A beta-lactamase complex capable of degrading multiple antibiotics, and immobilization method and application thereof

Abstract

The present application belongs to the technical field of complex enzyme. The present application provides a beta-lactamase complex enzyme capable of degrading various antibiotics, and a fixing method and application thereof. The preparation method of the complex enzyme comprises the following steps: extracting gene fragments of CTX-M type broad-spectrum beta-lactamase and VIM type metal-beta-lactamase from Klebsiella pneumoniae; obtaining the target gene fragments through PCR amplification, and constructing into a basic vector to obtain a recombinant vector; transforming the recombinant vector into host cells for heterologous expression; and through protein induction, SDS-PAGE protein gel electrophoresis and protein purification, the complex enzyme is obtained. The complex enzyme successfully degrades penicillins such as ampicillin, carbapenems such as imipenem, cephalosporins such as cefotaxime and other beta-lactams. The complex enzyme gene is successfully expressed in Escherichia coli, and the optimal fixing condition is obtained. The beta-lactamase complex enzyme is fixed under the optimal fixing condition, and can be repeatedly used.

Description

Technical Field

[0001] This invention belongs to the field of complex enzyme technology, specifically relating to a β-lactamase complex enzyme capable of degrading multiple antibiotics, its immobilization method, and its application. Background Technology

[0002] β-Lactamases are a class of enzymes that inactivate the β-lactam ring of β-lactam antibiotics. They catalyze the hydrolysis of β-lactams, breaking their amide bonds. They can also catalyze the hydrolysis of their N-acyl derivatives, thus causing the β-lactams to lose their antibacterial activity. The production of β-lactamases is related to stimulation by β-lactam antibiotics produced by microorganisms in nature, leading to antibiotic resistance. Therefore, resistance in β-lactamase-producing bacteria existed before the discovery and use of β-lactam antibiotics. The increasingly widespread and extensive use, and even abuse, of β-lactam antibiotics has accelerated the formation and complexity of resistance in β-lactamase-producing bacteria. With the continuous emergence of new types of penicillins and cephalosporins, the problem of drug resistance caused by β-lactamases is becoming increasingly serious in clinical practice.

[0003] β-lactam antibiotics are widely used in human medicine and animal husbandry due to their broad antibacterial spectrum, high activity, and low sensitization. While commonly used clinically, β-lactam antibiotics can be hydrolyzed by bacterial β-lactamases, rendering them inactive. Untreated β-lactam antibiotics can enter wastewater treatment plants through medical wastewater and other sources. If improperly treated, they can accumulate in water bodies, leading to antibiotic resistance in bacteria, disrupting ecosystems, and threatening human health. Therefore, the degradation and treatment of wastewater containing β-lactam antibiotics has become a pressing environmental issue.

[0004] Currently, methods for treating this type of wastewater include physical adsorption, chemical oxidation, and biodegradation. Biodegradation is considered the preferred method due to its high environmental compatibility, specificity, high efficiency, and relatively low cost. However, existing technologies lack enzymes with good stability, high catalytic activity, and the ability to effectively degrade β-lactam antibiotics. Summary of the Invention

[0005] To address the problems existing in the background art, the present invention provides a β-lactamase complex enzyme capable of degrading multiple antibiotics, its immobilization method, and its application.

[0006] To achieve the above objectives, in a first aspect, the present invention provides a β-lactamase complex enzyme capable of degrading multiple antibiotics, the preparation method of which is as follows:

[0007] Gene fragments of CTX-M type broad-spectrum β-lactamase and VIM type metallo-β-lactamase were extracted from Klebsiella pneumoniae;

[0008] The target gene fragment was obtained by PCR amplification and constructed into a basic vector to obtain a recombinant vector;

[0009] The recombinant vector was transformed into host cells for heterologous expression;

[0010] The β-lactamase complex enzyme capable of degrading multiple antibiotics was obtained by protein induction, SDS-PAGE protein gel electrophoresis, and protein purification.

[0011] Furthermore, the base vector is the pET-28a(+) plasmid.

[0012] Furthermore, the host cell is Escherichia coli BL21(DE3).

[0013] Secondly, the present invention provides a method for immobilizing the above-mentioned β-lactamase complex enzyme capable of degrading multiple antibiotics, comprising the following:

[0014] The β-lactamase complex enzyme was mixed with sodium alginate solution to obtain a mixture;

[0015] The mixture is added dropwise to a CaCl2 solution to form microspheres, which are then washed and dried to obtain the immobilized enzyme.

[0016] Furthermore, the final concentration of the sodium alginate solution is 1.5%, and the ratio of sodium alginate to β-lactamase complex enzyme is 5:1.

[0017] Furthermore, the final concentration of the CaCl2 solution is 2.5%.

[0018] Furthermore, the fixed time is 10 minutes.

[0019] Furthermore, the immobilized enzyme is reused at least four times.

[0020] Thirdly, the present invention provides an application of the above-mentioned β-lactamase complex enzyme capable of degrading multiple antibiotics, wherein the β-lactamase complex enzyme is treated by an immobilization method to obtain an immobilized enzyme, which is used in the treatment of wastewater containing β-lactam antibiotics.

[0021] Furthermore, the immobilized enzyme is used to degrade β-lactam antibiotics in wastewater to reduce environmental pollution and prevent the spread of antibiotic resistance.

[0022] This application has the following beneficial effects:

[0023] This invention relates to a complex β-lactamase that can degrade various antibiotics, successfully degrading penicillins such as ampicillin, carbapenems such as imipenem, cephalosporins such as cefotaxime, and other β-lactams such as aztreonam. The complex enzyme gene of this invention was successfully expressed in *E. coli*, and optimal immobilization conditions were obtained: a final concentration of 1.5% sodium alginate, a sodium alginate to complex enzyme solution ratio of 5:1, 2.5% CaCl2, and immobilization for 10 min. Immobilization of the β-lactamase complex enzyme under optimal conditions showed that it could be reused multiple times. Attached Figure Description

[0024] Figure 1 This refers to the extraction of pET28a plasmid in Example 1 of the present invention;

[0025] Figure 2 This refers to the PCR preliminary experiment results in Example 1 of the present invention;

[0026] Figure 3 This is the result of optimizing the PCR annealing temperature conditions in Example 1 of the present invention;

[0027] Figure 4 This is the result of PCR template concentration optimization in Example 1 of the present invention;

[0028] Figure 5 This is the result of optimizing the number of PCR cycles in Example 1 of the present invention;

[0029] Figure 6 The results of double enzyme digestion of pET-28a plasmid in Example 1 of this invention;

[0030] Figure 7 The results of double enzyme digestion of the gene in Example 1 of this invention;

[0031] Figure 8 The results of positive colony screening in Example 1 of this invention;

[0032] Figure 9 The results before and after induction in Example 1 of this invention;

[0033] Figure 10 This refers to the gradient elution results in Example 1 of the present invention;

[0034] Figure 11 The results show the relative enzyme activity at the optimal temperature for the four complex enzymes in Example 1 of this invention.

[0035] Figure 12 The results show the relative enzyme activity of the four complex enzymes in Example 1 of this invention, based on their temperature tolerance.

[0036] Figure 13 The optimal pH results for the four complex enzymes in Example 1 of this invention;

[0037] Figure 14 The results show the relative enzyme activity of the four composite enzymes in Example 1 of this invention, based on their temperature tolerance.

[0038] Figure 15 The relative activities of enzymes with different sodium alginate concentrations in Example 2 of this invention;

[0039] Figure 16 The relative activity of enzymes with different sodium alginate to enzyme volume ratios in Example 2 of this invention;

[0040] Figure 17 The relative activities of enzymes with different CaCl2 concentrations in Example 2 of this invention;

[0041] Figure 18 The relative activities of enzymes at different reaction times in Example 2 of this invention;

[0042] Figure 19 This represents the relative activity of enzymes in different reaction rounds in Example 2 of the present invention. Detailed Implementation

[0043] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the raw materials used in the embodiments and comparative examples of this application are all commercially available.

[0044] Example 1: A method for preparing a β-lactamase complex enzyme capable of degrading multiple antibiotics.

[0045] 1.2 Experimental Materials

[0046] 1.2.1 Strains: The strains and expression hosts were preserved in the laboratory.

[0047] 1.2.2 Experimental reagents: peptone, yeast extract, kanamycin, agarose, glycerol, sodium chloride, sterile water, 6×OrangeLoadingBuffer, 1kb DNA ladder, 2000bp DNA ladder.

[0048] 1.2.3 Experimental instruments: PCR instrument, clean bench, ice box, plasmid small-scale rapid extraction kit, UV gel electrophoresis apparatus, electrophoresis apparatus, electronic analytical balance, centrifuge.

[0049] 1.3 Experimental Methods

[0050] 1.3.1 pET-28a plasmid extraction: Plasmid extraction was performed using a rapid plasmid extraction kit. Add 500 μL of WB equilibration buffer to adsorption column AC (place the adsorption column in the collection tube). Add 500 μL of primary water to another adsorption column AC. Place both adsorption columns AC symmetrically in a centrifuge and centrifuge at 12000 rpm for 1 min. After centrifugation, discard the waste liquid in the collection tube, reassemble the collection tube and adsorption column, and take 2 mL of overnight culture. Centrifuge at 12000 rpm. Centrifuge for 30 seconds. After centrifugation, discard the supernatant, collect the precipitate, and resuspend the precipitate by pipetting with 250 μL of solution P1. Shake thoroughly until completely suspended. Add 250 μL of solution P2 to the adsorption column AC, and gently invert 4-7 times to ensure complete cell lysis. Then add 350 μL of solution P3, and immediately and carefully invert 4-7 times to mix thoroughly until white flocculent matter appears. Centrifuge at 12000 rpm for 5 minutes. After centrifugation, carefully transfer the supernatant to a new adsorption column AC (place the adsorption column in a collection tube), and centrifuge again at 12000 rpm for 30 seconds. Discard the waste liquid, add 500 μL of wash buffer WB containing anhydrous ethanol, and centrifuge at 12000 rpm for 30 seconds. Discard the waste liquid, add 500 μL of wash buffer WB containing anhydrous ethanol, centrifuge at 12000 rpm for 30 seconds, place the adsorption column into a new collection tube, centrifuge at 12000 rpm for 2 minutes, place the collection tube at room temperature for 3 minutes to allow the adsorption material to dry completely, remove the adsorption column AC and place it into a clean centrifuge tube, place at room temperature for 10 minutes, add 50 μL of elution buffer EB to the intermediate membrane of the adsorption column, heat the EB elution buffer in a 65°C water bath for 2 minutes, centrifuge at 12000 rpm, collect the liquid, label it with the name and date, and store it in a -20°C freezer for subsequent use.

