CN112680427B - Dehalogenase HldD1 and coding gene and application thereof - Google Patents

Abstract

The invention discloses a dehalogenase HldD1 and a coding gene and application thereof. The invention provides a protein which is shown as a sequence 2 in a sequence table. Experiments prove that the dehalogenase HldD1 obtained by prokaryotic expression of escherichia coli by adopting the gene sequence (sequence 1) has high activity and stronger organic solvent stability under the treatment conditions of not more than 20% of ethanol, 20% of acetonitrile and 40% of DMSO respectively.

Description

A kind of dehalogenase HldD1 and its encoding gene and application

technical field

The invention belongs to the field of biotechnology, in particular to a dehalogenase HldD1 and its encoding gene and application.

Background technique

Halogenated organic compounds have a wide range of applications in many fields. Among them, due to their extensive use in industrial enterprises and daily necessities, they have become an important class of environmental pollutants. These halogenated organic compounds continue to cause widespread environmental problems in the biosphere for many years due to their very stable chemical bonds. Halogenated organics are the most used and studied class of organics, mainly due to their relatively simple synthetic routes. However, halogenated organics have poor biodegradability for many years due to their generally high stability and even toxicity to microorganisms that may degrade them. Excessive exposure to these halogenated pollutants can lead to serious environmental and health consequences. In nature, the biodegradation ability to decompose these pollutants is limited, so for many years, human beings are still investing a lot of manpower and financial resources in the restoration and treatment of polluted environments. Among them, microbial remediation technology is the main means of environmental pollution control caused by halogenated organic matter. In recent years, the development of this technology has received increasing attention from researchers.

Microbial dehalogenation is mainly achieved by using strains to induce production and release dehalogenases. Different dehalogenases have different specific substrates, including chloro, bromo, iodo organics and different chain lengths, and the substrate preference depends on the respective catalytic mechanisms of different dehalogenases. The main catalytic mechanism of dehalogenation reaction is nucleophilic substitution. According to the type of catalytic substrate, dehalogenases can be divided into halogen acid dehalogenase (EC3.8.1.2, Haloacid dehalogenase, HAD), halogenated alkane dehalogenase ( EC3.8.1.5, Haloalkane dehalogenase, HLD), 4-chlorobenzoyl-CoA dehalogenase, halogenated acrylic acid dehalogenase, etc. The two most common dehalogenases are halogen acid dehalogenase and halogenated alkane dehalogenase, both of which are hydrolytic dehalogenases.

Dehalogenase not only plays a key role in the degradation of pesticides, chemical warfare agents, PCDD/Fs and other pollutants, but also is widely used in many fields such as industry and animal husbandry. Therefore, the development of novel dehalogenases with excellent properties has important application value and social significance.

SUMMARY OF THE INVENTION

In order to develop a dehalogenase with excellent properties, the present invention provides the following technical solutions:

An object of the present invention is to provide a dehalogenase HldD1 and its encoding gene.

The present invention provides a protein, which is a dehalogenase HldD1 or a recombinant dehalogenase HldD1 thereof, specifically any of the following (a1)-(a5):

(a1) the protein shown in sequence 2 in the sequence listing;

(a2) the protein shown in positions 1-308 of SEQ ID NO: 2 in the sequence listing;

(a3) a fusion protein obtained by linking a tag to the N-terminus or/and C-terminus of the protein of (a2);

(a4) A protein with dehalogenase activity obtained by substituting and/or deleting and/or adding any one of (a1)-(a3) one or several amino acid residues;

(a5) A protein having 98% or more identity with any one of (a1) to (a3) and having dehalogenase activity.

The specific labels are shown in Table 1.

Table 1 Sequences of tags

Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL HA 9 YPYDVPDYA

Nucleic acid molecules encoding the above proteins are also within the scope of the present invention.

The above-mentioned nucleic acid molecule is the DNA molecule shown in any one of the following (b1)-(b4):

(b1) the DNA molecule shown in sequence 1 in the sequence listing;

(b2) the DNA molecule shown in positions 1-924 of SEQ ID NO: 1 in the sequence listing;

(b3) A DNA molecule that is more than 95% identical to the nucleotide sequence defined in (b1) or (b2) and encodes the above-mentioned protein;

(b4) A DNA molecule that hybridizes to the nucleotide sequence defined in (b1) or (b2) under stringent conditions and encodes the above-mentioned protein.

