Method for guiding efficient and versatile encapsulation of enzymes by seeds and its use

Abstract

The application belongs to the technical field of metal organic framework (MOF) immobilized enzyme, and particularly relates to a method for wrapping enzyme by seed orientation and application thereof. The application discloses a method for synthesizing enzyme@MOF composite by seed orientation, and based on the characteristics of MOF crystal growth, the seed orientation synthesis method is innovatively proposed. The addition of seed can greatly promote the synthesis efficiency of enzyme@MOF composite and greatly increase the encapsulation efficiency of enzyme. Meanwhile, the seed orientation synthesis method has no requirement on the chemical properties of enzyme / protein, and does not need to destructively modify the enzyme / protein to improve the encapsulation efficiency, so that the seed orientation synthesis method has good universality. The seed orientation synthesis method solves the problems of low universality and low efficiency in the existing enzyme@MOF composite synthesis method, and has an expected application prospect in the fields of biological catalysis, biological sensing, biological medicine and the like.

Description

Technical Field

[0001] This invention belongs to the field of metal-organic framework (MOF) immobilized enzyme technology, specifically relating to a method for efficiently and universally encapsulating enzymes through seed crystal guidance and its application. Background Technology

[0002] Enzymes are highly selective and active natural catalysts with the potential to improve catalytic efficiency, selectivity, and environmental sustainability, and are widely used in industrial catalysis, chemical synthesis, and biotechnology. However, enzymes are unstable after leaving cells and are easily affected by pH, temperature, most organic solvents, and inhibitors, resulting in loss of activity and significantly impacting their extracellular applications. Therefore, improving enzyme stability is a critical issue that urgently needs to be addressed. To address this, researchers have proposed a strategy of immobilizing enzymes using metal-organic frameworks (MOFs). This strategy leverages the diversity of MOFs, their high porosity and specific surface area, good stability, easily tunable pore size, and mild synthesis conditions to provide effective protection for enzymes.

[0003] MOF-immobilized enzymes can be achieved through several methods, including surface biobinding and permeation, and encapsulation. Surface biobinding and permeation involve synthesizing the MOF first and then immobilizing the enzyme, while encapsulation involves simultaneous MOF synthesis and enzyme immobilization. Studies have shown that directly encapsulating enzymes within MOFs provides excellent protection, making it a key research area. One-pot synthesis is the primary method for MOF enzyme encapsulation, with solution-phase synthesis methods mainly including co-precipitation and biomimetic mineralization. Co-precipitation relies on the spontaneous nucleation of MOFs, encapsulating the enzyme precipitated on the MOF surface during nucleation and growth. However, due to the slow nature of spontaneous nucleation, this method is time-consuming and has low encapsulation efficiency. In biomimetic mineralization, the enzyme participates in the nucleation process, significantly improving the synthesis efficiency of the enzyme@MOF complex and enabling rapid and effective encapsulation. However, this method has certain requirements regarding enzyme properties—enzymes with negatively charged surfaces are more likely to aggregate metal ions, thus promoting MOF complex formation. To address the issue of the method's versatility, current research primarily focuses on complex, destructive modifications to the enzyme to induce the aggregation of metal ions around it, thereby promoting crystal nucleation. However, this method involves complex preparation processes, hindering its widespread application. Therefore, it is necessary to develop a universal, efficient enzyme@MOF synthesis method that does not have specific requirements regarding enzyme properties. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, this invention provides a highly efficient and universal method for encapsulating enzymes through seed crystal guidance, thereby solving the problem that existing methods for synthesizing enzyme@MOF complexes in solution phases (coprecipitation method, biomimetic mineralization method and its improved methods) are difficult to simultaneously achieve universality, synthesis efficiency and non-destructiveness to the enzyme.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0006] This invention provides a method for synthesizing a highly efficient and universal enzyme@MOF complex through seed crystal guidance. The method is characterized by the addition of seeds corresponding to the MOF during the enzyme encapsulation process to skip the slow nucleation stage and achieve rapid synthesis of the enzyme@MOF complex.

[0007] Introducing seed crystals during enzyme@MOF synthesis serves as a nucleus for enzyme attachment and MOF crystal growth. The presence of seed crystals skips the slow nucleation stage in MOF crystal synthesis, significantly improving the efficiency of enzyme encapsulation within MOFs, thus greatly promoting enzyme@MOF synthesis efficiency. This invention provides a universal and efficient method for synthesizing enzyme@MOF complexes.

