Modified SapB protein, method for preparing SapB protein and its modified form, and aqueous solution containing SapB protein or its modified form

Abstract

The present invention provides: a modified form of a SapB protein; a method for preparing a SapB protein and a modified form thereof; and an aqueous solution in which a SapB protein or a modified form thereof is dissolved.

Description

[Technical Field]

[0001] The present invention relates to a modified SapB protein, a method for preparing the SapB protein and its modified form, and an aqueous solution in which the SapB protein or its modified form is dissolved. [Background technology]

[0002] Surfactants are useful substances used in various industrial applications. The SapB protein was discovered as a protein with surfactant activity (Non-Patent Document 1). The SapB protein is generated by cleavage from its precursor, RamS. SapB is a part of RamS (SapB precursor) and is encoded by the ramS gene. The ramS gene exists in an operon that includes ramC, ramS, ramA, ramB, and ramR.

[0003] It has been revealed that the SapB protein contains dehydroalanine and lanthionine bonds (Non-Patent Document 2). This modified peptide can now be totally synthesized (Non-Patent Document 3). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Mol Microbiol. 1998;30(3):595-602 [Non-patent document 2] Proc Natl Acad Sci US A. 2004;101(31):11448-11453 [Non-patent document 3] J Org Chem. 2018;83(14): 7528-7533 Summary of the Invention

[0005] The present invention provides modified SapB protein, methods for preparing SapB protein and modified SapB protein, and aqueous solutions of SapB protein and modified SapB protein.

[0006] The present inventors have found that extraction of SapB protein from SapB-expressing cells at room temperature in the presence of urea or under heated conditions improves the recovery rate of native SapB protein. The present inventors also discovered a novel modified SapB with a chemically modified N-terminus during the purification process. Specifically, the present inventors extracted SapB protein from SapB-expressing cells under heated conditions and in the presence of urea, resulting in high yields of the novel modified SapB with a chemically modified N-terminus. They also found that the resulting modified SapB possessed excellent surfactant and emulsifying properties. Furthermore, the novel modified SapB with a chemically modified N-terminus was stable against some degradation treatments. Furthermore, the present inventors have found that the poorly water-soluble SapB protein and its modified forms exhibit high solubility in alkaline buffers. Furthermore, the present inventors have found that the modified SapB protein exhibits high solubility in buffers at pH 6 or higher.

[0007] According to the present invention, the following inventions are provided. (1) A method for preparing a SapB protein, comprising: Providing cells that produce SapB protein; Cells (a) under a temperature condition equal to or greater than a first predetermined temperature and in the absence of urea; (b) at a temperature below a second predetermined temperature and in the presence of urea; or (c) Under a temperature condition equal to or higher than a third predetermined temperature and in the presence of urea {wherein the first predetermined temperature is 40°C or higher}, thereby obtaining a SapB protein or a modified SapB protein in which the amino group of the N-terminal amino acid is modified or protected; A method comprising: (2) The method according to (1) above, wherein the incubation is carried out (a) under a temperature condition equal to or higher than a first predetermined temperature and in the absence of urea. (3) The method according to (1) above, wherein the incubation is carried out under temperature conditions (b) below a second predetermined temperature and in the presence of urea. (4) The method according to (1) above, wherein the incubation is carried out under temperature conditions of (c) a third predetermined temperature or higher and in the presence of urea. (5) A modified form of the isolated SapB protein, in which the amino group of the N-terminal amino acid is modified or protected. (6) A modified form of the SapB protein according to (5) above, wherein the modification or protection is modification or protection with an amide group. (7) A modified form of the SapB protein according to (5) or (6) above, wherein the modification or protection is by an NH2-C(O)- group. (8) A modified SapB protein according to any one of (5) to (7) above, wherein the SapB protein is derived from actinomycetes. (9) A structure of formula (I): [ka] A modified SapB protein according to any one of (5) to (8) above, which has the following structure: (10) A composition comprising a modified SapB protein according to any one of (5) to (9) above. (11) A surfactant comprising the modified SapB protein according to any one of (5) to (9) above or the composition according to (10) above. (12) An emulsifier comprising the modified SapB protein according to any one of (5) to (9) above or the composition according to (10) above. (13) A method for preparing a composition comprising an emulsion from a water-soluble composition and a fat-soluble composition, the method comprising mixing a modified SapB protein described in any one of (5) to (9) above or the composition described in (10) above with the water-soluble composition and the fat-soluble composition. (14) A composition comprising an emulsion containing a lipid-soluble solute molecule and a modified SapB protein according to any one of (5) to (9) above. (15) The composition according to (14) above, which is a cosmetic, a household detergent, or a food or beverage. (16) A composition comprising an isolated SapB protein or a modified form thereof and an aqueous solution, A composition, wherein the isolated SapB protein or a modified form thereof is dissolved in an aqueous solution, and the aqueous solution is alkaline. (17) The composition according to claim 14, wherein the modified form of the isolated SapB is the modified form according to any one of (5) to (9) above. (18) The composition according to (16) or (17) above, wherein the aqueous solution has a pH of 8 or higher. (19) The composition according to any one of (16) to (18) above, wherein the aqueous solution has a pH in the range of 8 to 11. (20) The composition according to any one of (16) to (19) above, wherein the concentration of the isolated SapB protein or a modified form thereof is 0.1% w / w or more in terms of the weight of the SapB protein portion. (21) An aqueous composition comprising a modified form of an isolated SapB protein and having a pH of 6 or higher. (22) The composition according to (21) above, wherein the modified form of the isolated SapB protein is the modified form according to any one of claims 5 to 9.

[0008] (31) A SapB expression vector containing a gene expression cassette having part or all of the SapB operon region. (32) A SapB expression vector comprising a gene expression cassette having a part or all of the SapB operon region, wherein the part or all of the SapB operon region comprises at least a gene encoding the SapB protein, and which is capable of at least expressing the SapB protein in actinomycetes. (33) A cell containing the SapB expression vector according to (31) or (32) above. (34) The cell according to (33) above, which is an actinomycete. (35) A composition comprising the SapB expression vector according to (31) or (32) above. (36) A composition comprising the cells according to (33) or (34) above. (37) The SapB expression vector according to (31) or (32), the cell according to (33) or (34), or the composition according to (35) or (36), for use in expressing the SapB protein. (38) A method for preparing a SapB protein, comprising: Introducing the SapB expression vector according to (31) or (32) into a cell; Culturing the resulting cells to produce the SapB protein; A method comprising: (39) A method for preparing SapB protein, comprising culturing the cell according to (33) or (34) above to produce SapB protein. (40) A method for preparing a modified SapB protein, comprising: Introducing the SapB expression vector according to (31) or (32) into a cell; Culturing the resulting cells to produce the SapB protein; heating the obtained SapB protein in the presence of urea to obtain a modified SapB protein; A method comprising: (41) A method for preparing a modified SapB protein, comprising: Culturing the cell according to (33) or (34) above to produce the SapB protein; heating the obtained SapB protein in the presence of urea to obtain a modified SapB; A method comprising:

