Sinonovacula constriceta glycoprotein nanosome Sc-NZ and a preparation method thereof
By subjecting razor clam glycoprotein to ultra-high pressure and heat treatment, a nanozyme Sc-NZ with peroxidase-like and laccase-like activities was prepared, solving the problems of poor biocompatibility and environmentally unfriendly preparation process of existing nanozymes, and achieving high efficiency, stable catalytic performance and wide application.
Patent Information
- Application Number
- CN202610495403.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-04-15
- Publication Date
- 2026-07-10
AI Technical Summary
The application of existing nanozymes in food safety and biomedicine is limited, mainly due to the complex synthesis process, the need for toxic chemical reagents, poor biocompatibility, and limited catalytic activity.
By subjecting razor clam glycoprotein to ultra-high pressure treatment combined with heat treatment, a nanozyme Sc-NZ with dual catalytic activities of peroxidase and laccase-like properties was prepared. This green physical modification method avoids chemical modification.
The prepared razor clam glycoprotein nanozyme Sc-NZ exhibits efficient and stable dual catalytic activity, possesses fluorescence properties, broadens its application scenarios, and is environmentally friendly in its preparation process.
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Figure CN122356253A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of nanozyme technology, and in particular to a clam glycoprotein nanozyme Sc-NZ and its preparation method. Background Technology
[0002] Nanozymes are a class of nanomaterials with enzyme-like catalytic activity. Compared to natural enzymes, they offer advantages such as lower cost, higher stability, and simpler preparation processes, demonstrating significant application potential in analytical testing, disease diagnosis, and environmental remediation. Currently, a wide variety of nanozymes have been reported, primarily including metal-based nanozymes, carbon-based nanozymes, and metal-organic framework materials. However, these artificially synthesized nanozymes generally suffer from complex synthesis processes, the need for toxic chemical reagents, poor biocompatibility, and limited catalytic activity, restricting their widespread application in fields with high safety requirements, such as food safety and biomedicine.
[0003] In recent years, the development of novel nanozymes using biomass resources has become a research hotspot. Marine shellfish resources are abundant, especially the razor clam (Sinocyclocheilus spp.), an important economically farmed shellfish, whose processing generates a large number of byproducts. Previous studies have shown that when subjected to environmental stresses such as high temperatures and heavy metals, the razor clam produces a series of glycoproteins in response to external damage, suggesting that its glycoprotein molecular backbone may possess unique physicochemical properties and potential catalytic active centers. Furthermore, shellfish proteins contain metallic minerals such as zinc, iron, and copper, as well as non-metallic elements such as carbon and nitrogen, making them ideal precursors for the preparation of multifunctional nanozymes. However, there are currently no reports on the preparation of nanozymes with high efficiency and bifunctional catalytic activity (peroxidase-like and laccase-like) using natural razor clam glycoproteins through physical modification. At the same time, traditional methods of protein modification, such as chemical modification, heat treatment, or enzymatic hydrolysis, often struggle to precisely control their spatial conformation, easily leading to the destruction of active centers or non-specific aggregation, thus resulting in the loss of catalytic function.
[0004] Therefore, there is an urgent need to develop a solution that can address the common problems of poor biocompatibility and environmentally unfriendly preparation processes of existing nanozymes. Summary of the Invention
[0005] This invention provides a razor clam glycoprotein nanozyme Sc-NZ and its preparation method. The razor clam glycoprotein nanozyme Sc-NZ has the advantages of being prepared through green physical modification, high activity, high stability, and multifunctionality.
[0006] The objective of this invention is achieved through the following technical solution: A type of razor clam glycoprotein nanozyme, Sc-NZ, is a cluster of nanoparticles obtained by ultra-high pressure treatment of the purified product from razor clams, with a particle size distribution of 30-50 nm.
[0007] As a preferred option, the razor clam glycoprotein nanozyme Sc-NZ has fluorescent properties, exhibiting a characteristic fluorescence emission peak at 520 nm under excitation light of 370 nm.
