JP2019122301A - Method for producing keratin fraction - Google Patents

Abstract

To provide a water-soluble keratin fraction of a polymer useful as materials for various products.SOLUTION: A method for producing a keratin fraction includes hydrolyzing a substrate containing keratin with M23A subfamily protease.SELECTED DRAWING: None

Description

  The present invention relates to a method of producing keratin fragments.

  Keratin is a protein constituting animal hair, nails, skin and the like. BACKGROUND ART Keratin has been attracting attention and used as a raw material of various products such as feeds, fertilizers, surfactants, films, fibers, cosmetics, medical products, and as research subjects. Natural keratin is a poorly soluble polymeric substance with a crosslinked structure. Therefore, in conventional industrial use of keratin, solubilized or reduced-molecularized keratin is used as a raw material. Solubilized or low molecular weight keratins are generally keratins by hydrolyzing natural keratins with acid, alkali, enzymes or the like, or by reductive cleavage of keratin disulfide bonds to thiol groups to form reduced keratins, etc. Manufactured by solubilization.

  Patent Document 1 discloses that the reduced keratin is produced by limiting hydrolysis of a reduced keratin with a proteolytic enzyme such as trypsin, having an average molecular weight of 3,000 to 30,000, and 4 to 16 cysteines per 100 amino acid residues. Keratin fragments are disclosed. The keratin fragments have a strength suitable for processing into films, fibers, etc., have crosslinkable thiol groups, and are less toxic to the human body, less irritating, and therefore materials of films, fibers and cosmetic groups It is also disclosed that it is suitable as a material or the like.

  Patent Document 2 discloses solubilization of a keratin protein into an α-helix region or a fragment thereof having the epitope region for an immune reaction with an antibody having an epitope in the α-helix region of keratin. Have been described. In this procedure, proteases such as urea and chymotrypsin are used for solubilization of keratin, but optimization of proteolysis conditions and termination of degradation reaction at appropriate timing so that the epitope is maintained in the solubilized fragment Is required.

JP-A-6-116300 Unexamined-Japanese-Patent No. 2-6756

  The present invention relates to a method for producing keratin fragments, for producing high-molecular keratin fragments useful as raw materials and research samples of various products such as feeds, fertilizers, surfactants, films, fibers, cosmetics and medical products.

  The inventors have found that hydrolysis of keratin by the M23A subfamily protease produces a polymeric water-soluble keratin fragment in which the α-helix region (rod region) is maintained. Furthermore, in the keratinolytic reaction by the M23A subfamily protease, since the keratin is not decomposed into small fragments over time, the rod region is maintained without stopping the enzymatic reaction. It has been found that high molecular weight, water-soluble keratin fragments can be obtained.

Thus, the present invention provides a method of producing keratin fragments, comprising hydrolyzing a substrate comprising keratin with M23A subfamily protease.
The present invention also provides a keratin fragmentation agent comprising M23A subfamily protease as an active ingredient.

  According to the present invention, polymeric water-soluble keratin fragments having rod regions useful as raw materials for various products and as a research sample can be produced. Since the M23A subfamily protease can produce keratin fragments from any of the keratin water soluble fraction and the water insoluble or poorly soluble fraction, according to the present invention, efficient utilization of natural keratin resources is required. it can. In addition, since the M23A subfamily protease does not fragment keratin to a size smaller than a certain size, according to the present invention, high molecular weight keratin fragments can be obtained without having to go through the step of stopping the enzyme reaction. Therefore, according to the present invention, it is possible to efficiently and conveniently produce high molecular weight keratin fragments useful for industrial use.

  Furthermore, the keratin fragments obtained by the method of the present invention are water soluble, have a rod region, and retain the epitope of anti-keratin antibody. Therefore, the keratin fragments of the present invention can be quantified and analyzed by various methods using antibodies, UV and the like. Furthermore, since the keratin fragments obtained by the method of the present invention mainly have a rod region, they can be used as research samples for clarifying the function of the rod region or head or tail region of keratin. .

Image detected by western blotting of keratin solution extracted from collar and sleeve dirt. SDS-PAGE image of a trypsin or BLP treated keratin solution. The SDS-PAGE image of the supernatant of the reaction solution of collar sleeve stain and BLP. The SDS-PAGE image and the detection image by western blotting of the keratin solution extracted from the dead corneum. SDS-PAGE image of keratin solution treated with M23 family enzyme. SDS-PAGE image of BLP-treated keratin solution. SDS-PAGE image of enzyme-treated stratum corneum insoluble fraction. Amino acid sequence of keratin 10 Bold letters indicate the peptide sequences identified by mass spectrometry of Example 5.

  In one aspect, the invention provides a method of producing keratin fragments using the M23A subfamily protease.

  Keratin is a protein constituting animal hair, nails, skin and the like. An α-helix region (rod region) is present in the central portion of the keratin molecule, and a head and tail region having a non-helix structure are present at the N and C terminal portions, respectively. Epidermal keratin is rich in glycine and serine in the head and tail regions (protein nucleic acid enzyme 38 (16) 1993, p 2711-2722). Regions rich in glycine such as the head and tail regions of this keratin have a flexible structure and are reported to be more hydrophobic (Korge. BP et. Al, Proc. Natl. Acad. Sci. Vol. 89, 1992, p 910-914, Molecular Biology of the Skin The Keratinocyte, 1993, p 164). On the other hand, the rod region of keratin is considered to be structurally more stable and more hydrophilic compared to full length keratin including flexible and hydrophobic head and tail regions.

  The M23A subfamily protease is a MEROPS database [http: // merops. sanger. ac. uk] in the proteolytic enzyme family described in the present invention, a protease belonging to the M23A subfamily, which is a subfamily of metalloproteases belonging to the M23 family. The MEROPS database classifies the enzymes into families or subfamilies based on the methods described in the following articles: Nucleic Acids Res, 1999, 27: 325-331, J Struct Biol, 2001, 134: 95-102. , Nucleic Acids Res, 2016, 44: D343-D350. In release 11.0 of the MEROPS database, the M23A subfamily includes beta-lytic metalloproteinases (BLPs) (MEROPS ID: M23.001), LasA protein (Las A protein; LAS), also called staphylolysin. Three proteases, (MEROPS ID: M23.002) and Aeromonas hydrophila proteinase (AhP) (MEROPS ID: M23.003), which are also called Mername-AA 291 peptidase, exist as Holotypes.

  Preferably, the M23A subfamily protease in the present specification is at least one selected from the group consisting of β-lytic metalloprotease (BLP), LasA protein (LasA or LAS), and Aeromonas hydrophila proteinase (AhP). Say In the present invention, any one of BLP, LAS and AhP may be used, and any two or three of them may be used in combination. Preferably, the M23A subfamily protease used in the present invention comprises BLP, more preferably BLP.

  [beta] -lytic metalloprotease (BLP), LasA protein (LasA or LAS), Aeromonas hydrophila proteinase (AhP) are enzymes having the activity of degrading glycine-glycine bond in the peptide sequence. Preferred examples of BLP include a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2. Another example of BLP is an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 2, preferably H22, D36, H121 and H123 in the amino acid sequence shown in SEQ ID NO: 2 Included are polypeptides comprising an amino acid sequence having the corresponding amino acid residue and having glycine-glycine bond degradation activity in the peptide sequence. A preferred example of LAS is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4. Another example of LAS is an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 4, preferably H23, D36, H120 and H122 in the amino acid sequence shown in SEQ ID NO: 4 Included are polypeptides comprising an amino acid sequence having the corresponding amino acid residue and having glycine-glycine bond degradation activity in the peptide sequence. A preferred example of AhP is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6. Another example of AhP is an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 6, preferably H21, D34, H118 and H120 of the amino acid sequence shown in SEQ ID NO: 6 Included are polypeptides comprising an amino acid sequence having the corresponding amino acid residue and having glycine-glycine bond degradation activity in the peptide sequence.

