Atrazine-degrading enzyme and related biomaterials and applications thereof

Abstract

The present invention discloses an atrazine-degrading enzyme, related biomaterials, and applications thereof. The invention docks the atrazine-resistant protein ME with an atrazine ligand small molecule based on its three-dimensional structural model to obtain binding sites. Amino acid mutations are predicted using amino acid mutagenesis technology. Based on the molecular docking and mutagenesis results, an enzyme with degradation potential, MP, is screened to obtain the enzyme. MP significantly enhances atrazine degradation performance and can be used in bioremediation of atrazine residues, demonstrating promising application potential.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to an atrazine-degrading enzyme and related biological materials and applications thereof. Background Art

[0002] Atrazine, a triazine herbicide with high weed control efficacy, is widely used in agricultural production. Atrazine enters the plant body through weed roots and leaves and rapidly travels to the meristem, destroying the plant's chloroplasts and inhibiting photosynthesis to kill the weeds. However, atrazine is a long-residual herbicide with an environmental half-life of 60-150 days. It is difficult to degrade in both water and soil and can easily cause phytotoxicity in sensitive crops. Studies have shown that atrazine is toxic to aquatic animals and plants, posing a threat to human health. Therefore, addressing the problem of atrazine residues in the environment is of great significance to human health and environmental stability.

[0003] Biodegradation is currently the primary approach to addressing pesticide residues. Biodegradation primarily comes from various microorganisms, which are low-cost, environmentally friendly, highly adaptable, and mutagenic, offering broad application prospects. Currently, atrazine-degrading microorganisms have been isolated from many different genera, primarily bacteria. However, studies on atrazine-degrading enzymes are limited, and the binding mechanism and site of action of these enzymes with the atrazine drug ligand small molecule are unclear.

[0004] Molecular docking is a method for drug design based on the characteristics of receptors and the interaction between receptors and drug molecules. It is a theoretical simulation method that mainly studies the interaction between molecules (such as ligands and receptors) and predicts their binding mode and affinity. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to degrade atrazine and / or how to prepare a product of degrading atrazine.

[0006] In order to solve the above technical problems, the present invention first provides a mutant protein, which can be the following protein A1), A2) or A3):

[0007] A1) The amino acid sequence is the protein of sequence 2 in the sequence listing;

[0008] A2) a protein derived from A1) or having more than 80% identity with the protein shown in A1) and having the same function as the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, obtained by substitution and / or deletion and / or addition of one or more amino acid residues;

[0009] A3) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0010] In order to solve the above technical problems, the present invention further provides any of the following applications of the mutant protein:

[0011] M1. Application in the preparation of products for degrading atrazine;

[0012] M2. Application in the preparation of products that degrade herbicides;

[0013] M3. Application in the preparation of herbicide residue soil bioremediation products.

[0014] In the above application, the herbicide may be atrazine.

[0015] In order to solve the above technical problems, the present invention also provides a biomaterial related to the mutant protein described above, which can be any of the following:

[0016] B1) a nucleic acid molecule encoding the mutant protein described above;

[0017] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0018] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0019] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0020] B5) a transgenic plant cell line containing the nucleic acid molecule described in B1) or a transgenic plant cell line containing the expression cassette described in B2);

[0021] B6) transgenic plant tissue containing the nucleic acid molecule described in B1) or transgenic plant tissue containing the expression cassette described in B2);

[0022] B7) a transgenic plant organ containing the nucleic acid molecule described in B1) or a transgenic plant organ containing the expression cassette described in B2);

[0023] B8) a nucleic acid molecule that promotes or increases the gene expression of the mutant protein according to claim 1;

[0024] B9) An expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line containing the nucleic acid molecule described in B8).

[0025] In the above application, the nucleic acid molecule in B1) may be the gene encoding the protein shown in b1), b2) or b3) below:

[0026] b1) a DNA molecule whose coding sequence of the coding strand is the nucleotide sequence 3 in the sequence listing;

[0027] b2) A DNA molecule that hybridizes with the DNA molecule defined in b1) and encodes a protein having the same function.

[0028] In order to solve the above technical problems, the present invention further provides any of the following applications of the biomaterial described above:

[0029] N1. Application in the preparation of products for degrading atrazine;

[0030] N2. Application in the preparation of products that degrade herbicides;

[0031] N3. Application in the preparation of herbicide residue soil bioremediation products.

[0032] In the above application, the herbicide may be atrazine.

[0033] In order to solve the above technical problems, the present invention also provides a method for preparing a herbicide-degrading product and / or a herbicide-residue soil bioremediation product, comprising expressing the mutant protein or the biomaterial described above.

[0034] In the above method, the herbicide may be atrazine.

