In-situ co-encapsulated laccase and mediator

Abstract

The invention discloses an in-situ co-encapsulation laccase and a mediator. According to the in-situ co-encapsulated laccase disclosed by the invention, a covalent organic framework synthesized by 2, 5-bis (2-methoxyethoxy) terephthaloyl hydrazine and benzenetricarboxaldehyde is taken as a carrier, and laccase and a mediator are loaded in the carrier. According to the invention, the enzyme and the mediator are co-encapsulated in the covalent organic framework in situ under mild conditions (aqueous solution and room temperature) to prepare the high-performance biocatalyst, the combination of high affinity, high stability and high loading of the laccase and the carrier is realized, the biocatalytic activity of the laccase is improved, and the method can be used for removing pollutants.

Description

Technical Field

[0001] The invention belongs to the technical field of catalysts, and particularly relates to in-situ co-encapsulation of laccase and a mediator. Background Art

[0002] Laccase (Lac) is a typical copper-containing polyphenol oxidase. Due to the polysaccharide protein and catalytic copper clusters in its structure, it has high catalytic activity and high specificity. Currently, this enzyme has been widely used in chemical processes and industrial uses such as bioremediation, dye decolorization, and wastewater detoxification. However, the contradiction between its fragile three-dimensional structure and the harsh actual operating conditions (temperature, acidity and alkali, and recyclability) has severely limited its large-scale industrial application.

[0003] Enzyme immobilization, i.e. spatial immobilization of specific enzymes using solid support materials or carriers, has shown great potential in improving enzyme stability and reuse. Different from porous materials such as metal organic frameworks (MOFs), hydrogen bonded organic frameworks (HOFs), and carbon nanotubes, covalent organic frameworks (COFs) have unique covalent bond support, crystallinity, and good biocompatibility, making them ideal immobilization platforms in the strategy of enzyme immobilization; currently, this material has been effectively used as a candidate carrier for a variety of enzymes.

[0004] Although COFs are regarded as an advantageous carrier for enzyme immobilization, there are still problems and challenges in using COFs to achieve enzyme immobilization. First, due to the harsh synthesis conditions of COFs, it is very rare to use a simple "one-pot method" to simultaneously complete enzyme immobilization; second, the biocatalytic efficiency of the prepared materials is not enough to be comparable to that in the natural state. COFs can tightly fix the enzyme while it is difficult to take into account its own flexibility, which can also lead to subtle changes in the optimal conformation of the enzyme. At the same time, the internalization of the enzyme will increase the distance between the substrate and the active center at the microscopic level, thereby reducing the biocatalytic efficiency of the material.

[0005] Mediators are small molecules with low redox potentials that easily gain or lose electrons. Under the oxidation of enzymes, they form stable intermediates with high redox potentials, which contribute to high redox potentials and shorten the distance between substrates and the active center of enzymes, thereby improving biocatalytic efficiency. However, currently, mediators are mostly added to the reaction solution together with enzymes, which also makes their loading capacity low and difficult to reuse. Therefore, the present invention designs the use of COFs to co-encapsulate enzymes and mediators in situ and thereby enhance the activity of biocatalysts. Summary of the invention

[0006] One of the purposes of the present invention is to provide an in-situ co-encapsulated laccase and mediator, using a covalent organic framework synthesized from 2,5-bis(2-methoxyethoxy)terephthaloylhydrazide and trimesicaldehyde as a carrier, in which the laccase and the mediator are loaded.

[0007] Furthermore, the mediator is syringaldehyde.

[0008] Furthermore, the usage ratio of the mediator to the laccase is 12-20 mg / g.

[0009] The second object of the present invention is to provide a method for preparing the above-mentioned in situ co-encapsulated laccase, comprising the following steps: mixing a laccase aqueous solution with a mediator, adding acetic acid for incubation; then adding a trimesaldehyde aqueous solution and acetic acid for incubation; finally adding a 2,5-bis(2-methoxyethoxy)terephthaloylhydrazide aqueous solution and catalyst acetic acid, allowing the reaction to stand at room temperature, collecting the precipitate generated by the reaction, and obtaining the in situ co-encapsulated laccase.

