An antifreeze peptide from fish scales and its method and application based on bioinformatics screening

Abstract

The present invention relates to the technical field of polypeptides, and particularly relates to an antifreeze peptide from fish scales, a method for screening the same based on bioinformatics, and an application thereof. The amino acid sequence of the antifreeze peptide from fish scales is: IAQPQEKAPDPY; the sites where it forms hydrogen bonds with ice crystals include GLU6, ASP10, and TVR12. 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 Pagellus acarne; the optimal conformation peptide sequence obtained by screening the peptide sequence and the ice crystal prism plane through molecular docking software; under three-dimensional analysis, the antifreeze peptide sequence obtained by screening from the optimal conformation peptide sequence with the number of formed hydrogen bonds as an index. The antifreeze peptide provided by the present invention 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 a scale antifreeze peptide and a method and application for screening the same based on bioinformatics. 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 the frozen or sub-freezing state. They can non-colligatively lower the freezing point of solutions, 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 are obtained through bioenzymolysis technology, with low production costs and stable performance. Compared with the limited number and expensive price of antifreeze proteins, antifreeze peptides have more application prospects.

[0004] Porgy is an important economic fish in China and is widely distributed in Chinese waters. During the production of canned foods and fish products, a large amount of debris and useless scales will be generated. Scale waste is 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 shortcomings and deficiencies of the prior art, the present invention provides a scale antifreeze peptide with excellent antifreeze properties, which is a new scale antifreeze peptide;

[0007] Correspondingly, the present invention also provides a method for screening scale antifreeze peptides based on bioinformatics, which screens out highly active antifreeze peptides from Dentex tumifrons scales 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 a scale antifreeze peptide 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 a first aspect, the present invention provides a squamation antifreeze peptide, and its amino acid sequence is: IAQPQEKAPDPY.

[0012] Optionally, the sites where the squamation antifreeze peptide of Dentex tumifrons forms hydrogen bonds with ice crystals include GLU6, ASP10, and TVR12.

[0013] TVR12 forms 4 hydrogen bonds with ice crystals.

[0014] Optionally, it has an α-helix structure.

[0015] In a second aspect, the present invention also provides a screening method for the squamation antifreeze peptide in any of the above solutions, which includes the following steps:

[0016] S1 The peptide sequence obtained by mass spectrometry analysis of the crude extract of the squamation antifreeze peptide of Dentex tumifrons;

[0017] S2 The optimal conformation peptide sequence obtained by screening the peptide sequence and the ice crystal facet through a molecular docking software;

[0018] S3 Under three-dimensional analysis, using the number of formed hydrogen bonds as an index, the antifreeze peptide sequence screened from the optimal conformation peptide sequence.

[0019] After obtaining the optimal conformation peptide sequence screened by the present invention, directly using the number of hydrogen bonds formed by the peptide sequence and ice crystals as an index, the antifreeze peptide sequence can be screened; the most important chemical bond for the interaction between the antifreeze peptide and ice crystals 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 of the peptide sequence and ice crystals (referring to the interaction energy of the peptide docking with ice crystals; 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 screening.

[0020] Among them, the molecular docking software can be the molecular docking software Hex8.0.0.

[0021] Among them, three-dimensional analysis uses three-dimensional analysis software, including PyMOL, Discovery studio, and VMD.

[0022] Optionally, the screening method further includes the following steps: before step S2, the peptide sequence is a peptide sequence with collagen source obtained by screening through a protein database. The present invention filters out the peptide sequences without collagen source. Since the Dentex tumifrons squamation polypeptide of the present invention is a polypeptide with collagen source, it can filter out the influence of residues such as instruments as non-Dentex tumifrons squamation polypeptides on the screening results. The protein database is preferably the Cryoprotect database.

[0023] 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 100 simulation times selected 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.

[0024] Optionally, the preparation of the crude antifreeze peptide extract includes the following steps: The fish scale slurry solution made from the scales of Pagellus acarne is adjusted to a weak alkaline pH value, and trypsin is added for enzymatic hydrolysis at 36-38 °C for 4-6 h to obtain a degradation product.

[0025] The concentration of the mixture of Pagellus acarne scales and ultrapure water is 1.57% (w / v).

[0026] Adjust the pH to 8.0.

[0027] The enzyme-substrate ratio of trypsin is 5.89% (w / w).

[0028] 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 scale degradation product. The ice affinity adsorption method can improve the purity of the antifreeze peptide in the obtained crude antifreeze peptide extract.

[0029] 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 of the Cryoprotect database is only 88.28%, the present invention can overcome the technical problem that the screening accuracy of the Cryoprotect database is only 88.28% through the combination of steps S2 and S3.

