A polypeptide having skin barrier repair activity and use thereof

Abstract

The application belongs to the technical field of biotechnology and cosmetics, and discloses a polypeptide with skin barrier repair activity and application thereof. The polypeptide is an enzymatic fragment derived from Bacillus subtilis neutral metalloproteinase A7UAM1, and the amino acid sequence is shown as SEQ ID NO:1. The mutant is a site-directed mutant of the wild-type A7UAM1, and is preferably a H142A / E143A double mutant. Compared with the wild-type enzyme, the proteolytic activity of the polypeptide and the mutant is reduced by greater than or equal to 90%, but the activity of promoting keratinocytes to express filaggrin (FLG) and loricrin (LOR) is not reduced or is significantly improved, and the application realizes the decoupling of the functions of 'gentle exfoliation' and 'active barrier repair'. The polypeptide or the mutant can be applied to the preparation of cosmetics or drugs with the effects of skin barrier repair, anti-sensitivity and anti-photoaging, and is especially suitable for sensitive skin and post-medical aesthetic repair.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and cosmetics technology, specifically relating to a polypeptide or mutant with skin barrier repair activity and its application. Background Technology

[0002] The skin barrier is mainly composed of the stratum corneum, the outermost layer of the epidermis. Its integrity depends on the keratinized desmosome junctions between keratinocytes and the normal expression of keratinized capsule proteins such as filaggrin (FLG), lobelin (LOR), and inner laminarin (IVL). When the skin barrier is damaged, transepidermal water loss increases, and external irritants can easily penetrate, leading to dry skin, sensitivity, inflammation, and even accelerated aging.

[0003] Traditional chemical exfoliants (such as glycolic acid and salicylic acid) non-selectively damage the stratum corneum structure through proton-mediated processes. While they can promote exfoliation and improve skin roughness, they also downregulate the expression of FLG and LOR, inhibit aquaporin-3 (AQP3), and activate inflammatory pathways such as NF-κB and IL-17, leading to further impairment of the skin barrier function. This dilemma of "exfoliation inevitably damaging the skin barrier" has long remained unresolved.

[0004] In recent years, enzymatic exfoliants have attracted attention due to their mild and highly specific effects. Neutral metalloproteinases (EC 3.4.24.-) derived from Bacillus subtilis have been reported to possess keratin hydrolytic activity. The applicant's previous research purified a neutral metalloproteinase from the fermentation product of Bacillus subtilis CGMCC No. 13932. LC-MS / MS identification (see the appendix LC-MS / MS protein identification list for the identification results) showed that its amino acid sequence had 100% sequence identity with the neutral metalloproteinase (EC 3.4.24.-) with UniProt database accession number A7UAM1 (i.e., SEQ ID NO:2). LC-MS / MS identification results showed that this protein was the most abundant protein component in the fermentation broth (LFQ intensity 5.69 × 10⁻⁶). 9 It has an abundance approximately 73 times higher than the second most abundant subtilisin, with a sequence coverage of 20.2%. For ease of description, this enzyme will be referred to as "A7UAM1 enzyme" below. It has been confirmed that it can significantly upregulate the expression of FLG, LOR, K5, and K14 at extremely low concentrations without activating inflammatory pathways. However, this enzyme also possesses proteolytic activity, and while hydrolyzing keratinized desmosomes, it may potentially affect barrier structures outside of desmosomes, limiting its widespread application in sensitive skin and other vulnerable populations.

[0005] Therefore, developing a peptide or mutant that retains or enhances barrier repair activity while significantly reducing proteolytic activity, thereby decoupling the functions of "gentle exfoliation" and "active barrier repair," has significant clinical application value and market prospects. Summary of the Invention

[0006] The present invention aims to solve the problem that acid or protease exfoliants in the prior art may damage the skin barrier while exerting their effects, and provides a polypeptide and its mutant with skin barrier repair activity.

[0007] The polypeptide is derived from a polypeptide fragment of Bacillus subtilis neutral metalloproteinase (EC 3.4.24.-) or a mutant thereof, a composition containing the polypeptide or the mutant, and its use in the preparation of cosmetics with skin barrier repair, soothing and anti-photoaging effects, or in the preparation of drugs for the treatment of diseases related to skin barrier dysfunction.

[0008] The technical solution adopted in this invention is as follows:

[0009] This invention provides a polypeptide with skin barrier repair activity.

[0010] The polypeptide is any one of the following (a) to (d):

[0011] (a) A polypeptide with an amino acid sequence as shown in SEQ ID NO:1;

[0012] (b) A derivative polypeptide having at least 85% sequence identity with SEQ ID NO:1 and having the activity of promoting the expression of filaggrin and / or pachyphyll in keratinocytes;

[0013] (c) A derivative polypeptide obtained by substituting, deleting or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:1, and having the activity of promoting the expression of filaggrin and / or pachymase in keratinocytes.

[0014] (d) A truncated form of any of the polypeptides in (a) to (c) above, which contains at least 15 consecutive amino acid residues and retains the activity of promoting the expression of filaggrin and / or pachyphyll.

[0015] Preferably, the polypeptide is derived from an enzymatic fragment of Bacillus subtilis neutral metalloproteinase A7UAM1, with a molecular weight of 1 kDa to 10 kDa and an isoelectric point of 5.0 to 9.0.

[0016] The polypeptide is a small molecule polypeptide composed of 33 amino acids that can promote the expression of filaggrin (FLG) and lobelin (LOR) in keratinocytes. Its molecular weight is about 3.8 kDa and its isoelectric point is about 6.5. It is a functional polypeptide fragment obtained by Glu-C proteolytic hydrolysis of the C-terminal region of the neutral metalloproteinase A7UAM1 of Bacillus subtilis. It is not a natural intact protein. Experiments have shown that this polypeptide can significantly promote the expression of skin barrier-related proteins at low concentrations (0.1 μM), while having extremely low proteolytic activity (relative enzyme activity <0.1%) and good safety.

[0017] The amino acid sequence is as follows:

[0018] Glu-Tyr-Tyr-Asp-Asp-Tyr-Ser-Gly-Ser-Ser-Leu-Tyr-Phe-Asp-Asp-Tyr-Ala-Gly-Asn-Thr-Lys-Tyr-Phe-Asp-Asp-Tyr-Ser-Asn-Thr-Lys-Tyr-Phe-Asp

[0019] The peptide promotes FLG and LOR expression, repairs the skin barrier, has a proteolytic activity of <0.1%, and is extremely safe.

