Modified form of sapb protein, method for preparing sapb protein and modified form thereof, and aqueous solution in which sapb protein or modified form thereof is dissolved

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

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

[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.

[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).

[0004] Mol Microbiol. 1998;30(3):595-602Proc Natl Acad Sci USA. 2004;101(31):11448-11453J Org Chem. 2018;83(14): 7528-7533

[0005] The present invention provides modified forms of SapB protein, methods for preparing SapB protein and modified forms thereof, and aqueous solutions in which SapB protein or modified forms thereof are dissolved.

[0006] The present inventors have found that extraction of SapB protein from SapB protein-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 have also discovered a novel modified SapB in which the N-terminus is chemically modified during the purification process of SapB protein. Specifically, the present inventors have found that extraction of SapB protein from SapB protein-expressing cells under heated conditions and in the presence of urea results in high yields of novel modified SapB in which the N-terminus is chemically modified, and that the resulting modified SapB has excellent surfactant and emulsifying properties. Furthermore, the novel modified SapB in which the N-terminus is chemically modified is stable against some degradation treatments. The present inventors have further found that the poorly water-soluble SapB protein and its modified forms exhibit high solubility in alkaline buffer solutions. The present inventors have further found that modified SapB protein exhibits high solubility in buffer solutions of pH 6 or higher.

[0007] The present invention provides the following: (1) A method for preparing SapB protein, comprising: providing cells that produce SapB protein; and incubating the cells (a) at a temperature equal to or higher 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) at a temperature 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 SapB protein or a modified SapB protein in which the amino group of the N-terminal amino acid is modified or protected. (2) The method described in (1) above, wherein the incubation is carried out (a) at a temperature equal to or higher than the first predetermined temperature and in the absence of urea. (3) The method described in (1) above, wherein the incubation is carried out (b) at a temperature below the 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 an isolated SapB protein, wherein the amino group of the N-terminal amino acid is modified or protected. (6) The modified form of the SapB protein according to (5) above, wherein the modification or protection is with an amide group. (7) The modified or protected form of an isolated SapB protein, wherein the modification or protection is with an NH ２ (9) A modified SapB protein according to any one of (5) to (7) above, wherein the SapB protein is modified or protected by a -C(O)- group. (10) A modified SapB protein according to any one of (5) to (7) above, wherein the SapB protein is derived from an actinomycete. (11) A modified SapB protein according to any one of (5) to (7) above, wherein the SapB protein is derived from an actinomycete. (12) A modified SapB protein according to any one of (5) to (7) above, wherein the SapB protein is modified or protected by a -C(O)- group. (13) A modified SapB protein according to any one of (5) to (7) above, wherein the SapB protein is derived from an actinomycete. (14) A modified SapB protein according to any one of (5) to (7) above, wherein the SapB protein has a structure represented by the following formula (I): (10) A composition comprising the 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). (12) An emulsifier comprising the modified SapB protein according to any one of (5) to (9) above or the composition according to (10). (13) A method for preparing a composition comprising an emulsion from a water-soluble composition and a fat-soluble composition, the method comprising mixing the modified SapB protein according to any one of (5) to (9) above or the composition according to (10) with the water-soluble composition and the fat-soluble composition. (14) A composition comprising an emulsion comprising a fat-soluble solute molecule and the 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, wherein the isolated SapB protein or modified form thereof is dissolved in the aqueous solution, and the aqueous solution is alkaline. (17) The composition according to claim 14, wherein the modified form of isolated SapB is the modified form according to any 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 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 of (16) to (19) above, wherein the concentration of the isolated SapB protein or modified form thereof is 0.1 wt / wt % or higher, calculated in terms of the weight of the SapB protein portion. (21) An aqueous composition comprising a modified form of isolated SapB protein and having a pH of 6 or higher. (22) The composition according to (21) above, wherein the modified isolated SapB protein is a modified form according to any one of claims 5 to 9.

