A CuZrO2NPs hydrolytic nanozyme, its preparation method and application

Abstract

The present application relates to CuZrO2 NPs hydrolysis nano-enzyme and its preparation method and application, first adopt the coprecipitation method synthesis CuZr (OH) 2 NPs, high temperature calcination, make the intramolecular dehydration synthesis CuZrO2 NPs.The application of CuZrO2 NPs hydrolysis nano-enzyme in proteolysis, mix CuZrO2 NPs with ultrapure water, make CuZrO2 NPs disperse uniformly, then add protein solution, water bath reaction, realize the proteolysis.CuZrO2 NPs still has hydrolysis effect for heat denatured and surfactant denatured protein, and has good recovery rate and wide pH activity range, the selected CuZrO2 NPs is applied to the hydrolysis of insoluble soybean protein and periplaneta americana protein.CuZrO2 NPs has great potential in biomedical and proteomics applications, further widening the possibility of developing nano-protease based on bimetallic oxide nanomaterials.

Description

Technical Field

[0001] This invention belongs to the field of enzyme research and development and production, specifically relating to a CuZrO2NPs hydrolytic nanozyme, its preparation method and application. Background Technology

[0002] Plant protein is a high-quality protein resource with higher sustainability compared to animal protein, and it has been widely used in various fields of the food industry. During processing, especially after enzymatic hydrolysis, some insoluble plant proteins can form insoluble protein aggregates. Soybeans, due to their high protein content, are one of the most important agricultural products worldwide. It is well known that soy protein is hydrophobic, and certain anti-nutritional factors it carries make it difficult for proteases in the digestive tract to hydrolyze, affecting digestion and potentially causing allergies. Enzymatic hydrolysis of soy protein not only effectively improves its solubility, emulsification, and foaming properties, but also reduces its allergenic effects. Furthermore, the essential amino acids in the insoluble soy peptide aggregates formed after enzymatic hydrolysis meet adult recommended amino acid standards, indicating high nutritional value. Studies have shown that soy protein is difficult to hydrolyze effectively in its natural state and can only be digested by various natural proteases after heating. However, heating easily alters the structure of proteins, affecting their function. Therefore, exploring methods to more effectively hydrolyze insoluble soy protein to improve its utilization rate in food processing has broad research value and market development potential.

[0003] Enzymes, as natural biocatalysts, possess high specificity and play a crucial role in biochemical reactions. However, the low stability, complex synthesis, and high cost of natural enzymes limit their application in biomedicine and food processing. Nanozymes are a class of nanomaterials with potential protease-like activity. The unique specific surface area at the nanoscale gives nanomaterials unique surface properties and volume effects, and they can mimic the structure or function of natural enzymes, thus exhibiting excellent catalytic activity. Therefore, nanozymes have the potential to become substitutes for natural enzymes. Research reports on nanozymes mainly focus on the following material types: gold nanoparticle-based nanozymes, polymer nanozymes, surfactant assemblies, peptide assemblies, metal and metal oxide nanoparticles, and metal-organic frameworks (MOFs).

[0004] Xu et al. (Xu JH, Ji NN, Guo MX, et al. CuCoO2Nanoparticles as a Nanoprotease for Selective Proteolysis with High Efficiency at Room Temperature[J]. ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 2023, 62(31)) discovered that CoCuO2NPs bimetallic oxide nanoparticles have the effect of hydrolyzing BSA and proposed a mechanism for the selective cleavage of hydrophobic residue peptide bonds by CoCuO2NPs. They proposed that this bimetallic oxide nanomaterial can be regarded as composed of two undetermined metal elements, A and B (the Lewis acids of the two elements must be different), and that its metal atoms can perform oxidative addition on hydroxyl groups in aqueous solution, thereby forming active centers for hydroxyl attachment on the surface. Xu et al. believe that Cu 2+ It has a low valence positive charge, while Co + With a high valence positive charge, after doping with two metals, the metal with the high valence positive charge can act as the carbonyl group of the Lewis acid-polarized peptide bond, while the -OH adsorbed on the surface of the low valence metal has a relatively strong nucleophilicity, thus promoting the entire peptide bond hydrolysis mechanism. Hydrolysis experiments of CoCuO2NPs on different protein substrates revealed that although literature reports that CoCuO2NPs exhibit good hydrolytic performance on BSA, OVA, and bovine blood γ-globulin, when the substrate is changed to a more complex insoluble plant protein (soybean protein) and an animal drug protein (American cockroach protein), the material did not show significant hydrolytic activity. This suggests that CoCuO2NPs has significant limitations in the application of hydrolyzing actual proteins. Summary of the Invention

[0005] The purpose of this invention is to provide a CuZrO2NPs hydrolytic nanozyme, its preparation method and application. This CuZrO2NPs hydrolytic nanozyme exhibits excellent performance in protein hydrolysis reactions and can be used for the enzymatic hydrolysis of soybean protein and American cockroach protein.

