Hydrolyase gene ipcH, the protein encoded thereby, and applications thereof

Abstract

The present invention discloses a hydrolase gene ipcH, the protein encoded thereby, and their applications. The full length of the hydrolase gene ipcH of the present invention is 1983 bp, and the sequence is as shown in SEQ ID NO.1. Its encoded product, hydrolase IpcH, contains 660 amino acids, and the sequence is SEQ ID NO.2. IpcH can degrade the carbamate insecticide isoprocarb. The hydrolase IpcH can be used to degrade the residues of isoprocarb in water bodies and soil, and has very important theoretical and application values.

Description

Technical Field

[0001] The present invention belongs to the fields of environmental microbiology and agriculture, and relates to a hydrolase gene ipcH, a protein encoded thereby, and applications thereof, and particularly relates to a hydrolase gene ipcH for degrading the monocyclic carbamate pesticide isoprocarb and applications thereof. Background Art

[0002] Carbamate pesticides are a class of pesticides with the functional group CH3-NH-O-C=O-, and have the characteristics of broad-spectrum insecticidal activity, high efficacy, strong selectivity, and simple synthesis, and are widely used as insecticides, nematicides, acaricides, and herbicides. Isoprocarb is one of the commonly used carbamate insecticides, and can achieve the insecticidal effect by inhibiting the activity of acetylcholinesterase, and can be effectively used as an insecticide and a herbicide. Isoprocarb is a contact and fast-acting pesticide, and is mainly used for controlling rice leafhoppers and planthoppers. Among them, isoprocarb is still active in the pesticide market and has a stable output. Therefore, from the perspective of usage, isoprocarb is one of the most widely used insecticides.

[0003] Due to the stable chemical properties and large usage amount of isoprocarb, isoprocarb will remain and accumulate in the environment and organisms, causing environmental pollution, affecting non-target ecological groups, and causing ecological interference. Isoprocarb has neurotoxicity, irreversibly inhibits acetylcholinesterase, causes the accumulation of acetylcholine, and endangers human health. The risks of isoprocarb in terms of blood, liver, developmental toxicity, and enzyme activity inhibition have been reported in several organisms. For example, isoprocarb will inhibit the expression of genes related to the neurodevelopmental signaling pathway in zebrafish embryos, induce oxidative stress responses in zebrafish embryos, and at the same time cause developmental defects in the hindbrain of zebrafish embryos and a decrease in motor ability; isoprocarb affects the growth, development, reproduction, and regeneration processes of earthworms by inducing oxidative stress and neurotoxicity. Therefore, due to the high toxicity of isoprocarb to humans and animals, isoprocarb is classified as moderately hazardous by the World Health Organization (WHO). Isoprocarb has received extensive attention due to its high detection rate in the environment and its liver and developmental toxicity.

[0004] Obtaining isoprocarb-degrading strains and degrading genes has the following effects in controlling isoprocarb residues in the environment: (1) for eliminating isoprocarb residues, the insecticide, in soil and water; (2) constructing genetically engineered strains through gene research, and then studying the enzymatic properties, which is of great significance for the remediation of isoprocarb pollution. Therefore, the degrading gene has very important theoretical and application values in eliminating isoprocarb residues, the insecticide. Summary of the Invention

[0005] The object of the present invention is to provide an isoprocarb hydrolase gene ipcH;

[0006] Another object of the present invention is to provide a protein IpcH encoded by this gene;

[0007] Another object of the present invention is to provide the application of the gene;

[0008] The object of the present invention is achieved by the following technical solutions:

[0009] A hydrolase gene ipcH, whose nucleotide sequence is SEQ ID NO.1.