[0008] (31) A SapB expression vector comprising a gene expression cassette having a 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 expressing at least the SapB protein in actinomycetes. (33) A cell comprising 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 cell according to (33) or (34) above. (37) The SapB expression vector according to (31) or (32) above, the cell according to (33) or (34) above, or the composition according to (35) or (36) above, for use in expressing the SapB protein. (38) A method for preparing the SapB protein, comprising: introducing the SapB expression vector according to (31) or (32) above into cells; and culturing the obtained cells to produce the SapB protein. (39) A method for preparing the SapB protein, comprising culturing the cell according to (33) or (34) above to produce the SapB protein. (40) A method for preparing a modified SapB protein, comprising: introducing the SapB expression vector according to (31) or (32) above into cells; culturing the resulting cells to produce the SapB protein; and heating the resulting SapB protein in the presence of urea to obtain a modified SapB protein. (41) A method for preparing a modified SapB protein, comprising: culturing the cell according to (33) or (34) above to produce the SapB protein; and heating the resulting SapB protein in the presence of urea to obtain a modified SapB protein.

[0009] The method for preparing the SapB protein of the present invention is suitable for recovering the poorly water-soluble 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.

[0010] FIG. 1 shows the emulsifying activity of SapB having the new chemical modification obtained in this example. FIG. 2A shows high-performance liquid chromatographs of native SapB protein and SapB having the new chemical modification obtained in this example. FIG. 2B shows high-performance liquid chromatographs of native SapB protein and SapB having the new chemical modification obtained in this example (modified SapB protein) in solutions incubated for different periods of time in the presence of different concentrations of urea. FIG. 3A shows the separation of SapB protein by liquid chromatography and the chromatographs extracted by MS for each. FIG. 3B shows the mass spectrometry spectrum of native SapB and the mass spectrometry spectrum of SapB having the new chemical modification obtained in this example. FIG. 3C shows native SapB (top row) and NH ２ Figure 3D shows the enlarged spectra of the m / z range from 130 to 160 of the peaks of SapB modified with -C(O)-. The upper panel shows native SapB, ​​and the lower panel shows NH ２Figure 4 shows enlarged views of the m / z region around 960 to 1190 in the cleaved spectra of the peaks of SapB modified with -C(O)-. Figure 4 shows the results of SDS-PAGE of suspensions (left panel) and centrifugal supernatants (right panel) containing modified forms of SapB protein in buffers with different pH values. The arrowheads in the figures indicate the bands of modified SapB protein. Figure 5 shows high-performance liquid chromatographs of centrifugal supernatants containing native SapB protein in buffers with different pH values. The arrowheads in the figures indicate the peaks of modified SapB protein. Figure 6 shows high-performance liquid chromatographs of centrifugal supernatants containing modified SapB protein in buffers with different pH values. The arrows in the figures indicate the peaks of modified SapB protein. 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 product). The arrows in the figure indicate the position of the SapB protein band. Figure 8 shows the relationship between SapB concentration and surface tension (mN / m) measured by the Wilhelmy method. Figure 9 shows the effect of surface treatment by SapB coating. Specific Description of the Invention

[0011] <Definitions> As used herein, "isolating" or "isolation" refers to separating a specific substance or molecule from at least one other substance or molecule. For example, when cell production is induced, isolation may include separating the specific substance from the cells. 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 separation of specific molecules based on affinity. While isolation is a different 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 polypeptides and proteins. 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) and 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-type. 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 treated as an amino acid in biochemistry and is treated as such herein. Furthermore, a dehydroamino acid with an unnatural structure called dehydroalanine is found in the SapB protein, and dehydroalanine is also classified as an amino acid. Dehydroalanine has the following structure: 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 structure HOOC-CH(NH ２ )-CH ２ -S-CH ２ -CH(NH ２ )-COOH, where the carbons on either side of S are β-carbons. In the SapB protein, before translational modification, what were serine and cysteine ​​residues undergo dehydration condensation between the side chains to form a structure of two alanine residues with a thioether bond at the β-carbon.

[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 into particles to form micelles. It is known that the formation of micelles by an amphiphilic substance stabilizes the dispersion 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 amino acids 22 to 42 of RamS, a protein having an amino acid sequence registered in UniProtKB / Swiss-Prot under registration number O88038, and may be a homologue thereof, such as an orthologue. 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 totally 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 amino acids 22 to 42 thereof is TGSRASLLLC GDSSLSITTC N (SEQ ID NO: 2) (wherein the underlined amino acids are chemically modified, with the third S and the tenth C linked via a lanthionine bond between their β-carbons, the thirteenth S and the twentieth C linked via a lanthionine bond between their β-carbons, and the sixth S and the sixteenth S being 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 such as an actinomycete (e.g., a bacterium) and expressing it. The SapB protein may 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 that includes ramC, ramS, ramA, ramB, and ramR. ramC, ramS, ramA, and ramB may be driven by the promoter ramCSABp, and ramR may be driven by the promoter ramRp.Therefore, the SapB protein may be produced by introducing the operon into a microorganism (eg, a bacterium) such as an actinomycete (eg, Streptomyces).

