N-acyl homoserine lactone degrading enzyme as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological enzymes, and discloses an N-acyl homoserine lactone degrading enzyme as well as a preparation method and application thereof. Wherein the amino acid sequence of the N-acyl homoserine lactone degrading enzyme is as shown in SEQ ID NO. 1. The N-acyl homoserine lactone degrading enzyme disclosed by the invention is derived from a co-epiphytic bacterium Pseudomonas onchidi XY-99 of a marine invertebrate organism, i.e., calf stone, has wide signal molecule degradation activity, can block a quorum sensing pathway of pathogenic bacteria and inhibit the generation of virulence factors and the formation of a biofilm, and can be used for preparing a product for preventing and controlling pseudomonas aeruginosa.

Description

Technical Field

[0001] This invention relates to the field of bioenzyme technology, and particularly to a... N -Acylhomoserine lactone degrading enzyme, its preparation method and application. Background Technology

[0002] Quorum sensing (QS) is a communication mechanism widely found in bacteria that regulates the expression of specific genes based on population density, including processes such as swarming, biofilm formation, and virulence factor expression. N N-acyl homoserine lactones (AHLs) are the most common bacterial QS signaling molecules. Their chemical structure consists of two parts: a homoserine lactone ring (HSL) and acyl side chains of varying lengths, such as those found in the common foodborne pathogen *Pseudomonas aeruginosa*. Pseudomonas aeruginosa ) and Erwinia carrot soft rot, which causes soft rot diseases in carrots, potatoes, etc. Pectobacterium carotovorum subsp. carotovorum All of them use AHLs as signaling molecules.

[0003] Quorum quenching (QQ) is a mechanism evolved in nature that disrupts the signaling molecules produced by bacteria, thereby blocking their quorum sensing effects. Compared to traditional treatments for bacterial infections, such as antibiotics, QQ aims to inhibit the production of pathogen virulence factors, thus reducing their pathogenicity. This method exerts less selective pressure on pathogens, avoiding problems such as bacterial resistance caused by antibiotic overuse, and is conducive to green and sustainable development. Quorum quenching can be divided into two types: QQ enzymes that degrade signaling molecules and QQ inhibitors that are structural analogs of signaling molecules. Quorum inhibitors specifically bind to the synthases or receptor proteins of QS signaling molecules, hindering the synthesis or recognition of signaling molecules, thereby blocking the regulation of the QS pathway. The other type utilizes QQ enzymes to continuously degrade AHL signaling molecules, reducing their concentration below a threshold to interfere with bacterial quorum sensing. QQ enzymes can destroy the structure of AHL-like signaling molecules, rendering them biologically inactive, and are currently one of the main means of disrupting microbial quorum sensing. Common QQ enzymes are mainly divided into AHL acyltransferases and AHL lactoneases. Acyltransferases mainly act on the amide bonds of the side chains, while lactoneases mainly act on the lactone ring structure. However, the currently disclosed types of QQ enzymes and their substrate selection range are still relatively limited, which is not conducive to their application in the prevention and control of pathogenic bacteria (such as PAO1) to alleviate the problem of antibiotic resistance caused by the use of antibiotics.

[0004] Therefore, existing technologies still need improvement and development. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the object of the present invention is to provide a N This study aims to provide a novel marine-derived α-acylhomoserine lactone degrading enzyme, its preparation method, and its applications, thereby inhibiting the production of PAO1 virulence factor and biofilm formation in Pseudomonas aeruginosa. N -Acylhomoserine lactone degrading enzymes can play a role in the prevention and control of Pseudomonas aeruginosa, thereby alleviating the problem of drug resistance in pathogens caused by the use of existing antibiotics.

[0006] The technical solution of the present invention is as follows: Firstly, providing a N -Acylhomoserine lactone degrading enzyme, the N The amino acid sequence of the α-acylhomoserine lactone degrading enzyme is shown in SEQ ID NO.1.

[0007] Secondly, a nucleic acid is provided, said nucleic acid encoding as described in the first aspect. N -Acylhomoserine lactone degrading enzyme.

[0008] In a preferred embodiment, the nucleotide sequence of the nucleic acid is shown in SEQ ID NO.2.

[0009] Thirdly, a recombinant vector is provided, the recombinant vector expressing the expression described in the first aspect. N -Acylhomoserine lactone degrading enzyme.

