A polypeptide mimetic enzyme, an immobilized mimetic enzyme and its application in AFB1 degradation

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a polypeptide mimetic enzyme, an immobilized mimetic enzyme and its application in AFB1 degradation. In view of the problems existing in the degradation of aflatoxin B1 by mimetic enzymes at this stage, the present invention provides a polypeptide mimetic enzyme, the raw materials include polypeptides and copper ions; the amino acid sequence of the polypeptide, as shown in SEQ ID No.1, is specifically Ac‑ICVHLHLHVHI‑CONH2; the N-terminus of the polypeptide is acetylated and the C-terminus is amidated. In order to further improve the stability and applicability of enzyme mimics under harsh conditions, a simple fixation method for manufacturing organic-inorganic hybrid nanoflowers using a metal phosphate crystalline compound as an inorganic component and a promising polypeptide mimetic enzyme as an organic component is also provided to prepare an immobilized enzyme for the degradation of aflatoxin B1 under harsh conditions.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a polypeptide mimetic enzyme, an immobilized mimetic enzyme and applications thereof in AFB1 degradation. Background Art

[0002] AFB1 is one of the most toxic aflatoxins known and is recognized as a strong carcinogen. For humans, high levels of AFB1 exposure can lead to aflatoxicosis, which manifests as severe liver damage and can even be fatal. Long-term low-dose exposure is closely associated with the development of liver cancer, especially in people infected with the hepatitis B virus. In addition, AFB1 can cause immunosuppression and growth retardation in children. In animals, AFB1 can also cause liver damage, decreased immunity, growth stagnation and reduced reproductive capacity, which is particularly worrying in livestock because contaminated feed can directly affect the health and productivity of animals. In order to reduce the harm of aflatoxins, strict standards for aflatoxin content in food and feed have been established around the world, and crop management and storage recommendations have been made to prevent mold growth and toxin contamination.

[0003] At present, the methods used to remove AFB1 at home and abroad can be divided into physical methods, chemical methods and biological methods. Physical methods mainly use adsorbent adsorption, high temperature heating, irradiation and other methods to remove AFB1. Chemical methods mainly use some strong oxidants, strong acids and strong bases to destroy the toxic sites of AFB1. However, these two methods will affect product quality and destroy nutritional components, and are obviously not suitable for food detoxification. Compared with traditional physical and chemical methods, enzyme degradation in biological methods removes or degrades AFB1 by biocatalysis. Its conditions are mild, specific, safe and effective, and the by-products are less toxic. Therefore, it is regarded as a more promising strategy for degrading AFB1. However, the separation and purification of natural enzymes is time-consuming and laborious. At the same time, it also faces challenges such as poor stability, difficult recycling, and limited reusability in complex environments, which seriously limit its practical application. Mimetic enzymes are artificial biocatalysts inspired by natural enzymes. Compared with natural enzymes, mimetic enzymes have advantages such as higher catalytic efficiency, simpler structure, and higher stability. These advantages also help mimetic enzymes become catalysts that are more widely used in practical industrial applications. Nowadays, many mimetic enzymes of various materials have been reported, such as magnetic ferroferric oxide, carbon nanotubes, gold nanoparticles, metal organic frameworks and peptides. Although these mimetic enzymes have high catalytic activity, they have disadvantages such as poor substrate selectivity, poor stability and poor reusability. Moreover, some nanomaterials may also have potential toxicity to the environment and organisms, and the preparation of high-quality and high-performance nanomaterials also requires complex synthesis processes. These disadvantages make them challenging in practical applications. Among them, peptides, as an ideal biological molecule, have a very high similarity in structure and chemical properties with natural enzymes; not only that, the structure of peptides is simpler, easier to modify and study the catalytic mechanism, and the preparation process is also more efficient and lower cost; these characteristics have laid the foundation for peptide mimetic enzymes to be widely constructed and used to degrade AFB1; but under harsh conditions such as high temperature, the degradation effect is not good, and conventional peptide mimetic enzymes are degraded under homogeneous conditions and are not easy to separate. Summary of the invention

[0004] In view of the problems existing in the degradation of aflatoxin B1 by mimetic enzymes at this stage, the present invention provides a polypeptide mimetic enzyme. In order to further improve the stability and applicability of enzyme mimics under harsh conditions, a simple fixation method for manufacturing organic-inorganic hybrid nanoflowers using metal phosphate crystalline compounds as inorganic components and promising polypeptide mimetic enzymes as organic components is provided to prepare immobilized enzymes for the degradation of aflatoxin B1 under harsh conditions.

