A beta-lactamase immobilized sponge and a preparation method and application thereof

Abstract

The application discloses a kind of β-lactamase immobilized sponge and its preparation method and application, belong to environmental engineering technical field, the present application utilizes polydopamine coated melamine sponge as carrier, by crosslinking method β-lactamase crosslinking enzyme aggregate is fixed on the surface of the carrier, obtain β-lactamase immobilized sponge (MS@β-lactamase-CLEAs), to remove antibiotic in waste water efficiently.The stability and catalytic activity of β-lactamase are enhanced by the method of the application, the MS@β-lactamase-CLEAs of the application can quickly and efficiently hydrolyze β-lactam antibiotics in waste water, significantly reduce its concentration;Compared with traditional treatment method, the present application has higher processing efficiency and good reusability, provides a kind of feasible solution for the treatment of antibiotic pollution wastewater, has wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of environmental engineering, and particularly relates to a β-lactamase immobilized sponge as well as a preparation method and application thereof. BACKGROUND

[0002] With the development of the pharmaceutical industry and the widespread use of antibiotics, antibiotic pollution has become an important problem of global water pollution. Antibiotics are one of the most widely used drugs in the world. The production of antibiotics has various ways according to different types, such as penicillin is biosynthesized by microbial fermentation, sulfonamides and quinolones can be produced by chemical synthesis, and semi-synthetic antibiotics are various derivatives prepared by chemical, biological or biochemical methods on the basis of biologically synthesized antibiotics. According to the chemical structure, antibiotics can be divided into quinolone antibiotics, β-lactam antibiotics, macrolide antibiotics, aminoglycoside antibiotics, etc. According to the use, antibiotics can be divided into antibacterial antibiotics, antifungal antibiotics, antitumor antibiotics, antiviral antibiotics, livestock antibiotics, agricultural antibiotics and other microbial drugs (such as ergot alkaloids produced by Claviceps purpurea, which have the effect of contracting the uterus), etc. Thousands of tons of antibiotics are used in poultry farming and human medical treatment every year. Most of the antibiotics cannot be completely metabolized in the human and animal body, and are excreted in the form of original shape and active metabolites through feces. Environmental pollution and ecological toxicological effects of antibiotics are one of the major environmental problems facing the world. Traditional wastewater treatment methods such as sedimentation, filtration and chemical oxidation often cannot effectively remove antibiotics in water, leading to their residues and biological accumulation in the environment, which poses a potential threat to the ecosystem and human health. Therefore, it is urgent to develop efficient and sustainable wastewater treatment technologies.

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

[0004] The present application provides a beta-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 antibiotic-polluted wastewater treatment.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical solutions.

[0006] One of the technical solutions of the present application is:

[0007] A preparation method of a beta-lactamase immobilized sponge, wherein a polydopamine-coated melamine sponge is used as a carrier, and beta-lactamase cross-linked enzyme aggregates (CLEAs) are immobilized on the surface of the carrier by cross-linking method to obtain the beta-lactamase immobilized sponge.

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

[0009] Beta-lactamase and bovine serum albumin are respectively dissolved in a phosphate buffer solution to obtain a beta-lactamase solution and a bovine serum albumin solution, the beta-lactamase solution and the bovine serum albumin solution are uniformly mixed, and then saturated ammonium sulfate solution is added for pre-assembly, followed by adding glutaraldehyde and the polydopamine-coated melamine sponge for cross-linking reaction, and after the cross-linking reaction is completed, washing is performed to obtain the beta-lactamase immobilized sponge (MS@beta-lactamase-CLEAs).

[0010] Melamine sponge is a kind of porous material with good physical and chemical properties and large specific surface area, which can provide a superior immobilization environment. Based on this, the present application cross-links beta-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 beta-lactamase, but also realize rapid hydrolysis of antibiotics in wastewater. The present application 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 antibiotic-polluted wastewater treatment.

[0011] The melamine sponge is used as a carrier, has excellent physical and chemical properties and a large specific surface area, and provides an ideal environment for the immobilization of beta-lactamase. By using saturated ammonium sulfate solution as a precipitant, the concentration of the enzyme can be effectively increased, and the rapid immobilization of the enzyme on the surface of the melamine sponge can be promoted, thereby significantly improving the efficiency of enzyme immobilization. In addition, glutaraldehyde as a crosslinking agent enhances the binding force between the enzyme and the carrier, forms a stable enzyme-carrier complex, and thus 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 the enzyme to maintain high activity in multiple uses, ensuring the rapid hydrolysis and removal of antibiotics in wastewater.

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

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

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

[0015] For example, the preparation method of the beta-lactamase solution with a concentration of 25mg / mL is to dissolve 25mg of beta-lactamase in 1mL of phosphate buffer with pH=7.4, stir uniformly, and obtain a beta-lactamase solution with a concentration of 25mg / mL.

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

[0017] For example, the preparation method of the bovine serum albumin solution with a concentration of 30mg / mL is to dissolve 30mg of bovine serum albumin in 1mL of phosphate buffer with pH=7.4, stir uniformly, and obtain a bovine serum albumin solution with a concentration of 30mg / mL.

[0018] The crosslinking reaction time is 1-4h. For example, the crosslinking reaction time is 1h, 2h, 3h or 4h, preferably the crosslinking reaction time is 3h.

[0019] The washing is to wash with phosphate buffer with pH=7.4 for three times. The purpose of washing is to remove the physically adsorbed beta-lactamase on the beta-lactamase immobilized sponge and the free enzyme not fixed 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 the amount of glutaraldehyde added is 60 mM.

