Acetylcholinesterase and application thereof in detection of organophosphorus pesticide

Abstract

The invention discloses acetylcholin esterase and application thereof in detection of organophosphorus pesticides, and belongs to the technical field of protein engineering. Amino acid on a substrate binding pocket of the N267D mutant (AnAChE-N267D) is systematically modified through rational design of a crystal structure (PDB ID 9L2A) based on Aspergillus niger source acetylcholin esterase, and a novel enzyme mutant with higher catalytic activity and pesticide sensitivity is obtained. The relationship between enzymatic properties and structures of the mutants is studied, and the potential of detecting organophosphorus pesticides when the mutants are fixed on a paper-based carrier is explored. The research result not only expands the molecular modification strategy of AnAChE, but also provides important theoretical basis and technical support for developing efficient and stable enzyme preparations for organophosphorus pesticide detection.

Description

Technical Field

[0001] The present invention relates to the technical field of protein engineering, in particular to acetylcholinesterase and its application in detecting organophosphorus pesticides. Background Art

[0002] Acetylcholinesterase (AChE) is a key serine hydrolase. Microbial AChE, primarily derived from bacteria and fungi, has attracted widespread attention in recent years due to its unique biological properties. The research team's previously discovered AChE from Aspergillus niger GZUF36(An) exhibits high specificity for the substrate acetylthiocholine (ATCh). Compared to animal-derived AChE, microbial AChE exhibits higher catalytic activity, is amenable to genetic manipulation for a sustainable supply, is easily scalable, and exhibits superior stability in extreme environments. These advantages broaden its application prospects in industry and biomedicine.

[0003] Natural enzymes, as effective biocatalysts, promote most biochemical reactions in organisms by reducing the activation energy of certain chemical reactions. However, natural enzymes suffer from poor stability in extreme environments, catalytic activity that is sensitive to the environment (pH and temperature), high preparation and purification costs, and low recycling and reuse efficiency, limiting their large-scale application in industrial production. Natural AChE also suffers from low catalytic efficiency and limited substrate selectivity in practical applications. Therefore, designing and modifying AChE to enhance its functional properties is crucial. Summary of the Invention

[0004] The purpose of the present invention is to provide an acetylcholinesterase and its application in detecting organophosphorus pesticides to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is an acetylcholinesterase, the amino acid sequence of the acetylcholinesterase is shown as SEQ ID NO.21.

[0007] The second technical solution of the present invention is a DNA molecule encoding the acetylcholinesterase, and the nucleotide sequence of the DNA molecule is shown in SEQ ID NO.22.

[0008] The third technical solution of the present invention is an expression vector, comprising the aforementioned DNA molecule.

[0009] The fourth technical solution of the present invention is a recombinant strain, comprising the expression vector.

[0010] The fifth technical solution of the present invention is the method for preparing the acetylcholinesterase, which comprises utilizing the recombinant strain to express the acetylcholinesterase, and isolating and purifying the acetylcholinesterase.

[0011] The sixth technical solution of the present invention is an immobilized enzyme, which can be obtained by immobilizing the acetylcholinesterase on a carrier.

[0012] The seventh technical solution of the present invention is the use of the acetylcholinesterase or the immobilized enzyme in the detection of organophosphorus pesticides.

[0013] The eighth technical solution of the present invention is a method for detecting organophosphorus pesticides, wherein the sample to be tested is dropped onto the immobilized enzyme, the reaction is incubated, and a solution containing the organophosphorus pesticide is used as a control. The presence of the organophosphorus pesticide in the sample to be tested can be determined based on the change in color intensity value.

[0014] Based on the above technical solution, the present invention has the following technical effects:

[0015] This study systematically modified the amino acids in the substrate-binding pocket of Aspergillus niger-derived acetylcholinesterase (AnAChE-N267D) through rational design based on the crystal structure (PDB ID 9L2A) of the N267D mutant. The goal was to obtain novel enzyme mutants with enhanced catalytic activity and pesticide sensitivity. The relationship between the enzymatic properties and structure of the mutants was studied, and their potential for detecting organophosphorus pesticides when immobilized on a paper-based support was explored. This research not only expands the molecular modification strategy of AnAChE but also provides important theoretical basis and technical support for the development of efficient and stable enzyme preparations for organophosphorus pesticide detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 These are the 11 amino acids that make up the substrate binding pocket structure of AnAChE-N267D.

[0017] Figure 2 (a) crude enzyme activity of the mutant (U / mg); (b) enzyme activity of the mutant after purification (U / mg).

[0018] Figure 3 The SDS-PAGE electrophoresis diagrams of AnAChE-N267D and AnAChE-N267D-A101G before and after purification.

[0019] Figure 4Effects of buffer solutions of different pH values ​​on the enzyme activity of AnAChE-N267D and its mutant AnAChE-N267D-A101G; uppercase and lowercase letters indicate significant differences (p<0.05). Note: The highest enzyme activity of AnAChE-N267D (8.60 U / mg) was taken as 100% relative enzyme activity.

[0020] Figure 5 Effects of different temperatures on the enzyme activity of AnAChE-N267D and its mutant AnAChE-N267D-A101G; different uppercase / lowercase letters indicate significant differences (p<0.05). Note: The highest enzyme activity of AnAChE-N267D (8.60 U / mg) was taken as 100% relative enzyme activity.

[0021] Figure 6 Residual enzyme activities of AnAChE-N267D and its mutant AnAChE-N267D-A101G after incubation at different temperatures (20°C, 30°C, 40°C, 50°C and 60°C) for 2 h; different uppercase / lowercase letters indicate significant differences (p<0.05).

[0022] Figure 7 The residual enzyme activity of AnAChE-N267D and its mutant AnAChE-N267D-A101G after incubation in 10 mM metal ion solution for 3 hours; different uppercase / lowercase letters indicate significant differences (p < 0.05). Note: The enzyme activity of AnAChE-N267D without metal ion treatment (8.60 U / mg) is 100%.

[0023] Figure 8 The residual enzyme activity of AnAChE-N267D and its mutant AnAChE-N267D-A101G after 2 hours of incubation in a 1% (v / v) surfactant solution; different uppercase and lowercase letters indicate significant differences (p < 0.05). Note: The enzyme activity of AnAChE-N267D without surfactant treatment (8.60 U / mg) is 100%.

[0024] Figure 9 The changes in the enzyme activity of AnAChE-N267D and its mutant AnAChE-N267D-A101G after treatment with different organic solvents for 5 hours; different uppercase and lowercase letters indicate significant differences (p < 0.05). Note: The enzyme activity of AnAChE-N267D without organic solvent treatment (8.60 U / mg) is 100%.

[0025] Figure 10This is the double reciprocal equation diagram of the substrate kinetics of AnAChE-N267D and its mutant AnAChE-N267D-A101G.

