An antifreeze peptide from the scales of Dentex tumifrons, and its screening method and application

Abstract

The present invention relates to the technical field of polypeptides, and particularly to an antifreeze peptide from the scales of Dentex tumifrons, a screening method therefor, and an application thereof. The antifreeze peptide from the scales of Dentex tumifrons has an amino acid sequence of: NAITDVVPAPK; the screening method is as follows: the peptide sequence obtained by mass spectrometry analysis of the crude extract of the antifreeze peptide from the scales of Dentex tumifrons; the optimal conformation peptide sequence obtained by screening the peptide sequence and the ice crystal prism plane through a molecular docking software; under three-dimensional analysis, the antifreeze peptide sequence obtained by screening from the optimal conformation peptide sequence with the number of hydrogen bonds formed as an index. The antifreeze peptide provided by the present invention fills the blank of the antifreeze peptide from the scales of Dentex tumifrons, and has high antifreeze activity, and can be used as an application in products such as antifreeze agents. The screening method of the present invention simplifies the cumbersome steps such as separation and purification, and the method is simple.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptides, and particularly to an antifreeze peptide from the scales of Dentex tumifrons, a screening method therefor, and an application thereof. Background Art

[0002] Cryopreservation is the most widely used storage method in the fields of food and biomedicine. However, foods will inevitably grow during cryopreservation and recrystallize during low-temperature transportation.

[0003] Antifreeze proteins are a class of proteins that can protect organisms from damage in a frozen or sub-frozen state. They can non-colligatively lower the freezing point of a solution, effectively inhibit the growth and recrystallization of ice crystals, and inhibit the low-temperature damage caused by freezing. Antifreeze peptides are the active domain parts of antifreeze proteins and have a unique cryoprotective ability for organisms similar to antifreeze proteins. Antifreeze peptides are a class of small-molecule proteins or protein hydrolysates. Since the content of natural-source antifreeze proteins is very low, specific polypeptide chain domain fragments can be obtained through biolytical technology, with low production costs and stable performance. Compared with the limited quantity and expensive price of antifreeze proteins, antifreeze peptides have a more promising application prospect.

[0004] Porgy is an important economic fish in China and is widely distributed in Chinese waters. During the production process of canned foods and fish products, a large amount of debris and useless scales will be generated. The scale scraps are usually treated as waste, which not only wastes resources but also pollutes the environment. Porgy scales are rich in collagen and can be used as a source for the production of active peptides. Developing and preparing antifreeze peptides from porgy scales not only makes full use of resources, improves the added value of fish processing, but also conforms to the concept of green, coordinated and sustainable development, and promotes the development of the fish processing industry. Compared with traditional mammalian-source collagen antifreeze peptides, fish-source collagen antifreeze peptides are safer. Currently, there has been no report on the process of preparing antifreeze peptides from the scales of Dentex tumifrons. Summary of the Invention

[0005] (I) Technical Problems to be Solved

[0006] In view of the above-mentioned disadvantages and deficiencies of the prior art, the present invention provides an antifreeze peptide from the scales of Dentex tumifrons, which has excellent antifreeze properties and is a new scale antifreeze peptide;

[0007] Correspondingly, the present invention also provides a screening method for an antifreeze peptide from the scales of Dentex tumifrons, which screens a highly active antifreeze peptide from the scales of Dentex tumifrons with a specific amino acid sequence through molecular docking with ice crystals, and the method is simple, efficient, convenient and low-cost.

[0008] Correspondingly, the present invention also provides an application of an antifreeze peptide from the scales of Dentex tumifrons as an antifreeze product.

[0009] (II) Technical Solutions

[0010] To achieve the above object, the main technical solutions adopted by the present invention include:

[0011] In the first aspect, the present invention provides an antifreeze peptide from the scales of Dentex tumifrons, and its amino acid sequence is: NAITDVVPAPK.

