Application of tin (II) oxide as an artificial metalloproteinase

Abstract

The invention discloses the application of stannous oxide as an artificial metalloprotease, which is prepared by a simple ultrasonic method. The artificial metalloprotease of the invention has a simple synthesis method, is easy to use, and has a fast hydrolysis reaction rate, high stability and good circulation performance.

Description

Technical Field

[0001] The present invention relates to the technical field of artificial proteases, and in particular to the application of stannous oxide as an artificial metalloprotease for hydrolyzing proteins. Background Art

[0002] Selective hydrolysis and cleavage of peptide bonds in proteins is often used for protein structure / function / folding analysis, protein engineering, and the design of targeted protein-cleaving drugs. However, the natural enzymes used are extremely unstable and have a limited range of applications. Currently, there are reports of using artificial metalloproteases such as metal-organic frameworks (MOFs), polyoxometalates (POMs), and metalloxoclusters (MOCs) to replace natural enzymes for protein hydrolysis. However, problems such as long reaction times and high temperatures still exist. Summary of the Invention

[0003] In order to solve the problems existing in current artificial metalloproteases, the present invention provides a new type of artificial metalloprotease. The present invention uses low-valent metal oxide stannous oxide for protein hydrolysis. The artificial protease can efficiently hydrolyze protein at 30°C, has high stability, and can be recycled.

[0004] The present invention also provides a method for preparing a SnO material as an artificial metalloprotease, comprising the following steps:

[0005] (1) adding stannous chloride dihydrate and sodium hydroxide into deionized water respectively and dissolving them under ultrasonication at room temperature, and then mixing them uniformly to obtain a reaction solution;

[0006] (2) transferring the reaction solution into an ultrasonic cell disruptor and sonicating to obtain a mixed solution;

[0007] (3) The mixed solution is centrifuged, washed and dried to obtain the SnO material as an artificial metalloprotease.

[0008] Preferably, in step (1), in the reaction solution, the usage ratio of stannous chloride dihydrate, sodium hydroxide and deionized water is 0.65 g:0.34 g:30 mL.

[0009] Preferably, in step (2), the ultrasonic frequency is 400W and the reaction time is 10 to 20 minutes.

[0010] Preferably, the particles obtained in step (3) are washed by centrifugation with deionized water.

[0011] Compared with the prior art, the beneficial effects of the present invention are embodied in:

[0012] 1. The present invention uses simple low-valent metal oxide SnO to hydrolyze bovine serum albumin. The hydrolysis reaction rate is fast and can be carried out at 30°C, with good hydrolysis effect.

[0013] 2. The artificial metalloprotease of the present invention undergoes heterogeneous catalytic hydrolysis, and the separation method is simple and the protein can be reused.

[0014] 3. The artificial metalloprotease of the present invention is prepared by ultrasonic method, the raw materials are simple and cheap, the preparation method and subsequent treatment are simple, and the cost is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the SEM image of the artificial metalloprotease obtained in Example 1 of the present invention.

[0016] Figure 2 This is an SDS-PAGE image of the artificial metalloprotease obtained in Example 1 of the present invention used to hydrolyze bovine serum albumin at pH 5.

[0017] Figure 3 This is an SDS-PAGE image of the artificial metalloprotease obtained in Example 1 of the present invention hydrolyzing bovine serum albumin for five cycles at pH 5. DETAILED DESCRIPTION

[0018] 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.

[0019] Example 1

[0020] In this embodiment, SnO particles were synthesized according to the following steps:

[0021] (1) 0.65 g of SnCl2·2H2O was added to 15 mL of deionized water and fully dissolved by ultrasonication at room temperature. 0.34 g of NaOH was added to 15 mL of deionized water and fully dissolved by ultrasonication at room temperature. The two solutions were then mixed to obtain a reaction solution.

[0022] (2) The reaction solution was transferred into an ultrasonic cell disruptor and ultrasonicated at 400 W for 10 minutes to obtain a mixed solution.

[0023] (3) The mixed solution was centrifuged and washed three times with deionized water, and then dried to obtain SnO particles.

[0024] Figure 1 This is a scanning electron microscope (SEM) image of SnO particles. It can be seen from the figure that the particles are square, smooth, and uniform in size.

[0025] Example 2

[0026] Protein hydrolysis experiment: 1.5 mg of bovine serum albumin was dissolved in 5 mL of 200 mM phosphate buffer at pH 5, and then 0.2 g of the SnO material obtained in Example 1 was dispersed in the solution. After reacting at 30°C for 30 min, the supernatant was discarded by centrifugation. 60 μL of 10% SDS was added to the precipitate and thoroughly mixed by ultrasonication. After waiting for 20 min, SDS can desorb the peptides adsorbed on the surface of the SnO material into the solution. 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 SnO artificial metalloproteinase, bovine serum albumin has obvious hydrolysis bands, while there is no hydrolysis band in the pure bovine serum albumin solution within the same reaction time, which proves the excellent protein hydrolysis ability of SnO particles.

[0027] Example 3

[0028] Cyclic hydrolysis experiment of protein at pH = 5: First, the experiment in Example 2 was carried out, and then the precipitate after the reaction was once and sampled was washed with 2 mol / L potassium chloride solution 3 times and deionized water 2 times until the excess SDS and impurities attached to the particles were cleaned. The steps in Example 2 were carried out again. After five cycles, the cyclic hydrolysis results were observed by SDS-PAGE gel electrophoresis. Figure 3 As shown. Figure 3 It can be seen that SnO artificial metalloprotease still maintains protease activity after hydrolyzing bovine serum albumin five times, proving the good recycling performance of SnO particles.

[0029] In summary, the SnO particles prepared by the simple ultrasonic method in the present invention can efficiently hydrolyze bovine serum albumin and have good stability and recyclability, thereby providing a new idea for the development of artificial metalloproteases.

Claims

1. Application of stannous oxide as an artificial metalloproteinase to hydrolyze bovine serum protein, the preparation method of the stannous oxide comprising the following steps: (1) adding stannous chloride dihydrate and sodium hydroxide into deionized water respectively and dissolving them by ultrasonication at room temperature, and then mixing them uniformly to obtain a reaction solution; (2) transferring the reaction solution into an ultrasonic cell disruptor and sonicating to obtain a mixed solution; (3) The mixed solution is centrifuged, washed and dried to obtain the SnO material as an artificial metalloprotease.

2. The use according to claim 1, wherein: In step (1), in the reaction solution, the ratio of stannous chloride dihydrate, sodium hydroxide and deionized water is 0.65 g:0.34 g:30 mL.

3. The use according to claim 1, wherein: In step (2), the ultrasonic frequency is 400W and the reaction time is 10-20 min.

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