CN112574972B - Bacillus belgii AiiA-homologous lactonase, gene and application thereof - Google Patents

Abstract

The invention discloses a Bacillus beiLeisi AiiA-homologous lactonase, a gene and an application thereof. The invention firstly discloses a Bacillus belgii AiiA-homologous lactonase, the amino acid sequence of which is shown in SEQ ID NO. 1. The invention further discloses application of the Bacillus belgii AiiA-homologous lactonase in treatment of domestic water. The Bacillus belgii AiiA-homologous lactonase can obviously inhibit the early proliferation, biofilm formation and virulence factor release of pseudomonas aeruginosa, and can effectively prevent and treat microbial pollution of domestic water.

Description

A kind of Velez bacillus AiiA-homologous lactonase and its gene and application

technical field

The invention relates to the field of genetic engineering, in particular to a Bacillus velei AiiA-homologous lactonase and its gene and application.

Background technique

With the widespread use of water dispensers and bottled drinking water, bacterial biofilms in water dispensers and tap water pipes cause bacterial contamination, directly affect the quality of drinking water, and pose a threat to public health, often causing secondary bacterial infections. Pseudomonas aeruginosa is a typical opportunistic pathogen that forms biofilms in water bodies and is one of the most important key factors monitored in water purification systems. At present, in addition to thorough disinfection of instruments and water supply, the common method to prevent and control Pseudomonas aeruginosa on biofilms is to use chemical drugs such as preservatives or antibiotics. Excessive and irregular use may affect food quality and cause drug resistance problems . Therefore, using bioactive, non-polluting, non-resistance, and non-toxic enzymes as substitutes to block bacterial biofilm formation has become a feasible strategy to solve this problem.

Quorum Sensing (hereinafter referred to as QS) is a phenomenon that regulates population behavior by sensing the concentration of its own inducers, including sporulation, biofilm and virulence expression. QS is regulated by the threshold properties of signal initiation, the relative specificity of signal receptors, and the cascade nature of regulatory processes. Pseudomonas aeruginosa has a typical hierarchical QS system, in which the LasI-LasR system senses the self-inducer homoserine lactone (hereinafter referred to as AHL), and regulates the downstream RhlI-RhlR and PqsABCDE-PqsR by affecting the transcription and translation of related genes path. Therefore, interfering with AHLs levels will lead to quorum-quenching (hereinafter referred to as QQ), and affect QS responses such as biofilm formation and virulence expression of Pseudomonas aeruginosa. So far, the research on QQ enzymes has mainly focused on enzymes that degrade AHL signal molecules, including AHL lactonase, AHL acyltransferase and AHL oxidoreductase, among which AHL degrading enzymes are mainly isolated from Bacillus owned. QQ enzymes come from different bacterial species, and AHL lactonases come from different Bacillus. However, due to the sensitivity of QQ enzyme to temperature and pH, the complex environment in MBR may inactivate the enzyme, and the application of QQ enzyme is limited.

Therefore, providing a QQ enzyme that can effectively degrade AHL, significantly inhibit the early proliferation, biofilm formation and virulence factor release of Pseudomonas aeruginosa, and prevent and control microbial pollution of domestic water has important application value.

Contents of the invention

The first object of the present invention is to provide a Bacillus Velez AiiA-homologous lactonase.

The second object of the present invention is to provide a Bacillus Velez AiiA-homologous lactonase gene.

The third object of the present invention is to provide a recombinant expression vector, cell line, engineering bacterium or host bacterium of the Bacillus velaisi AiiA-homologous lactonase gene.

The fourth object of the present invention is to provide a recombinant strain of Bacillus velaisi AiiA-homologous lactonase gene.

The fifth object of the present invention is to provide a method for preparing Bacillus velaisi AiiA-homologous lactonase.

The sixth object of the present invention is to provide the application of the above-mentioned Bacillus Velez AiiA-homologous lactonase in domestic water treatment.

