Recombinant esterase, engineering bacterium and application of recombinant esterase in degradation of carbaryl

Abstract

The invention discloses a recombinant esterase, an engineering bacterium and application of the recombinant esterase in degradation of carbaryl, the coding gene of the recombinant esterase provided by the invention is cloned and expressed in escherichia coli, the engineering bacterium E.coli BL21 (DE3)-pET-28a (+)-GE003489 is constructed, and the recombinant esterase subjected to induced expression has relatively good hydrolysis capability on carbaryl. A proper amount of recombinant esterase is used for catalytic hydrolysis of carbaryl, the substrate concentration is 0.1 g / L, the catalysis time is 1 h, and the substrate conversion rate is 90.5%. The method can be used for removing carbaryl pollution in the environment and has potential application value.

Description

(I) Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and specifically relates to bacteria derived from Pseudomonas (…). Pseudomonas Recombinant esterases (sp.), engineered bacteria, and their application in the degradation of carbaryl. (II) Background Technology

[0002] Carbaryl, also known as carbaryl carbamate, scientifically named 1-naphthyl-N-methylcarbamate, is a widely used carbamate insecticide. Its main insecticidal mechanism is the inhibition of acetylcholinesterase in insects. Carbaryl has contact, stomach, and weak fumigation effects, meaning that insects will die from contact with the pesticide or from ingesting sprayed plants. Carbaryl has been widely used in: agriculture – for controlling lepidopteran pests (such as leafrollers), coleopteran pests (such as scarab beetles), and hemiptera pests (such as aphids) on various crops including rice, cotton, and fruit trees; forestry – for controlling forest pests; and public health – for controlling sanitary pests such as mosquitoes and flies.

[0003] However, carbaryl also has certain drawbacks: it is highly toxic to bees, causing their death when used during flowering, threatening pollinating insects and the ecological balance; it is also highly toxic to aquatic organisms, posing a risk of water pollution. It also poses a threat to human health; large-scale exposure can cause typical carbamate pesticide poisoning symptoms such as tearing, constricted pupils, muscle tremors, nausea, and vomiting.

[0004] Compared to traditional physical and chemical treatment methods, microbial degradation is a novel pathway for pesticide degradation. Its advantages include low input, non-toxicity, no residue, and no secondary pollution. The treatment effect is also relatively significant, making it a recognized low-cost and environmentally friendly method for removing pollutants. Microorganisms can utilize pesticides as their sole carbon or nitrogen source for growth. They produce degrading enzymes that hydrolyze and oxidize pesticides. The main mode of microbial pesticide degradation is enzymatic degradation, where compounds enter bacteria through specific pathways and, under the action of various enzymes, undergo a series of physiological and biochemical reactions, ultimately partially or completely degrading the pesticide.

[0005] Currently, the following gene sequences for carbaryl hydrolase have been reported both domestically and internationally: mcb A (from) Pseudomonas sp.XWY-1); car H (from) Agrobacterium sp. XWY-2), cehA (from) Rhizobium sp. AC100). pch A (from) Pseudomonas sp. PS21); cah A (from) Arthrobacter (sp. RC100), the hydrolysis product is α-naphthol. However, the amino acid sequences of these enzymes are relatively long, which is not conducive to their artificial synthesis and application. (III) Summary of the Invention

[0006] The purpose of this invention is to provide a recombinant esterase, an engineered bacterium, and its application in the hydrolysis of carbaryl. This esterase has a short amino acid sequence, is easy to synthesize and express artificially, and has high hydrolytic activity, and can be used to enhance the degradation of residual carbaryl in the environment.

[0007] The technical solution adopted in this invention is:

[0008] This invention provides a strain derived from Pseudomonas ( Pseudomonas A recombinant esterase (denoted as XQest) of sp., the amino acid sequence of which is shown in SEQ ID No. 2.

[0009] This invention relates to the encoding gene of the recombinant esterase, the nucleotide sequence of which is shown in SEQ ID No. 1.

