Artificial antioxidant enzyme as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biocatalysts, and particularly relates to artificial antioxidant enzyme as well as a preparation method and application thereof. Mn is doped in the process of preparing a titanium oxygen cluster through solvothermal reaction to prepare the MnTi6 artificial antioxidant enzyme with the amorphous nano-porous structure, and the MnTi6 artificial antioxidant enzyme prepared through the method has the excellent effect and the broad-spectrum antioxidant characteristic due to doping of Mn, and has the good application prospect in the field of biological catalysis.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysts, and specifically relates to an artificial antioxidant enzyme and a preparation method and application thereof. Background Art

[0002] In biological systems, endogenous antioxidant enzymes, including superoxide dismutase (SOD) and catalase (CAT), help maintain cellular redox balance by neutralizing ROS and converting them into harmless byproducts. However, these natural antioxidants have limitations, such as high antigenicity, instability, and delivery challenges, which hinder their direct application in the treatment of diseases related to oxidative stress. Therefore, chemists and material scientists are actively involved in mimicking the mechanisms of natural antioxidant enzymes to create new biocatalytic reactive oxygen scavenging materials for antioxidant treatment. In recent years, inspired by the structure and function of natural enzymes, people have paid much attention to the use of catalytic materials containing metal oxides and metal-coordination structures to scavenge ROS and protect cells. Titanium (Ti) has the advantages of low density, good compatibility with human tissues, and low immune rejection in biomaterial applications. It is a biosafe metal material, but there are few reports on artificial antioxidant enzymes with Ti as the active center. In addition, the preparation of artificial antioxidant enzymes by simulating the active center of the natural antioxidant enzyme manganese superoxide dismutase (MnSOD) has also become a research hotspot. However, these single-metal-based artificial enzymes have limited ROS scavenging capabilities and insufficient broad-spectrum properties, and are unable to address a variety of ROS that are commonly found in organisms, which emphasizes the necessity of designing broad-spectrum ROS scavenging materials. Summary of the invention

[0003] In order to solve the above problems, the present invention prepares an artificial antioxidant enzyme with a bimetallic active site having an electronically regulated active center by integrating Mn / Ti catalytic sites, which is referred to as MnTi6 artificial antioxidant enzyme in this article. It has excellent efficacy and broad-spectrum antioxidant properties and has good application prospects in the field of biocatalysis.

[0004] In the first aspect, the present invention provides a MnTi6 artificial antioxidant enzyme, which is a Mn-doped titanium oxide cluster formed by a solvothermal reaction of a titanium source, a manganese source and 4-aminobenzoic acid, and has an amorphous nanoporous structure, and Mn-O and Mn-Ti coordination structures exist in the MnTi6 artificial antioxidant enzyme.

[0005] Furthermore, the MnTi6 artificial antioxidant enzyme has cascade SOD-CAT catalytic activity.

[0006] As used herein, the cascade SOD-CAT catalytic activity involves first converting superoxide anion (•O2 -) is converted into hydrogen peroxide (H2O2), which is then converted into oxygen (O2) and water (H2O) by catalase (CAT)-like activity.

[0007] Generally speaking, the mass ratio of Mn and Ti and the amount of Mn incorporated in the MnTi6 artificial antioxidant enzyme are not particularly limited, as long as the MnTi6 artificial antioxidant enzyme has the structure described herein. In certain embodiments, the mass percentage of Ti in the MnTi6 artificial antioxidant enzyme can be 10-25%, and the amount of Mn incorporated can be 5-10% by mass.

[0008] As used herein, titanium oxide clusters are a type of metal oxide clusters formed by connecting titanium (Ti) atoms and oxygen (O) atoms through bridging oxygen (O²⁻) or hydroxyl groups (OH⁻). They are usually composed of multiple titanium atom centers connected by oxygen bridges to form a multi-nuclear structure, and can be further modified by organic or inorganic ligands. The synthesis of titanium oxide clusters usually involves a hydrolysis-condensation reaction, that is, the titanium precursor is hydrolyzed under appropriate conditions and forms a Ti-O-Ti bridge bond through hydroxyl condensation, and finally assembles into a titanium oxide cluster. In the present invention, the titanium oxide cluster formed by a solvothermal reaction under the regulation of the ligand 4-aminobenzoic acid is referred to as Ti6.

