Multifunctional polypeptide mimetic enzyme catalysts and uses thereof

Abstract

The application discloses a multifunctional polypeptide mimetic enzyme catalyst and application thereof, and belongs to the technical field of mimetic enzyme catalysts. The multifunctional polypeptide mimetic enzyme catalyst comprises a co-assembly of polypeptide and chlorinated hematin; the amino acid sequence of the polypeptide is shown in any one of SEQ ID NO:1 to SEQ ID NO:6, wherein the N terminal of the polypeptide is acetyl modification, and the C terminal is amide modification. The polypeptide mimetic enzyme catalyst has high stability, can be repeatedly used, and can simultaneously catalyze multiple chemical reactions (including phenolic oxidative coupling reaction, phenolic oxidation reaction and ester hydrolysis reaction), and has wide application prospects.

Description

Technical Field

[0001] This invention belongs to the field of enzyme-mimicking catalyst technology, specifically relating to a multifunctional polypeptide enzyme-mimicking catalyst and its applications. Background Technology

[0002] The oxidation, coupling, and hydrolysis reactions of phenolic compounds have important applications in pollutant degradation, chemical synthesis, and biodetection. For example, the coupling reaction of guaiacol can be used for the colorimetric detection of phenolic compounds, and the hydrolysis reaction of p-nitrophenol acetate can be used to assess esterase activity or detect organophosphorus pesticides. Currently, the commonly used catalysts for these reactions are natural enzymes, including laccases, oxidases, and esterases.

[0003] Natural laccases can directly catalyze the oxidation of phenols and the coupling reaction of guaiacol using molecular oxygen; natural peroxidases rely on hydrogen peroxide to catalyze the oxidation of phenols; and natural esterases can catalyze the hydrolysis of substrates such as p-nitrophenol acetate. However, natural enzymes share common drawbacks: first, they have poor thermal and storage stability, easily becoming inactive in high-temperature, acidic, alkaline, or organic solvent environments; second, their catalytic functions are specific, and a single enzyme cannot simultaneously perform multiple reactions such as oxidation, coupling, and hydrolysis, requiring the use of multiple enzymes in combination, increasing system complexity and cost; and third, extraction and purification are difficult, enzyme preparations are expensive, and recycling and reuse are difficult.

[0004] To address the aforementioned issues, various artificial enzyme mimics have been developed in this field. Existing enzyme mimics include metalloporphyrin complexes, metal-organic frameworks, and nano-oxides, which to some extent mimic the single function of laccase or peroxidase. However, these mimics typically possess only one catalytic activity and lack the ability to simultaneously mimic multiple enzymes (laccase, peroxidase, esterase). Furthermore, there are no reports of a single catalyst simultaneously catalyzing phenol oxidation, guaiacol coupling, and the hydrolysis of p-nitrophenol acetate. In addition, existing enzyme mimics are mostly inorganic or small organic molecule systems with limited controllability, and their stability still needs improvement.

[0005] Peptide self-assembly technology offers a new approach to constructing biomimetic catalysts. By co-assembling peptides with functional groups such as porphyrins, supramolecular structures with specific catalytic activities can be formed. However, existing catalysts based on peptide-porphyrin co-assemblies are limited to mimicking either peroxidase or laccase, failing to achieve multifunctional integration and not simultaneously covering multiple phenolic reaction types such as oxidation, coupling, and hydrolysis.

[0006] Therefore, it is both practically necessary and valuable to develop a multifunctional catalyst that is highly stable, reusable, and capable of simultaneously catalyzing a variety of phenolic reactions (including laccase-catalyzed oxidative coupling reactions and esterase-catalyzed hydrolysis reactions). Summary of the Invention

[0007] To address the problems existing in the prior art, the present invention aims to provide a multifunctional polypeptide-mimicking enzyme catalyst and its applications.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A multifunctional polypeptide-mimicking enzyme catalyst, comprising a co-assembly of a polypeptide and heme chloride; the amino acid sequence of the polypeptide is shown in any one of SEQ ID NO:1 to SEQ ID NO:6, wherein the N-terminus of the polypeptide is modified with an acetyl group and the C-terminus is modified with an amide group.