In order to achieve the above object, the present invention adopts following technical scheme:

The present invention provides a kind of Bacillus Velez AiiA-homologous lactonase, named after AiiA _DH82 , derived from Bacillus Velez DH82 bacterial strain, said Bacillus Velez AiiA-homologous lactonase is produced by A protein composed of the amino acid sequence shown in the sequence listing SEQ ID NO.1.

The enzyme is composed of 250 amino acids, about 28.10kDa, the isopotential point is at pH5.34, the optimum temperature is 18°C, and the optimum pH value is 7. The enzyme has no transmembrane signal peptide, belongs to cytoplasmic enzyme, and is a kind of hydrophilic protein. Contains two zinc ion binding domains (LactonaseB, Metallo-beta-Lactonase superfamily), with lactamase activity.

The present invention also provides the gene encoding the above-mentioned lactonase. The present invention clones the gene encoding the above-mentioned lactonase by PCR method, the full length is 753bp, and its nucleotide sequence is shown in SEQ ID NO.2.

The present invention also provides a recombinant vector comprising the above-mentioned lactonase gene, preferably a pET28a vector. The lactonase gene of the present invention is inserted between suitable restriction sites of the expression vector, so that its nucleotide sequence is operably linked with the expression control sequence. As a most preferred embodiment of the present invention, it is preferred that the protease gene is inserted between the NdeI and XhoI restriction enzyme sites on the plasmid pET28a carrier, so that the nucleotide sequence is located downstream of the T7 promoter and is protected by it. Regulated, the recombinant plasmid pET28a-AiiA _DH82 was obtained.

The present invention also provides a recombinant bacterium comprising the above-mentioned lactonase gene, preferably Escherichia coli BL21-AiiA _DH82 .

The present invention also provides a method for preparing the above-mentioned lactonase, comprising the following steps:

S1. Transform engineering bacteria with the above-mentioned recombinant expression vector to obtain recombinant strains;

S2. Cultivate the recombinant strain and induce the expression of the recombinant protease; and

S3. Recovering and purifying the expressed Bacillus velei AiiA-homologous lactonase.

The present invention also provides the application of the above-mentioned lactonase in domestic water treatment.

Compared with the prior art, the present invention has the following outstanding advantages:

The present invention clones the AiiA-homologous lactonase gene from the Bacillus velei DH82 strain, and the AiiA-homologous lactonase gene encoded by it has the following advantages: AiiA _DH82 can effectively degrade AHL and significantly inhibit Pseudomonas aeruginosa The early proliferation, biofilm formation and release of virulence factors can prevent and control microbial pollution of domestic water. Using this bioactive, non-polluting, non-resistant, non-toxic enzyme as a substitute in the microfiltration process is a more reasonable strategy for domestic water treatment considering food safety and quality control.

Description of drawings

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

Figure 1 is the optimal temperature of Bacillus velei AiiA-homologous lactonase

Figure 2 is the optimal pH value of Bacillus velei AiiA-homologous lactonase

Fig. 3 is the bioinformatics analysis and protein purification of Bacillus velaisi AiiA-homologous lactonase. Among them: Panel A: predicted 3D structure of AiiA _DH82 .

Panel B: SDS-PAGE analysis of AiiA _DH82 enzyme preparation.

The first line: protein labeling;

The second road: extraction of non-loaded pET28a vector;

The third way: crude AiiA _DH82 enzyme extraction;

The fourth track: extraction of purified AiiA _DH82 enzyme.

Target protein bands in the gel are marked with red boxes.

Figure 4 shows the activity of Bacillus veleisi AiiA-homologous lactonase on the degradation of AHLS.

Wherein: the degradation activity of AiiA _DH82 is determined by the relative fluorescence intensity of the reporter operon (LuxR-P _luxI-lacO -RFP). AHLs (yellow C6-HSL and cyan 3-O-C12-HSL) treated with AiiA _DH82 were added to the experimental group, compared with AIIA3DHB as a positive control, the control check of untreated AHLs (CK. Results of statistical analysis Significant differences marked with * (significant difference of p-value<0.01 is marked as ***, significant difference of 0.01<p-value of 0.05 is marked as **).

