (I ′) has confirmed bands at 94 kDa, 43 kDa, and 37 kDa as representative values. This also confirms that the enzyme is a heterotrimeric heme protein.

  The hydrazine synthases (II) and (II ′) of the present invention were proteins in which only amino acid sequences with unknown functions were specified. In the present invention, the unknown characteristics of these proteins have been found. In addition, these enzymes alone have a certain hydrazine synthesis activity. That is, the present invention describes that “the subunit protein is at least one protein of the following (A ′), (B ′) or (C ′) and is heterogeneous with the following protein (2a) and the following protein (2b): Hydrazine synthase (II) for synthesizing hydrazine which is a dimeric heme protein. (Ii) Protein (2a) is a protein consisting of an amino acid sequence represented by SEQ ID NO: 4, and protein (2b) is a protein comprising an amino acid sequence represented by SEQ ID NO: 5; (B ′) In the above (b ′), at least one protein consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added to the protein represented by the corresponding SEQ ID NO. And a protein having hydrazine synthesizing activity by the constitution of hydrazine synthase (II); (ha ′) the above (ii ′) Thus, at least one protein can also be achieved as a “protein consisting of an amino acid sequence having 55% or more identity with the sequence number of the protein represented by the corresponding sequence number”. The present invention can also be achieved as a hydrazine synthase (II ′) for synthesizing hydrazine, which is a heterodimeric heme protein derived from Anammox bacteria.

[Enzyme reaction conditions]
The present invention is a hydrazine synthesis method for synthesizing hydrazine from a substrate (A) and a substrate (B) using the aforementioned specific enzyme catalyst. This synthesis method can be generally performed in a state in which a substrate and an enzyme are dissolved in an aqueous solution.

When carrying out the synthesis method of the present invention, the substrate is generally used in a state dissolved in an aqueous solution. In the aqueous solution, the substrate (A) is an ammonium ion (NH ₄ ⁺ ) and the substrate (B). It is dissolved as hydroxylamine (NH ₂ O) obtained by conversion or the like if necessary.

  The concentration of these substrates in the aqueous solution is not particularly limited as long as the reaction proceeds, and is, for example, about 50 to 500 μmol / L as ammonium ions and about 50 to 1000 μmol / L as hydroxylamine. When the concentration is too low, the reaction does not proceed easily, and the hydrazine synthesis activity decreases. On the other hand, even if the concentration is higher than a certain concentration, the reaction rate may not be improved.

  In the hydrazine synthesis method of the present invention, hydrazine synthase (I) or (I ′) and hydrazine synthase (II) or (II ′) are present (dissolved) in the aqueous solution in which the reaction is performed. To be done. The concentrations of these enzymes are preferably 0.1 to 5 μmol / L and 1 to 50 μmol / L in the reaction solution, respectively. If the enzyme concentration is too low, a sufficient synthesis rate cannot be obtained, and if the concentration is too high, the synthesis rate may not increase.

  The pH of the solution for carrying out the method of the present invention is not limited as long as the reaction proceeds, but it is preferably 6 to 9, more preferably 6.5 to 8.5 as the optimum pH at which the enzyme of the present invention reacts suitably. It is preferably near neutral.

  The temperature at which the method of the present invention is performed may be any number of times as long as the enzyme reaction proceeds without causing protein denaturation or the like, but is preferably performed at 20 ° C to 50 ° C, more preferably 25 ° C to 40 ° C. .

  In order to stabilize the pH and allow the reaction of the present invention to proceed stably, a buffer solution that does not inhibit the enzyme reaction can be used. As the buffer, a MOPS buffer, a Tris hydrochloric acid buffer, a phosphate buffer, or the like is preferably used.

[Evaluation Method] Detection of Hydrazine Hydrazine produced by the present invention can be detected by a known hydrazine detection method. For example, a detection method by an optical method using the fact that hydrazine and DMBA undergo a diazo coupling reaction and the product exhibits a characteristic absorbance at a wavelength of 470 nm, which will be described later in Examples, may be mentioned.

