Acidic serine keratinase genes and uses thereof

Abstract

This invention discloses an acidic serine keratinase gene and its applications, belonging to the field of genetic engineering technology. This invention screened a novel serine keratinase from the acidic hot spring environment of Niujie, Yunnan. This keratinase naturally possesses characteristics of acid resistance, good thermal stability, and high enzyme activity, allowing it to adapt well to the acidic microenvironment of the skin and temperature fluctuations throughout the product's production, storage, and transportation processes. Furthermore, it eliminates the need for complex stabilization treatments, simplifying the production process, reducing production costs, and extending the product's shelf life. This achieves green and efficient preparation, fundamentally solving key problems of existing keratinases in skin applications, such as irritation, poor stability, and difficulty in storage. It is particularly suitable for the gentle exfoliation of human keratin plugs, demonstrating outstanding application value and broad prospects in the daily chemical skincare field.

Description

Technical Field

[0001] This invention relates to the acid serine keratinase gene and its applications, belonging to the field of genetic engineering technology. Background Technology

[0002] Serine keratins, with their active sites containing a serine (Ser), histidine (His), and aspartic acid (Asp) catalytic triplet, represent the most promising category within the keratinase family. In terms of origin, these enzymes are primarily isolated from microbial strains such as Bacillus subtilis, Beauveria bassiana, and Bacillus licheniformis. They possess both disulfide bond reduction and peptide hydrolysis activities, efficiently breaking down the dense intramolecular bonds of keratin molecules, degrading insoluble keratin into soluble peptides or amino acids, while causing minimal damage to collagen and elastin, demonstrating natural biocatalytic advantages. Compared to enzymes derived from animals and plants, microbial serine keratins are easier to produce industrially through fermentation, and their enzymatic properties can be optimized through genetic engineering.

[0003] Currently, in daily chemical skincare, the mainstream exfoliating and conditioning ingredients fall into two categories: one is chemical exfoliants, such as glycolic acid, salicylic acid, and their derivatives. However, these substances are highly irritating, and long-term use can easily damage the skin barrier. Furthermore, their chemical synthesis process consumes a large amount of energy and generates high pollution, making green production impossible. The other category is biological enzymes, which are significantly milder than chemical exfoliants. However, their enzymatic stability is poor, and they are easily deactivated by pH and temperature: the acidic environment of the skin surface (pH 4.5-6.0) significantly reduces enzyme activity, and temperature fluctuations during production, storage, and use can also lead to deactivation. Naturally derived serine keratinases are rare, and those that do not meet the comprehensive requirements of acid resistance, heat resistance, and high enzyme activity are generally difficult to find simultaneously. Therefore, screening for keratinases that are acid-resistant, heat-resistant, and have high catalytic activity is crucial. Although existing technologies provide some new keratinases, such as the acidic keratinase screened by patent CN117286126A, these are only applicable to the wool processing field and are difficult to apply to the skin environment, limiting their application scenarios. Summary of the Invention

[0004] In view of the above-mentioned prior art, the present invention provides an acidic heat-stable serine keratinase TVG06-16620 screened from acidic hot springs in Niujie, Yunnan Province, and its encoding gene, including the expression vector of the gene, recombinant strain, and the keratin plug removal effect of the enzyme.

[0005] This invention screened a novel serine keratinase from acidic hot springs in Niujie, Yunnan Province. After being ligated into the expression vector pET-28a, it was transformed into *Escherichia coli* BL21(DE3) for induced expression. Its enzymatic properties were studied, and the optimal dosage for dissolving human keratin plugs was achieved through formulation optimization. This invention is of great significance for understanding the characteristics and efficacy of the microbial-derived acidic, heat-stable serine keratinase TVG06-16620.

[0006] This invention is achieved through the following technical solution:

[0007] This invention provides a serine keratinase TVG06-16620, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] The present invention also provides a gene encoding serine keratinase TVG06-16620.

[0009] In one embodiment, the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0010] The present invention also provides a recombinant vector containing a gene encoding an acidic, thermostable serine keratinase TVG06-16620 and a complete coding reading frame sequence.

[0011] In one embodiment, the carrier is preferably pET-28a.

[0012] The present invention also provides a recombinant strain expressing the serine keratinase TVG06-16620 or containing the recombinant vector.

