An organophosphate hydrolase mutant and its use in degrading tributyl phosphate

Abstract

This invention discloses an organophosphate hydrolase mutant and its application in the degradation of tributyl phosphate. The mutant is obtained by mutating serine at position 395 (SEQ ID NO.2) to phenylalanine (S395F), phenylalanine at position 476 (F476S), and glutamic acid at position 201 (E201F). This invention also discloses the gene encoding the mutant, the recombinant vector, the recombinant genetically engineered bacteria, and their applications. Experiments show that this mutant has a high efficiency in degrading tributyl phosphate, capable of degrading tributyl phosphate in aqueous solution at room temperature, demonstrating high degradation efficiency and promising application prospects.

Description

Technical Field

[0001] This invention relates to enzyme mutants, specifically to an organophosphate hydrolase mutant and its application in the degradation of tributyl phosphate. Background Technology

[0002] Tributyl phosphate (TBP), as an emerging flame retardant, poses environmental risks. Due to its difficulty in natural degradation, it can persist in water, sediment, and soil for extended periods and easily accumulate in organisms, amplifying through the food chain. Residual TBP in organisms can have various toxic effects. Traditional physicochemical treatment methods have drawbacks such as limited degradation efficiency and the potential for secondary pollution. In contrast, the application of bioenzymatic degradation of this compound offers advantages such as high selectivity, mild conditions, and environmental friendliness.

[0003] Currently, with the rapid development of molecular biology and bioinformatics analysis technologies, functional bacterial identification and screening, functional gene mining, and bioenzyme expression technologies have achieved relatively mature results in the bioremediation of various pollutants. Conventional methods generally involve screening, isolating, and purifying strains with degradation effects on specific pollutants from environmental samples containing pollutants, mining genes with pollutant degradation functions through whole-genome sequencing, and expressing the required enzymes in specific vectors. However, this method has low screening efficiency, and the original strains and enzymes have weak degradation effects on pollutants. For example, patent CN111394335A discloses a phosphodiesterase Sy-PTE and its application in degrading organophosphorus flame retardants. This enzyme has good salt tolerance and a broad substrate spectrum, but lacks research and evolution studies on the enzyme, failing to improve its degradation efficiency. Chinese patent CN110643534A discloses a strain of *Sphingosphospira* capable of degrading triphenyl phosphate, achieving a triphenyl phosphate degradation rate of 98.77% within 24 hours, but with a relatively long degradation cycle. Meanwhile, none of the aforementioned microbial remediation technologies have evolved or modified the enzymes that play a key role in the degradation of pollutants; they have only involved the discovery and recombination of original enzymes, and the selectivity and catalytic efficiency of the biological enzymes have not been further improved. Therefore, developing a high-efficiency haloalkyl dehalogenase mutant for the degradation of tributyl phosphate is of great significance for environmental bioremediation. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide an organophosphate hydrolase mutant with high degradation efficiency and its application in the degradation of tributyl phosphate, overcoming the shortcomings of the existing technology in the low degradation efficiency of tributyl phosphate.

[0005] Technical solution: The organophosphate hydrolase mutant of the present invention is obtained by combined mutation of the amino acid sequence shown in SEQ ID NO.2 at positions 395, 476 and 201; the serine at position 395 is mutated to phenylalanine S395F, the phenylalanine at position 476 is mutated to serine F476S, and the glutamic acid at position 201 is mutated to phenylalanine E201F.

[0006] The gene encoding an organophosphate hydrolase mutant described in this invention.

[0007] The recombinant vector of this invention contains the aforementioned gene. There are no limitations on the recombinant vector of this invention, as long as it can maintain its replication or autonomous replication in various host cells of prokaryotic and / or eukaryotic cells. The vector can be any conventional vector in the art, such as various plasmids, bacteriophages, or viral vectors, preferably using the pET28a(+) plasmid as the expression vector and *E. coli* as the expression host (*E. coli* C43 cells or *E. coli* BL21).

[0008] The recombinant genetically engineered bacteria of the present invention comprises the recombinant vector.

[0009] The application of the organophosphate hydrolase mutant, gene, recombinant vector, or recombinant genetically engineered bacteria described in this invention in the catalytic degradation of tributyl phosphate.

[0010] Preferably, the catalytic degradation of tributyl phosphate produces dibutyl phosphate and monobutyl phosphate.