[0009] Based on the above scheme, the molar ratio of the polypeptide to heme chloride is 1:10 to 1:80.

[0010] Based on the above scheme, the molar ratio of the polypeptide to heme chloride is 1:80.

[0011] Based on the above scheme, the polypeptide-mimicking enzyme catalyst has laccase-like activity, peroxidase-like activity and esterase-like activity.

[0012] The above-mentioned multifunctional polypeptide-mimicking enzyme catalysts are used as alternative catalysts for laccase-like, peroxidase-like, and esterase-like chemical reactions.

[0013] Based on the above scheme, the chemical reaction is at least one of phenolic oxidative coupling reaction, phenolic oxidation reaction, and ester substrate hydrolysis reaction.

[0014] Based on the above scheme, the phenolic oxidative coupling reaction is the guaiacol coupling reaction, the phenolic oxidation reaction is the catechol oxidation reaction, and the ester substrate hydrolysis reaction is the p-nitrophenol acetate hydrolysis reaction.

[0015] Based on the above scheme, the phenolic oxidative coupling reaction and phenolic oxidation reaction are carried out by mixing the polypeptide-mimicking enzyme catalyst with phenols, adding 0.8~8mM hydrogen peroxide, and reacting at pH 5.0~9.0 and temperature 25~60℃ for 3~180min.

[0016] Based on the above scheme, the hydrolysis reaction of the ester substrate is carried out by mixing the polypeptide-mimicking enzyme catalyst with the ester substrate.

[0017] Based on the above scheme, in the reaction system, the final concentration of the polypeptide in the polypeptide-mimicking enzyme catalyst is 0.4 mM, and the final concentration of heme chloride is 5 μM.

[0018] Advantages of the technical solution of the present invention: (1) Multifunctionality: The polypeptide-mimicking enzyme catalyst has laccase-like activity, peroxidase-like activity and esterase-like activity, and can catalyze various types of phenolic reactions such as phenol oxidation, guaiacol coupling reaction and hydrolysis of p-nitrophenol acetate, overcoming the defect of the single function of natural enzymes.

[0019] (2) Good stability: The polypeptide mimic enzyme can still retain 23.7% of its catalytic activity after being treated at 80°C for 1 hour, while natural horseradish peroxidase retains only 7.2% of its activity under the same conditions, indicating that the polypeptide mimic enzyme of the present invention has significantly better thermal stability than natural enzymes.

[0020] (3) Low cost and batch preparation: The polypeptide has a small molecular weight, making it suitable for batch preparation by solid-phase synthesis. It has good reproducibility and lower cost than natural enzymes.

[0021] Therefore, the polypeptide-mimicking enzyme catalyst of the present invention has high stability, is reusable, and can simultaneously catalyze multiple chemical reactions (including phenolic oxidative coupling reaction, phenolic oxidation reaction and ester hydrolysis reaction), and has broad application prospects. Attached Figure Description

[0022] Figure 1 The molecular structural formulas are for 6 polypeptides; Figure 2 The conversion rate of guaiacol by polypeptide mimic enzymes; Figure 3 The conversion rate of guaiacol by polypeptide mimicry enzymes at different pH values; Figure 4 The conversion rate of guaiacol by polypeptide mimicry enzymes under different H2O2 conditions; Figure 5 The conversion rate of guaiacol by polypeptide mimicry enzymes at different temperatures; Figure 6 The residual activity of peptide-mimicking enzymes and natural horseradish peroxidase after high-temperature treatment is shown. Detailed Implementation

[0023] The terminology used in this invention, unless otherwise specified, generally has the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. The following embodiments are merely illustrative and are not intended to limit the scope of the invention in any way.

[0024] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the experimental materials, reagents, and chemicals used in the following embodiments can be obtained through general channels.

[0025] Example 1

[0026] A multifunctional polypeptide-mimicking enzyme catalyst, wherein the catalyst is co-assembled from polypeptides and heme chloride.