[0010] Fourthly, a method as described in the first aspect is provided. N The preparation method of α-acylhomoserine lactone degrading enzyme includes the following steps: The encoding will be as described in the first aspect. N The gene for α-acylhomoserine lactone degrading enzyme was inserted into an expression vector to obtain a recombinant vector; The recombinant vector was transferred into host cells to obtain a recombinant bacterial strain; The recombinant strain was cultured and its protein expressed. The protein expression product was then purified to obtain... N -Acylhomoserine lactone degrading enzyme.

[0011] In a preferred embodiment, the expression vector is pET28a or pUCP18.

[0012] In a preferred embodiment, the host cell is Escherichia coli or Pseudomonas aeruginosa.

[0013] In a preferred embodiment, the recombinant vector further includes a promoter PrpsJ.

[0014] Fifthly, a method as described in the first aspect is provided. NApplication of α-acylhomoserine lactone degrading enzyme in the preparation of products for the prevention and control of Pseudomonas aeruginosa.

[0015] The preferred technical solution is that the product is obtained through the... N -Acylhomoserine lactone degrading enzymes inhibit the swarming movement of Pseudomonas aeruginosa.

[0016] The preferred technical solution is that the product is obtained through the... N -Acylhomoserine lactone degrading enzymes inhibit the production of virulence factors in Pseudomonas aeruginosa.

[0017] The preferred technical solution is that the product is obtained through the... N -Acylhomoserine lactone degrading enzymes inhibit biofilm formation in Pseudomonas aeruginosa.

[0018] Beneficial effects: Compared with the prior art, the advantages of the present invention are as follows: (1) The present invention provides N The α-acylhomoserine lactone degrading enzyme (PoAiiA) is derived from marine bacteria, providing an important research foundation for the subsequent discovery of quorum sensing quenching enzymes in marine microorganisms.

[0019] (2) The present invention provides N -Acylhomoserine lactone degrading enzyme (PoAiiA) has broad substrate selectivity and can degrade a variety of signaling molecules, both long and short, providing greater flexibility in practical applications.

[0020] (3) The present invention provides N α-Acylhomoserine lactone degrading enzyme (PoAiiA) can significantly inhibit the production of virulence factors and biofilm formation in Pseudomonas aeruginosa, laying a theoretical foundation for the prevention and control of Pseudomonas aeruginosa. Attached Figure Description

[0021] Figure 1 This is an SDS-PAGE electrophoresis image of PoAiiA expressed in vitro in Example 1 of this invention.

[0022] Figure 2 This is a graph showing the degradation range of PoAiiA in Example 2 of the present invention.

[0023] Figure 3 This is the mass spectrum of the PoAiiA degradation compound in Example 2 of the present invention.

[0024] Figure 4 This is a schematic diagram of the shuttle heterologous expression vector in Embodiment 3 of the present invention.

[0025] Figure 5 This is a diagram showing the results of PoAiiA inhibiting the cluster movement of Pseudomonas aeruginosa in Example 3 of the present invention.

[0026] Figure 6 This is a diagram showing the results of PoAiiA inhibiting the production of pyophytin by Pseudomonas aeruginosa in Example 3 of the present invention.

[0027] Figure 7 This is an electron microscope scan of the biofilm destruction of the heterologous expression strain in Example 3 of the present invention. Detailed Implementation

[0028] This invention provides N The invention relates to acylhomoserine lactone degrading enzymes, their preparation methods, and applications. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below.

[0029] The constantly changing marine environment, including factors such as salinity, temperature, osmotic pressure, and oligotrophic conditions, has enabled marine microorganisms to develop unique metabolic pathways to adapt to this environment. Over the past few decades, researchers have unearthed a rich variety of bioactive substances from marine bacteria. Given that current research on quorum sensing quenching primarily focuses on non-marine environments, and considering that the proportion of QQ-active bacteria in marine and other aquatic environments is far higher than that in the rhizosphere soil bacteria of agricultural crops, marine bacteria possess abundant QQ enzyme resources waiting to be explored. As a green and highly effective antibacterial agent, it has broad development prospects.

[0030] Based on this, the embodiments of the present invention provide a N -Acylhomoserine lactone degrading enzyme, the N The amino acid sequence of the α-acylhomoserine lactone degrading enzyme is shown in SEQ ID NO.1.

