A novel trichlorophenol-degrading enzyme and its application

Abstract

The present invention provides a novel trichlorophenol degrading enzyme, the amino acid sequence of which is shown in any one of SEQ ID NO.2~SEQ ID NO.5. The present invention also provides the application of the novel trichlorophenol degrading enzyme in degrading trichlorophenol and repairing trichlorophenol-contaminated environments, as well as a method for degrading trichlorophenol or repairing trichlorophenol-contaminated environments. The present invention uses the AlphaFold2 artificial intelligence model to construct the three-dimensional structure of the degradation enzyme TcpA, and analyzes its structure, and finally combines the mutation technology to screen out Tyr490His, Tyr490Arg, Ala494Ile, Ala494Ile-Tyr490Arg mutant enzymes with better degradation effects; the optimized mutant enzyme of the present invention can efficiently and quickly degrade trichlorophenol, and can also be used to repair natural environments such as water bodies contaminated by trichlorophenol, and efficiently and quickly remove the residual amount of such pesticides in water bodies.

Description

Technical Field

[0001] The invention relates to the technical field of biodegradation, in particular to a novel trichlorophenol degrading enzyme and application thereof. Background Art

[0002] Trichlorophenol is an important chemical raw material, commonly used as an intermediate in chemical dyes, bactericides, preservatives and pharmaceutical synthesis. It includes 2,3,4-trichlorophenol, 2,3,5-trichlorophenol, 2,3,6-trichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol and 3,4,5-trichlorophenol. Trichlorophenol has a long half-life in the environment and has been listed as a persistent organic pollutant by the International POPs Convention. my country's "Sanitary Standards for Drinking Water (GB 5749-2022)" and "Surface Water Environmental Quality Standards (GB 3838-2002)" also clearly define the limit value of 2,4,6-trichlorophenol in water (0.2 mg / L). At the same time, trichlorophenol has also been found to be toxic to aquatic organisms and mammals, and its residues in the environment will pose a serious threat to human health. Exploring efficient removal methods for trichlorophenol is of great significance to protecting the environment and human health safety.

[0003] Microbial degradation is an important way to remove trichlorophenol from the environment. However, trichlorophenol itself has antibacterial activity, and the high electronegative chlorine atoms make it difficult for the substituents on the benzene ring structure to be oxidized, which makes the degradation rate of the trichlorophenol-degrading strains currently screened relatively low. In recent years, with the development of machine learning technology, artificial intelligence models can be used to quickly and accurately analyze and predict the structure of degradation enzymes, and on this basis, the catalytic sites can be determined, and the molecular mechanism of degradation enzymes catalyzing pollutant degradation can be clarified, providing a theoretical basis for the structural modification of degradation enzymes and the improvement of catalytic efficiency.

[0004] In the early stage, the laboratory screened a strain of halogenated phenol efficient degradation bacteria Cupriavidus taiwanensis X1, and cloned and expressed the key enzyme TcpA for halogenated phenol degradation (authorized patent ZL 201811292459.8). In the present invention, an attempt was made to use the AlphaFold2 artificial intelligence model to construct a three-dimensional model of the degradation enzyme TcpA, determine the catalytic structure site, and further use the mutation method to obtain a trichlorophenol degradation enzyme with higher degradation efficiency, which is used to quickly and efficiently degrade trichlorophenol and repair the environment polluted by trichlorophenol residues. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a novel trichlorophenol degrading enzyme with higher degradation efficiency and application thereof.

[0006] The present invention adopts the following technical solutions to solve the above technical problems:

[0007] A novel trichlorophenol degrading enzyme, whose amino acid sequence is shown in any one of SEQ ID NO.2 to SEQ ID NO.5.

[0008] As one of the preferred embodiments of the present invention, the enzyme is obtained by optimizing and improving the structure of the original trichlorophenol degrading enzyme TcpA as shown in SEQ ID NO.1.

[0009] As one of the preferred embodiments of the present invention, the structural optimization and improvement is achieved by mutation.

[0010] As one of the preferred embodiments of the present invention, the SEQ ID NO.2 sequence corresponds to the Tyr490His mutant enzyme, the SEQ ID NO.3 sequence corresponds to the Tyr490Arg mutant enzyme, the SEQ ID NO.4 sequence corresponds to the Ala494Ile mutant enzyme, and the SEQ ID NO.5 sequence corresponds to the Ala494Ile-Tyr490Arg mutant enzyme.

[0011] An application of the novel trichlorophenol degrading enzyme in degrading trichlorophenol and repairing a trichlorophenol-contaminated environment.

[0012] As one of the preferred embodiments of the present invention, the polluted environment includes water and soil.