[0011] Preferably, the application method is as follows: using wet bacterial cells obtained by fermentation culture of recombinant genetically engineered bacteria containing the gene encoding organophosphate hydrolase mutant as a catalyst, using tributyl phosphate as a substrate, and using a Tris-HCl solution with pH 8.0-10.0 (preferably pH 8.5) to form a reaction system, and carrying out the reaction at 300-500 rpm (preferably 400 rpm) and 25-37℃ (preferably 35℃). After the reaction is completed, a reaction solution containing dibutyl phosphate and monobutyl phosphate is obtained. The reaction solution is then separated and purified to obtain dibutyl phosphate and monobutyl phosphate.

[0012] Preferably, the Tris-HCl solution system is a 50 mM aqueous solution of tris(hydroxymethyl)aminomethane (Tris), with the pH adjusted to 8.0-10.0 (preferably pH 8.5) using hydrochloric acid (HCl).

[0013] Preferably, the catalyst dosage is 10-40 g / L (preferably 20 g / L) buffer solution based on the weight of wet bacterial cells, and the initial substrate concentration is 10-100 mg / L (preferably 100 mg / L).

[0014] More preferably, the wet bacterial cells are prepared as follows: Recombinant engineered bacteria containing the gene encoding a phosphate ester hydrolase mutant are inoculated into LB broth containing a final concentration of 60 µg / mL kanamycin and cultured at 37°C for 8 h to obtain a seed culture; then, the seed culture is inoculated at a volume concentration of 2% into sterile LB liquid medium containing a final concentration of 60 µg / mL kanamycin and cultured at 37°C for approximately 8-12 h until the bacterial cell concentration OD 600 is 0.4-0.8; then, isopropyl thio-β-D-galactoside (IPTG) is added to the culture medium at a final concentration of 0.1-1.0 mM (preferably 0.5 mM), and expression is induced at 20°C for 12 h. The cells are then collected by centrifugation at 4°C and 4000 rpm for 10-20 min; the LB liquid medium consists of 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride, with deionized water as the solvent.

[0015] Preferably, the organophosphate hydrolase mutant is used for catalysis in whole-cell form, crude enzyme solution from cell disruption, or isolated and purified enzyme form, or the organophosphate hydrolase mutant is prepared into an immobilized enzyme or an enzyme in immobilized cell form using immobilization technology for catalysis.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0017] (1) This invention utilizes directed evolution and semi-rational design methods to modify the phosphoester hydrolase st-PTE, finding that positions 395, 476, and 201 are key sites affecting enzyme degradation efficiency. Site-directed saturation and iterative saturation mutagenesis techniques were used to modify these three sites, and mutants S395F / F476S / E201F were obtained through 96-well plate screening. m and K cat The values ​​were 0.13 mM and 3.8 s, respectively. -1 .

[0018] (2) The phosphate hydrolase mutant obtained in this invention can degrade the pollutant tributyl phosphate under ambient temperature conditions in the aqueous phase, so that 86.6% of 100 mg / L tributyl phosphate is degraded in 4 h.

[0019] (3) The phosphodiester hydrolase mutant of the present invention is based on the amino acid of the phosphodiester hydrolase st-PTE shown in SEQ ID NO.2. It is modified by site-directed saturation mutagenesis and iterative saturation mutagenesis to change its amino acid sequence, thereby changing the protein structure and function. Then, the phosphodiester hydrolase mutant with the above mutation is obtained by targeted screening. Attached Figure Description

[0020] Figure 1 This is the chemical reaction formula for the phosphate ester hydrolase catalyzing tributyl phosphate according to the present invention.

[0021] Figure 2 This is a chromatogram of tributyl phosphate after gas chromatography detection.

[0022] Figure 3 The degradation rate of tributyl phosphate catalyzed by the st-PTE mutant is shown.

[0023] Figure 4 The change in the concentration of tributyl phosphate catalyzed by the st-PTE mutant over time.

[0024] Figure 5 The degradation rate of tributyl phosphate catalyzed by st-PTE and its mutants at different pH values ​​(reaction time 2 hours) was determined.

