CN114985010B - Bionic protease and preparation method and application thereof - Google Patents

Bionic protease and preparation method and application thereof Download PDF

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CN114985010B
CN114985010B CN202210538765.5A CN202210538765A CN114985010B CN 114985010 B CN114985010 B CN 114985010B CN 202210538765 A CN202210538765 A CN 202210538765A CN 114985010 B CN114985010 B CN 114985010B
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protease
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hydrolysis
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CN114985010A (en
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许宙
许珂宇
程云辉
夏利伟
陈茂龙
文李
丁利
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Changsha University of Science and Technology
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
    • B01J31/16—Catalysts comprising hydrides, coordination complexes or organic compounds containing coordination complexes
    • B01J31/22—Organic complexes
    • B01J31/2204—Organic complexes the ligands containing oxygen or sulfur as complexing atoms
    • B01J31/2208—Oxygen, e.g. acetylacetonates
    • B01J31/2226—Anionic ligands, i.e. the overall ligand carries at least one formal negative charge
    • B01J31/223—At least two oxygen atoms present in one at least bidentate or bridging ligand
    • B01J31/2239—Bridging ligands, e.g. OAc in Cr2(OAc)4, Pt4(OAc)8 or dicarboxylate ligands
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/12—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • C08G83/008—Supramolecular polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2531/00—Additional information regarding catalytic systems classified in B01J31/00
    • B01J2531/40—Complexes comprising metals of Group IV (IVA or IVB) as the central metal
    • B01J2531/48—Zirconium

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Abstract

The invention discloses a bionic protease and a preparation method and application thereof, wherein the preparation method comprises the following steps: and carrying out coordination reaction on a zirconium source and pyromellitic acid in a solvent to obtain the bionic protease. The bionic protease has excellent catalytic activity, and the preparation method has simple steps and mild conditions, so that the bionic protease and the preparation method can be applied to proteolysis.

Description

Bionic protease and preparation method and application thereof
Technical Field
The invention relates to protease, in particular to bionic protease, and a preparation method and application thereof.
Background
Proteases catalyze the hydrolysis of peptide bonds to amino and carboxyl groups and have been widely used in the fields of food processing, biosensing, environmental protection, biomedical and the like. The protease has mild catalysis condition, high efficiency and specificity, and thus has wide and huge application in industry. The proportion of proteolytic enzymes is statistically the largest, about 75%, of all industrial enzyme preparations. However, the inherent disadvantages of natural enzymes, such as variability, high cost, laborious preparation and difficult recovery, have greatly limited their practical use.
To overcome these drawbacks, researchers have been working on the search for artificial enzyme mimics. To date, the use of metal ions as lewis acid catalysts to hydrolyze proteins is the most common method of constructing artificial proteases. However, this approach has a significant limitation because many transition metals and lanthanides form gels under neutral and alkaline conditions. To avoid gel formation, organic ligands are often added to form metal complexes.
Wherein the Co (III) complex [ Co (trien) OH (H) 2 O)] 2+ Is one of the most studied metal ion complexes capable of rapidly hydrolyzing peptide bonds. Metal-substituted Polyoxometalates (POMs) are also commonly reported to have protease-like activity, which is a class of polyoxometalate compounds formed from transition metal ions linked by oxygen, modified to have various chemical and physical properties. In addition, zr (IV) substituted POM (Zr-POMs) possess good protease-like activity due to the large coordination number, flexible geometry, high oxygen affinity and Lewis acidity of Zr (IV).
Li et al in 2014 reported a Copper-based metal organic framework material (Copper-based metal organic frame material, cu-MOF) with protease-like activity that was able to catalyze the hydrolysis of peptide bonds in bovine serum albumin (bovineserum albumin, BSA) and casein. Because of the large surface area and porous structure of the MOF, cu-MOF has a higher affinity for proteins than native trypsin and Cu (II) complexes.