[0010] Furthermore, the usage ratio of the laccase, trimesaldehyde and 2,5-bis(2-methoxyethoxy)terephthaloylhydrazide is 12.5 mg:0.04 mM:0.06 mM.

[0011] In one embodiment of the present invention, 0.1 mL of laccase aqueous solution was first mixed with the mediator syringaldehyde, and 10 μL of acetic acid was added for incubation; then 0.9 mL of trimesaldehyde aqueous solution and 10 μL of acetic acid were added for incubation; finally, 0.9 mL of 2,5-bis(2-methoxyethoxy)terephthaloylhydrazide aqueous solution and 40 μL of acetic acid were added, and the reaction was allowed to stand at room temperature, and the precipitate generated by the reaction was collected to obtain the in situ co-encapsulated laccase Lac-SA@BT-BMTH-COF.

[0012] The third object of the present invention is to provide the use of the above-mentioned in situ co-encapsulated laccase and mediator in catalytic degradation of pollutants.

[0013] Furthermore, the pollutants are tetracycline compounds and sulfonamide drugs.

[0014] The present invention successfully co-encapsulated Lac and SA in COF through an in-situ encapsulation strategy under mild conditions (room temperature, water), and synthesized Lac-SA@BT-BMTH-COF. The COF framework provides good support for Lac, greatly improving its tolerance to temperature and pH while having both reusability and storage stability. Enzyme kinetic studies have shown that this strategy can significantly improve the biocatalytic efficiency of the material (about twice that of traditional in-situ encapsulation strategies and 1.5 times that of natural enzymes). The biocatalytic reactor constructed in this way has shown faster pollutant removal efficiency and a wider spectrum of enzyme-catalyzed substrates in environmental remediation. In particular, for tetracyclines and sulfonamides, the removal rates can reach up to 82.0% and 86.6% respectively within 24 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1Infrared spectra of Lac, Lac@BT-BMTH-COF and their synthetic monomers BT and BMTH.

[0016] Figure 2 PXRD patterns of Lac@BT-BMTH-COF and Lac-SA@BT-BMTH-COF.

[0017] Figure 3 Laser confocal microscopy image of Lac-SA@BT-BMTH-COF.

[0018] Figure 4 The changes in the appearance of the composite material before and after the introduction of SA.

[0019] Figure 5 X-ray photoelectron spectra of Lac@BT-BMTH-COF (A, B) and Lac-SA@BT-BMTH-COF (C, D).

[0020] Figure 6 Relative activities of Lac-SA@BT-BMTH-COF and free Lac at different temperatures (A), pH (B), storage time (C), and after multiple cycles (D).

[0021] Figure 7 Michaelis-Menten curves (A, C, E) and Lineweaver-Burk double reciprocal plots (B, D, F) of Lac, Lac@BT-BMTH-COF, and Lac-SA@BT-BMTH-COF.

[0022] Figure 8 The removal capacity of TC with different mediator dosages (A), and the comparison of TC removal capacity between Lac@BT-BMTH-COF and Lac-SA@BT-BMTH-COF (B).

[0023] Fig. 9 The removal capacity of SMZ with different mediator dosages (A) and the comparison of the removal capacity of SMZ between Lac@BT-BMTH-COF and Lac-SA@BT-BMTH-COF (B). DETAILED DESCRIPTION

[0024] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0026] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0027] Comparative Example 1

[0028] BT-BMTH-COF in situ encapsulation of enzymes

[0029] 0.06mM 2,5-bis(2-methoxyethoxy)terephthaloylhydrazide (BMTH) and 0.08mM trimesic acid (BT) were dispersed in 0.9mL water and sonicated until completely dissolved. Then, 0.2mL 25mg / mL laccase aqueous solution (Lac) and 10μL acetic acid were dropped into the BT solution, incubated for 5 minutes, BMTH solution was added and 50μL acetic acid was added again as a catalyst, and a large amount of flocculent precipitate was formed after standing at room temperature (~30°C) for 20min. Subsequently, the flocculent precipitate was collected by centrifugation, washed three times with water, and freeze-dried to obtain the in situ encapsulated enzyme Lac@BT-BMTH-COF.