[0013] In one embodiment, the host cells of the recombinant strain include, but are not limited to, Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Lactobacillus, or yeast.

[0014] In one embodiment, the recombinant strain is *Escherichia coli*. Escherichia coli BL21(DE3) is the host.

[0015] In one embodiment, the recombinant strain contains the recombinant vector, and the gene encoding the acidic thermostable serine keratinase TVG06-16620 is fully expressed in the recipient cells through promoter manipulation, thereby conferring the recipient bacteria with the ability to produce the acidic thermostable serine keratinase TVG06-16620.

[0016] The present invention also provides a method for preparing the serine keratinase TVG06-16620, wherein the recombinant strain is fermented to induce the expression of serine keratinase TVG06-16620, and the serine keratinase TVG06-16620 is collected.

[0017] In one embodiment, the method further includes collecting bacterial cells from the fermentation broth, breaking down and purifying the serine keratinase TVG06-16620 enzyme protein.

[0018] The present invention also provides the application of the serine keratinase TVG06-16620 in dissolving corneal plugs.

[0019] In one embodiment, the application is carried out at 20°C to 55°C for at least 3-5 minutes.

[0020] The present invention also provides the use of the serine keratinase TVG06-16620 in the preparation of products that facilitate keratin exfoliation and / or dissolution of keratin plugs.

[0021] In one embodiment, the product is a daily chemical product, including but not limited to rinse-off cleaning products or leave-on skin care products.

[0022] In one embodiment, the rinse-off cleaning product includes facial cleanser, facial cleanser, shower gel, or exfoliating scrub; the leave-on skincare product includes toner, serum, body lotion, or face mask.

[0023] Beneficial effects:

[0024] (1) The present invention provides serine keratinase TVG06-16620, which contains 289 amino acids, has a theoretical molecular weight of 32kDa, a theoretical isoelectric point of 4.4, a measured isoelectric point of 3.0, an optimal temperature of 45~50℃, an optimal pH of 5.0, and the enzyme activity remains basically stable after being placed at room temperature for 60 min in a buffer solution of pH 4.0~pH 6.

[0025] (2) The serine keratinase TVG06-16620 provided by the present invention has good thermal stability. After being kept at 55°C for 60 min, the enzyme activity is retained by more than 85%.

[0026] (3) The serine keratinase TVG06-16620 provided by the present invention has significant keratin exfoliation effect and can be used in the preparation of daily chemical products related to exfoliation, rinsing and retention.

[0027] (4) The serine keratinase TVG06-16620 provided by the present invention has significant keratin plug dissolving ability and can be used in the preparation of daily chemical products for rinsing and retention of keratin plugs in pores. Attached Figure Description

[0028] Figure 1 The three-dimensional structure diagram of the predicted serine keratinase is shown.

[0029] Figure 2The results of SDS-PAGE validation of serine keratinase TVG06-16620 are shown.

[0030] Figure 3 Enzyme activity of serine keratinase TVG06-16620 at different temperatures.

[0031] Figure 4 Temperature stability of serine keratinase TVG06-16620.

[0032] Figure 5 The enzyme activity of serine keratinase TVG06-16620 at different pH values.

[0033] Figure 6 pH stability of serine keratinase TVG06-16620.

[0034] Figure 7 Schematic diagram of the stability of serine keratinase TVG06-16620 under different conditions.

[0035] Figure 8 This diagram illustrates the stability of papain under different conditions.

[0036] Figure 9 This is a schematic diagram showing the change rate of different components on the exfoliated protein in pigskin.

[0037] Figure 10 A schematic diagram illustrating the effects of different amounts of serine keratinase TVG06-16620 on dissolving human keratin plugs.

[0038] Figure 11 A schematic diagram illustrating the dissolution rate of human keratin plugs by different amounts of serine keratinase TVG06-16620. Detailed Implementation

[0039] The present invention will be further described below with reference to embodiments.

[0040] 1. Strains and vectors:

[0041] E. coli Escherichia coli BL21(DE3) was purchased from Qingke Biotechnology Co., Ltd.; expression vector pET-28a was purchased from TransGen Biotechnology Co., Ltd.

[0042] 2. Culture medium: LB medium / L: 10g tryptone, 5g yeast extract, 10g sodium chloride, pH is naturally 7.0, usually no additional adjustment is needed. Add 2.0% (w / v) agar to the liquid medium.

