JP7257662B2 - Compounds, methods for producing compounds, and uses thereof - Google Patents

Description

The present invention relates to compounds, methods for producing compounds, and uses thereof.

In vivo, it is known that there are various groups of compounds having a tandem structure in which organic groups of the same type (e.g., aminopropyl groups) are continuously bonded (e.g., Non-Patent Document 1 ~5, 8 and 10). In recent years, there have also been reports on the chemical properties of various compound groups having a tandem structure in which organic groups of the same type are continuously bonded, and methods for synthesizing the compound groups (e.g., non-patent literature 6, 7 and 9).

Recent studies have revealed that these compounds have various activities. For example, Non-Patent Document 9 shows that such compounds are cytotoxic to prostate cancer cells.

Kroger et al., Proc. Natl. Acad. Sci. USA, Vol.97, No.26, 2000, p14133-14138 Matsunaga et al., ChemBioChem, 8, 2007, p1729-1735 Bridoux et al., Rapid Commun. Mass Spectrom, 25, 2011, p877-888 Bridoux et al., Organic Geochemistry, 47, 2012, p9-21 Bridoux et al., Organic Geochemistry, 48, 2012, p9-20 Menzel et al., Chem. Commun., 2003, p2994-2995 Annenkov et al., ARKIVOC, 2009, xiii, p116-130 Knott, FEBS Letters, 583, 209, p3519-3524 Valasinas et al., Bioorganic and Medicinal Chemistry, 11, 2003, p4121-4131 Oshima et al., Methods and Protocols, Methods in Molecular Biology, Vol.720, 2011, p81-111

Since the organic groups attached to the ends of the tandem structure and/or the length of the tandem structure differ depending on the species, each species has its own biosynthetic pathway to form its own compounds. believed to have. Finding an unknown biosynthetic pathway is expected to lead to the development of new technologies such as compounds with new structures and new methods for producing compounds.

An object of one aspect of the present invention is to discover new genes involved in the biosynthesis of compounds having a tandem structure, and to provide new techniques for compounds having a tandem structure based on the findings.

The method for producing a compound of the present invention includes (i) a culturing step of culturing a bacterium of the genus Bacillus, or a transformant into which the BC5213 gene of a bacterium of the genus Bacillus or a homologous gene thereof has been introduced, or ( ii) A method for producing a compound represented by the following formula (I), comprising a reaction step of contacting a BC5213 protein of a bacterium belonging to the genus Bacillus or a homologous protein thereof with a substrate.

In the above formula (I), R is hydrogen or any organic group, and n is an integer of 2 or more.

In the method for producing a compound of the present invention, in the above formula (I), R is H ₂ N-(CH ₂ ) ₄ -, H ₂ N-(CH ₂ ) ₃ -, NH ₁₀ C ₅ -, or It may be an organic group represented by NH ₁₀ C ₆ —.

In the method for producing a compound of the present invention, the transformant may be one into which the BC5214 gene of a bacterium belonging to the genus Bacillus or a homologous gene thereof has been introduced.

In the method for producing a compound of the present invention, the reaction step includes: and may be in contact with each other.

The method for adding an aminopropyl group of the present invention includes (i) a culturing step of culturing a bacterium of the genus Bacillus, or a transformant into which a BC5213 gene of a bacterium of the genus Bacillus or a homologous gene thereof has been introduced, or , (ii) a reaction step of contacting the BC5213 protein of a bacterium belonging to the genus Bacillus or its homologue protein with a substrate.

In the method for adding an aminopropyl group of the present invention, the transformant may be one into which the BC5214 gene of a bacterium belonging to the genus Bacillus or its homologue gene has been introduced.

In the method for adding an aminopropyl group of the present invention, in the reaction step, the BC5213 protein of the bacterium belonging to the genus Bacillus, or a homologous protein thereof, the substrate, the BC5214 protein of the bacterium belonging to the genus Bacillus, or its may be brought into contact with homologous proteins.

The transformant of the present invention has introduced therein the BC5213 gene of Bacillus bacterium or its homologue gene.

The transformant of the present invention may further contain the BC5214 gene of Bacillus bacterium or its homologue gene.

The silica polymerization agent of the present invention contains a compound represented by the following formula (I) as an active ingredient.

In the above formula (I), R is hydrogen or any organic group, and n is an integer of 2 or more.

In the silica polymerizing agent of the present invention, in the above formula (I), R is H ₂ N-(CH ₂ ) ₄ -, H ₂ N-(CH ₂ ) ₃ -, NH ₁₀ C ₅ -, or NH ₁₀ It may be an organic group represented by C ₆ —.

The compound of the present invention is a compound represented by the following formula (I).

In the above formula (I), R is hydrogen or any organic group, and n is an integer of 16 or more.