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

[0022] (1) A melamine sponge with a size of 2*2*2 cm 3 is washed with anhydrous ethanol and water under ultrasonic condition for 5 min, respectively, and ultrasonic washing is performed for three times, respectively, and after drying in a drying oven, the washed melamine sponge is treated in a 2 mg / mL dopamine solution at room temperature for 12 h to obtain a polydopamine coated melamine sponge, which is ready for use;

[0023] (2) 25 mg of beta-lactamase and 30 mg of bovine serum albumin are respectively dissolved in 1 mL of phosphate buffer solution with pH = 7.4 to obtain a beta-lactamase solution and a bovine serum albumin solution, the beta-lactamase solution and the bovine serum albumin solution are mixed uniformly, then 5 mL of saturated ammonium sulfate solution is added, and mixed gently, pre-assembled for 1 h, 60 mM glutaraldehyde is added, and the polydopamine coated melamine sponge is added immediately, and crosslinking reaction is carried out at room temperature for 3 h, after the crosslinking reaction is completed, the beta-lactamase is recovered by gentle squeezing, and the beta-lactamase immobilized sponge is washed with phosphate buffer solution with pH = 7.4 for three times to remove the physically adsorbed beta-lactamase on the beta-lactamase immobilized sponge and the free enzyme in the solution which is not fixed, thereby obtaining the beta-lactamase immobilized sponge.

[0024] The second technical solution of the present application is:

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

[0026] The third technical solution of the present application is:

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

[0028] The method of the present application enhances the stability and catalytic activity of beta-lactamase, and the MS@beta-lactamase-CLEAs of the present application can quickly and efficiently hydrolyze beta-lactam antibiotics in wastewater, significantly reducing their concentration. Compared with traditional treatment methods, for example, using activated carbon adsorption method, although this method can remove part of the 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 cost. The present application has higher treatment efficiency and good reusability, providing a feasible solution for the treatment of antibiotic-polluted wastewater, and has broad application prospects.

[0029] Illustratively, the antibiotic is penicillin.

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

[0031] The present application uses melamine sponge as a carrier, and the beta-lactamase cross-linked enzyme aggregates (CLEAs) are fixed on the surface of the carrier by cross-linking method, 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, which can maintain high catalytic efficiency in a wide range of temperature and pH; second, the excellent physical properties of melamine sponge make it easier to recover and reuse the enzyme, reducing the operating cost. In addition, this technology not only can quickly remove antibiotics in wastewater, improve water quality, meet environmental protection requirements, but also promotes the recycling of resources, has good economic and social benefits. Through this innovation, the application of biological catalysis technology in wastewater treatment field is promoted, which makes positive contribution to the realization of sustainable development goal. BRIEF DESCRIPTION OF DRAWINGS

[0032] The accompanying drawings, which form a part of this application, are included to provide a further understanding of the application, and are incorporated in and constitute a part of this application. The illustrations, together with their description, serve to explain the application without limiting it. In the drawings:

[0033] Figure 1 A flow chart for preparing the beta-lactamase immobilized sponge of the present application and a working principle diagram;

[0034] Figure 2 The activity determination results of beta-lactamase;

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

[0036] Figure 4Pictures for characterization of MS, MS@ β-lactamase-CLEAs and CLEAs, wherein A: SEM image of MS, B: SEM image of MS@ β-lactamase-CLEAs, C: SEM image of CLEAs, D: TG-DSC picture of MS and MS@ β-lactamase-CLEAs;

[0037] Figure 5 Effect of different β-lactamase dosage on residual activity of β-lactamase-CLEAs in MS@ β-lactamase-CLEAs;

[0038] Figure 6 Effect of different cross-linking time on residual activity of β-lactamase-CLEAs in MS@ β-lactamase-CLEAs;

[0039] Figure 7 Effect of different glutaraldehyde addition amount on residual activity of β-lactamase-CLEAs in MS@ β-lactamase-CLEAs;

[0040] Figure 8 Effect of different temperature on recovery activity of free enzyme and cross-linked enzyme aggregates (CLEAs);

[0041] Figure 9 Effect of different pH on recovery activity of free enzyme and cross-linked enzyme aggregates (CLEAs);

[0042] Figure 10 Hydrolysis results of MS@ β-lactamase-CLEAs on antibiotics in wastewater. DETAILED DESCRIPTION

[0043] Various example embodiments of the present application will now be described in detail with reference to the drawings. Such description, however, is to be considered in all aspects as illustrative and not restrictive, understanding that the scope of the application will be determined by the appended claims.

[0044] It is to be understood that the terms used in the present specification are merely used to describe particular embodiments and are not intended to limit the present application otherwise. In addition, for numerical ranges in the present application, it is to be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range within the stated range and between any stated value or stated range and any other stated value or stated range is also encompassed. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0045] Unless otherwise indicated, all technical and scientific terms have the same meaning as those one of ordinary skill in the art of the disclosure would understand. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. All documents mentioned herein are incorporated by reference to disclose and describe in further detail the methods and / or materials associated with the documents. In the case of conflict between the present specification and any incorporated document, the present specification controls.

[0046] Many modifications and variations of the present disclosure described in the detailed description of the specification can be made without departing from the scope or spirit of the present disclosure, which will be apparent to those skilled in the art. Other embodiments resulting from the present disclosure will be apparent to the skilled person. The present specification and examples are merely illustrative.

[0047] As used herein, "comprise", "comprising", "have", "having", "include", "including", "contain", "containing", and the like, are open-ended terms that are intended to mean including, but not limited to.

[0048] Unless otherwise specified, the room temperature in the present disclosure is 25±2℃.

[0049] Each raw material used in the embodiments of the present disclosure is obtained by commercial purchase. As an example, β-lactamase is purchased from Shanghai Lingne Science and Technology Development Co., Ltd.; bovine serum albumin is purchased from Shanghai Aladdin Biochem Technology Co., Ltd.

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

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

[0052] 1. β-lactamase activity determination:

[0053] β-lactamase uses nitrocefin as a substrate, which can hydrolyze the amide bond between the carbonyl carbon and the nitrogen group on the β-lactam ring. After nitrocefin is hydrolyzed by β-lactamase, there is a significant color change from yellow to red, and the absorbance is determined at 482 nm by ultraviolet spectrophotometry.

