Dechlorinase DehA and DNA and application thereof

Abstract

The invention discloses a dechlorination enzyme DehA and DNA and application thereof, and relates to the technical field of sewage treatment, the amino acid sequence of the dechlorination enzyme DehA is shown as SEQ ID No.1, the dechlorination enzyme DehA can degrade hexachlorobutadiene into pentachlorobutadiene with low toxicity, the degradation efficiency of the dechlorination enzyme DehA to 520 mg / L hexachlorobutadiene within 30 min is 60%, and the degradation rate is high.

Description

Technical Field

[0001] The present invention relates to the technical field of sewage treatment, in particular to a dechlorinase DehA, a DNA thereof and an application thereof. Background Art

[0002] In the chemical industry, hexachlorobutadiene (HCBD) is an important component in the production of pesticides, herbicides, transformer oils and heat transfer fluids. It is widely used, making it one of the more common volatile organochlorine pollutants in groundwater sampling.

[0003] HCBD is highly toxic and carcinogenic. Due to its high chemical stability and low biodegradability, it can remain in the natural environment for a long time and accumulate in organisms, making HCBD a new type of persistent organic pollutants (POPs).

[0004] Currently, HCBD is usually treated by photodegradation or physicochemical degradation. However, these treatment methods will produce many problems such as highly toxic intermediates and high energy consumption, thereby causing secondary pollution to the environment and waste of resources. Summary of the Invention

[0005] The main purpose of the present invention is to propose a dechlorinase DehA and its DNA and application, aiming to solve the problems of the current method for degrading HCBD producing highly toxic intermediates and high energy consumption.

[0006] To achieve the above object, the present invention provides a dechlorinase DehA, the amino acid sequence of the dechlorinase DehA is shown in SEQ ID No. 1.

[0007] The present invention provides a DNA molecule, which is used to encode the aforementioned dechlorinase DehA.

[0008] In one embodiment, the nucleotide sequence of the DNA molecule is shown as SEQ ID No. 2.

[0009] In one embodiment, the source of the DNA molecule comprises Agrobacterium sp. BJ-04 strain.

[0010] The present invention provides a recombinant plasmid, which comprises the DNA molecule.

[0011] The present invention provides a host cell, which comprises the nucleotide sequence of the DNA molecule or the recombinant plasmid.

[0012] The present invention provides an application of the dechlorinase DehA in degrading halogenated organic matter.

[0013] In one embodiment, the halogenated organic compound comprises hexachlorobutadiene, trichloroethylene, dichloromethane, tetrachloroethylene or dichloroethane.

[0014] In one embodiment, the temperature at which the dechlorinase DehA degrades halogenated organic matter is 20°C to 40°C.

[0015] In one embodiment, the dechlorinase DehA degrades halogenated organic matter at a pH of 5.0 to 9.0.

[0016] The invention provides a dechlorinase DehA, DNA thereof and application thereof. The dechlorinase DehA can degrade hexachlorobutadiene into pentachlorobutadiene with lower toxicity. The dechlorinase DehA has a degradation efficiency of 60% for 520 mg / L hexachlorobutadiene within 30 minutes, and has a relatively fast degradation rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 This is a diagram showing the results of agarose gel electrophoresis detection of PCR amplified dehA products in Example 1 provided by the present invention;

[0019] Figure 2 This is a diagram showing the results of agarose gel electrophoresis detection of the linearized product of the PCR amplified vector pET28a in Example 1 provided by the present invention;

[0020] Figure 3 This is a diagram showing the results of enzyme digestion product detection by agarose gel electrophoresis in Example 1 provided by the present invention;

[0021] Figure 4 SDS-PAGE electrophoresis of DehA purified in Example 1 provided by the present invention;

[0022] Figure 5 The product diagram of HCBD catalyzed by dechlorinase DehA detected by GC-MS in Example 2 provided by the present invention;

[0023] Figure 6 The activity change diagram of dechlorinase DehA catalyzing HCBD at different pH values ​​in Example 3 provided by the present invention;

[0024] Figure 7 The activity change diagram of dechlorinase DehA catalyzing HCBD at different temperatures in Example 3 provided by the present invention;

[0025] Figure 8 This is the Lineweaver-Burk double reciprocal plot of the dechlorinase DehA in Example 3 provided by the present invention.

