CN117210362A

CN117210362A - Carbendazim degrading bacteria and degrading enzyme and application thereof

Info

Publication number: CN117210362A
Application number: CN202311174985.5A
Authority: CN
Inventors: 夏菠; 庞立; 易有金; 张懿婷
Original assignee: Hunan Agricultural University
Current assignee: Hunan Agricultural University
Priority date: 2023-09-13
Filing date: 2023-09-13
Publication date: 2023-12-12

Abstract

The invention discloses a carbendazim degrading bacterium, the strain of which is named Pantoea dispersa DJL-B and is preserved in the Guangdong province microorganism strain collection center, and the preservation number is GDMCC No:62372. the invention belongs to the technical field of environment-friendly biology, and discloses a method for preparing a bacterial strain capable of rapidly degrading carbendazim, which is identified as Pantoea dispersa DJL-B by taking carbendazim as a unique carbon source and screening out a bacterial strain capable of rapidly degrading carbendazim from soil polluted by carbendazim. In addition, the invention also separates a specific protease from the thallus broken liquid or fermentation supernatant of the carbendazim degrading bacteria.

Description

Carbendazim degrading bacteria and degrading enzyme and application thereof

Technical Field

The invention belongs to the technical field of environment-friendly biology, and particularly relates to carbendazim degrading bacteria, degrading enzyme thereof and application thereof.

Background

Carbendazim (Carbenazem) is a broad-spectrum benzimidazole bactericide with molecular formula of C ₉ H ₉ N ₃ O ₂ The white fine crystal powder has good heat stability and is commonly used for preventing and treating various fungal diseases of crops such as rice, fruit trees, vegetables and the like. Carbendazim is stable in acidic solutions and can form soluble salts; and slowly disintegrates in alkaline solutions. Carbendazim has low solubility in water, and has higher solubility in organic solvents such as ethyl acetate, acetone, chloroform and the like. It is generally prepared and sold in industry in the form of wettable powder or suspension concentrates and the like.

Due to the large usage amount, high usage frequency, good stability and strong migration capability in environmental media of the carbendazim, the problem that the residue of the carbendazim in many common foods exceeds the standard is caused, and the adverse effects on human health and ecosystems are caused. The research shows that the accumulated residue of carbendazim can interfere the normal hormone level in human and animals, and influence the normal physiological metabolism, endocrine system, immune system and reproductive function of the organism. The world health organization classifies carbendazim as a chemical hazard and as a human carcinogen. Biodegradation is the most main way to remove the organic pesticide residue at present. Therefore, the research on the aspects of the restoration of the soil with the excessive carbendazim, the degradation technology of the carbendazim in fruits and vegetables and the like is very necessary and urgent.

Disclosure of Invention

In order to solve the problems in the prior art, the invention takes carbendazim as the only carbon source, screens out a strain which can rapidly degrade the carbendazim from the contaminated soil of the carbendazim, and is named Pantoea dispersa DJL-B after identification and is preserved in the microorganism strain preservation center of Guangdong province, and the preservation number is GDMCC No:62372. in addition, the invention also separates and obtains a specific protease from the thallus broken liquid or fermentation supernatant of the carbendazim degrading bacteria, adopts an affinity chromatography technology and a flight mass spectrum detection to separate, purify and identify the structure of the protease, and can hydrolyze benzimidazole rings in carbendazim molecules, thereby realizing the degradation of the carbendazim. The carbendazim degrading bacteria Pantoea dispersa DJL-B and the carbendazim degrading enzyme provided by the invention are expected to play an important role in degrading the carbendazim in fruits and vegetables, waste water, polluted soil or liquid foods, and have important significance for protecting the environment and guaranteeing public health safety.

In one aspect, the invention provides a carbendazim degrading bacterium, the strain of which is named Pantoea dispersa DJL-B and is deposited in the Guangdong province microorganism strain collection, and the deposit number is GDMCC No:62372.

in addition, the invention also provides application of the carbendazim degrading bacteria in degrading fruits and vegetables, waste water, polluted soil or liquid food.