[0009] The method for preparing the SapB protein of the present invention is suitable for recovering the water-insoluble SapB protein expressed in cells. The modified SapB protein of the present invention is stabilized by the modification and can be resistant to amino acid degradation from the N-terminus. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows the emulsifying activity of the new chemically modified SapB obtained in this example. [Figure 2A] FIG. 2A shows high-performance liquid chromatographs of the native SapB protein and SapB with the new chemical modifications obtained in this example. [Figure 2B] Figure 2B shows high-performance liquid chromatographs of native SapB protein and SapB with the new chemical modifications obtained in this example (modified SapB protein) in solutions incubated for different periods of time in the presence of different concentrations of urea. [Figure 3A] Figure 3A shows the separation of SapB proteins by liquid chromatography and the chromatograms extracted by each MS. [Figure 3B] FIG. 3B shows the mass spectrometry spectrum of native SapB and the mass spectrometry spectrum of SapB with the new chemical modifications obtained in this example. [Figure 3C] FIG. 3C shows an enlarged view of the area around m / z 130 to 160 of the spectrum in which the peaks of native SapB (top panel) and SapB modified with NH 2 —C(O) − (bottom panel) were split. [Figure 3D] FIG. 3D shows an enlarged view of the area around m / z 960 to 1190 of the spectrum in which the peaks of native SapB (top panel) and SapB modified with NH 2 —C(O) − (bottom panel) were split. [Figure 4] Figure 4 shows the results of SDS-PAGE of suspensions (left panel) and supernatants (right panel) containing modified SapB proteins in buffers with different pH values. The arrowheads in the figure indicate the bands of modified SapB proteins. [Figure 5] Figure 5 shows high-performance liquid chromatographs of the centrifuged supernatants of buffers with different pH values ​​containing native SapB protein. The arrowheads in the figure indicate the peaks of modified SapB protein. [Figure 6]Figure 6 shows high-performance liquid chromatographs of the supernatants of the centrifuged buffers containing modified SapB protein at different pH levels. The arrows in the figure indicate the peaks of the modified SapB protein. [Figure 7] Figure 7 shows the expression of various SapB proteins. Lane 1 corresponds to SEQ ID NO: 9, lane 2 corresponds to SEQ ID NO: 7, lane 3 corresponds to SEQ ID NO: 5, lane 4 corresponds to SEQ ID NO: 6, lane 5 corresponds to SEQ ID NO: 8, and lane 6 corresponds to 0.1% SapB protein (lyophilized). The arrows in the figure indicate the positions of the SapB protein bands. [Figure 8] FIG. 8 shows the relationship between the SapB concentration and the surface tension (mN / m) determined by the Wilhelmy method. [Figure 9] Figure 9 shows the effect of surface treatment by SapB coating. Specific Description of the Invention

[0011] <Definition> As used herein, "isolating" or "isolation" refers to separating a specific substance or molecule from at least one other substance or molecule. For example, isolation may include isolating a specific substance from cells when cell production is induced. Isolation may also be followed by purification, such as separation of liquid and solid phases by filtration, solution exchange by dialysis, separation by molecular size (e.g., gel filtration), separation using a hydrophobic column, or affinity-based separation of specific molecules. While isolation is a distinct concept from purity, an isolated specific substance may have a purity of, for example, 50% w / w or more, 60% w / w or more, 70% w / w or more, 80% w / w or more, 90% w / w or more, or 95% w / w or more. When a specific substance is present in a solvent, purity can be determined as the weight / weight percentage of the specific substance relative to the total solute.

[0012] As used herein, the "N-terminus" refers to the amino terminus of a polypeptide or protein. Polypeptides and proteins are polymers formed by dehydration condensation of amino acids having amino and carboxyl groups, and polypeptides and proteins have a carboxyl group and an amino group at both ends. Generally, the end on the carboxyl group side is referred to as the C-terminus, and the end on the amino group side is referred to as the N-terminus. The N-terminal amino group is an amino group that can form a peptide backbone, and is therefore different from the amino group in the side chain of lysine (or ornithine) or the guanidino group in the side chain of arginine. As used herein, the N-terminal amino acid refers to the amino acid at the tip of the N-terminus (the first amino acid). As used herein, the terminal amino group refers to an amino group bonded to the alpha carbon of the terminal amino acid.

[0013] As used herein, "amino acid" refers to α-amino acids, which are the building blocks of biological proteins. α-Amino acids have at least an amino group and a carboxyl group on the α-carbon, and undergo dehydration condensation between the amino and carboxyl groups to form peptide bonds to form polypeptides or proteins. Examples of amino acids include alanine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, arginine, cysteine, glutamine, glycine, proline, tyrosine, aspartic acid, asparagine, glutamic acid, and serine. While D- and L-amino acids exist (with the exception of glycine, which has no asymmetric carbon and therefore does not have optical isomers), naturally occurring amino acids are primarily L-amino acids. The side chain of an amino acid refers to the portion of the amino acid other than the amino group, carboxyl group, and hydrogen bonded to the α-carbon. While proline is typically classified as an imino acid, it is considered an amino acid in biochemistry and is treated as such herein. In addition, the SapB protein contains a dehydroamino acid with an unnatural structure called dehydroalanine, which is also classified as an amino acid. Dehydroalanine has the following structure: [ka] It has.

[0014] As used herein, a "lanthionine bridge" or "lanthionine bond" refers to a bridge or bond between two alanine residues that form a thioether bond between their β-carbons. Two alanine residues that form a thioether bond at their β-carbons have the chemical formula HOOC-CH(NH)-CH-S-CH-CH(NH)-COOH, where the carbons on either side of S are β-carbons. In the SapB protein, before translational modification, the side chains of what were previously serine and cysteine ​​residues undergo dehydration condensation to form a structure of two alanine residues that form a thioether bond at their β-carbons.

[0015] As used herein, "protection" refers to chemical modification (also simply referred to as "modification") in which a reactive group is modified with a protecting group to convert it into an inactive functional group. Removing the protecting group from a protected functional group is called deprotection. When it is not necessary to remove the protecting group, the product can be used for applications with the protecting group still attached.

[0016] As used herein, "emulsion" refers to a dispersed solution containing a dispersoid and a dispersion medium, both of which are liquids. Emulsions are also referred to as emulsions or emulsions. As used herein, emulsification refers to the process of emulsifying two separate liquids. As used herein, a substance with emulsifying properties is referred to as an emulsifier. When immiscible liquids, such as water and oil, are mixed with an amphiphilic substance (also known as a surfactant), one of the liquids can associate in a granular form to form micelles. It is known that the formation of micelles by an amphiphilic substance stabilizes a dispersed system of droplets. Examples of emulsions include oil-in-water (O / W) emulsions and water-in-oil (W / O) emulsions. Examples of dispersion media include aqueous solvents (e.g., water, e.g., distilled water).

[0017] As used herein, the term "SapB protein" refers to a protein produced by excision from the region from positions 22 to 42 of RamS, a protein having an amino acid sequence registered in UniProtKB / Swiss-Prot under registration number O88038, and its homologs, such as orthologues. The SapB protein was discovered by Welly et al. (Cell 1991;65(4):641-650), and it was subsequently revealed that the SapB protein contains dehydroalanine and lanthionine bonds (Proc Natl Acad Sci U S A. 2004;101(31):11448-53). This modified peptide can be fully synthesized (J. Org. Chem. 2018;83(14):7528-7533). The amino acid sequence registered in UniProtKB / Swiss-Prot under registration number O88038 is MNLFDLQSME TPKEEAMGDV ETGSRASLLL CGDSSLSITT CN (SEQ ID NO: 1), and the amino acid sequence of the region from 22nd to 42nd is TG S RA S LLL C GD S SL S ITT CSapB is SEQ ID NO: 2 (SEQ ID NO: 2) {wherein the underlined amino acids have chemical modifications, the 3rd S and the 10th C are linked by a lanthionine bond between their β-carbons, the 13th S and the 20th C are linked by a lanthionine bond between their β-carbons, and the 6th S and the 16th S are dehydroalanine}. SapB can be obtained, for example, by introducing a nucleic acid encoding the amino acid sequence of SEQ ID NO: 1 into a microorganism (e.g., a bacterium) such as an actinomycete and expressing it. The SapB protein can have surfactant activity (Mol. Microbiol. 1998;30(3):595-602). As mentioned above, SapB is part of RamS (the SapB precursor) and is encoded by the ramS gene. The ramS gene is present in an operon containing ramC, ramS, ramA, ramB, and ramR. ramC, ramS, ramA, and ramB can be driven by the ramCSABp promoter, and ramR can be driven by the ramRp promoter. Therefore, the SapB protein may be produced by introducing the operon into a microorganism (eg, a bacterium) such as an actinomycete (eg, Streptomyces).

[0018] [ka]

[0019] Herein, cells into which the SapB operon has been introduced are referred to as SapB operon region-integrated cells. The SapB operon may be exogenous or heterologous to the introduced cell. The SapB protein may be produced by introducing only the ramS gene into a microorganism (e.g., a bacterium). Of course, the SapB protein can also be obtained by total chemical synthesis.

[0020] <SapB protein modified with a novel modification> According to the present disclosure, a modified SapB protein is provided. The modified SapB protein is isolated. Furthermore, the modified SapB protein has a novel chemical modification.

