Atrazine degrading enzyme as well as related biological material and application thereof

Abstract

The invention discloses an atrazine degrading enzyme as well as a related biological material and application thereof. The method comprises the following steps: docking atrazine resistant protein ME with atrazine ligand small molecules based on a three-dimensional structure model of the atrazine resistant protein ME to obtain a binding site, predicting amino acid mutation through an amino acid mutation technology, and screening according to molecular docking and mutation results to obtain enzyme MP with degradation potential. The MP can obviously enhance the atrazine degradation performance, can be applied to bioremediation of atrazine residues, and has good application potential.

Description

Technical Field

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

[0002] Atrazine is a triazine herbicide with high weed control efficiency and is widely used in agricultural production. Atrazine enters the plant body through the roots and leaves of weeds and is quickly transmitted to the meristem, destroying the plant chloroplasts and inhibiting photosynthesis to kill weeds. However, atrazine is a long-residual herbicide with a half-life in the environment of generally 60-150 days. It is difficult to degrade in both water and soil, and it is easy to cause phytotoxicity to sensitive crops in the next crop. Studies have found that atrazine is toxic to aquatic animals and plants, threatening human health. Therefore, solving the problem of atrazine residues in the environment is of great significance to human health and environmental homeostasis.

[0003] Biodegradation is currently the main method to solve the problem of pesticide residues. The degradation source mainly comes from various microorganisms, which are low-cost, environmentally friendly, highly adaptable and mutagenic, and have broad application prospects. At present, atrazine-degrading microorganisms have been isolated from many different genera, mainly bacteria. There are few studies on atrazine-degrading enzymes, and the binding mode and action site of the enzymes with the atrazine drug ligand small molecules are not clear.

[0004] Molecular docking is a method of 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, wherein the mutant protein may be the following protein A1), A2) or A3):

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

[0008] A2) a protein derived from A1) or having more than 80% identity with the protein shown in A1) and having the same function, obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 2 in the sequence list;

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

[0010] In order to solve the above technical problems, the present invention also 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 soil bioremediation products for herbicide residues.

[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 biological material related to the mutant protein described above, and the biological material can be any one 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, a recombinant vector, a recombinant microorganism or a 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 a gene encoding the protein shown in b1), b2) or b3) below:

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

[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 also provides any of the following applications of the above-mentioned biomaterial:

[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 soil bioremediation products for herbicide residues.

[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 product for degrading herbicides and / or a product for bioremediation of herbicide residues in soil, comprising expressing the mutant protein or the biomaterial as 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 on 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 connection ability between the resistance protein and the target herbicide ligand small molecule according to the docking result, selecting the three-dimensional structure (conformation) of the resistance protein with strong connection ability for alanine scanning analysis 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 a 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 degrading enzyme for the target herbicide based on the determination results.

[0043] In the above method, the three-dimensional structure of the known antibody protein can be obtained by 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 the 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-residue 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 solution:

[0048] The 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 a ME protein with an atrazine drug small molecule, characterized in that the steps are as follows:

[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 to 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) selects a wide and tight pocket;

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

[0065] Furthermore, in the 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 discloses an application of atrazine degrading enzyme MP in the degradation and restoration of long-residue herbicides.

[0068] It can be seen from the above technical scheme that, compared with the existing atrazine-degrading bacteria, the beneficial effects achieved by the present invention are as follows: the atrazine-resistant gene me obtained from Bacillus verticillioides in the present invention, the ME protein is docked with the atrazine ligand small molecule to obtain a binding site, and the atrazine-degrading enzyme MP is obtained by amino acid mutation technology. The atrazine-degrading performance can be significantly enhanced, and it is applied in the bioremediation of atrazine residues, and has good application potential. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0070] Figure 2 This is the three-dimensional structure of ME protein.

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

[0072] Figure 4 For the detection of mutant enzyme activity 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 the degradation rates of atrazine by mutant genes. 1 is MET2>PRO; 2 is MET2>TYR; 3 is LEU143>PRO; 4 is LEU143>PHE; PHE150>ARG; 5 is MET2>GLN; PHE150>TRP; 6 is MET2>TYR; LEU143>MET; PHE150>TRP.

