CN104694514A - Dehalogenase DhmB, encoding gene and application thereof - Google Patents
Dehalogenase DhmB, encoding gene and application thereof Download PDFInfo
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Abstract
The invention discloses a dehalogenase DhmB, an encoding genes and application thereof. The dehalogenase DhmB is a protein in the following (a) or (b); (a) an amino acid sequence is a protein of a sequence 2 in a sequence table; (b) a protein associated with dehalogenation is obtained by the amino acid sequence, which is shown in the sequence 2 of the sequence table and subjected to replacement and/or missing and/or adding of one or a plurality of amino acid residues. The dehalogenase DhmB is easy in purification, good in stability, resistant to high-medium temperature, applicable to the optimum temperature of 65 DEG C, resistant to organic solvents and capable of degrading organohalogen compounds with low-carbon chains, classic pesticides and chemical toxicant and the like and has wide industrial application prospects.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of dehalogenase DhmB and encoding gene thereof and application.
Background technology
There is the halide-containings such as a large amount of halohydrocarbon, halogenated aromatic acid, halogenated-chain acid and halohydrin in nature.These halogenated organic matters can be used as organic solvent, sterilant, medical pesticide intermediate etc.Halogenated organic matters generally has environmental toxicity, and generally more stable, not easily by fast degradation, and is easily accumulated in man and animal fatty tissue by food chain, causes great threat to the health of man and animal.
The mechanism of action of dehalogenase (Dehalogenase, EC 3.8.1): can carbon-halogen bond cracking in catalysis organohalogen compound, makes halogen atom discharge from compound.Dehalogenase by removing the chlorine in organochlorine pesticide, thus can eliminate its toxicity, and this has significant application value to the residue problem of administering China's once a large amount of used organochlorine pesticide (dichlorophenoxyacetic acid, Trichlorophenol, phenyl-hexachloride etc.).Dehalogenase can be applicable to grain, veterinary antibiotics, the removing of chloride pesticide residue and detection in tealeaves and Chinese patent medicine; Dehalogenase also can be applicable to the detoxification treatment of chloride toxic agent (yperite), and this leaves over the harmless process of chemical weapons for some, and protection of the environment safety is significant.
The current dehalogenase found owing to having very large defect, therefore Development of New Generation height vigor in catalytic activity, enzyme stability, productive rate etc., and the dehalogenase of high stability and high expression has significant application value and social effect.
Summary of the invention
An object of the present invention is to provide a kind of protein.
Protein provided by the invention is following protein a) or b):
A) aminoacid sequence is the protein of sequence 2 in sequence table;
B) by the aminoacid sequence shown in sequence in sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation obtain and the protein relevant to degraded halogen compounds.
The replacement of one or several amino-acid residue above-mentioned and/or disappearance and/or be added to the replacement and/or disappearance and/or interpolation that are no more than 10 amino-acid residues.
Another object of the present invention is to provide the biomaterial relevant to above-mentioned protein.
The biomaterial that provided by the invention and above-mentioned protein is relevant is following B1)-B5) in any one:
B1) to encode the nucleic acid molecule of above-mentioned protein;
B2) containing B1) expression cassette of described nucleic acid molecule;
B3) containing B1) recombinant vectors of described nucleic acid molecule or containing B2) recombinant vectors of described expression cassette;
B4) containing B1) recombinant bacterium of described nucleic acid molecule or containing B2) recombinant bacterium of described expression cassette or containing B3) recombinant bacterium of described recombinant vectors;
B5) containing B1) clone of described nucleic acid molecule or containing B2) clone of described expression cassette or containing B3) clone of described recombinant vectors.
In above-mentioned biomaterial, described clone does not comprise the reproductive material of plant and animal.