[0027] The amino acid sequence of the polypeptide is any one of SEQ ID NO:1 to SEQ ID NO:6, wherein the N-terminus of the polypeptide is modified with an acetyl group and the C-terminus is modified with an amide group. The molar ratio of the polypeptide to heme chloride is 1:80.

[0028] Polypeptide P1: Ac-IIIHH-NH2 (SEQ ID NO:1); Polypeptide P2: Ac-IIIGHH-NH2 (SEQ ID NO:2); Polypeptide P3: Ac-IIIGGH-NH2 (SEQ ID NO:3); Polypeptide P4: Ac-IIIHGH-NH2 (SEQ ID NO:4); Peptide P5: Ac-IIIHGGH-NH2 (SEQ ID NO:5); Polypeptide P6: Ac-IIIIHGH-NH2 (SEQ ID NO:6).

[0029] The above six polypeptide molecular structures are shown in [link to structural formula]. Figure 1 .

[0030] The multifunctional polypeptide-mimicking enzyme catalyst is prepared by the following method: The peptide and heme chloride were dissolved in PBS buffer (pH 7.0) at a molar ratio of 1:80 and incubated at room temperature for 24 hours to obtain the final product.

[0031] The polypeptide can be prepared using conventional methods in the art, such as by commissioning a specialized biotechnology company for synthesis or by using the Fmoc solid-phase polypeptide synthesis method. The obtained polypeptide is purified by high-performance liquid chromatography (HPLC) to a purity greater than 98%, and its molecular weight is confirmed by mass spectrometry. When using the Fmoc solid-phase synthesis method, the following steps can be followed: (1) Resin deprotection: The Fmoc protecting group on the solid resin is removed to expose the amino groups on the resin for subsequent coupling reactions. Deprotection is preferably performed using a 20% (volume fraction) piperidine / DMF solution under alkaline conditions to remove the Fmoc protecting group.

[0032] (2) Amino acid coupling: After activating the carboxyl group of the amino acid to be coupled, it undergoes a condensation reaction with the free amino group on the resin to form a peptide bond. HBTU and HOBt are used as carboxyl activating reagents, DMF is used as the reaction solvent, and DIEA (2 mol / L) is used as the acid-binding agent. The coupling reaction is completed under microwave-assisted conditions. The deprotection and coupling steps are repeated sequentially according to the target peptide sequence until all amino acid residues are linked. Depending on the needs of the target product, the N-terminus and / or C-terminus of the peptide can be protected or deprotected.

[0033] (3) Pyrolysis and Resin Removal: After the polypeptide chain synthesis is completed, the solid resin is collected by filtration, and a lysis buffer is added for lysis to release the target polypeptide from the solid support. The lysis buffer is prepared by trifluoroacetic acid (TFA), triisopropylsilane (TIS), and deionized water in a volume ratio of 95:2.5:2.5. The lysis time is 4 hours. After lysis, the resin is removed by filtration, the filtrate is collected, and most of the lysis buffer is removed by rotary evaporation to obtain the concentrated product.

[0034] (4) Peptide purification: Pre-cooled anhydrous diethyl ether was added to the concentrated product to precipitate the target peptide. The precipitate was collected by centrifugation at 4°C and washed repeatedly with pre-cooled anhydrous diethyl ether to fully remove residual trifluoroacetic acid. After washing, the precipitate was dissolved in deionized water and freeze-dried to obtain the target peptide powder, which was then sealed and stored at -20°C for later use.