In the compounds of the present invention, in the above formula (I), R is H ₂ N-(CH ₂ ) ₄ -, H ₂ N-(CH ₂ ) ₃ -, NH ₁₀ C ₅ -, or NH ₁₀ C ₆ It may be an organic group represented by -.

According to one aspect of the present invention, a new method for producing a compound having a tandem structure can be provided.

According to one aspect of the present invention, compounds with aminopropyl groups can be prepared.

According to one aspect of the present invention, a transformant that can be used to produce a compound having a tandem structure can be provided.

According to one aspect of the present invention, a new silica polymerization agent can be provided.

According to one aspect of the present invention, new compounds can be provided.

1 is a mass spectrum of long-chain polyamines derived from Bacillus cereus ATCC 14579 strain obtained by atmospheric pressure chemical ionization mass spectrometry. (a) is a mass spectrum of long-chain polyamines derived from Bacillus cereus strain YH64 obtained by atmospheric pressure chemical ionization mass spectrometry. (b) is a mass spectrum obtained by enlarging the peak of (a). Mass spectra of long-chain polyamines from Bacillus cereus ATCC 14579 strain obtained by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. (a) shows the chromatogram of the negative control. (b) is a chromatogram of polyamine-extracted fractions from E. coli into which the gene of interest has been introduced.

One embodiment of the present invention is described below, but the present invention is not limited thereto. The present invention is not limited to the configurations described below, and can be modified in various ways within the scope of the claims, and the technical means disclosed in different embodiments and examples. Embodiments and examples obtained by appropriate combinations are also included in the technical scope of the present invention. Also, all of the documents mentioned in this specification are incorporated herein by reference. In this specification, when "A to B" is described with respect to a numerical range, the description means "A or more and B or less".

The present inventors have discovered that Bacillus bacteria have unique biosynthetic pathways for synthesizing unique compounds. Specifically, the present inventor found that Bacillus bacterium produces a group of compounds having a tandem structure and has genes involved in the synthesis of the group of compounds. Based on this, we succeeded in providing a new technique for compounds having a tandem structure. The details of the present invention are described below.

[1. Compound of the present invention]
The compound of the present embodiment is a novel compound discovered by the present inventors, and can be used as an active ingredient of the silica polymerizing agent described below.

Compounds of this embodiment are compounds represented by the following formula (I):

(In formula (I) above, R is hydrogen or any organic group, and n is an integer of 16 or greater).

In the above formula (I), R is H ₂ N—(CH ₂ ) ₄ —, H ₂ N—(CH ₂ ) ₃ —, NH ₁₀ C ₅ —, or NH ₁₀ C ₆ —. may be a base.

In formula (I) above, the lower limit of n may be 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30. On the other hand, in the above formula (I), the upper limit of n may be 55, 60, 70, 80, 90 or 100.

[2. Method for producing compound]
The method for producing the compound of the present embodiment includes (i) a culturing step of culturing a bacterium of the genus Bacillus, or a transformant into which the BC5213 gene of a bacterium of the genus Bacillus or a homologous gene thereof has been introduced, or (ii) Bacillus and a reaction step of contacting the BC5213 protein of the genus bacterium or its homologue protein with a substrate.

The compound produced by the above production method is a compound represented by the following formula (I).

In the above formula (I), R is hydrogen or any organic group, and n is an integer of 2 or more. According to the above configuration, it is possible to provide a new method for producing a compound having a tandem structure (in other words, a structure in which aminopropyl groups are continuously bonded).

In the above formula (I), R is H ₂ N—(CH ₂ ) ₄ —, H ₂ N—(CH ₂ ) ₃ —, NH ₁₀ C ₅ —, or NH ₁₀ C ₆ —. may be a base.

In formula (I) above, the lower limit of n may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. On the other hand, in the above formula (I), the upper limit of n may be 55, 60, 70, 80, 90 or 100.

<A. Culture process>
In the culturing step in the method for producing the compound of the present embodiment, a Bacillus bacterium or a transformant into which the BC5213 gene of the Bacillus bacterium or its homologue gene has been introduced is cultured. The transformant may further have a Bacillus BC5214 gene or its homolog gene introduced therein.

The bacteria belonging to the genus Bacillus are not particularly limited as long as they are classified into the genus Bacillus. Specific examples of bacteria classified into the genus Bacillus include Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, and Bacillus megaterium.

Specific examples of the Bacillus cereus include Bacillus cereus ATCC 14579 strain, Bacillus cereus ATCC 10987 strain, Bacillus cereus YH64 strain, Bacillus cereus YH221 strain, and Bacillus cereus E33L.

