Beta-lactamase derived from soil metagenome and application thereof

Abstract

The application discloses a beta-lactamase and a gene for coding the beta-lactamase, and further discloses an expression cassette, a recombinant carrier, a recombinant cell or a recombinant bacteria, and a construction method of the recombinant bacteria, and further discloses the application of the beta-lactamase, the gene for coding the beta-lactamase, the expression cassette, the recombinant carrier, the recombinant cell or the recombinant bacteria, and the construction method of the recombinant bacteria in removing beta-lactam antibiotic pollution in the environment. The beta-lactamase gene is transformed into escherichia coli, so that the escherichia coli can hydrolyze beta-lactam antibiotics in the environment, including ampicillin, cefazolin, cefotaxime, imipenem and amikacin, and has great application value in eliminating beta-lactam antibiotic pollution in the environment.

Description

Technical Field

[0001] This invention relates to the fields of bioinformatics and biotechnology, and in particular to β-lactamases derived from soil metagenomics and their applications. Background Technology

[0002] β-lactam antibiotics primarily work by binding to transpeptidase, blocking the synthesis of mature cell walls and leading to cell autolysis and death. Since human cells lack cell walls, while both Gram-negative and Gram-positive bacteria do, β-lactam antibiotics possess advantages such as low toxicity, broad applicability, and strong antibacterial activity, making them the most widely used antibiotics in clinical practice, accounting for more than half of all antibiotic use globally. The wide range and large dosage of β-lactam drugs result in a high probability of their presence in natural aquatic environments. Numerous domestic and international studies have reported the detection of various antibiotics in natural aquatic environments, including oceans, rivers, lakes, and groundwater. The research group of Ying Guangguo at the Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, obtained the first inventory of antibiotic usage and emissions in China. The report predicted the environmental concentrations of 36 common antibiotics in various watersheds, with amoxicillin, a β-lactam antibiotic, showing the highest concentration. Sources of antibiotics in the environment mainly include domestic sewage, medical wastewater, and wastewater from animal feed and aquaculture. Currently, no effective removal methods have been found for β-lactam antibiotic pollution in the environment. β-lactamases are hydrolases that inactivate β-lactam antibiotics (such as penicillin, cephalosporins, monocyclic β-lactam antibiotics, and carbapenems) by cleaving the β-lactam ring, thus converting them into non-antibacterial substances. They have great potential for application in antibiotic degradation.

[0003] When screening functional enzymes, traditional isolation and culture methods can only search for target genes from less than 1% of culturable microorganisms, resulting in a very small candidate resource library. Emerging metagenomic technologies, however, do not rely on microbial isolation and culture, and can provide a much richer gene resource library for screening target genes. Summary of the Invention

[0004] Purpose of the invention: The first objective of this invention is to provide a β-lactamase derived from soil metagenomics.

[0005] A second object of the present invention is to provide a gene encoding the β-lactamase.

[0006] A third objective of the present invention is to provide an expression cassette, recombinant vector, recombinant cell, or recombinant bacterium containing the gene for said β-lactamase.

[0007] A fourth objective of this invention is to provide the application of the aforementioned β-lactamase, the gene encoding the β-lactamase, the expression cassette, the recombinant vector, the recombinant cell or recombinant bacteria, and the method for constructing the recombinant bacteria in removing β-lactam antibiotic contamination from the environment. Technical solution: To achieve the above objectives, this invention provides a β-lactamase with the amino acid sequence shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0008] The present invention also provides a gene encoding one of the β-lactamases, the nucleotide sequence of which is shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0009] The present invention also provides expression cassettes, recombinant vectors, recombinant cells or recombinant bacteria containing the gene for the β-lactamase described above.

[0010] The present invention also provides a method for constructing the recombinant bacteria, comprising the following steps:

[0011] (1) The β-lactamase gene described in claim 2 is ligated with an expression vector to obtain a recombinant plasmid;

[0012] (2) Transform the recombinant plasmid obtained in step (1) into the host bacteria to obtain the product.

[0013] Preferably, the expression vector is pET-28a(+).

[0014] Preferably, the host bacterium is Escherichia coli.