[0025] Figure 6 The degradation rate of tributyl phosphate catalyzed by st-PTE and its mutants at different temperatures (reaction time 2 hours). Detailed Implementation

[0026] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0027] Example 1: Construction of pET28a(+)-st-PTE plasmid

[0028] From Sphingolipids Sphingobium The wild-type phosphoester hydrolase st-PTE (PDB ID: 5HRM) gene of sp. TCM1 was synthesized by Genewiz (Suzhou) Co., Ltd. and constructed in the pET28a(+) vector. The constructed plasmid was transformed into competent cells. E. coli DH5α was transformed and the resulting mixture was evenly spread onto LB agar plates and incubated upside down at 37°C for 16 h. A single colony was picked and inoculated into 5 mL of sterile LB liquid medium and incubated at 37°C and 150 rpm for 8-12 h. Then, plasmid extraction was performed using a column-based plasmid extraction kit. E. coli The pET28a(+)-st-PTE plasmid was extracted from DH5α and used as a template for iterative saturation mutagenesis.

[0029] Example 2: Construction of a site-directed saturated library of phosphodiesterases

[0030] Based on the conclusion of Example 1, primers were designed based on the gene sequence of phosphodiesterase st-PTE (nucleotide sequence shown in SEQ ID NO.1 and amino acid sequence shown in SEQ ID NO.2) recorded in GenBank (see Table 1). Site-directed saturation mutagenesis was performed on the parental st-PTE gene (nucleotide sequence SEQ ID NO.1) using primers L139X-F / L139X-R, E201X-F / E201X-R, V274X-F / V274X-R, V275X-F / V275X-R, F476X-F / F476X-R, and S395X-F / S395X-R, respectively. Using pET-28a(+) as the expression vector, mutant plasmids carrying the target gene were obtained. These mutant plasmids were then transformed into *E. coli* BL21(DE3) to obtain recombinant bacteria containing the phosphodiesterase mutant gene, denoted as *E. coli* BL21(DE3)-L139X (referred to as mutant L139X) and *E. coli* BL21(DE3)-L139X (referred to as mutant L139X). E201X (denoted as mutant E201X), E. coli BL21(DE3)-V274X (denoted as mutant V274X), E. coli BL21(DE3)-V275X (denoted as mutant V275X), E. coli BL21(DE3)-F476X (denoted as mutant F476X), E. coli BL21(DE3)-S395X (denoted as mutant S395X).

[0031] Table 1: Primer Design Table for Site-Specific Saturation Mutant Library Construction of Phosphoester Hydrolase

[0032]

[0033] The PCR amplification system is as follows: 50 µL reaction volume.

[0034] ddH2O: 30 µL;

[0035] 10×Buffer: 5 µL;

[0036] dNTP: 5 µL;

[0037] MgSO4: 3 µL;

[0038] DMSO: 2 µL;

[0039] Upstream primer (50 µM): 1.5 µL;

[0040] Downstream primer (50 µM): 1.5 µL;

[0041] KOD enzyme: 1 μL;

[0042] Template DNA (plasmid): 1 µL;

[0043] The PCR reaction conditions were as follows: pre-denaturation at 95℃ for 3 min, followed by temperature cycling at 95℃ for 20 s, 55℃ for 10 s, and 72℃ for 30 s for a total of 30 cycles, with a final extension at 72℃ for 10 min, and a termination temperature of 4℃. After verification by 1% agarose gel electrophoresis, 1 µL of DpnI and 5 µL of buffer were added to the PCR product, and the sample was digested at 37℃ for 2 h to remove the template plasmid DNA. After inactivation at 65℃ for 10 min, the product was purified using a PCR cleanup kit and transformed into E. coli BL21(DE3) competent cells. The cells were plated on LB agar plates containing kanamycin (60 µg / mL) and incubated overnight at 37℃ to obtain a phosphatase mutant library. At this point, many single colonies with different mutations appeared on the LB agar plates. These single colonies were used for subsequent screening of the mutant library.

[0044] The parental strain was constructed using the same method: E. coli BL21(DE3)-st-PTE WT.