[0020] Secondly, the present invention also provides a mutant of Bacillus subtilis neutral metalloproteinase.

[0021] Compared with wild-type Bacillus subtilis neutral metalloproteinase (SEQ ID NO:2), the mutant has at least 90% reduced proteolytic activity, and its activity in promoting the expression of filaggrin and / or pachypodium in keratinocytes is neither reduced nor increased compared with the wild type.

[0022] The wild-type Bacillus subtilis neutral metalloproteinase is a full-length enzyme protein sequence of 561 amino acids, as shown in SEQ ID NO:2. It originates from Bacillus subtilis strain CGMCC No. 13932, with the corresponding UniProt accession number A7UAM1. LC-MS / MS identification confirmed the protein (see attached LC-MS / MS identification results statistics). The protein sequence coverage was 20.2%, and the LFQ intensity was 5.69 × 10⁻⁶. 9 This enzyme is the most important protein component in the fermentation broth. It has two key functions: first, it can gently break down old, dead skin cells (proteolytic activity); second, it can activate skin cells to produce barrier proteins (FLG and LOR). This invention is based on site-directed mutagenesis of this sequence, significantly reducing its proteolytic activity while preserving or even enhancing its barrier repair function.

[0023] Preferably, the mutant contains an amino acid substitution in the HEXXH zinc-binding motif corresponding to thermophilic protease.

[0024] More preferably, the mutant contains one or more amino acid substitutions selected from H142A, E143A, and H146A.

[0025] Most preferably, the mutant contains a double mutation of H142A and E143A.

[0026] In this invention, the amino acid sequence of the wild-type Bacillus subtilis neutral metalloproteinase is shown in SEQ ID NO:2 (corresponding to UniProt accession number A7UAM1, derived from Bacillus subtilis CGMCC No. 13932 strain).

[0027] The wild-type CAC at positions 424-426 (approximately) was mutated to GCC, and the GAA at positions 427-429 (approximately) was mutated to GCC, resulting in amino acid changes to H142A and E143A, thus obtaining the nucleotide sequence of the mutant H142A / E143A.

[0028] The nucleotide sequence encoding the preferred mutant H142A / E143A of the present invention is shown in SEQ ID NO:3.

[0029] An isolated nucleic acid molecule encoding the aforementioned polypeptide with skin barrier repair activity, or a mutant thereof.

[0030] A recombinant expression vector comprising the aforementioned nucleic acid molecules.

[0031] A host cell comprising the above-described recombinant expression vector, or having the described nucleic acid molecule integrated into its genome.

[0032] A composition comprising the peptide or the mutant described herein, and a cosmetically acceptable excipient or a pharmaceutically acceptable carrier.

[0033] The composition also contains one or more skin-care active ingredients selected from the following: niacinamide, panthenol, ceramide, asiaticoside, hyaluronic acid, vitamin C, vitamin E, allantoin, and dipotassium glycyrrhizate.

[0034] The use of the polypeptide or the mutant in the preparation of cosmetic or topical skin preparations having any of the following effects:

[0035] (a) Promotes skin barrier repair;

[0036] (b) Upregulate filaggrin and / or lipogrin expression;

[0037] (c) Reduce transepidermal water loss;

[0038] (d) Improves sensitive skin;

[0039] (e) Repair photo-aged skin;

[0040] (f) Repairing skin after cosmetic surgery.

[0041] The use of the polypeptide or mutant in the preparation of a medicament for the treatment of: (g) diseases or conditions associated with skin barrier dysfunction.

[0042] Beneficial effects

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] (1) Functional decoupling: For the first time, the functional separation of the "proteolytic activity" and "barrier repair activity" of Bacillus subtilis neutral metalloproteinase A7UAM1 was achieved. The polypeptide fragment (SEQ ID NO:1) and the H142A / E143A double mutant of the present invention retained FLG and LOR upregulation activities (up to 28.4%) that were comparable to or better than those of the wild type, while the proteolytic activity was reduced by 99.2%.

[0045] (2) Significantly improved safety: Due to the significant reduction in protein hydrolysis activity, the peptides and mutants of the present invention have reduced non-specific cleavage of proteins other than keratinized desmosomes, and their safety for sensitive skin is far superior to that of wild-type enzymes and chemical skin resurfacing agents.

[0046] (3) Small molecular weight, better transdermal absorption: The molecular weight of the active polypeptide fragment (about 3.8 kDa) is much smaller than that of the full-length enzyme (about 56.8 kDa), making it easier to penetrate the stratum corneum to exert its effects.

[0047] (4) Clear structure, meeting the requirements for new raw material registration: It has a clear amino acid sequence and meets the technical requirements for the registration of new cosmetic raw materials at home and abroad.

[0048] (5) Controllable production costs: It can be produced by chemical synthesis (peptides) or genetic engineering fermentation (mutants), which is suitable for industrial production. Attached Figure Description

[0049] Figure 1 This is a flowchart of the screening process for active polypeptide fragments in Example 1 of the present invention.

[0050] Figure 2 This is a dose-response curve of how each enzyme fragment promotes the expression of FLG and LOR in keratinocytes in Example 1 of the present invention.

[0051] Figure 3This is a comparison diagram of the proteolytic activity of wild-type and various mutants in Example 2 of the present invention.

[0052] Figure 4 This is a comparison of FLG and LOR mRNA expression levels in wild-type and mutant keratinocytes after treatment in Example 2 of the present invention.

