An engineered enzyme capable of degrading multiple bactericides

Abstract

This invention discloses an engineered enzyme capable of degrading various fungicides, belonging to the field of agricultural technology. This engineered enzyme is a double mutant obtained by site-directed mutagenesis of serine (S) at position 13 and glutamic acid (E) at position 66 of the wild-type CpGST. This double mutant significantly enhances the degradation capacity against various structurally different fungicides such as azoxystrobin, pyraclostrobin, oxadiazon, oxadixyl, and fluopyram, and has significant application value in the field of pesticide residue bioremediation.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an engineered enzyme that can degrade a variety of bactericides. Background Technology

[0002] In agricultural production, chemical fungicides are widely used to control fungal diseases in crops. However, their long-term and extensive use has led to pesticide residues in agricultural products and the farmland environment, posing a serious threat to food safety, the ecological environment, and human health. Currently, technologies for controlling fungicide residues mainly fall into three categories: physical adsorption, chemical oxidation degradation, and biodegradation. Physical methods can only achieve phase transfer of pollutants and cannot completely degrade them, easily causing secondary pollution. Chemical oxidation methods involve harsh reaction conditions, easily damaging the quality of agricultural products and posing a risk of secondary pollution from byproducts. In contrast, bio-enzymatic degradation methods have advantages such as mild reaction conditions, strong degradation specificity, and environmental friendliness, making them the core research direction for the green management of pesticide residues.

[0003] Glutathione S-transferase (GST) is a key detoxification enzyme widely present in organisms. It catalyzes the binding reaction of glutathione (GSH) with exogenous toxic substances, thereby achieving metabolic detoxification. CN117327675A discloses a papaya-derived glutathione S-transferase CpGST and its encoding gene. This enzyme exhibits degradation activity against the dithiocarbamate fungicide thiram without the need for exogenous GSH addition. However, further research by the inventors revealed that this natural wild-type CpGST has an extremely narrow substrate spectrum, exhibiting degradation ability only against thiram. It lacks effective degradation activity against mainstream fungicides widely used in agricultural production, such as methoxyacrylates, succinate dehydrogenase inhibitors (SDHI), and imidazoles. This fails to meet the need for synergistic management of multiple fungicide residues in fields and agricultural products, severely limiting its large-scale application in the field of pesticide residue bioremediation.

[0004] Therefore, the urgent technical problem to be solved in this field is to engineer wild-type CpGST through rational design to broaden its substrate degradation spectrum and obtain engineered enzymes that can simultaneously and efficiently degrade multiple types of bactericides with different structures. Summary of the Invention

[0005] One objective of this invention is to provide a glutathione S-transferase mutant, the amino acid sequence of which is as follows: MADEVVLLDFWPAPFGMRIRIALAEKGIHYEYKEENLRNKSPLLLQMNPVHKKIPVLIHNGKPICASLIQIQYIDEVWSDKAPLLPSDPYQRAQARFWADYVDKKMYEAGR RVWTTKGEEQEGAKKEFIEILKTLEGELGEKPYFGGESFGYVDLTFIPFYTWFSVYESFGKMSIEAECPKLFSWVKRCLEKESVSKSLPDQDKVYGFVLELRKALGI (SEQ ID NO.1).

[0006] This invention involves site-directed mutagenesis of residues S13 and E66 in wild-type glutathione S-transferase (CpGST), resulting in the double-mutant engineered enzyme CpGST-S13A / E66A. The S13A / E66A double mutation replaces two large, highly polar residues in the active site with alanine (A) residues with very small side chains, significantly eliminating steric hindrance in the binding cavity and optimizing its hydrophobic microenvironment. This "spatial relaxation" and "shape reshaping" allows bactericide molecules with a large coplanar aromatic system to bind more deeply and stably, achieving better geometric complementarity with residues such as Ala66 and Pro55, thereby stabilizing the pre-catalytic transition state conformation.

[0007] A second objective of this invention is to provide a gene encoding the aforementioned mutant glutathione S-transferase.

