A method for preparing and using a nano-enzyme mimic catalyst for protein hydrolysis

Abstract

The application relates to the technical field of biological catalysis and nanomaterials, and discloses a preparation method and application of a nano-enzyme catalyst for protein hydrolysis. 12 The mass ratio of the transition metal phosphomolybdate to the ZIF-8 and the specific structure of the catalyst show a hydrolysis activity which is significantly higher than that of a conventional proportion composite catalyst and a single component under mild conditions (such as pH 5.0 and 40 DEG C), the catalyst has stable structure, is easy to separate and recycle, and can be repeatedly used. The application effectively solves the technical problems of high cost and poor stability of an existing protease catalyst, low efficiency and easy dissolution of a heterogeneous enzyme catalyst, and has a good application prospect in the field of functional peptide preparation.

Description

Technical Field

[0001] This invention relates to the fields of biocatalysis and nanomaterials technology, and in particular to a method for preparing and applying a nano-enzyme-like catalyst for protein hydrolysis. Background Technology

[0002] Enzymatic hydrolysis of proteins is a crucial step in obtaining functional peptides in the food, pharmaceutical, and feed industries. However, the poor stability, high cost, and difficulty in recycling and reusing natural proteases limit their large-scale application. Traditional chemical catalysts or heterogeneous enzyme-mimicking catalysts generally exhibit low (typically below 50%) hydrolysis efficiency for proteins under mild conditions (e.g., neutral pH, 60-80°C), failing to meet the demands of high-efficiency production. Furthermore, many porous composite catalysts loaded with active components often experience leaching of the active component or dissolution of the support during the reaction due to the instability of the support structure, leading to a rapid decline in catalyst activity and hindering stable recycling. To address these challenges, the development of efficient, stable, and reusable protein hydrolysis nano-enzyme-mimicking catalysts has significant industrial value.

[0003] Enzyme nanoparticles, as a class of nanomaterials with enzyme-like catalytic activity, have shown great potential in the fields of biotechnology and environmental science due to their advantages such as ease of production, cost-effectiveness, long storage time, and excellent environmental stability. Metal-organic frameworks (MOFs), with their tunable structure and high specific surface area, are widely used as carriers or active components of nanozymes. MOFs, especially zeolite imidazole frameworks (ZIFs), provide an excellent platform for mimicking the active sites of natural enzymes due to their unique pore structure and tunable microenvironment. For example, ZIF-8 has been shown to possess inherent carbonic anhydride enzyme-like activity and exhibits esterase and acetylcholinesterase activities, attributed to its biomimetic structural units. In protein hydrolysis, several nanomaterials have been explored for mimicking protease activity. For example, Cu2O / SBA-3 nanozymes exhibit highly efficient hydrolytic activity against proteins under physiological conditions. Furthermore, LaCoO3 nanoparticles exhibit selective hydrolytic ability against casein at room temperature, with catalytic efficiency significantly superior to many nanoproteases that require high temperatures to hydrolyze peptide bonds. These studies demonstrate the great potential of nanozymes in mimicking the activity of natural proteases and achieving protein hydrolysis.

[0004] The ordered microporous structure of MOFs provides adsorption sites for proteins, while the Brønsted acidity of PTA synergistically promotes the targeted activation of peptide bonds. This synergistic effect of spatial confinement and acidic sites promises to achieve efficient protein hydrolysis under mild conditions, with the expectation of satisfactory hydrolysis conversion rates and stable recycling cycles. However, combining heteropolyacids (such as phosphotungstic acid, PTA) with MOFs (such as ZIF-8) to improve catalytic performance remains challenging. Traditional PTA@MOF composite catalysts are prone to structural collapse and dissolution in protein hydrolysis buffer systems, resulting in limited improvements in catalytic efficiency. This instability is mainly due to the insufficient stability of MOF chemical bonds under hydrolysis conditions. To overcome the limitations of existing technologies, developing a highly efficient, stable, and reusable protein hydrolysis nano-enzyme-like catalyst under mild conditions has significant industrial value. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a protein hydrolysis nano-enzyme catalyst with high catalytic activity, stable structure, and easy separation and recovery, its preparation method, and its application in the efficient hydrolysis of proteins under mild conditions.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing a nano-enzyme-like catalyst for protein hydrolysis includes the following steps: S1. Prepare an aqueous solution of phosphomolybdic acid and an aqueous solution of a transition metal salt, and mix them at a predetermined molar ratio under ultrasonication to obtain a suspension; subject the suspension to hydrothermal treatment at 40℃~100℃ for 60~180 minutes, and then centrifuge, wash, and dry to obtain a transition metal phosphomolybdate solid catalyst (denoted as PMo). 12 ); S2. At room temperature, 2-methylimidazole, zinc acetate, and the aforementioned transition metal phosphomolybdate solid catalyst (PMo) are reacted. 12 The nano-enzyme catalyst (PMo) was dispersed in an aqueous phase at a mass ratio of 1:2:3 to 21 and reacted under hydrothermal conditions for 30 to 60 minutes, followed by aging for 30 to 90 minutes to obtain a crude product. After washing and drying, the crude product was obtained. 12 @ZIF-8).

