A mutant of pyrethroid-hydrolyzing enzyme PytH, its encoding gene and use

Abstract

The application belongs to the technical field of bioengineering, and provides a mutant of pyrethroid hydrolase PytH, a coding gene thereof and purposes, and the amino acid sequence of the mutant is shown as SEQ ID NO. 4. The mutant PytH-M5 provided in the application has a significantly improved degradation efficiency of various pyrethroid pesticides compared with the wild type PytH, can be used for removing pyrethroid pesticide residues in soil and water, can also be used for constructing pyrethroid pesticide-resistant transgenic crops, and has very important theoretical and application values in the fields of pesticide residue bioremediation and environmental protection.

Description

Technical Field

[0001] This invention relates to a mutant of pyrethroid-hydrolyzing carboxylesterase (PytH), its encoding gene, and its uses, belonging to the field of bioengineering technology. This mutant can be used to remove pyrethroid insecticides from crops and soil, remediate polluted water bodies, and produce green and pollution-free agricultural products. Background Technology

[0002] Pyrethroid pesticides, developed in the 1970s, are a class of synthetic insecticides that mimic the chemical structure of natural pyrethroids. They possess advantages such as high efficiency, broad spectrum, low toxicity, and low residue, and have now replaced organophosphate pesticides as one of the most widely used pesticide categories globally. According to the Food and Agriculture Organization of the United Nations (FAO), the global annual production of pyrethroid pesticides exceeds 100,000 tons, widely used in agricultural pest control, sanitary pest control, and wood preservation. However, the widespread use of pyrethroid pesticides has also brought about a series of environmental problems. Although these pesticides have low acute toxicity to mammals, they are highly toxic to non-target organisms such as fish and bees. Furthermore, some pyrethroid pesticides are chemically stable and have long residual periods in soil and water, posing a potential threat to ecosystems and human health through the food chain. Therefore, developing efficient and safe technologies for removing pyrethroid pesticide residues is of significant practical importance.

[0003] Microbial enzymatic degradation is a green and efficient method for removing pesticide residues from the environment. Biodegradation methods have advantages such as mild reaction conditions, no secondary pollution, and high selectivity, and have received increasing attention in recent years. PytH is derived from Sphingosine monophosphate JZ-1 (… Sphingobium A novel pyrethroid pesticide hydrolase, isolated and identified from sp. JZ-1, belongs to the alpha / beta hydrolase superfamily. It catalyzes the hydrolysis of ester bonds in various pyrethroid pesticides, breaking them down into detoxified acid and alcohol components. The enzyme has a molecular weight of approximately 31 kDa, an optimal pH of approximately 7.5, an optimal temperature of approximately 37°C, and a broad substrate spectrum. It can hydrolyze various pyrethroid pesticides, including permethrin, cypermethrin, deltamethrin, lambda-cyhalothrin, deltamethrin, bifenthrin, and cypermethrin.

[0004] However, the hydrolytic activity of wild-type PytH enzyme against pyrethroid pesticides still needs to be improved, limiting its efficiency in practical bioremediation applications. Therefore, modifying PytH using protein engineering techniques to obtain mutant enzymes with higher catalytic activity and stronger degradation efficiency has important theoretical and applied value for the bioremediation of pyrethroid pesticide residue pollution. Summary of the Invention

[0005] The purpose of this invention is to provide a mutant of the pyrethroid hydrolase PytH, its encoding gene, and its uses. The mutant has a significantly higher degradation efficiency for various pyrethroid pesticides than the wild-type enzyme, thus solving the problem that the hydrolytic activity of the wild-type PytH enzyme against pyrethroid pesticides needs to be improved in the prior art.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] This invention provides a mutant of pyrethroid hydrolase PytH, the amino acid sequence of which is shown in SEQ ID NO.4.

[0008] The present invention also provides the coding gene of the above-mentioned mutant, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0009] The present invention also provides a recombinant expression vector comprising the above-mentioned coding gene.

[0010] Preferably, the recombinant expression vector is obtained by inserting the coding gene between the Nde I and Xho I restriction sites of pET-29a(+).

