阅读说明：本技术 一种纳米酶溶液及粉末的制备方法 (Preparation method of nano enzyme solution and nano enzyme powder ) 是由 张杨 张丹丹 陈晴 王蒙蒙 任群翔 于 2021-09-26 设计创作，主要内容包括：本发明公开了一种纳米酶溶液及粉末的制备方法,纳米酶溶液的制备方法包括：配制含有二价锰离子的金属源溶液；按照二价锰离子和转铁蛋白的质量比为(0.12-0.65):1,将转铁蛋白加入金属源溶液中,在第一温度下搅拌第一时间,得到混合溶液；调节混合溶液的pH至10-12后,在第二温度下搅拌第二时间,即得到纳米酶溶液。本公开通过转铁蛋白介导的生物矿化过程制备得到氧化锰纳米酶,制备方法简单易操作,所得纳米酶结构形态可控,且具有优良的多酶活性,包括过氧化物酶活性、抗坏血酸氧化酶活性、过氧化氢酶活性和超氧化物歧化酶活性,在生物传感、有害物质去除等方面具有一定的应用前景,可为纳米酶的进一步产业化提供支撑。(The invention discloses a preparation method of nano enzyme solution and powder, wherein the preparation method of the nano enzyme solution comprises the following steps: preparing a metal source solution containing divalent manganese ions; adding transferrin into a metal source solution according to the mass ratio of divalent manganese ions to transferrin (0.12-0.65):1, and stirring at a first temperature for a first time to obtain a mixed solution; and (3) adjusting the pH value of the mixed solution to 10-12, and stirring the mixed solution at a second temperature for a second time to obtain the nano enzyme solution. The manganese oxide nanoenzyme is prepared through a transferrin-mediated biomineralization process, the preparation method is simple and easy to operate, the structural form of the obtained nanoenzyme is controllable, and the nanoenzyme has excellent multienzyme activity including peroxidase activity, ascorbic acid oxidase activity, catalase activity and superoxide dismutase activity, has a certain application prospect in the aspects of biosensing, harmful substance removal and the like, and can provide support for further industrialization of the nanoenzyme.)

1. A preparation method of a nano enzyme solution is characterized by comprising the following steps:

step (1): preparing a metal source solution containing divalent manganese ions;

step (2): adding transferrin into a metal source solution according to the mass ratio of divalent manganese ions to transferrin (0.12-0.65):1, and stirring at a first temperature for a first time to obtain a mixed solution;

and (3): and (3) adjusting the pH value of the mixed solution to 10-12, and stirring the mixed solution at a second temperature for a second time to obtain the nano enzyme solution.

2. The method for preparing nanoenzyme solution according to claim 1, wherein the step (1) is specifically as follows:

dissolving manganese dichloride in pure water to obtain a metal source solution containing divalent manganese ions.

3. The method for preparing nanoenzyme solution according to claim 1, wherein the concentration of the divalent manganese ion in the metal source solution in the step (1) is 6.00 x 10^-3-6.50×10^-3mol/L。

4. The method for preparing nanoenzyme solution according to claim 3, wherein the concentration of the divalent manganese ion in the metal source solution in the step (1) is 6.35X 10^-3mol/L。

5. The method for preparing nanoenzyme solution according to claim 1, wherein the first time in the step (2) is shorter than the second time in the step (3);

the first temperature in the step (2) is equal to the second temperature in the step (3).

6. The method for preparing nanoenzyme solution according to claim 5, wherein the first temperature is 20-30 ℃ and the first time is 20-40 min;

the second temperature is 20-30 ℃, and the second time is 2-4 h.

7. The method for preparing nanoenzyme solution according to claim 1, wherein the step (2) is as follows:

adding the transferrin into the metal source solution according to the mass ratio of the divalent manganese ions to the transferrin of 0.63:1, and stirring for 30min at 20-30 ℃ to obtain a mixed solution.

8. The method for preparing nanoenzyme solution according to claim 1, wherein the step (3) is specifically as follows:

and (3) adjusting the pH value of the mixed solution to 11, and stirring for 2 hours at the temperature of 20-30 ℃ to obtain the nano enzyme solution.

9. A preparation method of nano enzyme powder is characterized by comprising the following steps:

step (1): dialyzing the nanoenzyme solution prepared according to any one of claims 1 to 8, and centrifuging to collect the product;

step (2): and (5) freeze-drying the product to obtain nano enzyme powder.

10. The method for preparing nanoenzyme powder according to claim 9, wherein the dialysis time in the step (1) is 48 to 72 hours.

Technical Field

The invention relates to the field of nano enzyme preparation, and more particularly relates to a preparation method of nano enzyme solution and nano enzyme powder.

Background

The nano enzyme is a nano material with enzymatic characteristics, can catalyze the substrate of the enzyme and the mediated biochemical reaction thereof under physiological conditions to generate catalytic reaction similar to natural enzyme, has the characteristics of enzymatic reaction kinetics and the like, and belongs to a novel artificial mimic enzyme.

