4-chlorocatechol 1, 2-dioxygenase CatA as well as coding gene catA and application thereof

Abstract

The invention discloses a 4-chlorocatechol 1, 2 dioxygenase CatA as well as a coding gene catA and application of the 4-chlorocatechol 1, 2 dioxygenase CatA. The nucleotide sequence of the catA is SEQ ID NO.1, and the amino acid sequence of the catA is SEQ ID NO.2; the enzyme capable of degrading the 4-chlorocatechol and the catechol at the same time has good stability in a high temperature range and a wide pH range, the gene sequence of the enzyme is remarkably different from that of a known functional gene, and the enzyme has great significance in construction of genetically engineered bacteria for efficiently degrading chlorinated aromatic pollutants and development of a novel biological enhancement treatment process and preparation. The method has important significance, a brand new technical approach is provided for solving the environmental problem of intractable chlorinated organic pollution, and social and economic benefits are remarkable.

Description

Technical Field

[0001] This invention relates to the fields of environmental microbiology and agricultural technology. Specifically, it relates to a 4-chlorocatechol 1,2-dioxygenase, CatA, and its encoding gene. catA Applications. Background Technology

[0002] Aromatic compounds are a class of recalcitrant pollutants commonly found in industrial wastewater from papermaking, petrochemicals, pesticides, pharmaceuticals, and dyes. Among them, chlorinated aromatic compounds, such as chlorophenols, exhibit higher biotoxicity, teratogenicity, and carcinogenicity due to the introduction of highly electronegative and sterically hindered chlorine atoms onto the benzene ring, making them typical representatives of persistent organic pollutants in the environment. Their efficient degradation and treatment pose a serious challenge to the environmental field.

[0003] In the aerobic metabolic pathways of microbial degradation of chlorinated aromatic compounds, these compounds are typically first converted into chlorinated catechols as key intermediate metabolites. Among them, 4-chlorocatechol is a core intermediate in the degradation pathways of many important pollutants (such as 5-chlorosalicylic acid, 2,4-dichlorophenoxyacetic acid, 4-chlorophenol, and 2,4-dichlorophenol, etc.). The ring-opening reaction of the benzene ring in catechol intermediates is the rate-limiting step in the entire degradation process, directly determining the flux and degradation efficiency of the entire metabolic pathway. The key enzyme catalyzing this step is catechol 1,2-dioxygenase (EC 1.13.11.1), which, with the participation of molecular oxygen, catalyzes the cleavage of the benzene ring of catechol between two adjacent hydroxyl groups (ortho-cleavage), generating kojic acid, thereby opening the benzene ring structure and allowing it to enter subsequent central metabolism. Over the years, various catechol 1,2-dioxygenases from different microbial strains have been isolated, purified, and characterized, and their corresponding encoding genes (usually named...) catA The genes were also cloned and sequenced. However, further research revealed the following prominent technical defects and limitations when these known enzymes were applied to the degradation of chlorinated aromatic pollutants: I. Low or Complete Absence of Catalytic Activity To Chlorinated Substrates: Most known catechol 1,2-dioxygenases exhibit high catalytic efficiency and specificity for their natural substrates—catechins. However, this high specificity leads to extremely low affinity and catalytic rates for structurally similar chlorinated substrates, such as 4-chlorocatechol. The molecular mechanism lies in the fact that the spatial structure of the enzyme's active site cannot effectively accommodate chlorine atoms, or the strong electron-withdrawing effect of chlorine atoms reduces the electron cloud density of the catechol ring, making it difficult for the enzyme to effectively bind to and catalyze its ring-opening.

[0004] Second, insufficient enzyme stability makes it difficult to adapt to practical application environments: Many known catechol 1,2-dioxygenases exhibit a sharp decline in enzyme activity and stability under conditions deviating from their optimal range. For example, in actual industrial wastewater treatment scenarios, pH values ​​may fluctuate significantly (leaning towards acidity or alkalinity), temperatures may be high, or the wastewater may contain inhibitors such as heavy metals and organic solvents. Known enzymes are easily inactivated under these harsh conditions, resulting in their effectiveness in practical applications being far lower than under ideal laboratory conditions.