[0006] To obtain superior nano-protein hydrolysates, the inventors not only explored CuZrO2NPs but also investigated ZnZrO2NPs, another compound with a similar structure. No research in this field has yet documented the use of these two bimetallic oxide nanomaterials, CuZrO2NPs and ZnZrO2NPs, for protein hydrolysis. Besides the valence differences between metal elements promoting peptide bond hydrolysis, the electron-withdrawing effect of highly electronegative elements, and the role of Cu... 2+ and Zn 2+ For Zr 4+The generation of oxygen vacancies due to substitution increases the Lewis acidity of nanomaterials, and Lewis acidity is closely related to the hydrolytic performance of nanozymes. Based on this, the inventors hypothesized that CuZrO2NPs and ZnZrO2NPs might exhibit excellent performance in protein hydrolysis reactions. Therefore, this invention uses SDS-PAGE gel electrophoresis to verify whether these two bimetallic oxide nanomaterials possess protein hydrolysis activity. Ultimately, a bimetallic oxide—CuZrO2NPs—was discovered as a novel nanoprotein hydrolase. Further investigation of the enzymatic hydrolysis mechanism using LC-MS / MS revealed that it selectively acts on the amino and carboxyl terms of hydrophobic residues, facilitating peptide bond cleavage. This also verified that CuZrO2NPs, as a novel nanoprotein hydrolase, possesses good stability and recovery rate. Finally, CuZrO2NPs were applied to the hydrolysis of soybean protein and American cockroach protein. This invention further expands the application potential of bimetallic oxide nanomaterials in the field of protein hydrolysis, especially the hydrolysis of insoluble plant proteins.

[0007] The objective of this invention is achieved through the following technical solution: This invention provides a method for preparing CuZrO2NPs hydrolytic nanozymes. First, CuZr(OH)2NPs are synthesized by coprecipitation, and then calcined at high temperature to achieve intramolecular dehydration and synthesize CuZrO2NPs.

[0008] The preparation method includes the following steps: ZrOCl2·8H2O and CuCl2 were mixed and dissolved in deionized water, then stirred vigorously in a water bath to obtain a mixed solution. A KOH aqueous solution was prepared for later use. The resulting mixed solution was then precipitated with KOH solution until the pH reached 11, indicating the reaction was complete. The solid-liquid products were collected, centrifuged, and the supernatant was discarded to obtain the solid product. The surface of the solid product was then repeatedly washed with hot deionized water to remove chloride ions (Cl). - The obtained solid product was dried in an oven to obtain uncalcined CuZrO2NPs, i.e., CuZr(OH)2NPs; CuZr(OH)2NPs was ground into fine particles and then calcined in a muffle furnace. After annealing, the final product CuZrO2NPs was obtained, which is the CuZrO2NPs hydrolytic nanozyme.

[0009] Preferably, the preparation method specifically comprises: mixing 0.5 M ZrOCl2·8H2O and 0.05 M CuCl2, dissolving in 50.00 mL of deionized water, and then stirring vigorously in an 80 ℃ water bath for 30 min to obtain a mixed solution; preparing 70-80 mL of 1 M KOH aqueous solution for later use, and then precipitating the obtained mixed solution with KOH until the pH value reaches 11, which is considered as the reaction being completed; collecting the solid-liquid product, centrifuging at 3000 rpm for 3 min, then discarding the supernatant to obtain the solid product; and then repeatedly washing the surface with hot deionized water to remove chloride ions (Cl). - The obtained solid product was dried in an oven at 100 °C for 12 h to obtain uncalcined CuZrO2NPs, i.e., CuZr(OH)2NPs; CuZr(OH)2NPs were ground into fine particles and then calcined in a muffle furnace at 400 °C for 4 h. After annealing, the final product CuZrO2NPs was obtained, which is the CuZrO2NPs hydrolytic nanozyme.

[0010] The present invention also provides a CuZrO2NPs hydrolytic nanozyme prepared by the aforementioned preparation method.

[0011] The application of the CuZrO2NPs hydrolytic nanozyme in proteolytic hydrolysis.

[0012] The application described herein utilizes the CuZrO2NPs hydrolytic nanozyme to enzymatically hydrolyze proteins as follows: the CuZrO2NPs hydrolytic nanozyme is mixed with ultrapure water to disperse the CuZrO2NPs hydrolytic nanozyme evenly, then a protein solution is added, and the mixture is reacted in a water bath to achieve protein enzymatic hydrolysis.

[0013] In the aforementioned application, the protein solution is prepared by mixing protein and solvent, and during enzymatic hydrolysis, the weight ratio of CuZrO2NPs hydrolytic nanozyme to protein is (2~10):8. Preferably, the weight ratio of CuZrO2NPs hydrolytic nanozyme to protein during enzymatic hydrolysis is 4:8.

[0014] The water bath reaction was carried out at a temperature of 60 °C for 24 h.

[0015] The application of the CuZrO2NPs hydrolytic nanozyme in the preparation of an enzymatic hydrolysant for protein hydrolysis, wherein the enzymatic hydrolysant includes the CuZrO2NPs hydrolytic nanozyme.

[0016] The protein in question is an insoluble plant protein or an animal drug protein.

[0017] The insoluble plant protein mentioned is soybean protein.

[0018] The animal drug protein mentioned is American cockroach protein.

[0019] The solvent is 0.25~1% wtSDS solution, ultrapure water, or 1% ammonium bicarbonate solution; when the protein is an insoluble plant protein, the solvent can be 1% wtSDS solution; when the protein is an animal drug protein, the solvent can be 1% ammonium bicarbonate solution; when the protein is a soluble protein, the solvent can be ultrapure water.

[0020] When the protein is soy protein, the solvent is preferably 1% wt SDS.