  In the present specification, "at least 80% identity" with respect to a nucleotide sequence or amino acid sequence is 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, still more preferably It refers to an identity of 97% or more, more preferably 98% or more, and even more preferably 99% or more.

  In the present specification, identity between nucleotide sequences or amino acid sequences can be calculated by the Lippman-Person method (Lipman-Pearson method; Science, 1985, 227: 1435-41). Specifically, analysis using Unit size to compare (ktup) as 2 using a homology analysis (Search homology) program of genetic information processing software Genetyx-Win (Ver. 5.1.1; software development) It can be calculated by

  In the present specification, the “corresponding position” on the amino acid sequence and nucleotide sequence refers to the conservation that the target sequence and the reference sequence (eg, the amino acid sequence shown in SEQ ID NO: 2) are present in each amino acid sequence or nucleotide sequence It can be determined by aligning the amino acid residues or nucleotides so as to give the greatest homology. Alignment can be performed using known algorithms, the procedures of which are known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Thompson, J. D. et al, 1994, Nucleic Acids Res., 22: 4673-4680) with default settings. Alternatively, Clustal W2 or Clustal omega, which is a revised version of Clustal W, can be used. Clustal W, Clustal W2 and Clustal omega are, for example, Japanese DNA data operated by the European Bioinformatics Institute (European Bioinformatics Institute: EBI [www.ebi.ac.uk/index.html]) and the National Institute of Genetics It can be used on the bank (DDBJ [www.ddbj.nig.ac.jp/Welcome-j.html]) website.

  The position of the amino acid residue or nucleotide of the target sequence aligned at the position corresponding to any position of the reference sequence by the above alignment is regarded as the “corresponding position” at any position. When the amino acid residue at the "corresponding position" on the target sequence is the same as that of the reference sequence, it is regarded as the "corresponding amino acid residue" to the amino acid residue of the reference sequence. For example, if the amino acid residue at a position corresponding to H22 of the amino acid sequence of SEQ ID NO: 2 on the target sequence is H, it is an amino acid residue corresponding to H22 of the amino acid sequence of SEQ ID NO: 2.

  The above-mentioned M23A subfamily protease can be extracted or prepared from a microorganism containing it or a culture thereof. For example, BLP can be extracted or prepared from Lysobacter sp. (NBRC 12725 or NBRC 12726), Achromobacter lyticus M497-1, Lysobacter sp. IB-9374, Lysobacter gummosus DSMZ 6980, etc., or cultures thereof , Pseudomonas aeruginosa PA01, Pseudomonas aeruginosa ATCC 10145, Pseudomonas aeruginosa FRD1 etc., or an extract thereof can be prepared from a culture thereof, and AhP is Aeromonas hydrophila subsp. Hydrophila ATCC 7966, Or can be extracted or prepared from the culture. The above microorganism can be purchased from a public microorganism preservation organization. The microorganism containing the M23A subfamily protease may be cultured under appropriate conditions using a medium containing assimilable carbon sources, nitrogen sources, metal salts, vitamins and the like. From the thus obtained microorganism or culture solution, the enzyme can be collected and prepared by a general method, and further, the required enzyme form can be obtained by lyophilization, spray drying, crystallization and the like. For example, recovery and preparation of the enzyme from the culture may be performed by separation of the recombinant microorganism by centrifugation or filtration, precipitation of the enzyme in the supernatant or filtrate by adding a salt such as ammonium sulfate, or an organic solvent such as ethanol. It can be carried out using a usual method such as precipitation by addition, concentration using an ultrafiltration membrane or the like, desalting, purification using various chromatographies such as ion exchange or gel filtration, and the like.

  Alternatively, the M23A subfamily protease can be produced by chemical synthesis or biological methods using the amino acid sequence described above. For example, by culturing a microorganism transformed to express the chemically synthesized M23A subfamily protease gene based on the amino acid sequence, the M23A subfamily protease can be obtained by preparing an enzyme of interest from the microorganism or the culture. . As such a transformed microorganism, for example, a microorganism obtained by introducing the M23A subfamily protease gene operably linked to the control region into the genome of a host cell or into a plasmid, a target gene at an appropriate position And the like.

  In the present specification, “operably linking” the target gene and the control region means that the control region and the target gene are on DNA so that the function of controlling gene expression by the control region acts on the target gene. It says to arrange. As a method of operably linking a target gene and a control region, there is a method of linking the target gene downstream of the control region.

  In the present specification, the “control region” of a gene has a function of controlling the expression in the cell of a gene downstream of the region, and preferably has a function of constitutively expressing or highly expressing a downstream gene. It is an area. Specifically, it can be defined as a region located upstream of the coding region in the gene and having a function to interact with RNA polymerase to control transcription of the gene. Preferably, the control region of a gene in the present specification refers to a region of about 200 to 600 nucleotides upstream of the coding region in the gene. The control region includes a transcription initiation control region and / or a translation initiation control region of a gene, or a region from the transcription initiation control region to the translation initiation control region. The transcription initiation regulatory region is a region including a promoter and a transcription initiation point, and the translation initiation regulatory region is a site corresponding to the Shine-Dalgarno (SD) sequence that forms a ribosome binding site with the initiation codon (Shine, J., Dalgarno , L., Proc. Natl. Acad. Sci. USA., 1974, 71: 1342-1346).

  An expression vector containing the M23A subfamily protease gene is capable of stably retaining the gene, capable of maintaining replication in a host microorganism, and capable of stably expressing the M23A subfamily protease, comprising the M23A subfamily. It can be produced by incorporating a protease gene. Such vectors include pUC18, pBR322, pHY300 PLK, etc. when using E. coli as a host, and when using B. subtilis as host, pUB110, pHSP64 (Sumitomo et al., Biosci. Biotechnol. Biocem., 1995, 59: 2172-). 2175) or pH Y 300 PLK (Takara Bio).

  Transformation of a host microorganism can be performed using a protoplast method, a competent cell method, an electroporation method or the like. The host microorganism is not particularly limited, but includes gram-positive bacteria such as Bacillus (eg Bacillus subtilis), gram-negative bacteria such as Escherichia coli, and fungi such as Streptomyces, Saccharomyces (yeast) and Aspergillus (mold). Be A preferred host is a microorganism of the same or the same genus as that of the M23A subfamily protease gene introduced into the host, E. coli, or Bacillus bacteria, more preferably a Bacillus bacteria. Furthermore, Bacillus bacteria are preferred which produce less protease than the introduced M23A subfamily protease. Preferred examples include Bacillus subtilis strains in which one or more genes selected from aprE, nprB, nprE, vpr, mpr, epr and wprA of Bacillus subtilis have been deleted or inactivated, more preferably aprX, aprE, nprB, Examples include eight-fold deletion strains in which eight genes of nprE, vpr, mpr, epr and wprA have been deleted.

