Antibacterial peptide and preparation method thereof

Abstract

The present disclosure provides a polypeptide having antibacterial activity, comprising: an amino acid sequence having 85% to 100% sequence identity with respect to an amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 6; or an amino acid sequence obtained by deleting, substituting, inserting and / or adding one or several amino acid residues to the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 6.

Description

Technical Field

[0001] The present invention relates to an antimicrobial peptide and a method for preparing the same. Background Art

[0002] Antimicrobial peptides can be produced by a variety of organisms including bacteria (e.g., lactic acid bacteria, etc.), animals (e.g., insects, mammals, etc.), and plants, and play an important role in the defense of organisms. Since antimicrobial peptides have a higher rapid effect compared to antibiotics and are decomposed into amino acids by digestive enzymes, etc. without remaining in the environment, it is considered difficult to generate drug-resistant bacteria. Antimicrobial peptides are expected as a countermeasure against drug-resistant bacteria that have become a serious social problem today.

[0003] Prior Art Documents

[0004] Patent Documents

[0005] Patent Document 1: U.S. Patent No. 4,584,199 Specification

[0006] Patent Document 2: U.S. Patent No. 3,295,989 Specification

[0007] Patent Document 3: International Publication No. WO89 / 12399

[0008] Patent Document 4: International Publication No. WO2004 / 029082 Summary of the Invention

[0009] Problems to be Solved by the Invention

[0010] As an antibacterial peptide produced by lactic acid bacteria (Lactococcus lactis strain ATCC-11454), Nisin A is known. Nisin A shows high antibacterial activity against many Gram-positive bacteria including heat-resistant spore-forming bacteria, is stable to acids, and is decomposed into amino acids by digestive enzymes in the intestine. Therefore, Nisin A is used practically as a food preservative in more than 50 countries. However, Nisin A is unstable in the neutral to alkaline range and also has low thermal stability in the neutral range. Therefore, the use of Nisin A is limited to food preservatives and the like for which the pH is often adjusted to the acidic range for commercial sale. In addition, since Nisin A contains unusual amino acids (e.g., lanthionine, etc.) generated by post-translational modification in cells, and a cross-linked structure of monosulfide bonds using lanthionine, etc., it is difficult to synthesize by chemical synthesis or genetic recombination techniques, and it is difficult to synthesize by in vitro translation. Further reports indicate that Nisin A is ineffective against Gram-negative bacteria (e.g., Escherichia coli) and fungi (e.g., Candida). U.S. Patent No. 4584199 (Patent Document 1), U.S. Patent No. 3295989 (Patent Document 2), International Publication No. 89 / 12399 (Patent Document 3), and International Publication No. 2004 / 029082 (Patent Document 4) disclose the application of Nisin in food preservation.

[0011] Under such circumstances, an antibacterial peptide with excellent stability in the neutral to alkaline range and excellent thermal stability is desired. The present invention has been completed in view of the above circumstances, and its object is to provide a polypeptide having antibacterial activity with excellent stability in the neutral to alkaline range and excellent thermal stability, and a method for producing the same.

[0012] Means for Solving the Problem

[0013] The present invention relates to the following items of examples.

[0014] [1] A polypeptide having antibacterial activity, comprising:

[0015] An amino acid sequence having 85% or more and 100% or less sequence identity with any one of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 6, or

[0016] An amino acid sequence in which 1 or several amino acid residues are deleted, substituted, inserted, and / or added with respect to any one of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 6.

[0017] [2] A nucleic acid comprising a base sequence encoding the polypeptide having antibacterial activity described in [1].

[0018] [3] The nucleic acid described in [2], which comprises any one of the base sequences of SEQ ID NO: 7 to SEQ ID NO: 12.

[0019] [4] A vector comprising the nucleic acid described in [2] or [3].

[0020] [5] An in vitro translation template molecule comprising the nucleic acid described in [2] or [3].

[0021] [6] The in vitro translation template molecule described in [5], which is RNA.

[0022] [7] A transformant comprising the nucleic acid described in [2] or [3], or the vector described in [4].

[0023] [8] A method for preparing a polypeptide having antibacterial activity, comprising:

[0024] (A) A step of synthesizing the polypeptide having antibacterial activity described in [1] by chemical synthesis;

[0025] (B) A step of translating the polypeptide having antibacterial activity described above using the in vitro translation template molecule described in [5] or [6];

[0026] (C) A step of culturing the transformant described in [7]; or

[0027] (D) A step of culturing at least one lactic acid bacterium selected from the group consisting of Deposit Numbers NITE BP-03198, NITE BP-03200, and NITE BP-03201.

[0028] [9] A combination of polypeptides having antibacterial activity, comprising a combination of a first polypeptide and a second polypeptide, or a combination of a third polypeptide and a fourth polypeptide, wherein

[0029] The above-mentioned first polypeptide comprises an amino acid sequence having 85% or more and 100% or less sequence identity to the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence in which 1 or several amino acid residues are deleted, substituted, inserted, and / or added relative to the amino acid sequence of SEQ ID NO: 3;

[0030] The above-mentioned second polypeptide comprises an amino acid sequence having 85% or more and 100% or less sequence identity to the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence in which 1 or several amino acid residues are deleted, substituted, inserted, and / or added relative to the amino acid sequence of SEQ ID NO: 4;

[0031] The above-mentioned third polypeptide comprises an amino acid sequence having 85% or more and 100% or less sequence identity to the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence in which 1 or several amino acid residues are deleted, substituted, inserted, and / or added relative to the amino acid sequence of SEQ ID NO: 13;

[0032] The above-mentioned 4th polypeptide comprises: an amino acid sequence having a sequence identity of more than 85% and 100% or less with respect to the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence in which 1 or several amino acid residues are deleted, substituted, inserted and / or added with respect to the amino acid sequence of SEQ ID NO: 14.

[0033]

[10] A combination of nucleic acids, which comprises the respective base sequences encoding the polypeptides with antibacterial activity described in [9].

[0034] The above-mentioned combination of nucleic acids comprises a combination of a 1st nucleic acid and a 2nd nucleic acid, or a combination of a 3rd nucleic acid and a 4th nucleic acid.

[0035] The above-mentioned 1st nucleic acid comprises a base sequence encoding the above-mentioned 1st polypeptide.

[0036] The above-mentioned 2nd nucleic acid comprises a base sequence encoding the above-mentioned 2nd polypeptide.

[0037] The above-mentioned 3rd nucleic acid comprises a base sequence encoding the above-mentioned 3rd polypeptide.

[0038] The above-mentioned 4th nucleic acid comprises a base sequence encoding the above-mentioned 4th polypeptide.

[0039]

[11] The combination of nucleic acids described in

[10] , wherein

[0040] The above-mentioned 1st nucleic acid comprises the base sequence of SEQ ID NO: 9.

[0041] The above-mentioned 2nd nucleic acid comprises the base sequence of SEQ ID NO: 10.

[0042] The above-mentioned 3rd nucleic acid comprises the base sequence of SEQ ID NO: 15.

[0043] The above-mentioned 4th nucleic acid comprises the base sequence of SEQ ID NO: 16.

[0044]

[12] A vector, which comprises the combination of nucleic acids described in

[10] or

[11] .

[0045]

[13] An in vitro translation template molecule, which comprises the combination of nucleic acids described in

[10] or

[11] .

[0046]

[14] The in vitro translation template molecule described in

[13] , which is RNA.

[0047]

[15] A transformant, which comprises the combination of nucleic acids described in

[10] or

[11] , or the vector described in

[12] .