[0021] Compared with the prior art, the advantages of the present invention are as follows: This invention designs nano-hydrolytic enzymes based on bimetallic oxide nanoparticle materials. By comparing the SDS-PAGE results of enzymatic incubation experiments of CuZrO2NPs and ZnZrO2NPs, the proteolytic activity of CuZrO2NPs was verified, thus discovering CuZrO2NPs as a novel nano-proteolytic enzyme. Furthermore, its enzymatic mechanism was investigated, revealing that CuZrO2NPs selectively cleave peptide bonds of hydrophobic residues, maintain hydrolytic activity even after heat denaturation and surfactant denaturation of BSA, and exhibit a wider pH range than natural proteases. Finally, the screened CuZrO2NPs were applied to the hydrolysis of insoluble soybean protein and American cockroach protein. In summary, this invention reveals the great potential of CuZrO2NPs in biomedical and proteomics applications, further expanding the possibilities for developing nano-proteases based on bimetallic oxide nanomaterials. Attached Figure Description

[0022] Figure 1 Here are the characterization images of CuZrO2NPs of this invention; (a) Transmission electron microscopy image at 20 nm; (b) Transmission electron microscopy image. Figure 10 nm; (c) X-ray powder diffraction pattern; (d) Fourier transform infrared spectrum; (e) X-ray photoelectron energy spectrum; (f) X-ray photoelectron energy spectrum of Zr element.

[0023] Figure 2 These are electrophoretic patterns of BSA hydrolyzed by CuZrO2NPs and ZnZrO2NPs under different catalytic amounts: (a) CuZrO2NPs; (b) ZnZrO2NPs.

[0024] Figure 3This is a diagram of the cleavage sites of CuZrO2NPs on BSA; (a) Peptides generated by CuZrO2NPs covering the BSA sequence, with red arrows indicating cleavage sites generated by CuZrO2NPs and red underlines indicating peptides generated by CuZrO2NPs; (b) Three-dimensional structure of BSA; (c) Hydrophilicity and hydrophobicity of BSA, with blue indicating hydrophilic amino acids, gray indicating hydrophobic amino acids, and red indicating cleavage sites of hydrophobic amino acids generated by the hydrolysis of CuZrO2NPs (12 cleavage sites).

[0025] Figure 4 It refers to CuZrO2NPs hydrolyzed and thermally denatured BSA. "BSA" refers to natural BSA after being hydrolyzed by nanomaterials; "thermally denatured BSA" refers to natural BSA that has undergone thermal denaturation treatment before being hydrolyzed by nanomaterials; "control group" refers to natural BSA that has not undergone any treatment.

[0026] Figure 5 It is CuZrO2NPs hydrolyzed SDS and denatured BSA.

[0027] Figure 6 The following are examples of BSA hydrolysis under different pH and buffer solutions: (a) PB buffer; (b) Tris-HCl buffer.

[0028] Figure 7 The process involves enzymatic hydrolysis of soy protein using CuZrO2NPs: (a) soy protein was dissolved in 1% wt SDS, followed by hydrolysis with 2 mg, 4 mg, and 6 mg of CuZrO2NPs; (b) soy protein was dissolved in different concentrations of SDS, followed by hydrolysis with 2 mg of CuZrO2NPs. "SDS-C" indicates that the soy protein was dissolved only in SDS buffer; "SDS" indicates that the soy protein was first dissolved in SDS buffer, followed by enzymatic hydrolysis using nanomaterials.

[0029] Figure 8 The study involved the enzymatic hydrolysis of American cockroach protein by CuZrO2NPs under different catalytic concentrations.

[0030] Figure 9 This is the XRD pattern of CuCoO2NPs.

[0031] Figure 10 These are transmission electron micrographs of CuCoO2NPs: (a) 100 nm; (b) 50 nm.

[0032] Figure 11 It is BSA that is enzymatically hydrolyzed by CuCoO2NPs.

[0033] Figure 12 These are examples of CuCoO2NPs enzymatically hydrolyzing soybean protein under different catalytic concentrations.

[0034] Figure 13 The results show the enzymatic hydrolysis of American cockroach protein by CuCoO2NPs under different catalytic concentrations. Detailed Implementation

[0035] The present invention will now be described in detail with reference to the accompanying drawings and embodiments: 1. Materials and Methods 1.1 Materials and Reagents CuZrO2NPs and ZnZrO2NPs were synthesized in the laboratory; soybean protein (Shanghai Aladdin Biochemical Technology Co., Ltd.); bovine serum albumin (Shanghai Beyotime Biotechnology Co., Ltd.); denaturing, non-reducing 5× protein loading buffer (Shanghai Yamei Biomedical Technology Co., Ltd.); SWE rapid high-resolution electrophoresis buffer powder (Wuhan Sewell Biotechnology Co., Ltd.); P0105-3 lower gel A solution, P0105-4 lower gel B solution, P0105-1 upper gel A solution, P0105-2 upper gel B solution, P0105-5 modified coagulant (Beijing Lanbolide Trading Co., Ltd.); R250 Coomassie Brilliant Blue (Beijing Lanjieke Technology Co., Ltd.); Prestained Protein Marker VII (8-195) kDa (Wuhan Sewell Biotechnology Co., Ltd.); Sodium hydroxide (analytical grade, Sinopharm Chemical Reagent Co., Ltd.); Potassium hydroxide (laboratory grade, Shanghai Aladdin Biochemical Technology Co., Ltd.); Ammonium bicarbonate, anhydrous ethanol, glacial acetic acid, hydrochloric acid (Xilong Scientific Co., Ltd.); Methanol (analytical grade, Sinopharm Chemical Reagent Co., Ltd.); Sodium dihydrogen phosphate, anhydrous copper chloride (chemically pure, Shanghai Maclean Biochemical Technology Co., Ltd.); American cockroach protein and ultrapure water were prepared in the laboratory.