[0031] Specifically, the inventors previously isolated marine bacteria from the gills of the manatee, a marine invertebrate, in the waters of Dapeng Nature Reserve in Shenzhen, Guangdong Province. Pseudooceanicola onchidii Preliminary studies on the cell activity of XY-99 have shown that it has the ability to degrade C6-HSL signaling molecules. Further research revealed that XY-99 contains... N α-Acylhomoserine lactone degrading enzyme (SEQ ID NO.1) can significantly inhibit the production of virulence factors in Pseudomonas aeruginosa, inhibit biofilm formation, and has a wide degradation range for signaling molecules and broad substrate recognition, which has obvious advantages in practical applications.

[0032] Based on the same inventive concept, embodiments of the present invention provide a nucleic acid, wherein the nucleic acid encodes as described above. N -Acylhomoserine lactone degrading enzyme.

[0033] In some embodiments, the nucleotide sequence of the nucleic acid is shown in SEQ ID NO.2.

[0034] Based on the same inventive concept, embodiments of the present invention provide a recombinant vector, wherein the recombinant vector expresses the expression described above. N -Acylhomoserine lactone degrading enzyme.

[0035] Based on the same inventive concept, embodiments of the present invention provide a method as described above. N The preparation method of α-acylhomoserine lactone degrading enzyme includes the following steps: Encode as described above N The gene for α-acylhomoserine lactone degrading enzyme was inserted into an expression vector to obtain a recombinant vector; The recombinant vector was transferred into host cells to obtain a recombinant bacterial strain; The recombinant strain was cultured and its protein expressed. The protein expression product was then purified to obtain... N -Acylhomoserine lactone degrading enzyme.

[0036] In some implementations, the encoding is as described above. N Methods for developing genes for α-acylhomoserine lactone degrading enzymes include: using marine bacteria Pseudooceanicola onchidii Using the XY-99 genome as a template, the gene encoding the above-mentioned genome was amplified. N Genes of α-acylhomoserine lactone degrading enzymes.

[0037] In some implementations, the encoding will be as described above. N The step of inserting the gene for α-acylhomoserine lactone degrading enzyme into the expression vector is carried out by enzyme digestion recombination or homologous recombination.

[0038] In some embodiments, the expression vector is a shuttle heterologous expression vector.

[0039] In some embodiments, the expression vector is pET28a or pUCP18.

[0040] In some embodiments, the host cell is Escherichia coli or Pseudomonas aeruginosa.

[0041] In some more specific embodiments, the Escherichia coli is BL21(DE3) or DH5α.

[0042] In some more specific embodiments, the Pseudomonas aeruginosa is PAO1.

[0043] In some embodiments, the recombinant vector further includes a promoter PrpsJ.

[0044] In some more specific embodiments, the nucleotide sequence of the promoter PrpsJ is shown in SEQ ID NO.9.

[0045] Based on the same inventive concept, embodiments of the present invention provide a method as described above. N Application of α-acylhomoserine lactone degrading enzyme in the preparation of products for the prevention and control of Pseudomonas aeruginosa.

[0046] In some embodiments, the product is delivered via the N -Acylhomoserine lactone degrading enzymes inhibit the swarming movement of Pseudomonas aeruginosa.

[0047] In some embodiments, the product is delivered via the N -Acylhomoserine lactone degrading enzymes inhibit the production of virulence factors in Pseudomonas aeruginosa.

[0048] In some more specific embodiments, the virulence factor is pyocyanin.

[0049] In some embodiments, the product is delivered via the N -Acylhomoserine lactone degrading enzymes inhibit biofilm formation in Pseudomonas aeruginosa.

[0050] The present invention will be further described below through specific embodiments.

[0051] Example 1: N Cloning of α-acylhomoserine lactone degrading enzyme PoAiiA 1. Genome analysis Marine bacteria Pseudooceanicola onchidii XY-99 was isolated on March 17, 2017, from the gills of the marine invertebrate *Schizothorax stink bug* in the waters of Dapeng Nature Reserve, Shenzhen, Guangdong Province (114.61°E, 22.55°N) (International Journal of Systematic and Evolutionary Microbiology, 70(2), 1224–1230. DOI:10.1099 / ijsem.0.003905.). The inventors previously conducted preliminary studies on its bacterial activity, indicating that XY-99 bacteria have the ability to degrade C6-HSL signaling molecules (Frontiers in Microbiology, 15, 1353711. DOI:10.3389 / FMICB.2024.1353711.). Through whole-genome sequence annotation, combined with local amino acid BlastP search and comparison with reported lactonease sequences, XY-99 was screened for... NAn acylhomoserine lactone degrading enzyme, named PoAiiA, has the amino acid sequence shown in SEQ ID NO.1 and the nucleotide sequence shown in SEQ ID NO.2. Domain analysis indicates that PoAiiA belongs to the α / β hydrolase family and has the highest similarity (26% identity) to the previously reported lactone enzyme QlcA.

