JP7034724B2 - Method for producing keratin fragments - Google Patents

Description

The present invention relates to a method for producing a keratin fragment.

Keratin is a protein that makes up animal hair, nails, and skin. Conventionally, keratin has been attracting attention and used as a material for various products such as feeds, fertilizers, surfactants, films, fibers, cosmetics, and medical products, and as a research target. Natural keratin is a sparingly soluble polymer substance with a crosslinked structure. Therefore, in the conventional industrial use of keratin, solubilized or low molecular weight keratin is used as a material. Solubilized or low molecular weight keratin generally produces keratin by hydrolyzing natural keratin with an acid, alkali, enzyme, etc., or by reducing and cleaving the disulfide bond of keratin to a thiol group to produce reduced keratin. Manufactured by solubilization.

Patent Document 1 has an average molecular weight of 3,000 to 30,000 and 4 to 16 cysteines per 100 amino acid residues, which are produced by limitingly hydrolyzing reduced keratin with a proteolytic enzyme such as trypsin. A keratin fragment has been disclosed. Since the keratin fragment has suitable strength when processed into a film, fiber, etc., has a crosslinkable thiol group, and is less toxic and irritating to the human body, it is a material for films and fibers and a cosmetic group. It is also disclosed that it is suitable as a material or the like.

Patent Document 2 describes solubilizing a keratin protein into an α-helix region or a fragment thereof having an epitope region for an immune reaction with an antibody having an epitope in the α-helix region of keratin. Are listed. In this procedure, proteases such as urea and chymotrypsin are used to solubilize keratin, but proteolysis conditions are optimized and the degradation reaction is stopped at an appropriate timing so that the epitope is maintained in the solubilized fragment. Is required.

Japanese Unexamined Patent Publication No. 6-116300 Japanese Unexamined Patent Publication No. 2-6756

The present invention relates to a method for producing a keratin fragment for producing a polymer keratin fragment useful as a raw material for various products such as feed, fertilizer, surfactant, film, fiber, cosmetics, and medical products and as a research sample.

The present inventor has found that hydrolysis of keratin by the M23A subfamily protease produces a high molecular weight water-soluble keratin fragment in which the α-helix region (rod region) is maintained. Further, in the keratin decomposition reaction by the M23A subfamily protease, the present inventors did not decompose keratin into small fragments over time, so that the rod region was maintained even if the enzymatic reaction was not stopped. It has been found that a high molecular weight water-soluble keratin fragment can be obtained.

Therefore, the present invention provides a method for producing a keratin fragment, which comprises hydrolyzing a substrate containing keratin with an M23A subfamily protease.
The present invention also provides a keratin fragmentation agent containing an M23A subfamily protease as an active ingredient.

According to the present invention, it is possible to produce a polymer water-soluble keratin fragment having a rod region useful as a material for various products and as a research sample. Since the M23A subfamily protease can generate a keratin fragment from either a water-soluble fraction or a water-insoluble or sparingly soluble fraction of keratin, according to the present invention, it is possible to efficiently utilize natural keratin resources. can. Further, since the M23A subfamily protease does not fragment keratin to a size smaller than a certain level, according to the present invention, a high molecular weight keratin fragment can be obtained without a step of stopping the enzymatic reaction. Therefore, according to the present invention, it becomes possible to efficiently and easily produce a polymer keratin fragment useful for industrial use.

Furthermore, the keratin fragment obtained by the method of the present invention is water-soluble, has a rod region, and retains an epitope of an anti-keratin antibody. Therefore, the keratin fragment of the present invention can be quantified and analyzed by various methods using an antibody, UV, or the like. Furthermore, since the keratin fragment obtained by the method of the present invention mainly has a rod region, it can be used as a research sample for clarifying the function of the rod region, or the head or tail region of keratin. ..

Western blotting detection image of keratin solution extracted from collar sleeve stains. SDS-PAGE image of trypsin or BLP treated keratin solution. SDS-PAGE image of the supernatant of the reaction solution between the dirt on the collar sleeve and BLP. SDS-PAGE image of keratin solution extracted from heel keratin and detection image by Western blotting. SDS-PAGE image of keratin solution treated with M23 family of enzymes. SDS-PAGE image of BLP-treated keratin solution. SDS-PAGE image of enzyme-treated keratin insoluble fraction. Amino acid sequence of keratin 10. Bold type indicates the peptide sequence identified by mass spectrometry in Example 5.

In one aspect, the present invention provides a method for producing a keratin fragment using an M23A subfamily protease.

Keratin is a protein that makes up animal hair, nails, and skin. An α-helix region (rod region) is present in the central portion of the keratin molecule, and a head and tail regions 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, p2711-2722). Glycine-rich regions such as the head and tail regions of this keratin have been reported to have a flexible structure and are more hydrophobic (Korge.BP et.al, Proc.Natur.Acad.Sci.Vol. 89, 1992, p910-914, Molecular Biology of the Skin The Keratinicite, 1993, p164). On the other hand, the rod region of keratin is considered to be structurally more stable and highly hydrophilic than the full-length keratin including the flexible and hydrophobic head and tail regions.

The M23A subfamily protease is a MEROPS database [http: // merops. sanger. ac. In the proteolytic enzyme family described in uk], it refers to a protease belonging to the M23A subfamily, which is a subfamily of metalloproteases belonging to the M23 family. The MEROPS database classifies enzymes into families or subfamilies based on the methods described in the following papers: 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 β-lytic metalloproteinase (BLP) (MEROPS ID: M23.001), Las A protein (LAS), also known as Staphylolysin. (MEROPS ID: M23.002) and Aeromonas hydrophila proteinase (AhP) (MEROPS ID: M23.003), also called Mername-AA291 peptidase, are present as Holotypes.