[0009] In a second aspect, the present invention provides a method for preparing the MnTi6 artificial antioxidant enzyme as described herein, wherein the MnTi6 artificial antioxidant enzyme is prepared by a solvothermal reaction of a titanium source, a manganese source and 4-aminobenzoic acid.

[0010] Specifically, the preparation method is: adding a manganese source to a solvent containing a titanium source and 4-aminobenzoic acid, reacting at 90-120° C. for 60-90 hours, collecting the reaction product, washing and drying, and obtaining the MnTi6 artificial antioxidant enzyme.

[0011] Furthermore, the titanium source includes titanium tetraisopropoxide, titanium tetrachloride or titanate.

[0012] Preferably, the titanium source is titanium tetraisopropoxide.

[0013] Furthermore, the manganese source includes manganese nitrate tetrahydrate, manganese chloride hexahydrate or manganese sulfate hexahydrate.

[0014] Preferably, the manganese source is manganese nitrate tetrahydrate.

[0015] Furthermore, the solvent includes isopropanol, methanol, ethanol, toluene, acetonitrile or N,N-dimethylformamide.

[0016] Preferably, the solvent is isopropanol.

[0017] In a third aspect, the present invention provides the use of the MnTi6 artificial antioxidant enzyme as described herein in the preparation of a preparation for scavenging active oxygen.

[0018] Furthermore, the removing active oxygen comprises removing active oxygen through cascade SOD-CAT catalytic activity.

[0019] Advantageous Effects of the Invention

[0020] The present invention prepares an artificial MnTi6 antioxidant enzyme with an amorphous nanoporous structure by incorporating Mn in the process of preparing titanium oxide clusters using a solvent thermal reaction, wherein the incorporation of Mn can form coordination with oxygen in the titanium oxide cluster by replacing the position of Ti, thereby leading to the formation of an amorphous nanoporous structure; in addition, it is also found that there is electron transfer from the Mn site to the Ti site, which can enable MnTi6 to overcome the challenges associated with multi-electron reactions in the process of scavenging reactive oxygen species (ROS). The MnTi6 artificial antioxidant enzyme prepared by the present invention has excellent efficacy and broad-spectrum antioxidant properties, and has good application prospects in the field of biocatalysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The structural characterization of the MnTi6 biocatalyst with an amorphous nanoporous structure prepared in the embodiment is shown: (a) scanning electron microscope (SEM) images of Ti6 and MnTi6; (b) and (c) atomic-level high-angle annular dark field scanning transmission electron microscope (HAADF-STEM) images of MnTi6; (d) energy dispersive X-ray spectroscopy (EDS) elemental surface distribution map of O, Ti, and Mn; and (e) EDS energy spectrum map of MnTi6; (f) the mass of Mn and Ti in MnTi6 obtained by X-ray photoelectron spectroscopy (XPS) and inductively coupled plasma optical emission spectroscopy (ICP-OES) ratio; (g) zeta potential and hydrodynamic size distribution; (h) Raman spectra of Ti6 and MnTi6; (i) XPS full spectrum scans of Ti6 and MnTi6; (jl) High-resolution XPS spectra of O1s, Ti2p and Mn2p of Ti6 and MnTi6; (m) K-edge XANES spectra of manganese (Mn) in MnTi6, manganese foil, manganese oxide (MnO) and manganese dioxide (MnO2); (n) Average valence state of manganese (Mn) in MnTi6, manganese oxide (MnO) and manganese dioxide (MnO2); (o) Fourier transform (FT) of the extended EXAFS spectrum in R space.

