Phenylalanine-manganous-manganic oxide nano-enzyme as well as preparation method and application thereof

Abstract

The invention provides a preparation method and application of phenylalanine-manganese nano-enzyme and iron and cobalt doped nano-enzyme, and relates to the technical field of biological catalysis. The phenylalanine-manganese nano-enzyme is a nano-scale material formed through a self-assembly process, and combines the biocompatibility of phenylalanine and the catalytic performance of manganous-manganic oxide. The preparation method of the nano enzyme comprises the following steps: mixing L-phenylalanine, a water-soluble manganese source and sodium hydroxide in a water phase, and forming a nano structure with a specific structure and function through self-assembly. The nano-enzyme obtained through water bath heating has good biocompatibility and biological stability, and is suitable for application in the field of biomedicine. By doping iron, cobalt and other transition metal elements, the catalytic performance of the nano-enzyme can be further optimized, and the selectivity and catalytic efficiency of the nano-enzyme to a specific substrate are improved. The novel nano laccase has wide application potential in the fields of biological catalysis, organic synthesis, environmental governance and the like, and can be used for catalyzing various chemical reactions including oxidation, reduction, addition, elimination and the like. The phenylalanine-manganese nano-enzyme and the iron and cobalt doped nano-enzyme thereof provided by the invention are novel biocatalysts with high efficiency, stability, high crystallinity and wide application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of biocatalysis, and in particular to a preparation method and application of a phenylalanine-manganese tetraoxide nanozyme and an iron- and cobalt-doped nanozyme. Background Art

[0002] Nanozymes are nanoscale catalysts with enzymatic activity, which are usually composed of metal nanoparticles, metal oxide nanoparticles or organic polymer nanocomposites. The catalytic activity of nanozymes originates from the active sites on their surfaces, which can specifically bind to substrates and promote chemical reactions. They have broad application prospects in biological detection, medical diagnosis, environmental monitoring and catalysis. However, existing nanozyme catalysts have problems such as poor stability and low catalytic efficiency, which limit their wide application. Developing nanozymes with simple synthesis, high activity and good stability is an important development direction.

[0003] At present, nanozymes mainly include organometallic nanozymes, composite nanozymes, organic nanozymes and quantum dot nanozymes. However, the synthesis and modification process of some nanomaterials may be complicated and costly, and there are potential biological toxicity and environmental stability issues. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a preparation method and application of phenylalanine-manganese tetraoxide nanozyme and iron- and cobalt-doped nanozyme. The phenylalanine-manganese tetraoxide nanozyme prepared by the preparation method provided by the present invention has strong stability and high catalytic activity.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The invention provides a phenylalanine-manganese tetraoxide nanozyme, which is a nanoscale complex obtained by self-assembly of phenylalanine and a manganese tetraoxide source in a water bath.

[0006] The phenylalanine-manganese tetraoxide nanozyme has a cubic crystal structure, in which Mn 3+ and Mn 2+ The ions are located in tetrahedral and octahedral coordination environments.

[0007] The present invention provides a method for preparing the phenylalanine-manganese tetraoxide nanozyme described in the above technical solution, comprising the following steps: Phenylalanine, a water-soluble manganese tetraoxide source, sodium hydroxide and water are mixed and heated in a water bath for a self-assembly reaction to obtain phenylalanine-manganese tetraoxide nanozyme.

[0008] Preferably, the ratio of phenylalanine to manganese tetraoxide source is 1:1 to 1:5. Preferably, the water-soluble manganese tetraoxide source is a water-soluble manganese tetraoxide salt.

[0009] Preferably, the temperature of the self-assembly reaction is 60° C. and the time is 2 h.

[0010] The present invention provides the application of the phenylalanine-manganese tetraoxide nanozyme described in the above technical scheme or the phenylalanine-manganese tetraoxide nanozyme obtained by the preparation method described in the above technical scheme in biosensing, environmental remediation, organic synthesis, bleaching or wastewater treatment.