[0035] In order to solve the above technical problems, the present invention also provides a method for screening and obtaining a target herbicide-degrading enzyme, which may include the following steps:

[0036] A1) constructing the three-dimensional structure of the target herbicide (known) resistance protein;

[0037] A2) based on the three-dimensional structure, selecting the active cavity of the (known) resistance protein as a docking pocket, and performing molecular docking between the (known) resistance protein and the target herbicide ligand small molecule to obtain a docking result, wherein the molecular docking is performed using Autodock;

[0038] A3) determining the binding ability of the resistance protein to the target herbicide ligand small molecule based on the docking results, and performing alanine scanning analysis on the three-dimensional structure (conformation) of the resistance protein with strong binding ability to obtain mutation sites;

[0039] A4) performing saturation mutagenesis on the mutation site to obtain a mutant protein sequence;

[0040] A5) connecting the coding sequence of the mutant protein sequence to an expression vector to obtain a recombinant vector, and expressing the recombinant vector to obtain the recombinant protein;

[0041] A6) performing enzyme activity assay and target herbicide degradation ability assay on the recombinant protein to obtain assay results;

[0042] A7) Screening and obtaining a target herbicide-degrading enzyme based on the assay results.

[0043] In the above method, the three-dimensional structure of the known antibody protein can be obtained by statistically evaluating the overall performance of non-bonded interactions between different atoms in the target structure using online software SAVES 6.0, Procheck, Ramachandran plot and / or ERRAT program.

[0044] The purpose of the present invention is to provide an atrazine-degrading enzyme and application thereof.

[0045] The present invention explores its binding mode through molecular docking technology, and screens out efficient degradation enzymes through computer simulation and virtual amino acid mutation technology, in order to provide new microbial resources for the degradation of long-residual herbicides.

[0046] The present invention aims to provide an atrazine-degrading enzyme, which can be applied in the bioremediation of long-residue herbicides.

[0047] In order to achieve the above object, the present invention adopts the following technical solutions:

[0048] The present invention provides an atrazine-degrading enzyme MP, which is derived from the mutation of an atrazine-resistant gene me.

[0049] Furthermore, the ME protein translated from the resistance gene me can bind to the atrazine drug small molecule.

[0050] Furthermore, the ME protein is docked with the atrazine ligand molecule, and the main binding sites are MET1, MET2, PRO147, and PHE150.

[0051] The present invention provides a method for docking an ME protein with an atrazine drug small molecule, characterized by the following steps:

[0052] Construct a ME receptor protein model and evaluate its reliability;

[0053] The ME receptor protein model was docked with the atrazine ligand small molecule;

[0054] Based on the docking results, ligands with tighter binding are screened;

[0055] Alanine (ALA) scanning of amino acid residues and saturation mutagenesis were performed;

[0056] Constructing a recombinant vector for the mutant and introducing it into the host bacteria;

[0057] Extract enzyme solution and measure enzyme activity;

[0058] The atrazine degradation ability of the mutant enzyme was determined;

[0059] Verification of mutant protein expression.

[0060] Furthermore, the reliability evaluation in step (1) is: Verify 3D>93%;

[0061] Furthermore, the ME receptor protein in step (2) needs to remove water molecules, remove metal ions, and add hydrogen atoms in PymMol;

[0062] Furthermore, the active position coordinates of the molecular docking in step (2) are: X=0.447, Y=6.988, Z=6.72;

[0063] Furthermore, the ME receptor protein in step (2) is selected to have a wide and tight pocket;

[0064] Furthermore, the screening criteria of step (3) are: selecting the highest CDOCKER-Energy allocator;

[0065] Furthermore, in step (4), the scanning range of ALA amino acid is Calculate Mutation Energy (Binding) ≤ 0.4 nm;

[0066] Furthermore, in step (5), the prokaryotic expression vector is pGEX-4T-1 and the Escherichia coli is BL21.

[0067] The invention provides an application of the atrazine-degrading enzyme MP in the degradation and repair of long-residue herbicides.

[0068] As can be seen from the above technical solution, compared with existing atrazine-degrading bacteria, the present invention achieves the following beneficial effects: the atrazine-resistant gene me, obtained from Bacillus verticillioides, is docked with the atrazine ligand small molecule by docking the ME protein to obtain a binding site, and the atrazine-degrading enzyme MP is obtained through amino acid mutation technology. This significantly enhances its atrazine-degrading performance and has great potential for application in the bioremediation of atrazine residues. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1Evaluation of the ME protein three-dimensional model. A is the Verify_3D score for the ME protein model, with the 3D / 1D value on the ordinate and the amino acid residues on the abscissa. B is the Pull-type conformational analysis of the ME protein model, with the rotation phi (φ) of the bond between the α-carbon atom and the nitrogen atom on the ordinate and the rotation psi (ψ) of the bond between the α-carbon atom and the carbonyl carbon atom within the peptide bond in the polypeptide protein three-dimensional structure on the abscissa. C is the error value of the ME-binding protein model residues calculated by ERRAT, with the error value on the ordinate and the amino acid residues on the abscissa.

[0070] Figure 2 The three-dimensional structure of ME protein.

[0071] Figure 3 The docking of ME protein with atrazine. A is the 3D molecular docking diagram, B is the overall molecular docking package diagram, C is the 3D molecular docking detail expansion diagram, and D is the 2D molecular docking diagram.

[0072] Figure 4 Activity assay for mutant enzymes docked with atrazine. Note: 1: MET2 > PRO; 2: MET2 > TYR; 3: LEU143 > PRO; 4: LEU143 > PHE; PHE150 > ARG; 5: MET2 > GLN; PHE150 > TRP; 6: MET2 > TYR; LEU143 > MET; PHE150 > TRP.

