Self-assembled supramolecular peptide nanoscale enzyme material, and preparation method and application thereof

Abstract

This invention belongs to the field of biomaterial preparation technology, specifically disclosing a self-assembled supramolecular peptide nanozyme material, its preparation method, and its application. The preparation method includes the following steps: S1, dissolving β-alanyl-L-histidine in ultrapure water and stirring until homogeneous to obtain solution A; S2, dissolving heme chloride in dimethyl sulfoxide and stirring until homogeneous to obtain solution B; S3, mixing solution A and solution B until homogeneous, adjusting the pH, heating in a water bath, incubating at room temperature, centrifuging, discarding the precipitate, and retaining the supernatant; S4, lyophilizing and grinding the supernatant obtained in S3 to obtain the self-assembled supramolecular peptide nanozyme material. This invention discloses a self-assembled supramolecular peptide nanozyme material, its preparation method, and its application. This supramolecular peptide nanozyme, when applied to food quality and safety testing, can achieve visual and sensitive colorimetric detection of biogenic amines in spoiled food.

Description

Technical Field

[0001] This invention belongs to the field of biomaterial preparation technology, specifically relating to a self-assembled supramolecular peptide nanoenzyme material, its preparation method, and its application. Background Technology

[0002] During processing, transportation, storage, and sales, food is susceptible to microbial contamination, leading to protein decomposition and the production of volatile biogenic amines. This can cause food spoilage and threaten food safety. While traditional methods for detecting volatile biogenic amines offer high accuracy, they suffer from drawbacks such as complex operation, reliance on specialized equipment, and high costs, making it difficult to meet the demands for real-time, high-throughput, and low-cost food quality monitoring. Therefore, developing a simple, efficient, and economical biogenic amine detection technology is of significant practical importance.

[0003] Natural enzymes, due to their highly efficient catalytic activity, are widely used in pollutant degradation and colorimetric sensing. However, natural enzymes have inherent drawbacks such as limited sources, poor stability, susceptibility to environmental factors such as temperature, high cost, and low reusability, which limit their large-scale application in practical scenarios. Nanozymes, as alternatives to natural enzymes, mimic the catalytic active sites of natural enzymes and possess 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, making it difficult to meet the stringent requirements of food testing in terms of biocompatibility and safety. Given the limitations of existing biogenic amine detection methods and the safety concerns of nanozyme materials, there is an urgent need for a self-assembled nanozyme material and detection technology based on biocompatible supramolecular peptides to meet the demands of food quality and safety monitoring for real-time performance, cost-effectiveness, and safety. Summary of the Invention

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

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing self-assembled supramolecular peptide nanozyme materials includes the following steps:

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

[0009] S2. Dissolve heme chloride in dimethyl sulfoxide and stir until homogeneous to obtain solution B;

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

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

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

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

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

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

[0016] Preferably, in S3, the centrifugation speed is 10,000 rpm and the centrifugation time is 10 min.

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

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

[0019] This invention also provides the application of the self-assembled supramolecular peptide nanozyme material as described above or the self-assembled supramolecular peptide nanozyme material prepared by the preparation method above in the detection of biogenic amines.

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

[0021] This invention discloses a self-assembled supramolecular peptide nanozyme material. Using biocompatible β-alanyl-L-histidine and heme chloride as raw materials, it self-assembles into a nanofiber structure under mild conditions through precise proportioning, avoiding the use of toxic reagents and complex processes in traditional nanomaterial preparation, thus reducing costs. This material mimics the active site of natural horseradish peroxidase, achieving highly efficient peroxidase-like catalytic activity through the coordination and non-covalent interaction between the histidine side chain and heme iron ions, completing the colorimetric reaction within 200 seconds. This material is suitable for detecting volatile biogenic amines such as putrescine, cadaverine, and histamine in various foods including meat, fish, and dairy products. It combines advantages such as green safety, high catalytic efficiency, high detection sensitivity, and low cost, providing a real-time, economical, and reliable innovative solution for food quality and safety monitoring. Furthermore, it has the potential to expand into the detection of multiple pollutants such as mycotoxins and pesticide residues, demonstrating significant technological advancement and industrial application value.