[0045] Example 3: Screening of phosphodiesterase mutant libraries

[0046] Screening of the phosphodiesterase mutant library was performed using phosphodiesterase st-PTE as a reference. Single colony clones (from the mutant library constructed in Example 2) were picked and cultured in 1 mL deep 96-well plates. 400 µL of LB medium containing a final concentration of 60 µg / mL kanamycin was added beforehand. Two parental strains were also picked as controls in the last two wells of the 96-well plate. The 1 mL 96-well plate was incubated at 37°C for 8 h as seed culture. Then, 100 µL of the seed culture was added to a new 2 mL deep 48-well plate and cultured in sterile TB medium containing a final concentration of 60 µg / mL kanamycin. After incubation at 37°C for 8 h, IPTG at a final concentration of 0.5 mM was added, and expression was induced at 20°C for 12 h. The plates were then centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and the wet cells were collected for further screening.

[0047] A 500 µL reaction mixture (50 mM Tris-HCl buffer, final concentration 100 mg / L tributyl phosphate) was added to each well, the bacterial cells were resuspended, and then the mixture was reacted at 35 °C and 250 rpm for 4 h. Extraction was performed with 500 µL of n-hexane, followed by centrifugation at 1,2000 rpm for 1 min. 300 µL of the organic phase solution was then analyzed by gas chromatography.

[0048] Gas chromatography conditions: Shimadzu-GC-2030 and Agilent HP-5 column; gas program: hold at 80℃ for 2 min, ramp to 250℃ at 25℃ / min, hold for 2 min, ramp to 280℃ at 50℃ / min; retention time of tributyl phosphate: 7.80 min. Figure 2 ).

[0049] The screened mutant S395F / F476S / E201F achieved a degradation rate of 86.6% for tributyl phosphate after 4 hours under optimal conditions. Figure 3 ).

[0050] Example 4: Degradation kinetics of tributyl phosphate by st-PTE mutant S395F / F476S / E201F

[0051] Based on the conclusions of Example 3, the mutants obtained from the above screening were... E. coli BL21(DE3)-st-PTES395F / F476S / E201F (nucleotide sequence shown in SEQ ID NO.3, amino acid sequence shown in SEQ ID NO.4) was inoculated into 10 mL of sterile test tubes containing LB medium with a final concentration of 60 µg / mL kanamycin. The tubes were cultured at 37 ℃ and 150 rpm for 6–8 h in a shaker. Then, 1% of the inoculum was added to 2 L Erlenmeyer flasks, which were then inoculated with 1 L of sterile TB medium containing a final concentration of 60 µg / mL kanamycin. After culturing at 37 ℃ for 12 h, IPTG with a final concentration of 0.5 mM was added. The cells were induced to express at 20 ℃ for 16 h. After centrifugation at 4000 rpm for 30 min, the supernatant was discarded, and the wet cells were collected. Resuspend the cells in 20 mL of 50 mM Tris-HCl (pH=8.5) buffer. Take 10 μL of the supernatant to determine the cell concentration (OD600). Dilute with 50 mM Tris-HCl (pH=8.5) buffer to OD600=10. Add 5 mL of the diluted supernatant to each 10 mL glass reaction flask, add tributyl phosphate to a final concentration of 100 mg / L, add a magnetic stir bar, and mix thoroughly. React at 35 ℃ and 400 rpm for 240 min. Take samples at 10 min, 30 min, 60 min, 120 min, 240 min, and 360 min, extract with 500 µL of n-hexane, centrifuge at 1,2000 rpm for 1 min, and take 300 µL of the organic phase solution for gas chromatography analysis.

[0052] The degradation kinetics of tributyl phosphate by the st-PTE mutants S395F / F476S / E201F were obtained. Figure 4As the reaction proceeds, tributyl phosphate is degraded, with a degradation rate of 77.3% at 120 min.

[0053] Example 5: Optimal pH screening for the degradation of tributyl phosphate by the st-PTE mutant S395F / F476S / E201F