[0043] 3. Reagents required for enzyme activity detection: Folin reagent (Merida), anhydrous sodium carbonate (Aladdin), trichloroacetic acid (Maclean), sodium hydroxide (Xihua), concentrated hydrochloric acid (Huachengda), Tris (Maclean), casein (Aladdin).

[0044] Example 1: Recombinant expression of serine keratinase TVG06-16620

[0045] The serine keratase TVG06-16620 DNA sequence (SEQ ID NO.2) and plasmid pET-28a-TVG06-16620 used in this invention were both synthesized by Shanghai Diwin Biotechnology Co., Ltd. Plasmid pET-28a-TVG06-16620 was transformed into *E. coli* BL21(DE3). Cloned strains were picked and cultured in LB broth containing 100 μg / mL kanamycin at 37°C and 220 rpm for recovery. Then, a 1% (v / v) inoculum was transferred to a conical flask containing 200 mL of LB broth, and kanamycin was added to a final concentration of 100 μg / mL. The flask was then cultured at 25°C and 220 rpm until the logarithmic growth phase (OD200). 600 The concentration was 0.6-0.8. At this point, IPTG was added to a final concentration of 1 mM as an inducer to induce expression. The mixture was cultured overnight at 30℃ and 220 rpm for 16 h to obtain a fermentation broth expressing serine keratinase TVG06-16620. The OD600 value at this point was 67.

[0046] Example 2: Preparation and purification of serine keratinase TVG06-16620 enzyme solution

[0047] The fermentation broth obtained in Example 1 was centrifuged at 4°C and 6000 rpm for 10 min, the supernatant was discarded and the bacterial cells were collected. The bacterial cells were then resuspended in 50 mM acetate-sodium acetate buffer to obtain a cell suspension with an OD600 value of 40. The cell suspension was sonicated for 30 min at 30% power using a cell sonicator. After the sonication, the suspension was centrifuged at 12000 rpm for 30 min to obtain a turbid enzyme solution. The enzyme solution was precipitated by salting out with 40% ammonium sulfate for pretreatment. Then, the solution was desalted by ultrafiltration to obtain a clear yellow enzyme solution. 0.5% activated carbon (model 701) was added to the enzyme solution and stirred at 200 rpm for 30 min in a 50°C water bath. The solution was then centrifuged at 12000 rpm for 10 min. The supernatant was filtered through a 0.22 μm sterile filter membrane to obtain a clear and colorless serine keratinase TVG06-16620 enzyme solution.

[0048] Example 3: Detection method for keratinase activity and determination of specific activity of serine keratinase TVG06-16620

[0049] The enzyme activity detection method is based on GB / T 23527.1-2023, mainly referring to the national standard for detecting enzyme activity in neutral protease preparations, with appropriate modifications for the enzyme activity detection of the enzyme solution in this invention. The specific detection method is as follows:

[0050] (1) First, prepare 0.4M sodium carbonate solution, 0.4M trichloroacetic acid solution, 20 g / L sodium hydroxide solution, 1M hydrochloric acid solution, and 50mM acetate-sodium acetate buffer (pH 5.0±0.05).

[0051] (2) Prepare a 10 g / L casein substrate solution: Weigh 1.00 g of casein and add it to 80 mL of acetate-sodium acetate buffer. Heat in a boiling water bath for 30 min and stir until dissolved. After cooling to room temperature, adjust the pH to 5.0 with 1 M hydrochloric acid solution or 20 g / L sodium hydroxide solution and then make up to 100 mL with acetate-sodium acetate buffer. Store at 4℃ and it is recommended to use within 3 days.

[0052] (3) Preparation of L-tyrosine standard stock solution (100 μg / mL): Weigh 0.100 g of L-tyrosine, dissolve it in 1 M hydrochloric acid solution and make up to 100 mL. Shake well to obtain a stock solution concentration of 1 mg / mL. Then dilute it 10 times with 0.1 M hydrochloric acid solution to obtain a stock solution of 100 μg / mL.

[0053] (4) Use Folin reagent mixed with water in a 1:2 ratio.

[0054] Plotting the standard curve: L-tyrosine standard solutions were prepared according to Table 1.