[0008] Preferably, the enzyme includes cytochrome C (Cyt c), hemoglobin (HB), and myoglobin (MB).

[0009] Preferably, the MOF is a ZIF-8 metal-organic framework.

[0010] This invention synthesizes an enzyme@ZIF-8 biocomposite material under ambient temperature and pressure using a seed-guided strategy. Compared to conventional one-pot synthesis, the addition of seed crystals significantly improves the synthesis efficiency of the enzyme@ZIF-8 biocomposite and greatly increases the enzyme encapsulation efficiency. Using cytochrome C (Cyt c) as an example, the residual amount of Cyt c in the supernatant before and after the reaction was compared using UV-Vis spectra to calculate the final enzyme content encapsulated within ZIF-8. It was found that with 7 mg of seed crystals added and a reaction time of 4 hours, the encapsulation efficiency of Cyt c reached 93.3%; while without seed crystals and with a reaction time of 4 hours, the encapsulation efficiency of Cyt c was only 11%, and the amount of product collected after centrifugation was very small, insufficient for subsequent experiments. This is because the seed crystals act as nuclei in the MOF crystal synthesis process, allowing crystal growth. The presence of seed crystals skips the slow nucleation stage in MOF crystal synthesis, entering a rapid crystal growth stage, thus enabling the enzyme to be encapsulated into the MOF more quickly and effectively. By adjusting the amount of seed crystals, the effect of seed crystals on promoting MOF encapsulation of enzymes was better demonstrated—a higher amount of seed crystals resulted in higher encapsulation efficiency and shorter synthesis time. Furthermore, the seed crystals, acting as nuclei, occupy the core position of the enzyme@ZIF-8 biocomplex, and the enzyme is successfully encapsulated by settling onto the surface of the continuously growing seed crystals. Based on this mechanism, the enzyme distribution is closer to the surface of the enzyme@MOF complex, making it easier to contact the catalytic substrate and thus exhibiting better activity. Cytochrome C (Cyt c) encapsulated by the seed crystal method showed up to 5.6 times the activity of free cytochrome C. Based on this, a visual colorimetric sensing system for detecting hydrogen peroxide was constructed using Cyt c@ZIF-8. This system demonstrated higher sensitivity and a wider linear detection range for hydrogen peroxide detection than free Cyt c, and also showed a lower detection limit observed by the naked eye.

[0011] As a preferred embodiment of the present invention, the above-described method for guiding a highly efficient and universal synthase@MOF complex via seed crystals includes the following steps:

[0012] S1. Seed crystals were prepared by reacting 2-methylimidazolium solution and zinc acetate solution with stirring at room temperature.

[0013] S2. After mixing the seed solution and the enzyme solution, add 2-methylimidazole solution and zinc acetate solution, and then prepare the enzyme@MOF complex by allowing it to stand at room temperature.

[0014] Preferably, in step S1, the concentration of the 2-methylimidazole solution is 2000-3000 mM, the concentration of the zinc acetate solution is 30-50 mM, and the volume ratio of the 2-methylimidazole solution to the zinc acetate solution is 1:1.

[0015] Preferably, in step S1, the stirring reaction time is 12-36 hours.

[0016] Preferably, in step S2, the concentration of the seed solution is 4-6 mg / mL, the concentration of the enzyme solution is 0.5-2 mg / mL, the concentration of the 2-methylimidazole solution is 1200-1600 mM, the concentration of the zinc acetate solution is 90-110 mM, and the volume ratio of the seed solution, enzyme solution, 2-methylimidazole solution and zinc acetate solution is 1:1:2:2.

[0017] Preferably, in step S2, the reaction time is 3-5 hours.

[0018] Preferably, in steps S1 and S2, after the reaction is completed, the product is collected by centrifugation, washed, and dried.