[0010] This invention also provides a recombinant plasmid containing the recombinant esterase encoding gene (preferably pET-28a(+)-GE003489), and a recombinant genetically engineered bacterium obtained by transformation of the recombinant plasmid (preferably...). E. coli BL21(DE3)-pET-28a(+)-GE003489).

[0011] Furthermore, the present invention also provides an application of the recombinant esterase in the hydrolysis of carbaryl, specifically: using purified enzyme solution extracted from wet cell lysis obtained by induced fermentation of engineered bacteria containing the recombinant esterase encoding gene as a catalyst, carbaryl as a substrate, and a buffer solution with pH 7.0-9.0 as a reaction medium, the hydrolysis reaction is carried out under the conditions of 20-60 °C and 120-240 rpm (preferably 37 °C and 200 rpm).

[0012] Preferably, the reaction medium is a 50 mM phosphate (PB) buffer solution with pH 7.4; the amount of substrate added is 0.1-1 g / L based on the total volume of the reaction system, preferably 0.1 g / L; and the amount of catalyst used is 80-200 mg / L based on the protein content, preferably 100 mg / L.

[0013] Further, the catalyst is prepared as follows: (1) wet bacterial cells are resuspended in pre-cooled pH 7.0, 50 mM phosphate buffer to obtain a bacterial suspension; the bacterial suspension is sonicated at 0-4 ℃ and 350-450 W for 10-15 min, with a working interval of 2 s and a 3 s interval during sonication; the obtained lysate is centrifuged at 4 ℃ and 5500-8000 rpm for 30 min, and the supernatant is collected; (2) the Ni-NTA affinity chromatography column is equilibrated with pH 7.4, 50 mM PB buffer containing 10-20 mM imidazole, and the supernatant obtained in step (1) is loaded at a rate of 1 ml / min. After loading, the column is first equilibrated with pH 7.4, 50 mM PB buffer containing 20-40 mM imidazole. The column was washed with mM PB buffer until the UV absorption reached a stable baseline. The target protein was then eluted in one step with 50mM PB buffer at pH 7.4 containing 250-300mM imidazole. The eluent containing the target protein was collected, dialyzed, and concentrated to obtain the purified enzyme solution of recombinant esterase.

[0014] Preferably, the dialysis and concentration method is as follows: using a dialysis bag with a molecular weight cutoff of 3500, dialyze in a pH 7.4, 50 mMPB buffer to remove salt ions from the eluent, and then concentrate the retentate to 1 / 10 of the original volume with polyethylene glycol (20000). The concentrate is then taken to obtain the purified enzyme solution of recombinant esterase.

[0015] Preferably, the wet bacterial cell concentration in the bacterial suspension is 0.5-35 g / L (preferably 10 g / L). The ultrasonic disruption conditions are 4 ℃, 400 W, and 15 min.

[0016] Preferably, the wet bacterial cells are obtained according to the following steps: Engineered bacteria containing the recombinant esterase encoding gene are inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured in a constant temperature shaker at 37 ℃ and 200 rpm for 12-16 h to obtain a seed culture; then, the seed culture is transferred to LB liquid medium containing 50 μg / mL kanamycin at a volume concentration of 1% and cultured in a constant temperature shaker at 37 ℃ and 200 rpm until the OD 600 reaches 0.6-0.8; IPTG is added to a final concentration of 0.1 mM, and the culture is induced in a constant temperature shaker at 24 ℃ and 180-200 rpm for 14 h; after fermentation, the culture is centrifuged at 4 ℃ and 5500-8000 rpm for 15 min to obtain wet bacterial cells.

[0017] Compared with existing technologies, the beneficial effects of this invention are mainly reflected in:

[0018] This invention identifies a novel esterase gene, GE003489, from Pseudomonas bacteria capable of hydrolyzing carbaryl. This gene is 948 bp in length (from start codon to stop codon) and encodes 315 amino acids. The encoded esterase is named XQest. Compared to existing esterases, this recombinant esterase has a shorter amino acid length, which is beneficial for artificial synthesis and exogenous expression.