[0011] The present invention provides a phenylalanine-manganese tetraoxide nanozyme (F-Mn 3 O 4 ), which is obtained by self-assembly of raw materials including phenylalanine and a water-soluble manganese tetraoxide source in a water bath. In the present invention, phenylalanine and a water-soluble manganese tetraoxide source self-assemble into a morphologically stable structure in an aqueous phase at 60°C. Compared with other nanozymes, F-Mn 3 O 4 As a self-assembled macromolecule, it well mimics the chemical properties of natural enzymes and the active site with manganese ions as the active center. Compared with natural enzymes, F-Mn 3 O 4 As a nanozyme, F-Mn 3 O 4 As a metal complex, it has better tolerance to the environment and higher stability. The phenylalanine-manganese tetraoxide nanozyme provided by the present invention has strong stability, high catalytic activity, and high crystallinity, and still maintains high enzyme catalytic activity under high temperature, acidic, alkaline and high ionic strength environments.

[0012] The present invention provides a method for preparing the phenylalanine-manganese tetraoxide nanozyme described in the above technical scheme, comprising the following steps: mixing phenylalanine, a water-soluble manganese tetraoxide source, sodium hydroxide and water, and heating in a water bath for self-assembly reaction to obtain the phenylalanine-manganese tetraoxide nanozyme. In the present invention, during the self-assembly reaction, the prepared phenylalanine-manganese tetraoxide nanozyme is highly stable, has high catalytic activity and high crystallinity under heating in a water bath at 60°C. The phenylalanine-manganese tetraoxide nanozyme still maintains high laccase catalytic activity under high temperature, acidic, alkaline and high ionic strength environments. The present invention adopts a one-pot preparation method, which has a simple process and a high yield; moreover, the present invention performs self-assembly in an aqueous phase system, which is safe and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 shows the Mn prepared in Example 1 3 O 4 ,Fe-Mn 3 O 4 ,Co-Mn 3 O 4A three-dimensional typical score diagram of the response sensor array and a curve diagram of the discriminant factor 1 versus the compound concentration were drawn by adding different concentrations of acid; Figure 2 shows the Mn prepared in Example 1 3 O 4 ,Fe-Mn 3 O 4 ,Co-Mn 3 O 4 Three-dimensional typical score diagram of the response sensor array drawn by adding mixtures of different molar ratios; Figure 3 shows the Mn prepared in Example 1 3 O 4 ,Fe-Mn 3 O 4 ,Co-Mn 3 O 4 Three-dimensional typical scoring diagram and blind sample discrimination results of laccase mimics in blind sample tests; FIG. 4 is a typical three-dimensional scoring diagram of seven serum samples (5, 10 μM) using a colorimetric sensor array in Example 1. DETAILED DESCRIPTION

[0014] The invention provides a phenylalanine-manganese nanozyme and an iron- and cobalt-doped nanozyme, which are obtained by mixing phenylalanine, a water-soluble manganese tetraoxide source, sodium hydroxide and water, heating the mixture in a water bath and then self-assembling the mixture.

[0015] The present invention provides a method for preparing the phenylalanine-manganese tetraoxide nano-laccase described in the above technical solution, comprising the following steps: Phenylalanine, a water-soluble manganese source, sodium hydroxide and water are mixed and heated in a water bath for self-assembly reaction to obtain phenylalanine-manganese tetraoxide nano-laccase.

[0016] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0017] In the present invention, the water-soluble manganese source is preferably a water-soluble manganese salt, more preferably including one or more of manganese sulfate, manganese chloride, and manganese nitrate. In the present invention, the water-soluble manganese source is preferably used in the form of a water-soluble manganese source aqueous solution, and the concentration of the water-soluble manganese source is preferably 15 mmol / L. In the present invention, the molar ratio of the water-soluble manganese source to phenylalanine is preferably 1:1.

[0018] In an embodiment of the present invention, the mixture of phenylalanine, a water-soluble manganese source and water is preferably prepared by adding an aqueous solution of phenylalanine and an aqueous solution of a water-soluble manganese source into water to obtain a primary mixture; the concentration of the aqueous solution of phenylalanine is preferably 100 mmol / L; the concentration of the aqueous solution of the water-soluble manganese source is preferably 100 mmol / L; and the concentration of the sodium hydroxide is preferably 1 mmol / L.