[0005] The technical solution of this application is as follows:

[0006] The first purpose of the present application is to protect a polypeptide mimetic enzyme, the raw materials of which include polypeptide and copper ions;

[0007] The amino acid sequence of the polypeptide is shown in SEQ ID No. 1, specifically Ac-ICVHLHLHVHI -CONH2;

[0008] The N-terminus of the polypeptide is acetylated, and the C-terminus is amidated.

[0009] According to some embodiments of the present application, the polypeptide molecular structure is:

[0010]

[0011] And / or, the concentration ratio of copper ions to polypeptides is 1-3:1.

[0012] The second purpose of the present application is to protect the preparation method of the above-mentioned polypeptide mimetic enzyme, in which the polypeptide is incubated with copper ions at a pH of 6-10 in the dark overnight.

[0013] According to some embodiments of the present application, the incubation is performed under a phosphate buffer system.

[0014] The third purpose of the present application is to protect the use of the above-mentioned polypeptide mimetic enzyme or the polypeptide mimetic enzyme prepared by the above-mentioned preparation method in the degradation of AFB1 aflatoxin B1.

[0015] The fourth object of the present application is to protect an immobilized mimetic enzyme, wherein the immobilized mimetic enzyme is in the shape of a flower and comprises an inorganic component and an organic component;

[0016] The inorganic component includes a metal phosphate crystalline compound;

[0017] The organic component includes the above-mentioned polypeptide mimetic enzyme or the polypeptide mimetic enzyme prepared by the above-mentioned preparation method.

[0018] According to some embodiments of the present application, the mass ratio of inorganic components to organic components is 0.8-3.5;

[0019] According to some preferred embodiments of the present application, the mass proportions of inorganic components and organic components are 0.8, 1.7, 2.6, and 3.5, respectively.

[0020] The metal in the metal phosphate crystals is derived from copper sulfate or copper nitrate, and the phosphate radical is derived from phosphate buffer.

[0021] According to some embodiments of the present application, the phosphate buffer is selected from disodium hydrogen phosphate or potassium dihydrogen phosphate.

[0022] The fifth purpose of the present application is to protect the preparation method of the above-mentioned immobilized mimetic enzyme, which can be achieved by mixing the polypeptide mimetic enzyme with phosphate buffer, copper sulfate or copper nitrate and incubating the mixture.

[0023] The sixth purpose of the present application is to protect the use of the above-mentioned immobilized mimetic enzyme or the immobilized mimetic enzyme prepared by the above-mentioned preparation method in the degradation of AFB1 aflatoxin B1.

[0024] Based on the analysis of the laccase structure and the idea of ​​​​mimicking enzymes, a polypeptide sequence (ICVHLHLHVHI) derived from the active domain of laccase was designed, in which hydrophobic amino acids (I: isoleucine, V: valine, L: leucine) and hydrophilic amino acids (C: cysteine, H: histidine) are arranged alternately, and can form a molecular aggregate with a clear and stable structure and physical and chemical properties through intermolecular non-covalent bond interactions. 2+ The key role of Cu in the active conformation is to add Cu to the designed peptide mimetic enzyme. 2+ , using peptides and Cu 2+ The binding of proteins to form aggregates was used to obtain a stable three-dimensional structure, and its structure and catalytic activity, as well as its role in the degradation of AFB1, were studied.

[0025] Beneficial effects of the present invention

[0026] The present invention designs a peptide sequence based on the active center of natural laccase, and efficiently combines with copper ions to simulate the structure of the binding site of copper ions and amino acid residues in laccase, achieving a high degree of consistency with natural laccase not only in three-dimensional structure but also in oxidation activity. The simulated enzyme peptide sequence is short and simple, highly active and stable. When the concentration of the polypeptide simulated enzyme is 0.5 mg / mL and the concentration of AFB1 is 0.5 μg / mL, the degradation rate reaches 100% in 3 hours, indicating that this polypeptide simulated enzyme has a significant advantage in degrading AFB1.

