Elastase inhibitory peptide and preparation method thereof

Abstract

The invention belongs to the field of functional polypeptides, and discloses an elastinase inhibitory peptide and a preparation method thereof. The elastase inhibitory peptide comprises nine types of oligopeptides from crayfish, and the amino acid sequences of the oligopeptides are respectively GASGPAGPRGPVGVA, IDGF, GAAGPAGPRGPAGPA, SGPAGPA, GPAGPRGPVGVA, SGPAGPRGPVGVA, VGPQGASGPLGPA and IGPA. The invention further discloses a preparation method of the elastase inhibitory peptide. The elastinase inhibitory peptide disclosed by the invention can be chemically synthesized and can also be directionally prepared from fish gelatin enzymolysis products. The elastase inhibitory peptide disclosed by the invention has good binding force with elastase and has remarkable elastase inhibitory activity.

Description

Technical Field

[0001] The present invention belongs to the field of functional polypeptides, and particularly relates to elastase inhibitory peptides and a preparation method thereof. Background Art

[0002] Matrix Metalloproteinases (MMPs) play a crucial role in skin photoaging. Ultraviolet (UV) irradiation is the most important exogenous factor leading to skin photoaging, and MMPs play a key role in this process. UV irradiation induces the overexpression of MMPs in dermal fibroblasts, and these enzymes are mainly responsible for degrading extracellular matrix components such as collagen, elastin, and fibronectin. Specifically, the overproduction of MMP-1, MMP-3, and MMP-9 leads to the excessive degradation of these proteins, thus accelerating skin photoaging. It is worth noting that in addition to directly degrading collagen and elastin, MMPs are also involved in skin photoaging through other mechanisms. MMPs can inhibit the synthesis of collagen by affecting the binding of transforming growth factor-β (TGF-β) to its receptor, further exacerbating the formation of skin laxity and wrinkles. Therefore, the role of matrix metal proteinases in skin photoaging is not limited to directly degrading collagen and elastin, but also includes regulating the expression of related genes through multiple signaling pathways, thus comprehensively affecting the structure and function of the skin. The research on MMPs provides potential targets for the development of anti-photoaging drugs. By inhibiting the activity of MMPs or their upstream regulatory factors, it may effectively slow down the process of skin photoaging.

[0003] At present, matrix metalloproteinase inhibitors (MMPIs) mainly include: (1) Peptidomimetic compounds: These inhibitors are designed based on the structure of peptides and inhibit MMPs by mimicking the interaction between peptides and related enzymes. For example, batimastat (BB-94) is a typical peptidomimetic MMP inhibitor. Its structure mimics collagenase and can chelate the zinc ion of MMP to inhibit its activity. (2) Non-peptidic MMPIs: Although they do not have the relevant characteristics of peptides, their structures are similar to peptides and can mimic the interaction between peptides and related enzymes. For example, Prinomastat and Rebimastat are both non-peptidic MMPIs, and they showed inhibitory effects on various MMPs in preclinical studies. (3) Tetracycline derivatives: These compounds are obtained through chemical modification and can not only inhibit MMP activity but also inhibit the production of MMPs. For example, doxycycline hyclate and COL-3 and other tetracycline derivatives have been proven to effectively inhibit the activity of MMPs. (4) α2-macroglobulin: This is a naturally occurring protein that can inhibit the activity of MMPs. (5) Tissue inhibitors of metalloproteinases (TIMPs): This is a group of low-molecular-weight glycoproteins that can specifically inhibit different types of MMPs. TIMPs inhibit their activity by binding to MMPs to form complexes. (6) Proteolytic products. For example, Chinese Patent CN112500452A, "A Bioactive Polypeptide IQIVQNEGR and Its Preparation Method and Application", discloses a bioactive polypeptide IQIVQNEGR with antioxidant and anti-aging effects, which can resist and block the damage caused by oxygen free radicals to the body, delay the aging of the body, and reduce oxidative stress damage. Chinese Patent CN109293737A, "A Tetrapeptide for Anti-Skin Aging and Its Use", discloses a tetrapeptide GLPY for anti-skin aging, which can reduce the apoptosis of fibroblasts caused by photo-damage by inhibiting the activity of elastase. Chinese Patent CN101575362A, "Cyclic Pentapeptide Human Elastase Inhibitor and Its Preparation Method", discloses a class of cyclic pentapeptide human elastase inhibitors, which are composed of five amino acids selected from aryl amino acids, aliphatic amino acids, and cyclic amino acids, and show good activity against human elastase. Patent CN115651079A, "A Class of Elastase-Inhibiting Peptides and Their Preparation Methods and Applications", discloses three functional peptides with elastase inhibitory activity, and their amino acid sequences are Phe-Phe-Pro-Gly-Ala-Gly (FFPGAG), Phe-Pro-Gly-Ile-Gly (FPGIG), and Gly-Ala-Gly-Gln-Pro-Phe-Pro-Ile (GAGQPFPI).Protein hydrolysates have the advantages of low cost, safety, non-toxicity, simple production process, environmental friendliness, etc., and have been widely used in the food industry. Therefore, exploring elastase inhibitors from food proteins is one of the current research hotspots in the industry.