[0022] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0023] Figure 1 The absorbance statistics of the self-assembled supramolecular peptide nanoenzyme materials provided in Examples 1-5 are shown in the figure.

[0024] Figure 2 The morphology characterization image of the self-assembled supramolecular peptide nanozyme material provided in Example 3 is shown with a scale bar of 5 μm.

[0025] Figure 3 The results show the peroxidase-like catalytic activity of the self-assembled supramolecular peptide nanozyme material provided in Example 3, wherein... Figure 3 In the figure, A represents the Michaelis-Menten curves with a fixed hydrogen peroxide concentration and varying concentrations of 3,3',5,5'-tetramethylbenzidine. Figure 3 B in the figure represents a double reciprocal plot obtained by fixing the hydrogen peroxide concentration and changing the concentration of 3,3',5,5'-tetramethylbenzidine. Figure 3 In the figure, C represents the Michaelis-Menten curves with a fixed concentration of 3,3',5,5'-tetramethylbenzidine and varying hydrogen peroxide concentrations. Figure 3 D in the figure is a double reciprocal plot with a fixed concentration of 3,3',5,5'-tetramethylbenzidine and a varying concentration of hydrogen peroxide.

[0026] Figure 4 The experimental parameters for the catalytic colorimetric reaction of 3,3',5,5'-tetramethylbenzidine with the self-assembled supramolecular peptide nanozyme material provided in Example 3 are shown below. Figure 4 In this context, A represents the experimental results of the catalytic system at different reaction times; Figure 4B in the figure represents the experimental results when different amounts of 3,3',5,5'-tetramethylbenzidine were added to the catalytic system. Figure 4 In the figure, C represents the experimental results when different amounts of hydrogen peroxide were added to the catalytic system. Figure 4 D in the figure represents the experimental results when different amounts of self-assembled supramolecular peptide nanozyme materials are added to the catalytic system.

[0027] Figure 5 The sensitivity test results for the self-assembled supramolecular peptide nanozyme material provided in Example 3 are as follows: Figure 5 In the figure, A represents the UV-Vis absorption spectrum of the catalytic system based on self-assembled supramolecular peptide nanozyme material in the presence of different concentrations of histamine. Figure 5 B in the figure represents the linear regression curve between histamine concentration and absorbance at 652 nm. Detailed Implementation

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

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

[0030] Source of experimental materials:

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

[0032] Example 1

[0033] A method for preparing self-assembled supramolecular peptide nanozyme materials includes the following steps:

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

[0035] S2. Dissolve 30 μM heme chloride in 1.2 mL of dimethyl sulfoxide and stir until homogeneous to obtain solution B;

[0036] S3. Mix solution A and solution B thoroughly, wherein the molar ratio of β-alanyl-L-histidine in solution A to heme chloride in solution B is 5 mol: 1 mol. Adjust the pH of the solution to 7.0, heat in a water bath at 60°C for 1 h, incubate at room temperature for 12 h, centrifuge at 10000 rpm for 10 min, discard the precipitate and keep the supernatant.

[0037] S4. The supernatant obtained in S3 is freeze-dried at -60℃ for 24 hours and then ground to obtain a self-assembled supramolecular peptide nanoenzyme material.

[0038] Example 2

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

[0040] S2. Dissolve 30 μM heme chloride in 1.2 mL of dimethyl sulfoxide and stir until homogeneous to obtain solution B;

[0041] S3. Mix solution A and solution B thoroughly, wherein the molar ratio of β-alanyl-L-histidine in solution A to heme chloride in solution B is 7.5 mol: 1 mol. Adjust the pH of the solution to 7.0, heat in a water bath at 60°C for 1 h, incubate at room temperature for 12 h, centrifuge at 10000 rpm for 10 min, discard the precipitate and keep the supernatant.

[0042] S4. The supernatant obtained in S3 is freeze-dried at -60℃ for 24 hours and then ground to obtain a self-assembled supramolecular peptide nanoenzyme material.