[0012] Optionally, the sites where the antifreeze peptide from the scales of Dentex tumifrons forms hydrogen bonds with ice crystals include THR4, ASP5, ALA9, and LYS11.

[0013] Optionally, it has an α-helix structure.

[0014] In the second aspect, the present invention also provides a screening method for the antifreeze peptide from the scales of Dentex tumifrons in any of the above solutions, which includes the following steps:

[0015] S1 The peptide sequence obtained by mass spectrometry analysis of the crude extract of the antifreeze peptide from the scales of Dentex tumifrons;

[0016] S2 The optimal conformation peptide sequence obtained by screening the peptide sequence and the ice crystal prism surface through a molecular docking software;

[0017] S3 Under three-dimensional analysis, the antifreeze peptide sequence obtained by screening from the optimal conformation peptide sequence with the number of hydrogen bonds formed as an index.

[0018] After obtaining the optimal conformation peptide sequence screened by the present invention, the antifreeze peptide sequence can be directly screened with the number of hydrogen bonds formed between the peptide sequence and the ice crystal as an index; the most important chemical bond for the interaction between the antifreeze peptide and the ice crystal is the hydrogen bond, and the peptide sequence with antifreeze activity can be directly screened through the number of hydrogen bonds. The present invention also uses the total docking energy between the peptide sequence and the ice crystal (referring to the interaction energy of the peptide docking with the ice crystal; under the premise of the same polypeptide sequence, the higher the absolute value of the docking energy, the more stable the system) to assist in the screening.

[0019] Among them, the molecular docking software can be the molecular docking software Hex8.0.0.

[0020] Among them, three-dimensional analysis uses three-dimensional analysis software, including PyMOL software.

[0021] Optionally, the screening method further includes the following steps: before step S2, the peptide sequence is a peptide sequence with a collagen source obtained by screening through a protein database. The present invention filters out the peptide sequences without a collagen source. Since the polypeptide from the scales of Dentex tumifrons in the present invention is a polypeptide with a collagen source, the influence of residues such as instruments, which are non-polypeptides from the scales of Dentex tumifrons, on the screening results can be filtered out. The protein database is preferably the Cryoprotect database.

[0022] Optionally, the screening method further includes the following steps: Before step S2, the peptide sequence is modeled by the PEP-FOLD 3.5 polypeptide structure prediction and modeling program in the RPBS structural biology database, with the number of simulation times selected as 100 and the model sorted according to sOPEP; the modeling result with the highest score is used as the final structure, and the peptide sequence with a secondary structure is screened out.

[0023] Optionally, the preparation of the crude antifreeze peptide extract includes the following steps: The fish scale slurry solution made from the fish scales of Pagellus acarne is adjusted to a weakly alkaline pH value, and trypsin is added for enzymatic hydrolysis at 36-38°C for 4-6 hours to obtain the degradation product.

[0024] Optionally, the preparation of the crude antifreeze peptide extract includes the following steps: The crude antifreeze peptide extract is obtained by ice affinity adsorption of the Pagellus acarne fish scale degradation product. The ice affinity adsorption method can improve the purity of the antifreeze peptide in the obtained crude antifreeze peptide extract.

[0025] Optionally, the screening method further includes the following steps: Before step S2, the peptide sequence is obtained by preliminary screening through an antifreeze polypeptide database. Among them, the antifreeze polypeptide database is preferably the Cryoprotect database, and the polypeptide sequences without cryoprotective activity are filtered out; although the screening accuracy rate of the Cryoprotect database is only 88.28%, the present invention can overcome the technical problem that the screening accuracy rate of the Cryoprotect database is only 88.28% through the combination of steps S2 and S3.

[0026] Optionally, the screening method further includes the following steps: Before step S2, the peptide sequence is screened through a polypeptide toxicity database to obtain a non-toxic peptide sequence with a hydrophilicity > 0. In order to simplify the screening of subsequent steps, the present invention can preferentially screen for hydrophilic and non-toxic sequences through the ToxinPred database. Since the antifreeze peptide functions in a solution system, the screening for hydrophilicity and non-toxicity can accurately remove the antifreeze active polypeptides that are difficult to dissolve or insoluble in water.

