Preparation method and application of metal mimic enzyme

Abstract

The invention relates to the field of peptide-based metal mimic enzyme biological materials, in particular to preparation and application of a metal mimic enzyme of metal nanofiber peptide. On one hand, the invention discloses the metal nanofiber peptide, and the sequence of the polypeptide is Ac-HCHMGLKLKLKL-NH2. The polypeptide and a metal ion Cu < 2 + > are co-assembled to form the metal nanofiber peptide. The metal nanofiber peptide can be used for degrading PE as a metal mimic enzyme, and compared with the prior art, the metal mimic enzyme has the characteristics of simplicity in preparation, stable activity, low price and the like.

Description

Technical Field

[0001] The invention relates to the field of peptide-based metallo-mimetic enzyme biomaterials, and in particular to a preparation method of a metallo-mimetic enzyme and application thereof in PE degradation. Background Art

[0002] Polyethylene (PE) is a polymer made from ethylene monomers. Plastic products made from PE are widely used in daily life due to its good chemical stability and low cost. According to statistics, the global production of synthetic plastics has reached 360 million tons, of which polyolefins such as polyethylene, polypropylene, and polystyrene account for more than 60% of the total plastic production. However, the vast majority of polyethylene plastic waste has caused environmental pollution due to its inability to degrade naturally and the lack of appropriate treatment methods.

[0003] At present, the treatment technologies for plastic waste worldwide mainly include raw material recycling, mechanical recycling and chemical thermal treatment. However, the reuse of plastic waste cannot solve the problem of poor waste degradation and recycling efficiency. In addition, plastic thermal degradation is a technology that is difficult to operate and consumes a lot of energy, and the incineration of plastic waste is often incomplete combustion, which will produce a large amount of toxic and harmful gases, thus causing secondary pollution to the ecological environment. White pollution caused by the abuse of plastics such as polyethylene has become a global problem. Therefore, the research on green and environmentally friendly degradation methods of PE plastics is of great significance to reducing environmental pollution and maintaining human health.

[0004] Bioenzyme degradation is a mild, environmentally friendly and safe method for PE degradation. Laccase has been proven to have the ability to oxidatively degrade PE. However, natural laccase also has many disadvantages, such as poor stability and susceptibility to environmental (temperature, pH) changes; secondly, the structure of natural enzymes is complex, and it is difficult to modify and regulate their catalytic performance; in addition, natural laccase needs to be extracted, separated and purified before application, so the process is complicated, time-consuming and the application cost is high, which limits the widespread application of natural enzymes.

[0005] Mimic enzymes are artificial biocatalysts inspired by natural enzymes. Compared with natural enzymes, mimic enzymes have advantages such as higher catalytic efficiency, simpler structure, and higher stability, making them ideal substitutes for natural enzymes. Peptide mimic enzymes are artificial mimics with enzyme catalytic activity formed by the rational selection and arrangement of amino acids. Compared with mimic enzymes constructed from other materials, peptide mimic enzymes have more obvious advantages. First, they are closer to natural enzymes in terms of material sources and have good homology. Secondly, the peptide chains in peptide mimic enzymes mainly form a certain structure through non-covalent forces, and complete catalysis with the help of non-covalent interactions with substrates. The catalytic substrate mechanism is similar to that of natural enzymes. Peptide mimic enzymes have smaller molecular weights, and peptides can obtain catalytic activity by simulating the structural characteristics of natural enzymes. Peptide mimic enzymes are more stable than natural enzymes, especially in high temperature environments, and have better solubility in solutions. Summary of the invention

[0006] In view of some problems existing in PE degradation at present, the present invention provides a method for preparing a metal mimetic enzyme and its application in PE degradation. The bionic idea of ​​mimetic enzyme is proposed, and the natural enzyme laccase is used as the simulation object to construct a PE plastic degradation mimetic enzyme based on the structural characteristics and catalytic properties of the natural enzyme.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows: A metal mimetic enzyme, the raw materials of which include a polypeptide sequence and copper ions; The polypeptide sequence is: Ac-HCHMGLKLKLKL-NH2 The N-terminus of the polypeptide is acetylated, and the C-terminus is amidated.

[0008] Preferably, the Cu 2+ The concentration ratio to the polypeptide is 1-3:1.

[0009] In the method for preparing the metallo-mimetic enzyme, the polypeptide and copper ions are incubated in the dark at a pH of 6.0-10.0 for 24 hours.

[0010] Furthermore, the incubation is carried out in a buffer system; preferably, the buffer system is a 4-hydroxyethylpiperazineethanesulfonic acid (Hepes) system.

