Self-assembled supramolecular peptide nano-enzyme material as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological material preparation, and particularly discloses a self-assembled supramolecular peptide nano-enzyme material as well as a preparation method and application thereof. The preparation method comprises the following steps: S1, dissolving beta-alanyl-L-histidine in ultrapure water, and uniformly stirring to obtain a solution A; s2, dissolving hemin in dimethyl sulfoxide, and uniformly stirring to obtain a solution B; s3, uniformly mixing the solution A and the solution B, adjusting the pH value of the solution, heating in a water bath, incubating at room temperature, centrifuging, discarding precipitate and retaining supernate; and S4, freeze-drying and grinding the supernate obtained in the step S3 to obtain the self-assembled supramolecular peptide nano-enzyme material. The invention discloses a self-assembled supramolecular peptide nano-enzyme material as well as a preparation method and application thereof. The supramolecular peptide nano-enzyme is applied to food quality safety detection and can realize visual sensitive colorimetric detection of biogenic amines in rotten food.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterial preparation, and in particular relates to a self-assembling supramolecular peptide nanoenzyme material and a preparation method and application thereof. Background Art

[0002] During food processing, transportation, storage, and distribution, microbial contamination can lead to protein breakdown, producing volatile biogenic amines, which can cause food spoilage and threaten food safety. While traditional methods for detecting volatile biogenic amines offer high accuracy, they are complex, require specialized equipment, and are expensive, making them difficult to meet the demands of real-time, high-throughput, and low-cost food quality monitoring. Therefore, developing a simple, efficient, and cost-effective method for detecting biogenic amines is of great practical significance.

[0003] Natural enzymes are widely used in pollutant degradation and colorimetric sensing due to their efficient catalytic activity. However, inherent drawbacks such as limited availability, poor stability, susceptibility to environmental factors such as temperature, high cost, and low reusability restrict their large-scale application in practical scenarios. Nanozymes, as alternatives to natural enzymes, mimic the catalytic active sites of natural enzymes and offer advantages such as structural diversity, strong environmental adaptability, and high stability, making them a research hotspot in the field of enzyme catalysis.

[0004] Currently, materials used to construct nanozymes often rely on harsh chemical synthesis conditions or toxic precursors, and their biocompatibility and safety struggle to meet the stringent requirements of food testing. Given the limitations of existing biogenic amine detection methods and the safety concerns of nanozyme materials, there is an urgent need for self-assembling nanozyme materials and detection technologies based on biocompatible supramolecular peptides to meet the real-time, cost-effective, and safe requirements of food quality and safety monitoring. Summary of the Invention

[0005] The present invention aims to provide a self-assembling supramolecular peptide nanozyme material, a preparation method and application thereof. The supramolecular peptide nanozyme has low toxicity and good peroxidase catalytic activity. When applied to food quality and safety testing, it can realize visual and sensitive colorimetric detection of biogenic amines in spoiled food.

[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0007] A method for preparing a self-assembling supramolecular peptide nanozyme material comprises the following steps:

[0008] S1. Dissolve β-alanyl-L-histidine in ultrapure water and stir evenly to obtain solution A.

[0009] S2. Dissolve hemin in dimethyl sulfoxide and stir evenly to obtain solution B;

[0010] S3. Mix solution A and solution B evenly, adjust the pH of the solution, heat in a water bath, incubate at room temperature, centrifuge, discard the precipitate and retain the supernatant;

[0011] S4. The supernatant obtained in S3 is freeze-dried and ground to obtain a self-assembled supramolecular peptide nanozyme material.

[0012] Preferably, in S3, the molar ratio of β-alanyl-L-histidine in solution A to hemin in solution B is 5-15:1.

[0013] Preferably, in S3, the pH of the solution is adjusted to 4.0-8.0.

[0014] Preferably, in S3, the water bath heating temperature is 40° C. to 70° C., and the heating time is 0.5 h to 2 h.

[0015] Preferably, in S3, the room temperature incubation time is 10 h to 20 h.

