An Antarctic krill antifreeze peptide AFP, its screening method, preparation method and application

Abstract

This invention discloses an Antarctic krill antifreeze peptide AFP, its screening method, preparation method, and applications, belonging to the field of bioactive peptide technology, and particularly relating to an Antarctic krill antifreeze peptide AFP, the amino acid sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2. The Antarctic krill antifreeze peptide AFP provided by this invention exhibits good spatial stability, strong ice crystal binding ability, and antifreeze protection, and can be used as a potential novel antifreeze functional peptide in fields such as food cold chain and cryopreservation of biological samples. The screening method provided by this invention predicts antifreeze proteins using a model, and after obtaining peptides through simulated hydrolysis, it again uses the model to predict and screen stable peptides. It ranks peptides by scoring the number of existing motifs, and finally determines the antifreeze peptides with stable binding by the number of hydrogen bonds formed by molecular docking with ice. This provides a valid reference for the standardized screening of antifreeze peptides.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, and particularly relates to an Antarctic krill antifreeze peptide AFP, its screening method, preparation method and application. Background Technology

[0002] Cryopreservation is a common method for preserving food, biological products, and viable cells. However, low-temperature freezing easily induces ice crystal formation, leading to cell membrane damage and protein denaturation, which seriously affects product quality and bioactivity. To alleviate these problems, antifreeze proteins and their functional fragments—antifreeze peptides—have attracted increasing attention. Antifreeze peptides have advantages such as small molecular weight, stable structure, and the ability to be obtained through artificial synthesis or enzymatic hydrolysis. They can effectively inhibit ice crystal growth and protect cell structure, showing potential applications in the field of cryogenics.

[0003] Antarctic krill (Euphausia superba, ES) is a typical polar cryogenically adapted organism. Its proteins maintain high activity and stable structure even under extreme conditions, making them a potential source of natural antifreeze peptides. Previous studies have shown that antifreeze-active peptides can be obtained by hydrolyzing Antarctic krill proteins. However, research in this field is still in its early stages and faces the following challenges: First, the complex structure of Antarctic krill proteins and the unclear distribution of antifreeze-active regions make it difficult to efficiently identify key peptides using traditional screening methods, necessitating high-throughput screening and molecular simulation techniques. Second, the activity of antifreeze peptides depends on specific amino acid sequences and spatial conformations, making them susceptible to interference from external environmental factors. Furthermore, Antarctic krill resources are mainly distributed in polar waters, resulting in high collection and processing costs and transportation limitations, hindering their large-scale development and application. Currently, there is a lack of systematic extraction and preparation processes, as well as standardized activity evaluation systems. Therefore, there is an urgent need to develop an efficient and reproducible technical pathway for the identification and functional verification of antifreeze peptides to improve the screening efficiency of Antarctic krill antifreeze peptides and promote their application in cryogenic protection and other fields. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes an Antarctic krill antifreeze peptide AFP, along with its screening, preparation, and applications. The Antarctic krill antifreeze peptide AFP provided by this invention exhibits good spatial stability, strong ice crystal binding ability, and antifreeze protection, and can be used as a potential novel antifreeze functional peptide in fields such as food cold chain and cryopreservation of biological samples.

[0005] To achieve the above objectives, the present invention provides an Antarctic krill antifreeze peptide AFP, the amino acid sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0006] This invention also provides a method for screening the Antarctic krill antifreeze peptide AFP, comprising the following steps:

[0007] 1) The Antarctic krill whole protein genome was screened for non-redundant whole protein genomes based on sequence similarity clustering;

[0008] 2) Based on the antifreeze protein prediction model, the non-redundant whole proteome obtained in step 1) is predicted to obtain the antifreeze protein sequence, and then CD-HIT clustering is performed to obtain the non-redundant protein sequence.

[0009] 3) The non-redundant protein sequence obtained in step 2) was digested using Expasy Peptide Cutter, and the Trypsin model was used for simulation to obtain peptides with a length of 10 to 14 amino acids.

[0010] 4) Screen for stable sequences with high antifreeze activity, stable physicochemical properties, and stable secondary structure among peptides of 10-14 amino acid length obtained in step 3);

[0011] 5) The stable sequences obtained in step 4) are scored as antifreeze motifs, and the top 5 antifreeze motifs are selected as the Antarctic krill antifreeze peptides AFP.