In above-mentioned biomaterial, B1) described nucleic acid molecule is following 1) or 2) or 3) DNA molecular:
1) its nucleotide sequence is the DNA molecular shown in sequence 1 in sequence table;
2) with 1) DNA sequence dna that limits at least has 70%, at least have 75%, at least have 80%, at least have 85%, at least have 90%, at least have 95%, at least have 96%, at least have 97%, at least have 98% or at least have the DNA molecular of 99% homology and code for said proteins;
3) under strict conditions with 1) or 2) DNA sequence dna that limits hybridizes and the DNA molecular of code for said proteins;
Described stringent condition can be as follows: at 50 DEG C, 7% sodium lauryl sulphate (SDS), 0.5MNa
3pO
4hybridize with in the mixing solutions of 1mM EDTA, at 50 DEG C, rinsing in 2 × SSC, 0.1%SDS; Also can be at 50 DEG C, at 7%SDS, 0.5M Na
3pO
4hybridize with in the mixing solutions of 1mM EDTA, at 50 DEG C, rinsing in 1 × SSC, 0.1%SDS; Also can be at 50 DEG C, 7%SDS, 0.5M Na
3pO
4hybridize with in the mixing solutions of 1mM EDTA, at 50 DEG C, rinsing in 0.5 × SSC, 0.1%SDS; Also can be at 50 DEG C, 7%SDS, 0.5M Na
3pO
4hybridize with in the mixing solutions of 1mM EDTA, at 50 DEG C, rinsing in 0.1 × SSC, 0.1%SDS; Also can be at 50 DEG C, 7%SDS, 0.5M Na
3pO
4hybridize with in the mixing solutions of 1mM EDTA, at 65 DEG C, rinsing in 0.1 × SSC, 0.1%SDS; Also can be at 65 DEG C, in the solution of 6 × SSC, 0.5%SDS, hybridization, respectively washes film once at 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS.
In above-mentioned biomaterial, containing B3) recombinant bacterium of described recombinant vectors is by containing B1) recombinant vectors of described nucleic acid molecule imports in Host Strains and obtains;
Described containing B1) recombinant vectors of described nucleic acid molecule is that the nucleic acid molecule of the above-mentioned protein of coding is inserted expression vector, obtains the carrier of expressing above-mentioned protein.
Above-mentioned protein is also belonging to protection scope of the present invention as the application in dehalogenase.
Above-mentioned relevant biological material also belongs to protection scope of the present invention preparing the application in dehalogenase.
Above-mentioned protein or the application of above-mentioned relevant biological material in degraded halogen compounds also belong to protection scope of the present invention.
In above-mentioned application, described halogen is chlorine and/or bromine.
A further object of the invention is to provide a kind of recombinant bacterium.
Recombinant bacterium provided by the invention is imported in Host Strains by the encoding gene of dehalogenase to obtain; The aminoacid sequence of described dehalogenase is as shown in sequence in sequence table 2.
In above-mentioned recombinant bacterium, the encoding gene of described dehalogenase imports Host Strains by recombinant vectors; Described recombinant vectors is that the nucleic acid molecule of the above-mentioned protein of coding is inserted expression vector, obtains the carrier of expressing above-mentioned protein; The coding gene sequence of described dehalogenase is as shown in sequence in sequence table 1.
In above-mentioned recombinant bacterium, described Host Strains is specially e. coli bl21 (DE3).
In above-mentioned recombinant bacterium, described expression vector is specially pET-28a (+).
Last object of the present invention is to provide a kind of expression method of enzyme.
The expression method of enzyme provided by the invention comprises cultivates above-mentioned recombinant bacterium, obtains above-mentioned dehalogenase.
Prove by experiment: the easy purifying of dehalogenase DhmB of the present invention, good stability, high temperature in tolerance, optimum temperuture 65 DEG C, tolerance organic solvent, the organohalogen compound of low carbon chain of can not only degrading, classical agricultural chemicals (as DTT and phenyl-hexachloride etc.) of can also degrading, the toxic chemical agent such as yperite of also degrading.There is wide prospects for commercial application.
Accompanying drawing explanation
Fig. 1 is that the SDS-PAGE of dehalogenase DhmB analyzes purifying figure.Wherein, swimming lane 1 is standard molecular weight; Swimming lane 2 is DhmB protein expression; Swimming lane 3 penetrates peak for wash-out; Swimming lane 4 is DhmB purification result.
Fig. 2 is that dehalogenase DhmB studies different halogenated substrates catalytic hydrolysis.
Fig. 3 is the catalyticing research of different dehalogenase to different substrate.
Fig. 4 is CD (circular dichroism spectrum) the result figure of dehalogenase.
Fig. 5 is the thermostability of dehalogenase DhmB.
Fig. 6 is the optimum temperuture of dehalogenase DhmB.
Fig. 7 is the tolerance to organic solvent of dehalogenase DhmB.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The bacterial strain number that manganese in following embodiment is oxidized yellow Salmonella (Huangia manganoxydans) is 8047, and preserving number is CGMCC No.6697, is be disclosed in CN 103805528 A at publication number.
PET-28a (+) is the product of Novagen company, and catalog number is 69865-3.