Bacillus bacteria synthesize the compound represented by the above formula (I). Synthesis of the compound is thought to involve, for example, aminopropyltransferase contained in the body of bacteria belonging to the genus Bacillus. The BC5213 protein encoded by the BC5213 gene has high identity with aminopropyltransferase, and these proteins are considered to have aminopropyltransferase activity. Here, the above-mentioned "aminopropyl group transferase" intends an enzyme that transfers an aminopropyl group from an aminopropyl group donor (eg, decarboxylated S-adenosylmethionine) to a substrate. More specifically, the above-mentioned "aminopropyl group transferase" refers to an enzyme that transfers an aminopropyl group from an aminopropyl group donor (for example, decarboxylated S-adenosylmethionine) to an aminopropyl group of a substrate. Intend.

More specifically, the BC5213 gene or its homologous gene is (1) a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 3 or 5, or (2) from the nucleotide sequence represented by SEQ ID NO: 3 or 5 hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to a polynucleotide comprising aminopropyltransferase activity (or tandem structure-forming activity), or aminopropyltransferase-assisting activity (or a polynucleotide that encodes a protein having a tandem structure formation assisting activity).

The BC5213 protein encoded by the BC5213 gene does not lose its function even if the 2nd to 73rd amino acids counted from the amino terminus are deleted. A polynucleotide consisting of the base sequence represented by SEQ ID NO: 3 corresponds to a polynucleotide encoding the full-length BC5213 protein. On the other hand, the polynucleotide consisting of the base sequence represented by SEQ ID NO: 5 corresponds to the polynucleotide encoding the BC5213 protein lacking the 2nd to 73rd amino acids counted from the amino terminus.

More specifically, the BC5214 gene or its homologous gene is (3) a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 7, or (4) a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 7. It hybridizes with a polynucleotide consisting of a complementary base sequence under stringent conditions, and has aminopropyltransferase activity (or tandem structure-forming activity) or aminopropyltransferase-assisting activity (or tandem It may be a polynucleotide that encodes a protein having structure formation assisting activity).

As used herein, the term "stringent conditions" refers to conditions under which double-stranded polynucleotides specific to a so-called base sequence are formed and non-specific double-stranded polynucleotides are not formed. Say. In other words, conditions for hybridization between highly identical nucleic acids, for example, at a temperature in the range from the melting temperature (Tm value) of perfectly matched hybrids to a temperature 15°C, preferably 10°C, more preferably 5°C lower. It can also be said.

For example, to give an example, in a buffer solution consisting of 0.25 M Na ₂ HPO ₄ , pH 7.2, 7% SDS, 1 mM EDTA, 1× Denhardt's solution, the temperature is 60 to 68° C., preferably 65° C., more preferably 65° C. is hybridized at 68° C. for 16-24 hours, and further incubated in a buffer containing 20 mM Na ₂ HPO ₄ , pH 7.2, 1% SDS and 1 mM EDTA at a temperature of 60-68° C., preferably 65° C. More preferably, there is a condition in which washing is performed twice for 15 minutes at 68°C.

Other examples include 25% formamide, more stringent 50% formamide, 4x SSC (sodium chloride/sodium citrate), 50 mM Hepes pH 7.0, 10x Denhardt's solution, 20 μg/mL denatured salmon sperm DNA. After prehybridization is performed overnight at 42°C in a hybridization solution, a labeled probe is added and hybridization is performed by incubating at 42°C overnight. The washing solution and temperature conditions for subsequent washing are about "1 x SSC, 0.1% SDS, 37°C", and more severe conditions are about "0.5 x SSC, 0.1% SDS, 42°C". As a more severe condition, it can be carried out at about "0.2×SSC, 0.1% SDS, 65° C.". Thus, the stricter the washing conditions for hybridization, the higher the specificity of hybridization. However, the combinations of conditions of SSC, SDS and temperature described above are examples, and those skilled in the art will recognize the above or other factors that determine the stringency of hybridization (e.g., probe concentration, probe length, hybridization reaction, etc.). time, etc.), it is possible to achieve the same stringency as above. This is described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory (2001).

Whether or not the polynucleotide encodes a protein having aminopropyltransferase activity (or tandem structure-forming activity) can be determined by introducing the polynucleotide into a host (e.g., E. coli), It can be determined by confirming whether the amount of the compound having the propyl group transferred increases or whether a new compound having the aminopropyl group transferred is synthesized. When the amount of a compound to which an aminopropyl group has been transferred increases in the host and/or when a new compound to which an aminopropyl group has been transferred is synthesized, the polynucleotide has an aminopropyl group. It can be determined to be a polynucleotide encoding a protein having transferase activity (or tandem structure-forming activity).