[0052] 2. Extraction of genomic DNA Marine bacteria XY-99 were inoculated into SG liquid medium and cultured overnight at 30°C on a shaker. After centrifugation to remove the supernatant, the bacterial cells were collected. The cells were washed with an appropriate amount of STE buffer, and the supernatant was removed by repeated centrifugation. A 2 mg / mL lysozyme solution prepared with STE buffer was added to fully suspend the cells, and the suspension was incubated in a 37°C water bath for 30 min until it became translucent. An appropriate amount of 6% (w / v) SDS solution was added, and the suspension was gently inverted to mix. The suspension was then incubated in a 37°C water bath until the liquid became clear. An appropriate amount of 3 M sodium acetate solution (pH=4.8) was added, and the suspension was briefly inverted to mix. Then, an appropriate amount of phenol:chloroform:isoamyl alcohol (25:24:1; v / v / v) solution was added, and the mixture was continuously mixed for 5 min before centrifugation at 13000 rpm for 10 minutes. Min to separate the layers; take the supernatant, add an equal volume of anhydrous ethanol, and mix until white flocculent DNA precipitates; pick out the flocculent precipitate, wash 1-2 times with 70% (v / v) alcohol, centrifuge to remove most of the liquid; after air drying at room temperature, dissolve the genomic DNA in an appropriate amount of TE solution, add an appropriate amount of RNase solution to remove RNA impurities, and set aside for later use.

[0053] The SG liquid culture medium consists of: 5 g soluble starch, 5 g glucose, 1 g yeast extract, 1 g tryptone, 1 g peptone, 17 g artificial sea salt, dissolved in tap water to a final volume of 1 L, and pH adjusted to 7.2–7.4.

[0054] 3. Construction of protein expression vectors and protein purification Design primer pairs: P1: 5' -GGAATTCC ATATG ACCAACCCCGCGACATCCCC- 3' (SEQ ID NO. 3); P2: 5' -CCG CTCGAG GGCCGCAGGGGCTTTCGGCC-3' (SEQ ID NO. 4).

[0055] The underlined part indicates the restriction site of XbaI (TCTAGA) and XhoI (CTCGAG).

[0056] PCR reaction system: 5 μL each of primer pairs P1 and P2 (50 pmol / L), 5 μL of total DNA template, 50 μL of PrimeSTAR Max Premix (2×), and ddH2O to bring the total to 100 μL.

[0057] PCR conditions: PoAiiA gene amplification conditions: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 68℃ annealing for 30 s, 72℃ extension for 20 s, 28 cycles; 72℃ final extension for 5 min.

[0058] The amplified DNA fragment and vector were then digested using NdeI and XhoI restriction endonucleases. The PoAiiA gene was ligated into the vector pET28a using T4 ligase to construct the protein expression plasmid pET28a-PoAiiA. The constructed plasmid was introduced into *E. coli* BL21(DE3) competent cells and inducing protein expression by incubation at 16°C with shaking at 180 rpm for 12–16 h using isopropyl thiogalactoside (IPTG, final concentration 0.2 mM). Cells were then collected by centrifugation at 4°C and 6000 rpm, resuspended in protein buffer, and sonicated for 30–45 min at low temperature (30% amplitude, 5 s disruption time, 5 s interval, 30–45 min). After disruption, the cells were centrifuged at 4°C and 10000 rpm for 30 min, and the supernatant (crude enzyme solution) was collected. The crude enzyme solution was loaded into a pre-equilibrated Ni-NTA column at a flow rate of 0.5–1.0 mL / min. After loading, non-specifically bound impurities were eluted sequentially with imidazole buffers of different concentrations (5 mmol / L, 20 mmol / L, 50 mmol / L), followed by elution of the target protein with 250 mmol / L imidazole buffer. The obtained target protein fraction was concentrated and replaced by ultrafiltration to obtain a lactonease extract with high purity and concentration.