[0019] The recombinant esterase encoding gene provided by this invention was cloned and expressed in Escherichia coli to construct engineered bacteria. E. coli The recombinant esterase expressed by BL21(DE3)-pET-28a(+)-GE003489 exhibits good hydrolytic ability for carbaryl. Using an appropriate amount of recombinant esterase to catalyze the hydrolysis of carbaryl at a substrate concentration of 0.1 g / L and a catalytic time of 1 h, the substrate conversion rate was 90.5%. This enzyme has potential application value for removing carbaryl contamination from the environment. (iv) Description of the attached drawings

[0020] Figure 1 Example 1: Agarose gel electrophoresis image of the PCR amplification product of the target gene. Lane 1: Target gene.

[0021] Figure 2 Agarose gel electrophoresis of recombinant plasmid PCR amplification products; Lane 1: pET-28a(+)-GE003489 recombinant plasmid; Lane 2: pET-28a(+) empty plasmid; Lane 3: amplified fragment of the target gene GE003489.

[0022] Figure 3 engineered bacteria E. coli SDS-PAGE image of the expression product BL21(DE3)-pET-28a(+)-GE003489; Marker; Lane 1: empty plasmid pET-28a(+); Lane 2: without IPTG induction; Lane 3: with IPTG induction; Lane 4: recombinant esterase purified by affinity.

[0023] Figure 4 HPLC chromatogram at the initial time 0 of the hydrolysis of 0.1 g / L carbaryl catalyzed by recombinant esterase.

[0024] Figure 5 HPLC chromatogram of the hydrolysis of 0.1 g / L carbaryl catalyzed by recombinant esterase for 20 min.

[0025] Figure 6 HPLC chromatogram of the hydrolysis of 0.1 g / L carbaryl catalyzed by recombinant esterase for 40 min.

[0026] Figure 7 Phylogenetic tree of esterase XQest and carbaryl hydrolase as reported in the literature.

[0027] Figure 8 Esterase XQest and other enzymes with the same function have been reported. ceh A ( Rhizobium Pairwise alignment of the amino acid sequences of the esterase encoded by the sp. AC100 gene. (V) Detailed Implementation Methods

[0028] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto: the experimental methods described in the following embodiments are conventional methods unless otherwise specified; the reagents and biological materials described are commercially available unless otherwise specified.

[0029] The Pseudomonas aeruginosa used in the embodiments of the present invention ( Pesudomonas sp.) zjut126 is deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M2017635, and has been published in patent application CN109321487A.

[0030] Example 1: Amplification of the target gene GE003489 and construction of recombinant genetically engineered bacteria

[0031] 1. Pseudomonas ( Pseudomonas Extraction of genomic DNA from sp.) zjut126.

[0032] Pseudomonas zjut126 was inoculated into seed culture medium and cultured at 30 °C and 200 rpm for 12–16 h to obtain seed culture. The seed culture was then transferred to fermentation medium at a volume concentration of 1% and cultured at 30 °C and 200 rpm for 12 h. The fermentation broth was centrifuged, and the wet cells were collected. Genomic DNA of the strain was extracted using the Ezup column-based bacterial genomic DNA extraction kit (product number SK8255).

[0033] Seed culture medium: K2HPO4 0.8 g, KH2PO4 3.0 g, NaCl 1.0 g, MgSO4 0.4 g, trace element solution 10.0 mL, add 800 mL deionized water, adjust pH to 7.0, then make up to 1 L with deionized water, sterilize at 121℃ for 20 min;

[0034] Trace element solution: CoCl2 0.1 g / L, MnSO4 0.5 g / L, FeSO4 7H2O 0.1 g / L, CuSO4 0.1 g / L, ZnSO4 7H2O 0.1 g / L, H3BO3 0.01 g / L, Al2(SO4)3 12H2O 0.01 g / L, Na2MoO4 2H₂O 0.01g / L, EDTA 2Na 1.0 g / L, solvent is deionized water; preparation method: accurately weigh 1.0 g EDTA Dissolve 2Na in 800mL of deionized water, then add the remaining components in sequence, and finally add deionized water to make up to 1 L;

[0035] Fermentation medium: 15.0 g tryptone, 5.0 g yeast extract, 0.5 g NaCl, 1.0 g K2HPO4, 1.0 g MgSO4, 0.8 g KH2PO4, add 800 mL deionized water, adjust pH to 7.0, then bring the volume to 1 L with deionized water, and sterilize at 121 ℃ for 20 min.

