Preparation method of hydrogel immobilized composite biological enzyme, product and application thereof

Abstract

The application discloses a preparation method of a hydrogel immobilized composite enzyme, and a product and application thereof. Due to the protection of bacterial cell walls, the removal efficiency of resistant genes in traditional processes is low. The application utilizes polyacrylamide hydrogel to jointly immobilize laccase, hydrolase and nuclease, so that the enzymes are effectively coated inside the carrier, the problems of easy loss and easy reduction of activity of free enzymes are avoided, and the reusability is good, and no secondary pollution is generated. The laccase is used to reduce the concentration of sulfadiazine, the hydrolase is used to hydrolyze the bacterial cell walls to release intracellular ARG, and the nuclease is used to degrade the released intracellular ARG, so that the purpose of quickly eliminating ARG in water is achieved. The method has high removal efficiency on ARG and sulfadiazine, is simple, efficient and safe, is suitable for remediation treatment of aquaculture wastewater containing antibiotic and ARG pollution, and solves the problems of poor environmental stability of water-soluble enzymes and difficult recycling.

Description

TECHNICAL FIELD

[0001] The application relates to a preparation method of a hydrogel immobilized composite biological enzyme and application thereof, and in particular to a method for degrading antibiotics and reducing antibiotic resistance genes in aquaculture wastewater by using a polyacrylamide hydrogel immobilized composite biological enzyme, and belongs to the field of biological environmental protection. BACKGROUND

[0002] During the process of livestock and poultry breeding, due to the overuse of antibiotics and due to the fact that the antibiotics cannot be completely metabolized in the animal body, the antibiotics are enriched in animal manure. The antibiotics remaining in the animal manure accelerate the spread and enrichment of antibiotic resistance genes (ARGs) by exerting selective pressure on microorganisms. The enriched antibiotics and ARGs enter the water environment and finally endanger human health. Therefore, how to repair the antibiotic pollution in the aquaculture wastewater while blocking the spread of ARGs has become the most urgent demand for developing healthy breeding. The intracellular ARG of microorganisms is the most stable form of ARG, and has the problems of stability and difficulty in removal. So far, methods including filtration, physical adsorption, chemical oxidation, microbial degradation, enzyme degradation and the like have been widely used for antibiotic and ARG pollution repair. Enzyme degradation technology has attracted more and more attention due to its mild reaction and no secondary pollution. Laccase can strengthen the removal of antibiotics through its electron extraction and electron transfer to the substrate, and has the advantages of high repair efficiency and no secondary pollution; hydrolases including proteases, amylases and lysozymes can effectively accelerate the hydrolysis of macromolecular substances, and also have the effect of accelerating the hydrolysis of bacterial cells; lysozyme is a hydrolytic enzyme that can specifically act on N-acetylmuramic acid in the cell wall of gram-positive bacteria; nucleases can hydrolyze and decompose DNA. However, free enzymes are prone to loss, which leads to reduced activity, cannot be reused and causes secondary pollution. Meanwhile, there is no research on how to simultaneously exert the synergistic removal effect of the above enzymes on antibiotic resistance. Therefore, it is necessary to improve the operational stability and reusability of free enzymes by using free enzyme immobilization technology, and to strengthen the removal of antibiotic resistance by using composite biological enzymes, so as to effectively reduce the antibiotic resistance in aquaculture pond wastewater. SUMMARY

[0003] The application aims to provide a preparation method of a hydrogel immobilized composite biological enzyme with good reusability and no secondary pollution, and application thereof, so as to strengthen the removal of ARGs.

[0004] Technical Solution: To solve the above-mentioned technical problems, the present invention provides a method for preparing hydrogel-immobilized composite bioenzymes, comprising the following steps: adding laccase, nuclease and hydrolase to enzyme-free water and mixing them; using an immobilizing agent to prepare hydrogel-immobilized composite enzymes; after preparation, immersing the hydrogel-immobilized composite enzymes in ultrapure water; washing them with a washing solution; placing the prepared hydrogel-immobilized composite enzymes in a freeze dryer; and after drying, grinding them into small particles to obtain the hydrogel-immobilized composite enzyme material.

[0005] The concentrations of the hydrolase, laccase, and nuclease are 0.5-1.5 g / L, 0.2-0.5 g / L, and 200-500 U / L, respectively; the hydrolase includes lysozyme, α-amylase, and protease, and the mass ratio of the three enzymes is 1:1:1.

[0006] The nucleases mentioned include, but are not limited to, DNase I; other nucleases may also be used in this invention.

