Application of lanthanum cobaltate nanoparticles as artificial nanoproteases

Abstract

The invention discloses the application of lanthanum cobaltate nanoparticles as artificial nanoprotease, which can be used to hydrolyze protein. The artificial nanoprotease of the invention has strong hydrolysis ability and fast hydrolysis rate, and the preparation method is simple to operate, highly repeatable and low in cost.

Description

Technical Field

[0001] The invention relates to the field of artificial nanoproteases, and in particular to the application of lanthanum cobaltate nanoparticles as artificial nanoproteases. Background Art

[0002] Proteins have excellent stability under physiological conditions due to their stable peptide bonds. The selective cleavage of amide bonds is of great significance for the determination of protein structure in proteomics and biotechnology. Trypsin is a proteolytic enzyme that is often used to selectively hydrolyze peptide bonds in proteins with a high reaction rate. However, trypsin is very sensitive to pH conditions and temperature, the length of the hydrolyzed peptide segment is short, the cost is high, and it is unstable. Therefore, it is necessary to develop artificial proteases.

[0003] Among the existing artificial proteases, nanoproteases have the advantages of good stability, good biosafety, low price, and large-scale preparation. IV 、Zr IV and Hf IV Substituted polyoxometalates (POMs), metal oxygen clusters (MOCs) and metal-organic frameworks (MOFs) mostly have problems of high reaction temperature, long reaction time or complex preparation. Summary of the invention

[0004] Based on the problems existing in the above-mentioned prior art, the main purpose of the present invention is to propose an application of lanthanum cobaltate nanoparticles as artificial nanoprotease, which has a strong hydrolysis effect and can be used for rapid hydrolysis of proteins such as bovine serum albumin at room temperature. The hydrolysis is selective hydrolysis, that is, it does not randomly hydrolyze any position of the protein, but only hydrolyzes specific positions.

[0005] The method for preparing lanthanum cobaltate nanoparticles of the present invention comprises the following steps:

[0006] (1) fully dissolving cobalt nitrate hexahydrate, lanthanum nitrate hexahydrate and citric acid monohydrate in deionized water and ethanol, stirring and ultrasonicating to obtain a mixed solution;

[0007] (2) placing the mixed solution in an oven to dry and grind to obtain a solid powder;

[0008] (3) calcining the solid powder to obtain lanthanum cobaltate nanoparticles used as artificial nanoprotease.

[0009] Preferably, in step (1), the usage ratio of cobalt nitrate hexahydrate, lanthanum nitrate hexahydrate, citric acid monohydrate, deionized water and ethanol is 0.73 g: 1.08 g: 1.05 g: 5.0 mL: 10.0 mL.

[0010] Preferably, in step (1), the ultrasonication time is 10 minutes.

[0011] Preferably, in step (2), the drying is performed at 90° C. for 24 hours.

[0012] Preferably, in step (3), the calcination temperature is 600° C., the calcination time is 5 hours, and the heating rate is 2° C. / min.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] 1. The present invention uses LaCoO3 nanoprotease as an artificial nanoprotease, which can hydrolyze proteins such as bovine serum albumin, with low reaction temperature, short reaction time, strong hydrolysis ability and fast hydrolysis rate.

[0015] 2. The present invention uses cobalt nitrate hexahydrate and lanthanum nitrate hexahydrate as raw materials to prepare LaCoO3, and the preparation method is simple to operate, highly repeatable and low in cost.

[0016] 3. The reaction between the artificial nanoprotease of the present invention and the protein is a heterogeneous reaction, which is conducive to the recovery and reuse of the artificial nanoprotease and will not cause pollution to the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the X-ray diffraction pattern of the LaCoO3 nanoparticles synthesized in Example 1 of the present invention.

[0018] Figure 2 This is a scanning electron microscope image of the LaCoO3 nanoparticles synthesized in Example 1 of the present invention.

[0019] Figure 3 This is the sodium dodecyl sulfate-polyacrylamide gel electrophoresis diagram of the LaCoO3 nanoparticles synthesized in Example 1 of the present invention. DETAILED DESCRIPTION

[0020] The present invention provides an application of lanthanum cobaltate nanoparticles as artificial nanoprotease. In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further elaborated in detail below in conjunction with examples and drawings, but the embodiments of the present invention are not limited thereto.