[0048]

[16] A method for preparing a combination of polypeptides with antibacterial activity, which comprises:

[0049] (A) A step of synthesizing each of the polypeptides in the combination of polypeptides described in [9] by a chemical synthesis method.

[0050] (B) The step of separately translating the polypeptides in the above-mentioned polypeptide combination with an in vitro translation template molecule described in

[13] or

[14] ,

[0051] (C) The step of culturing the transformant described in

[15] , or

[0052] (D) The step of culturing the lactic acid bacterium with the deposit number NITE BP-03200.

[0053]

[17] An antibacterial composition, comprising:

[0054] The polypeptide having antibacterial activity described in [1], or a combination of polypeptides having antibacterial activity described in [9], and

[0055] An additive.

[0056] Advantages of the Invention

[0057] According to the present invention, it is possible to provide a polypeptide having antibacterial activity with excellent stability in the neutral to alkaline range and excellent thermal stability, and a method for producing the same. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 A schematic diagram for explaining the antibacterial activity evaluation test.

[0059] Figure 2 A photograph showing the results of the antibacterial activity evaluation test of the polypeptide involved in the example. DETAILED DESCRIPTION OF THE INVENTION

[0060] Hereinafter, the mode for carrying out the present invention (hereinafter described as "the present embodiment") will be described in detail. In addition, the present invention is not limited to the following embodiments. In the present specification, the expression of "mode A to Z" means the upper and lower limits of the range (that is, A or more and Z or less). In the case where there is no unit description in A and only the unit is described in Z, the unit of A and the unit of Z are the same. In the present specification, the expression of "comprising ~" is an expression including the concepts of "consisting of ~" and "consisting only of ~".

[0061] [Polypeptide Having Antibacterial Activity]

[0062] The polypeptide having antibacterial activity according to the present embodiment is a polypeptide having antibacterial activity including the following:

[0063] An amino acid sequence having 85% or more and 100% or less sequence identity with any one of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 6, or

[0064] ​​An amino acid sequence in which one or several amino acid residues are deleted, substituted, inserted, and / or added relative to any one of SEQ ID NO:1 to SEQ ID NO:6. Hereinafter, there are cases where a polypeptide having antibacterial activity is simply expressed as "polypeptide".

[0065] In the present embodiment, "antibacterial activity" means the property of inhibiting the proliferation of a target microorganism. The above antibacterial activity can be evaluated by the antibacterial activity evaluation test described in the examples below.

[0066] The target microorganism may be pathogenic or harmful. The target microorganism may be a Gram-positive bacterium, a Gram-negative bacterium, or a fungus. For example, Lactobacillus sakei, Streptococcus uberis, and Staphylococcus aureus are listed as Gram-positive bacteria, Escherichia coli is listed as a Gram-negative bacterium, and Candida albicans is listed as a fungus.

[0067] The polypeptide according to the present embodiment may contain an amino acid sequence having a sequence identity of, for example, 85% or more and 100% or less relative to any one of SEQ ID NO:1 to SEQ ID NO:6. In one aspect of the present embodiment, the lower limit value of the above sequence identity may be 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The polypeptide according to the present embodiment may consist only of the amino acid sequence of any one of SEQ ID NO:1 to SEQ ID NO:6.

[0068] "Sequence identity" in the present embodiment means the ratio (%) of identical amino acid residues to all overlapping amino acid sequences in the optimal alignment (preferably, the algorithm takes into account introducing gaps into one or both of the sequences for optimal alignment) when comparing two amino acid sequences using a mathematical algorithm well-known in the technical field. The "sequence identity" of an amino acid sequence can be easily confirmed by those skilled in the art. For example, NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) can be applied. The sequence identity of a base sequence can also be confirmed by the same method as above.

[0069] The polypeptide involved in this embodiment may include an amino acid sequence in which 1 or several amino acid residues are deleted, substituted, inserted, and / or added relative to any one of SEQ ID NO: 1 to SEQ ID NO: 6.

[0070] "1 or several" may be, for example, 1 to 10 depending on the position and type of amino acid residues in the three-dimensional structure of the protein. The polypeptide involved in this embodiment may be a polypeptide comprising the following amino acid sequence: an amino acid sequence in which 1 or more and 10 or less amino acid residues are deleted, substituted, inserted, and / or added relative to any one of SEQ ID NO: 1 to SEQ ID NO: 6. The upper limit value of the number of amino acid residues deleted, substituted, inserted, and / or added may be 9 or less, may be 8 or less, may be 7 or less, may be 6 or less, may be 5 or less, may be 4 or less, may be 3 or less, may be 2 or less.

[0071] In one aspect of this embodiment, as the "amino acid sequence in which 1 or several amino acid residues are deleted, substituted, inserted, and / or added", for example, an amino acid sequence having a sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more relative to the amino acid sequence before deletion, substitution, insertion, and / or addition can be exemplified.

[0072] An example of substitution, deletion, insertion, and / or addition of an amino acid residue is a conservative mutation in which the function of the polypeptide remains normal. A representative type of conservative mutation is a conservative substitution. Conservative substitutions are mutations such as those shown below. In the case where the substitution site is an aromatic amino acid, it is a mutation in which Phe, Trp, and Tyr are substituted with each other. In the case where the substitution site is a hydrophobic amino acid, it is a mutation in which Leu, Ile, and Val are substituted with each other. In the case of a polar amino acid, it is a mutation in which Gln and Asn are substituted with each other. In the case of a basic amino acid, it is a mutation in which Lys, Arg, and His are substituted with each other. In the case of an acidic amino acid, it is a mutation in which Asp and Glu are substituted with each other. In the case of an amino acid having a hydroxyl group, it is a mutation in which Ser and Thr are substituted with each other. Substitutions regarded as conservative substitutions include: substitution of Ala with Ser or Thr, substitution of Arg with Gln, His, or Lys, substitution of Asn with Glu, Gln, Lys, His, or Asp, substitution of Asp with Asn, Glu, or Gln, substitution of Cys with Ser or Ala, substitution of Gln with Asn, Glu, Lys, His, Asp, or Arg, substitution of Glu with Gly, Asn, Gln, Lys, or Asp, substitution of Gly with Pro, substitution of His with Asn, Lys, Gln, Arg, or Tyr, substitution of Ile with Leu, Met, Val, or Phe, substitution of Leu with Ile, Met, Val, or Phe, substitution of Lys with Asn, Glu, Gln, His, or Arg, substitution of Met with Ile, Leu, Val, or Phe, substitution of Phe with Trp, Tyr, Met, Ile, or Leu, substitution of Ser with Thr or Ala, substitution of Thr with Ser or Ala, substitution of Trp with Phe or Tyr, substitution of Tyr with His, Phe, or Trp, and substitution of Val with Met, Ile, or Leu.

[0073] The amino acid sequences of SEQ ID NOs: 1 and 2 are each the amino acid sequence of the mature form of a polypeptide predicted from the genomic sequence of Lactiplantibacillus plantarum NITE BP-03198.

[0074] The amino acid sequences of SEQ ID NOs: 3 and 4, and the amino acid sequence of SEQ ID NO: 5 (X in the amino acid sequence is Ala) are each the amino acid sequence of the mature form of a polypeptide predicted from the genomic sequence of Lactiplantibacillus plantarum NITE BP-03200.

[0075] The amino acid sequence of SEQ ID NO: 5 (where X in the amino acid sequence is Val) and the amino acid sequence of SEQ ID NO: 6 are each the amino acid sequence of the mature form of a polypeptide predicted from the genomic sequence of Lactiplantibacillus plantarum NITE BP-03201.