[0010] The target gene was found by combining various protein purification methods and genome sequencing. First, ammonium sulfate precipitation, DEAE-Sepharose Fast Flow ion exchange chromatography column, Q-Sepharose Fast Flow ion exchange chromatography column, and Sephadex-200 gel chromatography were used to gradually purify the isoprocarb hydrolase in the crude enzyme solution of strain D-6. The protein electrophoresis pattern of the protein still having isoprocarb degradation activity after Sephadex-200 gel chromatography (see Figure 3 ), a single target protein has been purified, and it is preliminarily determined that the size of the isoprocarb hydrolase protein of this strain is about 73.0 kDa. This band was sent to Shanghai OE Biotech Co., Ltd. for peptide fingerprint analysis; then the genome of strain D-6 was extracted by the high-salt method, and the DNA sample was sent to Ling'en Biotech Co., Ltd. for genome sequencing. Through homology analysis of the peptide sequence and the amino acid sequence in strain D-6, it was found that the similarity with the amino acid sequence of ORF6004582 on scaffold 20 in the genome reached 73%. Therefore, ORF6004582 was analyzed, and the corresponding coding gene was named ipcH.

[0011] The protein IpcH encoded by the hydrolase gene ipcH has an amino acid sequence of SEQ ID NO.2. The hydrolase gene ipcH was synthesized by Beijing Tsingke Biotechnology Co., Ltd.

[0012] A recombinant expression vector containing the hydrolase gene ipcH.

[0013] The recombinant expression vector is preferably obtained by ligating the hydrolase gene ipcH between the NdeI and XhoI sites of pET-29a(+).

[0014] A genetically engineered bacterium containing the hydrolase gene ipcH, and the genetically engineered strain preferably uses E. coli BL21(DE3) as the starting strain.

[0015] The application of the hydrolase gene ipcH in degrading isoprocarb.

[0016] The application of the recombinant expression vector containing the hydrolase gene ipcH in degrading isoprocarb.

[0017] Application of the hydrolase protein IpcH in the degradation of isoprocarb.

[0018] Application of the hydrolase protein IpcH in the preparation of reagents for removing isoprocarb from water bodies and soil or for removing isoprocarb in soil and water bodies.

[0019] The beneficial effects of the present invention are as follows:

[0020] 1. A strain capable of degrading isoprocarb was isolated from the enrichment solution with isoprocarb degradation effect in the present invention, named Rhodococcus sp. D-6. On this basis, the present invention successfully cloned the hydrolase gene ipcH from strain D-6 by combining a variety of protein purification techniques and strain genome sequencing. By performing blastp online amino acid sequence analysis and homology comparison in NCBI (the UniProtKnowledgeBase / SwissProt databases), it was found that this gene is a new gene, with a full length (from the start codon to the stop codon) of 1983 bp and can encode 660 amino acids.

[0021] 2. The hydrolase IpcH provided by the present invention can completely degrade 50 mg / L of isoprocarb within 2 h. In addition, IpcH can also be used to construct a genetically engineered strain of isoprocarb for removing isoprocarb residues in water bodies and soil, which has very important theoretical and application values. Description of the Drawings

[0022] Figure 1 HPLC chromatogram of the degradation of isoprocarb by strain D-6 and MS chromatogram of the metabolite;

[0023] A: Isoprocarb standard; B: HPLC chromatogram of the sample taken at 5 h for the degradation of isoprocarb by D-6; C: MS chromatogram of isoprocarb; D: MS chromatogram of 2-isopropylphenol.

[0024] Figure 2 Strategy diagram for the cloning of the hydrolase gene ipcH.

[0025] Figure 3 Detection diagram of SDS-PAGE for different steps of purifying the hydrolase IpcH from strain D-6;

[0026] Lane 1: Protein marker; Lane 2: Broken solution of strain D-6; Lane 3: Purified by ammonium sulfate precipitation; Lane 4: Purified by DEAE-Sepharose anion exchange chromatography; Lane 5: Purified by Q-Sepharose cation exchange chromatography; Lane 6: Purified by Superdex G-200 chromatographic column.

[0027] Figure 4Expression strategy diagram of hydrolase gene ipcH in BL21(pET-29a).

