Preparation method of dephosphorized artificial enzyme

Abstract

The invention belongs to the technical field of artificial enzyme synthesis, and provides a preparation method of a dephosphorized artificial enzyme. The preparation method comprises the following steps: mixing a biomass raw material, a diamine compound and water, and carrying out hydrothermal reaction to obtain a nano stock solution; and mixing the nano stock solution with cysteine, and diluting to obtain the product. The preparation method comprises the following steps: firstly, carrying out a reaction on a biomass raw material and a diamine compound by utilizing a hydrothermal reaction to form a biochar material containing acylamino on the surface; then, cysteine is added; carboxyl and amino in cysteine have hydrogen bond interaction with a main functional group acylamino on the biochar material, so that sulfydryl on cysteine is distributed on the surfaces of nano-particles to form more active sites, and due to the fact that the sulfydryl and the biochar material are combined together through hydrogen bonds, the binding force between cysteine and the biochar material is stronger, and cysteine is not prone to falling off. The activity of the dephosphorized artificial enzyme is stable, and the antibacterial property is stable.

Description

Technical Field

[0001] The invention relates to the technical field of artificial enzyme synthesis, in particular to a method for preparing an artificial dephosphorylation enzyme. Background Art

[0002] Dephosphatase refers to a biological enzyme that hydrolyzes phosphate esters to generate free phosphate groups and hydroxylated molecules. Biological enzymes are divided into natural enzymes and artificial enzymes. The concept of artificial enzymes was first proposed by Breslow, and his work showed that enzymatic functions can be realized by connecting metal catalytic groups to hydrophobic substances to achieve macromolecular cavities. In the prior art, researchers have disclosed artificial dephosphorylation enzymes, which are sulfur-nitrogen co-doped nanoparticles (SN-CNP) with phosphatase activity that mimics cysteine ​​proteins. The synthesis method of the artificial dephosphorylation enzyme is as follows: the first step is to prepare beet powder: wash the beet root (Beet), peel it, and pulp it, then place the pulp in a vacuum drying oven at 60°C to dry it, and then powder it. The second step: 3g beet powder, 2g sulfur powder and 2g L-cysteine, 24mL ethylenediamine and 36mL deionized water are placed in a 100mL high-pressure reactor and reacted at 200°C for 5 hours to synthesize SN-CNPs. The liquid product is collected by filtration and centrifuged. The yellow filtrate was collected as the original product, and its concentration was about 50 mg / mL. The artificial dephosphorylase prepared by the above method is volatile, which will cause the loss of sulfhydryl groups, which are the center of the activity of the artificial dephosphorylase. This will cause the activity of the artificial dephosphorylase to disappear within three weeks, and the antibacterial activity will be greatly reduced accordingly. Summary of the invention

[0003] In view of this, the object of the present invention is to provide a method for preparing an artificial dephosphorylation enzyme. The artificial dephosphorylation enzyme obtained by the preparation method provided by the present invention has stable activity and stable antibacterial properties.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for preparing an artificial dephosphorylation enzyme, comprising the following steps:

[0006] The biomass raw material, the diamine compound and water are mixed and subjected to a hydrothermal reaction to obtain a nano stock solution;

[0007] The nano stock solution and cysteine ​​are mixed and diluted to obtain the dephosphorylation artificial enzyme.

[0008] Preferably, the biomass raw material includes one or more of fruits, vegetables and polysaccharides;

[0009] The fruit comprises one or more of apple, sugar cane, grape and pear, and the fruit is fresh fruit or rotten fruit;

[0010] Said vegetables include beetroot;

[0011] The polysaccharide comprises cellulose and / or chitosan;

[0012] The diamine compound includes ethylenediamine or o-phenylenediamine.

[0013] The diamine compound includes ethylenediamine or o-phenylenediamine.

[0014] Preferably, when the diamine compound is ethylenediamine, the solid-liquid ratio of the biomass raw material to ethylenediamine is 1 g:5 mL to 1 g:30 mL;

[0015] When the diamine compound is o-phenylenediamine, the material-liquid ratio of the biomass raw material to o-phenylenediamine is 1 g:1.25 mL to 1 g:2 mL.