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

β-Litic metalloprotease (BLP), LasA protein (LasA or LAS), Aeromonas hydrophilaproteinase (AhP) are enzymes that have the activity of degrading the 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 set forth in SEQ ID NO: 2, preferably in the amino acid sequences H22, D36, H121 and H123 set forth in SEQ ID NO: 2. Examples thereof include a polypeptide consisting of an amino acid sequence having a corresponding amino acid residue and having a glycine-glycine bond-degrading activity in the peptide sequence. Preferred examples of LAS include 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 set forth in SEQ ID NO: 4, preferably in the amino acid sequences H23, D36, H120 and H122 set forth in SEQ ID NO: 4. Examples thereof include a polypeptide consisting of an amino acid sequence having a corresponding amino acid residue and having a glycine-glycine bond-degrading activity in the peptide sequence. Preferred examples of AhP include 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 with the amino acid sequence set forth in SEQ ID NO: 6, preferably in the amino acid sequences H21, D34, H118 and H120 set forth in SEQ ID NO: 6. Examples thereof include a polypeptide consisting of an amino acid sequence having a corresponding amino acid residue and having a glycine-glycine bond-degrading activity in the peptide sequence.

As used herein, "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 97% or more, more preferably 98% or more, and even more preferably 99% or more identity.

As used herein, the identity between nucleotide sequences or amino acid sequences can be calculated by the Lipman-Pearson method (Science, 1985, 227: 1435-41). Specifically, the homology analysis (Search homology) program of the genetic information processing software Genetyx-Win (Ver. 5.1.; Software development) is used, and the analysis is performed with Unit size to homology (ktup) as 2. Can be calculated by

As used herein, the "corresponding position" on an amino acid sequence and a nucleotide sequence is a preservation in which the target sequence and the reference sequence (eg, the amino acid sequence represented by 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 to give maximum homology. Alignment can be performed using known algorithms and the procedure is known to those of skill in the art. For example, alignment can be done by using the Clustal W Multiple Alignment Program (Thompson, J.D. et al, 1994, Nucleic Acids Res., 22: 4673-4680) with default settings. Alternatively, a revised version of Clustal W, Clustal W2 or Clustal omega, can be used. Crystal W, Crystal W2 and Crystal omega are, for example, Japanese DNA data operated by the European Bioinformatics Institute (EBI [www.ebi.ac.uk/index.html]) and the National Institute of Genetics. It can be used on the website of the Bank (DDBJ [www.ddbj.nig.ac.jp/Welcome-j.html]).

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

The M23A subfamily proteases listed above can be extracted or prepared from the microorganisms containing them or their cultures. 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 a culture thereof, and LAS , Pseudomonas aeruginosa PA01, Pseudomonas aeruginosa ATCC 10145, Pseudomonas aeruginosa FRD1, etc., or can be extracted or prepared from its culture, AhP is Aeromonas hydrophila subsp. Hydrophila ATCC 7966, Aeromonas hydrophila (Chester) Stanier (ATCC) , Or its culture can be extracted or prepared. The above microorganisms can be purchased from public microorganism storage institutions. The microorganism containing the M23A subfamily protease may be cultured under appropriate conditions using a medium containing a carbon source, a nitrogen source, a metal salt, a vitamin and the like that can be assimilated. From the microorganism or culture solution thus obtained, an enzyme can be collected and prepared by a general method, and further, a required enzyme form can be obtained by freeze-drying, spray-drying, crystallization and the like. For example, the recovery and preparation of the enzyme from the culture may be performed by separating the recombinant microorganism by centrifugation or filtration, precipitating the enzyme in the supernatant or filtrate by adding a salt such as ammonium sulfate, or using an organic solvent such as ethanol. It can be carried out by using ordinary methods such as precipitation by addition, concentration and desalting using an ultrafiltration membrane, purification using various chromatographies such as ion exchange and gel filtration, and the like.

Alternatively, the M23A subfamily protease can be produced by chemical synthesis or biological techniques utilizing the amino acid sequences described above. For example, the M23A subfamily protease can be obtained by culturing a microorganism transformed to express the M23A subfamily protease gene chemically synthesized based on the amino acid sequence and preparing the desired enzyme from the microorganism or the culture. .. Such transformed microorganisms include, for example, a microorganism obtained by introducing an M23A subfamily protease gene operably linked to a control region into the genome or plasmid of a host cell, a target gene at an appropriate position. Examples thereof include microorganisms into which an expression vector incorporating the above is introduced.

In the present specification, "operably linking" a target gene and a control region means that the control region and the target gene are linked on DNA so that the function of controlling gene expression by the control region acts on the target gene. It means to arrange. Examples of the method for operably linking the target gene and the control region include a method of linking the target gene downstream of the control region.

As used herein, the "regulatory region" of a gene has a function of controlling the intracellular expression of a gene downstream of the region, and preferably has a function of constitutively expressing or highly expressing the downstream gene. It is an area. Specifically, it can be defined as a region that exists upstream of the coding region in the gene and has a function of interacting with RNA polymerase to control transcription of the gene. Preferably, the control region of a gene as used herein refers to a region of about 200 to 600 nucleotides upstream of the coding region of the gene. The regulatory 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 control region is a region containing a promoter and a transcription initiation site, and the translation initiation control region is a site corresponding to the Shine-Dalgarno (SD) sequence forming a ribosome binding site together with the start codon (Shine, J., Dalgarno). , L., Proc. Natl. Acad. Sci. USA., 1974, 71: 1342-1346).