  When the method for synthesizing hydrazine by the enzymatic reaction of the present invention is carried out in an aqueous solution, the produced hydrazine in the aqueous solution is distilled, separated by a separation method using an inclusion complex of hydrazines (see JP-A-1-192711), etc. By concentrating, hydrazine having high purity can be obtained.

  Hydrazine obtained by the production method of the present invention includes a fuel propellant and the like, a fuel cell (hydrogen substitute material or hydrogen storage material), a foaming agent at the time of plastic molding, an air bag initiator, an oxygen scavenger, It can also be used as an anticorrosive.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to a following example, unless the summary is changed.

[Evaluation item]
The measuring operation of N ₂ H ₄ concentration, SDS-PAGE, and evaluation method of tricine SDS-PAGE are shown below.

1. Measurement operation of N ₂ H ₄ concentration (measurement of generated N ₂ H ₄ )
<Hydrazine concentration determination reagent>
・ 4% dimethylaminobenzaldehyde solution (DMBA solution)
0.4 g of p-Dimethylaminobenzaldehyde (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 10 mL of 99% ethanol to obtain a DMBA solution having a concentration of 4% by weight.
-1 mol / L sodium carbonate solution Sodium carbonate (manufactured by Dojin Chemical Laboratory Co., Ltd.) was dissolved in pure water and adjusted so as to have a concentration of 1 mol / L.
Enzyme reaction terminator A mixed solution of 20% (w / v) trichloroacetic acid (manufactured by Wako Pure Chemical Industries, Ltd.) and 2 mmol / L EDTA · 2Na (manufactured by Dojin Chemical Laboratory) was used as an enzyme reaction terminator. .
<Operation method>
(1) The reaction was stopped by denaturing the protein by adding 200 μL of trichloroacetic acid to 300 μL of the reaction-terminated solution. Thereafter, the denatured protein was removed by centrifugation (14000 rpm, 10 min, 4 ° C.).
(2) 200 μL was taken from the supernatant of (1) and transferred to another eppen.
(3) 1 mL of 4% DMBA solution was added to (2) and mixed. (By adding DMBA to the reaction solution, a diazo coupling reaction with N ₂ H _{4 is performed,} and the absorbance A ₄₇₀ (absorbance at a wavelength of 470 nm) of the coupling product is measured to obtain the N ₂ H ₄ concentration. Can do.)
(4) 20min incubation at room temperature, was measured A _470.

2. SDS-PAGE
The molecular mass of enzyme (I) having a large apparent molecular mass was measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

3. Tricine SDS-PAGE
The molecular mass of enzyme (II) with a small apparent molecular mass was measured by tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

[Matters related to strains]
“Anamox Asahi BRW” (hereinafter, sometimes simply abbreviated as “BRW”)
Anammox Asahi BRW, which is an Anammox bacterium, has been acclimatized in the School of Biology and Life Sciences, Kimigatsu Gakuen Sojo University, and this Anammox sludge (BRW sludge) was prepared and used.
“Anamox KSU-1” (hereinafter, sometimes simply abbreviated as “KSU-1”)
In addition, Anammox KSU-1, which is an Anammox bacterium, is acclimatized in the Faculty of Biological Life Sciences of the school corporation Kimigasu Gakuen Sojo University, and this Anamox sludge (KSU-1 sludge) was prepared and used.

[Purification of hydrazine synthase (I ′), (II ′)]
A method for purifying hydrazine synthases (I ′) and (II ′) of BRW strain and KSU-1 strain is shown below.
"Reagents and equipment"
<Anamox sludge>
・ BRW sludge ・ KSU-1 sludge

<Hydrophobic column chromatography>
・ TOYOPEAL Butyl-650M

<Gel filtration column chromatography>
・ Superdex 200pg
・ Superdex 75pg

<Purification buffer>
Cell-free extract preparation buffer 100 mmol / L Tris-HCl (pH 8.0), 20% (w / v) glycerin, 1 mmol / LPMSF, and 1 mmol / L EDTA were mixed to prepare.
-TOYOPEAL Butyl equilibration buffer 50mmol / L Tris-HCl (pH 8.0), 10% (w / v) glycerol, and 40% saturated ammonium sulfate were mixed and prepared.
-TOYOPEAL Butyl effluent buffer 50 mmol / L Tris-HCl (pH 8.0) and 10% (w / v) glycerin were mixed and prepared.
Gel filtration buffer Prepared by mixing 20 mmol / L Tris-HCl (pH 8.0) and 200 mmol / L NaCl.