[0011] The present invention also provides a genetically engineered bacterium containing the above-mentioned encoding gene.

[0012] Preferably, the expression strain of the genetically engineered bacteria is Escherichia coli BL21(DE3).

[0013] The present invention also provides the application of the above-mentioned mutant, the above-mentioned recombinant expression vector, or the above-mentioned genetically engineered bacteria in the degradation of pyrethroid pesticides.

[0014] Preferably, the application includes the removal of pyrethroid pesticide residues from soil and water.

[0015] Preferably, the pyrethroid pesticides include permethrin, cypermethrin, deltamethrin, lambda-cyhalothrin, deltamethrin, bifenthrin, and cypermethrin.

[0016] This invention also provides the application of the above-mentioned encoding gene in the construction of transgenic crops resistant to pyrethroid pesticides.

[0017] The beneficial effects of this invention are as follows:

[0018] This invention uses wild-type PytH enzyme as a starting template and, through directed evolution technology, multiple rounds of error-prone PCR and efficient screening, obtained the mutant PytH-M5 with significantly enhanced enzyme activity. The mutant PytH-M5 contains mutations at three amino acid sites: glycine at position 30 is mutated to aspartic acid (G30D), glycine at position 70 is mutated to serine (G70S), and proline at position 266 is mutated to threonine (P266T).

[0019] Compared with the wild-type PytH, the mutant PytH-M5 exhibits 2.1-fold, 1.8-fold, 2.3-fold, 1.6-fold, 2.5-fold, and 1.9-fold increased specific enzyme activities against permethrin, cypermethrin, deltamethrin, fenvalerate, and bifenthrin, respectively. The mutant PytH-M5 and its encoding gene can be used for the removal of pyrethroid pesticide residues from soil and water bodies, and can also be used to construct pyrethroid-resistant transgenic crops, possessing significant theoretical and applied value in the fields of pesticide residue bioremediation and environmental protection. Attached Figure Description

[0020] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0021] Figure 1 Homology modeling analysis for wild-type PytH protein and PytH-M5 mutant: A. Three-dimensional structure of wild-type PytH protein; B. Three-dimensional structure of PytH-M5 mutant.

[0022] Figure 2 This is a schematic diagram of the construction of the PytH-M5 recombinant expression vector.

[0023] Figure 3 SDS-PAGE electrophoresis patterns of wild-type PytH and mutant PytH-M5.

[0024] Figure 4 To investigate the effects of different pH and temperature conditions on the activity of PytH-M5 enzyme, we have: A. Relative enzyme activity (%) under different pH conditions, and B. Relative enzyme activity (%) under different temperature conditions. Detailed Implementation

[0025] Unless otherwise specified, all experimental materials used in the examples were commercially available products. Unless otherwise specified, all molecular biology procedures used in the examples were performed in accordance with *Molecular Cloning: A Laboratory Manual* (3rd edition, Science Press).

[0026] 1. Directed evolution screening of PytH mutants with significantly increased enzyme activity

[0027] 1.1 Synthesis of wild-type PytH gene

[0028] According to data collected by GenBank Sphingobium Specific primers were designed based on the PytH gene sequence (accession number FJ688006) of sp. JZ-1. Using the genomic DNA of this strain as a template, PCR amplification was performed using the Phanta® Max Super-Fidelity DNA Polymerase kit (Novizan, P515) with primers 1 (SEQ ID NO.5) and 2 (SEQ ID NO.6). The PCR primer sequences used are as follows:

[0029] Primer 1, forward primer:

[0030] 5'-ATGACCGTCACCGATATCATC-3' (SEQ ID NO.5),

[0031] Primer 2, reverse primer:

[0032] 5'-TCACGGTAGATCCAGCTCGGTC-3' (SEQ ID NO. 6).

[0033] The amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 1 min, for a total of 30 cycles; final extension at 72℃ for 5 min. The obtained PCR products were bidirectionally sequenced by Beijing Qingke Biotechnology Co., Ltd.; the resulting sequences were assembled and compared with the GenBank database. The obtained wild-type PytH gene was 843 bp in length, with the nucleotide sequence shown in SEQ ID NO.1, encoding 281 amino acids, the amino acid sequence of which is shown in SEQ ID NO.2. The PCR amplification product was cloned into the pUC19 vector, the recombinant plasmid was named pUC-pytH, and transformed into E. coli DH5α for preservation.