[0073] Figure 5 The results of multiple comparisons of atrazine degradation rates by mutant genes are shown. 1: MET2 > PRO; 2: MET2 > TYR; 3: LEU143 > PRO; 4: LEU143 > PHE; PHE150 > ARG; 5: MET2 > GLN; PHE150 > TRP; 6: MET2 > TYR; LEU143 > MET; PHE150 > TRP.

[0074] Figure 6 Protein expression and Western Blot analysis of the atrazine-doped mutant enzyme MET2>PRO. The left panel shows protein expression of the atrazine-doped mutant enzyme MET2>PRO; the right panel shows Western Blot analysis of the atrazine-doped mutant enzyme MET2>PRO. M: Prestained protein marker; 1: Total protein before induction; 2: 20°C supernatant; 3: 20°C pellet; 4: 37°C supernatant; 5: 37°C pellet. DETAILED DESCRIPTION

[0075] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0076] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0077] Example 1. Screening of key mutation sites for the interaction between ME protein and atrazine ligand small molecule

[0078] 1. Construction and evaluation of ME receptor protein model

[0079] The amino acid sequence of the atrazine resistance protein (ME) from Fusarium verticillioide was obtained by gene cloning and sequencing (SEQ ID 1 in the sequence listing). The ME protein sequence was analyzed using NCBI online software (https: / / www.ncbi.nlm.nih.gov); template-free Robbeta de novo modeling was used to construct a three-dimensional structural model of the ME protein.

[0080] Sequence 1:

[0081] MMIHILTNPSVYKKLMTELDDATLAGQLSEMPQYDEVVENCPYYIACVKESMRLNPSAPNIFPRIAPPGGLEICGQFVPGGTEVTCNPWLVHRDPNIYGDDAEIFKPERWLDEDKAKVYNKYSMGFGYGARVCLGQDVARMELYKGPLQFLRSFKVEWVDEKNRGTYVVKGGVSYFENMNITIQRREVVA.

[0082] At the same time, the three-dimensional structural model of the constructed ME protein was evaluated for three-dimensional reliability using the online software SAVES 6.0 and Procheck (https: / / saves.mbi.ucla.edu). Generally, the average score of the side chain amino acid VERIFY 3D model must be above 0.2 for more than 80% to be reasonable. In the three-dimensional structural model of the ME protein constructed in the present invention, 93.65% of the side chain amino acid residues have an average score > 0.2. Figure 1 As shown in A, the model is reasonably applicable.

[0083] Ramachandran plot is a diagram that describes the dihedral angles ψ and A visualization method to determine whether the protein is in a reasonable region can also reflect whether the conformation of the protein is reasonable. The present invention uses Procheck (https: / / saves.mbi.ucla.edu) to generate a Ramachandran plot (Psi / Phi Ramachandran plot) to further evaluate the three-dimensional reliability of the constructed ME protein model ( Figure 1 Middle B), where three red areas ( Figure 1 A, B, and L in (B) represent the optimal region of the estimated amino acid structure. The proportion of amino acids in this region is proportional to the overall confidence of the conformation, and the number of residues in the optimal region is shown to be 89.9%; Figure 1 The four yellow areas in B ( Figure 1 a, b, l and p in B represent the reasonable region of protein skeleton structure, showing that 10.1% of amino acid residues are located in the additional reasonable region; light yellow ( Figure 1 The ~a, ~b, ~l and ~p of B are barely acceptable areas, and the white area is unreasonable; Figure 1 As shown in B, the model conformation has no amino acids and residues in the barely accepted and unreasonable regions, and 100% of the Φ and ψ dihedral angles are within the reasonable range, which conforms to the stereo energy rule. Therefore, the three-dimensional structural model of the ME protein constructed in the present invention is reasonable.

[0084] Finally, the ERRAT program (https: / / saves.mbi.ucla.edu) was used to statistically evaluate the overall performance of non-bonded interactions between different atoms in the target structure (the three-dimensional structural model of the ME protein constructed in the present invention). The region where the error value is within the confidence interval is considered to be a reasonable region, and the percentage of the reasonable region is required to be greater than 50%. Figure 1 The results of C show that the error value of the three-dimensional structural model of the ME protein obtained by analysis is 98.32%, which indicates that the non-bonded interactions in the three-dimensional structural model of the ME protein constructed by the present invention are reasonable as a whole. Therefore, the three-dimensional structural model of the ME protein constructed by the present invention can be further determined ( Figure 2 ) is a high-quality model.

[0085] 2. Autodock molecular docking

[0086] The Simulation-Minimization (QM / MM) module of Discovery studio 4.5 (related literature: Yan Junjie. Preparation of porcine reproductive and respiratory syndrome virus monoclonal antibodies and establishment of indirect ELISA method [D]. Jinan University, 2021. DOI: 10.27167 / d.cnki.gjinu.2019.000236.) was used to optimize the energy of atrazine drug ligand small molecules, and CHARMm was selected as the force field.

[0087] The three-dimensional structural model of the ME protein constructed in step 1 was preprocessed using PymMol 3.8 software (https: / / www.lfd.uci.edu / ~gohlke / pythonlibs / #pymol), hydrogenation, dehydration, removal of metal ions, charge calculation, and the processed protein was saved in pdb format.

