阅读说明：本技术 一种MOFs固载酶及其制备方法和应用 (MOFs immobilized enzyme and preparation method and application thereof ) 是由 任建军 倪申生 龚磊 李春雨 赵建 牛东泽 张晋 于 2021-08-28 设计创作，主要内容包括：本发明涉及一种MOFs固载酶及其制备方法和应用,属于材料与生物工程交叉技术领域。MOFs固载酶的制备方法,包括：将等量的红霉素降解酶与MOFs材料溶解于tris-HCl,调整溶液pH为6.0～8.0,25～35℃搅拌2～3小时,冷冻干燥得到MOFs固载酶ereB@Cu-BTC。MOFs固定化酶具有显著的稳定性和可回收性,可以有效降解含红霉素的菌渣、污水、环境中的水体,有利于在产业化中的放大应用。(The invention relates to a MOFs immobilized enzyme, a preparation method and an application thereof, belonging to the technical field of crossing of materials and biological engineering. The preparation method of the MOFs immobilized enzyme comprises the following steps: dissolving the same amount of erythromycin degradation enzyme and MOFs material in tris-HCl, adjusting the pH of the solution to 6.0-8.0, stirring for 2-3 hours at 25-35 ℃, and freeze-drying to obtain the MOFs immobilized enzyme ereB @ Cu-BTC. The MOFs immobilized enzyme has remarkable stability and recoverability, can effectively degrade bacterium residues containing erythromycin, sewage and water in the environment, and is favorable for large-scale application in industrialization.)

1. A preparation method of MOFs immobilized enzyme is characterized by comprising the following steps: dissolving the same amount of erythromycin degradation enzyme and MOFs material in tris-HCl, adjusting the pH of the solution to 6.0-8.0, stirring for 2-3 hours at 25-35 ℃, and freeze-drying to obtain the MOFs immobilized enzyme ereB @ Cu-BTC.

2. The process according to claim 1, wherein said MOFs is Cu-BTC.

3. The process for preparing MOFs immobilized enzymes according to claim 1, wherein said erythromycin degrading enzyme is ereB enzyme.

4. The method for preparing MOFs immobilized enzymes according to claim 1, wherein HCl and tris are used to adjust the pH of the solution to 6.0-8.0.

5. The method for preparing MOFs immobilized enzymes according to claim 1, wherein said specific preparation method of MOFs materials is as follows:

dissolving copper nitrate trihydrate, glacial acetic acid and trimethylamine in ethanol, and stirring for 1 hour at room temperature by using a magnetic stirrer;

adding trimesic acid into the solution, and stirring the solution for 1 hour at 30 ℃ by using a magnetic stirrer, wherein the molar ratio of the copper nitrate trihydrate, the glacial acetic acid, the trimethylamine to the trimesic acid is 3: 3: 1: 1;

adding into a reaction kettle, and heating for 24 hours at 85 ℃;

centrifuging, recovering and fixing the product, and washing for 3 times by using ethanol;

treating the product with ethanol at 30 deg.C for 12 hr;

and (4) carrying out freeze drying on the product at-65 to-45 ℃ to obtain a final product.

6. The MOFs immobilized enzyme prepared by the method according to any one of claims 1 to 5.

7. The use of the MOFs immobilized enzymes of claim 6 for degrading erythromycin-containing bacterial residues, sewage, water bodies in the environment.

Technical Field

The invention relates to a MOFs immobilized enzyme, a preparation method and an application thereof, belonging to the technical field of crossing of materials and biological engineering.

Background

In recent years, as the yield of erythromycin is continuously increased, the content of erythromycin mushroom dregs is also continuously increased, and the harm to human is also increased day by day, firstly, the microorganisms are excessively propagated by the surplus nutrient substances in the mushroom dregs, and secondly, the drug-resistant microorganisms are enriched to change the flora structure of the original environment, so that more safe methods are urgently needed for treating the antibiotic mushroom dregs. The conventional erythromycin mushroom residue treatment methods mainly comprise incineration treatment, anaerobic digestion technical treatment, feed treatment, fertilizer treatment, mushroom residue purification and energy treatment, and the treatment methods are high in treatment cost and cause other environmental problems. The method for treating the erythrocin fungi residues by the biological method has the advantages of low treatment cost, no secondary pollution and the like.