[0034] The nucleotide sequence of the wild-type PytH gene is as follows:

[0035] ATGACCGTCACCGATATCATCCTGATCCACGGCGCCTTGAACCGCGGCGCCTGCTATGACGCGGTCGTCCCGCTTCTCGAAGCGCGCGGCTACCGCGTCCATGCGCCCGACCTGACCGGCCATACGCCCGGCGATGGCGGCCATTTGTCGGTCGTCGACATGGAGCATTATACCCGCCCAGTCGCTGACATCCTGGCACGGGCCGAGGGGCAGTCGATCCTTCTGGGGCACAGCTTGGGCGGTGCATCCATCTCGTGGCTGGCGCAGCACCATCCCGACAAGGTGGCCGGGCTGATCTACCTGACCGCGGTCCTCACCGCGCCCGGTATAACGCCGGAAACCTTCGTCCTGCCCGGCGAGCCCAACCGGGGCACGCCGCACGCGCTGGACCTGATCCAGCCGGTCGACGAGGGACGTGGGCTACAGGCGGATTTCTCGCGACTGGAACGGCTCCGCGAAGTTTTCATGGGCGATTATCCCGGCGAGGGAATGCCGCCTGCCGAACAGTTCATCCAGACCCAGTCGACCGTGCCCTTTGGCACGCCCAATCCGATGGAGGGGCGCGCGCTGGAAATCCCACGCCTCTATATCGAGGCGCTGGACGATGTCGTGATTCCGATCGCCGTGCAGCGTCAGATGCAGAAGGAGTTCCCCGGTCCGGTCGCGGTCGTGTCGCTGCCGGCCAGCCATGCGCCCTATTACTCGATGCCCGAACGGCTGGCCGAGGCGATCGCCGATTTCGCCGATGCCCCGGCCGAGTATCGCCAGACGGCGACGAAGGCTGGGCCTGATCGACCAGCTGGAGCGGACGGGGGTCGAGCCGACCGAGCTGATCTACCGTGA(SEQ ID NO. 1);

[0036] The amino acid sequence of wild-type PytH protein is as follows:

[0037] MTVTDIILIHGALNRGACYDAVVPLLEARGYRVHAPDLTGHTPGDGGHLSVVDMEHYTRPVADILARAEGQSILLGHSLGGASISWLAQHHPDKVAGLIYLTAVLTAPGITPETFVLPGEPNRGTPHALDLIQPVDEGRG LQADFSRLERLREVFMGDYPGEGMPPAEQFIQTQSTVPFGTPNPMEGRALEIPRLYIEALDDVVIPIAVQRQMQKEFPGPVAVVSLPASHAPYYSMPERLAEAIADFADAPAEYRQTATKAGPDRPAGADGGRADRADLP (SEQ ID NO.2).

[0038] 1.2 Construction of PytH gene mutation library

[0039] Using plasmid pUC-pytH as a template, error-prone PCR was performed using the StarMut random mutation kit (Kangrun Biotechnology, T115). The primers used were the same as above: primer 1 (SEQ ID NO. 5) and primer 2 (SEQ ID NO. 6). The pytH gene mutated during amplification due to random base mismatches, and a random mutation library was constructed based on this mutation.

[0040] Error-prone PCR amplification systems (50 μL) are shown in Table 1.

[0041] Table 1. Error-prone PCR amplification system (50 μL)

[0042]

[0043] Commonly misused PCR amplification procedures:

[0044] a. Pre-denaturation at 95℃ for 2 min;

[0045] b. Denaturation at 94℃ for 0.5 min, annealing at 52℃ for 1.0 min, extension at 72℃ for 1.0 min, for 30 cycles;

[0046] c. Extend at 72°C for 10 minutes, then cool to room temperature.