[0021] In one embodiment of the present disclosure, the modified isolated SapB protein has a modified or protected amino group. In a preferred embodiment, the amino group is the amino group of the N-terminal amino acid. In particular, in a preferred embodiment, the amino group may be the amino group attached to the α-carbon of the N-terminal amino acid.

[0022] The SapB protein may be derived from a microorganism. In a preferred embodiment, the SapB protein may be derived from an actinomycete. In a preferred embodiment, the SapB protein may be derived from an actinomycete of the genus Streptomyces. In a preferred embodiment, the SapB protein may be derived from any of the microorganisms (e.g., bacteria or actinomycetes) listed in Table 1 below.

[0023] [Table 1] TIFF0007818015000005.tif217139TIFF0007818015000006.tif221141

[0024] For example, in a preferred embodiment, the SapB protein can be derived from any of Nos. 1 to 100 in Table 1. In one embodiment, the SapB protein can be derived from any of Nos. 1 to 38 and 41 in Table 1. In one embodiment, the SapB protein can be derived from any of Nos. 1 to 31 in Table 1. In one embodiment, the SapB protein can be derived from any of Nos. 1 to 20 and 22 to 25 in Table 1. In one embodiment, the SapB protein can be derived from any of Nos. 1 to 12 and 20 in Table 1. In these embodiments, the SapB protein can be produced in the bacteria corresponding to the same number.

[0025] In one embodiment, the SapB protein is derived from any of the following actinomycetes of the genus Streptomyces: S. coelicolor, S. griseus, S. albus, S. scabies, and S. avermitilis. In one embodiment, the SapB protein has the amino acid sequence of any of SEQ ID NOS: 2 to 9 (particularly SEQ ID NOS: 2 to 7, preferably SEQ ID NOS: 2, 5, 6, 7, 8, and 9), or a sequence corresponding to said amino acid sequence. Here, "having a sequence corresponding to a certain amino acid sequence" refers to an amino acid sequence of a region that corresponds upon alignment with another amino acid sequence having an amino acid sequence with at least 80% or more, 85% or more, 90% or more, or 95% or more sequence identity.

[0026] After being translated as a protein, the SapB protein undergoes post-translational modifications. (a) two amino acids of the SapB protein corresponding to serine 6 and serine 16 of SEQ ID NO: 2 are converted to dehydroalanine; (b) the two amino acids of the SapB protein corresponding to the third serine and the tenth cysteine ​​of SEQ ID NO: 2 form a lanthionine bond between their β carbons; and (c) The two amino acids of the SapB protein corresponding to the serine at position 13 and the cysteine ​​at position 20 of SEQ ID NO: 2 form a lanthionine bond between their β carbon atoms. In a preferred embodiment, the SapB protein may have any one, two, or preferably all of the modifications (a) to (c) above.

[0027] In a preferred embodiment, the SapB protein has two amino acids, corresponding to serine 6 and serine 16 of SEQ ID NO: 2, converted to dehydroalanine, two amino acids, corresponding to serine 3 and cysteine ​​10 of SEQ ID NO: 2, formed with a lanthionine bond between their β-carbons, and two amino acids, corresponding to serine 13 and cysteine ​​20 of SEQ ID NO: 2, formed with a lanthionine bond between their β-carbons.

[0028] In a preferred embodiment, when aligned with the SapB protein derived from S. coelicolor, the amino acids of the aligned SapB protein corresponding to serine 6 and serine 16 of SEQ ID NO: 2 are converted to dehydroalanine, a lanthionine bond is formed between the β-carbons of serine 3 and cysteine ​​10 of SEQ ID NO: 2, and a lanthionine bond is formed between the β-carbons of serine 13 and cysteine ​​20 of SEQ ID NO: 2. In a preferred embodiment, the SapB protein is derived from a peptide having the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 9 (particularly, SEQ ID NOs: 2 to 7, preferably SEQ ID NOs: 2, 5, 6, 7, 8, and 9), and may have one, two, or preferably all of the modifications (a) to (c) above.

[0029] [ka]

[0030] In the above alignment, the amino acid sequences of SapB before post-translational modification are aligned, and corresponding amino acid sequences are aligned at the same position. In the above alignment, the S and C that form the lanthionine bond are boxed. The bold S becomes dehydroalanine after post-translational modification. In the above alignment, missing amino acids are indicated by "-". As such, the modification sites of SapB are highly conserved across species.

[0031] In the present invention, the modified SapB protein has the amino group linked to the α-carbon of the N-terminal amino acid chemically modified (also referred to simply as "modified") or protected. This modification or protection can confer resistance to, for example, Edman degradation. Edman degradation can be performed under conditions suitable for amino acid sequence analysis. Such modification or protection of the N-terminus can be beneficial in improving protein stability. Meanwhile, the surfactant and emulsifying properties of the modified SapB protein can be maintained.

[0032] In a preferred embodiment, the modification or protection of the N-terminus may be modification or protection with an amide group, and in a more preferred embodiment, modification or protection with an NH2-C(O)- group.

[0033] In a preferred embodiment, the modified SapB protein of the present disclosure has the structure of formula (I): [ka] It has.

[0034] In one aspect of the present disclosure, a composition comprising a modified SapB protein according to the present disclosure is provided. In one embodiment of the present disclosure, the composition may comprise a native SapB protein and a modified SapB protein. The composition may have, for example, a surface tension-reducing effect, an emulsifying effect, a demulsifying effect, a penetrating effect, a dispersing effect, or an anti-redeposition effect. Therefore, the composition may be used as a surfactant. The composition may also be used as an emulsifier. The composition may contain an aqueous solvent (e.g., water). In addition to the aqueous solvent (e.g., water), the composition may also contain excipients (e.g., bulking agents, thickeners, pH adjusters, flavorings, osmotic pressure adjusters, salts, etc.).

[0035] In one embodiment of the present disclosure, the composition comprises a native SapB protein and a modified SapB protein, and the molar ratio thereof can be, but is not limited to, 99:1 to 1:99. The molar ratio of the modified SapB protein can be increased by changing one or more conditions selected from the group consisting of extending the incubation time, increasing the heating temperature, and increasing the urea concentration.

[0036] In one aspect of the invention, 1. A method for preparing a SapB protein, comprising: Providing a cell that produces the SapB protein; Incubating the cells at a temperature equal to or higher than a first predetermined temperature or in the absence of urea, thereby obtaining (or extracting) the SapB protein; A method comprising: is provided. In one aspect of the invention, 1. A method for preparing a SapB protein, comprising: Providing a cell that produces the SapB protein; incubating the cells at a temperature below a second predetermined temperature or in the presence of urea, thereby obtaining (or extracting) the SapB protein; A method comprising: is provided. In one aspect of the invention, 1. A method for preparing a SapB protein, comprising: Providing a cell that produces the SapB protein; incubating the cells at a temperature equal to or higher than a third predetermined temperature or in the presence of urea, thereby obtaining (or extracting) the SapB protein; A method comprising: is provided. That is, in one aspect of the present invention, 1. A method for preparing a SapB protein, comprising: Providing a cell that produces the SapB protein; Cells (a) under a temperature condition equal to or greater than a first predetermined temperature and in the absence of urea; (b) at a temperature below a second predetermined temperature and in the presence of urea; or (c) Under a temperature condition equal to or higher than a third predetermined temperature and in the presence of urea thereby obtaining (or extracting) the SapB protein; A method comprising: is provided. In the above, the cell may be a protein-producing cell.

[0037] In one embodiment, the conditions for extracting the SapB protein from cells (extraction conditions or treatment conditions) are: (a) under a temperature condition equal to or greater than a first predetermined temperature and in the absence of urea; (b) at a temperature below a second predetermined temperature and in the presence of urea; or (c) Under a temperature condition equal to or higher than a third predetermined temperature and in the presence of urea It can be either of the following. When extraction conditions are (a), native (unmodified) SapB protein can be recovered. When extraction conditions are (b), native (unmodified) SapB protein can be recovered. When extraction conditions are (c), modified SapB protein can be recovered. When extraction conditions are at a higher temperature and in the presence of a higher concentration of urea, the amount of modified SapB protein recovered can be increased. Extending the extraction time can increase this yield. The resulting extract can contain a mixture of native SapB protein and modified SapB protein. When extraction conditions are (a), extending the extraction time (incubation time) can increase the yield of native SapB protein. When extraction conditions are (c), extending the extraction time (incubation time) can increase the yield and the amount of modified SapB protein recovered can be increased. Thus, the method for preparing SapB protein of the present disclosure can yield SapB protein and / or modified SapB protein. In one embodiment, the method for preparing SapB protein of the present disclosure can yield a mixture of SapB protein and modified SapB protein. In either embodiment, incubation is preferably carried out in an aqueous solvent (preferably water). Treatment in the absence of urea can be carried out, for example, in a first aqueous solution described below, and treatment in the presence of urea can be carried out, for example, in a second aqueous solution described below. When extraction conditions are below a second predetermined temperature and in the absence of urea, only native SapB protein can be obtained.