[0059] The molecular weight and purity of the target protein were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the results are as follows: Figure 1 As shown, the concentration of the enzyme extract was determined using the BIOFORD method. The obtained enzyme extract was stored at –80°C with 50% glycerol and DTT to a final molar concentration of 0.01 mmol / L.

[0060] Protein buffer: Tris-HCl 50 mmol / L, NaCl 500 mmol / L, pH=7.8; Replacement buffer: Tris-HCl 25 mmol / L, NaCl 20 mmol / L, pH=7.8; Store at 4℃.

[0061] Example 2: In vitro enzyme activity detection of PoAiiA protein 1. Reaction system In vitro lactonease activity reaction system (50 μL): 10 μM of signal molecules AHLs were added to 100 mmol / L PIPES buffer (pH 7.0), followed by 2 μM PoAiiA. The reaction system was incubated in a 30℃ water bath for 12 h. After the reaction, the mixture was centrifuged at 13000 rpm for 20 min, and the supernatant was used for subsequent colorimetric detection. For mass spectrometry detection, an equal volume of ethyl acetate was added for extraction three times after the reaction. The organic phase extracts were combined, evaporated to dryness under reduced pressure, and dissolved in an appropriate amount of methanol for later use.

[0062] The signal molecules included: C6-HSL, C8-HSL, C10-HSL, C12-HSL, C14-HSL, 3-oxo-C6-HSL, 3-oxo-C8-HSL, 3-oxo-C10-HSL, 3-oxo-C12-HSL, and 3-oxo-C14-HSL; the 3-oxo-C8-HSL signal molecule was used as a representative for mass spectrometry detection.

[0063] 2. Detection Method Prepare the chromogenic assay strains by overnight culturing *Vibrio CV026* and *Vibrio VIR24* on LB liquid medium, and then adjusting the bacterial culture to OD using LB liquid medium. 600 The value is approximately 0.2-0.3. Using an EP tube with a small hole in the cap as the culture container, take 40 μL of the supernatant after the reaction, add 600 μL of *Pseudomonas violaceus* bacterial culture, and incubate with shaking at 750 rpm for 16-18 h. Then, remove the supernatant by centrifugation, retain the bacterial precipitate, add an appropriate amount of DMSO to extract the purple pigment, centrifuge again at high speed, and measure the OD of the supernatant. 550 The absorbance, after converting the degradation rate, is as follows: Figure 2 As shown.

[0064] The samples used for mass spectrometry detection were separated by HPLC and then introduced into the mass spectrometer to detect the molecular weights of the substrates and products. HPLC elution conditions: 0-10 min injection: 90%-70% Phase A (ddH2O + 0.1% formic acid) and 10%-30% Phase B (acetonitrile); 10-25 min linear elution: 70%-0% Phase A and 30%-100% Phase B; 25-30 min isocratic elution: 0% Phase A and 100% Phase B; column temperature: 30℃; flow rate: 1 mL / min; column specifications: Waters XSelect CSHTM C18 column (5 μm; 4.6 × 250 mm); the mass spectrometer used was an Agilent 6120 single quadrupole LC-MS system equipped with an atmospheric pressure electrospray ionization (API-ES) source. The mass spectrometry detection results of the PoAiiA degradation of the 3-oxo-C8-HSL signal molecule are as follows: Figure 3 As shown.

[0065] Example 3: Heterologous expression of the PoAiiA gene 1. Construction of shuttle recombination carrier Design primer pairs: P3: 5' - AATTGGAGTCTGAGGTCAA ATGACCAACCCCGCGACATC- 3' (SEQ ID NO. 5); P4: 5' - GACGGCCAGTGCCAAGCTT TCAGGCCGCAGGGGCTTTCG- 3' (SEQ ID NO. 6); P5: 5' - GCTCGGTACCCGGGGATCC CGTGGTTGCCAAGATCATCG- 3' (SEQ ID NO. 7); P6: 5'-TTGACCTCAGACTCCAATTT-3' (SEQ ID NO. 8).

[0066] Total DNA from marine bacteria XY-99 prepared in Example 1 was used as a template for PCR. Primer pair P3 / P4 was used to amplify the coding sequence. N The functional gene PoAiiA, an acylhomoserine lactone degrading enzyme, is amplified by primer pair P5 / P6, which is used to amplify the promoter PrpsJ (SEQ ID NO.9) encoding the ribosomal subunit S10 gene. The underlined region indicates the homologous arm region used for homologous recombination.