Figure 5 shows the bacterial blocking effect of Bacillus velei AiiA-homologous lactonase on Pseudomonas aeruginosa.

1.5mg/mL AiiA _DH82 was inoculated in the bacterial culture solution for treatment, and the bacterial density, growth curve, biofilm accumulation, pyocyanin and rhamnolipid were measured by microplate at 600nm, 580nm, 520nm and 620nm, respectively. amount released.

(A) Growth curve of Pseudomonas aeruginosa (red is enzyme-treated bacteria culture, black is negative control);

(B) Biofilm formed by Pseudomonas aeruginosa (light yellow is enzyme-treated biofilm, yellow is untreated biofilm, orange is biofilm added with AHLs);

(C) Released anthocyanins and (D) released rhamnolipids (enzyme-treated light cyan bacterial culture, untreated cyan bacterial culture, navy supplemented with AHLs bacterial culture).

Error bars are used to determine standard deviation. Statistical analysis results are marked with significant difference (indicated by *) (significant difference of p<0.01 is marked as ***, significant difference of 0.01<p<0.05 is marked as **).

Fig. 6 shows the antifouling performance of Bacillus velaisi AiiA-homologous lactonase on Pseudomonas aeruginosa.

1.5mg/mL AiiA _DH82 was mixed with bacterial culture solution and pumped continuously through 0.22μm PVDF filter membrane for 3 days.

Panel A: Pictures of filter membranes.

Panel B: Biofilm and permeability after treatment.

Permeability was determined by the flow rate (g/min) of sterile water through the treated PVDF membrane per minute. Biofilm biomass was measured by UV absorbance at 600 nm. Error bars represent standard deviation.

Panel C: SEM images of biomass accumulation after PVDF membrane filtration for 3 days.

(a) New PVDF membrane under 1000X magnification;

(b) AiiA _DH82- treated membrane at 1 kilofold magnification;

(c) Untreated membrane under 1000X magnification;

(d) The new PVDF membrane at 15,000 times magnification;

(e) AiiA _DH82- treated membrane at 15,000X magnification;

(f) Untreated membrane at 15,000X magnification.

Detailed ways

Test materials and reagents

1. Strains and vectors

Bacillus Velez DH82 strain (GenBank: MK203035) was isolated from seawater samples at a depth of 6,000 meters in the Yap Trench in the Western Pacific Ocean, provided by the Third Institute of Oceanography, Xiamen, China.

Pseudomonas aeruginosa PAO1 was provided by Xiamen University (Xiamen).

Escherichia coli DH5α and BL21(DE3) viable cells were purchased from Transgen (Beijing, China).

2. Enzymes and other reagents

Active cells with isopropyl-β-D-thiogalactoside (IPTG) and kanamycin were purchased from Transgen (Beijing, China)

Restriction enzymes and ligases were purchased from Dalian Gaochuan Biotechnology Co., Ltd.

Plasmid Extraction Mini Kit (Cat.GMK5999) and Gel Extraction Kit (Cat.D2500-02) were purchased from Promega.

N-(β-ketohexyl)-DL homoserine lactone (C6-(L)-HSL, Cat.K3255) and (3-oxodecanoyl)-L-homoserine lactone (3-oxo-C12 -(L)-HSL, Cat.09139) was purchased from Sigma Aldrich, USA.

N-acyl homoserine lactone hydrolase (PDB: 3DHB) of Bacillus thuringiensis (GenBank: AY943832) was provided by Xiamen University (Xiamen).

The AHL reporter operon of LuxR-P _luxI-lacO -RFP was provided by Xiamen University (Xiamen).

All strains were cultured in Luria Bertani (LB) medium.

The present invention will be further described below in conjunction with the embodiments. It should be understood that these examples are only for the purpose of illustration, and do not limit the protection scope of the present invention.