[0051] 1.3.2 PCR Preliminary Experiment: 1.3.2.1 Plate Streaking: Wear disposable PE gloves and rubber gloves, disinfect hands with 75% alcohol, and perform the operation in a clean bench. Light the alcohol lamp, hold the inoculation loop in your right hand, and heat the metal wire of the inoculation loop in the outer flame of the alcohol lamp until it is red-hot. Heat the tip of the metal rod as well. After the metal wire of the inoculation loop cools down, hold the EP tube containing the bacterial strain in your right hand, open the EP tube, and bring the metal wire of the inoculation loop against the solution on the EP cap for rapid cooling. Insert the metal wire into the bottom of the EP tube to pick up the bacterial culture from the bottom of the tube. Heat the opening of the EP tube and close it. Take a clean plate with your left hand, and use your thumb and forefinger to control the cap and the rest of the plate to streak the strain. Divide the plate into 3 regions. Streak each region continuously, tightly, and without connecting them. Connect the end of region 1 to the beginning of region 2, the end of region 2 to the beginning of region 3, and the end of region 3 to region 1. After streaking, the inoculation loop was sterilized by flame. The bacterial strain and date were recorded on the plate, and the plate was inverted and placed in a 37°C oven for overnight incubation. The next day, the plate was stored in a -4°C refrigerator.

[0052] 1.3.2.2 Preliminary Experimental Procedure: In a clean bench, take one sterile PCR tube and pipette 100 μL of sterile water. Take the original strain from 1.3.2.1 and streak it onto a plate. Use a pipette tip to pick up a single colony of suitable size from the plate. Place the pipette tip in the PCR tube and vortex to dissolve the picked colony in the sterile water. Close the PCR tube and vortex thoroughly. Take another sterile PCR tube and aliquot 50 μL from the previous PCR tube, label it, and incubate the aliquoted PCR tube in a 100°C water bath for 5 min. After the water bath, ... In a clean bench, prepare a 6-fold mixture according to Table 1.3.2.2.1, and mix thoroughly by shaking. Take three sterile PCR tubes and aliquot 19 μL of the mixture into each tube. Add 1 μL of sterile water to one PCR tube as a negative control, and add 1 μL of the plasmid extracted in 1.3.1 to the other as a positive control. Add 1 μL of colony solution that has been bathed in water to the other PCR tube. When adding the 1 μL solution, if the amount is too small, first coat the PCR tube wall with the solution, then close the PCR tube and shake thoroughly to mix. After adding the samples, centrifuge symmetrically for 5 seconds to mix thoroughly. Then, place all six PCR tubes into a PCR instrument and set up the PCR program according to Table 1.3.2.2.2 for PCR. After PCR, perform nucleic acid electrophoresis.

[0053] Table 1.3.2.2.1 Colony PCR System

[0054] name volume enzymes 10μL upper primer 2.8µL lower primer 2.8µL Genome template single colony sterile water 8.2μL

[0055] Table 1.3.2.2.2 PCR Procedure

[0056] step parameter 1. Pre-variation 94.0℃, 3min 2 Transgender 94℃，10s 3 Annealing 52℃，30s 4 extensions 72℃, 1min 5 2-4 30 cycles 6 extensions 72.0℃, 5min

[0057] 1.3.3 PCR Condition Optimization: 1.3.3.1 PCR Annealing Temperature Optimization: Based on the results of the preliminary experiments, the annealing temperature in the PCR program was optimized. Annealing temperature gradients of 50℃, 51℃, 52℃, 53℃, and 54℃ were designed. In a clean bench, six PCR tubes were used, and a 19µL system was prepared according to the method in 1.3.2.1. One PCR tube was added to 1µL of sterile water and labeled "negative" as a negative control. 3µL of the plasmid extracted in 1.3.1 was added to a sterile PCR tube and diluted with 12µL of sterile water. 1µL of the plasmid dilution was then added to the remaining five PCR tubes, labeled 1, 2, 3, 4, and 5. These tubes were then placed into the PCR instrument according to the settings in Table 1.3.2.2.2, and their annealing temperatures were set accordingly. After PCR amplification, nucleic acid electrophoresis was performed.

[0058] 1.3.3.2 Optimization of PCR Template Concentration Conditions: In a clean bench, take one sterile PCR tube and prepare a 7-fold system according to the method in 1.3.2.1. Take six new sterile PCR tubes, and aspirate 19 μL of the system from each PCR tube. Take one tube and add 1 μL of sterile water as a negative control and label it. Take one PCR tube and add 3 μL of the plasmid extracted in 1.3.1. Add 12 μL of sterile water to dilute and obtain the plasmid dilution. Then perform serial dilutions to obtain dilution rates of 10, 20, 50, 100, and 100. Take 1 μL of each diluted template concentration and add it to the remaining five PCR tubes. Mix them thoroughly in a centrifuge and place them in a PCR instrument. Set the program according to Table 1.3.2.2.2 for PCR amplification. After amplification, perform nucleic acid electrophoresis.

[0059] 1.3.3.3 Optimization of PCR Cycle Numbers: In a clean bench, take four sterile PCR tubes and prepare a 7-fold system according to Table 1.3.2.2.1. Dispense 19 μL of the system into each tube. Take one PCR tube and add 1 μL of sterile water to the 19 μL system to prepare a 20 μL PCR sample, labeled "Negative" as the negative control. Take one PCR tube and add 3 μL of the plasmid extracted in 1.3.1, then dilute with 12 μL of sterile water. Add 1 μL of plasmid dilution to the remaining PCR tubes, labeled "1, 2, 3". Based on the results of the preliminary experiment, design cycle number gradients of 30X, 35X, and 40X. Place the prepared PCR samples sequentially into the PCR instrument, set the program according to Table 1.3.2.2.2, and set the corresponding cycle numbers sequentially. After PCR amplification, perform nucleic acid electrophoresis.

[0060] 1.3.4 Double digestion of pET-28a plasmid: Place the plasmid and enzyme preparation that have undergone nucleic acid electrophoresis in an ice box. In a clean bench, take one sterile PCR tube, add the reagents according to Table 1.3.4 into the PCR tube, mix well and label the mixture. Place the mixed PCR tube in a water bath at 37°C for 30 min. Then digest the pET-28a(+) expression vector with EcoR1 enzyme. Verify the success of plasmid digestion using DNA gel electrophoresis.

[0061] Table 1.3.4 Plasmid digestion system

[0062] name volume 10×Fast Diggest Buffer 2μL EcoR1 0.5μL plasmid 5µL

[0063] 1.3.5 Double Enzyme Digestion: Place the PCR amplified product and the required enzyme preparation in an ice box. In a clean bench, take two sterile PCR tubes, add the reagents according to Table 1.3.5 to the PCR tubes, mix well, and label. Prepare a separate PCR sample without the amplified gene as a control. Place the PCR tubes in a 37°C water bath for 30 minutes. Perform nucleic acid electrophoresis to verify whether the double enzyme digestion was successful.

[0064] Table 1.3.5 Gene double enzyme digestion system

[0065] name volume PCR amplification of gene products 10µL Enzyme 1 0.5μL enzyme 2 0.5μL Buffer 5μL sterile water 9μL

[0066] 1.3.6 Transformation: 1.3.6.1 Transformation Plate Preparation: Prepare three plates: two without kanamycin and one with kanamycin. Take sterile LB solid medium and heat it in a microwave oven until it is completely melted into a transparent, bubble-free liquid. In a laminar flow hood, take three clean plates. Pour the appropriate amount of melted LB solid medium directly onto two of them. Take a 50 mL centrifuge tube, pour in 20 mL of LB solid medium, and then add 20 μL of 100 mg / mL kanamycin. Mix well and pour an appropriate amount into the other plate. Mark K with a marker. Half-open the three plates near an alcohol lamp, keep the ventilation open, turn on the UV lamp, leave a small gap in the laminar flow hood, and wait for the plates to completely solidify. Invert the plates to place them.

[0067] 1.3.6.2 Transformation Procedure: Place 100 μL of competent Escherichia coli BL21(DE3) cells on ice until completely thawed. In a clean bench, add 10 μL of the plasmid extracted in 1.3.2 to the 100 μL of competent cells and incubate on ice for 30 min. Then, incubate in a 2°C water bath for 90 s. Immediately transfer to ice and cool for 5 min. In the clean bench, slowly add 700 μL of antibiotic-free sterile LB medium along the tube wall, allowing it to mix naturally. After mixing, incubate at 37°C and 180 rpm on a shaker for 1 h. After incubation, pipette an appropriate volume of the bacterial culture onto one agar plate containing kanamycin and one agar plate without kanamycin as described in 1.3.4.1. Seal the plates and mark them. Take another agar plate without kanamycin and spread an appropriate amount of LB medium onto it as a negative control. Incubate overnight in a 37°C oven, inverted. After the bacteria have grown, store the plates in a refrigerator at 4°C.