[0026] Figure 11 The protein secondary structures of AnAChE-N267D and AnAChE-N267D-A101G were determined by circular dichroism spectroscopy; (a) circular dichroism spectrum; (b) relative content of secondary structure.

[0027] Figure 12 Thermal denaturation curves of AnAChE-N267D and AnAChE-N267D-A101G in the range of 25-95°C.

[0028] Figure 13 Molecular docking of AnAChE-N267D (a) and AnAChE-N267D-A101G (b) with the substrate ATCh, respectively.

[0029] Figure 14 Simulation of the conformational changes of AnAChE-N267D in aqueous solution. (a) Root mean square deviation (RMSD); (b) root mean square fluctuation (RMSF); (c) radius of gyration (Rog); and (d) solvent accessible area (SASA).

[0030] Figure 15 Simulation of the conformational changes of AnAChE-N267D-A101G in aqueous solution: (a) root mean square deviation (RMSD); (b) root mean square fluctuation (RMSF); (c) radius of gyration (Rog); and (d) solvent accessible area (SASA).

[0031] Figure 16 Optimization of pesticide detection conditions. (a) Effect of immobilized enzyme (FP&CS / GA@AnAChE-N267D-A101G) activity on the inhibition rate of the pesticide (0.1 mg / mL dichlorvos); (b) Effect of the co-incubation time of dichlorvos and immobilized enzyme on the inhibition rate; (c) Effect of the reaction time of immobilized enzyme and substrate on the inhibition rate.

[0032] Figure 17 This is the inhibition curve of dichlorvos on the immobilized enzyme FP&CS / GA@AnAChE-N267D-A101G.

[0033] Figure 18 Comparison of the inhibition rates of different concentrations (0.01-0.2 mg / mL) of dichlorvos on immobilized enzyme (FP&CS / GA@AnAChE-N267D-A101G) and commercial enzyme (AChE). DETAILED DESCRIPTION

[0034] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0035] An embodiment of the present invention provides an acetylcholinesterase, the amino acid sequence of the acetylcholinesterase is shown as SEQ ID NO.21.

[0036] The embodiment of the present invention also provides a DNA molecule encoding the acetylcholinesterase, and the nucleotide sequence of the DNA molecule is shown as SEQ ID NO.22.

[0037] An embodiment of the present invention further provides an expression vector comprising the DNA molecule.

[0038] An embodiment of the present invention also provides a recombinant strain, comprising the expression vector.

[0039] In some specific embodiments, the recombinant strain is AnAChE-N267D-A101G.

[0040] The embodiment of the present invention also provides a method for preparing the acetylcholinesterase, which comprises using the recombinant strain to express the acetylcholinesterase, and isolating and purifying the acetylcholinesterase to obtain the acetylcholinesterase.

[0041] The embodiment of the present invention further provides an immobilized enzyme, which can be obtained by immobilizing the acetylcholinesterase on a carrier.

[0042] The embodiment of the present invention also provides the use of the acetylcholinesterase or the immobilized enzyme in detecting organophosphorus pesticides.

[0043] In some specific embodiments, the organophosphorus pesticide comprises dichlorvos.

[0044] An embodiment of the present invention also provides a method for detecting organophosphorus pesticides. The sample to be tested is added dropwise to the immobilized enzyme, and the reaction is incubated. A solution containing the organophosphorus pesticide is used as a control. The presence of the organophosphorus pesticide in the sample to be tested can be determined based on the change in color intensity.

[0045] In some specific embodiments, the incubation reaction conditions are: enzyme activity 0.086 U / mg, incubation time 8 min.

[0046] Escherichia coli TOP10 was used to clone mutant plasmids, and E. coli BL21 was used as the expression host for target protein expression. Both were purchased from the laboratory. Plasmid pGEX4T-1-N267D was a mutant plasmid template constructed in our laboratory (construction method is described in CN117264928A). The amino acid sequence of AChE from Aspergillus niger GZUF36(An) is described in CN117264928A.

[0047] Example 1

[0048] 1.1 Construction of mutation library

[0049] 1.1.1 Site-directed saturation mutagenesis was performed on the substrate binding pocket amino acid sites selected based on the structural information of AChE from Aspergillus niger GZUF36(An). The primer sequences used are shown in Table 1.

[0050] Table 1 Primer sequences

[0051]

[0052]

[0053] 1.1.2 Extract the plasmid from E. coli BL21 carrying the plasmid and perform PCR amplification of the target gene. The PCR amplification reaction system is shown in Table 2.

[0054] Table 2 PCR amplification reaction system

[0055]

[0056] PCR reaction conditions: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 3 s, annealing at 63°C for 1 min, extension at 72°C for 12 min, 18 cycles; final extension at 72°C for 10 min, 4°C forever.

[0057] After amplification, perform agarose gel electrophoresis and place the gel on a gel imager to observe the target band.

[0058] 1.1.3 Purification and digestion of PCR products

[0059] After PCR amplification, 5 μL of the 50 μL product was subjected to 1% agarose gel electrophoresis for verification. The remaining product was purified using a DNA purification kit to remove the enzymes and buffer introduced during the PCR amplification process. To the purified product, 5 μL of 10× buffer and 1 μL of Dpn I restriction enzyme (10 U / μL) were added. The centrifuge tube was gently shaken to thoroughly mix the system and incubated in a 37°C water bath for 1 hour to remove the template plasmid.

[0060] 1.2 Transformation, screening and sequencing of recombinant strains

[0061] The PCR-amplified and purified digested product was transformed into E. coli BL21 competent cells and cultured on LB solid medium containing ampicillin (50 μg / mL) for 12-16 hours. A single colony was picked from the LB plate on a sterile operating table and inoculated into a 96-deep-well plate. The 96-deep-well plate was first added with 600 μL of LB liquid medium (Amp, 50 μg / mL). The deep-well plate was placed in a shaker with constant temperature shaking (37°C, 200 rpm) for 12 hours. After 12 hours, a 1% inoculum was inoculated into a new 96-deep-well plate containing 500 μL of LB liquid medium (Amp, 50 μg / mL). After constant temperature incubation at 37°C for 2-3 hours, the inducer IPTG was added to the 96-deep-well plate to a final concentration of 2 mM. The target protein was expressed by shaking on a constant temperature shaker (16°C, 200 rpm) for 16 hours. To screen the target strain, 10 μL of cell culture medium was added to a 96-well PCR plate, followed by the addition of 10 μL of the substrate acetylthiocholine (10 mM), 10 μL of DTNB (10 mM), and 170 μL of buffer (10 mM PBS, pH 7.3). After reacting at room temperature for 10 minutes, the absorbance at 405 nm was measured using a microplate reader. Positive mutants obtained through the initial screening (determination of whole-cell enzyme activity) were cultured and induced for expression in 50 mL of LB liquid medium (Amp, 50 μg / mL). After crushing and centrifugation, the enzyme activity of the supernatant (crude enzyme) was measured for rescreening. In this screening process, a high absorbance value represents a high activity of acetylcholinesterase.