[0053] Figure 5 The images show H&E staining of the stratum corneum morphology of the 3D epidermal model after treatment in Example 3 of this invention, where: A is the negative control (PBS), B is Example 1 (SEQ ID NO:1), C is Example 2 (wild-type WT), D is Example 6 (H142A / E143A), and E is Comparative Example 1 (salicylic acid). H&E staining shows that after treatment with Example 6 (H142A / E143A double mutant) and Example 1 (SEQ ID NO:1 peptide), the stratum corneum exhibits mild and controllable desquamation, with a dense and intact stratum corneum, and normal granular and basal layers, resulting in an "excellent" safety rating. Wild-type enzyme treatment resulted in more pronounced desquamation, posing a higher potential risk to sensitive skin. Salicylic acid (Comparative Example 1) treatment resulted in irregular stratum corneum shedding, thinning of the granular layer, and visible vacuolated cells in the basal layer, causing significant skin damage. These results confirm that Example 6, while maintaining excellent barrier repair activity, provides the mildest and safest treatment for the stratum corneum. Detailed Implementation

[0054] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Experimental methods not specified in the embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0055] Example 1

[0056] The fermentation process begins with Bacillus subtilis fermentation, followed by enzyme protein purification, restriction digestion, HPLC separation, cell viability screening, and structural identification, ultimately yielding an active polypeptide fragment (SEQ ID NO:1). The procedure is as follows: Figure 1 As shown, the screening process for active polypeptide fragments in Example 1 of this invention includes the following steps:

[0057] First, Bacillus subtilis CGMCC No.13932 was inoculated into an optimized fermentation medium (20 g / L soybean meal, 5 g / L corn flour, 0.3 g / L NaCl, pH 7.2) and fermented at 37°C and 240 rpm for 48 hours. The fermentation broth was centrifuged (4,000 × g, 15 min) to remove bacterial cells, then filtered through a 0.45 μm microfiltration membrane, concentrated 10 times by a 10 kDa ultrafiltration membrane, purified by Source 15Q anion exchange chromatography and Superdex 200 gel filtration chromatography to obtain A7UAM1 wild-type enzyme protein with a purity >95%.

[0058] The purified A7UAM1 enzyme was mixed with Glu-C protease at a ratio of 1:100 (w / w) and enzymatically digested at 37°C for 0.5, 1, 2, 4, 8, and 24 hours, respectively, for a total of 6 time points. The enzymatic digest products were separated by reversed-phase high-performance liquid chromatography (RP-HPLC) using a C18 column (4.6×250 mm, 5 μm) with gradient elution (5%-60% B, 60 min) using 0.1% trifluoroacetic acid-water as mobile phase A and 0.1% trifluoroacetic acid-acetonitrile as mobile phase B). The elution peaks were detected at wavelengths of 214 nm and 280 nm, and a total of 23 elution peaks were collected.

[0059] Primary human epidermal keratinocytes were treated with various components at concentrations ranging from 0.01 to 10 μM for 48 hours. Activity screening was performed by detecting the expression levels of FLG and LOR mRNA using qPCR. The results showed that the component with a retention time of approximately 28.5 min (number F-23) exhibited the strongest activity, with an EC50 < 0.1 μM, and its FLG expression level was 2.3 times that of the control group, while its LOR expression level was 2.1 times that of the control group. The active component was further analyzed by LC-MS / MS de novo sequencing and Edman degradation assay to verify the N-terminal sequence, ultimately yielding the polypeptide SEQ ID NO:1. This polypeptide consists of 33 amino acids, has a molecular weight of approximately 3.8 kDa, and an isoelectric point of approximately 6.5.

[0060] The amino acid sequence is as follows:

[0061] Glu-Tyr-Tyr-Asp-Asp-Tyr-Ser-Gly-Ser-Ser-Leu-Tyr-Phe-Asp-Asp-Tyr-Ala-Gly-Asn-Thr-Lys-Tyr-Phe-Asp-Asp-Tyr-Ser-Asn-Thr-Lys-Tyr-Phe-Asp (SEQ ID NO: 1).

[0062] A peptide with skin barrier repair activity (SEQ ID NO:1) can be directly prepared by solid-phase chemical synthesis, achieving a purity of over 95%, a molecular weight of approximately 3.8 kDa, and an isoelectric point of approximately 6.5. This peptide fragment is derived from the Glu-C enzymatic hydrolysis product of the neutral metalloproteinase A7UAM1 from Bacillus subtilis. Activity test results are as follows: Figure 2As shown, the dual Y-axis dose-response curves (X-axis: logarithmic concentration, Y-axis: relative mRNA expression) illustrate the dose-response relationship of FLG and LOR mRNA expression levels after primary human epidermal keratinocytes were treated with different concentrations (0.001-10 μM) of SEQ ID NO:1 peptide and wild-type A7UAM1 enzyme for 48 hours. The x-axis represents sample concentration (μM), expressed on a logarithmic scale, ranging from 0.001 μM to 10 μM; the y-axis represents the relative expression level of FLG or LOR mRNA, with the negative control group (PBS-treated group) as a 1.0-fold baseline.

[0063] The results showed that after treating primary human epidermal keratinocytes with the peptide at a concentration of 0.1 μM for 48 hours, the expression level of FLG mRNA was 2.3 ± 0.2 times that of the control group (PBS treatment group) (P < 0.01), and the expression level of LOR mRNA was 2.1 ± 0.1 times that of the control group (P < 0.01), which was comparable to that of wild-type A7UAM1 enzyme (0.015 μM) (P > 0.05). Meanwhile, the peptide exhibited extremely low proteolytic activity (relative enzyme activity < 0.1%), almost completely losing its non-specific cleavage ability against proteins other than keratinized desmosomes. In the 3D epidermal model validation, 0.0001% of SEQ ID NO:1 peptide upregulated FLG expression by 12.5% ​​(P < 0.05) and LOR expression by 22.1% (P < 0.05), without inhibiting AQP3 or activating the IL-8 / TNF-α inflammatory pathway, demonstrating superior safety compared to the wild-type enzyme. Thermal stability testing showed that its Tm value was 56.3±0.5°C, significantly better than the wild type (51.2±0.4°C). After accelerated storage at 40°C for 4 weeks, the purity retention rates in aqueous solution and gel reached 95.6% and 94.9%, respectively, demonstrating good formulation compatibility. This example confirms that the active polypeptide fragment obtained through restricted enzymatic hydrolysis successfully achieved functional decoupling between "gentle exfoliation" and "active barrier repair," and its small molecular weight and superior transdermal absorption make it suitable as a cosmetic ingredient.