Recently, it has been found that Zr-MOF has proteolytic enzyme-like activity, and that Zr 6 [Zr 6 (μ 3 -O) 4 (μ 3 -OH) 4 ]The Zr-MOF formed by self-assembly of nodes and carboxylic acid ligands is one of the most widely used MOFs at present, and has the function of selectively hydrolyzing dipeptides and proteins under physiological conditions as a heterogeneous and recyclable artificial enzyme, and has excellent recoverability and excellent stability. It has a strong Zr (IV) -O bond,thus having very excellent thermal and chemical stability. In addition, zr-MOFs are simple to prepare, inexpensive and therefore are often used as catalysts for some chemical reactions. However, zr-MOF has low catalytic activity and cannot meet the requirements of industrial production.
Disclosure of Invention
The invention aims to provide a bionic protease, a preparation method and application thereof, wherein the bionic protease has excellent catalytic activity, and the preparation method has simple steps and mild conditions, so that the bionic protease and the preparation method can be applied to proteolysis.
In order to achieve the above object, the present invention provides a preparation method of a biomimetic protease, comprising: and carrying out coordination reaction on a zirconium source and pyromellitic acid in a solvent to obtain the bionic protease.
Preferably, the zirconium source is selected from ZrOCl 2 ·8H 2 O、ZrCl 4 、Zr(NO 3 ) 2 .6H 2 At least one of O.
Preferably, the molar ratio of the zirconium source to the pyromellitic acid is 1:08-1.2.
Preferably, the coordination reaction satisfies at least the following conditions: the reaction is carried out in a closed environment at the temperature of 140-160 ℃ for 20-30h.
Preferably, the molar ratio of the zirconium source to the solvent is 28mmol:350-400mL.
Preferably, the solvent is selected from at least one of N, N-dimethylacetamide, formic acid, acetic acid, ethanol and hydrochloric acid.
Preferably, the solvent comprises N, N-dimethylacetamide and formic acid in a volume ratio of 2-3:1.
The invention also provides a bionic protease which is prepared by the preparation method.
The invention also provides an application of the bionic protease in catalyzing protein hydrolysis.
Preferably, the method of application is: taking the bionic protease as a catalyst, and carrying out hydrolysis reaction on the protein in a dispersing agent;
wherein the dosage mole ratio of the protein, the bionic protease and the dispersing agent is 20mmol:1.5-2.5mmol:900-1100mL;
preferably, the hydrolysis reaction satisfies at least the following conditions: the reaction temperature is 50-70 ℃ and the reaction time is 3-6h;
preferably, the protein is selected from at least one of soy isolate protein, surimi protein and casein;
preferably, the dispersing agent is at least one of water, phosphate buffer, tris solution and sodium trimethylsilylpropionate solution.
As described above, in recent years, with the rapid development of nanotechnology, various functional nanomaterials have been found to have protease-like activity as well. However, synthetic nanoenzymes also have problems and drawbacks, such as: the lower activity and specificity are two major obstacles limiting the application of nano-enzymes to replace natural enzymes, and the targeted improvement of selectivity is the key direction of research.
Since the catalytic reaction of the enzyme mainly occurs on the surface, surface modification to improve selectivity may reduce catalytic activity; the nanometer enzymes are more, but the current research on enhancing the nanometer enzyme activity mainly focuses on nanometer materials with peroxidase-like activity, and next to oxidase activity, there are few other types of enzyme activity enhancing strategies. The enhancement strategy of the activity of other types of enzymes is further researched, so that the activity level of the nano enzyme is integrally improved; accurate regulation and control of the enhancement of various enzyme activities of the nano material: such as AuNP, have both peroxidase, oxidase and catalase activities. Hybridization of nanoenzymes also produces products with two or more enzymatic activities. An activity enhancement strategy may have an effect on a variety of enzyme activities, but in practice only a single catalytic activity is often required. Therefore, the enhancement of enzyme activity should also take into account that single regulation is also a major drawback of nanoenzymes.