[0012] Preferably, the antifreeze protein prediction model in step 2) consists of AFP-LSE, Targetfreeze, CryoProtect, and Miyata's model.

[0013] Preferably, the antifreeze sequence scoring in step 5) specifically involves:

[0014] Step 4) The number of antifreeze feature motifs contained in the property-stable sequence is the antifreeze motif score;

[0015] The antifreeze characteristic motif is one of the following: threonine-any amino acid-threonine, glutamic acid-glutamic acid, glycine-proline-any amino acid, threonine-any amino acid-any amino acid-any amino acid-alanine-any amino acid-any amino acid-any amino acid-alanine-any amino acid-any amino acid-alanine-any amino acid-any amino acid.

[0016] This invention also provides a method for preparing the Antarctic krill antifreeze peptide AFP, comprising the following steps:

[0017] According to the amino acid sequence of the Antarctic krill antifreeze peptide AFP, the amino protecting groups of the amino acids are removed, the carboxyl groups of the next amino acid to be linked are activated, and peptide bonds are formed by coupling. The process is repeated, and then the peptide is eluted with a deprotecting agent to obtain the Antarctic krill antifreeze peptide AFP.

[0018] This invention also provides the application of the Antarctic krill antifreeze peptide AFP in the preparation of antifreeze agents.

[0019] This invention also provides the application of the Antarctic krill antifreeze peptide AFP in food freezing protection.

[0020] The present invention also provides an antifreeze agent comprising the Antarctic krill antifreeze peptide AFP.

[0021] The present invention also provides a food cryoprotectant comprising the Antarctic krill antifreeze peptide AFP.

[0022] The present invention also provides the application of the antifreeze agent in the freezing protection of food.

[0023] Compared with the prior art, the present invention has the following advantages and technical effects:

[0024] This invention provides an Antarctic krill antifreeze peptide, AFP, which exhibits good steric stability, strong ice crystal binding ability, and antifreeze protection. It can be used as a potential novel antifreeze functional peptide in fields such as food cold chain and cryopreservation of biological samples. The Antarctic krill antifreeze peptide AFP of this invention has a defined amino acid sequence, can be obtained through artificial synthesis, and possesses high antifreeze activity, filling a gap in the field of Antarctic krill antifreeze peptides.

[0025] This invention provides a method for screening Antarctic krill antifreeze peptide AFP, including protein sequence collection, antifreeze protein prediction, hydrolysis simulation, peptide activity and stability analysis, multidimensional screening scoring, and final in vitro antifreeze activity verification of screened peptides. Combining multi-model synergistic prediction and multi-parameter evaluation, this method can efficiently obtain structurally stable antifreeze peptides with strong potential antifreeze activity and significant interaction with ice crystals, providing technical support for the discovery of novel antifreeze peptides. Compared to traditional methods, the screening method of this invention predicts antifreeze proteins through models, and after obtaining peptides through simulated hydrolysis, it again uses models for prediction to screen stable peptides with α-helices. Existing motif scoring is used for ranking, and finally, the number of hydrogen bonds between molecular docking and ice is used to determine the antifreeze peptides with stable binding. The method is simple to operate, fast in screening, highly accurate, and has good reproducibility and scalability, providing a valid reference for the standardized screening of antifreeze peptides. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1The image shows a high-performance liquid chromatogram of the Antarctic krill antifreeze peptide AFP, where A is the amino acid sequence shown in SEQ ID NO.1 and B is the amino acid sequence shown in SEQ ID NO.2.

[0028] Figure 2 The image shows a secondary mass spectrum of the Antarctic krill antifreeze peptide AFP, where A is the amino acid sequence shown in SEQ ID NO.1 and B is the amino acid sequence shown in SEQ ID NO.2.

[0029] Figure 3 This is a schematic diagram of the secondary structure of Antarctic krill antifreeze peptide AFP, where A is the Antarctic krill antifreeze peptide AFP with the amino acid sequence shown in SEQ ID NO.1, and B is the Antarctic krill antifreeze peptide AFP with the amino acid sequence shown in SEQ ID NO.2. In the figure, red represents α-helix, blue represents β-sheet, and gray represents random coil.