[0062] Perform two controls at the same time:

[0063] Control 1: Replace the plasmid DNA with an equal volume of sterile double-distilled water. All other procedures remain the same. No colonies should grow on the LB plate containing Amp. Control 2: Replace the plasmid DNA with an equal volume of sterile double-distilled water. Spread 5 μL of the bacterial solution on the LB plate containing Amp. Numerous colonies should be visible.

[0064] 1.3 Expression of recombinant proteins

[0065] 50 μL of bacterial suspension of different mutants was inoculated into 50 mL of LB liquid medium (with an ampicillin content of 100 μg / mL) from the glycerol tube, and cultured at 37°C, 180 rpm for 12 h to obtain seed solution. 2% of the inoculum was inoculated into 100 mL of LB liquid medium (with an ampicillin content of 100 μg / mL), and cultured at 37°C, 180 rpm until OD 600=0.6~0.8. After adding IPTG to a final concentration of 0.1mM, induce expression at 16℃, 180rpm for 20h. Use a high-speed refrigerated centrifuge (6790g, 4℃, 10min) to collect the cells, resuspend the cells in 15mL of physiological saline, and centrifuge (6790g, 4℃, 10min). Discard the supernatant, weigh the wet cells, and resuspend them in lysis buffer at a ratio of cell mass: lysis buffer volume = 1:20 (15mL). After resuspension, use an ultrasonic cell disruptor in an ice-water bath to disrupt the cells (power 300w, ultrasonic time 5s, interval time 8s, working time 30min). After the cells are disrupted, centrifuge (4℃, 6790g, 30min) and collect the supernatant. The collected supernatant is the crude enzyme solution. Store the crude enzyme solution in a refrigerator at 4℃ until used.

[0066] 1.4 The crude enzyme solution was collected by centrifugation and filtered through a 0.45 μm sterile filter membrane. The purified enzyme was then purified by column chromatography and then subjected to SDS-PAGE. Protein concentration was determined by the BCA method, and acetylcholinesterase activity was determined using the Ellman method (Ellman et al., 1961).

[0067] 1.5 Determination of enzyme properties

[0068] Determine the optimal reaction pH and pH tolerance, optimal reaction temperature and temperature tolerance, metal ion tolerance, surfactant tolerance, organic solvent tolerance, kinetic parameters, and circular dichroism spectrum of acetylcholinesterase.

[0069] 1.6 Molecular docking

[0070] The 3D structure of ATCh was obtained from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ), and after energy minimization by Chem3D, it was converted to pdbqt format using AutoDockTools. The crystal structure of AnAChE-N267D was also converted to a pdbqt file after dehydration, hydrogenation, and charge calculation. The structure of AnAChE-N267D-A101G was obtained by homology modeling and subjected to the same treatment. The docking parameters were set as follows: center coordinates (x = -35.496, y = 4.108, z = 3.203), box size The exhaustiveness setting was 1000, and 10 conformations were generated. The docking was repeated three times using AutoDockVina, and the lowest energy conformation was selected to draw the interaction maps of AnAChE-N267D and AnAChE-N267D-A101G with ATCh using PyMol.

[0071] 1.7 Molecular dynamics simulation

[0072] The simulations of AnAChE-N267D and AnAChE-N267D-A101G in aqueous solution were performed using GROMACS 2022.4. AnAChE-N267D and AnAChE-N267D-A101G were described using the aber14sb_parmbsc1 force field. The water molecules were described using an explicit three-point water model. AnAChE-N267D and AnAChE-N267D-A101G were dissolved in a cubic water box at a distance from the edge of the water box. A protein periodic boundary was applied, and counterions were added to neutralize the system charge, rendering it electrically neutral. Energy minimization was performed using the steepest descent method to make the system structure more rational. The system was then heated (500 ps, ​​0-300 K) while the protein was constrained. A constant temperature and pressure simulation lasting 1 ns was performed under the Berendsen barostat to bring the system density to 1.0 g / cm. 3 , during which the protein remains constrained. After the system reaches NPT equilibrium, all constraints are removed and a 100 ns classical molecular dynamics (MD) simulation is performed. Three independent replicates are performed for each system. In order to strike a balance between computational accuracy and resource consumption, the cutoff radius for van der Waals forces and electrostatic interactions is set to In addition, hydrogen bonds were constrained using the lincs algorithm. After the simulation, the stability analysis of the simulation trajectory was performed using the built-in components in GROMACS (RMSD, RMSF, Rog and SASA).

[0073] 1.8 Data Analysis

[0074] All experimental results in this example were repeated three times, and the results are expressed as mean ± standard deviation (SD). SPSS26 was used for one-way analysis of variance, and Tukey test was used to analyze the significance of the differences. Origin 2021 was used for drawing figures. Different uppercase and lowercase letters in each experimental data graph indicate significant differences (p < 0.05).

[0075] 2 Experimental results

[0076] 2.1 Saturation mutagenesis of substrate binding pocket amino acids

[0077] The inventors previously obtained the crystal structure of acetylcholinesterase (AnAChE) from Aspergillus niger. After rationally designing and performing site-directed mutagenesis on the catalytic triad (H270, S34, N267), a mutant AnAChE-N267D (N267D) with a 2-fold increase in enzyme activity was screened out. In order to further improve the catalytic efficiency of N267D, the present invention modified its substrate binding pocket based on the structure of N267D. First, the tool Deepsite was used to identify the amino acid residues of potential ligand binding of N267D. After excluding highly conserved amino acid residues, the remaining 11 amino acids (G143, N144, A101, Y102, D33, D35, I139, Y269, N268, L190, F191) were respectively modified using N267D as a template ( Figure 1 ) and single-site saturation mutagenesis. Degenerate NKK primers were designed for each of the 11 sites, and full-plasmid PCR amplification was performed using the plasmid pGEX4T-1-N267D as a template to obtain mutant plasmids. The mutant plasmids were transformed into the E. coli BL21 strain, and the recombinant strains were cultured and expressed in 96-well plates.

[0078] 2.2 Screening and purification of mutants

[0079] The initial screening of mutants was performed by measuring the whole-cell enzyme activity of the recombinant strain using iodinated acetylthiocholine as a substrate. The higher the absorbance value, the stronger the enzyme activity of the mutant. The initial screening results showed that the enzyme activity of mutants G143E, N144E, N144A, and A101G was higher than that of N267D, while mutations at sites such as L190, F191, Y102, Y35, D33, I139, and Y269 had a negative impact on enzyme activity, which may be due to the high conservation of these amino acid residues (Xie et al., 2024). The mutants with improved enzyme activity obtained in the initial screening (G143E, N144E, N144A, and A101G) were expanded and cultured, induced for expression, and then crushed and centrifuged. The enzyme activity of the supernatant was measured for rescreening. The results are as follows: Figure 2 As shown in (a), the crude enzyme specific activities of G143E, N144E, N144A and A101G were 1.34 times, 1.16 times, 1.25 times and 1.09 times that of N267D, respectively.