[0076] [Combination of polypeptides having antibacterial activity]

[0077] The combination of polypeptides having antibacterial activity according to this embodiment is:

[0078] A combination of polypeptides having antibacterial activity, which comprises a combination of a first polypeptide and a second polypeptide, or a combination of a third polypeptide and a fourth polypeptide,

[0079] The above-mentioned first polypeptide comprises: an amino acid sequence having a sequence identity of 85% or more and 100% or less with respect to the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence in which 1 or several amino acid residues are deleted, substituted, inserted and / or added with respect to the amino acid sequence of SEQ ID NO: 3,

[0080] The above-mentioned second polypeptide comprises: an amino acid sequence having a sequence identity of 85% or more and 100% or less with respect to the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence in which 1 or several amino acid residues are deleted, substituted, inserted and / or added with respect to the amino acid sequence of SEQ ID NO: 4,

[0081] The above-mentioned third polypeptide comprises: an amino acid sequence having a sequence identity of 85% or more and 100% or less with respect to the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence in which 1 or several amino acid residues are deleted, substituted, inserted and / or added with respect to the amino acid sequence of SEQ ID NO: 13,

[0082] The above-mentioned fourth polypeptide comprises: an amino acid sequence having a sequence identity of 85% or more and 100% or less with respect to the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence in which 1 or several amino acid residues are deleted, substituted, inserted and / or added with respect to the amino acid sequence of SEQ ID NO: 14. Here, the "combination of polypeptides" can be in the form of a mixture of two polypeptides as the object, can be in the form that the two polypeptides as the object are each independent and used together when used, or can be in the form that the two polypeptides as the object are tandemly bound via a linker peptide (for example, 6×Gly) or a linker compound to form one molecule.

[0083] The amino acid sequences of SEQ ID NO: 13 and 14 are each the amino acid sequence of the mature form of a polypeptide predicted from the genomic sequence of Lactiplantibacillus plantarum NITE BP-03200.

[0084] The polypeptide involved in this embodiment has antibacterial activity against a wide range of microbial species. For example, it has antibacterial activity against Gram-positive bacteria, Gram-negative bacteria, fungi, etc. The antibacterial activity of the polypeptide can be measured, for example, according to the method of the antibacterial activity test described later (refer to Experiment 3). The polypeptide involved in this embodiment has high pH stability and has antibacterial activity not only in the acidic range (e.g., pH 4.0), but also in the neutral range (e.g., pH 7.4) to the alkaline range (e.g., pH 10.0). The polypeptide involved in this embodiment has high thermal stability and has antibacterial activity, for example, after heat treatment in the neutral range (temperatures such as 80°C, 100°C, 121°C, etc.). As described above, the polypeptide involved in this embodiment is a polypeptide of natural origin with high antibacterial activity, has a wide available pH range, and is stable to heating, etc., so it has high versatility as an antibacterial component.

[0085] The polypeptide involved in this embodiment may contain a signal peptide (leader peptide) and may be a precursor polypeptide of the above-mentioned polypeptide. The precursor polypeptide is cleaved by a peptidase intracellularly or extracellularly and can become the above-mentioned polypeptide (mature polypeptide). The precursor polypeptide usually contains an amino acid sequence for cleavage by a specific peptidase. As precursor polypeptides of the polypeptides each composed of the amino acid sequences of SEQ ID NOs: 1 to 6, polypeptides each composed of the amino acid sequences of SEQ ID NOs: 25 to 30 are listed (Table 1). As precursor polypeptides of the polypeptides each composed of the amino acid sequences of SEQ ID NOs: 13 and 14, polypeptides each composed of the amino acid sequences of SEQ ID NOs: 31 and 32 are listed (Table 1). The polypeptide involved in this embodiment may be a secreted polypeptide or a membrane-bound polypeptide.

[0086] In other aspects of this embodiment, the above-mentioned polypeptide may further contain a peptide serving as a tag for purification. As a tag for purification, for example, a polyhistidine sequence (6×His) etc. are listed.

[0087] [Antibacterial composition]

[0088] The antibacterial composition involved in this embodiment is an antibacterial composition containing the following:

[0089] The above-mentioned polypeptide having antibacterial activity, or a combination of the above-mentioned polypeptides having antibacterial activity, and

[0090] Additives.

[0091] The above-mentioned antibacterial composition may be a proliferation inhibitor or a bactericide for bacteria and other microorganisms. As additives contained in the above-mentioned antibacterial composition, for example, surfactants, preservatives, antiseptics, pH regulators, thickeners, excipients, and fragrances are listed. The additives may be contained singly as 1 kind, or may be contained in combination of 2 or more kinds.

[0092] The surfactant can be an anionic surfactant, a nonionic surfactant or an amphoteric surfactant. As the anionic surfactant, N-acyl amino acid salts, α-olefin sulfonates, N-acyl sulfonates, alkyl sulfates (e.g., sodium lauryl sulfate, etc.), sulfates of glycerol fatty acid esters, etc. are cited. As the nonionic surfactant, polyoxyethylene alkyl ethers, polyoxyethylene-polyoxypropylene block copolymers, polyoxyethylene hydrogenated castor oil, polyoxyethylene ethers of glycerol esters, alkyl alkanolamides, sucrose fatty acid esters, glycerol fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, glycerol organic acid fatty acid esters, polyglycerol fatty acid esters, calcium stearoyl lactate, sodium stearoyl lactate, polyoxyethylene sorbitan fatty acid esters, alkyl glycosides, etc. are cited. As the amphoteric surfactant, alkyl betaine surfactants, amine oxide surfactants, imidazolinium betaine surfactants are cited. As specific examples thereof, 2-alkyl-N-carboxymethyl-N-hydroxyethyl imidazolinium betaine, lauryl dimethylaminoacetic acid betaine, coconut oil alkyl betaine (coconut oil alkyl dimethylaminoacetic acid betaine), stearoyl dimethylaminoacetic acid betaine, sodium stearoyl dimethyl betaine, coconut oil fatty acid amide alkyl betaine, palm oil fatty acid amide propyl betaine, lauric acid amide propyl betaine, ricinoleic acid amide propyl betaine, stearoyl dihydroxyethyl betaine, etc. are cited.

[0093] As preservatives or antiseptics, p-hydroxybenzoates such as sodium benzoate, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, butyl p-hydroxybenzoate, isopropyl p-hydroxybenzoate, propyl p-hydroxybenzoate, isobutyl p-hydroxybenzoate, benzyl p-hydroxybenzoate, etc., alcohols such as phenoxyethanol, ethanol, etc., or sorbic acid, benzoic acid, dehydroacetic acid, propionic acid or their salts, salts such as sodium chloride, etc., ethylenediaminetetraacetate salts, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, alkyl diaminethylglycine hydrochloride, etc. are cited.

[0094] As pH regulators, acids / bases, buffers, etc. such as acetic acid, hydrochloric acid, sulfuric acid, nitric acid, citric acid, phosphoric acid, malic acid, gluconic acid, maleic acid, succinic acid, glutamic acid, pyrophosphoric acid, tartaric acid, sodium hydroxide acetate, potassium hydroxide, sodium hydroxide, sodium acetate, sodium carbonate, potassium carbonate, sodium citrate, sodium hydrogen citrate, phosphoric acid, sodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, etc. are cited.

[0095] As thickeners, organic binders such as methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose or their salts, pullulan, gelatin, carrageenan, sodium alginate, xanthan gum, sodium polyacrylate, gum arabic, guar gum, locust bean gum, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, etc., and inorganic binders such as thickening anhydrous silicic acid, bentonite, etc. are cited.