[0026] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0027] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0028] It should be noted that, in the embodiments, those without specifying specific conditions are carried out according to conventional conditions or the conditions recommended by the manufacturer. Reagents used or instruments without specifying manufacturers are conventional products that can be purchased commercially. In addition, the technical solutions between the various embodiments can be combined with each other, but must be based on those of ordinary skill in the art being able to realize the basis. When the combination of the technical solutions is mutually contradictory or cannot be realized, it should be considered that the combination of such technical solutions does not exist, nor is it within the scope of protection claimed in the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise belong to the scope of protection of the present invention.

[0029] The chemical industry produces a large number of halogenated organic compounds. They are generally environmentally toxic, have relatively stable chemical properties, and are difficult to degrade quickly. They are released into the environment for various reasons and are common persistent pollutants in soil, groundwater, and surface water.

[0030] In the chemical industry, hexachlorobutadiene (HCBD) is an important component in the production of pesticides, herbicides, transformer oils and heat transfer fluids. It is widely used, making it one of the more common volatile organochlorine pollutants in groundwater sampling.

[0031] HCBD is highly toxic and carcinogenic. Due to its high chemical stability and low biodegradability, it can remain in the natural environment for a long time and accumulate in organisms, making HCBD a new type of persistent organic pollutants (POPs).

[0032] Currently, HCBD is usually treated by photodegradation or physicochemical degradation. However, these treatment methods will produce many problems such as highly toxic intermediates and high energy consumption, thereby causing secondary pollution to the environment and waste of resources.

[0033] In view of this, the present invention provides a dechlorinase DehA, the amino acid sequence of the dechlorinase DehA is shown in SEQ ID No. 1.

[0034] SEQ ID No. 1:

[0035] MTKIEHIVFDIGKVLIHYDPHIPYSRLIPDADERKWFFENVCTHDWNLEQDRGRGWEDAEALLMEQFPEREEHIRAFRKFWHEMVSHSYDDSVAIMTGLIDKGHDVTMLTNFASDTFREAQKMFPFLTLPRGVTVSGDVKLLKPDVAIYELHAREFGLDPAASIFIDDTLVNVEGAKAAGWQAVHFTGAEKLKQDLRSYGVDA.

[0036] In the technical solution of the present invention, the dechlorinase DehA can degrade hexachlorobutadiene into pentachlorobutadiene with lower toxicity. The degradation efficiency of the dechlorinase DehA on 520 mg / L hexachlorobutadiene within 30 minutes is 60%, and the degradation rate is relatively fast.

[0037] Bacterial enzymes catalyze dehalogenation reactions in diverse ways, with varying dehalogenation mechanisms, including reductive dehalogenation, oxidative decomposition dehalogenation, hydrolytic dehalogenation, sulfidodehalogenation, intermolecular displacement dehalogenation, dehydrohalogenation, and hydration dehalogenation. Dehalogenation is generally the initial and key step in the mineralization of halogenated organic matter. Dehalogenation often reduces the toxicity of halogenated aliphatic compounds. Haloalkane dehalogenases (i.e., dechlorinases) utilize a hydrolytic dehalogenation mechanism, requiring no oxygen, energy, or coenzymes / prosthetic groups. This represents a simple route for bioreduction and biodegradation, with significant environmental and biotechnological significance.

[0038] The present invention provides a DNA molecule for encoding the aforementioned dechlorinase DehA. The dehalogenase gene dehA provided by the present invention has a protein sequence that is 23% identical to the haloacid dehalogenase from Bacteroides thetaiotaomicron VPI-548 and is a new dechlorinase.