[0035] Example 2

[0036] Oxidative coupling of guaiacol catalyzed by peptide-mimicking enzyme catalyst In a PBS buffer system (pH 7, 0.02M), guaiacol, a peptide mimic enzyme (P1-P6) catalyst, or a heme chloride stock solution with the same concentration as the mimic enzyme catalyst system were added sequentially, and hydrogen peroxide was added to initiate the reaction. The peptide mimic enzyme catalyst solution used in the reaction had a peptide concentration of 4 mM and a heme concentration of 50 μM, while the heme chloride stock solution used as a control had a concentration of 50 μM. Guaifenesin was first dissolved in ethanol to aid dissolution, and then diluted with water to prepare a guaiacol solution; wherein the final volume fraction of ethanol in the solution was 1% (v / v), and the final concentration of guaiacol was 1.6 mM. In 0.7 mL of PBS buffer, 100 μL of the peptide mimic enzyme catalyst solution or heme chloride stock solution, 100 μL of the 1.6 mM guaiacol solution, and finally 100 μL of the 8 mM hydrogen peroxide solution were added sequentially. The final reaction system contained a peptide concentration of 0.4 mM, a heme chloride concentration of 5 μM, an H₂O₂ concentration of 0.8 mM, and an initial guaiacol concentration of 0.16 mM. The absorption peak of the product at 470 nm was detected in real-time using a UV-Vis spectrometer. The reaction was carried out at 25 °C using a matching temperature control kit, with readings taken every 30 s. The conversion rate (ε) was calculated using the molar absorptivity of tetraguaiacol. 470 =2.66×10 4 L·mol -1 ·cm -1 The concentration C at different times was obtained by conversion. t Concentration C at a specific time point t Divide by the initial concentration C0 to obtain the guaiacol reaction conversion rate at the corresponding time point.

[0037] Table 1. Conversion rate of guaiacol catalyzed by polypeptide-mimicking enzyme at 3 minutes.

[0038] The conversion rates of the above-mentioned polypeptide mimic enzymes (P1-P6) catalyst and heme chloride as a control group at 3 minutes are shown in Table 1. Figure 2 The substrate conversion curves for guaiacol in the first 210 s are shown. The use of peptide-mimicking enzyme catalysts with different amino acid sequences (P1-P6) significantly improved the catalytic performance of free heme, enabling the detection of tetraguaiacol in a shorter time. Peptide P5 (Ac-I3HGGH-NH2) showed the highest performance improvement.

[0039] Example 3

[0040] The oxidation of catechol by peptide-mimicking enzyme catalysts To evaluate the catalytic degradation performance of the peptide-heme co-assembly system of this invention on phenolic pollutants, degradation experiments were conducted using catechol as a model pollutant. Solutions of peptides P1–P6 and heme chloride were prepared separately. The final concentration of heme chloride was 50 μmol / L. Peptides P1–P6 were mixed with heme chloride and incubated at room temperature for more than one day to form the corresponding peptide-heme co-assembly systems. A 1 mM aqueous solution of catechol was prepared. In 0.7 mL of PBS buffer (pH 7, 0.02 M), 100 μL of peptide-mimicking enzyme catalyst solution or heme chloride stock solution (control), 100 μL of 1 mM catechol solution, and finally 100 μL of 8 mM hydrogen peroxide solution were added sequentially. The final working concentrations of each component were as follows: peptide mimic enzyme (P1~P6) 0.4 mM, heme chloride 5 μM, H2O2 0.8 mM, and catechol 0.1 mM. The reaction was stopped by adding 100 μL of 10 mM sodium thiosulfate after reacting at 25°C for 180 min.

[0041] The characteristic absorption peak of catechol is located at 276 nm. The concentration of catechol in the reaction solution was determined by reversed-phase high-performance liquid chromatography (RP-HPLC) under the following conditions: a C18 column (4.6 mm × 250 mm, 5 μm); a mobile phase consisting of methanol and 0.2% acetic acid aqueous solution at a ratio of 50:50 (V / V); a flow rate of 0.8–1.0 mL / min; a UV detection wavelength of 276 nm; a column temperature maintained at room temperature or 38 °C; an injection volume of 20 μL; and a single analysis time of 10 minutes.

[0042] Table 2. Degradation rate of catechol catalyzed by peptide-mimic enzyme catalyst at 180 minutes.

[0043] The results are shown in Table 2. The peptide-based enzyme catalysts constructed using different amino acid sequences (P1-P6) significantly improved the catalytic efficiency of the oxidation reaction of phenolic substances. Among them, peptide P5 showed the greatest performance improvement.