[0027] In a third aspect, the present invention also provides the application of the Pagellus acarne fish scale antifreeze peptide in any of the above solutions as an antifreeze product.

[0028] The present invention uses the methods of peptidomics and molecular docking to simplify the cumbersome steps such as separation and purification, saves the use of expensive instruments and consumables, and efficiently and conveniently screens out highly active antifreeze peptides with specific amino acid sequences from the Pagellus acarne fish scale hydrolysate. The controllable enzymatic hydrolysis preparation of antifreeze peptides from food-derived by-products reduces costs and is conducive to the large-scale production and application of antifreeze peptides, providing a theoretical support for their application in food, health products, and cryogenic tissue engineering.

[0029] (III) Beneficial effects

[0030] The beneficial effects of the present invention are as follows:

[0031] 1. The antifreeze peptide from the scales of Dentex tumifrons provided by the present invention fills the gap of the antifreeze peptide from the scales of Dentex tumifrons, and it has high antifreeze activity and can be used in products such as antifreeze agents.

[0032] 2. The screening method for a new antifreeze peptide provided by the present invention screens the optimal conformation through the molecular docking of ice crystals and polypeptides. On this basis, taking the number of hydrogen bonds formed between the peptide sequence and ice crystals as an index, the antifreeze peptide sequence can be screened, which simplifies the cumbersome steps such as separation and purification, and the method is simple.

[0033] The present invention further screens peptide sequences with no toxicity, hydrophilicity > 0, peptide sequences with cryoprotective activity, sequences with secondary or higher structures, and collagen-derived peptide sequences through the corresponding database; improving the screening efficiency and yield of high-activity peptide sequences. Description of the Drawings

[0034] Figure 1 It is the total ion current chromatogram obtained by mass spectrometry analysis in Example 3;

[0035] Figure 2 It is the secondary mass spectrum of the antifreeze peptide in Example 1;

[0036] Figure 3 It is the secondary structure model diagram of the antifreeze peptide in Example 1;

[0037] Figure 4 It is the schematic diagram of the molecular docking structure between the antifreeze peptide and ice crystals described in Example 3;

[0038] Figure 5 It is the result of the determination of the low-temperature protection activity of different samples on bacteria in Example 4;

[0039] Figure 6 It is the thermal hysteresis activity value determined for different samples in Example 4;

[0040] Figure 7 It is the ice crystal content generated by the determination of different samples in Example 4. Detailed Embodiments

[0041] In order to better explain the present invention for easy understanding, the present invention will be described in detail below through specific embodiments.

[0042] The antifreeze peptide from the scales of Dentex tumifrons proposed in the embodiment of the present invention is from the degradation product of the scales of Dentex tumifrons, and it is a new polypeptide with high antifreeze activity, which can be directly used as an additive for antifreeze agents and applied to various products. This antifreeze peptide can form hydrogen bonds at 4 sites with ice crystals, making its antifreeze activity extremely strong.

[0043] To better understand the above technical solutions, exemplary embodiments of the present invention will be described in more detail below. Although the exemplary embodiments of the present invention are shown below, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a clearer and more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0044] Example 1

[0045] The antifreeze peptide from the scales of Pagellus acarne provided in this example has the amino acid sequence: NAITDVVPAPK.

[0046] The sites where the antifreeze peptide from the scales of Pagellus acarne in this example forms hydrogen bonds with ice crystals include the amino acid sites of THR4, ASP5, ALA9, and LYS11, and hydrogen bonds can be formed simultaneously at the four sites.

[0047] The hydrophilicity of the antifreeze peptide from the scales of Pagellus acarne in this example is 0, and the molecular weight is 1123.62 Da; it has a secondary α-helix structure, and the total energy required for docking with ice crystals is -237.50 Kcal / moL.