[0039] In some embodiments of the present invention, the nucleotide sequence of the DNA molecule is shown as SEQ ID No. 2.

[0040] SEQ ID No. 2:

[0041] .

[0042] In some embodiments of the present invention, the source of the DNA molecule includes Agrobacterium sp. BJ-04 strain. The cycle of expressing the dechlorinase DehA using a DNA molecule derived from the Agrobacterium sp. BJ-04 strain is shorter, making the dechlorinase DehA easy to obtain. The Agrobacterium sp. BJ-04 strain is deposited with the China Center for Type Culture Collection, Wuhan University, with the deposit number CCTCC No: M 2022213, and the deposit date is March 8, 2022.

[0043] The present invention provides a recombinant plasmid, which comprises the DNA molecule.

[0044] Specifically, the method for obtaining the dechlorinase using a recombinant plasmid containing the DNA molecule includes: using a restriction endonuclease to cut the plasmid vector and the target gene, and then connecting the two with a ligase to form a recombinant plasmid. The recombinant plasmid is introduced into host cells (such as Escherichia coli), and the selected host cells are cultured under suitable conditions to induce expression of the dechlorinase gene. The dechlorinase is then purified by centrifugation, chromatography, or other methods.

[0045] The present invention provides a host cell comprising the nucleotide sequence of the DNA molecule or the recombinant plasmid. The host cell is preferably Escherichia coli.

[0046] The present invention provides an application of the dechlorinase DehA in degrading halogenated organic matter.

[0047] In the technical solution provided by the present invention, the degradation of halogenated organic matter by dechlorinase DehA belongs to a biological degradation method. Compared with physical and chemical methods, this method does not introduce other harmful substances.

[0048] In some embodiments of the present invention, the halogenated organic compounds include hexachlorobutadiene, trichloroethylene, dichloromethane, tetrachloroethylene, or dichloroethane. Dechlorinase DehA has catalytic activity against hexachlorobutadiene, tetrachloroethylene, trichloroethylene, dichloroethane, and dichloromethane, and thus can degrade the halogenated organic compounds.

[0049] In some embodiments of the present invention, the temperature at which the dechlorinase DehA degrades halogenated organic matter is 20°C to 40°C. The temperature at which the dechlorinase DehA degrades halogenated organic matter can be 20°C, 30°C, or 40°C. When the degradation temperature is within an appropriate range, the relative enzyme activity of the dechlorinase DehA is high, and the degradation efficiency of the dechlorinase DehA is high. Preferably, the temperature at which the dechlorinase DehA degrades halogenated organic matter is 30°C.

[0050] In some embodiments of the present invention, the pH of the dechlorinase DehA during degradation of halogenated organic matter is 5.0 to 9.0. The pH of the dechlorinase DehA during degradation of halogenated organic matter can be 5, 7, or 9. When the degradation pH is within the appropriate range, the relative enzymatic activity of the dechlorinase DehA is high, and the degradation efficiency of the dechlorinase DehA is high. Preferably, the pH of the dechlorinase DehA during degradation of halogenated organic matter is 7.

[0051] The technical solutions of the present invention are further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0052] Example 1 Cloning of the dehalogenase gene dehA

[0053] The genome of the hexachlorobutadiene-degrading strain Agrobacterium sp. BJ-04 (strain deposit number CCTCC No: M2022213) was used as a template, and the dehA gene was amplified by PCR using primers dehA-F (sequence shown in SEQ ID No. 3) and dehA-R (sequence shown in SEQ ID No. 4).

[0054] SEQ ID No.3: 5'-aggagatataccatgACCAAGATTGAGCATATCG-3',

[0055] SEQ ID No. 4: 5'-tgcggccgcaagcttAGCATCGACACCGTAGGATC-3'.

[0056] The PCR reaction system was as follows: 5x fast pfu buffer 10 mL, dNTP mixture 1 mL (10 mM each), dehA-F 1 mL, dehA-R 1 mL, template 1 mL, fast pfu 0.5 mL, and ddH2O 35.5 mL.