[0027] At the same time, the immobilized mimetic enzyme was obtained by synthesizing the mimetic enzyme-copper phosphate inorganic hybrid nanoflower (Peptide-NF) through a one-step precipitation method. The synthesis process of this method is simple, and the immobilized enzyme produced has a large specific surface area while still maintaining high activity and high stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the relative activity of the peptide mimic enzyme when incubated under different copper ion concentration conditions;

[0029] Figure 2 This is the UV absorption spectrum of the peptide mimetic enzyme combined with Congo red;

[0030] Figure 3 The circular dichroism (CD) spectrum of the peptide mimetic enzyme;

[0031] Figure 4 This is the infrared spectrum (FTIR) of the peptide mimetic enzyme;

[0032] Figure 5is the degradation rate of AFB1 at different concentrations by peptide mimetic enzyme;

[0033] Figure 6 This is a graph showing the degradation rate of AFB1 by the peptide mimic enzyme over time at 50°C and pH 9 when the peptide mimic enzyme concentration is 0.5 mg / mL and the AFB1 concentration is 0.5 μg / mL;

[0034] Figure 7 To avoid Cu 2+ Co-incubated peptide self-assembly, with Cu 2+ High performance liquid chromatography (HPLC) graph of AFB1 degradation by co-incubated peptide mimetic enzyme and control group;

[0035] Figure 8 The following are scanning electron microscope (SEM) images of immobilized mimetic enzymes at different scales; the scale of A is 50 μm; the scale of B is 10 μm; the scale of C is 3 μm; the scale of D is 1 μm; the scale of E is 500 nm; and the scale of F is 200 nm.

[0036] Fig. 9 It is the energy spectrum of the immobilized simulated enzyme; A is the collected SEM image, and B, C, D, E, and F correspond to Cu element, C element, N element, P element, and O element, respectively.

[0037] Fig.10 This is a comparison of the degradation rates of AFB1 by the peptide mimetic enzyme and the immobilized mimetic enzyme after being incubated at different temperatures for 1 hour;

[0038] Fig.11 This is the degradation rate of AFB1 after five repeated uses of peptide mimetic enzyme and immobilized mimetic enzyme. DETAILED DESCRIPTION

[0039] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments. Unless otherwise specified, the technical means used in the specific embodiments of the present invention are methods well known to those skilled in the art.

[0040] Example 1: Preparation of polypeptide mimetic enzyme and exploration of catalytic activity:

[0041] The peptide freeze-dried powder was taken out of the refrigerator in advance and placed at room temperature for 30 minutes, then 20μ acetonitrile and 20μ water were added dropwise to dissolve the peptide, and then PB buffer (20mM, pH8) containing CuCl2 (0mM, 4mM, 6mM, 8mM, 10mM, 12mM) was added dropwise to make the final concentration of the peptide 4mM. The above solution was mixed evenly by vortex oscillator and incubated at room temperature in the dark for 24h.

[0042] We investigated whether the designed peptide mimic enzyme had laccase oxidation activity by producing a color reaction through the oxidative coupling of the phenolic compounds 2,4-dichlorophenol (2,4-DP) and 4-aminotriptyline (4-AP). The oxidation products of 2,4-DP and 4-AP have an ultraviolet absorption peak at 510nm, so the effect of copper ion concentration on the oxidation activity of the peptide mimic enzyme can be evaluated by measuring the absorbance. 2+ The co-incubated peptide mimic enzyme and 2,4-DP / 4-AP were mixed and reacted for 30 minutes, so that the final concentration of the peptide mimic enzyme in the reaction system was 0.2mM and the final concentration of 2,4-DP / 4-AP was 0.5mM. Figure 1 As shown, when the peptide mimic enzyme does not bind to Cu 2+ When co-incubated, it was found that it had no obvious activity, but with Cu 2+ Incubation, the peptide mimic enzyme showed obvious oxidative activity, and with the increase of Cu 2+ As the concentration increases, the activity of the peptide mimic enzyme also increases. 2+ The activity reached the highest when the concentration of Cu and peptide reached 2:1. 2+ When the concentration was 2.5 and 3 times of the peptide concentration, the activity decreased. 2+ The optimal binding ratio with the peptide is 2:1.