As can be seen, there are a number of difficulties in the modification of artificial synthetases, in this context, as shown in FIG. 1-1 and FIGS. 1-2, introducing carboxyl groups on the basis of Zr-MOFs structure to mimic the catalytic active center of natural zinc metalloproteases, zr with Lewis acidity 4+ Site to mimic Zn in native zinc metalloprotease 2+ The carboxylic acid group is used for simulating the action of glutamic acid in the active center of the natural metalloprotease, so that the bionic protease (Zr-MOF protease) is constructed, has high-activity protease-like function, can hydrolyze various industrial proteins with high efficiency, and can be applied to the food industry.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate the invention and together with the description serve to explain, without limitation, the invention. In the drawings:
FIG. 1-1 is a central graph of the catalytic activity of a natural zinc metalloprotease;
FIGS. 1-2 are graphs of Zr-MOF catalytic activity centers of biomimetic design;
FIG. 2 is a graph showing the characteristics of Zr-MOF and biomimetic protease, wherein (A) is a PXRD graph of Zr-MOF and biomimetic protease, (B) is FTIR of Zr-MOF and biomimetic protease, (C) is SEM of Zr-MOF, and (D) is an SEM graph of biomimetic protease;
FIG. 3 is a high performance liquid chromatogram showing the hydrolysis of diglycolamine;
FIG. 4 is a SDS-PAGE diagram of hydrolyzed soy protein isolate;
FIG. 5 is a SDS-PAGE diagram of hydrolyzed surimi protein;
FIG. 6 is a SDS-PAGE diagram of hydrolyzed casein;
FIG. 7 is a SDS-PAGE of three proteins without biomimetic protease.
Detailed Description
The following describes specific embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
The invention provides a preparation method of bionic protease, which comprises the following steps: and carrying out coordination reaction on a zirconium source and pyromellitic acid in a solvent to obtain the bionic protease.
In the above preparation method, the kind of the zirconium source is not particularly limited, but in order to further enhance the catalytic activity of the prepared biomimetic protease, it is preferable that the zirconium source is selected from ZrOCl 2 ·8H 2 O、ZrCl 4 、Zr(NO 3 ) 2 .6H 2 At least one of O.
In the above preparation method, the amount of each material is not specifically required, but in order to further improve the catalytic activity of the prepared biomimetic protease, preferably, the molar ratio of the amounts of the zirconium source and pyromellitic acid is 1:08-1.2.
In the above preparation method, the conditions of the coordination reaction are not particularly required, but in order to further improve the catalytic activity of the resulting biomimetic protease, it is preferable that the coordination reaction satisfies at least the following conditions: the reaction is carried out in a closed environment at 140-160 ℃ for 20-30h.
In the above preparation method, the amount of the solvent may be selected within a wide range, but in order to enable the respective materials to be sufficiently dispersed, thereby improving the reaction yield, it is preferable that the zirconium source, the solvent are used in a molar ratio of 28mmol:350-400.
The kind of the solvent may be selected within a wide range on the basis of the above embodiment, but in order to further improve the reaction yield, it is preferable that the solvent is at least one selected from the group consisting of N, N-dimethylacetamide, formic acid, acetic acid, ethanol and hydrochloric acid; further preferably, the solvent comprises N, N-dimethylacetamide and formic acid in a volume ratio of 2-3:1.
The invention also provides a bionic protease which is prepared by the preparation method.
The invention also provides an application of the bionic protease in catalyzing protein hydrolysis.
In the above application, the specific method for the catalytic application of the biomimetic protease may be various, but in order to further improve the catalytic effect, preferably, the method for the application is as follows: taking the bionic protease as a catalyst, and carrying out hydrolysis reaction on the protein in a dispersing agent; wherein the dosage mole ratio of the protein, the bionic protease and the dispersing agent is 20mmol:1.5-2.5mmol:900-1100mL.
In the above application, the conditions of the hydrolysis reaction may be selected within a wide range, but in order to further enhance the effect of hydrolysis, it is preferable that the hydrolysis reaction satisfies at least the following conditions: the reaction temperature is 50-70 ℃ and the reaction time is 3-6h;
in the above application, the kind of the protein may be selected in a wide range, but in view of the application prospect in the market, it is preferable that the protein is selected from at least one of soybean isolated protein, surimi protein and casein.
In the above application, the kind of the dispersant may be selected within a wide range, but in order to further enhance the effect of hydrolysis, it is preferable that the dispersant is at least one of water, phosphate buffer, tris (hydroxymethyl) aminomethane solution and sodium trimethylsilylpropionate solution.
The invention will be described in detail below by way of examples. In the following examples, proteins and their degradation products were separated by SDS-polyacrylamide gel electrophoresis in ultrapure water, and finally the separated bands were stained with Coomassie brilliant blue, thereby analyzing the degradation of the proteins by the biomimetic protease.
