Beta-lactamase immobilized sponge as well as preparation method and application thereof

Abstract

The invention discloses a beta-lactamase immobilized sponge and a preparation method and application thereof, and belongs to the technical field of environmental engineering.Polydopamine coated melamine sponge is used as a carrier, beta-lactamase cross-linked enzyme aggregate is immobilized on the surface of the carrier through a cross-linking method, and the beta-lactamase immobilized sponge is prepared. The beta-lactamase immobilized sponge (MS (at) beta-lactamase-CLEAs) is obtained, so that the antibiotics in the wastewater can be efficiently removed. By adopting the method disclosed by the invention, the stability and the catalytic activity of the beta-lactamase are enhanced, and the MS-coated beta-lactamase-CLEAs disclosed by the invention can be used for quickly and efficiently hydrolyzing beta-lactam antibiotics in wastewater and remarkably reducing the concentration of the beta-lactam antibiotics; compared with a traditional treatment method, the method has higher treatment efficiency and good reusability, a feasible solution is provided for treatment of wastewater polluted by antibiotics, and the method has wide application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental engineering, and in particular relates to a beta-lactamase immobilized sponge and a preparation method and application thereof. Background Art

[0002] With the development of the pharmaceutical industry and the widespread use of antibiotics, antibiotic pollution has become an important issue of global water pollution. Antibiotics are one of the most widely used and used drugs in the world. There are many ways to produce antibiotics according to their types. For example, penicillin is biosynthesized by microbial fermentation, and sulfonamides and quinolones can be produced by chemical synthesis. There are also semi-synthetic antibiotics, which are various derivatives made by modifying the molecular structure of antibiotics obtained by biosynthesis using chemical, biological or biochemical methods. According to the chemical structure, they can be divided into: quinolone antibiotics, β-lactam antibiotics, macrolides, aminoglycoside antibiotics, etc.; according to the use, they can be divided into antibacterial antibiotics, antifungal antibiotics, antitumor antibiotics, antiviral antibiotics, animal antibiotics, agricultural antibiotics and other microbial drugs (such as ergot alkaloids produced by ergot fungi with pharmacological activity, which have the effect of contracting the uterus). Every year, tens of thousands of tons of antibiotics are used in poultry and livestock farming and human medical treatment. Most antibiotics cannot be completely metabolized in humans and animals, and are excreted from the body through feces in the form of prototypes and active metabolites. Environmental pollution and ecotoxicological effects of antibiotics are one of the major environmental issues facing the world. Traditional wastewater treatment methods such as sedimentation, filtration and chemical oxidation are often unable to effectively remove antibiotics from water, resulting in their residues and bioaccumulation in the environment, posing a potential threat to ecosystems and human health. Therefore, it is urgent to develop efficient and sustainable wastewater treatment technologies.

[0003] As a green and environmentally friendly treatment method, enzyme catalysis has attracted attention due to its mild reaction conditions, high selectivity and high catalytic efficiency. β-lactamase is a class of enzymes that can hydrolyze β-lactam antibiotics and has good catalytic properties. However, free enzymes have disadvantages such as poor stability, easy inactivation, and difficulty in reuse in practical applications. Therefore, enzyme immobilization technology has emerged to improve the stability and catalytic efficiency of enzymes. Existing enzyme immobilization methods mainly include physical adsorption, covalent bonding and cross-linking. Among them, the cross-linking method can form stable enzyme aggregates (CLEAs), which enhance the thermal stability and durability of the enzyme while maintaining enzyme activity. However, traditional immobilization carriers such as polymers and ceramic materials still have certain limitations in terms of enzyme immobilization efficiency and reusability. Summary of the invention

[0004] The present invention proposes a β-lactamase immobilized sponge and a preparation method and application thereof, aiming to solve the problems of low enzyme immobilization efficiency, unstable enzyme activity and unsatisfactory wastewater treatment effect in the prior art, thereby providing an efficient and sustainable solution for the treatment of antibiotic-contaminated wastewater.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] One of the technical solutions of the present invention:

[0007] A method for preparing a β-lactamase immobilized sponge comprises taking a polydopamine-coated melamine sponge as a carrier, fixing β-lactamase cross-linked enzyme aggregates (CLEAs) on the surface of the carrier by a cross-linking method, and obtaining the β-lactamase immobilized sponge.

[0008] Furthermore, the preparation method of the β-lactamase immobilized sponge comprises the following steps: washing the melamine sponge (MS), drying it, and treating it in a dopamine solution to obtain a polydopamine-coated melamine sponge;

[0009] β-lactamase and bovine serum albumin are respectively dissolved in phosphate buffer to obtain a β-lactamase solution and a bovine serum albumin solution, the β-lactamase solution and the bovine serum albumin solution are mixed evenly, and then a saturated ammonium sulfate solution is added for pre-assembly, and then glutaraldehyde and the polydopamine-coated melamine sponge are added to perform a cross-linking reaction. After the cross-linking reaction is completed, the sponge is washed to obtain the β-lactamase immobilized sponge (MS@β-lactamase-CLEAs).

[0010] As a porous material, melamine sponge has good physical and chemical properties and a large specific surface area, and can provide a more superior immobilization environment. Based on this, the present invention cross-links β-lactamase on the surface of melamine sponge, and uses saturated ammonium sulfate solution and glutaraldehyde as precipitant and cross-linking agent respectively, which can not only improve the stability and catalytic performance of β-lactamase, but also achieve rapid hydrolysis of antibiotics in wastewater. The present invention solves the problems of low enzyme immobilization efficiency, unstable enzyme activity and unsatisfactory wastewater treatment effect in the prior art, and provides an efficient and sustainable solution for the treatment of antibiotic-contaminated wastewater.

[0011] The present invention utilizes melamine sponge as a carrier, has excellent physicochemical properties and a large specific surface area, and provides an ideal environment for the immobilization of beta-lactamase. By using a saturated ammonium sulfate solution as a precipitant, the concentration of the enzyme can be effectively increased, and its rapid immobilization on the surface of the melamine sponge is promoted, thereby significantly improving the immobilization efficiency of the enzyme. In addition, glutaraldehyde, as a cross-linking agent, enhances the binding force between the enzyme and the carrier, forms a stable enzyme-carrier complex, and then improves the activity stability of the enzyme, and reduces the activity loss caused by changes in the external environment. This immobilization method not only maintains the catalytic performance of the enzyme, but also enables it to maintain high-efficiency activity during multiple uses, ensuring the rapid hydrolysis and removal effect of antibiotics in wastewater.