[0028] Figure 5 Electrophoresis pattern of hydrolase IpcH protein;

[0029] Lane 1 is protein marker; Lane 2 is purified hydrolase IpcH protein.

[0030] Figure 6 HPLC pattern of isoprocarb degradation catalyzed by hydrolase IpcH and MS pattern of metabolites;

[0031] A: Liquid phase diagram of catalytic reaction system; B: MS pattern of isoprocarb; C: MS pattern of 2-isopropylphenol Detailed implementation method

[0032] Example 1 Isolation and screening of isoprocarb-degrading strain D-6

[0033] 1.1 Enrichment, domestication and isolation of isoprocarb-degrading strain D-6

[0034] The soil sample was collected from the sludge in the wastewater discharge system of a pesticide factory that had been producing isoprocarb for a long time, and a strain with isoprocarb degradation function was obtained through enrichment culture and isolation. 3 g of the sludge sample was added to 100 mL of MSM liquid medium containing 200 mg / L isoprocarb. It was incubated at a constant temperature of 30 °C on a shaker at 160 rpm. Every 5 days, it was transferred to fresh MSM liquid medium containing 50 mg / L isoprocarb with an inoculation amount of 5%. It was passaged continuously three times.

[0035] Basic salt medium formula (MSM): K2HPO4·3H2O 1.5 g; KH2PO4 0.5 g; NH4NO3 1.0 g; NaCl 0.5 g; MgSO4·7H2O 0.2 g, add deionized water to make up to 1 L. 15.0 g of agar was added to each liter of the solid medium.

[0036] 1.2 Purification, screening and identification of the degrading strain

[0037] Use high performance liquid chromatography to detect whether isoprocarb in the third-generation enrichment solution is degraded and whether new metabolites are generated. For the enrichment solution with degradation effect, at a dilution factor of 10 -1 to 10 -6Dilute it by multiples and coat it on the MSM solid medium containing 200 mg / L isoprocarb, and culture it in an incubator at 30 °C for 5 days. Select single colonies with different morphologies grown on the plate into an LB liquid test tube, place it in a shaker at 30 °C and 160 rpm for 5 days. Then, take 1 mL of the bacterial liquid and centrifuge it at 5,000 rpm for 3 min in a sterilized 1.5 mL centrifuge tube. After washing the bacteria 3 times with sterilized MSM liquid medium, transfer the strain to the MSM liquid medium containing 200 mg / L isoprocarb and culture it in a shaker at 30 °C and 160 rpm for 5 days. Use HPLC to detect and identify whether each single colony has the function of degrading isoprocarb.

[0038] A strain of isoprocarb-degrading bacterium was isolated and screened through enrichment and domestication, named D-6. After growing on an LB solid plate for 3 days, the colony of strain D-6 is orange-yellow, with an irregular edge and a convex and dry surface; strain D-6 is a Gram-positive bacterium.

[0039] Phylogenetic analysis of 16S rRNA gene sequence: Using the total DNA of the strain as a template, PCR amplification was carried out using the universal primers 27F / 1492R for 16S rRNA gene. The 27F primer is 5′-AGAGTTTGATCCTGGCTCAG-3′, and the 1492R primer is 5′-TACGGCTACCTTGTTACGACTT-3′.

[0040] Amplification reaction system (50 μL):

[0041]

[0042] The procedure used is as follows:

[0043]

[0044] Electrophoresis detection of the PCR product was carried out on a 1.0% agarose gel. After recovering the PCR product, it was ligated with the pMD19-T vector, and then transformed into E. coli DH5α competent cells. After culturing, positive clones were selected and sent to Tsingke Company for sequencing. Through comparative analysis of the obtained 16S rRNA gene in the EZtaxon database (https: / / www.ezbiocloud.net / ), the sequence similarity between strain D-6 and Rhodococcus pyridinivorans DSM 44555 T (GenBank accession number: KF410365) is 100%, and strain D-6 was identified as Rhodococcus sp.