[0013] A method for degrading trichlorophenol or repairing a trichlorophenol-polluted environment utilizes the novel trichlorophenol-degrading enzyme.

[0014] As one of the preferred embodiments of the present invention, pure enzyme solution is directly used.

[0015] The advantages of the present invention compared to the prior art are:

[0016] (1) The present invention discovered TcpA enzyme in the early stage, and constructed the three-dimensional structure of the degradation enzyme TcpA using the AlphaFold2 artificial intelligence model, and used the AutoDock4.2 software to perform molecular docking between the protein TcpA and the small molecule 2,4,6-trichlorophenol. Based on the molecular docking results of the degradation enzyme TcpA and 2,4,6-trichlorophenol, the key amino acids Tyr490 and Ala494 (amino acids Tyr490 and Ala494 in the amino acid sequence of TcpA) were iteratively saturated mutagenesis. Point mutations verified the molecular docking results, and mutant enzymes with better degradation effects were screened. Among them, the enzymatic activities of Tyr490His, Tyr490Arg, Ala494Ile, and Ala494Ile-Tyr490Arg mutant enzymes were increased by 1.47, 1.81, 1.73, and 1.99 times, respectively, compared with the original TcpA. The optimized mutant enzymes of the present invention can efficiently and quickly degrade trichlorophenol, and can also be used to repair natural environments such as water bodies contaminated by trichlorophenol, and efficiently and quickly remove the residues of such pesticides in water bodies.

[0017] (2) The present invention enriches the degradation enzyme resource library and has great application value in the bioremediation of water bodies, soil, etc. contaminated by trichlorophenol residues, and provides new materials and methods for breaking the existing bottleneck of controlling environmental persistent organic pollutant residue pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The degradation kinetics of 6 trichlorophenols by pure TcpA enzyme in Experimental Example 1 (Figure A shows the results with 2,3,4-TCP as substrate; Figure B shows the results with 2,4,5-TCP as substrate; Figure C shows the results with 3,4,5-TCP as substrate; Figure D shows the results with 2,4,6-TCP as substrate; Figure E shows the results with 2,3,6-TCP as substrate; Figure F shows the results with 2,3,5-TCP as substrate);

[0019] Figure 2 It is the three-dimensional structure diagram of TcpA protein predicted by AlphaFold2 in Experimental Example 3;

[0020] Figure 3 is the quality pull-type diagram evaluation result in Experimental Example 3;

[0021] Figure 4 It is a comparison of the binding sites of FADH2 and 2,4,6-trichlorophenol with TcpA in Experimental Example 3 (in the figure, Figure A is the Surface semi-transparent display mode; Figure B is the ribbon display mode);

[0022] Figure 5It is the analysis of the binding mode of FADH2 and TcpA in Experimental Example 3 (in the figure, Figure A is the molecular docking diagram of FADH2 and TcpA, and Figure B is the interaction relationship diagram of FADH2 and active pocket amino acids);

[0023] Figure 6 The binding mode analysis of FADH2-TcpA complex and 2,4,6-trichlorophenol in Experimental Example 3 (Figure A is a molecular docking diagram of 2,4,6-trichlorophenol and FADH2-TcpA complex, and Figure B is a diagram of the interaction between 2,4,6-trichlorophenol and active pocket amino acids);

[0024] Figure 7 It is the sequencing peak graph in Experimental Example 3 (in the figure, Figure A is the sequencing peak graph of the original enzyme of TcpA, Figure B is the sequencing peak graph of the Tyr490His mutant enzyme, Figure C is the sequencing peak graph of the Tyr490Arg mutant enzyme, Figure D is the sequencing peak graph of the Ala494Ile mutant enzyme, and Figure E is the sequencing peak graph of the Ala494Ile-Tyr490Arg mutant enzyme);

[0025] Figure 8 The degradation kinetics results of 2,4,6-trichlorophenol by pure TcpA enzyme and pure mutant enzymes in Experimental Example 4 (Figure A is the result corresponding to pure TcpA enzyme; Figure B is the result corresponding to pure Tyr490Arg enzyme; Figure C is the result corresponding to pure Ala494Ile enzyme; Figure D is the result corresponding to pure Tyr490His enzyme);

[0026] Fig. 9 This is the degradation kinetic result of 2,4,6-trichlorophenol by the iterative mutant enzyme Ala494Ile-Tyr490Arg in Experimental Example 4. DETAILED DESCRIPTION

[0027] The following is a detailed description of the embodiments of the present invention. The embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are provided, but the protection scope of the present invention is not limited to the following embodiments. Meanwhile, unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0028] The culture medium formulas involved in the following embodiments and experimental examples are as follows:

[0029] LB medium (Luria-Bertani Medium): 10 g / L tryptone, 5 g / L NaCl, 5 g / L yeast extract powder, adjusted to pH 7.0.