[0096] As excipients, crystalline cellulose, powdered cellulose, potato starch, corn starch, light anhydrous silicic acid, hydrous silicon dioxide, silicon dioxide, precipitated calcium carbonate, anhydrous calcium hydrogen phosphate, magnesium oxide, calcium lactate, calcium silicate, magnesium aluminum metasilicate, synthetic hydrotalcite, synthetic aluminum silicate, lactose, white sugar, D-mannitol, erythritol, glucose, fructose, talc, dextrin, cyclodextrin, etc. are listed.

[0097] As fragrances, for example, mint, aloe vera juice, etc. are listed.

[0098] The polypeptide having antibacterial activity and the antibacterial composition according to this embodiment are applied to inhibit the proliferation of bacteria and other microorganisms, or to sterilize the microorganisms.

[0099] [Nucleic acid encoding a polypeptide having antibacterial activity]

[0100] The nucleic acid according to this embodiment is a nucleic acid containing a base sequence encoding the above-mentioned polypeptide having antibacterial activity. The nucleic acid can be DNA or RNA. The nucleic acid may contain a start codon on the 5'-end side and a stop codon on the 3'-end side, or may not contain them. The nucleic acid may contain an intron sequence or may not contain it. An example of the nucleic acid according to this embodiment contains the base sequence of any one of SEQ ID NO: 7 to SEQ ID NO: 12. The base sequences of SEQ ID NO: 7 to 12 each encode the amino acids of SEQ ID NO: 1 to SEQ ID NO: 6 respectively. Needless to say, but in the case where the nucleic acid is RNA, the base sequence of the above RNA is the base sequence in which thymine in the corresponding DNA base sequence is replaced by uracil.

[0101] In one aspect of this embodiment, the base sequences of SEQ ID NO: 17 and 18 are sequences derived from the genomic sequence of Lactiplantibacillus plantarum (NITE BP-03198), and respectively encode precursor polypeptides composed of the amino acid sequences of SEQ ID NO: 25 and 26. The base sequences of SEQ ID NO: 7 and 8 respectively encode polypeptides composed of the amino acid sequences of SEQ ID NO: 1 and 2.

[0102] The base sequences of SEQ ID NO: 19 to 21 are sequences derived from the genomic sequence of Lactiplantibacillus plantarum (NITEBP-03200), and respectively encode precursor polypeptides composed of the amino acid sequences of SEQ ID NO: 27 to 29. The base sequences of SEQ ID NO: 9 to 11 respectively encode polypeptides composed of the amino acid sequences of SEQ ID NO: 3 to 5. In addition, in NITE BP-03200, the base shown as y in the base sequences of SEQ ID NO: 11 and 21 is cytosine, and the amino acid residue shown as X in the amino acid sequences of SEQ ID NO: 5 and 29 is alanine.

[0103] The base sequences of SEQ ID NOs: 21 and 22 are sequences derived from the genomic sequence of Lactiplantibacillus plantarum (NITE BP-03201), and encode precursor polypeptides composed of the amino acid sequences of SEQ ID NOs: 29 and 30, respectively. The base sequences of SEQ ID NOs: 11 and 12 encode polypeptides composed of the amino acid sequences of SEQ ID NOs: 5 and 6, respectively. In addition, in NITE BP-03201, the base shown as y in the base sequences of SEQ ID NOs: 11 and 21 is thymine, and the amino acid residue shown as X in the amino acid sequences of SEQ ID NOs: 5 and 29 is valine.

[0104] The nucleic acid is not limited to the base sequences possessed by the above-mentioned Lactobacillus NITE BP-03198, NITE BP-03200, and NITE BP-03201, and may also be a nucleic acid containing a base sequence in which the codons encoding each amino acid in the coding region are replaced with other equivalent codons encoding the same amino acid. The nucleic acid according to the present embodiment may also be a nucleic acid containing a base sequence with an altered codon usage in a manner that enhances the expression of the polypeptide according to the present embodiment.

[0105] In one aspect of the present embodiment, the above nucleic acid may further contain a base sequence encoding a peptide serving as a purification tag.

[0106] The nucleic acid according to the present embodiment can be obtained by chemical synthesis or by PCR or the like using the genomic sequences of Lactobacillus NITE BP-03198, NITE BP-03200, and NITE BP-03201 as templates.

[0107] [Combination of Nucleic Acids Encoding a Combination of Polypeptides with Antibacterial Activity]

[0108] The combination of nucleic acids according to the present embodiment is a combination of nucleic acids containing the base sequences encoding the respective combinations of the above-mentioned polypeptides with antibacterial activity.

[0109] The above combination of nucleic acids includes a combination of a first nucleic acid and a second nucleic acid, or a combination of a third nucleic acid and a fourth nucleic acid.

[0110] The above first nucleic acid contains a base sequence encoding the above first polypeptide.

[0111] The above second nucleic acid contains a base sequence encoding the above second polypeptide.

[0112] The above third nucleic acid contains a base sequence encoding the above third polypeptide.

[0113] The above-mentioned fourth nucleic acid contains a base sequence encoding the above-mentioned fourth polypeptide. Here, the "combination of nucleic acids" can be in the form of a mixture of two nucleic acids that are the objects, can be in the form where the two nucleic acids that are the objects are independent of each other and are used together, or can be in the form where the two nucleic acids that are the objects are tandemly bound via a nucleic acid encoding a linker peptide (e.g., 6×Gly) to form one molecule.

[0114] In one aspect of the present embodiment, it is preferred that the above-mentioned first nucleic acid contains the base sequence of SEQ ID NO: 9, the above-mentioned second nucleic acid contains the base sequence of SEQ ID NO: 10, the above-mentioned third nucleic acid contains the base sequence of SEQ ID NO: 15, and the above-mentioned fourth nucleic acid contains the base sequence of SEQ ID NO: 16.

[0115] The base sequences of SEQ ID NOs: 23 and 24 are sequences derived from the genomic sequence of Lactiplantibacillus plantarum (NITE BP-03200), and encode precursor polypeptides composed of the amino acid sequences of SEQ ID NOs: 31 and 32, respectively. The base sequences of SEQ ID NOs: 15 and 16 encode polypeptides composed of the amino acid sequences of SEQ ID NOs: 13 and 14, respectively.

[0116] The nucleic acids constituting the above-mentioned combination of nucleic acids can be DNA or RNA. The nucleic acid may contain a start codon on the 5'-end side and a stop codon on the 3'-end side, or may not contain them. The nucleic acid may contain an intron sequence or may not contain it. Needless to say, but in the case where the nucleic acid is RNA, the base sequence of the above-mentioned RNA is the base sequence in which thymine in the base sequence of the corresponding DNA is replaced by uracil.

[0117] The nucleic acids constituting the above-mentioned combination of nucleic acids are not limited to the base sequences possessed by the above-mentioned Lactobacillus NITE BP-03200, and can also be nucleic acids containing base sequences in which codons encoding each amino acid in the coding region are replaced by other equivalent codons encoding the same amino acid. This nucleic acid can also be a nucleic acid containing a base sequence with an altered codon usage in a manner to enhance the expression of the encoded polypeptide.

[0118] In one aspect of the present embodiment, the nucleic acids constituting the above-mentioned combination of nucleic acids may further contain a base sequence encoding a peptide or a linker peptide that serves as a purification tag.

[0119] The nucleic acids constituting the above-mentioned combination of nucleic acids can be obtained by chemical synthesis or can be obtained by PCR or the like using the genomic sequence of Lactobacillus NITE BP-03200 as a template.

