A highly active polyphenol oxidase and a method for preparing the same

Abstract

The application discloses a kind of high-activity polyphenol oxidase and preparation method thereof, belong to nano mimic enzyme synthesis technical field.The polyphenol oxidase of the application is with copper ion as active center, 2-methyl imidazole and guanylate disodium as organic ligand.Preparation method thereof includes the following steps: guanylate disodium solution is added to copper chloride solution under stirring condition, to obtain first mixed solution;First mixed solution is slowly added to 2-methyl imidazole solution under stirring condition, to obtain second mixed solution;Second mixed solution is centrifuged after ultrasonic at room temperature, to obtain brown precipitate, and the polyphenol oxidase is prepared by washing and drying brown precipitate.The mimic enzyme prepared by the application has higher catalytic performance, better stability and feasibility of large-scale preparation compared with natural enzyme, and will have broad application prospect in the field of biosensor.

Description

Technical Field

[0001] This invention relates to the field of nano-mimetic enzyme synthesis technology, specifically to a highly active polyphenol oxidase and its preparation method. Background Technology

[0002] Enzymes, as one of the most important biomolecules regulating biological growth, are biocatalysts with high catalytic efficiency, high substrate specificity, and high biocompatibility. Most are composed of proteins, with a small portion consisting of RNA, and they play a significant role in fields such as biosensing, medical diagnostics, and agro-food. However, the preparation and purification of most natural enzymes are time-consuming and labor-intensive, and they suffer from poor stability, easily denaturing and losing activity under harsh environments (high temperature, strong acid, strong alkali), greatly limiting their wider application. Therefore, finding a low-cost, highly stable enzyme analog to replace natural enzymes is of great significance.

[0003] Nanozymes are artificial enzymes inspired by natural enzymes, possessing both enzymatic activity and the unique properties of nanomaterials. Compared to natural enzymes, they are lower in preparation cost, easier to store, and more stable. Notably, nanozymes also possess the characteristic of tunable activity, which is absent in natural enzymes, making them highly promising for applications in biosensors, environmental remediation, and the food industry. The specific catalytic activities of nanozymes, such as peroxidase-like (POD-like), oxidase-like (OXD-like), hydrolase-like (EC3-like), catalase-like (CAT-like), superoxide dismutase-like (SOD-like), and polyphenol oxidase-like (PPO-like), have attracted widespread attention. Among these, polyphenol oxidase-like nanozymes have experienced rapid development in recent years due to their unique environmental friendliness and have become the preferred choice for various biosensing, food detection, and industrial processing applications.

[0004] Copper-based mimetic enzymes are a type of polyphenol oxidase, comprising a copper ion active center and a ligand. Existing copper-based mimetic enzymes mostly employ a single ligand. For example, Ke Zhimin et al. synthesized three copper-based laccase mimics—CH-Cu, Cu-Cys, and CA-Cu—using amino acids or peptides as ligands; Liang Hao et al. synthesized a Cu / GMP copper-based laccase mimic using GMP nucleotides as a ligand; and Sun Yue synthesized a two-dimensional nanozyme, MIZ-Cu, with laccase activity, using 2-methylimidazole as an organic ligand and divalent copper as the metal active center. While these synthesized copper-based mimetic enzymes exhibit superior performance compared to natural laccase, further research revealed that using a single ligand results in low coordination synthesis yields and requires further improvement in the coordination reaction rate with copper ions. Therefore, developing a polyphenol oxidase-like enzyme with high yield and better performance is of great significance. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide a highly active polyphenol oxidase and its preparation method.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a polyphenol oxidase, wherein the polyphenol oxidase uses copper ions as the active center and 2-methylimidazole and disodium guanylate as organic ligands.

[0008] The polyphenol oxidase of the present invention uses a combination of 2-methylimidazole and disodium guanylate as organic ligands, which effectively improves the synthesis yield compared with a single ligand; moreover, it has a significant synergistic effect in improving the catalytic activity of the polyphenol oxidase.

[0009] A second aspect of the present invention provides a method for preparing the above-mentioned polyphenol oxidase, comprising the following steps:

[0010] A copper chloride solution was added to a disodium guanylate solution under stirring to obtain a first mixture; the first mixture was then slowly added to a 2-methylimidazole solution under stirring to obtain a second mixture.