[0008] As a preferred option, the razor clam glycoprotein nanozyme Sc-NZ exhibits both peroxidase-like and laccase-like activities.
[0009] This invention also provides a method for preparing razor clam glycoprotein nanozyme Sc-NZ, which includes the following steps: S01. Take the soft tissue of razor clam, homogenize and defatted it, centrifuge and collect the precipitate to obtain defatted powder. The defatted powder is extracted by alkaline extraction and alcohol precipitation. The pH is adjusted to 4.0-5.0, the precipitate is collected and dried to obtain crude glycoprotein. S02. After enzymatic hydrolysis of the crude glycoprotein, it is sequentially subjected to anion exchange chromatography and gel filtration chromatography to collect the active components and obtain purified razor clam natural glycoprotein. S03. Add deionized water or phosphate buffer to the obtained razor clam natural glycoprotein to prepare a solution, place it in an ultra-high pressure device, and treat it at 60-90℃ and 400-1000MPa for 10-20 minutes to obtain the razor clam glycoprotein nanozyme Sc-NZ.
[0010] Preferably, in step S01, the degreasing agent is selected from either n-hexane or acetone.
[0011] Preferably, in step S01, the alkaline extraction and alcohol precipitation method includes the following steps: using Tris-HCl buffer solution with pH 8.0-10.0 as the extraction solvent, mixing at a material-to-liquid ratio of 1:5-1:20 (w / v), and stirring and extracting at 4-25℃ for 12-24 hours.
[0012] Preferably, in step S02, the enzymatic hydrolysis process specifically involves hydrolyzing papain at 40-60℃ and pH 6.0-7.0 for 2-4 hours.
[0013] Preferably, in step S02, the column chromatography purification includes DEAE-Sepharose FastFlow ion exchange chromatography and Sephadex G-100 gel filtration chromatography.
[0014] Preferably, in step S03, the concentration of the solution is 0.5-5.0 mg / mL, and the solvent is water or a phosphate buffer solution with a pH of 6.0-8.0.
[0015] This invention also provides applications of the razor clam glycoprotein nanozyme Sc-NZ, specifically including the following (1), (2), or (3): (1) Preparation of a biosensor for hydrogen peroxide detection; (2) Preparation of a biosensor for the detection of oxidized phenolic substrates; (3) Preparation of a biosensor for glucose detection.
[0016] Compared with the prior art, the advantages or beneficial effects of the technical solution of this application include: 1. This study is the first to discover and confirm that a natural glycoprotein derived from razor clams possesses highly efficient dual catalytic functions of peroxidase and laccase after ultra-high pressure synergistic heat treatment. The resulting Sc-NZ exhibits both peroxidase and laccase activities, and its peroxidase-like catalytic efficiency and substrate affinity are superior to those of natural HRP. Furthermore, it possesses inherent fluorescent properties not found in natural enzymes and most synthetic nanozymes.
[0017] 2. Compared to natural enzymes, Sc-NZ has excellent acid-base stability and thermal stability, and combines catalytic and fluorescent properties, enabling it to be used in more demanding environmental conditions and broadening its application scenarios.
[0018] 3. The entire preparation process mainly adopts ultra-high pressure physical field modification, without involving any toxic or harmful chemical reagents, which is in line with the concept of green chemistry and sustainable development. Attached Figure Description
[0019] Figure 1 The ultraviolet absorption spectra of the Sc-NZ catalyzed TMB-H2O2 colorimetric system obtained under different processing pressures.
[0020] Figure 2 The ultraviolet absorption spectra of the Sc-NZ catalyzed TMB-H2O2 colorimetric system obtained at different processing temperatures.
[0021] Figure 3 Transmission electron microscopy (TEM) image of Sc-NZ after rapid depressurization at 600 MPa and 80 °C for 15 min.
[0022] Figure 4 The parameter plot shows that the Km value of Sc-NZ determined using TMB as a substrate is 0.24 mM.
[0023] Figure 5 The parameter plot for determining the Vm value of Sc-NZ using TMB as a substrate is 0.24 mM.