The formula of non denatured nickel post binding buffer liquid I: 0.1M Tris – H
2sO
4, 0.1M NaCl, 0.01MImidazole, 0.5mM EDTA, 1mM BAM, 1mM PMSF; PH is 8.2.
The formula of non denatured nickel post elution buffer II: 0.1M Tris-H2SO4,0.1M NaCl, 0.5MImidazole, 0.5mM EDTA, 1mM BAM, 1mM PMSF, pH are 8.2.
The acquisition of embodiment 1, DhmB albumen and encoding gene thereof
Be oxidized the genomic dna of yellow Salmonella (Huangia manganoxydans) 8047CGMCC No.6697 for template with manganese, carry out pcr amplification with primer DhmBF and DhmBR, obtain pcr amplification product.Primer sequence following (sequence of underscore is restriction enzyme site):
DhmBF：5′-G
GAATTCCATATGATGCCCGCTGTCTACGTCTTC-3′；
DhmBR：5′-CCC
AAGCTTTCAGTCCGCGCGCAGGCTGATC-3′。
Reclaim pcr amplification product, be cloned into pGEM-T carrier, obtain recombinant vectors, deliver order-checking by after recombinant vectors transformation of E. coli DH5 α.
Sequencing result shows: the unnamed gene shown in this sequence, as shown in sequence in sequence table 1, is DhmB by the nucleotide sequence of this pcr amplification product, and the protein designations of this genes encoding is that the aminoacid sequence of DhmB, DhmB is as shown in sequence in sequence table 2.
The acquisition of embodiment 2, dehalogenase DhmB and functional verification thereof
One, dehalogenase obtains
1, the acquisition of recombinant vectors
With restriction enzyme, to the pcr amplification product obtained in above-described embodiment 1 and pET-28a (+) carrier, double digestion is carried out to Nde I and Hind III, connect, obtain recombinant vectors pET-DhmB, and deliver order-checking.
Sequencing result shows: pET-DhmB, for the DNA between the Nde I of pET-28a (+) carrier and Hind III site being replaced with the DhmB gene in sequence table shown in sequence 1, keeps the constant recombinant vectors obtained of other sequences of pET-28a (+) carrier.The aminoacid sequence of the albumen of DhmB genes encoding is as shown in sequence in sequence table 2.
2, the acquisition of recombinant bacterium
With calcium chloride chemical transformation, the recombinant vectors pET-DhmB obtained in step 2 is converted in e. coli bl21 (DE3), obtain recombinant bacterium, with the LB substratum containing kalamycin (50 μ g/ml), screening and culturing is carried out to recombinant bacterium, picking list bacterium colony, extracts plasmid and carries out bacterium colony PCR checking.To be the recombinant bacterium of DhmB gene of 660bp containing size, called after BL21-pET-DhmB.
3, the acquisition of dehalogenase DhmB
Single bacterium colony access of picking BL21-pET-DhmB contains in the LB substratum of kalamycin (50 μ g/ml), in 37 DEG C of incubated overnight.Overnight culture is inoculated in the LB substratum that 1L contains kalamycin (50 μ g/ml), 37 DEG C of thermal agitations (200rpm) are cultivated, to the OD of fermented liquid
600value reaches about 0.6-0.8, then in fermentation system, add IPTG (final concentration 0.5mM), cultivates 6-8 hour again under 30 DEG C of conditions.After fermentation, centrifugal 15 minutes of 5000rpm, abandons supernatant, collects thalline; With the resuspended thalline of non denatured nickel post binding buffer liquid I, ultrasonic disruption (ultrasonic 5s, the power of interval 5s, 40w, 30 circulations) 12,000rpm centrifugal 15min afterwards.Collection supernatant liquor is the crude enzyme liquid containing target protein DhmB.
4, the purifying of dehalogenase DhmB
Affinity chromatography is adopted to carry out purifying, with HiTrap chelating HP column (nickel post) the purifying supernatant of 1ml loading amount in the AKTA FPLC system of Amersham company.Non denatured nickel post binding buffer liquid I is for balancing purifying cylinder and loading.Albumen applied sample amount is 10ml, and flow velocity is set to 1ml/min.With 20% non denatured nickel post elution buffer (the i.e. mixing solutions of non denatured nickel post binding buffer liquid I and non denatured nickel post elution buffer II, the volume ratio of non denatured nickel post binding buffer liquid I and non denatured nickel post elution buffer II is 80:20) wash, remove the foreign protein of non-specific binding.With 80% non denatured nickel post elution buffer (the i.e. mixing solutions of non denatured nickel post binding buffer liquid I and non denatured nickel post elution buffer II, the volume ratio of non denatured nickel post binding buffer liquid I and non denatured nickel post elution buffer II is 20:80) carry out wash-out, collect the elutriant containing elution peak, obtain the dehalogenase DhmB after purifying, detect the purity of dehalogenase DhmB in elutriant with SDS-PAGE.Result is as shown in Figure 1: dehalogenase DhmB size is about 25kDa.