[0036] 1.2 Instruments and Equipment AUW120D electronic balance (SHIMADZU Corporation, Japan); PHS-3CpH precision pH meter (Shanghai Instrument & Electronics Scientific Instruments Co., Ltd., China); SX2 muffle furnace (Shanghai Yiheng Scientific Instruments Co., Ltd., China); JJ-1 precision booster electric stirrer (Shanghai Shangpu Instrument Equipment Co., Ltd., China); TS-2000A decolorizing shaker (Haimen Qilinbei Instrument Manufacturing Co., Ltd., China); SCI-RPro LCD CNC rocker shaker (Scilogex Corporation, USA); TGL-16L high-speed centrifuge (Shanghai Anting Scientific Instrument Factory, China); electrophoresis apparatus (BIO-RAD Corporation, USA); XH-C vortex mixer (Jintan Baitaxinbao Instrument Factory, China); VERTEX70 & ALPHA Fourier transform infrared spectrometer (Bruker Instruments GmbH, Germany); JEM-2100F transmission electron microscope (Nippon Electron Ltd., Japan); Ultima IV X-ray diffractometer (Rigaku Corporation, Japan); UltiMate 3000 RSLCnano nano-level liquid chromatography-tandem Q Exactive HF mass spectrometer (Thermo Scientific, USA).

[0037] 1.3 Sample Preparation 1.3.1 Synthesis of CuZrO2NPs CuZr(OH)2NPs were synthesized by coprecipitation, and CuZrO2NPs were synthesized by intramolecular dehydration after high-temperature calcination.

[0038] Mix 0.5 M ZrOCl₂·8H₂O with 0.05 M CuCl₂ and dissolve in 50.00 mL of deionized water. Stir vigorously in an 80°C water bath for 30 min. Prepare approximately 75 mL of 1 M KOH aqueous solution. Then, precipitate the resulting mixture with KOH until the pH reaches 11, indicating the reaction is complete. Collect the solid-liquid product and centrifuge at 3000 rpm for 3 min. Discard the supernatant to obtain the solid product. Wash the surface repeatedly with hot deionized water to remove chloride ions (Cl⁻). - The obtained solid product was dried in an oven at 100 °C for 12 h to obtain uncalcined CuZrO2NPs. These were then ground into fine particles and calcined in a muffle furnace at 400 °C for 4 h. After annealing, the final product, CuZrO2NPs, was obtained, which is the CuZrO2NPs hydrolytic nanozyme. The synthesized CuZrO2NPs were then characterized and analyzed using Fourier transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and transmission electron microscopy (TEM).

[0039] 1.3.2 Synthesis of ZnZrO2NPs This paper references the method of Moons et al. (Moons J, de Azambuja F, Mihailovic J, et al. Discrete Hf18 Metal-oxo Cluster as a Heterogeneous Nanozyme for Site-Specific Proteolysis[J]. ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 2020,59(23):9094-9101) to synthesize ZnZr(OH)2NPs via a hydrothermal method, followed by high-temperature calcination to achieve intramolecular dehydration and synthesize ZnZrO2NPs.

[0040] A suitable amount of ZrOCl2·8H2O and ZnO powder were mixed and dissolved in deionized water, then rapidly stirred in a 50 ℃ water bath for 1 h. 0.4 M NaOH solution was added dropwise to the clarified solution until no more precipitate formed. The reaction system was then sonicated for 45 min. After sonication, the solid-liquid product was placed in a 100 mL high-pressure reactor lined with fluoropolymer (ensuring the liner volume did not exceed 2 / 3), and heated in a muffle furnace to 120–130 ℃ for 12 h. The solid-liquid product was collected, centrifuged at 3000 rpm for 3 min, and the supernatant was discarded to obtain the solid product. The surface was repeatedly washed with hot deionized water to remove chloride ions (Cl). - The solid product was dried in an oven at 100 °C for 12 h to remove moisture, yielding uncalcined ZnZrO2NPs. Finally, the uncalcined ZnZrO2NPs were calcined in a muffle furnace at 550 °C for 6 h, and after annealing, pure ZnZrO2NPs were obtained.

[0041] 1.3.3 Preparation of soybean protein solution Accurately weigh 8.00 mg of soybean protein powder, add 1.00 mL of 1% wtSDS solution to prepare an 8 mg / mL soybean protein solution, sonicate for 10 min, centrifuge at 8000 r / min for 10 min, and take the supernatant for later use.

[0042] 1.3.4 Preparation of American cockroach protein solution Accurately weigh 100.00 mg of American cockroach protein, add 1.00 mL of 1% ammonium bicarbonate solution to prepare a 100 mg / mL American cockroach protein solution, sonicate for 10 min, centrifuge at 8000 r / min for 10 min, and take the supernatant for later use.

[0043] 1.3.5 Preparation of bovine serum albumin solution Accurately weigh 8.00 mg of bovine serum albumin (BSA), add 1.00 mL of ultrapure water to prepare an 8 mg / mL bovine serum albumin solution, sonicate for 10 min, centrifuge at 8000 r / min for 10 min, and collect the supernatant for later use.