[0005] III. Known enzyme genes exhibit high homology and functional convergence: Currently reported enzymes capable of efficiently degrading catechols... catA Genes typically share high homology (greater than 80%) among their sequences, and the enzymes they encode are also quite similar in catalytic properties. Further research on them is unlikely to yield unexpected new functions that break through existing technological bottlenecks.

[0006] Therefore, obtaining a novel catechol 1,2-dioxygenase that can effectively overcome the shortcomings of the existing technologies is of great significance for constructing genetically engineered bacteria that can efficiently degrade chlorinated aromatic pollutants, developing novel bio-enhanced treatment processes and preparations, and will also provide a brand-new technical means to solve the environmental problem of stubborn chlorinated organic pollution. Summary of the Invention

[0007] Therefore, the technical problem to be solved by the present invention is to provide a 4-chlorocatechol 1,2-dioxygenase CatA and its encoding gene. catA This approach can effectively solve the problems of low catalytic activity, insufficient enzyme stability, and functional homogeneity of existing catechol 1,2-dioxygenases for chlorinated substrates.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: A 4-chlorocatechol 1,2 dioxygenase gene, catA, has the nucleotide sequence SEQ ID NO.1.

[0009] The above-mentioned 4-chlorocatechol 1,2 dioxygenase gene catA was used in the construction of transgenic engineered bacteria that degrade catechol and 4-chlorocatechol.

[0010] The above-mentioned 4-chlorocatechol 1,2 dioxygenase gene catA is used in the preparation of formulations that degrade or remove catechol, 4-chlorocatechol and related chlorinated aromatic hydrocarbon pollutants from soil and water.

[0011] A 4-chlorocatechol 1,2-dioxygenase, CatA, is encoded by the nucleotide sequence of the 4-chlorocatechol 1,2-dioxygenase gene catA as described in claim 1, and its amino acid sequence is SEQ ID NO.2.

[0012] The above-mentioned 4-chlorocatechol 1,2 dioxygenase CatA is used in the preparation of enzyme preparations that degrade 4-chlorocatechol and catechol.

[0013] The above-mentioned 4-chlorocatechol 1,2 dioxygenase CatA is used in the preparation of formulations for removing catechols, 4-chlorocatechol and related chlorinated aromatic hydrocarbon pollutants from soil and water.

[0014] The technical solution of the present invention achieves the following beneficial technical effects: 1. The present invention provides a novel 4-chlorocatechol 1,2-dioxygenase CatA and its encoding gene. catA This is the first publicly disclosed gene capable of simultaneously degrading 4-chlorocatechol and catechol, especially exhibiting high catalytic efficiency for 4-chlorocatechol. The protein it encodes can ring-open and degrade both 4-chlorocatechol and catechol, and its amino acid sequence homology with the currently reported catechol 1,2-dioxygenase genes is only 55.5%. 2. The 4-chlorocatechol 1,2-dioxygenase CatA provided by this invention can degrade 0.1 mM of 4-chlorocatechol within 10 min and 0.2 mM of catechol within 1 min. Furthermore, in vitro enzyme activity experiments demonstrate that CatA maintains over 70% of its maximum enzyme activity within a relatively high temperature range (30-50℃) and a wide pH range (pH 7-10). 3. The 4-chlorocatechol and catechol 1,2 dioxygenase genes of the present invention catA The transgenic engineered bacteria used in this study have great potential for degrading 4-chlorocatechol and catechols, and can be applied in the preparation of formulations that degrade or remove catechols, 4-chlorocatechol, and related chlorinated aromatic hydrocarbon pollutants from soil and water. The encoded 4-chlorocatechol 1,2-dioxygenase CatA shows promising application in degrading 4-chlorocatechol and related chlorinated aromatic hydrocarbon pollutants from soil and water, and will provide a novel technological approach to solving the environmental problem of persistent chlorinated organic pollution, with significant social and economic benefits. Attached Figure Description