[0022] Figure 2 The O2 of the materials obtained in the examples and comparative examples is shown. - and H2O2 removal performance tests: (a) O2 removal of TiO2, Ti6, CeTi6, CuTi6, MnTi6 and VTi6- Removal performance; (b) H2O2 removal performance of TiO2, Ti6, CeTi6, CuTi6, MnTi6 and VTi6;

[0023] Figure 3 The ROS scavenging activity and kinetics of MnTi6 are shown: (a) Schematic diagram of the cascade superoxide dismutase-catalase (SOD-CAT) catalytic activity of MnTi6; (b) The scavenging rates of 2,2'-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) cation radical (ABTS·+) by Ti6 and MnTi6 at different times; (c) The scavenging rates of superoxide anions (·O2 - )-like superoxide dismutase (SOD) activity; (d) dynamic scavenging activity of hydrogen peroxide (H2O2) at different times; (e) dynamic generation characteristics of oxygen (O2) at different times; (f) cyclic catalytic ability of MnTi6 after adding hydrogen peroxide (H2O2); (g) typical Michaelis-Menten curve and double reciprocal plot of the kinetic constants of MnTi6 with hydrogen peroxide (H2O2) as substrate; (h) maximum reaction rate (V) of MnTi6 compared with other recently reported nanobiocatalysts for scavenging reactive oxygen species max ) and transformation number (TON) values; (i) V of MnTi6 max and the Michaelis constant (K m ) values; (j) in situ Fourier transform infrared (FTIR) spectrum of MnTi6 during catalase (CAT) catalysis and (k) the corresponding contour map. DETAILED DESCRIPTION

[0024] The present invention synthesizes MnTi6 with an amorphous and nanoporous structure by a simple solvothermal method. By uniformly dissolving the metal salt in an isopropanol solution and allowing it to react and precipitate rapidly under high temperature and high pressure, the formation and growth of the crystal nucleus are inhibited, thereby achieving the co-precipitation of a uniform amorphous structure. In the process of producing the invention, first, the inventor successfully synthesized a titanium material without manganese through exploration and named it Ti6. Commercial TiO2 was also purchased and its performance was tested as a control group of Ti6. At the same time, MTi6 (M represents metal Ce / Cu / Mn / V) material was prepared using metal salts of cerium (Ce) / copper (Cu) / manganese (Mn) / vanadium (V), and its antioxidant enzyme-like performance was tested for comparison, and it was found that MnTi6 had the best antioxidant enzyme-like performance. Since MnTi6 exhibits the best ability to scavenge multiple free radicals, the inventor also attempted to synthesize a control group without titanium using a similar method, but the yield was extremely low.

[0025] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0026] Materials and Reagents

[0027] Manganese(II) nitrate tetrahydrate (Mn(NO3)2•4H2O), titanium(IV) isopropoxide, titanium(IV) sulfate (Ti(SO4)2), and 4-aminobenzoic acid were purchased from Aladdin Reagents. Ultrapure water (18.2 MΩ•cm) used in the experiments was prepared by a Milli-Q Academic system (Millipore Corporation, Billerica, MA, USA). All chemical reagents were used as received without further purification.

[0028] Example: Synthesis of MnTi6 artificial antioxidant enzyme

[0029] MnTi6 with amorphous and nanoporous structure was synthesized by a facile solvothermal method. Briefly, manganese(II) nitrate tetrahydrate (3514.14 mg, 14 mmol) was added to an isopropanol solution (24 mL) containing 4-aminobenzoic acid (768.4 mg, 5.60 mmol) and titanium(IV) isopropoxide (103.6 μL, 0.35 mmol). After stirring at room temperature for 30 min, the orange slurry was transferred to a polytetrafluoroethylene-lined autoclave, heated to 100 °C and maintained for 72 h. The brown solid was collected, washed with isopropanol, and then dried under vacuum overnight.

[0030] Comparative Example 1: Synthesis of Ti6

[0031] This comparative example prepared a Ti6 titanium oxide cluster material without Mn incorporation. Briefly, titanium (IV) isopropoxide (103.6 μL, 0.35 mmol) was added to an isopropanol solution (6.0 ml) containing 4-aminobenzoic acid (192.1 mg, 1.40 mmol). After stirring at room temperature for 30 minutes, the orange slurry was transferred to a sealed glass tube, heated to 100 ° C and maintained for 77 hours. The bright yellow crystalline product was collected, washed with isopropanol, and then dried overnight under vacuum.