[0045] 1.3 Detection of isoprocarb concentration

[0046] Ultraviolet spectrophotometer detection: Take 1 mL of the sample to be detected, centrifuge at 12,000×g for 3 min, scan the upper layer liquid within 200 - 400 nm, and detect the change in the content of isoprocarb by the characteristic absorption peak and response value at 260 nm.

[0047] HPLC detection: Take 1 mL of the sample to be detected, centrifuge at 12,000×g for 3 min, take the upper layer liquid through an aqueous phase filter membrane, and use high performance liquid chromatography (HPLC) to detect the content of isoprocarb. HPLC detection conditions: Mobile phase ratio: methanol: water: acetic acid = 80:19.8:0.2; Column temperature: 40 °C; Mobile phase flow rate: 1.0 mL / min; Detection wavelength: 260 nm; Injection volume: 20 μL.

[0048] 1.4 Degradation characteristics and degradation product analysis of degradation strain D-6

[0049] Study on degradation characteristics: Inoculate strain D-6 into 100 mL of LB liquid medium at an inoculation amount of 1%, place it in a constant temperature shaker at 30 °C and 180 rpm for culture, and take samples every 2 h to measure OD 600 , set three replicates for the treatment to detect the growth of the strain. Inoculate the strain into LB liquid medium at an inoculation amount of 1%, culture it in a constant temperature shaker at 30 °C and 180 rpm until the logarithmic growth phase, centrifuge at 5000×g for 5 min to collect the cells, resuspend them with MSM liquid medium, centrifuge and repeat twice, wash the cells thoroughly, and then resuspend them with MSM liquid medium to an OD 600 of about 1.0 as the strain seed liquid for standby. Add strain D-6 to the MSM liquid medium containing 200 mg / L of isoprocarb, and adjust the final OD 600 of strain D-6 to 0.2, and culture it in a constant temperature shaker at 30 °C and 180 rpm. Take samples regularly every 1 h, and use HPLC to determine the change in the content of isoprocarb during the degradation of isoprocarb by strain D-6.

[0050] The experimental results show that strain D-6 can degrade isoprocarb, and it can degrade 200 mg / L of isoprocarb after 14 h of culture. The HPLC-MS analysis results show that the first step of isoprocarb degradation is the hydrolysis of isoprocarb to produce 2-isopropylphenol ( Figure 1 ).

[0051] Example 2 Cloning and functional verification of isoprocarb hydrolase gene (see the strategy map in Figure 2 )

[0052] 2.1 Sequencing analysis of total bacterial genomic DNA

[0053] 2.1.1 Extraction of total bacterial genomic DNA

[0054] The strain D-6 was cultured in LB liquid medium, and the cells were collected by centrifugation to verify the degradation ability of the strain to isoprocarb. The total genomic DNA of the strain D-6 was extracted by the high-salt combined with CTAB method and stored at -20 °C.

[0055] 2.1.2 Genome draft sequencing and result analysis

[0056] The extracted total DNA was sequenced by Ling'en Biotechnology Co., Ltd.: 1. The library was constructed by using the Illumina TruSeq TM NanoDNA Sample Prep Kit method; 2. The sequencing data was analyzed.

[0057] 2.1.3 Genome draft sequencing result analysis

[0058] Genome sequencing result: The size of the genome draft of the strain D-6 is 5,313,989 bp, with 73 scaffolds, and the G+C content is 67.5%.

[0059] 2.2 Protein purification of the crude enzyme solution from the broken cells of strain D-6

[0060] 2.2.1 Preparation of the cell lysate of the strain

[0061] The strain D-6 was inoculated into LB medium and cultured at 30 °C until the OD 600 was approximately 2.0. The cells were collected by centrifugation at 6,000 × g for 5 min, resuspended in Tris-HCl buffer (pH 7.4), washed twice, and then resuspended in 15 mL of Tris-HCl buffer. Under ice bath conditions, the cells were broken by an ultrasonic disruptor. After the disruption was completed, the mixture was centrifuged at 4 °C and 12,000 rpm for 30 min, and the supernatant was collected and filtered through a 0.22 μm aqueous filter to obtain the crude enzyme solution, which was stored at 4 °C.