The above stringent conditions can be hybridized at 65°C with a solution of 6×SSC, 0.5% SDS, and then washed once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS each.

Expression cassettes, recombinant vectors or recombinant microorganisms containing the above nucleic acid molecules all belong to the protection scope of the present invention.

The above-mentioned expression cassette can be composed of a promoter capable of starting the expression of the DNA molecule, the above-mentioned nucleic acid molecule, and a transcription termination sequence. In one embodiment of the present invention, the promoter of the above-mentioned nucleic acid molecule in the expression cassette is a T7 promoter , the terminator is the T7 terminator.

The above-mentioned recombinant vector can be a recombinant expression vector or a recombinant cloning vector.

The recombinant expression vector can be constructed using existing expression vectors. The expression vector may also contain the 3' untranslated region of the exogenous gene, i.e., containing the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The poly(A) signal directs the addition of poly(A) to the 3' end of the mRNA precursor. When using the gene to construct a recombinant expression vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added before its transcription initiation nucleotide, which can be used alone or with other promoters. Combined use; in addition, enhancers, including translation enhancers or transcription enhancers, can also be used when using the gene of the present invention to construct a recombinant expression vector. In order to facilitate the identification and screening of transgenic cells, the expression vector used can be processed, such as adding genes for luminescent compounds (GFP gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin marker) markers, kanamycin markers, etc.).

In one embodiment of the present invention, the recombinant vector is pET-D1, which is to replace the DNA molecule containing the HldD1 gene shown in sequence 1 with the pET-21a(+) vector containing the multiple cloning site NdeI (CATATG) To the DNA between HindIII (AAGCTT), the recombinant vector obtained by keeping the other sequences of the pET-21a(+) vector unchanged (refer to the ClonExpress MultiS One Step Cloning Kit of Novozymes for the replacement method), the vector contains the sequence The DNA molecule shown in 1 is used, and the recombinant protein HldD1 shown in sequence 2 is expressed by using the T7 promoter of the vector to promote the DNA molecule shown in sequence 1.

The above-mentioned recombinant bacteria are obtained by introducing the aforementioned DNA molecules or the aforementioned expression cassettes or the aforementioned recombinant vectors into host bacteria. In one embodiment of the present invention, the host bacteria is Escherichia coli.

Another object of the present invention is to provide the following applications.

The present invention provides the application of the protein in the first object as a dehalogenase;

Or the present invention provides the nucleic acid molecule in the first object, or the application of the expression cassette, recombinant vector or recombinant microorganism in the preparation of dehalogenase.

The above-mentioned dehalogenase is specifically a haloalkane dehalogenase.

The present invention also provides a method for preparing a dehalogenase, comprising the steps of: inducing and culturing the recombinant microorganism in the first objective to obtain a dehalogenase.

In an embodiment of the present invention, the induction culture is:

(a2) adding IPTG to the recombinant microorganism culture system to a final concentration of 0.5mM (the OD ₆₀₀ value of the culture system reaches about 0.6-0.8 when IPTG is added), and cultured at 16°C for 18 hours;

(a2) Ultrasonic (such as ultrasonic 5s, intermittent 5s, power of 40w, 50 cycles) disrupts the cells obtained in step (a1) and centrifugation (such as 6,500rpm for 20min), and obtain dehalogenase from the supernatant obtained by centrifugation.

In step (a2), after the centrifugation, a step of purifying the supernatant (eg, nickel column affinity chromatography purification and desalting purification) is further included.

In an embodiment of the present invention, the supernatant is specifically purified according to a method comprising the following steps:

The first step: carry out nickel column affinity chromatography purification (such as purification with 1ml HiTrap chelating HP column (nickel column) in the AKTA FPLC system of Amersham company), including the following steps: use native nickel column binding buffer Ⅰ Equilibrate and purify the column and load the sample (wherein, the protein loading amount can be 10ml, and the flow rate can be set to 1ml/min); wash with non-denaturing nickel column binding buffer Ⅰ to remove non-specifically bound impurity proteins; The non-denaturing nickel column elution buffer was eluted with a linear gradient of 0%-100%, and the eluate containing the elution peak was collected.

The composition of the native nickel column binding buffer I is as follows: 20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 7.5.

The composition of the native nickel column elution buffer II is as follows: 20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 7.5.