[0030] 0.01M PBS phosphate buffered saline: Pour the dry powder into pure water and stir thoroughly to dissolve (pH 7.2~7.4), and finally dilute to 2000mL for use.

[0031] The above solutions were sterilized by high temperature steam at 121°C for 20 min.

[0032] 1 mol / L IPTG solution: weigh 0.24 g IPTG in 10 mL ddH2O, shake thoroughly to dissolve, filter the solution through a 0.22 um filter membrane, and store it in aliquots at -20°C for later use.

[0033] 50mg / mL kanamycin solution: weigh 0.05g kanamycin into a 2mL EP tube, add 1mL sterile water to dissolve, filter through a 0.22um filter membrane, and store at -20℃ for later use.

[0034] Lysis Bufer buffer: weigh 1.56 g of anhydrous sodium dihydrogen phosphate and 3.508 g of sodium chloride in 200 mL of ddH2O and shake thoroughly to dissolve (pH 7-8).

[0035] Imidazole buffer: weigh 0.034 g, 0.085 g, 0.17 g, 0.51 g, and 0.85 g into 25 mL Lysis Bufer buffer and shake thoroughly to dissolve (pH 7-8) to form imidazole buffers of different concentrations: 20 mM, 50 mM, 100 mM, 300 mM, and 500 mM.

[0036] Example 1

[0037] The novel trichlorophenol degrading enzyme of this embodiment is a Tyr490His mutant enzyme of the original trichlorophenol degrading enzyme TcpA (sequence shown in SEQ ID NO.1), and the amino acid sequence is shown in SEQ ID NO.2.

[0038] Example 2

[0039] The novel trichlorophenol degrading enzyme of this embodiment is a Tyr490Arg mutant of the original trichlorophenol degrading enzyme TcpA (sequence shown in SEQ ID NO.1), and the amino acid sequence is shown in SEQ ID NO.3.

[0040] Example 3

[0041] The novel trichlorophenol degrading enzyme of this embodiment is an Ala494Ile mutant of the original trichlorophenol degrading enzyme TcpA (sequence shown in SEQ ID NO.1), and the amino acid sequence is shown in SEQ ID NO.4.

[0042] Example 4

[0043] A novel trichlorophenol degrading enzyme in this embodiment is an Ala494Ile-Tyr490Arg mutant enzyme of the original trichlorophenol degrading enzyme TcpA (sequence shown in SEQ ID NO.1), and the amino acid sequence is shown in SEQ ID NO.5.

[0044] Experimental Example 1

[0045] This experimental example is used to explore the degradation kinetics of trichlorophenol-degrading enzyme TcpA (original enzyme).

[0046] 1. Experimental Methods

[0047] 1. Expression and purification of TcpA

[0048] (1) Express the degradation enzyme TcpA, collect the cells by centrifugation at 5°C and 8000 r / min for 10 min, and resuspend them in PBS. Use an ultrasonic disruptor to disrupt the bacterial solution. The instrument working procedure is as follows: amplitude 35, work 2s, stop 2s, a total of 20min, until the solution becomes clear. After the disruption is completed, centrifuge at 8°C and 9000 r / min for 20min. The supernatant is the crude enzyme solution of the degradation enzyme.

[0049] (2) The 2,4,6-trichlorophenol monooxygenase expressed by fusion with the pET30a-TcpA vector contains a His tag and can be affinity purified by Ni-NTA resin. The specific steps are as follows:

[0050] ① Balance: Use 5 column volumes of binding buffer to balance the Ni Smart Beads 6FF gravity column;

[0051] ② Loading: Take 40 mL of 2,4,6-trichlorophenol monooxygenase crude enzyme solution, flow it through the polysaccharide Ni column at a flow rate of 0.8 mL / min, and wash the column with 10 times the column volume of buffer;

[0052] ③ Elution: Elute the target protein with 3 column volumes containing 20mM, 50mM, 100mM, 300mM, and 500mM imidazole, and collect the effluent;

[0053] ④ Regeneration: Clean and regenerate the Ni column in the following order: add 3 times the column volume of LysisBuffer, 3 times the column volume of deionized water, and finally store the filler in 20% alcohol.

[0054] All the above steps were carried out in a chromatography cabinet at 4°C.