[0068] 1.3.7 Screening of positive colonies: In a clean bench, take one sterile PCR tube and prepare a 13-fold volumetric system according to Table 1.3.2.2.1. Then take 12 more sterile PCR tubes and aliquot the system into 12 tubes, 9 μL per tube. Take the transformed plate from 1.3.6, select ten single colonies, and label them. Take 10 PCR tubes and add 100 μL of sterile water to each. Use a pipette tip to pick up a single colony and place it in the PCR tube, shaking to dissolve it in the sterile water. Label the PCR tubes with the corresponding colony sequence and place them in boiling water. After the water bath, in a clean bench, 1 μL of each PCR tube was added to a 19 μL system, inverted and mixed thoroughly. The corresponding colony sequence was then marked. In two other PCR tubes containing 19 μL of system, 1 μL of plasmid and 1 μL of primary water were added as positive and negative control groups, respectively. All 12 PCR tubes were symmetrically placed in a centrifuge to mix thoroughly. The tubes were then placed in a PCR instrument, and the PCR program was set according to Table 1.3.2.2.2. After the PCR program was completed, the PCR products were subjected to nucleic acid electrophoresis.

[0069] 1.3.8 Nucleic Acid Electrophoresis: 1.3.8.1 Preparation of Nucleic Acid Gel (1.0%): Weigh 0.2 g (or 0.4 g) of agarose using an analytical balance and place it in a 250 mL Erlenmeyer flask. Measure 20 mL (or 40 mL) of 0.5 × TAE using a graduated cylinder. Stir well and heat in a microwave oven until the agarose is completely dissolved into a clear solution. Add 2 μL of ethidium bromide nucleic acid staining solution (0.5 mg / mL), shake well, pour into a mold, insert a multi-well comb vertically, and place stably to allow solidification.

[0070] 1.3.8.2 Nucleic Acid Electrophoresis Procedure: Take the solidified nucleic acid gel from 3.5.1, remove the comb, and place the nucleic acid gel into the nucleic acid electrophoresis tank. Add 0.5×TAE grade I water to the electrophoresis tank to dilute it 100 times until the nucleic acid gel is submerged. Use a pipette with a volume of 10μL to add samples in the following order: compressed sample, nucleic acid marker, negative control grade I water, positive control plasmid, single colony. The marker volume is 2µL, and the other volumes are 5µL. Turn on the electrophoresis apparatus, set the voltage to 130V and the time to 25min. After electrophoresis, remove the nucleic acid gel and place it in a UV gel irradiator for observation and photography.

[0071] 1.3.9 Preservation: Take a 15mL centrifuge tube, add 3mL of sterilized LB medium, add 3μL of 100mg / mL kanamycin, pick a single colony from the transformed plate and transfer it to the single colony. Take another 15mL centrifuge tube and add 3mL of LB medium as a negative control. Incubate overnight at 37℃ and 180rpm in a shaker. In a laminar flow hood, aliquot the overnight culture into two 2mL sterilized centrifuge tubes and centrifuge at 12000rpm for 3min. In the laminar flow hood, first aspirate 200μL of supernatant, discard the remaining supernatant, and use the remaining 200μL of supernatant to mix the precipitate by pipetting. Then, transfer the entire solution to a 1.5mL centrifuge tube containing 60% glycerol. Store the glycerol tube at -20℃ for subsequent experiments.

[0072] 1.4 Experimental Results

[0073] 1.4.1 pET28a plasmid extraction: as follows Figure 1 As shown, the nucleic acid electrophoresis marker is clear and the separation is good, indicating that there is no problem with the DNA electrophoresis. The first lane is the negative control group, and no band appears. The second lane shows a clear band, which is about 1000bp in size, consistent with the size of the target band. Conclusion: Plasmid extraction was successful.

[0074] 1.4.2 Preliminary PCR experiment for the target fragment: such as Figure 2 As shown, the nucleic acid electrophoresis markers were clear and the separation was good, indicating successful electrophoresis. The first lane was the positive control group for pET28a plasmid, showing a clear band, indicating the experiment was reliable. The second lane was the negative control group, showing no band, proving the experiment was reliable. The third lane showed a band larger than the plasmid. Conclusion: PCR amplification was successful.

[0075] 1.4.3 PCR Condition Optimization: 1.4.3.1 PCR Annealing Temperature Optimization: (e.g.) Figure 3As shown, the nucleic acid electrophoresis markers were clear and the separation was good. Lane 1, the negative control, showed no bands, proving the experiment's reliability. Annealing temperatures for lanes 2-6 were 50℃, 51℃, 52℃, 53℃, and 54℃, respectively. With increasing temperature, the band brightness did not change significantly, but lane 4 had the fewest extraneous bands, corresponding to an annealing temperature of 52℃. Conclusion: The optimal annealing temperature is 52℃.

[0076] 1.4.3.2 Optimization of PCR template concentration conditions: From Figure 4 It can be seen that the marker is clear and the separation is good. The first lane is the negative control group with no band. DNA electrophoresis is normal. As the template concentration decreases, the PCR amplification effect gradually deteriorates and the bands gradually become lighter. The PCR amplification effect is best when the template concentration is diluted by 10. Conclusion: The optimal template dilution for this gene is 10.

[0077] 1.4.3.3 Optimization of PCR cycle number conditions: such as Figure 5 As shown, the marker separation was good. Lane 1, the negative control, had no band and showed normal results on nucleic acid electrophoresis. Lanes 2-4 all showed bands consistent with the target band, with the band in lane 3 being particularly prominent. The corresponding cycle number was 35. Conclusion: The optimal cycle number for this gene is 35.

[0078] 1.4.4 double digestion of pET28a plasmid: as follows Figure 6 As shown, the Maker assay showed good separation. Lane 1 was the plasmid before enzyme digestion, with some extraneous bands. Lane 2 was the plasmid after enzyme digestion. Both lanes 1 and 2 showed obvious bands at around 8000 bp. Conclusion: Double enzyme digestion of the plasmid was successful.

[0079] 1.4.5 Double enzyme digestion of genes: such as Figure 7 As shown, the Marker separation was good, DNA electrophoresis was normal, lane 1 contained the product of gene digestion, and a band appeared above 2000 bp, consistent with the known target gene fragment. Conclusion: Double gene digestion was successful.

[0080] 1.4.6 Screening for positive colonies: such as Figure 8 As shown, the nucleic acid electrophoresis marker was clear and the separation was good. The DNA electrophoresis was successful. The first lane was the negative control group and no band appeared, indicating that the DNA electrophoresis operation was correct. The second lane showed a band that was close to 1000 bp and was consistent with the target gene. Lanes 3-12 had no bands, indicating that the 10 selected single colonies were not positive colonies.

[0081] 2. Protein expression

[0082] 2.1 A gene fragment expressing β-lactamase was extracted from Klebsiella pneumoniae and ligated into the more stable pET-28a plasmid, then transferred to Escherichia coli for heterologous expression. Heterologous expression was performed in four E. coli strains containing different complex enzyme genes. Experimental conditions: 3 mL of 1 / 1000 kanamycin-containing LB medium was added to 15 mL centrifuge tubes, and the mixture was incubated at 37°C and 180 rpm on a shaker. When the OD235 value reached approximately 0.6-0.8, 1 / 1000 of the inducing agent IPTG (1 mol / mL) was added, and the mixture was induced overnight at 25°C and 180 rpm on a shaker. Target expression bands were observed on the protein electrophoresis map using SDS-PAGE polyacrylamide gel electrophoresis.

[0083] 2.2 Experimental Materials

[0084] 2.2.1 Experimental Materials: Activated bacterial solution: Stranding of bacterial strains: Take autoclaved LBA medium and heat it in a microwave oven until the solid medium melts into a transparent liquid. Dispense 20 mL into 50 mL centrifuge tubes in a laminar flow hood. Add 1 / 1000 of 100 mg / mL kanamycin and shake well. Take a clean plate, open the lid near an alcohol lamp flame, pour in an appropriate amount of medium, and gently shake to distribute it evenly. Half-open the lid, maintain ventilation, turn on the UV lamp, leaving only a small gap in the laminar flow hood, and wait for the solid medium to solidify. Burn the wire of the inoculation loop until the wire turns red. After the wire returns to room temperature, streak the transformed strain on a plate, label the strain name and date, and incubate overnight in a 37°C oven. The next day, store in a 4°C refrigerator. Then, pick single colonies and incubate overnight in a kanamycin-resistant medium at 37°C. Preserve the strain using 60% glycerol tubes and label them.

[0085] 2.2.2 Experimental Apparatus: Clean bench, ultraviolet spectrophotometer, centrifuge, ultrasonic cell disruptor, shaker

[0086] 2.2.3 Preparation of solutions used in the experiment: (1) Preparation of LB medium: Weigh 10g of peptone, 5g of yeast powder and 10g of sodium chloride with an analytical balance and add them to a 1L conical flask. Measure 1L of primary water with a graduated cylinder and add it to the conical flask. Mix it evenly with a glass rod. Dispense 100mL into 250mL conical flasks, seal them with a special sealing film and tie them tightly with a rubber band. Sterilize them in an autoclave at 120℃.

[0087] (2) Preparation of 100mg / mL kanamycin: Weigh 3g of kanamycin using an analytical balance and place it in a 50mL centrifuge tube. Place the tube and a conical flask containing sterile water in a clean bench and sterilize them by turning on the UV lamp for 3 minutes. Pour the sterile water into a plate, open the plate halfway, and use a pipette with a range of 1000μL to add 30mL of sterile water to the centrifuge tube containing kanamycin. Shake well and wait for it to dissolve completely. Use a 50mL syringe with a 0.25μm filter membrane to draw up the kanamycin solution and filter it into a new 50mL centrifuge tube. Finally, aliquot each 1mL of the solution into sterilized 1.5mL centrifuge tubes, put them into sample bags, mark the date and name with a marker, and store them in a -20℃ refrigerator for long-term storage.