[0018]

[0019] Herein, a cell into which the SapB operon has been introduced is referred to as a SapB operon region-integrated cell. 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] <Modified SapB protein having 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]

[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 aspect, 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 aspect, 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" means 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. These post-translational modifications include: (a) conversion of two amino acids in the SapB protein corresponding to serine 6 and serine 16 of SEQ ID NO: 2 to dehydroalanine; (b) formation of a lanthionine bond between the β-carbons of two amino acids in the SapB protein corresponding to serine 3 and cysteine ​​10 of SEQ ID NO: 2; and (c) formation of a lanthionine bond between the β-carbons of two amino acids in the SapB protein corresponding to serine 13 and cysteine ​​20 of SEQ ID NO: 2. 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 in it corresponding to the serine at position 6 and the serine at position 16 of SEQ ID NO: 2 converted to dehydroalanine, two amino acids in it corresponding to the serine at position 3 and the cysteine ​​at position 10 of SEQ ID NO: 2 formed with a lanthionine bond between their beta carbons, and two amino acids in it corresponding to the serine at position 13 and the cysteine ​​at position 20 of SEQ ID NO: 2 formed with a lanthionine bond between their beta 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]

[0030] In the above alignment, the amino acid sequence of SapB before post-translational modification is aligned, and the corresponding amino acid sequences are aligned at the same position. In the above alignment, S and C, which form a 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 simply referred to as "modified") or protected. This modification or protection can confer resistance to Edman degradation, for example. Edman degradation can be performed under conditions suitable for amino acid sequence deciphering, for example. 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 N-terminus may be modified or protected by an amide group, and in a more preferred embodiment, the N-terminus may be modified or protected by an NH ２ It is modified or protected by a —C(O)— group.

[0033] In a preferred embodiment, the modified SapB protein of the present disclosure has the structure of formula (I): 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 aspect 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 contain excipients (e.g., bulking agents, thickeners, pH adjusters, fragrances, osmotic pressure adjusters, salts, etc.).

[0035] In one aspect of the present disclosure, the composition comprises a native SapB protein and a modified SapB protein, and the abundance ratio (molar ratio) thereof can be, but is not limited to, 99:1 to 1:99. The abundance 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 present invention, there is provided a method for preparing SapB protein, the method comprising: providing cells that produce SapB protein; and incubating the cells at a temperature equal to or greater than a first predetermined temperature or in the absence of urea, thereby obtaining (or extracting) the SapB protein. In one aspect of the present invention, there is provided a method for preparing SapB protein, the method comprising: providing cells that produce SapB protein; and incubating the cells at a temperature equal to or greater than a second predetermined temperature or in the presence of urea, thereby obtaining (or extracting) the SapB protein. In one aspect of the present invention, there is provided a method for preparing SapB protein, the method comprising: providing cells that produce SapB protein; and incubating the cells at a temperature equal to or greater than a third predetermined temperature or in the presence of urea, thereby obtaining (or extracting) the SapB protein. That is, one aspect of the present invention provides a method for preparing SapB protein, comprising: providing cells that produce SapB protein; and incubating the cells under: (a) a temperature condition equal to or higher than a first predetermined temperature and in the absence of urea; (b) a temperature condition equal to or lower than a second predetermined temperature and in the presence of urea; or (c) 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. In the above-mentioned case, the cells may be protein-producing cells.