[0067] PCR reaction system: 5 μL (50 pmol) each of primer pairs P3 and P4 (P5 and P6), 5 μL of total DNA template, 50 μL of PrimeSTAR MaxPremix (2×), and ddH2O to bring the total to 100 μL.

[0068] PCR conditions: Promoter amplification conditions: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 60℃ annealing for 30 s, 72℃ extension for 5 s, 28 cycles; 72℃ for 5 min. PoAiiA gene amplification conditions: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 66℃ annealing for 30 s, 72℃ extension for 20 s, 28 cycles; 72℃ for 5 min.

[0069] The promoter (PrpsJ), PoAiiA gene, and shuttle heterologous expression vector (pUCP18) were ligated using homologous recombinase at 50°C for 15 min. The resulting mixture was transformed into *E. coli* DH5α competent cells. Positive clones were selected and sequenced for verification, thus constructing the recombinant heterologous expression vector. A schematic diagram of the plasmid is shown below. Figure 4 As shown, it is named pUCP18-PrpsJ-PoAiiA.

[0070] 2. Construction of heterologous expression lines Preparation of competent cells of *Pseudomonas aeruginosa*: *Pseudomonas aeruginosa* PAO1 was inoculated into LB liquid medium and cultured at 37°C with shaking at 200 rpm until mid-logarithmic growth (OD1). 600 (≈ 0.6-0.8). Centrifuge 1 mL of bacterial culture at 4℃, 10000 g for 5 min, and discard the supernatant. Resuspend the bacterial cells in pre-cooled 300 mmol / L sucrose solution and repeat the washing three times, centrifuging at 4℃, 10000 g for 5 min each time, to thoroughly remove ionic components from the culture medium. Finally, resuspend the bacterial cells in 50 μL of pre-cooled 300 mM sucrose solution to obtain a high-concentration competent cell suspension.

[0071] Electroporation method: Add 1-5 μg of the constructed recombinant heterologous expression vector pUCP18-PrpsJ-PoAiiA (volume not exceeding 5 μL) to 50 μL of competent cells, gently mix, and transfer to a pre-chilled 1 mm electroporation cuvette. Electroporate using an electroporator with the following parameters: voltage 2.5 kV, capacitance 25 μF, resistance 200 Ω (time constant approximately 4.5-5.5 ms). Immediately after electroporation, add 1 mL of LB liquid medium to the cuvette, gently pipette to mix, and transfer to a 1.5 mL sterile centrifuge tube. Incubate the transformation mixture at 37°C and 200 rpm for 1 hour with shaking. Then, spread a portion of the bacterial culture onto LB agar plates containing the appropriate antibiotic (carbenicillin 100 μg / mL) and incubate at 37°C for 16-24 hours. Transfect the resulting single clones onto LB agar plates containing carbenicillin.

[0072] Thus, the heterologous expression strain PAO1-PrpsJ-PoAiiA was obtained. Furthermore, the shuttle heterologous expression vector pUCP18 was transformed into the above competent cells using the same procedure to obtain the control strain PAO1-pUCP18.

[0073] 3. Virulence factor assay Cluster movement was detected as follows: PAO1-PrpsJ-PoAiiA and PAO1-pUCP18 were cultured in LB liquid medium until OD100. 600 The concentration was 1.0. 1 μL of bacterial suspension was spotted in the center of each semi-solid aggregation motility medium plate. The semi-solid medium consisted of 0.8% (w / v) nutrient broth, 0.5% (w / v) glucose, and 0.5% (w / v) agar (BD Biosciences, USA). After incubating the plates upright in a 30°C incubator for 24 hours, the diameter of the aggregation motility region was quantitatively analyzed using ImageJ software. The inhibitory effect of PoAiiA on the aggregation motility of *Pseudomonas aeruginosa* was as follows: Figure 5 As shown.

[0074] The yield of pyocyanin was determined as follows: PAO1-PrpsJ-PoAiiA and PAO1-pUCP18 were cultured in LB liquid medium until OD200. 600 The concentration was 1.0. After centrifugation, the supernatant of the culture was collected. 1 mL of the supernatant was mixed with 0.5 mL of chloroform, shaken thoroughly, and allowed to stand to separate into layers. The chloroform phase was collected. Subsequently, the chloroform phase was reverse-extracted with 0.3 mL of hydrochloric acid (0.2 mol / L) to obtain an acidic aqueous phase containing pyocyanin. The absorbance (OD) of this aqueous phase at 520 nm was measured. 520 The pyocyanin yield was calculated by multiplying the measured absorbance value by a conversion factor of 32.01. The results showed that heterologous expression of PoAiiA in Pseudomonas aeruginosa PAO1 significantly inhibited pyocyanin production. Figure 6 As shown.