[0016] Preferably, the material-liquid ratio of the biomass raw material and water is 1g:12mL to 1g:60mL.

[0017] Preferably, the temperature of the hydrothermal reaction is 180-220° C., and the time is 4-5 hours.

[0018] Preferably, after the hydrothermal reaction, the method further comprises: cooling the obtained hydrothermal reaction liquid and filtering it, and the obtained filtrate is the nano stock solution.

[0019] Preferably, the mass ratio of the biomass raw material to cysteine ​​is 1:1 to 1:7.5.

[0020] Preferably, the nano stock solution and cysteine ​​are mixed under stirring conditions, the stirring temperature is room temperature, the pressure is normal pressure, and the time is 5 to 12 hours.

[0021] Preferably, the dilution multiple is 200 to 1000 times.

[0022] Preferably, the artificial dephosphorylation enzyme is used in the form of an artificial dephosphorylation enzyme dispersion, and the concentration of the artificial dephosphorylation enzyme dispersion is 0.25 mg / mL.

[0023] The invention provides a method for preparing an artificial dephosphorylation enzyme, comprising the following steps: mixing a biomass raw material, a diamine compound and water, and performing a hydrothermal reaction to obtain a nano stock solution; mixing the nano stock solution and cysteine, and diluting the mixture to obtain the artificial dephosphorylation enzyme.

[0024] The present invention first utilizes a hydrothermal reaction to react a biomass raw material with a diamine compound to form a biochar material having an amide group on the surface; then cysteine ​​is added; the carboxyl group and the amino group in the cysteine ​​interact with the amide group, the main functional group on the biochar material, through hydrogen bonds, so that the sulfhydryl group on the cysteine ​​is distributed on the surface of the nanoparticles to form more active sites. Moreover, because the two are bonded together through hydrogen bonds, the binding force between the cysteine ​​and the biochar material is stronger and not easy to fall off, so that the activity of the artificial dephosphorylation enzyme is stable and the antibacterial property is stable.

[0025] Furthermore, the biomass raw material includes one or more of fruits, vegetables and polysaccharides; the fruits include one or more of apples, sugar canes, grapes and pears, and the fruits are fresh fruits or rotten fruits; the vegetables include beetroots; the polysaccharides include cellulose and / or chitosan; and the diamine compounds include ethylenediamine or o-phenylenediamine. In particular, the use of rotten fruits as biomass raw materials can further reduce the preparation cost of the artificial dephosphorylation enzyme.

[0026] Further, the nano stock solution and cysteine ​​are mixed under stirring, and the stirring temperature is preferably room temperature, the pressure is preferably normal pressure, and the time is preferably 5 to 12 hours. After adding cysteine, the present invention controls the stirring temperature to be room temperature and the pressure to be normal pressure. Compared with the prior art, the chemical properties of cysteine ​​are unstable, and it is oxidized by oxygen in the air to cysteine ​​dimers (cystine, the dimerization reaction is accelerated under heating conditions) in neutral or weakly alkaline solutions, resulting in a decrease in the utilization rate of cysteine; the method of the present invention does not cause a large amount of cysteine ​​to be oxidized to produce cystine. Therefore, the method provided by the present invention has a high utilization rate of cysteine ​​and reduces costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a scanning electron microscope photograph of the biochar material in the nano-solution obtained in Example 1. DETAILED DESCRIPTION

[0028] The present invention provides a method for preparing an artificial dephosphorylation enzyme, comprising the following steps:

[0029] The biomass raw material, the diamine compound and water are mixed and subjected to a hydrothermal reaction to obtain a nano stock solution;

[0030] The nano stock solution and cysteine ​​are mixed and diluted to obtain the dephosphorylation artificial enzyme.

[0031] In the present invention, unless otherwise specified, the raw materials used in the present invention are preferably commercially available products.

[0032] The invention mixes biomass raw materials, diamine compounds and water, and performs hydrothermal reaction to obtain nano stock solution.

