Esterase for degrading pyrethroid and construction of genetically engineered bacterium of esterase

Abstract

The invention discloses an esterase Est10 and a recombinant bacterium secreting the esterase Est10. The esterase contains 482 amino acids, and has an amino acid sequence as shown in SEQ ID NO. 1. The esterase gene is connected with an expression vector pET28a (+), and E.coli BL21 (DE3) is used as a host bacterium to construct a recombinant genetic engineering bacterium E.coli BL21 (DE3)-pET-28a (+)-est10. The genetically engineered bacterium and the esterase can be used for bioremediation of soil, water bodies and other environments polluted by pesticides, and can also be applied to reduction and removal of pesticide residues in agricultural products.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering and the field of residual pesticide reduction, and in particular to an esterase for degrading pyrethroids and a method for constructing the gene recombinant bacteria and obtaining the recombinant enzyme. Background Art

[0002] Pyrethroid pesticides are one of the most widely used insecticides, used to control a variety of pests in agricultural and forestry crops such as fruits, vegetables, and grains, as well as in households. The large-scale use of pyrethroids has led to their long-term presence in the environment and gradual accumulation in agricultural and forestry crops, seriously endangering ecological and environmental safety and food safety. At the same time, pyrethroids can also enter the human body through various pathways such as ingestion, respiration, and skin through media such as air, dust, water, and food, which may cause a series of toxic damage.

[0003] Esterase is a general term for hydrolases that catalyze the breaking of ester bonds. It has a wide source, high catalytic activity, mild reaction conditions, and does not require the participation of coenzymes. It can be used for the separation of chiral compounds and drug synthesis in the pharmaceutical industry, for improving the flavor of brewed food in the food industry, and for the removal of pyrethroid pesticides in the environment and food.

[0004] Ester bond cleavage is the first step in the degradation of pyrethroid pesticides. In this process, esterase plays an extremely important role. Microorganisms have fast reproduction speed and short culture cycle, and are one of the important sources of esterase. Starting from pyrethroid-degrading microorganisms, finding esterase genes and heterologously expressing them, constructing genetically engineered bacteria, and obtaining esterases, and further investigating the degradation ability of esterases on pyrethroids are important means to expand the resources of pyrethroid-degrading enzymes, which are of great significance in environmental bioremediation and the reduction of pesticides in food and human body. Summary of the invention

[0005] The purpose of the present invention is to provide a protein encoded by the esterase gene, which can effectively degrade pyrethroid pesticides. The amino acid sequence of the esterase is SEQ ID NO.1.

[0006] The enzyme protein consists of 482 amino acids, does not contain a signal peptide, has a molecular weight of 52.882 KDa, and an isoelectric point pI of 4.91.

[0007] The object of the present invention also includes providing a recombinant strain expressing the above esterase.

[0008] The invention also provides a method for producing esterase by using the recombinant strain.

[0009] The purpose of the present invention is achieved through the following solutions: The esterase gene of the present invention est10 Derived from a strain of Bacillus subtilis ( Bacillus subtilis )J6.

[0010] The upstream primer 5'-ATGACAAAACTTACCGTTCAAACCC-3' and the downstream primer 5'-TTATGCTTGAAACAGGATGCGGC-3' were designed. PCR amplification was performed using the genomic DNA of Bacillus subtilis J6 as a template. After agarose gel electrophoresis detection, the gene fragment was recovered and the fragment was TA-ligated with the pMD18T vector to construct a cloning vector, which was transferred into Escherichia coli DH5α. The bacterial solution was spread on LB solid medium containing 100 mg / L ampicillin, and white single colonies were obtained by blue-white screening. The bacterial cells were collected to extract the plasmid, and the cloning vector was PCR amplified using primers. Sequencing and comparison analysis were performed at the same time to obtain positive clone strains.