[0011] The invention also provides application of the metallo-mimetic enzyme in PE degradation.

[0012] The invention also provides application of the metal mimetic enzyme in PE degradation products.

[0013] As one of the preferred embodiments of the present invention, the degradation products of PE include but are not limited to enzyme preparations, flocculants, plastic additives, and feed additives.

[0014] The present invention provides a method for designing a metallo-mimetic enzyme as a PE degradation mimetic enzyme, comprising the following steps: (1) Analysis of the active domain of the natural enzyme laccase: Laccase is a multi-copper oxidase, and there are three types of copper in the active center structure: Type I copper has a conservative triangular coordination and forms a lateral ligand with two histidines and one cysteine; Type II copper is coordinated with two histidines and is adjacent to a type III copper ion; Type III copper and type II copper together form a trinuclear copper cluster, in which molecular oxygen is reduced and water is released.

[0015] (2) Design of peptide sequence: Based on the structural characteristics and catalytic mechanism of natural enzymes, the present invention designed a polypeptide sequence Ac-HCHMGLKLKLKL-NH2.

[0016] The polypeptide sequence contains amino acids in the copper ion binding site, including histidine, cysteine, and methionine (H, C, M); the self-assembly driving sequence LKLKLKL, in which hydrophobic leucine (L) and hydrophilic lysine (K) are arranged alternately; glycine (G) can be used as a spacer molecule to separate the amino acids in the active site. The N-terminus of the peptide chain is acetylated, and the C-terminus of the peptide chain is amidated to eliminate the unfavorable electrostatic interactions between the charged end groups to stabilize the co-assembled structure, making it more conducive to forming dense mesh fibers.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention designs peptide sequences based on the principle of bionic enzyme simulation, and forms metallo-mimetic enzymes after the polypeptides and copper ions are co-assembled. The metallo-mimetic enzymes have similar structures and activities to natural laccases. The enzyme-mimetic peptide sequence is short and simple, highly active and stable, and inexpensive, which plays an important role in promoting the wide application of enzyme-mimetic degradation of PE.

[0018] The polypeptide sequence contains active site amino acids (histidine, cysteine, methionine), self-assembly driving sequences, and active site spacer molecules, which enable it to co-assemble with copper to form a structure similar to the active center of a natural enzyme and have PE degradation activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the relative activity of the metallo-mimetic enzyme when incubated under different copper ion concentrations; Figure 2 is a transmission electron microscopy image of the metalloenzyme, where Figure 2 (a) Peptide alone without copper addition, Figure 2 (b) is the image after copper and peptide co-assembly; Figure 3 It is the UV-visible absorption spectrum of the metallo-mimetic enzyme combined with Congo red; Figure 4 is the circular dichroism spectrum of the metallo-mimetic enzyme; Figure 5 is the infrared spectrum of the metallo-mimetic enzyme; Figure 6 This is the infrared spectrum of PE after degradation by metallo-mimetic enzyme; Figure 7 This is a scanning electron micrograph of the metalloenzyme degrading PE film. Figure 7 (a) is the control group, Figure 7 (b) is the treatment group, Figure 7 (c) is the enlarged image of the PE film after degradation. DETAILED DESCRIPTION

[0020] 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.

[0021] If no specific techniques or conditions are specified in the examples, all are carried out according to conventional methods or the techniques or conditions described in the literature in the field, or according to the product instructions. If no manufacturer is specified for the reagents and instruments used, all are conventional products that can be purchased through regular channels.

[0022] The present invention provides a structure analysis method of a metallo-mimetic enzyme, comprising: transmission electron microscope ultraviolet spectrum analysis, circular dichroism spectrum analysis, Fourier transform infrared spectrum analysis and the like.

[0023] The present invention provides the application of metal mimetic enzyme in the degradation of PE film: monitoring the structural change of PE film before and after catalysis by infrared spectrum; monitoring the surface morphology change of PE film before and after catalysis by the metal mimetic enzyme by scanning electron microscope, and studying the application effect.

[0024] Example 1: Preparation of metallo-mimetic enzymes and detection of catalytic activity The lyophilized peptide was taken out of the refrigerator in advance and allowed to return to room temperature. Then 20 μL of acetonitrile and 20 μL of water were added dropwise to dissolve the peptide, and then Hepes buffer (50 mM, pH 8) containing CuCl2 (0 mM, 4 mM, 6 mM, 8 mM, 10 mM, 12 mM) was added dropwise to make the final concentration of the peptide 4 mM. The solution was fully shaken and mixed, then ultrasonically dispersed for 1 minute, and incubated at 25 ° C for 24 hours in the dark to form a uniform and stable nanofiber peptide metallo-mimetic enzyme.