[0075] 4. Electron scanning microscopy measurement Heterogeneous expression strains were cultured in biofilm form. Sterile flat toothpicks were cut to 2 cm in length and aseptically fixed to the inner wall of a 12-well plate, with the bottom end approximately 0.3 cm from the bottom of each well. PAO1-PrpsJ-PoAiiA and PAO1-pUCP18, cultured overnight in LB broth, were adjusted to OD using fresh LB broth. 600The concentration was 0.6, and then 3 mL was inoculated into each well of a 12-well plate. A 22 mm glass coverslip was carefully placed on each toothpick as a substrate for biofilm formation. The 12-well plate was then incubated at 37°C for 48 hours to promote biofilm formation. After incubation, the culture medium was carefully aspirated from the wells, and phosphate buffer containing 2.5% (v / v) glutaraldehyde was added for initial fixation of the biofilm. After fixation, the samples were washed three times with PBS buffer for 10 min each time to completely remove residual fixative. Subsequently, a gradient ethanol dehydration treatment was performed: 30% (v / v), 50% (v / v), 70% (v / v), 80% (v / v), 90% (v / v), and 95% (v / v) ethanol solutions were used sequentially, with each gradient dehydration treatment lasting 15-30 minutes; finally, the samples were treated twice more with anhydrous ethanol for 15-30 minutes each time to ensure complete dehydration. After dehydration, the samples underwent critical point drying, and were then fixed to an aluminum sample stage using conductive carbon tape. A 5 nm thick gold layer was sputtered onto the surface. High-resolution field emission scanning electron microscopy (SU8220, Hitachi) was used to observe and image the biofilm samples under 5 kV accelerating voltage and high vacuum conditions. The results showed that heterologous expression of PoAiiA in Pseudomonas aeruginosa PAO1 inhibited biofilm formation, resulting in a significantly thinner biofilm. Figure 7 As shown.

[0076] The above results indicate that N α-Acylhomoserine lactone degrading enzyme PoAiiA can significantly inhibit the production of virulence factors in Pseudomonas aeruginosa and inhibit biofilm formation.

[0077] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A kind N -Acylhomoserine lactone degrading enzyme, characterized in that... The N The amino acid sequence of the α-acylhomoserine lactone degrading enzyme is shown in SEQ ID NO.

1.

2. A nucleic acid, characterized in that, The nucleic acid encoding is as described in claim 1. N -Acylhomoserine lactone degrading enzyme.

3. The nucleic acid according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid is shown in SEQ ID NO.

2.

4. A recombinant vector, characterized in that, The recombinant vector expresses the expression as described in claim 1. N -Acylhomoserine lactone degrading enzyme.

5. A device as described in claim 1 N The method for preparing α-acylhomoserine lactone degrading enzyme is characterized by, Including the following steps: The encoding as described in claim 1 N The gene for α-acylhomoserine lactone degrading enzyme was inserted into an expression vector to obtain a recombinant vector; The recombinant vector was transferred into host cells to obtain a recombinant bacterial strain; The recombinant strain was cultured and its protein expressed. The protein expression product was then purified to obtain... N -Acylhomoserine lactone degrading enzyme.

6. The preparation method according to claim 5, characterized in that, The expression vector is pET28a or pUCP18; The host cell is either Escherichia coli or Pseudomonas aeruginosa; The recombinant vector also includes the promoter PrpsJ.

7. A device as described in claim 1 N Application of α-acylhomoserine lactone degrading enzyme in the preparation of products for the prevention and control of Pseudomonas aeruginosa.

8. The application according to claim 7, characterized in that, The product is obtained through the N -Acylhomoserine lactone degrading enzymes inhibit the swarming movement of Pseudomonas aeruginosa.

9. The application according to claim 7, characterized in that, The product is obtained through the N -Acylhomoserine lactone degrading enzymes inhibit the production of virulence factors in Pseudomonas aeruginosa.

10. The application according to claim 7, characterized in that, The product is obtained through the N -Acylhomoserine lactone degrading enzymes inhibit biofilm formation in Pseudomonas aeruginosa.

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