  The resulting transformant may be cultured under appropriate conditions using a medium containing assimilable carbon sources, nitrogen sources, metal salts, vitamins and the like. From the thus obtained microorganism or culture, the enzyme can be collected and prepared by a general method, and further, the required enzyme form can be obtained by lyophilization, spray drying, crystallization and the like. For example, recovery and preparation of the enzyme from the culture involves separation of the recombinant microorganism by centrifugation or filtration, precipitation of the enzyme in the supernatant or filtrate by addition of a salt such as ammonium sulfate or an organic solvent such as ethanol It can be carried out using a conventional method such as precipitation by itself, concentration using an ultrafiltration membrane or the like, desalting, purification using various chromatography such as ion exchange or gel filtration, or the like.

  Alternatively, the M23A subfamily protease can be prepared from an enzyme composition or the like containing it. For example, BLP can be prepared from achromopeptidase. Achromopeptidase is a lytic enzyme from Lysobacter enzymogenes and contains BLP. Achromopeptidase is commercially available from Wako Pure Chemical Industries, Ltd. and the like.

  In the method of producing a keratin fragment of the present invention, a substrate containing keratin is hydrolyzed with M23A subfamily protease.

  Any material containing keratin can be used as a substrate material comprising keratin for use in the method of the present invention. Examples of keratin-containing substances include natural keratin-containing substances such as animal hair, nails, skin, horns, wrinkles, wrinkles, feathers and epithelial cells, preferably birds and feathers such as chickens, and Cattle hair, horse hair, goat hair, wool, alpaca, mohair, animal hair such as angora and cashmere, human skin and its exfoliates, animal skin and the like. Among them, more preferable examples include human skin and exfoliates thereof and animal hides, and further preferable examples include human-derived skin and wrinkles, pig skin, sheepskin, lambskin, goatskin, deerskin, horsehide, Cordovan etc. are mentioned. Other examples of keratin-containing substances include processed products of the above-mentioned natural keratin-containing substances, such as duvets, woolen duvets, muttons, woolen fabrics, leather goods, and clothing such as down jackets, sweaters, etc. . As a raw material of the keratin-containing substrate used in the method of the present invention, any one or more of the above-mentioned keratin-containing substances can be used. However, the raw material of the matrix containing the said keratin is not limited to these.

  The substance containing keratin may be used as a substrate containing keratin in the method of the present invention as it is or, if necessary, appropriately subjected to grinding or pulverization, defatting treatment, or solubilization treatment with an activator or denaturant. Can. Alternatively, a keratin fraction separated from the keratin-containing substance may be used as a substrate containing keratin in the method of the present invention. In order to separate the keratin fraction from the substance containing keratin, for example, the substance containing keratin is pulverized or pulverized, defatted, or solubilized with an active agent or denaturant, followed by filtration, chromatography, solvent It may be separated by extraction, lyophilization, salting out, TCA precipitation, protein precipitation methods such as acetone precipitation, or the like. The keratin fraction used as a substrate comprising keratin in the method of the present invention may be a water soluble fraction or a water insoluble or poorly soluble fraction. Preferably, the keratin fraction is a fraction containing keratin containing a glycine rich region, more preferably a fraction containing keratin 10 (ACCESSION_AAH34697, SEQ ID NO: 26) or a fragment having the glycine rich region and a rod region. It is a minute.

  In the present specification, the glycine rich region has a sequence (-Gly-Gly-) in which at least two or more glycines are continuous in a region consisting of any 20 consecutive amino acids in a protein having a molecular weight of 25,000 or more, And a region having 10 or more glycines.

  Alternatively, reduced keratin obtained by reducing the keratin-containing substance may be used as a substrate containing keratin. The reduced keratin can be prepared from the above-mentioned keratin-containing substance according to a known method (for example, the method described in Patent Document 1). For example, after a substance containing keratin is reduced with a reducing agent in the presence of a protein denaturing agent or in the presence of a protein denaturing agent and a surfactant to remove insolubles, salting out of the obtained aqueous solution, dialysis The reduced keratin can be prepared by the like. The reducing agent may be any one capable of reducing the disulfide bond of keratin to convert it into a thiol group. For example, thiol compounds such as 2-mercaptoethanol, thioglycolic acid, dithiothreitol, dithioerythritol, etc .; Organophosphorus compounds such as propyl phosphine and tributyl phosphine; and inorganic compounds having a reducing ability such as sodium hydrogen sulfite. Examples of protein denaturants include urea and thiourea.

  In the method of the present invention, the M23A subfamily protease used for hydrolysis of the substrate containing keratin is preferably at least one selected from the group consisting of BLP, LAS and AhP. More preferably, the M23A subfamily protease used in the present invention is BLP.

  For hydrolysis of the keratin-containing substrate, an enzyme composition containing M23A subfamily protease can also be used. Preferably, the enzyme composition contains at least one selected from the group consisting of BLP, LAS and AhP. More preferably, the enzyme composition contains BLP.

  The content of the keratin-containing substrate in the reaction solution of the hydrolysis reaction is preferably 0.001 to 50% by mass, more preferably 0.01 to 20% by mass, and still more preferably 0.01 to 5% by mass. is there.

  The amount of M23A subfamily protease in the reaction solution of the hydrolysis reaction is preferably 0.000001 to 10% by mass, more preferably 0.0001 to 1% by mass, and still more preferably 0.001 to 0.1% by mass. is there.

  The temperature conditions of the hydrolysis reaction of the substrate containing the keratin by the M23A subfamily protease are preferably 0 to 90 ° C., more preferably 20 to 50 ° C. Further, the time of the hydrolysis reaction is preferably 1 minute or more, more preferably 30 minutes or more. As shown in the examples below, the M23A subfamily proteases do not fragment keratin into smaller than a certain size, even after prolonged reaction. Therefore, in the method of the present invention, it is not necessary to limit the upper limit of the time of hydrolysis reaction. From the economical point of view, the time of the hydrolysis reaction is preferably 1 to 3000 minutes, more preferably 1 to 600 minutes, still more preferably 1 to 60 minutes, and still more preferably 30 to 60 minutes.

  The hydrolysis produces keratin fragments. The generated keratin fragments can be recovered from the reaction solution by any method. In a preferred embodiment, the method for producing keratin fragments of the present invention further comprises recovering the keratin fragments obtained by the hydrolysis. As a method for recovering keratin fragments from the reaction solution, a general procedure for polypeptide isolation or purification, for example, filtration, centrifugation, concentration, microfiltration, ultrafiltration, separation by chromatography, etc., lyophilization And protein precipitation methods such as salting out, TCA precipitation, and acetone precipitation.

  As mentioned above, the M23A subfamily protease does not fragment keratin to a size smaller than a certain extent. Therefore, in the method for producing a keratin fragment of the present invention, after the hydrolysis of the keratin-containing substrate by the M23A subfamily protease, the treatment for stopping the enzyme reaction may not be performed. However, in the method of the present invention, it is also possible to carry out treatment to stop the enzyme reaction after the hydrolysis. As the treatment for stopping the enzyme reaction, for example, addition or reaction of reaction terminator (for example, reducing agent such as 1, 10-phenanthroline, 2-mercaptoethanol, dithiothreitol, chelating agent such as EDTA) to the reaction liquid The inactivation of the enzyme by heating the solution and the removal of the enzyme from the reaction solution can be mentioned. As a method of removing the enzyme from the reaction solution, a method of immobilizing the enzyme on a carrier in advance, performing a hydrolysis reaction of the substrate using the enzyme immobilized on the carrier, and removing the enzyme together with the carrier from the reaction solution after the reaction Can be mentioned.