<Sonication>
Astrason ULTRASONIC PROCESSOR

<Concentration>
Centrifugal Filter YM-50

<Absorbance measurement>
Spectrophotometer MPS-2400 (manufactured by SHIMADZU)

<Centrifuge>
Centrifuge Avanti HP-25 (manufactured by BECKMAN)

"Example 1"
Purification step 1 [Preparation of cell-free extract]
BRW sludge was put into a beaker as Anammox sludge, Cell-free extract preparation buffer was added, and the cell membrane of Anammox was disrupted by ultrasonic waves. Furthermore, the cell membrane was destroyed by grinding with a mortar and sonication. Thereafter, the Anammox sludge whose cell membrane was destroyed was centrifuged (4 ° C., 12000 rpm, 60 min, JA14) to obtain a cell-free extract (cell free extract: supernatant after centrifugation).

Purification step 2 [Salting out with ammonium sulfate]
Ammonium sulfate was added to the cell-free extract for salting out. After mixing with the cell-free extract, it was added so that the ammonium sulfate concentration became a 40% saturated solution, and after addition, the mixture was stirred with a stirrer for 30 minutes. After stirring, centrifugation (4 ° C., 12000 rpm, 30 min, JA14) was performed, and the supernatant was used for purification by hydrophobic column chromatography as the next step.

Purification step 3 [Purification by hydrophobic column chromatography]
Washed TOYOPEARL Butyl-650M was packed in an open column, and equilibrated with 50 mmol / L Tris-HCl (pH 8.0), 40% saturated ammonium sulfate, and 10% glycerin. Cell-free extract was purified using this column. For washing, 50 mmol / L Tris-HCl (pH 8.0), 20% glycerin, and 40% saturated ammonium sulfate were used. Linear gradient elution was performed using 50 mmol / L Tris-HCl (pH 8.0), 40% saturated ammonium sulfate, 50 mmol / L Tris-HCl (pH 8.0), and 10% glycerin. Monitoring was performed while measuring the spectrum of each fraction with a spectrophotometer.

(Purified liquid properties: hydrophobic column chromatography)
The ammonium sulfate supernatant was gradientized by hydrophobic column chromatography, the spectrum of each fraction was measured, and the results of a graph of the absorbance of heme (A ₄₀₈ ) and the absorbance of protein (A ₂₈₀ ) are shown in FIG.

  As shown in FIG. 2, when the BRW sludge was fractionated by hydrophobic column chromatography, Fraction No. Major peaks appeared at 52, 55, 64 and 72. The spectra of these peaks were measured respectively.

  When the spectrum of the peaks (fraction Nos. 52, 55, 64, 72) by hydrophobic column chromatography was measured, No. 2 in FIG. 52 (indicated by the arrow “ii”) is the hydrazine synthase (II ′) and No. 5 in FIG. The spectrum of the second peak of 64 (indicated by an arrow “i”) corresponds to hydrazine synthase (I ′).

Purification step 4 [Purification by gel filtration column chromatography (1)]
Each peak and its periphery (No. 51 to 55, No. 63 to 66) were separately subjected to gel filtration column chromatography (flow rate 1 ml / min), and eluted hydrazine synthase (II ′) and hydrazine synthase (I ') Peaks were collected separately and concentrated using a Centrifugal Filter YM-50.

Purification step 5 [Purification by gel filtration column chromatography (2)]
After the purification step 4, purification was further performed by gel filtration column chromatography. Then, storing the place A ₄₁₉ (absorbance at a wavelength of 419 nm) is high, and concentrated using a Centrifugal Filter YM-50 again.
As a result, a purified liquid (I′-1) of hydrazine synthase (I ′) and a purified liquid (II′-1) of hydrazine synthase (II ′) were obtained.