[0007] As a preferred technical solution, in step S1, the transition metal salt is selected from one of MnCl2, ZnCl2, FeCl2, FeCl3, CuCl2, and MoCl6.

[0008] As a preferred technical solution, in step S1, the molar ratio of phosphomolybdic acid to transition metal salt is 0.5 to 2:1.

[0009] As a preferred technical solution, in step S2, the aging time is 30 to 90 minutes.

[0010] The present invention also discloses a nano-enzyme-like catalyst for protein hydrolysis, which is prepared by the preparation method described above.

[0011] As a preferred technical solution, it includes zeolite imidazole framework material ZIF-8 and transition metal phosphomolybdate supported thereon.

[0012] As a preferred technical solution, the transition metal in the transition metal phosphomolybdate is selected from at least one of Mn, Zn, Fe, Cu, and Mo.

[0013] The present invention also discloses the application of the above-mentioned nano-enzyme-like catalyst in protein hydrolysis.

[0014] As a preferred technical solution, the protein is selected from at least one of casein, walnut protein, corn protein, and soy protein.

[0015] As a preferred technical solution, the conditions for the protein hydrolysis reaction in application are: protein mass concentration of 0.1% to 10%, catalyst mass concentration of 0.2 to 5 mg / mL, pH 4.0 to 9.0, and temperature of 40℃ to 80℃.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) High catalytic activity PMo nano-enzyme catalyst constructed via a two-step method 12 @ZIF-8 achieves synergy between the Brønsted acidic site of phosphomolybdic acid and the Lewis acidic site of zinc ions in the ZIF-8 support, and utilizes the micropore confinement effect of ZIF-8 to enrich protein substrates, thereby exhibiting significantly higher hydrolytic catalytic activity for proteins than single components under mild conditions.

[0017] (2) The structure is stable and easy to recycle. The composite material formed by the optimized preparation process has a stable structure and is not easily dissolved or collapsed in the aqueous reaction system. Solid-liquid separation and catalyst recovery can be achieved through simple centrifugation. Experiments show that the catalyst can maintain high catalytic activity even after repeated use.

[0018] (3) Mild conditions and wide applicability The optimal reaction conditions (pH 4.0~9.0, temperature 40℃~80℃) are close to neutral and moderate. It does not require strong acids, strong bases or high temperature and high pressure, has low energy consumption, and simple equipment requirements. It is suitable for the efficient hydrolysis of a variety of animal and plant proteins and has good industrial application potential.

[0019] (4) Environmental friendliness and safety potential Preparing catalyst precursors from metal salts of low-toxicity or essential trace elements such as zinc and iron provides a feasible path for developing protein hydrolysis catalysts with good biocompatibility. Attached Figure Description

[0020] Figure 1 The transition metal phosphomolybdate Mn-PMo obtained in Example 1 of this invention 12 The morphology SEM image shows that the overall structure is a dense and rough flocculent or sponge-like accumulation formed by the primary aggregation of a large number of nanoparticles. Figure 2 The image shows the morphology SEM image of ZIF-8 obtained in Example 1 of this invention, which exhibits a highly regular rhombic dodecahedral (polyhedral) crystal morphology. Figure 3 The nano-enzyme-mimicking catalyst Mn-PMo obtained in Example 1 of this invention 12 The SEM image of @ZIF-8 shows the morphology of spherical particles with uniform size and good dispersion.

[0021] Figure 4 The nano-enzyme-like catalyst Mn-PMo obtained in the embodiments of this invention 12 The XRD pattern of ZIF-8 shows that ZIF-8 retains its original crystal structure and phases, while PTA (i.e., Mn-PMo)... 12 PTA was successfully incorporated into the ZIF-8 framework as a guest component, demonstrating that PTA was successfully loaded onto ZIF-8, forming PTA@ZIF-8 (i.e., Mn-PMo). 12 @ZIF-8) catalyst. Detailed Implementation

[0022] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to specific embodiments.