[0015] Figure 1 This is the ultraviolet scan spectrum of the degradation of 5-chlorosalicylic acid by the strain ZSF-9 of this invention; Figure 2 This is an SDS-PAGE gel image of the recombinant protein CatA of the present invention (where M: protein marker; 1: crude enzyme solution for CatA induction; 2: purified CatA). Figure 3 The image shows the UV scan of the purified protein CatA degrading 4-chlorocatechol and catechol in this invention (where A represents CatA degrading 4-chlorocatechol; B represents CatA degrading catechol). Figure 4The image shows the HPLC detection of 4-chlorocatechol and catechins by the purified protein CatA of this invention (where A represents CatA degradation of 4-chlorocatechol, and B represents CatA degradation of catechins). Figure 5 The mass spectra of the metabolites of 4-chlorocatechol after CatA degradation in this invention are shown by LC / MS (where A is the mass spectrum of 4-chlorocatechol and B is the mass spectrum of the 1,2-dioxygenated ring-opening product of 4-chlorocatechol). Figure 6 The images show the LC / MS mass spectra of the catechol metabolites after CatA degradation in this invention (where A is the catechol mass spectrum and B is the mass spectrum of the 1,2-dioxygenated ring-opening product of catechol). Figure 7 This is a graph showing the effect of pH on CatA enzyme activity according to the present invention; Figure 8 This is a graph showing the effect of temperature on CatA enzyme activity according to the present invention; Figure 9 This diagram shows the effect of metal ions and chemical reagents on the activity of CatA in this invention. Detailed Implementation

[0016] The specific embodiments of the present invention will be further described in detail below with reference to examples.

[0017] This invention provides a novel and highly efficient 4-chlorocatechol 1,2-dioxygenase gene, catA. The protein encoded by this gene not only ring-opens 1,2-dioxygenase to form adipoderic acid, but also ring-opens 4-chlorocatechol 1,2-dioxygenase. It has significant application value in the degradation and removal of chlorinated aromatic compounds. Furthermore, the enzyme encoded by this gene can be used to prepare enzyme preparations for soil and water remediation, and for degrading residual catechols, 4-chlorocatechol, and related chlorinated aromatic hydrocarbon pollutants in the environment. Relevant experimental data are as follows: I. Isolation of strains that efficiently degrade 5-chlorosalicylic acid 1.1 Enrichment, Isolation and Purification of Highly Efficient 5-Chlorosalicylic Acid Degrading Strains Samples were collected from soil and activated sludge that had been contaminated with salicylic acid and chlorosalicylic acid for a long period of time. 5.0 g of soil sample was added to 100 ml of basal salt culture medium, and 200 mg / L of 5-chlorosalicylic acid was added to each medium. The samples were then incubated at 30℃ and 180 rpm. -1 After culturing for 5 days, the culture medium was transferred to the same fresh culture medium at a 5% inoculum, and this process was repeated. Samples were taken periodically and analyzed using a UV scanner to determine whether 5-chlorosalicylic acid (5-CHA) in the enriched solutions had degraded. The results showed that one of the enriched solutions efficiently degraded 5-CHA, completely degrading 200 mg / L of 5-CHA within 5 days.

[0018] The enriched solution with degradation effect was serially diluted, and 10% of the solution was taken. -4 ~10 -7 0.1 mL of each dilution of the enrichment solution was spread onto solid LB agar plates containing 200 mg / L 5-chlorosalicylic acid. After incubation at 30°C for 3–5 days, single colonies with different morphologies were picked and further purified using the streak plating method. The purified single colonies were then inoculated into basal salt medium containing 100 mg / L 5-chlorosalicylic acid and incubated at 30°C and 180 rpm. -1 The samples were cultured on a shaker for 3 days, and the degradation effect was verified by UV scanning. A strain ZSF-9 was obtained that can efficiently degrade 5-chlorosalicylic acid. Strain ZSF-9 was able to completely degrade 200 mg / L of 5-chlorosalicylic acid within 24 hours. The UV scanning results are shown below. Figure 1 .

[0019] The basic salt medium formula is: 1.5 g K₂HPO₄·3H₂O; 0.5 g KH₂PO₄; 1.0 g NH₄NO₃; 0.5 g NaCl; 0.2 g MgSO₄·7H₂O, with deionized water added to a final volume of 1 L, pH 7.0. For solid medium, add 15.0 g agar.