[0011] The second mixture was ultrasonicated and centrifuged at room temperature to obtain a brown precipitate. The brown precipitate was washed and dried to prepare a polyphenol oxidase.

[0012] In the preparation method of the polyphenol oxidase of the present invention, the order of addition of each raw material is based on the experimentally determined priority of the polyphenol oxidase catalytic performance. Since the copper chloride solution is blue, directly mixing the three components makes it impossible to determine whether disodium guanylate and 2-methylimidazole are completely dissolved, and the ligands will form uneven coordination with copper ions if they are not completely dissolved. Therefore, the present invention dissolves the three components separately and then mixes them in sequence.

[0013] Preferably, the solvent used to prepare the disodium guanylate solution, copper chloride solution, and 2-methylimidazole solution is a methanol-water mixture; in the methanol-water mixture, the volume ratio of methanol to water is 1:3.

[0014] Preferably, the concentration of the disodium guanylate solution is 0.005-0.015 g / ml; the concentration of the copper chloride solution is 0.01-0.02 g / ml; and the concentration of the 2-methylimidazole solution is 0.08-0.09 g / ml.

[0015] Preferably, the volume ratio of the disodium guanylate solution, copper chloride solution, and 2-methylimidazole solution added is 1:2:2.

[0016] Preferably, the ultrasonic time is 1 hour and the ultrasonic power is 500W.

[0017] To improve the efficiency of coordination synthesis, this invention employs ultrasonic treatment on the mixed raw materials. The power of the ultrasonic treatment is crucial; too low an ultrasonic power will increase the synthesis time and reduce the yield in the same amount of time, while too high an ultrasonic power will cause the coordination of the outer rings to be completed prematurely, resulting in uneven activity. Through experimental optimization, it was found that the coordination synthesis efficiency is optimal when the ultrasonic power is 500W.

[0018] Preferably, vacuum drying is used, with a drying temperature of 60°C and a drying time of 8 hours.

[0019] The beneficial effects of this invention are:

[0020] This invention, for the first time, utilizes copper ions as the active center and 2-methylimidazole and disodium guanylate as ligands to coordinate with copper ions, and prepares a polyphenol oxidase-like enzyme via ultrasonic synthesis. The preparation method provided by this invention is fast, mild, simple, and easy to implement. Compared with natural enzymes, the prepared mimic enzyme exhibits higher catalytic performance, better stability, and feasibility for large-scale preparation, showing broad application prospects in the field of biosensors. Attached Figure Description

[0021] Figure 1 Scanning electron microscope image of MI-Cu-GMP NPs prepared in Example 1 of this invention.

[0022] Figure 2 (A) XPS spectrum of MI-Cu-GMP NPs; (B) High-resolution XPS spectrum of Cu 2p obtained on MI-Cu-GMP NPs.

[0023] Figure 3 (A) Effect of 2,4-DP concentration on the activity of laccase mimic enzymes of MI-Cu-GMP NPs; (B) Lineweaver-Burk plot of the effect of 2,4-DP concentration on the activity of laccase mimic enzymes of MI-Cu-GMP NPs.

[0024] Figure 4 The activity of 2,4-DP was detected using MI-Cu-GMP NPs, Cu-GMP, and Cu-MI, respectively.

[0025] Figure 5 (A) Comparison of 2,4-DP activity at 30℃ and 70℃; (B) Comparison of 2,4-DP activity at pH 5.5 and 8.0; (C) Comparison of 2,4-DP activity on the first and seventh day of storage; (D) Comparison of 2,4-DP activity at ionic strengths of 0 mM and 600 mM. Detailed Implementation

[0026] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0027] Terminology Explanation:

[0028] The term "room temperature" as used in this invention refers to a temperature of 15-30°C.

[0029] Drying refers to drying under vacuum until the material is cured.

[0030] MI: 2-Methylimidazole.

[0031] GMP: Disodium guanylate.

[0032] 4-AP: 4-Aminoantipyrine.

[0033] 2,4-DP: 2,4-dichlorophenol.

[0034] As described in the background section, the preparation and purification of natural laccases are currently costly, and they are prone to denaturation and loss of activity under harsh environments (high temperature, strong acid, strong alkali). Moreover, existing methods for preparing laccase mimics are mostly complex and time-consuming. Therefore, developing a simpler and higher-performance laccase mimic is of great significance.