[0062] 2.2.2 Determination of the enzyme activity of the crude enzyme solution of the strain

[0063] 1 mL enzyme activity reaction system: 50 μL of the crude enzyme solution of strain D-6 was added to 930 μL of Tris-HCl buffer (pH 7.4), and 20 μL of isoprocarb (200 mg / L). The reaction was carried out in a water bath at 30 °C for 15 min. After the reaction was completed, it was boiled in boiling water for 10 min, and the concentration of isoprocarb was detected by HPLC. The definition of one enzyme activity unit (U): In this experiment, 1 enzyme activity unit (U) was defined as the amount of enzyme (mg) required to reduce 1 μmol of isoprocarb per minute.

[0064] The enzyme activity determination experiment showed that the specific enzyme activity of the crude enzyme solution of strain D-6 for degrading isoprocarb could reach 48.90 U / mg

[0065] 2.2.3 Ammonium sulfate fractional precipitation

[0066] After the volume of the crude enzyme solution was quantified, fractional precipitation was carried out at ammonium sulfate saturations of 0-20%, 20-40%, 40-60%, 60-80%, and 80-100%, respectively. Ammonium sulfate was ground into powder in advance and placed in an oven to dry the moisture, and the mass of ammonium sulfate to be added was calculated according to the corresponding components. The crude enzyme solution and a magnetic rotor were placed in a triangular flask, and the triangular flask was placed in a beaker in an ice bath. Ammonium sulfate was slowly and evenly added to make the saturation reach 20%. The crude enzyme solution was centrifuged at 12,000 rpm for 20 min in a pre-cooled centrifuge. The precipitate was added to 3 mL of 20 mM Tris-HCl buffer (pH 7.4) and dissolved on ice. The above steps were repeated until the ammonium sulfate saturation reached 100%. The precipitation time for each level of ammonium sulfate was controlled at 40 min. After the precipitates at each level were completely dissolved, dialysis was carried out overnight in 20 mM Tris-HCl buffer (pH 7.4) at 4°C. The cut-off molecular weight of the dialysis bag was 10 kDa. After the dialyzed enzyme solution was subjected to protein quantification and enzyme activity detection, the activity recovery (%) and protein content (%) of each precipitate were calculated, and the fraction with high enzyme activity was selected for further purification.

[0067] 2.2.4 DEAE-Sepharose fast flow ion column chromatography

[0068] The DEAE-Sepharose fast flow anion column was equilibrated with 20 mM Tris-Hcl (pH 7.4) buffer for 2-3 column volumes. The enzyme solution with the highest enzyme activity obtained in the previous step was slowly added to the chromatography column. After elution with 20 mM Tris-Hcl (pH 7.4) buffer, gradient elution was carried out with 20 mM Tris-Hcl (pH 7.4) buffer containing different concentrations of NaCl (0.20 M, 0.25 M, 0.3 M, and 0.35 M), and one tube was collected every 3 mL. The enzyme activity of the collected enzyme solution was detected, the enzyme solutions with the highest activity were combined, dialyzed overnight, and finally ultrafiltration concentrated with a 10 kDa ultrafiltration tube at 4°C and 3,000×g.

[0069] 2.2.5 Q-Sepharose fast flow ion column chromatography

[0070] The Q-Sepharose fast flow anion column was equilibrated with 20 mM Tris-Hcl (pH 7.4) buffer for 2 - 3 column volumes. The enzyme solution concentrated in the previous step was added to the chromatography column. After elution with 20 mM Tris-Hcl (pH 7.4) buffer, gradient elution was carried out with 20 mM Tris-Hcl (pH 7.4) buffer containing different concentrations of NaCl (0.20 M, 0.25 M, 0.3 M, and 0.35 M). One tube was collected every 3 mL, and the enzyme activity was measured for each tube. The enzyme solution with activity was dialyzed overnight, and finally, the enzyme solution with activity was collected by ultrafiltration concentration using a 10 kDa ultrafiltration tube at 4 °C and 3,000 × g.