[0044] 1.3.6 Preparation of Sample Solution Accurately weigh 2.00 mg each of CuZrO2NPs and ZnZrO2NPs, add 300 μL of ultrapure water, sonicate for 10 min, and shake on a shaker for 30 min to ensure uniform dispersion of the materials. Add 100 μL of each protein solution as needed, mix well, and incubate in a water bath at 60℃ for 24 h. Centrifuge at 10000 r / min for 10 min, and collect the supernatant for SDS-PAGE gel electrophoresis.

[0045] To investigate the enzymatic hydrolysis effects of different catalytic amounts of CuZrO2NPs and ZnZrO2NPs on proteins, following the sample solution preparation method, different catalytic amounts (2.00~10.00 mg) of CuZrO2NPs and ZnZrO2NPs were mixed with protein solutions for reaction. An equal volume of ultrapure water was added to the protein solution. Untreated materials were used as a control group. After centrifugation, the supernatant was collected for SDS-PAGE gel electrophoresis detection.

[0046] To investigate the effect of thermal denaturation on the hydrolysis effect, the inventors heated the protein at 100 °C for 30 min to denature it, and then hydrolyzed it using CuZrO2NPs material, following the same method for preparing the sample solution. A thermal denaturation control group and a normal control group were also set up. The thermal denaturation control group consisted of protein that was not heated and hydrolyzed using CuZrO2NPs. The normal control group consisted of protein solution with an equal volume of ultrapure water added, without hydrolysis using the material.

[0047] To investigate the effect of surfactants on hydrolysis, the inventors added different concentrations of SDS solution to the protein and prepared corresponding protein solutions. Referring to the preparation method of the sample solution, CuZrO2NPs were mixed with the protein solution for reaction. Only an equal amount of ultrapure water was added to the protein, and then it was mixed with CuZrO2NPs as a control group. After centrifugation, the supernatant was taken for SDS-PAGE gel electrophoresis detection.

[0048] In addition, the effects of pH and buffer type on the hydrolysis efficiency were investigated. CuZrO2NPs were hydrolyzed in water, PB buffer (pH=4.0~9.0), and Tris-HCl buffer (pH=4.0~9.0), respectively. After centrifugation, the supernatant was collected for SDS-PAGE gel electrophoresis.

[0049] 1.4 SDS-PAGE gel electrophoresis detection 1.4.1 Preparation of SDS-PAGE gels: Prepare 10% separating gel and 5% stacking gel according to the instructions of the SDS-PAGE gel rapid preparation kit, and then pour the gels.

[0050] 1.4.2 Preparation of electrophoresis buffer: Mix SWE rapid high-resolution electrophoresis buffer (dry powder) with 1.0 L of pure water to obtain the buffer.

[0051] 1.4.3 Preparation of staining solution: Accurately weigh 0.25 g of Coomassie Brilliant Blue, add 45 mL of methanol, 10 mL of glacial acetic acid and 45 mL of ultrapure water, stir to dissolve and obtain Coomassie Brilliant Blue R-250 staining solution.

[0052] 1.4.4 Preparation of decolorizing solution: Prepare the solution by mixing methanol, glacial acetic acid and ultrapure water in a volume ratio of 4.5:1:4.5.

[0053] 1.4.5 Sample loading: Take 40 uL of the sample solution from section 1.3.6, add 10 uL of 5×SDS-PAGE protein loading buffer, mix well, incubate at 100 ℃ for 10 min, and centrifuge at 6000 r / min for 1 min.

[0054] Sample loading: 5 μL for MARKER sample loading, 20 μL for sample solution loading.

[0055] 1.4.6 Electrophoresis: Voltage 200 V. After electrophoresis, cut off the edge of the gel.

[0056] 1.4.7 Staining and destaining: Coomassie brilliant blue staining method: Take an appropriate amount of staining solution and cover it on the PAGE gel after electrophoresis, shake for 20~30 min.

[0057] Decolorization: Place the stained PAGE gel in the decolorization solution and shake overnight until the gel turns transparent.

[0058] 2 Results and Discussion 2.1 Characterization of CuZrO2NPs The synthesized CuZrO2NPs were characterized and analyzed by Fourier transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and transmission electron microscopy (TEM).

[0059] Transmission electron microscopy (TEM) of CuZrO2NPs (Figures 1-a and 1-b) shows that the synthesized CuZrO2NPs have a particle size distribution of 2–5 nm, exhibiting nanoscale spherical particles. XRD patterns of CuZrO2NPs (Figure 1-c) show strong absorption peaks at 2θ = 28.42, 40.60, 50.24, 58.70, 66.40, 73.76, and 87.70. The peaks at 2θ = 28.42, 50.24, and 58.70 conform to the PDF card lines of ZrO2, while those at 2θ = 40.60, 66.40, and 87.70 correspond to Cu doping. The infrared spectrum of the material (see...) Figure 1 -d) Several characteristic medium-to-strong absorption peaks can be observed: 3390 (approximately 3400), 1558, 1362, 1328, 853, 752, 583, 534, 426 cm⁻¹ -1 3400 cm -1 The absorption peaks are rounded and blunt, consistent with the characteristic of broad and strong absorption peaks formed by associated hydroxyl groups that form hydrogen bonds due to the decrease in bond force constant, and are particularly prominent at 1362 and 1328 cm⁻¹. -1 The left and right absorptions suggest the presence of some free hydroxyl groups, hence the values ​​at 3400, 1362, and 1328 cm⁻¹. -1 The absorption peaks around the left and right are attributed to OH and Zr-OH; the characteristic wavenumber of Cu-O is approximately 500 cm⁻¹. -1 Mainly between 400 and 600 cm -1 The range is therefore 583~534 cm. -1 The absorption peaks around 400 cm⁻¹ belong to Cu-O; the absorption range of Zr-O is generally 400–700 cm⁻¹. -1 Therefore, 752, 426 cm -1 The absorption peaks around 400° and 500° are attributed to the presence of Zr-O, and it cannot be ruled out that some of the peaks around 400° and 500° are also attributed to Zr-O. The full spectrum of the material (see Figure 1-e) shows that the synthesized CuZrO2NPs contain Cu, Zr, and O elements.

