Bionic enzyme and application thereof in degrading aflatoxin

Abstract

The application belongs to the technical field of biology and particularly relates to a kind of biomimetic enzyme and its application in degrading aflatoxin.The raw material of the biomimetic enzyme includes polypeptide and divalent copper ion, the sequence of the polypeptide is shown in SEQ ID No.1, specifically: Ac-HCHLKLKLKL-CONH2, the N terminal of the polypeptide is acetylated, and the C terminal is amidated.The biomimetic enzyme is constructed by sequencing design and metal coordination to construct an active site, is stable in catalyzing AFB1 rupture in wide pH and temperature, has the advantages of good tolerance, high catalytic efficiency, and has potential application advantages in the application in grain and feed purification.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a biomimetic enzyme and its application in the degradation of aflatoxin. Background Technology

[0002] Aflatoxin (AF) is a class of mycotoxins produced by Aspergillus flavus (… Aspergillus flavus ) and parasitic aspergillus ( A. parasiticus Aflatoxin is a secondary metabolite produced by fungi such as B1, B2, G1, and G2 on high-starch or high-oil substrates such as grains, nuts, and animal feed. Among these, AFB1 is the most toxic and is classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC). Long-term consumption of food containing aflatoxin can lead to hepatocyte necrosis, liver failure, and significantly increase the risk of liver cancer; it also has serious toxic effects on livestock, thus threatening human food safety and public health.

[0003] Currently, methods for removing or degrading aflatoxin mainly fall into three categories: physical, chemical, and biological. Physical methods, such as adsorbents (activated carbon, kaolin), heating, and steam treatment, are simple and easy to implement, but often involve nutrient loss and high processing costs. Chemical methods, such as ozone, hydrogen peroxide, and alkaline reagent treatment, have high degradation efficiency, but may leave harmful chemical residues in food, and subsequent processing is more complicated and requires harsh operating conditions. In contrast, biodegradation methods utilize microorganisms or enzymes to specifically break down the molecular structure of aflatoxin under mild conditions, without producing secondary pollution, and are increasingly becoming a research hotspot.

[0004] In biodegradation, natural enzymes such as aflatoxin oxidase, cyclooxygenase, flavin-dependent monooxygenase, and laccase have been shown to effectively convert AFB1 into products with significantly reduced toxicity. However, these natural enzymes suffer from a series of drawbacks, including high preparation costs, cumbersome purification procedures, poor storage and application stability, sensitivity to pH and temperature, and susceptibility to proteolytic degradation, which limit their large-scale industrial application. Especially in complex matrices such as grain and feed processing, enzyme activity often decreases significantly, and they are difficult to reuse.

[0005] To overcome the shortcomings of natural enzymes, research on biomimetic enzyme catalysts has gradually emerged. Biomimetic enzyme catalysts refer to simplified polypeptide sequences designed from the amino acid residues of the active site of natural enzymes through chemical synthesis or solid-phase peptide synthesis techniques. Metal ions or small molecule cofactors are introduced to mimic the three-dimensional structure and acid-base properties of the enzyme's catalytic site. These mimics combine the catalytic efficiency of enzymes with the stability of small molecule materials, maintaining activity over a wide pH and temperature range. Biomimetic enzyme catalysts not only possess a high degree of structural tunability by precisely controlling the electronic environment of the catalytic center and its substrate binding ability through alterations in amino acid sequence, chain length, or cofactor coordination, but also achieve controllable purity and low-cost mass production thanks to mature solid-phase peptide synthesis technology. Furthermore, they exhibit excellent tolerance to organic solvents, temperature, and pH conditions and are not easily degraded by natural proteases, thus allowing for repeated use. Summary of the Invention

[0006] This invention provides a biomimetic enzyme catalyst based on functionalized peptides for the efficient degradation of aflatoxin. Through sequencing design and metal coordination to construct biomimetic sites, it stably catalyzes the cleavage of AFB1 over a wide range of pH and temperature conditions, exhibiting advantages such as good tolerance and high catalytic efficiency, and showing potential application advantages in grain and feed purification.

[0007] The technical solution of this invention is as follows:

[0008] In a first aspect, the present invention provides a biomimetic enzyme, the biomimetic enzyme comprising polypeptides and divalent copper ions;

[0009] The sequence of the polypeptide is shown in SEQ ID No. 1, specifically: Ac-HCHLKLKLKL-CONH2;

[0010] The peptide was acetylated at its N-terminus and amidated at its C-terminus.

[0011] The sequence of the polypeptide is: Ac-His Cys His Leu Lys Leu Lys Leu Lys Leu-CONH2.

[0012] As a specific embodiment of this application, the molecular structure of the polypeptide is shown below. Figure 13 .