[0030] Figure 4 The diagrams show the molecular docking of Antarctic krill antifreeze peptide AFP with ice crystals. In this diagram, A represents the molecular docking of the Antarctic krill antifreeze peptide AFP with the amino acid sequence shown in SEQ ID NO.1 with ice crystals; B represents the binding site of the Antarctic krill antifreeze peptide AFP with the amino acid sequence shown in SEQ ID NO.1 with ice crystals; C represents the molecular docking of the Antarctic krill antifreeze peptide AFP with the amino acid sequence shown in SEQ ID NO.2 with ice crystals; and D represents the molecular docking of the Antarctic krill antifreeze peptide AFP with the amino acid sequence shown in SEQ ID NO.2 with ice crystals.

[0031] Figure 5 The study investigated the effect of Antarctic krill antifreeze peptide AFP on ice crystal growth morphology. In the PBS group, A represented 0s, B 10s, C 20s, D 30s, E 40s, F 0s, G 10s, H 20s, I 30s, J 40s, K 0s, L 10s, M 20s, N 30s, and O 40s. The PBS group represented the control group treated with PBS. The AFP-1 group represented the experimental group treated with the Antarctic krill antifreeze peptide AFP (amino acid sequence shown in SEQ ID NO.1), and the AFP-2 group represented the experimental group treated with the Antarctic krill antifreeze peptide AFP (amino acid sequence shown in SEQ ID NO.2).

[0032] Figure 6To illustrate the effect of Antarctic krill antifreeze peptide AFP on ice crystal size, the PBS group represents the control group treated with PBS, the AFP-1 group represents the experimental group treated with Antarctic krill antifreeze peptide AFP with the amino acid sequence shown in SEQ ID NO.1, and the AFP-2 group represents the experimental group treated with Antarctic krill antifreeze peptide AFP with the amino acid sequence shown in SEQ ID NO.2.

[0033] Figure 7 The figure shows the effect of Antarctic krill antifreeze peptide AFP on ice crystal growth rate. The PBS group represents the control group treated with PBS, the AFP-1 group represents the experimental group treated with Antarctic krill antifreeze peptide AFP with the amino acid sequence shown in SEQ ID NO.1, and the AFP-2 group represents the experimental group treated with Antarctic krill antifreeze peptide AFP with the amino acid sequence shown in SEQ ID NO.2. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0038] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0039] The AFP-LSE used in this invention can be found in the reference USMAN M, KHAN S, LEE JA. AFP-LSE: Antifreeze Proteins Prediction Using Latent Space Encoding of Composition of k-Spaced Amino Acid Pairs[J]. Scientific Reports, 2020, 10(1):7197.; the Targetfreeze used can be found in the reference HE X, HAN K, HU J, et al. TargetFreeze: Identifying Antifreeze Proteins via a Combination of Weights using Sequence Evolutionary Information and Pseudo Amino Acid Composition[J]. The Journal of Membrane Biology, 2015, 248(6):1005-1014.; the CryoProtect used can be found in the reference PRATIWI R, MALIK AA, SCHADUANGRAT N, et al. CryoProtect: A Web Server for Classifying Antifreeze Proteins from Nonantifreeze. Proteins[J].JournalofChemistry,2017:9861752,15pages,2017. It can be found that the Miyata's model used can be found in the reference MIYATAR, MORIWAKI Y, TERADAT, et al.Prediction and analysis of antifreezeproteins[J].Heliyon,2021,7(9):e07953.

[0040] Example 1

[0041] Screening for Antarctic krill antifreeze peptide AFP:

[0042] 1) A total of 153,359 Antarctic krill whole protein sequences were collected from the KrillDB2 database as a screening dataset. The CD-HIT tool was used for sequence similarity clustering to screen out non-redundant whole protein sequences.

[0043] 2) Based on the antifreeze protein prediction models AFP-LSE, Targetfreeze, CryoProtect and Miyata'smodel, the non-redundant whole proteome was predicted, resulting in 3990 antifreeze protein sequences. Then, the CD-HIT tool was used for clustering, resulting in 610 non-redundant protein sequences.

[0044] 3) The Expasy Peptide Cutter tool was used to simulate trypsin digestion of representative protein sequences. The Trypsin model was selected for simulation, with lysine and arginine residues as the cleavage sites, resulting in peptides of 10-14 amino acids in length.