[0054] The absorbance of the reaction product is determined by colorimetry to obtain the activity standard curve of β-lactamase. As shown in Figure 2 The absorbance corresponding to different concentrations (0, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL) of β-lactamase increases proportionally. The absorbance of 6.25-100 μg / mL β-lactamase is linearly correlated with the concentration of β-lactamase, and the linear equation is A = 0.0056C + 0.0019 (R2= 0.9999), where A is the absorbance and C is the concentration of β-lactamase. -1The linear correlation (y = 0.0077x + 0.0924, R 2 The experimental results show that the β-lactamase activity detection method established in the present application is effective.

[0055] 2. Penicillin concentration determination:

[0056] The change of penicillin concentration is calculated by iodometric method. The product penicilloic acid of penicillin antibiotics hydrolyzed by alkali can react with iodine (1 mol of penicilloic acid can react with 8 mol of iodine atoms, i.e. penicillin: I2 = 1:4 (molar ratio)), and the content of penicillin can be calculated according to the amount of iodine consumed.

[0057] In the present application, the degradation rate of penicillin and the penicillin concentration are two different concepts. The degradation rate of penicillin refers to the degree of degradation of penicillin in the reaction process, which is usually expressed in percentage; and the penicillin concentration refers to the specific concentration value of penicillin in the solution. The calculation formula of the degradation rate of penicillin (%) is: degradation rate of penicillin (%) = (initial concentration of penicillin solution - concentration of penicillin solution after reaction) / initial concentration of penicillin solution x 100%. Wherein, 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 measured concentration of penicillin after the reaction.

[0058] In the present application, the testing process and calculation formula of residual activity are as follows: dilute CLEAs to 100 μg / mL to obtain a dilution solution, take 300 μL of the dilution solution and mix with 300 μL of cefoxitin with a concentration of 50 μg / mL, and react 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 a UV spectrophotometer. The calculation formula of residual activity (%) is: residual activity (%) = (total β-lactamase activity in β-lactamase-CLEAs) / (total β-lactamase activity used for preparation of β-lactamase-CLEAs) x 100%.

[0059] In an embodiment of the present application, the preparation method of cross-linked enzyme aggregate (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 solution with 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 uniformly, 5 mL of saturated ammonium sulfate solution is added, and the mixture is gently mixed and pre-assembled for 1 h, 60 mM glutaraldehyde is added, and the cross-linking reaction is carried out at room temperature for 3 h, after the cross-linking reaction is completed, the cross-linked enzyme aggregate is recovered by centrifugation, and the cross-linked enzyme aggregate is washed with phosphate buffer solution with pH = 7.4 for three times to obtain CLEAs. The CLEAs are diluted to 1 mg / mL, and the free enzyme is diluted to 100 μg / mL. First, the free enzyme and the CLEAs are incubated at 4-65°C for 60 minutes, and the absorbance (A) at each temperature is recorded. Subsequently, the two forms of enzymes are incubated in different pH buffer solutions (pH 4-10) for 60 minutes, and 300 μL of the diluted solution is mixed with 300 μL of cefalotin with a concentration of 50 μg / mL 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 a UV spectrophotometer. In order to evaluate the recovery activity at different pH values, the recovery activity is calculated using the formula: recovery activity (pH) = (A 样品 / A pH 7) × 100%, wherein A 样品 is the absorbance at a specific pH value, and A pH 7 is the absorbance at pH 7. At the same time, for the recovery activity at different temperatures, the recovery activity is calculated using the formula: recovery activity (temperature) = (A 样品 / A 4℃ ) × 100, wherein A 4℃ is the absorbance at 4°C. Through these steps, the recovery activity of the free enzyme and the CLEAs under different temperature and pH conditions can be systematically evaluated.

[0060] It should be noted that the details not described in the present application are all conventional operation means in the art, and are not the focus of the present application.

[0061] The technical solutions of the present application are further described below through typical embodiments.

[0062] Embodiment 1

[0063] (1) A 2*2*2 cm 3The melamine sponge was washed with anhydrous ethanol and water for 5 min under ultrasonic condition, respectively, and washed ultrasonically for three times, respectively. The water in the melamine sponge was absorbed by filter paper, and the dried melamine sponge was obtained by drying in a 60°C drying box for 2 h. The dried melamine sponge was treated in a 2 mg / mL dopamine solution (the solvent of the dopamine solution was 10 mM pH = 8.5 HCl-Tris) at room temperature for 12 h. After the end, the unreacted dopamine was removed by washing with purified water for three times to obtain a polydopamine coated melamine sponge, which was ready for use.

[0064] (2) 25 mg of β-lactamase and 30 mg of bovine serum albumin were dissolved in 1 mL of phosphate buffer solution with pH = 7.4, respectively, to obtain a β-lactamase solution (25 mg / mL) and a bovine serum albumin solution (30 mg / mL). After mixing 1 mL of the β-lactamase solution and 1 mL of the bovine serum albumin solution uniformly, 5 mL of saturated ammonium sulfate solution was added, and mixed gently. After pre-assembling for 1 h, 60 mM glutaraldehyde was added, and the polydopamine coated melamine sponge was added immediately. The crosslinking reaction was carried out at room temperature for 3 h. After the end of the crosslinking reaction, the β-lactamase was recovered by gentle squeezing, and the β-lactamase immobilized sponge was washed with phosphate buffer solution with pH = 7.4 for three times to remove the physically adsorbed β-lactamase on the β-lactamase immobilized sponge and the free enzyme in the solution which was not fixed, so as to obtain a β-lactamase immobilized sponge (MS@β-lactamase-CLEAs).