[0019] The present invention has no particular limitation on the mixing, as long as the raw materials can be mixed evenly, such as stirring and mixing.

[0020] In the present invention, the temperature of the self-assembly reaction is preferably 30-80 ° C, more preferably 40-80 ° C, further preferably 30-70 ° C, and most preferably 60 ° C; the time of the self-assembly reaction is preferably 0.5-3h, more preferably 1-2.5h, and more preferably 1.5-2h. After completing the self-assembly reaction, the present invention preferably further comprises solid-liquid separation of the reaction system obtained by the self-assembly reaction, washing the obtained solid product with water and drying it to obtain phenylalanine-manganese nanolaccase. There is no special limitation on the solid-liquid separation method in the present invention, and a solid-liquid separation method well known to those skilled in the art can be used, such as filtration or centrifugation. In the present invention, the water for washing preferably includes distilled water, and the number of washings is preferably 2-4 times, and more preferably 3 times. In the present invention, the drying temperature is preferably 20-60 ° C, and more preferably 40 ° C; the present invention does not specifically limit the drying time, and it can be dried to constant weight; the drying is preferably carried out in an oven.

[0021] The present invention provides the use of the phenylalanine-manganese nanolaccase described in the above technical solution or the phenylalanine-manganese nanolaccase obtained by the preparation method of the above technical solution in biosensing, environmental remediation, organic synthesis, bleaching or wastewater treatment. The phenylalanine-manganese nanolaccase provided by the present invention has stable morphology, high crystallinity, high catalytic activity, and still maintains high catalytic activity under high temperature, extreme pH and high ionic strength environments, and has good application prospects in biosensing, environmental remediation, organic synthesis, bleaching and wastewater treatment.

[0022] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Example 1 1.5 mL of 100 mmol / L phenylalanine aqueous solution and 1.5 mL of 100 mmol / L MnCl 2 The aqueous solution was mixed with 6.9 mL of water, and then 100 μL of 1 mmol / L NaOH aqueous solution was added, followed by 1.5 mL of 100 mmol / L phenylalanine aqueous solution, 1.35 mL of 100 mmol / L MnCl 2 Aqueous solution, 0.15mL FeCl 3 or CuCl 2 and 6.9 mL of water, then 100 μL of 1 mmol / L NaOH aqueous solution was added for self-assembly reaction at 60 °C for 2 h, and then centrifuged. The solid product was washed twice with distilled water and three times with alcohol, and then dried in an oven at 40 °C to constant weight to obtain phenylalanine-manganese nanolaccase (black powder, abbreviated as F-Mn 3 O 4 )); Among them, phenylalanine and MnCl 2 The molar ratio = 1:1.

[0024] Comparative Example 1 The natural laccase disclosed in the document “ Guan, M.; Wang, M.; Qi, W.; Su, R.; He, Z. Biomineralization-inspired copper-cystine nanoleaves capable of laccase-like catalysis for the colorimetric detection of epinephrine. Front. Chem. Sci. Eng. 2021, 15, 310-318”.

[0025] Comparative Example 2 Natural laccase disclosed in the literature “ Sun, H.; Zhou, Y.; Ren, J.; Qu, X. Carbon nanozymes: enzymatic properties, catalytic mechanism, and applications. Angew. Chem. Int. Edit. 2018, 57, 9224–9237.”

[0026] Natural laccase disclosed in the literature “Chen, Z.; Wang, Z.; Ren, J.; Qu, X. Enzyme mimicry for combating bacteria and biofilms. Accounts Chem. Res. 2018, 51, 789-799.”

[0027] Natural laccase disclosed in the literature “ Jiang, D.; Ni, D.; Rosenkrans, ZT; Huang, P.; Yan, X.; Cai,W. Nanozyme: new horizons for responsive biomedical applications. Chem.Soc.Rev. 2019, 48, 3683-3704.”