The keratin fragments produced by the method of the present invention preferably have a molecular weight of about 25,000 to 50,000, more preferably about 37,000 to 50,000. As used herein, the "molecular weight" of a protein or polypeptide can be calculated, for example, by SDS-PAGE. A preferred molecular weight measurement procedure is described below: Prepare the migration sample by mixing the sample solution with 2 × Laemmli Sample Buffer in an equal amount with 50 mM dithiothreitol and heating at 100 ° C. for 5 minutes. The electrophoresis sample the Any kD ^TM Mini PROTEAN (TM) TGX ^TM precast gels and electrophoresis buffer (25 mM Tris, 192 mM glycine, 0.1% (w / v) SDS, pH8.3) to electrophoresis using . At this time, the molecular weight markers using the Precision Plus Protein ^TM 2-color standard. The gel after electrophoresis is stained with CBB or ruby. The value obtained by substituting the mobility of the target protein or polypeptide into the formula of a calibration curve that can be prepared from the molecular weight and mobility of the molecular weight marker is taken as the molecular weight of the target protein or polypeptide. Here, the mobility (Rf value) of the sample is defined as the distance from the top of the gel (or the position to which the sample is applied) to the preceding dye (bromophenol blue) in the lane in which the sample is flowed is 1. The relative value of the distance from the top of the gel (or the position to which the sample was applied) to the band of the sample is taken.

  The keratin fragments produced by the method of the present invention are water soluble. Therefore, it can be recovered in a soluble state without using a hydrophilic solubilizer or denaturant. Therefore, keratin fragments produced by the method of the present invention are difficult to analyze if they contain solubilizers or denaturants (eg analysis by dynamic light scattering, circular dichroism (CD) spectroscopy, absorbance Analysis by measurement of

  Keratin fragments produced by the method of the present invention have a rod region. Preferably, the keratin fragments produced by the method of the invention consist mainly of the rod area. As an example of the rod area | region contained in the keratin fragment | piece produced | generated by the method of this invention, the area | region which consists of an amino acid sequence shown by amino acid numbers 157-450 of sequence number 26 is mentioned. Examples of keratin fragments produced by the method of the present invention include keratin fragments comprising the amino acid sequence shown by amino acid numbers 157 to 450 of SEQ ID NO: 26 and having a molecular weight of about 37,000 to 50,000. . Since the rod region of keratin is used as an epitope of anti-keratin antibody (see Patent Document 2), the keratin fragment of the present invention having a rod region is useful for analysis using an antibody. In addition, the keratin fragments of the present invention, which are mainly composed of rod regions, are structurally more stable, and thus are useful for analysis using X-rays, for example, X-ray crystal structure analysis. In conventional X-ray analysis of keratin, it is difficult to obtain highly accurate data because the orientation in full-length keratin crystal is incomplete (polymer, 53, (2), 2004, p74-78). Thus, keratin fragments produced by the method of the present invention are useful as tools for the analysis of keratins.

  As described above, M23A subfamily protease can be used to produce keratin fragments. In other words, the M23A subfamily protease can be used as an active ingredient for keratinization. Thus, in a further aspect, the present invention provides keratin fragmentation agents comprising the M23A subfamily protease. In a further aspect, the invention provides the use of the M23A subfamily protease for the production of a keratin fragmentation agent. In a further aspect, the invention provides the use of the M23A subfamily protease for keratinization. In a further aspect, the invention provides a kit for keratin fragmentation comprising the M23A subfamily protease.

  In a further aspect of the invention described above, the keratin fragmentation is achieved by hydrolysis of the substrate comprising keratin by the M23A subfamily protease. The kind of the substrate containing keratin, the amount of the substrate and the enzyme used for the hydrolysis reaction, and the conditions of the hydrolysis reaction are the same as in the method for producing the keratin fragment of the present invention described above. In the above-mentioned further aspect of the present invention, the M23A subfamily protease is preferably at least one selected from the group consisting of BLP, LAS and AhP, more preferably BLP. Alternatively, an enzyme composition containing M23A subfamily protease can also be used for keratinization. In a further embodiment of the invention as described above, said keratin fragmentation produces high molecular weight, water soluble keratin fragments, preferably having a molecular weight of about 25,000 to 50,000, more preferably about 37,000 to 50,000. Be done. The keratin fragments have a rod area, preferably mainly consisting of a rod area. An example of the keratin fragment includes a keratin fragment comprising the amino acid sequence shown by amino acid numbers 157 to 450 of SEQ ID NO: 26 and having a molecular weight of about 37,000 to 50,000.

  The kit for keratinization fragmentation of the present invention may further contain other reagents, solvents and the like that may be contained in a reaction solution for hydrolysis of a substrate containing keratin, as necessary. The M23A subfamily protease in the kit may be in the form of a premix mixed with a reagent or solvent for the reaction solution.

  The present invention also includes the following substances, production methods, uses, methods and the like as exemplary embodiments. However, the present invention is not limited to these embodiments.

[1] A method for producing a keratin fragment, comprising hydrolyzing a substrate containing keratin with M23A subfamily protease.
[2] The method according to [1], which preferably comprises hydrolyzing the keratin-containing substrate with an enzyme composition containing M23A subfamily protease.
[3] The method according to [1] or [2], preferably, wherein the M23A subfamily protease is at least one selected from the group consisting of β-lytic metalloprotease, LasA protein, and Aeromonas hydrophila proteinase .
[4] The method according to any one of [1] to [3], wherein the molecular weight of the keratin fragment is preferably 25,000 to 50,000, more preferably 37,000 to 50,000.
[5] Preferably, the method according to any one of [1] to [4], wherein the keratin fragment is water soluble.
[6] The keratin fragment is
Preferably having a rod area,
More preferably, it comprises the amino acid sequence shown by amino acids 157 to 450 of SEQ ID NO: 26,
The method according to any one of [1] to [5].
[7] Preferably, the substrate containing keratin is selected from the group consisting of animal hair, nails, skin, horns, eyebrows, eyelids, epithelial cells and feathers, their processed products, and keratin fractions separated therefrom The method according to any one of [1] to [6], which is at least one of
[8] Preferably, the method according to any one of [1] to [7], wherein the keratin contained in the substrate is a water-soluble keratin.
[9] Preferably, the method according to any one of [1] to [7], wherein the keratin contained in the substrate is a water insoluble or poorly soluble keratin.
[10] The method according to any one of [1] to [7], wherein the keratin-containing substrate preferably contains any of keratin 1 to 20, more preferably keratin 10.
[11] Preferably, the method according to any one of [1] to [10], further comprising recovering keratin fragments obtained by the hydrolysis.
[12] Preferably, the method according to any one of [1] to [11], wherein the treatment for stopping the enzyme reaction is not performed after the hydrolysis.

[13] A keratin fragmentation agent comprising M23A subfamily protease as an active ingredient.
[14] The keratin fragmentation agent according to [13], which preferably contains an enzyme composition containing the M23A subfamily protease as an active ingredient.
[15] The keratin according to [13] or [14], preferably, wherein the M23A subfamily protease is at least one selected from the group consisting of β-lytic metalloprotease, LasA protein, and Aeromonas hydrophila proteinase. Fragmentation agent.
[16] The keratin fragmentation according to any one of [13] to [15], wherein the molecular weight of the fragmentation keratin is preferably 25,000 to 50,000, more preferably 37,000 to 50,000. Agent.
[17] Preferably, the keratin fragmentation agent according to any one of [13] to [16], wherein the fragmented keratin is water soluble.
[18] The fragmented keratin is
Preferably having a rod area,
More preferably, it comprises the amino acid sequence shown by amino acids 157 to 450 of SEQ ID NO: 26,
The keratin fragmentation agent according to any one of [13] to [17].
[19] Preferably, the keratin fragmentation agent according to any one of [13] to [18], wherein the keratin is a water-soluble keratin.
[20] The keratin fragmentation agent according to any one of [13] to [18], which is preferably a water-insoluble or poorly soluble keratin.
[21] The keratin fragmentation agent according to any one of [13] to [18], wherein the keratin preferably contains any of keratin 1 to 20, more preferably keratin 10.

