Salicylic acid decarboxylase mutant and application thereof in degrading salicylic acid

Abstract

The application discloses a salicylic acid decarboxylase mutant and application thereof in degrading salicylic acid. The mutant is obtained by site-directed modification on salicylic acid decarboxylase NahG, and specifically, valine at the 46th position, tyrosine at the 380th position and leucine at the 382nd position in the amino acid sequence shown in SEQ ID NO. 2 are simultaneously mutated into cysteine, phenylalanine and phenylalanine. The mutant shows significantly improved catalytic efficiency, and can efficiently and completely decarboxylate salicylic acid into catechol under mild conditions. The application also provides a coding gene of the mutant, a recombinant vector and an engineering bacterium. The mutant can be used for efficient bioremediation of salicylic acid pollution, and the characteristic of directional generation of catechol can effectively open and accelerate the naphthalene degradation metabolic pathway, and has important application value in the fields of environmental pollution treatment and biological catalysis.

Description

Technical Field

[0001] This invention relates to environmental remediation microbial technology, specifically to a salicylic acid decarboxylase mutant and its application in the degradation of salicylic acid. Background Technology

[0002] Salicylic acid (SA), a widely distributed organic pollutant in the environment, requires efficient and thorough degradation for effective environmental pollution control. Currently, biodegradation and advanced oxidation processes (AOPs) have become the mainstream technologies for salicylic acid degradation. Conventional methods are mainly divided into two categories: biological methods, which involve screening and isolating functional strains with degradation capabilities from polluted environmental samples and optimizing culture conditions to improve degradation efficiency; and chemical methods, which utilize the synergistic effect of oxidants and ultraviolet light to generate free radicals, thereby achieving the oxidative decomposition of salicylic acid.

[0003] However, existing technologies have significant limitations. Biological screening methods are inefficient, and the original strains have limited ability to degrade high concentrations of salicylic acid. Chemical methods are prone to producing toxic byproducts or are too costly. Furthermore, insufficient molecular modification of key degradation enzymes and a lack of in-depth understanding of the selective regulation of free radical reactions make it difficult to achieve efficient and complete degradation of salicylic acid. For example, Chinese patent CN112358980A discloses a strain of Acinetobacter louvelii NL1, isolated from sludge in the ancient Grand Canal of Yangzhou. This strain achieved a 91.4% degradation rate of 30 mg / L salicylic acid after 14 hours of cultivation at 28 ℃ and exhibited antibiotic resistance. However, this strain is only suitable for degrading low concentrations of salicylic acid; the reaction rate decreases significantly at high concentrations, making it unsuitable for industrial wastewater treatment. Chinese patent CN119059629A discloses a method for treating bismuth hyposalicylate wastewater, achieving a salicylic acid removal rate of over 95% through pH adjustment and oxidation processes. However, this method relies on a specific wastewater system, and advanced oxidation processes such as UV / NaCl easily generate byproducts with potential carcinogenic, teratogenic, and mutagenic risks, resulting in insufficient environmental safety. Furthermore, existing biological methods largely rely on the optimization of original strains or simple culture conditions, without targeted evolutionary modification of key enzymes for salicylic acid degradation, making it difficult to achieve breakthroughs in catalytic efficiency and substrate adaptability. Therefore, developing modified enzyme preparations with low byproducts and high adaptability is of great significance for the large-scale treatment of salicylic acid pollution. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a salicylic acid decarboxylase mutant with high degradation activity, high substrate specificity, thorough degradation and low by-product accumulation, which can be adapted to the naphthalene degradation metabolic pathway. This overcomes the shortcomings of salicylic acid, such as low degradation efficiency, weak substrate affinity, incomplete degradation and easy accumulation of by-products. It achieves efficient and thorough conversion of salicylic acid to break through the rate-limiting bottleneck in the naphthalene degradation pathway, reduces the interference of by-products on the metabolic process, and further improves the overall degradation efficiency and conversion rate of naphthalene.

[0005] Technical solution: The salicylate decarboxylase mutant of the present invention is obtained by combined mutation of the 46th, 380th and 382nd positions of the amino acid sequence shown in SEQ ID NO.2; the valine at position 46 is mutated to cysteine ​​(V46C), the tyrosine at position 380 is mutated to phenylalanine (Y380F), and the leucine at position 382 is mutated to phenylalanine (L382F).

[0006] The gene of the salicylic acid decarboxylase mutant described in this invention.