The glycylglycine and its hydrolysate were measured by PITC pre-column derivatization liquid chromatography.
Derivatization of amino acids: accurately measuring 200 mu L of a sample solution, and placing the sample solution in a 1.5mL centrifuge tube; 100 mu L of triethylamine acetonitrile solution and 100 mu L of PITC acetonitrile solution are respectively added, evenly mixed and placed at 25 ℃ for 1 hour; adding 400 mu L of normal hexane into a centrifuge tube, uniformly mixing, and standing at 25 ℃ for 10min; taking the PITC-AA solution of the lower layer, and passing through a water film of 0.45 mu m; 200. Mu.L of filtrate was taken, diluted with 800. Mu.L of water, shaken well and 10. Mu.L of sample was introduced.
The chromatographic column is Venusil of Agela companyAmino acid analytical column (4.6X105 mm,5 μm), mobile phase A was 0.1mol/L acetic acid-sodium acetate buffer pH6.5, mobile phase B was pure acetonitrile solution. The elution flow rate was set at 1.0mL/min, the detection wavelength was 254nm, the column temperature was 40℃and the loading was 10. Mu.L. Adopting a gradient elution mode, wherein the elution condition is 0-2 min and 100% A; 2-15 min,85% A; 15-17 min,100% A; 17-18 min,100% A.
Example 1
Bionic protease (Zr-MOF- (COOH) 2 ) Is prepared from
Taking equal molar amount of ZrOCl 2 8H2O and pyromellitic acid ligand 27.9mmol was added to a mixture of 270mL of N, N-Dimethylacetamide (DMA) and 108mL of formic acid. After magnetic stirring for one hour, the solution was transferred to a 500mL polytetrafluoroethylene-lined reactor liner, the reactor liner was placed in a high-pressure reactor, the lid was screwed on, and the reactor liner was heated at 150℃for 24 hours. After the reaction was completed, the obtained precipitate was washed three times with DMF and acetone, respectively, and stored for use.
Example 2
The procedure of example 1 was followed, with the only difference that: zrOCl 2 ·8H 2 O is changed into ZrCl with equimolar quantity 4 。
Example 3
The procedure of example 1 was followed, with the only difference that: zrOCl 2 ·8H 2 O is changed to Zr (NO) 3 ) 2 .6H 2 O。
Comparative example 1
Preparation of ZR-MOF:
5.20mmol of zirconium tetrachloride is weighed into 300mL of DMF, 9mL of acetic acid is added after magnetic stirring is carried out for 1h, 5.18mmol of terephthalic acid is added after continuous stirring is carried out for 1h, after magnetic stirring is carried out for 2h until the terephthalic acid is uniformly dispersed, the solution is transferred into a 500mL polytetrafluoroethylene lining reaction kettle inner container, the reaction kettle inner container is placed into a high-pressure reaction kettle, a cover is screwed, and the reaction kettle inner container is heated for 24h at 120 ℃. After the reaction was completed, cooled to room temperature, the supernatant was discarded, and the resulting precipitate was washed three times with methanol and three times with DMF.
Detection example 1
1) The products of example 1 and comparative example 1 were subjected to PXRD characterization, the characterization results are shown in section a of fig. 2, and from the figures, the X-ray diffraction (XRD) patterns of the ZR-MOF and carboxylic acid functionalized derivative biomimetic proteases thereof are matched with the simulated patterns, which proves that the ZR-MOF and the biomimetic proteases have the same crystal structure.
2) The products of example 1 and comparative example 1 were subjected to FTIR characterization, the characterization results are shown in section B of FIG. 2, from which it is seen that the absorption bands of ZR-MOF and biomimetic protease are mostly concentrated at 1400cm -1 And 1584cm -1 This is attributable to the stretching vibration of the coordinated terephthalic acid binding molecules. 1740-1700 cm -1 Corresponds to both symmetrical and asymmetrical stretches of c=o groups, indicating the presence of free-COOH groups in the biomimetic protease, successfully demonstrating the success of MOF preparation. Furthermore, compared to ZR-MOF, the absorption peak of biomimetic proteases has shifted blue, which may also be due to free-COOH groups.