[0038] In one embodiment, the first predetermined temperature, the second predetermined temperature, and the third predetermined temperature (hereinafter collectively referred to simply as "predetermined temperatures") can each independently be temperatures included in any one or more temperature ranges selected from the group consisting of 0 to 10°C, 10 to 20°C, 20 to 30°C, 30 to 40°C, 40 to 50°C, 50 to 60°C, 60 to 70°C, 70 to 80°C, 80 to 90°C, 90 to 95°C, and 95 to 100°C. In one embodiment, the predetermined temperature can be included in, for example, one, two, or three temperature ranges selected from the group consisting of 30 to 50°C, 40 to 60°C, and 50 to 70°C. In one embodiment, the first and third predetermined temperatures can each independently be room temperature, 37°C, 40°C, 50°C, or 60°C, or any temperature between these two temperatures (e.g., room temperature and 40°C). In one embodiment, the second predetermined temperature can be 0 to 10°C, 10 to 20°C, 20 to 30°C, 30 to 40°C, 40 to 50°C, 50 to 60°C, or 60 to 70°C. In a preferred embodiment, the first or third predetermined temperature (more preferably, the first and third predetermined temperatures) can be equal to or higher than the second predetermined temperature. In a preferred embodiment, the treatment temperatures for (a) and (c) can be equal to or higher than the treatment temperature for (b).

[0039] In one embodiment, the incubation time can be 30 minutes or more, 60 minutes or more, 120 minutes or more, 180 minutes or more, or 240 minutes or more. The incubation time (and its upper and lower limits) can be appropriately determined depending on the yield of SapB protein and its modified forms. In one embodiment, the incubation time can be 30 to 240 minutes.

[0040] In one embodiment, the modified SapB protein can also be obtained by incubating extracted SapB protein under the condition (c) above.

[0041] The obtained SapB protein and its modified forms can be purified. The obtained composition containing the SapB protein and its modified forms can be subjected to dialysis. Solvent exchange is possible by dialysis. For example, when urea is removed from a solution, dialysis can be performed using a solution that does not contain urea as the external dialysis solution. The obtained composition containing the SapB protein and its modified forms can be an aqueous solution. The obtained aqueous solution can have an alkaline pH.

[0042] In one aspect of the present invention, a modified SapB protein according to the present disclosure can be used to prepare a composition comprising an emulsion from a water-soluble composition and a fat-soluble composition. In a preferred embodiment, the water-soluble composition can contain an aqueous solvent. The emulsion can be obtained by mixing the water-soluble composition and the fat-soluble composition in the presence of a modified SapB protein according to the present disclosure. The modified SapB protein can be added to either or both of the water-soluble and fat-soluble compositions during or before mixing in an amount sufficient for emulsion formation. SapB protein can be used to form emulsions in the same manner as other surfactants. An amount sufficient for emulsion formation can be, for example, an amount sufficient to achieve a concentration equal to or greater than the critical micelle concentration (CMC) (e.g., a concentration greater than the CMC, such as 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, 2 times or more, 3 times or more, 5 times or more, 10 times or more, 20 times or more, 30 times or more, 50 times or more, or 100 times or more of the CMC). Those skilled in the art can determine the amount sufficient to form an emulsion as appropriate. Furthermore, those skilled in the art can appropriately form an emulsion. The concentration of SapB does not prevent SapB from precipitating, but is preferably an amount that does not cause SapB to precipitate. In a preferred embodiment, the emulsion may be an O / W emulsion. In a preferred embodiment, the emulsion may be a W / O emulsion. In a preferred embodiment, the fat-soluble composition may contain a fat-soluble solvent.

[0043] In a preferred embodiment, the water-soluble composition and the fat-soluble composition can be food compositions. In this embodiment, the water-soluble composition can contain, for example, water, and the fat-soluble composition can contain an edible oil. The resulting emulsion can also be mixed with other food compositions before use. In a preferred embodiment, the water-soluble composition and the fat-soluble composition can be compositions suitable for cosmetic compositions. The resulting emulsion can also be mixed with other cosmetic compositions before use. In these embodiments, the resulting modified SapB protein can be used at a concentration and / or amount that does not exhibit biotoxicity.

[0044] In a preferred embodiment, the water-soluble composition and the fat-soluble composition can be used for surface treatment. By applying the water-soluble composition and the fat-soluble composition to the surface of an object or the like, the surface can be made hydrophilic. Therefore, in the present disclosure, the water-soluble composition and the fat-soluble composition can be used to improve the wettability of a surface or to make a surface hydrophilic. In one embodiment, by applying the water-soluble composition and the fat-soluble composition to a hydrophilic surface of an object or the like, the water-soluble composition can reduce the wettability of the surface or make the surface hydrophobic. This is because the water-soluble composition and the fat-soluble composition of the present disclosure are amphiphilic, and when the surface is hydrophilic, the hydrophilic portion of the active ingredient comes into contact with the surface, exposing the hydrophobic portion. Therefore, in the present disclosure, the water-soluble composition and the fat-soluble composition can be used to make a surface hydrophobic.

[0045] In a preferred embodiment, the water-soluble composition and the fat-soluble composition can be used in various types of products, such as household detergents, for example, laundry detergents (including clothing detergents), fabric softeners, fabric finishers, fabric softeners, antistatic agents, hand sanitizers, kitchen detergents, household cleaners, furniture cleaners, bath detergents (or toiletry products) such as toothpaste, toothpaste, body shampoos, hand soaps, facial cleansers, shampoos, dry shampoos, and hair care products such as hair rinses; pharmaceuticals such as topical skin preparations containing medicinal agents, quasi-drugs such as medicated cosmetics, skin care cosmetics such as emulsions, lotions, creams, serums, sunscreens, and daytime moisturizers; makeup cosmetics such as foundations, lipsticks, makeup bases, eye shadows, and mascaras; and hair care cosmetics such as hair treatments. Thus, the present disclosure provides any of the above products comprising a SapB protein or a modified SapB protein. In the above-mentioned products, for example, the SapB protein or a modified SapB protein may be contained in the form of an emulsion (e.g., a W / O emulsion or an O / W emulsion). In the above-mentioned products, for example, the SapB protein or a modified SapB protein may be dissolved in a solution. In the above-mentioned products, for example, the SapB protein or a modified SapB protein may be dispersed in a solution. In addition to the uses listed above, the water-soluble and oil-soluble compositions of the present disclosure can be used to wet an object, to penetrate an object with a target substance, to foam an object, for lubrication (e.g., lubrication between solids), for antistatic purposes, for rust prevention, and for sterilization or antibacterial purposes.

[0046] In a preferred embodiment, the water-soluble composition and the fat-soluble composition may further contain, in addition to the SapB protein or a modified SapB protein, other surfactants (e.g., cationic surfactants, anionic surfactants, nonionic surfactants, amphoteric surfactants, etc.).

