Application of Cuprous Ferrite as Artificial Protease

Abstract

The present invention discloses the use of cuprous ferrite as an artificial protease, which is prepared by a simple hydrothermal method. The artificial protease of the present invention not only has a simple synthesis method, but also can efficiently hydrolyze protein with a fast hydrolysis speed and a wide pH range.

Description

Technical Field

[0001] The invention belongs to the technical field of artificial proteases, and particularly relates to the application of cuprous ferrite as an artificial protease. Background Art

[0002] The primary protease used in proteomics research is trypsin, but this natural enzyme is limited in its ability to hydrolyze proteins. For example, its activity decreases or even inactivates at room temperature or low pH, making it unsuitable for practical applications. Currently, there are reports of using artificial proteases, such as high-valent metal-organic frameworks or metal oxygen clusters, to replace natural enzymes for protein hydrolysis. However, these studies still suffer from issues such as slow hydrolysis rates and high reaction temperatures. Summary of the Invention

[0003] In order to expand the types of proteases used in proteomics research, the present invention utilizes the synergistic effect of two metal ions in bimetallic oxides to provide a new type of artificial protease for hydrolyzing proteins. This artificial protease can not only hydrolyze proteins efficiently, but also maintain high activity at room temperature and low pH.

[0004] The artificial protease of the present invention is a submicron cuprous ferrite material, and its preparation method comprises the following steps:

[0005] (1) dissolving ferrous chloride and copper nitrate in deionized water respectively, and then mixing them uniformly by stirring at room temperature to obtain a mixed solution;

[0006] (2) adding the sodium hydroxide solution dropwise to the mixed solution obtained in step (1) under stirring, and continuing to stir evenly to obtain a reaction solution;

[0007] (3) The reaction solution obtained in step (2) is transferred to a reactor for hydrothermal reaction. The obtained product is washed and dried to obtain a submicron CuFeO2 material as an artificial protease.

[0008] Preferably, the usage ratio of ferrous chloride, copper nitrate and sodium hydroxide is 7.5 mmol:7.5 mmol:55-80 mmol.

[0009] Preferably, the stirring speed in step (1) and step (2) is 400-700 rpm.

[0010] Preferably, the temperature of the hydrothermal reaction in step (3) is 100-120° C. and the reaction time is 24 h.

[0011] Preferably, the washing in step (3) is performed by thoroughly washing with deionized water to remove the remaining sodium hydroxide and sodium chloride.

[0012] Compared with the prior art, the beneficial effects of the present invention are embodied in:

[0013] 1. The present invention uses submicron-sized CuFeO2 bimetallic oxide to specifically hydrolyze proteins, utilizing the synergistic effect of two metals, one for fixing the peptide bonds of proteins and the other for nucleophilic attack, thereby achieving efficient protein hydrolysis.

[0014] 2. The artificial protease of the present invention has high hydrolysis efficiency and is applicable to a wider pH range than trypsin.

[0015] 3. The protein hydrolysis reaction of the artificial protease of the present invention is a heterogeneous reaction, the catalyst is easy to separate and recover, and the environmental pollution is small.

[0016] 4. The method for synthesizing the artificial protease of the present invention is convenient, has simple steps, and uses cheap raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a SEM image of the artificial protease obtained in Example 1 of the present invention.

[0018] Figure 2 This is an SDS-PAGE image of bovine serum albumin hydrolyzed by the artificial protease obtained in Example 1 of the present invention.

[0019] Figure 3 This is an SDS-PAGE image of the artificial protease obtained in Example 1 of the present invention hydrolyzing bovine serum albumin under different pH conditions. DETAILED DESCRIPTION

[0020] The present invention will be further described in detail below with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.

[0021] Example 1

[0022] This embodiment synthesizes submicron CuFeO2 particles according to the following steps:

[0023] (1) Dissolve 1.4911 g (7.5 mmol) of FeCl2·4H2O in 20 mL of deionized water and 1.812 g (7.5 mmol) of Cu(NO3)2·3H2O in 20 mL of deionized water. Mix the two solutions and stir at 400 rpm for 1 min at room temperature to obtain a mixed solution.

[0024] (2) Weigh 2.2 g (55 mmol) of sodium hydroxide and dissolve it in 20 mL of deionized water. Add the sodium hydroxide solution dropwise to the mixed solution over 2 minutes, and stir at 400 rpm for 10 minutes at room temperature to obtain a reddish-brown reaction solution.

[0025] (3) The reaction solution was transferred into a 50 mL hydrothermal reactor and subjected to hydrothermal reaction at 100 °C for 24 h. The mixture was then filtered, washed thoroughly with deionized water, and vacuum-dried at room temperature for 12 h to obtain brown submicron artificial protease particles.

[0026] Example 2

[0027] Protein hydrolysis experiment: 200mM, pH=5 phosphate buffer was used to dissolve bovine serum albumin to obtain 5mL of a 5mg / mL solution. 0.1g of the material obtained in Example 1 was dispersed in the solution. After reacting at room temperature for 30min, the supernatant was centrifuged and the hydrolysis results were observed by SDS-PAGE gel electrophoresis. Figure 2 As shown. Figure 2 It can be seen that in the presence of the artificial protease obtained in Example 1, bovine serum albumin has obvious hydrolysis bands, while bovine serum albumin without the protease is not hydrolyzed at the same time, proving that the CuFeO2 particles have excellent protein hydrolysis ability.

[0028] Example 3

[0029] Protein hydrolysis experiment at different pH: bovine serum albumin was dissolved in 200 mM phosphate buffer at pH 5, 6, 7, and 8 to obtain 5 mL of 5 mg / mL solution at different pH values. Five portions of 0.1 g of the material obtained in Example 1 were dispersed in each of the five solutions. The mixture was reacted at room temperature for 30 min, then centrifuged and the supernatant was removed. The hydrolysis results were observed by SDS-PAGE gel electrophoresis. Figure 3 As shown. Figure 3 It can be seen that the protease can hydrolyze protein in the pH range of 5-8, and the pH range of hydrolysis is relatively wide.

[0030] In summary, the submicron CuFeO2 particles prepared by the present invention through a simple hydrothermal synthesis method can efficiently hydrolyze bovine serum albumin.

Claims

1. Application of submicron cuprous ferrite as an artificial protease to hydrolyze bovine serum albumin, the preparation method of the submicron cuprous ferrite comprising the following steps: (1) Dissolving ferrous chloride and copper nitrate in deionized water respectively, and then mixing them uniformly by stirring at room temperature to obtain a mixed solution; (2) adding the sodium hydroxide solution dropwise to the mixed solution obtained in step (1) under stirring, and continuing to stir evenly to obtain a reaction solution; (3) The reaction solution obtained in step (2) is transferred to a reactor for hydrothermal reaction. The obtained product is washed and dried to obtain a submicron CuFeO2 material as an artificial protease.

2. The use according to claim 1, wherein: The dosage ratio of ferrous chloride, copper nitrate and sodium hydroxide is 7.5 mmol:7.5 mmol:55~80 mmol.

3. The use according to claim 1, wherein: The temperature of the hydrothermal reaction in step (3) is 100-120°C and the reaction time is 24 h.

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