[0055] (3) Add the eluate containing the target protein to a 30kd ultrafiltration tube, centrifuge at 4°C, 4000 r / min for 25 min, and discard the lower layer of waste liquid; add 15 mL, 10 mmol / L PBS buffer, centrifuge at 4°C, 4000 r / min for 25 min, discard the lower layer of waste liquid, and repeat this step once; remove the purified target protein from the ultrafiltration tube.

[0056] 2. Degradation enzyme TcpA concentration determination

[0057] Draw a BSA standard curve. Prepare 0ug / mL, 22ug / mL, 66ug / mL, 110ug / mL, 154ug / mL, and 198ug / mL BSA solutions, take 1mL of each concentration solution in a 2mL centrifuge tube, number them in order from low to high concentration, add 1mL of Coomassie Brilliant Blue G250 solution to each tube, shake and mix, place at room temperature for 5 minutes, add 200uL to the ELISA plate, and use an ELISA instrument to measure the OD595 absorbance of different concentrations of BSA; draw a standard curve based on the measured values ​​and the concentration of the BSA solution.

[0058] Determine the protein content of different samples. Take 1uL of the supernatant of the sample to be tested and add it to 249uL of deionized water, aspirate 100uL, then add 1mL of Coomassie Brilliant Blue G250 solution, shake and mix, leave at room temperature for 5 minutes, and then use an enzyme reader to measure the OD595 absorbance value, and calculate the protein content of the sample to be tested according to the linear equation of the drawn standard curve.

[0059] 3. Degradation performance determination

[0060] Using "2,3,4-trichlorophenol, 2,3,5-trichlorophenol, 2,3,6-trichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol and 3,4,5-trichlorophenol" as substrates, substrate solutions with molar concentrations of 0.025, 0.05, 0.075, 0.1 and 0.125 mmol / L were prepared respectively. The activity properties of the enzyme were measured by measuring the residual amounts of the six trichlorophenols. The reaction was fitted with the Michaelis-Menten equation and plotted using the Linewear-Burk method. The Michaelis constant Km and affinity 1 / Km were calculated.

[0061] 2. Testing conditions

[0062] HPLC: type 2695 (Waters, USA);

[0063] Detector: Photodiode array (PDA) detector;

[0064] Detection wavelength: 280nm;

[0065] Chromatographic column: Agilent 5 HC-C18 (2) 250×4.6 mm;

[0066] Column temperature: 30°C;

[0067] Injection volume: 10uL;

[0068] Flow rate: 1 mL / min;

[0069] Mobile phase: formic acid water (A): acetonitrile (B) = 30:70.

[0070] 3. Experimental Results

[0071] The pure TcpA enzyme was diluted to 100ug / mL (10mM PBS, pH=7.2~7.4), and different molar concentrations (0.0125, 0.025, 0.05, 0.075, 0.1mmol / L) of 2,3,4-TCP, 2,4,5-TCP, 3,4,5-TCP, 2,4,6-TCP, 2,3,6-TCP, and 2,3,5-TCP were degraded at 37°C. Different substrate concentrations [S] were selected to determine the corresponding reaction rate v, and 1 / v and 1 / [S] were used as the horizontal and vertical coordinates for plotting. The results are shown in the figure. Figure 1 shown.

[0072] The Michaelis constant Km is a characteristic constant of the enzyme, reflecting the affinity between the enzyme and the substrate. 1 / Km is usually used to represent the affinity. The smaller 1 / Km is, the weaker the affinity between the enzyme and the substrate is. Figure 1 , the 1 / Km of pure TcpA enzyme and six trichlorophenols can be summarized as shown in Table 1. As shown in Table 1, the 1 / Km values ​​of pure TcpA enzyme for 2,3,4-TCP, 2,4,5-TCP, 3,4,5-TCP, 2,4,6-TCP, 2,3,6-TCP, and 2,3,5-TCP are 72.75, 38.119, 22.782, 14.877, 7.803, and 1.773, respectively. By comparison, it was found that pure TcpA enzyme had the best affinity for 2,3,4-TCP, followed by 2,4,5-TCP, and the affinity for 3,4,5-TCP, 2,4,6-TCP, and 2,3,6-TCP gradually weakened, and the affinity for 2,3,5-TCP was the weakest.

[0073] Table 1 1 / Km of pure TcpA enzyme and six trichlorophenols

[0074]

[0075] Experimental Example 2

[0076] The experimental example is used to specifically determine the affinity of trichlorophenol-degrading enzyme TcpA (original enzyme) to 6 trichlorophenols.

[0077] 1. Experimental Methods

[0078] 1. Reagent preparation

[0079] PBS (10X), pH 7.4 was diluted 10 times with deionized water to prepare running buffer 1: containing 2mM potassium dihydrogen phosphate, 137mM sodium chloride, 10mM sodium dihydrogen phosphate, 2.7mM potassium chloride, pH adjusted to 7.4, and filtered through a 0.22 um filter membrane before use.