[0074] Figure 6 The left figure shows the protein expression and Western Blot analysis of the mutant enzyme MET2>PRO docked with atrazine. The right figure shows the Western Blot analysis of the mutant enzyme MET2>PRO docked with atrazine. M: pre-stained protein marker; 1: total protein before induction; 2: 20℃ supernatant; 3: 20℃ precipitate; 4: 37℃ supernatant; 5: 37℃ precipitate. DETAILED DESCRIPTION

[0075] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0076] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[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 atrazine resistance protein (ME) in Fusarium verticillioide was obtained by gene cloning and sequencing (sequence 1 in the sequence list). The protein sequence of ME was analyzed using NCBI online software (https: / / www.ncbi.nlm.nih.gov); template-free Robbeta de novo modeling was selected to construct the three-dimensional structure model of 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 by using 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 should be above 0.2 for more than 80% to be reasonable. The three-dimensional structural model of the ME protein constructed in the present invention has an average score of 93.65% of the side chain amino acid residues > 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 protein conformation is reasonable. The present invention uses Procheck (https: / / saves.mbi.ucla.edu) to generate a Psi / Phi Ramachandran plot to further evaluate the three-dimensional structural model of the constructed ME protein. Figure 1 B), where three red areas ( Figure 1 A, B, and L in B) represent the optimal region for estimating the amino acid structure. The proportion of amino acids in this region is proportional to the overall credibility 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) are reasonable regions of the protein backbone structure, showing that 10.1% of the 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 area; Figure 1 As shown in B, the model conformation has no amino acids and residues in the barely acceptable and unreasonable regions, and 100% of the Φ and ψ dihedral angles are within a reasonable range, which conforms to the stereo energy rule. Therefore, the three-dimensional structural model of the ME protein constructed by the present invention is reasonable.

[0084] Finally, the ERRAT program (https: / / saves.mbi.ucla.edu) was used to 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 area where the error value is within the confidence interval is considered to be a reasonable area, and the percentage of the reasonable area 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 monoclonal antibodies against porcine reproductive and respiratory syndrome virus 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 the atrazine drug ligand small molecule, 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 other 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) through Autodock (https: / / autodock.scripps.edu). The default parameters saved the best top 10 ligand conformations, which were ranked by CDOCKER-Energy score, and the highest distribution body was selected for analysis.

[0089] 3. Molecular docking conformation analysis

[0090] The protein (three-dimensional structural model of ME protein) and substrate (atrazine drug small molecule) were docked by 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. Pymol3.8 (https: / / www.lfd.uci.edu / ~gohlke / pythonlibs / #pymol) was used to draw the binding map of ME protein and atrazine. 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 -3.69 Kcal / 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-terminal end of the ring and a π-alkyl reaction at the center of the ring; MET2 is connected to the atrazine drug ligand small molecule through the chlorine atom at the other end of the ring and the 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 the chlorine atom above the ring, and at the same time has a π-alkyl reaction with the center of the ring; PHE150 is connected to the chlorine atom by a halogen bond ( Figure 3 C and D).

[0091] 4. Amino Acid Mutation

[0092] The connection ability between ME protein and atrazine small molecule was determined according to the docking results. Based on this, the most stable (most tightly connected) ME protein conformation in docking with atrazine small molecule was subjected to alanine (ALA) scanning analysis (Discovery Studio4.5). The results are shown in Table 1, and 14 mutation sites of ME protein were obtained. Among them, the mutation energy between -0.5 and +0.5 represents that the site has no effect on the affinity of ME protein and atrazine small molecule, and the mutation result is marked as NETURAL; when the mutation energy is >0.5, it indicates that these mutation points will reduce the affinity between ME protein and atrazine small molecule and weaken the interaction ability, and the result is marked as DESTABILIZING; when the mutation energy is <-0.5, it indicates that these mutation sites play a key role in the binding of ME protein and atrazine small molecule, and the result is marked as 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 except ALA. As shown in Table 2, when MET2>TYR, MET2>GLN:PHE150>TRP, MET2>TYR:LEU143>MET:PHE150>TRP, the mutation energy is minimal and the mutation is relatively stable. When these sites are 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 6 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 of 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 by PHE and the 150th amino acid residue PHE of sequence 1 is replaced by ARG to obtain a mutant ME protein, which is 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 (sequence 4, sequence 6, sequence 8, sequence 10, sequence 12, sequence 14) and the coding sequence of the ME wild-type protein (sequence 2) were connected to 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 (named as the wild-type strain) were obtained.

