CN112574972A - Bacillus belgii AiiA-homologous lactonase, gene and application thereof - Google Patents

Abstract

The invention discloses a Bacillus velesi AiiA-homologous lactonase and its gene and application. The present invention firstly discloses Bacillus velesi AiiA-homologous lactonase, the amino acid sequence of which is shown in SEQ ID NO.1. The invention further discloses the application of Bacillus velesi AiiA-homologous lactonase in domestic water treatment. The Bacillus velesi AiiA-homologous lactonase of the invention can significantly inhibit the early proliferation, biofilm formation and release of virulence factors of Pseudomonas aeruginosa, and can effectively prevent and control the microbial pollution of domestic water.

Description

Bacillus belgii AiiA-homologous lactonase, gene and application thereof

Technical Field

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

Background

With the widespread use of water dispensers and barreled drinking water, bacterial biofilms in water dispensers and tap water pipelines cause bacterial contamination, directly affect the quality of drinking water, pose a threat to public health, and often cause secondary bacterial infections. Pseudomonas aeruginosa is a typical pathogenic bacterium, forms a biofilm in a water body, and is one of the most important monitored key factors in a purified water system. At present, except for thoroughly disinfecting instruments and water supply, the common method for preventing and treating pseudomonas aeruginosa on a biological membrane is to use chemical drugs such as antiseptic or antibiotic, and the like, so that the excessive and irregular use can influence the food quality and cause the drug resistance problem. Therefore, blocking the formation of bacterial biofilms using biologically active, non-polluting, non-resistant, non-toxic enzymes as an alternative is a viable strategy to solve this problem.

Quorum Sensing (QS) is a phenomenon that regulates population behavior by Sensing autoinducer concentration, including sporulation, biofilm, and virulence expression. QS is regulated by the threshold nature of signal initiation, the relative specificity of signal receptors, and the nature of the cascade of regulatory processes. P.aeruginosa has a typical class QS system, in which the LasI-LasR system senses the Autoinducer Homoserine Lactone (AHL) and regulates the downstream RhlI-RhlR and PqsABCDE-PqsR pathways by affecting the transcription and translation of the relevant genes. Thus, interference with AHLs levels results in quenching (QQ) of the population and affects QS responses such as biofilm formation and virulence expression in P.aeruginosa. To date, research on QQ enzymes has focused primarily on enzymes that degrade AHL signal molecules, including three classes of AHL lactonases, AHL acyltransferases, and AHL oxidoreductases, where AHL degrading enzymes are primarily isolated from bacillus. The QQ enzymes are from different bacterial species, with AHL lactonase from different bacilli. However, the use of QQ enzymes is limited due to their sensitivity to temperature and pH, the complex environment in MBRs may inactivate the enzymes.

Therefore, the QQ enzyme which can effectively degrade AHL, remarkably inhibit early proliferation, biofilm formation and virulence factor release of pseudomonas aeruginosa and prevent and treat microbial contamination of domestic water has important application value.

Disclosure of Invention

The first purpose of the invention is to provide a Bacillus beijerinckii AiiA-homologous lactonase.

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

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

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

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

The sixth purpose of the invention is to provide the application of the Bacillus belgii AiiA-homologous lactonase in the treatment of domestic water.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a Bacillus belgii AiiA-homologous lactonase named AiiA_DH82Derived from Bacillus belgii DH82 strain, wherein the Bacillus belgii AiiA-homologous lactonase is a protein consisting of an amino acid sequence shown in a sequence table SEQ ID NO. 1.

The enzyme consists of 250 amino acids, has a kDa of about 28.10, an equipotential point at pH5.34, an optimum temperature of 18 ℃ and an optimum pH of 7. The enzyme does not have a transmembrane signal peptide, and belongs to a cytoplasmic enzyme, a hydrophilic protein. Contains two zinc ion binding domains (lactosseb, Metallo-beta-Lactonase superfamily) and has lactamase activity.

The present invention also provides a gene encoding the above lactonase. The invention clones the gene for coding the lactonase by a PCR method, the total length is 753bp, and the nucleotide sequence is shown as SEQ ID NO. 2.