"Example 2"
In Example 1, except that KSU-1 sludge was used instead of BRW sludge as Anammox sludge, a purified solution (I ′) of KSU-1 hydrazine synthase (I ′) according to the purification steps 1 to 5 described above. -2) and a purified solution (II'-2) of hydrazine synthase (II '). In Example 2, since the purification step 3 was performed at a flow rate slower than that in Example 1 (purification at a low speed), the peak of the fraction obtained by this purification was as shown in FIG. Therefore, the fractions in the purification steps 4 and 5 correspond to the first peak, and the fraction containing hydrazine synthase (II ′) (indicated by “ii” in FIG. 3) is No. 185-194, the fraction corresponding to the second peak and containing hydrazine synthase (I ′) was No. The purification step was performed as 251 to 271 (indicated by “i” in FIG. 3).

[Properties of hydrazine synthase (I ')]
<Spectroscopic properties>
The peak at 408 nm in the oxidized form (shown by a solid line in FIG. 5) shifted to 419 nm in the reduced form (shown by a dotted line in FIG. 5). In the reduced form, peaks appeared in the α band at 523 nm and the β band at 553 nm, respectively.

<Properties understood from SDS-PAGE>
The purified liquids (I′-1) and (I′-2) were measured for molecular mass by SDS-PAGE, and both KSU-1 and BRW-derived purified liquids had bands at 94 kDa, 43 kDa, and 37 kDa. Appeared and confirmed to be a heterotrimer. (A measurement result is shown in FIG. 7.)

[Properties of hydrazine synthase (II ′)]
<Spectroscopic properties>
The oxidized spectrum of the purified liquid (II′-1) and the reduced spectrum reduced with dithionite were measured. The result is shown in FIG. In the oxidized state (shown by a solid line in FIG. 6), the Sole absorption band was at 420 nm, and in the present invention, a peak around 350 nm characteristic of NaxLS, which is hydrazine synthase (II ′), appeared. When dithionite was added as a reducing agent (indicated by a dotted line in FIG. 6), peaks of α and β bands peculiar to the reduced form of heme protein did not appear around 550 nm. This was difficult to reduce and was a characteristic phenomenon of NaxLS.

<Properties understood by Tricine SDS-PAGE>
The purified liquids (II′-1) and (II′-2) were measured for molecular mass by Tricine SDS-PAGE. As a result, both KSU-1 and BRW-derived purified liquids had bands at positions of 14 kDa and 11 kDa. It was observed. (A measurement result is shown in FIG. 8.)

<Measurement of enzyme concentration in purified solution>
The protein concentration in the purified solution was measured as a weight concentration with Pierce BCA Protein Assay Kit (manufactured by Thermo Scientific), and the value was divided by the molecular mass determined from the amino acid sequence of the enzyme to calculate the molar concentration of the enzyme.

[Test for measuring activity of hydrazine synthase]
<Purified solution containing enzyme>
・ Purified liquid (I'-1)
BRW-derived enzyme-containing purified solution obtained from Example 2 (96.2 μmol / L)
・ Purified liquid (II'-1)
BRW-derived enzyme-containing purified solution obtained from Example 2 (432 μmol / L)
・ Purified liquid (I'-2)
KSU-1-derived enzyme-containing purified solution obtained from Example 1 (106 μmol / L)
・ Purified liquid (II'-2)
KSU-1 derived enzyme-containing purified solution obtained from Example 1 (600 μmol / L)

<Substrate>
・ 100mmol / L hydroxylamine solution ・ 100mmol / L ammonium chloride solution

<Preparation of solution system>
-1 mol / L glucose and oxygen consumer agent It adjusted so that the density | concentration in a solution might be a mixed solution which is 10 micromol / L glucose oxidase and 150 micromol / L catalase. By adding together with glucose, the oxygen in the solution is consumed and the reaction is carried out in an anaerobic state.
・ 50 mmol / L MOPS (pH 7.4)
MOPS buffer (manufactured by Dojin Chemical Laboratory)

"Example 3, Comparative Examples 1-3"
[Hydrazine synthesis reaction]
<Reaction solution composition>
As a pH buffer, MOPS buffer that does not affect the hydrazine quantification method was used. Hydroxylamine hydrochloride and ammonium chloride were used as substrates. As the hydrazine synthase, two types of enzyme purified solutions, the purified solution (I′-1) and the purified solution (II′-1), were used. Table 2 shows the composition of the solution used in the test, and the adjustment method will be described later.