[0045] 4) Screen for stable sequences with high antifreeze activity, stable physicochemical properties, and stable secondary structure among peptides of 10–14 amino acid length. First, input peptides of 10–14 amino acid length into antifreeze prediction models (AFP-LSE and Miyata's model) to screen for antifreeze peptides. Then, use the Expasy ProtParam Cutter tool to predict the stability of antifreeze peptides. Sequences with an instability index of less than 40 are considered physicochemically stable antifreeze peptides. Use I-TASSER to predict the secondary structure of antifreeze peptides and retain structurally stable antifreeze peptides. Antifreeze peptides with stable physicochemical properties and structurally stable antifreeze peptides are collectively referred to as stable sequences.

[0046] 5) Scoring of cryoresistant motifs based on stable sequences:

[0047] The score for antifreeze motifs is as follows: the number of antifreeze characteristic motifs (threonine-any amino acid-threonine, glutamic acid-glutamic acid, glycine-proline-any amino acid, threonine-any amino acid-any amino acid-any amino acid-alanine-any amino acid-any amino acid-any amino acid-alanine-any amino acid-any amino acid-any amino acid-alanine-any amino acid-any amino acid) contained in the stable sequence is the score for antifreeze motifs.

[0048] The top 5 antifreeze motifs by score were selected as the Antarctic krill antifreeze peptides AFP.

[0049] The amino acid sequence of the Antarctic krill antifreeze peptide AFP is shown in SEQ ID NO.1 and SEQ ID NO.2.

[0050] SEQ ID NO.1: CGAFEELYGGTCR. It can form 4 hydrogen bonds with ice crystals. The hydrogen bond formation sites include Glu, Gly, Thr and Arg, each forming one hydrogen bond with ice crystals. It also has an α-helical structure and a hydrophilicity of -0.10.

[0051] SEQ ID NO.2: IAIETATAALNSLK. It can form three hydrogen bonds with ice crystals, with the hydrogen bond sites including Thr, Ala, and Asn. Each hydrogen bond forms one hydrogen bond with the ice crystal, and it has an α-helix structure with a hydrophilicity of 0.764.

[0052] Example 2

[0053] Fmoc solid-phase synthesis method for synthesizing Antarc krill antifreeze peptide AFP:

[0054] Following the amino acid sequence of the Antarctic krill antifreeze peptide AFP shown in SEQ ID NO.1, the Fmoc-protected column and the amino protecting groups of the amino acids were removed using hexahydropyridine. The carboxyl group of the next amino acid to be linked was activated using an activator (benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate ester HBTU and N,N-diisopropylethylamine DIEA mixed at a mass ratio of 0.9:1.5). The amino and carboxyl groups were coupled to form a peptide bond. This process was repeated until the amino acid sequence shown in SEQ ID NO.1 was obtained. Then, the peptide was eluted from the column using the deprotectant TFA to obtain the Antarctic krill antifreeze peptide AFP with a molecular weight of 1405.56 Da.

[0055] Following the amino acid sequence of the Antarctic krill antifreeze peptide AFP shown in SEQ ID NO.2, the Fmoc-protected column and the amino protecting groups of the amino acids were removed using hexahydropyridine. The carboxyl group of the next amino acid to be linked was activated using an activator (a mixture of benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate ester (HBTU) and N,N-diisopropylethylamine (DIEA) at a mass ratio of 0.9:1.5). The amino and carboxyl groups were coupled to form a peptide bond. This process was repeated until the amino acid sequence shown in SEQ ID NO.2 was obtained. Then, the peptide was eluted from the column using the deprotectant TFA to obtain the Antarctic krill antifreeze peptide AFP with a molecular weight of 1415.63 Da.

[0056] like Figure 1 China A and Figure 1 Figure B shows the high-performance liquid chromatograms of the Antarctic krill antifreeze peptide AFP, which are the sequences shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; Figure 2 China A and Figure 2 Figure B shows the secondary mass spectra of the Antarctic krill antifreeze peptide AFP, which contains the sequences shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. Figure 3 China A and Figure 3 Figure B shows a schematic diagram of the secondary structure of the Antarctic krill antifreeze peptide AFP with the sequence shown in SEQ ID NO.1 and SEQ ID NO.2.