[0060] 2.2 Verification of the enzymatic hydrolysis effect of CuZrO2NPs and ZnZrO2NPs nanozymes To investigate whether the synthesized CuZrO2NPs and ZnZrO2NPs have an enzymatic hydrolytic effect on BSA, different catalytic amounts of CuZrO2NPs and ZnZrO2NPs were reacted with BSA. After incubation, the supernatant was used for SDS-PAGE experiments. The results showed that, compared with the control group, the characteristic band (66.4 kDa) of CuZrO2NPs in the BSA bands under the three different catalytic amounts became thinner, forming new bands, indicating a certain distribution of other small molecule kDa, forming proteins and peptides of different sizes (Figure 2-a). However, the characteristic band of ZnZrO2NPs disappeared in the BSA bands under the three different catalytic amounts (Figure 2-b). This indicates that the CuZrO2NPs bimetallic oxide nanomaterials have a certain proteolytic activity on BSA and can be used as a novel nano-proteolytic enzyme. ZnZrO2NPs bimetallic oxide nanomaterials do not have proteolytic activity against BSA and are not suitable as a novel nano-protein hydrolase.

[0061] 2.3 Mechanism of Enzymatic Hydrolysis of CuZrO2NPs Nanoparticles 2.3.1 LC-MS / MS Analysis To further investigate the cleavage sites of CuZrO2NPs on BSA, the peptides observed in SDS-PAGE were analyzed by LC-MS / MS.

[0062] LC-MS / MS conditions: (1) Pre-column: 75 µm 2 cm, 3 µm particle size, 100 Å pore size (2) Analytical column: 75 µm 25 cm, 1.9 µm particle size, 100 Å pore size (3) Mobile phase A: 0.1% FA / 3% DMSO / 97% H2O (4) Mobile phase B: 0.1% FA / 3% DMSO / 97% ACN (5) Flow rate: 300 nL / min (6) Elution gradient: 0-5 min, 6%-11%B; 5-20 min, 11%-15%B; 20-43 min, 15%-25%B; 43-62.5 min, 25%-47%B; 62.5-63 min, 47%-90%B; 63-65 min, 90%-90%B.

[0063] Mass spectrometry conditions: Mass spectrometry data acquisition was performed in DDA mode. MSI full scan settings were: resolution 60K@200m / z, scan range 350-1500m / z, AGC target set to 3E. 6 The maximum injection time was set to 30 ms. The precursor ion selection window was set to 1, 4 Da, selecting the top 20 precursor ions for fragmentation, and the HCD collision energy was set to 28%. The MS2 scan parameters were set as follows: resolution 15K@200m / z, AGC target set to 1E. 5 The maximum injection time is set to 50 ms. The dynamic exclusion time is set to 30 seconds.

[0064] As shown in Figure 3-a, the material generates a total of 12 cleavage sites for BSA, of which 8 are concentrated at the amino or carboxyl termini of hydrophobic residues (as shown in Figure 3-c), namely the carboxyl terminus of leucine (L), the amino terminus of proline (P), and the N-terminus and C-terminus of alanine (A). This indicates that CuZrO2NPs facilitate peptide bond cleavage of hydrophobic residues.

[0065] 2.3.2 Effect of thermal denaturation on hydrolysis efficiency BSA was denatured by heating at 100 °C for 30 min, and then hydrolyzed with the material. The results are as follows: Figure 4 As shown, compared with the control group, the electrophoretic bands of thermally denatured BSA treated with CuZrO2NPs shifted significantly towards lower molecular weights, and new bands also appeared. These results indicate that CuZrO2NPs still possess the ability to decompose thermally denatured BSA. However, compared with natural BSA, the bands in the electrophoretic lanes of thermally denatured BSA were lighter. This may be because heat denaturation causes protein precipitation, reducing the concentration of BSA in the reaction system and decreasing the contact efficiency between the protein and the nanomaterials.

[0066] 2.3.3 Effect of surfactant denaturation on hydrolysis efficiency Different concentrations of SDS were added to BSA, and the materials were then hydrolyzed. The results are shown in Figure 5. Even after the addition of SDS, CuZrO2NPs still exhibited hydrolytic activity against BSA, with better hydrolysis results observed when the SDS concentration was between 0.25% and 1% wt. SDS is commonly used to solubilize insoluble proteins, which are difficult to hydrolyze by natural proteases. This suggests that CuZrO2NPs have the potential to hydrolyze insoluble proteins.