[0013] As a specific embodiment of this application, the concentration ratio of divalent copper ions to polypeptides is 1-3.5:1.

[0014] As a specific embodiment of this application, the biomimetic enzyme has a β-sheet structure.

[0015] Secondly, this application provides a method for preparing the above-mentioned biomimetic enzyme, in which the polypeptide is dissolved in acetonitrile, a buffer solution containing divalent copper ions is added, and then the mixture is incubated in the dark.

[0016] As a specific embodiment of this application, the incubation also includes a buffer solution.

[0017] As a specific embodiment of this application, the incubation buffer includes phosphate buffer solution (PBS), tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution (Tris-HCl), and 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution (Hepes).

[0018] As a preferred embodiment of this application, Hepes buffer solution is preferred.

[0019] In a specific embodiment of this application, the pH of the buffer solution is 8.0 ± 0.5.

[0020] As a specific embodiment of this application, the incubation time is ≥10h.

[0021] In a preferred embodiment of this application, the incubation time is 10-20 hours.

[0022] Thirdly, a catalyst is provided, comprising the above-described biomimetic enzyme or a biomimetic enzyme prepared using the above-described preparation method.

[0023] Fourthly, the application of the above-mentioned catalyst in the degradation of aflatoxin is provided.

[0024] The amino acid sequence of the polypeptide consists of two parts: a core catalytic module (HCH) and a module that promotes hydrophobic effects and secondary structure formation (LKLKLKL). This minimalist biomimetic design retains the metal catalytic function while significantly reducing molecular complexity. This design concept is highly related to the catalytic mechanism of natural metalloenzymes. Taking laccase as an example, it is a typical multi-copper oxidase that can efficiently catalyze substrate oxidation reactions. In the natural system, the active site of laccase is formed by the synergistic effect of copper ions in different valence states. Its active site contains T1 copper (coordinated by two histidine and cysteine) and T2 / T3 trinuclear cluster (coordinated by three histidines). Based on the analysis of the structure of the copper active site of natural laccase, this invention has developed a biomimetic design approach based on polypeptides, designing the polypeptide sequence HCHLKLKLKL, which consists of a core catalytic module (HCH) and a module that promotes hydrophobic effects and secondary structure formation (LKLKLKL). The catalytic motif is composed of the amino acid "H–C–H", in which histidine (H) and cysteine ​​(C) can co-complex Cu through the imidazole ring and thiol group. 2+ Reconstructing Cu in natural enzymes 2+The high affinity between the coordinating amino acid and the "LKLKLKL" uses alternating hydrophobic leucine (L) and hydrophilic lysine (K), which can not only form an amphiphilic β-sheet structure with one side hydrophobic and the other side charged in the aqueous phase, but also spatially orient the coordinating segment, so that the side chains of the three coordinating residues are tightly aggregated in three dimensions, further stabilizing the stereoconformation of the copper-peptide complex.

[0025] Beneficial effects of the present invention

[0026] Based on the structural characteristics of the active site of natural enzymes, this invention constructs a peptide-copper-based laccase biomimetic enzyme catalyst. This biomimetic enzyme catalyst has the advantages of simple preparation, high catalytic activity, and good stability, and will promote the widespread application of biomimetic enzymes in the degradation of aflatoxin.

[0027] The biomimetic enzyme exhibits excellent stability and high catalytic performance over a wide pH and temperature range, and can rapidly and efficiently degrade a variety of aflatoxins (AFB1, AFB2, AFG1, AFG2). Moreover, its catalytic efficiency is higher than that of natural laccase, giving it significant advantages in applications. Attached Figure Description

[0028] Figure 1 The Fourier transform infrared spectrum of the biomimetic enzyme;

[0029] Figure 2 The circular dichroism chromatogram of a biomimetic enzyme;

[0030] Figure 3 Transmission electron microscopy image of a biomimetic enzyme;

[0031] Figure 4 The graph shows the degradation curves of AFB1 by the biomimetic enzyme under different pH buffer conditions.

[0032] Figure 5 The graphs show the degradation curves of AFB1 by multiple biomimetic enzymes under different temperature conditions.

[0033] Figure 6 Figure 1 shows the degradation of different concentrations of AFB1 by multiple biomimetic enzymes at different times.

[0034] Figure 7 The graph shows the relative degradation rate of AFB1 by the biomimetic enzyme under different oxidizing media.

[0035] Figure 8 The graph shows the degradation rate of aflatoxin by the biomimetic enzyme at different time points.

[0036] Figure 9 Linear fitting plot of biomimetic enzyme degrading aflatoxin at different times;

[0037] Figure 10Liquid phase diagram of biomimetic enzyme degradation of different aflatoxins;

[0038] Figure 11 This is a comparison chart of the degradation rates of different aflatoxins by biomimetic enzymes.