The invention also provides a recombinant vector containing the lactonase gene, preferably a pET28a vector. The lactonase gene of the present invention is inserted between suitable restriction sites of an expression vector so that its nucleotide sequence is operably linked to an expression control sequence. As a most preferred embodiment of the present invention, it is preferable that the protease gene is inserted between NdeI and XhoI restriction sites in the plasmid pET28a vector so that the nucleotide sequence is located downstream of and under the control of the T7 promoter to obtain the recombinant plasmid pET28a-AiiA_DH82。

The invention also provides a recombinant bacterium containing the lactonase gene, preferably Escherichia coli BL21-AiiA_DH82。

The invention also provides a method for preparing the lactonase, which comprises the following steps:

s1, transforming engineering bacteria by using the recombinant expression vector to obtain a recombinant strain;

s2, culturing the recombinant strain, and inducing the expression of the recombinant protease; and

s3, recovering and purifying the expressed AiiA-homologous lactonase of the Bacillus belgii.

The invention also provides the application of the lactonase in domestic water treatment.

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

the AiiA-homologous lactonase gene is cloned from a Bacillus belgii DH82 strain, and the encoded AiiA-homologous lactonase has the following advantages: AiiA_DH82Can effectively degrade AHL, obviously inhibit the early proliferation, biofilm formation and virulence factor release of pseudomonas aeruginosa, and can prevent and treat the microbial pollution of the domestic water. In the microfiltration process, the enzyme with biological activity, no pollution, non-resistance and no toxicity is used as a substitute, and is a more reasonable strategy for treating domestic water in consideration of food safety and quality control.

Drawings

The invention is further described with reference to the following figures and specific examples.

FIG. 1 shows the optimum temperature of the AiiA-homologous lactonase from Bacillus belgii

FIG. 2 shows the optimum pH of the Bacillus belgii AiiA-homologous lactonase

FIG. 3 shows the bioinformatics analysis and protein purification of Bacillus belgii AiiA-homologous lactonase. Wherein: group A: AiiA_DH82Predicted 3D structure of (1).

Group B: AiiA_DH82SDS-PAGE analysis of enzyme preparations.

The first way is that: protein labeling;

and a second step: extracting a non-load pET28a vector;

and a third step: coarse AiiA_DH82Enzyme extraction;

and a fourth step: purified AiiA_DH82And (4) enzyme extraction.

The bands of target protein in the gel are marked with red boxes.

FIG. 4 shows the activity of Bacillus belgii AiiA-homologous lactonase on AHLS degradation.

Wherein: by reporting the operon (LuxR-P)_luxI-lacO-RFP) relative fluorescence intensity determination AiiA_DH82The degradation activity of (3). Experimental groups were added with AiiA separately_DH82Control examination of untreated AHLs (CK. statistical analysis results marked as a marked by a significant difference in p-value) compared to AIIA3DHB as a positive control for treated AHLs (yellow C6-HSL and cyan 3-O-C12-HSL) (CK. statistical analysis results<0.01 is labeled as<The significant difference in p-value was marked as 0.05 x).

FIG. 5 shows the bacterial blockade of Pseudomonas aeruginosa by the Bacillus belgii AiiA-homologous lactonase.

1.5mg/mL of AiiA_DH82Inoculating into bacteria culture solution, treating, and measuring bacterial density, growth curve, biofilm accumulation, and release amount of pyocyanin and rhamnolipid at 600nm, 580nm, 520nm and 620nm with microplate.

(A) A pseudomonas aeruginosa growth curve (red is cultured by enzyme-treated bacteria, and black is negative control);

(B) a biological membrane formed by pseudomonas aeruginosa (light yellow is an enzyme treatment biological membrane, yellow is an untreated biological membrane, orange is an AHLs added biological membrane);

(C) released anthocyanins and (D) released rhamnolipids (enzyme treated light cyan bacteria culture, untreated cyan bacteria culture, navy supplemented AHLs bacteria culture).