[0037] In one aspect, the conditions for extracting SapB protein from cells (extraction conditions or treatment conditions) can be any of the following: (a) a temperature equal to or higher than a first predetermined temperature and in the absence of urea; (b) a temperature below a second predetermined temperature and in the presence of urea; or (c) a temperature equal to or higher than a third predetermined temperature and in the presence of urea. 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 yield of modified SapB protein 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 the extraction condition is (a), extending the extraction time (incubation time) can increase the yield of native SapB protein. When the extraction condition is (c), extending the extraction time (incubation time) can increase the yield and the amount of modified SapB protein recovered. Therefore, the method for preparing SapB protein of the present disclosure can obtain SapB protein and / or modified SapB protein. In one aspect, the method for preparing SapB protein of the present disclosure can obtain a mixture of SapB protein and modified SapB protein. In either aspect, incubation is preferably carried out in an aqueous solvent (preferably water). Furthermore, 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 the extraction conditions are a temperature below the second predetermined temperature and in the absence of urea, only the native SapB protein can be obtained.

[0038] In one aspect, 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 aspect, the predetermined temperature can be included in one, two, or three temperature ranges selected from the group consisting of, for example, 30 to 50°C, 40 to 60°C, and 50 to 70°C. In one embodiment, the first predetermined temperature and the third predetermined temperature 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 one preferred embodiment, the first predetermined temperature or the third predetermined temperature (more preferably, the first predetermined temperature and the third predetermined temperature) can be a temperature equal to or higher than the second predetermined temperature. In one preferred embodiment, the treatment temperatures of (a) and (c) can be a temperature equal to or higher than the treatment temperature of (b).

[0039] In some embodiments, the incubation time may 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 lower and upper limits) may be appropriately determined depending on the yield of SapB protein and its modified forms. In some embodiments, the incubation time may 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 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 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 to form an emulsion. SapB protein can be used to form emulsions in the same manner as other surfactants. An amount sufficient to form an emulsion 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, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 3 times, 5 times, 10 times, 20 times, 30 times, 50 times, or 100 times the CMC). A person skilled in the art can appropriately determine the amount sufficient for forming an emulsion. Furthermore, a person skilled in the art can appropriately form an emulsion. The concentration of SapB does not prevent SapB from precipitating, but preferably is an amount that does not cause SapB to precipitate. In a preferred embodiment, the emulsion may be an O / W type emulsion. In a preferred embodiment, the emulsion may be a W / O type 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 may be food compositions. In this embodiment, the water-soluble composition may contain, for example, water, and the fat-soluble composition may contain an edible oil. The obtained emulsion may also be mixed with other food compositions before use. In a preferred embodiment, the water-soluble composition and the fat-soluble composition may be compositions suitable for cosmetic compositions. The obtained emulsion may also be mixed with other cosmetic compositions before use. In these embodiments, the obtained modified SapB protein may 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 finishing agents, 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 SapB protein or a modified version of 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 composition and fat-soluble composition of the present disclosure can be used for wetting an object, for allowing a target substance to penetrate an object, for foaming 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 has 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 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 increase extraction efficiency. The first aqueous solution is preferably free of or 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. The urea concentration in the second aqueous solution may, in some embodiments, be, for example, 1-8 M, 2-7 M, 3-6 M, for example, 4-5 M, e.g., 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 carried out, 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, 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. Incubation in the second aqueous solution at a low temperature for a short period of time can yield SapB protein, while incubation at a high temperature for a long period of time can yield a mixture of SapB protein and modified SapB protein. The resulting SapB protein can be purified as necessary. Purification can be achieved by filtering 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 can also be achieved by dialysis. Dialysis can replace the solvent with an external dialysis solution. In this manner, the method of the present disclosure can prepare SapB protein, a modified SapB protein, or a mixture thereof. In the above method, the first aqueous solution can 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 for the second aqueous solution can 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] In one aspect of the present invention, a method for preparing a modified SapB protein according to the present disclosure is provided. 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)) having 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)) comprises 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 SapB protein-producing cells 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 more, 0.5 M or more, preferably 1 M or more, more preferably 2 M or more, even more preferably 3 M or more, and particularly preferably 4 M or more, 5 M or more, 6 M or more, 7 M or more, or 8 M or more. Here, the higher the urea concentration in the treatment, the higher 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 heated conditions. Heating conditions can be, for example, 40°C or more, 50°C or more, 60°C or more, 70°C or more, 80°C or more, or 90°C or more. Heating conditions can be, for example, 70°C to 90°C, for example, about 80°C. The obtained 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 be used to remove impurities. 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 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. Note that 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 making appropriate modifications to the expression cassette.