[0043] Example 2: Structural characterization of peptide mimetic enzymes

[0044] Study on the peptide mimic enzyme in Cu 2+ The CD, FTIR and UV spectra before and after binding with Congo red are shown in the figure. Figure 2-4 As shown. Congo red is one of the most commonly used amyloid-specific dyes. Amyloids are characterized by their ability to bind Congo red. After incubation, 20 μ of the peptide mimetic was added to the Congo red solution (25 μM) and the UV absorption spectrum was measured between 400-650 nm. The results are shown in Figure 2 Show, with Cu 2+ The UV absorption intensity of the co-incubated peptide mimic enzyme is higher than that of the non-incubated peptide mimic enzyme. 2+ The UV absorption intensity of the co-incubated peptide is strong. Figure 3 ) was measured in the range of 185-260nm. After deducting the background signal of PB buffer, the results showed that 2+The secondary structure signal of the co-incubated peptide mimic enzyme is enhanced. According to the calculation on the BESTSEL website, the proportion of α-helix and β-fold structure of the peptide mimic enzyme co-incubated with copper is 44.4%, and the proportion of β-fold structure is 55.6%, while the proportion of α-helix and β-fold of the peptide secondary structure without copper is 52.1%, and 47.9%. Therefore, the addition of copper ions enhances the β-fold structure of the peptide mimic enzyme, which means that the copper ions bind to the peptide and complete self-assembly to make the mimic enzyme structure more stable. In the infrared spectrum ( Figure 4 ), the peptide is at 1623 cm in the amide I and II regions before and after binding with copper ions. -1 and 1541cm -1 This is consistent with the above results, but the signal of the peptide mimic enzyme bound to copper ions is significantly stronger than that of the peptide without copper ions. 2+ It interacts strongly with peptides and forms enzyme mimics.

[0045] Example 3: Degradation of AFB1 by Peptide Mimic Enzyme

[0046] Aflatoxin B1 (AFB1) stock solution (1 mg / mL) was prepared with methanol and diluted with water to a series of concentrations of 50 μg / mL, 10 μg / mL, 5 μg / mL, 2 μg / mL, and 1 μg / mL as AFB1 stock solution. AFB1 stock solutions of different concentrations were mixed with peptide mimetic enzyme (5.5 mg / mL) at 50°C and 120 rpm for 6 h, so that the final concentrations of AFB1 in the reaction system were 5 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.2 μg / mL, and 0.1 μg / mL, respectively, and the final concentration of peptide mimetic enzyme was 0.5 mg / mL. The effect of different concentrations of AFB1 on the activity of peptide mimetic enzymes was investigated by high performance liquid chromatography (mobile phase: acetonitrile: water (55:45), chromatographic column: 4.6×250mm 5μm, injection volume and flow rate were controlled at 20μL and 0.6mL / min, UV detector wavelength was 365nm, and running time was 8min). Figure 5 As shown in Figure 2, when the concentration was 0.5 μg / mL, AFB1 was completely degraded, so the degradation time was further optimized on this basis. The final concentration of AFB1 was set to 0.5 μg / mL and reacted with the peptide mimetic enzyme. The results are shown in Figure 2. Figure 6 As shown, it was found that the degradation rate reached 71% within 1 hour, the degradation rate of AFB1 within 2.5 hours was 98%, and it was completely degraded in three hours. This shows that this peptide mimetic enzyme has excellent laccase oxidation activity and has significant advantages in degrading AFB1 compared with the reported aflatoxin degrading enzymes.

[0047] In addition, three groups of control experiments were designed. The first group used CuCl2 solution to degrade AFB1; the second group used a polypeptide (amino acid sequence: Ac-ICIHIHMHIHI-CONH2) to replace the polypeptide of the present application; and the third group used a polypeptide (amino acid sequence: Ac-ICIHIHMHIHI-CONH2) to degrade AFB1 without CuCl2 solution. 2+ The co-incubated peptides degraded AFB1. Figure 7 The results showed that the peptide (Ac-ICIHIHMHIHI-CONH2) did not react with Cu 2+ The peptides co-incubated with CuCl2 solution had no obvious AFB1 degradation activity, but 2+ The co-incubated polypeptides showed significant activity, which not only verified the successful construction of the laccase mimetic of the present invention, but also further proved that it had high activity in AFB1 degradation.

