CN109456986A - Dioxygenase Snpd and its encoding gene and application to fragrant phenoxy phenoxy propionic acid herbicide intermediate with chiral selectivity - Google Patents
Dioxygenase Snpd and its encoding gene and application to fragrant phenoxy phenoxy propionic acid herbicide intermediate with chiral selectivity Download PDFInfo
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Abstract
The invention discloses the dioxygenase Snpd to fragrant phenoxy phenoxy propionic acid herbicide intermediate with chiral selectivity and its encoding gene and applications.A kind of double oxygenation gene snpd with stereoselectivity, nucleotide sequence are as follows: SEQ ID NO.1, the dioxygenase Snpd of coding, amino acid sequence are as follows: SEQ ID NO.2.The present invention passes through genome sequencing and gene comparison method successful clone to a α-ketoglutaric acid dependent form chiral selectivity dioxygenase gene snpd, the gene is first disclosed can degrade S-2- (1- naphthoxy) propionic acid and S-2- (2,4- dichlorophenoxy) propionic acid, S-2- (4- hydroxyphenoxy) propionic acid and herbicide 2, the dioxygenase of 4- dichlorphenoxyacetic acid intermediate has very important theoretical value and application prospect to the high optically pure R type herbicide of production.
Description
Technical field
The invention belongs to biotechnologies, the low isomers living applied to fragrant phenoxy phenoxy propionic acid herbicide intermediate
Zymetology split with the synthesis field of the effective isomers of optical voidness, be related to a kind of α-ketoglutaric acid dependent form, low S- isomers living
Dioxygenase Snpd and its encoding gene and application with chiral selectivity.
Background technique
Chirality is the essential attribute of three-dimensional molecular, if as soon as compound cannot be overlapped with its mirror image, which claims
For chipal compounds.Chiral pesticide is the important component part of chipal compounds, and Chiral pesticide is in global agrochemical market at present
Account for about 28%, and China's Chiral pesticide usage amount is higher, accounts for about 40%.The same each enantiomter of Chiral pesticide is not only to target
The weeding and pest-killing activity of mark biology has enantio-selectivity, and the poison of enantio-selectivity can be also generated to non-target organism
Property.For example, the activity of weeding of herbicide isopropyl methoxalamine, mainly from S- enantiomer, R- enantiomer but has mutagenesis to mouse
Effect.However, major part Chiral pesticide is still in the form of racemic modification at present due to difficulty, cost of production technology etc.
Production and use cause ecological environment so that largely remaining in the environment without the enantiomer that herbicide pesticide is active and toxicity is high
It seriously threatens.Therefore, the preparation of optical homochiral pesticide in recent years is just being increasingly subject to the concern of people.Chiral pesticide is gone at present
Racemization method mainly has physical method, chemical method and bioanalysis: 1) physical method mainly has selection absorption Split Method and crystallisation.It should
Class method production efficiency is low, narrow application range.2) chemical method mainly has chemical resolution, asymmetric syntheses etc..The operation of such method
It is cumbersome, a large amount of chiral sources and reagent need to be consumed, it is at high cost.3) bioanalysis is to utilize biological enzyme energy Stereoselective catalytic racemization
A certain enantiomer monomer in body, promotes the monomer to react, and is converted to other compounds, and then right in realization raceme
Reflect the separation of body monomer.Compared to physical method and chemical method, bioanalysis has more advantage.Biological enzyme is selected with High level of stereoselectivity
Property, it is optionally converted into optical homochiral compound, with product optical purity is high, side reaction is few, reaction condition is mild
And environmental pollution it is small the advantages that, have huge development potentiality and application value.
Fragrant phenoxy phenoxy propionic acid herbicide is to prevent and kill off one of important herbicide type of gramineae weed, and mechanisms are
By inhibiting the activity of acetyl-CoA carboxylase in gramineae plant body, and then inhibit the synthesis of plant body fat acid, thus
The growth and development for influencing plant, achievees the purpose that weeding.There are two types of optical isomer, active almost all collection for such herbicide tool
In in R- isomers, S- isomers is then inactive or active extremely low and toxic side effect is big.It is existing a large number of studies show that, fragrant phenoxy
Phenoxy propionic acid herbicide can generate acute and chronic harm to the non-target animals and plants of aquatic animals and plants and land.Therefore, chirality is utilized
The biological enzyme efficient separating or conversion racemic modification of specificity obtain optical voidness R- type virtue phenoxy phenoxy propionic acid herbicide intermediate
With highly important scientific meaning and application prospect, and it is crucial for obtaining optically pure R- type intermediate.However, can using with
It splits or the biological enzyme of the chiral specificity of conversion racemic modification virtue phenoxy phenoxy propionic acid herbicide intermediate has not been reported.
Acquisition has the degrading genes of chiral specificity and enzyme can be by existing fragrant phenoxy phenoxy propionic acid herbicide intermediate
For enzymatic and transformation technology, efficient, less toxic, the high single R- type intermediate of optical voidness of production, for producing R- type virtue phenoxy
Phenoxy propionic acid herbicide (such as cover grass energy, clodinafop-propargyl, fenoxapropPethyl, efficient fluazifop, Quizalotop-ethyl).