[0057] The PCR amplification program was as follows: 95°C for 5 min, 95°C for 30 s, 56°C for 30 s, 72°C for 1 min, 30 cycles, and 72°C for 8 min.

[0058] The results of electrophoresis detection of PCR products are shown in Figure 1 . Figure 1 In the figure, M represents DNA Marker DL 2000, and lane 1 represents the PCR product. It was found that the size of the electrophoresis band in lane 1 was consistent with the expected size of the dehA gene (856 bp).

[0059] Take a single activated DH5a / pET28a colony, suspend it in 50 μL of sterile water, incubate it in a metal bath at 100°C for 10 minutes, cool it on ice, and centrifuge it at 12,000 rpm. Take the DNA in the supernatant as a template and use primers pET28a-F (sequence shown in SEQ ID No. 5) and pET28a-R (sequence shown in SEQ ID No. 6) to PCR amplify the pET28a vector backbone.

[0060] SEQ ID No.5: 5'-aagcttgcggccgcactc-3',

[0061] SEQ ID No. 6: 5'-catggtatatctccttcttaaagttaaacaaaattat-3'.

[0062] The PCR reaction system was as follows: 5x fast pfu buffer 10 mL, dNTP mixture 1 mL (10 mM each), pET28a-F 1 mL, pET28a-R 1 mL, template 4 mL, fast pfu 0.5 mL, and ddH2O 32.5 mL.

[0063] The reaction conditions for PCR amplification of the vector were as follows: 95°C for 5 min, 95°C for 30 s, 60°C for 1 min, 72°C for 3 min, 30 cycles, and 72°C for 8 min.

[0064] The results of electrophoresis detection of PCR amplified vector pET28a backbone are shown in Figure 2 . Figure 2 In the figure, M represents DNA Marker DL 2000, and lane 1 represents the PCR product. It was found that the size of the band in lane 1 was equal to that of the vector pET28a (5369 bp).

[0065] Recombinant plasmid digestion and identification: The PCR-amplified dehA gene fragment and the pET28a linearized vector were subjected to electrophoresis, and the corresponding bands were cut and recovered from the gel. The mixture was then mixed in a 2:1 ratio. Onestep enzyme (Nanjing Novozyme Biotechnology Co., Ltd.), buffer, and sterile water were added. The reaction was incubated at 37°C for 30 minutes and immediately placed on ice to obtain the recombinant product. The specific ligation system is shown in Table 1.

[0066] Table 1 Ligation reaction system

[0067] Components Volume (μL) Linearized vector 2 Insert 4 5×CEⅡ buffer 2 EXnaseⅡ 1 Sterile water 1

[0068] The ligation product was transformed into DH5a and spread on LB plates containing 50 μg / mL. The cells were cultured at 37°C overnight. The transformants were picked and inoculated into LB liquid medium containing 50 mg / mL Kan. The cells were cultured overnight. The cells were collected and the plasmids were extracted. The cells were identified by double enzyme digestion with XbaI and HindIII and detected by agarose gel electrophoresis. The results were as follows: Figure 3 shown. Figure 3 In the figure, lane 1 represents pET28a-dehA, lane 2 represents pET28a-dehA / XbaI+HindIII, and M represents DNA marker DL 15000. Two bands can be observed in lane 2: the larger band is consistent with the size of the vector pET28a, and the smaller band is consistent with the size of the gene dehA.

[0069] The plasmids verified to be correct by enzyme digestion were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequence comparison of the determination results was performed using Blast software. The results showed that the gene dehA was successfully cloned into pET28a, and the recombinant plasmid was named pET28a-dehA.