[0055] Table 1 L-Tyrosine Standard Solutions

[0056]

[0057] Prepare different L-tyrosine standard solutions according to Table 1 above. Take 1.00 mL of each standard solution of different concentrations into a separate centrifuge tube, add 5.00 mL of sodium carbonate solution and 1.00 mL of Folin-Ciocalteu reagent, mix well, incubate at 45℃ for 20 min, cool to room temperature, and measure the absorbance at 680 nm using a UV spectrophotometer. Finally, plot a standard curve with the absorbance value on the ordinate and the L-tyrosine concentration on the abscissa. Calculate the amount of tyrosine (μg) when the absorbance is 1, which is the absorbance constant K value. Keep the K value within the range of 95-100.

[0058] Sample determination: The casein substrate solution was preheated in a 45℃ constant temperature water bath for 5 min. Other reagent solutions were added sequentially according to the procedures in Table 2, and the absorbance at 680 nm was measured.

[0059] Table 2 Operating Steps

[0060]

[0061] The enzyme activity of the sample is calculated according to Formula 1:

[0062] ;

[0063] X — Enzyme activity of the sample, expressed in units of enzyme activity (U / L); one unit of enzyme activity (U) is defined as the amount of protease required to hydrolyze casein to produce 1 μg of tyrosine in 1 minute under certain temperature and pH conditions.

[0064] C – The concentration of L-tyrosine produced by protease hydrolysis, as obtained from the standard curve, in micrograms per milliliter (μg / mL).

[0065] V — the volume of the sample, in milliliters (mL);

[0066] 4 — Total volume of the reaction reagents, in milliliters (mL);

[0067] n—the dilution factor of the sample;

[0068] 10 — Reaction time, in minutes (min).

[0069] The enzyme activity of the TVG06-16620 enzyme solution obtained in Example 2 was detected using the modified enzyme activity method described above. The enzyme activity result was 35500 U / L. Subsequently, the total protein content of the TVG06-16620 enzyme solution was detected using the Beyotime Total Protein Assay Kit (BCA method), and the result was 1.2 mg / mL. The specific activity of the serine keratinase TVG06-16620 was calculated to be 29583 U / g. The SDS-PAGE electrophoresis image of the enzyme solution is shown below. Figure 2 As shown, M: protein standard; TVG06-16620: total protein expressed in the serine keratinase TVG06-16620 enzyme solution after BL21(DE3) / pET-28a induction; the results showed that the molecular weight of TVG06-16620 protein was 32kDa.

[0070] Example 4: Determination of the optimal temperature and thermal stability of serine keratinase TVG06-16620

[0071] The TVG06-16620 enzyme solution obtained in Example 2 was placed in PBS buffer at pH 5.0 and incubated at 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 70℃, and 80℃ for 5 min, respectively, according to the enzyme activity assay method in Example 3. The enzyme activity at each temperature was then measured. The results are as follows: Figure 3As shown, the optimal temperature range for serine keratinase TVG06-16620 is 45℃~50℃, and the enzyme activity can reach 45790 U / L.

[0072] Temperature stability determination: The TVG06-16620 enzyme solution prepared according to the method in Example 2 was incubated in PBS buffer at pH 5.0 for 5 min, 10 min, 20 min, 30 min, and 60 min, respectively. Enzyme activity was measured at each time point, following the method described in Example 3. The enzyme activity retention rate was calculated with the initial enzyme activity as 100%. The results are as follows: Figure 4 As shown, after incubation at 35℃ and 45℃ for 60 min, the enzyme activity retention rate was 100%. After incubation at 55℃ and 65℃ for 60 min, the enzyme activity retention rates were 85% and 70%, respectively. After incubation at 75℃ and 85℃ for 60 min, the enzyme activity retention rates were 40% and 23%, respectively. Therefore, the serine keratinase TVG06-16620 enzyme solution exhibits good thermal stability under conditions of 35℃-55℃, with an enzyme activity retention rate of over 85%.