[0136] The mutant gene sequence was synthesized by Shanghai Sangon Biotechnology Co., Ltd., and then PCR amplified using primers 1F: 5'-GGGCTGGCAAGCCACGTTTGGTG-3' and 1R: 5'-CCGGGAGCTGCATGTGTCAGAGG-3', and then connected to the T vector to transform Escherichia coli for propagation, and then the propagation plasmid was extracted, and the expression vector pGEX-4T-1 plasmid (purchased from Shanghai Sangon Biotechnology Co., Ltd.) was extracted at the same time, and the target fragment and the vector were double-digested with restriction endonucleases (EcoRI and NotI, purchased from Shanghai Sangon Biotechnology Co., Ltd.), respectively. The enzyme digestion reaction solution was placed in a 37°C incubator for incubation for 3h, electrophoresis detection, gel cutting and purification of the target fragment, and the target gene was connected 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 1-2min, 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 min, heat shock in a 42°C water bath for 90 s, place on ice for 5-10 min, add 800 μL LB liquid (containing kanamycin) culture medium, culture in a shaking incubator at 37°C and 200 rpm for 1 h, then spread on a kanamycin (30 μg / mL) plate, invert the plate in a 37°C incubator for overnight culture, and 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 PCR-positive recombinant plasmids 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 6 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), the bacterial cells were collected and ultrasonically disrupted to extract the enzyme solution.

[0140] 3. Determination of the activity of mutant enzymes

[0141] After the enzyme solution was extracted, 3 μL of reduced coenzyme II (NADPH) was added to the EP tube, 10 mg / L atrazine was used as the substrate, 5 μL of the substrate was added, the phosphate buffer was supplemented to 90 μL, 10 μL of the purified enzyme solution was added, and the timing was started immediately. After reacting in a 40°C water bath for 4 minutes, the absorbance at 340 nm was recorded with a micro-spectrophotometer and calculated 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); n is the dilution factor. The activity of the mutant enzyme docked with atrazine was maintained at 40℃ for 4 minutes. Figure 4 As shown in the figure, when the 1st mutant protein MET2>PRO, the 6th mutant protein MET2>TYR;LEU143>MET;PHE150>TRP occurred, their enzyme activities were 1.12 and 1.12 times of the wild-type ME protein, respectively. However, when the 5th mutation: MET2>GLN, the enzyme activity was greatly reduced compared with the wild-type ME protein, only 23% of the wild-type ME protease activity; the 2nd mutant protein (MET2>TYR), the 3rd mutant protein (LEU143>PRO), and the 4th mutant protein (LEU143>PHE;PHE150>ARG) maintained 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 (obtained 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 cultivation. 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 represent) and wild-type strain ( Figure 5The degradation rates of atrazine by mutant engineering bacteria expressing mutation 1 protein were the highest at 93.17%, and those by mutant engineering bacteria expressing mutation 2 protein, mutation 3 protein, mutation 4 protein and mutation 5 protein all decreased to varying degrees. The degradation rate of mutant engineering bacteria expressing mutation 3 protein (LEU143>PRO) was the lowest at 17.56%, and the degradation rate of mutant engineering bacteria expressing mutation 1 protein increased, which was 1.14 times that of wild-type strains, and there was a significant difference between mutant engineering bacteria expressing mutation 1 protein and wild-type strains expressing ME protein. Therefore, it was found that when there was a 1:MET2>PRO mutation in ME protein, the enzyme (i.e., MP protein) had a stronger degradation effect on atrazine, which was of great significance for subsequent gene improvement and bioremediation.

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

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

[0148] SDS-PAGE protein expression detection: Take a single colony and inoculate it on a LB solid plate containing kanamycin, and culture it at 37℃, 180r until the OD value is 0.6-0.8. Heat shock the recombinant plasmid at 42℃ into E. coli competent BL21 cells, and spread it on a plate containing 50μg / mL ampicillin for overnight culture; pick a single clone colony for liquid culture; when the OD value reaches 0.6, induce it with 0.5mM IPTG, culture it overnight at 20℃ and 6h at 37℃, and the one without the addition of inducer is the negative control; centrifuge it at 4000rpm for 10min; use ultrasound to fully dissolve the collected bacteria. Centrifuge again to collect the supernatant and precipitate, prepare samples for the supernatant and precipitate proteins, and prepare for gel detection.