[0012] The step of cleaning the melamine sponge is: cleaning the melamine sponge with anhydrous ethanol and water respectively under ultrasonic conditions, and ultrasonic cleaning each for three times.

[0013] Exemplarily, the size of the melamine sponge is 2*2*2cm 3 .

[0014] The concentration of the β-lactamase solution is 10-30 mg / mL, preferably the concentration of the β-lactamase solution is 20-30 mg / mL. Exemplarily, the concentration of the β-lactamase solution is 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL or 30 mg / mL, more preferably the concentration of the β-lactamase solution is 25 mg / mL.

[0015] Exemplarily, a β-lactamase solution with a concentration of 25 mg / mL is prepared by dissolving 25 mg of β-lactamase in 1 mL of phosphate buffer at pH=7.4, and stirring the mixture to obtain a β-lactamase solution with a concentration of 25 mg / mL.

[0016] The concentration of the bovine serum albumin solution is 30 mg / mL.

[0017] Illustratively, a method for preparing a bovine serum albumin solution with a concentration of 30 mg / mL is as follows: 30 mg of bovine serum albumin is dissolved in 1 mL of phosphate buffer with a pH of 7.4, and stirred evenly to obtain a bovine serum albumin solution with a concentration of 30 mg / mL.

[0018] The cross-linking reaction time is 1-4 hours. Exemplarily, the cross-linking reaction time is 1 hour, 2 hours, 3 hours or 4 hours, and preferably the cross-linking reaction time is 3 hours.

[0019] The washing is: washing three times with a phosphate buffer solution of pH = 7.4. The purpose of washing is to remove the physically adsorbed β-lactamase on the β-lactamase immobilized sponge and the unimmobilized free enzyme in the solution.

[0020] The amount of glutaraldehyde added is 40-80 mM. Exemplarily, the amount of glutaraldehyde added is 40 mM, 60 mM or 80 mM, preferably 60 mM.

[0021] Preferably, the preparation method of the β-lactamase immobilized sponge is:

[0022] (1) Change the size to 2*2*2cm 3 The melamine sponge was cleaned with anhydrous ethanol and water for 5 minutes under ultrasound, respectively, and ultrasonically cleaned three times, and then dried in a drying oven, and then treated in a 2 mg / mL dopamine solution at room temperature for 12 hours to obtain a polydopamine-coated melamine sponge for standby use;

[0023] (2) 25 mg of β-lactamase and 30 mg of bovine serum albumin were respectively dissolved in 1 mL of phosphate buffer at pH = 7.4 to obtain a β-lactamase solution and a bovine serum albumin solution. The β-lactamase solution and the bovine serum albumin solution were mixed evenly, and then 5 mL of saturated ammonium sulfate solution was added, and the mixture was gently mixed. The mixture was preassembled for 1 hour, and 60 mM glutaraldehyde was added. The polydopamine-coated melamine sponge was immediately added and cross-linked at room temperature for 3 hours. After the cross-linking reaction was completed, the β-lactamase was gently squeezed to recover the β-lactamase, and the β-lactamase immobilized sponge was washed three times with phosphate buffer at pH = 7.4 to remove the β-lactamase physically adsorbed on the β-lactamase immobilized sponge and the free enzyme not immobilized in the solution to obtain the β-lactamase immobilized sponge.

[0024] The second technical solution of the present invention:

[0025] The present invention also provides a beta-lactamase immobilized sponge prepared according to the method.

[0026] The third technical solution of the present invention:

[0027] The present invention also provides application of the beta-lactamase immobilized sponge in hydrolyzing beta-lactam antibiotics in wastewater.

[0028] The method of the present invention enhances the stability and catalytic activity of β-lactamase, and the MS@β-lactamase-CLEAs of the present invention can quickly and efficiently hydrolyze β-lactam antibiotics in wastewater, significantly reducing their concentration. Compared with traditional treatment methods, such as the use of activated carbon adsorption, although this method can remove some antibiotics, it has limitations in treatment efficiency and selectivity, and it is difficult to achieve complete removal of antibiotics during the treatment process, and the reusability of activated carbon is poor, resulting in high treatment costs. The present invention has higher treatment efficiency and good reusability, provides a feasible solution for the treatment of antibiotic-contaminated wastewater, and has broad application prospects.

[0029] Exemplarily, the antibiotic is penicillin.

[0030] Compared with the prior art, the present invention has the following advantages and technical effects:

[0031] The present invention uses melamine sponge as a carrier, and fixes β-lactamase cross-linked enzyme aggregates (CLEAs) on the carrier surface by cross-linking, which significantly improves the hydrolysis efficiency of antibiotics in wastewater. This method has multiple advantages: first, the stability and catalytic activity of the immobilized enzyme are enhanced, and it can maintain efficient catalysis in a wider temperature and pH range; second, the excellent physical properties of melamine sponge make the recovery and reuse of enzymes more convenient, reducing operating costs. In addition, this technology can not only quickly remove antibiotics from wastewater, improve water quality, and meet environmental protection requirements, but also promote the recycling of resources, with good economic and social benefits. Through this innovation, the application of biocatalytic technology in the field of wastewater treatment has been promoted, making a positive contribution to achieving sustainable development goals. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0033] Figure 1 The present invention is a flow chart for preparing the β-lactamase immobilized sponge and a working principle diagram;

[0034] Figure 2 It is the result of β-lactamase activity determination;

[0035] Figure 3 The reusability performance of MS@β-lactamase-CLEAs prepared in Example 1;

[0036] Figure 4Characterization pictures of MS, MS@β-lactamase-CLEAs and CLEAs, where A: SEM image of MS, B: SEM image of MS@β-lactamase-CLEAs, C: SEM image of CLEAs, D: TG-DSC images of MS and MS@β-lactamase-CLEAs;

[0037] Figure 5 The effect of different β-lactamase dosages on the residual activity of β-lactamase-CLEAs in MS@β-lactamase-CLEAs;

[0038] Figure 6 The effect of different cross-linking times on the residual activity of β-lactamase-CLEAs in MS@β-lactamase-CLEAs;

[0039] Figure 7 The effect of different glutaraldehyde addition amounts on the residual activity of β-lactamase-CLEAs in MS@β-lactamase-CLEAs;

[0040] Figure 8 The effects of different temperatures on the recovery of free enzymes and cross-linked enzyme aggregates (CLEAs);

[0041] Fig. 9 The effects of different pH on the recovery activity of free enzyme and cross-linked enzyme aggregates (CLEAs);

[0042] Fig.10 The hydrolysis results of antibiotics in wastewater by MS@β-lactamase-CLEAs. DETAILED DESCRIPTION

[0043] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0044] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0045] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0046] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0047] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0048] Unless otherwise specified, the room temperature in the present invention is 25±2°C.