The second step: the eluate obtained in the first step was purified by a desalting column Hitrap Q (Pharmacia) (5 mL), and the desalting buffer was: 50 mM Gly-NaOH, pH 8.2.

The application of the protein or the nucleic acid molecule or the expression cassette, the recombinant vector or the recombinant microorganism in the first object in removing the halogen group on the halogenated organic matter is also within the scope of the present invention.

Or, the application of the protein or the nucleic acid molecule or the expression cassette, the recombinant vector or the recombinant microorganism in the first objective in the treatment of halogenated organic pollution is also within the scope of protection of the present invention.

Still another object of the present invention is to provide a method for removing halogen groups from halogenated organics.

The method provided by the present invention includes the following steps: using the protein in the first objective to catalyze the halogenation of organic compounds to achieve the removal of halogen groups on the halogenated organic compounds.

In the above method, the temperature of the catalysis is 15-40°C, and the optimum temperature of the catalysis is specifically 40°C;

The pH value of the catalysis is 7.0 to 9.0, wherein the optimum pH value of the catalysis is specifically 8.2.

The above halogenated organic substances can be 1,3-dibromopropane, 1,3-dichloropropane, 1,2-dibromoethane, 1-bromo-2-methylpropane, 1-chloropentane, 1-bromo Hexane or bromocyclohexane, the most suitable halogenated organic substance in the embodiment of the present invention is 1,3-dibromopropane.

Experiments show that the dehalogenase HldD1 obtained by prokaryotic expression of Escherichia coli using the gene sequence (sequence 1) of the present invention has the characteristics of high activity and strong stability.

Description of drawings

Figure 1 is the electrophoresis of HldD1 protein expression; M, protein molecular weight marker (Fermentas, SM0431); 1. hldD1 gene recombinant expression strain pET-D1 [BL21(λDE3)] induced expression at 16 ℃ whole bacterial solution; 2. hldD1 gene recombinant The supernatant of the expression strain pET-D1[BL21(λDE3)] induced expression at 16°C; detected by 12.5% SDS-PAGE.

Figure 2 shows the relative enzymatic activity of HldD1 protein on different substrates.

Figure 3 is the determination of the optimum temperature of HldD1 protein.

Figure 4 is the determination of the thermal stability of HldD1 protein.

Figure 5 is the determination of the organic solvent stability of HldD1 protein.

Figure 6 is the pH stability assay of HldD1 protein.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1. Expression and activity assay of HldD1 protein

1. Acquisition of the gene encoding HldD1 protein

Using the genomic DNA of the new strain D1 isolated from the Indian Ocean deep-sea sediment samples as a template, PCR amplification was performed with D1F and D1R primers:

D1F: 5’-AAGAAGGAGAATATACATATGGCCATCGACGCCCCTGCGCACC-3’

D1R: 5'-TCGAGTGCGGCCGCAAGCTTGCCGAGCCCGAACTTCTCCAG-3'

The PCR amplification product was recovered and cloned into the pET21a(+) vector (Novagen) to obtain the recombinant vector pET-D1. The recombinant vector was transformed into Escherichia coli BL21 (DE3) and then sent for sequencing.

The sequencing results show that: the vector contains the nucleotide shown in sequence 1, and the gene in sequence 1 is named HldD1, the coding region of the gene is the 1-924th position of sequence 1, and the protein encoded by this gene is named HldD1, The amino acid sequence of HldD1 is the 1-308th position of SEQ ID NO:2.

In the DNA molecule shown in sequence 1, positions 1-924 are the coding region of the HldD1 gene, and positions 925-966 are the stop codon of the plasmid vector pET21a(+) (Novagen) and the coding sequence containing 6 his-tag sequences before it. Sequence and AAGCTTGCGGCCGCACTCGAG sequence (including three restriction endonuclease recognition sites (HindⅢ(AAGCTT), NotⅠ(GCGGCCGC), XhoⅠ(CTCGAG))) total 42 nucleotides.

The protein shown in sequence 2 is a recombinant protein HldD1, wherein the 1-308th position is the amino acid sequence of HldD1 protein, and the 309th-321st position is the amino acid residues containing 6 His tags expressed at the 925th-966th position of sequence 1; 316-321 are 6 His tags.

Sequence 1 can be synthesized artificially.