[0016] Preferably, in S3, the centrifugal speed is 10000 rpm and the centrifugal time is 10 min.

[0017] Preferably, in S4, the freeze-drying temperature is -50°C to -80°C, and the freeze-drying time is 24h to 48h.

[0018] The present invention also provides a self-assembling supramolecular peptide nanozyme material prepared by the preparation method.

[0019] The present invention also provides the use of the self-assembling supramolecular peptide nanozyme material or the self-assembling supramolecular peptide nanozyme material prepared by the preparation method in the detection of biogenic amines.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects:

[0021] The present invention discloses a self-assembling supramolecular peptide nanoenzyme material, which uses β-alanyl-L-histidine and hemin with excellent biocompatibility as raw materials, and forms a nanofiber structure by self-assembly in a precise ratio under mild conditions, thereby avoiding the use of toxic reagents and complex processes in the preparation of traditional nanomaterials and reducing costs. The material simulates the active center of natural horseradish peroxidase, and with the help of the coordination effect and non-covalent interaction between the histidine side chain and the heme iron ion, achieves efficient peroxidase-like catalytic activity, and can complete the color development reaction within 200s. The material is suitable for the detection of volatile biogenic amines such as putrescine, cadaverine, and histamine in a variety of foods such as meat, fish, and dairy products. It has the advantages of being green and safe, highly efficient in catalysis, sensitive in detection, and low in cost. It provides a real-time, economical, and reliable innovative solution for food quality and safety monitoring. At the same time, it is expected to be expanded to the field of multi-pollutant detection such as mycotoxins and pesticide residues, and has significant technological advancement and industrial application value.

[0022] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Statistical graphs of absorbance of the self-assembled supramolecular peptide nanozyme materials provided in Examples 1 to 5;

[0024] Figure 2 This is a morphology characterization image of the self-assembled supramolecular peptide nanozyme material provided in Example 3, with a scale of 5 μm;

[0025] Figure 3 The peroxidase-like catalytic activity results of the self-assembled supramolecular peptide nanozyme material provided in Example 3, wherein: Figure 3 A in the figure is the Michaelis-Menten curve under the condition of fixed hydrogen peroxide concentration and changing 3,3',5,5'-tetramethylbenzidine concentration. Figure 3 B in the figure is a double reciprocal graph obtained by fixing the concentration of hydrogen peroxide and changing the concentration of 3,3',5,5'-tetramethylbenzidine. Figure 3 C in the figure is the Michaelis-Menten curve under fixed 3,3',5,5'-tetramethylbenzidine concentration and changing hydrogen peroxide concentration. Figure 3 D in the figure is a double reciprocal graph obtained by fixing the concentration of 3,3',5,5'-tetramethylbenzidine and changing the concentration of hydrogen peroxide;

[0026] Figure 4 The experimental parameter results of the self-assembled supramolecular peptide nanozyme material provided in Example 3 for catalyzing the colorimetric reaction of 3,3',5,5'-tetramethylbenzidine, wherein: Figure 4 A in the figure is the test result of the catalytic system at different reaction times; Figure 4B in the figure is the test result when different amounts of 3,3',5,5'-tetramethylbenzidine are added to the catalytic system. Figure 4 C in the figure is the test result when different amounts of hydrogen peroxide are added to the catalytic system. Figure 4 D in the figure is the experimental result when different amounts of self-assembled supramolecular peptide nanozyme materials are added to the catalytic system;

[0027] Figure 5 The sensitivity test results of the self-assembled supramolecular peptide nanozyme material provided in Example 3, wherein Figure 5 A in the figure is the UV-visible absorption spectrum of the catalytic system based on the self-assembled supramolecular peptide nanozyme material in the presence of different concentrations of histamine. Figure 5 B in FIG is the linear regression curve between histamine concentration and absorbance value at 652 nm. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0029] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0030] Source of test materials:

[0031] In the present invention, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in this field and can be purchased through commercial channels.

[0032] Example 1

[0033] A method for preparing a self-assembling supramolecular peptide nanozyme material comprises the following steps:

[0034] S1. Dissolve 300 μM β-alanyl-L-histidine in 3 mL of ultrapure water and stir well to obtain solution A.

