L-β-lysine homopolymer, method for producing the same, and antibacterial agent containing the polymer

Abstract

To provide a novel polyamino acid and / or a salt thereof, and develop an antibacterial agent having better bactericidal action which contains novel polyamino acid thereof and / or a salt thereof.SOLUTION: It is found that Streptoalloteichus hindustanus being a type of Actinomycetes synthesizes ε-poly-L-β-lysine being a novel amino acid homopolymer, and ε-poly-L-β-lysine is a cationic polymer and has excellent bactericidal ability against bacteria, yeast and mold. On the basis of the findings, novel polyamino acid and / or a salt thereof is provided, and an antibacterial agent which has better antibacterial action and contains novel polyamino acid thereof and / or a salt thereof is developed.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to an L-β-lysine homopolymer (hereinafter also referred to as ε-poly-L-β-lysine) and / or a salt thereof, a process for producing them, and an antibacterial agent containing the polymer and / or a salt thereof as an active ingredient. [Background technology]

[0002] Conventionally, ε-poly-L-α-lysine (hereinafter also referred to as EPL) produced by the actinomycete Streptomyces albulus has been widely known (Patent Document 1). EPL is a membrane-bound nonribosomal peptide. It is also known that it is biosynthesized by EPL synthase (Pls), a peptide synthase similar to NRPS, and that its structure is such that the ε-amino group and α-carboxyl group of lysine are connected by a peptide bond. In addition, EPL is a biodegradable amino acid homopolymer produced by bacteria. Furthermore, EPL has been widely known as an antibacterial agent. This was clear (Patent Document 2).

[0003] However, the antibacterial effect of EPL is not necessarily sufficient against all bacteria. Therefore, it is desirable to further enhance the antibacterial effect of EPL. there was.

[0004] In addition, Streptoalloteichus hindustanus, a type of actinomycete, has been reported to produce poly-D-diaminobutanoic acid (Poly-D-Dab) with antibacterial activity (Non-patented). 1), it was unclear whether EPL or other polyamino acids like Poly-D-Dab were produced. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication No. 59-20359 [Patent Document 2] Japanese Patent Application Publication No. 61-243010 [Non-patent literature]

[0006] [Non-Patent Document 1] The Journal of Antibiotics, 1988, 2, 3, 849-854 [Non-patent document 2] Chem. Sci., 2015, 6, 6385-6391 [Non-patent document 3] Anal. Chem., 1997, 69, 5146-5151 Summary of the Invention [Problem to be solved by the invention]

[0007] In view of the above, it has been desired to provide a novel polyamino acid and / or a salt thereof, and to develop an antibacterial agent containing the novel polyamino acid and / or a salt thereof and having a more excellent antibacterial activity. [Means for solving the problem]

[0008] The present inventors have found that actinomycetes other than the EPL-producing bacterium Streptomyces albulus synthesize amino acid homopolymers that are different from EPL. In addition, Pls-like peptide synthetases are known to synthesize a variety of unknown amino acid homopolymers. Genome mining revealed that this is also distributed in other microorganisms, and Streptoalloteichus hindustanus, a type of actinomycete, produced a novel amino acid homopolymer, ε-poly-L-β-lysine. We discovered how to synthesize gin.

[0009] Furthermore, ε-poly-L-β-lysine is a cationic polymer that is effective against bacteria, yeasts, and molds. It was found to have excellent antibacterial properties against

[0010] In view of such circumstances, the present invention provides a novel polyamino acid and / or a salt thereof, and develops an antibacterial agent having a more excellent antibacterial effect, which contains the novel polyamino acid and / or a salt thereof.