[0047] In one aspect of the present invention, a method for preparing a SapB protein according to the present disclosure is provided. The method for preparing a SapB protein according to the present disclosure may include culturing cells, such as microorganisms (e.g., bacteria (e.g., actinomycetes)), that have a gene encoding RamS or a SapB protein in an expressible state to produce the SapB protein. In one embodiment, the cells, such as microorganisms (e.g., bacteria (e.g., actinomycetes)), contain a gene expression cassette that contains part or all of the SapB operon region. In this manner, cells that produce SapB can be obtained. The method for preparing a SapB protein according to the present disclosure may include recovering cells (e.g., bacterial cells) from the culture medium. The source of the SapB protein and the SapB protein-producing cells may be the same or different. The method for preparing a SapB protein according to the present disclosure may include incubating the bacterial cells in an aqueous solution (first aqueous solution). Here, the aqueous solution may be water, for example, distilled water. Incubation of the bacterial cells in the aqueous solution can be carried out at, for example, 0°C or higher, room temperature, room temperature or higher, 30°C or higher, 35°C or higher, 40°C or higher, 50°C or higher, 60°C or higher, 70°C or higher, or 80°C or higher, or at a temperature between any two of these temperatures. Incubating the bacterial cells in the aqueous solution at a higher temperature can improve extraction efficiency. The first aqueous solution preferably does not contain or is substantially free of urea. The method for preparing a modified SapB protein according to the present disclosure may include incubating the bacterial cells in an aqueous solution containing urea (second aqueous solution). Incubation in the aqueous solution containing urea can be carried out without incubation in the first aqueous solution or, preferably, after incubation in the first aqueous solution. The urea concentration in the second aqueous solution can be, for example, 0.2M or higher, 0.5M or higher, 1M or higher, 2M or higher, 3M or higher, 4M or higher, 5M or higher, 6M or higher, 7M or higher, or 8M or higher. In some embodiments, the urea concentration in the second aqueous solution may be, for example, 1 to 8 M, 2 to 7 M, 3 to 6 M, for example, 4 to 5 M, for example, about 4 M, or about 5 M.As described below, incubation in the second aqueous solution at a high temperature for a long period of time promotes the conversion of SapB protein to a modified form. Therefore, incubation in the second aqueous solution can be performed, for example, at a temperature at which urea does not precipitate, such as 90°C or less, preferably 80°C or less, 70°C or less, 60°C or less, 50°C or less, 40°C or less, 35°C or less, or 30°C or less, and preferably at room temperature. The incubation time in the second aqueous solution can be, for example, 4 hours or less, 3 hours or less, 2 hours or less, 1 hour or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, or 20 minutes or less, for example, 5 to 20 minutes. By performing incubation in the second aqueous solution at a low temperature for a short period of time, SapB protein can be obtained, while by performing incubation at a high temperature for a long period of time, a mixture of SapB protein and modified SapB protein can be obtained. The obtained SapB protein can be purified as needed. Examples of purification include filtration through filter paper (e.g., Advantech No. 2 and No. 5C) and / or a filter to remove solid matter (e.g., bacterial debris). Filter filtration can also be used to remove contaminants. Purification also includes dialysis. Dialysis can replace the solvent with an external dialysis solution. In this way, the method of the present disclosure can prepare SapB protein, modified SapB protein, or a mixture thereof. In the above method, the first aqueous solution may comprise or consist of ultrapure water (e.g., an 18.2 MΩ cm aqueous solution, e.g., Milli-Q water) or distilled water. The first aqueous solution may further comprise an additive such as a salt. The solvent of the second aqueous solution may comprise or consist of ultrapure water (e.g., an 18.2 MΩ cm aqueous solution, e.g., Milli-Q water) or distilled water. The second aqueous solution may further comprise an additive such as a salt.

[0048] One aspect of the present invention provides a method for preparing a modified SapB protein according to the present disclosure. The method for preparing a modified SapB protein according to the present disclosure may include culturing a microorganism (e.g., a bacterium (e.g., an actinomycete)) that has an expressible gene encoding RamS or a SapB protein to produce the SapB protein. In one embodiment, the microorganism (e.g., a bacterium (e.g., an actinomycete)) contains a gene expression cassette having part or all of the SapB operon region. The method for preparing a modified SapB protein according to the present disclosure may include recovering the bacterial cells from the culture medium. The source of the SapB protein and the cells that produce the SapB protein may be the same or different. The method for preparing a modified SapB protein according to the present disclosure may include extracting SapB from the bacteria that produced the SapB protein in the presence of urea. The urea concentration can be, for example, 0.2 M or higher, 0.5 M or higher, preferably 1 M or higher, more preferably 2 M or higher, even more preferably 3 M or higher, and particularly preferably 4 M or higher, 5 M or higher, 6 M or higher, 7 M or higher, or 8 M or higher. Here, a higher urea concentration in the treatment can increase the conversion efficiency from the native form to the modified form. In some embodiments, the urea concentration can be, for example, 1 to 8 M, 2 to 7 M, 3 to 6 M, 3 to 7 M, or 3 to 8 M, for example, 4 to 5 M, 4 to 6 M, 4 to 7 M, or 4 to 8 M, for example, about 4 M or about 5 M. Extraction can be performed at room temperature or under heating. Heating conditions can be, for example, 40°C or higher, 50°C or higher, 60°C or higher, 70°C or higher, 80°C or higher, or 90°C or higher. Heating conditions can be, for example, 70 to 90°C, for example, about 80°C. The resulting modified SapB protein can be purified as needed. Examples of purification include filtration through filter paper (e.g., Advantech No. 2 and No. 5C) and / or a filter to remove solid matter (e.g., bacterial debris). Filter filtration can also be used to remove contaminants. Purification also includes dialysis. Dialysis can replace the solvent with the external dialysis solution. Hereinafter, the modified product obtained by the above method may be simply referred to as the modified product.In the method for preparing a modified SapB protein according to the present disclosure, a mixture of the SapB protein and its modified form may be obtained.

[0049] The expression cassette contains a gene encoding the SapB protein operably linked to a regulatory sequence. The gene encoding the SapB protein can be expressed in cells by being operably linked to a regulatory sequence. The regulatory sequence is, for example, a promoter. Any promoter can be used as long as it can express SapB in a SapB-expressing cell. The SapB operon originally contains a gene encoding the SapB protein operably linked to a regulatory sequence. Those skilled in the art can construct a system suitable for expressing the SapB protein by appropriately modifying the expression cassette.

[0050] In a preferred embodiment, the urea concentration in a composition containing a SapB protein or a modified form thereof may be less than 1%, less than 0.1%, or less than 0.01% (w / w), or below the detection limit. Urea in the composition can be removed by conventional methods, such as dialysis.

[0051] In a preferred embodiment, in a composition comprising a SapB protein or a composition comprising a modified SapB protein, the concentration of the SapB protein, in terms of the weight of the SapB protein moiety, is, for example, 0.001 wt / wt% or more, 0.01 wt / wt% or more, 0.02 wt / wt% or more, 0.03 wt / wt% or more, 0.04 wt / wt% or more, 0.05 wt / wt% or more, 0.06 wt / wt% or more, 0.07 wt / wt% or more, 0.08 wt / wt% or more, 0.09 wt / wt% or more, 0.1 wt / wt% or more, 0.11 wt / wt% or more, 0.12 wt / wt% or more, 0.13 wt / wt% or more, 0.14 wt / wt% or more, 0.15 wt / wt% or more, % or more by weight, 0.16% or more by weight, 0.17% or more by weight, 0.18% or more by weight, 0.19% or more by weight, 0.2% or more by weight, 0.3% or more by weight, 0.4% or more by weight, 0.5% or more by weight, 0.6% or more by weight, 0.7% or more by weight, 0.8% or more by weight, 0.9% or more by weight, 1.0% or more by weight, 1.1% or more by weight, 1.2% or more by weight, 1.3% or more by weight, 1.4% or more by weight, 1.5% or more by weight, 1.6% or more by weight, 1.7% or more by weight, 1.8% or more by weight, 1.9% or more by weight, or 2% or more by weight.

[0052] In a preferred embodiment, a composition comprising a SapB protein or a composition comprising a modified SapB protein may be an aqueous composition and may contain a pH buffer. An aqueous composition is a composition containing water. In one embodiment, water is included as a solvent. In a preferred embodiment, a composition comprising a SapB protein or a composition comprising a modified SapB protein may have a pH of 5 or higher, 6 or higher, 7 or higher, preferably 8 or higher, more preferably 9 or higher, and even more preferably 10 or higher. Increasing the pH is preferable because it increases the amount of SapB protein or its modified form dissolved in aqueous solution. The resulting composition comprising a SapB protein or a composition comprising a modified SapB protein may contain, but may not contain, or may be substantially free of, trifluoroacetic acid (TFA). In a preferred embodiment, a composition comprising a SapB protein or a composition comprising a modified SapB protein does not contain, or is substantially free of, trifluoroacetic acid (TFA). In one embodiment, a composition comprises a SapB protein or a modified form thereof.