[0007] The immobilizing agent includes acrylamide, N,N′-methylenebisacrylamide, and K2S2O8.

[0008] The amount of immobilizing agent added to each 20 mL enzyme solution is 1.065 g acrylamide, 0.1 g N,N′-methylenebisacrylamide and 0.122 g K2S2O8.

[0009] The present invention also provides a hydrogel-immobilized composite bioenzyme obtained by the preparation method described above.

[0010] This invention also provides the application of the aforementioned hydrogel-immobilized composite bioenzyme in the degradation of antibiotics in aquaculture wastewater and the removal of antibiotic resistance genes from sewage.

[0011] The present invention also provides a method for degrading antibiotics in aquaculture wastewater and removing antibiotic resistance genes from sewage by hydrogel-immobilized composite bioenzymes, comprising the following steps: adding hydrogel-immobilized composite bioenzymes to aquaculture wastewater containing antibiotics, placing it in a shaker, and reacting in the dark.

[0012] Wherein, the antibiotic is sulfadiazine, the antibiotic resistance gene is sulI, sulII or sulIII, the concentration of the antibiotic in the wastewater is 200 μg / L, and the mass-to-volume ratio of the hydrogel-immobilized composite bioenzyme to the wastewater is 5-20 mg / L.

[0013] Reaction mechanism: firstly, the hydrolytic enzyme (protease, amylase and lysozyme) is used to effectively destroy the bacterial cell wall, improve the release rate of intracellular ARG, and then the nuclease is used to quickly reduce the released ARG, so as to achieve the purpose of reducing intracellular ARG and free ARG. At the same time, the presence of laccase can effectively reduce the concentration of antibiotics, thereby reducing the antibiotic selective pressure and effectively reducing the enrichment efficiency of ARG. In view of the problem that the biological enzyme is easily affected by the external environment and cannot be reused in the actual repair process, the enzyme immobilization technology is adopted, the stability of the enzyme is improved by using polyacrylamide hydrogel immobilized enzyme technology, and the stability and catalytic efficiency of the biological enzyme in the actual application are improved. By combining the functions of laccase, nuclease and hydrolytic enzyme, the functions of the enzymes are coupled, so as to achieve the purpose of reducing the antibiotic resistance in aquaculture wastewater.

[0014] Beneficial effects: compared with the prior art, the present application has the following obvious advantages:

[0015] 1. The polyacrylamide hydrogel is used to combine laccase, hydrolytic enzyme and nuclease, so that the enzyme is effectively coated inside the carrier, the problems of easy loss and low activity of free enzyme are avoided, and the enzyme has good reusability and does not produce secondary pollution.

[0016] 2. The laccase in the present application has high catalytic degradation ability to antibiotics, which can significantly reduce the selective pressure of antibiotics on microorganisms in wastewater.

[0017] 3. The hydrolytic enzyme (protease, alpha-amylase and lysozyme) has the functions of destroying the bacterial cell wall and has good hydrolysis and lysis effect, so that the intracellular antibiotic resistance gene is released to the extracellular, and finally the released intracellular ARG is degraded under the action of nuclease, which provides a new idea for removing ARG in aquaculture wastewater.

[0018] 4. The method of the present application has high removal efficiency for ARG and sulfadiazine, and the whole process is simple, efficient and safe, and is suitable for repair treatment of aquaculture wastewater contaminated with antibiotics and ARG.

[0019] 5. The present application uses the emerging enzyme immobilization technology to solve the problems of poor environmental stability and difficult recycling of water-soluble enzymes. Several biological enzymes are fixed on the carrier by physical or chemical methods to form insoluble enzyme derivatives, thereby having good reusability.

[0020] 6. The laccase reduces the selective pressure of antibiotics, the hydrolytic enzyme makes the bacterial cell wall rupture and releases the intracellular ARG, so that the intracellular ARG of bacteria can be quickly released to the extracellular, the nuclease degrades the released ARG, reduces the risk of ARG enrichment and horizontal transfer, and comprehensively plays the synergistic degradation advantages of several enzymes. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Preparation flow chart of polyacrylamide hydrogel immobilized composite biological enzyme;

[0022] Figure 2 Physical diagram of polyacrylamide hydrogel immobilized composite biological enzyme;

[0023] Figure 3 Contact angle of polyacrylamide hydrogel immobilized composite biological enzyme;

[0024] Figure 4 Effect diagram of polyacrylamide hydrogel immobilized composite biological enzyme on removal of sulfa ARGs (sulI, sulII and sulIII) in aquaculture wastewater after 24h cultivation; the respective concentrations of the hydrolytic enzyme, laccase and DNase I are 1.5g / L, 0.5g / L and 500U / L;

[0025] Figure 5 Effect diagram of polyacrylamide hydrogel immobilized composite biological enzyme on cyclic removal of sulfa resistance genes and sulfadiazine after 24h cultivation; the respective concentrations of the hydrolytic enzyme, laccase and DNase I are 1.5g / L, 0.5g / L and 500U / L.