[0043] Example 3

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

[0045] S2. Dissolve 30 μM heme chloride in 1.2 mL of dimethyl sulfoxide and stir until homogeneous to obtain solution B;

[0046] S3. Mix solution A and solution B thoroughly, wherein the molar ratio of β-alanyl-L-histidine in solution A to heme chloride in solution B is 10 mol: 1 mol. Adjust the pH of the solution to 7.0, heat in a water bath at 60°C for 1 h, incubate at room temperature for 12 h, centrifuge at 10000 rpm for 10 min, discard the precipitate and keep the supernatant.

[0047] S4. The supernatant obtained in S3 is freeze-dried at -60℃ for 24 hours and then ground to obtain a self-assembled supramolecular peptide nanoenzyme material.

[0048] Example 4

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

[0050] S2. Dissolve 30 μM heme chloride in 1.2 mL of dimethyl sulfoxide and stir until homogeneous to obtain solution B;

[0051] S3. Mix solution A and solution B thoroughly, wherein the molar ratio of β-alanyl-L-histidine in solution A to heme chloride in solution B is 12.5 mol: 1 mol. Adjust the pH of the solution to 7.0, heat in a water bath at 60°C for 1 h, incubate at room temperature for 12 h, centrifuge at 10000 rpm for 10 min, discard the precipitate and keep the supernatant.

[0052] S4. The supernatant obtained in S3 is freeze-dried at -60℃ for 24 hours and then ground to obtain a self-assembled supramolecular peptide nanoenzyme material.

[0053] Example 5

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

[0055] S2. Dissolve 30 μM heme chloride in 1.2 mL of dimethyl sulfoxide and stir until homogeneous to obtain solution B;

[0056] S3. Mix solution A and solution B thoroughly, wherein the molar ratio of β-alanyl-L-histidine in solution A to heme chloride in solution B is 15 mol: 1 mol. Adjust the pH of the solution to 7.0, heat in a water bath at 60°C for 1 h, incubate at room temperature for 12 h, centrifuge at 10000 rpm for 10 min, discard the precipitate and keep the supernatant.

[0057] S4. The supernatant obtained in S3 is freeze-dried at -60℃ for 24 hours and then ground to obtain a self-assembled supramolecular peptide nanoenzyme material.

[0058] The effectiveness of the self-assembled supramolecular peptide nanozyme materials provided in Examples 1-5 above was verified through the following experiments.

[0059] 1. The catalytic activity of the self-assembled supramolecular peptide nanozyme materials provided in Examples 1-5 was determined using the following method: 1 mg of the self-assembled supramolecular peptide nanozyme material provided in Examples 1-5 was 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, respectively. The UV-Vis absorption peak at 652 nm was recorded using a UV spectrophotometer. The results are as follows: Figure 1 .

[0060] Depend on Figure 1 It is known that the catalytic activity of the self-assembled 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 heme chloride in solution B was 10:1, the catalytic activity basically reached a stable state.

[0061] 2. The morphology of the self-assembled supramolecular peptide nanozyme material provided in Example 3 was characterized, and the results are as follows: Figure 2 .

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

[0063] 3. The peroxidase-like catalytic performance of the self-assembled supramolecular peptide nanozyme material provided in Example 3 was determined. The experimental method is as follows: The self-assembled supramolecular peptide nanozyme material was added to a buffer solution of 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide, and the absorbance of the corresponding system at 652 nm was recorded. By changing the concentration of TMB (0-30 mM) at a fixed hydrogen peroxide concentration (130 mM), or by changing the concentration of hydrogen peroxide (0-150 mM) at a fixed TMB concentration (22 mM), the kinetic analysis of the nanozyme based on TMB and hydrogen peroxide as substrates was performed using the self-assembled supramolecular peptide nanozyme material (5 mg / mL). The initial reaction rate at different substrate concentrations was calculated according to the Lambert-Beer law. The data were further fitted using the Michaelis equation to calculate typical parameters of enzyme activity, including the Michaelis constant (K). m ), maximum reaction rate (V) max ) and specific activity (SA). Among them, specific activity is the core indicator for measuring enzyme purity and catalytic efficiency, defined as the ability of a 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] Use the following formula (2) to fit the data:

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

[0068] Specific vitality is 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] The initial reaction rate was determined by fixing one substrate concentration and changing the concentration of another substrate, and the Michaelis-Menten equation curves for 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide were plotted. The results are as follows: Figure 3 .