[0030] 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. To simplify the screening of subsequent steps, the present invention can preferentially screen sequences with hydrophilicity and non-toxicity through the ToxinPred database. Since the antifreeze peptide acts in a solution system, the screening of hydrophilicity and non-toxicity can accurately remove the antifreeze active polypeptides that are difficult to dissolve or insoluble in water.

[0031] In a third aspect, the present invention also provides the application of the Pagellus acarne scale antifreeze peptide in any of the above solutions as an antifreeze product.

[0032] The present invention simplifies the cumbersome steps such as separation and purification by using the methods of peptidomics and molecular docking, 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 enzymatic hydrolysate of the scales of Dentex tumifrons. The controllable enzymatic preparation of antifreeze peptides from food-derived waste materials reduces costs and is conducive to the large-scale production and application of antifreeze peptides, providing theoretical support for their application in food, health products, and cryogenic tissue engineering.

[0033] (III) Beneficial effects

[0034] The beneficial effects of the present invention are as follows:

[0035] 1. The antifreeze peptide from the scales of Dentex tumifrons provided by the present invention fills the blank of antifreeze peptides from the scales of Dentex tumifrons, and it has high antifreeze activity and can be used in products such as antifreeze agents.

[0036] 2. The new method for screening antifreeze peptides provided by the present invention combines biological information, screens the optimal conformation through the molecular docking of ice crystals and polypeptides, and on this basis, using the number of hydrogen bonds formed between the peptide sequence and ice crystals as an index, the antifreeze peptide sequence can be screened. It has the advantages of simplifying the cumbersome steps such as separation and purification, being simple in method, low in production cost, fast in screening speed, and high in accuracy.

[0037] The present invention further screens peptide sequences with no toxicity, hydrophilicity > 0, cryoprotective activity, sequences with secondary or higher structures, and collagen-derived peptide sequences through the corresponding database; improving the screening efficiency and yield of highly active peptide sequences. Description of the drawings

[0038] Figure 1 Total ion chromatogram obtained from mass spectrometry analysis in Example 3;

[0039] Figure 2 Secondary mass spectrum of the antifreeze peptide in Example 1;

[0040] Figure 3 Secondary structure model diagram of the antifreeze peptide in Example 1;

[0041] Figure 4 Schematic diagram of the molecular docking structure between the antifreeze peptide and ice crystals described in Example 3;

[0042] Figure 5 Results of the determination of the cryoprotective activity of different samples on bacteria at low temperature in Example 4;

[0043] Figure 6 Thermal hysteresis activity values measured for different samples in Example 4;

[0044] Figure 7 Content of ice crystals generated from the determination of different samples in Example 4. Detailed implementation mode

[0045] For better explaining the present invention and facilitating understanding, the present invention will be described in detail below through specific implementation modes.

[0046] The antifreeze peptide from the scales of Dentex tumifrons provided by the embodiment of the present invention is derived from the degradation product of the scales of Dentex tumifrons, and 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 four sites with ice crystals, making its antifreeze activity extremely strong, and the thermal hysteresis activity is between 0.2 and 0.4 °C.

[0047] To better understand the above technical solutions, the 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 described herein. On the contrary, these embodiments are provided to enable a clearer and more thorough understanding of the present invention and to be able to convey the scope of the present invention completely to those skilled in the art.

[0048] Example 1

[0049] The amino acid sequence of the antifreeze peptide from the scales of Dentex tumifrons provided in this example is: IAQPQEKAPDPY.

[0050] The more hydrogen bonds are bound, the stronger the antifreeze activity. The antifreeze peptide from the scales of Dentex tumifrons in this example forms six hydrogen bonds with ice crystals, and the corresponding sites include the GLU6, ASP10 and TVR12 amino acid sites;

[0051] TVR12 in the antifreeze peptide from the scales of Dentex tumifrons in this example can bind to ice crystals to form four hydrogen bonds. Based on its special amino acid composition and the secondary structure as Figure 3 shown, the antifreeze peptide in this example is given strong antifreeze activity, and its thermal hysteresis activity is between 0.2 and 0.4. Its low-temperature protection activity against bacteria is as high as 89.97%.

[0052] The hydrophilicity of the antifreeze peptide from the scales of Dentex tumifrons in this example is 0.36, and the molecular weight is 1355.672 Da; it has a secondary α-helix structure, and the total energy required for docking with ice crystals is -252.2 Kcal / moL.

[0053] The secondary mass spectrometry diagram of the antifreeze peptide from the scales of Dentex tumifrons in this example is as Figure 2 shown.

[0054] The antifreeze peptide provided in this example can be synthesized by solid-phase synthesis method, or can be extracted from the degradation product of the scales of Dentex tumifrons and purified by designing special purification steps according to special properties such as the molecular weight size of IAQPQEKAPDPY.