Whether the polynucleotide encodes a protein having aminopropyltransferase-assisting activity (or tandem structure formation-assisting activity) depends on the polynucleotide and another polynucleotide (e.g., BC5213 gene or BC5214 gene). ) is introduced into a host (e.g., E. coli), and whether or not the amount of a compound to which an aminopropyl group has been transferred increases in the host, or whether a new compound to which an aminopropyl group has been transferred is synthesized. It can be determined by confirming whether or not When the amount of a compound to which an aminopropyl group has been transferred increases in the host and/or when a new compound to which an aminopropyl group has been transferred is synthesized, the polynucleotide has an aminopropyl group. It can be determined to be a polynucleotide encoding a protein having transferase assisting activity (or tandem structure formation assisting activity). The term "aminopropyl transferase-assisting activity (or tandem structure formation-assisting activity)" refers to, for example, regulating the function of the aminopropyl transferase by forming a complex with the aminopropyl transferase ( For example, activity that activates) is contemplated.

Examples of the host for the transformant cultured in the culture step include Escherichia coli, Bacillus subtilis, microalgae, and yeast, but the present invention is, of course, not limited to these hosts. Among these hosts, Bacillus subtilis and Escherichia coli are preferred because they have the advantage of rapid growth. A transformant used in the method for producing the compound of the present invention can be produced by introducing a small number of genes. Therefore, any organism (eg, microorganism) can be used as a host. As a method for introducing a gene into a host, a well-known method may be used.

The medium used in the culture step is not particularly limited as long as it is a medium in which the bacteria of the genus Bacillus or the transformant can grow.

<B. Reaction process>
In the reaction step in the method for producing the compound of the present embodiment, the BC5213 protein of Bacillus or its homologous protein is brought into contact with the substrate. In the reaction step, the Bacillus BC5213 protein or its homologous protein may be brought into contact with the substrate and the Bacillus BC5214 protein or its homologous protein. The contact between substances may be performed in vitro or in vivo.

More specifically, the BC5213 protein or its homologous protein is (5) a protein consisting of the amino acid sequence shown in SEQ ID NO: 4 or 6, or (6) 1 or 1 in the amino acid sequence shown in SEQ ID NO: 4 or 6 consists of an amino acid sequence in which several amino acids are deleted, substituted or added, and has aminopropyltransferase activity (or tandem structure-forming activity) or aminopropyltransferase-assisting activity (or tandem structure-forming activity) auxiliary activity).

The BC5213 protein does not lose its function even if the 2nd to 73rd amino acids counted from the amino terminus are deleted. A protein consisting of the amino acid sequence shown in SEQ ID NO: 4 corresponds to the full length of the BC5213 protein. On the other hand, the protein consisting of the amino acid sequence shown in SEQ ID NO: 6 corresponds to the BC5213 protein lacking the 2nd to 73rd amino acids counted from the amino terminus.

More specifically, the BC5214 protein or its homologous protein is (5) a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, or (6) one or several amino acids in the amino acid sequence shown in SEQ ID NO: 8 consists of a deleted, substituted or added amino acid sequence, and has aminopropyltransferase activity (or tandem structure-forming activity) or aminopropyltransferase-assisting activity (or tandem structure-forming assisting activity) It may be a protein having

In this embodiment, the position of deletion, substitution or addition in the amino acid sequence is not particularly limited. In addition, in the present embodiment, the number of amino acids intended by "one or several amino acids" is not particularly limited. , 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 amino acid.

In the reaction step in the method for producing a compound of the present embodiment, the BC5213 protein of Bacillus or its homologue protein is brought into contact with a substrate.

The substrate is not particularly limited, and is a specific compound, a mixture of specific compounds, a crushed product of a living organism (e.g., microorganisms (e.g., E. coli, Bacillus)), or an organism (e.g., microorganisms (e.g., E. coli, It may be a solution containing crushed Bacillus bacteria)). More specifically, the substrate may be a compound obtained by removing one or more aminopropyl groups from the compound of formula (I). More specifically, the substrate is a compound obtained by removing one or more aminopropyl groups from the compound of formula (I), wherein R is, for example, H ₂ N—(CH ₂ ) ₄ -, H ₂ N-(CH ₂ ) ₃ -, NH ₁₀ C ₅ -, or NH ₁₀ C ₆ -.

The above reaction step may be carried out, for example, in a solvent. The solvent is not particularly limited as long as it does not inactivate the BC5213 protein of the Bacillus bacterium or its homologue protein, the substrate, and the BC5214 protein or its homologue protein. Water can be mentioned as such a solvent.

[3. Transformant]
The BC5213 gene of Bacillus bacterium or its homologous gene is introduced into the transformant of this embodiment. In addition, the transformant of the present embodiment may further have the BC5214 gene of Bacillus spp. or its homologue gene introduced therein. The transformant can be used for producing a compound having a tandem structure.

Descriptions of the genes, hosts, etc. described in the above [Method for producing a compound] are omitted here.