[0047] Error-prone PCR products were double-digested with restriction endonucleases Nde I (Takara, 1161A) and Xho I (Takara, 1094S). The digestion system is shown in Table 2.

[0048] Table 2. Double enzyme digestion reaction system

[0049]

[0050] After digestion at 37℃ for 12 h, the mutated gene fragments were separated and recovered by agarose gel electrophoresis and ligated with the pET-29a(+) vector that had undergone the same digestion using T4 DNA ligase (Thermo Fisher Scientific, 15224041) to construct a mutant library. The enzyme ligation system is shown in Table 3.

[0051] Table 3. Enzyme ligation system

[0052]

[0053] The obtained ligation product was transformed into E. coli BL21(DE3) competent cells, plated on LB agar plates containing 100 mg / L kanamycin, and incubated overnight at 37°C to obtain a PytH random mutant library.

[0054] 1.3 High-throughput screening of PytH gene mutation libraries

[0055] Transformants from the PytH random mutant library were selected as single clones and inoculated into 96-well deep-well plates containing 100 mg / L kanamycin and 0.4 mM cypermethrin in LB liquid medium. The plates were incubated at 37°C and 200 rpm for 24 h. The cultured bacterial suspension was centrifuged, and the cell pellet was collected. The pellet was washed twice with 50 mM PBS buffer (pH 7.5) and resuspended in 50 mM PBS reaction buffer (pH 7.5) containing 0.4 mM cypermethrin. The reaction was incubated at 30°C for 2 h. After the reaction was complete, an equal volume of acetonitrile was added to terminate the reaction. The supernatant was centrifuged, and the residual amount of cypermethrin was detected by gas chromatography (GC). The degradation rate of each clone was calculated using the reaction solution without inoculated strains as a control.

[0056] Positive clones with significantly higher degradation rates than wild-type PytH were selected, and plasmids were extracted and sequenced to analyze amino acid sequence mutations. After multiple rounds of screening and validation, a mutant with significantly increased enzyme activity was finally obtained and named PytH-M5.

[0057] 1.4 PytH-M5 mutant sequence analysis

[0058] The nucleotide sequence of the gene encoding the mutant PytH-M5 is as follows:

[0059] ATGACCGTCACCGATATCATCCTGATCCACGGCGCCTTGAACCGCGGCGCCTGCTATGACGCGGTCGTCCCGCTTCTCGAAGCGCGCG ACTACCGCGTCCATGCGCCCGACCTGACCGGCCATACGCCCGGCGATGGCGGCCATTTGTCGGTCGTCGACATGGAGCATTATACCCGCCCAGTCGCTGACATCCTGGCACGGGCCGAG A G C CAGTCGATCCTTCTGGGGCACAGCTTGGGCGGTGCATCCATCTCGTGGCTGGCGCAGCACCATCCCGACAAGGTGGCCGGGCTGATCTACCTGACCGCGGTCCTCACCGCGCCCGGTATAACGCCGGAAACCTTCGTCCTGCCCGGCGAGCCCAACCGGGGCACGCCGCACGCGCTGGACCTGATCCAGCCGGTCGACGAGGGACGTGGGCTACAGGCGGATTTCTCGCGACTGGAACGGCTCCGCGAAGTTTTCATGGGCGATTATCCCGGCGAGGGAATGCCGCCTGCCGAACAGTTCATCCAGACCCAGTCGACCGTGCCCTTTGGCACGCCCAATCCGATGGAGGGGCGCGCGCTGGAAATCCCACGCCTCTATATCGAGGCGCTGGACGATGTCGTGATTCCGATCGCCGTGCAGCGTCAGATGCAGAAGGAGTTCCCCGGTCCGGTCGCGGTCGTGTCGCTGCCGGCCAGCCATGCGCCCTATTACTCGATGCCCGAACGGCTGGCCGAGGCGATCGCCGATTTCGCCGATGCCCCGGCCGAGTATCGCCAGACGGCGACGAAGGCTGGGCCTGATCGA A C G GCTGGAGCGGACGGGGGTCGAGCCGACCGAGCTGATCTACCGTGA (SEQ ID NO: 3), Note: the mutation site is underlined.