[0044] Example 4

[0045] Peptide-mimicking enzyme catalysts for the catalytic hydrolysis of nitrophenol acetate p-Nitrophenol acetate was added to a PBS buffer system (pH 7, 0.02 M), and the reaction was directly triggered after the addition of a peptide-mimicking enzyme. The final reaction system contained a peptide concentration of 0.4 mM, a heme concentration of 5 μM, and an initial p-nitrophenol acetate concentration of 1 mM. After reacting for 60 min at 25 °C in a water bath, the absorption peak of the product at 405 nm was detected in real time using a UV-Vis spectrometer. The conversion rate was obtained using a standard curve of p-nitrophenol.

[0046] Table 3. Conversion rate of p-nitrophenol acetate catalyzed by peptide-mimicking enzyme at 60 min.

[0047] The results are shown in Table 3. The peptide-mimicking enzyme catalysts constructed using different amino acid sequences (P1-P6) significantly improved the hydrolysis efficiency of p-nitrophenol acetate. Among them, peptide P5 showed the greatest performance improvement.

[0048] Example 5

[0049] Effects of reaction conditions on the oxidative coupling of guaiacol catalyzed by peptide-mimicking enzyme catalyst Guaiacin and peptide mimicry were added sequentially to buffer solutions with different pH values ​​(pH 5, 7, or 9), and hydrogen peroxide was added to initiate the reaction. The effect of pH value on the oxidative coupling of guaiacin catalyzed by the peptide mimicry catalyst was determined. In the final reaction system, the peptide concentration was 0.4 mM, the heme chloride concentration was 5 μM, the H2O2 concentration was 0.8 mM, and the initial concentration of guaiacin was 0.16 mM. A control group was used, catalyzing the oxidative coupling of guaiacin with only heme chloride (final concentration in the reaction system was 5 μM). The reaction temperature was 25℃. The absorption peak of the product at 470 nm in each group was detected in real time using a UV-Vis spectrometer, and read every 30 s. The real-time conversion rate was calculated based on the percentage decrease in absorbance within the first 3 minutes. The results are as follows: Figure 3 As shown, using guaiacol as a model, the optimal pH condition for the reaction catalyzed by polypeptide mimic enzymes P1 (Ac-I3HH-NH2) and P5 (Ac-I3HGGH-NH2) is pH 9.

[0050] The effect of hydrogen peroxide concentration on the catalytic performance of the peptide-mimicking enzyme P5 was determined at different actual working concentrations (0.8 mM or 8 mM). In the final reaction system, the peptide concentration was 0.4 mM, the heme chloride concentration was 5 μM, the H₂O₂ concentration was 0.8 mM or 8 mM, the initial guaiacol concentration was 0.16 mM, the reaction temperature was 25 °C, and the pH was 7. The experimental method was the same as above, and the results are as follows. Figure 4 As shown, using guaiacol as a model, the optimal hydrogen peroxide concentration for the polypeptide mimic enzyme P5 (Ac-I3HGGH-NH2) catalyzed reaction is 8 mM.

[0051] The effect of reaction temperature on the catalytic performance of the peptide-mimicking enzyme P5 was determined at different temperatures (25℃, 40℃, and 60℃). In the final reaction system, the peptide concentration was 0.4 mM, the heme chloride concentration was 5 μM, the H₂O₂ concentration was 0.8 mM, and the pH was 7. The reaction was carried out in a water bath at the corresponding temperatures. All samples were preheated at the same temperature before being added sequentially. After 3 min of reaction, the absorbance change was measured and converted to conversion rate. The results are as follows: Figure 5 As shown, using guaiacol as a model, the optimal temperature for the reaction catalyzed by the polypeptide mimic enzyme P5 (Ac-I3HGGH-NH2) is 40℃.