[0080] G143E, N144E, N144A and A101G were purified by GST gravity chromatography column and then the specific enzyme activity was determined ( Figure 2 In (b), it was found that compared with N267D, the specific enzyme activities of N144E, N144A and A101G mutants were increased by 72.38%, 37.18% and 101.38%, respectively. Before GST purification, it was found that the G143E mutant had the highest specific enzyme activity ( Figure 2(a)), but the highest enzyme activity after purification was A101G ( Figure 2 (b) This is because the G143E mutation increased protein expression, but excess impurities were removed after purification. Because the A101G mutation at position 101 was found to significantly enhance enzyme activity after purification, the A101G mutant (designated AnAChE-N267D-A101G) was selected for subsequent research. By comparing the mutant with the premutation site (AnAChE-N267D), the molecular mechanism underlying the enhanced enzyme activity induced by the A101G mutation was explored.

[0081] The amino acid sequence of A101G is (SEQ ID NO.21): MTGIPTVTARPWTQRRPRAENSTTNPTYFFTFGDSYSQTGFSASGTQPSASNPMGNPD LGIGTTNGPNWIGYLTTTENASLVLSYNLAAGGATIDNALVPGYPGDLASQFRLFEDVYADKPASAPWSAEDAVFGVWIGINDIGNAYYSTDAETYTPKLISRLESLVEEVYKNGGR KFLFLNVPPTSRSPLFLEQGEEVVKQHAEYLSVYNENLEGMVDDFTKKKGDVTTVLYDSWSFMTKILDDPTAYGFPDATCINDDGTSCIWWDNYHPGMKYHLLQAEDMKPKLRKLGGW.

[0082] The nucleotide sequence of the DNA encoding A101G is (SEQ ID NO.22): ATGACCGGTATCCCGACCGTTACCGCGCGTCCGTGGACCCAGCGTCCGCGTGCGGAAAACAGCACCACCAACCCGACCTACTTCTTCACCTTCGGTGATAGCTACAGCCAGACCGGTTTCAGCGCGTCTGGTACTCAGCCGAGCGCGAGCAACCCGATGGGTAACCCGGATCTGGGTATCGGCACCACCACCAACGGTCCGAACTGGATCGGTTACCTGACCACCACCGAAAACGCCAGCCTGGTCCTGAGCTACAACCTGGCAGCAGGCGGCGCGACCATCGATAACGCGCTGGTTCCGGGTTATCCGGGTGATCTGGCGAGCCAGTTCCGTCTGTTTGAAGATGTTTACGCTGATAAACCGGCTAGCGCACCGTGGAGCGCTGAAGATGCAGTTTTCGGTGTTTGGATCGGTATTAACGATATCGGTAACGCATACTATAGCACTGATGCGGAAACCTATACCCCGAAACTGATCTCTCGTCTGGAATCTCTGGTTGAAGAAGTTTATAAAAACGGTGGCCGTAAATTCCTGTTCCTGAACGTTCCGCCGACCAGCCGTTCTCCGCTGTTCCTGGAACAGGGTGAAGAAGTAGTTAAACAGCACGCTGAATACCTGTCTGTTTATAACGAAAACCTGGAAGGCATGGTTGATGATTTCACTAAGAAAAAAGGTGATGTTACTACCGTTCTGTACGATTCTTGGTCTTTCATGACCAAAATTCTGGATGATCCGACCGCATACGGTTTCCCGGATGCTACCTGTATTAACGATGATGGTACCAGCTGCATCTGGTGGGATAACTACCATCCGGGTATGAAATATCACCTGCTGCAGGCGGAAGATATGAAACCGAAACTGCGTAAACTGGGCGGCTGG。

[0083] Figure 3SDS-PAGE electrophoresis diagrams of AnAChE-N267D and its mutant AnAChE-N267D-A101G before and after purification. From Figure 3 it can be found that after the crude enzyme solutions of AnAChE-N267D and AnAChE-N267D-A101G were purified by GST gravity chromatography column, electrophoretically pure AnAChE-N267D and AnAChE-N267D-A101G were obtained. The protein molecular weight of the pure enzyme of AnAChE-N267D was approximately 30 kDa (lane B), and the molecular weight of AnAChE-N267D-A101G was also around 30 kDa (lane D). From Figure 3 it was found that the mutation of alanine (A) at position 101 to glycine (G) did not cause a change in the protein molecular weight, and the effect of a single amino acid mutation on the protein molecular weight was small.

[0084] 2.3 Optimal reaction pH and pH tolerance

[0085] Figure 4 showed that when 2 ≤ pH ≤ 5, the relative enzyme activities of AnAChE-N267D and its mutant AnAChE-N267D-A101G were both lower than 5%. This may be because the acidic environment damaged the tertiary structure of the enzyme, resulting in almost undetectable enzyme activity. When 5 < pH ≤ 7, the enzyme activity of AnAChE-N267D increased with the increase of pH. When pH = 7, the relative enzyme activity of AnAChE-N267D reached the maximum value, and pH 7 was the optimal reaction pH value of AnAChE-N267D. When pH > 7, the enzyme activity of AnAChE-N267D gradually decreased with the increase of pH, and the alkaline environment inhibited the activity of AnAChE-N267D. When 5 ≤ pH ≤ 8, the enzyme activity of AnAChE-N267D-A101G increased with the increase of pH, and the enzyme activity reached the maximum value at pH = 8, and pH 8 was the optimal reaction pH value of AnAChE-N267D-A101G. When pH > 8, the relative enzyme activity of AnAChE-N267D-A101G gradually decreased with the increase of pH. After the alanine (A) at position 101 of AnAChE-N267D was mutated to glycine (G), the optimal pH value changed from 7 to 8, shifting towards the alkaline environment, and the mutation improved the efficiency of the enzyme in the alkaline industry.

[0086] Such as Figure 4The results showed that when the pH was <7, after incubation for 2 hours in buffer solutions of different pH values, the relative enzyme activity of AnAChE-N267D increased with increasing pH value, reaching the highest activity (97.12%) at pH 7, indicating that AnAChE-N267D was most stable in a buffer solution of pH 7. At pH 8, the relative enzyme activity was 76.23% of its initial activity, and then gradually decreased with increasing pH, reaching a residual activity of only 47.9% at pH 10. At pH values ​​of 5 to 8, the residual enzyme activity of AnAChE-N267D was above 70%, indicating that AnAChE-N267D can maintain good stability in weakly acidic and weakly alkaline environments. When the pH is ≤ 6, the relative enzyme activity of AnAChE-N267D-A101G gradually increases with increasing pH. When the pH is greater than 6, the residual enzyme activity of AnAChE-N267D-A101G significantly increases, and AnAChE-N267D-A101G has the highest stability (99.63%) at pH = 7. At pH = 8, the relative enzyme activity of AnAChE-N267D-A101G is 90.07% of its initial activity. The mutation at the A101G site significantly enhances stability in alkaline environments (pH 8). When the pH is greater than 8, the residual enzyme activity of AnAChE-N267D-A101G gradually decreases.