[0032] Comparative Example 2: Synthesis of CeTi6

[0033] CeTi6 material was synthesized as described in the Examples using cerium (IV) sulfate tetrahydrate instead of manganese (II) nitrate tetrahydrate.

[0034] Comparative Example 3: Synthesis of CuTi6

[0035] CuTi6 material was synthesized as described in the Examples using anhydrous cupric chloride instead of manganese (II) nitrate tetrahydrate.

[0036] Comparative Example 4: Synthesis of VTi6

[0037] Using vanadyl acetylacetonate instead of manganese (II) nitrate tetrahydrate, VTi6 material was synthesized as described in the Examples.

[0038] Performance Testing

[0039] 1. Structural characterization

[0040] Scanning electron microscopy (SEM) images were acquired by Apreo S HiVoc from Thermo Fisher Scientific (FEI). A gold coating of approximately 1 nm thick was deposited on the sample surface. Transmission electron microscopy (TEM) tests were performed using a Tecnai G2F20S-TWIN, operating at 200 kV. Raman spectroscopy was measured by XploRA PLUS (Horiba, France) to evaluate molecular vibrations and crystal defects within the sample. X-ray photoelectron spectroscopy (XPS) was performed on a K-Alpha™+ X-ray photoelectron spectrometer system (Thermo Fisher Scientific) using a 180° hemispherical double focusing analyzer equipped with a 128-channel detector to examine the valence state and electronic structure of MnTi6. Inductively coupled plasma optical emission spectrometer (ICP-OES, Thermo Fisher ICAPPRO) was used to analyze the elemental content. Scanning transmission electron microscopy (STEM) images and energy dispersive X-ray spectroscopy (EDX) elemental mapping were acquired by a cesium-corrected STEM (FEI Titan Cubed Themis G2 300). K-edge X-ray absorption (XAS) spectra were collected at beamline station BL07A1 in Taiwan Province, China. The radiation was generated by a scanning silicon (111) double-crystal monochromator. Data processing and analysis of X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure spectroscopy (EXAFS) were performed using Athena software. In this study, in situ Fourier transform infrared (FTIR) measurements were performed using an infrared spectrometer (Thermo Fisher Scientific, iS50FTIR) equipped with an in situ spectral cell (Shanghai Yuanfang Technology Co., Ltd., SPECEL-III).

[0041] 2. ABTS + Clear test 7.4 mmol of 2,2'-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) (ABTS) and 2.6 mmol of potassium persulfate (K2S2O8) were mixed in a 1:1 volume ratio and the reaction was allowed to stand overnight to oxidize ABTS to ABTS +The above solution was then added to phosphate buffer solution (PBS, pH = 7.4) to a total volume of 1 ml and a material concentration of 5 μg / ml. After 5 minutes of reaction, the absorbance of the solution was tested at 734 nm to evaluate the biocatalyst's effect on ABTS. + · Clearing ability.

[0042] 3. O2 - Clear Test

[0043] Dissolve 1 mg of potassium superoxide (KO2) in 1 ml of dimethyl sulfoxide solution (DMSO, containing 3 mg / ml of 18-crown-6-ether) to generate and stabilize •O2 - Then, Ti6 and MnTi6 were dispersed into the above KO2 / DMSO solution with a final concentration of 50 μg / mL. After 5 minutes of reaction, the remaining •O2 - The solution was captured by nitroblue tetrazolium (NBT)-DMSO solution (10 μl, 10 mg / ml). The absorbance of the solution was measured at 680 nm and then mixed with •O2 - to determine its effect on •O2 - cleaning ability.