[0007] This invention also relates to a recombinant vector constructed from the gene encoding the salicylate decarboxylase mutant, and recombinant genetically engineered bacteria prepared by transformation of the recombinant vector. The recombinant vector of this invention is not limited, 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 pET22b(+) plasmid as the expression vector, and *Escherichia coli* as the expression host (*E. coli* C43(DE3) cells or *E. coli* BL21(DE3)).

[0008] The present invention relates to the application of salicylic acid decarboxylase mutants, genes, recombinant vectors, or recombinant genetically engineered bacteria in the catalytic degradation of salicylic acid to produce catechols.

[0009] The application method is as follows: using wet bacterial cells obtained by fermentation culture of recombinant genetically engineered bacteria containing the gene encoding salicylic acid decarboxylase mutant as a catalyst, salicylic acid as a substrate, and Tris-SO4 solution with pH 8.0-10.0 as a reaction system, the reaction is carried out at 300-600 rpm (preferably 600 rpm) and 25-37℃ (preferably 25℃). After the reaction is completed, a reaction solution containing catechol is obtained. The reaction solution is then separated and purified to obtain catechol.

[0010] Furthermore, the Tris-SO4 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 sulfuric acid (H2SO4).

[0011] Furthermore, the amount of catalyst used is 10-40 g / L buffer (preferably 20 g / L) based on the weight of the wet bacterial cells, and the initial concentration of the substrate is 1-20 mM (preferably 5 mM).

[0012] Further, the wet bacterial cells are prepared as follows: recombinant engineered bacteria containing the gene encoding a salicylic acid decarboxylase mutant are inoculated into LB broth containing ampicillin at a final concentration of 100 µg / mL and cultured at 37°C for 8 h to obtain a seed culture; then, the seed culture is inoculated into sterile LB liquid medium containing ampicillin at a volume concentration of 2% and cultured at 37°C for about 8-12 h to achieve a bacterial cell concentration OD600 of 0.4-0.8; then, isopropyl thio-β-D-galactoside (IPTG) at a final concentration of 0.1-1.0 mM (preferably 0.5 mM) is added to the culture medium, and expression is induced at 20°C for 16 h. After centrifugation at 4°C and 4000 rpm for 10-20 min, the wet bacterial cells are collected; LB liquid medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, solvent is deionized water.

[0013] Furthermore, the salicylate decarboxylase mutant described in this invention can perform catalysis in whole-cell form, or it can be catalyzed using crude enzyme solution from cell disruption or purified enzyme. In addition, specific immobilization techniques can be used to prepare salicylate decarboxylase into immobilized enzymes or immobilized cellular enzymes.

[0014] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0015] (1) This invention utilizes directed evolution and semi-rational design methods to modify salicylate decarboxylase NahG, discovering that positions 46, 380, and 382 are key sites affecting salicylate decarboxylase activity. Site-directed saturation and iterative saturation mutagenesis techniques were used to screen for mutants V46C / Y380F / L382F, whose K... m and K cat The values ​​were 1.67 mM and 0.32 s, respectively. -1 By precisely modifying these key sites in the active site, the technical pain points of existing enzymes in the degradation of salicylic acid—low efficiency, incomplete degradation, and easy generation of byproducts—are effectively overcome.

[0016] (2) The salicylic acid decarboxylase mutant obtained in this invention can degrade the pollutant salicylic acid under ambient temperature conditions in an aqueous phase, and the degradation rate of 5 mM salicylic acid converted in 2 h is 100%.

[0017] (3) The salicylate decarboxylase mutant of the present invention is obtained by modifying NahG as shown in SEQ ID NO.2, thereby changing the protein structure and function and enabling the targeted conversion of salicylate into catechol, which is beneficial to upstream metabolism. More importantly, by combining the "targeted conversion of salicylate into catechol" with the overall efficiency improvement of the naphthalene degradation pathway, a salicylate decarboxylase mutant with significantly improved salicylate degradation efficiency, low by-product accumulation and targeted generation of catechol was constructed. This achieved precise targeted conversion of key intermediate products in naphthalene degradation, effectively breaking through the rate-limiting bottleneck in the naphthalene degradation pathway and providing key enzymatic support for the efficient degradation of naphthalene. Attached Figure Description

[0018] Figure 1 This is the chemical reaction formula for the decarboxylation of salicylic acid to catechol catalyzed by salicylic acid decarboxylase.

[0019] Figure 2 This is a chromatogram of the substrate salicylic acid after liquid chromatography detection.

[0020] Figure 3 This is a chromatogram of the degradation product catechol after liquid chromatography detection.