[0011] That is, the present invention is as follows. [1] ε-poly-L-β-lysine and / or a salt thereof having the structure of formula (I): [ka] (In formula (I), n is any integer.) [2] The ε-poly-L-β-lysine and / or its derivatives according to [1], wherein n=6 to 25 in the formula (I). Salt. [3] A method for producing ε-poly-L-β-lysine and / or a salt thereof according to [1] or [2], comprising the steps of culturing a bacterium belonging to the genus Streptoalloteichus and separating ε-poly-L-β-lysine and / or a salt thereof from the culture medium. [4] The method according to [3], wherein the bacterium belonging to the genus Streptoalloteichus is Streptoalloteichus hindustanus. [5] An antibacterial agent comprising the ε-poly-L-β-lysine and / or a salt thereof according to [1] or [2]. . [6] The antibacterial agent according to [5], which is targeted against bacteria belonging to any genus selected from the group consisting of the genera Cladosporium, Aspergillus, Penicillium, and Alternaria. [Effects of the Invention]

[0012] The present invention provides a novel polyamino acid and / or a salt thereof, and also develops an antibacterial agent containing the novel polyamino acid and / or a salt thereof, which has a low minimum inhibitory concentration (MIC) and a broad antibacterial spectrum. It was emitted. [Brief explanation of the drawings]

[0013] [Figure 1] This shows the results of high-performance liquid chromatography (HPLC) performed to identify the structures of stereoisomers of hydrolyzed monomers of ε-poly-L-β-lysine. The upper figure shows a reaction scheme for labeling ε-poly-L-β-lysine with D- or L-FDLA, and the lower figure shows a spectrum of the measurement results obtained by HPLC. [Figure 2] 1 is a survival curve graph for evaluating the antibacterial activity of ε-poly-L-β-lysine. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention provides ε-poly-L-β-lysine and / or a salt thereof having the structure of formula (I). [ka] (In formula (I), n is any integer.)

[0015] The degree of polymerization of ε-poly-L-β-lysine is not particularly limited, but is preferably 8 to 27, that is, n=6 to 25 in formula (I). ε-poly-L-β-lysine exhibits the effects of the present invention. As long as the amino acid substitution is not impaired, it may be present at any position. The weight average molecular weight of ε-poly-L-β-lysine is not particularly limited, but is preferably in the range of 1000 to 4000 when measured by, for example, the GPC-LALLS method. Like EPL, ε-poly-L-β-lysine is a polymer consisting of the ε-amino group and the α-carboxyl group of L-β-lysine. It is a structure in which groups are bonded by peptide bonds.

[0016] The ε-poly-L-β-lysine may be isolated, purified and / or synthetic. In the present invention, "isolation" or "purification" means separating the polyamino acid of interest of the present invention, ε-poly-L-β-lysine and / or a salt thereof, from at least one contaminant that is normally present in the natural environment, or The purpose is to increase the purity of lysine and / or its salts. "Isolation" or "purification" can be performed by any method known to those skilled in the art.

[0017] In the present invention, the term "synthesize" refers to the synthesis of monomer units of ε-poly-L-β-lysine by a method known to those skilled in the art. The chemical synthesis method is to polymerize the ε-poly-L-β-lysine by any known chemical synthesis method. The chemical synthesis method is not particularly limited, but an example thereof is the method described in Non-Patent Document 2. Specifically, this method involves protecting the β-amino group of the ε-poly-L-β-lysine monomer unit with a protecting group (such as a tert-butoxycarbonyl (Boc) group), polymerizing the monomer unit with a condensing agent, and then removing the protecting group.

[0018] The salt of ε-poly-L-β-lysine is not particularly limited, but examples thereof include hydrochloric acid, sulfuric acid, phosphoric acid, and Examples include salts of at least one inorganic acid selected from hydrobromic acid, and salts of at least one organic acid selected from acetic acid, propionic acid, fumaric acid, malic acid, and citric acid.

[0019] Even if not specifically stated in the present specification, the compound may be a compound having a structure with ε-poly-L-β-lysine. The term may include salts of ε-poly-L-β-lysine or may be salts of ε-poly-L-β-lysine itself.

[0020] The ε-poly-L-β-lysine may be biodegradable, and is preferably biodegradable. For example, the amino group at the ε position and the carboxyl group at the α position of L-β-lysine can cleave the peptide bond. It can be decomposed by bacteria. Biodegradability is the property of being completely decomposed by oxidation or hydrolysis in the environment or by microorganisms, and producing only natural by-products (carbon dioxide, water, amino acids, methane, biomass, etc.). Specific methods for measuring biodegradability include the dissolved organic carbon (DOC) method and the biomass method. The chemical oxygen consumption (BOD) method is an example of such a method. Being biodegradable means that 50% or more of the ε-poly-L-β-lysine is decomposed by the above method.

