Amidated polypeptide and application thereof

Abstract

The invention provides an amidated polypeptide and application thereof. The amidated polypeptide disclosed by the invention is obtained by amidating and modifying the C tail end of polypeptide; wherein the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1. The amidated polypeptide has excellent antioxidant activity, tyrosinase inhibitory activity and melanin inhibitory activity, is remarkably superior to products obtained by modification through other methods (such as acetylation, palmitoylation and the like), is suitable for preparing antioxidant products, tyrosinase inhibitors and melanin inhibitors, and can be used for preparing anti-oxidation products, tyrosinase inhibitors and melanin inhibitors. The method is also suitable for preparing products with the whitening effect, such as food, cosmetics or medicines with the whitening effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide modification, and more specifically, relates to amidated polypeptides and applications thereof. Background Art

[0002] Tyrosinase, also known as polyphenol oxidase, catechol oxidase, aged cheese enzyme, etc., is composed of multiple subunits, each of which contains two metal copper ions. These two copper ions are covalently bound to the imino groups of three histidine residues and fixed on the active center. In addition, there is an endogenous bridge group that links the two copper ions together to form the active center of tyrosinase. Tyrosinase plays a vital role in the synthesis of melanin. First, it hydroxylates monophenol into diphenol, then oxidizes o-diphenol into o-diquinone, and o-diquinone finally reacts to form melanin. Therefore, tyrosinase plays a key role in the process of melanin production.

[0003] Currently, whitening products on the market often reduce the production of melanin by inhibiting the activity of tyrosinase, thereby achieving a whitening effect. For example, a patent application with publication number CN116731105A provides a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1, which has the activity of inhibiting tyrosinase and can be used as a raw material for whitening products, but its effect is limited. Summary of the invention

[0004] The present invention aims to provide an amidated polypeptide by amidating the amino acid sequence of the polypeptide shown in SEQ ID NO: 1, thereby significantly reducing the IC value of the polypeptide to inhibit tyrosinase. 50 value, thereby improving its whitening effect.

[0005] The first object of the present invention is to provide an amidated polypeptide.

[0006] The second object of the present invention is to provide the use of the amidated polypeptide in the preparation of whitening products.

[0007] The third object of the present invention is to provide the use of the amidated polypeptide in the preparation of antioxidant products.

[0008] The fourth object of the present invention is to provide the use of the amidated polypeptide in the preparation of tyrosinase inhibitors.

[0009] The fifth object of the present invention is to provide the use of the amidated polypeptide in the preparation of a melanin inhibitor.

[0010] The sixth object of the present invention is to provide related biological materials of the amidated polypeptide.

[0011] The seventh objective of the present invention is to provide the use of the above-mentioned related biomaterials in the preparation of whitening products.

[0012] The eighth objective of the present invention is to provide the use of the above-mentioned related biological materials in the preparation of antioxidant products.

[0013] The ninth objective of the present invention is to provide the use of the above-mentioned related biological materials in the preparation of tyrosinase inhibitors.

[0014] The tenth objective of the present invention is to provide the use of the above-mentioned related biological materials in the preparation of melanin inhibitors.

[0015] The above-mentioned purpose of the present invention is achieved through the following technical solutions: The present invention provides an amidated polypeptide, which is obtained by amidating the C-terminus of the polypeptide, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1. SEQ ID NO: 1: AHYYD.

[0016] The effects of the amidated polypeptides are as follows: (1) they have excellent antioxidant activity and are suitable for the preparation of antioxidant products; (2) they can effectively inhibit tyrosinase and are suitable for the preparation of tyrosinase inhibitors; and (3) they can significantly inhibit the production of melanin and are suitable for the preparation of melanin inhibitors.

[0017] In summary, the amidated polypeptides are also suitable for preparing whitening products. Therefore, the use of the amidated polypeptides in preparing whitening products, antioxidant products, tyrosinase inhibitors, and melanin inhibitors should be within the protection scope of the present invention.

[0018] Based on this, the present invention also provides related biological materials of the above-mentioned amidated polypeptides and their use in the preparation of whitening products, antioxidant products, tyrosinase inhibitors, and melanin inhibitors. The related biological materials are nucleic acid molecules that can express the amidated polypeptides, or recombinant DNA, expression cassettes, transposons, vectors or host cells containing the nucleic acid molecules.

[0019] In addition, the present invention also provides a product comprising the amidated polypeptide and / or its related biological materials.

[0020] Preferably, the relevant biological material is a nucleic acid molecule capable of expressing the amidated polypeptide, or a recombinant DNA, an expression cassette, a transposon, a vector or a host cell containing the nucleic acid molecule.