[0088] The active cavity of the ME receptor protein was selected as the docking pocket, and the docking position coordinates were: X = 0.447, Y = 6.988, Z = 6.72. The remaining parameters were the default values. The three-dimensional structural model of the receptor ME protein was flexibly docked with the atrazine drug ligand small molecule (the 3D molecular structure of atrazine was searched on the Pubchem website and saved as an SDF format file) using Autodock (https: / / autodock.scripps.edu). The best top 10 ligand conformations were saved with the default parameters, ranked by CDOCKER-Energy score, and the highest-scoring ligand was selected for analysis.

[0089] 3. Molecular Docking Conformational Analysis

[0090] The protein (three-dimensional structural model of ME protein) and substrate (atrazine drug small molecule) were docked using the CDOCKER algorithm in Discovery Studio 4.5 (Related literature: Yan Junjie. Preparation of porcine reproductive and respiratory syndrome virus monoclonal antibodies and establishment of indirect ELISA method [D]. Jinan University, 2021. DOI: 10.27167 / d.cnki.gjinu.2019.000236.), and the docking results and interacting amino acid sites of the two were obtained. The binding map of ME protein and atrazine was drawn using Pymol3.8 (https: / / www.lfd.uci.edu / ~gohlke / pythonlibs / #pymol). The molecular docking 3D map showed that the ME protein and atrazine drug small molecule were connected by hydrogen bonds, carbon-hydrogen bonds, π-hydrogen bonds, halogen atoms and π-alkyl interactions ( Figure 3A and B in the figure). The amino acid residues at the action site of the ME protein are MET1, MET2, PRO147 and PHE150, with a binding energy of -3.69Kcal / mol. Among them, the MET1 amino acid residue (the first amino acid on the ME protein chain) is connected to the small molecule atrazine through a hydrogen bond at the N-terminus of the ring and a π-alkyl interaction at the center of the ring; MET2 is connected to the atrazine drug ligand small molecule through a chlorine atom at the other end of the ring and a π hydrogen bond of the ring; PRO147 is connected to the atrazine drug ligand small molecule through a carbon-hydrogen bond on the left side of the ring and a chlorine atom above the ring, and at the same time has a π-alkyl interaction with the center of the ring; PHE150 is bound to the chlorine atom through a halogen bond ( Figure 3 C and D).

[0091] 4. Amino acid mutation

[0092] The docking results determined the binding capacity between the ME protein and the atrazine small molecule. Based on this, alanine (ALA) scanning analysis (Discovery Studio 4.5) was performed on the most stable (tightly connected) ME protein conformation docked with the atrazine small molecule. The results are shown in Table 1. Fourteen mutation sites in the ME protein were identified. Mutation energies between -0.5 and +0.5 indicate that the site has no effect on the binding affinity between the ME protein and the atrazine small molecule, and the mutation results are labeled NETURAL. Mutation energies > 0.5 indicate that these mutations reduce the affinity between the ME protein and the atrazine small molecule and weaken the interaction ability, and the results are labeled DESTABILIZING. Mutation energies < -0.5 indicate that these mutations play a key role in the binding of the ME protein to the atrazine small molecule, and the results are labeled STABILIZING.

[0093] Table 1. ALA scan results of docking with atrazine

[0094]

[0095] The three key amino acids MET2 (methionine), LEU143 (leucine), and PHE150 (phenylalanine) were mutated (indicated by ">") to 19 amino acids other than ALA. As shown in Table 2, when MET2>TYR, MET2>GLN:PHE150>TRP, and MET2>TYR:LEU143>MET:PHE150>TRP, the mutation energy was minimal and the mutation was relatively stable. When these sites were mutated, a higher affinity between the enzyme and the substrate may be generated, making the enzyme more effective on atrazine. When MET2>PRO, MET2>PRO:PHE150>GLY, MET2>PRO:LEU143>PHE:PHE150>ARG, the maximum mutation energy is 4.67Kcal / mol, 5.89Kcal / mol, and 6.34Kcal / mol, respectively. At this time, the mutation is unstable. If this type of mutation occurs, the interaction between the enzyme and the substrate may be weakened, and the binding performance to atrazine will also be reduced.

[0096] Table 2. Saturation mutation sites for docking with atrazine

[0097]

[0098]

[0099] Example 2. Obtaining ME mutant protein

[0100] 1. Construction of mutant recombinant bacteria

[0101] The six mutants docked with atrazine in Example 1 were used to construct vectors to form recombinant plasmids.

[0102] The six ME protein mutants are:

[0103] The amino acid residue MET2 (methionine) of the ME protein is mutated to proline PRO, that is, the second amino acid residue MET in sequence 1 in the sequence list is replaced with PRO to obtain a mutant ME protein, which is named mutant 1 protein (MP protein); the amino acid sequence of the MP protein is shown in sequence 3 in the sequence list, and the encoding nucleotide sequence of the MP protein is shown in sequence 4 in the sequence list.

[0104] The amino acid residue MET2 (methionine) of the ME protein is mutated to tyrosine TYR, that is, the second amino acid residue MET in sequence 1 in the sequence list is replaced with TYR to obtain a mutant ME protein, which is named Mutant 2 protein (MET2>TYR); the amino acid sequence of the Mutant 2 protein is shown in Sequence 5 in the sequence list, and the encoding nucleotide sequence of the Mutant 2 protein is shown in Sequence 6 in the sequence list.