[0024] Figure 6 The parameter graph shows that the Km value of natural horseradish peroxidase (HRP) was 0.24 mM when TMB was used as a substrate.
[0025] Figure 7 The parameter graph shows that the Vm value of natural horseradish peroxidase (HRP) was 0.24 mM when TMB was used as a substrate.
[0026] Figure 8 The image shows the three-dimensional fluorescence spectrum of Sc-NZ.
[0027] Figure 9 The absorbance diagrams for the colorimetric systems corresponding to different concentrations of H2O2 are shown.
[0028] Figure 10 The linear relationship between absorbance A and H2O2 concentration C of the colorimetric system is shown.
[0029] Figure 11 The absorbance graphs for the detection systems at different glucose concentrations are shown.
[0030] Figure 12 To determine the linear relationship between absorbance A and glucose concentration C in the detection system. Figure 13 Effect of catalytic system temperature (20-90℃) on the catalytic activity of Sc-NZ Figure 14 The effect of temperature (20-90℃) on the catalytic activity of natural HRP. Detailed Implementation
[0031] The following detailed description of the embodiments of this application, in conjunction with the accompanying drawings, will provide a thorough understanding of how this application uses technical means to solve technical problems and achieve corresponding technical effects, enabling its implementation. The embodiments of this application and the various features within them can be combined with each other without conflict, and all resulting technical solutions are within the protection scope of this application.
[0032] It should be clearly stated that the embodiments described below are merely some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.
[0033] Example 1: Extraction and purification of natural glycoproteins from razor clams Take 500g of fresh razor clam soft tissue, wash and cut it into small pieces, add twice the volume of pre-cooled homogenate (50mM Tris-HCl, pH 8.0) for homogenization. Add an equal volume of n-hexane to the homogenate for defatting, stir for 2 hours, centrifuge (8000rpm, 20min), discard the organic phase, collect the precipitate and freeze-dry to obtain defatted powder. Take 100g of defatted powder, add 50mM NaOH solution (pH 10.0) at a material-to-liquid ratio of 1:15, and extract at 4℃ for 24 hours with stirring. Centrifuge (10000rpm, 30min) to collect the supernatant, adjust the pH to 4.5 with 1M HCl, and let stand overnight. Centrifuge to collect the precipitate, freeze-dry to obtain crude glycoprotein. Dissolve the crude product in 20mM PBS (pH 7.0), add papain (enzyme-to-protein ratio 1:50), and enzymatically hydrolyze in a 50℃ water bath for 3 hours. After boiling for 10min to inactivate, centrifuge to collect the supernatant. The supernatant was passed through a DEAE-Sepharose FF column and eluted with a linear gradient of 0-1M NaCl to collect the active component. It was then subjected to Sephadex G-100 gel filtration chromatography, and the main peak was collected. The purified natural glycoprotein from the razor clam, denoted as Sc-GP, was obtained by dialyzing and lyophilization.
[0034] Example 2: Specific steps for modifying Sc-GP based on the synergistic thermal effect of ultra-high pressure under different pressures to obtain the scallop glycoprotein nanozyme Sc-NZ.
[0035] The Sc-GP obtained in Example 1 was prepared into a 2 mg / mL solution using 10 mM PBS at pH 7.0. The samples were divided into 6 groups and placed in an ultra-high pressure apparatus. The treatment temperature was set to 80℃, the holding time to 15 min, and the treatment pressures were 0.1 MPa, 400, 600, 800, and 1000 MPa, respectively. Immediately after treatment, the samples were removed to measure the enzyme activity of the TMB-H2O2 system, and the absorbance at 652 nm was detected. The results are as follows: Figure 1 As shown, when the pressure is ≥400 MPa, the samples begin to exhibit significant enzyme-like activity. Furthermore, as the pressure increases, the activity first increases and then stabilizes, reaching its optimal level in the 800-1000 MPa range, which is more than four times that of the atmospheric pressure treatment group. This indicates that ultra-high pressure is a key factor in inducing enzyme activity.