Error bars are used to determine the standard deviation. Statistical analysis results are indicated as significant differences (indicated by @) (significant differences with p <0.01 are labeled as ×, significant differences with 0.01< p <0.05 are labeled as ×).

FIG. 6 shows the antifouling properties of Bacillus belgii AiiA-homologous lactonase against Pseudomonas aeruginosa.

1.5mg/mL AiiA_DH82Mixing with the bacterial culture solution, and continuously pumping 0.22 μm PVDF filter membrane for 3 days.

Group A: filter membrane pictures.

Group B: biofilm and permeability after treatment.

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

Group C: scanning electron microscopy images of biomass accumulation after 3 days of PVDF membrane filtration.

(a) A new PVDF membrane at 1 thousand fold magnification;

(b) AiiA at 1 Kfold magnification_DH82A treated membrane;

(c) untreated membrane at 1 thousand fold magnification;

(d) a new PVDF membrane amplified by 1.5 ten thousand times;

(e) AiiA at 1.5 ten thousand times magnification_DH82A treated membrane;

(f) untreated membrane at 1.5 ten thousand times magnification.

Detailed Description

Test materials and reagents

1. Bacterial strain and carrier

Bacillus beleisi strain DH82 (GenBank: MK203035) was isolated from a 6000 m deep seawater sample from Yapu Seawa, Western Pacific, and was provided by the third Marine institute of China Xiamen.

Pseudomonas aeruginosa PAO1 was supplied by xiamen university (xiamen).

Coli DH5 alpha and BL21(DE3) active cells were purchased from Transgen (Beijing, China).

2. Enzymes and other reagents

Active cells for isopropyl-. beta. -D-thiogalactoside (IPTG) and kanamycin were purchased from Transgen (Beijing, China)

Restriction enzymes and ligases were purchased from Dagaochuan biotechnology, Inc.

Plasmid extraction mini kit (Cat. GMK5999) and gel extraction kit (Cat. D2500-02) were purchased from Promega.

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

N-acylhomoserine lactonohydrolase (PDB:3DHB) of Bacillus thuringiensis (GenBank: AY943832) was supplied by Xiamen university (Xiamen).

LuxR-P_luxI-lacOAHL reporting operon for RFP is provided by building gate university (building gate).

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

The present invention is further described below in conjunction with embodiments, it being understood that these examples are for illustrative purposes only and do not limit the scope of the present invention.

Description of the drawings: the molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions.

The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

EXAMPLE 1 cloning of the AiiA-homologous lactonase Gene of Bacillus belgii

(1) Extraction of Bacillus belgii genomic DNA

The Bacillus belgii culture was incubated at 37 ℃ with shaking at 180rpm for 12 hours and centrifuged at 8000rpm for 10min to give bacterial particles. 100ng of genomic DNA of DH82 strain was extracted from the pellet by CTAB method.

(2) Cloning of the Bacillus belgii AiiA-homologous lactonase Gene

Amplification of aiiA from genomic DNA by PCR_DH82Of (2) a

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

The primer sequence is as follows:

forward primer aiiA-F: 5'-ATGACAGTAAAGAAGCTTTATTTCGTCC-3', respectively;

reverse primer aiiA-R: 5'-TTATATATATTCGAACACTTTACATCCCC-3' are provided.

The Bacillus belgii DH82 genomic DNA (100ng) was used as template according to the Takara PrimeSTAR kit protocol.

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

3) Sequence determination

After the PCR product was purified, the product was sent to Xiamen platinum Biotech Co., Ltd for sequence determination, and the sequence was as follows (shown in SEQ ID NO: 2):