[0049] All raw materials used in the examples of the present invention were purchased from commercial sources. For example, β-lactamase was purchased from Shanghai Linen Technology Development Co., Ltd.; and bovine serum albumin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0050] The preparation flow chart and working principle diagram of the β-lactamase immobilized sponge of the present invention are shown in Figure 1 .

[0051] In the embodiments of the present invention, the methods for determining the β-lactamase activity and the penicillin concentration are as follows:

[0052] 1. β-lactamase activity assay:

[0053] β-lactamase uses nitrocefin as a substrate and can hydrolyze the amide bond between the carbonyl carbon and the nitrogen group on its β-lactam ring. After nitrocefin is hydrolyzed by β-lactamase, an obvious color change will occur from yellow to red. The absorbance is measured at 482nm using an ultraviolet spectrophotometer.

[0054] The absorbance of the reaction product was measured by colorimetry to obtain the activity standard curve of β-lactamase. Figure 2 As shown in the figure, the absorbance corresponding to different concentrations (0, 20μg / mL, 40μg / mL, 60μg / mL, 80μg / mL, 100μg / mL) of β-lactamase increased proportionally. -1The range is linearly correlated (y=0.0077x+0.0924, R 2 =0.9937). The experimental results show that the β-lactamase activity detection method established in the present invention is effective.

[0055] 2. Determination of penicillin concentration:

[0056] The change in penicillin concentration is calculated by the iodine titration method. Penicillin antibiotics are hydrolyzed by alkali, and the product of penicillin thiazole acid can react with iodine (1 mol of penicillin thiazole acid can react with 8 mol of iodine atoms, that is, penicillin: I2 = 1:4 (molar ratio)). The content of penicillin can be calculated based on the amount of iodine consumed.

[0057] In the present invention, the degradation rate of penicillin and the concentration of penicillin are two different concepts. The degradation rate of penicillin refers to the degree to which penicillin is degraded during the reaction, usually expressed as a percentage; while the concentration of penicillin refers to the specific concentration value of penicillin in the solution. The calculation formula of the degradation rate of penicillin (%) is: the degradation rate of penicillin (%) = (initial concentration of penicillin solution - concentration of penicillin solution after reaction) / initial concentration of penicillin solution × 100%. Among them, the initial concentration of penicillin solution refers to the concentration of penicillin at the beginning of the reaction, and the concentration after reaction refers to the concentration of penicillin measured after the reaction is completed.

[0058] In the present invention, the test process and calculation formula of the residual activity are as follows: CLEAs are diluted to 100 μg / mL to obtain a dilution, 300 μL of the dilution is mixed with 300 μL of 50 μg / mL nitrocefin, and reacted at room temperature for 10 minutes. After the reaction is completed, the absorbance of the reaction mixture is measured at a wavelength of 482 nm using an ultraviolet spectrophotometer. The calculation formula of the residual activity (%) is: Residual activity (%) = (total β-lactamase activity in β-lactamase-CLEAs) / (total β-lactamase activity used for the preparation of β-lactamase-CLEAs) × 100%.

[0059] In an embodiment of the present invention, the preparation method of cross-linked enzyme aggregates (CLEAs) is as follows: 25 mg of β-lactamase and 30 mg of bovine serum albumin are respectively dissolved in 1 mL of phosphate buffer at pH = 7.4 to obtain a β-lactamase solution and a bovine serum albumin solution, the β-lactamase solution and the bovine serum albumin solution are mixed evenly, 5 mL of saturated ammonium sulfate solution is added, and the mixture is gently mixed, pre-assembled for 1 hour, 60 mM glutaraldehyde is added, and the cross-linking reaction is carried out at room temperature for 3 hours. After the cross-linking reaction is completed, the cross-linked enzyme aggregates are recovered by centrifugation, and the cross-linked enzyme aggregates are washed three times with a phosphate buffer at pH = 7.4 to obtain CLEAs. CLEAs are diluted to 1 mg / mL, and free enzymes are diluted to 100 μg / mL. First, the free enzymes and CLEAs are incubated in the range of 4-65°C for 60 minutes, respectively, and the absorbance (A) at each temperature is recorded. Subsequently, the two forms of enzyme were incubated in different pH buffers (pH 4-10) for the same 60 minutes, and 300 μL of the dilution was mixed with 300 μL of 50 μg / mL nitrocefin and reacted at room temperature for 10 minutes. After the reaction, the absorbance of the reaction mixture was measured at a wavelength of 482 nm using an ultraviolet spectrophotometer. In order to evaluate the recovery activity at different pH values, the recovery activity was calculated using the formula, recovery activity (pH) = (A 样品 / A pH 7) × 100%, where A 样品 is the absorbance at a specific pH value, A pH 7 is the absorbance at pH 7. At the same time, for the recovery activity at different temperatures, the recovery activity was calculated using the formula: recovery activity (temperature) = (A 样品 / A 4℃ )×100, where A 4℃ is the absorbance at 4°C. Through these steps, the recovery activities of free enzymes and CLEAs under different temperature and pH conditions can be systematically evaluated.

[0060] It should be pointed out that the matters not described in detail in the present invention are conventional operating means in the art and are not the focus of the present invention.

[0061] The technical solution of the present invention is further described below through typical embodiments.