<Reaction procedure>
(1) MOPS buffer (50 mmol / L, pH 7.4) was put in a vial bottle in advance and degassed by blowing argon gas for 30 minutes or more.
(2) 900 μL of MOPS buffer (50 mmol / L, pH 7.4) degassed to Eppen according to Table 2, 10 μL of oxygen consumer (glucose oxidase 10 μmol / L, catalase 150 μmol / L), purified solution (II′-2) ) (432 μmol / L) and 10 μL of purified solution (I′-1) (96.2 μmol / L) were added.
In addition, what extracted the refinement | purification liquid (II'-1) as a blank, what extracted the refinement | purification liquid (I'-1), and what extracted both the refinement | purification liquid (I'-1) (II'-1) Were prepared, and Milli-Q water was added so that the total amount of the reaction solution was 1000 μL.
(3) 10 μL each of 100 mmol / L NH ₂ OH solution and NH ₄ Cl solution were placed on the back of the eppen lid.
(4) In order to make the inside of a solution anaerobic, 50 microliters of glucose was added to each.
(5) The lid of the Eppen was closed, and after 3 minutes, the mixture was mixed by inversion and reacted at 35 ° C. for 1 hour.
The reaction solution obtained after the reaction according to (1) to (5) was used as the reaction completion solution.
(6) After completion of the reaction, the N ₂ H ₄ concentration was measured.

[Hydrazine purification concentration]
Table 3 shows the amount of hydrazine produced, which is the test result of the hydrazine synthesis reaction.

As a result of the synthesis reaction, hydrazine was not synthesized at all in Comparative Example 3 in which neither hydrazine synthase (II ′) nor hydrazine synthase (I ′) was added. In addition, hydrazine was not produced at all in Comparative Example 1, which was only hydrazine synthase (I ′). In Comparative Example 2 in which only hydrazine synthase (II ′) was added as an enzyme, it was synthesized slightly, but there was no significant difference from Comparative Example 3, and the range of variations in measured values was within the range. Unlike the other reaction solutions, hydrazine was clearly produced in the reaction solution to which both hydrazine synthase (I ′) and hydrazine synthase (II ′) were added.
Since 16 μmol / L hydrazine was produced in 1 hour, its activity value was (hydrazine production amount) / (enzyme (I ′) concentration) · reaction time = 16.0 / 0.962 · 1 = 16.6 / From the hour, 16.6 / 60 min = 0.28 / min.

Example 4
In Example 3 described above, the purified liquid (I′-2) was used instead of the purified liquid (I′-1), and the purified liquid (II′-2) was used instead of the purified liquid (II′-1). Conducted an experiment with an enzyme derived from the KSU-1 strain in the same manner as Example 4.
In the test results using the KSU-1 strain, the activity value was 0.71 / min.

"Examples 5-7"
[NH ₂ OH concentration dependence]
An experiment was conducted to examine whether a change in the amount of hydrazine produced occurred by changing the concentration of hydroxylamine (NH ₂ OH) as a substrate. The experiment was performed according to Example 3, and only the concentration of hydroxylamine was adjusted to the concentration shown in Table 4.