[0060] Sequencing analysis results showed that compared with the wild-type PytH gene (SEQ ID NO.1), the coding gene of the mutant PytH-M5 (SEQ ID NO.3) had a total of 5 nucleotide mutations: guanine (G) at position 89 was mutated to adenine (A), guanine (G) at position 208 was mutated to adenine (A), guanine (G) at position 210 was mutated to cytosine (C), cytosine (C) at position 796 was mutated to adenine (A), and adenine (A) at position 798 was mutated to guanine (G).

[0061] The aforementioned nucleotide mutations resulted in alterations at three amino acid sites in the PytH protein: glycine (Gly, G) at position 30 was mutated to aspartic acid (Asp, D), glycine (Gly, G) at position 70 was mutated to serine (Ser, S), and proline (Pro, P) at position 266 was mutated to threonine (Thr, T). The amino acid sequence of the mutant PytH-M5 is as follows:

[0062] MTVTDIILIHGALNRGACYDAVVPLLEAR D YRVHAPDLTGHTPGDGGHLSVVDMEHYTRPVADILARAE S QSILLGHSLGGASISWLAQHHPDKVAGLIYLTAVLTAPGITPETFVLPGEPNRGTPHALDLIQPVDEGRGLQADFSRLERLREVFMGDYPGEGMPPAEQFIQTQSTVPFGTPNPMEGRALEIPRLYIEALDDVVIPIAVQRQMQKEFPGPVAVVSLPASHAPYYSMPERLAEAIADFADAPAEYRQTATKAGPDR T AGADGGRADRADLP (SEQ ID NO.4), Note: Mutation sites are underlined.

[0063] Homology modeling analysis was performed on the three-dimensional structures of the wild-type PytH protein and the PytH-M5 mutant. All three mutation sites are spatially located at or near the active site of the enzyme (see Figures A and B in Figure 1). The aspartic acid at position 30 is located on the enzyme molecule surface, near the entrance to the substrate-binding pocket. The G30D mutation increases the negative charge density in this region, potentially enhancing the affinity for the potentiometric sites in the ester bonds of pyrethroid pesticides. The serine at position 70 is located in the flexible loop region near the catalytic triplet (Ser78-Asp202-His230). The G70S mutation introduces a polar hydroxyl group, which may improve the induced fit efficiency of enzyme-substrate binding through a hydrogen bond network. The threonine at position 266 is located near the C-terminus of the enzyme molecule. The P266T mutation replaces the cyclic proline with a threonine with a hydroxyl side chain, increasing the flexibility and polarity of this region. This may expand the spatial conformation of the substrate-binding pocket, facilitating the accommodation of pyrethroid substrate molecules with different structures. The synergistic effect of the three mutations enhanced the overall catalytic activity of PytH-M5 against pyrethroid pesticides.

[0064] 2. High-efficiency expression and purification of PytH-M5 mutant in Escherichia coli

[0065] 2.1 PCR amplification of the PytH-M5 mutant gene

[0066] Using the recombinant plasmid containing the PytH-M5 mutant gene obtained in Example 1 as a template, PCR amplification was performed using primers 3 (SEQ ID NO. 7) and 4 (SEQ ID NO. 8), with Nde I and Xho I restriction sites introduced at both ends of the primers, respectively. PrimeSTAR was used. ® PCR was performed using GXL DNA Polymerase (Takara, R050Q).

[0067] Primer 3, forward primer:

[0068] 5'-CGCG CATATG ACCGTCACCGATATCATC-3' (SEQ ID NO.7), the underlined part is the Nde I restriction site.

[0069] Primer 4, reverse primer:

[0070] 5'-CGCG CTCGAG TCACGGTAGATCAGCTCGGTC-3' (SEQ ID NO.8), the underlined part is the Xho I restriction site.

[0071] The PCR amplification system (50 μL) is shown in Table 4.

[0072] Table 4. PCR amplification system (50 μL)

[0073]

[0074] PCR amplification procedure:

[0075] a. Pre-denaturation at 98℃ for 3 min;

[0076] b. Denaturation at 98℃ for 0.5 min, annealing at 55℃ for 0.5 min, extension at 72℃ for 1.0 min, for 30 cycles;

[0077] c. Extend at 72°C for 10 minutes, then cool to room temperature.