An expression vector containing the M23A subfamily protease gene is a vector capable of stably retaining the gene, maintaining replication in a host microorganism, and stably expressing the M23A subfamily protease, in the M23A subfamily. It can be produced by incorporating a protease gene. Examples of such a vector include pUC18, pBR322, pHY300PLK and the like when Escherichia coli is used as a host, and pUB110 and pHSP64 (Sumitomo et al., Biosci. Biotechnol. Biocem., 1995, 59: 2172-) when Bacillus subtilis is used as a host. 2175) or pHY300PLK (Takara Bio) and the like.

Transformation of the host microorganism can be carried out by using a protoplast method, a competent cell method, an electroporation method or the like. The host microorganism is not particularly limited, and examples thereof include gram-positive bacteria such as Bacillus (for example, Bacillus subtilis), gram-negative bacteria such as Escherichia coli, Streptomyces, Saccharomyces (yeast), and fungi such as Aspergillus (mold). Will be. A preferred host is a microorganism of the same species or genus as the bacterium from which the M23A subfamily protease gene is introduced into the host, Escherichia coli, or a bacterium of the genus Bacillus, and more preferably a bacterium of the genus Bacillus. Further, Bacillus bacteria having low production of proteases other than the introduced M23A subfamily protease are preferable. Preferred examples are Bacillus subtilis strains in which one or more genes selected from the Bacillus subtilis aprE, nprB, nprE, vpr, mpr, epr and wprA have been deleted or inactivated, more preferably aprX, aprE, nprB, etc. Eight-fold deletion strains in which eight genes of nprE, vpr, mpr, epr and wprA have been deleted can be mentioned.

The obtained transformant may be cultured under appropriate conditions using a medium containing a carbon source, a nitrogen source, a metal salt, a vitamin and the like that can be assimilated. From the microorganism or culture thus obtained, an enzyme can be collected and prepared by a general method, and further, the required enzyme form can be obtained by freeze-drying, spray-drying, crystallization and the like. For example, for recovery and preparation of enzymes from cultures, separation of recombinant microorganisms by centrifugation or filtration, precipitation of enzymes in supernatants or filtrates by adding salts such as ammonium sulfate, or addition of organic solvents such as ethanol. This can be carried out by using ordinary methods such as precipitation, concentration and desalting using an ultrafiltration membrane, purification using various chromatographies such as ion exchange and gel filtration, and the like.

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

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

Any substance containing keratin can be used as a raw material for the substrate containing keratin used in the method of the present invention. Examples of keratin-containing substances include natural keratin-containing substances such as animal hair, claws, skin, horns, hoofs, scales, feathers, epithelial cells, preferably the feathers of birds such as chickens, and Examples thereof include cow hair, horse hair, goat hair, wool, alpaca, mohair, angora, animal hair such as cashmere, human skin and its exfoliated material, animal skin, and the like. Among these, more preferable examples include human skin and its exfoliated material, and animal skin, and further preferable examples are human-derived skin and dirt, pig skin, sheepskin, lambskin, goatskin, deerskin, horsehide, and the like. Examples include cordovan. Other examples of keratin-containing substances include processed products of the above-mentioned natural keratin-containing substances, such as duvets, wool duvets, moutons, woolen fabrics, leather products, and clothing such as down jackets and sweaters. .. As the raw material of the substrate containing keratin used in the method of the present invention, any one or more of the above-mentioned substances containing keratin can be used. However, the raw material of the substrate containing the keratin is not limited to these.

The keratin-containing substance may be used as a substrate containing keratin in the method of the present invention as it is or by appropriately pulverizing or pulverizing it, degreasing it, or solubilizing it with an activator or denaturing agent as necessary. Can be done. Alternatively, the keratin fraction separated from the keratin-containing substance may be used as the keratin-containing substrate in the method of the present invention. To separate the keratin fraction from the keratin-containing substance, for example, the keratin-containing substance is pulverized or pulverized, degreased, or solubilized with an activator or modifier, and then filtered, chromatographed, or solvent. Separation may be performed by a protein precipitation method such as extraction, freeze-drying, salting out, TCA precipitation, or acetone precipitation. The keratin fraction used as a substrate containing keratin in the method of the present invention may be a water-soluble fraction or a water-insoluble or sparingly soluble fraction. Preferably, the keratin fraction is a fraction containing keratin containing a glycine-rich region, and more preferably a fraction containing keratin 10 (ACCESSION_AAH34697, SEQ ID NO: 26) or a fragment having a glycine-rich region and a rod region thereof. Minutes.

As used herein, the glycine-rich region has a sequence (-Gly-Gly-) in which glycine is at least 2 consecutive in a region consisting of 20 consecutive amino acids in a protein having a molecular weight of 25,000 or more. And a region having 10 or more glycine.

Alternatively, the reduced keratin obtained by reducing the substance containing the keratin may be used as a substrate containing the keratin. The reduced keratin can be prepared from the above-mentioned substance containing keratin according to a known method (for example, the method described in Patent Document 1). For example, a substance containing keratin is reduced with a reducing agent in the presence of a protein denaturing agent or a protein denaturing agent and a surfactant to remove insoluble matters, and then salting out and dialysis from the obtained aqueous solution. Reduced keratin can be prepared by the above. The reducing agent may be any one that has the effect of reducing the disulfide bond of keratin and converting it into a thiol group. For example, a thiol compound such as 2-mercaptoethanol, thioglycolic acid, dithiothreitol, or dithioerythritol; Organic phosphorus compounds such as propylphosphin and tributylphosphine; inorganic compounds having a reducing ability such as sodium hydrogen sulfite, and the like can be mentioned. Examples of the protein modifier include urea, thiourea and the like.