[0036] 2. Amplification of the target gene

[0037] Using the genomic DNA extracted in step 1 as a template, the target gene was amplified. Specific primers with plasmid homologous arms were designed as follows:

[0038] Upstream primer: CTGGTGCCGCGCGGCAGCCATATGAAACGATTCCTCCTCGGTCTG;

[0039] Downstream primer: GGTGGTGGTGGTGGTGCTCGAGTCAGCGACCGGCCACCTGGGCG.

[0040] Table 1. PCR reaction procedure for target gene amplification

[0041]

[0042] After the PCR reaction, the amplification products were detected by agarose gel electrophoresis. A clear band was observed at approximately 1000 bp (see...). Figure 1 This is consistent with the expected size (948bp).

[0043] 3. Construction of recombinant Escherichia coli

[0044] pET28a(+) was linearized using reverse PCR. The primers for reverse PCR are as follows:

[0045] Upstream primer: GAGCACCACCACCACCACCACCACTG;

[0046] Downstream primer: GCGACCCATTTGCTGTCCACCAGTC.

[0047] Table 2 Reverse PCR reaction procedure

[0048]

[0049] Recombination reaction: Take 1 μL of linearized pET-28a(+) product, add 1 μL of the target gene fragment, 5 μL of DNAAssembly Mix, and finally add ddH2O to bring the volume to 10 μL. Set the PCR instrument to 58 ℃ for 20 min and then quickly place it on ice to cool.

[0050] Take 10 μL of the above recombinant reaction product and mix it with 100 μL of... E. coli After gently mixing BL21(DE3) competent cells, they were incubated on ice for 30 min, then transferred to a 42 ℃ water bath and heat-shocked for 90 s. The cells were then immediately placed on ice, and 900 μL of LB liquid medium was added. The cells were incubated at 37 ℃ and 200 rpm for 45-60 min. After centrifugation at 5000 rpm for 5 min, the supernatant was removed, and the cells were resuspended in physiological saline. The bacterial resuspension was spread onto LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37 ℃. Single colonies were randomly selected from several plates and inoculated into LB liquid medium containing 50 μg / mL kanamycin, and incubated overnight at 37 ℃. Recombinant plasmids were extracted using a plasmid DNA mini-scale kit, with the pET28a(+) empty vector as a control, and plasmid PCR verification was performed (see [link to relevant documentation]). Figure 2 A band of approximately 1000 bp was observed in lane 3, consistent with expectations. The recombinant plasmid was then sequenced, and the sequencing results confirmed the presence of recombinant *E. coli*. E. coli BL21(DE3)-pET-28a(+)-GE003489 was successfully constructed, and the nucleotide sequence of the recombinant esterase is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0051] LB liquid medium: 5.0 g yeast extract, 10.0 g tryptone, 10.0 g sodium chloride, add 800 mL deionized water, adjust pH to 7.0, then bring the volume to 1 L with deionized water, and sterilize at 121 °C for 20 min.

[0052] LB solid medium: Add 20 g / L agar powder to LB liquid medium.