[0054] Based on the conclusions of Example 3, the mutants obtained from the above screening were... E. coli BL21(DE3)-S395F / F476S / E201F (nucleotide sequence shown in SEQ ID NO.3, amino acid sequence shown in SEQ ID NO.4) was inoculated into 10 mL of sterile test tubes containing LB medium with a final concentration of 60 µg / mL kanamycin. The cells were cultured at 37 °C and 150 rpm for 6–8 h. Then, 1% of the inoculum was added to 2 L Erlenmeyer flasks, which were then inoculated with 1 L of sterile TB medium containing a final concentration of 60 µg / mL kanamycin. After culturing at 37 °C for 12 h, IPTG with a final concentration of 0.5 mM was added. Expression was induced at 20 °C for 16 h. The cells were then centrifuged at 4 °C and 4000 rpm for 30 min, the supernatant was discarded, and the wet cells were collected. Add 20 mL of 50 mM Tris-HCl (pH=8.5) buffer to the wet bacterial cells to resuspend the cells, measure the OD600 of the resuspended cells, and adjust the OD600 to 10 using 50 mM Tris-HCl (pH=8.5) buffer. Add 1 mL of the resuspended mixture to each of five 2 mL sterile centrifuge tubes, centrifuge at 4000 rpm for 10 min at 4 °C, discard the supernatant, and add 900 μL of buffer solutions of different pH values ​​(pH=6 disodium hydrogen phosphate-citrate buffer, pH=7 sodium phosphate buffer, pH=8 disodium hydrogen phosphate-citrate buffer, pH=8.5 Tris-SO4 buffer, and pH=10 borate buffer) to each of the five centrifuge tubes to resuspend the bacterial culture. Take 500 μL of the supernatant and add it to each of five 5 mL glass reaction flasks, then add tributyl phosphate to each flask to a final concentration of 100 mg / L. Mix well with a magnetic stirrer and react at 35 °C for 400 rpm for 2 h. Extract with 500 µL of n-hexane, then centrifuge at 1,2000 rpm for 1 min, and take 300 µL of the organic phase solution for gas chromatography detection.

[0055] Experimental results are as follows Figure 5 As shown, the st-PTE mutant S395F / F476S / E201F maintains good activity against tributyl phosphate in the alkaline range, with the best activity at pH 8.5, and the degradation rate reaches 79.6% after 120 min of reaction.

[0056] Example 6: Screening for the optimal temperature for the degradation of tributyl phosphate by the st-PTE mutant S395F / F476S / E201F

[0057] Based on the conclusions of Example 3, the mutants obtained from the above screening were... E. coli BL21(DE3)-S395F / F476S / E201F (nucleotide sequence shown in SEQ ID NO.3, amino acid sequence shown in SEQ ID NO.4) was inoculated into 10 mL of sterile test tubes containing LB medium with a final concentration of 60 µg / mL kanamycin. The cells were cultured at 37 °C and 150 rpm for 6–8 h. Then, 1% of the inoculum was added to 2 L Erlenmeyer flasks, which were then inoculated with 1 L of sterile TB medium containing a final concentration of 60 µg / mL kanamycin. After culturing at 37 °C for 12 h, IPTG with a final concentration of 0.5 mM was added. Expression was induced at 20 °C for 16 h. The cells were then centrifuged at 4 °C and 4000 rpm for 30 min, the supernatant was discarded, and the wet cells were collected. Resuspend the wet bacterial cells in 20 mL of 50 mM Tris-HCl (pH=8.5) buffer. Measure the OD600 of the resuspended cells and adjust the OD600 to 10 using 50 mM Tris-HCl (pH=8.5) buffer. Transfer 1 mL of the diluted supernatant to five 5 mL glass reaction flasks. Add tributyl phosphate to each flask to a final concentration of 100 mg / L. Add magnetic stir bar and mix thoroughly. React at 20 ℃, 25 ℃, 35 ℃, 45 ℃, and 55 ℃ at 400 rpm for 120 min, respectively. Extract with 500 µL of n-hexane, centrifuge at 1,2000 rpm for 1 min, and analyze 300 µL of the organic phase solution using gas chromatography.

[0058] Experimental results are as follows Figure 6 As shown, the st-PTE mutants S395F / F476S / E201F exhibit good activity in the temperature range of 20℃-55℃, with a wide suitable temperature range. The activity is best at 35℃, with a degradation rate of 77.2%. Therefore, the st-PTE mutants can efficiently degrade tributyl phosphate under ambient and mesophilic conditions without the need for external temperature control equipment, saving energy and demonstrating green and low-carbon characteristics.