3) SEM characterization of the products of example 1 and comparative example 1, characterization of the product of example 1, see part D of fig. 2, characterization of the product of comparative example 1, see part C of fig. 2, from which it can be seen that ZR-MOF presents a more regular pentagon, about 100nm long; the prepared bionic protease is round and has a rough surface, and the average particle size under an electron microscope is about 150nm long.
4) The products of examples 2-3 were characterized in the same manner as described above, with characterization results substantially identical to those of example 1.
Application example 1
Hydrolysis of the glycylglycine:
the biomimetic protease (0.20 mmol) prepared in example 1 was taken into a centrifuge tube containing 950mL of ultrapure water and stirred (magnetic stirrer shaking) at 25℃for 30 minutes to disperse the MOF particles homogeneously. Then 50mL of 4mmol/L of diglycerin (2.0 mmol) was added. The reaction mixture was kept at 60℃for reaction at pH 7.0, and after completion of the reaction, the reaction was centrifuged at 15000rpm for 20 minutes to remove Zr-MOF (i.e., biomimetic protease).
Detecting hydrolysis conditions of the glycylglycine in different reaction time periods, specifically referring to fig. 3, wherein part a represents a high performance liquid chromatogram after glycylglycine is hydrolyzed for different time periods, part B represents a graph of change of glycylglycine concentration and glycine concentration with hydrolysis time, and part C represents a graph of ln [ GG ] as a function of reaction time; gly represents glycine, gly-Gly and GG each represent glycylglycine.
As can be seen from fig. 3: at 60 ℃ and pH 7.0, the data show that within 3 hours, the glycylglycine hydrolyzes rapidly, and the reaction rate also slows down gradually as glycylglycine concentration decreases. The reaction rate constant of bionic proteinase hydrolyzed N-glycylglycine is 2.57×10 as shown in the C part of the figure -5 s -1 。
The reaction rate constant of the hydrolyzed glycylglycine was substantially identical to that of the biomimetic protease prepared in example 1, by changing the biomimetic protease prepared in example 1 to the products of example 2 and example 3 in the same manner.
According to the same method, the bionic protease is removed or replaced by ZrCl with equal molar quantity 4 Or MOF-808, the hydrolysis reaction rate constants were finally measured, and the specific results are shown in Table 1.
In addition, the rate constant of the uncatalyzed hydrolysis of the glycylglycine at 60℃was 7.4X10 -9 s -1 This indicates that the reaction rate of hydrolysis of the diglycol is increased by 3.47×10 when the biomimetic protease is used 3 Multiple times.
TABLE 1
Catalyst No catalyst ZrCl 4 MOF-808 Bionic protease
Reaction rate constant 7.4×10 -9 s -1 5.55×10 -7 s -1 2.63×10 -5 s -1 2.57×10 -5 s -1
Application example 2
The biomimetic protease (2.0 mmol) prepared in example 1 was taken and added to a beaker containing 1L of ultra pure water and stirred (magnetic stirrer shaking) at room temperature for 30 minutes to disperse the MOF particles homogeneously. Then 20mmol of the isolated soy protein sample was added. The reaction mixture was kept at 60℃for reaction, and after the completion of the reaction, it was centrifuged at 15000rpm for 20 minutes to remove the Zr-MOF. The protein samples were tested for hydrolysis over different reaction time periods, see in particular FIGS. 4-6. FIG. 4 is a SDS-PAGE diagram of hydrolyzed soy protein isolate; FIG. 5 is a SDS-PAGE diagram of hydrolyzed surimi protein; FIG. 6 is a SDS-PAGE diagram of hydrolyzed casein; FIG. 7 is a SDS-PAGE of three proteins without biomimetic protease.