[0019] This invention also provides the application of the above-described method for synthesizing highly efficient and universal synthase@MOF complexes via seed crystal guidance in the synthesis of MOF immobilized enzymes.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] This invention discloses a highly efficient and versatile method for encapsulating enzymes using seed-guided synthesis. Based on the characteristics of MOF crystal growth, a seed-guided synthesis method is innovatively proposed. First, the addition of seeds significantly promotes the synthesis efficiency of enzyme@MOF complexes, greatly increasing the encapsulation efficiency. Second, the seed-guided synthesis method has no requirements on the chemical properties of the enzyme / protein and does not require destructive modification of the enzyme / protein to achieve the effect of improving encapsulation efficiency, thus possessing good versatility. Furthermore, enzymes / proteins such as cytochrome C, hemoglobin, and myoglobin, due to their positive surface charge, have relatively low encapsulation efficiency using conventional methods. However, the seed-guided method of this invention significantly improves the encapsulation efficiency of these enzymes / proteins compared to synthesis methods without seeds. For example, the encapsulated cytochrome C exhibits 5.6 times higher catalytic activity than free cytochrome C. Based on this, a simple visual colorimetric sensing system for detecting hydrogen peroxide was successfully constructed, exhibiting high sensitivity and a wide linear detection range. Therefore, this invention solves the problems of low versatility and low efficiency in current enzyme@MOF complex synthesis methods. This method is a simple, feasible, non-destructive, efficient, and universal method for synthesizing enzyme@MOF complexes. It shows promising application prospects in fields such as biocatalysis, biosensing, and biomedicine. Attached Figure Description

[0022] Figure 1The process flow diagram for seed-guided synthase@MOF complex;

[0023] Figure 2 SEM image of the seed crystal;

[0024] Figure 3 (A) UV-Vis spectra of the supernatant: (a) Supernatant before encapsulation (Cyt c content is equivalent to the amount added during synthesis), (b) Reacting for 2 hours without seed crystals, (c) Reacting for 4 hours without seed crystals, (d) Reacting for 2 hours with seed crystals, (e) Reacting for 4 hours with seed crystals; The embedded image is a photograph of the reaction supernatant, and the brownish-red color is the characteristic color of cytochrome C; (B) XRD patterns of Cyt c@ZIF-8 and pure ZIF-8; (C) SEM image of Cyt c@ZIF-8.

[0025] Figure 4 The following are UV-Vis spectra: (A) Hemoglobin (HB); (B) Myoglobin (MB); (C) XRD patterns of HB@ZIF-8, MB@ZIF-8 and ZIF-8; (D) SEM images of HB@ZIF-8 and MB@ZIF-8.

[0026] Figure 5 The results show the effects of reaction time and seed dosage on encapsulation efficiency (encapsulation efficiency was calculated by measuring the difference in Cyt c concentration in the supernatant before and after encapsulation using UV-Vis spectroscopy at 408 nm).

[0027] Figure 6 The relative bioactivities of free Cyt c, Cyt c@ZIF-8 biocomplex, Cyt c with zinc ions mixture, Cyt c with 2-methylimidazole mixture, and Cyt c with pure ZIF-8 mixture.

[0028] Figure 7 Linear working curves for the detection of H2O2 by free Cyt c and Cyt c@ZIF-8. Detailed Implementation

[0029] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0030] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0031] Example 1: A method for seed-guided, highly efficient, and versatile synthase@MOF complexes

[0032] like Figure 1 As shown, the method includes the following steps:

[0033] (1) Synthesis of seed crystals: 20 mL of 2400 mM dimethylimidazole solution and 20 mL of 40 mM zinc acetate solution were mixed and stirred at an appropriate speed (400-500 rpm) for 24 h at room temperature using a magnetic stirrer. The resulting solution was centrifuged at 8000 rpm for 10 min to obtain a precipitate, which was then washed three times with deionized water and dried in a vacuum drying oven at 60 °C for 12 h to obtain a white powder. The morphology of the white powder was characterized using scanning electron microscopy (SEM), as shown below. Figure 2 As shown in the figure, the seed crystals possess the typical sodalite structure of ZIF-8, with a uniform particle size distribution ranging from 200 to 300 nm.

[0034] (2) Seed synthesis of enzyme@ZIF-8 complex: Prepare a 5 mg / mL seed solution from step (1), then add 1 mL of the seed solution to 1 mL of enzyme (cytochrome C, myoglobin, or hemoglobin) solution (1 mg / mL) (with a control without seed). After thorough mixing, add 2 mL of 2-methylimidazole solution (1400 mM) and 2 mL of zinc acetate solution (100 mM) sequentially. Then let the mixture stand at room temperature for 2-4 hours. After completion, collect the product by centrifugation at 8000 rpm for 10 minutes. The supernatant obtained after centrifugation is then measured using a UV-Vis spectrophotometer to determine the absorption peak at a specific wavelength (408 nm for cytochrome C, and 405 nm for myoglobin and hemoglobin) to measure the enzyme / protein content encapsulated in the MOF. The resulting precipitate was first washed three times with SDS solution (0.1 g / mL) to remove surface-adsorbed enzymes, then washed three times with deionized water, and finally vacuum dried at room temperature. The dried product was characterized by X-ray diffraction (XRD), SEM, etc. Figure 3 ,4).