Second, the acquisition of dehalogenase HldD1

1. Construction of recombinant vector

Using the pET21a(+) vector as a template, PCR amplification was performed with pET21aF and pET21aR primers:

pET21aF: 5'-AAGCTTGCGGCCGCACTC-3'

pET21aR: 5'-CATATGTATATCTCCTTC-3'

The 5385bp PCR amplification product was recovered and ligated with the artificially synthesized sequence 1 by the method of homologous recombination. The recombinant vector pET-D1 was obtained and sent for sequencing.

Sequencing results showed that pET-D1 replaced the DNA molecule containing HldD1 gene shown in sequence 1 with the DNA between NdeⅠ(CATATG) and HidⅢ(AAGCTT) containing multiple cloning sites in pET-21a(+) vector, keeping pET -21a(+) The recombinant vector obtained by keeping the other sequences of the vector unchanged, the vector contains the DNA molecule shown in sequence 1, and the recombinant protein HldD1 shown in sequence 2 is expressed by using the T7 promoter of the vector to promote the DNA molecule shown in sequence 1. .

2. Acquisition of recombinant bacteria

The recombinant vector pET-D1 obtained in step 1 was transformed into Escherichia coli BL21 (DE3) by the chemical transformation method of calcium chloride to obtain recombinant bacteria, and the recombinant bacteria were screened with LB medium containing ampicillin (100 μg/ml) Culture, pick a single colony, extract the plasmid and verify it (amplify with D1F and D1R primers, get 961bp as positive), and name the final positive recombinant bacteria as pET-D1[BL21(DE3)].

3. Obtainment of dehalogenase HldD1

A single colony of pET-D1[BL21(DE3)] was picked and transferred to LB medium containing ampicillin (100 μg/ml), and cultured at 37°C overnight. The overnight culture was inoculated into 100 mL of LB medium containing ampicillin (100 μg/ml), and incubated at 37°C with shaking (200 rpm) until the OD ₆₀₀ value of the fermentation broth reached about 0.6-0.8, and then IPTG (finally) was added to the fermentation system. 0.5 mM) and incubated at 16°C for another 18 hours. After fermentation, centrifuge at 5000 rpm for 15 minutes, discard the supernatant, and collect the bacteria; resuspend the bacteria with non-denaturing nickel column binding buffer I, and ultrasonically disrupt (ultrasonic 5s, intermittent 5s, 40w power, 50 cycles) after 12,000 Centrifuge at rpm for 15 min. The collected supernatant is the crude enzyme solution containing recombinant protein HldD1, and the band size of the target recombinant protein HldD1 is 40kD (this protein is the recombinant protein HldD1 shown in sequence 2) (Fig. 1).

4. Purification of dehalogenase HldD1

The composition of native nickel column binding buffer I is as follows: 20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, the solvent is water, pH 7.5.

The composition of native nickel column elution buffer II is as follows: 20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, the solvent is water, pH 7.5.

The desalting buffer composition was as follows: 50 mM Gly-NaOH, pH 8.2.

The supernatant was purified by nickel column affinity chromatography, and the supernatant was purified by HiTrapchelating HP column (nickel column) in 1 ml capacity in the AKTA FPLC system of Amersham Company. Equilibrate and purify the cartridge with non-denaturing nickel column binding buffer I and load the sample. The protein loading volume is 10 ml, and the flow rate is set to 1 ml/min; wash with non-denaturing nickel column binding buffer I to remove non-specifically bound impurities. Protein; use native nickel column elution buffer to elute with a linear gradient of 0%-100%, and collect the eluate containing the elution peak. The obtained eluate was purified by desalting column Hitrap Q (5 mL), and the concentration of purified recombinant protein HldD1 was 2.477 mg/mL.

3. Determination method of dehalogenase activity

1. Determination method of enzyme activity

Haloalkane dehalogenases catalyze the dehalogenation of haloalkanes to form halohydrin, halide ions and protons. The principle of the following colorimetric method is to use the phenol red indicator to measure the content of hydrogen ions in the reaction system, and the principle of the ion electrode method is to detect the content of halogen ions in the reaction system by measuring the conductivity.

R-CH ₂ Cl+H ₂ O→R-CH ₂ OH+HCl

The unit of dehalogenase activity is defined as the amount of enzyme required to catalyze 1 μmol of substrate per minute for dehalogenation reaction under optimal conditions.