[0105] The amino acid residue LEU143 (leucine) of the ME protein is mutated to proline PRO, that is, the 143rd amino acid residue LEU of sequence 1 in the sequence list is replaced with PRO to obtain a mutant ME protein, which is named mutant 3 protein (LEU143>PRO); the amino acid sequence of the mutant 3 protein is shown in sequence 7 in the sequence list, and the encoding nucleotide sequence of the mutant 3 protein is shown in sequence 8 in the sequence list.

[0106] The amino acid residue LEU143 (leucine) of the ME protein is mutated to phenylalanine PHE and the amino acid residue PHE150 (phenylalanine) of the ME protein is mutated to arginine ARG, that is, the 143rd amino acid residue LEU of sequence 1 in the sequence list is replaced with PHE and the 150th amino acid residue PHE of sequence 1 is replaced with ARG to obtain a mutant ME protein, named mutant 4 protein (LEU143>PHE; PHE150>ARG); the amino acid sequence of mutant 4 protein is shown in sequence 9 in the sequence list, and the encoding nucleotide sequence of mutant 4 protein is shown in sequence 10 in the sequence list.

[0107] The amino acid residue MET2 (methionine) of the ME protein is mutated to glutamine GLN and the amino acid residue PHE150 (phenylalanine) of the ME protein is mutated to tryptophan TRP, that is, the second amino acid residue MET of sequence 1 in the sequence list is replaced by GLN and the 150th amino acid residue PHE of sequence 1 is replaced by TRP to obtain a mutant ME protein, which is named mutant 5 protein (MET2>GLN; PHE150>TRP); the amino acid sequence of mutant 5 protein is shown in sequence 11 in the sequence list, and the encoding nucleotide sequence of mutant 5 protein is shown in sequence 12 in the sequence list.

[0108] The amino acid residue MET2 (methionine) of the ME protein is mutated to tyrosine TYR, and at the same time, the amino acid residue LEU143 (leucine) of the ME protein is mutated to methionine MET, and the amino acid residue PHE150 of the ME protein is mutated to tryptophan TRP; the mutant ME protein obtained by replacing the second amino acid residue MET of sequence 1 in the sequence list with tyrosine TYR, and at the same time replacing the 143rd amino acid residue LEU of sequence 1 with methionine MET, and replacing the 150th amino acid residue phenylalanine PHE of sequence 1 with tryptophan TRP is named mutant 6 protein (MET2>TYR; LEU143>MET; PHE150>TRP); the amino acid sequence of mutant 6 protein is shown in sequence 13 in the sequence list, and the encoding nucleotide sequence of mutant 6 protein is shown in sequence 14 in the sequence list.

[0109] Sequence 2 (5'-3'):

[0110] Atgatgatccacattctcaccaacccatctgtgtacaagaagctcatgactgagcttgatgacgctactctcgctggtcaactctccgagatgcctcagtacgatgaagtcgtcgagaactgcccctactacatcgcctgcgtcaaagaaagcatgcgtctcaatccctctgcaccaaacatcttcccccgcatcgctccaccaggcggtctcgagatctgcggtcaattcgttcccggaggcacagaagtgacttgcaacccgtggctcgtgcatcgtgacccgaacatctacggtgatgacgccgagatcttcaagcctgagcgttggttagatgaagacaaggccaaggtttacaacaagtacagtatgggctttggttatggtgctagagtgtgtctaggccaggatgtcgcgcgcatggagctttacaagggaccgctacagttcttgaggagcttcaaggttgaatgggttgatgagaagaatagaggaacgtatgttgttaagggcggtgtgagttactttgagaacatgaacatcacgatccagagacgagaagtcgtggcatga。

[0111] Sequence 3:

[0112] MPIHILTNPSVYKKLMTELDDATLAGQLSEMPQYDEVVENCPYYIACVKESMRLNPSAPNIFPRIAPPGGLEICGQFVPGGTEVTCNPWLVHRDPNIYGDDAEIFKPERWLDEDKAKVYNKYSMGFGYGARVCLGQDVARMELYKGPLQFLRSFKVEWVDEKNRGTYVVKGGVSYFENMNITIQRREVVA。

[0113] Sequence 4 (5'-3'):

[0114] Atgccgatccacattctcaccaacccatctgtgtacaagaagctcatgactgagcttgatgacgctactctcgctggtcaactctccgagatgcctcagtacgatgaagtcgtcgagaactgcccctactacatcgcctgcgtcaaagaaagcatgcgtctcaatccctctgcaccaaacatcttcccccgcatcgctccaccaggcggtctcgagatctgcggtcaattcgttcccggaggcacagaagtgacttgcaacccgtggctcgtgcatcgtgacccgaacatctacggtgatgacgccgagatcttcaagcctgagcgttggttagatgaagacaaggccaaggtttacaacaagtacagtatgggctttggttatggtgctagagtgtgtctaggccaggatgtcgcgcgcatggagctttacaagggaccgctacagttcttgaggagcttcaaggttgaatgggttgatgagaagaatagaggaacgtatgttgttaagggcggtgtgagttactttgagaacatgaacatcacgatccagagacgagaagtcgtggcatga。

[0115] Sequence 5:

[0116] MYIHILTNPSVYKKLMTELDDATLAGQLSEMPQYDEVVENCPYYIACVKESMRLNPSAPNIFPRIAPPGGLEICGQFVPGGTEVTCNPWLVHRDPNIYGDDAEIFKPERWLDEDKAKVYNKYSMGFGYGARVCLGQDVARMELYKGPLQFLRSFKVEWVDEKNRGTYVVKGGVSYFENMNITIQRREVVA。

[0117] Sequence 6 (5'-3'):

[0118] Atgtatatccacattctcaccaacccatctgtgtacaagaagctcatgactgagcttgatgacgctactctcgctggtcaactctccgagatgcctcagtacgatgaagtcgtcgagaactgcccctactacatcgcctgcgtcaaagaaagcatgcgtctcaatccctctgcaccaaacatcttcccccgcatcgctccaccaggcggtctcgagatctgcggtcaattcgttcccggaggcacagaagtgacttgcaacccgtggctcgtgcatcgtgacccgaacatctacggtgatgacgccgagatcttcaagcctgagcgttggttagatgaagacaaggccaaggtttacaacaagtacagtatgggctttggttatggtgctagagtgtgtctaggccaggatgtcgcgcgcatggagctttacaagggaccgctacagttcttgaggagcttcaaggttgaatgggttgatgagaagaatagaggaacgtatgttgttaagggcggtgtgagttactttgagaacatgaacatcacgatccagagacgagaagtcgtggcatga。

[0119] Sequence 7:

[0120] MMIHILTNPSVYKKLMTELDDATLAGQLSEMPQYDEVVENCPYYIACVKESMRLNPSAPNIFPRIAPPGGLEICGQFVPGGTEVTCNPWLVHRDPNIYGDDAEIFKPERWLDEDKAKVYNKYSMGFGYGARVCLGQDVARMEPYKGPLQFLRSFKVEWVDEKNRGTYVVKGGVSYFENMNITIQRREVVA。

[0121] Sequence 8 (5'-3'):

[0122] Atgatgatccacattctcaccaacccatctgtgtacaagaagctcatgactgagcttgatgacgctactctcgctggtcaactctccgagatgcctcagtacgatgaagtcgtcgagaactgcccctactacatcgcctgcgtcaaagaaagcatgcgtctcaatccctctgcaccaaacatcttcccccgcatcgctccaccaggcggtctcgagatctgcggtcaattcgttcccggaggcacagaagtgacttgcaacccgtggctcgtgcatcgtgacccgaacatctacggtgatgacgccgagatcttcaagcctgagcgttggttagatgaagacaaggccaaggtttacaacaagtacagtatgggctttggttatggtgctagagtgtgtctaggccaggatgtcgcgcgcatggagccgtacaagggaccgctacagttcttgaggagcttcaaggttgaatgggttgatgagaagaatagaggaacgtatgttgttaagggcggtgtgagttactttgagaacatgaacatcacgatccagagacgagaagtcgtggcatga。

[0123] Sequence 9:

[0124] MMIHILTNPSVYKKLMTELDDATLAGQLSEMPQYDEVVENCPYYIACVKESMRLNPSAPNIFPRIAPPGGLEICGQFVPGGTEVTCNPWLVHRDPNIYGDDAEIFKPERWLDEDKAKVYNKYSMGFGYGARVCLGQDVARMEFYKGPLQRLRSFKVEWVDEKNRGTYVVKGGVSYFENMNITIQRREVVA。

[0125] Sequence 10 (5'-3'):

[0126] Atgatgatccacattctcaccaacccatctgtgtacaagaagctcatgactgagcttgatgacgctactctcgctggtcaactctccgagatgcctcagtacgatgaagtcgtcgagaactgcccctactacatcgcctgcgtcaaagaaagcatgcgtctcaatccctctgcaccaaacatcttcccccgcatcgctccaccaggcggtctcgagatctgcggtcaattcgttcccggaggcacagaagtgacttgcaacccgtggctcgtgcatcgtgacccgaacatctacggtgatgacgccgagatcttcaagcctgagcgttggttagatgaagacaaggccaaggtttacaacaagtacagtatgggctttggttatggtgctagagtgtgtctaggccaggatgtcgcgcgcatggagttttacaagggaccgctacagcgcttgaggagcttcaaggttgaatgggttgatgagaagaatagaggaacgtatgttgttaagggcggtgtgagttactttgagaacatgaacatcacgatccagagacgagaagtcgtggcatga。

[0127] Sequence 11:

[0128] MQIHILTNPSVYKKLMTELDDATLAGQLSEMPQYDEVVENCPYYIACVKESMRLNPSAPNIFPRIAPPGGLEICGQFVPGGTEVTCNPWLVHRDPNIYGDDAEIFKPERWLDEDKAKVYNKYSMGFGYGARVCLGQDVARMELYKGPLQWLRSFKVEWVDEKNRGTYVVKGGVSYFENMNITIQRREVVA。

[0129] Sequence 12 (5'-3'):