In another aspect, the invention provides a carbendazim degrading enzyme, which is separated from a bacterial body broken liquid or fermentation supernatant of the carbendazim degrading bacteria, and belongs to aryl esterase.

Preferably, the amino acid sequence of the carbendazim degrading enzyme is as follows:

MSTFKTKDGVNLYFKDWGKGQPVLFSHGWPLDADMWDSQLNFLAERGYRVIAFDRRGFG

RSDQPWEGYDYDTFADDIHGLIEHLQLDEVTLVGFSMGGGDVSRYIGRYGTAKVKGLVLL

GAVTPIFGKTDDHPEGVESAVFDGIKAGLLKDRAQFIKEFATPFYGTNAGQTVSDGVLTQTL

NIALLASLKGTLDCVTAFSETDFRADIAKVDVPTLVIHGSNDQVVPFEATGKLVHEMIAGSQLKVYENGPHGFAVTHQDQLNADLLAFLQGN, i.e. SEQ ID NO:1.

preferably, the carbendazim degrading enzyme has a theoretical isoelectric point of 4.93.

In addition, the invention also provides application of the carbendazim degrading enzyme in degrading fruits and vegetables, waste water, polluted soil or liquid food.

Compared with the prior art, the invention has the beneficial effects that:

(1) The invention takes carbendazim as the only carbon source, a strain which can rapidly degrade the carbendazim is screened from the contaminated soil of the carbendazim, is identified and named as Pantoea dispersa DJL-B, and is preserved in the microorganism strain preservation center of Guangdong province, and the preservation number is GDMCC No:62372.

(2) The invention uses affinity chromatography technology to separate and purify the carbendazim degrading enzyme from the thallus broken liquid or fermentation supernatant of the carbendazim degrading bacteria Pantoea dispersa DJL-B, and analyzes the amino acid sequence and structure.

(3) The invention also provides application of the carbendazim degrading bacteria Pantoea dispersa DJL-B and the carbendazim degrading enzyme in degrading fruits and vegetables, waste water, polluted soil or liquid food, and has important significance for protecting environment and guaranteeing public health safety.

Drawings

FIG. 1 is a microscopic examination and colony morphology of carbendazim-degrading bacteria Pantoea dispersa DJL-B.

FIG. 2 shows a 16S-conserved sequence similarity analysis of carbendazim-degrading bacteria Pantoea dispersa DJL-B.

FIG. 3 shows the OD value change of different collecting pipes of the affinity chromatographic column eluent.

FIG. 4 shows an HPLC detection chart of the degradation effect of affinity chromatography purification enzyme on carbendazim; wherein A is fermentation supernatant and B is thallus broken liquid.

FIG. 5 is a diagram showing the result of SDS-PAGE gel electrophoresis detection of the separation and purification effects of degrading enzymes; wherein, 1 is thallus broken liquid without column purification, 2 is thallus broken eluent, 3 is protein Marker,4 is fermentation upper cleaning liquid, and 5 is fermentation supernatant without column purification.

FIG. 6 is a schematic diagram of a homology modeling architecture.

FIG. 7 shows a schematic diagram of the secondary structure of the protein Arylesterase.

FIG. 8 shows a graph of activity prediction of the protein Arylesterase; wherein a, catalytic site detection; and b, pocket detection.

FIG. 9 schematic representation of molecular docking of aryl esterase Arylesterase with carbendazim molecules.

Carbendazim degrading bacteria Pantoea dispersa DJL-B were deposited at the microorganism strain collection of Guangdong province at 4/11/2022 under the accession number of GDMCC No:62372, the preservation address is 5 buildings of No. 59 of No. 100 university of Mitrex, guangzhou City, and the preservation time is 2022, 4 and 11 days.

Detailed Description

The invention will be described in further detail with reference to the drawings and examples.