[0021] Figure 4 The liquid chromatogram is shown after the degradation of salicylic acid by NahG V46C / Y380F / L382F (reaction time 2h).

[0022] Figure 5 The yield of catechol, a salicylic acid product catalyzed by the NahG mutant.

[0023] Figure 6 The changes in the yield and degradation rate of salicylic acid to catechol catalyzed by NahG V46C / Y380F / L382F over time.

[0024] Figure 7 The yield of salicylic acid to catechol catalyzed by NahG V46C / Y380F / L382F at different pH values ​​(reaction time 4h).

[0025] Figure 8 The yield of salicylic acid to catechol catalyzed by NahG V46C / Y380F / L382F at different temperatures (reaction time 4h).

[0026] Figure 9 The image shows the SDS-PAGE electrophoresis results of NahG V46C / Y380F / L382F. Detailed Implementation

[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0028] Example 1: Construction of pET22b(+)-NahG plasmid

[0029] From *Pseudomonas putida* Pseudomonas putida The G7 salicylate decarboxylase NahG (PDB ID: 6BZ5) gene was synthesized by Genewiz (Suzhou) Co., Ltd. and constructed into the pET22b(+) 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 pET22b(+)-nahG plasmid was extracted from DH5α and used as a template for iterative saturation mutagenesis.

[0030] Example 2: Construction of a site-directed saturated library of salicylate decarboxylase

[0031] Based on the conclusion of Example 1, primers were designed based on the gene sequence of salicylate decarboxylase NahG (nucleotide sequence shown in SEQ ID NO.1, amino acid sequence shown in SEQ ID NO.2) (see Table 1). Site-directed saturation mutagenesis was performed on the parental NahG gene (nucleotide sequence SEQ ID NO.1) using primers V46X-F, A48X-F, L221X-F, G222X-F, V241X-F, L379X-F, Y380X-F, and L382X-F, respectively. Using pET-22b(+) 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 a halogenated alkane dehalogenase mutant gene, namely E. coli BL21(DE3)-V46X (denoted as mutant V46X), E. coli BL21(DE3)-A48X (denoted as mutant A48X), and E. coli BL21(DE3)-L221X (denoted as mutant L221X). E. coli BL21(DE3)-G222X (denoted as mutant G222X), E. coli BL21(DE3)-V241X (denoted as mutant V241X), E. coli BL21(DE3)-L379X (denoted as mutant L379X), E. coli BL21(DE3)-Y380X (denoted as mutant Y380X), E. coli BL21(DE3)-L382X (denoted as mutant L382X).

[0032] Table 1: Primer Design for Construction of Salicylate Decarboxylase Site-Directed Saturation Mutant Library

[0033]

[0034] The PCR amplification system is as follows: 50 µL reaction volume.

[0035] ddH2O: 30 µL;

[0036] 10×Buffer: 5 µL;

[0037] dNTP: 5 µL;

[0038] MgSO4: 3 µL;

[0039] DMSO: 2 µL;

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

[0041] Downstream primer (50 µM): 1.5 µL;

[0042] KOD enzyme: 1 μL;

[0043] Template DNA (plasmid): 1 µL;

[0044] 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 ampicillin (100 µg / mL) and incubated overnight at 37℃ to obtain a mutant library of salicylate decarboxylase. 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.

[0045] The parental strain was constructed using the same method: E. coli BL21(DE3)-NahG WT.

[0046] Example 3: Screening of salicylate decarboxylase mutant libraries

[0047] The screening of salicylate decarboxylase mutant libraries was based on salicylate decarboxylase NahG WT. 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 100 µg / mL ampicillin 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 100 µg / mL ampicillin beforehand. After incubation at 37°C for 8 h, IPTG was added at a final concentration of 0.5 mM, and expression was induced at 20°C for 16 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.

[0048] A 500 µL reaction mixture (50 mM Tris-SO4 buffer, final concentration 5 mM salicylic acid) was added to each well, the bacterial cells were resuspended, and then the mixture was incubated at 25 °C and 250 rpm for 4 h. 500 µL of methanol was added and mixed thoroughly, followed by centrifugation at 12000 rpm for 1 min. 300 µL of the solution was then analyzed by liquid chromatography.