[0008] In one embodiment of the present invention, the nucleotide sequence of the gene is as follows: (SEQ ID NO.2).

[0009] A third objective of this invention is to provide a recombinant vector carrying the aforementioned genes.

[0010] In one embodiment of the present invention, the recombinant vector is pET-30a.

[0011] The fourth objective of this invention is to provide engineered bacteria containing the aforementioned genes.

[0012] In one embodiment of the present invention, the engineered bacteria is Escherichia coli.

[0013] The fifth objective of this invention is to provide the application of the above-mentioned glutathione S-transferase mutant, the above-mentioned recombinant vector, or the above-mentioned engineered bacteria in the degradation of fungicides, wherein the fungicides include azoxystrobin, pyraclostrobin, oxadiazon, oxadixyl, and fluopyram.

[0014] Compared with the prior art, the present invention has the following significant advantages: 1. Significantly broadened substrate spectrum: The CpGST-S13A / E66A double mutant obtained in this invention, while retaining the activity of degrading thiram, has for the first time obtained a highly efficient degradation ability of methoxyacrylate fungicides, SDHI fungicides and oxazoline fungicides with very different structures, realizing a functional leap from "single target" to "broad spectrum" degradation.

[0015] 2. Outstanding degradation activity and catalytic efficiency: Experiments showed that this mutant achieved a 51.47% degradation rate of azoxystrobin within 2 hours, and 45.00% and 44.86% degradation rates of pyraclostrobin and azoxystrobin, respectively. Enzyme kinetic analysis revealed a catalytic efficiency (Kcat / Km) of 507.33 mM⁻¹·h⁻¹ for oxadiazine, demonstrating excellent substrate affinity and conversion ability.

[0016] 3. Precise and Clear Modification Mechanism: By site-directedly mutating two highly polar side chain residues, serine (S) at position 13 and glutamic acid (Glu, E) at position 66 of the active site, to alanine (Ala, A), steric hindrance was eliminated, and the hydrophobic binding pocket was reshaped. This rational design explains the structural basis for the enhanced enzyme activity at the atomic level, and the mutation strategy is highly targeted.

[0017] 4. Broad application prospects: This mutant does not require the addition of exogenous GSH and has mild reaction conditions, providing a core component for the development of broad-spectrum enzyme preparations for the purification of agricultural products and the remediation of farmland pollution, with huge industrialization potential. Attached Figure Description

[0018] Figure 1 The image shows an SDS-PAGE electrophoresis image of the CpGST-S13A / E66A mutant induced and purified in Escherichia coli BL21 (DE3) in Example 1. The molecular weight of the target protein band is approximately 26.2 kDa, which is consistent with the theoretical molecular weight.

[0019] Figure 2 The bar chart shows the degradation rate of the CpGST-S13A / E66A mutant against five target fungicides after 2 hours of incubation in Example 2.

[0020] Figure 3 The image shows the enzyme kinetic fitting curves of the CpGST-S13A / E66A mutant against five target fungicides in Example 3; where A is the enzyme kinetic curve of azoxystrobin substrate, B is the enzyme kinetic curve of pyraclostrobin substrate, C is the enzyme kinetic curve of oxadiazon substrate, D is the enzyme kinetic curve of oximazole substrate, and E is the enzyme kinetic curve of fluopyram substrate.

[0021] Figure 4 The diagram shows the molecular docking simulation of the CpGST-S13A / E66A mutant with five target fungicides in Example 4; where A is the docking pattern with azoxystrobin, B is the docking pattern with pyraclostrobin, C is the docking pattern with oxadiazon, D is the docking pattern with oxadiazon, and E is the docking pattern with fluopyram. Detailed Implementation

[0022] Addressing the technical bottlenecks of low degradation activity and narrow substrate specificity of wild-type glutathione S-transferase (CpGST) against fungicides, this invention constructs a CpGST-S13A / E66A double mutant using site-directed mutagenesis. This represents the first time that synergistic mutational modification at the S13 and E66 sites has been achieved, significantly improving the mutant's catalytic degradation efficiency and substrate compatibility for various fungicides. This invention provides methods for the discovery, site-directed mutagenesis, cloning, expression, and purification of this mutant, and also offers its application in the catalytic degradation of fungicides and the bioremediation of pesticide residues. It overcomes the limitations of existing single mutants with limited degradation effects, providing a new technical pathway and highly active material basis for the green remediation of pesticide residues, demonstrating significant innovation and application value.