[0021] Another aspect of the present invention provides a method for producing ε-poly-L-β-lysine and / or a salt thereof, the method comprising the steps of culturing a bacterium belonging to the genus Streptoalloteichus and separating ε-poly-L-β-lysine and / or a salt thereof from the culture medium.

[0022] Bacteria belonging to the genus Streptoalloteichus can produce ε-poly-L-β-lysine There is no particular limitation as long as it is a bacterium. Preferably, it is Streptoalloteichus hindustanus, more preferably Streptoalloteichus hindustanus NBRC15115, and more preferably ε-poly-L-β-lysine It is possible to produce

[0023] The step of culturing bacteria can be carried out by culturing bacteria, and the bacteria can produce ε-poly-L-β-lysine and / or a salt thereof. There are no particular limitations on the type of ε-poly-L-β-lysine as long as it can produce ε-poly-L-β-lysine and / or a salt thereof and release it into the culture medium. The method is not limited and can be carried out by methods known to those skilled in the art. A medium for culturing bacteria belonging to the genus Streptoalloteichus may contain, for example, 50 g glucose, 5 g yeast extract, 1 g polypeptone, 1.6 g NaHPO, 1.4 g KHPO, 0.5 g MgSO·7H0, 0.04 g ZnSO·7H0, and 0.03 g FeSO·7H0 per liter of water, with a pH of 6.8. The components of the medium are not particularly limited and can be added and / or changed as desired, and the amount of each component and the pH of the medium are not particularly limited and can be increased or decreased as desired. The culture conditions can be, for example, as follows: After the medium is sterilized by autoclaving, it is allowed to cool to room temperature, and the bacteria belonging to the genus Streptoalloteichus cultured on the slant medium are cultured for 1 hour. The bacteria were inoculated into 100 mL of medium in a Sakaguchi flask using a platinum loop, and cultured at 30°C for 24 hours with shaking (200 rpm). After that, the entire culture solution in the Sakaguchi flask was poured into 1 L of medium in a mini jar fermenter, and cultured for 24 hours at 30°C with an aeration rate of 3 L / min and an agitation speed of 400 rpm. Add 500mM MOPS-NaOH buffer (pH 6.5) containing 1 / 4 of the culture medium, and continue culturing for 24 hours. The culture conditions (volume, temperature, time, shaking method, aeration amount, stirring speed), and the equipment and reagents used for culture can be changed as desired. Furthermore, large-scale culture can be performed under any culture conditions and using any culture equipment and reagents. After the culture, contaminants such as cell debris present in the culture medium can be removed by any method known to those skilled in the art.

[0024] The step of separating ε-poly-L-β-lysine and / or a salt thereof from the culture medium by the above steps is There are no particular limitations on the separation method as long as it can separate ε-poly-L-β-lysine. For example, this can be done by the following method: Citric acid is added to the culture supernatant to a final concentration of 50 mM, and a mixture of acetone and methanol (acetone:methanol = 3:1) in an amount twice the volume of the culture supernatant is added to crystallize the mixture. The resulting precipitate is collected by centrifugation and resuspended in 50 mM bicarbonate. The fraction is dissolved in ammonium bicarbonate buffer (pH 8.0) and injected into a BioRex70 (weak cation exchanger, Bio-Rad) that has been pre-equilibrated with the buffer. The non-adsorbed fraction is washed away with the buffer, and then 50 mM ammonium bicarbonate buffer (pH 9.2) is passed through to elute the co-produced Poly-D-Dab. ε-Poly-L-β-lysine is then eluted with 0.5 M HCl. The fraction containing ε-Poly-L-β-lysine is identified and collected by the methyl orange method. The fraction is dried under reduced pressure to obtain ε-Poly-L-β-lysine hydrochloride.