[0078] 2.2 Construction of expression vectors and acquisition of recombinant engineered bacteria

[0079] The PCR products were purified and recovered using an agarose gel DNA recovery kit. The purified PCR products and the pET-29a(+) expression vector were double-digested with Nde I and Xho I, respectively. The digestion system is shown in Table 2.

[0080] After enzyme digestion at 37℃ for 12 h, the digested mutant gene fragment and the linearized pET-29a(+) vector were separated and recovered by agarose gel electrophoresis. The recovered mutant gene fragment and the linearized pET-29a(+) vector were ligated using T4 DNA ligase (Thermo Fisher Scientific, 15224041) to construct the recombinant expression vector pET29a-PytH-M5 (e.g., ...). Figure 2 (As shown). The recombinant expression vector was transformed into *E. coli* BL21(DE3) competent cells, plated on LB agar plates containing 100 mg / L kanamycin, and incubated overnight at 37°C. Positive clones were picked for colony PCR and enzyme digestion verification to obtain the recombinant engineered strain BL21 / PytH-M5. Simultaneously, the wild-type PytH recombinant expression vector pET29a-PytH and the recombinant engineered strain BL21 / PytH were constructed as controls using the same method.

[0081] 2.3 Induction and purification of PytH-M5 mutant

[0082] The recombinant engineered strain BL21 / PytH-M5 was inoculated into LB liquid medium containing 100 mg / L kanamycin and cultured at 37°C with shaking at 200 rpm until the OD600 reached 0.4-0.6. IPTG was then added to a final concentration of 0.5 mM, and expression was induced at 16°C for 12 h. After induction, the bacterial pellet was collected by centrifugation (8000 rpm, 10 min), washed twice with PBS buffer (50 mM, pH 7.5), and resuspended in lysis buffer (50 mM PBS, 300 mM NaCl, 10 mM imidazole, pH 7.5).

[0083] The bacterial suspension was sonicated in an ice bath (300 W, 1 s operation, 2 s interval, total 15 min), then centrifuged (12000 rpm, 30 min, 4℃) and the supernatant was collected. The supernatant was added to a Ni-NTA affinity chromatography column and incubated at 4℃ for 2 h. Impurities were washed sequentially with washing buffer (50 mM PBS, 300 mM NaCl, 20 mM imidazole, pH 7.5), and the target protein was eluted with elution buffer (50 mM PBS, 300 mM NaCl, 250 mM imidazole, pH 7.5).

[0084] Collect the eluent and dialyze overnight with PBS buffer (50 mM, pH 7.5), changing the dialysate three times during the process. Determine the concentration of the purified protein using the Bradford method, aliquot, and store at -80°C for later use. Simultaneously purify wild-type PytH protein as a control using the same method.

[0085] The purified PytH-M5 and wild-type PytH proteins were analyzed by SDS-PAGE electrophoresis, and the results are as follows: Figure 3 As shown in the figure. Lane M is the protein molecular weight marker (Takara, 3595Q), lane 1 is the crude enzyme for breaking down wild-type PytH cells, lane 2 is wild-type PytH (approximately 30.8 kDa), lane 3 is the crude enzyme for breaking down mutant PytH-M5 cells, and lane 4 is the mutant PytH-M5 (approximately 30.8 kDa). This indicates that the PytH-M5 mutant was successfully expressed and its molecular weight is consistent with that of the wild type.

[0086] 3. PytH-M5 mutant enzyme activity assay

[0087] 3.1 Enzyme activity assay method

[0088] Using different pyrethroid pesticides as substrates, the enzyme activities of wild-type PytH and mutant PytH-M5 were determined. The enzyme activity reaction system (3.0 mL) consisted of adding a final concentration of 100 μM of the pyrethroid pesticide substrate (permethrin, cypermethrin, deltamethrin, lambda-cyhalothrin, deltamethrin, or bifenthrin) and an appropriate amount of purified enzyme solution to 50 mM PBS buffer (pH 7.5), and reacting at 30 °C for 10 min. After the reaction, an equal volume of acetonitrile was added to terminate the reaction, and the supernatant was collected by centrifugation. The reduction in substrate concentration was detected by gas chromatography (GC).