[0065] Example 2

[0066] (1) The melamine sponge with a size of 2*2*2 cm 3 was washed with anhydrous ethanol and water for 5 min under ultrasonic condition, respectively, and washed ultrasonically for three times, respectively. The water in the melamine sponge was absorbed by filter paper, and the dried melamine sponge was obtained by drying in a 60°C drying box for 2 h. The dried melamine sponge was treated in a 2 mg / mL dopamine solution at room temperature for 12 h. After the end, the unreacted dopamine was removed by washing with purified water for three times to obtain a polydopamine coated melamine sponge, which was ready for use;

[0067] (2) 25 mg of β-lactamase and 30 mg of bovine serum albumin were respectively dissolved in 1 mL of phosphate buffer solution with pH = 7.4 to obtain a β-lactamase solution (25 mg / mL) and a bovine serum albumin solution (30 mg / mL), 1 mL of the β-lactamase solution and 1 mL of the bovine serum albumin solution were mixed uniformly, and then 5 mL of saturated ammonium sulfate solution was added and mixed gently, pre-assembled for 1 h, 60 mM glutaraldehyde was added, and then the polydopamine coated melamine sponge was immediately added, and crosslinking reaction was carried out at room temperature for 1 h, after the crosslinking reaction was completed, the β-lactamase was recovered by gentle squeezing, and the β-lactamase immobilized sponge was washed with phosphate buffer solution with pH = 7.4 for three times to remove the physically adsorbed β-lactamase on the β-lactamase immobilized sponge and the free enzyme in the solution which was not immobilized, and thus a β-lactamase immobilized sponge was obtained.

[0068] Example 3

[0069] (1) The melamine sponge with a size of 2*2*2 cm 3 was washed with anhydrous ethanol and water under ultrasonic condition for 5 min, respectively, and was ultrasonically washed for three times, respectively, the water in the melamine sponge was absorbed by filter paper, and the dried melamine sponge was dried in a 60°C drying box for 2 h, the dried melamine sponge was treated in a 2 mg / mL dopamine solution at room temperature for 12 h, and then was washed with pure water for three times to remove the unreacted dopamine, and thus a polydopamine coated melamine sponge was obtained and was ready for use;

[0070] (2) 25 mg of β-lactamase and 30 mg of bovine serum albumin were respectively dissolved in 1 mL of phosphate buffer solution with pH = 7.4 to obtain a β-lactamase solution (25 mg / mL) and a bovine serum albumin solution (30 mg / mL), 1 mL of the β-lactamase solution and 1 mL of the bovine serum albumin solution were mixed uniformly, and then 5 mL of saturated ammonium sulfate solution was added and mixed gently, pre-assembled for 1 h, 60 mM glutaraldehyde was added, and then the polydopamine coated melamine sponge was immediately added, and crosslinking reaction was carried out at room temperature for 2 h, after the crosslinking reaction was completed, the β-lactamase was recovered by gentle squeezing, and the β-lactamase immobilized sponge was washed with phosphate buffer solution with pH = 7.4 for three times to remove the physically adsorbed β-lactamase on the β-lactamase immobilized sponge and the free enzyme in the solution which was not immobilized, and thus a β-lactamase immobilized sponge was obtained.

[0071] Example 4

[0072] (1) The melamine sponge with a size of 2*2*2 cm 3The melamine sponge was washed with anhydrous ethanol and water for 5 min under ultrasonic condition respectively, and washed for three times respectively, the water in the melamine sponge was absorbed by filter paper, and the dried melamine sponge was obtained by drying in a 60°C drying box for 2 h, the dried melamine sponge was treated in a 2 mg / mL dopamine solution at room temperature for 12 h, and then washed with purified water for three times to remove the unreacted dopamine, to obtain the polydopamine coated melamine sponge, which was ready for use;

[0073] (2) 25 mg of β-lactamase and 30 mg of bovine serum albumin were dissolved in 1 mL of phosphate buffer solution with pH = 7.4 respectively to obtain a β-lactamase solution (25 mg / mL) and a bovine serum albumin solution (30 mg / mL), 1 mL of the β-lactamase solution and 1 mL of the bovine serum albumin solution were mixed uniformly, then 5 mL of saturated ammonium sulfate solution was added, and mixed gently, pre-assembled for 1 h, 60 mM glutaraldehyde was added, and the polydopamine coated melamine sponge was added immediately, and the crosslinking reaction was carried out at room temperature for 4 h, after the crosslinking reaction was completed, the β-lactamase was recovered by gentle squeezing, and the β-lactamase immobilized sponge was washed with phosphate buffer solution with pH = 7.4 for three times to remove the physically adsorbed β-lactamase on the β-lactamase immobilized sponge and the free enzyme in the solution which was not fixed, to obtain the β-lactamase immobilized sponge.

[0074] Example 5

[0075] (1) The melamine sponge with a size of 2*2*2 cm 3 was washed with anhydrous ethanol and water for 5 min under ultrasonic condition respectively, and washed for three times respectively, the water in the melamine sponge was absorbed by filter paper, and the dried melamine sponge was obtained by drying in a 60°C drying box for 2 h, the dried melamine sponge was treated in a 2 mg / mL dopamine solution at room temperature for 12 h, and then washed with purified water for three times to remove the unreacted dopamine, to obtain the polydopamine coated melamine sponge, which was ready for use;

[0076] (2) 10 mg of β-lactamase and 30 mg of bovine serum albumin were respectively dissolved in 1 mL of phosphate buffer solution with pH = 7.4 to obtain a β-lactamase solution (10 mg / mL) and a bovine serum albumin solution (30 mg / mL), 1 mL of the β-lactamase solution and 1 mL of the bovine serum albumin solution were mixed uniformly, and then 5 mL of saturated ammonium sulfate solution was added and mixed gently, pre-assembled for 1 h, 60 mM glutaraldehyde was added, and then the polydopamine coated melamine sponge was immediately added, and crosslinking reaction was carried out at room temperature for 3 h, after the crosslinking reaction was completed, the β-lactamase was recovered by gentle squeezing, and the β-lactamase immobilized sponge was washed with phosphate buffer solution with pH = 7.4 for three times to remove the physically adsorbed β-lactamase on the β-lactamase immobilized sponge and the free enzyme in the solution which was not immobilized, and thus a β-lactamase immobilized sponge was obtained.