[0004] Fish maw is a dried product of fish swim bladder. Fresh fish swim bladder is processed through a series of processes (such as removing fat, blood vessels, etc.), and then dried or baked to make fish maw. Due to its high protein (protein content up to 84.2%) and low fat characteristics, as well as rich nutritional components, fish maw is considered an ideal tonic. Fish maw is considered to have various effects such as tonifying the kidney and replenishing essence, nourishing tendons and vessels, stopping bleeding and dispersing stasis, and is often used to treat symptoms such as spermatorrhea due to kidney deficiency and postpartum wind spasm. Modern research also shows that fish maw has various biological activities such as anti-fatigue, scavenging free radicals, and antioxidant. In addition, fish maw is also considered to be able to improve the digestive and absorption function of the gastrointestinal tract and may be helpful in treating diseases such as peptic ulcer. However, so far, there has been no report on elastase inhibitors derived from fish maw. Summary of the Invention

[0005] The primary object of the present invention is to provide a class of protein peptides derived from fish maw with elastase inhibitory activity.

[0006] Another object of the present invention is to provide a preparation method of a class of protein peptides derived from fish maw with elastase inhibitory activity.

[0007] The object of the present invention is achieved by one of the following technical solutions:

[0008] The present invention provides a class of elastase inhibitory peptides, which include 9 oligopeptides, and their amino acid sequences are GASGPAGPRGPVGVA, IDGF, GAAGPAGPRGPAGPA, SGPAGPA, GPAGPRGPVGVA, SGPAGPRGPVGVA, VGPQGASGPLGPA, and IGPA respectively.

[0009] The present invention also provides a preparation method of the above elastase inhibitory peptides, which is obtained by separating and purifying from fish maw hydrolysate, and includes the following steps:

[0010] After crushing the fish maw, it is mixed with water according to the mass ratio, the initial pH is adjusted to 8.0 - 11.0, alkaline protease is added, after the enzymatic hydrolysis reaction, the temperature is raised for enzyme inactivation treatment, and the supernatant is taken by centrifugation to obtain fish maw hydrolysate.

[0011] Furthermore, the source of the fish maw is the swim bladder of Pangasianodon hypophthalmus.

[0012] The mass ratio of the fish maw to water is 1:9 - 1:15.

[0013] The addition amount of the alkaline protease is 1.0% - 2.5% of the mass of elastin.

[0014] The temperature of the enzymatic hydrolysis reaction is 45 - 55 °C, and the time of enzymatic hydrolysis is 12 - 18 h.

[0015] The temperature of the enzyme inactivation treatment is 80 - 95 °C, and the time is 15 - 30 min.

[0016] The present invention has the following advantages and beneficial effects compared with the prior art:

[0017] The present invention provides 9 new elastase inhibitory peptides, which have good binding ability with elastase, and the binding energy is lower than -6.0; and the inhibition rate of elastase reaches more than 60% at a concentration of 20 mM, showing significant elastase inhibitory activity. Brief Description of the Drawings

[0018] Figure 1 It is the secondary mass spectrum of GASGPAGPRGPVGVA in Example 1.

[0019] Figure 2 It is the secondary mass spectrum of IDGF in Example 1.

[0020] Figure 3 It is the secondary mass spectrum of GAAGPAGPRGPAGPA in Example 1.

[0021] Figure 4 It is the secondary mass spectrum of SGPAGPA in Example 1.

[0022] Figure 5 It is the secondary mass spectrum of GPAGPRGPVGVA in Example 1.

[0023] Figure 6 It is the secondary mass spectrum of SGPAGPRGPVGVA in Example 1.

[0024] Figure 7 It is the secondary mass spectrum of VGPQ in Example 1.

[0025] Figure 8 It is the secondary mass spectrum of GASGPLGPA in Example 1.