[0080] PBS (10X), pH 7.4 was diluted with deionized water and acetonitrile was added to prepare running buffer 2: containing 2mM potassium dihydrogen phosphate, 137mM sodium chloride, 10mM sodium dihydrogen phosphate, 2.7mM potassium chloride, 5% acetonitrile, pH adjusted to 7.4, and filtered through a 0.22um filter membrane before use.

[0081] 2. Chip processing

[0082] The ligand (TcpA) was diluted 20 times with the immobilization reagent (10mM sodium acetate, pH 4.5). First, the surface of the CM5 chip was activated with 400mM EDC and 100mM NHS at a flow rate of 10uL / min for 900s. Secondly, the diluted ligand was injected into the experimental channel (Fc4) at a flow rate of 30uL / min, and the fixed amount was about 25000RU. Finally, the chip was blocked with 1M ethanolamine at 10uL / min for 900s. The reference channel (Fc3) was only activated and blocked, and the ligand was not coupled. This process was performed in running buffer 1.

[0083] 3. Sample processing

[0084] Prepare solvent calibration products with different acetonitrile concentrations according to Table 2.

[0085] Table 2 Preparation of solvent calibration products with different acetonitrile concentrations

[0086]

[0087] Each analyte was first dissolved into a 10mM mother solution with acetonitrile, and then the analyte was gradiently diluted with running buffer 2. The diluted analytes were injected into the reference channel (Fc3) and the experimental channel (Fc4) at a flow rate of 30uL / min, with a binding time of 60s and a dissociation time of 60s. The temperature of the chip compartment and the sample compartment was selected to be 25°C, and a solvent calibration was performed every 30 cycles to determine the affinity between the analyte and the ligand. The binding and dissociation steps were all performed in running buffer 2.

[0088] 2. Experimental Results

[0089] KD is the equilibrium dissociation constant between the antibody and its antigen, i.e., koff / k on The KD is inversely proportional to the affinity. The lower the KD value, the higher the affinity between the protein and the small molecule. The experimental results were analyzed using the Biacore T200 Evaluation Software. The affnity 1:1 binding model was selected for fitting. The results are shown in Table 3.

[0090] It can be seen from Table 3 that the KD values ​​of pure TcpA enzyme for 2,3,4-TCP, 2,4,5-TCP, 3,4,5-TCP, 2,4,6-TCP, 2,3,6-TCP and 2,3,5-TCP are 3.01E-03, 3.43E-03, 4.21E-03, 6.39E-03, 7.29E-03 and 9.51E-03 respectively. By comparing the KD values, it is found that the affinity of pure TcpA enzyme to 2,3,4-TCP is the highest, followed by 2,4,5-Tcp, and gradually decreases with 3,4,5-Tcp, 2,4,6-Tcp and 2,3,6-Tcp, and the affinity to 2,3,5-Tcp is the lowest, and the effect is the worst.

[0091] Table 3 Affinity of TcpA to six trichlorophenols

[0092]

[0093] Experimental Example 3

[0094] The experimental example is used to describe the structural optimization of the trichlorophenol-degrading enzyme TcpA.

[0095] 1. Experimental Methods

[0096] 1. Protein treatment

[0097] The protein TcpA structure was modeled using Alphafold2, the structural reliability was determined using a pull-out diagram, hydrogen atoms were added using UCSF Chimera software, Amber99SB charges were assigned, and protonation states were assigned using the H++3.0 program. SiteMap was then used to predict the best small molecule binding site.

[0098] 2. Small molecule processing

[0099] The 2D structures of the small molecules FADH2 and 2,4,6-trichlorophenol were downloaded from the PubChem database, and then the small molecule structures were generated into three-dimensional structures using the chemical information software package RDKit, optimized using the MMFF94 force field, and then local charges were assigned using the AM1-BCC method using UCSF Chimera.

[0100] 3. Molecular docking

[0101] In this study, AutoDock4.2 software was used for molecular docking, and the predicted active site was set as the docking center. During the docking process, when docking FADH2, the center coordinates X, Y, and Z were -15.07, 16.14, and -3.01, respectively. The box size was set to a cube with a side length of 22.50 Å, and the Spacing step was set to 0.375. The maximum limit number of search conformations was set to 10,000, and a genetic algorithm was used for conformation sampling and scoring. The conformations were sorted and the optimal conformation was selected based on the docking score. When docking 2,4,6-trichlorophenol, the FADH2-TCPA complex was used as the receptor, and SiteMap was used to predict the best binding site. The center coordinates X, Y, and Z were -7.50, 14.84, and 16.14, respectively. The genetic algorithm was used to score the configuration and calculate the binding free energy.