[0011] According to the expression vector pET-28a (+) and the target gene sequence, select appropriate restriction sites and protective bases to design specific primers: upstream primer 5'-CATGCCATGGCCATGACAAAACTTACCGTTCAAACCC-3', downstream primer 5'-CCGCTCGAGTGCTTGAAACAGGATGCGGCGTTTT-3'. Use restriction endonucleases Nco I and XOt I was treated in a 37°C water bath for 2 h to express the expression vector pET-28a(+) and the target gene est10 Double enzyme digestion was performed, and the fragments were purified and recovered using a PCR purification kit. T4 ligase was used for ligation, and the competent cells BL21 (DE3) were transformed. The bacterial solution was spread on an LB plate containing kanamycin and cultured at 37°C for 10-12 h to obtain positive transformants, that is, genetically engineered bacteria. E. coli BL21(DE3)-pET-28a(+)- est10 .

[0012] The induction method for obtaining the secretion expression of esterase by genetically engineered bacteria is carried out according to the following steps.

[0013] Genetically engineered bacteria E. coli BL21(DE3)-pET-28a(+)- est10 The glycerol bacteria were inoculated into LB liquid culture medium containing 40 mg / L kanamycin and cultured overnight at 37°C with shaking to obtain a genetically engineered bacterial liquid.

[0014] The above bacterial solution was inoculated into LB liquid medium containing 40 mg / L kanamycin at a 1% inoculum volume and cultured at 37°C and 160 rpm until the OD 600was 0.8; IPTG was added to a final concentration of 1 mmol / L, and cultured at 20°C and 160 rpm for 16 h.

[0015] The beneficial effects of the present invention are mainly reflected in: providing an esterase gene derived from Bacillus subtilis J6 est10 The constructed recombinant bacteria can express the recombinant esterase Est10; the genetically engineered bacteria of the present invention are easy to culture, the esterase can catalyze the hydrolysis of pyrethroid pesticides, the reaction conditions are mild, and the bacteria has good application development potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 for est10 Electrophoresis of PCR amplification products, lane M is DNA Marker DL2000, lane 1 is amplified est10 .

[0017] Figure 2 Genetically engineered bacteria E. coli BL21(DE3)-pET-28a(+)- est10 HPLC chromatogram of degradation of highly effective cypermethrin.

[0018] Figure 3 The catalytic ability of Est10 on the hydrolysis of highly effective cypermethrin at different times. Implementation

[0019] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0020] Embodiment 1: est10 Acquisition of esterase genes.

[0021] Design upstream primer F: 5'-ATGACAAAACTTACCGTTCAAACCC-3', downstream primer R: 5'-TTATGCTTGAAACAGGATGCGGC-3'. Extract genomic DNA of Bacillus subtilis J6 and use this DNA as a template to perform PCR amplification under the action of Ex Taq polymerase (see Figure 1 ).

[0022] PCR amplification system (50 μL): 34.5 μL of sterile ultrapure water, 5 μL of 10×Ex Taq Buffer, 4 μL of dNTPMixture, 2 μL of upstream primer, 2 μL of downstream primer, 2 μL of template DNA, and 0.5 μL of Ex Taq polymerase.

[0023] PCR amplification conditions: 98°C 30 s; 98°C 10 s, 65°C 30 s, 72°C 30 s, 10 cycles, annealing temperature decreased by 1°C after each cycle; 98°C 10 s, 55°C 30 s, 72°C 30 s, 20 cycles; 72°C 2 min; end at 4°C.

[0024] Example 2: Engineered strains containing esterase genes est10 Build.

[0025] After gel electrophoresis was confirmed, the

[0022] gene fragment was recovered and TA-ligated with the pMD18T vector. The ligation system was as follows: 0.2 μL of pMD18-T vector, 4.8 μL of DNA template, and 5.0 μL of ligation buffer. The mixture was placed in a PCR instrument at 16°C for 10 h.

[0026] Take the ligation product and add DH5α, and place it on ice for 30 min. Then, heat it at 42℃ for 90 s, take it out and transfer it smoothly, place it on ice for 3min, add 500 µL LB medium, and culture it at 37℃ and 160 rpm for 1 h for blue-white screening. The steps of blue-white screening are as follows: take 8 µL 500 mmol / L IPTG and 32 µL 50 mg / mL X-gal, mix them evenly, and then spread them on LB plates containing 100 µg / mL Amp, place them for 30 min, take 200 µL of the transformed bacterial culture solution for coating, place it in a 37℃ incubator, adsorb it upright for 0.5~1 h, and then flip it upside down for 12~24 h. Collect the bacteria to extract the plasmid, use primers to amplify the cloning vector by PCR, and perform sequencing and comparison analysis at the same time to obtain positive clone strains.