[0120] [Vector]

[0121] The vector involved in this embodiment contains the above nucleic acid, or a combination of the above nucleic acids. A vector is a nucleic acid molecule that can amplify and maintain DNA. For example, expression vectors and cloning vectors are listed. In one example, the above nucleic acid is introduced into a host cell or the like by inserting it into an expression vector, and a polypeptide with antibacterial activity is expressed. The vector can express a polypeptide in the host cell by being introduced into the host cell. The expression vector may have a promoter sequence and a terminator sequence for expressing the integrated gene. The above vector can be constructed by integrating the above nucleic acid into the original vector according to the usual genetic engineering methods. The above vector may have a selectable marker sequence.

[0122] In one aspect of this embodiment, in the case where the above vector contains a combination of the above nucleic acids, a single vector may jointly contain two nucleic acids that make up the combination. It may be that the above vector contains a first vector and a second vector, the first vector contains the first nucleic acid, and the second vector contains the second nucleic acid. Or it may be that the above vector contains a first vector and a second vector, the first vector contains the third nucleic acid, and the second vector contains the fourth nucleic acid.

[0123] The vector can be, for example, a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a viral vector, a cosmid vector, a phage vector, an artificial chromosome vector, etc. For example, pBR322, pUC plasmid vectors, pET series plasmid vectors, etc. are listed. Specifically, in the case where Escherichia coli is used as the host cell, pUC19, pUC18, pUC119, pBluescriptII, pET32, etc. can be listed. In the case where mammalian cells are used as the host cell, for example, pRc / RSV, pRc / CMV, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, etc. can be listed.

[0124] The above-mentioned nucleic acid or combination of the above-mentioned nucleic acids integrated in the vector is inserted downstream of the promoter in a state where the promoter can function. As the promoter, any promoter can be used as long as it is an appropriate promoter of the host used in the expression of the corresponding gene. For example, in the case where the host is an animal cell, the SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, etc. are used. Among them, the CMV promoter, SRα promoter, etc. are preferred. In the case where the host is Escherichia coli, the T7 promoter, cspA promoter, trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, tac promoter, etc. are preferred. In the case where the host is a Bacillus bacterium, the SPO1 promoter, SPO2 promoter, penP promoter, etc. are preferred. In the case where the host is yeast, the Gal1 / 10 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, AOX1 promoter, etc. are preferred. In the case where the host is Aspergillus, the ENOA promoter, TAA promoter, GLA promoter, AGL promoter, etc. are preferred. In the case where the host is an insect cell, the polyhedrin promoter, P10 promoter, etc. are preferred. In the case where the host is a plant cell, the CaMV35S promoter, CaMV19S promoter, NOS promoter, etc. are preferred.

[0125] As an expression vector, in addition to the above, those having an enhancer, a cleavage signal, a poly-A addition signal, a drug resistance gene, a selection marker such as a nutritional requirement complementary gene, an origin of replication, etc. according to the purpose can be used.

[0126] [In vitro translation template molecule]

[0127] The in vitro translation template molecule according to this embodiment contains the above-mentioned nucleic acid or combination of the above-mentioned nucleic acids. Here, the "in vitro translation template molecule" means a nucleic acid molecule that becomes a template for translation in a cell-free protein synthesis system. The above in vitro translation template molecule can be DNA or RNA. The above in vitro translation template molecule can be single-stranded or double-stranded. The above in vitro translation template molecule can have a promoter sequence and a terminator sequence for expressing the integrated gene. Hereinafter, there are cases where the in vitro translation template molecule is expressed as a "translation template molecule".

[0128] In one aspect of the present embodiment, in the case where the in vitro translation template molecule contains the combination of the above nucleic acids, a single in vitro translation template molecule may jointly contain two nucleic acids constituting the combination. The in vitro translation template molecule may be the first translation template molecule and the second translation template molecule. The first translation template molecule contains the first nucleic acid, and the second translation template molecule contains the second nucleic acid. Or the in vitro translation template molecule may be the first translation template molecule and the second translation template molecule. The first translation template molecule contains the third nucleic acid, and the second translation template molecule contains the fourth nucleic acid.

[0129] The nucleic acid or the combination of nucleic acids integrated in the in vitro translation template molecule is inserted downstream of the promoter in a state where the promoter can function. As the promoter, the above promoters are exemplified.

[0130] [Transformant]

[0131] The transformant according to the present embodiment is a cell into which the above nucleic acid or vector has been introduced. The transformant can express the polypeptide according to the present embodiment.

[0132] As the host cell into which the vector has been introduced, cells of eukaryotes or prokaryotes can be used. More specifically, for example, bacteria, fungi, plant cells, animal cells, and insect cells are exemplified. The host cell may be yeast, Escherichia coli, Aspergillus, or mammalian cells. As the above mammals, humans, cows, horses, sheep, monkeys, pigs, mice, rats, hamsters, guinea pigs, rabbits, dogs, etc. are exemplified. In one aspect of the present embodiment, as the above host cell, for example, yeast (such as BG10, BG11, etc.), Escherichia coli (such as BL21, DH5α, etc.), HEK 293T cells, CHO cells, Expi293F cells, and ExpiCHO cells can be used.

[0133] As a method for introducing the above nucleic acid or vector into a host cell, chemical methods such as the calcium phosphate method, DEAE-dextran method, and cationic liposome method are exemplified; biological methods such as adenovirus vectors, vaccinia virus vectors, retrovirus vectors, and HVJ liposomes; physical methods such as electroporation, DNA direct injection, and gene gun. A suitable introduction method can be selected according to the host cell into which the above nucleic acid or vector is introduced.

[0134] The above vector can be maintained extrachromosomally or integrated into the chromosome in the host cell.

[0135] The transformant into which the above nucleic acid or vector has been introduced can be selected using a selection marker. For example, the nucleic acid according to this embodiment and a selection marker gene are simultaneously introduced into a host cell, and the host cell is cultured by a method corresponding to the property of the selection marker. In the case where the selection marker gene is a gene that confers resistance to a screening agent that exhibits lethal activity on the host cell, after the introduction operation of the nucleic acid, the host cell can be cultured in a medium supplemented with the screening agent.

[0136] [Method for preparing a polypeptide having antibacterial activity]

[0137] The method for preparing a polypeptide having antibacterial activity according to this embodiment is a method for preparing a polypeptide having antibacterial activity including the following:

[0138] (A) A step of synthesizing the above polypeptide having antibacterial activity by a chemical synthesis method,

[0139] (B) A step of translating the above polypeptide having antibacterial activity from the above in vitro translation template molecule,

[0140] (C) A step of culturing the above transformant, or

[0141] (D) A step of culturing at least one lactic acid bacterium selected from Deposit Numbers NITE BP-03198, NITE BP-03200, and NITE BP-03201.

[0142] The method for preparing a combination of polypeptides having antibacterial activity according to this embodiment is a method for preparing a combination of polypeptides having antibacterial activity including the following:

[0143] (A) Steps of synthesizing each of the polypeptides in the above combination of polypeptides by a chemical synthesis method,

[0144] (B) Steps of translating each of the polypeptides in the above combination of polypeptides from the above in vitro translation template molecule,

[0145] (C) A step of culturing the above transformant, or

[0146] (D) A step of culturing the lactic acid bacterium of Deposit Number NITE BP-03200.

[0147] Each of the polypeptides according to this embodiment, or each of the polypeptides in the combination of polypeptides, can be prepared by a general chemical synthesis method for proteins. As the chemical synthesis method for proteins, for example, the liquid phase method and the solid phase method are listed.

[0148] Each of the polypeptides according to this embodiment, or each of the polypeptides in the combination of polypeptides, can be prepared by translating the polypeptide from an in vitro translation template molecule using a general cell-free protein synthesis system. As the cell-free protein synthesis system, for example, the wheat germ cell-free protein synthesis system is listed.