[0030] Examples 1-5

[0031] BT-BMTH-COF in situ co-encapsulation of enzyme and mediator

[0032] 0.06 mM 2,5-bis(2-methoxyethoxy)terephthaloylhydrazide (BMTH) and 0.04 mM trimesic acid (BT) were dispersed in 0.9 mL of water and sonicated until completely dissolved. Then, 0.1 mL of 50 mg / mL laccase aqueous solution was mixed with different amounts of syringaldehyde (SA, the ratio of syringaldehyde to laccase was 12, 14, 16, 18, and 20 mg / g, respectively) solution and incubated with 10 μL of acetic acid for 5 min; then, BT solution was added and 10 μL of acetic acid was added again for incubation for 5 min; finally, BMTH solution and 40 μL of acetic acid were added, and after standing at room temperature (~30°C) for 20 min, a large amount of flocculent precipitate was formed. Subsequently, the flocculent precipitate was collected by centrifugation, washed three times with water, and freeze-dried to obtain the in situ co-encapsulated enzyme Lac-SA@BT-BMTH-COF.

[0033] Test Example 1

[0034] The in situ co-encapsulated enzyme with a dosage ratio of 14 mg / g of syringaldehyde to laccase was used for structural analysis.

[0035] FTIR results show that the broad peak of carboxyl in Lac (≈2900 cm -1 ,≈3400cm -1) appears in Lac@BT-BMTH-COF and at ≈1620 cm -1 A new peak (-CH=N-) appears at Figure 1 ). PXRD results show that the introduction of the mediator does not affect the crystallinity of the material ( Figure 2 ). In order to verify the distribution of Lac in the composite material, fluorescein isothiocyanate (FITC) was used for labeling and confocal laser scanning microscopy (CLSM) was used. The results showed that Lac-SA@BT-BMTH-COF exhibited a uniformly distributed fluorescent green ( Figure 3 In addition, due to the generation of free radicals, the appearance color of the composite material changed from white to yellow after the introduction of SA ( Figure 4 ). X-ray photoelectron spectroscopy (XPS) analysis shows that in the C1s high-resolution spectrum of Lac-SA@BT-BMTH-COF, four peaks with binding energies (BE) of 284.46, 285.90, 287.57, and 290.28 eV can be attributed to CC / CH, CN / CO, C=N, and C=O, respectively; CC / CH and CN / CO correspond to benzene ring, amino group, and carboxyl group, respectively, which come from the ligands of Lac-SA@BT-BMTH-COF and the free residues on Lac; the presence of C=N and C=O is the residual group after the amine-aldehyde condensation reaction; in addition, the high-resolution spectrum of N1s of Lac-SA@BT-BMTH-COF can be decomposed into three main peaks at 399.47, 399.93, and 402.95 eV, which are attributed to CN, C=N, and -NH2( Figure 5 ).

[0036] Test Example 2

[0037] The enzyme activity detection used in this test example is to determine the activity of Lac, Lac@BT-BMTH-COF and Lac-SA@BT-BMTH-COF by oxidizing 2,4-dichlorophenol (2,4-DP) in the presence of 4-aminoantipyrine (4-AP), and its product can be detected colorimetrically at UV510nm. Specifically: First, 2,4-DP (2.0 mg / mL, 250 μL) and 4-AP (2.0 mg / mL, 250 μL) solutions were added to 4 mL of acetate buffer (0.2M, pH=6.0), and then 500 μL of enzyme preparation (1.0 mg / mL, suspended water) was added to the above solution and mixed. The reaction was carried out at room temperature for 30 minutes, and the absorbance of the supernatant was measured at UV 510nm.

[0038] 1. Study on the enzymatic properties of immobilized laccase and free laccase