[0035] like Figure 3As shown in Figure A, under the condition of 4 hours of reaction with added seeds (curve e), the peak at 408 nm wavelength almost disappeared compared to before encapsulation (curve a), indicating that there was almost no Cyt c residue in the supernatant, meaning that the vast majority of Cyt c was encapsulated within the ZIF-8. Conversely, under the conditions of 2 hours and 4 hours of reaction without added seeds (curves b and c), a large amount of Cyt c remained in the supernatant, with only a slight reduction compared to the residue before encapsulation, and even far less than the reduction in residue achieved under the condition of 2 hours of reaction with added seeds. This demonstrates that the seed-directed synthesis method can effectively improve the encapsulation ability of MOFs for enzymes. Figure 3 The XRD results of B show that the Cytc@ZIF-8 composite material has the same Bragg diffraction pattern as pure ZIF-8, which proves that the biocomposite material has the same crystal form as pure ZIF-8. Figure 3 SEM results for C indicate that the Cytc@ZIF-8 composite material exhibits the same typical sodalite morphology as pure ZIF-8, and its particle size (800-1000 nm) is larger than that of the seed crystals (200 nm-300 nm). Figure 2 As shown in the figure, this demonstrates the successful implementation of the seed-guided synthesis method.

[0036] at the same time, Figure 4 This also demonstrates the feasibility, efficiency, and versatility of the seed-guided synthesis method.

[0037] Example 2: Effect of Seed Amount and Reaction Time on Packaging Efficiency

[0038] Using the synthesis method of Example 1, 1 mL of seed crystal solution (with concentrations of 0 mg / mL, 1 mg / mL, 3 mg / mL, 5 mg / mL, and 7 mg / mL, a total of 5 groups) was added to 1 mL of Cyt c (cytochrome C) solution (1 mg / mL). After thorough mixing, 2 mL of 2-methylimidazole solution (1400 mM) and 2 mL of zinc acetate solution (100 mM) were added sequentially, and the reaction was allowed to proceed at room temperature. Each group of seed crystal solution with different concentrations had reaction times of 0.5 h, 1 h, 2 h, and 4 h, for a total of 4 groups, resulting in a total of 20 experiments. After each reaction, the supernatant was centrifuged, and the UV-Vis absorbance was measured. The encapsulation efficiency was calculated by comparing the changes in absorbance before and after the reaction. The final measurement results are as follows: Figure 5 As shown, when the amount of seed crystals used is 3-7 mg / mL, the enzyme encapsulation effect reaches more than 70%, especially when the amount of seed crystals used is 5-7 mg / mL, the enzyme encapsulation effect is as high as 90% or more.

[0039] Example 3: Effect of Seed-Directed Method on Cytochrome C (Cyt c) Activity

[0040] Prepare PBS buffer (100 mM, pH 7.4), ABTS solution (2 mM), hydrogen peroxide solution (1 mM), Cytc@ZIF-8 solution (synthesized in Example 1), and free Cytc solution (the total amount of free Cytc was consistent with the amount of Cytc in Cytc@ZIF-8). First, mix 50 μL of Cytc@ZIF-8 or free Cytc solution with 50 μL of PBS (100 mM, pH 7.4). Then, add 100 μL of ABTS solution (2 mM) and 50 μL of H2O2 solution (1 mM), respectively. The increase in absorbance at 420 nm after 10 minutes of reaction was measured using a UV-Vis spectrophotometer to reflect biological activity. The relative activity of cytochrome C under various conditions was calculated using the activity of free cytochrome C as a standard.

[0041] like Figure 6 As shown, the activity of encapsulated cytochrome C was 5.6 times higher than that of free cytochrome C. Meanwhile, when free cytochrome C was mixed with each of the solutions required for the seed synthesis method and their activities were measured individually, no increase in activity was observed. These results indicate that the increased activity of encapsulated cytochrome C is due to its encapsulation within ZIF-8 via the seed synthesis method.

[0042] Example 4: Effect of a visual sensing system for detecting hydrogen peroxide and a seed-guided method on the activity of hydrogen peroxide.