[0025] We used the oxidative coupling of the phenolic compounds 2,4-dichlorophenol (2,4-DP) and 4-aminotriptyline (4-AP) to produce a color reaction to verify whether the designed metalloenzyme has laccase oxidation activity. 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 metalloenzyme 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 1.5:1. 2+ When the concentration was 2.5 and 3 times of the peptide concentration, the activity decreased. 2+ The optimal binding ratio to the peptide is 2:1.

[0026] Example 2: Structural Characterization of Metallomimetic Enzymes Peptide and Cu 2+ Combined with the structure before and after characterization. Figure 2 From the TEM image, it can be seen that the peptides in the presence of copper assemble and aggregate to form a nanofiber network structure, while in the absence of copper, the peptides are more dispersed and no obvious aggregation is formed; Figure 3 The UV-visible spectrum shows that the metallo-mimetic enzyme after co-assembly with copper has a stronger Congo red binding ability, a greater absorption intensity and a blue shift in the maximum absorption wavelength, indicating the formation of nanofiber peptides; Figure 4 From the circular dichroism spectrum, we can see that the presence of metal ions changes the structure of the peptide. When the peptide is alone, it forms a random coil structure (negative peak near 198 nm), and after the peptide and copper are co-assembled, it forms a β-folded secondary structure (positive peak near 195 nm and negative peak at 216 nm). Figure 5 From the FTIR graph, we can see that the metalloenzyme formed by the co-assembly of peptide and copper has a peak at 1674 cm -1 、1624 cm -1 and 1538 cm -1 There is a strong peak at , proving that the metalloenzyme has an antiparallel β-folded structure.

[0027] These results show that Cu 2+ It interacts strongly with peptides to form metallo-mimetic enzymes.

[0028] Example 3: Degradation of PE by metallo-mimetic enzymes PE was added to the metallo-mimetic enzyme solution and subjected to degradation reaction at pH 8 and 50°C for 4 days. The PET film after degradation reaction was washed with 1% SDS, distilled water and ethanol in turn, dried at room temperature, and its functional groups were characterized by infrared spectroscopy, and its microscopic morphology was characterized by scanning electron microscopy. Figure 6 and 7 .Depend on Figure 6 It can be seen that compared with the control group, the PE film treated with metal mimetic enzyme has a -1 A new obvious peak appeared at , which is the peak position of keto carbonyl peak, indicating the oxidative degradation of PE membrane by metal mimetic enzyme. Figure 7 (a) is the control group (no simulated enzyme added), Figure 7 (b) is the PE film after degradation by metal mimetic enzyme. Figure 7 (c) is the enlarged picture of the PE film after degradation. Figure 7 It can be clearly seen that compared with the smooth and uniform film of the control group, the PE film was obviously corroded after degradation by the simulated enzyme, and obvious etching aging and fragmentation occurred on the surface. These results all prove that the metal simulated enzyme has a good PE degradation effect.

Claims

1. A nanofiber peptide, characterized in that: The peptide sequence is: Ac-HCHMGLKLKLKL-NH2; Among them, H is histidine, C is cysteine, M is methionine, L is hydrophobic leucine, K is hydrophilic lysine, and G is glycine. The N-terminus of the polypeptide is acetylated and the C-terminus is amidated.

2. A metallo-mimetic enzyme, characterized in that: The polypeptide according to claim 1 is co-assembled with copper ions, and the concentration ratio of copper ions to polypeptides is 1-3:

1.

3. A method for preparing the metallo-mimetic enzyme according to claim 2, characterized in that: The polypeptide and copper ions were incubated in the dark at a pH of 6-10 for 24 hours.

4. The preparation method according to claim 3, characterized in that: The incubation was carried out in a buffer system.

5. The preparation method according to claim 4, characterized in that: The buffer system is a 4-hydroxyethylpiperazineethanesulfonic acid buffer system.

6. Use of the metallo-mimetic enzyme according to claim 2 in PE degradation.

7. The use according to claim 6, characterized in that: PE was co-reacted with the metallomimetic enzyme at 25-40°C.

8. The use according to claim 7, characterized in that: The co-reaction degradation system contains Hepes buffer.

9. Use of the metallo-mimetic enzyme according to claim 2 in the preparation of PE degradation products.

10. The use according to claim 9, characterized in that: The products are enzyme preparations, flocculants, plastic additives or feed additives.

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