[0036] Example 3: Modification of Sc-GP based on the synergistic thermal effect of ultra-high pressure under different temperature conditions The Sc-GP obtained in Example 1 was prepared into a 2 mg / mL solution using 10 mM PBS at pH 7.0. The ultra-high pressure treatment was set at 600 MPa for 15 min, and the treatment temperatures were 25, 40, 60, 70, 80, and 90 °C. Peroxidase-like activity was measured after treatment. The results are as follows: Figure 2As shown, at room temperature (25℃), high-pressure treatment alone has limited effect on improving enzyme activity. When the temperature rises above 60℃, enzyme activity begins to increase significantly, maintaining high activity within the range of 70-90℃, and reaching a peak at 80℃. This indicates that the thermal effect, combined with high pressure, works to induce conformational changes in glycoproteins, forming active sites.
[0037] Example 4: Morphological characterization of Sc-NZ The Sc-NZ sample prepared in Example 3 at 600 MPa, 80 °C, and 15 min was diluted and dropped onto a copper grid. It was then negatively stained with 2% phosphotungstic acid, and its morphology was observed using a transmission electron microscope. The results are as follows: Figure 3 As shown, Sc-NZ exhibits uniform clustered nanoparticles with particle sizes mainly distributed between 30-50 nm, demonstrating good dispersibility and no obvious aggregation.
[0038] Example 5: Kinetic analysis of peroxidase-like enzymes in Sc-NZ Using TMB as a substrate, the steady-state kinetic parameters of Sc-NZ and natural horseradish peroxidase (HRP) were determined under optimal reaction conditions (pH 4.0, 40℃). With the H₂O₂ concentration fixed and the TMB concentration varied, Lineweaver-Burk curves were fitted. The results are shown below. Figure 4 As shown, the Km value of Sc-NZ for TMB is 0.24 mM, V m The value is 2.2 × 10 -8 M -1 The Km value for HRP against TMB is 0.76 mM, and the Vm value is 4.57 × 10⁻⁶ mM. -8 M -1 It is evident that Sc-NZ has three times the affinity for TMB substrates as HRP.
[0039] The results showed that Sc-NZ had a significantly better affinity for the substrate (lower Km value) than natural HRP.
[0040] Example 6: Verification of Laccase-like Activity of Sc-NZ Under H2O2-free conditions, using 2,4-dichlorophenol (2,4-DCP) as a substrate, Sc-NZ and natural laccase were added at pH 6.5 and 50℃, respectively, and the absorbance of the reaction system at 510 nm was measured. The results showed that Sc-NZ could effectively catalyze the oxidation of 2,4-DCP, with a specific activity of approximately 65% that of natural laccase, confirming that it possesses laccase-like activity.
[0041] Example 7: Fluorescence performance characterization of Sc-NZ Sc-NZ solutions of different concentrations (0.1, 0.5, 1.0, 2.0, 4.0 mg / mL) were prepared, and their emission spectra were scanned using a fluorescence spectrophotometer at an excitation wavelength of 370 nm. The results are as follows: Figure 8 As shown, Sc-NZ exhibits a strong emission peak near 520 nm, and the fluorescence intensity shows a good linear relationship with concentration (in the range of 0.1-2.0 mg / mL). This property can be used for real-time monitoring of the distribution of nanozymes in organisms and the assembly of sensing interfaces.
[0042] Example 8: Sc-NZ-based colorimetric sensing of H2O2 and glucose H₂O₂ detection: Add 100 μL of acetate buffer (pH 4.0), 20 μL of sc-NZ solution (0.5 mg / mL), 20 μL of TMB solution (5 mM), and 20 μL of H₂O₂ standard solutions of different concentrations (0-200 μM) sequentially to a 96-well plate. After reacting for 10 min, measure the absorbance at 652 nm. Figure 9 and Figure 10 As shown, the results indicate that the absorbance and H2O2 concentration exhibit a good linear relationship in the range of 0.5-100 μM, with a detection limit of 0.3 μM.