[0149] Each polypeptide in the polypeptide or combination of polypeptides according to this embodiment can be prepared by culturing the above-mentioned transformant.

[0150] The culturing of the transformant can be carried out by the method of culturing the host cell that is the source. In the case where the transformant is a microorganism, for example, various media appropriately containing carbon sources, nitrogen sources, organic and inorganic salts, etc. commonly used in the culturing of microorganisms can be used for culturing. Here, as the carbon source, for example, glucose, dextrin, soluble starch, sucrose, etc. are listed. As the nitrogen source, for example, inorganic substances such as ammonium salts and nitrates, and organic substances such as corn steep liquor, peptone, casein, meat extract, soybean meal, and potato extract are listed. As the inorganic substances, for example, calcium chloride, sodium dihydrogen phosphate, and magnesium chloride are listed. Yeast extract, vitamins, growth promoting factors, etc. can be further added to the medium.

[0151] By culturing the transformant, a culture containing the polypeptide according to this embodiment is obtained. The polypeptide according to this embodiment can be accumulated, for example, in and / or outside the transformant (for example, in the culture supernatant of the transformant). The polypeptide according to this embodiment can be obtained from the transformant and / or its culture supernatant. The polypeptide according to this embodiment can be a polypeptide obtained by appropriately disrupting, dissolving, extracting, and purifying the transformant. Disruption, dissolution, extraction, etc. can be carried out by known methods. As such methods, for example, ultrasonic disruption method, dyno milling method, bead disruption, French press, lysozyme treatment are listed. These methods can be used alone for 1 type, or 2 types or more can be appropriately combined for use. Purification can be carried out by known methods used in the purification of polypeptides. As such methods, for example, ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration chromatography, isoelectric point precipitation are listed. These methods can be used alone for 1 type, or 2 types or more can be appropriately combined for use. The transformant can be recovered from the culture by centrifugation, etc. In the case where the polypeptide according to the present invention is accumulated in the culture supernatant, the culture supernatant can be obtained by centrifugation, etc., and the polypeptide according to the present invention can be recovered from the culture supernatant. The polypeptide according to this embodiment can be any fraction of the culture supernatant of the transformant.

[0152] The polypeptide according to this embodiment can also be obtained by culturing at least 1 kind of lactic acid bacterium selected from the deposit numbers NITE - BP - 03198, NITE BP - 03200, and NITE BP - 03201 in an appropriate medium such as MRS liquid medium. The polypeptide according to this embodiment can be recovered, extracted, and purified from the culture of the above-mentioned lactic acid bacterium by the same method as the culture of the transformant.

[0153] As bacteria having the gene of the polypeptide involved in the present embodiment, NITE-BP-03198, NITE-BP-03200, and NITE-BP-03201 are listed. NITE-BP-03198, NITE-BP-03200, and NITE-BP-03201 are bacteria internationally deposited based on the Budapest Treaty at the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (NPMD, Address: Room 122, 2-5-8 Kamigoura, Kisarazu-shi, Chiba-ken, 292-0818, Japan) under the deposit numbers NITE BP-03198 (Original deposit date: April 9, 2020), deposit number NITE BP-03200 (Original deposit date: April 9, 2020), and deposit number NITE BP-03201 (Original deposit date: April 9, 2020), respectively. All of the above bacteria are bacteria belonging to Lactiplantibacillus plantarum, which is a kind of lactic acid bacteria.

[0154] Example

[0155] Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples.

[0156] [Experiment 1: Prediction of Antimicrobial Peptides by Genome Analysis of Lactiplantibacillus plantarum (NITE BP-03198, BP-03200, or NITE BP-03201)]

[0157] For each of the three kinds of Lactiplantibacillus plantarum (NITE BP-03198, BP-03200, or NITE BP-03201), long-read sequence data were obtained using a PacBio RSII sequencer (Pacific Biosciences) and short-read sequences were obtained using a NovaSeq6000 sequencer (Illumina). The obtained short sequence data and long sequence data were subjected to hybrid assembly processing using Unicycler to obtain the genomic sequences of each of the three kinds of lactic acid bacteria. Thereafter, gene prediction was performed on the obtained genomic sequences using DFAST, retrieval of homologous sequences of known antimicrobial peptides was performed using HMMER, and retrieval of genes related to secondary metabolites was performed using antiSMASH to obtain base sequences and amino acid sequences that are candidates for antimicrobial peptides. The results are shown in Table 1. In Table 1, the expressions (y = c) and (y = t) respectively mean that the base indicated by y in the base sequence is cytosine and thymine. In Table 1, the expressions (X = A) and (X = V) respectively mean that the amino acid residue indicated by X in the amino acid sequence is alanine and valine.

[0158] [Table 1]

[0159]

[0160] [Experiment 2: Homology search results (amino acid sequence)]

[0161] In order to determine whether the antimicrobial peptides inferred from genome analysis are new peptides, a homology search with the amino acid sequences of known peptides included in BLAST was performed. (The query is the amino acid sequence of the sequence without the leader peptide. Database: All non-redundant GenBank CDS translations + PDB + SwissProt + PIR + PRF excluding environmental samples from WGS projects). As a result, the antimicrobial peptides inferred from genome analysis are mostly similar to hypothetical proteins, and their homology is 65% to 100%. Regarding Locus_29660, Locus_03310, and Locus_03320, it was inferred to be bacteriosin based on the gene sequence, but there are no reports on such peptides. Regarding Locus_32840, 4 amino acid residues are different from mutacin1140. Based on these results, the peptides inferred from genome analysis are considered to be new antimicrobial peptides.

[0162] [Experiment 3: Isolation / Purification of Antibacterial Substances]

[0163] Purification of active ingredients

[0164] Purification of the active ingredient is carried out in the following order. Each of the above-mentioned 3 lactic acid bacteria is inoculated into MRS medium and cultured under the conditions of 24 hours and 30 °C to obtain each culture solution. Each of the obtained culture solutions is centrifuged at 6000 g for 10 minutes to obtain each culture supernatant. Each of the obtained culture supernatants is further filtered through a cellulose acetate membrane with a pore size of 0.22 μm to remove the bacteria. By adding ammonium sulfate to 500 mL of the lactic acid bacteria culture supernatant to a final concentration of 80% and stirring overnight at 6 °C, the protein containing the antibacterial peptide is precipitated. By centrifuging at 13000 g for 60 minutes, the precipitated protein is recovered and redissolved in 50 mL of 50 mM sodium phosphate buffer (pH 5.6; buffer A). Subsequently, the solution of the redissolved protein is used for an AKTA pure Fraction Collecter (GE Healthcare) connected to Hitrap SP FF 1 mL (GE Healthcare) for fractionation. Regarding the mobile phase, buffer A is used for mobile phase A, and 50 mM sodium phosphate buffer (pH 5.6) containing 1 M NaCl is used for mobile phase B. The flow rate during fractionation is 1 mL / min. As the gradient condition, the initial mobile phase composition is 0% of mobile phase B, the final composition is 100% of mobile phase B, and the gradient time is set to 10 minutes. The fractions are recovered at 1 mL each.

[0165] Among them, the active fraction is further purified using an AKTA pure Fraction Collecter (GE Healthcare) connected to 1 mL RESOURCE PRC (GE Healthcare). Regarding the mobile phase, 0.1% formic acid aqueous solution is used for mobile phase A, and 0.1% formic acid and ethanol are used for mobile phase B. The flow rate during purification is 1 mL / min. As the gradient condition, the initial mobile phase composition is 0% of mobile phase B, the final composition is 100% of mobile phase B, and the gradient time is set to 10 minutes. The fractions are recovered at 1 mL each. The purified active fraction is stored at -30 °C. The antibacterial activities of the fractions obtained in each purification step are evaluated as follows.