[0070] The recombinant plasmid with correct sequencing was transformed into Escherichia coli BL21 (DE3) to obtain the recombinant strain BL21 (DE3) / pET28a-dehA. A single colony of the recombinant strain was inoculated into 5 mL of LB medium containing 50 μg / mL Kan and cultured on a shaker at 37 ° C for 12 to 14 hours. Then, 1% inoculum was inoculated into 200 mL of LB medium containing 50 μg / mL Kan and cultured on a shaker at 37 ° C until OD 600 The concentration of isopropyl-β-D-thiogalactopyranoside (IPTG) was 0.4-0.6, and then isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 0.1 mmol / L. The culture was induced by shaking at 16°C for about 16 hours. The IPTG-induced bacterial solution was collected by centrifugation, the supernatant was discarded, the cells were collected, PB buffer was added, and the solution was centrifuged at 4°C at a speed of 6000 rpm / min for 10 minutes, and the bacteria were washed 2-3 times. 20 mL of phosphate buffer (PB) was added to resuspend the solution and ultrasonically disrupted. The parameter settings were 5 s for the interval time, 3 s for the working time, 30 min for the whole process, and the power was 400-600 W (generally, the disruption can be completed in 10 minutes). After disruption, the solution was centrifuged at 12,000 rpm / min at 4°C for 15 minutes, and the supernatant was taken as a crude enzyme solution sample. Protein purification was performed by affinity chromatography (nickel column filler was Ni-NAT beads 6FF) to obtain purified dechlorinase DehA. The expression of the protein and the purification of the product were detected by electrophoresis (see Figure 4 ). Figure 4 M in the figure indicates a 180 kDa marker; lane 1 represents whole cells of E. coli BL21 (pET28a-dehA); lane 2 represents crude enzyme solution of E. coli BL21 (pET28a-dehA); lane 3 represents the flow-through; lane 4 represents the protein eluted in PB solution containing 40 mmol / L imidazole. The theoretical molecular weight is 23.35 kDa, and the size of the protein band detected by electrophoresis is consistent with the theoretical molecular weight. The purified protein sample shows a single band, indicating high purity.

[0071] Example 2 Identification of HCBD Degradation Products Catalyzed by DehA

[0072] Dissolve 10 μL of HCBD in 20 μL of dimethyl sulfoxide (DMSO). Add 50 μL of purified dechlorinase DehA and 0.155 μL of HCBD reagent (final HCBD concentration is 2 mM) to 500 μL with 50 mM pH 7.4 PB buffer. Use the purified dechlorinase DehA after inactivation as a control, and set up three replicates. After the above system is mixed evenly, after 30 minutes in a 37°C water bath (Eppendorf tubes are regularly inverted and mixed during the water bath), 500 μL of acetonitrile is added to terminate the reaction, and 1 mL of n-hexane is extracted. After standing and stratification, the upper organic phase is taken and dried with anhydrous sodium sulfate. After filtering, it is placed in a chromatographic bottle and placed in a -20°C refrigerator. Since the substance to be detected is volatile, it should be detected by gas chromatography-mass spectrometry (GC-MS) as soon as possible.

[0073] The GC-MS instrument used was an Agilent Technologies 7890B, with an HP-5 column and helium as the carrier gas at a flow rate of 1.0 mL / min. A 2 μL injection was performed in splitless mode (splitless time 0.5 min). The electron ionization mode was set to 70 eV. The column oven temperature was initially set at 60°C for 1 min, then increased to 100°C at 5°C / min, then to 10°C / min, held at 250°C for 3 min, and then increased to 300°C for 3 min. The temperatures of the inlet, quadrupole, and ion source were 280°C, 180°C, and 230°C, respectively. Analysis of the target compounds was performed using selected ion monitoring (SIM) mode.

[0074] The results of GC-MS showed that the product of HCBD catalyzed by DehA was pentachlorobutadiene (see Figure 5 ).