[0073] Example 5: Determination of the optimal pH and pH stability of serine keratinase TVG06-16620

[0074] Determination of optimal pH: The TVG06-16620 enzyme solution obtained in Example 2 was tested according to the enzyme activity determination method in Example 3 under the following conditions: acidic conditions (0.1M Na2HPO4-NaH2PO4, pH=3.0-7.0), neutral conditions (50mM Tris-HCl, pH=7.0-9.0), and alkaline conditions (50mM Gly-NaOH, pH=9.0-11.0). The highest enzyme activity was represented as 100%. The relative enzyme activity under other pH conditions was calculated to determine the optimal pH. The results showed that the optimal pH for serine keratinase TVG06-16620 was 5.0, and the relative enzyme activity was above 85% at pH 4.0-6.0. Figure 5 );

[0075] pH stability determination: The enzyme solution obtained in Example 2 was incubated in different buffer solutions with pH values ​​ranging from 3.0 to 11.0 at the optimal enzyme temperature of 45°C for 60 min according to the enzyme activity determination method in Example 3. The remaining enzyme activity was then measured and compared with the initial enzyme activity to calculate the enzyme activity retention rate, thus evaluating the stability of the enzyme solution under different pH conditions. After treatment with buffer solutions ranging from pH 3.0 to pH 11.0 at 45°C for 60 min, the enzyme activity retention rate remained 100% within the pH range of 4.0-6.0, indicating stability. Figure 6 This indicates that serine keratinase TVG06-16620 has excellent acid resistance.

[0076] Example 6: Stability of the serine keratinase TVG06-16620 formulation

[0077] The enzyme solution prepared using the method described in Example 2 yielded an enzyme solution with an enzyme activity of 50,000 U / L (TVG06-16620), which was used as the mother liquor. This mother liquor was added at a rate of 2% (based on the final volume percentage) to a formulation solution at pH 5, resulting in a formulation solution containing TVG06-16620 with an initial enzyme activity of 1000 U / L. This formulation solution used pure water as a base and was compounded with commonly used preservative components including 1% (v / v) hexanediol, 2.5% (v / v) ethylhexylglycerin, and 10% (v / v) glycerin to ensure the stability of the enzyme solution under aseptic conditions.

[0078] Papain (Pangbo Biotechnology), which is available on the market, was used as the control group. The initial enzyme activity was also set at 1000 U / L. The optimal pH of this enzyme is 6.5. The pH of the formula was adjusted to 6.5. Other test conditions were the same as those of the experimental group.

[0079] The prepared TVG06-16620 enzyme solution for the experimental group and the papain enzyme solution for the control group were subjected to an 8-week stability study, mainly including two aspects:

[0080] (1) Temperature stability: The samples were placed at 5℃, 25℃ and 45℃ respectively and stored statically;

[0081] (2) Light stability: The sample was placed in a light stability tester (Shanghai Yiheng, model LHH-400GP-UV), and the UV radiation energy was controlled at 200 W.hr / m. 2 The temperature is controlled at 25℃;

[0082] During the investigation, enzyme activity retention rate was used as the main evaluation index to comprehensively assess the stability of TVG06-16620 enzyme under different environmental conditions, providing a reference for its subsequent addition amount in cosmetics.

[0083] The results are as follows Figure 7 As shown, the enzyme activity of a 2% TVG06-16620 enzyme solution sample remained at 1000 U / L after 8 weeks of storage at 5℃, 25℃, and 45℃, showing virtually no loss. However, after 8 weeks of storage under UV conditions, the enzyme activity was 500 U / L, representing a 50% loss. Figure 8As shown, after 8 weeks of storage, the 1000 U / L papain enzyme activity was 695 U / L at 5°C, a loss of 30%; at 25°C and 45°C, the enzyme activities were 427 U / L and 300 U / L, respectively, with losses of 57% and 70%; and under UV conditions, the enzyme activity was 435 U / L, a loss of 56%. In summary, the serine keratinase TVG06-16620 enzyme solution of this invention exhibits stronger stability under both temperature and light conditions.

[0084] Example 7: Effect of serine keratinase TVG06-16620 on pigskin peeling

[0085] Using salicylic acid, lactobionic acid, and mandelic acid as references, the enzyme solution prepared in Example 2 was used to obtain an enzyme solution with an activity of 50,000 U / L. TVG06-16620 enzyme solution was used as the stock solution, and different addition amounts were used as experimental groups to evaluate its peeling effect on pigskin. Specific method: Fresh miniature pigskin from the back was taken, and 100 μL of different addition amounts of TVG06-16620 enzyme solution and other control samples were dripped onto different areas within a certain area. All groups were treated for 10 min, and pigskin tissue from the corresponding areas was collected. The tissue was homogenized using a cell homogenizer, and cell lysis buffer was added for lysis. The supernatant was collected by centrifugation, and the protein content was detected using the BCA method to measure the relative amount of cells peeled off.