[0044] 4. Catalase-like (CAT) test - Hydrogen peroxide (H2O2) removal

[0045] 10 mmol of hydrogen peroxide (H2O2) and 50 μg / mL of the biocatalyst were mixed in phosphate buffer solution (PBS, pH=7.4) to a total volume of 2 mL. Then, 100 μL of the above solution was mixed with titanium (IV) sulfate solution (100 μL, 13.9 mmol), and the absorbance at 405 nm was recorded every 10 minutes until 40 minutes. After the reaction reached 40 minutes, the absorbance of the solution at 405 nm was tested to evaluate the ability of the biocatalyst to scavenge H2O2.

[0046] 5. Catalase-like (CAT) test - oxygen (O2) generation assay

[0047] 200 mmol of hydrogen peroxide (H2O2) and 25 μg / mL of the biocatalyst were mixed in phosphate buffer solution (PBS, pH = 7.4) to a total volume of 20 mL, and then the oxygen concentration was measured every 5 seconds until 300 seconds using a dissolved oxygen meter (Shanghai Yidian Scientific Instrument Co., Ltd., JPSJ-605F). To analyze the biocatalytic kinetics of O2 generation, 25 μg / mL of the biocatalyst was mixed with different concentrations of hydrogen peroxide (H2O2) (50, 100, 150, 200, 250, and 300 mmol) in PBS to prepare a 20 mL solution, and then the O2 concentration was measured every 5 seconds until 100 seconds. The reaction rate was plotted against the corresponding H2O2 concentration and then fitted using the Michaelis-Menten equation (Equation (1)). In addition, the double reciprocal plot method (Lineweaver–Burk plot, Equation (2)) was used to determine the maximum reaction rate (V max ) and the Michaelis constant (K m In addition, the turnover number (TON, i.e., the maximum number of substrates converted per unit active catalytic center) was calculated according to formula (3), where [S] is the concentration of hydrogen peroxide (H2O2) and [E0] represents the molar concentration of the metal in the material.

[0048] (S1)

[0049] (S2)

[0050] (S3)

[0051] Among them, V max is the maximum reaction rate in the active oxygen catalytic reaction; K m It represents the affinity between the catalyst and the substrate (the smaller the value, the better the affinity); TON reflects the maximum number of substrates converted by a unit active catalytic atom, reflecting the intrinsic activity that is independent of the number of active centers.

[0052] 6. Detailed information on in-situ Fourier transform infrared (FTIR) measurements

[0053] In this study, in situ FTIR measurements were performed using an infrared spectrometer (Thermo Fisher Scientific, iS50 FTIR) equipped with an in situ spectral cell (Shanghai Yuanfang Technology Co., Ltd., SPECEL-III). We usually prepared a Nafion solution consisting of 210 μL of isopropanol, 750 μL of deionized water, and 40 μL of Nafion (perfluorosulfonic acid ion exchange resin, source leaf bio, 5% mass fraction, dissolved in water and 1-propanol). Then, a catalyst / Nafion solution with a concentration of 10 mg / mL was prepared. During the experiment, 40 μL of the catalyst solution was drop-coated on the surface of zinc selenide (ZnSe) crystals, allowed to dry, and then the crystals were mounted in the in situ cell. Next, 5 mL of 0.5 M hydrogen peroxide (H2O2) solution was added to the in situ cell, and in situ FTIR spectra were collected at specific time intervals, with the reaction time kept at 10 min.

[0054] Test Results

[0055] 1. Structural characterization

[0056] The nanoparticle morphology of MnTi6 was confirmed by scanning electron microscopy (SEM) and high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) images. Figure 1 As shown in ab. The images show that compared with the non-porous dense structure of Ti6, MnTi6 contains many nanopores, which are marked with yellow circles. These nanopores can increase the specific surface area, which is believed to be beneficial to improve the catalytic performance. Figure 1 As shown in Figure c, no obvious long-range ordered atomic arrangement was observed, proving the structural disorder of MnTi6. Scanning transmission electron microscopy-energy dispersive X-ray spectroscopy (STEM-EDS) showed that oxygen (O), titanium (Ti) and manganese (Mn) were well and evenly distributed in the nanostructure, without local aggregation of manganese, which confirmed the successful synthesis of MnTi6 ( Figure 1 d, e). Dynamic light scattering (DLS) and zeta potential showed that MnTi6 has good water dispersibility (zeta potential: -6.04 mV) and an average hydrodynamic size of about 900 nm ( Figure 1 g).