(1) Colorimetry:

Experimental group: The purified recombinant protein HldD1 obtained in the above two was diluted to 200 μg/mL with desalting buffer (50 mM Gly-NaOH, pH 8.2). Take 20 μL of the diluted enzyme, add it to 180 μL of colorimetric assay buffer, mix well and immediately react at the optimum temperature for 10 minutes, then immediately place the reaction system on ice to stop the reaction, and use a microplate reader to measure 540 nm absorbance value. The difference between the experimental group and the control group in which the substrate is self-hydrolyzed is compared to the standard curve (y=-0.4211x+0.846, x is the hydrogen ion concentration, y is the absorbance value at 540nm, the standard substance is concentrated hydrochloric acid HCl, diluted to different concentrations ) to get the measurement results. The experiment was repeated 3 times.

Colorimetric assay buffer: 1 mM HEPES, 1 mM EDTA, 20 mM Na2SO4, 10 mM reaction substrate, 20 μg/mL phenol red, pH 8.2.

The control group in which the above-mentioned substrates are autohydrolyzed: the difference from the experimental group is that no enzyme is added.

(2) Ion electrode method:

The purified recombinant protein HldD1 obtained in the above two steps was diluted to 200 μg/mL with desalting buffer. Take 200 μL of the diluted enzyme, add it to 1.8 mL of ion electrode assay buffer, mix well, react at the optimum temperature for 10 min, stop the reaction on ice, and use an ion analyzer to determine the control of the experimental group and the substrate autohydrolysis The difference in halide ion concentration before and after the reaction in the group was calculated to obtain the measurement result. The experiment was repeated 3 times.

Ion electrode assay buffer: 100 mM HEPES, 10 mM reaction substrate, pH 8.2.

The control group in which the above-mentioned substrates are autohydrolyzed: the difference from the experimental group is that no enzyme is added.

2. Substrate specificity of dehalogenase HldD1

Select 1,3-dibromopropane, 1,3-dichloropropane, 1,2-dibromoethane, 1-bromo-2-methylpropane, 1-chloropentane, 1-bromohexane, bromohexane Cyclohexane was used as the reaction substrate, and the colorimetric method was used to determine the reaction temperature at 40 °C; the enzyme activity when catalyzing the optimum substrate was defined as 100%, and the relative activity of catalyzing other substrates was calculated. Three replicates were designed for each experimental group.

The result is shown in Figure 2,

The optimal substrate for HldD1 was 1,3-dibromopropane, with a specific activity of 1612.44 U/mg, which was defined as 100% relative activity. HldD1 showed high specificity for carbon chain length. Except for the best substrate 1,3-dibromopropane, 1,2-dibromoethane with short carbon chain and similar length showed relatively high catalytic activity (38.9%); The long halogenated alkane 1-bromohexane, and bromocyclohexane with cyclic structure, have very low relative activities, only 10.5% and 21.6% of the optimal substrate, 1,3-dibromopropane, respectively. In addition, HdlD1 showed very high specificity for the type of substrate halogen. It has no obvious catalytic activity to 1,3-dichloropropane (0.6%) and 1-chloropentane (2,9%). At the same time, 1-bromo-2-methylpropane containing branched chains has obvious differences from 1,3-dibromopropane due to its steric structure, and its relative activity (10.5%) is significantly lower than that of 1,3-dibromopropane despite the same main chain length. optimum substrate.

3. Determination method of optimum temperature for dehalogenase HldD1

Experiments were carried out using a colorimetric method at reaction temperatures of 4°C, 15°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C; the substrate was 1,3-dibromopropane . The enzyme activity at the optimum temperature obtained by the determination was defined as 100%, and the relative activity at other reaction temperatures was calculated. Three replicates were designed for each experimental group.

The results are shown in Figure 3. The optimum temperature of the purified recombinant protein HldD1 was 40°C, and the specific activity was 1612.44 U/mg, which was defined as 100% relative activity. Under the condition of lower temperature, HldD1 hardly exhibits catalytic activity, and the relative activity is less than 5%. In the range of 15 °C to 40 °C, with the increase of the temperature in the set reaction conditions, the relative activity has been significantly improved. At temperatures above 40 °C, the relative viability decreased rapidly with further increase in temperature in the reaction conditions. When the reaction condition was 50℃, the specific activity of HldD1 was only 31.2% of that at 40℃. When the reaction temperature is higher than 50 °C, the relative activity gradually decreases until it has no catalytic activity.