[0077] Example 6

[0078] (1) The melamine sponge with a size of 2*2*2 cm 3 was washed with anhydrous ethanol and water under ultrasonic condition for 5 min, respectively, and was ultrasonically washed for three times, respectively, the water in the melamine sponge was absorbed by filter paper, and the dried melamine sponge was dried in a 60°C drying box for 2 h, the dried melamine sponge was treated in a 2 mg / mL dopamine solution at room temperature for 12 h, and then was washed with pure water for three times to remove the unreacted dopamine, and thus a polydopamine coated melamine sponge was obtained and was ready for use;

[0079] (2) 15 mg of β-lactamase and 30 mg of bovine serum albumin were respectively dissolved in 1 mL of phosphate buffer solution with pH = 7.4 to obtain a β-lactamase solution (15 mg / mL) and a bovine serum albumin solution (30 mg / mL), 1 mL of the β-lactamase solution and 1 mL of the bovine serum albumin solution were mixed uniformly, and then 5 mL of saturated ammonium sulfate solution was added and mixed gently, pre-assembled for 1 h, 60 mM glutaraldehyde was added, and then the polydopamine coated melamine sponge was immediately added, and crosslinking reaction was carried out at room temperature for 3 h, after the crosslinking reaction was completed, the β-lactamase was recovered by gentle squeezing, and the β-lactamase immobilized sponge was washed with phosphate buffer solution with pH = 7.4 for three times to remove the physically adsorbed β-lactamase on the β-lactamase immobilized sponge and the free enzyme in the solution which was not immobilized, and thus a β-lactamase immobilized sponge was obtained.

[0080] Example 7

[0081] (1) The melamine sponge with a size of 2*2*2 cm 3The melamine sponge was washed with anhydrous ethanol and water for 5 min under ultrasonic condition, respectively, and washed ultrasonically for three times, respectively. The water in the melamine sponge was absorbed by filter paper, and the dried melamine sponge was dried in a 60°C drying box for 2 h. The dried melamine sponge was treated in a 2 mg / mL dopamine solution at room temperature for 12 h. After the end, the unreacted dopamine was removed by washing with purified water for three times to obtain a polydopamine coated melamine sponge, which was ready for use.

[0082] (2) 20 mg of β-lactamase and 30 mg of bovine serum albumin were dissolved in 1 mL of phosphate buffer solution with pH = 7.4 to obtain a β-lactamase solution (20 mg / mL) and a bovine serum albumin solution (30 mg / mL). After mixing 1 mL of the β-lactamase solution and 1 mL of the bovine serum albumin solution uniformly, 5 mL of saturated ammonium sulfate solution was added, and mixed gently. After pre-assembly for 1 h, 60 mM glutaraldehyde was added, and the polydopamine coated melamine sponge was added immediately. The crosslinking reaction was carried out at room temperature for 3 h. After the end of the crosslinking reaction, the β-lactamase was recovered by gentle squeezing, and the β-lactamase immobilized sponge was washed with phosphate buffer solution with pH = 7.4 for three times to remove the physically adsorbed β-lactamase on the β-lactamase immobilized sponge and the free enzyme in the solution which was not fixed, thereby obtaining a β-lactamase immobilized sponge.

[0083] Example 8

[0084] (1) The melamine sponge with a size of 2*2*2 cm 3 was washed with anhydrous ethanol and water for 5 min under ultrasonic condition, respectively, and washed ultrasonically for three times, respectively. The water in the melamine sponge was absorbed by filter paper, and the dried melamine sponge was dried in a 60°C drying box for 2 h. The dried melamine sponge was treated in a 2 mg / mL dopamine solution at room temperature for 12 h. After the end, the unreacted dopamine was removed by washing with purified water for three times to obtain a polydopamine coated melamine sponge, which was ready for use;

[0085] (2) 30 mg of β-lactamase and 30 mg of bovine serum albumin were respectively dissolved in 1 mL of phosphate buffer solution with pH = 7.4 to obtain a β-lactamase solution (30 mg / mL) and a bovine serum albumin solution (30 mg / mL), 1 mL of the β-lactamase solution and 1 mL of the bovine serum albumin solution were mixed uniformly, and then 5 mL of saturated ammonium sulfate solution was added and gently mixed, pre-assembled for 1 h, 60 mM glutaraldehyde was added, and then the polydopamine coated melamine sponge was immediately added, crosslinking reaction was carried out at room temperature for 3 h, after the crosslinking reaction was completed, the β-lactamase was recovered by gently squeezing, and the β-lactamase immobilized sponge was washed with phosphate buffer solution with pH = 7.4 for three times to remove the physically adsorbed β-lactamase on the β-lactamase immobilized sponge and the free enzyme in the solution which was not immobilized, and the β-lactamase immobilized sponge was obtained.