[0035] Based on this, the present invention uses copper ions as the active center, and coordinates disodium guanylate and 2-methylimidazole with copper ions to prepare a copper-based laccase mimic enzyme with high catalytic activity and high stability via ultrasonic synthesis. Furthermore, this preparation method is fast, mild, yields high amounts, and is simple and easy to implement.

[0036] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0037] The test materials used in the embodiments and comparative examples of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions were performed according to conventional test methods or the supplier's recommended operating instructions.

[0038] Example 1: Preparation of copper-based laccase mimics (MI-Cu-GMP NPs)

[0039] Disperse 0.2 g of disodium guanylate in 20 mL of methanol / H2O (1:3, v / v), and sonicate the dispersion for 10 min to prepare a disodium guanylate solution.

[0040] 0.6 g of copper chloride was dispersed in 40 mL of methanol / H2O (1:3, v / v), and the dispersion was sonicated for 10 min to prepare a copper chloride solution.

[0041] 3.28 g of 2-methylimidazole was dispersed in 40 mL of methanol / H2O (1:3, v / v), and the dispersion was sonicated for 10 min to prepare a 2-methylimidazole solution.

[0042] Then, 20 mL of disodium guanylate solution was slowly added to 40 mL of copper chloride solution under stirring (500 rpm) to obtain the first mixture. The first mixture was then slowly added to 40 mL of 2-methylimidazole solution under stirring (500 rpm) to obtain the dark blue second mixture. The dark blue second mixture was sonicated at room temperature (500 W) for 1 h and then centrifuged (5000 rpm). The resulting brown precipitate was washed three times with ultrapure water and dried under vacuum at 60 °C for 8 h to prepare 420 mg of copper-based laccase mimic enzyme (denoted as MI-Cu-GMP NPs or MI-Cu-GMP).

[0043] The MI-Cu-GMP NPs prepared in this embodiment were structurally characterized using scanning electron microscopy, and the results are shown in the figure. Figure 1 The structure of MI-Cu-GMP NPs exhibits an irregular spherical shape.

[0044] The MI-Cu-GMP NPs prepared in this embodiment were analyzed using X-ray photoelectron spectroscopy, and the results are shown in the figure. Figure 2 A. Full-scan spectroscopy revealed that MI-Cu-GMP NPs contain Cu, O, N, C, and P elements. Since Cu is the active center, we further analyzed the oxidation state of copper ions within them; the results are shown below. Figure 2 B. The Cu 2p3 / 2 and Cu 2p1 / 2 electrons of Cu(II) show peaks at 934.7 eV and 954.6 eV, respectively, while the binding energies of Cu(I) are lower at 932.8 eV and 952.2 eV, indicating that some Cu... 2+ After coordination with a ligand, it is reduced to Cu. + .

[0045] Comparative Example 1: Preparation of Cu-GMP Laccase Mimic Enzyme

[0046] Disperse 0.2 g of disodium guanylate in 20 mL of methanol / H2O (1:3, v / v), and sonicate the dispersion for 10 min to prepare a disodium guanylate solution.

[0047] 0.6 g of copper chloride was dispersed in 40 mL of methanol / H2O (1:3, v / v), and the dispersion was sonicated for 10 min to prepare a copper chloride solution.

[0048] Then, 20 mL of disodium guanylate solution was slowly added to 40 mL of copper chloride solution under stirring (500 r / min) to obtain a mixture. The mixture was sonicated at room temperature (500 W) for 1 h and then centrifuged (5000 r / min). The precipitate was washed three times with ultrapure water and dried under vacuum at 60 °C for 8 h to prepare 25 mg of laccase mimic enzyme (Cu-GMP).

[0049] Comparative Example 2: Preparation of Cu-MI Laccase Mimic Enzyme

[0050] 0.6 g of copper chloride was dispersed in 40 mL of methanol / H2O (1:3, v / v), and the dispersion was sonicated for 10 min to prepare a copper chloride solution.

[0051] 3.28 g of 2-methylimidazole was dispersed in 40 mL of methanol / H2O (1:3, v / v), and the dispersion was sonicated for 10 min to prepare a 2-methylimidazole solution.

