Artificial nano-enzyme based on conformational engineering as well as preparation method and application of artificial nano-enzyme

Abstract

The invention discloses an artificial nano-enzyme based on conformation engineering as well as a preparation method and an application of the artificial nano-enzyme, polypeptide which is in a random conformation in a free state can be coupled to nano-particles in an alpha-helical conformation manner, so that the artificial nano-enzyme has a conformation-related function. The preparation method comprises the following steps: S1, adding 2, 2, 2-trifluoroethanol and a stabilizer into a polypeptide solution, and incubating; s2, in the presence of 2, 2, 2-trifluoroethanol, coupling the polypeptide with the nanoparticles; and S3, removing the 2, 2, 2-trifluoroethanol by a freeze drying method to obtain the artificial nano-enzyme based on conformation engineering. The method is simple and convenient to operate, the experiment time is shortened, and the reaction efficiency is improved; moreover, the experimental conditions are milder, the energy demand of a laboratory is effectively saved, and the cost requirements on experimental equipment and environment are lower.

Description

Technical Field

[0001] The present invention relates to an artificial nanozyme based on conformational engineering and a preparation method and application thereof, and belongs to the fields of molecular structure design, nanobiotechnology, biochemistry and structural biology. Background Art

[0002] As one of the biological macromolecules, natural proteins have complex three-dimensional spatial structures, and there is a close relationship between structure and function. In order to test hypotheses about basic aspects of protein folding and function, many methods have been developed that rely on obtaining backbone fragments and statistics from structural bioinformatics to sample foldable protein structures and sequences. In recent years, machine learning methods can design recognizable tertiary structures using 20 common amino acids, and generative adversarial networks can generate tertiary structures and sequences starting from only a rough sketch of the desired structure. Although some success has been achieved in protein design, it is still impossible to precisely control the conformation of macromolecules such as polymers, peptides, and nanoparticles.

[0003] A key challenge in nanoscience is to design the surface of nanoparticles (NPs) to achieve highly specific and reversible binding properties similar to those of proteins, especially in the case of non-functional groups. This design requires precise control of the interaction between the molecules on the surface of the nanoparticles and the target molecules to ensure their functionality and flexibility. To address this problem, scientists have proposed an innovative method - "conformation engineering", which gives new functions to conjugated molecules by precisely regulating the conformation of conjugated molecules on nanoparticles. This method can give nanoparticles more complex and specific binding capabilities without relying on traditional functional groups, and promote the application of nanotechnology in biomedicine, sensors and materials science. In the field of artificial enzyme design, this conformational engineering method is particularly important. The catalytic activity of enzymes is achieved and regulated by their overall protein topology and the solvation of specific residues located in their active sites. The synergistic effect of amino acids brought about by the conformation of proteins plays a very important role in achieving catalytic functions. By imitating the catalytic mechanism of natural enzymes, not only can efficient chemical reactions be achieved, but also researchers can deepen their understanding of the structure and function of natural proteins in order to design more artificial proteins.

[0004] Chinese patents CN110184265A and CN118307636A disclose a method for preparing nanozymes / artificial enzymes based on gold nanoparticles. This method simulates the charge network in the hydrolysis active site of natural enzymes by precisely regulating the conformation of polypeptides on gold nanoparticles, and effectively enhances the activity and stability of artificial enzymes in catalytic reactions. Among them, both patents use a method for conformational engineering on nanoparticles, namely, a method of inducing polypeptides to form ɑ-helical structures on gold nanoparticles using TFE. 2,2,2-Trifluoroethanol (TFE) is a common inducer of protein secondary structure, which can induce polypeptides (or proteins) to form specific secondary structures, especially ɑ-helical structures. This method first couples polypeptides on gold nanoparticles, and then adds TFE to induce the polypeptides into ɑ-helical structures. In the step of coupling polypeptides, the polypeptide solution needs to be added dropwise to the nanoparticle solution, and then TFE is added to induce at a specific temperature. The process is cumbersome, time-consuming and requires more energy.

[0005] Therefore, simplifying this method of conformational engineering on nanoparticles is a worthy consideration. Summary of the invention

[0006] Based on the defects existing in the above-mentioned prior art, the purpose of the present invention is to provide an artificial nanozyme based on conformational engineering and a preparation method and application thereof, which can simplify the operation steps, save reaction time and reduce energy consumption.