ATGACAGTAAAGAAGCTTTATTTCATCCCAGCAGGTCGTTGTATGTTAGATCAA TCTTCTGTTAATAGTACACTCACAGCGGGGAATTTATTGAACTTACCTGTATGGTGTT ATCTTTTGGAGACAGAAGAGGGGCCTATTTTAGTAGATACAGGTATGCCAGAAAGT GCGGTTCATAATGAAAATTTATTTGAAGGGACATTTGCAGAGGGACAGATTTTGCC GAAAATGACTGAAGAAGATAGAATCGTAACTATTTTAAAACGTGTAGGATATAAGC CGGAAGACCTTCTATATATTATTAGTTCTCACTTGCATTTTGATCATGCAGGAGGAAA TGGTGCTTTTTCGAATACGCCAATCATTATACAGCGTGCTGAATATGAGGCGGCACA ATATAGGGAGGAATATTTGAAAGAGTGTATACTGCCGAATTTAAACTATAAAATTATT GAAGGTGATTATGAAGTGGTACCTGGTGTTCAGTTATTGTATACACCAGGACATTCC CCAGGGCATCAGTCATTACTAATTGAGACGGAAAAATCTGGTCTTGTATTATTAACG ATTGATGCATCTTATACGAAAGAGAATTTTGAAGGTGAAGTGCCGTTTGCGGGGTTT GATTCGGAATTAGCCTTATCTTCAATTAAACGTTTAAAAGAAGTTGTGATGAAAGAG AAACCGATTGTTTTCTTTGGACATGATATAGAACAGGAAAAGGGATGTAAAGTGTT CCCTGAATATATATAA

example 2 construction of Bacillus beilesiensis AiiA-homologous lactonase engineering bacteria

The PCR product was digested with restriction enzymes NdeI and XhoI, respectively, and then ligated between multiple cloning sites on pET28a vector using Takara ligation kit (T4 ligase). Expression clones were driven by the T7 promoter, respectively, and the His tag coding sequence on the plasmid encoded histidine to the N-terminus of the target protein. The positive clone was amplified in E.coli DH5 alpha and transferred to E.coli BL21 for protein expression.

EXAMPLE 3 preparation of the Bacillus belgii AiiA-homologous lactonase

AiiA was induced by inoculating 0.4mM IPTG into bacterial cultures after 3 hours_DH82The bacterial colony particles were harvested after 20 hours of culture and dissolved in a dissolution buffer [300mM NaCl, 50mM NaH ]₂PO4(pH 7.4)]Resuspended and then eluted with imidazole elution buffer [300mM NaCl, 200mM imidazole, 50mM NaH ]₂PO4(pH 7.4)]And (6) washing. And (3) purifying the target protein by adopting high-affinity NI-NTA chromatography. The purified protein was further analyzed by SDS-PAGE.

Example 4 analysis of the partial Properties of AiiA-homologous lactonase from Bacillus beilis

(1)AiiA_DH82Bioinformatics analysis and expression of

The resulting sequences were analyzed using NCBI-BLAST software and the maximum likelihood tree was calculated using Poisson's correction using MEGA7.0 software. The three-dimensional structure of the enzyme was modeled using the Swiss model (https:// swisssmall. expasy. org.). bioinformatics information including the smart domain (http:// smart. embl-heidelberg. de /) was analyzed using TMHMM2.0(https:// services. healthech.dtu.dk/service. phpThmm-2.0).

Keeping the temperature at 18 deg.C, 25 deg.C, 37 deg.C, 45 deg.C, 55 deg.C and 65 deg.C for 45min in enzyme activity measuring system with pH of 7.5, adding appropriate amount of enzyme solution, and taking AHL as substrate. The temperature of the reaction was plotted against the enzyme activity (FIG. 1), and the optimum temperature for the enzyme-catalyzed reaction was found to be 18 ℃.

In a system for measuring activity at 28 ℃, the pH values of phosphate buffer solutions are changed to be 2, 4, 6, 7 and 8 respectively, the influence of the pH on the enzyme activity catalyzed by protease is measured, and the enzyme activity degradation capability is plotted (figure 2). As is clear from the figure, the optimum pH for the reaction catalyzed by the enzyme was 7.