[0023] Unless otherwise specified, all reagents used in the following examples were of analytical grade and purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0024] Example 1: Mn-PMo nano-enzyme catalyst 12 Preparation of ZIF-8 and its enzymatic hydrolysis of casein A phosphomolybdic acid aqueous solution and a MnCl2 aqueous solution were prepared and mixed at a molar ratio of 1:1 under ultrasonication to obtain a suspension. The suspension was hydrothermally treated at 100°C for 180 minutes, followed by centrifugation, washing, and drying to obtain a transition metal phosphomolybdate solid catalyst (denoted as Mn-PMo). 12 ).

[0025] At room temperature, 2-methylimidazole, zinc acetate and Mn-PMo 12 The product was dispersed in an aqueous phase at a molar ratio of 1:2:6 and reacted under hydrothermal conditions for 60 minutes, followed by aging for 90 minutes to obtain a crude product. After washing and drying, the nano-enzyme-like catalyst Mn-PMo was obtained. 12 @ZIF-8, its XRD pattern can be found here. Figure 4 .

[0026] The above-obtained Mn-PMo 12 @ZIF-8 was added to a reaction system containing 1% casein at a working concentration of 3 mg / mL. The hydrolysis reaction was carried out under stirring at pH 5.0 and a temperature of 40℃ for 5 hours. The hydrolysis rate of casein was 71.5% as determined by the o-phthalaldehyde method.

[0027] Example 2: Mn-PMo nano-enzyme catalyst 12 @ZIF-8 Reusability The reaction system after the hydrolysis reaction in Example 1 was centrifuged to collect Mn-PMo. 12 @ZIF-8 (after initial use) was washed three times with deionized water to remove adsorbed proteins and products from the surface. The washed nano-enzyme-like catalyst was then vacuum-dried at 60°C for 6 hours to obtain the recovered catalyst.

[0028] The recovered catalyst was subjected to the same conditions (1% casein, pH 5.0, 40℃, 5 h, catalyst concentration 3 mg / mL) for the next round of hydrolysis experiments. This process of "reaction-centrifugation-washing-drying-re-reaction" was repeated, and the hydrolysis rate was recorded each time.

[0029] The results showed that the hydrolysis rate was 71.5% in the first use (Example 1), 70.2% in the second use, 68.6% in the third use, and 66.3% in the fourth use, indicating that the catalyst maintained good catalytic activity after being used four times.

[0030] Example 3: Zn-PMo nano-enzyme catalyst 12 Preparation of ZIF-8 and its enzymatic hydrolysis of soybean protein A phosphomolybdic acid aqueous solution and a ZnCl2 aqueous solution were prepared and mixed at a molar ratio of 0.5:1 under ultrasonication to obtain a suspension. The suspension was then hydrothermally treated at 60°C for 120 minutes, followed by centrifugation, washing, and drying to obtain a zinc phosphomolybdate solid catalyst (denoted as Zn-PMo). 12 ).

[0031] At room temperature, 2-methylimidazole, zinc acetate and Zn-PMo 12The product was dispersed in an aqueous phase at a molar ratio of 1:2:3 and reacted under hydrothermal conditions for 30 minutes, followed by aging for 60 minutes to obtain a crude product. After washing and drying, the nano-enzyme-like catalyst Zn-PMo was obtained. 12 @ZIF-8.

[0032] The Zn-PMo obtained above 12 @ZIF-8 was added to a reaction system containing 0.1% soy protein at a working concentration of 1 mg / mL. The reaction was carried out under stirring at pH 4.0 and a temperature of 80℃ for 5 hours, and the hydrolysis rate of soy protein was 40.2%.

[0033] Example 4: Fe-PMo nano-enzyme catalyst 12 Preparation of ZIF-8 and its enzymatic hydrolysis of corn gluten A phosphomolybdic acid aqueous solution and a FeCl2 aqueous solution were prepared and mixed at a molar ratio of 2:1 under ultrasonication to obtain a suspension. The suspension was then hydrothermally treated at 60°C for 120 minutes, followed by centrifugation, washing, and drying to obtain a solid catalyst of iron phosphomolybdate (denoted as Fe-PMo). 12 ).

[0034] At room temperature, 2-methylimidazole, zinc acetate and Fe-PMo 12 The product was dispersed in an aqueous phase at a molar ratio of 1:2:12 and reacted under hydrothermal conditions for 60 minutes, followed by aging for 30 minutes to obtain a crude product. After washing and drying, the nano-enzyme-like catalyst Fe-PMo was obtained. 12 @ZIF-8.

[0035] The Fe-PMo obtained above 12 @ZIF-8 was added to a reaction system containing 1% corn protein at a working concentration of 5 mg / mL. The reaction was carried out under stirring at pH 9.0 and a temperature of 70°C for 5 hours, and the hydrolysis rate of corn protein was 52.4%.