[0007] The present invention is achieved by the following technical means:

[0008] One of the technical solutions of the present invention provides a method for preparing an artificial nanozyme based on conformational engineering, comprising the following steps:

[0009] S1. adding 2,2,2-trifluoroethanol (TFE) and a stabilizer to the polypeptide solution, incubating, and obtaining a mixed solution;

[0010] S2. in the presence of 2,2,2-trifluoroethanol, mixing the mixed solution with the nanoparticle solution to couple the polypeptide with the nanoparticles;

[0011] S3. Remove 2,2,2-trifluoroethanol by freeze-drying to obtain a polypeptide-nanoparticle conjugate, which is the artificial nanozyme based on conformational engineering.

[0012] Furthermore, the polypeptide in step S1 is characterized by containing two or more cysteines (C) and arranged on one side of the α-helix at a spacing of i, i+3 or i+4, i+7, so as to form a sulfur-metal bond with the metal nanoparticles to stabilize the induced α-helix structure.

[0013] α-helix is ​​one of the main forms of protein secondary structure, which refers to the regular spiral rise of the main chain of the polypeptide chain around the central axis. Every 3.6 amino acid residues spiral up one circle, translating upward 0.54nm, so the pitch is 0.54nm, and the distance between two amino acid residues is 0.15nm. The amino acid side chain R group extends to the outside of the spiral, and the carbonyl oxygen of the peptide bond of each peptide chain forms a hydrogen bond with the fourth NH, and the direction of the hydrogen bond is basically parallel to the long axis of the spiral. Since all peptide bonds in the peptide chain can form hydrogen bonds, the α-helix is ​​very stable.

[0014] Furthermore, the general formula of the polypeptide sequence is X1CX3DCX6HSCX 10 SWX 13 G, wherein C, D, H, S, W, G represent amino acids; X1, X3, X6 and X 10 is independently selected from any one of A (alanine), N (asparagine), D (aspartic acid), Q (glutamine), E (glutamic acid), G (glycine), H (histidine), I (isoleucine), L (leucine), M (methionine), F (phenylalanine), P (proline), S (serine), T (threonine), W (tryptophan), Y (tyrosine) or V (valine); X 13 Selected from R (arginine) or K (lysine). The sequence of the polypeptide solution is preferably NCLECLHSCGSWRG.

[0015] Furthermore, the concentration of the polypeptide solution is 10-50 μM, and the solvent is deionized water.

[0016] Furthermore, the stabilizer is trisodium citrate solution and sodium hydroxide solution; the concentration of the trisodium citrate solution is preferably 0.2M, and the concentration of the sodium hydroxide solution is preferably 0.2M.

[0017] In step S1, the role of 2,2,2-trifluoroethanol (TFE) is to induce the polypeptide to form an ɑ-helical structure. The polypeptide can form an ɑ-helical structure better and more easily in a free state. At the same time, the reaction process can be carried out at room temperature.

[0018] Furthermore, the nanoparticles in step S2 include but are not limited to Au, Pt, Pd, and Ag nanoparticles, preferably Au nanoparticles. The concentration of the nanoparticle solution is 60 to 100 nM; and the particle size of the nanoparticles is 3 to 20 nm.

[0019] Furthermore, the dosage relationship of the 2,2,2-trifluoroethanol, trisodium citrate solution, sodium hydroxide solution, polypeptide solution in step S1 and the nanoparticle solution in step S2 is (10-20) mL: (10-30) μL: (80-120) μL: (3-5) mL: (10-15) mL.

[0020] Furthermore, the incubation conditions in step S1 are: room temperature, 1 to 2 hours;

[0021] Furthermore, the coupling operation conditions in step S2 are: room temperature, 1 to 2 hours.