When both (a) BC5213 gene or its homologous gene and (b) BC5214 gene or its homologous gene are introduced into the host, (a) BC5213 gene or its homologous gene and (b) BC5214 The gene or its homologous gene may be inserted into separate expression vectors and separately introduced into the host, and (a) the BC5213 gene or its homologous gene and (b) the BC5214 gene or its homologous The genes may be introduced into the host together in a single expression vector.

From the viewpoint of efficiently producing a desired compound by making the expression level of each gene approximately the same, (a) the BC5213 gene or its homologous gene, and (b) the BC5214 gene or its homologous gene, It is preferable to introduce them into a host together in a state of being inserted into one expression vector. Also, in this case, it is more preferable to control the expression of both genes by a common promoter.

The expression vector used to prepare the transformant is not limited, and a commercially available expression vector suitable for the host may be used. Transformants may be produced according to well-known methods depending on the host.

[4. Method for adding aminopropyl group]
The method for adding an aminopropyl group of the present embodiment includes (i) a culturing step of culturing a Bacillus bacterium, or a transformant into which a BC5213 gene of a Bacillus bacterium or a homologous gene thereof has been introduced, or ( ii) a reaction step of contacting the BC5213 protein of the bacterium belonging to the genus Bacillus or its homologue protein with a substrate; According to the above configuration, a compound to which an aminopropyl group is added can be produced.

In the aminopropyl group addition method of the present embodiment, the aminopropyl group is transferred from the aminopropyl group donor to the substrate, and the aminopropyl group is added to the substrate. More specifically, in the aminopropyl group addition method of the present embodiment, the aminopropyl group is transferred from the aminopropyl group donor to the substrate, and the aminopropyl group is added to the aminopropyl group of the substrate.

The method for adding an aminopropyl group of the present embodiment is realized by the culturing step and the reaction step described in the above [Method for producing a compound]. Therefore, descriptions of those already described are omitted here.

In the culture step, the Bacillus bacterium or the transformant into which the BC5213 gene of the Bacillus bacterium or its homologue gene has been introduced may be cultured using a substrate-containing medium.

The transformant may further have a Bacillus BC5214 gene or its homolog gene introduced therein.

In the reaction step, the Bacillus BC5213 protein or its homologous protein may be brought into contact with the substrate and the Bacillus BC5214 protein or its homologous protein.

The substrate is not particularly limited, and is a specific compound, a mixture of specific compounds, a crushed product of a living organism (e.g., microorganisms (e.g., E. coli, Bacillus)), or an organism (e.g., microorganisms (e.g., E. coli, It may be a solution containing crushed Bacillus bacteria)). More specifically, the substrate may be a compound obtained by removing one or more aminopropyl groups from the compound of formula (I). More specifically, the substrate is a compound obtained by removing one or more aminopropyl groups from the compound of formula (I), wherein R is, for example, H ₂ N—(CH ₂ ) ₄ -, H ₂ N-(CH ₂ ) ₃ -, NH ₁₀ C ₅ -, or a compound represented by NH ₁₀ C ₆ -.

[5. Silica polymerization agent]
The silica polymerization agent of the present embodiment contains a compound represented by the following formula (I) as an active ingredient:

In the above formula (I), R is hydrogen or any organic group, and n is an integer of 2 or more. According to the above configuration, a new silica polymerization agent can be provided.

In formula (I) above, R is an organic compound represented by H ₂ N-(CH ₂ ) ₄ -, H ₂ N-(CH ₂ ) ₃ -, NH ₁₀ C ₅ -, or NH ₁₀ C ₆ - may be a base.

In formula (I) above, the lower limit of n may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. On the other hand, in the above formula (I), the upper limit of n may be 55, 60, 70, 80, 90 or 100.

As used herein, the term "silica polymerizing agent" is intended to produce silica by polymerizing silicic acid (or promoting the polymerization of silicic acid). When silicic acid is polymerized, silica can be efficiently polymerized at normal temperature, normal pressure, or in water by the compound contained in the above formula (I). The method for producing silica using the silica polymerizing agent of the present embodiment does not require strict conditions such as high temperature, acidic conditions, and organic solvents, as compared with conventional methods for producing silica by chemical synthesis.

The amount of the active ingredient in the silica polymerizing agent of the present embodiment is not particularly limited. % by weight, 0.1% to 100% by weight, 0.1% to 95% by weight, 0.1% to 90% by weight, 0.1 wt % to 80 wt %, 0.1 wt % to 70 wt %, 0.1 wt % to 60 wt %, 0.1 wt % to 80 wt %. 1 wt% to 50 wt%, 0.1 wt% to 40 wt%, 0.1 wt% to 30 wt%, 0.1 wt% to 20 wt% % by weight, or between 0.1% and 10% by weight.

The silica polymerization agent of the present embodiment may contain components other than active ingredients (such as pharmaceutically acceptable carriers). Ingredients other than the active ingredient can include excipients, lubricants, binders, disintegrants, solvents, solubilizers, suspending agents, tonicity agents, buffers and soothing agents. Other ingredients other than active ingredients may also include preservatives, antioxidants and stabilizers.