[0036] Example 5: Mo-PMo nano-enzyme catalyst 12 Preparation of ZIF-8 and its enzymatic hydrolysis of walnut protein A phosphomolybdic acid aqueous solution and a MoCl6 aqueous solution were prepared and mixed at a molar ratio of 0.5:1 under ultrasonication to obtain a suspension. The suspension was then hydrothermally treated at 40°C for 60 minutes, followed by centrifugation, washing, and drying to obtain a solid catalyst of molybdenum phosphomolybdate (denoted as Mo-PMo). 12 ).

[0037] At room temperature, 2-methylimidazole, zinc acetate and Mo-PMo 12The product was dispersed in an aqueous phase at a molar ratio of 1:2:21 and reacted under hydrothermal conditions for 30 minutes, followed by aging for 90 minutes to obtain a crude product. After washing and drying, the nano-enzyme-like catalyst Mo-PMo was obtained. 12 @ZIF-8.

[0038] The above-obtained Mo-PMo 12 @ZIF-8 was added to a reaction system containing 10% walnut protein at a working concentration of 5 mg / mL. The hydrolysis reaction was carried out under stirring at pH 9.0 and temperature 70℃ for 5 hours, and the hydrolysis rate of walnut protein was 64.1%.

[0039] Example 6: Comparison of protein hydrolysis performance of single-component catalysts Under the same hydrolysis conditions as in Example 1 (1% casein, pH 5.0, 40°C, 5 h, catalyst concentration 3 mg / mL), the conventional ZIF-8 catalyst and the pure Mn-PMo prepared by step S1 were tested respectively. 12 Protein hydrolysis performance of catalysts.

[0040] The results showed that when ZIF-8 catalyst was used alone, the casein hydrolysis rate was 15.9%; when Mn-PMo was used alone... 12 When catalyzed, the casein hydrolysis rate was 27.5%. The hydrolysis rates of both single-component catalysts were significantly lower than those of the Mn-PMo catalyst of this invention. 12 The 71.5% hydrolysis rate of the ZIF-8 composite catalyst demonstrates a significant synergistic effect between the ZIF-8 support and the transition metal phosphomolybdate active component in the composite material of this invention. This synergistic effect originates from the microporous confinement effect of ZIF-8, which enriches the protein substrate, and the combined effect of the Brønsted acidic sites of phosphomolybdate and the Lewis acidic sites of zinc ions.

[0041] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a nano-enzyme-like catalyst for protein hydrolysis, characterized in that, Includes the following steps: S1. Prepare an aqueous solution of phosphomolybdic acid and an aqueous solution of a transition metal salt, mix them under ultrasonication at a predetermined molar ratio to obtain a suspension; subject the suspension to hydrothermal treatment at 40℃~100℃ for 60~180 minutes, and obtain a solid catalyst of transition metal phosphomolybdate after centrifugation, washing and drying. S2. At room temperature, 2-methylimidazole, zinc acetate and the transition metal phosphomolybdate solid catalyst are dispersed in an aqueous phase at a mass ratio of 1:2:3-21. The mixture is reacted under hydrothermal conditions for 30-60 minutes, and then aged for 30-90 minutes to obtain a crude product. After washing and drying, the nano-enzyme-like catalyst is obtained.

2. The method according to claim 1, characterized in that: In step S1, the transition metal salt is selected from one of MnCl2, ZnCl2, FeCl2, FeCl3, CuCl2, and MoCl6.

3. The method according to claim 1, characterized in that: In step S1, the molar ratio of phosphomolybdic acid to transition metal salt is 0.5 to 2:

1.

4. The method according to claim 1, characterized in that: In step S2, the aging time is 30 to 90 minutes.

5. A nano-enzyme-like catalyst for protein hydrolysis, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 4.

6. The nano-enzyme-like catalyst according to claim 5, characterized in that: It contains the zeolite imidazole framework material ZIF-8 and the transition metal phosphomolybdate supported thereon.

7. The nano-enzyme-like catalyst according to claim 6, characterized in that: The transition metal in the transition metal phosphomolybdate is selected from at least one of Mn, Zn, Fe, Cu, and Mo.

8. The application of a nano-enzyme-like catalyst according to any one of claims 5 to 7 in protein hydrolysis.

9. The application according to claim 8, characterized in that: The protein is selected from at least one of casein, walnut protein, corn protein, and soy protein.

10. The application according to claim 8, characterized in that, In applications, the conditions for protein hydrolysis are: protein mass concentration 0.1%–10%, catalyst mass concentration 0.2–5 mg / mL, pH 4.0–9.0, and temperature 40℃–80℃.

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