[0022] The coupling mechanism of step S2 is: the thiol group on the polypeptide is covalently linked to the gold nanoparticles to form an Au-S bond. The stability of the Au-S bond makes the present invention have the excellent performance of not being easily denatured. After removing trifluoroethanol, the nanozyme still has a stable secondary structure and excellent catalytic activity. The present invention uses gold nanoparticles as a rigid skeleton, which can greatly improve the stability of the nanozyme, and its surface is easy to modify and has a large number of binding sites. The surface is composed of conformationally adjustable polypeptides and has a flexible and dynamic secondary structure. The conformational regulation of the nanozyme proposed by the present invention enables non-functional groups to have functions, which is an advanced simulation of natural enzymes.

[0023] Furthermore, the freeze drying in step S3 is carried out in a freeze dryer, and the duration and temperature are selected according to conventional operating techniques in the art, preferably for 72 hours at a temperature of -45 to -30°C.

[0024] The second technical solution of the present invention provides an artificial nanozyme based on conformational engineering prepared by the above method.

[0025] Furthermore, the artificial nanozyme based on conformational engineering is a polypeptide-nanoparticle conjugate having an α-helical structure.

[0026] A third technical solution of the present invention provides an application of the above-mentioned artificial nanozyme based on conformational engineering, in which the artificial nanozyme is used as a catalyst to catalyze ester hydrolysis or ester exchange reaction.

[0027] Compared with the prior art, the present invention has the following obvious outstanding substantive features and significant advantages:

[0028] (1) The present invention first uses TFE to induce the polypeptide to form an ɑ-helical structure, and then mixes the nanoparticles. This method has the same effect as the method described in patents CN110184265A and CN118307636A, which is to first couple the polypeptide to the nanoparticles and then add TFE for induction. Both methods can construct an ideal ɑ-helical structure on the nanoparticles and exert the corresponding function. Moreover, because the free polypeptide is easier to form an ɑ-helical structure in TFE, the reaction can be completed within a few minutes at room temperature. Compared with the existing induction method, this new method significantly reduces the reaction time and does not require a long incubation process. This advantage not only greatly improves the efficiency of the experiment, but also avoids the side effects and unstable factors that may be caused by high temperature or long-term treatment in the traditional method to the reaction system. By directly mixing the polypeptide with 2,2,2-trifluoroethanol (TFE), the tedious step of dripping the polypeptide is omitted, making the entire reaction process more concise and efficient, and also making this method more feasible and advantageous in large-scale experiments or industrial applications.

[0029] (2) The process of TFE-induced peptides can be carried out smoothly at room temperature, which not only eliminates the high-temperature treatment step in traditional methods, but also significantly reduces the energy consumption required for the experiment. The advantage of inducing peptide conformational transformation at room temperature is that it avoids side reactions and unstable factors that may be caused during high-temperature reactions, and maintains the stability of the structure and function of the peptide. This low-temperature operation not only makes the experiment more gentle, but also avoids the environmental burden caused by high energy consumption, thereby effectively saving the energy demand of the experiment. Similarly, the binding reaction between nanoparticles and induced peptides can also be carried out at room temperature. This feature greatly simplifies the reaction conditions and reduces the dependence on external heat sources. Compared with traditional reactions that require high-temperature catalysis or heating, this method reduces energy consumption, making the entire experimental process more energy-saving and environmentally friendly. By completing these key steps at room temperature, not only energy is saved, but also the thermal effects and thermal damage that may occur in the experiment are reduced, ensuring the efficiency and stability of the reaction.

[0030] (3) In addition, reducing high-temperature operations and complex thermal control systems also makes the requirements for experimental equipment simpler and more cost-effective. This energy-saving and consumption-reducing feature makes this method particularly advantageous in resource-limited laboratory environments and also provides a more sustainable solution for large-scale applications. In general, compared with existing induction methods, this method not only improves experimental efficiency through low-energy operation, but also achieves greener and more environmentally friendly resource utilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Circular dichroism characterization of the α-helical structure of the artificial enzyme prepared in Example 1 and Comparative Example 1;

[0032] Figure 2is the activity of the artificial enzyme prepared in Example 1 and Comparative Example 1;

[0033] The marks in the figure indicate:

[0034] a is the hydrolysis process of the artificial enzyme prepared by TFE induction before / after coupling;

[0035] b is the rate of the artificial enzyme prepared by TFE induction before / after coupling. DETAILED DESCRIPTION