In the method of the present invention, the M23A subfamily protease used for hydrolysis of the keratin-containing substrate 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.

An enzyme composition containing an M23A subfamily protease can also be used for hydrolysis of the keratin-containing substrate. 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 substrate containing the keratin 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 further preferably 0.01 to 5% by mass. be.

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, still more preferably 0.001 to 0.1% by mass. be.

The temperature condition for the hydrolysis reaction of the substrate containing the keratin by the M23A subfamily protease is preferably 0 to 90 ° C, more preferably 20 to 50 ° C. The time of the hydrolysis reaction is preferably 1 minute or longer, more preferably 30 minutes or longer. As shown in the examples below, the M23A subfamily protease does not fragment keratin to a size smaller than a certain degree even after long-term reaction. Therefore, in the method of the present invention, it is not necessary to limit the upper limit of the time of the hydrolysis reaction. From the viewpoint of economy, 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, still more preferably 30 to 60 minutes.

The hydrolysis produces keratin fragments. The generated keratin fragment can be recovered from the reaction solution by any method. In a preferred embodiment, the method for producing a keratin fragment of the present invention further comprises recovering the keratin fragment obtained by the hydrolysis. As a method for recovering a keratin fragment from a reaction solution, a general method for isolating or purifying a polypeptide, for example, filtration, centrifugation, concentration, precision filtration, ultrafiltration, chromatographic separation, freeze-drying, etc. , Protein precipitation methods such as salting out, TCA precipitation, and acetone precipitation, and the like can be used.

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

The keratin fragment produced by the method of the present invention preferably has 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. The preferred molecular weight measurement procedure is described below: Equivalently mix the sample solution with 2 × Laemmli Sample Buffer containing 50 mM dithiothreitol and heat at 100 ° C. for 5 minutes to prepare an electrophoretic sample. This migration sample is electrophoresed using Any kD ^TM miniprotian® TGX ^TM precast gel and electrophoresis buffer (25 mM Tris, 192 mM glycine, 0.1% (w / v) SDS, pH 8.3). .. At this time, Precision Plus ^ProteinTM 2-color standard is used as the molecular weight marker. After migration, the gel is stained with CBB or ruby. The value obtained by substituting the mobility of the target protein or polypeptide into the formula of the 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 the same when the distance from the upper end of the gel (or the position where the sample is applied) to the preceding dye (bromophenol blue) in the lane through which the sample was flown is 1. It is a relative value of the distance from the upper end of the gel (or the position where the sample is applied) to the band of the sample.

The keratin fragment produced by the method of the present invention is water-soluble. Therefore, it can be recovered in a soluble state without using a hydrophilic solubilizer or a denaturing agent. Therefore, the keratin fragment produced by the method of the present invention is difficult to analyze if it contains a solubilizer or a denaturing agent (for example, dynamic light scattering method, analysis by circular dichroism (CD) spectrum measurement, absorbance. Analysis by measurement) is possible.

The keratin fragment produced by the method of the present invention has a rod region. Preferably, the keratin fragment produced by the method of the invention consists primarily of a rod region. Examples of the rod region contained in the keratin fragment produced by the method of the present invention include a region consisting of the amino acid sequences represented by amino acid numbers 157 to 450 of SEQ ID NO: 26. Examples of the keratin fragment produced by the method of the present invention include a keratin fragment containing the amino acid sequence represented 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 an 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. Further, since the keratin fragment of the present invention mainly composed of a rod region is structurally more stable, it is useful for analysis using X-rays, for example, X-ray crystal structure analysis. In the conventional X-ray analysis of keratin, it was difficult to obtain highly accurate data because the orientation in the full-length keratin crystal was incomplete (Polymer, 53, (2), 2004, p74-78). As described above, the keratin fragment produced by the method of the present invention is useful as a tool for the analysis of keratin.

As described above, the keratin fragment can be produced using the M23A subfamily protease. In other words, the M23A subfamily protease can be used as an active ingredient for keratin fragmentation. Therefore, in a further aspect, the invention provides a keratin fragmentation agent containing an M23A subfamily protease. In a further aspect, the invention provides the use of M23A subfamily proteases for the production of keratin fragmentation agents. In a further aspect, the invention provides the use of M23A subfamily proteases for keratin fragmentation. In a further aspect, the invention provides a kit for keratin fragmentation containing an M23A subfamily protease.

In a further aspect of the invention described above, the keratin fragmentation is achieved by hydrolysis of a substrate containing keratin with an M23A subfamily protease. The type of the substrate containing the keratin, the amount of the substrate and the enzyme used in the hydrolysis reaction, and the conditions of the hydrolysis reaction are the same as in the case of the above-mentioned method for producing a keratin fragment of the present invention. In a further aspect of the invention described above, 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 an M23A subfamily protease can be used for keratin fragmentation. In a further aspect of the invention described above, the keratin fragmentation produces a polymeric water-soluble keratin fragment having a molecular weight of preferably about 25,000 to 50,000, more preferably about 37,000 to 50,000. Will be done. The keratin fragment has a rod region, preferably mainly composed of a rod region. Examples of the keratin fragment include a keratin fragment containing the amino acid sequence represented 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 keratin fragmentation kit of the present invention may further contain other reagents, solvents, etc. that may be contained in the reaction solution for hydrolysis of the substrate containing keratin, if 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, as exemplary embodiments, the following substances, manufacturing methods, uses, methods and the like. However, the present invention is not limited to these embodiments.