[0053] SEQ ID NO.1

[0054] ATGAAACGATTCCTCCTCGGTCTGGTTCTGCTGCTGGCGGTCGCCGCCGGCGTCCTCTACTTCGTCCCGGCTACCCTCCTCGCCAGCGTACGCACCGTCGAGCGCGGTCTCGCCGGTCTCAGCGAGCACAGCGTGCAGGTCGACAACCTGGAGATCGCCTACCTGGAAGGTGGCTCGGAGAAGAACCCGACCCTGTTGCTGATCCACGGCTTCGGCGCCGACAAGGACAACTGGCTGCGCTTCGCCCGGCCGCTGACCGAGCGCTACCATGTGGTCGCCCTCGACCTGCCCGGCTTCGGCGACAGCAGCAAGCCGCAACAGGCCAGCTACGACGTCGGCACCCAGGCCGAGCGAGTCGCCAATTTCGCCGCCGCCATCGGCGTGCGCCGCCTGCACCTGGCCGGCAACTCCATGGGCGGGCACATCGCCGCGCTCTACGCGGCGCGCCATCCGGAACAGGTGCTATCGCTGGCGCTGATCGACAACGCCGGGGTGATGCCGGCGCGCAAGAGCGAACTGTTCGAGGACCTGGAGCGCGGCGAGAATCCCCTGGTGGTGCGCCAGCCGGAAGACTTCCAGAAGCTGCTCGACTTCGTGTTCGTCCAGCAACCGCCGCTGCCGGCGCCGCTCAAGCGCTACCTCGGCGAACGCGCGGTAGCCGCGTCGGCGTTCAACGCGCAGATATTCGAACAACTGCGCCAGCGCTACATCCCGCTGGAGCCGGAACTGCCGAAGATCGAGGCACCGACCCTGCTGCTCTGGGGCGACCGCGACCGCGTGCTGGACGTCTCCAGCATCGAGGTGATGCGTCCGCTGCTGAAGCGGCCCAGCGTGGTGATCATGGAAAACTGCGGACACGTGCCGATGGTCGAACGCCCGGAGGAAACCGCGCAGCACTACCAGGCCTTCCTCGACGGCGTACGGAACGCCCAGGTGGCCGGTCGCTGA。

[0055] SEQ ID NO.2

[0056] MKRFLLGLVLLLAVAAGVLYFVPATLLASVRTVERGLAGLSEHSVQVDNLEIAYLEGGSEKNPTLLLIHGFGADKDNWLRFARPLTERYHVVALDLPGFGDSSKPQQASYDVGTQAERVANFAAAIGVRRLHLAGNSMGGHIAALYAARHPEQVLSLA LIDNAGVMPARKSELFEDLERGENPLVVRQPEDFQKLLDFVFVQQPPLPAPLKRYLGERAVAASAFNAQIFEQLRQRYIPLEPELPKIEAPTLLLWGDRDRVLDVSSIEVMRPLLKRPSVVIIMENCGHVPMVERPEETAQHYQAFLDGVRNAQVAGR.

[0057] Example 2: Expression and affinity chromatography purification of recombinant esterase

[0058] 1. Induction of recombinant esterase expression by engineered bacteria

[0059] Pick the recombinant Escherichia coli preserved on the plate in Example 1 E. coli A single colony of BL21(DE3)-pET-28a(+)-GE003489 was inoculated into LB liquid medium containing 50 μg / mL kanamycin resistance and cultured at 37 ℃ and 200 rpm for 12 h. A 1% (v / v) inoculum was then transferred to 50 mL of LB liquid medium containing 50 μg / mL kanamycin resistance and cultured at 37 ℃ and 200 rpm for 3 h until the OD 600 reached 0.6-0.8. Then, 50 μL IPTG was added to a final concentration of 0.1 mM, and the culture was induced at 24 ℃ and 200 rpm for 14 h to obtain the induction culture. After induction, the induction culture was analyzed by SDS-PAGE electrophoresis. The results are as follows: Figure 3 As shown in the figure, after IPTG induction, the recombinant E. coli expressed the target protein, with a size of approximately 34 kDa, which is consistent with the expected size (34.8 kDa).

[0060] 2. Affinity purification of recombinant esterase

[0061] (1) Ultrasonic disruption: Centrifuge the induction culture medium at 4 ℃ and 8000 rpm for 15 min, and collect the precipitate as wet bacterial cells. Resuspend the wet bacterial cells at 10 g / L in pre-cooled pH 7.0, 50 mM phosphate (PB) buffer. The bacterial suspension is ultrasonically disrupted at 4 ℃ and 400 W for 10 min, with a 2 s interval and a 3 s interval. Centrifuge the obtained disrupted liquid at 4 ℃ and 8000 rpm for 30 min, and collect the supernatant, which is the crude enzyme solution.