[0048] Example 4: Preparation of immobilized mimetic enzyme

[0049] In order to improve the stability of enzyme mimics under high temperature and harsh conditions, an immobilized mimic enzyme, namely mimic enzyme-copper phosphate hybrid nanoflower, was constructed. After adding 40 μL of copper sulfate solution (25 mM) to 1 ml of phosphate buffer solution (PBS, 10 mM, pH 7.4) containing 2 mg / mL peptide mimic enzyme, the mixture was incubated at 4°C for 24 h. After incubation, the precipitate was collected by centrifugation at 5000 g and 4°C for 5 minutes, and the immobilized mimic enzyme was washed three times with ultrapure water. The scanning electron microscopy and energy spectrum images taken are shown in Figure 8 , Fig. 9 , proving the successful construction of peptide mimetic enzyme-copper phosphate hybrid nanoflowers (immobilized mimetic enzyme); Figure 8 The result diagrams of the immobilized mimetic enzymes at different magnification sizes are given. It can be seen that the immobilized mimetic enzymes of the present application are flower-like structures. The increase in specific surface area not only improves the catalytic activity, but also increases the reuse rate; Fig. 9 The energy spectrum given can significantly observe the P element and Cu element belonging to copper phosphate and the C element, O element, and N element belonging to the polypeptide, further proving the successful combination of polypeptide mimetic enzyme-copper phosphate hybrid nanoflowers (immobilized mimetic enzyme).

[0050] Implementation effect example

[0051] The constructed immobilized mimetic enzyme (final concentration 0.5 μg / mL) and peptide mimetic enzyme (final concentration 0.5 μg / mL) were placed in different temperature environments (37°C, 50°C, 60°C, 70°C, 80°C, 90°C) for one hour, and then reacted with AFB1 to investigate the difference in temperature stability between the two. Fig.10) showed that after being kept at 70°C, the activity of the peptide mimic enzyme decreased significantly, while the degradation rate of the immobilized mimic enzyme on AFB1 remained at about 90%. When the temperature was raised to 90°C, the peptide mimic enzyme activity had almost no degradation activity for AFB1, while the immobilized mimic enzyme still had nearly half the degradation rate. This shows that the construction of the immobilized mimic enzyme greatly improved the stability of the peptide mimic enzyme.

[0052] After constructing the immobilized enzyme mimic, not only the stability is improved, but also the reuse rate is significantly improved. Fig.11 As shown, after 5 cycles of AFB1 degradation, the activity change of the immobilized mimetic enzyme in degrading AFB1 was much smaller than that of the peptide mimetic enzyme, further proving the high stability of the immobilized mimetic enzyme.

Claims

1. A polypeptide mimetic enzyme, characterized in that: The raw materials include peptides and copper ions; The amino acid sequence of the polypeptide is shown in SEQ ID No. 1, specifically Ac-ICVHLHLHVHI -CONH2; The N-terminus of the polypeptide is acetylated and the C-terminus is amidated; The polypeptide molecular structure is: ； The concentration ratio of copper ions to polypeptides is 1-3:

1.

2. A method for preparing the polypeptide mimetic enzyme according to claim 1, characterized in that: The peptides were incubated with copper ions at pH 8 overnight in the dark.

3. The preparation method according to claim 2, characterized in that: Incubations were performed in phosphate buffer.

4. Use of the polypeptide mimetic enzyme according to claim 1 or the polypeptide mimetic enzyme prepared by the preparation method according to claim 2 or 3 in the degradation of AFB1 aflatoxin B1.

5. An immobilized mimetic enzyme, characterized in that: The immobilized mimetic enzyme is in the shape of a flower and includes inorganic components and organic components; The inorganic component includes a metal phosphate crystalline compound; The organic component comprises the polypeptide mimetic enzyme according to claim 1 or the polypeptide mimetic enzyme prepared by the preparation method according to claim 2 or 3; The mass ratio of inorganic and organic components is 0.8-3.5; The metal in the metal phosphate crystals is derived from copper sulfate or copper nitrate, and the phosphate radical is derived from phosphate buffer.

6. The immobilized mimetic enzyme according to claim 5, characterized in that: The phosphate buffer is selected from disodium hydrogen phosphate or potassium dihydrogen phosphate.

7. A method for preparing the immobilized mimetic enzyme according to claim 5 or 6, characterized in that: The peptide mimetic enzyme is mixed with phosphate buffer, copper sulfate or copper nitrate and incubated.

8. Use of the immobilized mimetic enzyme according to claim 5 or 6 or the immobilized mimetic enzyme prepared by the preparation method according to claim 7 in the degradation of AFB1 aflatoxin B1.

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