[0088] (3) Preparation of Buffer A: Weigh 2.4g of Tris and 29.2g of sodium chloride using an electronic balance and put them into a 1L beaker. Measure 800mL of primary water using a graduated cylinder with a range of 1L. Add the primary water to the beaker and stir evenly with a glass rod rinsed with primary water. Open the pH meter, remove the pH meter probe, open the cap, rinse the probe with primary water and wipe it dry with paper. Insert the probe into the solution in the beaker without touching the beaker and measure the pH of the solution. Use a pipette with a range of 1000μL to add 30% hydrochloric acid and adjust the pH to 7.0. Pour the solution into a graduated cylinder, add primary water, and bring the solution to a final volume of 1L.

[0089] (4) Preparation of Buffer B: Weigh 2.4g of Tris, 29.2g of sodium chloride, and 34.03g of imidazole using an electronic balance and place them in a 1L beaker. Measure 700mL of primary water using a graduated cylinder and add it to the beaker. Stir well with a glass rod. Turn on the pH meter, remove the probe, open the cap, rinse and dry it with primary water, and measure the pH of the solution by inserting it into the liquid surface. Add 30% hydrochloric acid to the solution to adjust the pH to 7.0. Due to the imidazole, adjusting the pH will be more difficult and a larger amount needs to be added. Pour the pH-adjusted solution into a graduated cylinder and bring the volume to 1L with primary water.

[0090] (5) Filtration of Buffer A and Buffer B solutions: Place a microporous filter membrane with a pore size of 0.2 μm and a diameter of 50 mm at the core of the standard sand core filter and wet the filter membrane with primary water. Connect the sand core filter to a 500 mL circular glass funnel and clamp the connection with an aluminum alloy clamp. Connect the rubber tube of the vacuum pump to the air outlet of the sand core filter. Connect the conical flask that has been rinsed with primary water to the lower end of the sand core filter. Turn on the vacuum pump and first check for leaks with primary water. Then pour the pH-adjusted Buffer A or Buffer B into the circular glass funnel for vacuum filtration. Replace the filter membrane if necessary. Put the filtered solution into a special bottle and label it with the name and time.

[0091] (6) Preparation of 20 mmol / L tris-HCl: Weigh 2.42 g of Tris and dissolve it in 0.8 L of deionized water. Adjust the pH to 7.0 with 30% HCl and then bring the volume to 1 L with deionized water. Filter the solution using a 0.2 μm aqueous filter membrane.

[0092] (7) The protein gradient elution solution is prepared as shown in Table 2.2.3:

[0093] Table 2.2.3 Gradient Elution Solutions

[0094] (mMol / L) Buffer A (mL) Buffer B (mL) 5 49.5 0.5 10 49 1 20 48 2 50 45 5 100 40 10 200 30 20

[0095] 2.3 Experimental Methods

[0096] 2.3.1 Protein Induction: Start the laminar flow hood, turn on the UV lamp for 3 minutes, then turn it off. Turn on the laminar flow hood, turn on the lighting and ventilation, and light the alcohol lamp. Disinfect items placed in the laminar flow hood by spraying them with 95% ethanol (using a spray bottle filled with ethanol) both sides. Wear disposable PE gloves and rubber gloves. After disinfecting your hands, enter the laminar flow hood and wait for the alcohol to dry before starting the procedure. Take a 15mL centrifuge tube and add 3mL of LB medium near the flame of the alcohol lamp. Add 1μL of 1... 0.0 mg / mL carbamycin. Hold a streaked agar plate (within one week) in your left hand, and a 10.0 μL pipette in your left hand. Open the agar plate with your left thumb near an alcohol lamp. Use the pipette tip in your right hand to pick up a single colony of suitable size. Close the agar plate. Use your left hand to open a centrifuge tube with your thumb and forefinger. Sterilize the tube opening by flaming it with the outer flame of an alcohol lamp. Tilt the centrifuge tube to bring the liquid close to the opening. Insert the pipette tip into the liquid surface and shake it left and right to dissolve the picked colony in the culture medium. Sterilize the tube opening again by flaming it with the outer flame of an alcohol lamp. Close the centrifuge tube. Mark the date and bacterial species with a marker. Take another 15 mL centrifuge tube and add 3 mL of LB medium as a negative control. Mark the date. Incubate the tubes overnight at 37°C and 180 rpm on a shaker to obtain activated bacterial culture. Take 1.5 mL of the previous day's culture and transfer it to a 2 mL centrifuge tube. Aliquot the two tubes and store at 4°C. Take 3 μL of the bacteria from the previous day and transfer it to 3 mL of LB medium, then transfer the medium into a 15 mL centrifuge tube. Incubate the centrifuge tube at 37°C and 180 rpm in a shaker. Measure the OD235 value of the bacterial solution using a UV spectrophotometer. When the OD235 value reaches approximately 0.6-0.8, add 1 / 1000 of the inducing agent IPTG (1 mol / mL) and incubate overnight at 25°C and 180 rpm in a shaker. The next day, aliquot the induced bacterial solution into 1.5 mL 2 mL centrifuge tubes, dividing each tube into two separate tubes. Place both tubes symmetrically with the uninduced bacterial solution in a centrifuge and centrifuge at 12000 rpm for 5 min. Separate and retain the supernatant and precipitate. Label the induction status, bacterial species, and supernatant / precipitate, and store at -20°C.

[0097] 2.3.2 SDS-PAGE Protein Gel Electrophoresis: 2.3.2.1 Protein Sample Preparation: The supernatant and precipitate of different bacteria before and after induction, stored at -20℃, were removed. 160 μL of the supernatant was transferred to a 1.5 mL centrifuge tube using a 200 μL pipette. 1 mL of 20 mmol Tris-HCl (Ph=7.0) was added to the precipitate and the mixture was resuspended by pipetting. 160 μL of the solution was transferred to a 1.5 mL centrifuge tube using a 200 μL pipette. The supernatant and precipitate were labeled. Then, according to a bacterial culture to loading buffer ratio of 4:1, 40 μL of loading buffer was added to the 160 μL precipitate and supernatant. The mixture was vortexed until homogeneous. The prepared protein sample was placed in a sample bag, labeled with the bacterial species name, time, and induction status. The sample was stored at -20℃ for long-term preservation. The remaining supernatant without loading buffer was used for protein concentration measurement.

[0098] 2.3.2.2 Protein Concentration Measurement: Turn on the computer and the ultra-micro UV spectrophotometer. Click on the ND500 software on the computer, select protein measurement, and first use a 10μL pipette to draw 2μL of 20mMol Tris-HCl (pH=7.0) for zeroing. Then take the supernatant (2μL each time) without loading buffer from 2.3.1 to measure the protein concentration. After each measurement, wipe the sample inlet and cap of the ultra-micro UV spectrophotometer with lens paper before performing the next measurement. After the measurement is completed, observe and record the data.

[0099] 2.3.2.3 Protein Gel Preparation: Take 1.0 mm glass short and long plates, clean and dry them with primary water, install the short and long plates with their bottom ends aligned, and then install them in the clamping plate with the short plate facing outwards. Fix the long and short plates in place. Wet the sponge strip on the gel casting frame with primary water. Fix the fixed long and short plates onto the gel casting plate. Fill the space between the glass plates with primary water and let it stand for 5 minutes. Observe whether the liquid level drops and check whether the device leaks. After the leak is checked, pour out the primary water. Add the separating gel between the glass plates according to Table 2.3.2.3. Add TEMED last, and open the window for ventilation when adding it. Hold the gel above your nose. After adding the appropriate amount of water, add primary water between the plates until the liquid level is flush with the short plate. Gently shake to remove air bubbles, flatten the liquid surface, and let it stand on a flat surface. Wait for the separating gel to solidify and for clear separation lines to appear. Pour off the primary water and prepare the stacking gel according to Table 2.3.2.3. Fill the space between the glass plates with the separating gel and insert a 1.0mm comb. Slowly and evenly press the comb to avoid air bubbles. Let it stand on a flat surface and wait for the stacking gel to solidify. Remove the prepared protein gel, wrap it in a paper towel, moisten it with primary water, place it in a disposable PE glove, and store it in a refrigerator at 4°C.

[0100] Table 2.3.2.3 Protein Gel

[0101] Separating gel (12%) Concentrated gel (5%) Total volume Approximately 15 mL Approximately 7.5 mL 30% Acrylamide (29:1) 6.0mL 1.24mL 4×SDS solution PAGE stacking gel buffer solution 0 1.87mL 1.5 mol / L Tris-HCl (pH=8.8) 3.8mL 0 10% SDS 150µL 0 10% ammonium persulfate 150μL 75μL TEMED 6μL 3.5µL Grade I water 4.9mL 4.36mL

[0102] 2.3.2.4 Protein Gel Electrophoresis Procedure: Take the protein gel prepared in 2.3.3, and install two gel pieces on both sides of the clamp with electrodes, with the shorter gel plates facing inwards and the upper ends of the shorter gel plates tightly against the lower end of the protrusion of the clamp. Then, fasten the clips and place it in the electrophoresis tank, aligning the red and black electrodes. Fill the space between the two gel pieces with Running Buffer, remove the comb, and rinse the microsyringe with Running Buffer before loading the protein sample. The loading order is: 2 μL marker, 20 μL supernatant before induction, 20 μL supernatant after induction, 20 μL precipitate before induction, and 20 μL precipitate after induction. After loading, add Running Buffer to the tank until the liquid level covers the green gel strip at the lower end of the clamp. Connect the electrophoresis apparatus, aligning the red and black electrodes, turn on the electrophoresis apparatus, and set the parameters: voltage to 125V and time to 58 min. After electrophoresis, remove the protein gel, remove the short plate under running water, cut off both ends of the gel, remove the gel, place it in a glass dish, add an appropriate amount of Coomassie Brilliant Blue staining solution to cover the gel, cover it with another glass dish, and place it on a shaker for staining. After about an hour and a half, pour off the waste liquid, add primary water, and destain the gel. After destaining, observe and photograph the protein gel.