[0021] Example 1

[0022] This example synthesizes LaCoO3 nanoparticles according to the following steps:

[0023] (1) Weigh 0.73 g of cobalt nitrate hexahydrate, 1.08 g of lanthanum nitrate hexahydrate, and 1.05 g of citric acid monohydrate, respectively, and fully dissolve them in 5.0 mL of deionized water and 10.0 mL of ethanol, and stir and ultrasonicate for 10 minutes to obtain a mixed solution.

[0024] (2) The mixed solution was placed in an oven at 90° C. and dried for 24 hours, and then ground to obtain a solid powder.

[0025] (3) The solid powder was calcined at 600°C for 5 hours with a heating rate of 2°C per minute to obtain LaCoO3 nanoparticles.

[0026] Figure 1 is the X-ray diffraction (XRD) pattern of LaCoO3 nanoparticles. The XRD spectrum of LaCoO3 has obvious characteristic peaks at 23.2°, 32.9°, 33.3°, 40.6°, 47.5° and 58.9°, which are (012), (110), (104), (202), (024) and (214) crystal planes, respectively, which are consistent with the LaCoO3 standard card (JCPDS48-0123), indicating that high-purity LaCoO3 has been synthesized.

[0027] Figure 2 This is a scanning electron microscope (SEM) image of LaCoO3 nanoparticles. It can be seen that the synthesized nanoparticles are ellipsoidal in shape, with a smooth surface, uniform size, and are aggregated together. Most of the sizes are 50-100nm.

[0028] Example 2

[0029] LaCoO3 hydrolysis of bovine serum albumin (BSA): To study the hydrolysis of BSA, LaCoO3 powder (0.10 g) was placed in a centrifuge tube, 3.0 mL of buffer solution (the buffer solution consisted of a phosphate buffer solution with a pH of 5.0 and a concentration of 200 mM) was added, and 1.0 mL of a phosphate buffer solution containing 10.0 mg / mL BSA was added at room temperature, mixed thoroughly, and reacted on a shaker at room temperature for 30 minutes. Subsequently, the precipitate was left by centrifugation, and the supernatant was poured off to remove the BSA that was not adsorbed on the surface of the nanoparticles. A few drops of 1.0% SDS aqueous solution were added, and the nanoparticles were completely immersed in ultrasound. The role of the SDS aqueous solution is to denature BSA and its peptide fragments, thereby separating the particles from BSA and its peptide fragments. After one hour, the mixture was centrifuged to obtain the supernatant, which was mixed with sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) sample buffer and heated at 95°C for 5 minutes. Finally, the sample was loaded into a stacking gel for electrophoresis. The room temperature was about 20-25°C.

[0030] Figure 3 The SDS-PAGE diagram of LaCoO3 is shown in Figure 1. The first lane is the hydrolysis control sample of pure BSA without adding LaCoO3, and the second lane is the BSA and its peptide fragments hydrolyzed by LaCoO3. Figure 3As shown in the figure, under the same temperature and pH conditions, pure BSA is hardly hydrolyzed, while BSA with the addition of LaCoO3 nanoparticles is hydrolyzed into 7-8 peptide fragments, reflecting the excellent protease activity of LaCoO3.

[0031] In summary, the present invention can prepare artificial nanoprotease LaCoO3 in large quantities through a simple synthesis method, which can efficiently hydrolyze proteins such as BSA.

[0032] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of lanthanum cobaltate nanoparticles as artificial nanoprotease, characterized in that: Used for hydrolysis of bovine serum albumin.

2. The use according to claim 1, characterized in that: The method for preparing the lanthanum cobaltate nanoparticles comprises the following steps: (1) fully dissolving cobalt nitrate hexahydrate, lanthanum nitrate hexahydrate and citric acid monohydrate in deionized water and ethanol, stirring and ultrasonicating to obtain a mixed solution; (2) placing the mixed solution in an oven for drying and grinding to obtain a solid powder; (3) calcining the solid powder to obtain lanthanum cobaltate nanoparticles for use as artificial nanoprotease.

3. The use according to claim 2, characterized in that: In step (1), the usage ratio of cobalt nitrate hexahydrate, lanthanum nitrate hexahydrate, citric acid monohydrate, deionized water and ethanol is 0.73 g: 1.08 g: 1.05 g: 5.0 mL: 10.0 mL.

4. The use according to claim 2, characterized in that: In step (1), the ultrasonication time is 10 minutes.

5. The use according to claim 2, characterized in that: In step (2), the drying is performed at 90° C. for 24 hours.

6. The use according to claim 2, characterized in that: In step (3), the calcination temperature is 600°C, the calcination time is 5 hours, and the heating rate is 2°C / min.

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