[0055] Example 2

[0056] The preparation method of the crude antifreeze peptide from the scales of Dentex tumifrons provided in this example comprises the following steps:

[0057] S1 Obtaining the enzymatic hydrolysate

[0058] S11 Use a pulverizer to pulverize the scales of Dentex tumifrons into a flocculent shape. Weigh 15.7 g of the flocculent scales of Dentex tumifrons, add 1 L of deionized water, adjust the pH to 8.00, add 0.9247 g (the enzyme-substrate ratio is 5.89%) 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;

[0059] S12 Repeat step S11 twice, and mix the supernatants of the two times to obtain the enzymatic hydrolysate of the scales of Dentex tumifrons;

[0060] S2 Place the enzymatic hydrolysate of the scales of Dentex tumifrons in the container of the ice extraction device. The middle interlayer of the container circulates anhydrous ethanol driven by a low-temperature constant temperature bath, and the temperature is set at -6°C; after part of the enzymatic hydrolysate freezes, adjust the temperature to -4°C to make the high-activity antifreeze components adsorb and combine with the ice on the outer wall of the container. After continuous extraction for 5 h, discard the unfrozen solution, slowly wash the ice with an appropriate amount of deionized water around the ice, close the water outlet switch, turn off the refrigeration system, collect the liquid after the ice melts and freeze-dry it to obtain the crude antifreeze peptide.

[0061] Example 3

[0062] The screening method of the antifreeze peptide from the scales of Dentex tumifrons provided in this example comprises the following steps:

[0063] S1 Use a nano-liquid chromatography-quadrupole orbitrap mass spectrometer to identify the amino acid sequences of the polypeptides contained in the crude antifreeze peptide obtained in Example 2, and respectively obtain the total ion chromatogram of the 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 ;

[0064] S2 Screening of polypeptide sequences that are non-toxic and have a hydrophilicity > 0: Input the amino acid sequences of all the obtained polypeptides into the ToxinPred database, and eliminate the non-water-soluble and toxic polypeptide sequences;

[0065] S3 Screening of polypeptide sequences with cryoprotective activity: Input the obtained non-toxic polypeptide sequences with a hydrophilicity > 0 into the Cryoprotect database, and screen out the polypeptide sequences with cryoprotective activity;

[0066] Screening of polypeptide sequences with secondary or higher structures: Use the PEP-FOLD3.5 polypeptide structure prediction and modeling program in the RPBS structural biology database to model the peptide sequences screened for cryoprotective activity. Select 100 simulation times, and the model sorting is based on sOPEP. Use the modeling result with the highest score as the final structure, and select the polypeptide sequences with secondary structures;

[0067] Screening of collagen-derived polypeptide sequences: Input the polypeptide sequences with secondary or higher structures into the Uniprot database to analyze the polypeptide sources, and screen out the collagen-derived polypeptide sequences;

[0068] S6 Use the molecular docking software Hex8.0.0 to perform molecular docking on the ice crystal facet and the collagen-derived polypeptide sequences to obtain the polypeptide sequences with the optimal conformation and Etotal;

[0069] S7 Using the number of hydrogen bonds formed between the ice crystal facet and the polypeptide sequences as an indicator, screen out the antifreeze peptide sequences from the peptide sequences with the optimal conformation through PyMOL software.

[0070] The screening method of this example sequentially screens peptide sequences without toxicity, hydrophilicity > 0, peptide sequences with cryoprotective activity, sequences with secondary or higher structures, and collagen-derived peptide sequences through the corresponding databases, improving the screening efficiency, yield, and accuracy of high-activity peptide sequences, and can overcome the technical problem that the accuracy of screening in the Cryoprotect database is only 88.28%. Through this screening method, more than 10 antifreeze polypeptide sequences with a thermal hysteresis activity value above 0.2 are screened out. Therefore, the screening method of this example has the advantages of high accuracy and high efficiency.

[0071] In this example, the screened antifreeze peptide sequences are synthesized into corresponding polypeptides; by measuring their thermal hysteresis activities, comprehensively screen out antifreeze peptide sequences with high thermal hysteresis activities, low ice crystal formation amounts, high survival rates of bacterial cell protection, and the highest total energy of contact with ice crystals among peptide sequences of the same length.

[0072] The hydrophilicity of the antifreeze peptide from the scales of Pagellus acarne in Example 1 is 0.36, and the molecular weight is 1355.672 Da; it has a secondary α-helix structure, and the total energy required for docking with ice crystals is -252.2 Kcal / moL. It has 12 amino acid residues, and four of the amino acid residue sites can form six hydrogen bonds with ice crystals, and TVR12 can form four hydrogen bonds with ice crystals. Its thermal hysteresis activity is between 0.2 and 0.4. Its low-temperature protection activity for bacterial cells is as high as 89.97%, indicating that it has strong antifreeze activity and strong stability.