Example 1: gradient culture of carbendazim degrading bacteria, purification and identification of target bacteria

Soil samples were collected from three different areas, 25g each, from the field where the carbendazim was sprayed. Then, each soil sample was dissolved in 225mL of ultrapure water, and left to stand after sufficient shaking, 1mL of the supernatant was taken in a small conical flask, and 9mL of ultrapure water was added for dilution. 300mL of inorganic salt culture medium lacking a carbon source is prepared, and the culture medium is evenly filled into three conical flasks, and under the aseptic condition, carbendazim with different concentrations, namely 0.05g/100mL, 0.25g/100mL and 0.5g/100mL, is respectively added into each conical flask. These concentrations are 1, 5 and 10 times the effective concentration of carbendazim, respectively. The medium of each concentration was poured into four plates, and three soil dilutions, 0.1mL, were added to each of the three plates, and spread evenly. The remaining one plate served as a blank. All plates were incubated in a 28℃incubator and observed for colony growth. Standing the final domestication enrichment culture solution, and sequentially mixing with 10 ^-1 -10 ^-6 Concentration gradient dilution of 10 ^-4 、10 ^-5 、10 ^-6 200 mu L of dilution liquid of dilution gradient is coated on a separation and purification culture medium rich in 100mg/L carbendazim, and is cultured for 5d at 30 ℃, and three gradients are arranged in parallel. Colonies with different morphological characteristics are picked, streaked and separated on a separation and purification culture medium (containing 100mg/L carbendazim) and cultured at 30 ℃. This procedure was repeated until the plates appeared single colonies with good growth and stable passage, and then transferred to a medium more suitable for their growth and propagation for storage.

2. Purification of target bacteria

1 bacterial strain which can grow in 50mg/L carbendazim environment and shows high-efficiency degradation capacity is separated in an enrichment culture domestication mode, and is named as DJL-B. The degradation rate of the strain to carbendazim in 4d reaches more than 95.3%. To further identify the characteristics and classification status of the strain, it was transferred to LB agar medium and subjected to purification culture in an incubator at 37 ℃. Since the strain has resistance to carbendazim, 0.5 times of effective concentration (0.025 g/100 mL) of carbendazim is added into the culture medium as selective pressure to prevent mixed bacteria from polluting. After single round transparent colorless or pale yellow single bacterial drop appears on the flat plate, sealing the flat plate by using a sealing film, and transferring the flat plate into a refrigerator at the temperature of 4 ℃ for storage.

3. Identification of target bacteria

In order to identify the morphological characteristics and the classification status of DJL-B, the following experiments were performed. First, the microscopic examination and colony morphology of DJL-B were observed, and the results are shown in FIG. 1. The colony is round, transparent, colorless or pale yellow, has neat edges and soft texture; the cells were rod-shaped with rounded ends and gram-negative. Secondly, extracting the genomic DNA of DJL-B by using the kit, and carrying out PCR amplification of the 16S rRNA gene by using the genomic DNA as a template and using a universal primer 27F/1492R, wherein the 16S rDNA sequence of the DJL-B is as follows:

CGCCAATGCGGAGCTACACATGCAAGTCGAACGGCAGCACAGAAGAGCTTGCTCTTTG

GGTGGCGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCCGATGGAGGGGGATA

ACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCGAGACCAAAGTGGGGGACCTTC

GGGCCTCACACCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAATGGCTC

ACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGA

CACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGC

CTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCG

GGGAGGAAGGCGGTGAGGTTAATAACCTTGCCGATTGACGTTACCCGCAGAAGAAGCA

CCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAAT

TACTGGGCGTAAAGCGCACGCAGGCGGTCTGTTAAGTCAGATGTGAAATCCCCGGGCTT

AACCTGGGAACTGCATTTGAAACTGGCAGGCTTGAGTCTCGTAGAGGGGGGTAGAATTC

CAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCC

CCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATAC

CCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCT

TCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTC

AAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACG

CGAAGAACCTTACCTGGCCTTGACATCCAGAGAACTTAGCAGAGATGCTTTGGTGCCTT

CGGGAACTCTGAGACAGTGCTGCATGGCTGTCGTCAGCTCGTGTGTGAAATGTGGGTTA

GTCCCGCACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGCTCGGCCGGGAACTCAAAGGAGACTGCCGGGTGATA, i.e. SEQ ID NO:2. the amplified product was obtained by agarose gel electrophoresis analysis and had a size of about 1.5 kb. Then, the PCR product of the 16S rRNA gene was ligated with pGM-T vector to transform E.coli DH 5. Alpha. And positive clones were selected on LB agar medium containing ampicillin. The purpose of this is to improve the accuracy and reliability of the sequencing results. Positive clones were sequenced bi-directionally and the sequencing results were aligned homology to the nucleotide sequences registered in GenBank using Blast on NCBI website. Through comparison analysis, DJL-B (GenBank accession number OM 943751) was found to belong to Pantoea sp. To further confirm the phylogenetic relationship between this strain and other related strains, a corresponding appropriate amount of model strain was selected, and a phylogenetic evolutionary tree was established using the proximity method (based on similarity of 16S rRNA sequences), as shown in fig. 2. The strain has been deposited in the Guangdong province microorganism bacterial culture Collection (GDCCC) under the accession number 62372.

Example 2: separation and purification of carbendazim degrading enzyme

1. Preparation of carbendazim-agarose gel CL-4B chromatographic column

1.1 activation: accurately weighing 1.0g of agarose gel medium, sequentially adding 20-30mL of a 50%,70% dimethyl sulfoxide aqueous solution into the medium, sequentially stirring and cleaning for 5min, vacuum-pumping and filtering out cleaning liquid after each cleaning, adding epichlorohydrin, dimethyl sulfoxide and 5mL of 5mol/L sodium hydroxide solution with the volume ratio of 1.5:1 into the medium after gradient washing to prepare suspension, and placing the suspension into a constant-temperature oscillating box at 44 ℃ for oscillating reaction for 4h at 150 rpm. After the reaction was completed, the reaction mixture was poured into a sand core funnel and washed with 20 volumes of deionized water until no epoxy groups were detected in the eluate (epoxy groups were detected by sodium thiosulfate titration).

1.2 coupling: the activated agarose gel CL-4B is weighed and suspended in acetate buffer (pH 6.0) containing a certain concentration of carbendazim ligand (the coupling amount is generally 10-30mg ligand per g carrier), the coupling reaction is carried out for 20 hours at 37 ℃ and 110rpm, free ligand is removed by using a polyploid volume of deionized water, the solution is transferred into 1mol/L ethanolamine solution, and the solution is subjected to shaking incubation for 6 hours at 37 ℃ to remove residual active epoxy groups. After the reaction is completed, washing with a large amount of deionized water, filling the prepared affinity adsorbent into a chromatographic column, naturally settling, washing 10-20 column bed volumes with 1 x Phosphate Buffer Solution (PBS), soaking in 20% ethanol, and storing at 4 ℃ for later use.

2. Separation and purification of carbendazim degrading enzyme

2.1, taking bacterial liquid after activation culture, centrifuging at 4000rpm for 15min at 4 ℃, respectively recovering supernatant and precipitated bacterial bodies, and passing the supernatant through a microporous filter membrane with the thickness of 0.45 mu m to obtain extracellular crude enzyme liquid for later use. The bacteria were resuspended in bacterial protein preparation lysate (containing 0.2mmol/L trypsin inhibitor PMSF) and sonicated (power 400W,2 s/time, interval 6s, time 15 min) under ice bath conditions. After the completion of the ultrasonic treatment, the mixture is centrifuged for 20min at 12000rpm and 4 ℃, and the supernatant is collected, thus obtaining the intracellular crude enzyme liquid.

2.2 the coupled carbendazim-agarose gel CL-4B medium was loaded onto a chromatography column and the column was equilibrated with 1 XPhosphate buffer (PBS). Affinity chromatography: and loading the crude enzyme liquid into an affinity chromatography column, and controlling the outflow speed of the crude enzyme liquid to be not more than 1mL/min. Washing: after the crude enzyme solution is completely discharged, the crude enzyme solution is used as a washing impurity solution to be passed through a column to remove impurities, and the light absorption of the effluent liquid at the wavelength of 280nm is achievedAfter the degree value stabilized at baseline, the 1 XPBS buffer wash was stopped. Eluting: eluting the target protein with acetate buffer solution (containing 0.5mol/L NaCl) at pH4.0 and 0.1mol/L, and immediately collecting the effluent until OD ₂₈₀ The value was reduced to 0.00. And (3) dialysis: all the effluent was collected with a 1000D dialysis bag and dialyzed in ultrapure water for 20 hours to remove residual ions. Concentrating: concentrating the target protein liquid by adopting a freeze-drying mode to obtain the carbendazim degrading enzyme with higher concentration and purity. And (3) re-dissolving a proper amount of freeze-dried enzyme powder in ultrapure water for activity detection, and performing SDS-PAGE electrophoresis on the active components.