[0062] Example 1

[0063] (1) Change the size to 2*2*2cm 3The melamine sponge was cleaned with anhydrous ethanol and water for 5 minutes under ultrasound, respectively, and ultrasonically cleaned three times, the water in the melamine sponge was absorbed with filter paper, and dried in a drying oven at 60°C for 2 hours to obtain a dry melamine sponge. The dry melamine sponge was treated in a 2 mg / mL dopamine solution (the solvent of the dopamine solution was 10 mM HCl-Tris with a pH of 8.5, the same below) at room temperature for 12 hours. After the treatment, it was washed three times with purified water to remove unreacted dopamine to obtain a polydopamine-coated melamine sponge for use;

[0064] (2) 25 mg of β-lactamase and 30 mg of bovine serum albumin were dissolved in 1 mL of phosphate buffer at pH = 7.4, respectively, to obtain a β-lactamase solution (25 mg / mL) and a bovine serum albumin solution (30 mg / mL). After 1 mL of the β-lactamase solution and 1 mL of the bovine serum albumin solution were mixed evenly, 5 mL of saturated ammonium sulfate solution was added and gently mixed. The mixture was preassembled for 1 h, 60 mM glutaraldehyde was added, and polydopamine-coated melamine sponge was immediately added. The mixture was cross-linked at room temperature for 3 h. After the cross-linking reaction was completed, the β-lactamase was gently squeezed to recover the β-lactamase, and the β-lactamase immobilized sponge was washed three times with phosphate buffer at pH = 7.4 to remove the β-lactamase physically adsorbed on the β-lactamase immobilized sponge and the free enzyme not immobilized in the solution, thereby obtaining a β-lactamase immobilized sponge (MS@β-lactamase-CLEAs).

[0065] Example 2

[0066] (1) Change the size to 2*2*2cm 3 The melamine sponge was cleaned with anhydrous ethanol and water for 5 minutes under ultrasound, respectively, and ultrasonically cleaned three times, and the water in the melamine sponge was absorbed with filter paper, and dried in a drying oven at 60°C for 2 hours to obtain a dry melamine sponge. The dry melamine sponge was treated in a 2 mg / mL dopamine solution at room temperature for 12 hours. After the treatment, it was washed three times with purified water to remove unreacted dopamine to obtain a polydopamine-coated melamine sponge for use;

[0067] (2) 25 mg of β-lactamase and 30 mg of bovine serum albumin were dissolved in 1 mL of phosphate buffer at pH = 7.4, respectively, to obtain a β-lactamase solution (25 mg / mL) and a bovine serum albumin solution (30 mg / mL). After 1 mL of the β-lactamase solution and 1 mL of the bovine serum albumin solution were mixed evenly, 5 mL of saturated ammonium sulfate solution was added, and the mixture was gently mixed. The mixture was preassembled for 1 h, 60 mM glutaraldehyde was added, and a polydopamine-coated melamine sponge was immediately added. The mixture was cross-linked at room temperature for 1 h. After the cross-linking reaction was completed, the β-lactamase was gently squeezed to recover the β-lactamase, and the β-lactamase immobilized sponge was washed three times with phosphate buffer at pH = 7.4 to remove the β-lactamase physically adsorbed on the β-lactamase immobilized sponge and the free enzyme not immobilized in the solution, thereby obtaining a β-lactamase immobilized sponge.

[0068] Example 3

[0069] (1) Change the size to 2*2*2cm 3 The melamine sponge was cleaned with anhydrous ethanol and water for 5 minutes under ultrasound, respectively, and ultrasonically cleaned three times, and the water in the melamine sponge was absorbed with filter paper, and dried in a drying oven at 60°C for 2 hours to obtain a dry melamine sponge. The dry melamine sponge was treated in a 2 mg / mL dopamine solution at room temperature for 12 hours. After the treatment, it was washed three times with purified water to remove unreacted dopamine to obtain a polydopamine-coated melamine sponge for use;

[0070] (2) 25 mg of β-lactamase and 30 mg of bovine serum albumin were dissolved in 1 mL of phosphate buffer at pH = 7.4, respectively, to obtain a β-lactamase solution (25 mg / mL) and a bovine serum albumin solution (30 mg / mL). After 1 mL of the β-lactamase solution and 1 mL of the bovine serum albumin solution were mixed evenly, 5 mL of saturated ammonium sulfate solution was added, and the mixture was gently mixed. The mixture was preassembled for 1 h, 60 mM glutaraldehyde was added, and a polydopamine-coated melamine sponge was immediately added. The mixture was cross-linked at room temperature for 2 h. After the cross-linking reaction was completed, the β-lactamase was gently squeezed to recover the β-lactamase, and the β-lactamase immobilized sponge was washed three times with phosphate buffer at pH = 7.4 to remove the β-lactamase physically adsorbed on the β-lactamase immobilized sponge and the free enzyme not immobilized in the solution, thereby obtaining a β-lactamase immobilized sponge.

[0071] Example 4

[0072] (1) Change the size to 2*2*2cm 3The melamine sponge was cleaned with anhydrous ethanol and water for 5 minutes under ultrasound, respectively, and ultrasonically cleaned three times, and the water in the melamine sponge was absorbed with filter paper, and dried in a drying oven at 60°C for 2 hours to obtain a dry melamine sponge. The dry melamine sponge was treated in a 2 mg / mL dopamine solution at room temperature for 12 hours. After the treatment, it was washed three times with purified water to remove unreacted dopamine to obtain a polydopamine-coated melamine sponge for use;

[0073] (2) 25 mg of β-lactamase and 30 mg of bovine serum albumin were dissolved in 1 mL of phosphate buffer at pH = 7.4, respectively, to obtain a β-lactamase solution (25 mg / mL) and a bovine serum albumin solution (30 mg / mL). After 1 mL of the β-lactamase solution and 1 mL of the bovine serum albumin solution were mixed evenly, 5 mL of saturated ammonium sulfate solution was added, and the mixture was gently mixed. The mixture was preassembled for 1 h, 60 mM glutaraldehyde was added, and a polydopamine-coated melamine sponge was immediately added. The mixture was cross-linked at room temperature for 4 h. After the cross-linking reaction was completed, the β-lactamase was gently squeezed to recover the β-lactamase, and the β-lactamase immobilized sponge was washed three times with phosphate buffer at pH = 7.4 to remove the β-lactamase physically adsorbed on the β-lactamase immobilized sponge and the free enzyme not immobilized in the solution, thereby obtaining a β-lactamase immobilized sponge.