[0057] In addition, we deeply studied the detailed chemical structure of MnTi6. First, using Raman spectroscopy, the Ti-O-Ti structure of MnTi6 was determined based on the ν(Ti-O-Ti) peak at 614 cm⁻¹, as shown in Figure 1 h. The coordinated 4-aminobenzoic acid [ν(COO) at 1394 cm -1 、1524cm -1 and 1600cm -1The Raman peaks of A1g and B2g modes at 1000 cm⁻¹ and ν(CCO) at 857 cm⁻¹ of isopropanol were observed, which confirmed that MnTi6 has a similar coordination structure to Ti6. It is worth noting that a new Raman peak at 536 cm⁻¹ was observed, which was attributed to the stretching vibration of Mn-O, indicating the formation of Mn-O bonds. The surface composition and electronic structure of MnTi6 were studied by X-ray photoelectron spectroscopy (XPS). The full XPS spectrum ( Figure 1 i) shows that manganese (Mn), titanium (Ti), nitrogen (N) and oxygen (O) exist in MnTi6, which is consistent with the results of STEM-EDS. The specific contents of manganese and titanium are 7.19% and 17.94%, respectively, which is consistent with the results of inductively coupled plasma optical emission spectrometry (ICP-OES) ( Figure 1 f). Figure 1 As shown in Figure 6, the O 1s spectra of both MnTi6 and Ti6 show different signals related to organic oxygen and OM (metal). The binding energy of the O 1s peak of MnTi6 is reduced by 0.14 eV and 0.22 eV compared with Ti6. These results indicate that atomic-level Mn-O sites are formed in MnTi6. At the same time, the binding energy level of the Ti 2p peak in MnTi6 is reduced compared with Ti6 ( Figure 1 k). These observations suggest the presence of electron transfer from the electron-donating manganese to the titanium oxide substrate. High-resolution Mn 2p spectroscopy results ( Figure 1 l) demonstrated the existence of this electron transfer, and the spectrum can be deconvoluted into the 2p1 / 2 and 2p3 / 2 regions. The two main components in the spectrum, Mn³⁺ at 652.26 eV and 641.26 eV, and Mn²⁺ at 651.42 eV and 639.74 eV, fit well, indicating that the manganese valence is higher than that of the initial salt Mn(NO3)2·4H2O, and indicating that atomic-level Mn-O sites are formed in MnTi6. Subsequently, we performed X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) analysis to explore the local coordination environment of MnTi6. The K-edge spectrum of manganese (Mn) shows that the pre-edge peak of MnTi6 is located between the pre-edge peak positions of manganese oxide (MnO) and manganese dioxide (MnO2) ( Figure 1 m), and its average valence state of manganese is +2.22 ( Figure 1 n), indicating the presence of electron transfer from low-valence manganese atoms to titanium (Ti) atoms, which is consistent with our XPS analysis results. According to the k³-weighted Fourier transform extended X-ray absorption fine structure (EXAFS) spectrum, MnTi6 has major peaks at 1.66 Å and 2.31 Å, indicating the presence of manganese-oxygen (Mn-O) and manganese-titanium (Mn-Ti) structures ( Figure 1o). Overall, the STEM, XPS, and XAS data corroborate each other, confirming that the incorporation of Mn may coordinate with the oxygen in the titanium oxygen clusters by replacing the position of Ti, leading to the formation of an amorphous nanoporous structure. In addition, we also found that there is electron transfer from the Mn site to the Ti site, which may enable MnTi6 to overcome the challenges associated with multi-electron reactions in the process of scavenging reactive oxygen species (ROS).