[0015] The present invention also provides the application of the β-lactamase, the gene of the β-lactamase, the expression cassette, the recombinant vector, the recombinant cell or recombinant bacteria, and the method for constructing the recombinant bacteria in removing β-lactam antibiotic contamination from the environment.

[0016] Preferably, the β-lactam antibiotic is one or more of ampicillin, cefazolin, cefotaxime, imipenem, and aztreonam.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: Escherichia coli with β-lactamase gene can significantly improve resistance to β-lactam antibiotics, including ampicillin, cefazolin, cefotaxime, imipenem and aztreonam, and has great application prospects in eliminating β-lactam antibiotic pollution in the environment. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the pET-28a(+) restriction site of the present invention.

[0019] Figure 2 To assess the resistance of *E. coli* strain pET-28a / NO:1-9 constructed in this invention to cefazolin.

[0020] Figure 3 To assess the resistance of the pET-28a / NO:1-9 strain of Escherichia coli constructed in this invention to ampicillin.

[0021] Figure 4 To assess the resistance of pET-28a / NO:1-9 *Escherichia coli* strains constructed in this invention to cefotaxime.

[0022] Figure 5 To assess the resistance of *Escherichia coli* strain pET-28a / NO:1-9 constructed in this invention to aztreonam.

[0023] Figure 6 To assess the resistance of pET-28a / NO:1-9 *Escherichia coli* strains constructed in this invention to imipenem. Detailed Implementation

[0024] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0025] Escherichia coli BL21(DE3), purchased from Tiangen Biotech Co., Ltd.; plasmid pET-28a(+), preserved in our laboratory, carrying kanamycin resistance, chromatogram as shown. Figure 1 As shown.

[0026] Ampicillin, cefazolin, cefotaxime, and imipenem were all purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., while aztreonam was purchased from the National Institutes for Food and Drug Control.

[0027] Example 1: Mining and Identifying β-Lactamase Encoding Genes from Metagenomic Data

[0028] Soil samples were collected in 2017 from the primeval forest of the Yuanjiang County National Nature Reserve in Yunnan Province. Soil DNA was extracted, and Illumina paired-end sequencing was used to obtain reads. The specific steps are as follows:

[0029] (1) Sequencing data processing, using FastQC

[0030] (http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ) performs quality assessment on sequencing data, and uses Trimmomatic to remove adapters and cut low-quality data to obtain high-quality sequencing results (Q>20).

[0031] (2) The β-lactamase encoding gene was mined by using Fargene software to splice high-quality sequencing data and compare it with the built-in β-lactamase hidden Markov model to obtain the β-lactamase encoding gene sequence and the corresponding amino acid sequence.

[0032] (3) Analysis of amino acid sequences: The amino acid sequence similarity analysis of the target protein was performed by using the Blast engine provided by the National Center for Biotechnology Information (NCBI) to align the predicted β-lactamase amino acid sequence with all β-lactamase amino acid sequences in the CARD (Comprehensive Antibiotic Research Database). The analysis of conserved domains of the target protein amino acid sequence was performed using the NCBI CDD (Conserved Domain Database), and the comparison analysis between different amino acid sequences was performed using the software DNAMAN.

[0033] (4) Selection of β-lactamase encoding genes: Select β-lactamase encoding genes that have a conserved β-lactamase sequence domain (pfam00144) and have less than 70% similarity to known sequences in the database.

[0034] Example 2: Preparation of engineered Escherichia coli containing a β-lactamase encoding gene

[0035] (1) Preparation of competent E. coli BL21 cells

[0036] ① Streak competent E. coli BL21 cells on LB solid medium and incubate overnight at 37°C for 15 h.

[0037] ② Pick a single colony and inoculate it into 5 mL of LB liquid medium. Incubate overnight at 37°C with shaking at 220 rpm.

[0038] ③ Add 1 mL of seed culture to 50 mL of LB liquid medium, incubate at 37°C and 220 rpm for about 2 hours, measuring OD every half hour. 600 When OD600 Stop culturing when the pH reaches 0.35–0.4.