2.3 after passing the two crude enzyme solutions through the affinity chromatography column, the column was washed with 1 XPBS buffer to wash off the heteroproteins. Effluent OD of column to be affinity ₂₈₀ Values stabilized at baseline, indicating that the hybrid protein had been cleared. After passing through the column with 0.1mol/L acetate buffer (containing 0.5mol/L NaCl), the degrading enzyme combined with carbendazim molecule can be separated out and become free again and eluted due to the change of pH value in the column environment. As shown in fig. 3, at the beginning of elution, almost no protein was detected as the eluent did not completely infiltrate the affinity column; after 4 tubes were collected, the protein content of the eluate increased sharply. Therefore, the protein is eluted by the buffer solution with higher ionic strength, and the aim of fast eluting once and obtaining very concentrated protein peaks in a smaller eluting volume can be achieved.

2.4 analysis of the degradation Effect of affinity chromatography purified protein on carbendazim

In order to confirm that the protein purified by the carbendazim-agarose gel CL-4B affinity chromatography column is indeed a degrading enzyme acting on carbendazim, 200. Mu.L of the purified enzyme was placed in 5mL of Tris-HCl buffer pH7.2 and mixed uniformly, and the mixture was subjected to high performance liquid chromatography HPLC at 30℃for 4 hours. By comparing the negative control group with the treatment group treated by the degrading enzyme, the protein purified by the affinity chromatography is found to have a degradation effect on carbendazim, and the degradation effect of the thallus breaking liquid is obviously better than that of the fermentation supernatant, and the degradation rate reaches 44.7 percent, as shown in figure 4.

2.5SDS-PAGE gel electrophoresis detection of degrading enzyme separation and purification effect

2.5.1 glue preparation: 10% split gum and 5% concentrated gum were prepared. Two glass plates which are washed and dried in advance are aligned and fixed on a glue making frame, ultra-pure water is added into a gap and placed for a period of time to check the air tightness. Slowly pumping the pre-mixed separating gel along two glass gaps by using a pipetting gun, and standing for gel 30min in a 37 ℃ incubator after the gel surface rises to the middle height of the green sheets of the gel making frame. When a clear parting line appears between the water and the lower glue, indicating that the glue has polymerized and coagulated, pouring the upper water and sucking the water dry with filter paper, immediately inserting a sample comb after filling with concentrated glue, waiting for polymerization for 30min. Finally, the electrophoresis system was assembled, and after 1 XTris-glycine running buffer was added to the inside glass plate, the sample comb was carefully pulled out to prevent the generation of bubbles.

2.5.2 loading: according to the proportion of 4 mu L and 1 mu L, the protein sample liquid to be tested and 5 multiplied by protein loading buffer are mixed in an EP tube, and the mixture is placed in a constant temperature metal bath at 100 ℃ for heating for 8min to denature protein, and the mixture is subjected to micro-speed centrifugation. 12. Mu.L of the supernatant was aspirated with a microsyringe and added to each lane in sequence, and 4. Mu.L of protein Marker was added to the reserved lane as a reference.

2.5.3 electrophoresis: electrophoresis parameter setting: firstly, concentrating the gel at 80V and running for 17min; the gel was separated at 100V and run for 1.5h until the bromophenol blue indicator migrates near the bottom of the gel surface and the electrophoresis was terminated.

2.5.4 staining: after electrophoresis, the glass plate is removed, the film is gently peeled off and placed in a large petri dish with a diameter of 150mm, and a proper amount of 0.25% coomassie brilliant blue R-250 dye solution is injected for dyeing for 2.5 hours, and if necessary, overnight.