Explanation: For the molecular biology experimental methods not specifically described in the following examples, all refer to the specific methods listed in the book "Molecular Cloning Experiment Guide" (Third Edition) J. Sambrook, or follow the kit and product manual.

The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

Cloning of Example 1 Bacillus Veles AiiA-Homologous Lactonase Gene

(1) Extraction of Bacillus Velez genome DNA

The culture of Bacillus Velez was incubated at 37°C, shaken at 180rpm for 12 hours, and centrifuged at 8000rpm for 10min to obtain bacterial pellets. 100ng of genomic DNA of DH82 strain was extracted from the particles by CTAB method.

(2) Cloning of Bacillus Velez AiiA-homologous lactonase gene

The sequence of aiiA _DH82 was amplified from genomic DNA by PCR method

PCR primers were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

The primer sequences are:

Forward primer aiiA-F: 5′-ATGACAGTAAAGAAAGCTTTATTTCGTCC-3′;

Reverse primer aiiA-R: 5'-TTATATATATTCGAACACTTTTACATCCCC-3'.

According to the Takara PrimeSTAR kit operating instructions, the Bacillus beilesi DH82 genomic DNA (100ng) was used as a template.

PCR amplification program: pre-denaturation at 98°C for 10s; denaturation at 98°C for 10s, annealing at 58°C for 5s, extension at 72°C for 5s, and 32 cycles; final extension at 72°C for 10s. The PCR product was detected by 1% agarose gel electrophoresis, and a positive product with a purified fragment length of about 750 bp was recovered.

3) Sequence determination

After the PCR product was purified, it was sent to Xiamen Platinum Biotechnology Co., Ltd. for sequence determination. The sequence is as follows (as shown in SEQID NO: 2):

ATGACAGTAAAGAAAGCTTTATTTCATCCCAGCAGGTCGTTGTATGTTAGATCAATCTTCTGTTAATAGTACACTCACAGCGGGGAATTTATTGAACTTACCTGTATGGTGTTATCTTTTGGAGACAGAAGAGGGGCCTATTTTAGTAGATACAGGTATGCCAGAAAGTGCGGTTCATAATGAAAATTTATTTGAAGGGACATTTGCAGAGGGACAGATTTTGCCGAAAGTGA GTAGGATATAAGCCGGAAGACCTTCTATATATTATTAGTTCTCACTTGCATTTTGATCATGCAGGAGGAAATGGTGCTTTTTCGAATACGCCAATCATTATACAGCGTGCTGAATATGAGGCGGCACAATAGGGAGGAATATTTGAAAGAGTGTATACTGCCGAATTTAAACTATAAAATTATTGAAGGTGATTATGAAGTGGTACCTGGATACCCATTGA GACGGAAAAAATCTGGTCTTGTATTATTAACGATTGATGCATCTTATACGAAAGAGAATTTTGAAGGTGAAGTGCCGTTTGCGGGGTTTGATTCGGAATTAGCCTTATCTTCAATTAAACGTTTAAAAAGAAGTTGTGATGAAAGAGAAACCGATTGTTTTCTTTGGACATGATATAGAACAGGAAAAGGGATGTAAAGTGTTCCCTGAATATATAA

Example 2 Construction of Bacillus Velez AiiA-Homologous Lactonase Engineering Bacteria

The PCR products were digested with restriction endonucleases NdeI and XhoI, respectively, and then ligated between multiple cloning sites on the pET28a vector with Takara ligation kit (T4 ligase). Expression cloning is driven by T7 promoter respectively, and the His tag coding sequence on the plasmid encodes histidine to the N-terminus of the target protein. Positive clones were amplified in Escherichia coli DH5α and transferred to Escherichia coli BL21 for protein expression.