[0130] Atgcgaatccacattctcaccaacccatctgtgtacaagaagctcatgactgagcttgatgacgctactctcgctggtcaactctccgagatgcctcagtacgatgaagtcgtcgagaactgcccctactacatcgcctgcgtcaaagaaagcatgcgtctcaatccctctgcaccaaacatcttcccccgcatcgctccaccaggcggtctcgagatctgcggtcaattcgttcccggaggcacagaagtgacttgcaacccgtggctcgtgcatcgtgacccgaacatctacggtgatgacgccgagatcttcaagcctgagcgttggttagatgaagacaaggccaaggtttacaacaagtacagtatgggctttggttatggtgctagagtgtgtctaggccaggatgtcgcgcgcatggagctttacaagggaccgctacagtggttgaggagcttcaaggttgaatgggttgatgagaagaatagaggaacgtatgttgttaagggcggtgtgagttactttgagaacatgaacatcacgatccagagacgagaagtcgtggcatga。

[0131] Sequence 13:

[0132] MYIHILTNPSVYKKLMTELDDATLAGQLSEMPQYDEVVENCPYYIACVKESMRLNPSAPNIFPRIAPPGGLEICGQFVPGGTEVTCNPWLVHRDPNIYGDDAEIFKPERWLDEDKAKVYNKYSMGFGYGARVCLGQDVARMEMYKGPLQWLRSFKVEWVDEKNRGTYVVKGGVSYFENMNITIQRREVVA.

[0133] Sequence 14 (5'-3'):

[0134] .

[0135] The mutant nucleotide sequences of the six ME proteins (SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14) and the coding sequence of the ME wild-type protein (SEQ ID NO: 2) were ligated into the expression vector pGEX-4T-1 and transformed into competent Escherichia coli BL21. Positive clones were screened, and the plasmids were extracted for enzyme digestion verification. Six recombinant BL21 Escherichia coli expressing the six ME mutant proteins and a recombinant Escherichia coli BL21 / pGEX-4T-1-ME expressing the wild-type ME protein (designated as the wild-type strain) were obtained.

[0136] The mutant gene sequence was synthesized by Sangon Biotechnology Co., Ltd. (Shanghai), and then amplified by PCR using primers 1F: 5'-GGGCTGGCAAGCCACGTTTGGTG-3' and 1R: 5'-CCGGGAGCTGCATGTGTCAGAGG-3'. The resulting fragment was then ligated to a T vector to transform Escherichia coli for propagation. The amplified plasmid was then extracted, and the expression vector pGEX-4T-1 plasmid (purchased from Shanghai Sangon Biotechnology Co., Ltd.) was also extracted. The target fragment and the vector were double-digested with restriction endonucleases (EcoRI and NotI, purchased from Shanghai Sangon Biotechnology Co., Ltd.). The enzyme digestion reaction solution was incubated in a 37°C incubator for 3 h, detected by electrophoresis, and the target fragment was purified by gel cutting. The target gene was then ligated to the expression vector to obtain a recombinant plasmid. At the same time, the Escherichia coli BL21 competent cells (purchased from Shanghai Sangon Biotechnology Co., Ltd.) were thawed on ice for 1-2 min, and the water bath was heated to 42°C in advance. Add 10 μL of ligation product (recombinant plasmid) to the competent cells, shake gently, place on ice for 30 minutes, heat shock in a 42°C water bath for 90 seconds, place on ice for 5-10 minutes, add 800 μL of LB liquid (containing kanamycin) medium, culture in a shaking incubator at 37°C and 200 rpm for 1 hour, then spread on a kanamycin (30 μg / mL) plate, invert the plate and culture overnight in a 37°C incubator, pick white colonies for PCR detection and gel electrophoresis experiments.

[0137] The PCR primers were: 1F: 5'-GGGCTGGCAAGCCACGTTTGGTG-3' and 1R: 5'-CCGGGAGCTGCATGTGTCAGAGG-3'. The recombinant plasmids positive for PCR detection were sent to Shanghai Biotech Co., Ltd. for sequencing.

[0138] 2. Inducible expression of mutant enzymes

[0139] Referring to the method in the literature (Li Xun, Wang Ting, Wang Liangliang, et al. Expression, purification and directed evolution of Bacillus megaterium cytochrome P450 enzyme [J]. Chemistry and Industry of Forest Products, 2014, 34(06): 135-140.), the six recombinant BL21 Escherichia coli and wild-type strains in step 1 were induced with IPTG (when the bacterial concentration reached 0.8-1.0, 0.5 mmol / L IPTG was added for induction for 10 hours) and the bacterial cells were collected for ultrasonic disruption to extract the enzyme solution.

[0140] 3. Determination of Mutant Enzyme Activity

[0141] After the enzyme solution is extracted, add 3 μL of reduced coenzyme II (NADPH) to the EP tube, use 10 mg / L atrazine as the substrate, add 5 μL of the substrate, make up to 90 μL with phosphate buffer, add 10 μL of the purified enzyme solution, start the timer immediately, react in a 40°C water bath for 4 minutes, and record the absorbance at 340 nm with a microspectrophotometer. Calculate according to the following formula:

[0142] X = ΔA × Vtotal × n / (ε × Δt × Venzyme)

[0143] Where ΔA is the change in absorbance; ε is the absorbance coefficient 6.22*103L / (mol*cm); and n is the dilution factor. Figure 4 As shown, when mutation 1 (MET2>PRO) and mutation 6 (MET2>TYR; LEU143>MET; PHE150>TRP) occurred, their enzyme activities were 1.12 and 1.12 times those of the wild-type ME protein, respectively. However, when mutation 5 (MET2>GLN) occurred, the enzyme activity decreased significantly compared to the wild-type ME protein, reaching only 23% of the wild-type ME protease activity. Mutation 2 (MET2>TYR), mutation 3 (LEU143>PRO), and mutation 4 (LEU143>PHE; PHE150>ARG) retained 62.79%, 40.39%, and 61.08% of the wild-type ME protease activity, respectively.