[0020] 1.2 Identification of 5-chlorosalicylic acid degrading strains PCR amplification and 16S rRNA gene sequencing and analysis were performed on strain ZSF-9, which is capable of degrading 5-chlorosalicylic acid. The reaction system included 15 µL of Premix rTaq, 1 µL each of forward and reverse primers, 1 µL of template, and 12 µL of ddH2O. The PCR program was: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 52℃ annealing for 30 s, and 72℃ extension for 90 s, for 30 cycles. Universal primers 27F (5´AGAGTTTGATCMTGGCTCAG3´) and 1492R (5´TACGGYTACCTTGTTACGACTT3´) were used to amplify the 16S rRNA gene fragment of strain ZSF-9, yielding a 16S rRNA gene fragment of approximately 1.5 kb in length. The 16S rRNA gene sequence was compared and analyzed online on EzBioCloud (http: / / www.ezbiocloud.net / ). The results showed that the degrading strain ZSF-9 and... Pseudomonas alkylphenolica KL28 T The similarity is as high as 99.36%, and... Pseudomonas japonica NBRC 103040 T The similarity reached 99.22%, and... Pseudomonas huaxiensis WCHPs060044 TThe similarity was 99.07%, therefore, strain ZSF-9 was preliminarily identified as... Pseudomonas sp.

[0021] 1.3 Degradation experiments of strain ZSF-9 on other salicylic acid analogs The degradation of 4-chlorosalicylic acid, 3,6-dichlorosalicylic acid, 3,5-dichlorosalicylic acid, 2,5-chlorobenzoic acid, 2-chlorobenzoic acid, gentianic acid, catechol, 4-chlorocatechol, and salicylic acid by strain ZSF-9 was observed, and the results are shown in Table 1. Besides degrading 5-chlorosalicylic acid, strain ZSF-9 can also degrade 4-chlorocatechol and catechol. Since salicylic acid degradation generally involves the gentianic acid pathway and the catechol pathway, and strain ZSF-9 can degrade catechol but not gentianic acid, it can be concluded that strain ZSF-9 degrades 5-chlorosalicylic acid through the catechol pathway.

[0022] Table 1. Degradation of salicylic acid analogues by strain ZSF-9 Substrate ZSF-9 degradation ability 4-Chlorosalicylic acid － 3,6-Dichlorosalicylic acid － 3,5-Dichlorosalicylic acid － 2,5-Chlorobenzoic acid － 2-Chlorobenzoic acid － Gentian acid － Catechol ＋ 4-Chlorocatechol Salicylic Acid ＋－ (Note: "+" indicates biodegradable; "-" indicates non-biodegradable.) II. Cloning and Functional Verification Experiment of 4-Chlorocatechol 1,2-Dioxygenase Gene 2.1 Cloning experiment of 4-chlorocatechol 1,2-dioxygenase gene 2.1.1 Extraction and sequencing of total bacterial genomic DNA Total DNA was extracted from strain ZSF-9 using a modified high-salt extraction method. The genome framework of strain ZSF-9 was sequenced by Beijing Novogene Biotechnology Co., Ltd. using MPS Illumina technology. The genome sequencing results showed that the genome framework of strain ZSF-9 was 4.93 Mb in size and contained 45 contigs.

[0023] 2.1.2 Search for the 4-chlorocatechol 1,2-dioxygenase gene Based on gene functional annotation and amino acid sequence alignment analysis of strain ZSF-9, a sequence was found in the genome of strain ZSF-9 that corresponds to a previously reported catechol 1,2-dioxygenase (from strain ZSF-9). Acinetobacter baylyi ADP1 catechol 1,2-dioxygenase gene catA The highest homology (P07773.1) was only 55.5%, therefore the gene in strain ZSF-9 was named as... catA The nucleotide sequence is shown in SEQ ID NO.1, which is 933 bp in length and encodes 310 amino acids. The amino acid sequence is shown in SEQ ID NO.2.

[0024] 2.2 pET24b- catA Construction of expression carrier 2.2.1 PCR Amplification catA Using total DNA from strain ZSF-9 as a template, forward primer: 5'-TAAGAAGGAGATATACATATGACCGTGAAGATTGCTCACACTGC-3' (SEQ ID NO.3) and reverse primer: 5'-GTGGTGGTGGTGCTCGAGAGCGTCCTGCAACGCCCGTGGC-3' (SEQ ID NO.4) were used to specifically amplify the DNA from the ZSF-9 genomic DNA using PCR. catA Take 4 μL of PCR product and perform agarose gel electrophoresis.