[0050] In a preferred embodiment, the urea concentration in a composition containing SapB protein or a modified form thereof may be less than 1%, less than 0.1%, or less than 0.01% by weight, or below the detection limit. Urea in the composition can be removed by conventional methods, for example, by 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 trifluoroacetic acid (TFA), but may be free or substantially free of TFA. In a preferred embodiment, a composition comprising a SapB protein or a composition comprising a modified SapB protein is free of or substantially free of trifluoroacetic acid (TFA). In one embodiment, the composition comprises a SapB protein and its modified form.

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

[0054] In one aspect, the obtained composition comprising SapB protein or the obtained composition comprising a modified form of 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 SapB protein and / or its modified form at a concentration of 0.1 wt / wt% or higher, preferably 0.5 wt / wt% or higher, and more preferably 1 wt / wt% or higher, calculated as the weight of the SapB protein portion. In this aspect, preferably, the composition does not contain or is substantially free of trifluoroacetic acid (TFA). In one aspect, the composition comprises SapB protein and its modified form. The concentration of SapB protein and / or modified forms thereof may 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 forms thereof.

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

[0056] In a preferred embodiment, the composition of the present disclosure comprises a wild-type 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 wild-type 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 may be selected from the group consisting of glycine-HCl, acetate, phosphate, Tris, and glycine-NaOH. For example, the pH buffer may 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 may be used at pH 3-4, an acetate buffer at pH 3-6, a phosphate buffer at pH 5-8, a Tris buffer at pH 7-9, and a glycine-NaOH buffer 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.

[0060] Example 1: Preparation of actinomycetes with high SapB expression The SapB operon region (including ramC, ramS, ramA, ramB, and ramR) was cloned from Streptomyces coelicolor, and the fragment was 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, and SapB is produced by excision from the precursor. Genes that correspond to RamS in various actinomycetes and are predicted to encode SapB (hereinafter simply referred to as "SapB" or "SapB protein") were identified, and the SapB operon region of each was introduced into an expression vector in the same manner as described above. To overexpress the SapB protein, Streptomyces lividans 1326 was transformed with the above expression vector containing the gene encoding SapB to obtain a transformant (a strain with integrated SapB operon region and high expression of SapB protein).

[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 has the following structure due to post-translational modification (Proc Natl Acad Sci USA. 2004; 101(31): 11448-11453).

[0062]

[0063] The amino acid sequences of other SapB precursors also undergo similar post-translational modifications. {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 when the methyl groups of the two alanine side chains are linked by S as shown above.}

[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 (TSB medium (Becton Dickinson Bacto™ Tryptic Soy Broth (Soybean-Casein Digest Medium, product number: 211825) 5 mL, thiostrepton 50 μg / mL). Thereafter, 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 cultured under these culture conditions, and the expression of SapB protein was analyzed by SDS-PAGE after ultrasonic disruption. As a result, a band indicating the expression of SapB protein was confirmed (see FIG. 7 ). When the S. griseus SapB protein was subjected to Edman degradation, the first four amino acids from the N-terminus could be decoded, but the third amino acid, which contains a lysine bond, had a structure that could not be subjected to Edman degradation and could not be decoded. This result is consistent with the result predicted from the intramolecular cross-linked structure of S. griseus.

[0067] Example 2: Extraction and Recovery of SapB After the main culture, the culture medium was recovered and the bacterial cells were collected by centrifugation. SapB protein was extracted from the collected bacterial cells. Because SapB has low water solubility, extraction procedures were performed under the following conditions to increase the amount of extraction. (Condition 1) Extraction was performed by treating the bacterial cells in a 4 M urea solution (distilled water containing 4 M urea) under heating conditions at 80°C. The bacterial cells were then stirred in the urea solution for 2 hours; (Condition 2) Extraction was performed by stirring the bacterial cells in distilled water at 80°C for 2 hours, followed by cooling to room temperature, adding urea to a final concentration of 4 M, and stirring for 10 minutes; or (Condition 3) Extraction was performed by incubating the bacterial 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 (Advantech), and the permeate was recovered. Next, filtration was performed using filter paper No. 5C (Advantech), 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 yield of SapB protein extracted with water under condition 3 was compared with the amount of SapB protein extracted with a urea solution. Only under condition 3 was the culture medium supplemented with a synthetic medium (4.5% glucose, NH ４ Cl 0.94%, KH ２ P.O. ４ 0.14%, MgSO ４ ・6H ２ O 0.02%, KCl 0.07%, Na ２ SO ４ 0.09%, NaCl 0.2%, Trace Element 0.2% (FeCl ３ ・6H ２ O 1.35%, CuCl ２ ・6H ２ O 0.15%, ZnCl ２ 0.9% MnCl ２ ・4H ２ O 0.36%, Na ２ MoO ４ ・2H ２ O 0.06%, CoCl ２ 0.04%, H ３ BO ４ 0.03%)).