[0074] Example 5

[0075] (1) Change the size to 2*2*2cm 3 The melamine sponge was cleaned with anhydrous ethanol and water for 5 minutes under ultrasound, respectively, and ultrasonically cleaned three times, and the water in the melamine sponge was absorbed with filter paper, and dried in a drying oven at 60°C for 2 hours to obtain a dry melamine sponge. The dry melamine sponge was treated in a 2 mg / mL dopamine solution at room temperature for 12 hours. After the treatment, it was washed three times with purified water to remove unreacted dopamine to obtain a polydopamine-coated melamine sponge for use;

[0076] (2) 10 mg of β-lactamase and 30 mg of bovine serum albumin were dissolved in 1 mL of phosphate buffer at pH = 7.4, respectively, to obtain a β-lactamase solution (10 mg / mL) and a bovine serum albumin solution (30 mg / mL). After 1 mL of the β-lactamase solution and 1 mL of the bovine serum albumin solution were mixed evenly, 5 mL of saturated ammonium sulfate solution was added, and the mixture was gently mixed. The mixture was preassembled for 1 h, 60 mM glutaraldehyde was added, and a polydopamine-coated melamine sponge was immediately added. The mixture was cross-linked at room temperature for 3 h. After the cross-linking reaction was completed, the β-lactamase was gently squeezed to recover the β-lactamase, and the β-lactamase immobilized sponge was washed three times with phosphate buffer at pH = 7.4 to remove the β-lactamase physically adsorbed on the β-lactamase immobilized sponge and the free enzyme not immobilized in the solution, thereby obtaining a β-lactamase immobilized sponge.

[0077] Example 6

[0078] (1) Change the size to 2*2*2cm 3 The melamine sponge was cleaned with anhydrous ethanol and water for 5 minutes under ultrasound, respectively, and ultrasonically cleaned three times, and the water in the melamine sponge was absorbed with filter paper, and dried in a drying oven at 60°C for 2 hours to obtain a dry melamine sponge. The dry melamine sponge was treated in a 2 mg / mL dopamine solution at room temperature for 12 hours. After the treatment, it was washed three times with purified water to remove unreacted dopamine to obtain a polydopamine-coated melamine sponge for use;

[0079] (2) 15 mg of β-lactamase and 30 mg of bovine serum albumin were dissolved in 1 mL of phosphate buffer at pH = 7.4, respectively, to obtain a β-lactamase solution (15 mg / mL) and a bovine serum albumin solution (30 mg / mL). After 1 mL of the β-lactamase solution and 1 mL of the bovine serum albumin solution were mixed evenly, 5 mL of saturated ammonium sulfate solution was added, and the mixture was gently mixed. The mixture was preassembled for 1 hour, 60 mM glutaraldehyde was added, and a polydopamine-coated melamine sponge was immediately added. The mixture was cross-linked at room temperature for 3 hours. After the cross-linking reaction was completed, the β-lactamase was gently squeezed to recover the β-lactamase, and the β-lactamase immobilized sponge was washed three times with phosphate buffer at pH = 7.4 to remove the β-lactamase physically adsorbed on the β-lactamase immobilized sponge and the free enzyme not immobilized in the solution, thereby obtaining a β-lactamase immobilized sponge.

[0080] Example 7

[0081] (1) Change the size to 2*2*2cm 3The melamine sponge was cleaned with anhydrous ethanol and water for 5 minutes under ultrasound, respectively, and ultrasonically cleaned three times, and the water in the melamine sponge was absorbed with filter paper, and dried in a drying oven at 60°C for 2 hours to obtain a dry melamine sponge. The dry melamine sponge was treated in a 2 mg / mL dopamine solution at room temperature for 12 hours. After the treatment, it was washed three times with purified water to remove unreacted dopamine to obtain a polydopamine-coated melamine sponge for use;

[0082] (2) 20 mg of β-lactamase and 30 mg of bovine serum albumin were dissolved in 1 mL of phosphate buffer at pH = 7.4, respectively, to obtain a β-lactamase solution (20 mg / mL) and a bovine serum albumin solution (30 mg / mL). After 1 mL of the β-lactamase solution and 1 mL of the bovine serum albumin solution were mixed evenly, 5 mL of saturated ammonium sulfate solution was added, and the mixture was gently mixed. The mixture was preassembled for 1 h, 60 mM glutaraldehyde was added, and a polydopamine-coated melamine sponge was immediately added. The mixture was cross-linked at room temperature for 3 h. After the cross-linking reaction was completed, the β-lactamase was gently squeezed to recover the β-lactamase, and the β-lactamase immobilized sponge was washed three times with phosphate buffer at pH = 7.4 to remove the β-lactamase physically adsorbed on the β-lactamase immobilized sponge and the free enzyme not immobilized in the solution, thereby obtaining a β-lactamase immobilized sponge.

[0083] Example 8

[0084] (1) Change the size to 2*2*2cm 3 The melamine sponge was cleaned with anhydrous ethanol and water for 5 minutes under ultrasound, respectively, and ultrasonically cleaned three times, and the water in the melamine sponge was absorbed with filter paper, and dried in a drying oven at 60°C for 2 hours to obtain a dry melamine sponge. The dry melamine sponge was treated in a 2 mg / mL dopamine solution at room temperature for 12 hours. After the treatment, it was washed three times with purified water to remove unreacted dopamine to obtain a polydopamine-coated melamine sponge for use;

[0085] (2) 30 mg of β-lactamase and 30 mg of bovine serum albumin were dissolved in 1 mL of phosphate buffer at pH = 7.4, respectively, to obtain a β-lactamase solution (30 mg / mL) and a bovine serum albumin solution (30 mg / mL). After 1 mL of the β-lactamase solution and 1 mL of the bovine serum albumin solution were mixed evenly, 5 mL of saturated ammonium sulfate solution was added, and the mixture was gently mixed. The mixture was preassembled for 1 hour, 60 mM glutaraldehyde was added, and a polydopamine-coated melamine sponge was immediately added. The mixture was cross-linked at room temperature for 3 hours. After the cross-linking reaction was completed, the β-lactamase was gently squeezed to recover the β-lactamase, and the β-lactamase immobilized sponge was washed three times with phosphate buffer at pH = 7.4 to remove the β-lactamase physically adsorbed on the β-lactamase immobilized sponge and the free enzyme not immobilized in the solution, thereby obtaining a β-lactamase immobilized sponge.