[0086] Example 9

[0087] (1) The melamine sponge with a size of 2*2*2 cm 3 was washed with anhydrous ethanol and water under ultrasonic condition for 5 min respectively, and was ultrasonically washed for three times respectively, the water in the melamine sponge was absorbed by filter paper, and the dried melamine sponge was obtained by drying in a 60°C drying box for 2 h, the dried melamine sponge was treated in a 2 mg / mL dopamine solution at room temperature for 12 h, and then was washed with pure water for three times to remove the unreacted dopamine, and the polydopamine coated melamine sponge was obtained and was prepared for use;

[0088] (2) 25 mg of β-lactamase and 30 mg of bovine serum albumin were respectively dissolved in 1 mL of phosphate buffer solution with pH = 7.4 to obtain a β-lactamase solution (25 mg / mL) and a bovine serum albumin solution (30 mg / mL), 1 mL of the β-lactamase solution and 1 mL of the bovine serum albumin solution were mixed uniformly, and then 5 mL of saturated ammonium sulfate solution was added and gently mixed, pre-assembled for 1 h, 40 mM glutaraldehyde was added, and then the polydopamine coated melamine sponge was immediately added, crosslinking reaction was carried out at room temperature for 3 h, after the crosslinking reaction was completed, the β-lactamase was recovered by gently squeezing, and the β-lactamase immobilized sponge was washed with phosphate buffer solution with pH = 7.4 for three times to remove the physically adsorbed β-lactamase on the β-lactamase immobilized sponge and the free enzyme in the solution which was not immobilized, and the β-lactamase immobilized sponge was obtained.

[0089] Example 10

[0090] (1) The melamine sponge with a size of 2*2*2 cm 3The melamine sponge was washed with anhydrous ethanol and water for 5 min under ultrasonic condition respectively, and washed for three times respectively, the water in the melamine sponge was absorbed by filter paper, and the dried melamine sponge was obtained by drying in a 60℃ drying box for 2 h, the dried melamine sponge was treated in a 2 mg / mL dopamine solution at room temperature for 12 h, and then washed with purified water for three times to remove the unreacted dopamine, to obtain the polydopamine coated melamine sponge, which was ready for use;

[0091] (2) 25 mg of β-lactamase and 30 mg of bovine serum albumin were dissolved in 1 mL of phosphate buffer solution with pH = 7.4 respectively to obtain a β-lactamase solution (25 mg / mL) and a bovine serum albumin solution (30 mg / mL), 1 mL of the β-lactamase solution and 1 mL of the bovine serum albumin solution were mixed uniformly, then 5 mL of saturated ammonium sulfate solution was added, and mixed gently, pre-assembled for 1 h, 80 mM glutaraldehyde was added, and the polydopamine coated melamine sponge was added immediately, and the crosslinking reaction was carried out at room temperature for 3 h, after the crosslinking reaction was completed, the β-lactamase was recovered by gentle squeezing, and the β-lactamase immobilized sponge was washed with phosphate buffer solution with pH = 7.4 for three times to remove the physically adsorbed β-lactamase on the β-lactamase immobilized sponge and the free enzyme in the solution which was not fixed, to obtain the β-lactamase immobilized sponge.

[0092] Comparative Example 1

[0093] (1) The melamine sponge with a size of 2*2*2 cm 3 was washed with anhydrous ethanol and water for 5 min under ultrasonic condition respectively, and washed for three times respectively, the water in the melamine sponge was absorbed by filter paper, and the dried melamine sponge was obtained by drying in a 60℃ drying box for 2 h, the dried melamine sponge was treated in a 2 mg / mL dopamine solution at room temperature for 12 h, and then washed with purified water for three times to remove the unreacted dopamine, to obtain the polydopamine coated melamine sponge, which was ready for use;

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

[0095] Comparative Example 2

[0096] (1) The melamine sponge with a size of 2*2*2 cm 3 was washed with anhydrous ethanol and water under ultrasonic condition for 5 min, respectively, and the washing was performed for three times. The water in the melamine sponge was absorbed by filter paper, and the dried melamine sponge was obtained by drying in a 60°C drying box for 2 h. The dried melamine sponge was treated in a 2 mg / mL dopamine solution at room temperature for 12 h. After the treatment, the unreacted dopamine was removed by washing with purified water for three times, thereby obtaining the polydopamine-coated melamine sponge, which was ready for use.

[0097] (2) 25 mg of β-lactamase and 30 mg of bovine serum albumin were respectively dissolved in 1 mL of phosphate buffer solution with pH = 7.4 to obtain a β-lactamase solution (25 mg / mL) and a bovine serum albumin solution (30 mg / mL). After mixing 1 mL of the β-lactamase solution and 1 mL of the bovine serum albumin solution uniformly, 5 mL of saturated ammonium sulfate solution was added, and then mixed gently. After pre-assembly for 1 h, 20 mM glutaraldehyde was added, and then the polydopamine-coated melamine sponge was immediately added. After cross-linking reaction at room temperature for 3 h, the β-lactamase was recovered by gentle squeezing, and the β-lactamase immobilized sponge was washed with phosphate buffer solution with pH = 7.4 for three times to remove the physically adsorbed β-lactamase on the β-lactamase immobilized sponge and the free enzyme in the solution which was not immobilized, thereby obtaining the β-lactamase immobilized sponge.

[0098] Comparative Example 3

[0099] (1) The melamine sponge with a size of 2*2*2 cm 3The melamine sponge was washed with anhydrous ethanol and water for 5 min under ultrasonic condition respectively, and washed for three times respectively, the water in the melamine sponge was absorbed by filter paper, and the dried melamine sponge was obtained by drying in a 60°C drying box for 2 h. The dried melamine sponge was treated in a 2 mg / mL dopamine solution at room temperature for 12 h, and then washed with purified water for three times to remove the unreacted dopamine, to obtain a polydopamine coated melamine sponge, which was ready for use.

[0100] (2) 25 mg of β-lactamase and 30 mg of bovine serum albumin were dissolved in 1 mL of phosphate buffer solution with pH = 7.4 respectively to obtain a β-lactamase solution (25 mg / mL) and a bovine serum albumin solution (30 mg / mL). After mixing 1 mL of the β-lactamase solution and 1 mL of the bovine serum albumin solution uniformly, 5 mL of saturated ammonium sulfate solution was added, and mixed gently, pre-assembled for 1 h, 60 mM glutaraldehyde was added, and then the polydopamine coated melamine sponge was added immediately, and crosslinking reaction was carried out at room temperature for 5 h. After the crosslinking reaction was completed, the β-lactamase was recovered by gentle squeezing, and the β-lactamase immobilized sponge was washed with phosphate buffer solution with pH = 7.4 for three times to remove the physically adsorbed β-lactamase on the β-lactamase immobilized sponge and the free enzyme in the solution which was not fixed, to obtain a β-lactamase immobilized sponge.