[0052] Based on the above optimal conditions, the reaction process was carried out in a Tris-HCl buffer at pH 9, resulting in a final reaction system with a peptide concentration of 0.4 mM, a heme chloride concentration of 5 μM, an H₂O₂ concentration of 8 mM, and a pH of 9. The reaction was conducted in a water bath at 40°C to obtain the conversion rate of guaiacol coupling under the optimal reaction conditions. The results are shown in Table 4. This demonstrates that under the optimal reaction conditions, the catalytic efficiency of the peptide-mimicking enzyme P5 for phenolic oxidative coupling is significantly improved.

[0053] Table 4. Conversion rate of guaiacol conjugation catalyzed by polypeptide-mimicking enzyme at 3 minutes.

[0054] Example 6

[0055] Retention of peptide-mimicking enzyme activity after high-temperature treatment Compared to natural enzymes, peptide-mimicking enzymes generally retain their original catalytic activity better after high-temperature or extreme acid-base treatment. In this example, peptide-mimicking enzyme P5 and natural horseradish peroxidase solutions were incubated in a water bath at 80°C for 1 hour, then placed in a refrigerator at 4°C for 24 hours. Under the reaction conditions of Example 1, the reaction rate of catalytic oxidation of guaiacol by mimicking enzyme P5 and natural horseradish peroxidase at 3 min was re-measured, and divided by their original reaction rate to obtain the final catalytic rate residual rate. Figure 6 (and Table 5).

[0056] Table 5 Comparison of activity retention between simulated enzymes and natural enzymes

[0057] The results showed that the peptide-mimicking enzyme catalyst constructed using P5(Ac-I3HGGH-NH2) retained higher reactivity than natural enzymes after high-temperature treatment.

[0058] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A multifunctional polypeptide-mimicking enzyme catalyst, characterized in that, The multifunctional polypeptide-mimicking enzyme catalyst comprises a co-assembly of a polypeptide and heme chloride; the amino acid sequence of the polypeptide is shown in any one of SEQ ID NO:1 to SEQ ID NO:6, wherein the N-terminus of the polypeptide is modified with an acetyl group and the C-terminus is modified with an amide group.

2. The multifunctional polypeptide-mimicking enzyme catalyst according to claim 1, characterized in that, The molar ratio of the polypeptide to heme chloride is 1:10 to 1:

80.

3. The multifunctional polypeptide-mimicking enzyme catalyst according to claim 2, characterized in that, The molar ratio of the polypeptide to heme chloride is 1:

80.

4. The multifunctional polypeptide-mimicking enzyme catalyst according to claim 1, characterized in that, The polypeptide-mimicking enzyme catalyst exhibits laccase-like, peroxidase-like, and esterase-like activities.

5. The use of the multifunctional polypeptide-mimicking enzyme catalyst according to any one of claims 1 to 4 as an alternative catalyst for laccase-like, peroxidase-like, and esterase-like chemical reactions.

6. The application according to claim 5, characterized in that, The chemical reaction is at least one of phenolic oxidative coupling reaction, phenolic oxidation reaction, and hydrolysis reaction of ester substrate.

7. The application according to claim 6, characterized in that, The phenolic oxidative coupling reaction is a guaiacol coupling reaction, the phenolic oxidation reaction is an oxidation reaction of catechol, and the hydrolysis reaction of the ester substrate is a hydrolysis reaction of p-nitrophenol acetate.

8. The application according to claim 7, characterized in that, The phenolic oxidative coupling reaction and phenolic oxidation reaction are carried out by mixing a polypeptide-mimicking enzyme catalyst with phenols, adding 0.8-8 mM hydrogen peroxide, and reacting at pH 5.0-9.0 and temperature 25-60℃ for 3-180 min.

9. The application according to claim 7, characterized in that, The hydrolysis reaction of the ester substrate is carried out by mixing the polypeptide-mimicking enzyme catalyst with the ester substrate.

10. The application according to claim 8 or 9, characterized in that, In the reaction system, the final concentration of the polypeptide in the polypeptide-mimicking enzyme catalyst is 0.4 mM, and the final concentration of heme chloride is 5 μM.

Images