[0086] Example 9

[0087] (1) Change the size to 2*2*2cm 3 The melamine sponge was cleaned with anhydrous ethanol and water for 5 minutes under ultrasound, respectively, and ultrasonically cleaned three times, and the water in the melamine sponge was absorbed with filter paper, and dried in a drying oven at 60°C for 2 hours to obtain a dry melamine sponge. The dry melamine sponge was treated in a 2 mg / mL dopamine solution at room temperature for 12 hours. After the treatment, it was washed three times with purified water to remove unreacted dopamine to obtain a polydopamine-coated melamine sponge for use;

[0088] (2) 25 mg of β-lactamase and 30 mg of bovine serum albumin were dissolved in 1 mL of phosphate buffer at pH = 7.4, respectively, to obtain a β-lactamase solution (25 mg / mL) and a bovine serum albumin solution (30 mg / mL). After 1 mL of the β-lactamase solution and 1 mL of the bovine serum albumin solution were mixed evenly, 5 mL of saturated ammonium sulfate solution was added, and the mixture was gently mixed. The mixture was preassembled for 1 h, 40 mM glutaraldehyde was added, and a polydopamine-coated melamine sponge was immediately added. The mixture was cross-linked at room temperature for 3 h. After the cross-linking reaction was completed, the β-lactamase was gently squeezed to recover the β-lactamase, and the β-lactamase immobilized sponge was washed three times with phosphate buffer at pH = 7.4 to remove the β-lactamase physically adsorbed on the β-lactamase immobilized sponge and the free enzyme not immobilized in the solution, thereby obtaining a β-lactamase immobilized sponge.

[0089] Example 10

[0090] (1) Change the size to 2*2*2cm 3The melamine sponge was cleaned with anhydrous ethanol and water for 5 minutes under ultrasound, respectively, and ultrasonically cleaned three times, and the water in the melamine sponge was absorbed with filter paper, and dried in a drying oven at 60°C for 2 hours to obtain a dry melamine sponge. The dry melamine sponge was treated in a 2 mg / mL dopamine solution at room temperature for 12 hours. After the treatment, it was washed three times with purified water to remove unreacted dopamine to obtain a polydopamine-coated melamine sponge for use;

[0091] (2) 25 mg of β-lactamase and 30 mg of bovine serum albumin were dissolved in 1 mL of phosphate buffer at pH = 7.4, respectively, to obtain a β-lactamase solution (25 mg / mL) and a bovine serum albumin solution (30 mg / mL). After 1 mL of the β-lactamase solution and 1 mL of the bovine serum albumin solution were mixed evenly, 5 mL of saturated ammonium sulfate solution was added, and the mixture was gently mixed. The mixture was preassembled for 1 h, 80 mM glutaraldehyde was added, and a polydopamine-coated melamine sponge was immediately added. The mixture was cross-linked at room temperature for 3 h. After the cross-linking reaction was completed, the β-lactamase was gently squeezed to recover the β-lactamase, and the β-lactamase immobilized sponge was washed three times with phosphate buffer at pH = 7.4 to remove the β-lactamase physically adsorbed on the β-lactamase immobilized sponge and the free enzyme not immobilized in the solution, thereby obtaining a β-lactamase immobilized sponge.

[0092] Comparative Example 1

[0093] (1) Change the size to 2*2*2cm 3 The melamine sponge was cleaned with anhydrous ethanol and water for 5 minutes under ultrasound, respectively, and ultrasonically cleaned three times, and the water in the melamine sponge was absorbed with filter paper, and dried in a drying oven at 60°C for 2 hours to obtain a dry melamine sponge. The dry melamine sponge was treated in a 2 mg / mL dopamine solution at room temperature for 12 hours. After the treatment, it was washed three times with purified water to remove unreacted dopamine to obtain a polydopamine-coated melamine sponge for use;

[0094] (2) 25 mg of β-lactamase and 30 mg of bovine serum albumin were dissolved in 1 mL of phosphate buffer at pH = 7.4, respectively, to obtain a β-lactamase solution (25 mg / mL) and a bovine serum albumin solution (30 mg / mL). After 1 mL of the β-lactamase solution and 1 mL of the bovine serum albumin solution were mixed evenly, 5 mL of saturated ammonium sulfate solution was added, and the mixture was gently mixed. The mixture was preassembled for 1 h, 20 mM glutaraldehyde was added, and a polydopamine-coated melamine sponge was immediately added. The mixture was cross-linked at room temperature for 3 h. After the cross-linking reaction was completed, the β-lactamase was gently squeezed to recover the β-lactamase, and the β-lactamase immobilized sponge was washed three times with phosphate buffer at pH = 7.4 to remove the β-lactamase physically adsorbed on the β-lactamase immobilized sponge and the free enzyme not immobilized in the solution, thereby obtaining a β-lactamase immobilized sponge.

[0095] Comparative Example 2

[0096] (1) Change the size to 2*2*2cm 3 The melamine sponge was cleaned with anhydrous ethanol and water for 5 minutes under ultrasound, respectively, and ultrasonically cleaned three times, and the water in the melamine sponge was absorbed with filter paper, and dried in a drying oven at 60°C for 2 hours to obtain a dry melamine sponge. The dry melamine sponge was treated in a 2 mg / mL dopamine solution at room temperature for 12 hours. After the treatment, it was washed three times with purified water to remove unreacted dopamine to obtain a polydopamine-coated melamine sponge for use;

[0097] (2) 25 mg of β-lactamase and 30 mg of bovine serum albumin were dissolved in 1 mL of phosphate buffer at pH = 7.4, respectively, to obtain a β-lactamase solution (25 mg / mL) and a bovine serum albumin solution (30 mg / mL). After 1 mL of the β-lactamase solution and 1 mL of the bovine serum albumin solution were mixed evenly, 5 mL of saturated ammonium sulfate solution was added, and the mixture was gently mixed. The mixture was preassembled for 1 h, 100 mM glutaraldehyde was added, and a polydopamine-coated melamine sponge was immediately added. The mixture was cross-linked at room temperature for 3 h. After the cross-linking reaction was completed, the β-lactamase was gently squeezed to recover the β-lactamase, and the β-lactamase immobilized sponge was washed three times with phosphate buffer at pH = 7.4 to remove the β-lactamase physically adsorbed on the β-lactamase immobilized sponge and the free enzyme not immobilized in the solution, thereby obtaining a β-lactamase immobilized sponge.