[0166] Antibacterial activity test

[0167] The antibacterial activity of the antibacterial peptide is evaluated using the spot-on-lawn method in which 10 μL of the test solution is spotted at 13 points on the lawn of the test bacterium (on soft agar medium 11) ( Figure 1 ). Lactobacillus sakei (NBRC15893) is used as the test bacterium. The specific procedure is as follows. First, on an MRS plate containing 2% agar (agar medium 12), the test bacterium cultured overnight is adjusted to 4×10 7Mix and dilute and layer in a manner of the density of CFU / mL with Lactobacilli AOAC agar (soft agar medium 11). Pipette the test solution 13, and after overnight incubation at 30 °C, evaluate the size of the antibacterial zone 14 ( Figure 1 ). The larger the size of the antibacterial zone, the higher the antibacterial activity can be judged. In the above antibacterial activity test, the fraction with the highest antibacterial activity was used as the purified active fraction and applied in Experiments 4 and 5 described below.

[0168] [Experiment 4: Determination of the structure of the antibacterial peptide 1]

[0169] Take 30 μl of the ethanol solution (purified active fraction) containing the target antibacterial peptide, evaporate it to dryness, and then redissolve it with a 50% methanol solution to make it 10 μl. The determination of the redissolved purified active fraction was carried out by liquid chromatography-tandem mass spectrometry (LC / MS / MS) using electrospray ionization method at the interface. The liquid chromatography and the autosampler used Ultimate3000 RSLCnano (Thermo Fisher Scientific). Mobile phase A was an aqueous solution of 0.5% formic acid and 0.05% trifluoroacetic acid (TFA), and mobile phase B was an acetonitrile solution of 0.5% formic acid and 0.05% TFA. The flow rate during the analysis was 400 nL / min. As the gradient condition, the initial mobile phase composition was 5% of mobile phase B, the final composition was 90% of mobile phase B, and the gradient time was set to 40 minutes. As the analytical column, NTCC-360 / 100-3-125 (Nikkyotechnos) was used. The mass spectrometer used orbitrap elite (Thermo Fisher Scientific), and the MS spectrum of the full-length peptide was obtained in the full scan mode. In addition, the mass (theoretical value) of the peptide calculated from Experiment 1 above was compared with the spectral data (measured value) obtained by MS analysis, and when confirmed to exist, the putative sequence was recorded. The results are shown in the column of "Determination method 1" in Table 2.

[0170] [Experiment 5: Determination of the structure of the antibacterial substance 2]

[0171] Trypsin digestion

[0172] Regarding the digestion of the peptide as the target, it was carried out according to the conventional method. That is, 10 μl of the fraction with antibacterial activity (purified active fraction) was mixed with 10 μl of 2× sample buffer. After separation by Tris-Tricin SDS Page (both from Invitrogen), the band around a molecular weight of 3000 was cut out. At this time, the above-mentioned 2× sample buffer used "Novex Tricine SDS Sample Buffer". The gel for SDS Page used "Novex 10 to 20%, Tricine, 1.0 mm, MiniProtein Gels 15 well". For the cut gel, after reduction alkylation with iodoacetamide (Thermo Fisher Scientific), trypsin digestion was carried out at 37°C for 16 hours. The digestion used Sequencing Grade Modified Trypsin (Promega). Thereafter, the digested peptide was extracted by shrinking the above gel with acetonitrile. The extract was desalted and analyzed by MS. The MS analysis was carried out under the same conditions as in Experiment 4 above, but the gradient time was changed to 30 minutes, and the data acquisition method was changed to the Data Dependent Acquisition (DDA) mode to obtain MS spectra and MS / MS spectra.

[0173] Mascot search

[0174] The protein frameworks obtained by genomic analysis were converted into FASTA files, and each strain was read into MASCOT (Matrix Science). This database was compared with the MS / MS spectrum data and MS spectrum data obtained by measurement (tolerance: precursor 30 ppm, fragment 0.8 Da, maximum charge 2+). Antimicrobial peptide candidates were selected according to the following: those with high reliability of the score calculated by Proteome discoverer (Thermo Fisher Scientific), those that meet the condition of being a peptide (about 50 amino acid residues or less), and those that are not nucleoproteins, etc. The results are shown in the column of "Determination method 2" in Table 2. Regarding the case where an antimicrobial peptide with an internally consistent sequence was detected, it was expressed as +, and regarding the case where it was not detected, it was left blank. Even in the case of non-detection, it does not deny the inclusion of the peptide in the purified active fraction.

[0175] [Table 2]

[0176]

[0177] [Experiment 6: Antibacterial activity of synthetic peptides]

[0178] The chemically synthesized peptides (full-length sequences and partial sequences) used in this experiment were those synthesized by solid-phase synthesis purchased from Hokkaido System Science. For each of the chemically synthesized peptides, the antibacterial activity test was carried out in the same manner as in Experiment 3 above, and the minimum concentration at which an inhibition zone was formed ( Figure 2 ) was determined. At this time, the test solution was prepared by dissolving it in water so as to reach the target concentration. The results are shown in Table 3. In Table 3, the "alone" column represents the minimum concentration at which antibacterial activity is shown when the antibacterial peptide being targeted is applied alone. The "combination" column represents the minimum concentrations of the two antibacterial peptides being targeted when the two antibacterial peptides are combined in equimolar amounts and antibacterial activity is shown.

[0179] From the results in Table 3, it was found that the combined application of the polypeptide with sequence number 3 and the polypeptide with sequence number 4 showed stronger antibacterial activity than when each was applied alone. It was found that the antibacterial activity of the polypeptide with sequence number 13 and the polypeptide with sequence number 14 increased by combined application compared to when each was applied alone.

[0180] [Table 3]

[0181]

[0182] [Experiment 7: Stability test over a wide pH range]

[0183] Regarding the polypeptides of SEQ ID NOs: 1 to 5, 13 and 14 in Table 3, it was evaluated whether they have antibacterial activity in a wide pH range. As a comparative example, commercially available nisin A (manufactured by Sigma Aldrich) was used. First, each peptide was redissolved in the following buffer solutions at a concentration 10 times the minimum concentration shown in Table 3: sodium phosphate buffer (50 mM, pH 4), 1x phosphate buffered saline (pH 7.4), ammonium chloride - ammonia buffer (50 mM, pH 10.0). The test solutions prepared at each pH were incubated at 27°C for 24 hours. Then, Lactobacillus sakei (NBRC 15893) strain was used as the test bacterium, and the antibacterial activity test was carried out in the same manner as in Experiment 3, and the antibacterial activity of each test solution incubated at each pH was measured. As a control, a test solution in which the target peptide was diluted in water was used. The antibacterial activity was evaluated by the size of the spot (the size of the inhibition zone). Compared with the size of the spot of the control, those with the same size (the diameter of the spot is 80% or more) were designated as A, those with a smaller size but remaining antibacterial activity were designated as B. Those without an inhibition zone confirmed were designated as C. Peptides evaluated as A or B can be judged to have stability at the corresponding pH. The results are shown in Table 4 (in the column of pH stability). In the column of "Peptide SEQ ID NO or Substance Name" in Table 4, "SEQ ID NO 3 & SEQ ID NO 4" and "SEQ ID NO 13 & SEQ ID NO 14" respectively mean that the two target peptides were applied in an equimolar amount combination. From the results in Table 4, it was found that the polypeptides of SEQ ID NOs: 1 to 5, 13 and 14 have stable antibacterial activity in a wide pH range.