[0043] Prepare PBS buffer (100 mM, pH 7.4), ABTS solution (2 mM), hydrogen peroxide solutions of different concentrations (0.05 mM, 0.1 mM, 0.3 mM, 0.5 mM, 1 mM), Cytc@ZIF-8 solution, and free Cytc solution (the total amount of free Cytc was consistent with the amount of Cytc in Cytc@ZIF-8). First, mix 50 μL of Cytc@ZIF-8 or free Cytc solution with 50 μL of PBS (100 mM, pH 7.4). Then add 100 μL of ABTS solution (2 mM) and 50 μL of H2O2 solution, respectively. Measure the absorbance of the mixture at 420 nm after 10 minutes of reaction using a UV-Vis spectrophotometer to construct a linear fitting curve between hydrogen peroxide concentration and absorbance.

[0044] like Figure 7 As shown, compared with the detection of hydrogen peroxide using free cytochrome C, the detection of hydrogen peroxide using Cytc@ZIF-8 has better sensitivity and a higher linear detection range, and the visually observable detection limit is 0.1 mM, which is much lower than the 0.5 mM of free cytochrome C.

[0045] Comparative Example 1: Conventional one-pot synthesis of enzyme@MOF complex

[0046] 1 mL of enzyme (cytochrome C, myoglobin, or hemoglobin) solution (1 mg / mL), 2 mL of 2-methylimidazole solution (1400 mM), and 2 mL of zinc acetate solution (100 mM) were thoroughly mixed, and the mixture was allowed to stand at room temperature for 4 hours. After the reaction was complete, the product was collected by centrifugation at 8000 rpm for 10 minutes. The supernatant obtained after centrifugation was used to measure the absorption peak at specific wavelengths using a UV-Vis spectrophotometer (408 nm for cytochrome C, and 405 nm for myoglobin and hemoglobin) to determine the enzyme / protein content encapsulated within the MOF. The test results are as follows: Figure 3 (A) diagram and Figure 4 Figures (A) and (B) show the groups without seeds. Compared with the seed-directed synthesis method, this shows that the seed-directed synthesis method can effectively improve the encapsulation ability of MOFs for enzymes.

[0047] In MOF-encapsulated enzyme technology, MOF nucleation and growth are crucial steps. This invention addresses the nucleation problem from the perspective of MOF synthesis, innovatively proposing a seed-based method for synthesizing enzyme@MOF complexes. The seed-based method is a common method for synthesizing porous materials. MOF crystal synthesis mainly includes nucleation and growth stages. In the initial stage of MOF crystal synthesis, the nucleus forms slowly; when the nucleus reaches a critical size, crystal growth begins. For example... Figure 1 As shown, without the addition of seed crystals, the slow formation of nuclei leads to low enzyme encapsulation efficiency. However, by adding seed crystals during the MOF encapsulation process, the slow nucleation stage can be skipped, achieving rapid synthesis of the enzyme@MOF complex. Since the nucleus is provided by the seed crystals, this method avoids both the slow nucleation of co-precipitation methods and the stringent requirements on enzyme properties imposed by biomimetic mineralization methods. It combines the advantages of both methods, making it a highly promising and reliable encapsulation method.

[0048] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A method for guiding highly efficient and universal synthase@MOF complexes via seed crystals, characterized in that, In the process of encapsulating enzymes using MOF, the slow nucleation stage is skipped by adding MOF-corresponding seed crystals, thereby achieving the goal of rapidly synthesizing enzyme@MOF complexes. The method for seed-guided, highly efficient, and universal synthase@MOF complexes is as follows: S1. Seed crystals are prepared by reacting 2-methylimidazole solution and zinc acetate solution with stirring at room temperature for 12-36 hours; the concentration of 2-methylimidazole solution is 2000-3000 mM, the concentration of zinc acetate solution is 30-50 mM, and the volume ratio of 2-methylimidazole solution to zinc acetate solution is 1:

1. S2. After mixing the seed crystal solution and enzyme solution, add 2-methylimidazole solution and zinc acetate solution, and then prepare the enzyme@MOF complex by static reaction at room temperature; the concentration of the seed crystal solution is 4-6 mg / mL, the enzyme includes cytochrome C, hemoglobin, and myoglobin, the MOF is ZIF-8 metal-organic framework, and the static reaction time is 3-5 h; the concentration of the enzyme solution is 0.5-2 mg / mL, the concentration of the 2-methylimidazole solution is 1200-1600 mM, the concentration of the zinc acetate solution is 90-110 mM, and the volume ratio of the seed crystal solution, enzyme solution, 2-methylimidazole solution and zinc acetate solution is 1:1:2:

2.

2. The application of the method of claim 1 in the synthesis of MOF immobilized enzymes.

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