[0028] Stability test Figure 1 shows the Mn prepared in Test Example 1 3 O 4 ,Fe-Mn 3 O 4 ,Co-Mn 3 O 4 Multiple detection capabilities. Figure 1 As shown in a1-1a7, the LDA graph clearly shows that each concentration of DPAs can be well distinguished without cross-effects. At the same time, it can be seen that the clusters in the LDA graph are not randomly distributed, but arranged as the concentration of DPAs increases, as shown in Figure 1 As shown in b1, as the niacin concentration increases, the value of factor 1 decreases. In the range of niacin concentration from 0.01 to 10 μM and from 10 to 100 μM, the score of factor 1 shows a good linear relationship with the niacin concentration (e.g. Figure 1 As shown in b1), the quantification of other DPAs also achieved a linear range of 0.01-10 μM and 10-100 μM; Figure 2 is a graph showing the Mn prepared in Example 1. 3 O 4 ,Fe-Mn 3 O 4 ,Co-Mn 3 O 4Multiplex detection capability. The selected binary DPA mixtures (nicotinic acid / 2-pyridinecarboxylic acid; 2-pyridinecarboxylic acid / 2,3-pyridinedicarboxylic acid; 2,3-pyridinedicarboxylic acid / 3,4-pyridinedicarboxylic acid = 1:1, 1:3 and 3:2, with a total concentration of 500 μM) and ternary DPA mixtures (nicotinic acid / isonicotinic acid / 2-pyridinecarboxylic acid; nicotinic acid / 2-pyridinecarboxylic acid / 2,6-pyridinecarboxylic acid = 1:1:1 and 1:1:2; isonicotinic acid / 2-pyridinecarboxylic acid / 3,4-pyridinedicarboxylic acid = 3:3:1, with a total concentration of 500 μM) were used as target analytes. Figure 2 As shown in a and 2b, DPAs with different molar ratios can be well aggregated into different groups in the mixture, achieving 100% accuracy to separate and distinguish them, laying the foundation for the recognition and identification of complex real samples; Figure 3 is a diagram for evaluating the practicality of the sensor array. 3 O 4 ,Fe-Mn 3 O 4 ,Co-Mn 3 O 4 Blind sample tests and interference experiments were conducted as laccase mimics, and all 35 blind samples could be accurately classified; Figure 4 shows the Mn prepared in Example 1 3 O 4 ,Fe-Mn 3 O 4 ,Co-Mn 3 O 4 Human serum was collected from a healthy volunteer to simulate the actual biological fluid situation. After pre-treatment of the serum with methanol, the seven DPAs were added to the pre-treated serum and diluted 1000 times to obtain the final concentrations of the seven DPAs of 5 and 10 μM. Figure 4 As shown, all clusters were separated in a regular arrangement, where higher concentrations of spiked samples tended to be away from pure serum samples, indicating that the proposed sensor array had reliable discrimination ability.

Claims

1. A phenylalanine-manganese nanozyme, characterized in that: It is a nanoscale complex obtained by mixing phenylalanine, a water-soluble manganese source and sodium hydroxide in an aqueous phase through self-assembly, wherein the nanoenzyme has the activity of catalyzing oxidation reactions.

2. The phenylalanine-manganese nanozyme according to claim 1, characterized in that: The size of the nanozyme is 10-100 nm.

3. The method for preparing the phenylalanine-manganese nanozyme and the iron- and cobalt-doped nanozyme according to any one of claims 1 to 2, comprising the following steps: Phenylalanine, a water-soluble manganese source, sodium hydroxide and water are mixed and heated in a water bath for self-assembly reaction to obtain phenylalanine-manganese nanozyme. Iron-doped and cobalt-doped nanozymes are prepared by doping manganese tetraoxide with iron or cobalt in a ratio of 9:1 and then preparing the nanozyme according to the above method.

4. The preparation method according to claim 3, characterized in that: The ratio of the phenylalanine to the water-soluble manganese source is 1:1 to 1:

5.

5. The preparation method according to claim 3 or 4, characterized in that: The water-soluble manganese source is a water-soluble manganese salt.

6. The preparation method according to claim 3, characterized in that: The temperature of the self-assembly reaction is 60° C. and the time is 2 h.

7. Use of the phenylalanine-manganese nanozyme according to any one of claims 1 to 2 or the phenylalanine-manganese nanozyme obtained by the preparation method according to any one of claims 3 to 6 in biosensing, environmental remediation, organic synthesis, bleaching or wastewater treatment.

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