[0039] ④ Dispense 50 mL of bacterial culture into each 50 mL pre-cooled centrifuge tube, incubate on ice for 10 min, then centrifuge at 3000 rpm for 10 min at 4℃, collect the bacterial cells, and discard the supernatant.

[0040] ⑤ Add 30 mL of pre-cooled CaCl2-MgCl2 solution to each tube of bacterial cells, resuspend the bacterial cells (operate on ice), centrifuge at 3000 rpm for 10 min to collect the bacterial cells, and discard the supernatant.

[0041] ⑥ Resuspend the bacterial cells in 2 mL of pre-chilled 0.1 M glycerol-CaCl2 solution, mix well to obtain competent E. coli BL21 cells, aliquot the competent cells into pre-chilled 1.5 mL centrifuge tubes at 100 μL / tube, and store at -80 °C.

[0042] (2) Construction of recombinant plasmids pET-28a-classA-23, pET-28a-classA-53, pET-28a-classA-58, pET-28a-classA-67, pET-28a-classB-3, pET-28a-classB-5, pET-28a-classB-27, pET-28a-classB-67, and pET-28a-classD-4

[0043] The target gene, consisting of DNA sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, was directly synthesized by Nanjing GenScript Biotech Co., Ltd. The pET-28a(+) plasmid and the target gene SEQ ID NO: 1-9 were digested with enzymes, as shown in the following enzyme digestion system:

[0044] Table 1 Enzyme digestion system

[0045]

[0046] After the system is prepared, mix well and incubate at 37°C for 12 hours to improve enzyme digestion efficiency. Inactivate the restriction endonuclease by incubating at 65°C for 15 minutes. Check the digestion results by gel electrophoresis, and cut off the strips containing the target gene / pET-28a(+) plasmid. Recover the DNA using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit (TaKaRa). After obtaining the purified target gene and the linear pET-28a(+) plasmid backbone, the target gene was ligated to the linear pET-28a(+) plasmid backbone according to the following system. After ligation at 22℃ for 30-50 min, recombinant vectors pET-28a-classA-23 (SEQ ID NO: 1), pET-28a-classA-53 (SEQ ID NO: 2), pET-28a-classA-58 (SEQ ID NO: 3), pET-28a-classA-67 (SEQ ID NO: 4), pET-28a-classB-3 (SEQ ID NO: 5), pET-28a-classB-5 (SEQ ID NO: 6), pET-28a-classB-27 (SEQ ID NO: 7), pET-28a-classB-67 (SEQ ID NO: 8), and pET-28a-classD-4 (SEQ ID NO: 9) were obtained.

[0047] Table 2 Connection System

[0048]

[0049]

[0050] (3) The recombinant plasmids pET-28a-classA-23, pET-28a-classA-53, pET-28a-classA-58, pET-28a-classA-67, pET-28a-classB-3, pET-28a-classB-5, pET-28a-classB-27, pET-28a-classB-67, and pET-28a-classD-4 were transformed into E. coli BL21.

[0051] ① Take competent cells and thaw them in ice.

[0052] ②After melting, add 20μL of ligation product to the competent cell suspension, gently rotate the centrifuge tube to mix, and let stand in an ice bath for 30min.

[0053] ③ Heat shock at 42℃ for 90 seconds, followed by an ice bath for 2 minutes.

[0054] ④ Add 900 μL of LB antibiotic-free liquid medium and incubate at 37℃ and 180 rpm for 1 h to allow the cells to recover.

[0055] ⑤ Centrifuge at 4000 rpm for 5 min at 4℃, discard the supernatant, spread the precipitate on an LB agar plate containing 50 μg / mL kanamycin, place the plate at 37℃ until the liquid is absorbed, invert the plate, and incubate overnight at 37℃.

[0056] Following the conversion steps above, set up a blank control for transferring to an unloaded pET-28a.