[0033] In the present invention, the biomass raw material preferably includes one or more of fruits, vegetables and polysaccharides, and more preferably vegetables or polysaccharides. In the present invention, the fruit preferably includes one or more of apples, sugar cane, grapes and pears. In the present invention, the fruit is fresh fruit or rotten fruit. In the present invention, the vegetable preferably includes beetroot. In the present invention, the polysaccharide preferably includes cellulose and / or chitosan, and more preferably chitosan. In the present invention, when rotten fruit is used as the biomass raw material, after the hydrothermal reaction, the bacteria in the rotten fruit have been eliminated, and the remaining sugar can form a biochar material with an amide group like the diamine compound under hydrothermal conditions; moreover, using rotten fruit as the biomass raw material can turn waste into treasure and greatly reduce production costs.

[0034] In the present invention, the diamine compound preferably includes ethylenediamine or o-phenylenediamine, and more preferably ethylenediamine.

[0035] In the present invention, the water is preferably deionized water.

[0036] In the present invention, when the diamine compound is preferably ethylenediamine, the solid-liquid ratio of the biomass raw material to ethylenediamine is preferably 1g:5mL to 1g:30mL, specifically 1g:5mL, 1g:6mL, 1g:7mL, 1g:8mL, 1g:10mL, 1g:15mL, 1g:20mL, 1g:25mL or 1g:30mL. In the present invention, when the diamine compound is preferably o-phenylenediamine, the solid-liquid ratio of the biomass raw material to o-phenylenediamine is preferably 1g:1.25mL to 1g:2mL, specifically 1g:1.25mL, 1g:1.5mL, 1g:1.75mL or 1g:2mL.

[0037] In the present invention, the solid-liquid ratio of the biomass raw material and water is preferably 1g:12mL to 1g:60mL, specifically 1g:12mL, 1g:15mL, 1g:18mL, 1g:20mL, 1g:30mL, 1g:40mL, 1g:50mL or 1g:60mL.

[0038] In the present invention, the temperature of the hydrothermal reaction is preferably 180-220°C, more preferably 190-210°C, specifically 180°C, 190°C, 200°C, 210°C or 220°C; the time is preferably 4-5h, specifically 4h or 5h.

[0039] In the present invention, the hydrothermal reaction is preferably carried out in a high-pressure reactor. During the hydrothermal reaction, the high-pressure reactor is preferably in a closed state.

[0040] After the hydrothermal reaction, the present invention preferably further comprises: cooling the obtained hydrothermal reaction liquid and filtering it, and the obtained filtrate is the nano stock solution.

[0041] In the present invention, the nano stock solution includes biochar material, and the particle size of the biochar material is preferably 130 to 160 nm.

[0042] After obtaining the nano stock solution, the present invention mixes the nano stock solution with cysteine ​​and dilutes the mixture to obtain the dephosphorylation artificial enzyme.

[0043] In the present invention, the cysteine ​​is preferably L-cysteine. In the present invention, the mass ratio of the biomass raw material to cysteine ​​is preferably 1:1 to 1:7.5, specifically 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7 or 1:7.5.

[0044] In the present invention, the mixing of the nano stock solution and cysteine ​​is preferably carried out under stirring conditions, the stirring temperature is preferably room temperature, the pressure is preferably normal pressure, and the time is preferably 5 to 12 hours, specifically 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours.

[0045] In the present invention, the dilution multiple is preferably 200 to 1000 times.

[0046] In the present invention, the artificial dephosphorylation enzyme is used in the form of an artificial dephosphorylation enzyme dispersion liquid, and the concentration of the artificial dephosphorylation enzyme dispersion liquid is 0.25 mg / mL.

[0047] The preparation method of the artificial dephosphorylation enzyme provided by the present invention is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0048] Example 1

[0049] Step 1: Mix 1 g of beetroot and 6 mL of ethylenediamine in a high-pressure reactor, then add 12 mL of water, perform hydrothermal reaction at 200° C. for 5 h, filter, and obtain a nano stock solution.

[0050] Step 2: Add 1.5 g of cysteine ​​to the nano stock solution, stir for 5 hours at room temperature and pressure, and then dilute 200 times to obtain a dephosphorylation artificial enzyme dispersion.