[0064] Example 2

[0065] Preparation and performance verification of wild-type Bacillus subtilis neutral metalloproteinase (A7UAM1, SEQ ID NO:2). Bacillus subtilis CGMCC No. 13932 was inoculated into the optimized fermentation medium (20 g / L soybean meal, 5 g / L corn flour, 0.3 g / L NaCl, pH 7.2) and fermented at 37°C, pH 7.2, and 240 rpm for 48 hours. The wild-type enzyme obtained through this optimized process exhibited the highest barrier repair activity (FLG 2.1 times, LOR 2.2 times), which can be used as a benchmark for subsequent comparison of mutant efficacy. The fermentation broth was purified by centrifugation, ultrafiltration concentration, Source 15Q anion exchange chromatography, and Superdex 200 gel filtration chromatography to obtain wild-type enzyme protein with a purity >95%, yielding approximately 65 mg / L of fermentation broth.

[0066] LC-MS / MS identification confirmed that the protein was a neutral metalloproteinase (UniProt accession number A7UAM1), with a sequence coverage of 20.2% and an LFQ intensity of 5.69 × 10⁻⁶. 9 It is the most abundant protein component in the fermentation broth (approximately 73 times more abundant than subtilisin, the second most abundant). Its full-length amino acid sequence is shown in SEQ ID NO:2, which is 100% identical to the UniProt database entry A7UAM1. Its relative enzyme activity, determined by the FITC-casein method, was 100 ± 4.2%. Functional validation showed that after treating keratinocytes at a concentration of 0.000194% for 48 hours, the expression levels of FLG and LOR mRNA were 2.1 ± 0.2 times and 2.2 ± 0.1 times that of the control group, respectively (P < 0.01). In the 3D epidermal model, FLG and LOR protein expression were upregulated by 14.1% (P < 0.05) and 25.4% (P < 0.01), respectively, while AQP3 showed no significant change, and IL-8 and TNF-α showed no significant increase. This embodiment confirms that the wild-type A7UAM1 enzyme has significant barrier repair activity, but its proteolytic activity (relative enzyme activity of 100%) may pose a potential risk to sensitive skin, and further mutation modification is needed to reduce its enzyme activity.

[0067] Example 3

[0068] Construction and performance validation of the H142A single-point mutant. Using the wild-type nprE gene as a template, the H142A mutation (histidine at position 142 was mutated to alanine) was introduced via overlap extension PCR. The mutant gene was cloned into the pP43NMK expression vector, transformed into Bacillus subtilis BS168, and fermented at 37°C, pH 7.2, and 240 rpm for 48 hours. The H142A mutant protein with a purity >90% was obtained after purification, with a yield of approximately 62 mg / L fermentation broth.

[0069] The relative enzyme activity determined by the FITC-casein method was 8.2 ± 1.1%, approximately 8.2% of the wild type, indicating a 91.8% reduction in proteolytic activity. Barrier repair activity testing showed that after treating keratinocytes at a concentration of 0.000194% for 48 hours, the expression levels of FLG and LOR mRNA were 1.2 ± 0.1 times and 1.1 ± 0.1 times that of the control group, respectively (P < 0.05), approximately 58% and 52% of the wild-type activity, indicating a significant decrease in barrier repair activity. This example demonstrates that while the H142A single-point mutation can significantly reduce enzyme activity, it also impairs its ability to promote FLG / LOR expression, failing to achieve functional decoupling.

[0070] Example 4

[0071] Construction and performance validation of the E143A single-point mutant. Using the wild-type nprE gene as a template, the E143A mutation (glutamic acid at position 143 was replaced with alanine) was introduced via overlap extension PCR. The mutant gene was cloned into the pP43NMK expression vector, transformed into Bacillus subtilis BS168, and fermented at 37°C, pH 7.2, and 240 rpm for 48 hours. The E143A mutant protein with a purity >90% was obtained after purification, with a yield of approximately 60 mg / L fermentation broth.

[0072] The relative enzyme activity determined by the FITC-casein method was 5.6 ± 0.9%, approximately 5.6% of the wild type, indicating a 94.4% reduction in proteolytic activity. Barrier repair activity testing showed that after treating keratinocytes at a 0.000194% concentration for 48 hours, the expression levels of FLG and LOR mRNA were 1.3 ± 0.1 times and 1.2 ± 0.1 times that of the control group, respectively (P < 0.05), approximately 62% and 55% of the wild-type activity, indicating a significant decrease in barrier repair activity. This example demonstrates that the E143A single-point mutation can significantly reduce enzyme activity, but it also impairs barrier repair activity, failing to achieve functional decoupling.

[0073] Example 5

[0074] Construction and performance validation of the H146A single-point mutant. Using the wild-type nprE gene as a template, the H146A mutation (histidine at position 146 was mutated to alanine) was introduced via overlap extension PCR. The mutant gene was cloned into the pP43NMK expression vector, transformed into Bacillus subtilis BS168, and fermented at 37°C, pH 7.2, and 240 rpm for 48 hours. The H146A mutant protein with a purity >90% was obtained after purification, with a yield of approximately 58 mg / L fermentation broth.

[0075] The relative enzyme activity determined by the FITC-casein method was 42.3±3.8%, approximately 42.3% of the wild type, indicating a 57.7% reduction in proteolytic activity. Barrier repair activity testing showed that after treating keratinocytes at a concentration of 0.000194% for 48 hours, the expression levels of FLG and LOR mRNA were 1.8±0.2 times and 1.8±0.2 times that of the control group, respectively (P<0.05), approximately 85% and 81% of the wild-type activity, with no statistically significant difference compared to the wild type (P>0.05). This example demonstrates that the H146A mutation, while retaining high barrier repair activity, only reduced enzyme activity by approximately 57.7%, failing to achieve the expected target of ≥90% reduction in enzyme activity.

[0076] Example 6

[0077] Construction and Performance Verification of the H142A / E143A Double Mutant (Optimal Mutant of this Invention). Using the wild-type nprE gene as a template, the H142A and E143A double mutants were sequentially introduced using overlap extension PCR. The mutant gene was cloned into the pP43NMK expression vector, transformed into Bacillus subtilis BS168, and fermented for 48 hours under suitable conditions, such as 37°C, pH 7.2, and 240 rpm. The resulting double mutant protein, with a purity >90%, was purified to approximately 55 mg / L of fermentation broth.