[0098] Comparative Example 3

[0099] (1) Change the size to 2*2*2cm 3The melamine sponge was cleaned with anhydrous ethanol and water for 5 minutes under ultrasound, respectively, and ultrasonically cleaned three times, and the water in the melamine sponge was absorbed with filter paper, and dried in a drying oven at 60°C for 2 hours to obtain a dry melamine sponge. The dry melamine sponge was treated in a 2 mg / mL dopamine solution at room temperature for 12 hours. After the treatment, it was washed three times with purified water to remove unreacted dopamine to obtain a polydopamine-coated melamine sponge for use;

[0100] (2) 25 mg of β-lactamase and 30 mg of bovine serum albumin were dissolved in 1 mL of phosphate buffer at pH = 7.4, respectively, to obtain a β-lactamase solution (25 mg / mL) and a bovine serum albumin solution (30 mg / mL). After 1 mL of the β-lactamase solution and 1 mL of the bovine serum albumin solution were mixed evenly, 5 mL of saturated ammonium sulfate solution was added, and the mixture was gently mixed. The mixture was preassembled for 1 h, 60 mM glutaraldehyde was added, and a polydopamine-coated melamine sponge was immediately added. The mixture was cross-linked at room temperature for 5 h. After the cross-linking reaction was completed, the β-lactamase was gently squeezed to recover the β-lactamase, and the β-lactamase immobilized sponge was washed three times with phosphate buffer at pH = 7.4 to remove the β-lactamase physically adsorbed on the β-lactamase immobilized sponge and the free enzyme not immobilized in the solution, thereby obtaining a β-lactamase immobilized sponge.

[0101] Comparative Example 4

[0102] (1) Change the size to 2*2*2cm 3 The melamine sponge was cleaned with anhydrous ethanol and water for 5 minutes under ultrasound, respectively, and ultrasonically cleaned three times, and the water in the melamine sponge was absorbed with filter paper, and dried in a drying oven at 60°C for 2 hours to obtain a dry melamine sponge. The dry melamine sponge was treated in a 2 mg / mL dopamine solution at room temperature for 12 hours. After the treatment, it was washed three times with purified water to remove unreacted dopamine to obtain a polydopamine-coated melamine sponge for use;

[0103] (2) 5 mg of β-lactamase and 30 mg of bovine serum albumin were dissolved in 1 mL of phosphate buffer at pH = 7.4, respectively, to obtain a β-lactamase solution (5 mg / mL) and a bovine serum albumin solution (30 mg / mL). After 1 mL of the β-lactamase solution and 1 mL of the bovine serum albumin solution were mixed evenly, 5 mL of saturated ammonium sulfate solution was added and gently mixed. The mixture was preassembled for 1 h, 60 mM glutaraldehyde was added, and a polydopamine-coated melamine sponge was immediately added. The mixture was cross-linked at room temperature for 3 h. After the cross-linking reaction was completed, the β-lactamase was gently squeezed to recover the β-lactamase, and the β-lactamase immobilized sponge was washed three times with phosphate buffer at pH = 7.4 to remove the β-lactamase physically adsorbed on the β-lactamase immobilized sponge and the free enzyme not immobilized in the solution, thereby obtaining a β-lactamase immobilized sponge.

[0104] The reusability of MS@β-lactamase-CLEAs will affect its cost and application prospects. Taking the MS@β-lactamase-CLEAs prepared in Example 1 as an example, its reusability was tested. The specific method is: MS@β-lactamase-CLEAs is placed in 20mL of penicillin solution with a concentration of 200μg / mL, and reacted at room temperature for 25 minutes to evaluate its reusability. The reusability of MS@β-lactamase-CLEAs was verified by conducting 7 cycles of use experiments. After each cycle, the sponge was squeezed out and thoroughly rinsed with purified water to remove the reaction residues. The calculation formula for the degradation rate of penicillin (%) is: Penicillin degradation rate (%) = (initial concentration of penicillin solution-concentration of penicillin solution after reaction) / initial concentration of penicillin solution × 100%. The results are as follows Figure 3 As shown, in Example 1, the penicillin degradation rate of MS@β-lactamase-CLEAs during the recycling process was measured, and it was found that the degradation rate was still above 85% after being reused 7 times, indicating that the MS@β-lactamase-CLEAs prepared by the present invention has good reusability.

[0105] MS, MS@β-lactamase-CLEAs, and CLEAs were characterized by scanning electron microscopy (SEM) (Thermo Scientific Apreo 2C). The mass of MS and MS@β-lactamase-CLEAs was measured as a function of temperature and time using a simultaneous thermal analyzer (TGA / DSC 1), allowing for a comprehensive analysis of the thermal stability and reactivity of the materials. Figure 4 As shown, A: SEM image of MS, B: SEM image of MS@β-lactamase-CLEAs, C: SEM image of CLEAs, D: TG-DSC images of MS and MS@β-lactamase-CLEAs. Figure 4As shown in Figures A, B, and C, individual β-lactamase-CLEAs were effectively loaded into the sponge, taking advantage of the well-structured backbone of the sponge. The individual sponges have a smooth and flat surface, as well as a porous backbone, which provides a large surface area for the adsorption of the immobilized enzyme. In contrast, the surface of the modified sponge is rough and bumpy, indicating that the enzyme was successfully grafted into the sponge. Figure 4 As shown in D, in the range of 260℃-663℃, the mass loss of melamine sponge is less than that of melamine sponge@β-lactamase-cross-linked enzyme aggregates, suggesting that the cross-linked enzyme aggregates may have undergone more significant degradation in this temperature range, reflecting the poor thermal stability of β-lactamase. In the range of 663℃-800℃, the weight loss of melamine sponge is higher than that of melamine sponge@β-lactamase-cross-linked enzyme aggregates, indicating that the cross-linking method enhances the structural integrity and stability of the material at high temperatures. These results support the effectiveness of the present invention and show that the prepared composite material has excellent thermal stability in practical applications.

[0106] Optimization results and stability analysis of CLEAs in Example 1

[0107] The residual activity of β-lactamase-CLEAs in the MS@β-lactamase-CLEAs prepared in the above-mentioned embodiments and comparative examples was tested. The effects of different β-lactamase dosages on the residual activity of β-lactamase-CLEAs in MS@β-lactamase-CLEAs are shown in Figure 5 The results of the effects of different cross-linking times on the residual activity of β-lactamase-CLEAs in MS@β-lactamase-CLEAs are shown in Figure 6 The results of the effects of different glutaraldehyde addition amounts on the residual activity of β-lactamase-CLEAs in MS@β-lactamase-CLEAs are shown in Figure 7 . Figure 5 The enzyme activity in β-lactamase-CLEAs reached the maximum when the enzyme dosage was 25 mg. Excessive enzyme would lead to decreased activity of β-lactamase-CLEAs due to the destruction of the steric effect around the enzyme active site. Figure 6 The results showed that the enzyme activity increased with time, reached the maximum at 3 h, and began to decrease after 3 h. Figure 7 The effect of glutaraldehyde addition on enzyme activity was demonstrated. When the amount of glutaraldehyde added exceeded 60 mM, the excess glutaraldehyde would lead to partial enzyme inactivation, indicating that the residual activity was optimal when the glutaraldehyde addition was 60 mM. In summary, the optimal parameters for preparing β-lactamase-CLEAs were: β-lactamase content of 25 mg, cross-linking time of 3 h, and glutaraldehyde addition of 60 mM. At this dose, the maximum residual activity of β-lactamase-CLEAs was 29.6%.