[0026] Figure 9 It is the secondary mass spectrum of IGPA in Example 1. Detailed Description of the Invention

[0027] The following further illustrates the specific implementation of the present invention in conjunction with the accompanying drawings and embodiments. However, the implementation and protection of the present invention are not limited thereto. It should be particularly noted that, unless otherwise specified, the following are carried out according to conventional conditions or the conditions recommended by the manufacturer. Raw materials, reagents, etc. without indicating the manufacturer can be obtained as conventional products through commercial purchase.

[0028] The experimental methods for the test indexes involved in the embodiments of the present invention are as follows:

[0029] Determination of in vitro elastase inhibitory activity:

[0030] Slightly modified according to the method of (Liu Yang. Research on the anti-skin photoaging effect and mechanism of elastin peptides [D]. South China University of Technology, 2019.), 200 μL of sample solution, 50 μL of elastase (60 mU / mL), and 50 μL of L-N-succinyl-Ala-Ala-Ala-pNA (1 mmol / L) were added to the wells of a 96-well plate in sequence and mixed well. All substances were dissolved in 50 mM Tris-HCl buffer with a pH of 8.0. Each sample was analyzed in triplicate. The plate was incubated at 25 °C for 10 min in a full-wavelength scanner. The absorbance of the reaction product (p-nitroaniline) was recorded at 410 nm. Tris-HCl buffer was used as a blank control. The formula for calculating the elastase inhibitory activity is as follows:

[0031]

[0032] Where As0 is the absorbance of the sample group before the reaction, As is the absorbance of the sample group after the reaction; Ac0 is the absorbance of the blank control group before the reaction, and Ac is the absorbance of the blank control group after the reaction.

[0033] The following further elaborates the present invention in conjunction with embodiments.

[0034] Example 1

[0035] After the fish glue was crushed to 60 meshes, it was mixed with water at a mass ratio of 1:9, the initial pH was adjusted to 11.0, 1.0% of alkaline protease based on the mass of the fish glue was added, the temperature was raised to 45 °C, after enzymatic hydrolysis for 12 h, the temperature was raised to 80 °C to inactivate the enzyme for 30 min, and the supernatant was obtained by centrifugation, which was the fish glue hydrolysate. The fish glue hydrolysate was passed through an ultrafiltration membrane with a cut-off molecular weight of 3000 Da, and the permeate was taken for LC-MS / MS analysis.

[0036] The permeate was desalted through a C18 desalting column and analyzed by LC-MS / MS equipped with an online nano-ESI ion source. The whole system was an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific, MA, USA) in tandem with an EASY-nanoLC 1200. A total of 2 μL of the sample was loaded (C18 column: 20 cm × 75 μm i.d., 1.9 μm particle size), and the sample was separated with a 60-min gradient. The column flow rate was controlled at 350 nL / min, the column temperature was 40 °C, the electrospray voltage was 2 kV, and the gradient started from 4% of phase B, increased to 90% in a non-linear gradient at 54.5 min, increased to 99% within 0.5 min, and was maintained for 5 min.

[0037] The mass spectrometer was operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisitions. The mass spectrometry parameters were set as follows: (1) MS: scan range (m / z): 200 - 1500; resolution: 60,000; Normalized AGC target: 300%; maximum injection time: 25 ms; (2) HCD-MS / MS: resolution: 15,000; Normalized AGC target: 50%; maximum injection time: 22 ms; collision energy 30%; dynamic exclusion time: 30 s.

[0038] The primary structures of the main oligopeptides in the enzymatic hydrolysate permeate are shown in Table 1 and Figures 1 to 9 .

[0039] Table 1 Information on oligopeptides derived from Chayu obtained in Example 1

[0040]

[0041]

[0042] Among them, GASGPAGPRGPVGVA is derived from the Alpha-1 type I collagen of Chazhu fish (protein ID: A0A5N5MFS7); IDGF is derived from the glycerophosphocholine phosphodiesterase of Chazhu fish (protein ID: A0A5N5NV04); GAAGPAGPRGPAGPA is derived from the Alpha-1 type I collagen of Chazhu fish (protein ID: A0A5N5MFS7); SGPAGPA is derived from the Fibrillar collagen NC1 domain-containing protein of Chazhu fish (protein ID: A0A5N5KKB7); GPAGPRGPVGVA is derived from the Alpha-1 type I collagen of Chazhu fish (protein ID: A0A5N5MFS7); VGPQ is derived from the Fibrillar collagen NC1 domain-containing protein of Chazhu fish (protein ID: A0A5N5KZP1); GASGPLGPA is derived from the Fibrillar collagen NC1 domain-containing protein of Chazhu fish (protein ID: A0A5N5KZP1); IGPA is derived from the Alpha-1 type II collagen of Chazhu fish (protein ID: A0A5N5NM88).