[0078] SDS-PAGE showed that the mutant exhibited a single major band at approximately 42.5 kDa. FITC-casein assay revealed a relative enzyme activity of 0.8 ± 0.2%, only 0.8% of the wild type, indicating a 99.2% reduction in proteolytic activity. Barrier repair activity assays showed that after 48 hours of treatment of keratinocytes at a 0.000194% concentration, the expression levels of FLG and LOR mRNA were 2.4 ± 0.2 times and 2.3 ± 0.2 times that of the control group, respectively (P < 0.01), showing no statistically significant difference compared to the wild type (2.1 times and 2.2 times, respectively) (P > 0.05), with FLG expression slightly higher than the wild type (+14.3%). In 3D epidermal model validation, this double mutant upregulated FLG and LOR protein expression by 16.8% (P<0.05) and 28.4% (P<0.01), respectively, which was superior to the wild type (14.1% and 25.4%), and did not inhibit AQP3 or activate inflammatory pathways. Thermostability testing showed its Tm value was 52.8±0.5°C, essentially equivalent to the wild type (51.2°C). This embodiment confirms that the H142A / E143A double mutant, while reducing enzyme activity by 99.2%, completely retained or even enhanced the barrier repair activity of the wild-type enzyme, successfully achieving functional decoupling between "proteolytic activity" and "barrier repair promotion activity," making it the optimal mutant of this invention.

[0079] Those skilled in the art will understand that the above fermentation conditions (temperature, pH, rotation speed, time) can be routinely optimized according to the characteristics of the host strain and expression vector, and mutant proteins with the aforementioned activity can be obtained without creative effort. The conditions described in Example 6 above are merely examples and do not constitute a limitation of the present invention.

[0080] Example 7

[0081] Construction and performance validation of the H142A / E143A / H146A triple mutant. Using the H142A / E143A double mutant gene as a template, the H146A mutation was further introduced using overlap extension PCR. The mutant gene was cloned into the pP43NMK expression vector, transformed into Bacillus subtilis BS168, and fermented at 37°C, pH 7.2, and 240 rpm for 48 hours. The triple mutant protein with a purity >90% was obtained after purification, with a yield of approximately 50 mg / L fermentation broth.

[0082] The relative enzyme activity, as determined by the FITC-casein method, was <0.1%, indicating an almost complete loss of proteolytic activity (reduction of over 99.9%). Barrier repair activity testing showed that after treating keratinocytes at a concentration of 0.000194% for 48 hours, the expression levels of FLG and LOR mRNA were only 0.6±0.1 times and 0.6±0.1 times that of the control group, respectively (P<0.01), approximately 30% of the wild-type activity, indicating a significant decrease in barrier repair activity. This example demonstrates that excessive mutation leads to a significant change in the spatial conformation of the enzyme protein. Although proteolytic activity is almost completely lost, key functional sites promoting FLG / LOR expression are also disrupted, resulting in a loss of barrier repair activity. This suggests that the three zinc coordinating residues in the HEXXH motif do not act independently but synergistically maintain the protein's active conformation, rendering the triple mutation strategy infeasible.

[0083] Figure 3 This study compares the proteolytic activities of wild-type A7UAM1 enzyme and its mutants (H142A, E143A, H146A, H142A / E143A, H142A / E143A / H146A). The x-axis represents sample grouping, including Example 2: Wild-type (WT), Example 3: H142A, Example 4: E143A, Example 5: H146A, Example 6: H142A / E143A, and Example 7: H142A / E143A / H146A; the y-axis represents relative enzyme activity (%), with wild-type (Example 2) as 100%. The H142A / E143A double mutant (Example 6) showed a 99.2% reduction in proteolytic activity (relative enzyme activity of only 0.8%), one of the combinations with the largest reduction in enzyme activity among all mutants, significantly superior to the single-point mutants, laying a solid foundation for improved safety.

[0084] Comparative Example 1

[0085] A comparison of the barrier repair and safety of the chemical peeling agent salicylic acid. A commercially available 0.2% salicylic acid solution was used as a positive control. 3D epidermal models were treated for 24 hours using the same methods as in Examples 2-7. Results showed that the salicylic acid treatment group exhibited a 23.0% downregulation of FLG expression (P<0.01), a 20.5% downregulation of LOR expression (P<0.01), and an 18.4% downregulation of AQP3 expression (P<0.01), indicating that salicylic acid severely inhibited the expression of barrier-related proteins. Inflammatory factor detection showed a 33.9% increase in TNF-α levels (P<0.01). Morphological observation of the stratum corneum revealed irregular shedding of the stratum corneum, thinning of the granular layer, and a small number of vacuolated cells in the basal layer in the salicylic acid group. This comparative study demonstrates that while traditional chemical peeling agents have an exfoliating effect, they simultaneously inhibit barrier protein expression, suppress AQP3, and activate inflammation, leading to the dilemma of "exfoliation inevitably damaging the skin barrier."

[0086] Figure 4 This study compares the expression levels of FLG and LOR mRNA in primary human epidermal keratinocytes after 48 hours of treatment with wild-type A7UAM1 enzyme and various mutants. The x-axis represents sample grouping, including negative control (PBS), Example 2: wild-type (WT), Example 3: H142A, Example 4: E143A, Example 5: H146A, Example 6: H142A / E143A, Example 7: H142A / E143A / H146A, and Comparative Example 1: salicylic acid (0.2%). The y-axis represents relative mRNA expression levels (fold higher than the PBS control group, PBS=1.0). The H142A / E143A double mutant (Example 6) exhibited a 99.2% reduction in enzyme activity while completely preserving or even slightly outperforming the wild-type in upregulated FLG / LOR activity. It was the only mutant to achieve a significant reduction in enzyme activity combined with an increase in barrier activity, successfully realizing functional decoupling.

[0087] Comparative Examples 2-4 aimed to verify the effect of fermentation conditions on the barrier repair activity of wild-type A7UAM1 enzyme. The results showed that deviations from the optimized conditions described in Example 2 (high temperature, acidic pH, low dissolved oxygen) led to abnormal enzyme protein conformation and a significant decrease in barrier repair activity. The "significant reduction in enzyme activity without a decrease in barrier activity" achieved in this invention through site-directed mutagenesis (rather than altering fermentation conditions) represents a structure-determined functional decoupling.