[0052] Then, 40 mL of copper chloride solution was slowly added to 20 mL of 2-methylimidazole solution under stirring (500 r / min) to obtain a mixture. The mixture was sonicated at room temperature (500 W) for 1 h and then centrifuged (5000 r / min). The precipitate was washed three times with ultrapure water and dried under vacuum at 60 °C for 8 h to prepare 255 mg of laccase mimic enzyme (Cu-MI).

[0053] Experimental example:

[0054] 1. Catalytic performance evaluation

[0055] 1.1 Catalytic performance evaluation of MI-Cu-GMP NPs

[0056] The catalytic performance of the MI-Cu-GMP NPs prepared in Example 1 was determined by a colorimetric reaction of 4-AP and 2,4-DP. 4-AP (1 mg / mL) was used to react with 2,4-DP. -1 Mix 100 μL of 2,4-DP and 100 μL of different concentrations with MES buffer (30 mM, pH = 7.0, 700 μL), then add MI-Cu-GMP NPs (1 mg / mL). -1 The initial reaction rate was determined at room temperature (100 μL).

[0057] The catalytic performance of natural laccase was determined using the same method. Natural laccase is an existing product, and the natural laccase used in this experiment is described in the literature (Wang, Y., He, C., Li, W., Zhang, J., & Fu, Y. (2017). Catalytic performance of oligonucleotide-templated Pt nanozyme evaluated by laccase substrates. Catalysis Letters, 147, 2144-2152.).

[0058] See results Figure 3 As the concentration of 2,4-DP increases, the catalytic activity of the enzyme increases, and the catalytic reaction of MI-Cu-GMP NPs conforms to the Michaelis-Menten kinetic curve.

[0059] Calculate K using the Michaelis-Menten model m and V max The values ​​are shown in Table 1.

[0060] Table 1: K m and V max value

[0061]

[0062] The Km value of MI-Cu-GMP NPs is higher than that of the natural enzyme, indicating that MI-Cu-GMP NPs have a higher affinity for the substrate than natural laccase. The V0.05 of MI-Cu-GMP NPs... max This is 7.96 times that of natural laccase, indicating that the catalytic rate of MI-Cu-GMP NPs is significantly higher than that of natural laccase. This demonstrates the synthesis of a laccase mimic with high affinity and high activity for its substrate.

[0063] 1.2 Performance comparison of laccase mimics prepared with two ligands and one ligand

[0064] Using 2,4-DP as a substrate and 4-AP as a chromogenic agent, a red azo substance is produced under the catalytic action of the laccase mimic enzyme. The catalytic activity of the laccase mimic enzyme is characterized by measuring the absorbance of the red azo substance at a wavelength of 510 nm. Details are as follows:

[0065] 4-AP (1 mg mL) -1 100 μL) and 2,4-DP (1 mg mL) -1 Mix 100 μL of laccase mimic enzyme with MES buffer (30 mM, pH = 7.0, 700 μL), then add 1 mg / mL of laccase mimic enzyme.-1 100 μL was reacted at room temperature for 30 min, centrifuged, and the absorbance of the supernatant was measured at a wavelength of 510 nm.

[0066] The laccase mimics used here are MI-Cu-GMP NPs prepared in Example 1, Cu-GMP prepared in Comparative Example 1, and Cu-MI prepared in Comparative Example 2. The amount of laccase mimics added is kept constant, and the type of laccase mimic is used as a single variable.

[0067] See results Figure 4 The catalytic activity of MI-Cu-GMP NPs (absorbance 1.8) was higher than that of Cu-GMP (absorbance 0.6) and Cu-MI (absorbance 1.1). This demonstrates that the polyphenol oxidase prepared using dual ligands has higher activity than the enzyme prepared using a single ligand.

[0068] 2. Stability Performance Test

[0069] The stability of the MI-Cu-GMP NPs prepared in Example 1 was evaluated under different conditions (temperature, pH, storage time, ionic strength). Details are as follows:

[0070] (1) Temperature stability

[0071] The MI-Cu-GMP NPs (nanozymes) and natural laccase (natural enzymes) prepared in Example 1 were respectively prepared into enzyme solutions of 1 mg / ml. The enzyme solutions were incubated in water baths at 30℃ and 70℃ for 30 min, respectively. The enzyme activity under different temperature treatments was calculated according to the method for evaluating catalytic performance in 1.1. The enzyme activity of the nanozymes treated at 30℃ was used as the baseline (denoted as 100%), and the ratio of the enzyme activity of other treatments to the baseline was used as the relative activity.