[0043] Example 2

[0044] The nine kinds of oligopeptides (GASGPAGPRGPVGVA, IDGF, GAAGPAGPRGPAGPA, SGPAGPA, GPAGPRGPVGVA, SGPAGPRGPVGVA, VGPQGASGPLGPA, and IGPA) derived from Chazhu fish identified in Example 1 were chemically synthesized to obtain oligopeptides with a purity of 99.0%. The in vitro elastase inhibitory activities of the above nine kinds of oligopeptides were measured at concentrations of 10 mM and 20 mM respectively, and the results are shown in Table 2.

[0045] Table 2

[0046]

[0047]

[0048] As can be seen from Table 2, the above nine kinds of oligopeptides all have certain inhibitory activities against metalloelastase. Among them, IDGF, SGPAGPA, and VGPQ have the strongest inhibitory activities, with inhibition rates of 49.65%, 42.55%, and 40.26% at a concentration of 10 mM; and inhibition rates of 66.47%, 57.19%, and 50.83% at a concentration of 20 mM.

[0049] All of the above 9 kinds of oligopeptides were subjected to molecular docking with elastase, and the binding energies of the 9 kinds of oligopeptides with metalloelastase were evaluated, as shown in Table 3.

[0050] Table 3

[0051]

[0052] As can be seen from Table 3, the binding energies of the 9 kinds of oligopeptides with metalloelastase are all lower than -6.0, indicating that the 9 kinds of oligopeptides can bind tightly to metalloelastase.

[0053] Example 3

[0054] After the fish glue was crushed, it was mixed with water at a mass ratio of 1:9, the initial pH was adjusted to 8.0, 1.0%% alkaline protease was added, and after enzymatic hydrolysis at 45°C for 18 h, the temperature was raised to 95°C for 15 min to inactivate the enzyme, and the supernatant was taken by centrifugation, which was the elastase inhibitory peptide.

[0055] Example 4

[0056] After the fish glue was crushed, it was mixed with water at a mass ratio of 1:15, the initial pH was adjusted to 11.0, 2.5% alkaline protease was added, and after enzymatic hydrolysis at 55°C for 12 h, the temperature was raised to 80°C for 30 min to inactivate the enzyme, and the supernatant was taken by centrifugation, which was the elastase inhibitory peptide.

[0057] The above embodiments are preferred embodiments of the present invention and are only used to explain the present invention. However, the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A class of elastase inhibitory peptides, characterized in that, The elastase inhibitory peptides include 9 peptides with amino acid sequences of GASGPAGPRGPVGVA, IDGF, GAAGPAGPRGPAGPA, SGPAGPA, GPAGPRGPVGVA, SGPAGPRGPVGVA, VGPQ, GASGPLGPA, and IGPA, respectively.

2. The preparation method of the elastase inhibitory peptide according to claim 1, characterized in that, It is obtained by separation and purification from fish swim bladder hydrolysate, including the following steps: crushing the fish swim bladder and mixing it with water, adjusting the initial pH to 8.0 - 11.0, adding alkaline protease, performing an enzymatic hydrolysis reaction, then heating up for enzyme inactivation treatment, centrifuging to obtain the supernatant, which is the elastase inhibitory peptide of fish swim bladder hydrolysate.

3. The preparation method of the elastase inhibitory peptide according to claim 2, wherein The source of the fish swim bladder is the swim bladder of Pseudosciaena crocea.

4. The preparation method of the elastase inhibitory peptide according to claim 2, characterized in that, The mass ratio of the fish swim bladder to water is 1:9 - 1:

15.

5. The preparation method of the elastase inhibitory peptide according to claim 2, wherein, The addition amount of the alkaline protease is 1.0% - 2.5% of the mass of the elastase inhibitory peptide.

6. The preparation method of the elastase inhibitory peptide according to claim 2, wherein, The temperature of the enzymatic hydrolysis reaction is 45 - 55 °C, and the hydrolysis time is 12 - 18 h.

7. The preparation method of the elastase inhibitory peptide according to claim 2, wherein The temperature of the enzyme inactivation treatment is 80 - 95 °C, and the time is 15 - 30 min.

8. The preparation method of the elastase inhibitory peptide according to claim 2, characterized in that, The initial pH is adjusted to 11.

9. The preparation method of the elastase inhibitory peptide according to claim 2, characterized in that, The mass ratio of the fish swim bladder to water is 1:

9.

10. The preparation method of the elastase inhibitory peptide according to claim 2, characterized in that, The temperature of the enzymatic hydrolysis reaction is 45 °C, and the hydrolysis time is 12 h.

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