[0088] Comparative Example 2

[0089] Comparison of the activity of wild-type A7UAM1 enzyme produced under non-optimized fermentation conditions (high temperature 50°C). The fermentation temperature was increased from 37°C to 50°C, while other conditions remained the same as in Example 2. The fermentation broth was purified using the same process to obtain wild-type enzyme protein, with a significantly reduced yield of approximately 20 mg / L fermentation broth. The relative enzyme activity determined by the FITC-casein method was 45.1 ± 5.2%, approximately 45% of that under standard fermentation conditions. Barrier repair activity tests showed that after 48 hours of treatment of keratinocytes with this high-temperature fermentation product, the expression levels of FLG and LOR mRNA were 1.5 ± 0.2 times and 1.6 ± 0.2 times, respectively, compared to the control group (P < 0.05), significantly lower than the standard wild-type (2.1 times and 2.2 times). Thermostability tests showed that its Tm value decreased to 48.5 ± 0.6°C. This comparative example indicates that excessively high fermentation temperatures may lead to partial denaturation or misfolding of the enzyme protein, thereby impairing barrier repair activity.

[0090] Comparative Example 3

[0091] Comparison of wild-type A7UAM1 enzyme activity produced under unoptimized fermentation conditions (acidic pH 4.0). The fermentation pH was lowered from 7.2 to 4.0, while other conditions remained the same as in Example 2. The fermentation broth was purified using the same procedure to obtain wild-type enzyme protein, with a significantly reduced yield of approximately 15 mg / L. The relative enzyme activity was determined to be 28.5 ± 4.1% by the FITC-casein method. Barrier repair activity tests showed that after 48 hours of treatment of keratinocytes with the acidic pH fermentation product, the expression levels of FLG and LOR mRNA were only 1.1 ± 0.1 times and 1.2 ± 0.1 times that of the control group, respectively (P < 0.05), indicating a near-complete loss of barrier repair activity. This comparative example demonstrates that excessively low fermentation pH severely inhibits the proper expression and folding of neutral metalloproteinases.

[0092] Comparative Example 4

[0093] Comparison of the activity of wild-type A7UAM1 enzyme produced under unoptimized fermentation conditions (low dissolved oxygen, 100 rpm). The fermentation speed was reduced from 240 rpm to 100 rpm, while other conditions remained the same as in Example 2. The fermentation broth was purified using the same process to obtain wild-type enzyme protein, with a significantly reduced yield of approximately 18 mg / L fermentation broth. The relative enzyme activity was determined to be 33.6 ± 3.5% by the FITC-casein method. Barrier repair activity assays showed that after 48 hours of treatment of keratinocytes with this low dissolved oxygen fermentation product, the expression levels of FLG and LOR mRNA were 1.4 ± 0.1 times and 1.3 ± 0.1 times that of the control group, respectively (P < 0.05), significantly lower than the standard wild-type. This comparative example demonstrates that adequate oxygen supply plays a crucial role in promoting the efficient expression and proper folding of neutral metalloproteinases.

[0094] Table 1. Comparison of process parameters and performance of each embodiment / comparative example

[0095] Example 1 Purity > 95% <0.1 2.3 ± 0.2 2.1 ± 0.1 56.3 ± 0.5 excellent Example 2 65 mg / L 100 ± 4.2 2.1 ± 0.2 2.2 ± 0.1 51.2 ± 0.4 good Example 3 62 mg / L 8.2 ± 1.1 1.2 ± 0.1 1.1 ± 0.1 50.8 ± 0.5 good Example 4 60 mg / L 5.6 ± 0.9 1.3 ± 0.1 1.2 ± 0.1 50.5 ± 0.6 good Example 5 58 mg / L 42.3 ± 3.8 1.8 ± 0.2 1.8 ± 0.2 51.0 ± 0.4 good Example 6 55 mg / L 0.8 ± 0.2 2.4 ± 0.2 2.3 ± 0.2 52.8 ± 0.5 excellent Example 7 50 mg / L <0.1 0.6 ± 0.1 0.6 ± 0.1 49.5 ± 0.7 Difference Comparative Example 1 - N / A 0.8 ± 0.1 0.8 ± 0.1 N / A Difference Comparative Example 2 20 mg / L 45.1 ± 5.2 1.5 ± 0.2 1.6 ± 0.2 48.5 ± 0.6 generally Comparative Example 3 15 mg / L 28.5 ± 4.1 1.1 ± 0.1 1.2 ± 0.1 49.2 ± 0.5 Difference Comparative Example 4 18 mg / L 33.6 ± 3.5 1.4 ± 0.1 1.3 ± 0.1 50.1 ± 0.5 generally

[0096] Efficacy evaluation and results

[0097] I. Evaluation of Skin Barrier Repair Efficacy

[0098] 1.1 Detection of FLG and LOR mRNA expression in keratinocytes

[0099] Assay method: Primary human epidermal keratinocytes (KCs) were cultured in EpiLife™ medium (containing HKGS additive) at 37°C and 5% CO2. Cells in the logarithmic growth phase were seeded into 96-well plates (2 × 10⁻⁶ cells / wells). 4 Cells / well were cultured overnight and then synchronized for 12 hours using serum-free medium. The experimental groups were treated with samples from each example and comparative example (concentration: 0.000194% for Examples 1-7, 0.2% salicylic acid for Comparative Example 1, and 0.000194% for Comparative Examples 2-4). The positive control group was treated with wild-type A7UAM1 enzyme (0.000194%), and the negative control group was treated with an equal volume of PBS. Each group had three replicates. After incubation at 37°C for 48 hours, total RNA was extracted, reverse transcribed, and then detected by qPCR. The relative expression level was calculated using the 2^-ΔΔCt method, with the negative control group as a 1.0-fold baseline. The test results are shown in Table 2.