Two, the detection of dehalogenase DhmB character
1, the measuring method that enzyme is alive
The enzyme activity determination of dehalogenase DhmB adopts the colorimetric analysis improved.Concrete grammar is as follows: the dehalogenase DhmB (0.2mg/ml) getting the purifying of the 50ul that step one obtains joins in the survey damping fluid alive of 450ul, and 25 DEG C of reactions 30min, on ice termination reactions, 540nm measures absorbance.
Enzyme is lived definition: the amount that the enzyme per minute hydrolysis substrate of every milligram produces 1mM Hcl is defined as a Ge Meihuo unit.
Survey the preparation method of damping fluid of living: 1mM HEPES, 1mM EDTA, 20mM sulfate buffer, 10mM substrate, 20ug/ml are phenol red; PH 8.2.(do not have particular requirement, substrate all uses s-2 chloropropionic acid)
Make Hcl typical curve, by the measurement result of dehalogenase DhmB liquid and the comparison of Hcl typical curve, obtain the enzyme size alive of dehalogenase DhmB liquid.3 repetitions are established in experiment.
2, the suitableeest substrate
By dichloro acetic acid (2-chloroacetic acid), dibromoacetic acid (2-bromoacetic acid), bromoacetic acid (Bromoacetic Acid), s-2-chloropropionic acid (S-2-chloropropionic acid), R-2-Mono Chloro Acetic Acid (R-2-chloropropionic acid), 3-Mono Chloro Acetic Acid (3-chloroacetic acid), 3-bromo-propionic acid (3-chlooacetic acid), 4-chloro-benzoic acid (4-chlorobenzcic acid), DTT, phenyl-hexachloride and yperite simulant 2-chloroethylethyl thioether (2CEES) are as substrate, the Substratspezifitaet of the dehalogenase DhmB adopting the enzyme activity determination method determination step one of step 1 to obtain.3 repetitions are established in experiment.
Result is as shown in Figures 2 and 3: dehalogenase DhmB demonstrates high reactivity in the halogen compounds of low carbon chain (C≤3): dichloro acetic acid (100%), dibromoacetic acid (96%), bromoacetic acid (90%), s-2-chloropropionic acid (99%); Long chain active does not have short chain strong: 4-chloro-benzoic acid (22%); Tendentious preference (Fig. 2) is not had to the substrate of chlorine or bromine; There is a certain amount of Degradation to traditional agricultural chemicals such as DTT and phenyl-hexachloride, have detoxification (Fig. 3) to toxic chemical agent such as yperite simulant 2-chloroethylethyl thioether (2-CEES).
3, thermostability
Denaturation temperature (Tm value) definition of zymoprotein: be in the process of continuous warming, temperature corresponding during 50% albumen generation sex change.
The enzyme of the dehalogenase DhmB enzyme liquid of differing temps different time is lived and measures.Method for measuring: the Tm value measuring the dehalogenase DhmB after differing temps and different time process with circular dichroism spectrometer (CD (circular dichroism)).Put 10min on ice after process, then start to survey enzyme and live.3 repetitions are established in experiment.
Result is as shown in Figure 4 and Figure 5: the Tm value of dehalogenase DhmB 68.8 DEG C, and dehalogenase DhmB enzyme liquid processes below 50 DEG C, and enzyme is lived stable.
4, optimum temperuture
The dehalogenase DhmB obtained in comparison step one enzyme at different temperatures size alive, obtains the optimum temperuture of dehalogenase DhmB.The mensuration of optimum temperuture carries out enzymatic reaction at different temperatures.
Result is as shown in Figure 6: the enzyme of dehalogenase DhmB under 60-70 DEG C of condition is lived very high, and optimum temperuture is 65 DEG C, belongs to middle high temperature enzyme.
5, solvent stability
The solvent stability of the dehalogenase DhmB obtained in determination step one: by dehalogenase DhmB respectively at different solvents (methyl alcohol, ethanol, dimethyl sulfoxide (DMSO) DMSO and second eyeball), room temperature treatment 1 hour under different mass mark (10%-70%).Then the enzyme adopting the method for step 1 to measure dehalogenase is lived.