Currently, most of the biological treatment research focuses on screening and isolation of erythromycin-degrading bacteria, and research on erythromycin-degrading enzymes is less. Erythromycin is generally composed of a lactone ring, a erythromycin sugar, and an oxyaminosugar. In the process of degrading erythromycin, the hydrolysis of the lactone ring is catalyzed by erythromycin esterase, the enzyme catalysis plays a key role, and the activity of the erythromycin esterase is a factor directly related to the degradation efficiency of erythromycin. The existing erythromycin degrading enzyme ereB can effectively degrade erythromycin under mild conditions, but the application cost is high due to the fact that the enzyme cannot be recycled, and therefore popularization and application of the enzyme ereB are limited.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects of the prior art and provide the MOFs immobilized enzyme and the preparation method and the application thereof, wherein the MOFs immobilized enzyme has obvious stability and recoverability, can effectively degrade bacterial residues containing erythromycin, sewage and water in the environment, and is favorable for the large-scale application in industrialization.

In order to solve the technical problem, the invention provides a preparation method of MOFs immobilized enzyme, which comprises the following steps: dissolving the same amount of erythromycin degrading enzyme and MOFs material in tris-HCl, adjusting the pH to 6.0-8.0, stirring for 2-3 hours at 25-35 ℃, and freeze-drying to obtain the MOFs immobilized enzyme ereB @ Cu-BTC.

Preferably, the MOFs are Cu-BTC.

Preferably, the erythromycin-degrading enzyme is an ereB enzyme.

Preferably, the freeze drying temperature is-65 to-45 ℃.

Preferably, HCl and tris are used to adjust the pH of the solution to 6.0-8.0.

Preferably, the specific preparation method of the MOFs material is as follows:

dissolving copper nitrate trihydrate, glacial acetic acid and trimethylamine in ethanol, and stirring for 1 hour at room temperature by using a magnetic stirrer;

adding trimesic acid into the solution, and stirring the solution for 1 hour at 30 ℃ by using a magnetic stirrer, wherein the molar ratio of the copper nitrate trihydrate, the glacial acetic acid, the trimethylamine to the trimesic acid is 3: 3: 1: 1;

adding into a reaction kettle, and heating for 24 hours at 85 ℃;

centrifuging, recovering and fixing the product, and washing for 3 times by using ethanol;

treating the product with ethanol at 30 deg.C for 12 hr;

and (4) carrying out freeze drying on the product at-65 to-45 ℃ to obtain a final product.

The invention also provides the MOFs immobilized enzyme prepared by the method.

The invention also provides application of the MOFs immobilized enzyme in degrading bacterium residues containing erythromycin, sewage and water in the environment.

The invention achieves the following beneficial effects:

the Metal-Organic Frameworks (MOFs) have the characteristics of large specific surface area, adjustable porosity, designable functionality, good stability and the like, and have very good prospects as substrate materials for enzyme immobilization. The MOFs immobilized enzyme has remarkable stability and recoverability, can effectively degrade bacterium residues containing erythromycin, sewage and water in the environment, and is favorable for large-scale application in industrialization.

Drawings

FIG. 1 is a Cu-BTC material prepared;

FIG. 2 shows the enzyme activities of ereB @ Cu-BTC at different temperatures;

FIG. 3 is the enzyme activity of ereB @ Cu-BTC at different pH;

FIG. 4 is the degradation rate of ereB @ Cu-BTC application in erythromycin fermentation broth;

FIG. 5 is the degradation rate of ereB @ Cu-BTC in an erythromycin-containing water body.

Detailed Description

The present invention will be further described with reference to the following examples. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.