4. Determination of thermal stability of dehalogenase

The selected treatment temperatures were 40°C, 50°C, 60°C and 70°C; different groups were set, and the purified recombinant protein HldD1 was heat-treated at different temperatures for 30min, 60min and 90min respectively. The activity of the treated enzyme was determined by colorimetric method, and the substrate was 1,3-dibromopropane. The enzyme activity of the control group without heat treatment was defined as 100%, and the specific activity was 1612.44 U/mg, and the relative activities of different experimental groups were calculated. Three replicates were designed for each experimental group.

The results are shown in Figure 4. The relative activity of the enzyme did not decrease significantly (96.0%) after heat treatment at 40 °C until 90 min; the relative activity did not decrease within 60 min of heat treatment at 50 °C, and the heat treatment time was extended to 90 min. After treatment, the specific activity of the enzyme was still more than 80% of that of the control group; under the condition of 60 °C, the relative activity gradually decreased to 36.5% with the increase of heat treatment time; and at the heat treatment temperature of 70 °C, the specific activity of the enzyme decreased to 36.5%. The activity decreased rapidly to less than 30% of the specific activity before treatment within 30 min, and HldD1 gradually lost its activity with the further prolongation of treatment time.

5. Determination method of dehalogenase organic solvent stability

Set up different groups, and place the purified recombinant protein HldD1 in methanol aqueous solution, ethanol aqueous solution, DMSO aqueous solution and 10%, 20%, 30%, 40%, 50%, 60% and 70% by volume respectively In acetonitrile aqueous solution, after 1 h treatment at room temperature, its activity was determined by colorimetric method, the substrate was 1,3-dibromopropane, and the reaction temperature was 40 ℃. The relative activity of the control group without organic solvent treatment was defined as 100%, and the specific activity was 1612.44 U/mg, and the relative activity of the experimental group was calculated. Three replicates were designed for each experimental group.

The results are shown in Figure 5. It can be seen that HldD1 has the worst tolerance to methanol, and its relative activity rapidly drops to 22.6% under low concentration (10%-20%) treatment conditions; Under the condition of concentration treatment, the relative activity of the enzyme was almost unaffected, but with the further increase of the organic solvent concentration, its specific activity decreased rapidly. When the concentration of ethanol was higher than 40% and the concentration of acetonitrile was higher than 50%, HldD1 basically lost its catalysis. Vitality (relative viability below 10%). Under the condition of DMSO treatment, when its concentration is lower than 40%, the activity of the enzyme is relatively high, which can still keep more than 75% of the specific activity of the control group. With the further increase of DMSO concentration, the relative activity gradually decreases to 26.8%.

6. Determination method of pH stability of dehalogenase

Prepare citric acid-sodium citrate buffers at pH 5.0 and 6.0, Tris _{- H2SO4} buffers at pH 7.0 and 9.0, and sodium carbonate-sodium bicarbonate buffers at pH 10.0 and 11.0; separate the dehalogenases It was placed in the above buffer, treated at room temperature for 5 h, and then the activity was determined according to the ion electrode method. The substrate was 1,3-dibromopropane, and the reaction temperature was 40 °C. The enzyme activity of the untreated group, that is, the group left at room temperature for 5 hours under pH 8.2, was defined as 100%, and the specific activity was 1612.44 U/mg, and the relative activities of the enzymes treated under other pH conditions were calculated. Three replicates were designed for each experimental group.

Citric acid-sodium citrate buffer: the final concentration is 100 mM sodium citrate, and the volume is adjusted to the pH required for the experiment.

Tris-H ₂ SO ₄ buffer: the final concentration is 100 mM Tris in distilled water and adjusted to the pH required for the experiment.

Sodium carbonate-sodium bicarbonate buffer: the final concentration is 100 mM sodium bicarbonate, and the volume is adjusted to the pH required for the experiment.

The results are shown in Fig. 6. After treatment at pH 7.0 to pH 9.0, the catalytic activity can still be maintained at a high level, and its relative activity is above 80%. The tolerance of HldD1 was significantly reduced, in which the pH decreased to 5.0, the relative activity was 13.2%, and the pH increased to 11.0, the relative activity was 36%; the optimum pH was 8.2, and the relative activity was 100%.