[0108] Under the optimal preparation conditions (i.e., the preparation method of Example 1), the effects of temperature and pH on the enzyme activity of free β-lactamase and β-lactamase-CLEAs were investigated. 25 mg of β-lactamase and 30 mg of bovine serum albumin were dissolved in 1 mL of phosphate buffer at pH = 7.4, respectively, to obtain a β-lactamase solution and a bovine serum albumin solution. The β-lactamase solution and the bovine serum albumin solution were mixed evenly, and then 5 mL of saturated ammonium sulfate solution was added, gently mixed, pre-assembled for 1 hour, 60 mM glutaraldehyde was added, and cross-linked reaction was carried out at room temperature for 3 hours. After the cross-linking reaction was completed, the cross-linked enzyme aggregates were washed three times with a phosphate buffer at pH = 7.4 to obtain CLEAs.

[0109] The relative enzyme activity of free enzyme and cross-linked enzyme aggregates (CLEAs) was measured after incubation for 60 min in the range of 4-65°C. In addition, for both forms of the enzyme, the recovery activity at different pH values ​​was evaluated by setting the activity at pH 7 as 100% after incubation for 60 min in different pH buffers (pH 4-10). At the same time, the recovery activity was determined at different temperatures, setting the activity at 4°C as 100%. The results are shown in Figure 2. Figure 8 It was shown that at 35°C and 45°C, the restored activity of cross-linked enzyme aggregates (CLEAs) was significantly higher than that of free enzyme (i.e., individual β-lactamase). Fig. 9 The results showed that both acidic and alkaline conditions would lead to a decrease in the activity of free enzymes and cross-linked enzyme aggregates. However, it is worth noting that cross-linked enzyme aggregates showed higher activity under both acidic and alkaline conditions. Based on the above results, cross-linked enzyme aggregates showed better stability than free enzymes under different temperature and pH conditions, which supports their potential in industrial applications.

[0110] Application example: Antibiotic treatment results in wastewater

[0111] Taking the β-lactamase immobilized sponge prepared in Example 1 as an example, it was placed in a circulating flow device to treat wastewater containing penicillin. Samples were taken at different times (10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min) for concentration analysis.

[0112] like Fig.10 As shown in the reaction device with circulating flow, 400 mL of wastewater containing 80 mg of penicillin is treated, and after about 80 minutes of reaction, the degradation rate of penicillin reaches the maximum (about 90%). Specifically, the initial penicillin concentration is 0.2 mg / mL, and the remaining concentration after degradation is reduced to 0.02 mg / mL, indicating that the β-lactamase immobilized sponge of the present invention has good catalytic activity and application potential in the degradation of penicillin.

[0113] When treating wastewater containing antibiotics, traditional methods mainly include chemical degradation, biological degradation, physical treatment and thermal treatment. Chemical degradation usually uses acid or alkali to destroy the β-lactam ring of penicillin. Although the method is simple, it may cause secondary pollution and requires strict control of reaction conditions. Biodegradation uses specific microorganisms or enzymes (such as bacteria that produce β-lactamase) to treat penicillin wastewater. This method is relatively environmentally friendly, but the treatment efficiency may be affected by environmental conditions. Physical treatment uses adsorbents (such as activated carbon) to reduce the concentration of penicillin in water through physical adsorption. Its effect depends on the properties of the adsorbent and operating conditions. Thermal treatment destroys the molecular structure of penicillin by heating. Although it can effectively reduce the activity, it has the disadvantages of high energy consumption and possible impact on water quality. The above traditional methods have their own advantages and disadvantages. The selection of a suitable method requires comprehensive consideration of factors such as treatment efficiency, environmental impact and economy. Compared with traditional methods, β-lactamase immobilized sponge shows higher catalytic activity and application potential.

[0114] The above are only preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for preparing a β-lactamase immobilized sponge, characterized in that: The polydopamine-coated melamine sponge is used as a carrier, and the beta-lactamase cross-linked enzyme aggregates are fixed on the surface of the carrier by a cross-linking method to obtain the beta-lactamase immobilized sponge.

2. The method for preparing the β-lactamase immobilized sponge according to claim 1, characterized in that: The following steps are involved: The melamine sponge is cleaned, dried and then treated in a dopamine solution to obtain a polydopamine-coated melamine sponge; β-lactamase and bovine serum albumin are respectively dissolved in phosphate buffer to obtain β-lactamase solution and bovine serum albumin solution, the β-lactamase solution and the bovine serum albumin solution are evenly mixed and then added with saturated ammonium sulfate solution for pre-assembly, and then glutaraldehyde and the polydopamine-coated melamine sponge are added for cross-linking reaction, and after the cross-linking reaction is completed, washing is performed to obtain the β-lactamase immobilized sponge.

3. The method for preparing the β-lactamase immobilized sponge according to claim 2, characterized in that: The step of cleaning the melamine sponge is: cleaning the melamine sponge with anhydrous ethanol and water respectively under ultrasonic conditions, and ultrasonic cleaning each for three times.

4. The method for preparing the β-lactamase immobilized sponge according to claim 2, characterized in that: The concentration of the β-lactamase solution is 10-30 mg / mL.

5. The method for preparing the β-lactamase immobilized sponge according to claim 4, characterized in that: The concentration of the β-lactamase solution is 20-30 mg / mL.

6. The method for preparing the β-lactamase immobilized sponge according to claim 2, characterized in that: The concentration of the bovine serum albumin solution is 30 mg / mL.

7. The method for preparing the β-lactamase immobilized sponge according to claim 6, characterized in that: The cross-linking reaction time is 1-4h.

8. The method for preparing the β-lactamase immobilized sponge according to claim 7, characterized in that: The amount of glutaraldehyde added was 40-80 mM.

9. A β-lactamase immobilized sponge, characterized in that: Prepared according to the preparation method according to any one of claims 1 to 8.

10. Use of the β-lactamase immobilized sponge according to claim 9 in hydrolyzing β-lactam antibiotics in wastewater.

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