[0086] The results are as follows Figure 9 As shown, the rate of change in exfoliated protein content (%) = (C T / T) × 100%; C - protein content in the experimental group; T - protein content in the negative control group. BC: blank group (water); significance compared with the BC group is indicated by *, p-value < 0.05 is indicated as *; p-value < 0.01 is indicated as **; pH of all samples was adjusted to 5.0.

[0087] Sample information:

[0088] 1#: 2% salicylic acid, pH: 5.0;

[0089] #2: 2% mandelic acid, pH: 5.0;

[0090] 3#: 2% supramolecular acid-enzyme composition, pH: 5.0;

[0091] #4: C-Coffee Pore-Clearing Essence Water, pH: 5.0;

[0092] 5#: 2% supramolecular acid-enzyme composition + 2% keratinase TVG06-16620, pH: 5.0;

[0093] 6#: 1% keratinase TVG06-16620, pH: 5.0;

[0094] 7#: 2% keratinase TVG06-16620, pH: 5.0;

[0095] 8#: 4% keratinase TVG06-16620, pH: 5.0;

[0096] #9: 2% lactobionic acid, pH: 5.0;

[0097] Among them, the supramolecular acid-enzyme composition is disclosed in the paper "Development and Clinical Evaluation of a Supramolecular Acid-Enzyme Complex for Skin Exfoliation, Sebum Control, and Pore Refinement."

[0098] All samples (1#~9#) showed significant differences compared to group BC, indicating that these components or formulations could alter the content of exfoliated protein. Among the groups with added keratinase, 8# (4% keratinase TVG06-16620) showed a 120% change rate in exfoliated protein, while 5# (2% supramolecular acid-enzyme composition + 2% keratinase TVG06-16620), 6# (1% keratinase TVG06-16620), and 7# (2% keratinase TVG06-16620) showed relatively weaker effects with 52%, 17%, and 40% changes in exfoliated protein, respectively. Overall, under pH 5.0 conditions, all tested components or formulations significantly altered the content of exfoliated protein. After comprehensive comparison, 4% keratinase TVG06-16620 showed the best effect, followed by 2% supramolecular acid-enzyme composition + 4% keratinase TVG06-16620. This indicates that the keratinase TVG06-16620 has a good keratin exfoliation effect.

[0099] Example 8: Effect of serine keratinase TVG06-16620 on dissolving human keratin plugs

[0100] Samples of keratin plugs were collected from people at beauty salons and quickly rinsed 1-2 times with sterile saline solution to remove surface sebum and dirt.

[0101] Air dry at room temperature, then crush and sieve to obtain uniform horn plug powder. Store in a sealed container at -20℃ for later use.

[0102] Test group: The TVG06-16620 enzyme solution with an activity of 50000 U / L prepared in Example 2 was used as the stock solution. A 2% keratinase TVG06-16620 enzyme solution formulation was prepared (the preparation method of the basic enzyme solution formulation is the same as in Example 5);

[0103] Blank control group: Enzyme-free formulation matrix (preparation method as in Example 5, pH as in the test group).

[0104] Accurately weigh 5 mg of keratin plug powder and place it in a 1.5 mL centrifuge tube. Add 1 mL of test solution or control solution to each tube. Incubate at 37℃ to simulate human skin temperature. Observe the morphological changes of the keratin plug at different time points of 0, 1, 2, 3, 4, and 5 min, and calculate the keratin plug dissolution rate by measuring the content of soluble protein in the supernatant (BCA method).

[0105] ;

[0106] The results showed that the formulation containing 2% TVG06-16620 keratinase enzyme solution could significantly soften, disintegrate, and dissolve human keratin plugs. Figure 10 After soaking at 37℃ for 5 minutes, the keratin plug dissolution rate of the 2% TVG06-16620 enzyme solution reached over 75%, significantly higher than that of the blank control group. Figure 11 It exhibits excellent ability to degrade keratin plugs, so adding 2% of TVG06-16620 keratinase solution (enzyme activity of 1000U / L) can achieve a significant keratin plug dissolution effect.