[0062] (2) Ni-NTA affinity chromatography: The Ni-NTA affinity chromatography column (GXK 16 / 20 jacketed protein chromatography column, 1.6×20cm) was equilibrated with PB buffer containing 10mM imidazole at pH 7.4 and 50mM. The supernatant obtained in step (1) was loaded at a rate of 1 mL / min. The column was then washed with PB buffer containing 20mM imidazole at pH 7.4 and 50mM until the UV absorption reached a stable baseline. Finally, the target protein was eluted in one step with PB buffer containing 250mM imidazole at pH 7.4 and 50mM, for 10 column volumes. Collect the eluent containing the target protein. Dialyze the eluent in dialysis buffer (pH 7.4, 50 mM PB buffer) using a dialysis bag (molecular weight cutoff 3500) to remove salt ions. The retentate is then concentrated to 1 / 10 of its original volume using polyethylene glycol (20000). The concentrate is collected to obtain the purified enzyme solution of the recombinant esterase. The purified protein bands are shown in the figure. Figure 3 Lane 4. Protein content was determined using the Bradford method for subsequent hydrolysis reactions.

[0063] Example 3: Recombinant esterase catalyzes the hydrolysis of carbaryl to generate α-naphthol.

[0064] Catalytic system: 0.1 g / L of the substrate carbaryl was added to 50 mM PB buffer (pH 7.4) as the reaction medium, along with 50 μL of purified enzyme solution of the recombinant esterase prepared in Example 2 (the amount of purified enzyme solution added was 100 mg / L based on protein content). The total reaction system was 1 mL, and the reaction was carried out in a metal bath at 37 ℃ and 200 r / min for 1 h. The conversion rate of the substrate was 90.5%.

[0065] Samples were taken at 0, 20, and 40 min, and reversed-phase HPLC was used to detect substrate consumption and product formation. The results are as follows: Figure 4 , Figure 5 , Figure 6 As shown. The main product of the hydrolysis of carbaryl esterase is α-naphthol, and the reaction formula is as follows:

[0066]

[0067] The reversed-phase HPLC was performed using a Shimadzu LC-20AT chromatograph with a 4.6 × 250 mm, 5 μm C18 column. The mobile phase was acetonitrile:water:TFA = 60:40:0.1 (v / v / v), the flow rate was 1.0 mL / min, the detection wavelength was 210 nm, the column temperature was 30℃, and the injection volume was 10 μL.

[0068] Substrate conversion (C) is calculated using the following formula:

[0069]

[0070] Note: C s0 and C s denoted as substrate concentration at the initial reaction time and at time t, respectively.

[0071] Example 4: Amino acid sequence alignment of recombinant esterase XQest with NCBI database

[0072] The Blast+ program was used on the NCBI website to perform amino acid sequence alignment of the recombinant esterase XQest in the protein database. The selected database was Non-redundant protein sequences (nr), and other parameters were left at their default values.

[0073] Table 3 lists the top 5 most matching entries from the returned results. As can be seen from the table, the first 3 proteins have 100% amino acid sequence identity with the recombinant esterase XQest, and they all originate from... Pseudomonas The annotation is "alpha / beta fold hydrolase".

[0074] Table 3. Results of amino acid sequence alignment of esterase XQest in the NCBI protein database.

[0075]

[0076] Example 5: Evolutionary analysis of recombinant esterase XQest and reported carbaryl hydrolase.

[0077] The recombinant esterase XQest was combined with the previously reported carbaryl hydrolase gene. mcb A ( Pseudomonas sp.XWY-1), car H ( Agrobacterium sp. XWY-2), ceh A ( Rhizobium sp. AC100), pch A ( Pseudomonas sp. PS21) and cahA ( Arthrobacter A phylogenetic tree of the enzyme encoded by sp. RC100 was constructed using Mega 7.0, as follows: Figure 7 As shown, XQest and ceh The esterase encoded by gene A is most closely related.