Example 1

The invention provides a preparation method of MOFs immobilized enzyme, which comprises the following steps:

1. preparation of Cu-BTC Material

Cu (NO3) 2.3H 2O (0.435g, 1.8mmol), glacial acetic acid (0.62mL, 10.8mmol), and trimethylamine 0.50mL were dissolved in 12mL of ethanol at room temperature. After stirring at room temperature for 1H, H is added₃BTC (0.210g, 1.0mmol) was added to the dark blue solution. The mixture was stirred for an additional 2 hours to form a homogeneous solution. The mixture was then transferred to a teflon lined autoclave. The mixture was heated at 85 ℃ for 24 hours. The solid was recovered by centrifugation and washed three times with ethanol. Finally, the product was treated with ethanol at 30 ℃ for 12 h. Freeze drying at-65 deg.C to-45 deg.C to obtain Cu-BTC product (shown in figure 1).

Immobilization of ereB enzyme

Dissolving an equivalent amount of an erythromycin degradation enzyme ereB and a MOFs material in tris-HCl, adjusting the pH of the solution to 8.0 by adopting HCl and tris, stirring at 30 ℃ for 2h, and freeze-drying at-65 to-45 ℃ to obtain a final product ereB @ Cu-BTC.

Example 2

And (3) measuring the enzyme activity of the MOFs immobilized enzyme:

1. enzyme activity at different temperatures

Adding 1mL of erythromycin methanol solution with the concentration of 10g/L into 100mL of ultrapure water to prepare 100mg/L erythromycin solution; then 0.1g of prepared ereB @ Cu-BTC is added and respectively placed in water bath shaking tables with the temperature of 25 ℃, 35 ℃, 45, 55 and 65 ℃ for reaction for 10 minutes; the enzyme activity was measured at the different temperatures described above (see FIG. 2).

As can be seen from FIG. 2, the optimum reaction temperature for ereB @ Cu-BTC was 55 ℃.

2. Enzyme activity under different pH conditions

1mL of 10g/L erythromycin methanol solution was added to 100mL of Na2HPO4-NaH2PO4 buffer (pH =7, 7.5, 8), Tris-HCl (pH =8, 8.5, 9), Gly-NaOH (pH =9, 9.5, 10), respectively, to prepare 100mg/L erythromycin solutions at different pH; then 0.1g of prepared ereB @ Cu-BTC is added and placed in a water bath shaking table at the temperature of 55 ℃ for reaction for 10 minutes; the enzyme activity was measured at different pH as described above (see FIG. 3).

As can be seen from FIG. 3, the optimum reaction pH for ereB @ Cu-BTC was 8.0, and Tris-HCl was more preferable.

Example 3

The application of ereB @ Cu-BTC in the mushroom dregs is as follows:

taking 100mL of erythromycin mushroom residue, and determining the concentration of erythromycin to be about 650 mg/L; then 0.65g of the prepared ereB @ Cu-BTC was added and the mixture was left to react for 30min at room temperature under normal pressure. Stirring to promote catalysis, wherein the degradation rate reaches 80% in 15min (as shown in figure 4), centrifuging to collect ereB @ Cu-BTC, and repeating the steps to recycle the recovery rate to 70%.

The control group was prepared by adding an equal amount of unsupported ereB enzyme to the same system.

As can be seen from FIG. 4, the ereB @ Cu-BTC degradation rate in the mushroom dregs in about 15 minutes is greater than 80%, the effect is better, and the degradation efficiency and rate are better compared with those of free enzyme.

Example 4

The application of ereB @ Cu-BTC in the erythromycin-containing wastewater is as follows:

taking 100mL of pharmaceutical factory sewage, and determining the concentration of erythromycin to be about 350 mg/L; then 0.35g of prepared ereB @ Cu-BTC is added, the mixture is placed at normal temperature and normal pressure for reaction for 30min, the mixture is stirred to promote catalysis, the degradation rate reaches 80% after 20min (as shown in figure 5), the ereB @ Cu-BTC is collected in a centrifugal mode, and the recovery rate reaches 85%.

The control group was prepared by adding the same amount of unsupported ereB enzyme to the same system, and the degradation efficiency and rate were superior to the free enzyme.

As can be seen from FIG. 5, the degrading effect of ereB @ Cu-BTC in water is not as good as that in the mushroom dregs, and reaches 83% in 25 minutes.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.