[0101] Comparative Example 4

[0102] (1) The melamine sponge with a size of 2*2*2 cm 3 was washed with anhydrous ethanol and water for 5 min under ultrasonic condition respectively, and washed for three times respectively, the water in the melamine sponge was absorbed by filter paper, and the dried melamine sponge was obtained by drying in a 60°C drying box for 2 h. The dried melamine sponge was treated in a 2 mg / mL dopamine solution at room temperature for 12 h, and then washed with purified water for three times to remove the unreacted dopamine, to obtain a polydopamine coated melamine sponge, which was ready for use.

[0103] (2) 5 mg of β-lactamase and 30 mg of bovine serum albumin were respectively dissolved in 1 mL of phosphate buffer solution with pH = 7.4 to obtain a β-lactamase solution (5 mg / mL) and a bovine serum albumin solution (30 mg / mL), 1 mL of the β-lactamase solution and 1 mL of the bovine serum albumin solution were mixed uniformly, then 5 mL of saturated ammonium sulfate solution was added, and mixed gently, pre-assembled for 1 h, 60 mM glutaraldehyde was added, and then polydopamine coated melamine sponge was added immediately, and crosslinking reaction was carried out at room temperature for 3 h, after the crosslinking reaction was completed, the β-lactamase was recovered by gentle squeezing, and the β-lactamase immobilized sponge was washed with phosphate buffer solution with pH = 7.4 for three times to remove the physically adsorbed β-lactamase on the β-lactamase immobilized sponge and the free enzyme in the solution which was not immobilized, and thus the β-lactamase immobilized sponge was obtained.

[0104] The reusability of MS@β-lactamase-CLEAs affects its cost and application prospect. The reusability of the MS@β-lactamase-CLEAs prepared in Example 1 was tested. The specific method was as follows: the MS@β-lactamase-CLEAs were placed in 20 mL of penicillin solution with a concentration of 200 μg / mL, and the reaction was carried out at room temperature for 25 minutes to evaluate the reusability. The reusability of the MS@β-lactamase-CLEAs was verified by carrying out 7 cycles of use experiments. After the end of each cycle, the sponge was squeezed and washed thoroughly with purified water to remove the reaction residues. The calculation formula of the degradation rate (%) of penicillin was: the degradation rate (%) of penicillin = (the initial concentration of penicillin solution - the concentration of penicillin solution after reaction) / the initial concentration of penicillin solution × 100%. The results are shown in Figure 3 The degradation rate of penicillin during the recycling process of the MS@β-lactamase-CLEAs was determined by Example 1, and it was found that the degradation rate was still more than 85% after 7 times of reuse, indicating that the MS@β-lactamase-CLEAs prepared by the application had good reusability.

[0105] The 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 as a function of temperature and time was measured by a simultaneous thermal analyzer (TGA / DSC 1), so as to comprehensively analyze the thermal stability and reactivity of the materials. The results are shown in Figure 4 Figure 4 ​As shown in A, B and C, the sponge individual β-lactamase-CLEAs were effectively loaded into the sponge, which took advantage of the well-structured framework of the sponge. The single sponge has a smooth and flat surface, as well as a porous framework, which provides a large surface area for the adsorption of immobilized enzymes. In contrast, the modified sponge surface is rough and uneven, indicating that the enzyme is successfully grafted into the sponge. As shown in D, the mass loss of melamine sponge is less than that of melamine sponge @ β-lactamase-CLEAs in the range of 260-663 ℃, suggesting that the cross-linked enzyme aggregates may undergo more significant degradation in this temperature range, reflecting the poor thermal stability of β-lactamase. While in the interval of 663-800 ℃, the weight loss of melamine sponge is higher than that of melamine sponge @ β-lactamase-CLEAs, indicating that the cross-linking method enhances the structural integrity and stability of the material at high temperature. These results support the effectiveness of the present application, indicating that the prepared composite material has excellent thermal stability in practical applications. Figure 4 As shown in A, B and C, the sponge individual β-lactamase-CLEAs were effectively loaded into the sponge, which took advantage of the well-structured framework of the sponge. The single sponge has a smooth and flat surface, as well as a porous framework, which provides a large surface area for the adsorption of immobilized enzymes. In contrast, the modified sponge surface is rough and uneven, indicating that the enzyme is successfully grafted into the sponge. As shown in D, the mass loss of melamine sponge is less than that of melamine sponge @ β-lactamase-CLEAs in the range of 260-663 ℃, suggesting that the cross-linked enzyme aggregates may undergo more significant degradation in this temperature range, reflecting the poor thermal stability of β-lactamase. While in the interval of 663-800 ℃, the weight loss of melamine sponge is higher than that of melamine sponge @ β-lactamase-CLEAs, indicating that the cross-linking method enhances the structural integrity and stability of the material at high temperature. These results support the effectiveness of the present application, indicating 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 remaining activity of β-lactamase-CLEAs in MS @ β-lactamase-CLEAs prepared in each of the above examples and each of the comparative examples was tested, and the results of the effect of different β-lactamase dosages on the remaining activity of β-lactamase-CLEAs in MS @ β-lactamase-CLEAs are shown in Table 1. Figure 5 The results of the effect of different cross-linking times on the remaining activity of β-lactamase-CLEAs in MS @ β-lactamase-CLEAs are shown in Table 2. Figure 6 The results of the effect of different glutaraldehyde dosages on the remaining activity of β-lactamase-CLEAs in MS @ β-lactamase-CLEAs are shown in Table 3. Figure 7 . Figure 5 As shown in Table 1, when the enzyme dosage is 25 mg, the enzyme activity in β-lactamase-CLEAs reaches a maximum value. Excessive enzyme can lead to a decrease in the activity of β-lactamase-CLEAs, as the space effect around the enzyme active site is destroyed. Figure 6 As shown in Table 2, the enzyme activity increases with time, and the enzyme activity reaches a maximum value at 3 h, and then starts to decrease after 3 h. Figure 7 As shown in Table 3, the effect of glutaraldehyde dosage on enzyme activity is shown. When the glutaraldehyde dosage exceeds 60 mM, excessive glutaraldehyde can cause partial enzyme inactivation, indicating that the remaining activity is best when the glutaraldehyde dosage is 60 mM. In summary, the optimal parameters for preparing β-lactamase-CLEAs are: the content of β-lactamase is 25 mg, the cross-linking time is 3 h, and the glutaraldehyde dosage is 60 mM. Under this dosage, the maximum remaining activity of β-lactamase-CLEAs is 29.6%.