From fig. 4-6, it can be seen that the biomimetic protease can catalyze the hydrolysis of three proteins simultaneously, and shows a broad spectrum of catalytic activities, wherein the biomimetic protease has higher catalytic activity on soy protein and surimi protein, and lower catalytic activity on casein. As shown in FIG. 4, the bands of isolated soy protein become progressively lighter as the reaction proceeds, and almost disappear by 24 hours, indicating that hydrolysis has occurred. No new bands with lower molecular weight appeared during the reaction, probably because the biomimetic protease had a broad spectrum of cleavage sites, which directly hydrolyse the long peptides of the soy protein isolate into many shorter fragments, which could not be observed (because short polypeptides would tend to leak out of the SDS PAGE gel). As can be seen from FIG. 5, after 3h incubation, a new band with a molecular weight greater than 96kDa was generated in the electrophoresis pattern of the minced fillet protein, and the concentration increased and then decreased as the reaction proceeded, well demonstrating that the minced fillet protein was hydrolyzed in the presence of SDS-PAGE pattern and that the biomimetic protease had selective cleavage sites for this band. Similarly, the bands of casein also become shallower with increasing hydrolysis time, with bands having a molecular weight of about 26kDa hydrolyzing faster, and bands having a molecular weight of about 23kDa hydrolyzing slower (see FIG. 6). Control experiments showed that no hydrolysis of the three proteins was observed in the absence of biomimetic protease at 60 ℃ (see figure 7), confirming the catalysis of SDS-PAGE in proteolysis.
The results of the products of example 2 and example 3, which were obtained in the same manner, were substantially equivalent to the biomimetic protease obtained in example 1, with respect to the catalytic hydrolysis of soy protein isolate, surimi protein and casein.
The experiment shows that the bionic protease prepared by the invention can be used for hydrolyzing protein paper commonly used in the food industry; and the method has feasibility of being applied to the food industry, selects several proteins (casein, soy protein and minced fillet protein) commonly used in the food industry, and incubates the three proteins with bionic protease at 60 ℃ respectively, wherein the bionic protease can catalyze the hydrolysis of the three proteins at the same time within 3 hours and shows a broad-spectrum catalytic activity, and the bionic protease has higher catalytic activity on the soy protein and the minced fillet protein and lower catalytic activity on the casein. In conclusion, the bionic protease has good capability of catalyzing the hydrolysis of extremely stable peptide bonds in proteins.
Application example 3
The Zr-MOF obtained at the end of the test in application example 1 was stirred in methanol for 1 day to exchange water (Zr-MOF was dispersed in water during the experiment, so that moisture was present in the material after the test, water in methanol was exchanged for methanol after stirring), the process was repeated twice, and the used MOF was air-dried and activated at 150℃for 20 hours. Subsequently, the diglycolamine was hydrolyzed as described in application example 1. Catalytic hydrolysis was repeated 5 times, and the reaction rate constant for each time was recorded, and the results are shown in table 2.
TABLE 2
As can be seen from Table 2, the biomimetic protease prepared by the invention can be repeatedly catalyzed, and the catalytic efficiency of repeated hydrolysis for 5 times is not obviously reduced, so that the biomimetic protease can be recycled for at least 5 times.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.

Claims (7)

1. The application of the bionic protease in catalyzing protein hydrolysis is characterized in that the preparation method of the bionic protease comprises the following steps: carrying out coordination reaction on a zirconium source and pyromellitic acid in a solvent to prepare the bionic protease Zr-MOF;
the bionic protease is used for catalyzing hydrolysis reaction, and the protein is subjected to hydrolysis reaction in a dispersing agent;
the hydrolysis reaction satisfies at least the following conditions: the reaction temperature is 50-70 ℃ and the reaction time is 3-6h;
the dispersing agent is at least one of water, phosphate buffer solution, tris (hydroxymethyl) aminomethane solution and sodium trimethylsilyl propionate solution;
the protein is one or more of soybean isolated protein, surimi protein and casein.
2. The use according to claim 1, wherein the zirconium source is selected from ZrOCl 2 ∙8H 2 O、ZrCl 4 At least one of them.
3. The use according to claim 1, wherein the zirconium source and pyromellitic acid are used in a molar ratio of 1:08-1.2.
4. Use according to claim 1, wherein the coordination reaction satisfies at least the following conditions: the reaction is carried out in a closed environment at the temperature of 140-160 ℃ for 20-30h.
5. Use according to claim 1, wherein the zirconium source, solvent are used in a molar ratio of 28mmol:350-400mL.
6. The use according to any one of claims 1-5, wherein the solvent is selected from at least one of N, N-dimethylacetamide, formic acid, acetic acid, ethanol and hydrochloric acid.
7. The use according to claim 6, wherein the solvent comprises N, N-dimethylacetamide and formic acid in a volume ratio of 2-3:1.
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