Result is as shown in Figure 7: dehalogenase DhmB is in organic solvent, more stable.
Claims (10)
1. protein is following protein a) or b):
A) aminoacid sequence is the protein of sequence 2 in sequence table;
B) by the aminoacid sequence shown in sequence in sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation obtain and the protein relevant to degraded halogen compounds.
2. the biomaterial relevant to protein described in claim 1 is following B1)-B5) in any one:
B1) nucleic acid molecule of protein described in coding claim 1;
B2) containing B1) expression cassette of described nucleic acid molecule;
B3) containing B1) recombinant vectors of described nucleic acid molecule or containing B2) recombinant vectors of described expression cassette;
B4) containing B1) recombinant bacterium of described nucleic acid molecule or containing B2) recombinant bacterium of described expression cassette or containing B3) recombinant bacterium of described recombinant vectors;
B5) containing B1) clone of described nucleic acid molecule or containing B2) clone of described expression cassette or containing B3) clone of described recombinant vectors.
3. relevant biological material according to claim 2, is characterized in that: B1) described nucleic acid molecule for following 1) or 2) or 3) DNA molecular:
1) its nucleotide sequence is the DNA molecular shown in sequence 1 in sequence table;
2) with 1) DNA sequence dna that limits at least has 70%, at least have 75%, at least have 80%, at least have 85%, at least have 90%, at least have 95%, at least have 96%, at least have 97%, at least have 98% or at least have the DNA molecular of 99% homology and code for said proteins;
3) under strict conditions with 1) or 2) DNA sequence dna that limits hybridizes and the DNA molecular of code for said proteins.
4. the relevant biological material according to Claims 2 or 3, is characterized in that: containing B3) recombinant bacterium of described recombinant vectors is by containing B1) recombinant vectors of described nucleic acid molecule imports in Host Strains and obtains;
Described containing B1) recombinant vectors of described nucleic acid molecule is that the nucleic acid molecule of protein described in coding claim 1 is inserted expression vector, obtains the carrier of expressing protein described in claim 1.
5. protein described in claim 1 is as the application in dehalogenase.
6. in claim 2-4, arbitrary described relevant biological material is preparing the application in dehalogenase.
7. the application of arbitrary described relevant biological material in degraded halogen compounds in protein described in claim 1 or claim 2-4;
Described halogen is specially chlorine and/or bromine.
8. a recombinant bacterium is imported in Host Strains by the encoding gene of dehalogenase to obtain; The aminoacid sequence of described dehalogenase is as shown in sequence in sequence table 2.
9. recombinant bacterium according to claim 8, is characterized in that: the encoding gene of described dehalogenase imports Host Strains by recombinant vectors;
Described recombinant vectors is that the nucleic acid molecule of protein described in coding claim 1 is inserted expression vector, obtains the carrier of expressing protein described in claim 1;
The coding gene sequence of described dehalogenase is as shown in sequence in sequence table 1.
10. an expression method for enzyme, comprises the recombinant bacterium cultivated described in claim 8 or 9, obtains dehalogenase.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112680427A (en) * | 2019-10-18 | 2021-04-20 | 中国科学院微生物研究所 | Dehalogenase HldD1 and coding gene and application thereof |
| CN112680428A (en) * | 2019-10-18 | 2021-04-20 | 中国科学院微生物研究所 | Dehalogenase Had2CG4B and coding gene and application thereof |
| CN112680429A (en) * | 2019-10-18 | 2021-04-20 | 中国科学院微生物研究所 | Dehalogenase HadD14, and coding gene and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112680427A (en) * | 2019-10-18 | 2021-04-20 | 中国科学院微生物研究所 | Dehalogenase HldD1 and coding gene and application thereof |
| CN112680428A (en) * | 2019-10-18 | 2021-04-20 | 中国科学院微生物研究所 | Dehalogenase Had2CG4B and coding gene and application thereof |
| CN112680429A (en) * | 2019-10-18 | 2021-04-20 | 中国科学院微生物研究所 | Dehalogenase HadD14, and coding gene and application thereof |
| CN112680427B (en) * | 2019-10-18 | 2022-10-14 | 中国科学院微生物研究所 | Dehalogenase HldD1 and coding gene and application thereof |
| CN112680429B (en) * | 2019-10-18 | 2022-10-14 | 中国科学院微生物研究所 | Dehalogenase HadD14 and coding gene and application thereof |
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| CN104694514B (en) | 2017-11-28 |
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