[0078] In Clustal Omega, the esterase XQest (amino acid sequence shown in SEQ ID NO.2) and the previously reported seviin hydrolase gene were compared. ceh A ( Rhizobium The amino acid sequences of the esterase encoded by sp. AC100 were pairedly aligned, and the alignment results are shown below. Figure 8 esterase XQest and ceh The sequence identity of the esterase encoded by A is only 13.0%. Therefore, esterase XQest is a novel esterase that can hydrolyze carbaryl.

Claims

1. A species derived from Pseudomonas ( Pseudomonas A recombinant esterase of sp., characterized in that, The amino acid sequence of the recombinant esterase is shown in SEQ ID No.

2.

2. A recombinant genetically engineered bacterium containing the recombinant esterase encoding gene of claim 1.

3. The use of the recombinant esterase of claim 1 in the hydrolysis of carbaryl.

4. The application as described in claim 3, characterized in that, The application is as follows: using purified enzyme solution extracted from wet bacterial cells obtained by induced fermentation of engineered bacteria containing recombinant esterase encoding gene as a catalyst, using carbaryl as a substrate, and using a buffer solution with pH 7.0-9.0 as a reaction medium, the hydrolysis reaction is carried out at 20-60 ℃ and 120-240 rpm.

5. The application as described in claim 4, characterized in that, The reaction medium is a 50 mM phosphate buffer solution with a pH of 7.

4.

6. The application as described in claim 4, characterized in that, The amount of substrate added is 0.1-1 g / L based on the total volume of the reaction system, and the amount of catalyst used is 80-200 mg / L based on the protein content.

7. The application as described in claim 4, characterized in that, The catalyst was prepared as follows: (1) The wet bacterial cells were resuspended in pre-cooled pH 7.0, 50 mM phosphate buffer to obtain a bacterial suspension; the bacterial suspension was sonicated at 0-4 ℃ and 350-450 W for 10-15 min, with a working time of 2 s and an interval of 3 s during sonication; the obtained lysate was centrifuged at 4 ℃ and 5500-8000 rpm for 30 min, and the supernatant was collected; (2) The Ni-NTA affinity chromatography column was equilibrated with pH 7.4, 50 mM PB buffer containing 10-20 mM imidazole, and the supernatant obtained in step (1) was loaded at a rate of 1 ml / min. After loading, the column was first washed with pH 7.4, 50 mM PB buffer containing 20-40 mM imidazole until the UV absorption reached a stable baseline, and then washed with pH 7.4, 50 mM PB buffer containing 250-300 mM imidazole. The target protein was eluted in one step with mM PB buffer, and the eluent containing the target protein was collected, dialyzed, and concentrated to obtain the purified enzyme solution of recombinant esterase.

8. The application as described in claim 7, characterized in that, The dialysis and concentration method is as follows: using a dialysis bag with a molecular weight cutoff of 3500, dialyze in pH 7.4, 50 mM PB buffer to remove salt ions from the eluent. The retentate is then concentrated with polyethylene glycol to 1 / 10 of its original volume. The concentrate is then collected to obtain the purified enzyme solution of recombinant esterase.

9. The application as described in claim 7, characterized in that, The wet bacterial cell concentration in the bacterial suspension is 0.5-35 g / L, and the ultrasonic disruption conditions are 4 ℃, 400 W, and 15 min.

10. The application as described in claim 4, characterized in that, The wet bacterial cells were obtained according to the following steps: Engineered bacteria containing the recombinant esterase encoding gene were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured in a constant temperature shaker at 37 ℃ and 200 rpm for 12-16 h to obtain a seed culture; then, the seed culture was transferred to LB liquid medium containing 50 μg / mL kanamycin at a volume concentration of 1% and cultured in a constant temperature shaker at 37 ℃ and 200 rpm until the OD 600 reached 0.6-0.8; IPTG was added to a final concentration of 0.1 mM and induced in a constant temperature shaker at 24 ℃ and 180-200 rpm for 14 h; after fermentation, the cells were centrifuged at 4 ℃ and 5500-8000 rpm for 15 min to obtain wet bacterial cells.

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