[0026] Figure 6 Effect diagram of polyacrylamide hydrogel immobilized composite biological enzyme on removal of sulfa ARGs (sulI, sulII and sulIII) and sulfadiazine in aquaculture wastewater after 24h cultivation; the respective concentrations of the hydrolytic enzyme, laccase and DNase I are 1g / L, 0.35g / L and 350U / L;

[0027] Figure 7 Effect diagram of polyacrylamide hydrogel immobilized composite biological enzyme on removal of sulfa ARGs (sulI, sulII and sulIII) and sulfadiazine in aquaculture wastewater after 24h cultivation; the respective concentrations of the hydrolytic enzyme, laccase and DNase I are 0.5g / L, 0.2g / L and 200U / L. DETAILED DESCRIPTION

[0028] The technical solutions of the present application will be further described below in combination with the drawings.

[0029] The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained by commercial channels by those skilled in the art.

[0030] The experimental instruments and drugs used in the following examples are as follows:

[0031] Ultraviolet spectrophotometer (UV-754N; Shanghai Youke Instrument Co., Ltd.);

[0032] Liquid chromatography-mass spectrometry system (Agilent 1200-6410B; Agilent Technologies (China) Co., Ltd.);

[0033] Freeze dryer (Scientz-18N; Ningbo Xinzhi).

[0034] The aquaculture wastewater used in the experiment was taken from a livestock and poultry farm in Jiangsu Province, and the concentration of sulfadiazine was 0.2 mg / L.

[0035] Table 1. List of Experimental Reagents

[0036]

[0037] The concentration of sulfadiazine was determined using liquid chromatography-mass spectrometry (LC-MS / MS). Testing for the resistance gene was commissioned to Shanghai Qiyin Biotechnology Co., Ltd. The LC-MS / MS determination method for sulfadiazine, the detection method for the target gene, and the primers are disclosed in Chemical Engineering Journal, 350, 920-929.

[0038] Example 1: Preparation of polyacrylamide hydrogel composite immobilized biological enzymes

[0039] according to Figure 1 The steps shown illustrate the preparation of polyacrylamide hydrogel composite immobilized bio-enzymes. First, three bio-enzymes—laccase, hydrolases (protease, amylase, and lysozyme), and nuclease—were added to 20 mL of enzyme-free water. The concentrations of the hydrolases, laccase, and DNase I were 1.5 g / L, 0.5 g / L, and 500 U / L, respectively. The hydrolases included α-amylase, protease, and lysozyme, with a mass ratio of 1:1:1. Laccase was purchased from Nanjing Dulai Biotechnology Co., Ltd.; the hydrolases and amylases were purchased from Beijing Solarbio Technology Co., Ltd.; the lysozyme was purchased from Beijing Bio-Top Technology Co., Ltd.; and the nuclease DNase I was purchased from Beijing Bio-Laibo Co., Ltd. Next, 1.065 g of polyacrylamide, 0.1 g of N,N′-methylenebisacrylamide, and 0.122 g of K₂S₂O₈ were added to the above solution to obtain 20 mL of mixed solution. The resulting mixed solution was stirred thoroughly for 20 min under N₂ stripping to homogenize it. Then, the solution was heated in a 37°C water bath for 30 minutes until it completely gelled, generating a composite immobilized enzyme. Finally, the composite immobilized enzyme was immersed in ultrapure water for 24 hours to remove unreacted reagents adsorbed on the surface of the immobilized composite enzyme. Figure 2 a). Then place it in a freeze dryer and freeze-dry at -50°C for 24 hours. Figure 2b). After drying, grinding to obtain the desired polyacrylamide hydrogel immobilized composite enzyme powder Figure 2 c). As shown in Figure 2 d, the freeze-dried and ground polyacrylamide hydrogel immobilized composite enzyme is in powder form. By measuring the turbidity of the supernatant, the value is less than 1 NTU, indicating that the polyacrylamide hydrogel immobilized composite enzyme has good sedimentation performance in solution.