The nano-enzyme has the characteristics of high catalytic efficiency, high stability, multiple functions, low cost, large-scale production and the like, and has become a research hotspot crossing multiple disciplines since the first report in 2007, and the application research of the nano-enzyme relates to multiple fields of biomedicine, environmental protection, agriculture, safety detection and the like.

Most of the existing nano enzymes only show single or double enzyme activities, and the application prospect of the nano enzymes is greatly limited.

Therefore, how to provide a nanoenzyme with multiple enzyme activities is a technical problem to be solved urgently in the field.

Disclosure of Invention

The invention aims to provide a novel technical scheme of a preparation method of nano-enzyme with various enzyme activities.

According to a first aspect of the present invention, a method for preparing a nanoenzyme solution is provided.

The preparation method of the nano enzyme solution comprises the following steps:

step (1): preparing a metal source solution containing divalent manganese ions;

step (2): adding transferrin into a metal source solution according to the mass ratio of divalent manganese ions to transferrin (0.12-0.65):1, and stirring at a first temperature for a first time to obtain a mixed solution;

and (3): and (3) adjusting the pH value of the mixed solution to 10-12, and stirring the mixed solution at a second temperature for a second time to obtain the nano enzyme solution.

Optionally, the step (1) is specifically as follows:

dissolving manganese dichloride in pure water to obtain a metal source solution containing divalent manganese ions.

Optionally, the concentration of the divalent manganese ions in the metal source solution in the step (1) is 6.00 × 10^-3-6.50×10^-3mol/L。

Optionally, the concentration of the divalent manganese ions in the metal source solution in the step (1) is 6.35 × 10^-3mol/L。

Optionally, the first time in the step (2) is less than the second time in the step (3);

the first temperature in the step (2) is equal to the second temperature in the step (3).

Optionally, the first temperature is 20-30 ℃, and the first time is 20-40 min;

the second temperature is 20-30 ℃, and the second time is 2-4 h.

Optionally, the step (2) is specifically as follows:

adding the transferrin into the metal source solution according to the mass ratio of the divalent manganese ions to the transferrin of 0.63:1, and stirring for 30min at 20-30 ℃ to obtain a mixed solution.

Optionally, the step (3) is specifically as follows:

and (3) adjusting the pH value of the mixed solution to 11, and stirring for 2 hours at the temperature of 20-30 ℃ to obtain the nano enzyme solution.

According to a second aspect of the present invention, there is provided a method for preparing a nanoenzyme powder.

The preparation method of the nano enzyme powder comprises the following steps:

step (1): dialyzing the nano enzyme solution prepared by the method, and centrifuging to collect a product;

step (2): and (5) freeze-drying the product to obtain nano enzyme powder.

Optionally, the dialysis time in step (1) is 48-72 h.

The manganese oxide nanoenzyme is prepared through a transferrin-mediated biomineralization process, the preparation method is simple and easy to operate, the structural form of the obtained nanoenzyme is controllable, and the nanoenzyme has excellent multienzyme activity including peroxidase activity, ascorbic acid oxidase activity, catalase activity and superoxide dismutase activity, has a certain application prospect in the aspects of biosensing, harmful substance removal and the like, and can provide support for further industrialization of the nanoenzyme.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description, serve to explain the principles of the invention.

FIG. 1 shows Trf-Mn prepared in example 1₃O₄TEM photograph of (a).

FIG. 2 shows Trf-Mn prepared in example 2₃O₄TEM photograph of (a).

FIG. 3 shows Trf-Mn prepared in example 3₃O₄TEM photograph of (a).

FIG. 4 shows Trf-Mn prepared in example 3₃O₄Size distribution map of (a).

FIG. 5 shows Trf-Mn prepared in example 3₃O₄XRD pattern of nanocubes.

FIG. 6 shows Trf-Mn₃O₄POD activity of nanoenzyme.

FIG. 7 shows Trf-Mn₃O₄AAO activity of the nanoenzyme.

FIG. 8 shows Trf-Mn₃O₄CAT and SOD activity of the nano enzyme.

Detailed Description

Various exemplary embodiments of the present invention will now be described in detail. It should be noted that: the relative arrangement of the components and steps, the numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present invention unless specifically stated otherwise.

The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses.

Techniques, methods, and apparatus known to those of ordinary skill in the relevant art may not be discussed in detail but are intended to be part of the specification where appropriate.

In all examples shown and discussed herein, any particular value should be construed as merely illustrative, and not limiting. Thus, other examples of the exemplary embodiments may have different values.

The preparation method of the nano enzyme solution comprises the following steps:

step (1): preparing a metal source solution containing divalent manganese ions.

The step (1) may be specifically as follows:

dissolving manganese dichloride in pure water to obtain a metal source solution containing divalent manganese ions.