[0021] Preferably, the product is food, cosmetic or medicine.

[0022] More preferably, it also contains excipients acceptable to food, cosmetics or medicines.

[0023] Further preferably, the food is nutritious and healthy food.

[0024] The present invention has the following beneficial effects: The present invention performs amidation modification on a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1, and the obtained amidated polypeptide has excellent antioxidant activity, tyrosinase inhibitory activity, and melanin inhibitory activity, and is significantly superior to products modified by other methods (such as acetylation, palmitoylation, etc.). The amidated polypeptide is not only suitable for preparing antioxidant products, tyrosinase inhibitors, and melanin inhibitors, but also suitable for preparing products with whitening effects, such as foods, cosmetics or medicines with whitening effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the mass spectrometry identification result of T1.

[0026] Figure 2 This is the mass spectrometry identification result of T2.

[0027] Figure 3 This is the mass spectrometry identification result of T3.

[0028] Figure 4 This is the mass spectrometry identification result of T4.

[0029] Figure 5 This is the antioxidant activity test result of T1 to T3.

[0030] Figure 6 This is the safety test result diagram from T1 to T2.

[0031] Figure 7 This is a graph showing the test results of the inhibitory activity of T1-T2 on tyrosinase at the cellular level.

[0032] Figure 8 This is a graph showing the test results of the inhibitory activity of T1 to T2 on melanin. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0034] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0035] Example 1 Synthesis and identification of T1 to T4 1. Synthesis of T1~T4 In this example, Shanghai Aminlian Biotechnology Co., Ltd. was commissioned to assist in synthesizing the polypeptide T1 (AHYYD) with an amino acid sequence as shown in SEQ ID NO: 1, and three chemical modifications were performed on it, namely: (1) Performing amidation modification on its C-terminus to obtain amidated polypeptide T2 (AHYYD-NH2); (2) Acetylation modification was performed on its N-terminus to obtain acetylated peptide T3 (Ac-AHYYD); (3) Palmitoylation modification was performed on its N-terminus to obtain palmitoylated polypeptide T4 (Pal-AHYYD).

[0036] The structural formulas of T1 to T4 are as follows: T1: ; T2: ; T3: ; T4: .

[0037] 2. Purity Identification of T1~T4 The purity of T1~T4 was identified by HPLC.

[0038] Among them, the identification conditions of high performance liquid chromatography are: Chromatographic column: SHIMADZU Inertsil ODS-SP (4.6*250mm*5um); Mobile phase A: acetonitrile (containing 0.1% ( v / v ) trifluoroacetic acid); Mobile phase B: water (containing 0.1% ( v / v ) trifluoroacetic acid); Elution gradient: 0→25 min, 99% ( v / v ) → 50% ( v / v )A; Flow rate: 1 mL / min; Detection wavelength: 220 nm; Sample volume: 35 μL.

[0039] The results of purity identification showed that the purities of T1 to T4 were 98.51%, 98.63%, 98.63% and 99.51% respectively.

[0040] 3. Mass spectrometry identification of T1~T4 ESI-MS mass spectrometry was used to identify the molecular weights of T1~T4.

[0041] Among them, the identification conditions of ESI-MS mass spectrum are: Mobile phase A: water (containing 1% ( v / v )formic acid); Mobile phase B: acetonitrile (containing 1% ( v / v )formic acid); Flow rate: 0.2 mL / min; Running time: 4 min. Operation mode: positive ion mode; Scan range: 0~2000 Da.

[0042] The results of mass spectrometry identification are as follows Figures 1 to 4 As shown, Figure 1 This is the mass spectrometry identification result of T1. Figure 2 This is the mass spectrometry identification result of T2. Figure 3 This is the mass spectrometry identification result of T3. Figure 4 The mass spectrometry results of T4 are shown in Figure 1. It can be seen that the molecular ion peaks of T1 to T4 are m / z 668.76, 667.35, 710.82, and 907.06, respectively, with a single charge, which is consistent with the theoretical molecular weight value, indicating that T1 to T4 were successfully synthesized.

[0043] Example 2 Inhibitory activity of T1-T4 on tyrosinase in vitro 1. Solution preparation Sample solution: T1-T4 obtained in Example 1 were dissolved in PBS buffer (pH=6.8) to a final concentration of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL, respectively.

[0044] L-Tyrosine solution: Dissolve L-tyrosine in PBS buffer (pH=6.8) to a final concentration of 0.5 mg / mL.

[0045] Tyrosinase solution: Dissolve tyrosinase in PBS buffer (pH=6.8) to a final concentration of 500 U / mL.