Examples of the "excipient" include, but are not limited to, lactose, sucrose, D-mannitol, xylitol, sorbitol, erythritol, starch and crystalline cellulose.

Examples of the "lubricant" include, but are not limited to, magnesium stearate, calcium stearate, wax, talc and colloidal silica.

Examples of the "binder" include, but are not limited to, pregelatinized starch, methylcellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose and polyvinylpyrrolidone.

Examples of the above-mentioned "disintegrant" include, but are not limited to, starch, carboxymethylcellulose, low-substituted hydroxypropylcellulose, carboxymethylcellulose calcium, croscarmellose sodium and carboxymethylstarch sodium.

Examples of the "solvent" include, but are not limited to, water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil and tricaprylin.

Examples of the above-mentioned "solubilizing agent" include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate and sodium citrate. It is not limited to these.

Examples of the above-mentioned "suspending agent" include surfactants (e.g., stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate) and highly hydrophilic Molecules such as, but not limited to, polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose.

Examples of the above-mentioned "tonicity agent" include, but are not limited to, sodium chloride, glycerin and D-mannitol.

Examples of the "buffer" include, but are not limited to, phosphates, acetates, carbonates and citrates.

Examples of the above-mentioned "salting agent" include, but are not limited to, benzyl alcohol.

Examples of the above-mentioned "preservatives" include, but are not limited to, paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid and sorbic acid.

Examples of the above "antioxidant" include, but are not limited to, sulfites and ascorbic acid.

The above-mentioned "stabilizer" is not particularly limited as long as it is commonly used in the pharmaceutical field.

The amount of components other than the active ingredient in the silica polymerization agent of the present embodiment is not particularly limited, and may be, for example, 0% by weight to 99.999% by weight with respect to the silica polymerization agent, or 0% by weight. ~99.99 wt%, 0 wt% to 99.9 wt%, 5 wt% to 99.9 wt%, 10 wt% to 99.9 wt% %, may be 20% to 99.9% by weight, may be 30% to 99.9% by weight, and may be 40% to 99.9% by weight well, may be 50 wt% to 99.9 wt%, may be 60 wt% to 99.9 wt%, may be 70 wt% to 99.9 wt%, may be 80 wt% ~99.9% by weight, or 90% to 99.9% by weight.

An embodiment of the invention is described below.

<1. Extraction of long-chain polyamines from Bacillus cereus>
Bacillus cereus strain ATCC 14579 (obtained from ATCC) or Bacillus cereus strain YH64 (obtained from NBRC) was mixed with 0.6 mM CaCl ₂ , 0.03 mM MnCl ₂ , 0.05 mM ZnCl ₂ , 0.05 mM FeSO ₄ and 100 μg/ml. By culturing for 72 hours in R2A medium containing silicic acid (Si[OH] ₄ ), spores with silica (SiO ₂ ) accumulated on the surface were formed. The details of the composition of the R2A medium are described in Reference A (Reference A: Reasoner and Geldreich 1985 Appl. Environ. Microbiol. 49, 1).

After that, the spores were collected by centrifugation (2000 g, 20 minutes) and washed with pure water. After washing, an equal amount of concentrated nitric acid and four times the amount of concentrated sulfuric acid were added to the spore suspension placed in the test tube, and the spore suspension was heated with a gas burner in a fume hood to boil. After the temperature of the spore suspension was lowered to about room temperature, the spore suspension was subjected to ultracentrifugation (100,000 g, 15 minutes) to precipitate an acid-insoluble fraction.

The resulting precipitate was repeatedly washed with pure water until the pH became neutral. The washed precipitate was suspended in a 6 M ammonium fluoride aqueous solution and allowed to stand at room temperature for 30 minutes, thereby dissolving silica in the ammonium fluoride aqueous solution. The resulting solution was dialyzed overnight against a 200 mM ammonium acetate solution to remove ammonium fluoride from the solution. The dialyzed solution was freeze-dried to remove the solvent, and the remaining fraction was dissolved in ultrapure water. The solution obtained after dissolution was subjected to atmospheric pressure chemical ionization mass spectrometry (LTQ Orbitrap XL, manufactured by Thermo Fisher Scientific) or matrix-assisted laser desorption ionization time-of-flight mass spectrometry (device name: AXIMA-CFR plus, manufactured by Shimadzu Corporation). ) to identify the substances contained in the solution. As a result, it was confirmed that long-chain polyamine was present in the above solution.