The concentration of the divalent manganese ion in the metal source solution in the step (1) may be 6.00X 10^-3-6.50×10^-3mol/L。

Further, the concentration of the divalent manganese ion in the metal source solution in the step (1) is 6.35X 10^-3mol/L。

Step (2): adding transferrin into the metal source solution according to the mass ratio of the divalent manganese ions to the transferrin of (0.12-0.65):1, and stirring at a first temperature for a first time to obtain a mixed solution.

In order to obtain the nano enzyme more efficiently, the first time in the step (2) is less than the second time in the step (3);

the first temperature in step (2) is equal to the second temperature in step (3).

Further, the first temperature is 20-30 ℃, and the first time is 20-40 min;

the second temperature is 20-30 ℃, and the second time is 2-4 h.

In specific implementation, the step (2) may be specifically as follows:

adding the transferrin into the metal source solution according to the mass ratio of the divalent manganese ions to the transferrin of 0.63:1, and stirring for 30min at 20-30 ℃ to obtain a mixed solution.

And (3): and (3) adjusting the pH value of the mixed solution to 10-12, and stirring the mixed solution at a second temperature for a second time to obtain the nano enzyme solution.

When the method is specifically implemented, the step (3) can be specifically as follows:

and (3) adjusting the pH value of the mixed solution to 11, and stirring for 2 hours at the temperature of 20-30 ℃ to obtain the nano enzyme solution.

The present disclosure also provides a method for preparing nano enzyme powder, comprising the following steps:

step (1): and (3) dialyzing the nano enzyme solution prepared by the method, and centrifuging to collect a product.

The dialysis time in step (1) may be 48-72 h.

Step (2): and (5) freeze-drying the product to obtain nano enzyme powder.

The experimental procedures used in the examples below are conventional unless otherwise specified, the materials and reagents used therein are commercially available, and the equipment used in the experiments are well known to those skilled in the art without otherwise specified.

Example 1

Mixing MnCl₂Dissolving in pure water to give a solution with a concentration of 6.35X 10^-3mol/L; adding proper amount of Trf into the solution, and stirring at room temperature for 30min, wherein Mn is²⁺The mass ratio of/Trf is 0.126: 1; then, the pH value of the solution is adjusted to 11, and the solution is continuously stirred for 2 hours at room temperature to obtain brown manganese oxide nano enzyme solution. The obtained nano enzyme solution is dialyzed for 48 hours, and the product is collected by centrifugation and then is frozen and dried to obtain solid powder.

The nano-material was characterized by JEM-2100F Transmission Electron Microscope (TEM), as shown in FIG. 1, and the example obtained Trf-Mn₃O₄Is a random cross-linked network structure.

Example 2

Mixing MnCl₂Dissolving in pure water to give a solution with a concentration of 6.35X 10^-3mol/L; adding proper amount of Trf into the solution, and stirring at room temperature for 30min, wherein Mn is²⁺the/Trf mass ratio is 0.315: 1; the solution was then adjusted to pH 11 and continued at room temperatureStirring for 2h to obtain brown manganese oxide nano enzyme solution. The obtained nano enzyme solution is dialyzed for 48 hours, and the product is collected by centrifugation and then is frozen and dried to obtain solid powder.

The nanomaterial was characterized by JEM-2100F transmission electron microscope, as shown in FIG. 2, the Trf-Mn obtained in this example₃O₄The particle size is about 40 nm.

Example 3

Mixing MnCl₂Dissolving in pure water to give a solution with a concentration of 6.35X 10^-3mol/L; adding proper amount of Trf into the solution, and stirring at room temperature for 30min, wherein Mn is²⁺The mass ratio of/Trf is 0.63: 1; then, the pH value of the solution is adjusted to 11, and the solution is continuously stirred for 2 hours at room temperature to obtain brown manganese oxide nano enzyme solution. The obtained nano enzyme solution is dialyzed for 48 hours, and the product is collected by centrifugation and then is frozen and dried to obtain solid powder.

The nanomaterials were characterized by JEM-2100F transmission electron microscope, as shown in FIGS. 3 and 4, the example obtained Trf-Mn₃O₄Is 3D nanocubes with an average size of 10.1nm, D_[101]＝0.492nm。

The X-ray diffraction pattern (XRD) in FIG. 5 shows that the resulting Trf-Mn is₃O₄The nanocubes are in a highly crystalline state.

For Trf-Mn₃O₄The enzyme-like activity of (2) was evaluated, and as shown in FIGS. 6 to 8, the obtained Trf-Mn₃O₄The nanoenzyme has 4 kinds of enzyme activities including Peroxidase (POD) activity, ascorbate oxidase (AAO) activity, Catalase (CAT) activity and superoxide dismutase (SOD) activity.

Although some specific embodiments of the present invention have been described in detail by way of examples, it should be understood by those skilled in the art that the above examples are for illustrative purposes only and are not intended to limit the scope of the present invention. It will be appreciated by those skilled in the art that modifications may be made to the above embodiments without departing from the scope and spirit of the invention. The scope of the invention is defined by the appended claims.