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

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

[22] A keratin fragment containing the amino acid sequence represented 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 described in more detail with reference to examples. However, the technical scope of the present invention is not limited to these examples.

Table 1 shows a list of primers used in this example.

Reference Example 1 Preparation of protease (1) Preparation of culture supernatant containing BLP (1-1) Preparation of expression vector BLP gene (SEQ ID NO: 1) inserted into plasmid pUC57 (BLP / pUC57) was artificially produced by GenScript. Produced using a gene synthesis service. PCR reactions were performed using the primer pairs BLP_S237signal_F / BLP_S237signal_R (SEQ ID NOs: 9 and 10) and PrimeSTAR Max Premix (Takara Bio) using BLP / pUC57 as a template. The PCR reaction was similarly carried out 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 treated with DpnI (New England Biolabs). Subsequently, an In-Fusion reaction was carried out according to the protocol of In-Fusion and HD Cloning kit (Clontech).

Using the In-Fusion reaction solution, ECOS ^TM Compentent E. et al. col DH5 α (Nippon Gene, 310-06236) was transformed. The transformed cells were smeared on an LB plate containing ampicillin and cultured at 37 ° C. overnight. The colonies formed on the plate are inoculated into LB medium containing ampicillin, cultured overnight, and then the cells are collected and the plasmid (BLP / pHY) is extracted using the High Pure Plasmamid Isolation Kit (Roche). did. A PCR reaction was carried out 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 is referred to as E.I. It was transformed into coli HST08 Premium Competent Cells (Takara Bio). The transformed cells were smeared on an LB plate containing ampicillin and cultured at 37 ° C. overnight. The colonies formed on the plate are inoculated into LB medium containing ampicillin, cultured overnight, and then the cells are collected and the plasmid (BLP2 / pHY) is extracted using the High Pure Plasmamid Isolation Kit (Roche). did.

(1-2) Preparation of Enzyme-Producing Transformed Strain Bacillus subtilis Marburg No. 168 Strain (Bacillus subtilis Marburg No. 168 Strain: Nature, 390, 1997, p.249) was inoculated into 1 mL of LB medium at 30 ° C. and 200 rpm. The cells were shake-cultured overnight. 10 μL of this culture solution was inoculated into 1 mL of fresh LB medium and cultured at 37 ° C. and 200 rpm for 3 hours. The culture was centrifuged to collect pellets. Add 500 μL of SMMP [0.5 M shoe cloth, 20 mM disodium maleate, 20 mM magnesium chloride 6-hydrate, 35% (w / v) Antibiotic Medium 3 (Difco)] containing 4 mg / mL lysozyme (SIGMA) to the pellet. And incubated at 37 ° C. for 1 hour. The pellet was then collected by centrifugation and suspended in 400 μL SMMP. Mix 13 μL of the suspension, 2 μL of the plasmid BLP2 / pHY solution (10 mM Tris-HCl pH 8.5, 34.2 ng / μL) obtained in (1-1), and 20 μL of SMMP, add 100 μL of 40% PEG, and stir. After further adding 350 μL of SMMP, the mixture was shaken at 30 ° C. for 1 hour. 200 μL of this solution was added to DM3 regenerated agar medium containing tetracycline (15 μg / mL, SIGMA) [0.8% agar (Wako Pure Chemical Industries, Ltd.), 0.5% disodium succinate 6-hydrate, 0.5% casamino acid technical ( Difco), 0.5% yeast extract, 0.35% 1 potassium phosphate, 0.15% 2 potassium phosphate, 0.5% glucose, 0.4% magnesium chloride hexahydrate, 0.01% bovine serum Albumin (SIGMA), 0.5% carboxymethyl cellulose, 0.005% tripan blue (Merck) and amino acid mixture (10 μg / mL each of tryptophan, lysine and methionine);% is (w / v)%] and smeared 30 Incubated at ° C for 3 days to obtain the formed colonies.

(1-3) Enzyme production by transforming strain culture Tetracycline was added to the LB medium so as to have 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 next day, 400 μL of this culture medium was added to 2 × L-martose medium (2% trypton, 1% yeast extract, 1% NaCl, 7.5% maltose, 7.5 ppm manganese sulfate pentahydrate, 15 ppm tetracycline, 6 ppm zinc sulfate seven waters). Japanese product;% was inoculated into (w / v)%) 20 mL, cultured at 32 ° C. and 230 rpm for 2 days, and then the culture supernatant containing the enzyme produced from the cells was recovered by centrifugation.

(2) Preparation of culture supernatant containing LasA A LasA gene (SEQ ID NO: 3) inserted into a plasmid pUC57 (LasA / pUC57) was prepared using an artificial gene synthesis service manufactured by GenScript. Using LasA / pUC57 as a template and the primer pair LasA_F / LasA_CR (SEQ ID NOs: 15 and 16), a PCR reaction was performed according to the PrimeSTAR Max Premix (Takara Bio) protocol. The same PCR reaction was carried out using the plasmid pHY-S237 described in Example 7 of WO2006 / 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 treated with DpnI (New England Biolabs). Subsequently, an In-Fusion reaction was carried out according to the protocol of In-Fusion and HD Cloning kit (Clontech) to obtain a plasmid (LasA / pHY) solution.