7. Determination method of enzymatic reaction kinetic parameters of dehalogenase

The optimal substrate 1,3-dibromopropane was prepared with a colorimetric assay buffer, so that the optimal substrate concentrations were 0.1 mM, 0.15 mM, 0.2 mM, 0.3 mM, 0.5 mM, 1 mM, 2 mM, 3 mM and 5mM; with different optimal substrate concentrations as experimental groups, colorimetric experiments were carried out, the reaction temperature was 40 °C, and each substrate concentration was measured three times in parallel; the reciprocal 1/[S] of the substrate concentration was used as the horizontal The coordinates are plotted with the reciprocal 1/v of the reaction rate as the ordinate, the slope of the obtained straight line is K _m /V _max , the intercept of the obtained straight line is 1/V _max , and then according to the concentration of dehalogenase and the molecular weight, the enzymatic activity is calculated. Reaction kinetic parameters V _max , K _m , k _cat and k _cat /K _m .

Results: Using 1,3-dibromopropane as the substrate, the maximum reaction rate of the enzyme was determined as V _max =0.54mM/min, Michaelis constant K _m =0.95mM, conversion number k _cat =17.90s ^-1 , k _cat /K _m was 18.95 mM ^-1 ·s ^-1 .

SEQUENCE LISTING

<110> Institute of Microbiology, Chinese Academy of Sciences China Ocean Mineral Resources Research and Development Association (China Ocean Affairs Administration)