[0059] Example 7: Determination of Michaelis-Menten kinetic parameters for the degradation of tributyl phosphate by the st-PTE mutant S395F / F476S / E201F

[0060] Based on the conclusions of Example 3, the mutants obtained from the above screening were... E. coliBL21(DE3)-S395F / F476S / E201F (nucleotide sequence shown in SEQ ID NO.3, amino acid sequence shown in SEQ ID NO.4) was inoculated into 10 mL of sterile LB medium containing a final concentration of 60 µg / mL kanamycin and cultured at 37 ℃ and 150 rpm for 6–8 h. Then, 1% of the inoculum was added to 2 L Erlenmeyer flasks, which were then inoculated with 1 L of sterile TB medium containing a final concentration of 60 µg / mL kanamycin and cultured at 37 ℃ for 12 h. IPTG was then added to a final concentration of 0.5 mM, and expression was induced at 20 ℃ for 16 h. The cells were then centrifuged at 4 ℃ and 4000 rpm for 30 min, the supernatant was discarded, and the wet cells were collected. The wet cells were resuspended in 20 mL of 50 mM Tris-HCl (pH=8.5) buffer. The protein was sonicated at 4 ℃ for 10 min with 65% sonication power, and the sonication cycle was 2 s on and 6 s off. It was then centrifuged at 12000 rpm at 4 ℃ for 15 min. The supernatant was collected, and the protein was purified by affinity chromatography. The absorbance was measured using an A280 UV spectrophotometer to calculate the protein concentration. The protein was then diluted to a concentration of 2 μM with 50 mM Tris-HCl (pH=8.5) buffer. 1 mL of diluted protein solution was divided into ten 5 mL glass reaction flasks. Eight of these flasks contained tributyl phosphate at final concentrations of 5 mg / L, 10 mg / L, 20 mg / L, 40 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, and 300 mg / L, respectively. The mixture was magnetically stirred at 35 °C and 400 rpm for 20 min. Extraction was performed with 500 µL of n-hexane, followed by centrifugation at 12000 rpm for 1 min. 300 µL of the organic phase solution was collected and analyzed by gas chromatography. The enzyme reaction rate was measured, and a double reciprocal curve was plotted based on the reaction rate and the reciprocal of the substrate concentration to calculate the Michaelis kinetic parameters. The results showed that the Michaelis kinetic parameter K of the st-PTE mutant S395F / F476S / E201F on tributyl phosphate was... m and K cat The values ​​were 0.13 mM and 3.8 s, respectively. -1 .

Claims

1. An organophosphorus acid ester hydrolase mutant, characterized in that, The mutant was obtained by combining mutations at positions 395, 476, and 201 of the amino acid sequence shown in SEQ ID NO.2; the serine at position 395 was mutated to phenylalanine S395F, the phenylalanine at position 476 was mutated to serine F476S, and the glutamic acid at position 201 was mutated to phenylalanine E201F.

2. A gene encoding an organophosphate hydrolase mutant as described in claim 1.

3. A recombinant vector, characterized in that, It contains the gene described in claim 2.

4. The recombinant vector of claim 3, wherein, The recombinant vector is a plasmid.

5. A recombinant genetically engineered bacteria, characterized in that, It includes the recombinant vector as described in claim 3 or 4.

6. The use of the organophosphate hydrolase mutant of claim 1, the gene of claim 2, the recombinant vector of claim 3 or 4, or the recombinant genetically engineered bacteria of claim 5 in the catalytic degradation of tributyl phosphate.

7. Use according to claim 6, characterized in that, Catalytic degradation of tributyl phosphate produces dibutyl phosphate and monobutyl phosphate.

8. Use according to claim 6, characterized in that, The application method is as follows: using wet bacterial cells obtained by fermentation culture of recombinant genetically engineered bacteria containing the gene encoding organophosphate hydrolase mutant as a catalyst, using tributyl phosphate as a substrate, and using Tris-HCl solution with pH 8.0-10.0 to form a reaction system, the reaction is carried out at 300-500 rpm and 25-37℃. After the reaction is completed, a reaction solution containing dibutyl phosphate and monobutyl phosphate is obtained. The reaction solution is separated and purified to obtain dibutyl phosphate and monobutyl phosphate.

9. Use according to claim 8, characterized in that, The amount of catalyst used is 10-40 g / L buffer solution based on the weight of wet bacterial cells, and the initial concentration of the substrate is 10-100 mg / L.

10. Use according to claim 6, characterized in that, The organophosphate hydrolase mutant is used for catalysis in whole-cell form, crude enzyme solution from cell disruption, or isolated and purified enzyme form, or the organophosphate hydrolase mutant is prepared into an immobilized enzyme or an immobilized cell form using immobilization technology for catalysis.

Images