<Changes in hydrazine production due to changes in hydroxylamine concentration>
When the hydroxylamine addition amount was 1000 μmol / L, the amount of hydrazine produced was 16 μmol / L. In 500 μmol / L of Example 5 in which the amount of hydroxylamine added was halved, 28 μmol / L, which is 2.5 times, was generated. At 200 μmol / L of Example 6 in which the amount of hydroxylamine added was 1/5, 43 μmol / L hydrazine, which was about 4 times, was produced. At 100 μmol / L of Example 7 in which the amount of hydroxylamine added was 1/10, 31 μmol / L hydrazine, approximately three times as large, was produced. The most hydrazine was produced when hydroxylamine was added at 200 μmol / L (Example 6). In the case of 43 μmol / L, (hydrazine production amount) / {(HZS concentration) · reaction time) = 43. 0 / 0.962 · 1 = 44.7 / hour, and thus 44.7 / 60 min = 0.74 / min.
As described above, since the data of the KSU-1 strain is 0.71 / min, it was confirmed that the same activity value was obtained by adjusting the substrate concentration.

[Hydrazine synthase activity measurement test (reducing agent added system)]
In the hydrazine synthesis method of the present invention, a test method in which hydrazine synthesis activity is improved in the presence of a reducing agent by further mixing a reducing agent and the results are shown below.

<Purified solution containing enzyme>
・ Purified liquid (I'-3)
KSU-1 derived enzyme-containing purified solution (95 μmol / L) obtained by diluting the purified solution (I′-2) obtained from Example 2 with milli-Q water (ultra pure water)
・ Purified liquid (II'-3)
Escherichia coli by the plasmid (SEQ ID NOS: 14 and 15) corresponding to the so-called “NaxLS”, which is the protein (2a) or (2b) of the present invention, and another plasmid connected with the c-type heme protein maturation gene group. Competent cells prepared by the Hanahan method (E. coli BL21 (DE3) strain) were co-transformed to produce genetically modified E. coli that synthesizes NaxLS. An enzyme-containing purified solution (concentration: 1.32 mmol / L) of the enzyme corresponding to NaxLS was obtained from this genetically modified Escherichia coli according to the method for obtaining a purified solution (II′-2). This enzyme-containing purified solution was used as a purified solution (II′-3).

<Substrate>
-2 mmol / L NO saturated aqueous solution Prepared by dissolving sodium hydroxide (manufactured by Wako Pure Chemical Industries, Ltd.) to a concentration of 1 mol / L using milli-Q water after degassing by bubbling argon gas A saturated NO gas aqueous solution was prepared by aeration of NO gas into the prepared aqueous solution. The NO concentration in this saturated aqueous solution is about 2 mmol / L.

<Reducing agent>
-100 mmol / L dithionite solution Sodium dithionite (abbreviated as "dithionite": manufactured by Wako Pure Chemical Industries, Ltd.) was diluted with milli-Q water to prepare a dithionite solution having a dithionite concentration of 100 mmol / L. This was used as a reducing agent.

"Examples 8 to 11"
[Hydrazine synthesis reaction]
<Reaction solution composition>
As a pH buffer, MOPS buffer that does not affect the hydrazine quantification method was used. As the substrate, NO (adjusted to a saturated aqueous solution) and ammonium chloride were used. As the hydrazine synthase, two types of enzyme purified solutions, the purified solution (I′-3) and the purified solution (II′-3), were used. Furthermore, the amount of hydrazine synthesized when a dithionite solution was used as a reducing agent was evaluated. Table 5 below shows the composition of the hydrazine synthesis reaction solution used in the tests of Examples 8 to 11.

<Reaction procedure>
(1) MOPS buffer (50 mmol / L, pH 7.4) was put in a vial bottle in advance and degassed by blowing argon gas for 30 minutes or more.
(2) Each reagent, solution, etc. were mixed according to Table 5 below. First, MOPS buffer (50 mmol / L, pH 7.4) degassed in an eppen, oxygen consumer (glucose oxidase 10 μmol / L, catalase 150 μmol / L), purified solution (II′-3), purified solution (I′− 3) The dithionite solution was added respectively.
(3) A NO saturated solution and an NH ₄ Cl solution were placed on the back of the eppen lid, respectively.
(4) A glucose solution was added to make the solution anaerobic.
(5) The lid of the Eppen was closed, mixed by inversion and reacted at 35 ° C. for 10 minutes.
The reaction solution obtained after the reaction according to (1) to (5) was used as the reaction completion solution.