[0040] Example 2 verifies the resistance of polyacrylamide hydrogel immobilized composite enzyme to antibiotics and ARG in aquaculture wastewater

[0041] In order to verify the technical effect of the polyacrylamide hydrogel immobilized composite enzyme in Example 1, the polyacrylamide hydrogel immobilized composite enzyme was used to determine the concentration of antibiotics and ARG in the aquaculture wastewater sample, and the specific steps were as follows:

[0042] S1, add polyacrylamide hydrogel immobilized composite enzyme to 50 mL of aquaculture wastewater containing 200 μg / L of sulfadiazine, so that the final concentration of polyacrylamide hydrogel immobilized composite enzyme is 5 mg / L, and place it in a 30°C, 120 rpm shaker at pH 6 and a temperature of 25°C. Light reaction for 24 h to reduce antibiotics and ARG in water, complete water treatment; take the water sample after 0 h, 12 h and 24 h reaction for subsequent experiment;

[0043] S2, after the reaction in step S1 is completed, remove the supernatant, and continue to add 50 mL of the above aquaculture wastewater to the system to carry out the next batch of cyclic degradation experiment to verify the degradation performance of the prepared immobilized material. In this embodiment, the number of cycles is 3 times, and the degradation performance of the immobilized composite enzyme to sulfadiazine is verified. After 3 cycles, the contact angle of the polyacrylamide hydrogel immobilized composite enzyme is measured Figure 3 ), the wetting angle is about 30 degrees, indicating that the polyacrylamide hydrogel composite enzyme has good hydrophilicity and is easily penetrated by water.

[0044] S3, collect the effluent treated in steps S1 and S2. The collection process is as follows: rotate the liquid in the reactor uniformly, collect 30 mL of volume, use a 0.22 μm filter membrane to filter the bacteria in the water, collect the filter membrane and extract the bacterial genomic DNA on the filter membrane. The water sample after membrane filtration is used to analyze the concentration of sulfadiazine.

[0045] S4, DNA extraction was performed on the bacteria retained on the filter membrane using a DNA extraction kit. The A260 / A280 value was measured and calculated using a spectrophotometer, and both values were near 1.8, indicating that the DNA quality was qualified. The qualified DNA was entrusted to Shanghai Qiyin Biotechnology Co., Ltd. to detect the abundance of ARGs at 0h and 24h using fluorescent quantitative qPCR, and the removal efficiency λ(%) of the target ARG at 24h was calculated by formula (1).

[0046] λ(%) = (Gene abundance(Copies / mL) 0h -Gene abundance(Copies / mL) 24h ) / Gene

[0047] abundance(Copies / mL) 0h (1)

[0048] abundance(Copies / mL) 0h represents the absolute copy number of the target ARG before the reaction, and Gene

[0049] abundance(Copies / mL) 24h represents the absolute copy number of the target ARG after 24h of reaction,

[0050] S5, the concentration Ct of sulfadiazine remaining in the water sample after membrane filtration was determined using a liquid chromatograph-mass spectrometer, and the removal efficiency η(%) of the antibiotic was calculated. 磺胺嘧啶

[0051] η(%) = (200-Ct 磺胺嘧啶 ) / 200

[0052] Experimental results: After the polyacrylamide hydrogel immobilized composite enzyme reacted with aquaculture wastewater for 0, 12 and 24 hours in step S1, the concentrations of ARGs (sulI, sulII and sulIII) and sulfadiazine obtained are shown in Table 2.

[0053] Table 2

[0054]

[0055] According to the concentrations described in Table 2, the removal efficiency of polyacrylamide hydrogel immobilized composite enzyme on sulfadiazine and sulfonamide ARGs (sulI, sulII and sulIII) was calculated. Figure 4 ​The results show that the degradation rate of sulfadiazine is more than 70% at 12h, the degradation rate of sulfadiazine is 92.86% within 12-24h, and the degradation effect of sulfadiazine still maintains 98% after 24h. After 24h of reaction, the removal rate of sulI in the sample is 60%, and the removal efficiency of sulII and sulIII is the highest, which is close to 80%.

[0056] The processing result of step S2 can be obtained from Figure 5 It is found that after three consecutive reactions, the immobilized composite biological enzyme still maintains high removal efficiency for sulfonamide ARGs (sulI, sulII and sulIII) and sulfadiazine, and the removal efficiency does not decrease significantly (p>0.05). The removal efficiency for sulI, sulII and sulIII is 65%, 80% and 79% respectively, and the removal efficiency for sulfadiazine is 98%.