[0023] Specifically, the site-directed mutagenesis, cloning, expression, and purification of the glutathione S-transferase mutant CpGST-S13A / E66A includes the following steps: Step 1: Based on the wild-type glutathione S-transferase (CpGST) gene sequence, a site-directed mutagenesis primer for the S13 site was designed using bioinformatics analysis. Using the expression plasmid of the CpGST-E66A mutant as a template, the CpGST-S13A / E66A double mutant gene fragment was obtained by PCR amplification. Step 2: After enzyme digestion and circularization, the PCR amplification product was transformed into E. coli DH5α competent cells. The mutation was successfully confirmed by both PCR verification and gene sequencing, and the CpGST-S13A / E66A recombinant expression plasmid was constructed. Step 3: The recombinant expression plasmid was transformed into E. coli BL21(DE3) competent cells. After heterologous induction of expression, the protein was purified by Ni-NTA affinity chromatography to obtain high-purity and high-activity CpGST-S13A / E66A mutant protein.

[0024] Specifically, the application of the glutathione S-transferase mutant CpGST-S13A / E66A in the catalytic degradation of bactericides includes the following steps: Step 1: Prepare high-purity CpGST-S13A / E66A mutant protein, accurately prepare standard solutions of different types of bactericides and various reagents required for enzymatic reactions, and ensure the stability and repeatability of the reaction system. Step 2: Construct a standardized enzymatic reaction system, initiate the catalytic degradation reaction of the target fungicide by CpGST-S13A / E66A, detect the substrate reduction using high performance liquid chromatography (HPLC), calculate the degradation rate using the external standard method, and quantify the degradation activity of the mutant. Step 3: Determine the enzyme kinetic parameters of CpGST-S13A / E66A for different bactericides to clarify its binding ability and catalytic efficiency for various substrates; verify the binding mechanism between the mutant and the bactericide using molecular docking technology to elucidate the intrinsic principle of enhanced degradation activity of the mutant at the molecular level.

[0025] Those skilled in the art can adjust the process parameters appropriately based on the technical solutions disclosed herein to achieve the technical effects of the present invention. It should be specifically noted that all similar substitutions and improvements are obvious to those skilled in the art and are considered to be included within the scope of protection of the present invention. The methods and applications of the present invention have been described in detail through preferred embodiments. Those skilled in the art can modify, alter, or combine the methods and applications described herein without departing from the technical principles, spirit, and scope of the present invention to achieve the technical applications of the present invention.

[0026] Unless otherwise specified, the experimental methods used in the following examples are all conventional experimental methods in the field of molecular biology; unless otherwise specified, the experimental materials, reagents and consumables used in the following examples are all commercially available products. Example 1

[0027] Site-directed mutagenesis of CpGST, expression and purification of CpGST-S13A / E66A (1) Site-directed mutagenesis of CpGST, expression and purification of CpGST-S13A / E66A The following experiments were conducted using conventional molecular experimental methods.

[0028] The inventors, through molecular docking analysis, determined that residues F10, S13, K53, I54, E66, and S67 at the G site of CpGST are located close to the disulfide bond positions of the substrate, and then mutated these amino acid residues to alanine at the site. Furthermore, based on the CpGST-E66A mutant, the inventors designed the following site-directed mutagenesis primers for the S13 site using SnapGene software: S13 For:TCTGGCCAGCACCTTTTGGAATGAGAATCAGAATCG S13 Rev:AAAAGTGCTGGCCAGAAATCCAAGAGAACAA.