Using the obtained plasmid (LasA / pHY) solution, a Bacillus subtilis prsA gene expression-enhanced strain (prsA- produced in Example 1 of JP-A-2007-49986) was prepared in the same procedure as in (1-2) above. The Kc strain) was transformed to obtain a Bacillus subtilis transformant colony. Tetracycline was added to a 2 × L liquid medium to a final concentration of 15 ppm. Bacillus subtilis transformant colonies were inoculated into 5 mL of this medium, and then cultured overnight at 30 ° C. and 250 rpm. The pellet was collected from the culture medium and the plasmid LasA / pHY was extracted from the pellet. PCR reaction, plasmid digestion by 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). Was performed to obtain a plasmid (LasA2 / pHY).

The obtained plasmid (LasA2 / pHY) was used for transformation 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. Then, the obtained transformant was cultured in the same procedure as in (1-3), and the culture supernatant containing the enzyme produced from the cells was collected.

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

The obtained plasmid (AhP / pHY) was used for transformation in the same manner as in (1-2) above. At this time, 168 strains of Bacillus subtilis were used as the host. Then, the obtained transformant was cultured in the same procedure as in (1-3), and the culture supernatant containing the enzyme produced from the cells was collected.

(4) Preparation of culture supernatant containing ALE-1 ALE-1 glycylglycine endopeptidase (ALE-1) (MEROPS ID: M23.012; SEQ ID NO: 8), which is an M23B subfamily protease, was prepared. An ALE1 gene (SEQ ID NO: 7) inserted into a plasmid pUC57 (ALE1 / pUC57) was prepared using an artificial gene synthesis service manufactured by GenScript. PCR reactions were performed using ALE1 / pUC57 as a template and the primer pairs pHY-like6-just-F / pHY-like6-just-CHisR (SEQ ID NOs: 24 and 25) and PrimeSTAR Max Premix (Takara Bio). The same PCR reaction was carried out using the plasmid pHY-S237 described in Example 7 of WO2006 / 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 treated with DpnI (New England Biolabs). Subsequently, an In-Fusion reaction was carried out according to the protocol of In-Fusion and HD Cloning kit (Clontech) to obtain a plasmid (ALE1 / pHY) solution.

Using the obtained plasmid (ALE1 / pHY), 168 strains of Bacillus subtilis were transformed in the same manner as in (1-2) above to obtain Bacillus subtilis transformant colonies. Tetracycline was added to a 2 × L liquid medium to a final concentration of 15 ppm. Bacillus subtilis transformant colonies were inoculated into 5 mL of this medium, and then cultured overnight at 30 ° C. and 250 rpm. The pellet was collected from the culture medium and the plasmid ALE1 / pHY was extracted from the pellet. The extracted plasmid (ALE1 / pHY) was used for transformation in the same manner as in (1-2) above. 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 procedure as in (1-3), and the culture supernatant containing the enzyme produced from the cells was collected.

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

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

Reference example 3 SDS-PAGE
An equal volume of 2 × Laemmli Sample Buffer (Bio-Rad, # 161-0737) containing 50 mM dithiothreitol (Thermo Fisher Scientific) and a sample solution were mixed and heated at 100 ° C. for 5 minutes to prepare an electrophoretic sample. This migration sample was electrophoresed using Any kD ^TM Miniprotian® TGX ^TM precast gel (Bio-Rad) and 10-fold diluted 10x Tris / Glycine / SDS (Bio-Rad, 1610732). Precision Plus ^ProteinTM 2-color standard (Bio-Rad, # 161-0374) was used as the molecular weight marker. The gel after migration was CBB-stained or ruby-stained. 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 the calibration curve prepared from the molecular weight and mobility of the molecular weight marker was taken as the molecular weight of the target protein or polypeptide. The mobility (Rf value) of a sample is the upper end of the gel when the distance from the upper end of the gel (the position where the sample is applied) to the preceding dye (bromophenol blue) in the lane through which the sample is flowed is 1. It was 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) Recovery of cloth with dirt on collar sleeves A cloth (composition: 65% polyester, 35% cotton) sewn on the collar area of a shirt was prepared. By having an adult man wear this shirt during the daytime for 3 days, the cloth with dirt on the collar and sleeves was recovered.

(2) Preparation of keratin solution 2 × Laemmli Sample Buffer (Bio-Rad, # 161-0737) containing 50 mM Dithiothreitol (Thermo Fisher Scientific) by chopping the collar sleeve stain-attached cloth obtained in (1). After immersing in and heating at 100 ° C. for 10 minutes, the mixture was centrifuged and the supernatant was collected. Using this supernatant, SDS-PAGE was performed according to the procedure described in Reference Example 3 (however, additional 2 × Laemmli Sample Buffer was not added, heated, and stained). After shaking the gel after SDS-PAGE in deionized water, a gel having a molecular weight of 75,000 to 100,000 was cut out using a marker as a guide. The cut gel was crushed with a spatula and then shaken overnight in 20 mM Tris-HCl (pH 7.5). After shaking, the gel was removed using Atoprep MF (ATTO, 3521370). The solution after removing the gel was concentrated using Amicon Ultra Fractional Molecular Weight 10K (Merck Millipore) to obtain a keratin solution. The protein concentration of the obtained keratin solution was measured with Protein A280 using a Nanodrop ND-1000 Spectrophotometer (Thermo Fisher Scientific) and found to be 0.14 mg / mL.

(3) Evaluation of keratin fraction The keratin fraction contained in the keratin solution obtained in (2) was evaluated by Western blotting with an anti-keratin antibody. In Western blotting, the keratin solution is subjected to SDS-PAGE (unstained) according to the procedure described in Reference Example 3, and then the gel is subjected to a transblot Turbo ^TM system (Bio-Rad) and a transblot Turbo ^TM mini PVDF transfer pack (Bio-). It was transferred to a 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 the primary antibody, Stabilized Peroxidase Conjugated Goat Anti-Muse (H + L) as the secondary antibody, Thermo Fisher (H + L) (Thermo Fisher) GE Healthcare) was used. As a result of Western blotting, it was confirmed that the keratin solution prepared in (2) contained keratin 10 (FIG. 1).