[0046] 2. Experimental Grouping and Treatment Solvent background well (A1): Add 110 μL of PBS buffer (pH=6.8) to the 96-well plate and mix thoroughly.

[0047] Solvent reaction well (A2): Add 40 μL of L-tyrosine solution and 70 μL of PBS buffer (pH=6.8) to the 96-well plate and mix thoroughly.

[0048] Sample background well (A3): Add 40 μL of sample solution and 70 μL of PBS buffer (pH=6.8) into a 96-well plate and mix thoroughly.

[0049] Sample reaction well (A4): Add 40 μL of L-tyrosine solution, 40 μL of sample solution and 30 μL of PBS buffer (pH=6.8) to the 96-well plate and mix thoroughly.

[0050] After incubating each well at a constant temperature of 37°C for 10 min, 20 μL of tyrosinase solution was added to each well, and then mixed and reacted at 37°C for 10 min. The absorbance was measured at 475 nm, and the tyrosinase inhibition rate was calculated according to the formula “Tyrosinase inhibition rate (%) = [1-(A4-A3) / (A2-A1)] × 100%”.

[0051] 3. Experimental Results The results showed that T1-T3 inhibited tyrosinase with IC 50 The values ​​were 2.012±0.088 mM, 0.979±0.028 mM, and 3.430±0.017 mM, respectively, while T4 had no tyrosinase inhibitory activity. It can be seen that compared with the unmodified T1, the tyrosinase inhibitory activity of the amidated polypeptide T2 was significantly improved, the tyrosinase inhibitory activity of the acetylated polypeptide T3 was significantly reduced, and even the palmitoylated polypeptide T4 completely lost its tyrosinase inhibitory activity. This shows that the amidation modification of T1 by the present invention can significantly improve its tyrosinase inhibitory activity, while conventional modification methods (such as acetylation modification, palmitoylation modification, etc.) cannot improve its tyrosinase inhibitory activity, and may even inhibit its activity, indicating that it is precisely the present invention that specifically selects the amidation modification method for T1, which can significantly improve its tyrosinase inhibitory activity.

[0052] Example 3 Antioxidant activity of T1 to T3 According to the DPPH kit instructions of Grace Biotechnology Co., Ltd., the DPPH free radical scavenging ability of T1 to T3 obtained in Example 1 was measured. The results are as follows Figure 5 shown.

[0053] It can be seen that compared with the unmodified T1, the DPPH radical scavenging rate of the amidated polypeptide T2 is significantly improved, and the DPPH radical scavenging rate of the acetylated polypeptide T3 has no significant difference. This shows that the amidation modification of T1 in the present invention can significantly improve its antioxidant activity, while conventional modification methods (such as acetylation modification, etc.) cannot improve its antioxidant activity, indicating that it is precisely the present invention that specifically selects the amidation modification method for T1, which can significantly improve its antioxidant activity.

[0054] Example 4 Safety of T1-T2 1. Solution preparation Sample solution: In a clean bench, dissolve T1-T2 obtained in Example 1 in a 10% ( v / v ) fetal bovine serum and 1% ( v / v ) penicillin-streptomycin dual antibody DMEM / F-12 culture medium to make the final concentrations of 0, 15, 37.5, 75, 150, 300, and 600 μM, respectively, and then filter with a 0.22 μm filter membrane for sterilization.

[0055] 2. B16F10 cell culture B16F10 cells were inoculated in a medium containing 10% ( v / v ) fetal bovine serum and 1% ( v / v ) penicillin-streptomycin dual antibody DMEM / F-12 medium, cultured in an incubator at 37 ℃, saturated humidity, and 5% CO2 until the cells basically cover the bottom of the culture bottle, remove the waste liquid in the culture bottle, wash with PBS, digest with 1 mL of 0.25% trypsin for 1 min, add 2 mL of 10% ( v / v ) fetal bovine serum and 1% ( v / v ) penicillin-streptomycin dual antibody DMEM / F-12 medium to terminate the reaction, blow the bottom of the bottle with a pipette, transfer the liquid to a centrifuge tube, centrifuge at 1000 rpm for 5 min, remove the supernatant, and add 3 mL of 10% ( v / v ) fetal bovine serum and 1% ( v / v ) were resuspended in DMEM / F-12 medium containing penicillin-streptomycin dual antibodies, and subcultured at a ratio of 1:2. The experiment was carried out after the cell growth state was stable.