[0108] Under optimal preparation conditions (i.e., the preparation method of Example 1), the effects of temperature and pH on the activities 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 (pH 7.4) to obtain β-lactamase and bovine serum albumin solutions, respectively. The β-lactamase and bovine serum albumin solutions were mixed thoroughly, and 5 mL of saturated ammonium sulfate solution was added. The mixture was gently mixed and pre-assembled for 1 h. Then, 60 mM glutaraldehyde was added, and the cross-linking reaction was carried out at room temperature for 3 h. After the cross-linking reaction was completed, the cross-linked enzyme aggregates were washed three times with phosphate buffer (pH 7.4) to obtain CLEAs.

[0109] The relative enzyme activities of free enzymes and cross-linked enzyme aggregates (CLEAs) were measured after incubation for 60 minutes in the range of 4–65 °C. Furthermore, for both enzyme forms, recovery activities at different pH values ​​were evaluated after incubation for 60 minutes in different pH buffers (pH 4–10), with pH 7 activity set as 100%. Simultaneously, recovery activities were measured at different temperatures, with 4 °C activity set as 100%. Results are as follows: Figure 8 The results showed that at 35°C and 45°C, the recovery activity of cross-linked enzyme aggregates (CLEAs) was significantly higher than that of free enzymes (i.e., individual β-lactamases). Figure 9 The results showed that both acidic and alkaline conditions reduced the activity of both free enzymes and cross-linked enzyme aggregates. However, it is noteworthy that cross-linked enzyme aggregates exhibited higher activity under both acidic and alkaline conditions. In summary, these results demonstrate that cross-linked enzyme aggregates exhibit superior stability compared to free enzymes under various temperature and pH conditions, supporting their potential for industrial applications.

[0110] Application example: Results of antibiotic treatment 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, and 90 min) for concentration analysis.

[0112] like Figure 10 As shown, in a circulating reaction apparatus, 400 mL of wastewater containing 80 mg of penicillin was treated. After approximately 80 minutes of reaction, the degradation rate of penicillin reached its maximum (approximately 90%). Specifically, the initial penicillin concentration was 0.2 mg / mL, and the residual concentration after degradation decreased to 0.02 mg / mL, indicating that the β-lactamase immobilized sponge of this invention has good catalytic activity and application potential in the degradation of penicillin.

[0113] In the treatment of antibiotic-containing wastewater, traditional methods mainly include chemical degradation, biological degradation, physical treatment and thermal treatment. Chemical degradation usually uses acid or base to destroy the β-lactam ring of penicillin, although the method is simple, but may produce secondary pollution, and the reaction conditions need to be strictly controlled. Biological degradation uses specific microorganisms or enzymes (such as β-lactamase-producing bacteria) to treat penicillin wastewater, which 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, and its effect depends on the properties of the adsorbent and operating conditions. Thermal treatment destroys the structure of penicillin molecules by heating, although it can effectively reduce the activity, but it has the disadvantages of high energy consumption and may affect the water quality. The above traditional methods have their own advantages and disadvantages, and the selection of the appropriate method needs to consider the treatment efficiency, environmental impact and economy, etc. Compared with traditional methods, β-lactamase immobilized sponge shows higher catalytic activity and application potential.

[0114] The above, only for the preferred specific embodiments of the present application, but the scope of protection of the present application is not limited to this, any skilled in the art of the technical personnel in the technical range disclosed by the present application, can easily think of changes or replacement, should be covered in the scope of protection of the present application. Therefore, the scope of protection of the present application should be subject to the scope of protection of the claims.

Claims

1. The application of a β-lactamase immobilized sponge in the use of β-lactam antibiotics in hydrolyzed wastewater, characterized in that, The method for preparing the β-lactamase-immobilized sponge includes the following steps: The melamine sponge was cleaned, dried, and then treated in a dopamine solution to obtain polydopamine-coated melamine sponge. β-lactamase and bovine serum albumin were dissolved in phosphate buffer to obtain β-lactamase solution and bovine serum albumin solution, respectively. The β-lactamase solution and bovine serum albumin solution were mixed evenly and then saturated ammonium sulfate solution was added for pre-assembly. Glutaraldehyde and the polydopamine-coated melamine sponge were then added for cross-linking reaction. After the cross-linking reaction was completed, the sponge was washed to obtain the β-lactamase immobilized sponge. The concentration of the β-lactamase solution is 10-30 mg / mL; The cross-linking reaction takes 1-4 hours; The amount of glutaraldehyde added is 40-80 mM.

2. The application according to claim 1, characterized in that, The steps for cleaning the melamine sponge are as follows: the melamine sponge is cleaned with anhydrous ethanol and water under ultrasonic conditions, and ultrasonic cleaning is performed three times for each.

3. The application according to claim 1, characterized in that, The concentration of the β-lactamase solution is 20-30 mg / mL.

4. The application according to claim 1, characterized in that, The concentration of the bovine serum albumin solution is 30 mg / mL.

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