[0025] PCR amplification system: Primer star enzyme (5 U / μl) 0.5 μl 5× PCR Buffer II (Mg 2+ Plus) 10 μl dNTP Mixture (2.5 mM each) 2 μl Template DNA 10 ng Forward primer (20 μM) 1 μl Reverse primer (20 μM) 1 μl Sterile distilled water to 50 μl PCR amplification procedure: a. Denaturation at 98℃ for 3 min; b. Denaturation at 98℃ for 0.5 min, annealing at 60℃ for 0.5 min, extension at 72℃ for 1 min, for 30 cycles; c. 72℃ for 10 minutes, then cool to room temperature.

[0026] 2.2.2 Double enzyme digestion and homologous recombination of PCR products and plasmids The expression vector pET-24b(+) was used Nde I and Xho I Double enzyme digestion was performed, and the enzyme digestion system is as follows: 10 ×M Buffer 5 μL Nde I (10U·μL -1 2.0 μL Xho I (10 U·μL -1 2.0 μL DNA (purified PCR or plasmid) 30 μL ddH2O 11 μL Warm bath at 37°C for 30 minutes.

[0027] The enzyme digestion efficiency was detected by 0.75% agarose gel electrophoresis. The corresponding DNA fragments were recovered and ligated using homologous recombination. catA Add the double-digested pET-24b(+) to the homologous recombination system according to the One Step Cloning kit instructions, and react in a water bath at 37°C for 30 min.

[0028] 2.2.3 Enzyme-linked product transformation and screening experiment for positive transformants Take from -70℃ E. coli 100 µL of BL21(DE3) competent cells were thawed in the palm of the hand. 20 µL of homologous recombination product was added, and the tube was gently rotated to mix. The mixture was then placed on ice for 30 min. The centrifuge tube was then gently placed in a 42°C water bath for 60-90 s for heat shock, and then returned to ice for 10 min. 500-800 µL of LB medium was added, and the tube was placed on a 37°C shaker at 150 rpm for 45-60 min to recover. The tube was centrifuged at 5,000 rpm for 2 min, and a portion of the supernatant was discarded, leaving approximately 200 µL. The cells were mixed thoroughly by pipetting, and 100 µL of the bacterial culture was evenly spread onto an LB agar plate (100 mg / L kanamycin). The cells were incubated overnight at 37°C. Single colonies were picked, and plasmids were extracted and sequenced to verify whether the target gene was ligated into a vector and had six His-tags at the ends. This positive transformant, BL21(pET24b- catA )save.

[0029] 2.3 Assay of CatA Enzyme Activity 2.3.1 Expression and purification experiments of CatA in Escherichia coli The recombinant expression strain BL21(pET24b- catA Inoculate into liquid LB medium and incubate on a shaker until OD. 600nm The concentration was 0.4–0.6, and IPTG was added to a final concentration of 0.05 mM. Induction was performed at 16°C for 8 h. Cells were collected by centrifugation at 12000 rpm for 5 min, washed and resuspended in 50 mM PBS buffer (pH 7.4) at 4°C, and sonicated on ice for 8 min. The supernatant was collected after centrifugation at 12000 rpm for 30 min. Protein purification was performed using a cobalt affinity chromatography column, and the purification effect was assessed by SDS-PAGE electrophoresis.

[0030] After Ni 2+Following NTA affinity purification, CatA with high purity was successfully obtained in elution buffer with 100 mM imidazole. SDS-PAGE protein electrophoresis results showed that the purified CatA was approximately 34 kDa in size, with high purity, consistent with the theoretical value of 34.2 kDa, and the purified protein content was high. Figure 2 ).

[0031] 2.3.2 CatA in vitro enzyme activity assay Enzyme activity reaction system (1 ml): 50 mM PBS (pH 7.0), 0.1 mM FeSO4, appropriate amount of CatA, 0.2 mM catechol / 0.1 mM 4-chlorocatechol. The enzyme reaction was started from the time of enzyme addition. Qualitative detection was first performed using a UV spectrophotometer (200–320 nm). Then, the sample was processed, and quantitative detection was performed using HPLC.