[0036] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, several variations and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0037] All raw materials of the present invention have no particular limitation on their sources, and can be purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0038] The peptides were synthesized using solid phase synthesis;

[0039] Synthesis of gold nanoparticles: Place a round-bottom flask containing 98.35 mL of ultrapure water in a constant temperature heated magnetic stirrer, set the temperature to 25 ° C, and the speed to 20 rpm. Then add 1 mL of 25 mM tetrahydrate chloroauric acid solution and stir for 1 min, then add 770 μL of 39.47 mM sodium citrate solution and continue stirring for 1 min, and finally quickly add 380 μL of freshly prepared 39.47 mM sodium citrate solution containing 0.075% sodium borohydride by mass, and continue stirring at a constant speed for 5 min. After the preparation is completed, remove the round-bottom flask from the pot in the water bath and remove the stirring rotor from it, seal it and store it in the dark for 2 hours. A gold nanoparticle aqueous solution with a particle size of 3.6 nm and a concentration of 80 nM is obtained.

[0040] Example 1 An artificial enzyme based on gold nanoparticles (artificial enzyme prepared by TFE induction before coupling)

[0041] 2,2,2-trifluoroethanol (TFE) was used to induce the peptide to form an ɑ-helical structure on gold nanoparticles to construct an artificial enzyme based on gold nanoparticles:

[0042] 1): Add 15 mL of TFE to 4 mL of a peptide solution (named Triad6, sequence: NCLECLHSCGSWRG, solvent: deionized water), and add 20 μL of 0.2 M trisodium citrate solution and 100 μL of 0.2 M sodium hydroxide solution as stabilizers, and incubate at room temperature for 1-2 hours.

[0043] 2): Add 12 mL of a gold nanoparticle solution with a particle size of 3.6 nm (concentration of 80 nM) to the mixed solution in 1), place it in a multi-point heating magnetic stirrer (German IKA (15 points) RT 15), and react at room temperature for 1 hour to couple the polypeptide folded into an α-helical structure to the gold nanoparticles.

[0044] 3): The above solution was freeze-dried, the temperature of the freeze dryer was set to -45°C, and the time was set to 72 hours to obtain an artificial enzyme based on gold nanoparticles.

[0045] Comparative Example 1: Artificial enzyme prepared by TFE induction after coupling

[0046] Take 12mL gold nanoparticle aqueous solution (particle size is 3.6nm, concentration is 80nM) and add 20μL of 0.2M trisodium citrate solution, then add 4mL polypeptide solution (concentration is 20μM, containing 100μL 0.2MNaOH, solvent is deionized water, sequence is the same as Example 1) dropwise to the stirring gold nanoparticle aqueous solution, continue stirring for 1h (speed 600-700rpm, room temperature) to obtain a functionalized gold nano solution. The polypeptide functionalized gold nano solution is mixed with 15mL of pure TFE solution, induced at room temperature for 24h, then, the induced solution is quickly frozen into a solid state with liquid nitrogen, put into a freeze dryer, the vacuum degree is 0.07mbar, the temperature is -45°C, and frozen for 72h to obtain an artificial enzyme solid powder with TFE completely removed.

[0047] Example 2 Verification of artificial enzyme structure

[0048] The α-helix structure induced in Example 1 and Comparative Example 1 was characterized by circular dichroism spectroscopy. Figure 1 As shown, compared with the existing method of TFE induction after coupling, both methods can induce the polypeptide to form an ɑ-helical structure on gold nanoparticles, and the method of TFE induction before coupling shows more ideal ɑ-helical characteristics and has a better signal-to-noise ratio.

[0049] Example 3 Study on artificial enzyme activity

[0050] The determination method of artificial enzyme activity in this embodiment:

[0051] p-NPA (substrate, concentration of 5 mM) was dissolved in acetonitrile and then diluted in water to 2% (v / v) acetonitrile to prepare a fresh p-NPA stock solution. The hydrolysis reaction was carried out at pH 7, the final concentration of the artificial enzyme was 0.5 μM, and the temperature was 25°C. The substrate, pH 7 buffer, and artificial enzyme were mixed using an SFM-3000 / S stopped-flow mixer (Bio-logic, France) to start the hydrolysis reaction. The increase in absorbance was recorded at 400 nm using a MOS-500 spectrophotometer (Bio-logic, France), and the molar extinction coefficient ε was determined experimentally. 400nm =10.85cm -1 (mM -1 ) was used to quantify the yield of the product p-NP.