[0087] 2.4 Optimum reaction temperature and temperature stability

[0088] from Figure 5 The study found that the relative enzyme activity of AnAChE-N267D increased with increasing temperature from 0°C to 30°C, indicating that the rate of an enzyme-catalyzed reaction is temperature-dependent. The relative enzyme activity of AnAChE-N267D was highest between 30°C and 45°C, with no significant change. Therefore, the optimal reaction temperature for AnAChE-N267D is 30-45°C. Above 45°C, the relative enzyme activity of AnAChE-N267D decreased sharply, reaching almost no activity at 60°C. Below 45°C, the relative enzyme activity of AnAChE-N267D-A101G increased with increasing temperature, reaching its highest level at 45°C. The enzyme activity of AnAChE-N267D-A101G did not change significantly between 35°C and 45°C, and similarly, the activity at 30°C and 35°C was not significantly different. Therefore, the mutation at the A101G site did not change the enzyme's optimal temperature, which remained between 30 and 45°C. As the temperature continued to rise, the relative enzyme activity of AnAChE-N267D-A101G decreased significantly. Unlike AnAChE-N267D, AnAChE-N267D-A101G still had a detectable 20% relative enzyme activity at 60°C.

[0089] Figure 6 Figure 3. Residual enzyme activity of AnAChE-N267D and its mutant, AnAChE-N267D-A101G, after 2 h of incubation at different temperatures. When the temperature gradually increased from 20°C to 60°C, the residual enzyme activity of both AnAChE-N267D and its mutant, AnAChE-N267D-A101G, decreased with increasing temperature. After 2 h of incubation in buffer solution at 20°C, 30°C, 40°C, 50°C, and 60°C, AnAChE-N267D retained 83.2%, 72.48%, 70.67%, 62.27%, and 51.03% of its initial enzyme activity, respectively, while AnAChE-N267D-A101G retained 90.48%, 73.86%, 73.06%, 72.59%, and 59.79%, respectively. The residual enzyme activity of AnAChE-N267D-A101G was higher than that of AnAChE-N267D over a wide temperature range (20-60°C), and the mutation at the A101G site enhanced the stability within the range of 20-60°C. The enhanced thermal stability may be related to the change in the flexibility of the tertiary structure after the mutation.

[0090] 2.5 Metal ion tolerance

[0091] like Figure 7 As shown, AnAChE-N267D and its mutant AnAChE-N267D-A101G were respectively 2+ , Na + , Mn 2 + , Fe 2+ and Zn 2+ After incubation in the solution for 3 h, the residual enzyme activities of AnAChE-N267D and AnAChE-N267D-A101G decreased to varying degrees. + , Mn 2+ and Zn 2+ The enzyme activity showed no significant change. 2+ showed significant intolerance and AnAChE-N267D (54.02%) was more tolerant than AnAChE-N267D-A101G (51.20%), but was less tolerant to Fe 2+ On the contrary, AnAChE-N267D-A101G (95.33%) has a negative effect on Fe 2+ The tolerance of the A101G site was stronger than that of AnAChE-N267D (67.34%), and the mutation of the A101G site significantly enhanced the tolerance to Fe 2+ Tolerance of Mg 2+ , Ca 2+ and K+ It inhibited the enzyme activity of AnAChE-N267D (p>0.05), but activated the enzyme activity of AnAChE-N267D-A101G (p>0.05). The mutation of A101G site enhanced the activity of Mg 2+ , Ca 2+ and K + Tolerance of Fe 3+ It has an activating effect on both AnAChE-N267D and AnAChE-N267D-A101G. It does not significantly increase the enzyme activity of AnAChE-N267D (0.6%), but it increases the enzyme activity of AnAChE-N267D-A101G by 19.33%. 2+ and Fe 3+ tolerance.

[0092] 2.6 Surfactant tolerance

[0093] Figure 8The results showed that cationic surfactants cetyltrimethylammonium bromide (CTAB), N-hydroxysuccinimide (N-Hydroxysuccinimide), anionic surfactants sodium dodecyl sulfate (SDS) and nonionic surfactants such as Triton X-100, Tween 20, Tween 40, Tween 60 and Tween 80 all had different degrees of activation effects on the activity of AnAChE-N267D and AnAChE-N267D-A101G, and all surfactants had a significant activation effect on the activity of AnAChE-N267D-A101G, while only N-Hydroxysuccinimide, Tween 20, Tween 40, Tween 60 and Tween 80 had a significant activation effect on the activity of AnAChE-N267D. The relative enzyme activities of the A101G site were increased by 31.16%, 14.78%, 16.38%, 10.92%, and 13.05%, respectively. N-Hydroxysuccinimide showed the strongest activation effect on enzyme activity before and after the mutation. Mutation at the A101G site showed the same activation effect on surfactants (CTAB, N-Hydroxysuccinimide, SDS, Triton X-100, Tween 20, Tween 40, Tween 60, and Tween 80), but with enhanced activation, increasing enzyme activity by 24.16%, 4.94%, 15.42%, 16.66%, 10.42%, 8.3%, 9.39%, and 8.83%, respectively. CTAB showed the most significant enhancement in enzyme activation after the mutation.

[0094] 2.7 Organic solvent resistance

[0095] Figure 9 The results showed that after 5 hours of treatment with the organic solvents n-heptane and dichloromethane, the relative enzyme activity of AnAChE-N267D was 104.95% and 101.14%, respectively, indicating no significant increase in enzyme activity (p>0.05). However, after 5 hours of treatment with xylene, isooctane, acetone, acetonitrile, methanol, and dimethyl sulfoxide, the enzyme activity was inhibited to varying degrees. After treatment with dimethyl sulfoxide, the residual enzyme activity of AnAChE-N267D was 15.61%, indicating that dimethyl sulfoxide has a strong toxic effect on the enzyme activity of AnAChE-N267D.

[0096] After treatment with the hydrophobic organic solvents n-heptane and isooctane for 5 hours, the relative enzyme activity of AnAChE-N267D-A101G was enhanced. n-heptane and isooctane activated the enzyme activity of AnAChE-N267D-A101G. Xylene, dichloromethane, acetone, acetonitrile, methanol, and dimethyl sulfoxide all inhibited the enzyme activity of AnAChE-N267D-A101G. The inhibitory effect ranked as methanol > xylene > dichloromethane > acetonitrile > acetone > dimethyl sulfoxide. Methanol had a strong inhibitory effect on the enzyme activity of AnAChE-N267D-A101G, with the remaining enzyme activity being only 7.39% of its initial activity.