[0057] Example 3 verifies the resistance of different composite biological enzyme component concentrations to antibiotics and resistance genes in aquaculture wastewater

[0058] The operations of example 1 and example 2 are repeated, only the concentrations of hydrolytic enzyme, laccase and DNase I are changed, which are 1g / L, 0.35g / L and 350U / L respectively. The polyacrylamide hydrogel immobilized composite biological enzyme is prepared according to the steps in example 1. The removal efficiency of sulI, sulII, sulIII and sulfadiazine in the sample is measured according to the steps in example 2, and the concentrations of ARGs (sulI, sulII and sulIII) and sulfadiazine before and after reaction are measured as shown in table 3.

[0059] Table 3

[0060] 0h (Copies / mL) 12h (Copies / mL) 24h (Copies / mL) sul I 1 x 10 8 ± 1.5 x 10 7 ]]> - 4.8 x 10 7 ±7 x 10 6 ]]> sul II 5 x 10 7 ±1.5 x 10 7 ]]> - 3 x 10 7 ± 2 x 10 6 ]]> sul III 5.7 x 10 5 ± 1.2 x 10 5 ]]> - 2.5 x 10 5 ± 1.8 x 10 4 ]]> sulfadiazine 200 124±18.6 76±15.2

[0061] According to the concentrations described in table 3, the removal efficiency of polyacrylamide hydrogel immobilized composite biological enzyme for sulfadiazine and sulfonamide ARGs (sulI, sulII and sulIII) is calculated respectively. Figure 6 The results show that within 24h, the removal efficiency of sulI, sulII and sulIII in the sample is 52%, 40% and 56% respectively, and the removal efficiency of sulfadiazine is 62%.

[0062] Example 4 verifies the resistance of different composite biological enzyme component concentrations to antibiotics and resistance genes in aquaculture wastewater

[0063] The procedures of Example 1 and Example 2 were repeated, only changing the concentration of each of the hydrolytic enzyme, laccase and DNase I added, which were 0.5 g / L, 0.2 g / L and 200 U / L, respectively. The polyacrylamide hydrogel immobilized composite biological enzyme was prepared according to the procedure in Example 1. The procedure in Example 2 was repeated to determine the removal efficiency of sulI, sulII, sulIII and sulfadiazine in the sample, and the concentrations of ARGs (sulI, sulII and sulIII) and sulfadiazine before and after the reaction were measured as shown in Table 4.

[0064] Table 4

[0065]

[0066]

[0067] According to the concentrations described in Table 4, the removal efficiency of the polyacrylamide hydrogel immobilized composite biological enzyme on sulfadiazine and sulfonamide ARGs (sulI, sulII and sulIII) was calculated, respectively. Figure 7 The results show that within 24 hours, the removal efficiency of sulI, sulII and sulIII in the sample is 48%, 44% and 65%, respectively, and the removal efficiency of sulfadiazine is 55%.

Claims

1. The application of hydrogel-immobilized composite bioenzymes in the degradation of antibiotics in aquaculture wastewater and / or the removal of antibiotic resistance genes from wastewater, characterized in that, The preparation method of the hydrogel-immobilized composite enzyme includes the following steps: laccase, DNase I nuclease, and hydrolase are added to enzyme-free water and mixed thoroughly. An immobilization agent is used to prepare the hydrogel-immobilized composite enzyme. After preparation, the hydrogel-immobilized composite enzyme is immersed in ultrapure water, washed with a washing solution, and then placed in a freeze dryer. After drying, it is ground into small particles to obtain the hydrogel-immobilized composite enzyme material. The concentrations of the hydrolase, laccase, and DNase I nuclease are 1.5 g / L, 0.5 g / L, and 500 units / L, respectively. The hydrolase includes lysozyme, α-amylase, and protease, with a mass ratio of 1:1:

1. The immobilization agent includes acrylamide, N,N'-methylenebisacrylamide, and K2S2O8. The antibiotic is sulfadiazine, and the antibiotic resistance gene is... sulI , sulII or sulIII .

2. The application according to claim 1, characterized in that, Includes the following steps: The hydrogel-immobilized composite bio-enzyme was added to aquaculture wastewater containing antibiotics and placed in a shaker for reaction in the dark.

3. The application according to claim 2, characterized in that, The concentration of the antibiotic in the wastewater is 200 μg / L, and the mass-to-volume ratio of the hydrogel-immobilized composite bio-enzyme to the wastewater is 5-20 mg / L.

Images