[0027] According to the expression vector pET-28a (+) and the target gene sequence, select appropriate restriction sites and protective bases to design specific primers: upstream primer 5'-CATGCCATGGCCATGACAAAACTTACCGTTCAAACCC-3', downstream primer 5'-CCGCTCGAGTGCTTGAAACAGGATGCGGCGTTTT-3'. Use restriction endonucleases Nco I and XOt I was treated in a 37°C water bath for 2 h to express the expression vector pET-28a(+) and the target gene est10 Double enzyme digestion was performed, and the fragments were purified and recovered using a PCR purification kit. T4 ligase was used for ligation, and the competent cells BL21 (DE3) were transformed. The bacterial solution was spread on an LB plate containing 40 mg / L kanamycin and cultured at 37°C for 10-12 h to obtain positive transformants, that is, genetically engineered bacteria. E. coliBL21(DE3)-pET-28a(+)- est10 .

[0028] est10 Enzyme digestion system with expression vector pET-28a(+): purified and recovered est10 Fragment / pET-28a(+) 10.0 μL, 10×FD Buffer 5.0 μL, Nco I 1.0 μL, XOt I 1.0 μL, sterile ultrapure water 33.0 μL. Mix well and place in a 37℃ constant temperature water bath for 2 h.

[0029] est10 Connection system with expression vector pET-28a(+): enzyme digestion est10 8.0 μL of fragment, 4.0 μL of digested pET-28a(+), 2.0 μL of 10×T4 DNAligase Buffer, 1.0 μL of T4 DNAligase, 5.0 μL of sterile ultrapure water, and connected in a PCR instrument at 22°C for 1 h.

[0030] Example 3: Verification of genetically engineered bacteria E. coli BL21(DE3)-pET-28a(+)- est10 Degradation ability of cypermethrin.

[0031] The genetically engineered bacteria preserved in glycerol were inoculated into LB liquid medium containing 40 mg / L kanamycin for activation and cultured at 37°C and 160 rpm for 10-12 h.

[0032] The activated bacterial solution was transferred to LB liquid medium containing 40 mg / L kanamycin at a volume fraction of 1%, and cultured at 37°C and 160 rpm until OD 600 was 0.8; IPTG was added to a final concentration of 1 mmol / L, and cultured at 20°C and 160 rpm for 16 h.

[0033] 10 mg / mL of highly effective cypermethrin was added to the above culture medium to make the final concentration 20 mg / L, and cultured at 37°C and 160 rpm for 48 h. E. coli BL21(DE3)-pET-28a(+) was used as a negative control.

[0034] Take 1 mL of bacterial solution, add 4 mL of acetonitrile and ultrasonically extract for 30 min. After mixing, take 1.5 mL and centrifuge. Take the supernatant and filter it through a 0.22 μm organic microporous filter membrane. The filtrate is placed in an injection bottle and the concentration of highly effective chlorpyrifos is determined by HPLC.

[0035] The HPLC detection conditions were as follows: Gemini 100Å C18 column (5.0 µm, 150 mm×4.60 mm, id), mobile phase acetonitrile: ultrapure water = 83:17 ( v / v ), flow rate 1.0 mL / min, column temperature 25°C, UV detection wavelength 210 nm, injection volume 10 µL. The results showed that genetically engineered bacteria E. coli BL21(DE3)-pET-28a(+)- est10 The degradation rate of 20 mg / L cypermethrin was 30.37% (see Figure 2 ).

[0036] Example 4: Obtaining esterase Est10.

[0037] The genetically engineered bacteria are cultured, and the bacterial bodies are collected and crushed to obtain esterase.

[0038] The details are as follows.

[0039] 1) Seed solution preparation: The genetically engineered bacteria preserved in glycerol were inoculated into LB liquid medium containing 40 mg / L kanamycin and cultured at 37°C and 160 rpm for 10-12 h.