[0097] Isooctane inhibited the activity of AnAChE-N267D but activated the activity of AnAChE-N267D-A101G (p>0.05). The mutation shifted the isooctane effect on the enzyme from inhibition to positive activation (enzyme activity before and after mutation was 93.83% and 103.38% of the initial activity, respectively). This is likely due to changes in the tertiary structure of the esterase caused by the mutation. Furthermore, the A101G mutation significantly enhanced activation by n-heptane, with residual enzyme activity before and after mutation reaching 104.95% and 117.02% of the initial activity, respectively. Compared to AnAChE-N267D, AnAChE-N267D-A101G exhibited significantly enhanced tolerance to the organic solvent dimethyl sulfoxide (DMSO), with residual enzyme activity before and after mutation reaching 15.61% and 92.49% of the initial activity, respectively.

[0098] 2.8 Kinetic parameters

[0099] Table 3 Substrate kinetic parameters of AnAChE-N267D and AnAChE-N267D-A101G

[0100]

[0101] Depend on Figure 10 The Michaelis constant (K m ), maximum reaction rate (V max ), conversion number (K cat ) and catalytic efficiency (K cat / K m ) are shown in Table 3. K m As a characteristic constant of an enzyme, K is only related to the properties of the enzyme. m The value reflects the affinity of the enzyme to the substrate. m The larger the value, the lower the affinity for the substrate. As can be seen from Table 3, the K mThe value is 2.33mmol / L lower than that of AnAChE-N267D, indicating that the mutation of the A101G site increases the affinity for the substrate acetylthiocholine, which also explains the reason for the increase in enzyme activity. max K refers to the reaction rate when the enzyme is completely saturated with substrate molecules, also known as the maximum reaction rate. The maximum reaction rate of the mutant AnAChE-N267D-A101G (37.04 μmol / min / mg) is higher than that of AnAChE-N267D (28.57 μmol / min / g). The maximum reaction rate increases after the A101G site mutation. cat The size of K reflects the rate at which the enzyme converts the substrate. cat The larger the value, the higher the efficiency of the enzyme in converting the substrate. The conversion rate of AnAChE-N267D-A101G to acetylthiocholine (20.06s -1 ) was significantly higher than AnAChE-N267D (15.48s -1 ). K cat / K m It is the most important parameter for measuring enzyme catalytic efficiency. The catalytic efficiency of AnAChE-N267D-A101G is 42.11% higher than that of AnAChE-N267D. The mutation of the A101G site improves the catalytic efficiency.

[0102] 2.9 Circular Dichroism (CD) Spectra

[0103] like Figure 11 As shown in (a), AnAChE-N267D and AnAChE-N267D-A101G exhibit negative ellipticity at 222 nm and 208 nm, and a positive peak at 192 nm, characteristic of α-helices in the protein structure. The negative ellipticity at 218 nm and the positive peak at 196 nm in the CD spectra are characteristic of β-sheets. Furthermore, the peak shapes at 196 nm for AnAChE-N267D and AnAChE-N267D-A101G are significantly different, indicating a difference in β-sheet content before and after the AnAChE-N267D mutation.

[0104] like Figure 11 (b) is the information on the relative content of the secondary structures of AnAChE-N267D and AnAChE-N267D-A101G obtained by calculation after processing the CD spectrum data using CDNN software. Figure 11In (b), it was found that after the A101G site mutation, the α-helix content changed from 32.18% to 32.64%, while the β-sheet content decreased from 17.43% to 16.96%. This may be because the A101G site mutation caused some β-sheets to convert into α-helices, resulting in enhanced structural stability and rigidity of the mutant AnAChE-N267D-A101G. The β-turn content decreased from 16.93% to 16.77%. The reduction in β-turn content may affect the folding and function of the protein. The random coil content decreased from 33.47% to 33.73%. The increase in random coil content enhances the flexibility of AnAChE-N267D-A101G. The increase in protein structural flexibility makes the binding of the active center to the substrate more flexible, thereby increasing enzyme activity.

[0105] T m The value indicates the temperature at which the protein denatures by 50%. The larger the Tm value, the more stable the protein, and vice versa. Figure 2-14 As shown, the T of AnAChE-N267D m The value was 58.52℃, while the T of AnAChE-N267D-A101G was m The value is 59.94, and the mutation of A101G site leads to T m The value increased by 1.4℃. The mutation enhanced the stability, T m The increase indicates that the temperature stability is enhanced after mutation, and the temperature stability of the mutant is enhanced at 20-60℃.

[0106] 2.10 Molecular docking

[0107] from Figure 13 As can be seen, ATCh is docked into the bowl-shaped pocket of AnAChE-N267D and AnAChE-N267D-A101G, with the acyl group deeply embedded in the pocket and the choline head facing the solvent. The predicted binding free energy of ATCh for AnAChE-N267D-A101G (-6.74±0.14 kcal / mol) is greater than that of ATCh for AnAChE-N267D (-6.02±0.23 kcal / mol), indicating that AnAChE-N267D-A101G has a higher affinity for ATCh, which is consistent with the experimental results that the enzyme activity is enhanced after the A101G site mutation. According to the docking results, in AnAChE-N267D-A101G, the acyl group of ATCh is stabilized by hydrophobic interactions with I139, Y269 and Y35, the carbonyl group is stabilized by hydrogen bonds formed between the oxygen anion hole and amino acid residues (S34, G91 and N140), and the choline head is stabilized by hydrophobic interactions with Y269, N144, and Y102.

[0108] 2.11 Molecular dynamics simulation (MD)

[0109] Figure 14 (a) shows the RMSD values ​​of AnAChE-N267D from three independent 100 ns simulations. After 65 ns, the RMSD of AnAChE-N267D in aqueous solution fluctuates slightly, indicating that it gradually stabilizes. The average RMSD of AnAChE-N267D is 0.096 ± 0.011 nm, indicating that the structure of AnAChE-N267D in aqueous solution undergoes minimal changes.

[0110] Figure 14 (b) shows the RMSF of three independent 100ns simulations of AnAChE-N267D, among which the RMSF values ​​of the three simulations of amino acids Y102, R111 and 147 are quite different, which may be due to the greater flexibility of these residues.

[0111] Figure 14 (c) shows the Rog values ​​of AnAChE-N267D from three independent 100 ns simulations. The average Rog value of AnAChE-N267D is 1.80±0.005 nm. The Rog value of N267D is found to be the smallest at 65 ns, suggesting that the conformation of AnAChE-N267D gradually stabilizes after 65 ns.