[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), indicating that extraction with urea solution significantly increased the yield. Under condition 2, the yield was 109 mg / L (culture volume), indicating that heating followed by urea treatment at room temperature significantly increased the yield.

[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 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. A similar surfactant activity was observed with the protein obtained under condition 2. Generally, surfactants cause emulsification when their concentration is sufficiently higher than the CMC, and exhibit demulsifying activity at concentrations slightly above the CMC; similar characteristics were observed with 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 apparatus, and data was acquired 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 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 measurements were performed under the following conditions: Column: Cosmosil 5Ph-AR-300 (4.6 mm ID x 150 mm) Eluent: Solution A H ２ Solution B: Acetonitrile: 2-propanol (3:2) (0.1% TFA) Flow rate: 1 mL / min Gradient: 20-50% / 1-31 min Detection: 220 nm

[0075]

[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, it was believed that the sample extracted under condition 2 was native SapB, ​​while the sample extracted under condition 1 was a modified form in which native SapB had been 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 minutes. The reaction product was 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 minutes 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 the upper panel of Figure 3A). 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 at room temperature in the presence of urea without heating, it was suggested that modification required treatment of the SapB protein in the presence of urea under heated conditions. The difference was about 21.5 m / z. From this, it was concluded that SapB is NH ２ It was thought to have been modified with —C(O)—.

[0078] (3) LC-MS / MS Two peaks (natural SapB and NH) detected by the LC-MS were ２ The peaks of SapB modified with -C(O)- were fragmented at a collision energy of 40 eV using an Agilent 6545XT QTOF instrument. The results are shown in Figure 3C (showing m / z values ​​around 130 to 160) and Figure 3D (showing m / z values ​​around 960 to 1190).

[0079] As shown in Figure 3C, [M+H] ＋ The fragment peak at 145.0607 is NH ２ Only the cleavage of the peak of SapB modified with -C(O)- (SapB modified product) (lower panel in Figure 3C) was detected, which is due to the NH ２ —C(O)—NH ２ -CH(CH(OH)CH ３ This coincided with the b ion peak (theoretical value: 145.0613) derived from )-CO. This is because the amino group of the N-terminal threonine is NH ２ This indicates that the compound has been modified with —C(O)—. ＋ 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 fragmentation of the molecular ion peak of native SapB is [M+H] ＋ The b-ion peak series of NH = 966.5168 (theoretical value: 966.5195), 1023.5385 (theoretical value: 1023.5410), and 1138.5650 (theoretical value: 1138.5679) ２ When the molecular ion peak of SapB modified with -C(O)- (modified SapB) splits, [M+H] ＋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 to 57.0217 and 115.0255 to 115.0265, respectively. This result indicates that the 11th residue from the N-terminus of the SapB modified 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 is 43.0067 to 43.0083, and NH ２ -C(O) (theoretical value: 43.0058). Furthermore, [M+H] ＋ A product peak at 1061.4509 to 1061.4545 was commonly detected, which coincided with the y ion peak (theoretical value: 1061.4573) derived from the 11 residues on the C-terminal side. These results suggest that any of the 10 residues on the N-terminal side is NH ２ This indicates that the residue has been modified with —C(O)—.

[0082]

[0083] (5) Resistance of Modified SapB Protein to Degradation The protein was subjected to degradation. The degradation involved degradation of the N-terminal amino acid by Edman degradation. Edman degradation is a technique used in amino acid sequencing and was performed by 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 process is used to analyze the amino acid sequence. With native SapB, ​​it was possible to decode two amino acid sequences from the N-terminus by Edman degradation. However, with SapB extracted under Condition 1 of the present invention, the N-terminal amino acid could not be degraded, and the first amino acid could not be decoded. 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 Dissolution 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 buffers having different pH values ​​listed in Table 3 below to a concentration of 0.1% by weight (mg / mL).