[0075] Example 3 Enzymatic property test of dechlorinase DehA

[0076] (1) Establishment of enzyme activity determination method

[0077] The principle of the chloride ion electrode method is based on the fact that the Cl generated in the reaction system by dechlorinase in the dechlorination reaction - The concentration is accurately detected and displayed by the ion electrode probe, allowing the enzyme activity to be determined based on the rate of change in chloride ion concentration. A suitable dechlorinase activity assay was established. The enzyme reaction system is as follows: 15 mmol / L chloroolefin substrate and 200 μg of purified enzyme are added to a 5 mL reaction volume. The volume is filled to 5 mL with 50 mmol / L, pH 7.4, PB buffer. The reaction is shaken at 30°C and 200 rpm / min for 30 minutes. The reaction is quickly terminated on ice. Samples are taken and the released chloride ion concentration is measured using a chloride ion electrode to calculate the enzyme activity.

[0078] The enzyme activity unit (U) is defined as the amount of enzyme required to produce 1 μmol of chloride ions in 1 min at 30°C and pH 7.0.

[0079] (2) Optimal reaction pH of dechlorinase DehA

[0080] The pH of the reaction medium is a key factor affecting enzyme activity, because different pH can affect the dissociation state of amino acids on the peptide chain, thereby affecting the structure, catalytic activity and stability of the enzyme.

[0081] The optimal reaction pH of dechlorinase DehA was determined by measuring its activity in buffer solutions with different pH values. When measuring DehA enzyme, the activity measured at pH = 7.0 was taken as 100% and the relative activity was calculated. Figure 6 As shown in the figure, the optimal reaction pH of DehA enzyme is 7.0, and the reaction conditions of excessive acidity (pH 5.0) and excessive alkalinity (pH 12.0) are not suitable for the DehA enzyme-catalyzed reaction.

[0082] (3) Optimal reaction temperature of dechlorinase DehA

[0083] The results of the activity determination of DehA catalyzing HCBD at different temperatures are shown in Figure 2. Figure 7 When measuring DehA enzyme, relative activity is calculated based on the enzyme activity measured at a reaction temperature of 30°C as 100%. The optimal reaction temperature for DehA enzyme is 30°C. At 25 and 35°C, relative enzyme activity is high, maintaining around 80%. At 20-40°C, relative enzyme activity can be maintained above 60%.

[0084] (4) Effects of metal ions on dechlorinase DehA

[0085] Using various metal ion solutions with a final concentration of 1 mM, the Cu 2+ 、Co 2+ 、Mn 2+ 、Fe 2+ Mg 2+ and Zn 2+ The test method for the effect of dehalogenase DehA on the catalysis of HCBD was as shown in (1), except that the final concentration of each metal ion in the enzyme reaction system was set to 1mM. Using hexachlorobutadiene as substrate, the enzyme activity of the purified dehalogenase under the influence of different metal ions was determined after 30 minutes of reaction, and each group was repeated 3 times in parallel. The enzyme activity without metal ions was taken as 100% (control), and the relative activity under different metal ion conditions was calculated. The results are shown in Table 2. The results show that Mn 2+ 、Fe 2+ Mg 2+ and Co 2+It has a promoting effect on DehA enzyme, Co 2+ The promoting effect is the most obvious.

[0086] Table 2 Effects of metal ions on DehA enzyme activity

[0087] Type of metal ion Relative activity% none 100 <![CDATA[Cu 2+ ]]> 83.5 <![CDATA[Mn 2+ ]]> 108.2 <![CDATA[Fe 2+ ]]> 106.6 <![CDATA[Mg 2+ ]]> 125.3 <![CDATA[Zn 2+ ]]> 95.6 <![CDATA[Co 2+ ]]> 140.3

[0088] (4) Substrate range of dechlorinase DehA

[0089] Seven dechlorinase substrates were selected and the specific activity of DehA against these halogenated hydrocarbons was determined. The assay method was as follows: 2 mmol / L chlorinated olefins or chlorinated alkanes and 200 μg of purified enzyme were added to a 5 mL reaction system. The volume was made up to 5 mL with 50 mmol / L PB buffer, pH 7.4. The reaction was shaken at 30°C and 200 rpm for 30 minutes. The reaction was immediately terminated on ice, and samples were collected and analyzed using a chloride electrode. The results are shown in Table 3. DehA exhibited catalytic activity against not only hexachlorobutadiene but also tetrachloroethylene, trichloroethylene, dichloroethane, and dichloromethane. DehA exhibited the highest activity against trichloroethylene, followed by hexachlorobutadiene and dichloromethane. Its activity against dichloroethane and tetrachloroethylene was lower.