[0032] The results showed that after reacting with CatA for 10 min, the peak shape of 4-chlorocatechol detected by UV scanning changed, and the peak shape did not continue to change with time. After reacting with CatA for 1 min, the peak shape of the catechol UV absorption peak changed, and the peak shape did not continue to change with time. Therefore, it was concluded that the purified 4-chlorocatechol-1,2-dioxygenase CatA has the ability to degrade 4-chlorocatechol and catechol. Figure 3 The UV-scanned sample was freeze-dried, dissolved in an equal volume of methanol, filtered, and then analyzed by HPLC for changes in the substrate 4-chlorocatechol and catechol after the CatA enzymatic reaction. The HPLC results are shown below. Figure 4 The peak of 4-chlorocatechol was at 4.02 min. After degradation of 4-chlorocatechol by CatA, the substrate peak disappeared and a new product peak appeared at 2.46 min. Simultaneously, the peak of catechol appeared at 3.16 min, and the product peak appeared at 2.37 min after CatA degradation of catechol. Figure 4 ).

[0033] 2.3.3 LC / MS Identification of Enzyme-Catalyzed Reaction Products The samples after degradation of catechol and 4-chlorocatechol by catechol-1,2-dioxygenase CatA were freeze-dried, dissolved in methanol and filtered, and then detected by LC / MS to identify the products of catechol and 4-chlorocatechol conversion.

[0034] In the LC / MS results of the 4-chlorocatechol sample, the mass spectrometry-detected structure is as follows: Figure 5Because the mass spectrometry detection was performed in negative ion mode, the main peak mass-to-charge ratio in mass spectrum A was 143.0 m / z, which perfectly matches the molecular weight of 4-chlorocatechol, thus confirming it as 4-chlorocatechol. In mass spectrum B, the main peak mass-to-charge ratio of the product peak was 175.1 m / z, which perfectly matches the molecular weight of 3-chloroethylenedicarboxylic acid, the 1,2-dioxygenated ring-opening product of 4-chlorocatechol. Figure 5 B). LC / MS results of enzyme reaction samples using catechol as a substrate are as follows: Figure 6 Since the mass spectrometry detection was performed in negative ion mode, the main peak mass-to-charge ratio in the A-mass spectrum was 109.0 m / z, which perfectly matches the molecular weight of catechol. Mass spectrometry analysis of the product peak revealed that 141.0 m / z matched the molecular weight of adipedicarboxylic acid, the ring-opening product of catechol. Figure 6 B). The above results indicate that CatA can catalyze the ring-opening of 4-chlorocatechol and 1,2-dioxygenated catechol, respectively.

[0035] III. Enzymatic Characteristics Experiment of 4-Chlorocatechol 1,2-dioxygenase (CatA) 3.1 Effect of pH on CatA enzyme activity (experiment) The buffer solutions used were 20 mM citrate-sodium citrate buffer (pH 3.0–7.0), 20 mM Tris-HCl buffer (pH 7.0–9.0), and 20 mM glycine-sodium hydroxide buffer (pH 9.0–10.0). 0.2 mM 4-chlorocatechol substrate was added to each of the different pH buffers. Equal volumes of pure enzyme were added to the enzyme reaction system, and the reaction was terminated by heating to boiling after an appropriate reaction time (5.0 min). The residual amount of 4-chlorocatechol was quantitatively determined by HPLC. The enzyme activity was set to 100% at pH 7.0 with 20 mM Tris-HCl buffer, and the relative enzyme activity percentage under other pH conditions was calculated.

[0036] The relative enzyme activity of 4-chlorocatechol 1,2-dioxygenase CatA under different pH conditions is as follows: Figure 7 As shown in the figure. Data analysis indicates that 4-chlorocatechol 1,2-dioxygenase CatA exhibits the highest enzyme activity in 20 mM Tris-HCl buffer at pH 8.0. The enzyme activity of CatA in 20 mM Tris-HCl buffer (pH 7.0–9.0) and 20 mM glycine-sodium hydroxide buffer (pH 9.0–10.0) both reach over 70%, demonstrating a wide pH adaptation range for activity.