[0039] High activity retention, cyclic catalysis, and long storage are the remarkable characteristics of immobilized enzymes. In view of this, the evaluation of the enzyme activity retention ability of different materials under harsh conditions was carried out here. In order to evaluate the temperature tolerance of the composite material, Lac-SA@BT-BMTH-COF or laccase was dispersed in water and treated at different temperatures (30℃, 40℃, 50℃, 60℃, 70℃) for 1.0h, and the catalytic activity was measured after cooling to room temperature. In order to study the pH stability of the composite material, Lac-SA@BT-BMTH-COF or laccase was incubated in a series of pH buffers (pH=4, 5, 6, 7, 8) for 1.0h, and then the catalytic activity was measured. Similarly, to study its storage stability, Lac-SA@BT-BMTH-COF or laccase was dispersed in water and stored at 4℃ for 9d. During this period, its residual catalytic activity was measured at predetermined time intervals (1, 3, 5, 7, 9d). Finally, the reproducible catalytic ability of Lac-SA@BT-BMTH-COF was investigated by centrifugal recovery and residual activity determination. The temperature tolerance results showed that Lac-SA@BT-BMTH-COF maintained about 90% of its catalytic activity even at 70°C ( Figure 6 A) while the free enzyme has dropped to below 40%, which is due to the internalization of the enzyme, which allows the COFs template to provide structural protection similar to an "exoskeleton", thereby effectively limiting the spatial structural distortion of the enzyme. Similarly, the pH stability results show that the optimal pH for both Lac-SA@BT-BMTH-COF and free enzyme is 6, which also shows that the in situ co-encapsulation of COFs does not change the properties of the enzyme itself; at pH = 8, Lac-SA@BT-BMTH-COF can still retain ≥ 80% of its catalytic activity, while the free enzyme has dropped to below 70% ( Figure 6 In terms of long-term storage, the catalytic activity of both showed different degrees of loss. After 9 days, the relative catalytic activity of Lac-SA@BT-BMTH-COF still maintained about 85% of its activity, while the free enzyme only maintained 60% of its initial enzyme activity ( Figure 6 C). The results of repeated use showed that Lac-SA@BT-BMTH-COF could still retain more than 80% of its activity after 5 cycles of catalysis ( Figure 6 (middle D).

[0040] 2. Enzyme-catalyzed reaction kinetics

[0041] In order to determine the kinetic parameters of 2,4-DP oxidation by Lac@BT-BMTH-COF, Lac-SA@BT-BMTH-COF and Lac, the initial reaction rates (V) at different substrate concentrations (0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14, 0.16, 0.18, 0.20 mg / mL) were calculated according to formula (1).o ). Where ε is the product extinction coefficient (13.6mM -1 cm -1 ), L is the optical path length (1 cm), and T is the reaction time (5 min). The V value was plotted as a function of substrate concentration ([S]) and fitted to the Michaelis-Menten equation according to formula (2), and the kinetic parameters were further calculated by the Lineweaver-Burk double inverse equation. V max is the maximum reaction rate, K m Represents the affinity of the enzyme for the substrate. m The smaller the value, the higher the affinity of the enzyme for the substrate. The catalytic rate constant (K) is calculated according to formula (3): cat )([E]: enzyme concentration). The catalytic efficiency was calculated according to formula (4), and the catalytic efficiency (K cat / K m ) is used as a criterion for judging the catalytic activity of composite materials.

[0042] V0=[A 510 / (ε·L)] / T (1)

[0043] 1 / V0=K m / V max (1 / [S]+1 / [K m ]) (2)

[0044] K cat =V max / [E] (3)K cat / K m (4)

[0045] Table 1. Catalytic kinetic parameters

[0046]

[0047] K of a single fixed Lac@BT-BMTH-COF m The K value of Lac-SA@BT-BMTH-COF was 0.738 mM, which was much higher than that of free laccase, indicating that it had poor affinity for the substrate. m The value is 0.477mM, indicating that its affinity for the substrate is significantly improved compared with the single fixed material. At the same time, the biocatalytic efficiency of Lac@BT-BMTH-COF is 0.073mM -1 min -1 , which is about 30% lower than that of free Lac. In contrast, the Lac-SA@BT-BMTH-COF with the introduction of the mediator showed the expected results in terms of catalytic efficiency. Its biocatalytic efficiency was 0.144mM -1 min -1, which is about twice the catalytic efficiency of Lac@BT-BMTH-COF and 1.5 times that of the natural enzyme.