[0053] In one embodiment, a composition comprising a SapB protein or a composition comprising a modified SapB protein comprises a pH buffer and has a pH of 8 or greater, more preferably 9 or greater, and even more preferably 10 or greater. In this embodiment, the composition preferably does not contain or is substantially free of trifluoroacetic acid (TFA). In one embodiment, the composition comprises a SapB protein and a modified SapB protein.

[0054] In one embodiment, the resulting composition comprising the SapB protein or the resulting composition comprising a modified SapB protein comprises a pH buffer, has a pH of 8 or higher, more preferably 9 or higher, and even more preferably 10 or higher, and comprises the SapB protein and / or its modified form at a concentration of 0.1% w / w or higher, preferably 0.5% w / w or higher, and more preferably 1% w / w or higher, calculated as the weight of the SapB protein portion. In this embodiment, preferably, the composition does not contain or is substantially free of trifluoroacetic acid (TFA). In one embodiment, the composition comprises the SapB protein and its modified form. The concentration of the SapB protein and / or modified form thereof can be, for example, 0.1 wt / wt% or more, 0.2 wt / wt% or more, 0.3 wt / wt% or more, 0.4 wt / wt% or more, 0.5 wt / wt% or more, 0.6 wt / wt% or more, 0.7 wt / wt% or more, 0.8 wt / wt% or more, 0.9 wt / wt% or more, 1.0 wt / wt% or more, 1.1 wt / wt% or more, 1.2 wt / wt% or more, 1.3 wt / wt% or more, 1.4 wt / wt% or more, 1.5 wt / wt% or more, 1.6 wt / wt% or more, 1.7 wt / wt% or more, 1.8 wt / wt% or more, 1.9 wt / wt% or more, or 2 wt / wt% or more. Here, the concentration is calculated based on only the dissolved SapB protein and modified form thereof.

[0055] In some embodiments, the pH of the composition of the present disclosure may be 11 or less, 10 or less, or 9 or less. In some embodiments, the pH of the composition of the present disclosure may be 8 to 11. In some embodiments, the pH of the composition of the present disclosure may be 8 to 10. In some embodiments, the pH of the composition of the present disclosure may be 9 to 11. In some embodiments, the pH of the composition of the present disclosure may be 9 to 10. In some embodiments, the pH of the composition of the present disclosure may be 8.5 to 10.5. In some embodiments, the pH of the composition of the present disclosure may be 9 to 10.5.

[0056] In a preferred embodiment, the composition of the present disclosure comprises a native SapB protein and may have a pH of 8 or higher. In a preferred embodiment, the composition of the present disclosure comprises the above-described modified SapB protein and may have a pH of 6 or higher. Under these pH conditions, the native SapB protein and the above-described modified SapB protein may exhibit good solubility in aqueous solutions.

[0057] The pH range suitable for each pH buffer is well known, and those skilled in the art can select a pH buffer to adjust the solution to the desired pH. For example, the pH buffer can be selected from the group consisting of glycine-HCl, acetate, phosphate, Tris, and glycine-NaOH. For example, the pH buffer can be selected from the group consisting of BES, MOPS, TES, HEPES, DIPSO, TAPSO, Tricine, POPSO, HEPPSO, Bicine, TAPS, HEPPS, CHES, and CAPS. For example, a glycine-HCl buffer can be used at pH 3-4, an acetate buffer can be used at pH 3-6, a phosphate buffer can be used at pH 5-8, a Tris buffer can be used at pH 7-9, and a glycine-NaOH buffer can be used at pH 8-11.

[0058] In some embodiments, in the compositions of the present disclosure, the SapB protein and modified SapB protein are at least partially dissolved in the aqueous solution. In some preferred embodiments, in the compositions of the present disclosure, the SapB protein and modified SapB protein are completely dissolved in the aqueous solution. In some preferred embodiments, the SapB protein and modified SapB protein are suspended in the aqueous solution. In some preferred embodiments, the compositions of the present disclosure do not contain precipitates of the SapB protein and SapB protein.

[0059] In some embodiments, the compositions of the present disclosure have surface-active properties, and therefore can be used as surfactants or emulsifiers. [Example]

[0060] Example 1: Construction of actinomycetes with high SapB expression The SapB operon region (including ramC, ramS, ramA, ramB, and ramR) was cloned from Streptomyces coelicolor and introduced into an expression vector. ramS (having the amino acid sequence registered as O88038 in UniProtKB / Swiss-Prot) encodes the precursor of the SapB protein, which is produced by excision from the precursor. Genes predicted to encode SapB and correspond to RamS in various actinomycetes (hereafter referred to simply as "SapB" or "SapB protein") were identified, and the SapB operon region of each gene was introduced into an expression vector in the same manner as above. To overexpress the SapB protein, Streptomyces lividans 1326 was transformed with the expression vector containing the SapB-encoding gene to obtain transformants (strains with integrated SapB operon region and high SapB protein expression).

[0061] The RamS used in this example has the amino acid sequence of the SapB precursor of SEQ ID NO: 1, but the native structure of the SapB protein is modified after post-translation to have the following structure (Proc Natl Acad Sci USA. 2004; 101(31): 11448-11453).

[0062] [ka]

[0063] The amino acid sequences of other SapB precursors also undergo similar post-translational modifications. [ka] {Here, the amino acid sequence is represented according to the primary sequence encoded by the gene. In reality, post-translational modification occurs, where the OH group of serine (S) reacts with the SH group of cysteine ​​(C), resulting in the same structure as shown above, where the methyl groups of the two alanine side chains are linked by an S.}

[0064] In the above, Dha represents dehydroalanine, and the 3rd alanine and the 10th alanine form a lanthionine bridge, and the 13th alanine and the 20th alanine form a lanthionine bridge.

[0065] For seed culture, the SapB-expressing strain was cultured with shaking at 28°C for 3 days in a test tube containing 5 mL of TSB medium (Becton Dickinson Bacto™ Tryptic Soy Broth (Soybean-Casein Digest Medium, product number 211825), 50 μg / mL of thiostrepton. Subsequently, for main culture, 1000 μL of the seed culture was inoculated into a flask (TSB medium, 100 mL) and cultured with shaking at 28°C for 4 days.

[0066] A strain expressing SapB protein having the amino acid sequence set forth in SEQ ID NOS: 2 to 9 was cultivated under these culture conditions, and the expression of SapB protein was analyzed by SDS-PAGE after ultrasonic disruption. A band indicating SapB protein expression was confirmed (see Figure 7). When the S. griseus SapB protein was subjected to Edman degradation, the first four amino acids from the N-terminus were decoded, but the third amino acid, which contains a lysine bond, was not decoded because its structure is incompatible with Edman degradation. This result is consistent with the result predicted from the intramolecular cross-linking structure of S. griseus.

[0067] Example 2: Extraction and recovery of SapB After the main culture, the culture medium was collected and the cells were centrifuged to recover the SapB protein. Because SapB has low water solubility, the extraction procedure was carried out under the following conditions to increase the amount of SapB extracted. (Condition 1) Extraction was performed by treating the cells in a 4M urea solution (distilled water containing 4M urea) under heating at 80°C, followed by stirring the cells in the urea solution for 2 hours; (Condition 2) Extraction is performed by stirring the bacterial cells in distilled water at 80°C for 2 hours, then cooling to room temperature, adding urea to a final concentration of 4 M, and stirring for 10 minutes; or (Condition 3) Extraction is performed by incubating the cells in distilled water at 80°C.

[0068] After treatment under conditions 1 and 2, the liquid phase was recovered from the stirred urea solution. The recovered urea solution contained SapB protein. Filtration was performed using filter paper No. 2 (Advantec), and the permeate was recovered. Next, filtration was performed using filter paper No. 5C (Advantec), and the permeate was recovered. The solution was then concentrated using an evaporator. Dialysis was performed using distilled water as the external solution. The resulting concentrated solution was freeze-dried.