[0048] The secondary mass spectrometry diagram of the antifreeze peptide from the scales of Pagellus acarne in this example is as Figure 2 shown.

[0049] The antifreeze peptide provided in this example can be synthesized by solid-phase synthesis method, or can be extracted from the degradation products of Pagellus acarne scales and purified by designing special purification steps according to special properties such as the molecular weight size of NAITDVVPAPK.

[0050] Example 2

[0051] The preparation method of the crude extract of the antifreeze peptide from the scales of Pagellus acarne provided in this example comprises the following steps:

[0052] S1 Obtaining the enzymatic hydrolysate

[0053] S11 Use a pulverizer to pulverize the scales of Pagellus acarne into a flocculent state. Weigh 15.7 g of the flocculent scales of Pagellus acarne, add 1 L of deionized water, adjust the pH to 8.00, add 0.9247 g of trypsin, place it in an enzymatic hydrolysis at 37 °C for 5.00 h, then place it in a boiling water bath and boil for 10 min, centrifuge at 10000 rpm for 10 min, and take the supernatant;

[0054] S12 Repeat step S11 twice, and mix the supernatants of the two times to obtain the enzymatic hydrolysate of the scales of Pagellus acarne;

[0055] The S2 parrotfish scale hydrolysate was placed in a container of an ice extraction device. Anhydrous ethanol driven by a low-temperature constant temperature bath circulated in the middle sandwich layer of the container, and the temperature was set at -6°C. After part of the enzyme hydrolysate froze, the temperature was adjusted to -4°C to enable the high-activity antifreeze components to adsorb and bind to the ice on the outer wall of the container. After continuous extraction for 5 h, the unfrozen solution was discarded, and the ice was slowly washed with an appropriate amount of deionized water around it. The water outlet switch was closed, and the refrigeration system was turned off. After the ice melted, the liquid was collected and freeze-dried to obtain the crude extract of antifreeze peptides.

[0056] Example 3

[0057] The screening method of the parrotfish scale antifreeze peptide provided in this example has the following steps:

[0058] S1 The amino acid sequences of the polypeptides contained in the crude extract of antifreeze peptides obtained in Example 2 were identified using a nano-liquid chromatography-quadrupole orbitrap mass spectrometer, and the total ion chromatogram of mass spectrometry analysis as shown in Figure 1 and the secondary mass spectrometry diagram of the antifreeze peptide sequence of Example 1 as shown in Figure 2 were respectively obtained;

[0059] S2 Screening of polypeptide sequences that are non-toxic and have a hydrophilicity > 0: The amino acid sequences of all the obtained polypeptides were input into the ToxinPred database, and the non-water-soluble and toxic polypeptide sequences were excluded;

[0060] S3 Screening of polypeptide sequences with cryoprotective activity: The obtained polypeptide sequences that are non-toxic and have a hydrophilicity > 0 were input into the Cryoprotect database, and the polypeptide sequences with cryoprotective activity were screened out;

[0061] S4 Screening of polypeptide sequences with a secondary structure or higher: The PEP-FOLD3.5 polypeptide structure prediction and modeling program in the RPBS structural biology database was used to model the screened peptide sequences with cryoprotective activity. The simulation times were selected as 100, and the model sorting was based on sOPEP. The modeling result with the highest score was used as the final structure, and the polypeptide sequences with a secondary structure were selected;

[0062] S5 Screening of collagen-derived polypeptide sequences: The polypeptide sequences with a secondary structure or higher were input into the Uniprot database for analysis of the polypeptide source, and the collagen-derived polypeptide sequences were screened out;

[0063] S6 Molecular docking of the ice crystal prism plane and the collagen-derived polypeptide sequence was performed using the molecular docking software Hex8.0.0 to obtain the polypeptide sequence with the optimal conformation and Etotal;

[0064] S7 Using the number of hydrogen bonds formed between the ice crystal prism plane and the polypeptide sequence as an index, the antifreeze peptide sequence was screened from the peptide sequences with the optimal conformation through the PyMOL software.