The mass spectrum of the long-chain polyamine derived from the Bacillus cereus ATCC 14579 strain (corresponding to the above-mentioned "compound having a tandem structure") obtained by atmospheric pressure chemical ionization mass spectrometry is shown in FIG. A mass spectrum of the chain polyamine is shown in FIG. Also, FIG. 3 shows the mass spectrum of the long-chain polyamine derived from Bacillus cereus ATCC 14579 strain obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

FIG. 1 is a mass spectrum of long-chain polyamines derived from Bacillus cereus ATCC 14579 strain obtained by atmospheric pressure chemical ionization mass spectrometry. As shown in FIG. 1, multiple peaks were detected at m/z intervals of 57.058.各ピークのｍ／ｚの値は、545.57、602.63、659.69、716.74、773.80、830.86、887.92、944.98、1002.03、1059.09、116.15、1173.21、1230.27、1287.32、1344.38、1401.44、1458.50、1515.56、1572.61、1572.61、 1630.68, 1687.73 and 1743.79. From these peaks, the long chain polyamine from Bacillus cereus strain ATCC 14579 is H ₂ N—CH ₂ —CH ₂ —CH ₂ —CH ₂ —[—NH—CH ₂ —CH ₂ —CH ₂ —] _n —NH ₂ (n=8-29).

FIG. 2(a) is a mass spectrum of long-chain polyamine derived from Bacillus cereus strain YH64 obtained by atmospheric pressure chemical ionization mass spectrometry. (b) of FIG. 2 is a mass spectrum in which the peak of (a) of FIG. 2 is enlarged. As shown in FIG. 2, equally spaced peaks derived from long-chain polyamines were detected as in FIG. In FIG. 2, in addition to the same peak group (A in (b) of FIG. 2) detected in FIG. 1, there were three types of peak groups with different m / z values (( B, C and D in b). Since the interval between the peaks is 57.058 for all peak groups, it is considered that the structures of the repeating portions of the long-chain polyamines are common, but the terminal structures of the long-chain polyamines are different. From these peaks, the long-chain polyamine derived from Bacillus cereus YH64 strain is considered to have the following structure.
Common repeating structures: R-[-NH-CH ₂ -CH ₂ -CH ₂ -] _n -NH ₂ , (n=4-34),
Peak Group A: R=--CH ₂ --CH ₂ --CH ₂ --CH ₂ --NH ₂ ,
Peak Group B: R=--CH ₂ --CH ₂ --CH ₂ --NH ₂ ,
Peak group _C : R = _-C5H10N ,
Peak group D: R = _{-C6H10N .}

FIG. 3 is a mass spectrum of long-chain polyamines from Bacillus cereus ATCC 14579 strain obtained by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. As shown in FIG. 3, similarly to FIG. 1, equally spaced peaks derived from long-chain polyamines were detected. The m/z value of the largest observable peak is 3227, which corresponds to an n value of 55. From the mass of each peak group, the long-chain polyamine derived from Bacillus cereus ATCC 14579 strain is believed to have the following structure:
_H2N - _CH2 - _CH2 - _CH2 - _CH2 -[-NH- _CH2 - _CH2 - _CH2- ] _n - _NH2 .

<2. Example of gene disruption>
When the nucleotide sequence of the genome of Bacillus cereus ATCC 14579 strain was confirmed, BC5213 protein (Accession Number: NP_834876) that has high homology with aminopropyltransferase, and BC5214 protein that seems to be functionally related to the BC5213 protein (Accession Number: NP_834877) was found to exist.

Therefore, either the BC5213 gene or the BC5214 gene encoding these proteins is disrupted according to a well-known method (for example, the method described in Arnaud et al. 2004 Appl. Environ. Microbiol. 70, 6887). A gene-disrupted strain was constructed.

Next, the above-described <1. Extraction of long-chain polyamines from Bacillus cereus>, detection of long-chain polyamines derived from the gene-disrupted strain was attempted.

However, in the gene-disrupted strain in which either the BC5213 gene or the BC5214 gene was disrupted, the long-chain polyamine peak disappeared in the mass spectrum.

This revealed that the BC5213 gene and BC5214 gene are involved in biosynthesis of long-chain polyamines.

<3. Example of gene introduction into E. coli>
From the protein (SEQ ID NO: 4) encoded by the BC5213 gene (SEQ ID NO: 3) of Bacillus cereus ATCC 14579 strain, the protein (SEQ ID NO: 6 ) (SEQ ID NO: 5) and the BC5214 gene (SEQ ID NO: 7) located downstream of the BC5213 gene were amplified as one polynucleotide fragment by PCR. Genomic DNA of Bacillus cereus ATCC 14579 strain was used as a template for PCR, and oligo DNAs having the following sequences were used as primers (5′-AAGGAGATATACATATGGATGTGTGGGATGAAATTTCTCT-3′ (SEQ ID NO: 1), 5′-GGTGGTGGTGCTCGAGTCAATCGGCAGTAACAATTTCTCCT-3′ (SEQ ID NO: 2)).