[0069] To compare the yield, the amount of SapB protein extracted with water under condition 3 was compared with the amount of SapB protein extracted with urea solution. Only in condition 3 was the culture medium changed to synthetic medium (4.5% glucose, 0.94% NH4Cl, 0.14% KH2PO4, 0.02% MgSO4·6H2O, 0.07% KCl, 0.09% Na2SO4, 0.2% NaCl, 0.2% trace elements (1.35% FeCl3·6H2O, 0.15% CuCl2·6H2O, 0.9% ZnCl2, 0.36% MnCl2·4H2O, 0.06% Na2MoO4·2H2O, 0.04% CoCl2, 0.03% H3BO4).

[0070] When extracted with water, the yield was 8 mg / L (culture volume), whereas under condition 1 the yield was 174 mg / L (culture volume), and the yield was significantly increased by extraction with urea solution. Under condition 2, the yield was 109 mg / L (culture volume), and the yield was significantly increased by heating followed by urea treatment at room temperature.

[0071] The emulsifying activity of SapB obtained in Example 2 was confirmed. Oil and water were mixed in a 1:2 ratio in the presence of 1% w / w of SapB protein obtained under Conditions 1 or 2 and stirred. After 30 seconds, emulsifying activity was observed in the presence of the SapB protein obtained under Condition 1, as shown in the left panel of Figure 1. Thus, it was revealed that the obtained SapB exhibits good surfactant activity and is useful as an emulsifier. Similar surfactant activity was observed for the protein obtained under Condition 2. Generally, surfactants cause emulsification when their concentration is sufficiently higher than the CMC, and demulsification occurs at concentrations slightly above the CMC. Similar characteristics were observed for SapB.

[0072] Next, the critical micelle concentration (CMC) of the resulting SapB was estimated using the Wilhelmy method. The SapB solution was placed in the instrument, and data was collected after 2 hours to determine the CMC. As a result, the CMC was estimated to be 0.006% (see Figure 8).

[0073] Example 3: Analysis of the obtained SapB The two types of SapB proteins obtained in Example 2 were analyzed by HPLC, LC-MS, LC-MS / MS, and N-terminal amino acid analysis.

[0074] (1) HPLC SapB was dissolved in 100 mM Tris-HCl (pH 8.0) to a concentration of 0.1 mg / mL. HPLC measurement was performed under the following conditions. Column: Cosmosil 5Ph-AR-300 (4.6 mm I.D. x 150 mm) Eluent: Solution A HO (0.1% TFA) Solution B: Acetonitrile:2-propanol (3:2) (0.1% TFA) Flow rate: 1mL / min Gradient: 20-50% / 1-31 min Detection: 220 nm

[0075] [Table 2]

[0076] The results are shown in Figure 2A. As shown in Figure 2A, a peak with a delayed retention time was detected in the sample containing SapB protein extracted under condition 1 compared to SapB protein extracted under condition 2. Based on the LC-MS analysis results shown in Figures 3A and 3B, as described below, the sample extracted under condition 2 was native SapB, ​​while the sample extracted under condition 1 was considered to be a modified form of native SapB modified with a carboxamide group. Furthermore, the conversion efficiency of SapB protein to a modified form was examined by reacting the native SapB sample prepared under condition 2 with a urea solution. The urea concentration during heating was 1 M, 2 M, or 4 M, and the heating time was 0 to 240 min. The reaction products were then analyzed by HPLC. The results are shown in Figure 2B. As shown in Figure 2B, a modified form peak was observed in samples heated for 60 min or longer. In the composition obtained by heating for 240 minutes in the presence of 4 M urea, 40-60% of the native SapB protein was converted to the modified form. In other words, the native form can be efficiently converted to the modified form by directly reacting native SapB with a urea solution.

[0077] (2) LC-MS Next, liquid chromatography-mass spectrometry (LC-MS) was performed. When urea-extracted SapB was loaded onto a liquid chromatograph, two peaks were observed (see Figure 3A, upper panel). Fractions containing each of these peaks were subjected to mass spectrometry. The results are shown in Figure 3B. In the first fraction, a doubly charged ion peak was observed at 1013.9866 m / z (theoretical value: 1013.9884 m / z), and in the second fraction, a doubly charged ion peak was observed at 1035.4900 m / z (theoretical value: 1035.4914 m / z). The peak in the first fraction corresponded to the molecular weight of native SapB. The second peak was only observed in urea-extracted SapB. Since the second peak was barely observed when extracted in the presence of urea at room temperature without heating, it was suggested that treatment of the SapB protein in the presence of urea under heated conditions was necessary for modification. The difference in m / z was approximately 21.5. This suggests that SapB is modified with NH2-C(O)-.

[0078] (3) LC-MS / MS The two peaks detected by LC-MS (native SapB and NH-C(O)-modified SapB) were cleaved using an Agilent 6545XT QTOF instrument at a collision energy of 40 eV. The results are shown in Figure 3C (showing m / z values ​​around 130–160) and Figure 3D (showing m / z values ​​around 960–1190).

[0079] As shown in Figure 3C, [M+H] + The fragment peak at 145.0607 was detected only upon cleavage of the peak of SapB modified with NH2-C(O)- (SapB modified form) (lower panel of Figure 3C), which coincided with the b ion peak (theoretical value: 145.0613) derived from NH2-C(O)-NH2-CH(CH(OH)CH3)-CO. This indicates that the amino group of the N-terminal threonine was modified with NH2-C(O)-. Furthermore, [M+H] +A product peak at 133.0604 to 133.0605 was commonly detected, which coincided with the y ion peak (theoretical value: 133.0613) derived from the C-terminal asparagine residue.

[0080] As shown in Figure 3D, the molecular ion peak of native SapB splits into [M+H] + The b-ion peak series at 966.5168 (theoretical value: 966.5195), 1023.5385 (theoretical value: 1023.5410), and 1138.5650 (theoretical value: 1138.5679) correspond to [M+H] when the molecular ion peak of SapB modified with NH2-C(O)- (SapB modified product) is split. + The b ion peak series detected were 1009.5251 (theoretical value: 1009.5253), 1066.5462 (theoretical value: 1066.5468), and 1181.5717 (theoretical value: 1181.5737). The mass differences within each series were 57.0211–57.0217 and 115.0255–115.0265, respectively. This indicates that the 11th residue from the N-terminus of the SapB modification product is a glycine residue (theoretical value: 57.0215), and the 12th residue is an aspartic acid residue (theoretical value: 115.0269).

[0081] The mass difference between the two series was 43.0067-43.0083, which was consistent with NH2-C(O) (theoretical value: 43.0058). + The product peaks at 1061.4509–1061.4545 were commonly detected, which coincided with the y ion peak (theoretical value: 1061.4573) derived from the 11 residues on the C-terminus. These results indicate that one of the 10 residues on the N-terminus was modified with NH2-C(O)-.

[0082] [ka] TIFF0007818015000013.tif139142

[0083] (5) Resistance of modified SapB protein to degradation The protein was subjected to degradation. The degradation involved the degradation of the N-terminal amino acid by Edman degradation. Edman degradation is a technique used to decipher amino acid sequences and was performed using standard methods. In Edman degradation, an amino acid is reacted with phenylisothiocyanate, followed by acid treatment to liberate a phenylthiohydantoin derivative containing the first residue and the second amino acid. This allows for amino acid sequence analysis. While Edman degradation of native SapB allowed for the deciphering of two amino acid sequences from the N-terminus, the SapB extracted under Condition 1 of the present invention was unable to decipher the N-terminal amino acid, and therefore the first amino acid could not be deciphered. This was thought to be due to the resistance of the N-terminus to degradation. Based on the reaction mechanism of Edman degradation, it was determined that the urea modification occurred at the N-terminal amino group of SapB.

[0084] (6) Characteristics of lysis buffer The lyophilized product of native SapB obtained under Condition 2 in Example 2 or the dried product of modified SapB obtained under Condition 1 was suspended or dissolved in buffer solutions having different pH values ​​as shown in Table 3 below to a concentration of 0.1% by weight (mg / mL).

[0085] [Table 3]

[0086] After vigorous stirring, the solution was allowed to stand and the dissolution state of the native SapB protein was observed. The results are shown in Figure 4. As shown in Table 4, the alkaline buffer solution (pH 8-10) was clear and no precipitate was observed, whereas the acidic to neutral buffer solution (pH 3-7) was cloudy. Precipitation was observed at the bottom of the bottle, particularly at pH 3-5. In contrast, as shown in Table 4, the modified SapB protein solution became clear at pH 6 or higher and no precipitate was observed. This demonstrates that the modified SapB protein is soluble in aqueous solutions at pH 6 or higher.