[0103] 2.3.2.5 Protein Electrophoresis Image Processing: The protein electrophoresis gel images were processed. First, the protein gel images were blacked out using Photoshop software, and the excess parts were cropped. Protein markers and lanes were marked in the image. Image-J analysis was performed on the image after Photoshop processing, and the analysis data were plotted as a bar chart using GraphPad Prism. The optimized results were then analyzed to determine the optimal induction expression conditions.

[0104] 2.3.3 Protein Purification: 2.3.3.1 Bacterial Culture and Collection: In a clean bench, add 250 mL of sterilized LB medium to a 500 mL Erlenmeyer flask. Add 3 mL of pre-activated bacterial culture to the Erlenmeyer flask, along with 250 μL of 100 mg / mL kanamycin. Separately, add 3 mL of LB medium to a 15 mL centrifuge tube as a negative control. Place the Erlenmeyer flask and centrifuge tube in a shaker at 37℃ and 180 rpm for 4 hours. The negative control group serves as a reference. The OD235 value was measured using a UV spectrophotometer. When the value reached 0.6 to 0.8, the conical flask was removed and 250 μL of 1 mol / mL IPTG was added in a clean bench. The flask was then placed in a shaker at 25℃ and 180 rpm for overnight incubation. The induced bacterial culture was then aliquoted into 50 mL centrifuge tubes, with two tubes in total. The tubes were balanced using an electronic balance and placed symmetrically in a centrifuge. The tubes were centrifuged at 12000 rpm for 5 min. The supernatant was discarded, and the precipitate was retained. The precipitate was then stored in a refrigerator at -20℃.

[0105] 2.3.3.2 Cell Disruption: The collected bacterial precipitate was mixed by pipetting with 30 mL of Buffer A. The centrifuge tube was fixed in a beaker filled with ice and placed in an ultrasonic cell disruptor. The height of the platform was adjusted so that the metal probe was suspended in the solution, avoiding contact with the centrifuge tube wall. The parameters were set to 60% power and the cell disruptor was turned on. The cell disruption continued until the solution was clear and transparent. The solution was aliquoted into 2 mL centrifuge tubes and placed symmetrically into the centrifuge. The nuts were tightened and the centrifuge lid was closed. The parameters were set to 12000 rpm for 5 min. The supernatant was collected into a 50 mL centrifuge tube. 2 mL of the supernatant was then transferred into another 2 mL centrifuge tube, labeled, and stored in an ice box.

[0106] 2.3.3.3 Purification Steps: Turn on the peristaltic pump and UV detector. First, place the inlet tube of the peristaltic pump into Buffer A to rinse the elution column until the UV detector reading remains stable. Always keep the inlet tube of the peristaltic pump below the liquid surface to avoid air bubbles entering. After the reading stabilizes, turn off the peristaltic pump and carefully connect the inlet tube to the sample. Turn on the peristaltic pump to load the sample and observe the change in the UV detector reading. When the reading reaches its maximum value, collect the dripping solution using a 2mL centrifuge tube to obtain the breakthrough peak. After all the sample has been loaded, use Buffer A to... After rinsing until the UV spectrophotometer reading stabilizes, turn off the peristaltic pump. Place the peristaltic pump inlet into a 5 mL centrifuge tube, turn on the peristaltic pump, and observe the UV detector. When the reading changes, collect the dripping solution in a 2 mL centrifuge tube. After filling the tube, label it with numbers like 5-1, 5-2, etc. Record the labels and the corresponding UV reading changes in the logbook. When the UV detector reading first rises and then falls to form a complete peak or remains unchanged, switch to the next gradient elution, successively using 10 mmol / L, 20 mmol / L, 50 mmol / L, 100 mmol / L, and 200 mmol / L. After completing the gradient elution, use 500 mmol / L Buffer B to elute all the proteins. Once the readings stabilize, turn off the peristaltic pump and the UV detector, and place the inlet tube into Buffer A. For the eluted proteins, based on the data in the logbook, select centrifuge tubes corresponding to the peak or stability scores using UV values. One value should be selected for each gradient. Then, perform gel electrophoresis in the following order: Marker, proenzyme, breakthrough peak, 5, 10, 20, 50, 100, 200. After obtaining the electrophoresis pattern, select two values ​​on each side of the peak of the gradient that elutes the most proteins, and perform gel electrophoresis on the proteins in the five corresponding centrifuge tubes. Based on the electrophoresis pattern, select the gradient from the four bacteria with the highest concentration and purity of the enzyme for gel electrophoresis.

[0107] 2.4 Experimental Results

[0108] 2.4.1 Protein induction: such as Figure 9 As shown, the nucleic acid electrophoresis markers are clear and the separation is good, indicating successful DNA electrophoresis. The first lane shows the result before supernatant induction, and the second lane shows the result after supernatant induction. The second lane shows a band that is clearly close to 1000 bp, consistent with the target gene. The third lane shows the result before precipitation induction, and the fourth lane shows the result after precipitation induction. The fourth lane shows a band that is clearly close to 40 bp, consistent with the target gene, indicating successful PCR amplification.

[0109] 2.4.2 Protein purification: such as Figure 10As shown, the marker protein exhibits a wavy pattern but good separation. The target band appears near 40 kDa. Lane 1 contains the protein band of strain 1, lane 2 contains the protein band of strain 2, lane 3 contains the protein band of strain 3, and lane 4 contains the protein band of strain 4, all containing the target band. Conclusion: All four strains contain relatively pure target proteins.

[0110] 3. Enzymatic properties

[0111] 3.1 The enzymatic properties of the complex enzymes from four bacteria were investigated. The optimal temperature, temperature tolerance, optimal pH, and pH tolerance of the four complex enzymes were analyzed. Finally, the complex enzyme with better enzymatic properties was screened for subsequent immobilization and application. This experiment utilized the degradation of ampicillin by a β-lactamase complex enzyme to explore its enzymatic properties. The experimental results showed that the complex enzyme of CTX-33 and VIM-2 best met the requirements.

[0112] 3.2 Experimental Materials

[0113] 3.2.1 Experimental materials: purified enzyme solution.

[0114] 3.2.2 Experimental instruments: constant temperature water bath, dry thermostat, ultraviolet spectrophotometer.

[0115] 3.2.3 Preparation of solutions used in the experiment: (1) Preparation of 20 mmol / L tris-HCl: Weigh 2.42 g of Tris and dissolve it in 0.8 L of deionized water. Adjust the pH value to 7.0 with 30% HCl and then make up to 1 L with deionized water. Filter with a 0.2 μm aqueous filter membrane.

[0116] (2) Preparation of pH buffer: Prepare glycine-hydrochloric acid buffer (pH=3.0, 4.0, 5.0): Take 2.5 mL of 0.2 mol / L glycine solution into a 50 mL centrifuge tube, add 0.2 mol / L hydrochloric acid solution to pH=2.9-3.1, and add pure water to make up to 10 mL. Prepare pH 4.0 and 5.0 buffer solutions using the same method; prepare PBS buffer (pH 6.0): add 1.23 mL of 0.2 mol / L disodium hydrogen phosphate and 8.77 mL of 0.2 mol / L sodium dihydrogen phosphate to a 50 mL centrifuge tube; prepare Tris-HCl buffer solutions (pH 7.0, 8.0, 9.0): weigh 2.42 g of Tris into a 1000 mL reagent bottle, dissolve in 800 mL of pure water, adjust the pH to 7.0 with HCl, and add pure water to a final volume of 1000 mL; weigh 0.48 g of Tris into a 50 mL reagent bottle, dissolve in 16 mL of pure water, adjust the pH to 8.0 with HCl, and add pure water to a final volume of 20 mL; weigh 0.48 g... Dissolve Tris in 16 mL of pure water in a 50 mL reagent bottle, adjust the pH to 9.0 with HCl, and then add pure water to a final volume of 20 mL. Prepare glycine-sodium hydroxide buffer solutions (pH=10.0, 11.0): Take 2.5 mL of 0.2 mol / L glycine solution in a 50 mL centrifuge tube, and add 0.2 mol / L sodium hydroxide solution to a pH of 10.0. Prepare a pH=11.0 buffer solution using the same method.

[0117] (3) Preparation of 200 μg / mL ampicillin: Weigh 0.0100 g ampicillin into a 50 mL centrifuge tube, dissolve it in 50 mL of 20 mM Tris-HCl (pH=7.0), and place it on ice for later use.

[0118] 3.3 Experimental Methods

[0119] 3.3.1 Optimal Temperature: Five 2mL centrifuge tubes were used as a group, one as a positive control, one as a negative control, and three as parallel experimental groups. 960μL of 20mmol / L tris-HCl was added to the positive control, 960μL of 200μg / mL ampicillin was added to the negative control, and 960μL of 200μg / mL ampicillin was added to each of the parallel groups. The centrifuge tubes were preheated at the experimental temperature for 5 min. Then, 40μL of enzyme solution was added to the positive control, 40μL of 20mmol / L tris-HCl to the negative control, and 40μL of enzyme solution to each of the parallel groups. The mixture was thoroughly mixed by pipetting and incubated at the experimental temperature for 15 min. The OD235 was then measured using a UV spectrophotometer. The experiment was conducted at temperature gradients of 10℃, 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, 90℃, and 100℃, and the relative enzyme activities of the four complex enzymes were calculated to determine their optimal temperatures.