2.5.5 decoloring: the dyeing liquid is discarded, the glue surface is rinsed for several times by distilled water, the decoloring liquid is added until the glue surface is completely soaked, and finally, diffusion decoloring is carried out on a decoloring shaking table, and the decoloring liquid is replaced every 25 minutes until the protein strips are clearly visible.

2.5.6 gel image acquisition. The two enzyme solutions are subjected to carbendazim-agarose gel CL-4B affinity chromatography, dialysis and concentration, and then the separation and purification effects of degrading enzymes are detected by SDS-PAGE gel electrophoresis, as shown in figure 5. Lanes 1 and 5 show the cell disruption solution and fermentation supernatant, respectively, without column purification. As a result of electrophoresis, the strain DJL-B produced various enzymes, and the molecular weight was substantially 100kDa or less. Lanes 2 and 4 show the cell disruption eluate and the wash solution on fermentation, respectively. It can be found that the thallus broken liquid and the fermentation supernatant after column purification have bands at two identical positions (about 30kDa and 40 kDa) of the film, the protein bands of the thallus broken eluent are more abundant, and the target protein and other proteins are possibly interacted and combined together in the process of column passing affinity for simultaneous elution; secondly, the presence of hybrid proteins is possible. The analysis of the degradation effect of the purified enzyme can be combined, and the self-made carbendazim-agarose gel CL-4B affinity chromatographic column of the research has higher specific binding property, so that the protein with degradation effect on carbendazim can be effectively purified.

Example 3: identification of carbendazim degrading enzyme

3.1 Mass Spectrometry characterization

After SDS-PAGE electrophoresis and comparative analysis, the enzyme solution is selected to cut three distinct gel blocks with the molecular weight of about 60kDa, 40kDa and 30kDa (see box label in FIG. 5), and protein mass spectrometry is performed by MALDI-TOF-MS. The protein bands can be subjected to enzymolysis analysis to obtain peptide sequences, and sequence comparison is carried out in NCBI, uniProt and other databases, and analysis results show that the theoretical molecular weight (Mass) of three target bands is consistent with that of SDS-PAGE gel electrophoresis, wherein the protein A0A6A5BA66 is named as Arylesterase, the similarity of the peptide sequences and Alpha/beta hydrolase foldprotein is 95%, and the protein is Alpha/beta hydrolase; the A0A437T546 protein is named as Porin OmpC, the similarity of the peptide fragment sequence and the Membrane protein is 100%, and the protein is Porin; the peptide sequence of A0A6A5B7B7 has 90 percent of similarity with Glucose-6-phosphate isomerase, and is an isomerase. The currently accepted classical degradation pathway of carbendazim is that the alpha/beta hydrolase encoded by the gene MheI is responsible for the first degradation of carbendazim, which exerts the hydrolytic action of deamidating groups. The Arylesterase and the homonymous carbendazim hydrolase MheI encoded by the gene MheI are both catalytically active and belong to the alpha/beta hydrolase superfamily. From the above results, it is found that the carbendazim degrading enzyme Arylesterase plays an important role in the degradation of carbendazim.

3.2 analysis of primary Structure of protein

The primary structure and related physicochemical characteristics of the carbendazim degrading enzyme Arylesterase were evaluated using a ExPASy ProtParam Server (http:// web. Expasy. Org/protparam /) network database system. The results show that the theoretical isoelectric point of the Arylesterase is 4.93, the Arylesterase has 273 amino acids in total, the sequence only contains 1 cysteine Cys, the disulfide bond formation possibility does not exist, and the Instability Index (II) is calculated to be 16.89 and is classified into a stable category. The protein FASTA sequence is as follows: MSTFKTKDGVNLYFKDWGKGQPVLFSHGWPLDADMWDSQLNFLAERGYRVIAFDRRGFGRSDQPWEGYDYDTFADDIHGLIEHLQLDEVTLVGFSMGGGDVSRYIGRYGTAKVKGLVLLGAVTPIFGKTDDHPEGVESAVFDGIKAGLLKDRAQFIKEFATPFYGTNAGQTVSDGVLTQTLNIALLASLKGTLDCVTAFSETDFRADIAKVDVPTLVIHGSNDQVVPFEATGKLVHEMIAGSQLKVYENGPHGFAVTHQDQLNADLLAFLQGN, i.e. SEQ ID NO:1.