[0057] (4) Validation of recombinant plasmids pET-28a-classA-23, pET-28a-classA-53, pET-28a-classA-58, pET-28a-classA-67, pET-28a-classB-3, pET-28a-classB-5, pET-28a-classB-27, pET-28a-classB-67, and pET-28a-classD-4

[0058] Single clones were picked from the kanamycin LB agar plates and cultured overnight at 37°C in 4 mL of LB liquid containing 50 μg / mL kanamycin. 1 mL of the liquid was used for bacterial preservation, and the remainder was used to verify the constructed recombinant plasmid. After plasmid extraction, the plasmid was sent to Nanjing Genscript Biotech Co., Ltd. for sequencing. The sequencing results showed that the inserted fragment completely matched the target gene sequence.

[0059] Example 3: Resistance of Escherichia coli containing a β-lactamase encoding gene to β-lactam antibiotics

[0060] (1) Antibiotic preparation

[0061] Dissolve 22g of Mueller Hinton II (BD Difco, 212322) liquid medium in 1L of distilled water and autoclave at 121℃ for 10min. Dissolve 2g of IPTG in 8mL of water, and after the IPTG is completely dissolved, bring the volume to 10mL with water. Filter the solution through a 0.22μm disposable filter to sterilize, and aliquot into 1mL portions for storage at -20℃. Add IPTG solution to Mueller Hinton II (BD Difco, 212322) liquid medium until the final IPTG concentration is 0.3mmol.

[0062] Taking ampicillin as an example, dissolve 0.1184g of ampicillin in 20mL of phosphate buffer solution with pH=8. After the ampicillin is completely dissolved, filter the solution through a 0.22μm disposable filter to remove bacteria, and dispense it into 1mL portions for storage at -20℃.

[0063] (2) Preparation of 96-well plates for drug resistance testing

[0064] Taking ampicillin as an example, the specific preparation process is as follows:

[0065] ① Add 100 μL of MH II medium (containing 0.3 mmol IPTG) to each well of a 96-well plate;

[0066] ② Dilute the prepared ampicillin tenfold, add 100 μL of the diluted ampicillin solution to the first column of each row of a 96-well plate, and mix by pipetting until the final concentration in the first column of each row is 256 μg / mL.

[0067] ③ Using an eight-spin pipette, take 100 μL of culture medium with a final concentration of 256 μg / mL in the first column of each row, and continue to mix by pipetting in the second column of each row.

[0068] ④ Repeat the above steps, aspirate 100 μL from each well in column 2 and transfer it to column 3, mix well by aspiration, and so on, until the lowest concentration of 2 μg / mL is reached (column 8). At this point, there is 200 μL of culture medium in column 8. Aspirate 100 μL of the liquid and discard it to keep the volume of all wells at 100 μL.

[0069] For the preparation of 96-well plates for other antibiotics, the initial concentration and minimum concentration can be adjusted according to the actual situation. For example, it can be diluted from 32 μg / mL to 0.25 μg / mL; or from 512 μg / mL to 2 μg / mL, etc.

[0070] (3) Determination of drug resistance phenotype

[0071] ①On the first day, streak the bacteria on LB agar plates containing 50 μg / mL kanamycin and incubate overnight;

[0072] ② On the second day, pick colonies from the LB plate and transfer them to 3 mL of LB liquid medium (containing 50 μg / mL kanamycin). Shake for about 2 hours, and measure the OD every half hour. 600 When OD 600 Stop culturing when the value reaches 0.5;

[0073] ③ After diluting the bacterial suspension 1000 times, inoculate 100 μL of the adjusted bacterial suspension into each well of 100 μL of culture medium containing different concentrations of antibiotics.

[0074] ④ At the same time, a negative control was set up, and empty vector Escherichia coli that does not contain the β-lactamase encoding gene described in this invention was used as a negative control.

[0075] ⑤ Incubate the 96-well plate at a suitable temperature for 18–20 hours. Observe for bacterial growth and compare with a negative result to determine the fold increase in MIC (microbial resistance).

[0076] ⑥ The lowest antibiotic concentration at which bacteria will not grow is the MIC of the strain.

[0077] As shown in Table 3 and Figure 1-6 As shown, compared with the empty vector control pET-28a(+), the sequences encoding nine β-lactamase genes significantly improved the resistance of Escherichia coli to ampicillin, cefazolin, cefotaxime, aztreonam, and imipenem.