[0120] 3.3.2 Temperature Tolerance: Five 2mL centrifuge tubes were used as a group, one as a positive control, one as a negative control, and three as parallel experimental groups. 40μL of enzyme solution was added to the positive control, 40μL of 20mmol / L tris-HCl to the negative control, and 40μL of enzyme solution to each parallel group. The centrifuge tubes were stored at the experimental temperature for 1 hour. Then, 960μL of 20mmol / L tris-HCl was added to the positive control, 960μL of 200μg / mL ampicillin to the negative control, and 200μg / mL ampicillin to each parallel group. The mixture was thoroughly mixed and incubated at the optimal temperature for 15 minutes. The OD235nm was then measured using a UV spectrophotometer. The reaction was carried out at temperature gradients of 10℃, 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, 90℃, and 100℃, and the relative enzyme activities of the four complex enzymes were calculated.

[0121] 3.3.3 Optimal pH: Five 2mL centrifuge tubes were used as a group, one as a positive control, one as a negative control, and three as parallel experimental groups. The positive control was supplemented with 900μL of 20mmol / L tris-HCl, 60μL of experimental pH buffer, and 40μL of enzyme solution. The negative control was supplemented with 900μL of 200μg / mL ampicillin, 60μL of experimental pH buffer, and 40μL of 20mmol / L tris-HCl. The parallel groups were supplemented with 900μL of 200μg / mL ampicillin, 60μL of experimental pH buffer, and 40μL of enzyme solution. After mixing by pipetting, the centrifuge tubes were placed at the optimal temperature for 15 min, and the OD235nm was measured using a UV spectrophotometer. The reaction was carried out at pH gradients of 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and the relative enzyme activities of the four complex enzymes were calculated to determine their optimal pH.

[0122] 3.3.4 pH Tolerance: Five 2mL centrifuge tubes were used as a group, one as a positive control, one as a negative control, and three as parallel experimental groups. 40μL of enzyme solution and 60μL of pH buffer were added to the positive control, 40μL of 20mmol / L tris-HCl and 60μL of pH buffer were added to the negative control, and 40μL of enzyme solution and 60μL of pH buffer were added to the parallel experimental groups. The centrifuge tubes were incubated at the experimental temperature for 1 hour. Then, 900μL of 20mmol / L tris-HCl was added to the positive control, 900μL of 200μg / mL ampicillin was added to the negative control, and 900μL of 200μg / mL ampicillin was added to the parallel experimental groups. The mixtures were thoroughly mixed and incubated at the optimal temperature for 15 minutes. The OD at 235nm was then measured using a UV spectrophotometer. The experiment was conducted at pH gradients of 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and the relative enzyme activities of the four complex enzymes were calculated.

[0123] 3.4 Experimental Results

[0124] 3.4.1 Optimal Temperature: See Figure 11 The optimal temperatures for the four complex enzymes are 30℃, 40℃, 30℃, and 30℃, respectively.

[0125] 3.4.2 Temperature tolerance: see Figure 12 .

[0126] 3.4.3 Optimal pH: See Figure 13 The optimal pH values ​​for the four complex enzymes are 7, 8, 8, and 9, respectively.

[0127] 3.4.4 pH tolerance: see Figure 14 .

[0128] Example 2: Optimization of immobilization conditions.

[0129] 4.1 Using free enzyme solutions to treat wastewater results in unrecoverable enzymes after mixing with the wastewater, requiring the addition of new enzymes for each treatment, essentially making them disposable consumables with very high long-term operating costs. Enzymes directly exposed to the complex wastewater environment are prone to rapid denaturation and inactivation due to harsh conditions. The treated effluent contains inactivated or residual enzyme proteins, potentially causing secondary pollution and requiring additional separation steps to purify the effluent, further complicating the process. Furthermore, the reaction of free enzyme solutions is difficult to stop midway after initiation, resulting in poor control precision. In contrast, immobilized enzymes can be recovered from the treated wastewater after the reaction is complete through simple filtration, sedimentation, or magnetic separation, and then used for the next batch of wastewater treatment. While enzymes themselves are expensive, reusability drastically reduces the cost per treatment. Immobilizing enzymes on a carrier makes their structure more stable, better resistant to the impact of harmful substances (such as heavy metals and organic solvents), pH fluctuations, and temperature changes in wastewater, reducing inactivation and extending their lifespan. The treated effluent is free of enzyme proteins, resulting in purer water quality and eliminating the need for subsequent steps to remove residual enzymes.

[0130] For small-scale laboratory studies, using enzyme solutions directly may be more convenient. However, once applied to actual wastewater treatment projects, immobilized enzyme technology has an overwhelming advantage. It is a crucial step in moving enzyme technology from the laboratory to industrial applications, making it possible to treat wastewater efficiently, economically, and sustainably using enzymatic methods.

[0131] After enzymes are immobilized, their catalytic performance and environmental requirements change significantly. If we directly apply the optimal reaction conditions of free enzymes to immobilized enzymes, their catalytic efficiency is often far lower than expected. Therefore, optimizing the conditions for immobilized enzymes can not only improve catalytic efficiency but also extend their lifespan and improve process economy. Only under optimal conditions can immobilized enzymes achieve their fastest reaction rates, shorten processing time, and increase throughput per unit time. Inappropriate conditions will accelerate enzyme inactivation. By optimizing conditions, we can ensure that enzymes operate under mild and efficient conditions, thereby extending their operational stability, which is key to reducing long-term operating costs.

[0132] 4.2 Experimental Materials

[0133] 4.2.1 Experimental materials: purified enzyme solution.

[0134] 4.2.2 Experimental instruments: ultraviolet spectrophotometer, constant temperature water bath.

[0135] 4.2.3 Preparation of solutions used in the experiment:

[0136] (1) Preparation of sodium alginate solution: Prepare sodium alginate solutions of 0.5%, 1.0%, 1.5%, 2.0% and 2.5% respectively. Weigh 0.10g, 0.20g, 0.30g, 0.40g and 0.50g of sodium alginate into 50mL centrifuge tubes, add 20mL of pure water and stir until completely dissolved.

[0137] (2) Preparation of CaCl2 solution: Prepare 1.0%, 1.5%, 2.0%, 2.5% and 3.0% CaCl2 solutions. Weigh 0.20g, 0.30g, 0.40g, 0.50g and 0.60g of CaCl2 into 50mL centrifuge tubes respectively and dissolve them in 20mL of pure water.

[0138] pH buffer preparation: Prepare glycine-hydrochloric acid buffer (pH=3.0, 4.0, 5.0): Take 2.5 mL of 0.2 mol / L glycine solution into a 50 mL centrifuge tube, add 0.2 mol / L hydrochloric acid solution to pH=2.9-3.1, and add pure water to make up to 10 mL. Prepare pH 4.0 and 5.0 buffer solutions using the same method; prepare PBS buffer (pH 6.0): add 1.23 mL of 0.2 mol / L disodium hydrogen phosphate and 8.77 mL of 0.2 mol / L sodium dihydrogen phosphate to a 50 mL centrifuge tube; prepare Tris-HCl buffer solutions (pH 7.0, 8.0, 9.0): weigh 2.42 g Tris into a 1000 mL reagent bottle, dissolve in 800 mL of pure water, adjust the pH to 7.0 with HCl, and add pure water to a final volume of 1000 mL; weigh 0.48 g Tris into a 50 mL reagent bottle, dissolve in 16 mL of pure water, adjust the pH to 8.0 with HCl, and add pure water to a final volume of 20 mL; weigh 0.48 g... Dissolve Tris in 16 mL of pure water in a 50 mL reagent bottle, adjust the pH to 9.0 with HCl, and then add pure water to a final volume of 20 mL. Prepare glycine-sodium hydroxide buffer solutions (pH=10.0, 11.0): Take 2.5 mL of 0.2 mol / L glycine solution in a 50 mL centrifuge tube, and add 0.2 mol / L sodium hydroxide solution to a pH of 10.0. Prepare a pH=11.0 buffer solution using the same method.

[0139] (3) 200 μg / mL ampicillin: Weigh 0.0100 g of ampicillin into a 50 mL centrifuge tube, dissolve it in 50 mL of 20 mL Tris-HCl (pH=7.0), and place it on ice for later use.

[0140] 4.3 Experimental Methods

[0141] 4.3.1 Optimization of Sodium Alginate Concentration: 4.3.1.1 Preparation of Blank and Experimental Microspheres: The final concentrations of sodium alginate were 0.5%, 1%, 1.5%, 2%, and 2.5%, respectively. The volumes of sodium alginate and enzyme solution added per mL of different concentrations are given below: 0.5% SA: 833 μL 0.5% sodium alginate solution + 167 μL compound enzyme solution; 1.0% SA: 833 μL 1.0% sodium alginate solution + 167 μL compound enzyme solution; 1.5% SA: 833 μL 1.5% sodium alginate solution + 167 μL compound enzyme solution; 2.0% SA: 833 μL 2.0% sodium alginate solution + 167 μL compound enzyme solution; 2.5% SA: 833 μL 2.5% sodium alginate solution + 167 μL of compound enzyme solution; for the blank group, simply replace the mutant enzyme solution with Tris-HCl (pH=7.0) buffer.

[0142] Preparation of microspheres: Prepare 5 culture dishes and add 5 mL of 2.5% CaCl2 solution to each dish. Using a syringe, slowly drop the sodium alginate-enzyme mixture into each culture dish containing 2.5% CaCl2 solution from a distance of 1 mm from the liquid surface. After static immobilization for 10 min, rinse 3 times with 20 mM Tris-HCl (pH=7.0). Then store the microspheres in EP tubes, label them, and place them on ice for later use. The blank group is prepared in the same way.