[0048] Test Example 3

[0049] Evaluation of tetracycline hydrochloride (TC) removal capacity

[0050] TC was used to demonstrate the effect of mediators in improving the proximity of substrates and enzymes at the microscopic scale. 1.0 mL of 20 mg / L TC solution was added to 1.0 mL of sodium acetate buffer (0.2 M pH = 6.0), which contained 2.0 mg Lac or a solid-loaded material with an equal amount of enzyme (Lac@COFs, Lac-SA@COFs). The enzymatic hydrolysis system was reacted in a centrifuge tube. Under the initial conditions, no sacrificial agent or additive was added, and it was stirred at room temperature for 24 hours. The results showed that when only Lac was present, the removal rate of TC was only 32.0%, while after adding 12 mg / g SA, the removal rate of TC could reach 88.0%; by further increasing the dosage of SA to 16 mg / g, TC could be completely removed ( Figure 8 A). On this basis, free enzyme, Lac@BT-BMTH-COF, and Lac-SA@BT-BMTH-COF (containing the same amount of enzyme) were dispersed in acetic acid buffer and subjected to degradation reaction under the same conditions. Since COF has a high specific surface area and high porosity, the empty COF exhibits a good adsorption effect on TC, of ​​which BT-BMTH-COF can reach 42.0%; with the introduction of Lac, the porosity of the composite material will be significantly reduced, thereby affecting the adsorption capacity (reduced to 28.0%); compared with Lac@COFs, the introduction of the mediator can effectively shorten the distance between the active site and the substrate at the microscopic scale, and improve the removal effect of TC, Lac-SA@BT-BMTH-COF can be as high as 82.0% ( Figure 8 (B).

[0051] Test Example 4

[0052] Evaluation of sulfamethoxazole (SMZ) removal capacity

[0053] 1.0mL of 20mg / L SMZ solution was added to 1.0mL of sodium acetate buffer (0.2M pH=6.0), which contained 2.0mg Lac or solid-loaded materials with equal amounts of enzymes (Lac@COFs, Lac-SA@COFs). The enzymatic hydrolysis system was reacted in a centrifuge tube. Under the initial conditions, no sacrificial agent or additive was added, and it was stirred at room temperature for 24 hours. The results showed that when only Lac was present, SMZ had almost no degradation effect, but with the addition of SA, SMZ showed an obvious removal trend. When the dosage of SA increased to 16mg / g, SMZ could reach the maximum removal value ( Fig. 9 A in the middle). On this basis, free enzyme, Lac@BT-BMTH-COF, and Lac-SA@BT-BMTH-COF (containing the same amount of enzyme) were dispersed in acetic acid buffer and subjected to degradation reaction under the same conditions. The results show that for SMZ, Lac-SA@COFs exhibited a better removal efficiency than Lac@COFs materials, among which Lac@BT-BMTH-COF was 41.86%, which mainly relied on the adsorption effect provided by the porous structure in the COFs template; Lac-SA@BT-BMTH-COF could reach 86.56%. Compared with Lac@COFs, the introduction of the mediator can improve the redox potential of Lac and expand its catalytic substrate spectrum ( Fig. 9 (B).

Claims

1. An in situ co-encapsulation of laccase and mediator, characterized in that The covalent organic framework synthesized from 2,5-bis(2-methoxyethoxy)terephthaloylhydrazide and trimesicaldehyde is used as a carrier, in which laccase and a mediator are loaded.

2. The in situ co-encapsulated laccase and mediator according to claim 1, characterized in that The mediator is syringaldehyde.

3. The in situ co-encapsulated laccase and mediator according to claim 2, characterized in that: The dosage ratio of the mediator to the laccase is 12-20 mg / g.

4. The method for preparing the in situ co-encapsulated laccase and mediator according to any one of claims 1 to 3, characterized in that: The laccase aqueous solution is mixed with the mediator, and acetic acid is added for incubation; then, a trimesaldehyde aqueous solution and acetic acid are added for incubation; finally, a 2,5-bis(2-methoxyethoxy)terephthaloylhydrazide aqueous solution and catalyst acetic acid are added, and the reaction is allowed to stand at room temperature, and the precipitate generated by the reaction is collected to obtain an in situ co-encapsulated laccase.

5. The preparation method according to claim 4, characterized in that The dosage ratio of the laccase, trimesaldehyde and 2,5-bis(2-methoxyethoxy)terephthaloylhydrazide is 2.5 mg: 0.04 mM: 0.06 mM.

6. Use of the in situ co-encapsulated laccase and mediator according to any one of claims 1 to 3 in catalytic degradation of pollutants.

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