[0102] 4. Site-directed mutagenesis

[0103] According to the results of homology modeling and molecular docking, after determining the active pocket and active site of 2,4,6-trichlorophenol, point mutation was used for further verification. Using the TcpA protein expression vector pET-TcpA as a template, with Tyr490, Ala494, and Arg461 residues as mutation points, the expression vector pET30a-TcpA was PCR amplified using three pairs of overlapping primers (as shown in Table 4). The 50uL reaction system contained 1uL of expression plasmid pET30a-TcpA, 2uL of corresponding upstream and downstream primers, 25uL 2×TransStartFastPfu Fly PCR SuperMix, and 22uL Nuclease-free Water. The PCR reaction program was 94℃ denaturation for 20s, 55℃ annealing for 20s, and 72℃ extension for 1.5min, with 25 cycles. After the reaction, 10uL of PCR product was taken and the target band size was detected by 1% gel electrophoresis. 1uL DMT enzyme was added to the remaining PCR product and reacted at 37℃ for 1h to digest the methylated plasmid template (non-mutant plasmid template) in vitro. The DMT enzyme digestion product was transformed into DMT competent cells for in vivo degradation of the methylated plasmid template, and clones were screened using 50mg / L kanamycin resistance plates and identified by sequencing. The mutant expression plasmids pET30a-Ala490Ile, pET30a-Tyr490His and pET30a-Tyr490Arg with correct sequencing were extracted and transformed into E. coli BL21 (DE3) competent cells.

[0104] Then, using the expression plasmid pET30a-Ala494Ile as a template, iterative mutation primers were designed to iteratively mutate the expression plasmid pET30a-Ala494Ile. The mutation method was the same as the single mutation method, and the iterative mutation expression plasmid pET30a-Ala494Ile-Tyr490Arg was obtained. The expression plasmid was transformed into Escherichia coli BL21 (DE3) competent cells. The amplified expression vector was sent to Anhui General Biotechnology Company for sequencing to verify the mutated sequence. After sequencing was correct, the mutated protein was expressed.

[0105] Table 4 Site-directed mutagenesis primers

[0106]

[0107] 2. Experimental Results

[0108] 1. The three-dimensional structure of TcpA protein was constructed using AlphaFold2. The results are as follows Figure 2 As shown in Figure 2, the reliability of the three-dimensional structure is evaluated using the Pull-type diagram ( Figure 3 ), analysis showed that in the modeled structure, all amino acids fell within the core region (core) and the generally allowed region (allowed). The pull-out diagram evaluation results showed that the three-dimensional structure of TcpA modeled by Alphafold2 was reasonable and could be used for the next step of molecular docking research.

[0109] 2. The binding model and key role of FADH2 and TcpA proteins were analyzed by molecular docking experiments. Then 2,4,6-trichlorophenol was docked to the FADH2-TcpA complex. The study showed that both compounds can bind to TcpA, and the binding sites are obviously different, such as Figure 4 shown.

[0110] Specific analysis of the interaction between FADH2 and TcpA proteins Figure 5 As shown, FADH2 forms multiple and various types of interactions with TcpA, including hydrogen bonds, salt bridges, hydrophobic interactions, and polar van der Waals contacts, such as Figure 5 As shown in Figure A, the unsaturated N on the adenine ring of FADH2 forms a weak hydrogen bond with the aliphatic carbon chain of the arginine side chain at position 208, and forms a hydrogen bond with the backbone amide bond of aspartic acid at position 156. The amino group on the purine ring forms a hydrogen bond with the side chain of aspartic acid at position 156, one of the phosphate groups forms a salt bridge with the basic amino acid arginine at position 161, and arginine at position 161 also forms a hydrogen bond with another phosphate group. The amino group on the tricyclic ring of FADH2 forms a hydrogen bond with the side chain of asparagine at position 445, and the tricyclic ring forms more hydrophobic interactions with the surrounding hydrophobic amino acids. Polar amino acids form polar van der Waals contacts with FADH2.

[0111] The molecular docking results of 2,4,6-trichlorophenol and FADH2-TcpA complex are as follows Figure 6 As shown, the complex of trichlorophenol, TcpA and FADH2 is combined through hydrogen bonds, halogen bonds and CH-π interactions or alkane-π stacking. The chlorine at the position opposite to the hydroxyl group forms a Cl...O=C halogen bond with the backbone carbonyl group of tyrosine No. 490, the carbon chain of arginine No. 461 forms an alkane-π stacking (arene-H) with the charge center of the benzene ring, and the hydroxyl group forms a hydrogen bond with the side chain amino group of arginine No. 461 after protonation. Trichlorophenol forms a hydrophobic interaction with the hydrophobic amino acids alanine No. 494, isoleucine No. 360 and isoleucine No. 280, and forms a van der Waals contact with other polar amino acids. Thus, potential amino acid mutation sites are obtained.