[0056] 3. Experimental Grouping and Treatment Dosing sample hole (A 样 ): Melanoma cells (B16F10) in the logarithmic growth phase were seeded in a 96-well plate at a density of 3000 cells / well and 100 μL per well. After adaptive culture in a 37°C incubator for 24 h, the supernatant was discarded. 100 μL of sample solution was added. After culture in a 37°C incubator for 48 h, the supernatant was discarded. Under light-proof conditions, 110 μL of 10% ( v / v ) CCK-8 solution was incubated in a 37 ℃ incubator for 1 h, and the absorbance at 450 nm was measured.

[0057] Zero adjustment hole (A 调 ): Add 100 μL of 10% ( v / v ) fetal bovine serum and 1% ( v / v ) penicillin-streptomycin dual-antibody DMEM / F-12 medium, incubate in a 37 ℃ incubator for 24 h, discard the supernatant, add 100 μL of 10% ( v / v ) fetal bovine serum and 1% ( v / v ) penicillin-streptomycin dual antibody DMEM / F-12 medium, culture in a 37 ℃ incubator for 48 h, discard the supernatant, add 110 μL of 10% ( v / v ) CCK-8 solution was incubated in a 37 ℃ incubator for 1 h, and the absorbance at 450 nm was measured.

[0058] Blank well (A 空 ): Melanoma cells (B16F10) in the logarithmic growth phase were seeded in a 96-well plate at a density of 3000 cells / well and 100 μL per well. After adaptive culture in a 37°C incubator for 24 h, the supernatant was discarded and 100 μL of 10% ( v / v ) fetal bovine serum and 1% ( v / v ) penicillin-streptomycin dual antibody DMEM / F-12 medium, culture in a 37 ℃ incubator for 48 h, discard the supernatant, add 110 μL of 10% ( v / v ) CCK-8 solution was incubated in a 37 ℃ incubator for 1 h, and the absorbance at 450 nm was measured.

[0059] According to the formula "Cell viability (%) = [(A 样 -A 调 ) / (A 空 -A 调 )]×100%” to calculate the cell viability after the sample solution was applied to evaluate the safety of T1 to T2.

[0060] IV. Experimental Results The results are as follows Figure 6 As shown, compared with no addition of T1, T2, and only addition of unmodified T1, there is no significant difference in the activity of B16F10 cells after the action of 15-600 μM amidated polypeptide T2, indicating that the amidated polypeptide T2 of the present invention is non-toxic to cells and has high safety.

[0061] Example 5 Inhibitory activity of T1-T2 on tyrosinase at the cellular level 1. Solution preparation Sample solution: Dissolve T1-T2 obtained in Example 1 in a solution containing 10% ( v / v ) fetal bovine serum and 1% ( v / v ) penicillin-streptomycin dual antibody DMEM / F-12 culture medium to a final concentration of 37.5 and 75 μM, respectively, and then sterilized with a 0.22 μm syringe filter.

[0062] 2. Experimental Grouping and Treatment Sample measurement hole (A 测 ): Melanoma cells (B16F10) in logarithmic growth phase were collected and 2×10 5The cells were seeded at a density of 10 cells / well in a 6-well plate. After adaptive culture in a 37°C incubator for 24 h, the supernatant was discarded and washed twice with PBS solution (pH=7.4). 2 mL of sample solution was added and cultured in a 37°C incubator for 48 h. The supernatant was discarded and washed twice with PBS solution (pH=7.4) to remove non-adherent cells. 400 μL of lysis buffer (PMSF:RIPA volume ratio was 1:100) was added and lysed at 4°C for 30 min. The supernatant was collected by centrifugation.

[0063] Blank well (A 空 ): Melanoma cells (B16F10) in logarithmic growth phase were collected and 2×10 5 The cells were seeded at a density of 100 cells / well in a 6-well plate and cultured in a 37°C incubator for 24 h. The supernatant was discarded and the cells were washed twice with PBS solution (pH = 7.4). 2 mL of 10% ( v / v ) fetal bovine serum and 1% ( v / v ) DMEM / F-12 medium containing penicillin-streptomycin double antibodies was cultured in an incubator at 37°C for 48 h, and the supernatant was discarded. The cells were washed twice with PBS solution (pH=7.4) to remove non-adherent cells, and 400 μL of lysis buffer (PMSF:RIPA volume ratio of 1:100) was added, and the cells were lysed at 4°C for 30 min, and the supernatant was collected by centrifugation.

[0064] The protein concentration of the supernatant was determined by BCA kit and normalized. Then, 50 μL of protein solution was mixed with 50 μL of PBS solution (pH=7.4) and 50 μL of L-DOPA (1 mg / mL). After reacting at 37 °C for 30 min, the absorbance was measured at 475 nm. According to the formula “Tyrosinase activity (%) = A 测 / A 空 ×100%” to calculate the tyrosinase activity after the sample solution was applied.