AiiA_DH82Consists of 250 amino acids, has a size of about 28.10kDa and has an equipotential point at pH 5.34. Among amino acid residues, glutamic acid (Glu), glycine (Gly), and leucine (Leu) are contained in high amounts, exceeding 8% of the total composition. The average value of hydrophilicity (GRAVY) was-0.209, indicating AIIA_DH82Is a hydrophilic protein. The prediction results also show AiiA_DH82Has no transmembrane signal peptide, indicating AIIA_DH82Belongs to cytoplasmic enzymes. Like other known AHL-lactonases, AiiA_DH82Contains two zinc binding domains (lactosseb, Metallo-beta-Lactonase superfamily) and has lactamase activity. The three-dimensional modeling of the enzyme structure was simulated as shown in FIG. 1. Incubating at 18 deg.C for 20h under induction of 0.1mMIPT G, and extracting engineered AiiA from bacterial extract by Ni2 affinity chromatography_DH82As shown in FIG. 3B. The concentration was 15 mg/mL. AiiA_DH82The amino acid sequence is as follows (as shown in SEQ ID NO: 1):

MTVKKLYFIPAGRCMLDQSSVNSTLTAGNLLNLPVWCYLLETEEGPILVDTGMPE SAVHNENLFEGTFAEGQILPKMTEEDRIVTILKRVGYKPEDLLYIISSHLHFDHAGGNG AFSNTPIIIQRAEYEAAQYREEYLKECILPNLNYKIIEGDYEVVPGVQLLYTPGHSPGHQ SLLIETEKSGLVLLTIDASYTKENFEGEVPFAGFDSELALSSIKRLKEVVMKEKPIVFFG HDIEQEKGCKVFPEYI

example 5 evaluation of the Effect of Bacillus belgii AiiA-homologous lactonase

(1) In vitro evaluation of AHLs degradation Capacity

Reporter gene operon, LuxR-P_luxI-lacORFP for in vitro assessment of levels of AHLs in free state. As shown in FIG. 4, the engineered AiiA was evaluated using two typical QS signals C6-HSL or C12-HSL of Pseudomonas aeruginosa as substrates and AiiA of Bacillus thuringiensis as positive controls_DH82The degradation ability of (a). The untreated AHL of the control group served as negative controls and were labeled CK1 and CK 2. Engineering AiiA based on the comparison_DH82The degradation rate of C6-HSL (P ═ 0.00065) and C12-HSL (P ═ 0.021) has similar activity to that of positive control, and the degradation rate is similar to that of CKs groupSignificant difference, i.e. AiiA_DH82The QQ capacity of Pseudomonas aeruginosa is affected by affecting AHLs levels.

(2) Effect on disruption of Pseudomonas aeruginosa bacteria

As shown in FIG. 5A, the growth curve of P.aeruginosa PAO1 with or without AiiA_DH82There was no significant difference in the time, but in the first 2 hours of the lag phase, the slope of the observed bacterial growth curve was lower than in the absence of the bacillus beilis AiiA-homologous lactonase, indicating that the bacillus beilis AiiA-homologous lactonase inhibited the increase in bacterial biomass at low cell densities, and that the bacillus beilis AiiA-homologous lactonase had no effect on the bacteria after the cell density reached the log phase.

As shown in fig. 5B, AiiA_DH82The presence of (A) significantly inhibited the biofilm formation of Pseudomonas aeruginosa PAO1 (P0.0013), while exogenous C6-HSL and C12-HSL did not have a significant difference in the increase of bacterial biofilm biomass, indicating that AiiA_DH82The formation of the biofilm of pseudomonas aeruginosa PAO1 is blocked by degrading AHLs in bacterial culture, and the fact that the increase of biomass is not influenced once endogenous AHLs are generated at a threshold level by exogenous AHLs is verified.

Adding AiiA_DH82Thereafter, both pseudomonas aeruginosa PAO1 and rhamnolipid were significantly down-regulated in virulence factor expression, as shown in fig. 5C and 5D, with P values of 0.0000095 and 0.015, respectively. The results show that AiiA_DH82The QQ mediates AHLs level, inhibits the release of pseudomonas aeruginosa and rhamnolipid, and reduces the pathogenicity and potential danger of pseudomonas aeruginosa.