[0089] GC detection conditions: Electron capture detector (ECD), SPB-5 capillary column (30 m × 0.25 mm × 0.25 μm), injection port temperature 260℃, detector temperature 300℃. Column temperature program: initial temperature 100℃, hold for 2 min, ramp to 280℃ at 15℃ / min, hold for 8 min. Carrier gas: high-purity nitrogen (≥99.999%), constant flow mode, column flow rate 0.8 mL / min; ECD makeup gas: high-purity nitrogen, flow rate 30 mL / min. Splitless injection, injection volume 1.0 μL.

[0090] One unit of enzyme activity (U) is defined as the amount of enzyme required to catalyze the hydrolysis of 1.0 μmol of substrate per minute at pH 7.5 and 30°C. Specific enzyme activity (U / mg) is the enzyme activity divided by the protein concentration (mg / mL).

[0091] 3.2 Enzyme activity assay results

[0092] Under the same reaction conditions, the specific enzyme activities of wild-type PytH and mutant PytH-M5 against six pyrethroid pesticides were determined, and the experimental results are shown in Table 5.

[0093] Table 5. Specific enzyme activities of wild-type PytH and mutant PytH-M5 against different pyrethroid pesticides

[0094]

[0095] The results showed that, compared with the wild-type PytH, the mutant PytH-M5 exhibited 2.1-fold, 1.8-fold, 2.3-fold, 1.6-fold, 2.5-fold, and 1.9-fold increased specific enzyme activities against permethrin, cypermethrin, deltamethrin, deltamethrin, and bifenthrin, respectively. The mutant PytH-M5 also showed significantly enhanced hydrolytic activity against various pyrethroid pesticides, demonstrating good substrate broad-spectrum activity and practical application potential.

[0096] 4. Enzymatic characterization of PytH-M5 mutant

[0097] 4.1 Effect of different pH values ​​on PytH-M5 enzyme activity

[0098] The hydrolytic activity of PytH-M5 against cypermethrin was determined under different pH conditions (pH 4.0-10.0). The specific procedures were the same as described in 3.1. The slight difference was that the substrate concentration was 20 μM and the reaction time was 3 min. The treatment with the highest degradation efficiency was set as 100%, and the degradation efficiency of the other treatments was compared with that of the highest efficiency group to calculate the relative enzyme activity (%). The results are shown in Figure 4A. PytH-M5 and wild-type PytH both exhibited similar bell-shaped activity curves in the pH range of 4.0–10.0, and their optimal pH was 7.5 (relative enzyme activity of 100%), indicating that the mutation did not change the enzyme's acid-base preference. However, there were subtle differences between the two at specific activity levels: in the acidic region (pH 4.0–6.5), the relative enzyme activity of PytH-M5 was always slightly higher than that of wild-type PytH; in the neutral to weakly alkaline high-activity plateau region (pH 7.0–8.5), the activities of the two highly overlapped (both >80%), but PytH-M5 still maintained a slight advantage; while in the alkaline inactivation region (pH 9.0–10.0), although the activities of both decreased sharply. The results showed that mutations at positions 30, 70, and 266 of PytH did not alter the enzyme's acid-base preference, but slightly enhanced the enzyme's catalytic efficiency or structural stability under suboptimal pH conditions (especially acidic and alkaline conditions).

[0099] 4.2 Effect of different temperatures on PytH-M5 enzyme activity

[0100] The hydrolytic activity of PytH-M5 against cypermethrin was determined under different temperature conditions (20-60℃). Figure 4 As shown in Figure B, the optimal temperature for both PytH and PytH-M5 is 40℃ (relative enzyme activity of 100%). However, PytH-M5 is slightly higher than or equal to wild-type PytH across the entire temperature range (20-60℃): it exhibits higher activity in the low-temperature region (20-35℃) (e.g., 46.4% vs 42.5% at 20℃) and slower inactivation in the high-temperature region (45-60℃) (e.g., 76.3% vs 73.2% at 60℃). This indicates that the M5 mutation did not change the optimal temperature but instead slightly improved the catalytic stability and efficiency over a wide temperature range.