Reference Example 5 As a substrate for measuring enzyme activity, a FRET substrate [hereinafter referred to as FRET-GGGGG] (manufactured by PH Japan) in which pentaglycine is between the fluorescent group Nma and the quenching group Lys (Dnp) was used. Here, Nma refers to 2- (N-methylamino) benzoyl (Nma). Further, Lys (Dnp) refers to a substance having 2,4-dinitrophenyl (Dnp) in the side chain of lysine (Lys). 190 μL of the enzyme solution (enzyme, 20 mM Tris-HCl (pH 7.5)) obtained in Reference Example 1 (5) was added to a 96-well assay plate (AGC technoglass, 3881-096), and a FRET-GGGG solution (FRET-GGGGG solution) was further added. A 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 M200 (TECAN) at a temperature of 30 ° C., an excitation wavelength of 340 nm, and a measurement wavelength of 440 nm. Under the same reaction conditions, a reaction solution using 20 mM Tris-HCl pH 7.5 solution instead of the enzyme solution and FRETS-25-STD1 (Peptide Institute, 3720-v) dissolved in dimethyl sulfoxide instead of FRET-GGGGG. The fluorescence intensity was measured and a calibration curve was prepared. The activity of 1 unit (U) was defined as the amount of enzyme required to show a change in fluorescence intensity corresponding to the fluorescence intensity by 1 nmol FRETS-25-STD1 per minute.

Example 1 Decomposition of keratin by trypsin and BLP 1 μL of an 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 the enzyme was adjusted to 2 μg / mL. After allowing the reaction solution to stand at 30 ° C. for 3 hours, 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 band derived from keratin disappeared. The band of the trypsin-degraded keratin fragment was not detected, probably because the trypsin decomposed the keratin into fragments of various molecular weights, which was below the detection limit, or because the fragment became low molecular weight, and it was outside the gel. It was thought that this was due to the outflow. On the other hand, in the sample to which BLP was added, a band of keratin fragments was detected between the molecular weight 37,000 marker and the molecular weight 50,000 marker.

Example 2 Preparation of Keratin Fragment from Collar Sleeve Stain Adhering Cloth Three sheets of the collar sleeve stain adhering cloth obtained in Reference Example 4 (1) were immersed in 100 mL of 20 mM Tris-HCL (pH 7.5). After adding BLP prepared in Reference Example 1 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 recovered solution was concentrated 40-fold with an Amicon Ultra fractional molecular weight of 10 K (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. A band was detected between the molecular weight 37,000 marker and the molecular weight 50,000 marker in the sample to which BLP was added, as in Example 1. It was considered that this was a keratin fragment produced by BLP from water-soluble or insoluble keratin contained in the stain-attached cloth, keratin forming a complex with a protein present in keratinocytes, and the like. That is, it was shown that a water-soluble keratin fragment can be produced from a keratin-containing sample collected from a living body by BLP without adding a denaturing agent or an activator in advance and performing an operation such as heating.

Example 3 Evaluation of keratin-degrading activity by enzyme (1) Preparation of keratin solution The keratin-degrading activity of various enzymes prepared in Reference Example 1 was investigated. The keratin was carved from the human heel using a velvet smooth electric keratin remover diamond (Reckitt Benkeiser). To 5 mg of heel keratin, 1 mL of solubilizer (100 mM Tris-HCl (pH 8.5), 2% SDS, 25 mM DTT, 5 mM EDTA) was added, heated at 100 ° C. for 10 minutes, centrifuged, and above. Qing was recovered. The collected supernatant was added to a dialysis buffer (20 mM Tris-HCl (pH 7.5), 0.1% SDS) using 2 mL of Mini Dialysis kit 8 kDa cut-off (GE Healthcare, # 80-6484-32). On the other hand, it was dialyzed 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 SDS-PAGE (CBB staining) and Western blotting were performed according to the same procedure as in Reference Examples 3 to 4. Evaluated by. As a result of evaluation by SDS-PAGE and Western blotting, it was confirmed that the keratin solution prepared from the heel keratin contained keratin 10 (FIG. 4). Although multiple bands were detected in FIG. 4, these were considered to be complexed keratin 10, modified keratin 10, keratin 1, and other types of keratin, in addition to the monomeric keratin 10. Was done.

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

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

Example 4 Evaluation of Keratin Resolution of BLP To 100 μL of the keratin solution prepared in the same procedure as in Example 3, 1 μL of BLP (100 μg enzyme / mL) prepared in Reference Example 1 was added. The obtained solution was divided into two, and each was allowed to stand at 30 ° C. for 8 hours. Then, one solution is mixed with 2 × Laemmli Sample Buffer (Bio-Rad, # 161-0737) containing 25 mM Dithiothreitol (Thermo Fisher Scientific) in an equal amount, and then heated at 100 ° C. for 5 minutes 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. The sample after standing is mixed with 2 × Laemmli Sample Buffer (Bio-Rad, # 161-0737) containing 25 mM Dithiothreitol (Thermo Fisher Scientific) in an equal amount, and then heated at 100 ° C. for 5 minutes to stop the reaction. I let you. Each reaction solution was evaluated by SDS-PAGE (CBB staining, but no additional 2 × Laemmli Sample Buffer was added and heated) according to the procedure of Reference Example 3.