[0072] Depend on Figure 3 A and Figure 3 Calculations in B show that the maximum reaction rate V of 3,3',5,5'-tetramethylbenzidine is... max Michaelis constant K m The specific activity SA was 13.33 × 10⁻⁶. -7 Ms -1 9.013M and 16.00U / mg.

[0073] Depend on Figure 3 C and Figure 3 The calculations in the diagram show that the maximum reaction rate V of hydrogen peroxide is... max Michaelis constant K m The specific activity SA was 16.89 × 10⁻⁶. -7 Ms -1 69.99M and 20.26U / mg.

[0074] Overall, across both substrate concentration ranges, the inverse curves of the initial reaction rate versus substrate concentration both show a series of straight lines without intersection, consistent with a typical ping-pong reaction mechanism. This is because, during the catalytic process, the self-assembled supramolecular peptide nanozyme material binds to 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, such as... Figure 4 As shown in Figure A, the intensity of its UV absorption peak at 652 nm basically stabilizes at 200 s; therefore, 200 s was chosen as the catalytic time for subsequent experiments. Furthermore, the effects of the contents of 3,3',5,5'-tetramethylbenzidine, hydrogen peroxide, and self-assembled supramolecular peptide nanozyme material on the enzyme's catalytic activity were also investigated. The results are as follows: Figure 4 B- Figure 4 As shown in D, the final determination of the content of 3,3',5,5'-tetramethylbenzidine in 1 mL of the catalytic system was 22 mM; the content of hydrogen peroxide was 130 mM; and the content of the self-assembled supramolecular peptide nanozyme material was 5 mg / mL.

[0076] 5. Sensitivity test:

[0077] Histamine standard solutions of different concentrations were prepared using phosphate buffer as solvent. Under optimal conditions, these solutions were added to the 3,3',5,5'-tetramethylbenzidine colorimetric system catalyzed by the self-assembled supramolecular peptide nanozyme synthesized in Example 3. The wavelength measurement range was set to 450-800 nm, and the value of the UV-Vis absorption peak at 652 nm was recorded. Based on the following formula (4), a colorimetric response platform for histamine detection was constructed using the obtained data, and the results are as follows. Figure 5 .

[0078] Formula (4):

[0079] A = KC + b;

[0080] Where C represents the concentration of the target analyte, 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 B, as the histamine concentration increased from 1 mg / L to 200 mg / L, the UV absorption intensity of the nanozyme catalytic system at 652 nm gradually decreased. Using the UV absorption peak intensity at 652 nm as the ordinate and the histamine concentration as the abscissa, a linear fitting analysis was performed, revealing a good linear relationship between the two (y = 0.0079x + 2.054, R0). 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 and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions 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-assembled supramolecular peptide nanoenzyme material, characterized in that, Includes the following steps: S1. Dissolve β-alanyl-L-histidine in ultrapure water and stir until homogeneous to obtain solution A; S2. Dissolve heme chloride in dimethyl sulfoxide and stir until homogeneous to obtain solution B; S3. Mix solution A and solution B until homogeneous, 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 nanoenzyme material. In S3, the molar ratio of β-alanyl-L-histidine in solution A to heme chloride in solution B is 5~15:1; In S3, the pH of the adjusted solution is 4.0~8.0; In S3, the water bath heating temperature is 40℃~70℃, and the heating time is 0.5h~2h; In S3, the room temperature incubation time is 10h~20h; In S3, the centrifugation speed is 10000 rpm and the centrifugation time is 10 min; In S4, the freeze-drying temperature is -50℃ to -80℃, and the freeze-drying time is 24h to 48h.

2. The self-assembled supramolecular peptide nanozyme material prepared by the preparation method described in claim 1.

3. The application of the self-assembled supramolecular peptide nanozyme material as described in claim 2 in the detection of biogenic amines.

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