[0072] See results Figure 5 A. The results showed that MI-Cu-GMP NPs maintained extremely high activity after treatment at 70℃, while the natural enzymes were almost completely inactivated after treatment at 70℃.

[0073] (2) pH stability

[0074] The MI-Cu-GMP NPs (nanozymes) and natural laccase (natural enzymes) prepared in Example 1 were each prepared into enzyme solutions of 1 mg / ml. After the enzyme solutions were placed in environments of pH 5.5 and 8.0 for 30 min, the enzyme activity under different pH treatments was calculated according to the method for evaluating catalytic performance in 1.1. The enzyme activity of the nanozyme treated at pH 5.5 was used as the baseline (denoted as 100%), and the ratio of the enzyme activity of other treatments to the baseline was used as the relative activity.

[0075] See results Figure 5B. The results showed that MI-Cu-GMP NPs have a strong ability to adapt to different acids and bases, but the natural enzymes have extremely low activity at pH 5.5 and 8.0.

[0076] (3) Storage stability

[0077] The MI-Cu-GMP NPs (nanozymes) and natural laccase (natural enzymes) prepared in Example 1 were respectively prepared into enzyme solutions of 1 mg / ml. The enzyme activities were calculated on the first day and after 7 days of storage, respectively, according to the method for evaluating catalytic performance in 1.1. The enzyme activities of the nanozymes and natural enzymes on the first day were used as the baseline, and the ratio of the enzyme activity on the 7th day to the corresponding baseline was the relative activity.

[0078] See results Figure 5 C. The results showed that MI-Cu-GMP NPs retained more than 96% of their initial activity after seven days of storage, while the native enzyme retained only 28% of its initial activity.

[0079] (4) Salt ion stability

[0080] The MI-Cu-GMP NPs (nanozymes) and natural laccase (natural enzymes) prepared in Example 1 were each prepared into enzyme solutions of 1 mg / ml. 100 μL of 0 mM and 600 mM sodium chloride solutions were added to the enzyme solutions, respectively. The enzyme activity under different salt concentrations was calculated according to the method for evaluating catalytic performance in 1.1. The enzyme activity of the nanozymes treated with 0 mM sodium chloride was used as the baseline (denoted as 100%), and the ratio of the enzyme activity of other treatments to the baseline was used as the relative activity.

[0081] See results Figure 5 D. The results showed that the activity of MI-Cu-GMP NPs was increased and the activity of the native enzyme was inhibited in the presence of 600 mM salt ions.

[0082] The above demonstrates that the present invention synthesizes a nanozyme that can adapt to extreme environments (temperature, pH, ionic strength) and has excellent storage stability.

[0083] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A polyphenol oxidase-like enzyme, characterized in that, The polyphenol oxidase described uses copper ions as the active center and 2-methylimidazole and disodium guanylate as organic ligands.

2. The method for preparing the polyphenol oxidase according to claim 1, characterized in that, Includes the following steps: A copper chloride solution was added to a disodium guanylate solution under stirring to obtain the first mixture. The first mixture was slowly added to a 2-methylimidazole solution under stirring to obtain the second mixture; The second mixture was ultrasonicated and centrifuged at room temperature to obtain a brown precipitate. The brown precipitate was washed and dried to prepare a polyphenol oxidase.

3. The preparation method according to claim 2, characterized in that, The solvents used to prepare the disodium guanylate solution, copper chloride solution, and 2-methylimidazole solution are all methanol-water mixtures; in the methanol-water mixture, the volume ratio of methanol to water is 1:

3.

4. The preparation method according to claim 2, characterized in that, The concentration of the disodium guanylate solution is 0.005-0.015 g / ml; the concentration of the copper chloride solution is 0.01-0.02 g / ml; and the concentration of the 2-methylimidazole solution is 0.08-0.09 g / ml.

5. The preparation method according to claim 2, characterized in that, The volume ratio of the added disodium guanylate solution, copper chloride solution, and 2-methylimidazole solution is 1:2:

2.

6. The preparation method according to claim 2, characterized in that, The ultrasound duration was 1 hour, and the ultrasound power was 500W.

7. The preparation method according to claim 2, characterized in that, Vacuum drying was used, with a drying temperature of 60℃ and a drying time of 8 hours.

Images