[0035] S2. Dissolve 30 μM hemin in 1.2 mL dimethyl sulfoxide and stir well to obtain solution B.

[0036] S3. Mix solution A and solution B uniformly, wherein the molar ratio of β-alanyl-L-histidine in solution A to hemin in solution B is 5 mol:1 mol, adjust the pH of the solution to 7.0, heat in a 60° C. water bath for 1 h, incubate at room temperature for 12 h, centrifuge at 10,000 rpm for 10 min, discard the precipitate and retain the supernatant;

[0037] S4. The supernatant obtained in S3 was freeze-dried at -60°C for 24 hours and ground to obtain a self-assembled supramolecular peptide nanozyme material.

[0038] Example 2

[0039] S1. Dissolve 300 μM β-alanyl-L-histidine in 3 mL of ultrapure water and stir well to obtain solution A.

[0040] S2. Dissolve 30 μM hemin in 1.2 mL dimethyl sulfoxide and stir well to obtain solution B.

[0041] S3. Mix solution A and solution B uniformly, wherein the molar ratio of β-alanyl-L-histidine in solution A to hemin in solution B is 7.5 mol:1 mol, adjust the pH of the solution to 7.0, heat in a 60° C. water bath for 1 h, incubate at room temperature for 12 h, centrifuge at 10,000 rpm for 10 min, discard the precipitate and retain the supernatant;

[0042] S4. The supernatant obtained in S3 was freeze-dried at -60°C for 24 hours and ground to obtain a self-assembled supramolecular peptide nanozyme material.

[0043] Example 3

[0044] S1. Dissolve 300 μM β-alanyl-L-histidine in 3 mL of ultrapure water and stir well to obtain solution A.

[0045] S2. Dissolve 30 μM hemin in 1.2 mL dimethyl sulfoxide and stir well to obtain solution B.

[0046] S3. Mix solution A and solution B uniformly, wherein the molar ratio of β-alanyl-L-histidine in solution A to hemin in solution B is 10 mol:1 mol, adjust the pH of the solution to 7.0, heat in a 60° C. water bath for 1 h, incubate at room temperature for 12 h, centrifuge at 10,000 rpm for 10 min, discard the precipitate and retain the supernatant;

[0047] S4. The supernatant obtained in S3 was freeze-dried at -60°C for 24 hours and ground to obtain a self-assembled supramolecular peptide nanozyme material.

[0048] Example 4

[0049] S1. Dissolve 300 μM β-alanyl-L-histidine in 3 mL of ultrapure water and stir well to obtain solution A.

[0050] S2. Dissolve 30 μM hemin in 1.2 mL dimethyl sulfoxide and stir well to obtain solution B.

[0051] S3. Mix solution A and solution B uniformly, wherein the molar ratio of β-alanyl-L-histidine in solution A to hemin in solution B is 12.5 mol:1 mol, adjust the pH of the solution to 7.0, heat in a 60° C. water bath for 1 h, incubate at room temperature for 12 h, centrifuge at 10,000 rpm for 10 min, discard the precipitate and retain the supernatant;

[0052] S4. The supernatant obtained in S3 was freeze-dried at -60°C for 24 hours and ground to obtain a self-assembled supramolecular peptide nanozyme material.

[0053] Example 5

[0054] S1. Dissolve 300 μM β-alanyl-L-histidine in 3 mL of ultrapure water and stir well to obtain solution A.

[0055] S2. Dissolve 30 μM hemin in 1.2 mL dimethyl sulfoxide and stir well to obtain solution B.

[0056] S3. Mix solution A and solution B uniformly, wherein the molar ratio of β-alanyl-L-histidine in solution A to hemin in solution B is 15 mol:1 mol, adjust the pH of the solution to 7.0, heat in a 60° C. water bath for 1 h, incubate at room temperature for 12 h, centrifuge at 10,000 rpm for 10 min, discard the precipitate and retain the supernatant;

[0057] S4. The supernatant obtained in S3 was freeze-dried at -60°C for 24 hours and ground to obtain a self-assembled supramolecular peptide nanozyme material.