[0184] [Experiment 8: Thermal Stability Test]

[0185] Regarding the polypeptides with sequence numbers 1 to 5, 13, and 14 in Table 3, it was evaluated whether they also have antibacterial activity after heat treatment. As a comparative example, commercially available nisin A (manufactured by Sigma Aldrich) was used. First, each peptide was redissolved in water at a concentration 10 times the minimum concentration shown in Table 3. The prepared test solutions were each heat-treated at 80°C for 30 minutes, at 100°C for 30 minutes, or at 121°C for 15 minutes. The heat treatment at 121°C was carried out using an autoclave "LSX-700" manufactured by Tommy Seiko Co., Ltd. Then, Lactobacillus sakei (NBRC15893) strain was used as the test bacterium, and the antibacterial activity test was carried out in the same manner as in Experiment 3 to measure the antibacterial activity of the test solutions after each heat treatment. The sample solution without heat treatment was used as a control. The antibacterial activity was evaluated by the size of the dots (the size of the inhibition zone), and compared with the size of the dots of the control. Those with the same size (the diameter of the dots is 80% or more) were expressed as A, those with a smaller size but remaining activity were expressed as B. Those without confirmed dots were expressed as C. Peptides evaluated as A or B can be judged to have stability at the corresponding temperature. The results are shown in Table 4 (in the column of thermal stability). In the column of "peptide sequence number or substance name" in Table 4, "sequence number 3 & sequence number 4" and "sequence number 13 & sequence number 14" respectively mean that the two target peptides were applied in an equimolar amount combination. From the results in Table 4, it was found that the polypeptides with sequence numbers 1 to 5, 13, and 14 have antibacterial activity in the temperature range of 80°C to 121°C.

[0186] [Table 4]

[0187]

[0188] [Experiment 9: Antibacterial Activity Test of New Antibacterial Peptides]

[0189] Regarding the polypeptides with sequence numbers 1 to 5, 13, and 14 in Table 3, it was tested against which microorganisms they show antibacterial activity. As a comparative example, commercially available nisin A (manufactured by Sigma Aldrich) was used. First, each peptide was dissolved in MRS medium at a concentration ranging from 20 μM to 1000 μM. 10 μl of this was taken out and mixed with various test bacteria and 90 μl of the culture medium for the test bacteria, and cultured for 24 to 48 hours. After culturing, OD was measured for the number of bacteria 630 . Compared with the culture solution before culturing, OD 630In cases where turbidity increases or is visually observed, it can be judged that the test bacteria have proliferated. The results are shown in Table 5. In cases where the test bacteria have not proliferated, it is judged that there is antibacterial activity, which is expressed as + in Table 5. In the column of "Peptide Sequence Number or Substance Name" in Table 5, "Sequence Number 3 & Sequence Number 4" and "Sequence Number 13 & Sequence Number 14" respectively mean that two kinds of peptides to be targeted are applied in an equimolar amount combination. According to the results in Table 5, the polypeptides of Sequence Numbers 1 to 5, 13, and 14 are effective against Gram-positive bacteria, and some of the polypeptides are also effective against Gram-negative bacteria such as Escherichia coli and fungi such as Candida albicans.

[0190] [Table 5]

[0191]

Claims

1. A polypeptide having antibacterial activity, comprising: an amino acid sequence having a sequence identity of 85% or more and 100% or less with respect to any one of SEQ ID NOs: 1 to 6, or an amino acid sequence in which 1 or several amino acid residues are deleted, substituted, inserted and / or added with respect to any one of SEQ ID NOs: 1 to 6.

2. A nucleic acid comprising a base sequence encoding the polypeptide having antibacterial activity according to claim 1.

3. The nucleic acid according to claim 2, comprising any one of SEQ ID NOs: 7 to 12.

4. A vector comprising the nucleic acid according to claim 2.

5. An in vitro translation template molecule comprising the nucleic acid according to claim 2 or claim 3.

6. The in vitro translation template molecule according to claim 5, which is RNA.

7. A transformant comprising the nucleic acid according to claim 2 or claim 3, or the vector according to claim 4.

8. A method for preparing a polypeptide having antibacterial activity, the method comprising: (A) a step of synthesizing the polypeptide having antibacterial activity according to claim 1 by chemical synthesis, (B) a step of translating the polypeptide having antibacterial activity from the in vitro translation template molecule according to claim 5, (C) a step of culturing the transformant according to claim 7, or (D) a step of culturing at least one lactic acid bacterium selected from Deposit Numbers NITE BP-03198, NITE BP-03200 and NITE BP-03201.

9. A combination of polypeptides having antibacterial activity, comprising a combination of a first polypeptide and a second polypeptide, or a combination of a third polypeptide and a fourth polypeptide, wherein the first polypeptide comprises: an amino acid sequence having a sequence identity of 85% or more and 100% or less with respect to the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence in which 1 or several amino acid residues are deleted, substituted, inserted and / or added with respect to the amino acid sequence of SEQ ID NO: 3, the second polypeptide comprises: an amino acid sequence having a sequence identity of 85% or more and 100% or less with respect to the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence in which 1 or several amino acid residues are deleted, substituted, inserted and / or added with respect to the amino acid sequence of SEQ ID NO: 4, the third polypeptide comprises: an amino acid sequence having a sequence identity of 85% or more and 100% or less with respect to the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence in which 1 or several amino acid residues are deleted, substituted, inserted and / or added with respect to the amino acid sequence of SEQ ID NO: 13, the fourth polypeptide comprises: an amino acid sequence having a sequence identity of 85% or more and 100% or less with respect to the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence in which 1 or several amino acid residues are deleted, substituted, inserted and / or added with respect to the amino acid sequence of SEQ ID NO:

14.

10. A combination of nucleic acids, comprising the respective base sequences encoding the combination of polypeptides having antibacterial activity according to claim 9, The combination of nucleic acids includes a combination of a first nucleic acid and a second nucleic acid, or a combination of a third nucleic acid and a fourth nucleic acid, wherein, the first nucleic acid includes a base sequence encoding the first polypeptide, the second nucleic acid includes a base sequence encoding the second polypeptide, the third nucleic acid includes a base sequence encoding the third polypeptide, the fourth nucleic acid includes a base sequence encoding the fourth polypeptide.

11. The combination of nucleic acids according to claim 10, wherein, the first nucleic acid includes the base sequence of SEQ ID NO: 9, the second nucleic acid includes the base sequence of SEQ ID NO: 10, the third nucleic acid includes the base sequence of SEQ ID NO: 15, the fourth nucleic acid includes the base sequence of SEQ ID NO:

16.

12. A vector, which includes the combination of nucleic acids according to claim 10.

13. An in vitro translation template molecule, which includes the combination of nucleic acids according to claim 10 or claim 11.

14. The in vitro translation template molecule according to claim 13, which is RNA.

15. A transformant, which includes the combination of nucleic acids according to claim 10 or claim 11, or the vector according to claim 12.

16. A method for preparing a combination of polypeptides having antibacterial activity, the method comprising: (A) separately synthesizing each of the polypeptides in the combination of polypeptides according to claim 9 by chemical synthesis, (B) separately translating each of the polypeptides in the combination of polypeptides by the in vitro translation template molecule according to claim 13, (C) culturing the transformant according to claim 15, or (D) culturing Lactobacillus sp. with the deposit number NITE BP-03200.

17. An antibacterial composition, which includes: the polypeptide having antibacterial activity according to claim 1, or the combination of polypeptides having antibacterial activity according to claim 9, and an additive.

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