[0039] Figure 12 A comparison diagram of the degradation of aflatoxin by biomimetic enzymes and publicly disclosed patented polypeptide-mimicking enzymes;

[0040] Figure 13 This is a molecular structure diagram of a polypeptide. Detailed Implementation

[0041] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise specified, the technical means used in the specific embodiments of the present invention are all methods known to those skilled in the art.

[0042] Example 1: Preparation and structural characterization of biomimetic enzymes

[0043] I. Preparation of Bionic Enzymes

[0044] 1 mg of lyophilized peptide (the sequence of the peptide is: Ac-His Cys His Leu Lys Leu Lys LeuLys Leu-CONH2; or Ac-HCHLKLKLKL-CONH2; the N-terminus of the peptide is acetylated and the C-terminus is amidated; the molecular structure diagram of the peptide is shown in the figure) was prepared. Figure 13 Remove from the -80℃ freezer and allow to stand at room temperature for half an hour. Pre-dissolve in 20 μL of acetonitrile, shake thoroughly, add 20 μL of water, and then add dropwise Hepes buffer (25 mM, pH 8.0) containing different concentrations of CuCl2 (0 mM, 4 mM, 6 mM, 8 mM, 10 mM, 12 mM, 14 mM). Mix well and sonicate for 90 s to obtain a peptide solution with a final concentration of 4 mM. To ensure sufficient self-assembly of the peptide, incubate the peptide solution at room temperature for 12 hours.

[0045] II. To investigate the structure of the biomimetic enzyme, it was characterized by FTIR, CD, and TEM.

[0046] like Figure 1 As shown, the biomimetic enzyme appears in the infrared spectrum at 3483 cm⁻¹. -1 and 3388cm -1 A characteristic absorption peak appears, belonging to -NH2, and at 1669 cm⁻¹... -1 and 1342cm -1 The peak values ​​belong to C=O and CN, respectively, while 832cm -1 The band is caused by NH stretching. Furthermore, at 1623 cm⁻¹... -1 and 1538cm-1 Strong absorption peaks appeared nearby, corresponding to the amide I and amide II regions, respectively, indicating that the biomimetic enzyme formed a β-sheet structure.

[0047] The secondary structure of biomimetic enzymes was determined using a Chirascan circular dichroism spectrometer. For example... Figure 2 As shown, the biomimetic enzyme exhibits strong negative peaks near 198 nm and 218 nm, indicating that the biomimetic enzyme contains both random coil and β-sheet structures. This hybrid structure provides the molecule with sufficient flexibility to facilitate substrate binding and conformational adjustment, while also providing sufficient rigidity to ensure the stability of the active site, thus benefiting substrate catalysis.

[0048] Transmission electron microscopy (TEM) further revealed the morphological characteristics of the biomimetic enzyme. For example... Figure 3 As shown, the biomimetic enzyme aggregates and exhibits a slender, fibrous structure, a typical characteristic of β-sheet peptide assembly. The results are consistent with FTIR and CD.

[0049] Example 2: Optimal conditions for the degradation of aflatoxin by a biomimetic enzyme catalyst

[0050] Aflatoxin B1 (AFB1) is the most potent and widely harmful aflatoxin, exhibiting high carcinogenicity and acute toxicity. This study investigated the optimal reaction conditions using a biomimetic enzyme catalyst to target AFB1. In the experiment, AFB1 standard was dissolved in methanol to prepare a stock solution with a concentration of 1 mg / mL. In the degradation experiment, the biomimetic enzyme was added to a Hepes buffer solution containing aflatoxin and degraded for a certain period (5–90 min) under different temperatures (25–75 °C) and pH values ​​(5–10), with a final substrate concentration of 5 μg / mL. After the reaction, the samples were analyzed using HPLC. HPLC detection was performed using an Agilent Eclipse Plus C18 column (5 μm, 4.6 × 250 mm), with an acetonitrile-water solution (55:45 v:v) as the mobile phase, a column temperature of 40 °C, an injection volume of 20 μL, a flow rate of 0.6 mL / min, and a UV detector at a wavelength of 365 nm.

[0051] The percentage of aflatoxin degradation was calculated using the following equation:

[0052] Aflatoxin degradation rate (%) = [(C0 - C] t In the formula ) / C0]×100, C0 represents the concentration of aflatoxin in the control group, C t This indicates the concentration of aflatoxin in the experimental group.