[0078] pET-28a-classA-23 (SEQ ID NO: 1) increased the resistance of E. coli BL21 to the penicillin antibiotic ampicillin by 16-fold (MIC = 32 μg / mL) and to the penicillin antibiotic amoxicillin by 8-fold (MIC = 16 μg / mL). For cephalosporins, resistance to the third-generation cephalosporin cefotaxime increased by 2-fold (MIC = 0.125 μg / mL), resistance to the monocyclic β-lactam antibiotic aztreonam increased by 32-fold (MIC = 0.5 μg / mL), and resistance to the carbapenem antibiotic imipenem increased by 4-fold (MIC = 0.5 μg / mL).

[0079] pET-28a-class A-53 (SEQ ID NO: 2) increased the resistance of E. coli BL21 to the penicillin antibiotic ampicillin by 16-fold (MIC = 32 μg / mL) and to the penicillin antibiotic amoxicillin by 8-fold (MIC = 16 μg / mL). It also increased resistance to the cephalosporin antibiotic cefazolin by 2-fold (MIC = 4 μg / mL), to the cephalosporin antibiotic cefotaxime by 4-fold (MIC = 0.25 μg / mL), to the monocyclic β-lactam antibiotic aztreonam by 32-fold (MIC = 0.5 μg / mL), and to the carbapenem antibiotic imipenem by 2-fold (MIC = 0.25 μg / mL).

[0080] pET-28a-class A-58 (SEQ ID NO: 3) increased the resistance of E. coli BL21 to the penicillin antibiotic ampicillin by 16-fold (MIC = 32 μg / mL) and to the penicillin antibiotic amoxicillin by 16-fold (MIC = 32 μg / mL). It also increased resistance to the cephalosporin antibiotic cefazolin by 2-fold (MIC = 4 μg / mL), to the cephalosporin antibiotic cefotaxime by 4-fold (MIC = 0.25 μg / mL), to the monocyclic β-lactam antibiotic aztreonam by 1024-fold (MIC = 16 μg / mL), and to the carbapenem antibiotic imipenem by 4-fold (MIC = 0.5 μg / mL).

[0081] pET-28a-class A-67 (SEQ ID NO: 4) increased the resistance of E. coli BL21 to the penicillin antibiotic ampicillin by 8 times (MIC = 16 μg / mL) and to the penicillin antibiotic amoxicillin by 8 times (MIC = 16 μg / mL). It also increased resistance to the cephalosporin antibiotic cefotaxime by 4 times (MIC = 0.25 μg / mL), to the monocyclic β-lactam antibiotic aztreonam by 32 times (MIC = 0.5 μg / mL), and to the carbapenem antibiotic imipenem by 2 times (MIC = 0.25 μg / mL).

[0082] pET-28a-class B-3 (SEQ ID NO: 5) increased the resistance of E. coli BL21 to the penicillin antibiotic ampicillin by 8 times (MIC = 16 μg / mL) and to the penicillin antibiotic amoxicillin by 8 times (MIC = 16 μg / mL). It also increased resistance to the cephalosporin antibiotic cefazolin by 2 times (MIC = 4 μg / mL), to the cephalosporin antibiotic cefotaxime by 4 times (MIC = 0.25 μg / mL), to the monocyclic β-lactam antibiotic aztreonam by 32 times (MIC = 0.5 μg / mL), and to the carbapenem antibiotic imipenem by 4 times (MIC = 0.5 μg / mL).

[0083] pET-28a-class B-5 (SEQ ID NO: 6) increased the resistance of E. coli BL21 to the penicillin antibiotic ampicillin by 8 times (MIC = 16 μg / mL) and to the penicillin antibiotic amoxicillin by 8 times (MIC = 16 μg / mL). It also increased resistance to the cephalosporin antibiotic cefazolin by 4 times (MIC = 8 μg / mL), to the cephalosporin antibiotic cefotaxime by 4 times (MIC = 0.25 μg / mL), to the monocyclic β-lactam antibiotic aztreonam by 32 times (MIC = 0.5 μg / mL), and to the carbapenem antibiotic imipenem by 2 times (MIC = 0.25 μg / mL).