[0107] Example 9: Closed-patch test of serine keratinase TVG06-16620 enzyme solution on human skin

[0108] The human skin occlusive patch test was commissioned to a third-party testing organization, Shanghai Shengping Testing Technology Co., Ltd., and the testing standards referred to Chapter 7:2 Human Skin Patch Test of the "Cosmetic Safety Technical Specifications" (2015 Edition).

[0109] The specific steps are as follows:

[0110] 1. Test substance: 2% TVG06-16620 enzyme solution (1000U / L).

[0111] 2. Negative control: Blank control.

[0112] 3. Participants: A total of 30 participants, including 5 males and 25 females, aged 26 to 59 years, with a mean age of 44.7 ± 9.7 years, who met the criteria for voluntary inclusion.

[0113] 4. Patch Test Method: Select qualified patch test equipment and use the closed patch test method. Place 0.020mL-0.025mL (liquid) of the test substance in the patch test device, apply it to the flexor side of the subject's forearm with hypoallergenic adhesive tape, remove the test substance after 24 hours, and observe the skin reaction at 0.5, 24 and 48 hours after removal. Record the results according to the skin reaction grading standard in the current effective technical specifications.

[0114] Table 3 Summary of Human Skin Patch Test Results for 2% TVG06-16620 Enzyme Solution

[0115]

[0116] Table 4 Subject Information and Trial Results

[0117]

[0118] Note: Results of the first 30 valid subjects were obtained using systematic sampling.

[0119] The test results are shown in Table 4. The results of the human skin patch test showed that 0 out of 30 people experienced adverse skin reactions, indicating that the 2% TVG06-16620 enzyme solution is non-irritating and mild to human skin.

[0120] Comparative example:

[0121] The specific implementation method is the same as in Example 1, except that the enzymes TVG06-16620-23, TVG06-16620-49, and TVG06-16620-121 shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5 are also expressed. The enzyme activity of the enzyme solution was detected, and the results showed that the enzyme activity of TVG06-16620-23 was 21200 U / L, the enzyme activity of TVG06-16620-49 was 31340 U / L, and the enzyme activity of TVG06-16620-121 was 29000 U / L.

[0122] The temperature stability of the enzymes was verified according to the methods in Examples 3 and 4. The results showed that TVG06-16620-23, TVG06-16620-49, and TVG06-16620-121 lost 80%, 65%, and 88% of their enzyme activity, respectively, at 50°C. The optimal reaction temperatures for enzyme activity were 25°C, 45°C, and 25°C, respectively. The pH stability results showed that the relative enzyme activity of TVG06-16620-23, TVG06-16620-49, and TVG06-16620-121 remained at 30%-40% in the pH range of 3-6, at 56%-60% in the pH range of 7-9, and at 35%-40% in the pH range of 9-12.

[0123] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Serine keratinase TVG06-16620, characterized in that, The amino acid sequence is shown in SEQ ID NO.

1.

2. The gene encoding the serine keratinase TVG06-16620 of claim 1.

3. A recombinant vector, characterized in that, It contains the gene described in claim 2.

4. A recombinant strain, characterized in that, It expresses the serine keratinase TVG06-16620 of claim 1 or contains the recombinant vector of claim 3.

5. The recombinant strain of claim 4, wherein, Host cells include Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Lactobacillus, or yeast.

6. A recombinant E. coli bacterium, characterized in that, Using Escherichia coli BL21(DE3) as the host and pET-28a as the vector, the serine keratinase TVG06-16620 described in claim 1 was expressed.

7. Process for the preparation of the serine keratinase TVG06-16620 according to claim 1, characterized in that, The recombinant strain of claim 4 or 5 or the recombinant Escherichia coli of claim 6 is fermented to induce the expression of serine keratinase TVG06-16620, and the serine keratinase TVG06-16620 is collected.

8. The use of the serine keratinase TVG06-16620 of claim 1 in the preparation of products that facilitate keratin exfoliation and / or dissolution of keratin plugs.

9. The application according to claim 8, characterized in that, The products are daily chemical products, including rinse-off cleaning products or leave-on skin care products.

10. The application according to claim 9, characterized in that, The rinse-off type cleansing products include facial cleansers, facial cleansing creams, shower gels, or exfoliating scrubs; the leave-on type skincare products include toners, serums, body lotions, or face masks.

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