(3) AiiA on filter of drinking machine_DH82Test (2)

By AiiA_DH82Pseudomonas aeruginosa PAO1 was treated and the formation of bacterial biofilms and membrane permeability were determined. As shown in fig. 6, AiiA_DH82The treatment obviously inhibits the pollution of pseudomonas aeruginosa PAO1 on a PVDF membrane, and free AiiA is used in an experimental group_DH82The membrane permeability after the solution treatment is still high, and the membrane does not contain AiiA_DH82The membrane of (a) was blocked by a biofilm formed by pseudomonas aeruginosa PAO1 after 3 days, crystal violet staining of a plate and membrane flux measurements andSEM images of the fouling layer of the C plate also demonstrate AiiA_DH82The antifouling ability of (1). The results show that AiiA_DH82The treatment can be used as an effective anti-fouling strategy for the water purifier of the water dispenser.

In conclusion, the research clones AiiA homologous lactonase from a potential probiotic Bacillus beleister DH82 strain separated from 6000 m deep in Seattle sea ditch, performs heterologous expression on the AiiA homologous lactonase, and examines the capability of QQ in degrading AHL, and the water-polluted bacteria interruption and the antifouling of a filter membrane. AiiA was observed by degrading QS of AHLs mediated pathogenic bacteria_DH82The homologous lactonase has obvious inhibition effect on early proliferation, biofilm formation and virulence factor release of the pseudomonas aeruginosa, so that the pollution of the pseudomonas aeruginosa on a filter of a water dispenser is inhibited. The results show that AiiA_DH82The homologous lactonase can be used as an effective way for preventing and treating the microbial pollution of domestic water and reducing the infection of opportunistic pathogen pseudomonas aeruginosa.

Although specific embodiments of the invention have been described above, it will be understood by those skilled in the art that the specific embodiments described are illustrative only and are not limiting upon the scope of the invention, and that equivalent modifications and variations can be made by those skilled in the art without departing from the spirit of the invention, which is to be limited only by the appended claims.

Sequence listing

<110> university of Chinese

Fujian Huishi Biotech Co Ltd

<120> Bacillus belgii AiiA-homologous lactonase, gene and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 250

<212> PRT

<213> Bacillus belgii (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 belgii (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

gaaaatttat 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 (9)

1. A Bacillus belgii AiiA-homologous lactonase, characterized in that the amino acid sequence thereof is shown in SEQ ID NO. 1.

2. A Bacillus belgii AiiA-homologous lactonase gene encoding the Bacillus belgii AiiA-homologous lactonase according to claim 1.

3. The Bacillus belgii AiiA-homologous lactonase gene according to claim 2, wherein the nucleotide sequence thereof is represented by SEQ ID No. 2.

4. A recombinant expression vector, cell line, engineered bacterium or host bacterium comprising the bacillus belgii AiiA-homologous lactonase gene of claim 2.

5. A recombinant expression vector pET28a-DH82 comprising the Bacillus belgii AiiA-homologous lactonase gene according to claim 2, wherein the Bacillus belgii AiiA-homologous lactonase gene having the nucleotide sequence shown in SEQ ID No.2 is inserted between restriction enzyme sites on the plasmid pET28a so that the nucleotide sequence is located downstream of and under the control of the T7 promoter, to thereby obtain a recombinant expression vector pET28a-DH 82.

6. A recombinant strain comprising the bacillus belgii AiiA-homologous lactonase gene of claim 2.

7. A recombinant strain Escherichia coli BL21-DH82 comprising the Bacillus bleekii AiiA-homologous lactonase gene according to claim 2, wherein the recombinant expression vector pET28a-DH82 according to claim 5 is transformed into Escherichia coli BL21 to obtain a recombinant strain Escherichia coli BL21-DH 82.

8. A process for preparing the bacillus beijerinckii AiiA-homologous lactonase of claim 1, comprising the steps of:

s1, transforming engineering bacteria by using the recombinant expression vector of claim 4 to obtain a recombinant strain;

s2, culturing the recombinant strain, and inducing the expression of the recombinant protease; and

s3, recovering and purifying the expressed AiiA-homologous lactonase of the Bacillus belgii.

9. Use of a bacillus beijerinckii AiiA-homologous lactonase according to claim 1 in the treatment of domestic water.

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