[0025] Another method for producing the ε-poly-L-β-lysine of the present invention is to use a member of the genus Streptoalloteichus Using ε-poly-L-β-lysine synthase from bacteria, particularly Streptoalloteichus hindustanus, There are several ways to do this. Specifically, it can be carried out by a method known to those skilled in the art, for example, ε-poly-L-β-lysine By introducing a sequence encoding an ε-poly-L-β-lysine synthase into any cell by any method, the cell can express ε-poly-L-β-lysine, and the ε-poly-L-β-lysine can be recovered and separated by any method. The sequence encoding the ε-poly-L-β-lysine synthase is not particularly limited, but For example, the amino acid sequence represented by SEQ ID NO: 1 can be mentioned (Protein n ID: WP_073484487.1). The sequence encoding ε-poly-L-β-lysine synthase is As long as the function is not impaired, the amino acid sequence may have an identity of, for example, 80% or more, 90% or more, 95% or more, or 98% or more with the amino acid sequence shown in SEQ ID NO: 1.

[0026] The structure of ε-poly-L-β-lysine can be identified by methods known to those skilled in the art. . For example, high performance liquid chromatography (HPLC) was used to analyze the addition of ε-poly-L-β-lysine. This can be achieved by identifying the structure of the hydrolysate and / or by identifying the structure of the hydrolysate of the ε-poly-L-β-lysine derivative using nuclear magnetic resonance (NMR) analysis.

[0027] Another aspect of the present invention is to provide an antibacterial agent comprising ε-poly-L-β-lysine.

[0028] The antibacterial agent of the present invention may target any of bacteria, yeast, and mold, but is preferably effective against mold, and exhibits particularly excellent antibacterial activity. In this specification, "antibacterial" refers to any of the following: sterilization, which kills bacteria; sterilization, which reduces the number of bacteria; and bacteriostasis, which prevents or suppresses the proliferation and growth of bacteria.

[0029] When the antibacterial agent of the present invention is intended to target bacteria, the bacteria to be treated with the antibacterial agent are not particularly limited, but are preferably bacteria belonging to a genus selected from the group consisting of Gram-positive bacteria, such as Escherichia, Pseudomonas, and Salmonella, or Gram-negative bacteria, such as Bacillus, Staphylococcus, and Listeria. Specific examples of the type of bacteria include Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Bacillus cereus, Staphylococcus aureus, and Listeria monocytogenes. The bacteria to be treated with the antibacterial agent do not include bacteria belonging to the genus Streptoalloteichus, which synthesizes ε-poly-L-β-lysine, and in particular do not include Streptoalloteichus hindustanus.

[0030] When the antibacterial agent of the present invention is used against yeast, the yeast to be treated is not particularly limited, but is preferably a yeast belonging to a genus selected from the group consisting of the genera Candida and Pichia, for example, Candida albicans or Pichia anomala.

[0031] When the antibacterial agent of the present invention is used against fungi, the fungi to be treated are not particularly limited, but are preferably fungi belonging to any genus selected from the group consisting of the genera Cladosporium, Aspergillus, Penicillium, and Alternaria. Specific examples of the fungi include Cladosporium cladosporioides, Aspergillus brasiliensis, Penicillium verrucosum, and Alternaria alternata.

[0032] The content of ε-poly-L-β-lysine in the antibacterial agent of the present invention is as long as it can exert its antibacterial effect. For example, ε-poly-L-β-lysine is Therefore, it can be contained in the antibacterial agent at a concentration of 1 to 1000 μg / mL.

[0033] The conditions for using the antibacterial agent of the present invention are not particularly limited as long as it can exert its antibacterial effect, and it can be used under any temperature, humidity, application amount, time and pH.

[0034] The method for evaluating the antibacterial activity is not particularly limited, and can be any method known to those skilled in the art. For example, the minimum inhibitory concentration (MIC) of the antibacterial agent of the present invention can be measured, and the cell culture medium after administration of the antibacterial agent can be analyzed. Antibacterial activity can be evaluated by measuring the survival rate of bacteria.

[0035] The method for evaluating antibacterial activity by measuring MIC is, for example, to compare an antibacterial agent using ε-poly-L-β-lysine with a control sample (EPL) and to determine the effectiveness of the control sample. The antibacterial effect can be evaluated as being higher when the MIC is lower than that of the other antibacterial agents. In a method for evaluating the antibacterial effect by measuring the survival rate of bacteria after administration of an antibacterial agent, for example, an antibacterial agent using ε-poly-L-β-lysine is compared with a control sample (EPL) or the like, and a higher antibacterial effect can be evaluated by comparing the survival rate of bacteria with that of the control sample. [Example]

[0036] The present invention will be described in more detail below with reference to examples, but it goes without saying that the scope of the present invention is not limited to the examples.