[0071] 2.2.6 Superdex-200 Gel Filtration Chromatography

[0072] First, the Superdex G-200 molecular sieve column was equilibrated with 20 mM Tris HCl (pH 7.4) buffer containing 0.1 M NaCl for 2 - 3 column volumes. The enzyme solution after ultrafiltration was injected into the injection valve to start elution, and the flow rate was 0.4 mL min -1 One tube was collected every 0.5 mL. After the column elution was completed and rinsed clean, it was stored in 20% ethanol for later use. An appropriate amount of the collected enzyme solution was taken for enzyme activity detection.

[0073] 2.2.7 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

[0074] For the enzyme solution with activity detected after each step of purification, 30 μL was taken and 10 μL of 4× SDS-PAGE Loading Buffer was added. After boiling in water for 10 min, it was taken out and cooled to room temperature. With a sample loading volume of 20 μL, it was added to the SDS-PAGE sample wells in sequence. The power supply was turned on. When the bromophenol blue reached the bottom of the separation gel, the electrophoresis was completed. The gel was placed in a fresh-keeping box, and an appropriate amount of Coomassie Brilliant Blue R-250 staining solution was poured in and stained for 30 min. The staining solution was poured out, and an appropriate amount of decolorizing solution was added and slowly shaken on a shaker for decolorization for 8 - 10 h. During this period, the decolorizing solution was changed 2 - 3 times until the bands were clearly visible.

[0075] 2.2.8 Analysis of Hydrolytic Enzyme Peptide Fingerprint

[0076] The target protein obtained by SDS-PAGE detection ( Figure 3) The band where the target protein is located was cut and sent to Shanghai OE Biotech Co., Ltd. for LC-MS / MS proteomic identification. Through homologous alignment analysis of the peptide sequence with the amino acid sequence in the genome of strain D-6, an ORF annotated as hydrolase was found, named IpcH, with 660 amino acids. Its coding nucleotide is 1983bp. The nucleotide sequence of the coding gene ipcH is shown in SEQ ID NO.1, and the amino acid sequence of IpcH is shown in SEQ ID NO.2.

[0077] Example 3 Heterologous expression and verification of the hydrolase gene ipcH (see the strategy diagram in Figure 4 )

[0078] 3.1 Synthesis of the expression vector for the isoprocarb hydrolase gene ipcH

[0079]

[0080] The ipcH gene expression vector pET-ipcH was synthesized by Beijing Tsingke Biotechnology Co., Ltd., and the nucleotide sequence of the ipcH gene is as shown in SEQ ID NO.1.

[0081] 3.2 Construction and verification of the expression strain

[0082] The above-constructed expression vector was transferred into the Escherichia coli expression strain E. coli BL21(DE3). The transformant was picked into an LB liquid test tube containing 50 mg / L Km and cultured at 37°C with a shaking speed of 180 rpm. PCR was used to verify whether the obtained strain was correct, and the successfully transformed positive clone strain was named: E. coli BL21(DE3)-ipcH.

[0083] 3.3 Induced expression and purification of the expression vector

[0084] A single colony of E. coli BL21(DE3)-ipcH was selected in an LB liquid medium containing Km (50 mg / L) and cultured at 37°C with a shaking speed of 180 rpm for about 8 h. When the bacteria grew to the logarithmic phase, 1 mL of the bacterial solution was inoculated into 100 mL of LB liquid medium and cultured at 37°C with a shaking speed of 180 rpm until OD 600It was 0.4 - 0.6, and IPTG with a final concentration of 0.15 mM was added. It was induced and cultured at 180 rpm at 16°C for 10 h. After the induction ended, the bacterial solution was centrifuged at 8000×g for 3 min, the bacterial cells were retained, washed with Tris-HCl buffer and resuspended. The bacterial suspension was placed in an ice-water mixture and broken using an ultrasonic disruptor. The crude enzyme solution after disruption was stored at 4°C, centrifuged at 12000×g for 30 min, the upper clear enzyme solution was taken, filtered through a 0.22-μm aqueous filter, and the crude enzyme solution was stored in a 4°C refrigerator.