[0029] The expression plasmid of recombinant CpGST-E66A (nucleotide sequence: [insert nucleotide sequence here]) was extracted from DH5α cells and used as a template for PCR amplification. The PCR program was as follows: pre-denaturation at 95°C for 5 minutes; followed by 25 cycles, each cycle consisting of denaturation at 95°C for 10 seconds, annealing at 58°C for 15 seconds, and extension at 72°C for 30 seconds; after the cycles, a final extension at 72°C for 10 minutes was performed, and the mixture was stored at 16°C. The PCR reaction system is shown in Table 1. Green buffer and Dpn I were directly added to the PCR product. The resulting solution was incubated at 37°C for 3 hours to remove template DNA. Then, Exnase II was added and the mixture was incubated at 37°C for 30 minutes. The reaction solution was then heat-transferred to E. coli DH5α competent cells and plated. Single colonies were picked from the plates for sequencing to verify the success of site-directed mutagenesis.

[0030] Table 1

[0031] Green buffer (5.1 μL) and Dpn I (1 μL) were added directly to the PCR product. The resulting solution was incubated at 37ºC for 3 h to remove template DNA. Then, Exnase II (1 μL) was added and the solution was incubated at 37ºC for 30 min. The reaction solution was then heat-transferred to E. coli DH5α competent cells and plated. Single colonies were picked from the plates for sequencing to verify the success of site-directed mutagenesis.

[0032] (2) Construction of recombinant expression vectors and heterologous expression The recombinant vector was sequentially introduced into DH5α and BL21(DE3) competent cells. PCR was used to verify the fragment size, and after confirmation, CpGST was heterologously expressed using BL21(DE3). The resulting protein was purified using Ni-NTA, and SDS-PAGE analysis showed that the purified protein size was consistent with the predicted protein size of 26.2 kDa. Figure 1 (As shown). The content of purified protein was determined using the Coomassie brilliant blue colorimetric method. Example 2

[0033] Degradation activity of CpGST-S13A / E66A against various fungicides Using azoxystrobin, pyraclostrobin, oxadiazon, oxadixyl, and fluopyram as substrates, the degradation activities of wild-type GST (WT), S13A mutant, E66A mutant, and S13A / E66A mutant were evaluated. Stock solutions of each fungicide at 45 mM were prepared using DMSO.

[0034] The total volume of the reaction system was 100 μL, containing: 0.5 μL of bactericide stock solution (final concentration 0.18 mM) and 5 μL of purified CpGST-S13A / E66A enzyme solution (concentration 0.1203 mg / mL). A system without enzyme solution was used as a negative control. The reaction was carried out at room temperature (approximately 25°C) for 2 hours, and then terminated by adding an equal volume of pre-cooled methanol. The reaction solution was filtered through a 0.22 μm filter membrane and analyzed by high-performance liquid chromatography (HPLC).

[0035] HPLC conditions: C18 column (Eclipse XDB-C18, 4.6×250 mm); column temperature 25°C; detection wavelength 260 nm; flow rate 1 mL / min; mobile phase A: water, mobile phase B: acetonitrile; gradient elution program: 0-20 min, phase B linearly increases from 30% to 77%; 20-20.01 min, phase B decreases from 77% to 30%; 20.01-25 min, maintain phase B at 30%.

[0036] Table 2

[0037] The degradation rate was calculated using the external standard method, and the results are as follows: Figure 2 The wild-type enzymes showed no significant degradation activity or only extremely low degradation levels against azoxystrobin, pyraclostrobin, oxadiazon, oxadixyl, and fluopyram. The single mutants S13A and E66A showed a certain degree of improvement in the degradation ability of the above substrates compared with the wild-type enzymes. The S13A / E66A double mutant showed significantly higher degradation efficiency than the single mutant and wild-type enzymes for all tested substrates, with particularly significant improvement in azoxystrobin, oxadixyl, and fluopyram substrates, demonstrating that the double mutant enzyme has a broad-spectrum and high-efficiency degradation advantage against fungicides of different structural types.