[0101] 4.3 Dynamic Parameter Analysis

[0102] Kinetic parameters of wild-type PytH and mutant PytH-M5 were determined using cypermethrin as a substrate. The affinity (Km) and catalytic constant (kcat) were calculated using the Lineweaver-Burk double reciprocal plot method, and the results are shown in Table 6.

[0103] Table 6. Comparison of kinetic parameters between wild-type PytH and mutant PytH-M5

[0104]

[0105] The results showed that compared with the wild-type PytH, the mutant PytH-M5 exhibited a 34.5% decrease in affinity (Km) for cypermethrin, a 57.3% increase in catalytic constant (kcat), and a 2.4-fold increase in catalytic efficiency (kcat / Km). This indicates that the synergistic effect of the three amino acid mutations G30D / G70S / P266T not only enhanced the affinity between the enzyme and the substrate but also improved the enzyme's catalytic conversion efficiency.

[0106] 5. Application of PytH-M5 mutant in simulated soil remediation

[0107] Topsoil (0-20 cm) from farmland that had not been treated with pyrethroid pesticides was collected, sieved through a 2 mm sieve, air-dried, and sterilized. Cypermethrin was dissolved in acetone and mixed evenly into the soil to achieve a final concentration of 50 mg / kg. The soil was divided into three groups: (1) Control group: no enzyme added; (2) Wild-type PytH treatment group: purified wild-type PytH enzyme solution was added (final concentration 5 U / g soil); (3) PytH-M5 treatment group: purified PytH-M5 mutant enzyme solution was added (final concentration 5 U / g soil). Each group had three replicates. The soil moisture content was adjusted to 60% of the field capacity and incubated at a constant temperature of 25℃.

[0108] Samples were taken at 0, 1, 3, 5, 7, and 10 days after treatment. Cypermethrin residues in the soil were extracted with acetonitrile and detected by GC-ECD. The results are shown in Table 7.

[0109] Table 7. Degradation effect of PytH-M5 on cypermethrin residues in soil

[0110]

[0111] The results showed that, under the same enzyme dosage, PytH-M5 exhibited significantly higher degradation efficiency for cypermethrin in soil than the wild-type PytH. After 10 days of treatment, the residual rate of cypermethrin in the soil treated with PytH-M5 was only 3.1%, while it was 15.2% in the wild-type PytH treatment group and 91.5% in the control group. This demonstrates that the PytH-M5 mutant maintains highly efficient degradation activity in actual soil environments and shows promising potential for soil remediation applications.

[0112] The above embodiments do not limit the present invention in any way. All technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A mutant of the pyrethroid hydrolase PytH, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO.

4.

2. The coding gene of the mutant according to claim 1, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

3.

3. A recombinant expression vector, characterized in that, It includes the coding gene as described in claim 2.

4. The recombinant expression vector according to claim 3, characterized in that, The recombinant expression vector is obtained by inserting the coding gene between the Nde I and Xho I restriction sites of pET-29a(+).

5. A genetically engineered bacterium, characterized in that, It includes the coding gene as described in claim 2.

6. The genetically engineered bacterium according to claim 5, characterized in that, The expression strain of the genetically engineered bacteria is Escherichia coli BL21(DE3).

7. The application of the mutant of claim 1, the recombinant expression vector of claim 3, or the genetically engineered bacteria of claim 5 in the degradation of pyrethroid pesticides.

8. The application according to claim 7, characterized in that, The applications include the removal of pyrethroid pesticide residues from soil and water.

9. The application according to claim 7, characterized in that, The pyrethroid pesticides include permethrin, cypermethrin, deltamethrin, lambda-cyhalothrin, deltamethrin, bifenthrin, and cypermethrin.

10. The application of the encoding gene of claim 2 in the construction of transgenic crops resistant to pyrethroid pesticides.

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