[0073] Figure 1The total ion chromatogram of mass spectrometry is: the ion current signal generated by all ions after the separated components are ionized by the ion source, which is amplified and then made into a chromatogram with the elution time of the components.

[0074] Figure 2 The second-level mass spectrometry diagram of mass spectrometry: 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.

[0075] Figure 3 It is the visualized second-level structure diagram of the antifreeze peptide of the scales of Dentex tumifrons in Example 1.

[0076] Figure 4 It is the schematic diagram of the molecular docking of the antifreeze peptide of the scales of Dentex tumifrons in Example 1 and ice crystals in Step S7. The polypeptide in Example 1 has a secondary structure of α-helix (red), denoted as IY12. IY12 forms six hydrogen bonds (yellow) with ice crystals, and the corresponding docking site amino acids are GLU6, ASP10, and TVR12.

[0077] Example 4

[0078] This example provides a method for measuring the low-temperature protection activity of the antifreeze peptide of the scales of Dentex tumifrons obtained in Example 1, and uses the enzymatic hydrolysate in Example 2 (denoted as: EjAFPs) and the crude extract of the antifreeze peptide as controls (denoted as: I-EjAFPs). During the experiment, solutions with the same protein concentration are respectively prepared as samples.

[0079] The specific method is as follows:

[0080] Take 50 μL of Streptococcus thermophilus after secondary activation and inoculate it into 4 mL of M17 liquid medium. Culture for 4 h (37 °C, 180 rpm), centrifuge at 5000 rpm for 10 min, collect the bacterial sludge, wash it twice with physiological saline and then resuspend it in an equal volume of physiological saline to obtain the Streptococcus thermophilus bacterial solution.

[0081] Take 540 μL of the sample and mix it with 60 μL of the bacterial solution. Use physiological saline to replace the sample as the blank group. Pipette 50 μL of the mixture into 4 mL of M17 culture medium, culture 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 take another 50 μL to inoculate and culture for 7 h, and then measure the absorbance value A2. Calculate the survival rate according to formula (1).

[0082]

[0083] 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 .

[0084] This example obtained data as follows: Figure 5 shown:

[0085] The figure shows the cold stress protection ability of different cryoprotectants on Streptococcus thermophilus. The blank group is the normal saline group. It can be seen from the figure that compared with the blank group, the freezing protection rate of the enzymolysis solution EjAFPs on Streptococcus thermophilus has been significantly improved. The freezing protection rates of the crude antifreeze peptide extract I-EjAFPs and the screened pure peptide are higher than those of the previous step respectively, and its low-temperature protection activity on the bacteria is as high as 89.97%. It shows that the antifreeze peptide prepared in Example 1 has significantly stronger antifreeze activity.

[0086] Through step-by-step screening, a pure peptide sequence with high antifreeze activity was obtained, which can be used for future bacterial cryopreservation.

[0087] Example 5

[0088] This example provides the inhibitory effect of the antifreeze peptide from the scales of Dentex tumifrons obtained in Example 1 (denoted as IY12) on the ice crystal content, and uses the enzymolysis solution in Example 2 (denoted as: EjAFPs) and the crude antifreeze peptide extract as controls (denoted as: I-EjAFPs). During the experiment, solutions with the same protein concentration were respectively prepared as samples.

[0089] The specific methods for measuring the inhibitory effect and thermal hysteresis activity are as follows:

[0090] 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 placed into a differential scanning calorimeter at the same time, cooled at a rate of -2 °C / min to -30 °C, equilibrated for 5 min, and then heated at a rate of 2 °C / min to the retention temperature (-1.0 °C, -0.5 °C, -0.3 °C, 0 °C) to make 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.

[0091]

[0092] THA = T h -T0 (3)

[0093] 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 the sample crystallization. T h is the retention temperature, that is, a certain temperature in 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 maximum slope tangent and the minimum slope tangent at both ends of the inflection point of the heat flow curve.

[0094] The results of the thermal hysteresis activity determination in this embodiment 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.

[0095] Figure 6 In, 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 IY12 in Example 1 has been greatly improved compared with other control groups. The thermal hysteresis activity is between 0.2 and 0.4.

[0096] The ice crystal contents corresponding to different samples measured in this embodiment are as Figure 7 shown:

[0097] The blank group is the bovine serum albumin group (a protein without antifreeze activity). It can be seen from the figure that the control of the ice crystal content in the system of the purified peptide IY12 has been greatly improved compared with the control group, and the ice crystal content in the system can be significantly reduced, thereby exerting an antifreeze effect.

[0098] 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 it; 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 recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antifreeze peptide from fish scales, characterized in that, Its amino acid sequence is: IAQPQEKAPDPY.

2. Use of the fish scale antifreeze peptide as claimed in claim 1 in an antifreeze product.

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