[0052] The activity of the artificial enzyme prepared in Study Example 1 and Comparative Example 1 was measured by stopped-flow spectrometer. Figure 2 As shown, the method of polypeptide induction before coupling used in the present application has almost the same activity as the artificial enzyme prepared by the existing method, which proves that both methods can prepare artificial enzymes with catalytic activity.

[0053] In summary, the method of conformational engineering on nanoparticles described in this case shows significant advantages over existing preparation methods. This method can induce the peptide to form an ideal α-helical structure on the surface of gold nanoparticles, while ensuring the catalytic efficiency and enhancing the stability and controllability of the catalytic process. In addition, this method has the advantages of simple operation steps and simple process flow. It avoids the requirements of complex chemical modifications and multiple reaction conditions in traditional methods, making the synthesis process of artificial enzymes more efficient, low-cost, and easier to achieve in large-scale production. Compared with existing conformational engineering strategies, this method can optimize the surface conformation of nanoparticles in a shorter time, showing more significant application potential. Based on this, this method of conformational engineering on nanoparticles provides new ideas and technical paths for the development of new artificial enzymes.

[0054] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for preparing an artificial nanozyme based on conformational engineering, characterized in that: The following steps are involved: S1. adding 2,2,2-trifluoroethanol and a stabilizer to the polypeptide solution, incubating, and obtaining a mixed solution; S2. in the presence of 2,2,2-trifluoroethanol, mixing the mixed solution with the nanoparticle solution to couple the polypeptide with the nanoparticles; S3. 2,2,2-Trifluoroethanol was removed by freeze-drying to obtain artificial nanozyme based on conformational engineering.

2. The method for preparing an artificial nanozyme based on conformational engineering according to claim 1, characterized in that: The polypeptide in step S1 is characterized by containing two or more cysteines, and arranged on one side of the α-helix at a spacing of i, i+3 or i+4, i+7.

3. A method for preparing an artificial nanozyme based on conformational engineering according to claim 1 or 2, characterized in that: The general formula of the polypeptide sequence in step S1 is X1CX3DCX6HSCX 10 SWX 13 G, where X1, X3, X6 and X 10 are independently selected from any one of alanine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine; X 13 Selected from arginine or lysine.

4. The method for preparing an artificial nanozyme based on conformational engineering according to claim 1, characterized in that: The concentration of the polypeptide solution in step S1 is 10-50 μM.

5. The method for preparing an artificial nanozyme based on conformational engineering according to claim 1, characterized in that: The stabilizer in step S1 is trisodium citrate solution and sodium hydroxide solution.

6. The method for preparing an artificial nanozyme based on conformational engineering according to claim 1, characterized in that: In step S2, the nanoparticles are selected from any one of Au, Pt, Pd, and Ag nanoparticles; the concentration of the nanoparticle solution is 60 to 100 nM; and the particle size of the nanoparticles is 3 to 20 nm.

7. The method for preparing an artificial nanozyme based on conformational engineering according to claim 1, characterized in that: The dosage relationship of the 2,2,2-trifluoroethanol, trisodium citrate solution, sodium hydroxide solution, polypeptide solution in step S1 and the nanoparticle solution in step S2 is (10-20) mL: (10-30) μL: (80-120) μL: (3-5) mL: (10-15) mL.

8. The method for preparing an artificial nanozyme based on conformational engineering according to claim 1, characterized in that: The incubation conditions in step S1 are: room temperature, 1 to 2 hours; The coupling operation conditions in step S2 are: room temperature, 1 to 2 hours.

9. An artificial nanozyme based on conformational engineering, characterized in that: According to the preparation method according to any one of claims 1 to 8, the artificial nanozyme based on conformational engineering is a polypeptide-nanoparticle conjugate having an α-helical structure.

10. The use of the artificial nanozyme based on conformational engineering as claimed in claim 9, characterized in that: In the application, the artificial nanozyme acts as a catalyst to catalyze ester hydrolysis or ester exchange reaction.

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