[0065] The screening method of this embodiment sequentially screens peptide sequences with non-toxicity, hydrophilicity > 0, cryoprotective activity, sequences with secondary or higher structures, and collagen-derived peptide sequences through corresponding databases, improving the screening efficiency and yield of highly active peptide sequences. Through this screening method, more than 10 antifreeze polypeptide sequences with a thermal hysteresis activity value of more than 0.2 are screened. Thus, the screening method of this embodiment has the advantages of high accuracy and high efficiency.

[0066] In this embodiment, the antifreeze peptide sequences obtained by screening are synthesized into corresponding polypeptides; by measuring their thermal hysteresis activities, antifreeze peptide sequences with high thermal hysteresis activities, low ice crystal formation amounts, high bacterial cell protection survival rates, and the highest total energy of docking with ice crystals among peptide sequences of the same length are comprehensively screened to obtain more antifreeze peptide sequences.

[0067] The hydrophilicity of the antifreeze peptide from the scales of Pagellus acarne in Example 1 is 0, and its molecular weight is 1123.62 Da; it has a secondary α-helix structure, and the total energy required for docking with ice crystals is -237.50 Kcal / moL. It is formed by 11 amino acid residues and can form four hydrogen bonds. Its thermal hysteresis activity is between 0.2 and 1.3, and its low-temperature protection activity for bacterial cells is 86.17%. It can be seen that it has strong antifreeze activity and strong stability.

[0068] Figure 1 The total ion current chromatogram of mass spectrometry analysis is: the ion current signal generated by all ions after the separated components are ionized by the ion source, and the chromatogram obtained by amplifying and plotting against the elution time of the components.

[0069] Figure 2 The secondary mass spectrometry diagram of mass spectrometry analysis: Select the parent ion peptide segment, further dissociate it, and analyze the mass-to-charge ratio and intensity of the formed daughter ions, which is a proof that this peptide is separated in this mass spectrometry experiment.

[0070] Figure 3 It is the visualized secondary structure diagram of the model of the antifreeze peptide from the scales of Pagellus acarne in Example 1.

[0071] Figure 4 It is the schematic diagram of the molecular docking of the antifreeze peptide from the scales of Pagellus acarne in Example 1 with ice crystals in step S7. The polypeptide in Example 1 has a secondary structure of α-helix (red), and NK11 forms 4 hydrogen bonds with ice crystals (yellow). The corresponding docking site amino acids are THR4, ASP5, ALA9, and LYS11.

[0072] Example 4

[0073] This example provides a method for measuring the cryoprotective activity of the antifreeze peptides from the scales of *Diplodus argentatus* obtained in Example 1. Using the enzymatic hydrolysate of Example 2 (denoted as: EjAFPs) and the crude extract of antifreeze peptides as controls (denoted as: I-EjAFPs), during the experiment, solutions with the same protein concentration were prepared as samples respectively.

[0074] The specific method is as follows:

[0075] Take 50 μL of *Streptococcus thermophilus* after secondary activation and inoculate it into 4 mL of M17 liquid medium. Incubate for 4 h (37 °C, 180 rpm), centrifuge at 5000 rpm for 10 min, collect the bacterial pellet, wash it twice with physiological saline and then resuspend it in an equal volume of physiological saline to obtain the *Streptococcus thermophilus* bacterial solution.

[0076] Mix 540 μL of the sample with 60 μL of the bacterial solution, and use physiological saline to replace the sample as the blank group. Pipette 50 μL of the mixture into 4 mL of M17 culture medium, incubate for 7 h, and measure the absorbance value A1 at 600 nm. The remaining bacterial solution is frozen at -20 °C for 24 h and undergoes two freeze-thaw cycles at intervals of 2 h within the starting time. Then, thaw the bacterial solution in a water bath at 37 °C for 10 min, and again take 50 μL to inoculate and incubate for 7 h, and then measure the absorbance value A2. Calculate the survival rate according to formula (1).