[0038] The above results show that the S13A / E66A double mutant enzyme of the present invention effectively improves the enzyme's catalytic activity and substrate compatibility through site-directed mutagenesis modification, solving the technical problems of low degradation activity and narrow substrate spectrum of existing wild-type enzymes. It can be applied to fields such as bioremediation of fungicide-containing pollutants and development of pesticide degradation agents. Example 3

[0039] Enzyme kinetic parameter determination The enzyme kinetics of CpGST-S13A / E66A were determined using azoxystrobin, pyraclostrobin, oxadixyl, oxadixyl, and fluopyram as substrates. A series of reaction systems with different substrate concentrations (total volume 100 μL) were set up, each containing 5 μL of enzyme solution (0.02 U). After reacting for 1 hour at 28°C and pH 7.0, pre-cooled methanol was added to terminate the reaction. The amount of substrate decreased was detected by HPLC (under the same conditions as in Example 2), and the initial reaction rate was calculated.

[0040] Plotting substrate concentration on the x-axis and initial reaction velocity on the y-axis, nonlinear fitting of the Michaelis-Menten equation was performed using OriginPro 9.1 software to obtain enzyme kinetic curves. Figure 3 As shown in Table 3, the maximum reaction rate (Vmax), Michaelis constant (Km), and catalytic constant (Kcat) were calculated, and the results are shown in Table 3.

[0041] Table 3 Example 4

[0042] Molecular docking The three-dimensional structure of the CpGST-S13A / E66A mutant was predicted using the Phyre2 online server. Five fungicide molecules were coupled into the active pocket (G site) of the mutant using the CB-Dock server to obtain the lowest-energy binding conformation, such as... Figure 4 As shown.

[0043] Molecular docking results explained the differences in degradation activity at the structural level: the S13A / E66A double mutation formed a hydrophobic binding cavity with Ala13 and Ala66 as its core. For the azoxystrobin with the highest degradation rate (…),… Figure 4 (A) Its molecule can be inserted in optimal conformation. The aromatic ring forms a strong π-π stack with Tyr107 and Trp163, and the polar groups form a stable hydrogen bond network with Arg18, Lys52, etc. The binding is strong and the orientation is conducive to catalysis. Pyraclostrobin ( Figure 4 (B) and oxime ester ( Figure 4 The binding mode of C is similar, but the interaction network is slightly weaker. Fluopyram (with a lower degradation rate) Figure 4 (E) and oximazole ( Figure 4 The molecular structures of the two mutants (S13A and E66A) resulted in insufficient hydrogen bonding or steric hindrance, leading to poor binding stability and catalytic orientation, thus resulting in relatively low activity. This analysis confirmed at the atomic level that the S13A / E66A double mutation is the key structural basis for endowing the enzyme with new functions.

[0044] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A glutathione S-transferase mutant, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. The gene encoding the mutant glutathione S-transferase of claim 1.

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

2.

4. A recombinant vector carrying the gene of claim 2.

5. The recombinant vector according to claim 3, characterized in that, The recombinant vector is pET-30a.

6. Engineered bacteria containing the gene described in claim 2.

7. A method for preparing the glutathione S-transferase mutant according to claim 1, characterized in that, The preparation method includes the following steps: (1) Construct a recombinant expression vector containing the nucleic acid sequence shown in SEQ ID NO.2; (2) The recombinant expression vector is transformed into the host bacteria, cultured and purified to obtain the glutathione S-transferase mutant.

8. An enzyme preparation, characterized in that, The enzyme preparation contains the glutathione S-transferase mutant as described in claim 1 and excipients.

9. The application of the glutathione S-transferase mutant of claim 1, the recombinant vector of claim 4, or the engineered bacteria of claim 5 in the degradation of bactericides.

10. The application according to claim 8, characterized in that, The bactericide is selected from methoxyacrylate bactericides, SDHI bactericides, and oxazoline bactericides.

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