[22] A keratin fragment comprising the amino acid sequence shown by amino acid numbers 157 to 450 of SEQ ID NO: 26 and having a molecular weight of 37,000 to 50,000.

  Hereinafter, the present invention will be more specifically described using examples. However, the technical scope of the present invention is not limited to these examples.

  A list of primers used in this example is shown in Table 1.

Reference Example 1 Preparation of Protease (1) Preparation of Culture Supernatant Containing BLP (1-1) Preparation of Expression Vector The BLP gene (SEQ ID NO: 1) was inserted into plasmid pUC57 (BLP / pUC57) artificially prepared by GenScript Inc. Made using gene synthesis service. A PCR reaction was performed using BLP / pUC57 as a template and the primer pair BLP_S237signal_F / BLP_S237signal_R (SEQ ID NOS: 9 and 10) and PrimeSTAR Max Premix (Takara Bio). Similarly, PCR reaction was performed using the plasmid pHY-S237 described in Example 7 of WO2006 / 068148 A1 as a template and the primer pair vector-F / vector-sig-R (SEQ ID NOS: 11 and 12). Each PCR product was subjected to DpnI treatment with DpnI (New England Biolabs). Subsequently, In-Fusion reaction was performed according to the protocol of In-Fusion, HD Cloning kit (Clontech).

ECOS ^TM Competent E. using In-Fusion reaction E. coli DH5 alpha (Nippon Gene, 310-06236) was transformed. Transformed cells were plated on LB plates containing ampicillin and cultured at 37 ° C. overnight. Colonies formed on the plate are inoculated into LB medium containing ampicillin and cultured overnight, then the cells are recovered and plasmid (BLP / pHY) is extracted using High Pure Plasmid Isolation Kit (Roche) did. A PCR reaction was performed using the extracted BLP / pHY as a template and the primer pair ΔS237N_fw / ΔS237N_rv (SEQ ID NOS: 13 and 14). This PCR product was purified by E. coli. E. coli HST08 Premium Competent Cells (Takara Bio) were transformed. Transformed cells were plated on LB plates containing ampicillin and cultured at 37 ° C. overnight. The colonies formed on the plate are inoculated into LB medium containing ampicillin and cultured overnight, and then the cells are recovered and the plasmid (BLP2 / pHY) is extracted using High Pure Plasmid Isolation Kit (Roche). did.

(1-2) Preparation of an enzyme-producing transformant strain Bacillus subtilis 168 strain (Bacillus subtilis Marburg No. 168 strain: Nature, 390, 1997, p. 249) is inoculated in 1 mL of LB medium, and the mixture is cultured at 30 ° C. and 200 rpm. Shake culture overnight. 10 μL of this culture solution was inoculated in 1 mL of fresh LB medium and cultured at 37 ° C., 200 rpm for 3 hours. The culture was centrifuged to recover the pellet. 500 μL of SMMP (0.5 M sucrose, 20 mM disodium maleate, 20 mM magnesium chloride hexahydrate, 35% (w / v) Antibiotic Medium 3 (Difco)) containing 4 mg / mL lysozyme (SIGMA) was added to the pellet And incubated at 37.degree. C. for 1 hour. The pellet was then collected by centrifugation and suspended in 400 μL of SMMP. 13 μL of suspension, 2 μL of plasmid BLP2 / pHY solution (10 mM Tris-HCl pH 8.5, 34.2 ng / μL) obtained in (1-1), and 20 μL of SMMP are mixed, and 100 μL of 40% PEG is further added and stirred. After further adding 350 μL of SMMP, it was shaken at 30 ° C. for 1 hour. 200 μL of this solution was added to DM3 regeneration agar medium containing 0.8 μg of tetracycline (15 μg / mL, SIGMA) [0.8% agar (Wako Pure Chemical Industries, Ltd.), 0.5% disodium succinate hexahydrate, 0.5% casamino acid technical ( Difco) 0.5% yeast extract, 0.35% potassium monophosphate, 0.15% potassium diphosphate, 0.5% glucose, 0.4% magnesium chloride hexahydrate, 0.01% bovine serum Albumin (SIGMA), 0.5% carboxymethylcellulose, 0.005% trypan blue (Merck) and amino acid mixed solution (tryptophan, lysine, methionine each 10 μg / mL); smeared on (w / v)%] 30 Incubate at 3 ° C for 3 days to obtain formed colonies.

(1-3) Enzyme Production by Transformant Strain Culture Tetracycline was added to LB medium to a final concentration of 15 ppm. The Bacillus subtilis transformant colony obtained in (1-2) was inoculated into 5 mL of this medium, and then cultured overnight at 30 ° C. and 250 rpm. The following day, 400 μL of this culture broth was mixed with 2 × L-maltose medium (2% tryptone, 1% yeast extract, 1% NaCl, 7.5% maltose, 7.5 ppm manganese sulfate pentahydrate, 15 ppm tetracycline, 6 ppm zinc sulfate heptahydrate %; (W / v)%) was inoculated into 20 mL and cultured at 32 ° C and 230 rpm for 2 days, and then the culture supernatant containing the enzyme produced from the cells was collected by centrifugation.

(2) Preparation of Culture Supernatant Containing LasA The LasA gene (SEQ ID NO: 3) was inserted into plasmid pUC57 (LasA / pUC57) was prepared using an artificial gene synthesis service from GenScript. PCR reaction was performed according to the protocol of PrimeSTAR Max Premix (Takara Bio) using LasA / pUC57 as a template and the primer pair LasA_F / LasA_CR (SEQ ID NOS: 15 and 16). The PCR reaction was carried out similarly using the plasmid pHY-S237 described in Example 7 of WO 2006/068148 A1 as a template and the primer pair pHY_just_F / pHY_just_R_NEW (SEQ ID NOS: 17 and 18). Each PCR product was subjected to DpnI treatment with DpnI (New England Biolabs). Then, the plasmid (LasA / pHY) solution was obtained by performing In-Fusion reaction according to the protocol of In-Fusion, HD Cloning kit (Clontech).

  Using the obtained plasmid (LasA / pHY) solution, a Bacillus subtilis prsA gene expression-enhanced strain (prsA- prepared in Example 1 of JP-A-2007-49986) in the same manner as in (1-2) above. The Kc strain was transformed to obtain a Bacillus subtilis transformant colony. Tetracycline was added to 2 × L liquid medium to a final concentration of 15 ppm. A Bacillus subtilis transformant colony was inoculated into 5 mL of this medium, and then cultured overnight at 30 ° C. and 250 rpm. The pellet was recovered from the culture solution, and plasmid LasA / pHY was extracted from the pellet. PCR reaction, digestion of the plasmid with Dpn I, and ligation using the extracted plasmid LasA / pHY as a template and the primer pair LasA_Chis_n_F / LasA_Chis_n_R (SEQ ID NOS: 19 and 20) and KOD-Plus-Mutagenesis Kit (TOYOBO) To obtain a plasmid (LasA2 / pHY).