[0090] Table 3 Substrate range of dechlorinase DehA

[0091] substrate DehA specific activity (U / mg) Hexachlorobutadiene 0.80±0.43 Tetrachloroethylene 0.15±0.04 Trichloroethylene 6.25±0.74 Chloroform - dichloromethane 0.22±0.05 Hexachloroethane - Ethylene dichloride 0.17±0.07

[0092] Note: “-” means not detected.

[0093] (5) Kinetic parameters of dechlorinase DehA

[0094] Catalytic reaction system: Add hexachlorobutadiene at concentrations of 0.5 mmol / L, 1 mmol / L, 2 mmol / L, 5 mmol / L, 10 mmol / L, and 15 mmol / L (methanol as solvent) to a total volume of 5 mL. Add 200 μg of purified dechlorinase DehA and make up to 5 mL with 50 mmol / LPB buffer (pH 7.4). Shake the reaction at 30°C and 200 rpm / min for 30 min. Samples are taken and the change in chloride ion concentration in the supernatant is detected using a chloride ion electrode. According to the Lineweaver-Burk plot method, a double reciprocal curve for dechlorinase DehA is drawn with the reciprocal of the hexachlorobutadiene substrate concentration 1 / [S] as the abscissa and the reciprocal of the reaction rate 1 / [V] as the ordinate (see Figure 8 The kinetic parameters of dechlorinase DehA can be obtained based on the double reciprocal plot of dechlorinase DehA ( Figure 4As shown in Table 4, the Km value of dechlorinase DehA for hexachlorobutadiene is 1.57±0.08 mM, the maximum enzymatic reaction rate Vmax value is 37.72±1.01 μM / min, and the Kcat / Km value is 0.24±0.01 mM -1 S -1 .

[0095] Table 4 Kinetic parameters of dechlorinase DehA

[0096]

[0097] In summary, the dechlorinase DehA in the present invention can be used for bioremediation of organic polluted environments such as hexachlorobutadiene, trichloroethylene, and dichloromethane.

[0098] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of the present invention.

Claims

1. A dechlorinase DehA, characterized in that The amino acid sequence of the dechlorinase DehA is shown in SEQ ID No.

1.

2. A DNA molecule, characterized in that The DNA molecule is used to encode the dechlorinase DehA as claimed in claim 1.

3. The DNA molecule according to claim 2, characterized in that The nucleotide sequence of the DNA molecule is shown as SEQ ID No.

2.

4. The DNA molecule according to claim 3, characterized in that The sources of the DNA molecule include Agrobacterium sp. BJ-04 strain.

5. A recombinant plasmid, characterized in that: The recombinant plasmid comprises the DNA molecule as claimed in claim 3.

6. A host cell, characterized in that The host cell comprises the nucleotide sequence of the DNA molecule according to claim 3 or the recombinant plasmid according to claim 5.

7. Use of the dechlorinase DehA as claimed in claim 1 in degrading halogenated organic matter.

8. The use according to claim 7, characterized in that The halogenated organic compound includes hexachlorobutadiene, trichloroethylene, dichloromethane, tetrachloroethylene or dichloroethane.

9. The use according to claim 7, wherein the temperature at which the dechlorinase DehA degrades halogenated organic matter is 20°C to 40°C.

10. The use according to claim 7, wherein the pH of the dechlorinase DehA when degrading halogenated organic matter is 5.0 to 9.0.

Images