[0087] [Table 4]

[0088] Lyophilized native SapB protein was suspended or dissolved in various buffer solutions. As above, the solution was cloudy at pH 7 and clear at pH 8, with no precipitate observed.

[0089] The native SapB protein obtained above was suspended or dissolved in buffers of various pH levels, or the supernatants thereof were subjected to SDS-PAGE. The results are shown in Figure 4. As shown in Figure 4, when the suspension was loaded, the same amount of native SapB protein was observed in each lane. However, when the supernatant was loaded, no modified SapB was detected at pH 3 to 7, and the native SapB protein was detected at pH 8, with the amount significantly increasing at pH 9 to 10. This result indicates that the solubility of the native SapB protein in solution increases at alkaline pH.

[0090] The supernatants of the centrifuged buffers with various pH values ​​in which the native SapB protein obtained above was suspended or dissolved were analyzed by HPLC under the same conditions as in Example 3. The results are shown in Figure 5. As shown in Figure 5, a peak indicated by an arrowhead in the figure was strongly detected at pH 9 or 10. This peak corresponds to the dissolved native SapB protein. The same peak was also weakly detected at pH 8. This result is consistent with the SDS-PAGE results.

[0091] The SapB protein is poorly water-soluble and has traditionally been dissolved in trifluoroacetic acid (TFA), an organic acid. However, our results demonstrate that a TFA-free SapB protein solution can be prepared by dissolving the SapB protein in an alkaline buffer solution.

[0092] The supernatants of the centrifuged buffers of various pH levels in which the modified SapB protein obtained above was suspended or dissolved were analyzed by HPLC under the same conditions as in Example 3. The results are shown in Figure 6. As shown in Figure 6, a peak indicated by an arrowhead in the figure was strongly detected at pH 7 or higher. This peak corresponds to the dissolved modified SapB protein. The same peak was also weakly detected at pH 6.

[0093] Example 4: Hydrophilic modification of surfaces using SapB protein We coated a hydrophobic polycarbonate plate with native SapB protein and tested the wettability of the surface. Specifically, lyophilized native SapB protein was dissolved in a pH 10 buffer solution to a concentration of 0.1 wt% to obtain an aqueous SapB solution. The SapB solution was coated onto a portion of the polycarbonate plate and heated at 60°C for 5 minutes to evaporate the water, forming a SapB-coated area. Distilled water was then dropped onto both the untreated and coated areas of the polycarbonate plate. As shown in Figure 9, water formed droplets with a large contact angle in the untreated area, whereas in the coated area, water spread thinly across the surface. Similar results were obtained with surface treatment using modified SapB. These results demonstrate that surface treatment with SapB and its modified forms are both useful for hydrophilic surface modification.

[0094] Sequence Listing SEQ ID NO: 1: An example of the amino acid sequence of RamS from S. coelicolor SEQ ID NO: 2: An example of the amino acid sequence of the SapB protein of S. coelicolor SEQ ID NO: 5: An example of the amino acid sequence of the SapB protein of S. scabies SEQ ID NO: 6: An example of the amino acid sequence of the SapB protein of S. scabies SEQ ID NO: 7: An example of the amino acid sequence of the SapB protein of S. avermitilis SEQ ID NO: 8: An example of the amino acid sequence of the SapB protein of S. griseus SEQ ID NO: 9: An example of the amino acid sequence of the SapB protein of S. albus [Sequence table] Sequence listing information: Title of invention: Modified SapB protein, method for preparing SapB protein and its modified form, and aqueous solution containing SapB protein or its modified form (ja) Title of the invention: Modified form of SapB protein, method of preparing SapB protein and a modified form thereof, and aqueous solution dissolving SapB protein and a modified form thereof ( en ) Total number of arrays: 9 array: Sequence ID: 1 Length: 42 Molecular type: AA feature position / qualifier: - REGION, 1..42 > note, Amino acid sequence of RamS protein of S. coelicoler - source, 1..42 > mol_type, protein > organism, synthetic construct residue: MNLFDLQSME TPKEEAMGDV ETGSRASLLL CGDSSLSITT CN 42 Sequence ID: 2 Length: 21 Molecular type: AA feature position / qualifier: - REGION, 1..21 > note, Amino acid sequence of unmodified SapB protein of S. griseus - source, 1..21 > mol_type, protein > organism, synthetic construct residue: TGSRASLLLC GDSSLSITTC N 21 Sequence ID: 3 Length: 22 Molecular type: AA feature position / qualifier: - REGION, 1..22 > note, Amino acid sequence of unmodified SapB protein of S. griseus - source, 1..22 > mol_type, protein > organism, synthetic construct residue: TGSQASLLLC EYSSLSVVLC TP 22 Sequence ID: 4 Length: 22 Molecular type: AA feature position / qualifier: - REGION, 1..22 > note, Amino acid sequence of unmodified SapB protein of S. albus - source, 1..22 > mol_type, protein > organism, synthetic construct residue: TGSQVSLLVC EYSSLSVTLC TP 22 Sequence ID: 5 Length: 27 Molecular type: AA feature position / qualifier: - REGION, 1..27 > note, Amino acid sequence of unmodified SapB protein of S.scabies - source, 1..27 > mol_type, protein > organism, synthetic construct residue: TVEYLSVLSS LSVVNCTNST VSTLLCL 27 Sequence ID: 6 Length: 22 Molecular type: AA feature position / qualifier: - REGION, 1..22 > note, Amino acid sequence of unmodified SapB protein of S. scabies 2 - source, 1..22 > mol_type, protein > organism, synthetic construct residue: TGGPSSLSVL SCVSAASITL CL 22 Sequence ID: 7 Length: 24 Molecular type: AA feature position / qualifier: REGION, 1..24 > note, Amino acid sequence of unmodified SapB protein of S. avermitilis - source, 1..24 > mol_type, protein > organism, synthetic construct residue: TGGGGASTVS LLSCVSAASV LLCL 24 Sequence ID: 8 Length: 22 Molecular type: AA feature position / qualifier: - source, 1..22 > mol_type, protein > organism, Streptomyces griseus residue: TGSQVSLLVC EYSSLSVVLC TP 22 Sequence ID: 9 Length: 22 Molecular type: AA feature position / qualifier: - source, 1..22 > mol_type, protein > organism, Streptomyces albus residue: TGSQVSLLVC EYSSLSVVLC SP 22 END

Claims

1. 1. A method for preparing a SapB protein, comprising: Cultivating an actinomycete having a gene encoding the RamS or SapB protein in an expressible manner in a culture medium to allow the actinomycete to produce the SapB protein; recovering the actinomycete that produced the SapB protein from the culture medium; The recovered actinomycetes (a) under a temperature condition equal to or higher than a first predetermined temperature and in the absence of urea incubating in a first aqueous solution, wherein the first predetermined temperature is 0° C. or higher; wherein the SapB protein is derived from a portion of the RamS protein.

2. The method according to claim 1, wherein, when the SapB protein is aligned with the SapB protein derived from S. coelicolor, two amino acids in the SapB protein corresponding to the serine at position 6 and the serine at position 16 of SEQ ID NO: 2 are converted to dehydroalanine, two amino acids in the SapB protein corresponding to the serine at position 3 and the cysteine ​​at position 10 of SEQ ID NO: 2 form a lanthionine bond between their β carbons, and two amino acids in the SapB protein corresponding to the serine at position 13 and the cysteine ​​at position 20 of SEQ ID NO: 2 form a lanthionine bond between their β carbons.

3. The method according to claim 1 or 2, wherein the SapB protein is derived from an actinomycete.

4. The method described in claim 1 or 2, wherein the SapB protein is derived from a peptide having an amino acid sequence set forth in any one of SEQ ID NOs: 2, 5, 6, 7, 8, and 9.

5. 3. The method of claim 1, wherein the first predetermined temperature is 40°C or higher.

6. 3. The method of claim 1 or 2, further comprising purifying the obtained SapB protein to obtain a product comprising a composition comprising the isolated SapB protein and an aqueous solution, wherein the isolated SapB protein is dissolved in the aqueous solution, and the aqueous solution is alkaline.

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