[0100] Table 2. Expression levels of FLG and LOR mRNA in keratinocytes after treatment in each example / comparative example

[0101] negative control PBS 1.00 ± 0.05 1.00 ± 0.04 Example 1 SEQ ID NO:1 polypeptide 2.30 ± 0.20 2.10 ± 0.10 Example 2 Wild type (WT) 2.10 ± 0.20 2.20 ± 0.10 Example 3 H142A 1.20 ± 0.10 1.10 ± 0.10 Example 4 E143A 1.30 ± 0.10 1.20 ± 0.10 Example 5 H146A 1.80 ± 0.20 1.80 ± 0.20 Example 6 H142A / E143A 2.40 ± 0.20 2.30 ± 0.20 Example 7 H142A / E143A / H146A 0.60 ± 0.10 0.60 ± 0.10 Comparative Example 1 Salicylic acid (0.2%) 0.80 ± 0.10 0.80 ± 0.10 Comparative Example 2 Wild type (fermented at 50°C) 1.50 ± 0.20 1.60 ± 0.20 Comparative Example 3 Wild type (fermented at pH 4.0) 1.10 ± 0.10 1.20 ± 0.10 Comparative Example 4 Wild type (fermented at 100 rpm) 1.40 ± 0.10 1.30 ± 0.10

[0102] The results of FLG and LOR mRNA expression detection showed that after treatment in Examples 1 and 6 of this invention, the expression levels of FLG and LOR in keratinocytes were significantly upregulated (P<0.01), reaching 2.3-fold / 2.1-fold and 2.4-fold / 2.3-fold respectively compared to the control group, with effects comparable to or slightly better than the wild-type enzyme (2.1-fold / 2.2-fold). Example 6 was the only mutant that achieved "enzyme activity reduction ≥90% (actual reduction 99.2%) + no decrease or enhancement of barrier activity". From the dose-response curve ( Figure 2The results showed that both the SEQ ID NO:1 peptide and the H142A / E143A double mutant significantly upregulated FLG and LOR mRNA expression in a concentration range of 0.01 μM to 1 μM (approximately equivalent to 0.000038% to 0.0038%, w / v) (P<0.05), exhibiting a typical bell-shaped dose-response relationship. Based on considerations of enzyme activity and safety, the preferred concentration for use in cosmetics is 0.0001% to 0.001% (w / w). Those skilled in the art can determine the optimal concentration based on the target efficacy and product dosage form through routine cell activity screening experiments.

[0103] 1.2 Detection of FLG and LOR protein expression in 3D epidermal model (immunofluorescence staining)

[0104] Test method: After 24 hours of treatment with the 3D epidermal model (EpiSkin®), it was fixed, embedded, sectioned, and subjected to immunofluorescence staining. The mean fluorescence intensity was quantitatively analyzed using ImageJ software, with the negative control group as 100% baseline. The test results are shown in Table 3.

[0105] Table 3. Changes in FLG, LOR, and AQP3 protein expression in 3D epidermal models of various examples / comparative examples.

[0106] negative control PBS 100 ± 8 100 ± 7 100 ± 5 Example 1 SEQ ID NO:1 polypeptide 112.5 ± 9* 122.1 ± 8* 100.5 ± 4 Example 2 Wild type (WT) 114.1 ± 10* 125.4 ± 9** 101.2 ± 5 Example 6 H142A / E143A 116.8 ± 9* 128.4 ± 10** 102.0 ± 4 Comparative Example 1 Salicylic acid (0.2%) 77.0 ± 9** 79.5 ± 8** 81.6 ± 7**

[0107] *P<0.05, **P<0.01 vs negative control group

[0108] The results of immunofluorescence staining and quantitative analysis were highly consistent with the qPCR results. In the 3D epidermal model treated in Example 6, FLG and LOR fluorescence intensities were the strongest, increasing by 16.8% and 28.4% respectively compared to the negative control group, showing superior effects compared to the wild-type enzyme. AQP3 expression showed no significant change in any of the enzyme treatment groups, indicating that the peptides and mutants of the present invention do not inhibit aquaporin expression.

[0109] II. Safety Evaluation

[0110] 2.1 Protein hydrolysis activity assay

[0111] Test method: The proteolytic activity of each example and comparative sample was determined by the FITC-casein method, with wild type as 100% baseline. The test results are shown in Table 4.

[0112] Table 4. Protein hydrolysis activity of each example / comparative sample

[0113] Example 1 SEQ ID NO:1 polypeptide <0.1 Reduced by >99.9% Example 2 Wild type (WT) 100 ± 4.2 0% Example 3 H142A 8.2 ± 1.1 Reduced by 91.8% Example 4 E143A 5.6 ± 0.9 Reduced by 94.4% Example 5 H146A 42.3 ± 3.8 Reduced by 57.7% Example 6 H142A / E143A 0.8 ± 0.2 Reduced by 99.2% Example 7 H142A / E143A / H146A <0.1 Reduced by >99.9%

[0114] As can be seen from Table 4, the protein hydrolysis activity of Example 6 is extremely low, with a relative enzyme activity of only 0.8%, which is 99.2% lower than that of the wild-type enzyme, and its safety is excellent.

[0115] 2.2 Morphological evaluation of the stratum corneum of the 3D epidermal model (H&E staining)

[0116] Test method: After processing the 3D epidermal model, H&E staining was performed, and the morphology of the stratum corneum was observed under an optical microscope. The test results are shown in Table 5.

[0117] Table 5. Morphological evaluation of the stratum corneum in 3D epidermal models after processing of each embodiment / comparative example

[0118] negative control PBS Dense and complete No peeling normal Example 1 SEQ ID NO:1 polypeptide dense Gentle and controllable desquamation excellent Example 2 Wild type (WT) Mildly loose Obvious peeling good Example 6 H142A / E143A Dense and complete Controlled desquamation excellent Comparative Example 1 Salicylic acid (0.2%) irregular Irregular shedding Difference

[0119] As can be seen from Table 5, after treatment in Example 6, the stratum corneum exhibited mild and controllable desquamation, and the stratum corneum was dense and intact, with a safety rating of "excellent".

[0120] III. Stability Evaluation

[0121] 3.1 Determination of thermal stability (differential scanning calorimetry)

[0122] Test method: The midpoint temperature (Tm value) of thermal denaturation was determined using a differential scanning calorimeter. The test results are shown in Table 6.

[0123] Table 6. Thermal denaturation temperatures (Tm values) of each example / comparative sample

[0124] Example 1 SEQ ID NO:1 polypeptide 56.3 ± 0.5 +5.1°C Example 2 Wild type (WT) 51.2 ± 0.4 benchmark Example 6 H142A / E143A 52.8 ± 0.5 +1.6°C

[0125] Conclusion: Example 1 had the highest Tm value, reaching 56.3±0.5°C, which was 5.1°C higher than the wild-type enzyme; Example 6 had a Tm value of 52.8±0.5°C, which was 1.6°C higher than the wild-type enzyme, and both had better thermal stability than the wild-type enzyme.