  Using the obtained plasmid (LasA2 / pHY), transformation was performed in the same manner as in (1-2) above. At this time, a Bacillus subtilis prsA gene expression-enhanced strain (prsA-Kc strain prepared in Example 1 of JP-A-2007-49986) was used as a host. Subsequently, the obtained transformed strain was cultured in the same procedure as (1-3), and the culture supernatant containing the enzyme produced from the cells was recovered.

(3) Preparation of culture supernatant containing AhP An AhP gene (SEQ ID NO: 5) was inserted into a plasmid pUC57 (AhP / pUC57) was prepared using an artificial gene synthesis service of GenScript. PCR reaction was performed according to the protocol of PrimeSTAR Max Premix (Takara Bio) using AhP / pUC57 as a template and the primer pair 2F / 2R_bacillus-Chis (SEQ ID NOS: 21 and 22). A PCR reaction was performed in the same manner using plasmid pHY-S237 described in Example 7 of WO2006 / 068148 A1 as a template and primer pair vector-F / vector-R (SEQ ID NOS: 11 and 23). Each PCR product was subjected to DpnI treatment with DpnI (New England Biolabs). Then, the plasmid (AhP / pHY) solution was obtained by performing In-Fusion reaction according to the protocol of In-Fusion, HD Cloning kit (Clontech).

  Using the obtained plasmid (AhP / pHY), transformation was performed in the same manner as in (1-2) above. At this time, Bacillus subtilis strain 168 was used as a host. Subsequently, the obtained transformed strain was cultured in the same procedure as (1-3), and the culture supernatant containing the enzyme produced from the cells was recovered.

(4) Preparation of culture supernatant containing ALE-1 ALE-1 glycocylycine endopeptidase (ALE-1) (MEROPS ID: M23.012; SEQ ID NO: 8), which is a M23B subfamily protease, was prepared. The ALE1 gene (SEQ ID NO: 7) was inserted into plasmid pUC57 (ALE1 / pUC57) was constructed using an artificial gene synthesis service from GenScript. PCR reaction was performed using ALE1 / pUC57 as a template and the primer pair pHY-like6-just-F / pHY-like6-just-CHisR (SEQ ID NOS: 24 and 25) and PrimeSTAR Max Premix (Takara Bio). The PCR reaction was carried out similarly using the plasmid pHY-S237 described in Example 7 of WO 2006/068148 A1 as a template and the primer pair pHY_just_F / pHY_just_R_NEW (SEQ ID NOS: 17 and 18). Each PCR product was subjected to DpnI treatment with DpnI (New England Biolabs). Then, the plasmid (ALE1 / pHY) solution was obtained by performing In-Fusion reaction according to the protocol of In-Fusion, HD Cloning kit (Clontech).

  The obtained plasmid (ALE1 / pHY) was used to transform Bacillus subtilis 168 strain in the same manner as in (1-2) above to obtain a Bacillus subtilis transformant colony. Tetracycline was added to 2 × L liquid medium to a final concentration of 15 ppm. A Bacillus subtilis transformant colony was inoculated into 5 mL of this medium, and then cultured overnight at 30 ° C. and 250 rpm. The pellet was recovered from the culture solution, and the plasmid ALE1 / pHY was extracted from the pellet. Using the extracted plasmid (ALE1 / pHY), transformation was performed in the same manner as the above (1-2). At this time, Bacillus subtilis Dpr9 strain (Kao9 strain prepared in Examples 1 to 5 of JP-A-2006-174707) was used as a host. The obtained transformant was cultured in the same manner as in (1-3), and the culture supernatant containing the enzyme produced from the cells was collected.

(5) Preparation of Protease from Culture Supernatant The target protease was prepared from the culture supernatant obtained in (1) to (4). The culture supernatant was buffer exchanged with Buffer A using Amicon Ultra Fractionated molecular weight 10 K (Merck Millipore). The enzyme was prepared from the solution after buffer exchange using AKTA explorer 10S (GE Healthcare). First, the solution obtained by the buffer exchange was passed through column 1, and then the adsorbed components of column 1 were eluted using Buffer B. Among the eluted fractions, fractions in which the degradation activity of FRET-GGGGG (Reference Example 5) was observed were collected. Subsequently, the collected fraction solution is subjected to Size Exclusion Chromatography using column 2 equilibrated with a solution of 20 mM Tris-HCl (pH 7.5) and 200 mM NaCl, and a fraction solution in which the degradation activity of FRET-GGGGG is observed Was collected. The collected fraction was buffer-exchanged with 20 mM Tris-HCl (pH 7.5) solution using Amicon Ultra Fractionated molecular weight 10 K to obtain an enzyme solution containing the target protease. The buffer A, buffer B, column 1 and column 2 used for each culture supernatant were as shown in Table 2.

Reference Example 2 Concentration Measurement of Enzyme Solution A DC protein assay kit (Bio-Rad) was used to measure the concentration of the enzyme solution. BSA Standard Solution (WAKO) was used as a standard solution for protein amount calculation.

Reference Example 3 SDS-PAGE
The migration sample was prepared by mixing an equal amount of sample solution with 2 × Laemmli Sample Buffer (Bio-Rad, # 161-0737) containing 50 mM dithiothreitol (Thermo Fisher Scientific), and heating at 100 ° C. for 5 minutes. The electrophoresis sample was electrophoresed using the Any kD ^TM Mini PROTEAN (TM) TGX ^TM precast gels (Bio-Rad) and 10-fold diluted 10 × Tris / glycine / SDS (Bio-Rad, 1610732 ). The molecular weight marker Precision Plus Protein ^TM 2-color standard (Bio-Rad, # 161-0374) was used. The gel after electrophoresis was stained with CBB or ruby. Bio-Safe CBB G-250 stain (Bio-Rad, 161-0786) was used for CBB staining. One-step Ruby (APRO life Science, SP-4040) was used for ruby staining. The value obtained by substituting the mobility of the target protein or polypeptide into the formula of a calibration curve that can be prepared from the molecular weight and the mobility of the molecular weight marker was used as the molecular weight of the target protein or polypeptide. The mobility (Rf value) of the sample is the upper end of the gel when the distance from the upper end of the gel (the position to which the sample is applied) to the preceding dye (bromophenol blue) in the lane in which the sample was run is 1. It measured as a relative value of the distance from the sample to the band of the sample.

Reference Example 4 Preparation of Keratin Sample (1) Collection of Collar-Sleeved Dirt-Attached Fabric A region of a shirt collar was sewed on a fabric (composition: polyester 65%, cotton 35%). The collar and sleeve dirt-adhered cloth was recovered by allowing an adult man to wear this shirt for 3 days during the day.

(2) Preparation of Keratin solution The collar and sleeve soiling attachment cloth obtained in (1) is shredded, and 2 × Laemmli Sample Buffer (Bio-Rad, # 161-0737) containing 50 mM dithiothreitol (Thermo Fisher Scientific). And heated at 100 ° C. for 10 minutes, and centrifuged to recover the supernatant. Using this supernatant, SDS-PAGE was performed according to the procedure described in Reference Example 3 (however, addition of 2 × Laemmli Sample Buffer, heating, and staining were not performed). After shaking the gel after SDS-PAGE in deionized water, the gel having a molecular weight of 75,000 to 100,000 was excised using a marker as a guide. The excised gel was crushed with a spoonful and shaken overnight in 20 mM Tris-HCl (pH 7.5). The solution after shaking removed gel using Atprep MF (ATTO, 3521370). The solution after gel removal was concentrated using Amicon Ultra Fractionated molecular weight 10 K (Merck Millipore) to obtain a keratin solution. The protein concentration of the obtained keratin solution was 0.14 mg / mL as measured with Protein A 280 using a Nanodrop ND-1000 Spectrophotometer (Thermo Fisher Scientific).