[0149] Western Blot verification: The protein samples were processed and prepared. Concentrating gel 5% (H2O 2.975mL, Acrylamide / bis0.67mL, Tris-HCl 1.25mL, 10% SDS 50μL, TEMED 5μL, 10% APS 50μL), separation gel 12% (gel volume 5mL, H2O 1.6mL, 30% acrylamide mixture 2mL, 1.5mol / L Tris 1.3mL, 10% SDS 0.05mL, 10% APS 0.05mL, TEMED 0.002mL). The primary antibody was mouse anti-GST tag, the secondary antibody was goat anti-mouse, and the tag antibody (supplied from Shanghai Shenggong Biological Co., Ltd.) was used for verification.

[0150] from Figure 6It can be seen that the mutant enzyme MP (MET2>PRO) has an obvious band above 45kDa, and the molecular weight is close to the predicted protein molecular weight of 48.3kDa. To further determine whether the target protein is expressed, TMB colorimetric kit was used for colorimetric development, and the label antibody was used for detection according to the Western Blot steps. In summary, the target MP protein was detected to be expressed, and 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 the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A mutant protein, characterized in that: The mutant protein is the following protein A1), A2) or A3): A1) The amino acid sequence is the protein of sequence 2 in the sequence list; A2) a protein derived from A1) or having more than 80% identity with the protein shown in A1) and having the same function, obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 2 in the sequence list; A3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1) or A2).

2. Any of the following uses of the mutant protein according to claim 1: M1. Application in the preparation of products for degrading atrazine; M2. Application in the preparation of products that degrade herbicides; M3. Application in the preparation of soil bioremediation products for herbicide residues.

3. A biological material related to the mutant protein according to claim 1, characterized in that: The biological material is any one of the following B1) to B9): B1) a nucleic acid molecule encoding the mutant 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); 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); B6) transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2); 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); B8) A nucleic acid molecule that promotes or increases the gene expression of the mutant protein according to claim 1; B9) An expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the nucleic acid molecule described in B8).

4. The use according to claim 3, characterized in that: B1) The nucleic acid molecule is a gene encoding the protein shown in b1), b2) or b3) below: b1) The coding sequence of the coding strand is a DNA molecule of the nucleotide sequence 3 in the sequence list; b2) A DNA molecule that hybridizes with the DNA molecule defined in b1) and encodes a protein having the same function.

5. Any of the following uses of the biomaterial according to claim 3 or 4: N1. Application in the preparation of products for degrading atrazine; N2. Application in the preparation of products that degrade herbicides; N3. Application in the preparation of soil bioremediation products for herbicide residues.

6. The use according to claim 5, characterized in that: The herbicide is atrazine.

7. A method for preparing a product for degrading herbicides and / or a product for bioremediation of herbicide residues in soil, comprising expressing the mutant protein of claim 1 or the biomaterial of claim 3 or 4.

8. The method according to claim 7, characterized in that: The herbicide is atrazine.

9. A method for screening and obtaining a target herbicide-degrading enzyme, characterized in that: The method comprises the following steps: A1) constructing the three-dimensional structure of the target herbicide resistance protein; A2) based on the three-dimensional structure, selecting the active cavity of the known resistance protein as a docking pocket, and performing molecular docking on the resistance protein and the target herbicide ligand small molecule to obtain a docking result, wherein the molecular docking is performed using Autodock; A3) determining the connection ability between the resistance protein and the target herbicide ligand small molecule according to the docking result, selecting the three-dimensional structure of the resistance protein with strong connection ability for alanine scanning analysis to obtain mutation sites; A4) performing saturation mutagenesis on the mutation site to obtain a mutant protein sequence; 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 a recombinant protein; A6) performing enzyme activity assay and target herbicide degradation ability assay on the recombinant protein to obtain assay results; A7) Screening and obtaining a degrading enzyme for the target herbicide based on the determination results.

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