[Measurement of hydrazine concentration when NO is used as a substrate]
After the hydrazine synthesis reaction, in order to reduce and remove NO in the reaction solution, the solution was diluted with a 100 mmol / L dithionite solution and milliQ water so that the dithionite concentration in the solution was 25 mmol / L. This is because NO may interfere with the absorbance measurement when measuring the hydrazine concentration. Table 6 shows the results of measuring the hydrazine concentration in each solution of Examples 8 to 11 after this NO removal.

  The hydrazine synthetic molecular activity of the enzyme (I ′) in the table is obtained from the formula of (hydrazine production amount) / {(HZS concentration) · reaction time)} as in Examples 3 to 7 described above. is there. As shown in these, Examples 8 to 11 using NO as a substrate (B) and using a reducing agent showed very excellent hydrazine synthesis activity.

[Confirmation test of hydrazine synthesis]
In order to confirm that hydrazine was obtained by the synthesis method using the enzyme of the present invention, the following synthesis test was performed using isotopically labeled ammonium chloride.

As "Example 12" Substrate (B), except for using the isotope-labeled ammonium chloride ⁽¹⁵ NH ₄ Cl) is prepared analogously to Example 11, it was subjected to hydrazine synthesis reaction.
After the reaction, dithionite solution is mixed to reduce and remove NO, trichloroacetic acid is added and mixed, and centrifuged (15000 rpm, 4 ° C., 10 min) to hydrazine synthesis reaction using isotope-labeled ammonium chloride. A supernatant of the liquid was obtained.
1 mL of 1% DMBA was added to 200 μL of the supernatant, and a color reaction was performed for 20 minutes. After the color development reaction, a color reaction solution and an equivalent amount of CHCA solution were mixed, dried on a plate for TOF-MS, and subjected to mass spectrometry using MALDI TOF-MS. When performing mass spectrometry with MALDI TOF-MS, hydrazine is detected as a compound with DMBA. This compound with DMBA appears as 296 MW of ²⁹ N ₂ H ₄ -DMBA compound when isotope-labelled ammonium chloride is used and hydrazine is synthesized. On the other hand, ²⁸ N ₂ H ₄ -DMBA compound using ordinary hydrazine not labeled with isotope appears as 295 MW.

The results of mass spectrometry are shown in FIG. As a blank, hydrazine ²⁸ N ₂ H ₄ manufactured by Wako Pure Chemical Industries, Ltd. was used. Compared with m / z of ²⁸ N ₂ H ₄ , hydrazine synthesized using the HZS enzyme and NaxLS enzyme corresponding to the enzyme of the present invention had an increased ratio of 296 MW. This shows that it is ²⁹ N ₂ H ₄ using ammonium chloride labeled with isotope as a raw material.

  The present invention relates to a method for synthesizing hydrazine using an enzyme catalyst. Since the synthesis by the enzyme catalyst of the present invention does not require conventional high-temperature treatment, it is useful as a novel hydrazine synthesis method.

Claims (8)