[0112] This study used AutoDock's genetic algorithm to evaluate the affinity of small molecules and proteins, using binding free energy (ΔG) and inhibition constant (Ki) to reflect the binding size. As shown in Table 5. The ΔG of FADH2 is -11.19 kcal / mol, and the corresponding Ki value is 6.26 nM. The ΔG of 2,4,6-trichlorophenol is -4.92 kcal / mol, and the corresponding Ki value is 245.97 uM. From the various indicators of binding free energy, hydrogen bonds and van der Waals interactions play a leading role in the binding process. Among them, the electrostatic energy of FADH2 is -4.36 kcal / mol, indicating that FADH2 and TcpA have a strong charge attraction. From the binding mode, FADH2 also forms a salt bridge with TcpA. The FADH2 molecule has more rotatable bonds, so the torsion free energy is positive, resulting in a loss of binding energy. The configuration of trichlorophenol is relatively fixed and there are no rotatable bonds, so there is no loss of binding energy.

[0113] Table 5 Calculation of binding affinity between TcpA and small molecules

[0114]

[0115] 3. Site-directed mutagenesis is to regulate the properties and functions of proteins by changing the amino acid residues at specific positions in the protein sequence. It can enhance the activity of the enzyme and improve the recognition and binding ability with the substrate. In order to verify the molecular docking results and obtain a mutant enzyme with better effect, point mutations were made to Tyr at position 490 and Ala at position 494. The sequencing peaks are shown in the figure below. Figure 7 As shown, the sequencing results were correct.

[0116] Experimental Example 4

[0117] The experimental example is used to verify the degradation kinetics of the degradation enzyme after mutation.

[0118] 1. Experimental Methods

[0119] 1. Expression and purification of mutant enzymes

[0120] (1) Express the mutant enzyme, collect the cells by centrifugation at 5°C and 8000 r / min for 10 min, and resuspend them in PBS. Use an ultrasonic disruptor to disrupt the bacterial solution. The instrument working procedure is as follows: amplitude 35, work 2s, stop 2s, a total of 20min, until the solution becomes clear. After the disruption is completed, centrifuge at 8°C and 9000 r / min for 20min. The supernatant is the crude enzyme solution of the mutant enzyme.

[0121] (2) Affinity purification using Ni-NTA resin. The specific steps are as follows:

[0122] ① Balance: Use 5 column volumes of binding buffer to balance the Ni Smart Beads 6FF gravity column;

[0123] ② Loading: Take 40 mL of crude mutant enzyme solution and flow it through the polysaccharide Ni column at a flow rate of 0.8 mL / min, and wash the column with 10 column volumes of buffer;

[0124] ③ Elution: Elute the target protein with 3 column volumes containing 20mM, 50mM, 100mM, 300mM, and 500mM imidazole, and collect the effluent;

[0125] ④ Regeneration: Clean and regenerate the Ni column in the following order: add 3 times the column volume of LysisBuffer, 3 times the column volume of deionized water, and finally store the filler in 20% alcohol.

[0126] All the above steps were carried out in a chromatography cabinet at 4°C.

[0127] (3) Add the eluate containing the target protein to a 30kd ultrafiltration tube, centrifuge at 4°C, 4000 r / min for 25 min, and discard the lower layer of waste liquid; add 15 mL, 10 mmol / L PBS buffer, centrifuge at 4°C, 4000 r / min for 25 min, discard the lower layer of waste liquid, and repeat this step once; remove the purified target protein from the ultrafiltration tube.

[0128] 2. Determination of mutant enzyme concentration

[0129] Draw a BSA standard curve. Prepare 0ug / mL, 22ug / mL, 66ug / mL, 110ug / mL, 154ug / mL, and 198ug / mL BSA solutions, take 1mL of each concentration solution in a 2mL centrifuge tube, number them in order from low to high concentration, add 1mL of Coomassie Brilliant Blue G250 solution to each tube, shake and mix, place at room temperature for 5 minutes, add 200uL to the ELISA plate, and use an ELISA instrument to measure the OD595 absorbance of different concentrations of BSA; draw a standard curve based on the measured values ​​and the concentration of the BSA solution.