(3) Evaluation of Keratin Fraction The keratin fraction contained in the keratin solution obtained in (2) was evaluated by Western blotting using an anti-keratin antibody. For Western blotting, after being subjected to SDS-PAGE in the procedure described (unstained) keratin solution in Reference Example 3, the gel transblotted Turbo ^TM system (Bio-Rad) and transblotted Turbo ^TM Mini PVDF transfer pack (Bio- It transferred to the PVDF membrane using Rad, # 170-4156). Ibind Western Device (Thermo Fisher Scientific) was used for the antibody reaction. KRT10 monoclonal antibody (M01), clone 1H6 (Abnova) as primary antibody, Stabilized Peroxidase Conjugated Goat Anti-Mouse (H + L) as secondary antibody (Thermo Fisher Scientific, # 32430), ECL Select Western Blotting Detection Reagent (Detection reagent as detection reagent) GE Healthcare was used. Western blotting confirmed that the keratin solution prepared in (2) contained keratin 10 (FIG. 1).

Reference Example 5 Measurement of Enzymatic Activity As a substrate, a FRET substrate [hereinafter FRET-GGGGG] (made-to-order manufactured by P Japan Ltd.) in which between the fluorescent group Nma and the quenching group Lys (Dnp) is pentaglycine was used. Here, Nma refers to 2- (N-methylamino) benzoyl (Nma). In addition, Lys (Dnp) refers to one having 2,4-dinitrophenyl (Dnp) in the side chain of lysine (Lys). Add 190 μL of the enzyme solution (enzyme, 20 mM Tris-HCl (pH 7.5)) obtained in Reference Example 1 (5) to a 96-well assay plate (AGC Technoglass, 3881-096), and further add the FRET-GGGGG solution ( The reaction solution was prepared by adding 10 μL of 1 mM FRET-GGGGG, 100 mM Tris-HCl (pH 7.5). The fluorescence intensity of the reaction solution was measured over time using an infinite M 200 (TECAN) at a temperature of 30 ° C., an excitation wavelength of 340 nm, and a measurement wavelength of 440 nm. The reaction solution using FRETS-25-STD1 (Peptide Research Laboratories, 3720-v) dissolved in 20 mM Tris-HCl pH 7.5 instead of enzyme solution and dimethylsulfoxide instead of FRET-GGGGG under the same reaction conditions The fluorescence intensity was measured and a calibration curve was prepared. The activity of 1 unit (U) was the amount of enzyme required to show the change in fluorescence intensity corresponding to the fluorescence intensity with 1 nmol of FRETS-25-STD1 per minute.

Example 1 Degradation of Keratin by Trypsin and BLP 1 μL of the enzyme solution was added to 30 μL of the keratin solution prepared in Reference Example 4. The enzyme used was trypsin (SIGMA, T1426-100MG) or BLP prepared in Reference Example 1. The final concentration of enzyme was adjusted to 2 μg / mL. The reaction solution was allowed to stand at 30 ° C. for 3 hours, and then SDS-PAGE (ruby staining) was performed according to the procedure described in Reference Example 3.

  The results are shown in FIG. In the sample to which trypsin was added, the keratin-derived band had disappeared. The bands of trypsinized keratin fragments were not detected, probably because the keratin was degraded to fragments of various molecular weights by trypsin and fell below the detection limit, or the fragments were depolymerized, so that the gel could not be detected. It was thought that it was due to spillage. On the other hand, in the sample to which BLP was added, a keratin fragment band was detected between the molecular weight of 37,000 markers and the molecular weight of 50,000 markers.

Example 2 Preparation of Keratin Fragment from Collar and Sleeve Soiling Attachment Fabric Three collar and sleeve soiling attachment fabrics obtained in Reference Example 4 (1) were immersed in 100 mL of 20 mM Tris-HCL (pH 7.5). After BLP prepared in Reference Example 1 was added thereto so that the final concentration was 3 μg / mL, the mixture was stirred with a magnetic stirrer for 3 hours. The solution was centrifuged (8,000 G, 15 minutes) to recover the supernatant. The collected solution was concentrated 40-fold with Amicon Ultra Molecular Weight cut off 10K (Merck Millipore), and then centrifuged (10,000 G, 10 minutes) to recover the supernatant. Using this supernatant, SDS-PAGE (CBB staining) was performed according to the procedure described in Reference Example 3.

  The results are shown in FIG. As in Example 1, a band was detected between the molecular weight of 37,000 markers and the molecular weight of 50,000 markers from the sample to which BLP was added. This was considered to be a keratin fragment produced by BLP from a water-soluble or insoluble keratin contained in the soiling cloth and a keratin complexed with a protein present in keratinocytes. That is, it has been shown that water-soluble keratin fragments can be generated by BLP from keratin-containing samples collected from a living body without performing addition of denaturing agents and activators and operations such as heating in advance.

Example 3 Evaluation of Keratinolytic Activity by Enzyme (1) Preparation of Keratin Solution The keratinolytic activity of various enzymes prepared in Reference Example 1 was examined. From human eyebrows, the stratum corneum was scraped using Velvet smooth electric horny remover diamond (Rekit Benkiza). Add 1 mL of solubilizer (100 mM Tris-HCl (pH 8.5), 2% SDS, 25 mM DTT, 5 mM EDTA) to 5 mg of stratum corneum, heat at 100 ° C for 10 minutes, and then centrifuge. Qing was recovered. The collected supernatant was used in dialysis buffer (20 mM Tris-HCl (pH 7.5), 0.1% SDS) using Mini Dialysation kit 8 kDa cut-off 2 mL (GE Healthcare, # 80-6484-32). It dialyzed against overnight to obtain a keratin solution.

(2) Evaluation of Keratin Fraction The obtained keratin solution was diluted 20-fold with 20 mM Tris-HCl (pH 7.5), and subjected to SDS-PAGE (CBB staining) and Western blotting according to the same procedure as in Reference Examples 3 to 4. It evaluated by. As a result of evaluation by SDS-PAGE and western blotting, it was confirmed that the keratin solution prepared from the stratum corneum contained keratin 10 (FIG. 4). Although a plurality of bands were detected in FIG. 4, they were considered to be complexed keratin 10, modified keratin 10, keratin 1 and other types of keratin, etc. in addition to monomeric keratin 10 It was done.

(3) Keratinolysis by enzymes 1 μL of the enzyme solution was added to 30 μL of a keratin solution diluted 20 fold with 20 mM Tris-HCl (pH 7.5). As enzymes, BLP prepared in Reference Example 1, LasA, AhP and ALE-1, and Lysostaphin (Wako, # 120-04313) were used. The final concentration of enzyme was adjusted so that BLP was 1 μg / mL and LasA, AhP, ALE-1 and Lysostaphin were 10 μg / mL. The reaction solution was allowed to stand at 30 ° C. for 15 hours, and then evaluated by SDS-PAGE (CBB staining) according to the procedure of Reference Example 3.

  The results are shown in FIG. In the solution to which the M23A subfamily enzymes BLP, LasA or AhP were added, a band was detected between the molecular weight of 37,000 markers and the molecular weight of 50,000 markers, indicating that these enzymes degraded keratin . On the other hand, in the solution to which ALE-1 or Lysostaphin, which is an enzyme of the M23B subfamily, was added, no decomposition of keratin was observed. That is, it was shown that only enzymes of the M23A subfamily among the M23 family degrade keratin.