[0051] Figure 1 The atomic force microscope photo of the biochar material in the obtained nano-solution. Figure 1It can be seen that the average size of biochar materials is distributed between 150 and 160 nm.

[0052] Example 2

[0053] Step 1: Mix 0.2 g of chitosan and 6 mL of ethylenediamine in a high-pressure reactor, then add 12 mL of water, perform hydrothermal reaction at 200° C. for 5 h, filter, and obtain a nano stock solution.

[0054] Step 2: Add 1.5 g of cysteine ​​to the nano stock solution, stir for 5 hours at room temperature and pressure, and then dilute 200 times to obtain a dephosphorylation artificial enzyme dispersion.

[0055] Comparative Example 1

[0056] Mix beetroot, sulfur, ethylenediamine and cysteine ​​in a mass ratio of 3g:2g:24mL:2g, stir at 60°C for 2h, add 36mL of water, react at 200°C for 5h, take the liquid phase and dilute it 200 times to obtain the product.

[0057] The antibacterial data and the stability of the antibacterial activity of the artificial dephosphorylation enzymes obtained in Examples 1 to 2 and Comparative Example 1 are shown in Table 1.

[0058] Table 1 Antibacterial data of dephosphorylation artificial enzyme and stability of antibacterial activity

[0059]

[0060] It can be seen from Table 1 that the artificial dephosphorylation enzyme provided by the present invention increases the antibacterial stability and has an inhibitory effect on more bacteria, especially in Example 2, where the amount of chitosan used is only one-fifth of the amount of beetroot used in Example 1, and the antibacterial property and antibacterial stability of the obtained artificial dephosphorylation enzyme are better than those in Comparative Example 1.

[0061] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing an artificial dephosphorylation enzyme, characterized in that: The following steps are involved: The biomass raw material, the diamine compound and water are mixed and subjected to a hydrothermal reaction to obtain a nano stock solution; The nano stock solution and cysteine ​​are mixed and diluted to obtain the dephosphorylation artificial enzyme.

2. The preparation method according to claim 1, characterized in that: The biomass raw materials include one or more of fruits, vegetables and polysaccharides; The fruit comprises one or more of apple, sugar cane, grape and pear, and the fruit is fresh fruit or rotten fruit; Said vegetables include beetroot; The polysaccharide comprises cellulose and / or chitosan; The diamine compound includes ethylenediamine or o-phenylenediamine.

3. The preparation method according to claim 2, characterized in that: When the diamine compound is ethylenediamine, the material-liquid ratio of the biomass raw material to ethylenediamine is 1g:5mL to 1g:30mL; When the diamine compound is o-phenylenediamine, the material-liquid ratio of the biomass raw material to o-phenylenediamine is 1 g:1.25 mL to 1 g:2 mL.

4. The preparation method according to claim 1, characterized in that: The material-liquid ratio of the biomass raw material and water is 1g:12mL to 1g:60mL.

5. The preparation method according to claim 1, 2, 3 or 4, characterized in that: The temperature of the hydrothermal reaction is 180-220° C. and the time is 4-5 hours.

6. The preparation method according to claim 1, characterized in that: After the hydrothermal reaction, the method further comprises: cooling the obtained hydrothermal reaction liquid and filtering the obtained filtrate, and the obtained filtrate is the nano stock solution.

7. The preparation method according to claim 1, characterized in that: The mass ratio of the biomass raw material to cysteine ​​is 1:1 to 1:7.

5.

8. The preparation method according to claim 1 or 7, characterized in that: The nano stock solution and cysteine ​​are mixed under stirring conditions, the stirring temperature is room temperature, the pressure is normal pressure, and the time is 5 to 12 hours.

9. The preparation method according to claim 1, characterized in that: The dilution multiple is 200 to 1000 times.

10. The preparation method according to claim 1, characterized in that: The artificial dephosphorylation enzyme is used in the form of an artificial dephosphorylation enzyme dispersion liquid, and the concentration of the artificial dephosphorylation enzyme dispersion liquid is 0.25 mg / mL.

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