[0130] Determine the protein content of different samples. Take 1uL of the supernatant of the sample to be tested and add it to 249uL of deionized water, aspirate 100uL, then add 1mL of Coomassie Brilliant Blue G250 solution, shake and mix, leave at room temperature for 5 minutes, and then use an enzyme reader to measure the OD595 absorbance value, and calculate the protein content of the sample to be tested according to the linear equation of the drawn standard curve.

[0131] 3. Degradation performance determination

[0132] The experiment used 6 trichlorophenols as substrates and prepared substrate solutions with molar concentrations of 0.0125, 0.025, 0.05, 0.075, 0.1, 0.125, and 0.015 mmol / L, respectively. The activity properties of the enzyme were measured by measuring the residual amounts of the 6 trichlorophenols. The reaction was fitted with the Michaelis-Menten equation and plotted using the Linewear-Burk method. The Michaelis constant Km and affinity 1 / Km were calculated.

[0133] 2. Testing conditions

[0134] HPLC: type 2695 (Waters, USA);

[0135] Detector: Photodiode array (PDA) detector;

[0136] Detection wavelength: 280nm;

[0137] Chromatographic column: Agilent 5 HC-C18 (2) 250×4.6 mm;

[0138] Column temperature: 30°C;

[0139] Injection volume: 10uL;

[0140] Flow rate: 1 mL / min;

[0141] Mobile phase: formic acid water (A): acetonitrile (B) = 30:70.

[0142] 3. Experimental Results

[0143] (1) The mutated Tyr490His pure enzyme, Tyr490Arg pure enzyme and Ala494Ile pure enzyme were diluted to 100 g / mL (10 mM PBS pH = 7.2-7.4), and different molar concentrations (0.0125, 0.025, 0.05, 0.075, 0.1 mmol / L) of 2,4,6-trichlorophenol were degraded at 37°C. Different substrate concentrations [S] were selected to determine the corresponding reaction rate v, and a graph was plotted with 1 / v and 1 / [S] as the horizontal and vertical coordinates, as shown in the figure. Figure 8 shown.

[0144] The affinity (1 / Km value) of each mutant pure enzyme to 2,4,6-trichlorophenol is shown in Table 6: the degradation of Tyr490Arg pure enzyme, Ala494Ile pure enzyme and Tyr490His pure enzyme was 1.81, 1.73 and 1.47 times higher than that of the original TcpA pure enzyme, respectively. The degradation results verified that the molecular docking results were correct and that the mutant enzymes with better degradation effects than the original TcpA were obtained.

[0145] Table 6 Affinity of mutant pure enzymes for 2,4,6-trichlorophenol

[0146]

[0147] (2) Iterative mutagenesis is to gradually optimize the properties of the enzyme through multiple rounds of site-directed mutagenesis. In order to further obtain a mutant enzyme with better degradation effect, site-directed mutagenesis was performed on the expression plasmid pET30a-Ala494Ile to obtain the iterative mutant enzyme Ala494Ile-Tyr490Arg. The degradation effect of the iterative mutant enzyme Ala494Ile-Tyr490Arg was verified by degradation experiments, such as Fig. 9 As shown: the 1 / Km value of the iterative mutant enzyme is 29.613, which has a better degradation effect on 2,4,6-trichlorophenol than the single mutant Tyr490Arg pure enzyme, Ala494Ile pure enzyme and Tyr490His pure enzyme, and is 1.99 times that of the original TcpA pure enzyme. This shows that a mutant enzyme with better effect can be obtained through iterative mutation.

[0148] In summary, the optimized mutant enzymes Tyr490His, Tyr490Arg, Ala494Ile, and Ala494Ile-Tyr490Arg of the present invention can efficiently and quickly degrade trichlorophenol, and can also be used to repair natural environments such as water bodies contaminated by trichlorophenol, and efficiently and quickly remove the residues of such pesticides in water bodies.

[0149] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A novel trichlorophenol degrading enzyme, characterized in that: Its amino acid sequence is shown in any one of SEQ ID NO.2 to SEQ ID NO.

5.

2. An application of the novel trichlorophenol degrading enzyme as claimed in claim 1 in degrading 2,4,6-trichlorophenol and repairing a 2,4,6-trichlorophenol-contaminated environment.

3. The use according to claim 2, characterized in that: The polluted environment includes water and soil.

4. A method for degrading 2,4,6-trichlorophenol or repairing an environment polluted by 2,4,6-trichlorophenol, characterized in that: Utilize the novel trichlorophenol-degrading enzyme described in claim 1.

5. The method for degrading 2,4,6-trichlorophenol or repairing the environment polluted by 2,4,6-trichlorophenol according to claim 4, characterized in that: Use pure enzyme solution directly.

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