The preparation of embodiment 3 Veles bacillus AiiA-homologous lactonase

After 3 hours, 0.4mM IPTG was inoculated into the bacterial culture to induce the expression of AiiA _DH82 . After 20 hours of cultivation, the bacterial population particles were harvested, resuspended with lysis buffer [300mM NaCl, 50mM NaH ₂ PO4 (pH 7.4)], and then used Wash with imidazole elution buffer [300 mM NaCl, 200 mM imidazole, 50 mM NaH ₂ PO4 (pH 7.4)]. The target protein was purified by high-affinity NI-NTA chromatography. The purified protein was further analyzed by SDS-PAGE.

Example 4 Partial Property Analysis of Bacillus Velez AiiA-Homologous Lactonase

(1) Bioinformatics analysis and expression of AiiA _DH82

The obtained sequences were analyzed by NCBI-BLAST software, and the maximum likelihood tree was calculated by Poisson correction method by MEGA7.0 software. The three-dimensional structure of the enzyme was simulated using the Swiss model (https://swissmodel.expasy.org/). Using TMHMM2.0 (https://services.healthtech.dtu.dk/service.php?Tmhmm-2.0), the analysis included Bioinformatics information including Smart Domain (http://smart.embl-heidelberg.de/).

In the enzyme activity measurement system with a pH value of 7.5, the temperature was 18°C, 25°C, 37°C, 45°C, 55°C, and 65°C for 45 minutes, and an appropriate amount of enzyme solution was added, with AHL as the substrate. The temperature of the reaction was plotted against the enzyme activity (Figure 1), and the optimum temperature for the enzyme-catalyzed reaction was found to be 18°C.

In the activity measurement system at 28°C, the pH values of the phosphate buffer were changed to 2, 4, 6, 7, and 8 respectively, and the influence of pH on the enzyme activity catalyzed by protease was measured, and the degradation ability of the enzyme activity was plotted (Figure 2 ). It can be seen from the figure that the optimum pH value of the enzyme-catalyzed reaction is 7.

AiiA _DH82 consists of 250 amino acids, its size is about 28.10kDa, and its isopotential point is at pH5.34. Among amino acid residues, the contents of glutamic acid (Glu), glycine (Gly) and leucine (Leu) are relatively high, exceeding 8% of the total composition. The average value of hydrophilicity (GRAVY) was -0.209, indicating that AIIA _DH82 is a hydrophilic protein. The prediction results also showed that there was no transmembrane signal peptide on AiiA _DH82 , indicating that AIIA _DH82 was a cytoplasmic enzyme. Like other known AHL-Lactonases, AiiA _DH82 contains two zinc-binding domains (LactonaseB, Metallo-beta-Lactonase superfamily) with lactamase activity. Three-dimensional modeling of the enzyme structure was simulated, as shown in Figure 1. Induced by 0.1 mMIPT G and incubated at 18°C for 20 h, the target protein of engineered AiiA _DH82 was extracted from bacterial extracts by Ni2 affinity chromatography, as shown in Figure 3B panel. The concentration is 15mg/mL. The amino acid sequence of AiiA _DH82 is as follows (as shown in SEQ ID NO: 1):

MTVKKLYFIPAGRCMLDQSSVNSTLTAGNLLNLPVWCYLLETEEGPILVDTGMPESAVHNENLFEGTFAEGQILPKMTEEDRIVTILKRVGYKPEDLLYIISSHLHFDHAGGNGAFSNTPIIIQRAEYASAAQYREEYLKECILPNLNYKIIEGDYEVVPGVQLLYTPGHSPGHSPALVFETIDGFYKSGL SSIKRLKEVVMKEKPIVFFGHDIEQEKGCKVFPEYI

Example 5 Evaluation of the effect of Bacillus Velez AiiA-homologous lactonase

(1) In vitro evaluation of AHLs degradation ability

The reporter gene operon, LuxR-P _luxI-lacO -RFP, was used to assess the level of AHLs in the free state in vitro. As shown in Figure 4, two typical QS signals C6-HSL or C12-HSL of Pseudomonas aeruginosa were used as substrates, and AiiA of Bacillus thuringiensis was used as a positive control to evaluate the degradation ability of engineered AiiA _DH82 . Control untreated AHLs served as negative controls, labeled CK1 and CK2. According to the comparison results, the engineered AiiA _DH82 had similar activity to the positive control in the degradation rate of C6-HSL (P=0.00065) and C12-HSL (P=0.021), which was significantly different from the CKs group, indicating that AiiA _DH82 affected AHLs by Levels affect the QQ ability of Pseudomonas aeruginosa.