[0049] Liquid chromatography analysis conditions: Salicylic acid and catechol were separated by chromatography on a C18 column (250 mm × 4.6 mm, 5 µm) using 0.1% formic acid and methanol as the mobile phase. Gradient elution was performed as follows: 0-8.0 min, methanol concentration increased from 30% to 50%; 8.0-15.0 min, methanol concentration increased from 50% to 70%; 15.0-18.0 min, methanol concentration decreased from 70% to 30%; 18.0-20.0 min, methanol concentration was maintained at 30%. The flow rate was set at 1 mL / min. -1 The column temperature was 30℃, the detection wavelength was 280nm, and the injection volume was 5 μL.

[0050] Example 4: Degradation kinetics of salicylic acid by NahG mutation

[0051] Based on the conclusions of Example 3, the mutants obtained from the above screening were... E. coliBL21(DE3)-NahG V46C / Y380F / L382F (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 100 µg / mL ampicillin. The culture was carried out at 37 ℃ and 150 rpm for 6-8 h. Then, 1% of the inoculum was added to 2 L Erlenmeyer flasks and cultured with 1 L of sterile TB medium containing a final concentration of 100 µg / mL ampicillin. After culturing at 37 ℃ for 12 h, IPTG with a final concentration of 0.5 mM was added. Expression was induced 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-SO4 (pH=8.5) buffer. Take 10 μL of the supernatant to determine the cell concentration (OD600). Dilute with 50 mM Tris-SO4 (pH=8.5) buffer to OD600=10. Add 5 mL of the diluted supernatant to each 10 mL glass reaction flask, add 5 mM salicylic acid to the flask, add a magnetic stir bar, and mix thoroughly. React at 25 ℃ and 600 rpm for 120 min. Take 500 μL of the supernatant at 10 min, 20 min, 30 min, 60 min, and 120 min, add 500 µL of methanol, mix well, centrifuge at 12000 rpm for 1 min, and take 300 µL of the solution for analysis by liquid chromatography.

[0052] The degradation kinetics of salicylic acid by the NahG mutant V46C / Y380F / L382F were obtained. Figure 6 As the reaction proceeds, salicylic acid is degraded, and the concentration of the product catechol gradually increases.

[0053] Example 5: Screening for the optimal pH for salicylic acid degradation by NahG mutant

[0054] Based on the conclusion of Example 3, the mutant E. coli BL21(DE3)-NahG V46C / Y380F / L382F (nucleotide sequence shown in SEQ ID NO.3, amino acid sequence shown in SEQ ID NO.4) obtained above was inoculated into 10 mL of sterile test tubes containing LB medium with a final concentration of 100 µg / mL ampicillin. The culture was carried out at 37 ℃ and 150 rpm for 6-8 h. Then, 1% of the inoculum was added to a 2 L Erlenmeyer flask for culture. 1 L of sterile TB medium containing a final concentration of 100 µg / mL ampicillin was added beforehand. After culturing at 37 ℃ for 12 h, IPTG with a final concentration of 0.5 mM was added. After inducing expression at 20 ℃ for 16 h, the culture was centrifuged at 4 ℃ and 4000 rpm for 30 min. The supernatant was discarded and the wet cells were collected. Add 20 mL of 50 mM Tris-SO4 (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-SO4 (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 ℃, discard the supernatant, and add 900 μL of buffer solutions of different pH values ​​(pH=5 dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer, pH=6 dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer, pH=7 dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer, pH=8.5 Tris-SO4 buffer, and pH=10 glycine-sodium hydroxide 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 5 mM salicylic acid to each flask. Mix well with a magnetic stirrer and react at 25 ℃ for 600 rpm for 4 h. Take 500 μL of the reaction solution from each flask, mix with 500 µL of methanol, centrifuge at 12000 rpm for 1 min, and take 300 µL of the solution for analysis by liquid chromatography.

[0055] Experimental results are as follows Figure 7 As shown, the NahG mutant V46C / Y380F / L382F maintains good activity for the decarboxylation of salicylic acid in the pH range of 6-8.5, and the yield of catechol is ≥99% after 4 hours of reaction.

[0056] Example 6: Screening for the optimal temperature for salicylic acid degradation by NahG mutant V46C / Y380F / L382F