[0043] Glucose detection: Take 50 μL of glucose standard solutions of different concentrations, add 50 μL of glucose oxidase solution (0.5 mg / mL), and incubate at 37℃ for 30 min. Then add the above-mentioned mixed solution of TMB and Sc-NZ, and continue the reaction for 15 min before measuring the absorbance. Experimental results are as follows: Figure 11 and Figure 12 The results showed that glucose concentration exhibited a linear relationship with absorbance in the range of 2-200 μM, with a detection limit of 1.0 μM. Example 9: Stability evaluation of Sc-NZ After incubating Sc-NZ and natural HRP at different pH values (3.0-10.0) and temperatures (20-90℃) for 1 h, the residual peroxidase-like activities were measured. The results showed that Sc-NZ retained over 80% of its activity within the pH range of 4.0-9.0, while HRP activity decreased sharply at pH > 8.0. Regarding thermal stability, Sc-NZ retained over 54% of its initial activity after treatment at 90℃ for 1 h, while HRP was essentially inactivated at 60℃. Figure 13 and Figure 14 This indicates that Sc-NZ has excellent acid and alkali stability and thermal stability.
[0044] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A scallop glycoprotein nanozyme Sc-NZ, characterized in that, These are clustered nanoparticles obtained by treating the purified product from razor clams under ultra-high pressure, with a particle size distribution of 30-50 nm.
2. The *Sc-NZ* nanozyme derived from *Clam slug* according to claim 1, characterized in that, It exhibits fluorescence properties, with a characteristic fluorescence emission peak around 520 nm when excited by light at 370 nm.
3. The *Sc-NZ* nanozyme derived from *Clam slug* according to claim 1, characterized in that, It possesses both peroxidase-like and laccase-like activities.
4. A method for preparing a razor clam glycoprotein nanozyme Sc-NZ, characterized in that, Includes the following steps: S01. Take the soft tissue of razor clam, homogenize and defatted it, centrifuge and collect the precipitate to obtain defatted powder. The defatted powder is extracted by alkaline extraction and alcohol precipitation. The pH is adjusted to 4.0-5.0, the precipitate is collected and dried to obtain crude glycoprotein. S02. After enzymatic hydrolysis of the crude glycoprotein, it is sequentially subjected to anion exchange chromatography and gel filtration chromatography to collect the active components and obtain purified razor clam natural glycoprotein. S03. Add deionized water or phosphate buffer to the obtained razor clam natural glycoprotein to prepare a solution, place it in an ultra-high pressure device, and treat it at 60-90℃ and 400-1000 MPa for 10-20 minutes to obtain the razor clam glycoprotein nanozyme Sc-NZ.
5. The preparation method according to claim 4, characterized in that, In step S01, the degreasing agent is selected from either n-hexane or acetone.
6. The preparation method according to claim 4, characterized in that, In step S01, the alkaline extraction and alcohol precipitation method includes the following steps: using Tris-HCl buffer solution with pH 8.0-10.0 as the extraction solvent, mixing at a material-to-liquid ratio of 1:5-1:20 (w / v), and stirring and extracting at 4-25℃ for 12-24 hours.
7. The preparation method according to claim 4, characterized in that, In step S02, the enzymatic hydrolysis process specifically involves using papain to hydrolyze the enzyme at 40-60 °C and pH 6.0-7.0 for 2-4 h.
8. The preparation method according to claim 4, characterized in that, In step S02, column chromatography purification includes DEAE-Sepharose Fast Flow ion exchange chromatography and Sephadex G-100 gel filtration chromatography.
9. The preparation method according to claim 5, characterized in that, In step S03, the concentration of the solution is 0.5-5.0 mg / mL, and the solvent is water or phosphate buffer with pH 6.0-8.
0.
10. The application of the *Sc-NZ* glycoprotein nanozyme according to any one of claims 1-3 and the *Sc-NZ* glycoprotein nanozyme prepared by the preparation method according to any one of claims 4-9, characterized in that, Including the following (1), (2), or (3): (1) Preparation of a biosensor for hydrogen peroxide detection; (2) Preparation of a biosensor for the detection of oxidized phenolic substrates; (3) Preparation of a biosensor for glucose detection.