[0143] 4.3.1.2 Detection: The final concentration of the substrate (ampicillin) was fixed at 200 μg / mL. Prepare an EP tube rack and 1.5 mL EP tubes. Perform three parallel experiments and one blank experiment for each group, for a total of four groups (four EP tubes per group). Number and label the EP tubes. Pipette 600 μL of Tris-HCl (pH=7.0) buffer into the EP tube using a 1 mL pipette, then pipette 400 μL of 500 μg / mL ampicillin into the EP tube. Preheat in a 40℃ water bath for 5 min. After preheating, remove the EP tubes and take out the prepared microspheres from the blank and experimental groups. Quickly add 3 microspheres to each microsphere, and immediately return the tubes to the 40℃ water bath for 15 min. Measure and record the OD value for each group using a spectrophotometer at a wavelength of 235 nm. Repeat the above steps for each group. Perform calculations and processing on the obtained data.

[0144] 4.3.2 Optimization of the ratio of sodium alginate solution to mutant enzyme solution: 4.3.2.1 Preparation of blank and experimental group microspheres: The final concentration of sodium alginate was 1.5%. The ratio of sodium alginate solution to mutant enzyme solution was set to 1:1, 2:1, 3:1, 4:1 and 5:1, respectively. The following are different ratios of 1 mL of 1.5% sodium alginate and compound enzyme solution: 1.5% SA: enzyme solution = 1:1:500 μL 1.5% sodium alginate solution + 500 μL compound enzyme solution; 1.5% SA: enzyme solution = 2:1:666 μL 1.5% sodium alginate solution + 333 μL compound enzyme solution; 1.5% SA: enzyme solution = 3:1:750 μL 1.5% sodium alginate solution + 250 μL compound enzyme solution; 1.5% SA: enzyme solution = 4:1:800 μL 1.5% sodium alginate solution + 200 μL of compound enzyme solution; 1.5% SA: enzyme solution = 5:1:833 μL; 1.5% sodium alginate solution + 167 μL of compound enzyme solution; for the blank group, simply replace the mutant enzyme solution with Tris-HCl (pH=7.0) buffer.

[0145] Preparation of microspheres: Prepare 5 culture dishes and add 5 mL of 2.5% CaCl2 solution to each dish. Using a syringe, slowly drop the sodium alginate-enzyme mixture into each culture dish containing 2.5% CaCl2 solution from a distance of 1 mm from the liquid surface. After static immobilization for 10 min, rinse 3 times with 20 mM Tris-HCl (pH=7.0). Then store the microspheres in EP tubes, label them, and place them on ice for later use. The blank group is prepared in the same way.

[0146] 4.3.2.2 Detection: The final concentration of the substrate (ampicillin) was fixed at 200 μg / mL. Prepare an EP tube rack and 1.5 mL EP tubes. Perform three parallel experiments and one blank experiment for each group, for a total of four groups (four EP tubes per group). Number and label the EP tubes. Pipette 600 μL of Tris-HCl (pH=7.0) buffer into the EP tube using a 1 mL pipette, then pipette 400 μL of 500 μg / mL ampicillin into the EP tube. Preheat in a 40℃ water bath for 5 min. After preheating, remove the EP tubes and take out the prepared microspheres from the blank and experimental groups. Quickly add 3 microspheres to each microsphere, and immediately return the tubes to the 40℃ water bath for 15 min. Measure and record the OD value for each group using a spectrophotometer at a wavelength of 235 nm. Repeat the above steps for each group. Perform calculations and processing on the obtained data.

[0147] 4.3.3 Optimization of CaCl2 Concentration: 4.3.3.1 Preparation of Microspheres for Blank and Experimental Groups: The final concentration of sodium alginate was 1.5%, the final ratio of sodium alginate to compound enzyme solution was 5:1, and the final concentrations of CaCl2 were 1%, 1.5%, 2%, 2.5%, and 3%, respectively. For example, to prepare 1 mL of sodium alginate-enzyme mixture, use 833 μL of 1.5% sodium alginate solution + 167 μL of compound enzyme solution; for the blank group, simply replace the compound enzyme solution with 20 mM Tris-HCl (pH=7.0) buffer.

[0148] Preparation of microspheres: Prepare 5 culture dishes. Pour CaCl2 solutions of different concentrations into the culture dishes respectively. Using a syringe, slowly drip the sodium alginate-enzyme mixture into the culture dishes containing CaCl2 of different concentrations from a height of 1 mm above the liquid surface. After static immobilization for 10 min, rinse 3 times with 20 mM Tris-HCl (pH=7.0). Store the microspheres in EP tubes, label them, and place them on ice for later use. The blank group is prepared by repeating the above operation. Repeat the above operation for the experimental group and the blank group with different concentrations of CaCl2. After preparation, the microspheres are placed on ice for later use.

[0149] 4.3.3.2 Detection: The final concentration of the substrate (ampicillin) was fixed at 200 μg / mL. Prepare an EP tube rack and 1.5 mL EP tubes. Perform three parallel experiments and one blank experiment for each group, for a total of four groups (four EP tubes per group). Number and label the EP tubes. Pipette 600 μL of Tris-HCl (pH=7.0) buffer into the EP tube using a 1 mL pipette, then pipette 400 μL of 500 μg / mL ampicillin into the EP tube. Preheat in a 40℃ water bath for 5 min. After preheating, remove the EP tubes and take out the prepared microspheres from the blank and experimental groups. Quickly add 3 microspheres to each microsphere, and immediately return the tubes to the 40℃ water bath for 15 min. Detect and record the OD value for each group using a spectrophotometer at a wavelength of 235 nm. Repeat the above steps for each group. Perform calculations and processing on the obtained data.

[0150] 4.3.4 Optimization of the optimal immobilization time: 4.3.4.1 Preparation of microspheres for the blank and experimental groups: The final concentration of sodium alginate was 1.5%, the final ratio of sodium alginate to the compound enzyme solution was 5:1, and the CaCl2 concentration was 2.5%. Because the immobilization time was different, a timer was prepared to collect microspheres at 5 min, 10 min, 15 min, 20 min, and 30 min. The ratio of the sodium alginate-enzyme mixture remained unchanged; for example, to prepare 1 mL of sodium alginate-enzyme mixture, use 833 μL of 1.5% sodium alginate solution + 167 μL of enzyme. For the blank group, the enzyme was replaced with 20 mM Tris-HCl (pH=7.0) buffer.

[0151] 4.3.4.2 Detection: The final concentration of the substrate (ampicillin) was fixed at 200 μg / mL. Prepare an EP tube rack and 1.5 mL EP tubes. Perform three parallel experiments and one blank experiment for each group, for a total of four groups (four EP tubes per group). Number and label the EP tubes. Pipette 600 μL of Tris-HCl (pH=7.0) buffer into the EP tube using a 1 mL pipette, then pipette 400 μL of 500 μg / mL ampicillin into the EP tube. Preheat in a 40℃ water bath for 5 min. After preheating, remove the EP tube, take out the prepared microspheres from the blank and experimental groups, and quickly add 3 microspheres. Immediately after adding the microspheres, return the tube to the 40℃ water bath and react for 15 min. Detect and record the OD value for each group using a UV spectrophotometer at 235 nm. Repeat the above steps for each group. Perform calculations and processing on the obtained data.

[0152] 4.3.5 Reusability Experiment of Immobilized Composite Enzyme: 4.3.5.1 Preparation of Blank and Experimental Microspheres: From the above experiments, the optimal conditions for composite enzyme immobilization were: 1.5% sodium alginate, sodium alginate solution to composite enzyme solution ratio of 5:1, 2.5% CaCl2, and immobilization time of 10 min. The enzyme content in the prepared sodium alginate-enzyme mixture was fixed at 16.7%. To prepare 1 mL of sodium alginate-enzyme mixture, use 833 μL of 3% sodium alginate solution + 167 μL of enzyme.

[0153] Preparation of microspheres: Prepare a culture dish and pour 2.5% CaCl2 solution into it. At a distance of 1 mm from the liquid surface, slowly drip the above 1.5% sodium alginate-enzyme mixture into the culture dish containing 2.5% CaCl2 solution using a syringe. After static immobilization for 10 minutes, rinse three times with 20 mM Tris-HCl (pH=7.0). Then store the microspheres in EP tubes, label them, and place them on ice for later use. The blank group is prepared in the same way.

[0154] 4.3.5.2 Detection: The final concentration of the substrate (ampicillin) was fixed at 200 μg / mL. Prepare an EP tube rack and 1.5 mL EP tubes. Perform three parallel experiments and one blank experiment for each group, for a total of four groups (four EP tubes per group). Number and label the EP tubes. Pipette 600 μL of Tris-HCl (pH=7.0) buffer into the EP tube using a 1 mL pipette, then pipette 400 μL of 500 μg / mL ampicillin into the EP tube. Preheat in a 40℃ water bath for 5 min. After preheating, remove the EP tube and take out the prepared microspheres from the blank and experimental groups. Quickly add three microspheres to each microsphere, and immediately return the tube to the 40℃ water bath for 15 min. Measure and record the OD value for each group using a spectrophotometer at a wavelength of 235 nm. Repeat the above steps with the original three microspheres for each group. Perform calculations and processing on the obtained data.

[0155] 4.4 Experimental Results

[0156] 4.4.1 Sodium alginate concentration: such as Figure 15 As shown, the relative activity of the enzyme after immobilization with sodium alginate concentrations ranging from 0.5% to 2.5% roughly followed a normal distribution, with the highest relative activity observed at 1.5%. Under other sodium alginate concentrations, the relative enzyme activity of the composite enzyme solution was consistently above 50%. Therefore, the optimal sodium alginate concentration for immobilization of the composite enzyme solution is 1.5%.

[0157] 4.4.2 Volume ratio of sodium alginate to enzyme: (e.g.) Figure 16 The results showed that the relative activity of the immobilized enzyme was highest when the ratio of sodium alginate solution to the complex enzyme solution was 5:1, and the relative activity was also relatively high when the ratio was 3:1. Under the other sodium alginate solution to complex enzyme solution ratios, the relative enzyme activity of the immobilized complex enzyme solution was all above 60%. Therefore, the optimal ratio of sodium alginate solution to complex enzyme solution for immobilization is 5:1.