The resulting PCR product was purified, and pET-24b plasmid (Merck) cleaved with restriction enzymes NdeI and XhoI was reacted with the purified PCR product using InFusion HD Cloning Kit (Clontech) to obtain pET. The PCR product was inserted into the -24b plasmid. The solution after the reaction was used to transform E. coli host DH5α strain for cloning, and E. coli host HD5α for cloning into which the desired plasmid was introduced was selected. The desired plasmid was then purified from the cloning E. coli host HD5α.

E. coli Rosetta2(DE3)pLysS (Merck) was transformed with the resulting plasmid. The resulting transformants were cultured in 2xYT medium supplemented with 1% (w/v) glucose, 50 µg/ml kanamycin and 30 µg/ml chloramphenicol until _OD600 was approximately 0.5. Cultured at 37°C. After that, IPTG (Isopropyl beta-D-thiogalacto-pyranoside, Nacalai Tesque, Inc.) was added to the 2×YT medium to a final concentration of 1 mM. The transformants were further cultured at 28° C. for 6 hours to express the target proteins (BC5213 protein and BC5214 protein).

The cells were collected by centrifugation (12,000 g, 1 minute), suspended in a 5% trichloroacetic acid aqueous solution, and subjected to ultrasonication to disrupt the cells. After the solution containing the crushed cells was allowed to stand on ice for 30 minutes, it was subjected to centrifugation (12,000 g, 15 minutes), and the supernatant was collected as a polyamine extraction fraction. The collected fractions were subjected to C8 reversed phase chromatography using heptafluorobutyric acid as an ion pair reagent to separate polyamines with different chain lengths, and according to a previous report (Reference B: Moriya 2015 Polyamine 2, 15), polyamine post The presence of long-chain polyamines was confirmed by column derivatization and fluorescence detection.

FIG. 4 shows the detection results of long-chain polyamines by HPLC. (a) of FIG. 4 shows the chromatogram of the negative control (that is, the E. coli Rosetta2 (DE3) pLysS strain introduced with the pET-24b plasmid without the insert was subjected to the same operation as described above). . FIG. 4(b) is a chromatogram of the polyamine-extracted fraction of E. coli into which the gene of interest has been introduced.

As shown in FIG. 4(b), multiple continuous peaks (see inside the square frame) not present in the negative control were observed in the elution time range of 26-34 minutes. These are peaks of long-chain polyamines with different chain lengths, and the value of n corresponds to 4-9. The structure of the long-chain polyamine was identified by subjecting the eluate containing the peak appearing in the range _of 26 to 34 minutes to the above-described atmospheric pressure chemical ionization mass spectrometry. -CH ₂ -CH ₂ -CH ₂ -CH ₂ -[-NH-CH ₂ -CH ₂ -CH ₂ -] _n -NH ₂ (n=4-9).

From the above results, a compound having a tandem structure (long-chain polyamine) was artificially produced.

INDUSTRIAL APPLICABILITY The present invention can be suitably used in the field of producing compounds having a tandem structure, the medical field, and the field of producing silica.

Claims (4)

or
(ii) A method for producing a compound represented by the following formula (I), comprising a reaction step of contacting a BC5213 protein of a bacterium belonging to the genus Bacillus or a homologue protein thereof with a substrate;

(In formula (I) above, R is hydrogen or any organic group, and n is an integer of 9 or more and 55 or less)
The homologous gene of the BC5213 gene hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 3 or 5, and aminopropyl group transfer. A polynucleotide encoding a protein having enzymatic activity or aminopropyltransferase-assisting activity,
The homologous protein of the above BC5213 protein has deletion or substitution of within 30 amino acids for SEQ ID NO: 4 and within 20 amino acids for SEQ ID NO: 6 in the amino acid sequence shown in SEQ ID NO: 4 or 6. or a protein consisting of an added amino acid sequence and having aminopropyltransferase activity or aminopropyltransferase-assisting activity,
Production method. organic represented by the above formula (I), wherein R is H ₂ N—(CH ₂ ) ₄ —, H ₂ N—(CH ₂ ) ₃ —, NH ₁₀ C ₅ —, or NH ₁₀ C ₆ —; The production method according to claim 1, which is a group. 3. The production method according to claim 1 or 2, wherein the transformant is further introduced with the BC5214 gene of a bacterium belonging to the genus Bacillus or a homologous gene thereof,
The homologous gene of the BC5214 gene hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 7, and has aminopropyltransferase activity. or a polynucleotide encoding a protein having aminopropyltransferase-assisting activity,
Production method. 1 or 2. The manufacturing method according to 2,
The homologous protein of the BC5214 protein consists of an amino acid sequence in which up to 10 amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 8, and has aminopropyl transferase activity or aminopropyl group a protein having transferase-assisting activity,
Production method.

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