[0058] 2. Evaluation of reactive oxygen species scavenging activity

[0059] After verifying the chemical and electronic structure of MnTi6, its biocatalytic performance in removing ROS was systematically studied. First, all the materials obtained in the examples and comparative examples were subjected to O2 - Clear performance tests such as Figure 2 As shown in a. Commercial TiO2 does not show the ability to scavenge free radicals, while Ti6 shows excellent ·O2 - At the same time, other MTi6 (M represents metal Ce / Cu / Mn / V) materials also show O2 - Scavenging performance. It is worth noting that Cu and Mn are the metal active sites of natural SOD enzymes. However, Ti6, as a material without these active metals (Cu and Mn), still exhibits excellent SOD-like enzyme performance. Currently, there are few reports on the efficient SOD-like enzyme performance of titanium metal-related materials. Secondly, Figure 2 As shown in Figure b, we monitored the H2O2 scavenging ability of the obtained materials. Within 30 minutes, the H2O2 scavenging efficiency of MnTi6 alone reached 91%. H2O2 scavenging, as the first step of CAT performance, indicates the existence of CAT performance of MnTi6.

[0060] After the initial screening of the obtained materials, the performance of MnTi6 was investigated in detail. General resistance of biocatalyst (MnTi6) to active oxygen cascade reactions such as Figure 3 a. First, the 2,2'-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) free radical scavenging experiment was used to test the total free radical scavenging ability of the material. The results showed that compared with Ti6, MnTi6 had an improved scavenging ability for ABTS⁺· Figure 3 b). Due to the removal of superoxide anions (·O2 - ) is the initial step of the anti-ROS cascade reaction, so tetrazolium nitro blue (NBT) is used as a specific indicator to monitor the superoxide anion (·O2 - ) clearance rate to study its superoxide dismutase (SOD)-like activity (·O2 - Converted into hydrogen peroxide (H2O2). Figure 3As shown in Figure 3, both Ti6 and MnTi6 exhibited strong superoxide dismutase (SOD)-like activity, indicating that the titanium (Ti) site plays a key role in the SOD-like activity. - The clearance of ·O2 - The clearance rate can reach about 90%.

[0061] Afterwards, catalase (CAT)-like activity [converting hydrogen peroxide (H2O2) into oxygen (O2) and water (H2O2)], the second key step in the scavenging cascade system for reactive oxygen species, was monitored for Ti6 and MnTi6. In the time-dependent H2O2 scavenging experiment, the H2O2 content of MnTi6 was significantly reduced (6.54%) compared to the original Ti6 (94.39%) ( Figure 3 d), indicating that the Mn site plays a key role in the CAT-like activity. The generated oxygen was also tested and correlated with the decomposition of H2O2 ( Figure 3 e), MnTi6 showed a significant increase in oxygen (O2) concentration within 5 minutes, 27.26 mg / mL higher than Ti6. Importantly, after repeated addition of hydrogen peroxide (H2O2), MnTi6 showed good cycling durability (scavenging rate greater than 90% after 5 cycles) ( Figure 3 f). Using hydrogen peroxide (H2O2) as substrate, the steady-state catalytic kinetics of the simulated catalase (CAT) process was determined, and the maximum reaction rate (V max ) and conversion number (TON) ( Figure 3 g). After calculation, the V max and TON values ​​were 44.3 μM·s -1 and 1.26 s -1 (Table 1 and Figure 3 i). Subsequently, we will determine the V max The TON values ​​were compared with those of transition metal-based ROS scavenging materials (Table 1). The MnTi6 biocatalyst exhibited significant differences from the most advanced ROS scavenging nanobiocatalysts reported so far, including most manganese-based biocatalysts (Mn3O4 cubes, Mn3O4 hexagonal plates, etc.), as well as other metal-based biocatalysts (ultrasmall Cu 5.4 O clusters, Co3O4 nanoparticles, FeNzy and RuSA-CN, etc.) Figure 3 h and Table 1). In situ Fourier transform infrared spectroscopy (FTIR) was used to study the structural changes of the intermediates in the process of simulated catalase (CAT), and the results showed that* OOH and O2 - intermediates, and the reaction of hydrogen peroxide (H2O2) to oxygen (O2) ( Figure 3 j, k). Therefore, we demonstrated that the MnTi6 artificial antioxidant enzyme exhibited cascade SOD-CAT catalytic activity due to its Mn-O-Ti dual active sites.