[0085] Using the histidine tag on the pET-29a(+) vector as a marker, Ni 2+ -NTA affinity chromatography column was used to separate and purify the target protein IpcH, and the eluate with the highest purity was collected for overnight dialysis. SDS-PAGE protein electrophoresis was used to detect the purification effect, and the band size was consistent with the theoretically predicted size (73.0 kDa) ( Figure 5 ).

[0086] 3.4 Determination of IpcH Activity

[0087] A standard reaction system (1 mL) for the hydrolase IpcH was established: isoprocarb 50 mg / L, hydrolase IpcH 24.74 μg, and 20 mM Tris-HCl (pH 7.4) buffer was made up to 1 mL. The enzymatic reaction system was reacted at 30°C for 1 h. The enzyme reaction was terminated by boiling water bath for 10 min, and the change in isoprocarb content was detected by HPLC after filtration.

[0088] Definition of enzyme activity unit (U): In this experiment, 1 enzyme activity unit (U) was defined as: the amount of enzyme (mg) required to reduce 1 μmol of isoprocarb per minute. The degradation test showed that the purified IpcH could degrade 50 mg / L of isoprocarb within 2 h, and the enzymology test showed that the specific enzyme activity of IpcH against isoprocarb was 87.58 U / mg.

[0089] 3.5 Identification of Metabolites

[0090] The degradation products of isoprocarb after the enzymatic reaction were detected and analyzed by HPLC-MS technology. HPLC conditions: Mobile phase ratio: methanol: water: acetic acid = 80:19.8:0.2; column temperature: 40°C; mobile phase flow rate: 1.0 mL / min; detection wavelength: 260 nm, injection volume: 20 μL. The MS analysis ion source of the LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific) was ESI, and the positive ion detection mode was used. The liquid chromatography-mass spectrometry detection data was processed and analyzed using Xcalibur software.

[0091] Analysis of the HPLC-MS detection results indicated that IpcH could hydrolyze isoprocarb, and the generation of hydrolysis products was shown on the HPLC chromatogram. The MS results analysis identified the product as 2-isopropylphenol ( Figure 6 ).

Claims

1. A hydrolase gene ipcH , characterized in that The nucleotide sequence is SEQ ID NO.

1.

2. A hydrolase protein IpcH, characterized in that, The hydrolase gene according to claim 1 ipcH encoding, with the amino acid sequence of SEQ ID NO.

2.

3. A recombinant expression vector containing the hydrolase gene recited in claim 1 ipcH .

4. The recombinant expression vector according to claim 3, wherein The hydrolase gene described in claim 1 ipcH is inserted between the Nde I and Xho I sites to obtain 5. A genetically engineered bacterium containing the hydrolase gene described in claim 1 ipcH .

6. The genetically engineered bacterium according to claim 5, characterized in that, The genetically engineered bacterium uses E. coli BL21(DE3) as the starting strain.

7. The hydrolase gene according to claim 1 ipcH Use in degrading isoprocarb.

8. Use of the recombinant expression vector containing the hydrolase gene according to claim 3 or 4 ipcH in the degradation of isoprocarb.

9. Use of the hydrolase protein IpcH according to claim 2 in the degradation of isoprocarb.

10. Use of the hydrolase protein IpcH according to claim 2 in the preparation of a reagent for removing isoprocarb from water bodies and soil or in removing isoprocarb from water bodies and soil.

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