3.3 Secondary Structure of proteins and homology modeling analysis

Template search was performed on the protein sequence by SWISS-MODEL (http:// swissmodel. Expasy. Org /) on-line server, and the amino acid sequence similarity of Arylesterase and Pseudomonas fluorescens aryl esterase (PDB number: 3 HEA) derived from Pseudomonas fluorescens was found to be the highest and 72.32%. It is reported that the similarity between the amino acid sequence and the template exceeds 30%, so that the reliability of the model obtained by homologous modeling is high. Thus, the amino acid sequence of Arylesterase was introduced into Phyre ² (Protein Fold Recognition Server) in-line software, 3D modeling of protein sequences was performed using a method of remote homology detection. The homology modeling structure is shown in FIG. 6, indicating that 99% of the amino acids are modeled with a confidence level of greater than 90% (resolution) The protein model is reasonable to construct, achieves the experimental verification level of the protein structure, and can be used for subsequent protein transformation or related research. At the same time via Phyre ² It was found that the secondary structure of the protein Arylesterase containedThere was 45% alpha helix, 17% beta sheet and 6% transmembrane helix as shown in figure 7.

3.4 discussion of mechanism of action

By Phyre ² The three-stage structure model of the protein Arylesterase is deeply analyzed on line to obtain a predicted catalytic active site, and a large pocket is found at the position of the corresponding active site and is a reactive center, as shown in FIG. 8. The carbendazim structural formula was constructed using ChemDraw software and optimized using classical MM3 force field and semi-empirical PM6 force field to ensure that the molecules exist in the best active form. Molecular docking analysis was then performed on aryl esterase Arylesterase and carbendazim molecules using Autodock-Vina, and the results are shown in FIG. 9. The carbendazim molecule is located exactly in the active cavity of the aryl esterase and the affinity between the two is-5.6 kcal/mol, a smaller value indicating that the structure is more stable, indicating that this binding is advantageous.

The foregoing is a further detailed description of the invention in connection with the preferred embodiments, and it is not intended that the invention be limited to the specific embodiments described. It will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the invention, and these should be considered to be within the scope of the invention.

Claims

1. The carbendazim degrading bacteria are characterized in that: the strain is named Pantoea dispersa DJL-B and is deposited in the Guangdong province microorganism strain collection center, and the deposit number is GDMCC No:62372.

2. the use of the carbendazim degrading bacteria according to claim 1 for degrading carbendazim in fruits and vegetables, waste water, contaminated soil or liquid food.

3. A carbendazim degrading enzyme, characterized in that: a cell disruption solution or fermentation supernatant isolated from the carbendazim degrading bacterium of claim 1, which is an aryl esterase.

4. The carbendazim degrading enzyme according to claim 2, wherein: the amino acid sequence is as follows:

MSTFKTKDGVNLYFKDWGKGQPVLFSHGWPLDADMWDSQLNFLAERGYRVIAFDRRGFGRSDQPWEGYDYDTFADDIHGLIEHLQLDEVTLVGFSMGGGDVSRYIGRYGTAKVKGLVLLGAVTPIFGKTDDHPEGVESAVFDGIKAGLLKDRAQFIKEFATPFYGTNAGQTVSDGVLTQTLNIALLASLKGTLDCVTAFSETDFRADIAKVDVPTLVIHGSNDQVVPFEATGKLVHEMIAGSQLKVYENGPHGFAVTHQDQLNADLLAFLQGN, i.e. SEQ ID NO:1.

5. a carbendazim degrading enzyme according to claim 3, wherein: its theoretical isoelectric point is 4.93.

6. Use of a carbendazim degrading enzyme according to any one of claims 3 to 5 for degrading carbendazim in fruits and vegetables, waste water, contaminated soil or liquid food.