[0057] Example 3

[0058] Validation of the antifreeze properties of Antarctic krill antifreeze peptides:

[0059] Ice crystal morphology observation experiment: For the AFP-1 treated experimental group, the Antarctic krill antifreeze peptide AFP with the sequence shown in SEQ ID NO.1 prepared in Example 2 was dissolved in phosphate-buffered saline (PBS) to prepare a peptide solution with a concentration of 5 mg / mL. 2 μL of the peptide solution was added dropwise to a pre-chilled glass slide. For the AFP-2 treated experimental group, the Antarctic krill antifreeze peptide AFP with the sequence shown in SEQ ID NO.2 prepared in Example 2 was dissolved in phosphate-buffered saline (PBS) to prepare a peptide solution with a concentration of 5 mg / mL. 2 μL of the peptide solution was added dropwise to a pre-chilled glass slide. The control group used phosphate-buffered saline (PBS), with 2 μL added dropwise to a pre-chilled glass slide. The temperature was lowered to -30℃ at a rate of 20℃ / min and held at that temperature for 3 minutes. Then, it was reheated to the phase transition temperature at a rate of 10℃ / min and held at that temperature for 3 minutes. Next, it was reheated at a rate of 1℃ / min until the ice crystals began to melt. Then, it was lowered at a rate of 1℃ / min. This process was repeated until only a single tiny ice crystal remained in the field of view. Finally, the temperature was lowered at a rate of 0.5℃ / min and the growth morphology and rate of the ice crystals were recorded in real time.

[0060] like Figure 4 China A Figure 4 B, Figure 4 C and Figure 4 As shown in Figure D, this is a schematic diagram of the docking structure of the Antarctic krill antifreeze peptide AFP with ice crystal molecules, based on the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 of this invention.

[0061] like Figure 5 As shown, this invention uses cryomicroscopy to observe the growth morphology of a single ice crystal in the PBS group (control group) and (…). Figure 5 China A~ Figure 5 In the E group, ice crystals exhibit a flat and irregular shape and grow extremely rapidly, filling the entire field of view in just 30 seconds. In contrast, in the AFP-1 group ( Figure 5 Middle F~ Figure 5 (J) and AFP-2 group ( Figure 5 Middle K~ Figure 5 In the O (experimental group), the morphology of ice crystals changed significantly, with the size decreasing and the shape transforming into a well-defined hexagon.

[0062] like Figure 6 As shown, the AFP-1 and AFP-2 groups of Antarctic krill antifreeze peptides exhibited significant ice crystal growth inhibitory activity. At 30 s, the average ice crystal size in the PBS group increased to 55852.36 μm. 2The average ice crystal size in the AFP-1 group was only 7846.11 μm. 2 The average size of ice crystals in the AFP-2 group was only 10852.56 μm. 2 .

[0063] like Figure 7 As shown, the AFP-1 and AFP-2 groups of Antarctic krill antifreeze peptides exhibited significant inhibitory effects on ice crystal growth. In the AFP-1 group, the ice crystal growth rate in the AFP solution was 257.67 μm / s, significantly lower than the 1847.26 μm / s in the PBS group, demonstrating its superior antifreeze activity. In the AFP-2 group, the ice crystal growth rate in the AFP solution was 358.04 μm / s.

[0064] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An Antarctic krill antifreeze peptide AFP, characterized in that the amino acid sequence of the Antarctic krill antifreeze peptide AFP is as shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The method for preparing Antarctic krill antifreeze peptide AFP as described in claim 1, characterized in that, Includes the following steps: According to the amino acid sequence of the Antarctic krill antifreeze peptide AFP, the amino protecting groups of the amino acids are removed, the carboxyl groups of the next amino acid to be linked are activated, and peptide bonds are formed by coupling. The process is repeated, and then the peptide is eluted with a deprotecting agent to obtain the Antarctic krill antifreeze peptide AFP.

3. The application of the Antarctic krill antifreeze peptide AFP as described in claim 1 in the preparation of antifreeze agents.

4. The application of the Antarctic krill antifreeze peptide AFP as described in claim 1 in food freezing protection.

5. An antifreeze agent, characterized in that, It contains the Antarctic krill antifreeze peptide AFP as described in claim 1.

6. A food cryoprotectant, characterized in that, It contains the Antarctic krill antifreeze peptide AFP as described in claim 1.

7. The application of the antifreeze agent as described in claim 5 in the frozen food protection.

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