[0112] Figure 14 (d) shows the SASA of AnAChE-N267D from three independent 100 ns simulations. The average SASA value of AnAChE-N267D is 115.28 ± 1.93 nm. The SASA value of N267D is minimized at 65 ns, suggesting that the conformation gradually stabilizes after 65 ns. Changes in RMSD, rog, and SASA values ​​indicate that the conformation of AnAChE-N267D gradually stabilizes after 65 ns.

[0113] Figure 15 (a) shows the RMSD of AnAChE-N267D-A101G from three independent 100 ns simulations. After 60 ns, the RMSD value of AnAChE-N267D-A101G fluctuates slightly and gradually stabilizes in aqueous solution. The average RMSD value of AnAChE-N267D-A101G is 0.09±0.01 nm, indicating that the structure of AnAChE-N267D-A101G undergoes minimal changes in aqueous solution. Compared with the RMSD value of AnAChE-N267D (0.096±0.011 nm), the RMSD value of AnAChE-N267D-A101G is smaller, indicating that the conformation of AnAChE-N267D-A101G is more stable in aqueous solution.

[0114] Figure 15 (b) shows the RMSF of three independent 100ns simulations of AnAChE-N267D-A101G. The RMSF values ​​of the three simulations of amino acids Y102, R111 and 197 are quite different, which may be due to the high flexibility of these three amino acid residues. A looP99-P104 of AnAChE-N267D has undergone significant changes ( Figure 14 (b) ), while the RMSF value of looP99-P104 of AnAChE-N267D-A101G changed little, indicating that the A101G mutation reduced structural flexibility while increasing rigidity, making the structure more stable. The enhanced temperature stability after the A101G mutation is precisely due to the increased structural rigidity.

[0115] Figure 15 Middle (c) shows the Rog of AnAChE-N267D-A101G from three independent 100ns simulations. The average Rog of AnAChE-N267D-A101G is 1.79±0.005nm. It was found that after 60ns, the Rog value of N267D-A101G was the smallest, suggesting that the conformation gradually stabilized after 60ns.

[0116] Figure 15 Middle (d) shows the SASA of AnAChE-N267D-A101G from three independent 100 ns simulations. The average SASA value of AnAChE-N267D-A101G is 114.23±1.66 nm. After 60 ns, the SASA value of AnAChE-N267D-A101G is the smallest, and the conformation gradually stabilizes after 60 ns.

[0117] Changes in RMSD, Rog, and SASA values ​​indicate that the conformation of AnAChE-N267D-A101G gradually stabilized after 60 ns. In contrast, AnAChE-N267D only stabilized after 65 ns in aqueous solution, indicating that the conformation of AnAChE-N267D-A101G is more stable in aqueous solution. The enhanced conformational stability after the A101G mutation facilitates substrate binding and enhances enzyme activity.

[0118] This example uses rational design to molecularly modify the substrate binding pocket of acetylcholinesterase (AnAChE) from Aspergillus niger to improve enzyme activity. Through rational design, saturation mutations were performed on each of the 11 amino acids that comprise the substrate binding pocket of AnAChE-N267D. After vector recombinant expression was induced in Escherichia coli, a recombinant strain (AnAChE-N267D-A101G) with enhanced enzyme activity was successfully screened. The main research conclusions of this example are as follows:

[0119] (1) The amino acids constituting the substrate binding pocket of AnAChE-N267D were molecularly modified, and a mutant strain with the most significant increase in enzyme activity was obtained by screening, with the enzyme activity increased by 101.38%. Sequencing confirmed that the alanine (A) at position 101 was mutated to glycine (G), and the recombinant strain was named AnAChE-N267D-A101G.

[0120] (2) After the alanine (A) at position 101 of AnAChE-N267D was mutated to glycine (G), the optimal pH value shifted from 7 to 8, and the optimal pH value shifted toward an alkaline environment after the mutation. At the same time, the A101G mutation did not cause a change in the enzyme's optimal temperature, which remained at 30-45°C. The enzyme activity after incubation at pH 8 for 2 h was 76.23% and 90.07% of its initial activity before and after the mutation, respectively. The stability in an alkaline environment (pH 8) was significantly enhanced after the mutation, and the temperature stability in the range of 20-60°C was also improved.

[0121] (3) A101G mutation enhances Fe 2+ tolerance (the remaining enzyme activities before and after mutation were 67.34% and 95.33%, respectively) and Fe 3+ The enzyme was activated by the surfactants (relative enzyme activity increased by 0.6% and 19.33% before and after the mutation, respectively). Surfactants (CTAB, N-Hydroxysuccinimide, SDS, Triton X-100, Tween 20, Tween 40, Tween 60, and Tween 80) exhibited the same activation effect, but with enhanced activation, increasing enzyme activity by 24.16%, 4.94%, 15.42%, 16.66%, 10.42%, 8.3%, 9.39%, and 8.83%, respectively, after the mutation. The A101G mutation shifted the effect of the organic solvent isooctane on enzyme activity from inhibition to positive activation (enzyme activity was 93.83% and 103.38% of its initial activity before and after the mutation, respectively). The results of kinetic analysis showed that the A101G mutation increased the affinity (K) for the substrate acetylthiocholine (ATCh) by significantly enhancing the activation effect of n-heptane on the enzyme activity (104.95% and 117.02%) and the tolerance to dimethyl sulfoxide (15.61% and 92.49%). m decreased by 2.33mmol / L), catalytic efficiency (K cat / K m ) increased by 42.11%.

[0122] (4) CD analyzed the secondary structure and T mThe results showed that the α-helix content increased after the A101G mutation, and the structural stability and rigidity of the protein were enhanced. m The value increased by 1.4°C, indicating enhanced temperature stability. Molecular docking results revealed that AnAChE-N267D-A101G has a larger predicted binding free energy with the substrate ATCh and a higher affinity for the substrate, explaining the molecular mechanism of enhanced enzyme activity. MD simulation RMSD, Rog, and SASA values ​​revealed that the conformation of AnAChE-N267D-A101G gradually stabilized after 60 ns, while the conformation of AnAChE-N267D only stabilized after 65 ns in aqueous solution. The enhanced conformational stability of the A101G site mutation in aqueous solution is more conducive to substrate binding, thereby increasing enzyme activity.

[0123] Example 2

[0124] 1 Synthesis of immobilized carrier

[0125] A cellulose filter paper (FP) with a diameter of 10 cm was soaked in a 0.25 mg / mL chitosan (CS) solution at room temperature for 12 hours. After soaking, the FP was removed with tweezers and rinsed three times with deionized water to remove the CS that was not covalently cross-linked with the hydroxyl groups on the FP surface. It was then placed in a constant temperature incubator at 37°C and dried (FP&CS) for use. 8% (v / v) glutaraldehyde aqueous solution (GA) was used to soak the FP&CS filter paper at room temperature for 2 hours (FP&CS / GA). After being removed with tweezers, it was rinsed three times with deionized water to remove the unstable GA that was simply adsorbed on the FP&CS surface. After drying at 37°C, the immobilized enzyme carrier FP&CS / GA was obtained. It was placed in a dry environment at room temperature for use to prevent the filter paper from absorbing moisture.