(2) The effect on the interruption of Pseudomonas aeruginosa bacteria

As shown in Figure 5A, the growth curve of Pseudomonas aeruginosa PAO1 was not significantly different with or without the addition of AiiA _DH82 , but during the first 2 h of the lag phase, the observed slope of the bacterial growth curve was lower than that without B. In the case of the Bacillus AiiA-homologous lactonase, it was shown that the Bacillus Velez AiiA-homologous lactonase inhibited the increase in bacterial biomass at low cell densities, and when the cell density reached the logarithmic phase, Velez Bacillus AiiA-homolactonase has no effect on bacteria.

As shown in Figure 5B, the presence of AiiA _DH82 significantly inhibited P. aeruginosa PAO1 biofilm formation (P=0.0013), while exogenous C6-HSL and C12-HSL had no significant difference in the increase of bacterial biofilm biomass , which indicated that AiiA _DH82 blocked P. aeruginosa PAO1 biofilm formation by degrading AHLs in bacterial culture, verifying that exogenous AHLs did not affect the biomass increase once endogenous AHLs were produced at a threshold level.

After adding AiiA _DH82 , the expressions of virulence factors of pyocyanin PAO1 and rhamnolipid were both significantly down-regulated, as shown in Figure 5C and Figure 5D, with P values of 0.0000095 and 0.015, respectively. The results showed that the QQ of AiiA _DH82 mediated the level of AHLs, inhibited the release of Pseudomonas aeruginosa and rhamnolipids, and reduced the pathogenicity and potential danger of Pseudomonas aeruginosa.

(3) Test of AiiA _DH82 on water dispenser filter

Pseudomonas aeruginosa PAO1 was treated with AiiA _DH82 to measure bacterial biofilm formation and membrane permeability. As shown in Figure 6, AiiA _DH82 treatment significantly inhibited Pseudomonas aeruginosa PAO1 contamination on PVDF membranes _, and the experimental group still maintained high membrane permeability after treatment with free AiiA _DH82 solution, while the membranes without The antifouling ability of AiiA _DH82 was also demonstrated by the crystal violet staining and membrane flux assay results of panel A and the SEM image of the fouling layer of panel C after 3 days had been blocked by the biofilm formed by Pseudomonas aeruginosa PAO1. The results showed that AiiA _DH82 treatment could be used as an effective strategy for antifouling of drinking fountain water purifiers.

In summary, in this study, the AiiA homologous lactonase was cloned from a potential probiotic strain Bacillus beileici DH82 isolated at a depth of 6,000 m in the Yap Trench in the western Pacific Ocean, and its heterologous Expression, the ability of its QQ to degrade AHL, the bacterial interruption of water pollution and the antifouling of filter membrane were investigated. AiiA _DH82 homologous lactonase was observed to inhibit the early proliferation, biofilm formation and virulence factor release of Pseudomonas aeruginosa by degrading AHLs-mediated QS Contamination of water dispenser filters. The results show that AiiA _DH82 homologous lactonase can be used as an effective way to prevent and control microbial pollution of domestic water and reduce the infection of opportunistic pathogen Pseudomonas aeruginosa.

Although the specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments we have described are only illustrative, rather than used to limit the scope of the present invention. Equivalent modifications and changes made by skilled personnel in accordance with the spirit of the present invention shall fall within the protection scope of the claims of the present invention.

Sequence Listing

<110> Huaqiao University

  Fujian Huisheng Biotechnology Co., Ltd.