[0037] 3.2 Effect of Temperature on CatA Enzyme Activity (Experiment) Under optimal pH reaction conditions, 4-chlorocatechol 1,2-dioxygenase (CatA) was added to the enzyme activity system. Different temperature gradients from 4 to 80°C were set, and the reaction was carried out at different temperatures. The activity of the hydrolase was measured at regular intervals. With the enzyme activity at 30°C as 100%, the relative enzyme activity percentage under other temperature conditions was calculated.

[0038] The relative enzyme activity analysis results of CatA under different temperature conditions are as follows: Figure 8 As shown in the figure. Data analysis indicates that CatA exhibits the highest enzyme activity at 30℃, which is determined to be the optimal temperature for this enzyme. CatA maintains over 85% of its maximum catalytic activity at temperatures ranging from 30℃ to 50℃, and at 55℃, the enzyme activity still reaches 64.6% of its maximum activity, demonstrating a wide temperature range suitable for its activity. This indicates that CatA is a novel enzyme with strong heat resistance and good stability over a relatively high temperature range, which is beneficial for its subsequent applications.

[0039] 3.3 Effects of Metal Ions and Chemical Reagents on CatA Activity (Experiment) In an enzymatic reaction system with a known optimal reaction temperature (30℃) and pH (pH 8.0), 1.0 mM of metal ions (K+) were added. + Na + Mg 2+ Hg 2+ Mn 2+ Ni 2+ Co 2+ Ca 2+ Zn 2+ Fe 2+ Fe 3+ The enzyme was reacted with 5.0 mM of chemical reagents EDTA, SDS, PMSF, Urea, and Tween 80 at the optimal reaction temperature and pH. The relative percentage of enzyme activity with the addition of any metal ions or metal ion chelating agents was calculated, with 100% enzyme activity without the addition of any metal ions or chemical reagents.

[0040] The effects of metal ions and chemical reagents on the activity of CatA in degrading 4-chlorocatechol, such as Figure 9 As shown. K + Mg 2+ Mn 2+ Ni 2+ Fe 2+ Fe 3+ Tween 80 significantly improved the activity of CatA, among which Fe 2+ and Fe 3+ The most significant increase was in the activity against CatA, reaching 345% and 264%, respectively. The addition of Hg... 2+ Co2+ Zn 2+ EDTA, PMSF, SDS, and Urea all inhibited the ring-opening activity of 4-chlorocatechol 1,2-dioxygenase, with PMSF and SDS showing the most significant inhibition.

[0041] In summary, the 4-chlorocatechol 1,2-dioxygenase and its encoding gene of the present invention... catA Its gene sequence differs significantly from known functional genes, thus bringing novel catalytic properties. It can simultaneously and efficiently degrade catechols and 4-chlorocatechol, especially exhibiting high catalytic efficiency for 4-chlorocatechol. It also shows good stability over a wide range of temperatures and pH levels. This is of vital importance for constructing genetically engineered bacteria that efficiently degrade chlorinated aromatic pollutants and developing novel bio-enhanced treatment processes and formulations. It will also provide a new technological approach to solving the environmental problem of persistent chlorinated organic pollution.

[0042] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. A 4-chlorocatechol 1,2-dioxygenase gene catA, characterized in that, Its nucleotide sequence is SEQ ID NO.

1.

2. The application of the 4-chlorocatechol 1,2 dioxygenase gene catA as described in claim 1 in the construction of transgenic engineered bacteria that degrade catechols and 4-chlorocatechol.

3. The use of the 4-chlorocatechol 1,2 dioxygenase gene catA as described in claim 1 in the preparation of formulations that degrade or remove catechols, 4-chlorocatechols and related chlorinated aromatic hydrocarbon pollutants from soil and water.

4. A 4-chlorocatechol 1,2-dioxygenase, CatA, characterized in that, The amino acid sequence encoded by the nucleotide sequence catA of the 4-chlorocatechol 1,2 dioxygenase gene as described in claim 1 is SEQ ID NO.

2.

5. The use of the 4-chlorocatechol 1,2-dioxygenase CatA according to claim 4 in the preparation of enzyme preparations for degrading 4-chlorocatechol and catechol.

6. The use of the 4-chlorocatechol 1,2-dioxygenase CatA according to claim 4 in the preparation of formulations for removing catechols, 4-chlorocatechol and related chlorinated aromatic hydrocarbon pollutants from soil and water.

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