[0085]

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

[0087]

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

[0089] The native SapB protein obtained above was suspended or dissolved in buffers of various pH values, or the supernatants thereof, which were then 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 centrifugal 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 native SapB protein in solution increases at alkaline pH.

[0090] The supernatants obtained by centrifugation of the buffer solutions having 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 centrifuged supernatants of buffer solutions having various pH values ​​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 Surface Treatment with SapB Protein A polycarbonate plate with a hydrophobic surface was coated with native SapB protein, and the wettability of the treated surface was tested. Specifically, a lyophilized product of 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 aqueous solution was applied to a portion of the polycarbonate plate and heated at 60°C for 5 minutes to evaporate the water, forming a SapB-coated region. Distilled water was dropped onto both the SapB-untreated and SapB-coated regions of the polycarbonate plate. As shown in Figure 9, water formed droplets with a large contact angle in the SapB-untreated region, whereas water spread thinly across the surface in the SapB-coated region. Similar results were obtained with surface treatment using modified SapB. This demonstrates that surface treatment with SapB and its modified forms are both useful for hydrophilic surface treatment.

[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 from S. coelicolor SEQ ID NO: 5: An example of the amino acid sequence of the SapB protein from S. scabies SEQ ID NO: 6: An example of the amino acid sequence of the SapB protein from S. scabies SEQ ID NO: 7: An example of the amino acid sequence of the SapB protein from S. avermitilis SEQ ID NO: 8: An example of the amino acid sequence of the SapB protein from S. griseus SEQ ID NO: 9: An example of the amino acid sequence of the S. Example of amino acid sequence of SapB protein of S. albus [Sequence Listing] Sequence Listing Information: 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 ( 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 sequences: 9 Sequences: SEQ ID NO: 1 Length: 42 Molecule 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 Residues: MNLFDLQSME TPKEEAMGDV ETGSRASLLL CGDSSLSITT CN 42 SEQ ID NO: 2 Length: 21 Molecule 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 residues: TGSRASLLLC GDSSLSITTC N 21 ID: 3 length: 22 molecule 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 residues: TGSQASLLLC EYSSLSVVLC TP 22 ID: 4 length: 22 molecule 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 residues: TGSQVSLLVC EYSSLSVTLC TP 22 ID: 5 length: 27 Molecule 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 residues: TVEYLSVLSS LSVVNCTNST VSTLLCL 27 SEQ ID NO: 6 Length: 22 Mol 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 residues: TGGPSSLSVL SCVSAASITL CL 22 SEQ ID NO: 7 Length: 24 Mol 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 residues: TGGGGASTVS LLSCVSAASV LLCL 24 SEQ ID NO: 8 Length: 22 Mol_type: AA feature position / qualifier: - source, 1..22 > mol_type, protein > organism, Streptomyces griseus residues: TGSQVSLLVC EYSSLSVVLC TP 22 SEQ ID NO: 9 Length: 22 Mol_type: AA feature position / qualifier: - source, 1..22 > mol_type, protein > organism, Streptomyces albus residues: 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; A method comprising:

2. The method described in claim 1, wherein the SapB protein is derived from actinomycetes.

3. A method as described in claim 1 or 2, wherein the first predetermined temperature is 40°C or higher.

4. A method according to any one of claims 1 to 3, 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.

5. 1. An article of manufacture comprising a composition comprising an isolated SapB protein and an aqueous solution, A product in which the isolated SapB protein is dissolved in an aqueous solution, and the aqueous solution is alkaline.

6. 6. The product of claim 5, wherein the aqueous solution has a pH of 8 or greater.

7. 7. The product of claim 5 or 6, wherein the aqueous solution has a pH in the range of 8 to 11.

8. The product according to any one of claims 5 to 7, wherein the concentration of the isolated SapB protein is 0.1% w / w or more in terms of the weight of the SapB protein portion.

9. A product described in any one of claims 5 to 8, wherein the aqueous solution has a pH of 10 or more and contains SapB protein at a concentration of 0.5% w / w or more.