[0144] 4. Degradation activity of mutant enzymes on herbicides

[0145] Using the wild-type strain as a control, the degradation degree of atrazine (available from Weifang Runfeng Chemical Co., Ltd.) by the six mutant engineered bacteria (the six recombinant BL21 Escherichia coli in step 1) was determined after 7 days of culture. Figure 5 As shown, mutant bacteria expressing mutation 1 protein, mutation 2 protein, mutation 3 protein, mutation 4 protein or mutation 5 protein ( Figure 5 1-5 representatives) and wild-type strain ( Figure 5The degradation rates of atrazine in the mutant strains expressing mutation 1 (LEU143>PRO) showed significant differences (P<0.05). The degradation rate of atrazine by the mutant strain expressing mutation 1 (1:MET2>PRO) was the highest, at 93.17%. Mutants expressing mutations 2, 3, 4, and 5 all showed varying degrees of decrease. The degradation rate of the mutant strain expressing mutation 3 (LEU143>PRO) was the lowest, at 17.56%. The degradation rate of the mutant strain expressing mutation 1 increased, reaching 1.14 times that of the wild-type strain. Furthermore, the mutant strain expressing mutation 1 showed significant differences from the wild-type strain expressing the ME protein. Therefore, the finding that the presence of the 1:MET2>PRO mutation in the ME protein enhances the degradation of atrazine by the enzyme (i.e., the MP protein), has important implications for subsequent genetic modification and bioremediation.

[0146] Example 3. Analysis of mutant MP protein expression

[0147] Mutant enzyme protein expression and Western Blot experiment:

[0148] Protein expression assay by SDS-PAGE: Inoculate a single colony onto a solid LB plate containing kanamycin and incubate at 37°C for 180 rpm until the OD value reaches 0.6-0.8. Heat-shock the recombinant plasmid into competent E. coli BL21 cells at 42°C and plate onto a plate containing 50 μg / mL ampicillin for overnight incubation. Select a single colony and culture it in liquid culture. When the OD value reaches 0.6, induce with 0.5 mM IPTG and incubate at 20°C overnight and 37°C for 6 hours, respectively. A negative control without inducer was used. Centrifuge at 4000 rpm for 10 minutes. Solubilize the collected cells using ultrasound. Centrifuge again to collect the supernatant and precipitate. Prepare samples for both the supernatant and precipitate for analysis on gel.

[0149] Western Blot Verification: Protein samples were processed and prepared using a 5% stacking gel (2.975 mL of H₂O, 0.67 mL of acrylamide / bis, 1.25 mL of Tris-HCl, 50 μL of 10% SDS, 5 μL of TEMED, and 50 μL of 10% APS) and a 12% separating gel (5 mL of gel volume, 1.6 mL of H₂O, 2 mL of a 30% acrylamide mixture, 1.3 mL of 1.5 mol / L Tris, 0.05 mL of 10% SDS, 0.05 mL of 10% APS, and 0.002 mL of TEMED). The primary antibody was mouse anti-GST tag, and the secondary antibody was goat anti-mouse. Validation was performed using the tag antibody (purchased from Shanghai Sangon Biotechnology Co., Ltd.).

[0150] from Figure 6We can see a distinct band above 45kD for the mutant enzyme MP (MET2>PRO), which is close to the predicted protein molecular weight of 48.3kDa. To further confirm whether the target protein is expressed, we used TMB colorimetric kit to develop the protein, and followed the Western Blot procedure to detect it using the tag antibody. In summary, the target MP protein was detected to be expressed, and the MP was mostly expressed in an insoluble form ( Figure 6 The mutant protein MP docked with atrazine can be expressed normally.

[0151] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. Protein, characterized in that: The protein is the following protein: A1) The amino acid sequence is the protein of sequence 3 in the sequence listing; A2) The amino acid sequence is the protein of sequence 13 in the sequence listing; A3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1) or A2).

2. Use of the protein according to claim 1 in preparing a product for degrading atrazine.

3. The biomaterial related to the protein according to claim 1, characterized in that: The biological material is any one of the following: B1) a nucleic acid molecule encoding the protein according to claim 1; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

4. The biomaterial according to claim 3, characterized in that: B1) The nucleic acid molecule is a gene encoding the protein shown below: b1) A DNA molecule whose coding sequence of the coding strand is the nucleotide sequence 4 in the sequence listing; b2) The coding sequence of the coding strand is a DNA molecule having nucleotide sequence 14 in the sequence listing.

5. Use of the biomaterial according to claim 3 or 4 in preparing a product for degrading atrazine.

6. A method for preparing a product for degrading atrazine and / or a product for bioremediation of atrazine residue in soil, comprising expressing the protein of claim 1 or the biomaterial of claim 3 or 4.

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