<120> A Bacillus Velez AiiA-Homologous Lactonase and Its Gene and Application

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 250

<212> PRT

<213> Bacillus velezensis

<400> 1

Met Thr Val Lys Lys Leu Tyr Phe Ile Pro Ala Gly Arg Cys Met Leu

1 5 10 15

Asp Gln Ser Ser Val Asn Ser Thr Leu Thr Ala Gly Asn Leu Leu Asn

20 25 30

Leu Pro Val Trp Cys Tyr Leu Leu Glu Thr Glu Glu Gly Pro Ile Leu

35 40 45

Val Asp Thr Gly Met Pro Glu Ser Ala Val His Asn Glu Asn Leu Phe

50 55 60

Glu Gly Thr Phe Ala Glu Gly Gln Ile Leu Pro Lys Met Thr Glu Glu

65 70 75 80

Asp Arg Ile Val Thr Ile Leu Lys Arg Val Gly Tyr Lys Pro Glu Asp

85 90 95

Leu Leu Tyr Ile Ile Ser Ser His Leu His Phe Asp His Ala Gly Gly

100 105 110

Asn Gly Ala Phe Ser Asn Thr Pro Ile Ile Ile Gln Arg Ala Glu Tyr

115 120 125

Glu Ala Ala Gln Tyr Arg Glu Glu Tyr Leu Lys Glu Cys Ile Leu Pro

130 135 140

Asn Leu Asn Tyr Lys Ile Ile Glu Gly Asp Tyr Glu Val Val Pro Gly

145 150 155 160

Val Gln Leu Leu Tyr Thr Pro Gly His Ser Pro Gly His Gln Ser Leu

165 170 175

Leu Ile Glu Thr Glu Lys Ser Gly Leu Val Leu Leu Thr Ile Asp Ala

180 185 190

Ser Tyr Thr Lys Glu Asn Phe Glu Gly Glu Val Pro Phe Ala Gly Phe

195 200 205

Asp Ser Glu Leu Ala Leu Ser Ser Ile Lys Arg Leu Lys Glu Val Val

210 215 220

Met Lys Glu Lys Pro Ile Val Phe Phe Gly His Asp Ile Glu Gln Glu

225 230 235 240

Lys Gly Cys Lys Val Phe Pro Glu Tyr Ile

245 250

<210> 2

<211> 753

<212> DNA

<213> Bacillus velezensis

<400> 2

atgacagtaa agaagcttta tttcatccca gcaggtcgtt gtatgttaga tcaatcttct 60

gttaatagta cactcacagc ggggaattta ttgaacttac ctgtatggtg ttatcttttg 120

gagacagaag aggggcctat tttagtagat acaggtatgc cagaaagtgc ggttcataat 180

gaaaatttt ttgaagggac atttgcagag ggacagattt tgccgaaaat gactgaagaa 240

gatagaatcg taactatttt aaaacgtgta ggatataagc cggaagacct tctatatatt 300

attagttctc acttgcattt tgatcatgca ggaggaaatg gtgctttttc gaatacgcca 360

atcattatac agcgtgctga atatgaggcg gcacaatata gggaggaata tttgaaagag 420

tgtatactgc cgaatttaaa ctataaaatt attgaaggtg attatgaagt ggtacctggt 480

gttcagttat tgtatacacc aggacattcc ccagggcatc agtcattact aattgagacg 540

gaaaaatctg gtcttgtatt attaacgatt gatgcatctt atacgaaaga gaattttgaa 600

ggtgaagtgc cgtttgcggg gtttgattcg gaattagcct tatcttcaat taaacgttta 660

aaagaagttg tgatgaaaga gaaaccgatt gttttctttg gacatgatat agaacaggaa 720

aagggatgta aagtgttccc tgaatatata taa 753

Claims (1)

1. The application of the Bacillus belgii AiiA-homologous lactonase in preventing and treating microbial pollution of living water is characterized in that the amino acid sequence of the Bacillus belgii AiiA-homologous lactonase is shown in SEQ ID No. 1.

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