[0067] 2.3.4 Effects of pH and buffer type on hydrolysis efficiency CuZrO2NPs were used to hydrolyze BSA in water, PB buffer (pH 4.0–9.0), and Tris-HCl buffer (pH 4.0–9.0). The results are shown in Figure 6. The material exhibited the best hydrolytic effect on BSA in PB buffer (pH 7). While the hydrolytic activity of the material showed little difference in Tris-HCl buffer (pH 4–9), the effect was optimal in Tris-HCl buffer (pH 9).

[0068] In PB buffer (pH=4, 5), the small molecule bands essentially disappeared (see Figure 6-a), possibly because BSA reaches its isoelectric point at pH 4-5, resulting in precipitation. At pH=6, 8, and 9, distinct bands were observed in the 195 kDa region (see Figure 6-a), indicating that the enzymatic hydrolysis effect was less effective compared to pH=7. This may be because, at pH=7, positively charged BSA and negatively charged nanoparticles exhibit electrostatic attraction, thus exhibiting a high adsorption capacity for BSA; while at pH>7, in PB buffer, as pH increases, HPO4... 2- As the concentration of BSA increases, the compression of the electric double layer on the material surface increases the zeta potential. However, as the pH increases from 4.7 to 9.0, the zeta potential of BSA decreases significantly (from 0 to -35 mV) – at this point, both potentials are negative, resulting in some electrostatic repulsion. Therefore, the adsorption effect is low, and the enzymatic hydrolysis effect is not significant.

[0069] 2.4 Material recovery rate The weights of CuZrO2NPs before and after enzymatic hydrolysis were recorded, and the recovery rates were calculated. As shown in Table 1, the results indicate that the average recovery rate of CuZrO2NPs can reach 57.08% (RSD=4.96%, n=6).

[0070] Table 1 Recovery rates of CuZrO2NPs

[0071] 2.5 Enzymatic application of CuZrO2NPs Insoluble proteins cannot be directly broken down by traditional enzymes. LC-MS / MS results showed that CuZrO2NPs selectively cleaved hydrophobic residues and retained hydrolytic ability against surfactant-denatured proteins. Therefore, this study hypothesized that CuZrO2NPs could be further applied to the hydrolysis of insoluble proteins. The effect of different concentrations of SDS (0.25–1% wt) on the hydrolytic effect of soybean protein was investigated. The best hydrolysis effect was observed at an SDS concentration of 1% wt (see Figure 7-b), so 1% wt SDS was chosen to solubilize soybean protein. Subsequently, different amounts of the material were reacted with soybean protein, and the supernatant was analyzed by SDS-PAGE after incubation. The results are shown in Figure 7-a. Compared with the control group, the protein bands in the soybean protein lanes were significantly lighter and some bands disappeared under different catalytic concentrations of CuZrO2NPs, indicating that CuZrO2NPs have an enzymatic hydrolytic effect on soybean protein. This further verifies the potential of CuZrO2NPs to hydrolyze insoluble soybean protein.

[0072] Proteins are a crucial pharmacological basis for the efficacy of animal-derived drugs. To fully utilize the pharmacological effects of animal-derived drugs and enhance their medicinal value, enzymatic hydrolysis is commonly employed to convert proteins into smaller peptides or amino acids. Enzymatic hydrolysis verification experiments have shown that CuZrO2NPs have excellent hydrolytic effects. Therefore, this study further applied CuZrO2NPs to the hydrolysis of American cockroach protein. As shown in Figure 8, compared with the control group, the bands in the American cockroach protein lanes were all lighter under different catalytic concentrations of CuZrO2NPs, indicating that CuZrO2NPs have an enzymatic hydrolytic effect on American cockroach protein. This verifies the feasibility of constructing a new method for the hydrolysis of American cockroach protein based on CuZrO2NPs, providing a new approach for establishing quality control and content identification strategies for American cockroach medicinal materials.

[0073] To verify the enzymatic hydrolysis effect of other materials on proteins, the inventors of this application synthesized CuCoO2NPs and conducted enzymatic hydrolysis experiments on them with BSA, soy protein, and American cockroach protein, as detailed below: 2.6 Characterization and Enzymatic Application of CuCoO2NPs CuCoO2 NPs were synthesized according to the literature (Xu JH, Ji NN, Guo MX, et al. CuCoO2 Nanoparticles as a Nanoprotease for Selective Proteolysis with High Efficiency at Room Temperature[J]. ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 2023, 62(31)), and the materials were characterized by XRD and TEM. The XRD results (Figure 9) showed that the 2θ values ​​of the diffraction peaks were 15.6°, 31.7°, 37.8°, 39.8°, 42.2°, 57.2°, 61.9°, 65.2°, 66.9°, and 74.4°. This is consistent with the literature report and the CuCoO2 standard card (JCPDS:21-0256). TEM results (Figure 10) show that at a 100 nm imaging size, the material thickness ranges from 24 nm to 43 nm, consistent with the reported average thickness of 30 nm. Characterization results show that the synthesized CuCoO2NPs are consistent with the original literature.

[0074] To verify whether the synthesized CuCoO2NPs possess BSA hydrolytic activity as reported in the literature, 10 mg of CuCoO2NPs were reacted with 2.5 mg / mL BSA at room temperature for 3 h. After incubation, the supernatant was used for SDS-PAGE. The results are shown in Figure 11. Compared with the control group, the characteristic bands of BSA after incubation with CuCoO2NPs became thinner and lighter, and new bands were formed, indicating a certain distribution of other small molecules in kDa, forming proteins and peptides of different sizes. This confirms that the synthesized CuCoO2NPs also possess BSA hydrolytic activity as reported in the literature.