[0058] The effects of the self-assembling supramolecular peptide nanozyme materials provided in Examples 1-5 above were verified by the following experiments.

[0059] 1. The catalytic activity of the self-assembled supramolecular peptide nanozyme materials provided in Examples 1-5 was determined. The test method was as follows: 1 mg of the self-assembled supramolecular peptide nanozyme materials provided in Examples 1-5 were weighed and dissolved in 1 mL of phosphate buffer. 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide were added to construct the nanozyme catalytic system. The UV-visible absorption peak at 652 nm was recorded using an ultraviolet spectrophotometer. The results are shown in Figure 2. Figure 1 .

[0060] Depend on Figure 1 It can be seen that the catalytic activity of the self-assembling supramolecular peptide nanozyme increases with the increase of β-alanyl-L-histidine content. In Example 3, when the molar ratio of β-alanyl-L-histidine in solution A to hemin in solution B is 10:1, the catalytic activity basically reaches stability.

[0061] 2. The morphology of the self-assembled supramolecular peptide nanozyme material provided in Example 3 was characterized. Figure 2 .

[0062] Depend on Figure 2 It can be seen that the self-assembled supramolecular peptide nanozyme material synthesized in Example 3 is a nanofiber structure.

[0063] 3. Determine the peroxidase-like catalytic performance of the self-assembled supramolecular peptide nanozyme material provided in Example 3. The test method is as follows: add the self-assembled supramolecular peptide nanozyme material to a buffer solution of 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide, and record the absorbance of the corresponding system at 652nm. By changing the concentration of TMB (0-30mM), at a fixed hydrogen peroxide concentration (130mM), or changing the concentration of hydrogen peroxide (0-150mM), at a fixed TMB concentration (22mM), using the self-assembled supramolecular peptide nanozyme material (5mg / mL), the nanozyme based on TMB and hydrogen peroxide as substrates was kinetically analyzed. The initial reaction rate at different substrate concentrations was calculated according to the Lambert-Beer law. The data were further fitted with the Michaelis equation to calculate the typical parameters of the enzyme-like activity, including the Michaelis constant (K m ), maximum reaction speed (V max ) and specific activity (SA). Specific activity is the core indicator for measuring enzyme purity and catalytic efficiency, and is defined as the ability of unit mass of enzyme to catalyze substrate conversion per unit time.

[0064] The initial reaction rate at different substrate concentrations was calculated using the following formula (1):

[0065] V = A / εbt;

[0066] The following formula (2) is used to fit the data:

[0067] 1 / V=Km / (Vmax[S])+1 / Vmax;

[0068] The specific activity was calculated using the following formula (3):

[0069] Specific activity = Vmax / enzyme mass concentration;

[0070] Where V is the initial reaction rate, A is the absorbance, ε is the molar absorptivity, b is the optical path length, and [S] is the substrate concentration.

[0071] By fixing the concentration of one substrate and changing the concentration of the other substrate, the initial reaction rate is determined and the Michaelis-Menten curve of 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide is plotted. The results are shown in Figure 3 .

[0072] Depend on Figure 3 A and Figure 3 From the calculation of B in the figure, we can know that the maximum reaction rate V of 3,3',5,5'-tetramethylbenzidine max , Michaelis constant K m and specific activity SA were 13.33×10 -7 Ms -1 , 9.013M and 16.00U / mg.

[0073] Depend on Figure 3 C and Figure 3 From the calculation of D in the equation, we can see that the maximum reaction rate of hydrogen peroxide is V max , Michaelis constant K m and specific activity SA were 16.89×10 -7 Ms -1 , 69.99M and 20.26U / mg.

[0074] Overall, within the two substrate concentration ranges, the double reciprocal plots of the initial reaction rate versus substrate concentration are a series of straight lines with no intersection, consistent with the typical ping-pong reaction mechanism. This is because during the catalytic process, the self-assembled supramolecular peptide nanozyme material binds and reacts with hydrogen peroxide, the first substrate, releasing the active catalytic intermediate ·OH, which oxidizes TMB to ox-TMB.