Example 4 Evaluation of Keratinine Degradability of BLP To 100 μL of a keratin solution prepared by the same procedure as Example 3, 1 μL of BLP (100 μg enzyme / mL) prepared in Reference Example 1 was added. The resulting solution was divided into two and each was allowed to stand at 30 ° C. for 8 hours. Thereafter, one solution is mixed in an equal amount with 2 × Laemmli Sample Buffer (Bio-Rad, # 161-0737) containing 25 mM dithiothreitol (Thermo Fisher Scientific), and then heated for 5 minutes at 100 ° C. to react. I stopped it. The other solution was further added with 1 μL of the same BLP (100 μg enzyme / mL) and allowed to stand at 30 ° C. for 15 hours. After standing, the sample is mixed in an equal amount with 2 × Laemmli Sample Buffer (Bio-Rad, # 161-0737) containing 25 mM dithiothreitol (Thermo Fisher Scientific), and then heated at 100 ° C. for 5 minutes to stop the reaction. I did. Each reaction solution was evaluated by SDS-PAGE (CBB staining, but addition of additional 2 × Laemmli Sample Buffer and heating were not performed) according to the procedure of Reference Example 3.

  The results are shown in FIG. Addition of BLP reduced the molecular weight of keratin in solution. In the solutions 8 hours and 15 hours after BLP addition, bands of keratin fragments were detected between the 37,000 marker and the 50,000 marker. On the other hand, the molecular weight of keratin fragments did not change in the solution 8 hours and 15 hours after BLP addition. From these, it was suggested that BLP has keratinolytic activity and that further degradation of keratin fragments located between 37,000 and 50,000 molecular weight does not proceed by BLP.

Example 5 Solubilization of Keratinocyte-Insoluble Fraction of Keratinocyte-Derived Insoluble Fraction by M23A Subfamily Protease Using 2 cm of tape (Nichiban, CT-24), keratin was obtained from human neck by tape stripping. A solubilization solution (100 mM Tris-HCl (pH 8.5), 2% SDS, 25 mM DTT, 5 mM EDTA) is added to this keratin, heated at 100 ° C. for 10 minutes, centrifuged and the supernatant removed The precipitate was obtained. The insoluble fraction derived from keratinocytes was obtained by further performing the operations of addition of the solubilization solution, heating and centrifugation to the precipitate twice. 40 μL of 20 mM Tris-HCl (pH 7.5) was added to the obtained insoluble fraction, and 1 μL of the enzyme solution was further added. As enzymes, BLP, LasA and AhP prepared in Reference Example 1 were used. The final concentration of enzyme was adjusted to 1 μg / mL for BLP and 10 μg / mL for LasA and AhP. The reaction mixture was allowed to stand at 30 ° C. for 15 hours, and then the supernatant was subjected to SDS-PAGE (ruby staining) according to the procedure of Reference Example 3.

  The results are shown in FIG. In the solutions to which BLP, LasA and AhP were added, a band was detected between the molecular weight of 37,000 marker and the molecular weight of 50,000 marker, and these enzymes degraded keratin in the insoluble fraction of stratum corneum into keratin fragments. It was shown that it did.

As shown in FIG. 7, on SDS-PAGE of the BLP-added solution, two bands were detected between the 37,000 marker and the 50,000 marker. Among these, the mass analysis of the low molecular weight band was performed. For mass spectrometry, MALDI-TOF Mass Spec Analysis of Rivers Co., Ltd. was used. This mass spectrometry service used trypsin for protein digestion. As a result of mass spectrometry, it was found that the protein contained in the low molecular weight band is a fragment of keratin 10 (ACCESSION_AAH34697). Also, as shown in FIG. 8, all peptide sequences identified by mass spectrometry (bold) correspond to the region present between amino acid numbers 157 and 450 of the amino acid sequence of keratin 10 (SEQ ID NO: 26). The According to the information disclosed in Keratin, type I cytoskeletal 10 [UniProt Knowledgebase_P13645], in the amino acid sequence of keratin 10, amino acid numbers 1 to 145 in the amino acid sequence is a head region, amino acid numbers 146 to 456 is a rod region, amino acid numbers 457 to 584 Is the tail region. Therefore, it was shown that the keratin fragments obtained in this example include the rod region of keratin 10.
From the above, BLP and other M23A subfamily enzymes cleave amino acid regions 1 to 156 or amino acids 451 to 584 of the amino acid sequence of keratin 10 (SEQ ID NO: 26) to obtain a keratinous insoluble fraction. It has been found to produce fragments comprising the rod area of keratin 10 contained in

  While the embodiments of the present invention have been described above, it should be understood that they are not intended to limit the present invention to the particular embodiments described. Various other changes and modifications within the scope of the present invention will be apparent to those skilled in the art. The documents and patent applications cited herein are incorporated by reference as if they were fully described herein.

Claims (21)

  A method for producing a keratin fragment, comprising hydrolyzing a substrate containing keratin with M23A subfamily protease.   The method according to claim 1, comprising hydrolyzing the keratin-containing substrate with an enzyme composition containing M23A subfamily protease.   The method according to claim 1 or 2, wherein the M23A subfamily protease is at least one selected from the group consisting of β-lytic metalloprotease, LasA protein, and Aeromonas hydrophila proteinase.   The method according to any one of claims 1 to 3, wherein the molecular weight of the keratin fragment is 25,000 to 50,000.   5. The method of any one of claims 1 to 4, wherein the keratin fragments are water soluble.   6. The method according to any one of the preceding claims, wherein the keratin fragments have a rod area.   The keratin-containing substrate is at least one selected from the group consisting of animal hair, nails, skin, horns, spines, spines, epithelial cells and feathers, processed products thereof, and keratin fractions separated therefrom The method according to any one of claims 1 to 6.   The method according to any one of claims 1 to 7, wherein the keratin contained in the substrate is a water-soluble keratin.   The method according to any one of claims 1 to 7, wherein the keratin contained in the substrate is water insoluble or poorly soluble keratin.   The method according to any one of claims 1 to 7, wherein the substrate comprising keratin comprises keratin 10.   The method according to any one of claims 1 to 10, further comprising recovering the keratin fragments obtained by the hydrolysis.   The method according to any one of claims 1 to 11, wherein the treatment for stopping the enzymatic reaction is not performed after the hydrolysis.   A keratin fragmentation agent comprising M23A subfamily protease as an active ingredient.   The keratin fragmentation agent according to claim 13, wherein the enzyme composition containing the M23A subfamily protease is an active ingredient.   The keratin fragmentation agent according to claim 13 or 14, wherein the M23A subfamily protease is at least one selected from the group consisting of β-lytic metalloprotease, LasA protein, and Aeromonas hydrophila proteinase.   The keratin fragmentation agent according to any one of claims 13 to 15, wherein the molecular weight of the fragmented keratin is 25,000 to 50,000.   The keratin fragmentation agent according to any one of claims 13 to 16, wherein the fragmented keratin is water soluble.   The keratin fragmentation agent according to any one of claims 13 to 17, wherein the fragmented keratin has a rod region.   The keratin fragmentation agent according to any one of claims 13 to 18, wherein the keratin is a water-soluble keratin.   The keratin fragmentation agent according to any one of claims 13 to 18, wherein the keratin is water insoluble or poorly soluble keratin.   The keratin fragmentation agent according to any one of claims 13 to 18, wherein the keratin is keratin 10.