The results are shown in FIG. The addition of BLP reduced the molecular weight of keratin in the solution. A band of keratin fragments was detected between the molecular weight 37,000 marker and the molecular weight 50,000 marker in both the solutions 8 hours and 15 hours after the addition of BLP. On the other hand, the molecular weight of the keratin fragment did not change between the solutions 8 hours and 15 hours after the addition of BLP. These results suggest that BLP has keratin-degrading activity and that further degradation of keratin fragments located between 37,000 and 50,000 molecular weights is not promoted by BLP.

Example 5 Solubilization of Keratin in Insoluble Fractions Derived from Corneocytes by M23A Subfamily Protease Using 2 cm of tape (Nichiban, CT-24), keratin was obtained from the human neck by tape stripping. A solubilizing solution (100 mM Tris-HCl (pH 8.5), 2% SDS, 25 mM DTT, 5 mM EDTA) was added to this keratin, heated at 100 ° C. for 10 minutes, and then centrifuged to remove the supernatant. To obtain a precipitate. The insoluble fraction derived from keratinocytes was obtained by further performing the operation of adding the solubilizing solution to the precipitate, heating, and centrifuging twice. To the obtained insoluble fraction, 40 μL of 20 mM Tris-HCl (pH 7.5) was added, and 1 μL of the enzyme solution was further added. As the enzyme, BLP, LasA, and AhP prepared in Reference Example 1 were used. The final concentration of the enzyme was adjusted to 1 μg / mL for BLP and 10 μg / mL for LasA and AhP. After allowing the reaction solution to stand at 30 ° C. for 15 hours, SDS-PAGE (ruby staining) of the supernatant was performed according to the procedure of Reference Example 3.

The results are shown in FIG. In all solutions supplemented with BLP, LasA and AhP, a band was detected between the 37,000 and 50,000 markers, and these enzymes decomposed keratin in the insoluble fraction of the keratin into keratin fragments. It was shown that it did.

As shown in FIG. 7, two bands were detected between the molecular weight 37,000 marker and the molecular weight 50,000 marker on SDS-PAGE of the BLP-added solution. Of these, mass spectrometry was performed on the band on the small molecule side. MALDI-TOF Mass Spec Analysis of Leave a Nest Co., Ltd. was used for mass spectrometry. This mass spectrometry service used trypsin for protein digestion. As a result of mass spectrometry, it was found that the protein contained in the band on the small molecule side was a fragment of keratin 10 (ACCESSION_AAH34697). Further, as shown in FIG. 8, the peptide sequences (bold) identified by mass spectrometry correspond to the regions existing between amino acids 157 and 450 of the amino acid sequence of keratin 10 (SEQ ID NO: 26). rice field. According to the information published in Keratin, type I cytoskeleton 10 [UniProt Knowngebase_P13645], amino acids 1 to 145 in the amino acid sequence of keratin 10 are head regions, amino acids 146 to 456 are rod regions, and amino acids 457 to 584. Is the tail area. Therefore, it was shown that the keratin fragment obtained in this example contained the rod region of keratin 10.
From the above, BLP and other M23A subfamily enzymes cleave the amino acid regions 1 to 156 or the amino acid regions 451 to 584 of the amino acid sequence of keratin 10 (SEQ ID NO: 26) in the keratin insoluble fraction. It was found to produce a fragment containing the rod region of keratin 10 contained in.

Although embodiments of the present invention have been described above, it should be understood that these are not intended to limit the 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 (19)

A method for producing a keratin fragment, which comprises hydrolyzing a substrate containing keratin 10 with an M23A subfamily protease. The method according to claim 1, wherein the substrate containing keratin 10 is hydrolyzed with an enzyme composition containing an 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 hydrophilaproteinase. The method according to any one of claims 1 to 3, wherein the keratin fragment has a molecular weight of 25,000 to 50,000. The method according to any one of claims 1 to 4, wherein the keratin fragment is water-soluble. The method according to any one of claims 1 to 5, wherein the keratin fragment has a rod region. The substrate containing keratin 10 is at least one selected from the group consisting of animal hair, claws, skin, horns, hoofs, scales, epithelial cells and feathers, their processed products, and the keratin fraction separated from them. 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 sparingly soluble keratin. The method according to any one of claims 1 to 9 , further comprising recovering the keratin fragment obtained by the hydrolysis. The method according to any one of claims 1 to 10 , wherein the treatment for stopping the enzymatic reaction is not performed after the hydrolysis. A keratin fragmentation agent containing an M23A subfamily protease as an active ingredient , wherein the fragmented keratin comprises a fragmented keratin having a rod region of keratin 10 . The keratin fragmentation agent according to claim 12 , wherein the enzyme composition containing the M23A subfamily protease is used as an active ingredient. The keratin fragmentation agent according to claim 12 or 13 , wherein the M23A subfamily protease is at least one selected from the group consisting of β-lytic metalloprotease, LasA protein, and Aeromonas hydrophilaproteinase. The keratin fragmentation agent according to any one of claims 12 to 14 , wherein the fragmented keratin has a molecular weight of 25,000 to 50,000. The keratin fragmentation agent according to any one of claims 12 to 15 , wherein the fragmented keratin is water-soluble. The keratin fragmentation agent according to any one of claims 12 to 16 , wherein the keratin is a water-soluble keratin. The keratin fragmentation agent according to any one of claims 12 to 16 , wherein the keratin is water-insoluble or sparingly soluble keratin. The keratin fragmentation agent according to any one of claims 12 to 18 , wherein the keratin is keratin 10.

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