[0084] pET-28a-class B-27 (SEQ ID NO: 7) increased E. coli BL21 resistance to ampicillin by 8-fold (MIC = 16 μg / mL) and resistance to amoxicillin by 8-fold (MIC = 16 μg / mL). Resistance to cefazolin increased by 4-fold (MIC = 8 μg / mL), resistance to cefotaxime increased by 4-fold (MIC = 0.25 μg / mL), resistance to the monocyclic β-lactam antibiotic aztreonam increased by 32-fold (MIC = 0.5 μg / mL), and resistance to imipenem increased by 4-fold (MIC = 0.5 μg / mL).

[0085] pET-28a-class B-67 (SEQ ID NO: 8) increased E. coli BL21 resistance to ampicillin by 8-fold (MIC = 16 μg / mL) and resistance to amoxicillin by 8-fold (MIC = 16 μg / mL). Resistance to cefazolin increased by 2-fold (MIC = 4 μg / mL), resistance to cefotaxime increased by 4-fold (MIC = 0.25 μg / mL), resistance to the monocyclic β-lactam antibiotic aztreonam increased by 32-fold (MIC = 0.5 μg / mL), and resistance to imipenem increased by 2-fold (MIC = 0.25 μg / mL).

[0086] pET-28a-classD-4 (SEQ ID NO: 9) increased the resistance of E. coli BL21 to the penicillin antibiotic ampicillin by 32-fold (MIC = 64 μg / mL), to the penicillin antibiotic amoxicillin by 32-fold (MIC = 64 μg / mL), to the cephalosporin antibiotic cefazolin by 4-fold (MIC = 8 μg / mL), to the cephalosporin antibiotic cefotaxime by 4-fold (MIC = 0.25 μg / mL), to the monocyclic β-lactam antibiotic aztreonam by 32-fold (MIC = 0.5 μg / mL), and to the carbapenem antibiotic imipenem by 2-fold (MIC = 0.25 μg / mL).

[0087] Table 3. Resistance to β-lactamases (unit: μg / mL)

[0088] pET-28a NO: 9 NO.8 NO.7 NO.6 NO.5 NO.4 NO.3 NO.2 NO.1 Ampicillin 2 64 16 16 16 16 16 32 32 32 Amoxicillin 2 64 16 16 16 16 16 32 16 16 cefazolin 2 8 4 8 8 4 2 4 4 2 Cefotaxime 0.063 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.125 Aztreonam 0.016 0.5 0.5 0.5 0.5 0.5 0.5 16 0.5 0.5 Imipenem 0.125 0.25 0.25 0.5 0.25 0.5 0.25 0.5 0.25 0.5

Claims

1. A beta-lactamase, characterized in that, The amino acid sequence of which is shown as SEQ ID NO:

18.

2. A gene encoding a beta-lactamase, characterized in that, The nucleotide sequence of which is shown as SEQ ID NO:

9.

3. Expression cassette, recombinant vector or recombinant bacteria, characterized in that, The gene encoding beta-lactamase according to claim 2.

4. The method of constructing a recombinant bacterium according to claim 3, wherein, The method comprises the following steps: (1) connecting the gene encoding beta-lactamase according to claim 2 with an expression vector to obtain a recombinant plasmid; (2) transforming the recombinant plasmid obtained in step (1) into a host bacterium, and obtaining the same.

5. The method of constructing a recombinant bacterium according to claim 4, wherein The expression vector is pET-28a(+).

6. The method of constructing a recombinant bacterium according to claim 4, wherein The host bacterium is Escherichia coli.

7. The use of the beta-lactamase according to claim 1, the gene encoding beta-lactamase according to claim 2, the expression cassette according to claim 3, the recombinant vector or the recombinant bacterium according to claim 3, or the beta-lactamase obtained by the method for constructing the recombinant bacterium according to any one of claims 4-6 in removing the pollution of beta-lactam antibiotics in the environment, wherein the beta-lactam antibiotics are one or more of ampicillin, cefazolin, cefotaxime, imipenem and aztreonam.

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