[0075] Soy protein was dissolved in 1% wt SDS, and the enzymatic hydrolysis effect of different catalytic amounts of CuCoO2NPs on soy protein was investigated (the enzymatic hydrolysis method is described in section 1.3.6, "Sample Solution Preparation"). SDS-PAGE results (Figure 12) showed that, compared with the control, the bands of CuCoO2NPs at different catalytic amounts did not become lighter or thinner, nor did they show a trend of migrating to lower molecular weights to form new bands. Therefore, it is concluded that CuCoO2NPs have no significant hydrolytic activity on soy protein and are not suitable as a biotechnological tool for hydrolyzing insoluble soy protein.

[0076] American cockroach protein was dissolved in 1% ammonium bicarbonate, and the enzymatic hydrolysis effect of different catalytic amounts of CuCoO2NPs on American cockroach protein was investigated (the enzymatic hydrolysis method is described in section 1.3.6, sample solution preparation). SDS-PAGE results (Figure 13) showed that, compared with the control, the CuCoO2NPs bands at 2 mg catalytic amount showed a slight tendency to lighten, while the protein bands at 4 mg and 6 mg catalytic amounts did not become thinner or lighter, nor did they show a tendency to migrate to lower molecular weights to form new bands. Compared with CuZrO2NPs, the band change trend in each lane was more obvious (significantly lighter) under different catalytic amounts of CuZrO2NPs, indicating that the overall enzymatic hydrolysis effect of CuCoO2NPs on American cockroach protein is not as good as that of CuZrO2NPs. Therefore, CuZrO2NPs is more suitable as a biotechnological tool for constructing new methods for hydrolyzing American cockroach protein.

[0077] 3. Summary This study designed nano-hydrolytic enzymes based on bimetallic oxide nanoparticles. By comparing the SDS-PAGE results of enzymatic incubation experiments of CuZrO2NPs and ZnZrO2NPs, the proteolytic activity of CuZrO2NPs was verified, thus identifying CuZrO2NPs as a novel nano-proteolytic enzyme. Furthermore, its enzymatic mechanism was investigated, revealing that CuZrO2NPs selectively cleave peptide bonds of hydrophobic residues, maintain hydrolytic activity even under heat denaturation and surfactant-denatured BSA, and exhibit a wider pH range than natural proteases. Finally, the screened CuZrO2NPs were applied to the hydrolysis of insoluble plant proteins (soybean protein) and animal drug proteins (American cockroach protein). In summary, this study demonstrates the significant potential of CuZrO2NPs in biomedical and proteomics applications, provides a new method for the hydrolysis of insoluble plant proteins, and further expands the possibilities for developing nano-proteases based on bimetallic oxide nanomaterials.

[0078] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing CuZrO2NPs hydrolytic nanozyme, characterized in that: First, CuZr(OH)₂NPs are synthesized using a co-precipitation method, followed by high-temperature calcination to achieve intramolecular dehydration and synthesize CuZrO₂NPs; the specific steps include: ZrOCl2·8H2O and CuCl2 were mixed and dissolved in deionized water, then stirred in a water bath to obtain a mixed solution. A KOH aqueous solution was prepared for later use. The resulting mixed solution was then precipitated with KOH solution until the pH value reached 11. The solid-liquid product was collected, and the supernatant was removed by centrifugation to obtain a solid product. The surface of the solid product was then repeatedly washed with hot deionized water. The obtained solid product was placed in an oven to dry, resulting in uncalcined CuZrO2NPs, i.e., CuZr(OH)2NPs. The CuZr(OH)2NPs were ground into fine particles and then calcined in a muffle furnace. After annealing, the final product CuZrO2NPs was obtained, which is the CuZrO2NPs hydrolytic nanozyme.

2. A CuZrO2NPs hydrolytic nanozyme prepared by the preparation method described in claim 1.

3. The application of the CuZrO2NPs hydrolytic nanozyme as described in claim 2 in proteolytic hydrolysis.

4. The application according to claim 3, characterized in that: The method for enzymatically hydrolyzing proteins using the CuZrO2NPs hydrolytic nanozyme is as follows: the CuZrO2NPs hydrolytic nanozyme is mixed with ultrapure water to disperse the CuZrO2NPs hydrolytic nanozyme evenly, and then a protein solution is added. The mixture is then reacted in a water bath to achieve the enzymatic hydrolysis of the protein.

5. The application according to claim 4, characterized in that: The protein in question is an insoluble plant protein or an animal drug protein.

6. The application according to claim 5, characterized in that: The insoluble plant protein mentioned is soybean protein.

7. The application according to claim 5, characterized in that: The animal drug protein mentioned is American cockroach protein.

8. The application according to claim 4, characterized in that: The protein solution is prepared by mixing protein and solvent, and the weight ratio of CuZrO2NPs hydrolytic nanozyme to protein during enzymatic hydrolysis is (2~10):

8.

9. The application according to claim 4, characterized in that: The water bath reaction was carried out at a temperature of 60 °C for 24 h.

10. The application of the CuZrO2NPs hydrolytic nanozyme as described in claim 2 in the preparation of an enzymatic hydrolysant for protein hydrolysis, characterized in that: The enzymatic hydrolysant includes the CuZrO2NPs hydrolytic nanozyme.

Images