[0057] Based on the conclusions of Example 3, the mutants obtained from the above screening were... E. coliBL21(DE3)-V46C / Y380F / L382F (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 100 µg / mL ampicillin. The culture was carried out at 37 °C and 150 rpm for 6-8 h. Then, 1% of the inoculum was added to 2 L Erlenmeyer flasks and cultured with 1 L of sterile TB medium containing a final concentration of 100 µg / mL ampicillin. After culturing at 37 °C for 12 h, IPTG with a final concentration of 0.5 mM was added. After inducing expression at 20 °C for 16 h, the cells were 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-SO4 (pH=8.5) buffer. Measure the OD600 of the resuspended cells and adjust the OD600 to 10 using 50 mM Tris-SO4 (pH=8.5) buffer. Take 1 mL of the diluted supernatant and place it in five 5 mL glass reaction flasks. Add salicylic acid to each flask to a final concentration of 5 mM. Add magnetic stir bar and mix thoroughly. React at 20 ℃, 25 ℃, 35 ℃, 40 ℃, and 45 ℃ at 600 rpm for 4 h, respectively. Take 500 μL of the reaction solution, add 500 µL of methanol, mix well, and centrifuge at 12000 rpm for 1 min. Take 300 µL of the solution for analysis by liquid chromatography.

[0058] Experimental results are as follows Figure 8 As shown, the NahG mutants V46C / Y380F / L382F exhibit good activity within the temperature range of 20℃ to 45℃, demonstrating a wide suitable temperature range. Therefore, the NahG mutants can efficiently degrade salicylic acid under ambient and mesophilic conditions without the need for external temperature control equipment, saving energy and exhibiting green and low-carbon characteristics.

[0059] Example 7: Determination of Michaelis-Menten kinetic parameters for the degradation of salicylic acid by the NahG mutant V46C / Y380F / L382F

[0060] Based on the conclusions of Example 3, the mutants obtained from the above screening were... E. coliBL21(DE3)-V46C / Y380F / L382F (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 100 µg / mL ampicillin and cultured at 37 °C and 150 rpm for 6–8 h. Then, 1% of the inoculum was added to a 2 L Erlenmeyer flask and cultured in 1 L of sterile TB medium containing 100 µg / mL ampicillin. After 12 h of culture at 37 °C, IPTG was added to a final concentration of 0.5 mM, and expression was induced at 20 °C for 16 h. The flask was then centrifuged at 4 °C 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-SO4 (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. The protein 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 10 μM with 50 mM Tris-SO4 (pH=8.5) buffer. 1 mL of diluted protein solution was taken into ten 5 mL glass reaction flasks. Five of these flasks contained salicylic acid at final concentrations of 0.2 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, 5 mM, 10 mM, and 50 mM, respectively. The mixture was magnetically stirred at 25 ℃ and 600 rpm for 10 min. 500 μL of the reaction solution from each flask was added to 500 µL of methanol and mixed thoroughly. The mixture was then centrifuged at 12000 rpm for 1 min, and 300 µL of the solution was analyzed by liquid 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 NahG mutant V46C / Y380F / L382F to salicylic acid was... m and K cat The values ​​were 1.67 mM and 0.32 s, respectively. -1 .

Claims

1. A salicylate decarboxylase mutant, characterized in that, The mutant was obtained by combining mutations at positions 46, 380, and 382 of the amino acid sequence shown in SEQ ID NO.2; valine at position 46 was replaced by cysteine ​​V46C, tyrosine at position 380 was replaced by phenylalanine Y380F, and leucine at position 382 was replaced by phenylalanine L382F.

2. A gene characterized in that, The gene encodes the salicylic acid decarboxylase 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 according to claim 3, characterized in that, The recombinant vector is a vector that can maintain its replication or autonomously replicate in the host cells of prokaryotic and / or eukaryotic cells.

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

6. The use of the salicylic acid decarboxylase 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 salicylic acid to produce catechol.

7. The application 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 salicylic acid decarboxylase mutant as a catalyst, salicylic acid as a substrate, and Tris-SO4 solution with pH 8.0-10.0 as a reaction system, the reaction is carried out at 300-600 rpm and 25-37℃. After the reaction is completed, a reaction solution containing catechol is obtained. The reaction solution is then separated and purified to obtain catechol.

8. The application according to claim 7, characterized in that, The Tris-SO4 solution system consists of an aqueous solution of tris(hydroxymethyl)aminomethane with a concentration of (50±2) mM, and its pH is adjusted to the range of 8.0 to 10.0 using sulfuric acid.

9. The application according to claim 7, characterized in that, The catalyst dosage is 10 to 40 grams of buffer solution per liter based on the weight of the wet bacterial cells, and the initial substrate concentration is 1 to 20 mmol per liter.

10. The application according to claim 6, characterized in that, The salicylic acid decarboxylase mutant is selected from the following forms for catalysis: whole cells containing the salicylic acid decarboxylase, crude enzyme solution after cell disruption, isolated and purified enzyme, immobilized enzyme, or immobilized cells.

Images