[0126] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0127] SEQ ID NO:1 — Active polypeptide fragment

[0128] EYYDDYSGSSLYFDDYAGNTKYFDDYSNTKYFD

[0129] SEQ ID NO:2 - Full-length sequence of wild-type enzyme protein

[0130] MKKRLLLMLALALVLTGCTNNFTNNAANNINNNNNNTNNNTNTTNNTNNNTNNNTNNTNNNTNNNTNTNNNTNNTTTTNNTTTTTNSTTATSTTTTTNTTTSTTATSTTTTPAEEAAKETQKQLEALNNEKQAVSDYFGHGTSAPSGSETGKKKVTHKGTKEGQQLQKEGDLKVAAKAEKVAGTKPGVKVKVLKNSGQPGKDGKVTIDKVAAKTSQTKVEVKPTAESGTVSNDVKHVGTVKVESATMSGKVNVVDFPGVAPVADQNANFTGSTNWEIDGSGTVTFTDYGNNKGRVKNSKSSHFNVDKGVKGVTVAHEFGHVLGLTHSADNALYNFIENYQKNETSIFYVVNDAGKRTFSGSTTIDVAAVNAFSLYGSKQNGGIVAVASGDNEDYIYDLTNAGSDTYFFNYSNTYYDNNEKVVFGVTGTNAGDGSTYGIDASVASGDTYFFDPYTNNGNTAGTSVYFDDYTSGSSYFYDDQNTQSTYFDDYQSSSLYFDDYAGTKLYYDDYSGSSLYFDNYAGNTKYFDDFDGSNSLYYDDYSGSSLYFDDYAGTKLYYDDY

[0131] SEQ ID NO: 3 Nucleotide sequence of the Bacillus subtilis neutral metalloprotease H142A / E143A double mutant

[0132] atgaagaagc gtttgttact aatgttggcg ctagcgttgg tgttgacgggctgtacgaatattttaca ataatgcagc aaataataca aataataata aatacaataatacaatacaatacaataaatacaacaacaacaacaacaacaaca aaacacaaca acaacaagca caacggcaac atcaacaa acaacag cagaagcagcaaaagaaca aaaaaagcaat taggagcatt aaataatgaa aaaacaagcag ttcagatta ttggtcatggaacgctt ctgcaacctc agaacaggcaaagaaag ttaaaagttg cagcaaagc agaaaaagtt gcaggaaca aaccaggcgttaagttaaagttttaaaa atagcggcca accaggcaaa gatgtaag ttacgattgaatagtagcagcaaaaaaaaaaaaaaaagggaat agttgaagtt aaaccacag cagagaatcattacacaacaat gttgaaagtg siacaatgtc aggtaaagttaatgttgttgattttccagg cgttgcacca gttgcagatc aaaatgcaaa ttttacaggc agcacaatt gggaaattgatggcagcgga acagttacat ttacagatta tggtaataataaggcagag cgttaaagcgttacagttg cacatgaatt tggtcatgttcttggcttaacatagtgc agataatgcattatataatt ttatgaaaa ttatcaaaaa aatgaaacaa gcatttttatgttgttaat gatgcaggca aaagaacatt ttcaggcagc acaacgattg atgttgcagcagttaatgcatttagcttat acggcagcaa acaaaatggc ggcattgttg cagttgcaag cggcgataatgaagaattata tttatgattt aacaaatgca ggcagcgata catattttt taattatagc aatacatattatgataataa tgaaaaagtt gttttggcg ttacaggaac aaatgcaggcgatggcagca catatggcattgatgcaagc gttgcaagc gcgatacata ttttttgat ccatatacaa ataatggca tacagcaggcacaagcgtt atttgatga ttatacaagc ggcagcagct atttttatga tgatcaaaaat acacaaagcacatattttga tgattatca agcagcagcc tttatttga tgattatgca ggcacaaaac tttattgatgattatagc ggcagcagcc tttatttga taattatgca ggcaatacaa aatatttga tgattttgatggcagcaata gcctttatta tgatgattat agcggcagca gcctttatt tgataattat gcaggcaatacaaaatatt tgatgat

Claims

1. A polypeptide with skin barrier repair activity, characterized in that, The polypeptide is an enzymatic digestion fragment of neutral metalloproteinase A7UAM1 from Bacillus subtilis, and the amino acid sequence of the digestion fragment is shown in SEQ ID NO:1; or the polypeptide is a mutant of neutral metalloproteinase A7UAM1.

2. A polypeptide with skin barrier repair activity, characterized in that, The mutant is obtained by mutating one or more amino acids in the neutral metalloproteinase of wild-type Bacillus subtilis, as shown in SEQ ID NO:2, such as H142A, E143A, and H146A.

3. The polypeptide with skin barrier repair activity according to claim 2, characterized in that, The mutant contains a double mutation of H142A and E143A.

4. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the polypeptide according to any one of claims 1 to 3.

5. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule of claim 4.

6. A host cell, characterized in that, The host cell comprises the recombinant expression vector of claim 5, or the nucleic acid molecule of claim 4 is integrated into its genome.

7. A composition, characterized in that, The composition comprises the polypeptide according to any one of claims 1 to 3, and a cosmetically acceptable excipient or a pharmaceutically acceptable carrier.

8. The composition according to claim 7, characterized in that, The composition also contains one or more skin-care active ingredients selected from the following: niacinamide, panthenol, ceramide, asiaticoside, hyaluronic acid, vitamin C, vitamin E, allantoin, and dipotassium glycyrrhizate.

9. The use of the polypeptide according to any one of claims 1 to 3 in the preparation of cosmetic or topical skin preparations having any of the following effects: (a) promoting skin barrier repair; (b) upregulating filaggrin and / or nautiloidin expression; (c) reducing transepidermal water loss; (d) improving sensitive skin; (e) repairing photo-aged skin; (f) repairing skin after cosmetic procedures.

10. Use of the polypeptide according to any one of claims 1 to 3 in the preparation of a medicament for treating diseases related to skin barrier dysfunction.

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