[0065] 3. Experimental Results The results are as follows Figure 7 As shown, compared with the unmodified T1, the tyrosinase activity of B16F10 cells was significantly reduced after the amidated polypeptide T2 was treated with 37.5-75 μM, indicating that the amidated polypeptide T2 of the present invention can significantly inhibit the activity of tyrosinase and is suitable for preparing whitening products.

[0066] Example 6 Inhibitory activity of T1-T2 on melanin 1. Solution preparation Sample solution: Dissolve T1-T2 obtained in Example 1 in a solution containing 10% ( v / v ) fetal bovine serum and 1% ( v / v) penicillin-streptomycin dual antibody DMEM / F-12 culture medium to a final concentration of 37.5 and 75 μM, respectively, and then sterilized with a 0.22 μm syringe filter.

[0067] 2. Experimental Grouping and Treatment Sample measurement hole (A 测 ): Melanoma cells (B16F10) in logarithmic growth phase were collected and 2×10 5 The cells were inoculated at a density of 10 cells / well in a 6-well plate. After adaptive culture in a 37°C incubator for 24 h, the supernatant was discarded and washed twice with PBS solution (pH=7.4). 2 mL of sample solution was added. After culture in a 37°C incubator for 48 h, the supernatant was discarded and washed twice with PBS solution (pH=7.4) to remove non-adherent cells. The cells were then digested with trypsin, and the digestion products were collected and centrifuged at 1500 r / min for 10 min. 1 mL of 10% ( v / v ) DMSO-NaOH solution (1 mol / L) was added and the samples were extracted in a water bath at 80 °C for 1 h, and the absorbance at 450 nm was measured.

[0068] Blank well (A 空 ): Melanoma cells (B16F10) in logarithmic growth phase were collected and 2×10 5 The cells were seeded at a density of 100 cells / well in a 6-well plate and cultured in a 37°C incubator for 24 h. The supernatant was discarded and the cells were washed twice with PBS solution (pH = 7.4). 2 mL of 10% ( v / v ) fetal bovine serum and 1% ( v / v ) penicillin-streptomycin dual antibody DMEM / F-12 medium, cultured in a 37 ℃ incubator for 48 h, discarded the supernatant, washed twice with PBS solution (pH=7.4) to remove non-adherent cells, and then digested the cells with trypsin, collected the digestion products and centrifuged at 1500 r / min for 10 min, and added 1 mL of 10% ( v / v ) DMSO-NaOH solution (1 mol / L) was added and the samples were extracted in a water bath at 80 °C for 1 h, and the absorbance at 450 nm was measured.

[0069] According to the formula "Melanin content (%) = A 测 / A 空 ×100%” to calculate the melanin content after the sample solution was applied.

[0070] 3. Experimental Results The results are as follows Figure 8As shown, compared with the unmodified T1, the melanin content of B16F10 cells was significantly reduced after the amidated polypeptide T2 was treated with 37.5-75 μM, indicating that the amidated polypeptide T2 of the present invention can significantly inhibit the formation of melanin and is suitable for preparing whitening products.

[0071] In addition, combining the results of Example 5 and Example 6, it can be seen that the inhibitory effect of the amidated polypeptide T2 on tyrosinase activity is not dose-dependent, but the inhibitory effect on melanin content is dose-dependent, indicating that the inhibitory effect of the amidated polypeptide of the present invention on melanin content is not simply dependent on the inhibition of tyrosinase activity.

[0072] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An amidated polypeptide, characterized in that: The polypeptide is obtained by amidation modification of the C-terminus of the polypeptide; wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

2. Use of the amidated polypeptide according to claim 1 in the preparation of whitening products.

3. Use of the amidated polypeptide according to claim 1 in the preparation of antioxidant products.

4. Use of the amidated polypeptide according to claim 1 in the preparation of a tyrosinase inhibitor.

5. Use of the amidated polypeptide according to claim 1 in the preparation of a melanin inhibitor.

6. The biomaterial related to the amidated polypeptide according to claim 1, characterized in that: The related biological material is a nucleic acid molecule capable of expressing the amidated polypeptide, or a recombinant DNA, an expression cassette, a transposon, a vector or a host cell containing the nucleic acid molecule.

7. Use of the relevant biological material described in claim 6 in the preparation of whitening products.

8. Use of the relevant biological materials according to claim 6 in the preparation of antioxidant products.

9. Use of the relevant biological material according to claim 6 in the preparation of tyrosinase inhibitors.

10. Use of the relevant biological material according to claim 6 in the preparation of a melanin inhibitor.

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