[0053] The results are as follows Figure 4As shown, after reacting at 37℃ for 6 hours, the biomimetic enzyme catalyst exhibited a higher AFB1 degradation rate under alkaline conditions (pH 9.0). Subsequently, the effect of temperature on degradation was investigated under optimal pH conditions, and the results are as follows. Figure 5 At 65℃, the degradation rate of AFB1 can reach 100%. Furthermore, the effect of AFB1 concentration on the degradation rate was investigated, such as... Figure 6 When the substrate concentration was 1 μg / mL and 5 μg / mL, the degradation rate reached 100% after 90 minutes of reaction under optimal conditions, while the degradation rate was about 95% when the concentration was 10 μg / mL, indicating that the biomimetic enzyme catalyst has the ability to efficiently degrade AFB1.

[0054] Furthermore, this invention also investigated the effect of oxidizing media on the catalytic activity of biomimetic enzymes. Because natural enzymes have low redox potential, redox media are usually required to accelerate the catalytic reaction when oxidizing non-phenolic substrates. In the degradation experiment of aflatoxin AFB1 by the biomimetic enzyme, the effects of different oxidizing media were investigated. Specifically, 50 μL of oxidizing media stock solution (10 mM) was added to a reaction system with a total volume of 500 μL. The final concentration of the biomimetic enzyme in the reaction system was 0.2 mM, and the final concentration of the substrate AFB1 was 5 μg / mL. The degradation experiment was conducted at 65°C and 120 r / min on a shaker for 90 minutes. The results are as follows: Figure 7 As shown, the addition of oxidizing media AS (acetylsuccinone) and SA (syringaldehyde) and TBHQ (tert-butylhydroquinone) significantly improves the degradation rate of aflatoxin AFB1 by the biomimetic enzyme catalyst, increasing the catalytic activity by about 20%.

[0055] Example 3: Degradation of different aflatoxins by biomimetic enzymes

[0056] To further verify the broad degradation ability of biomimetic enzymes for multiple aflatoxins, degradation studies were conducted on three other aflatoxins (AFB2, AFG1, and AFG2) using biomimetic enzyme catalysts. Figure 8-11 As shown, using the same degradation conditions as in Example 2, the degradation efficiency of the biomimetic enzymes for aflatoxin was, in descending order: AFB1 > AFB2 > AFG1 > AFG2, with the B series showing higher degradation efficiency than the G series. We further investigated the degradation efficiency using first-order kinetics (-In(C...). t Analysis of the reaction time t ( / C0) showed that the biomimetic enzyme exhibited different degradation kinetics for different aflatoxins, such as Figure 9As shown, the degradation of aflatoxin by the biomimetic enzymes all conforms to first-order kinetics. AFB1 and AFB2 exhibit relatively fast degradation rates, AFG1 has a moderate degradation rate, while AFG2 is the slowest among the four toxins. Overall, the biomimetic enzyme catalysts demonstrate degradation activity for all aflatoxins, and the first-order kinetic parameters (rate and goodness of fit) for each toxin's degradation differ, providing data support and theoretical basis for subsequent targeted optimization of the degradation process and efficient removal of aflatoxins.

[0057] Comparative Example 1

[0058] Using the polypeptide-mimicking enzyme disclosed in Chinese Patent CN118388597A, under the same degradation conditions as in Example 2, the results showed that the degradation efficiency of AFB1 under low-temperature conditions (25~45℃) was significantly lower than that of the biomimetic enzyme in this patent. Figure 12 This further illustrates that the biomimetic enzyme of this application has a high aflatoxin degradation capacity over a wide temperature range, which is a significant advantage over the polypeptide mimic enzyme disclosed in patent CN118388597A.

Claims

1. A biomimetic enzyme, characterized in that, The biomimetic enzyme includes polypeptides and divalent copper ions; The sequence of the polypeptide is shown in SEQ ID No. 1, specifically: Ac-HCHLKLKLKL-CONH2; The peptide was acetylated at its N-terminus and amidated at its C-terminus; The biomimetic enzyme described above has a β-sheet structure.

2. The biomimetic enzyme according to claim 1, characterized in that, The concentration ratio of divalent copper ions to polypeptides is 1-3.5:

1.

3. A method for preparing the biomimetic enzyme according to claim 1 or 2, characterized in that, The polypeptide was incubated with divalent copper ions in acetonitrile solution in the dark.

4. The preparation method according to claim 3, characterized in that, The incubation also includes a buffer solution.

5. The preparation method according to claim 4, characterized in that, The pH of the buffer solution is 8.0 ± 0.

5.

6. The preparation method according to claim 4 or 5, characterized in that, The incubation time is ≥10h.

7. A catalyst, characterized in that, The biomimetic enzymes include those described in 1 or 2, or those prepared by the method described in any one of claims 3-6.

8. The use of the catalyst according to claim 7 in the preparation of aflatoxin degradation products.

9. The application according to claim 8, characterized in that, Used to degrade aflatoxin in food and / or feed.

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