[0040] 2) Induction of genetically engineered bacteria: The above seed solution was transferred to LB liquid medium containing 40 mg / L kanamycin at a volume fraction of 1%, and cultured at 37°C and 160 rpm until OD 600 was 0.8; IPTG was added to a final concentration of 1 mmol / L, and cultured at 20°C and 160 rpm for 16 h.

[0041] 3) Obtaining esterase Est10 The above culture medium was centrifuged at 4℃ and 8000 rpm for 10 min, the cells were collected, and pH 7.0 0.02 mol / L Tris-HCl solution was added to resuspend the cells, and the supernatant was discarded after centrifugation at 4℃ and 8000 rpm for 10 min. The cells were collected for later use. The cells were resuspended in pH 7.0 0.02 mol / L Tris-HCl solution again, and the cells were disrupted with an ultrasonic cell disruptor in an ice bath, and the cells were centrifuged at 4℃ and 10000 rpm for 10 min, and the supernatant was collected.

[0042] First, the Ni-IDA column was equilibrated with binding / washing buffer, the supernatant obtained by

[0042] was loaded, and the flow-through was collected and loaded again; it was eluted with binding / washing equilibration buffer (10 mM imidazole, 500 mM NaCl, 20 mM NaH2PO4·2H2O, pH 7.9~8.1) to remove impurities; it was then eluted with elution buffer (250 mM imidazole, 500 mM NaCl, 20 mM NaH2PO4·2H2O, pH 7.9~8.1), the initial flow-through was discarded, and the subsequent flow-through was collected and dialyzed at 4°C for 24~48 h using a dialysis bag with a molecular weight cutoff of 8000~14000Da to obtain the Est10 enzyme solution.

[0043] Example 4: Recombinant esterase catalyzes the hydrolysis of cypermethrin.

[0044] Take 2.4 mL of pH 7.0 0.02 mol / L Tris-HCl buffer, add 0.1 mL of 0.3 mg / mL highly effective cypermethrin-acetone solution, add 0.5 mL of the enzyme solution obtained in Example 4, and react in a 37°C constant temperature water bath shaker (160 rpm) for 1, 2, 4, and 24 h. After the reaction, add an equal volume of acetonitrile, ultrasonically extract for 30 min, mix well, take 1.5 mL and centrifuge (12000 rpm, 10 min), take the supernatant and filter it through a 0.22 μm organic microporous filter membrane, and place the filtrate in a sample injection bottle. Determine the concentration of the substrate highly effective cypermethrin by the method described in

[0036] , and calculate the degradation rate. Use an equal volume of Tris-HCl buffer instead of the enzyme solution as a control.

[0045] The recombinant esterase could catalyze the hydrolysis of 16.45%, 19.93%, 29.18% and 43.18% of highly effective cypermethrin in 1, 2, 4 and 24 h (see Figure 3 ).

[0046] The above examples are merely descriptions of the embodiments of the present invention, and are not intended to limit the scope of the present invention. Those skilled in the art may improve or modify the above descriptions, but all such improvements or modifications should fall within the scope of protection defined by the claims of the present invention.

Claims

1. An esterase Est10 having ester bond hydrolysis function, characterized in that: Derived from Bacillus subtilis ( Bacillus subtilis ) Esterase gene of J6 est10 Encoded, and its amino acid sequence is shown in SEQ ID NO.

1.

2. Containing the esterase gene according to claim 1 est10 The recombinant vector is pET-28a(+)- est10 .

3. The recombinant strain of the esterase gene according to claim 1.

4. A method for preparing esterase, characterized in that: The following steps are involved: (1) Transform the host cell Escherichia coli BL21 with the recombinant vector pET-28a(+) to obtain the recombinant strain E. coli BL21(DE3)-pET-28a(+)- est10 ; (2) Inducing the expression of esterase Est10; (3) Lyse the recombinant strain, collect the supernatant, and purify it with Ni-IDA agarose purification resin.

5. Application of the esterase according to claim 1 in the removal of ester-containing pesticides in the environment, crops and agricultural products.

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