[0075] 4. The optimal time for the self-assembled supramolecular peptide nanozyme material provided in Example 3 to catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine was tested. Figure 4 As shown in Figure A, the UV absorption peak intensity at 652 nm is basically stable at 200 s, so 200 s was selected as the catalytic time for subsequent experiments. In addition, the effects of the content of 3,3',5,5'-tetramethylbenzidine, hydrogen peroxide and self-assembled supramolecular peptide nanozyme materials on the catalytic activity of the enzyme were also explored. The results are shown in Figure 3. Figure 4 B- Figure 4 As shown in D in the figure, it was finally determined that the substance content of 3,3',5,5'-tetramethylbenzidine in 1 mL of the catalytic system was 22 mM; the substance content of hydrogen peroxide was 130 mM; and the substance content of the self-assembled supramolecular peptide nanozyme material was 5 mg / mL.

[0076] 5. Sensitivity test:

[0077] Using phosphate buffer as solvent, prepare histamine standard solutions of different concentrations. Under optimal conditions, add it to the 3,3',5,5'-tetramethylbenzidine color development system catalyzed by the self-assembled supramolecular peptide nanozyme synthesized in Example 3. The wavelength measurement range is set to 450-800nm, and the value of the ultraviolet-visible absorption peak at 652nm is recorded. According to the following formula (4), the obtained data is used to construct a colorimetric response platform for histamine detection. The results are as follows: Figure 5 .

[0078] Formula (4):

[0079] A=KC+b;

[0080] Wherein, C represents the concentration of the target, K represents the slope of the calibration curve, and A represents the absorbance at 652 nm.

[0081] Depend on Figure 5 A and Figure 5 As shown in Figure B, as the histamine concentration increases from 1 mg / L to 200 mg / L, the ultraviolet absorption intensity of the nanozyme catalytic system at 652 nm gradually decreases. With the ultraviolet absorption peak intensity at 652 nm as the ordinate and the histamine concentration as the abscissa, a linear fitting analysis was performed and it was found that the two had a good linear relationship (y = 0.0079x + 2.054, R 2 =0.9967), and the detection limit was further calculated to be 0.62 mg / L.

[0082] 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 the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a self-assembling supramolecular peptide nanozyme material, characterized in that: The following steps are involved: S1. Dissolve β-alanyl-L-histidine in ultrapure water and stir evenly to obtain solution A. S2. Dissolve hemin in dimethyl sulfoxide and stir evenly to obtain solution B; S3. Mix solution A and solution B evenly, adjust the pH of the solution, heat in a water bath, incubate at room temperature, centrifuge, discard the precipitate and retain the supernatant; S4. The supernatant obtained in S3 is freeze-dried and ground to obtain a self-assembled supramolecular peptide nanozyme material.

2. The preparation method according to claim 1, characterized in that In S3, the molar ratio of β-alanyl-L-histidine in solution A to hemin in solution B is 5 to 15:

1.

3. The preparation method according to claim 1, characterized in that In S3, the pH of the solution is adjusted to 4.0-8.

0.

4. The preparation method according to claim 1, characterized in that In S3, the water bath heating temperature is 40° C. to 70° C., and the heating time is 0.5 h to 2 h.

5. The preparation method according to claim 1, characterized in that In S3, the room temperature incubation time is 10 h to 20 h.

6. The preparation method according to claim 1, characterized in that In S3, the centrifugal speed is 10000 rpm, and the centrifugal time is 10 min.

7. The preparation method according to claim 1, characterized in that In S4, the freeze-drying temperature is -50°C to -80°C, and the freeze-drying time is 24h to 48h.

8. The self-assembled supramolecular peptide nanozyme material prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the self-assembling supramolecular peptide nanozyme material according to claim 8 or the self-assembling supramolecular peptide nanozyme material prepared by the preparation method according to any one of claims 1 to 7 in the detection of biogenic amines.

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