[0037] <Example 1: Cultivation of ε-poly-L-β-lysine-producing bacteria and method for separating ε-poly-L-β-lysine from the culture medium> (Cultivation of ε-poly-L-β-lysine-producing bacteria) A culture medium for the ε-poly-L-β-lysine-producing bacteria was prepared with the following composition: 50g of α-glucan per 1L of water. Glucose, 5 g yeast extract, 1 g polypeptone, 1.6 g NaHPO, 1.4 g KHPO, 0.5 g MgSO 7H, 0.04 g ZnSO 7H, and 0.03 g FeSO 7H were added to the culture medium to adjust the pH to 6.8. This culture medium was sterilized by autoclaving and allowed to cool to room temperature. One loopful of Streptoalloteichus hindustanus NBRC15115 cultured in a slant medium was inoculated into 100 mL of culture medium in a Sakaguchi flask and cultured at 30°C for 24 hours with shaking (200 rpm). The entire culture medium from the Sakaguchi flask was then poured into 1 L of culture medium in a mini jar fermenter and cultured at 3 L / min with agitation at 400 rpm for 24 hours at 30°C. Further, 500 mM 3-(N-morpholino)propanesulfonic acid (MOPS)-NaOH buffer (pH 6.5) containing 5% (NH4)2SO4 was added in an amount of 1 / 4 of the culture medium, and the culture was continued for 24 hours.

[0038] (ε-Poly-L-β-Lysine Separation Method) Citric acid was added to the culture supernatant to a final concentration of 50 mM, and a mixture of acetone and methanol (acetone:methanol = 3:1) in an amount twice the volume of the culture supernatant was added to allow crystallization. The resulting precipitate was collected by centrifugation and dissolved in 50 mM ammonium bicarbonate buffer (pH 8.0). The precipitate was then loaded onto a BioRex® 70 (weak cation exchanger, Bio-Rad) pre-equilibrated with the buffer. The non-adsorbed fraction was washed away with the buffer, and 50 mM ammonium bicarbonate buffer (pH 9.2) was then passed through to elute the co-produced Poly-D-Dab. ε-poly-L-β-lysine was then eluted with 0.5 M HCl. The ε-poly-L-β-lysine-containing fraction was identified by the methyl orange method, and ε-poly-L-β-lysine was recovered. The precipitate was dried under reduced pressure to yield approximately 90 mg of ε-poly-L-β-lysine hydrochloride.

[0039] <Example 2: Structural determination of isolated polyamino acid (ε-poly-L-β-lysine)> (Structure determination of constituent amino acid residues) The constituent residues of the polyamino acids separated and purified by the above production method were determined using the Marfey method, which is a method known to those skilled in the art. The purified amino acids are added to 6M HCl and heated at 100°C for 16 hours to completely hydrolyze them. This gave a monomer. Next, this monomer was derivatized by reacting it with L-FDLA (Nα-(5-Fluoro-2,4-dinitrophenyl)-L-leucinamide) in a sodium bicarbonate buffer at 37°C for 1 hour (Figure 1, upper panel). HPLC analysis was then performed under the following conditions: ·HPLC analysis conditions HPLC equipment: Shimadzu Prominence HPLC System Column: SunShell C18 (Chromanic Technologies, 2.6 μl, φ4.6 × 100) Column temperature: 40℃ Solvent A: MilliQ medium, 0.1% TFA Solvent B: Acetonitrile Flow rate: 0.2 ml / min Gradient: 25%-65%B Detection wavelength: A340 As a result, the hydrolysate derivatized with L-FDLA was found to be the L-FDLA derivatized L-β-lysine preparation. The retention time closely matched that of the polyamino acid, confirming that the polyamino acid was an amino acid homopolymer with L-β-lysine as the constituent residue (Figure 1, bottom).