<120> A dehalogenase HldD1 and its encoding gene and application

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 966

<212> DNA

<213> Artificial sequence

<400> 1

atggccatcg acgccctgcg cacccccgac gaacgatttc agaacctttc cggctgggat 60

tacgccccgc gctatattga cgacctgccg ggttatgagg ggctccgcat gcattatgtc 120

gacaaagggc cgaaggatgg cgtgacgttc ctgtgcattc atggtgagcc ctcatggtct 180

tatctcttcc gcaagatggc gccggtgttc ctggacgccg gtcaccgctt tgtggcggtc 240

gacatgttcg gcttcgggcg ctccgacaag ccggtggacg atgatgtcta cacctatcat 300

tttcaccgca atgccctcct cgcttttgtc gagcgcatgg acctgaacaa tgtctgtctc 360

gtcgtgcagg actggggcgg gctgctcggc ctcacgctgc cgctggaagc gccggcgcgg 420

tatacgcgcc ttatcgtcat gaataccggg cttcccgcgg gcgagaacgc gggcgaaggc 480

ttcgccgcct ggcgcgcgtt ctgtaaagcc aatcccgatc tcgatgttgg tgcgctgatg 540

aaacgcgcaa cgcccatcct taccgatgat gaagtcgccg cctatgccgc gcccttcccg 600

gatgtgaaat acaaagccgg cgtaagacgc tttccggaac tcgtcatgct caccggcggg 660

aaagatgagc cgttgacccc gtccgccagc gaaggcgttg aaacctcatt gaaagcgcac 720

aaattctggt cagaagactg gaccggcgac agcttcatgg ccatcggcat gcaggatcct 780

gttttgggtc cgcccgccat gcatatgctc cgcaaggtga ttaaaaactg ccccgagccg 840

atggaagtcg cggacggcgg ccacttcgtc caggaatggg gcgaaccgat tgcgaaagcg 900

gcgctggaga agttcgggct cggcaagctt gcggccgcac tcgagcacca ccaccaccac 960

cactga 966

<210> 2

<211> 321

<212> PRT

<213> Artificial sequence

<400> 2

Met Ala Ile Asp Ala Leu Arg Thr Pro Asp Glu Arg Phe Gln Asn Leu

1 5 10 15

Ser Gly Trp Asp Tyr Ala Pro Arg Tyr Ile Asp Asp Leu Pro Gly Tyr

20 25 30

Glu Gly Leu Arg Met His Tyr Val Asp Lys Gly Pro Lys Asp Gly Val

35 40 45

Thr Phe Leu Cys Ile His Gly Glu Pro Ser Trp Ser Tyr Leu Phe Arg

50 55 60

Lys Met Ala Pro Val Phe Leu Asp Ala Gly His Arg Phe Val Ala Val

65 70 75 80

Asp Met Phe Gly Phe Gly Arg Ser Asp Lys Pro Val Asp Asp Asp Val

85 90 95

Tyr Thr Tyr His Phe His Arg Asn Ala Leu Leu Ala Phe Val Glu Arg

100 105 110

Met Asp Leu Asn Asn Val Cys Leu Val Val Gln Asp Trp Gly Gly Leu

115 120 125

Leu Gly Leu Thr Leu Pro Leu Glu Ala Pro Ala Arg Tyr Thr Arg Leu

130 135 140

Ile Val Met Asn Thr Gly Leu Pro Ala Gly Glu Asn Ala Gly Glu Gly

145 150 155 160

Phe Ala Ala Trp Arg Ala Phe Cys Lys Ala Asn Pro Asp Leu Asp Val

165 170 175

Gly Ala Leu Met Lys Arg Ala Thr Pro Ile Leu Thr Asp Asp Glu Val

180 185 190

Ala Ala Tyr Ala Ala Pro Phe Pro Asp Val Lys Tyr Lys Ala Gly Val

195 200 205

Arg Arg Phe Pro Glu Leu Val Met Leu Thr Gly Gly Lys Asp Glu Pro

210 215 220

Leu Thr Pro Ser Ala Ser Glu Gly Val Glu Thr Ser Leu Lys Ala His

225 230 235 240

Lys Phe Trp Ser Glu Asp Trp Thr Gly Asp Ser Phe Met Ala Ile Gly

245 250 255

Met Gln Asp Pro Val Leu Gly Pro Pro Ala Met His Met Leu Arg Lys

260 265 270

Val Ile Lys Asn Cys Pro Glu Pro Met Glu Val Ala Asp Gly Gly His

275 280 285

Phe Val Gln Glu Trp Gly Glu Pro Ile Ala Lys Ala Ala Leu Glu Lys

290 295 300

Phe Gly Leu Gly Lys Leu Ala Ala Ala Leu Glu His His His His His

305 310 315 320

His

Claims (10)

1. A protein, which is any of the following (a1)-(a3): (a1) the protein shown in sequence 2 in the sequence listing; (a2) the protein shown in positions 1-308 of SEQ ID NO: 2 in the sequence listing; (a3) A fusion protein obtained by linking a tag to the N-terminus or/and C-terminus of the protein of (a2). 2. A nucleic acid molecule encoding the protein of claim 1. 3. nucleic acid molecule as claimed in claim 2, is characterized in that: described nucleic acid molecule is the DNA molecule shown in any one of following (b1)-(b2): (b1) the DNA molecule shown in sequence 1 in the sequence listing; (b2) The DNA molecule represented by positions 1-924 of SEQ ID NO: 1 in the sequence listing. 4. An expression cassette, a recombinant vector or a recombinant microorganism containing the nucleic acid molecule of claim 2 or 3. 5. the application of the described protein of claim 1 as dehalogenase; Or the application of the nucleic acid molecule of claim 2 or 3, or the expression cassette, recombinant vector or recombinant microorganism of claim 4, in the preparation of dehalogenase. 6. A method for preparing a dehalogenase, comprising the steps of: inducing and culturing the recombinant microorganism described in claim 5 to obtain a dehalogenase. 7. the application of the described protein of claim 1 or the described nucleic acid molecule of claim 2 or 3 or the described expression cassette of claim 4, recombinant vector or recombinant microorganism in removing the halogen group on halogenated organic matter; Or, the application of the protein described in claim 1 or the nucleic acid molecule described in claim 2 or 3 or the expression cassette described in claim 4, a recombinant vector or a recombinant microorganism in the treatment of halogenated organic pollution; The halogenated organic substance is 1,3-dibromopropane, 1,2-dibromoethane, 1-bromo-2-methylpropane, 1-bromohexane or bromocyclohexane. 8. a method for removing the halogen group on the halogenated organic matter, comprising the steps: use the protein catalysis halogenated organic matter of claim 1 to realize the removal of the halogenated organic matter on the halogenated organic matter; The halogenated organic substance is 1,3-dibromopropane, 1,2-dibromoethane, 1-bromo-2-methylpropane, 1-bromohexane or bromocyclohexane. 9. The method according to claim 8, wherein: The temperature of the catalysis is 15-40°C; The catalytic pH is 7.0 to 9.0. 10. The method according to claim 9, wherein: The temperature of the catalysis is 40°C; The catalytic pH was 8.2.

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