Using an isolated enzyme catalyst, hydrazine is synthesized from an ammonium salt substrate (A) and at least one substrate (B) selected from the group consisting of hydroxylamine, nitrate, nitrite and nitric oxide. A hydrazine synthesis method to be recovered ,
The subunit protein constituting the enzyme catalyst is at least one protein of (a) or (b) below,
The enzyme catalyst is hydrazine synthase (I) which is a heterotrimeric heme protein of the following protein (1a), protein (1b) and protein (1c);
A hydrazine synthesizing method comprising using hydrazine synthase (II) which is a heterodimeric heme protein with the following protein (2a) and protein (2b).
(A) Protein (1a) is represented by SEQ ID NO: 1, protein (1b) is SEQ ID NO: 2, protein (1c) is SEQ ID NO: 3, protein (2a) is SEQ ID NO: 4, and protein (2b) is SEQ ID NO: 5, respectively. (B) In the above (a), one or several amino acids are deleted, substituted or added to the protein represented by the corresponding SEQ ID NO. and to the amino acid sequence, and hydrazine synthase (I), proteins having a hydrazine synthesis activity by the configuration of (II) Recombinant hydrazine synthase (I) and recombinant hydrazine synthase (II), wherein the enzyme catalyst is a protein expressed from a gene encoding a subunit protein constituting at least one enzyme catalyst of (a) or (b) above The method for synthesizing hydrazine according to claim 1. Using enzyme catalysis, the substrate (A) consisting of ammonium salts, hydroxylamine, nitrate, and synthesizing at least one hydrazine from a substrate (B) is a substrate selected from the group consisting of nitrite and nitric oxide A method for synthesizing hydrazine to be recovered , comprising:
As the enzyme catalyst, hydrazine synthase (I ′) which is a heterotrimeric heme protein derived from Anammox,
Using hydrazine synthase (II ′), which is a heterodimeric heme protein derived from Anammox ,
The enzyme (I ′) and the enzyme (II ′) crush the Anammox cells, then centrifuge, salt the supernatant, centrifuge, and purify the resulting supernatant by hydrophobic column chromatography And
When the enzymes (I ′) and (II ′) are purified by the hydrophobic column chromatography, the first peak of heme protein is confirmed by monitoring the absorbance at a wavelength of 280 nm and the absorbance at a wavelength of 408 nm. And an enzyme obtained from the second peak fraction,
The molecular mass of the enzyme (I ′) measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis is 91.5-96.5 kDa, 40.5-45.5 kDa, 34.5-39.5 kDa, (II ′) was measured by tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis, the molecular mass was 11-17 kDa, 8-14 kDa,
When the enzyme (I ′) is in a reduced form, it has an absorption maximum in the α band, β band, and Sole band characteristic of c-type heme protein, and
A method for synthesizing hydrazine, characterized in that the enzyme (II ′) has a non-reducing property in which a peak at a wavelength of 419 nm in the absorption spectrum is observed before and after the addition of dithionite . As buffer, a pH 7.0-8.0, Tris-HCl buffer, hydrazine according to any one of claims 1 to 3, phosphate buffer, or by using a MOPS buffer for synthesizing hydrazine synthesis Method. The hydrazine synthesis method according to any one of claims 1 to 4 , wherein the substrate (B) is hydroxylamine. The hydrazine synthesis method according to any one of claims 1 to 5 , wherein the hydrazine synthesis method is performed in the presence of a reducing agent. The subunit protein constituting the enzyme catalyst is at least one protein of (a) or (b) below ,
The enzyme catalyst is hydrazine synthase (I) which is a heterotrimeric heme protein of the following protein (1a), protein (1b) and protein (1c);
The enzyme composition for hydrazine synthesis | combination containing the enzyme catalyst which is hydrazine synthetase (II) which is a hetero dimer heme protein with the following protein (2a) and protein (2b) .
(A) Protein (1a) is represented by SEQ ID NO: 1, protein (1b) is SEQ ID NO: 2, protein (1c) is SEQ ID NO: 3, protein (2a) is SEQ ID NO: 4, and protein (2b) is SEQ ID NO: 5, respectively. Protein consisting of amino acid sequence
(B) In (a) above, at least one protein comprises an amino acid sequence in which one or several amino acids are deleted, substituted or added to the protein represented by the corresponding SEQ ID NO. Protein having hydrazine synthesizing activity by the constitution of hydrazine synthase (I), (II) As an enzyme catalyst, hydrazine synthase (I ′), which is a heterotrimeric heme protein derived from Anammox,
Containing the Anammox bacteria-derived heterodimeric heme proteins der Ruhi hydrazine synthase (II'),
The molecular mass of the enzyme (I ′) measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis is 91.5-96.5 kDa, 40.5-45.5 kDa, 34.5-39.5 kDa, (II ′) was measured by tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis, the molecular mass was 11-17 kDa, 8-14 kDa,
When the enzyme (I ′) is in a reduced form, it has an absorption maximum in the α band, β band, and Sole band characteristic of c-type heme protein, and
An enzyme composition for synthesizing hydrazine, wherein the enzyme (II ′) contains an enzyme catalyst having a peak of a wavelength of 419 nm in an absorption spectrum which is hardly reduced before and after the addition of dithionite.