[0040] (Determination of polymer structure) The binding mode of L-β-lysine in the polyamino acid was determined by the following method. The purified β-lysine polymer was dissolved in 60% (v / v) ethanol and 1.6% (w / v) sodium bicarbonate. The resulting yellow precipitate was washed thoroughly with water, ethanol, and acetone in that order, and then dissolved in 100 ml of 6 M HCl. The hydrolysis was completed by heating at 20°C for 16 hours. To 7.5 ml of this hydrolysis solution, an equal amount of acetic acid was added. After adding ethyl acetate and mixing thoroughly, the recovered aqueous layer was evaporated to dryness under vacuum, dissolved in a small amount of dimethyl sulfoxide-d6, and subjected to two-dimensional NMR (HMQC spectrum with direct HC correlation) analysis. As a result, based on the chemical shift value of the amino group of the main product after hydrolysis (4.31 ppm), The inserted dinitrophenyl group is bound to the 3-amino group of L-β-lysine. In other words, the polyamino acid is an ε-poly-L-β-lysine in which L-β-lysines are linked by isopeptide bonds, just like EPL. It was confirmed to be lysine.

[0041] Example 3 To evaluate the antibacterial activity, the minimum inhibitory concentration (MIC) was measured (Table 1). [Table 1] Nutrient medium containing ε-poly-L-β-lysine (referred to as Poly-L-β-Lys in the table) (degree of polymerization 8-27) Gram-positive bacteria (Escherichia coli, Pseudomonas aeruginosa, or Salmonella enterica) ) or Gram-negative bacteria (Bacillus cereus, Staphylococcus aureus, or Listeria monocytogenes) were inoculated and cultured at 30°C for 48 hours. Yeast (Candida albicans or Pichia anomala) or mold (Cladosporium cladosporioides, Aspergillus brasiliensis, Penicilium verrucosum, or Alternaria alternata) were inoculated into YPD medium containing ε-poly-L-β-lysine (degree of polymerization: 8 to 27) and cultured at 30°C for 72 hours. EPL (referred to as Poly-L-Lys in the table) (degree of polymerization: 25 to 35) was used as a control. The ε-poly-L-β-lysine concentration was serially diluted, and the lowest concentration of ε-poly-L-β-lysine at which bacterial growth was inhibited was defined as the MIC. As a result, the MIC of ε-poly-L-β-lysine was comparable to that of EPL used as a control for all gram-positive bacteria, gram-negative bacteria, and yeast, and it exhibited antibacterial activity comparable to that of EPL against gram-positive bacteria, gram-negative bacteria, and yeast. Furthermore, the MIC of ε-poly-L-β-lysine was lower than that of EPL used as a control for all fungi. In other words, ε-poly-L-β-lysine was more effective than EPL, especially against fungi. It showed excellent antibacterial activity.

[0042] Example 4 To evaluate the antibacterial effect, the survival rate of bacteria after administration of the antibacterial agent was measured (Fig. 2). 1 x 10 cells in 10 mM HEPES-NaOH (pH 7.0) 7 Escherichia coli NBRC3301 The amino acid homopolymer of the present invention, ε-poly-L-β-lysine (referred to as Poly-L-β-Lys in the figure) (degree of polymerization: 8 to 27) was mixed with 10 μg / ml. (In the figure, it is indicated as Poly-L-Lys) (degree of polymerization 25 to 35) was used. The number of viable Escherichia coli cells exposed to each amino acid homopolymer was counted after 10, 20, and 30 minutes. The results showed that after 20 minutes, ε-poly-L- It was shown that the number of viable Escherichia coli cells was lower when β-lysine was used. That is, ε-poly-L-β-lysine showed a superior antibacterial activity to EPL. [Industrial Applicability]

[0043] The ε-poly-L-β-lysine of the present invention can be used as an antibacterial substance in the food industry and other fields. It can be used and is very useful in industry.

Claims

1. of bacteria belonging to the genus Streptoalloteichus Formula (I): 【Chemical 1】 (In formula (I), n is any integer.) in the synthesis of ε-poly-L-β-lysine having the structure The use, wherein the bacterium belonging to the genus Streptoalloteichus is Streptoalloteichus hindustanus.

2. The use according to claim 1, wherein in formula (I), n=6 to 25.

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