[0158] 4.4.3 CaCl2 concentration: such as Figure 17 As shown, the relative activity of the enzyme was highest when the CaCl2 concentration was 2.5%. At 2.0% and 3.0%, the relative enzyme activities of the two groups of immobilized complex enzymes were similar, approximately 95%. At 1.0% and 1.5%, the relative enzyme activities of the immobilized complex enzymes were similar, approximately 80%. That is, the optimal CaCl2 concentration for immobilization of the complex enzyme is 2.5%.

[0159] 4.4.4 Reaction time: e.g. Figure 18As shown, the relative activity of the enzyme was highest when the immobilization time was 10 min; the activity of the immobilized complex enzyme was relatively high when immobilized for 15 min, and slightly lower when immobilized for 5 min, 20 min, and 30 min. Overall, the relative activity of the immobilized complex enzyme was high, exceeding 85%. Therefore, the optimal immobilization time for the complex enzyme is 10 min.

[0160] 4.4.5 Reuse of immobilized enzymes: such as Figure 19 As shown, the relative activity of immobilized enzymes gradually decreases with increasing number of uses. The same batch of immobilized enzymes can be used 4-6 times. After more than 6 uses, the relative activity of the enzyme is too low, and the reaction efficiency is insufficient. That is, the optimal number of uses for immobilized enzymes is 4-6 times.

[0161] Example 3: Determination of enzyme activity reduction by four antibiotics: ampicillin, imipenem, aztreonam, and cefotaxime.

[0162] 5.1 Through enzymatic experiments, a well-expressed complex enzyme was screened. Its enzyme activity against four antibiotics—ampicillin, imipenem, aztreonam, and cefotaxime—was measured to determine its ability to degrade these four antibiotics. In particular, the enzyme's ability to degrade carbapenems such as imipenem and monocyclic β-lactamases such as aztreonam was investigated.

[0163] 5.2 Experimental Materials

[0164] 5.2.1 Experimental materials: crude enzyme solution.

[0165] 5.2.2 Experimental instruments: ultraviolet spectrophotometer, constant temperature water bath.

[0166] 5.2.3 Preparation of solutions used in the experiment: (1) Preparation of 20 mmol / L tris-HCl: Weigh 2.42 g of Tris and dissolve it in 0.8 L of deionized water. Adjust the pH value to 7.0 with 30% HCl and then make up to 1 L with deionized water. Filter with a 0.2 μm aqueous filter membrane.

[0167] (2) Preparation of pH buffer: Prepare glycine-hydrochloric acid buffer (pH=3.0, 4.0, 5.0): Take 2.5 mL of 0.2 mol / L glycine solution into a 50 mL centrifuge tube, add 0.2 mol / L hydrochloric acid solution to pH=2.9-3.1, and add pure water to make up to 10 mL. Prepare pH 4.0 and 5.0 buffer solutions using the same method; prepare PBS buffer (pH 6.0): add 1.23 mL of 0.2 mol / L disodium hydrogen phosphate and 8.77 mL of 0.2 mol / L sodium dihydrogen phosphate to a 50 mL centrifuge tube; prepare Tris-HCl buffer solutions (pH 7.0, 8.0, 9.0): weigh 2.42 g of Tris into a 1000 mL reagent bottle, dissolve in 800 mL of pure water, adjust the pH to 7.0 with HCl, and add pure water to a final volume of 1000 mL; weigh 0.48 g of Tris into a 50 mL reagent bottle, dissolve in 16 mL of pure water, adjust the pH to 8.0 with HCl, and add pure water to a final volume of 20 mL; weigh 0.48 g... Dissolve Tris in 16 mL of pure water in a 50 mL reagent bottle, adjust the pH to 9.0 with HCl, and then add pure water to a final volume of 20 mL. Prepare glycine-sodium hydroxide buffer solutions (pH=10.0, 11.0): Take 2.5 mL of 0.2 mol / L glycine solution in a 50 mL centrifuge tube, and add 0.2 mol / L sodium hydroxide solution to a pH of 10.0. Prepare a pH=11.0 buffer solution using the same method.

[0168] (3) Preparation of 500 μg / mL ampicillin: Weigh 0.0100 g ampicillin into a 50 mL centrifuge tube, dissolve it with 20 mL 20 mM Tris-HCl (pH=7.0), and place it on ice for later use.

[0169] (4) Preparation of 200 μg / mL imipenem: Weigh 0.0100 g imipenem into a 50 mL centrifuge tube, dissolve it in 50 mL of 20 mM Tris-HCl (pH=7.0), and place it on ice for later use.

[0170] (5) Preparation of 200 μg / mL aztreonam: Weigh 0.0100 g aztreonam into a 50 mL centrifuge tube, dissolve it with 20 mL 50 mL Tris-HCl (pH=7.0), and place it on ice for later use.

[0171] (6) Preparation of 200 μg / mL cefotaxime: Weigh 0.0100 g of cefotaxime into a 50 mL centrifuge tube, dissolve it in 50 mL of 20 mM Tris-HCl (pH=7.0), and place it on ice for later use.

[0172] 5.3 Experimental Methods

[0173] 5.3.1 Ampicillin Enzyme Activity Assay: In a 1 mL quartz cuvette, add 590 μL of 20 mM Tris-HCl (pH=7.0) buffer to an EP tube, then pipette 400 μL of 500 μg / mL ampicillin solution (final concentration fixed at 200 μg / mL). Add 10 μL of undiluted crude enzyme solution to the cuvette and mix quickly. Immediately monitor the absorbance value over time at 235 nm using a UV spectrophotometer until the absorbance value no longer changes.

[0174] 5.3.2 Imipenem enzyme activity assay: In a 1 mL quartz cuvette, add 890 μL of 20 mM Tris-HCl (pH=7.0) buffer to an EP tube, then pipette 100 μL of 200 μg / mL imipenem solution (final concentration fixed at 20 μg / mL). Add 10 μL of undiluted crude enzyme solution to the cuvette and mix quickly. Immediately monitor the absorbance value over time at 300 nm using a UV spectrophotometer until the absorbance value no longer changes.

[0175] 5.3.3 Assay of aztreonam enzyme activity: In a 1 mL quartz cuvette, add 700 μL of 20 mM Tris-HCl (pH=7.0) buffer to an EP tube, then pipette 100 μL of 200 μg / mL aztreonam solution (final concentration fixed at 20 μg / mL). Add 200 μL of undiluted crude enzyme solution to the cuvette and mix quickly. Immediately monitor the absorbance value over time at 320 nm using a UV spectrophotometer until the absorbance value no longer changes.

[0176] 5.3.4 Cefotaxime Enzyme Activity Assay: In a 1 mL quartz cuvette, add 865 μL of 20 mM Tris-HCl (pH=7.0) buffer to an EP tube, then pipette 125 μL of 200 μg / mL cefotaxime solution (final concentration fixed at 25 μg / mL). Add 10 μL of undiluted crude enzyme solution to the cuvette and mix quickly. Immediately monitor the absorbance value over time at 260 nm using a UV spectrophotometer until the absorbance value no longer changes.

[0177] 5.4 Experimental Results

[0178] Calculations show that this enzyme can degrade four types of antibiotics.

[0179] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A β-lactamase complex enzyme capable of degrading multiple antibiotics, characterized in that, Its preparation method is as follows: Gene fragments of CTX-M type broad-spectrum β-lactamase and VIM type metallo-β-lactamase were extracted from Klebsiella pneumoniae; The target gene fragment was obtained by PCR amplification and constructed into a basic vector to obtain a recombinant vector; The recombinant vector was transformed into host cells for heterologous expression; The β-lactamase complex enzyme capable of degrading multiple antibiotics was obtained by protein induction, SDS-PAGE protein gel electrophoresis, and protein purification.

2. The β-lactamase complex enzyme capable of degrading multiple antibiotics according to claim 1, characterized in that, The base vector is the pET-28a(+) plasmid.

3. The β-lactamase complex enzyme capable of degrading multiple antibiotics according to claim 1, characterized in that, The host cell was Escherichia coli BL21(DE3).

4. A method for immobilizing a β-lactamase complex enzyme capable of degrading multiple antibiotics as described in any one of claims 1-3, characterized in that, Includes the following: The β-lactamase complex enzyme was mixed with sodium alginate solution to obtain a mixture; The mixture is added dropwise to a CaCl2 solution to form microspheres, which are then washed and dried to obtain the immobilized enzyme.

5. The method for immobilizing a β-lactamase complex enzyme capable of degrading multiple antibiotics according to claim 4, characterized in that, The final concentration of the sodium alginate solution is 1.5%, and the ratio of sodium alginate to β-lactamase complex enzyme is 5:

1.

6. The method for immobilizing a β-lactamase complex enzyme capable of degrading multiple antibiotics according to claim 4, characterized in that, The final concentration of the CaCl2 solution is 2.5%.

7. The method for immobilizing a β-lactamase complex enzyme capable of degrading multiple antibiotics according to claim 4, characterized in that, The fixed time is 10 minutes.

8. The method for immobilizing a β-lactamase complex enzyme capable of degrading multiple antibiotics according to claim 4, characterized in that, The immobilized enzyme is reused at least four times.

9. The application of a β-lactamase complex enzyme capable of degrading multiple antibiotics as described in any one of claims 1-3, characterized in that, The β-lactamase complex enzyme was treated by immobilization to obtain an immobilized enzyme, which was used in the treatment of wastewater containing β-lactam antibiotics.

10. The application of the β-lactamase complex enzyme capable of degrading multiple antibiotics according to claim 9, characterized in that, The immobilized enzyme is used to degrade β-lactam antibiotics in wastewater to reduce environmental pollution and prevent the spread of antibiotic resistance.

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