[0077]

[0078] In the formula: A1 represents the OD of the bacterial solution before freezing 600 ; A2 represents the OD of the bacterial solution after freezing 600 .

[0079] This example obtained the data as Figure 5 shown below:

[0080] The figure shows the cryoprotective ability of different antifreeze agents against *Streptococcus thermophilus*. The blank group is the physiological saline group. It can be seen from the figure that compared with the blank group, the freeze protection rate of the enzymatic hydrolysate EjAFPs for *Streptococcus thermophilus* has been significantly improved. The freeze protection rates of the crude extract of antifreeze peptides I-EjAFPs and the purified peptides after screening have increased compared with the previous step. This shows that the antifreeze peptides prepared in Example 1 have significantly stronger antifreeze activity.

[0081] Through step-by-step screening, a pure peptide sequence with high antifreeze activity was obtained, which can be used for future cryoprotection of bacteria.

[0082] Example 5

[0083] This example provides the inhibitory effect of the antifreeze peptide from the scales of Pagellus acarne obtained in Example 1 (denoted as NK11), and uses the enzymatic hydrolysate of Example 2 (denoted as: EjAFPs) and the crude extract of antifreeze peptide as controls (denoted as: I-EjAFPs). During the experiment, solutions with the same protein concentration were respectively prepared as samples.

[0084] The specific methods for measuring the inhibitory effect and thermal hysteresis activity are as follows:

[0085] Take 5 μL of a 15 mg / mL sample into an aluminum sample pan. The reference group is a blank aluminum sample pan. Both are simultaneously placed in a differential scanning calorimeter and cooled to -30 °C at a rate of -2 °C / min, then equilibrated for 5 min, and then heated to the retention temperature (-1.0 °C, -0.5 °C, -0.3 °C, 0 °C) at a rate of 2 °C / min to keep the sample in a partially molten state and equilibrated for 5 min. Analyze the antifreeze activity of the sample according to the DSC heat flow curve. The ice crystal content (Φ) and THA are calculated according to formulas (2) and (3) respectively.

[0086]

[0087] THA = T h -T0 (3)

[0088] In the formula: Φ is the ice crystal content in the sample, △H f is the heat release enthalpy of the system during the continuous cooling process after staying at the retention temperature, △H m is the total heat release enthalpy of sample crystallization. T h is the retention temperature, that is, a certain temperature within the temperature range covered by the sample melting peak, and the sample is in a partially molten state; T0 is the starting temperature when the melted part of the system freezes again, which is determined by the intersection point of the tangents of the maximum slope and the minimum slope at both ends of the inflection point of the heat flow curve.

[0089] The results of measuring the thermal hysteresis activity in this example are as Figure 6 shown; when the antifreeze polypeptide adsorbs on the ice-water interface, it causes a change in the ice crystal growth trajectory, resulting in an increase in the vapor pressure at the ice-water interface, making a difference between the freezing point and the melting point of the solution, and the level of thermal hysteresis activity can be reflected by this temperature difference.

[0090] Figure 6 Among them, the blank group is the bovine serum albumin group (a protein without antifreeze activity). It can be seen from the figure that the thermal hysteresis activity of the pure peptide NK11 in Example 1 has been greatly improved compared with the control group.

[0091] The ice crystal content corresponding to different samples measured in this example is as Figure 7 shown:

[0092] The blank group is the bovine serum albumin group (protein without antifreeze activity). As can be seen from the figure, the control of the ice crystal content in the system for screening and purifying peptide NK11 has been greatly improved compared with the control group, and the ice crystal content in the system can be significantly reduced, thus exerting an antifreeze effect.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antifreeze peptide from the scales of Dentex tumifrons, characterized in that, Its amino acid sequence is: NAITDVVPAPK.

2. Use of the antifreeze peptide from the scales of Dentex tumifrons as claimed in claim 1 in an antifreeze product.

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