[0062] Table 1: Maximum reaction rates (V) of recently reported state-of-the-art ROS scavenging biocatalysts max ) and turnover number (TON). Turnover number (TON) = maximum reaction rate (V max ) / [E0], where [E0] is the molar concentration of the metal in the entire nanomaterial.

[0063] Biocatalysts <![CDATA[V max (μM·s -1 )]]> <![CDATA[[E0] (μM)]]> <![CDATA[TON (s -1 )]]> References <![CDATA[MnTi6]]> 44.30 35.16 1.26 The present invention RuTeNRs 0.98 196340 5.00E-06 1 <![CDATA[MnO2]]> 2.4 4965.52 4.83E-04 2 OxgeMCC-r 0.2 29.28 6.83E-03 3 <![CDATA[Mn3O4]]> 10.73 198.7 5.40E-02 4 <![CDATA[Mn3O4Cb]]> 5.3 65.45 0.08 5 <![CDATA[Mn3O4Ph]]> 5.8 65.45 8.86E-02 5 MC-1.0 24.82 118.19 0.21 4 Cu NCs 418.41 1820 0.23 6 F 1.22 4.82 0.25 7 <![CDATA[Cu 5.4 The]]> 3.92 15.04 0.26 8 Mf 21.75 65.45 0.33 5 <![CDATA[Ru SA -CN]]> 10.02 13.01 0.77 9 <![CDATA[Cu x The]]> 109.2 125.81 0.87 10

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[0075] It should be noted that the preferred embodiments of the present invention are given in the specification and drawings of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not used as additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features continue to be combined with each other to form various embodiments not listed above, which are all regarded as the scope of the present invention; further, for ordinary technicians in this field, they can be improved or transformed according to the above description, and all these improvements and transformations should belong to the protection scope of the claims attached to the present invention.

Claims

1. A MnTi6 artificial antioxidant enzyme, characterized in that: The MnTi6 artificial antioxidant enzyme is a Mn-doped titanium oxide cluster formed by a solvothermal reaction of a titanium source, a manganese source and 4-aminobenzoic acid, and has an amorphous nanoporous structure; Furthermore, there are Mn-O and Mn-Ti coordination structures in the MnTi6 artificial antioxidant enzyme.

2. The MnTi6 artificial antioxidant enzyme according to claim 1, characterized in that The MnTi6 artificial antioxidant enzyme has cascade SOD-CAT catalytic activity.

3. A method for preparing the MnTi6 artificial antioxidant enzyme as claimed in claim 1 or 2, characterized in that: The preparation method comprises the following steps: adding a manganese source to a solvent containing a titanium source and 4-aminobenzoic acid, reacting at 90-120° C. for 60-90 hours, collecting the reaction product, washing it, and drying it to obtain the MnTi6 artificial antioxidant enzyme.

4. The preparation method according to claim 3, characterized in that: The titanium source includes titanium tetraisopropoxide, titanium tetrachloride or titanate.

5. The preparation method according to claim 4, characterized in that: The titanium source is titanium tetraisopropoxide.

6. The preparation method according to claim 3, characterized in that: The manganese source includes manganese nitrate tetrahydrate, manganese chloride hexahydrate or manganese sulfate hexahydrate.

7. The preparation method according to claim 6, characterized in that: The manganese source is manganese nitrate tetrahydrate.

8. The preparation method according to claim 3, characterized in that: The solvent includes isopropanol, methanol, ethanol, toluene, acetonitrile or N,N-dimethylformamide.

9. The preparation method according to claim 8, characterized in that: The solvent is isopropanol.

10. Use of the MnTi6 artificial antioxidant enzyme as claimed in claim 1 or 2 in the preparation of a preparation for removing active oxygen.

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