[0126] 2 Immobilization of recombinant acetylcholinesterase

[0127] A 10-cm-diameter modified cellulose filter paper support (FP&CS / GA) was cut into 10-mm-diameter discs using a hole punch. 12 μL of a 3.06 mg / mL enzyme solution (AnAChE-N267D-A101G) was then dripped onto the 10-mm-diameter FP&CS / GA. After incubation at room temperature for 1.5 hours, the surface was rinsed three times with phosphate buffer (10 mM, pH 7.4). This process removed any AnAChE-N267D-A101G that was simply adsorbed on the FP&CS / GA surface, rather than covalently bound via a Schiff base reaction. After incubation at room temperature for 1.5 hours, the immobilized enzyme FP&CS / GA@AnAChE-N267D-A101G was successfully obtained.

[0128] Example 3

[0129] Application of immobilized enzymes in the detection of pesticide residues

[0130] AnAChE-N267D-A101G was fixed on cellulose filter paper according to the immobilization method provided in Example 2. 10 μL of 2,6-dichloroindophenol acetate (30 mg / mL) was added dropwise to an empty filter paper with a diameter of 10 mm, and adsorbed at room temperature for two hours to obtain a substrate-loaded paper. 50 μL of dichlorvos (0.1 mg / mL) was added dropwise to the immobilized enzyme, and the reaction was incubated at room temperature for 10 minutes. A buffer solution for dissolving pesticides was used as a control. The substrate paper was clamped with tweezers and covered on the incubated immobilized enzyme paper. After 3 minutes of reaction, the color average intensity value was evaluated using a colorimeter (Shenzhen Linshang Technology Co., Ltd., LS171). The color intensity analysis software LScolor, which is compatible with the colorimeter, was downloaded on an iPhone 15 mobile phone (China, MTLK3CH / A). The colorimeter and the mobile phone were connected via Bluetooth. The color intensity value was presented as visual data on the LScolor software, and the color intensity Δb value was read. The calculation formula for the pesticide inhibition rate is as follows:

[0131]

[0132] Where Δb0 represents the color intensity value without pesticide; Δb1 represents the color intensity value with pesticide added.

[0133] Figure 16 Panel (a) shows the inhibitory effect of dichlorvos on immobilized enzymes (FP&CS / GA@AnAChE-N267D-A101G) with varying activity levels. When the specific activity of the immobilized enzyme was 0.87 U / mg, the inhibitory effect of 0.1 mg / mL dichlorvos on the immobilized enzyme was poor, with an inhibition rate of only 13.29%. As the specific activity gradually decreased, the inhibitory effect of dichlorvos on the immobilized enzyme gradually increased, reaching a maximum inhibition rate of 78.78% when the specific activity dropped to 0.086 U / mg. The inhibition rate of dichlorvos on the immobilized enzyme also decreased with the continued decrease in specific activity, reaching 65.27% when the specific activity dropped to 0.052. This is because at low enzyme concentrations, the catalytic interaction between the enzyme and substrate is stronger than the inhibitory interaction between the enzyme and dichlorvos, and at low enzyme concentrations, the catalytic effect dominates.

[0134] Figure 16(b) shows the effect of different incubation times of dichlorvos and immobilized enzyme on the inhibition rate. When the incubation time is 3 minutes, the inhibition rate is only 55.08%. As the incubation time increases, the inhibitory effect of dichlorvos on the immobilized enzyme activity increases. When the incubation time is 8 minutes, the inhibition rate reaches 78.41%. When the incubation time is further extended, the inhibition rate increases slightly, but the increase in inhibition rate is not significant (p>0.05). This is because dichlorvos has fully reacted with the immobilized enzyme, and further extension of the incubation time will not increase its inhibition rate. To improve the speed of pesticide rapid detection and reduce time costs, the subsequent pesticide inhibition rate determination is selected with an incubation time of 8 minutes.

[0135] Figure 16 Panel (c) shows the effect of reaction time on the inhibition rate of the immobilized enzyme after incubation with dichlorvos for 8 minutes. When the reaction time was only 30 seconds, the inhibition rate reached 61.87%. As the reaction time increased, the inhibition rate gradually increased, reaching a maximum of 79.57% after 3 minutes. This may be due to insufficient reaction time, resulting in insufficient binding between the enzyme and substrate. Subsequent studies showed that the optimal reaction time was 3 minutes. As the reaction time continued to increase, the inhibition rate initially decreased and then remained constant (p>0.05). This may be due to the dynamic equilibrium of competitive inhibition between the enzyme, substrate, and pesticide.

[0136] Depend on Figure 17 The sensitivity (IC 50 ) was 0.055706327 mg / mL. The detection limit was 0.012072316 mg / mL. This example found that the immobilized enzyme FP&CS / GA@AnAChE-N267D-A101G has the potential to detect organophosphorus pesticides. Figure 18 This was also confirmed by the results that when the concentration of the immobilized enzyme was gradually reduced from 0.2 mg / mL, the blue color gradually became darker until it dropped to 0.01 mg / mL.

[0137] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the implementation methods of the present invention. A person skilled in the art will be able to make other variations or modifications based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. An acetylcholinesterase, characterized in that The amino acid sequence of the acetylcholinesterase is shown in SEQ ID NO.

21.

2. The DNA molecule encoding the acetylcholinesterase according to claim 1, characterized in that The nucleotide sequence of the DNA molecule is shown in SEQ ID NO.

22.

3. An expression vector, characterized in that Comprising the DNA molecule according to claim 2.

4. A recombinant strain, characterized in that The invention comprises the expression vector according to claim 3.

5. The recombinant strain according to claim 4, characterized in that The recombinant strain is AnAChE-N267D-A101G.

6. The method for preparing acetylcholinesterase according to claim 1, characterized in that: The acetylcholinesterase is expressed by the recombinant strain according to claim 4, and the acetylcholinesterase is separated and purified to obtain the acetylcholinesterase.

7. An immobilized enzyme, characterized in that The immobilized enzyme can be obtained by immobilizing the acetylcholinesterase according to claim 1 on a carrier.

8. Use of the acetylcholinesterase according to claim 1 or the immobilized enzyme according to claim 7 in detecting organophosphorus pesticides.

9. The use according to claim 8, characterized in that The organophosphorus pesticide includes dichlorvos.

10. A method for detecting organophosphorus pesticides, characterized in that: The sample to be tested is added dropwise to the immobilized enzyme according to claim 7, and the reaction is incubated. A solution containing an organophosphorus pesticide is used as a control, and the presence of the organophosphorus pesticide in the sample to be tested can be determined based on the change in color intensity.

Images