Summary of the invention
The albumen of the object of the present invention is to provide a kind of dioxygenase gene snpd and its coding with stereoselectivity
Matter and its application.The enzyme Spnd of gene coding can selective degradation S- virtue phenoxy phenoxy propionic acid herbicide (such as S- cover grass energy, S-
Clodinafop-propargyl, S- fenoxapropPethyl, S- efficient fluazifop, S- Quizalotop-ethyl) and S- napropamide S- type intermediate S-2- (1-
Naphthoxy) propionic acid etc..Therefore, dioxygenase Snpd can be applied to the life of the fragrant phenoxy phenoxy propionic acid herbicide of napropamide and part
Production. art removes the higher S- isomers of toxicity in specific manner, obtains low toxicity, high herbicidal activity, high optically pure R- isomers.
It is a further object of the present invention to provide the applications of the gene.
A kind of double oxygenation gene snpd with stereoselectivity, nucleotide sequence are as follows: SEQ ID NO.1.
The dioxygenase Snpd of α-ketoglutaric acid dependent form dioxygenase gene coding of the present invention, amino acid sequence
Are as follows: SEQ ID NO.2.
Contain the α-ketoglutaric acid dependent form dioxygenase gene snpd recombinant expression carrier.
The recombinant expression carrier, preferably by the NdeI of dioxygenase gene snpd insertion pET-28a (+)
Between the site XhoI, and retain the histidine tag purifying protein of N-terminal.
Genetic engineering bacterium containing the α-ketoglutaric acid dependent form dioxygenase gene snpd.
The preferred e. coli bl21 of expression bacterial strain (DE3) of the genetic engineering bacterium.
α-ketoglutaric acid dependent form dioxygenase of the present invention, recombinant expression carrier, genetic engineering bacterium in degradation or
Convert S-2- (1- naphthoxy) propionic acid, 2,4 dichlorophenoxyacetic acid, S-2- (2,4 dichloro benzene oxygroup) propionic acid and S-2- (4- hydroxyl
Phenoxy group) application in propionic acid.
Beneficial effects of the present invention are as follows:
1. the present invention passes through genome sequencing and gene comparison method successful clone to a α-ketoglutaric acid dependent form
Chiral selectivity dioxygenase gene snpd, the gene are the first disclosed S- napropamide intermediates S-2- that can degrade
(1- naphthoxy) propionic acid and fragrant phenoxy phenoxy propionic acid herbicide intermediate S-2- (2,4 dichloro benzene oxygroup) propionic acid, S-2- (4- hydroxyl
Phenoxyl) propionic acid and herbicide 2,4- dichlorphenoxyacetic acid intermediate, the full length gene is (from initiation codon to termination codon
Son) it is 891bp, encode 296 amino acid.
2. the pure enzyme of Snpd provided by the invention can in 1h S-2- (1- naphthoxy) propionic acid of degradable 0.2mM and fragrant oxygen
R-Phenoxypropionate herbicide intermediate S-2- (2,4 dichloro benzene oxygroup) propionic acid, S-2- (4- hydroxyphenoxy) propionic acid and herbicide
2,4- dichlorphenoxyacetic acid intermediates can be used for optimizing napropamide, 2- (2,4- dichlorophenoxy) propionic acid and 2- (4- hydroxy benzenes oxygen
Base) propionic acid synthesis technology, the high optically pure R type herbicide of production is with very important theoretical value and application prospect.
Detailed description of the invention
Fig. 1 bacterial strain B2 degradation R-2- (4- hydroxyphenoxy) propionic acid, the HPLC map of S-2- (4- hydroxyphenoxy) propionic acid.
Fig. 2 chiral selectivity dioxygenase Snpd degradation S-2- resulting product of (4- hydroxyphenoxy) propionic acid and substrate S-
The LC-MS/MS first order spectrum of 2- (4- hydroxyphenoxy) propionic acid.
Fig. 3 chiral selectivity dioxygenase Snpd converts fragrant phenoxy phenoxy propionic acid herbicide schematic diagram.
Expression strategy figure of Fig. 4 chiral selectivity dioxygenase gene snpd in BL21 (pET-29a (+)).
The SDS-PAGE of Fig. 5 chiral selectivity dioxygenase Snpd schemes.1: the BL21 of albumen marker, 2:IPTG induction
(SnaH) thick enzyme, 3: thick enzyme penetrates liquid, 4:50mM imidazole elution, 5:100mM imidazole elution, 6:150mM imidazole elution,
7:200mM imidazole elution, 8:250mM imidazole elution, 9:300mM imidazole elution.
Biomaterial preservation information
B2, classification naming are Sphingobium sp.strain B2, are preserved in China typical culture collection center, are protected
Hiding address is Wuhan, China university, and the deposit date is on October 16th, 2018, deposit number was CCTCC NO:M2018684.
Specific embodiment:
The determination of 1 bacterial strain B2 of embodiment degradation S-2- (4- hydroxyphenoxy) propionic acid metabolite
The determination of catabolite: bacterial strain B2 is seeded in 100mL LB liquid medium, and 30 DEG C, 180rpm/min, culture is extremely
Exponential phase, 6000rpm/mim are centrifuged 10min and collect thallus, and thallus is washed 2 times with sterilized base salt culture medium, are resuspended in 10ml
Basal salt media is seeded in 100mL inorganic salt liquid culture medium, adjusts OD600About 1.0 or so, 0.2mM S-2- is added
(4- hydroxyphenoxy) propionic acid, in 30 DEG C, 180rpm/min shaking table shaken cultivation.Every 3h timing sampling, timing sampling culture
Isometric methanol is added in liquid, and after oscillation mixes, 12000rpm/min is centrifuged 5min, and sample is filtered with 0.22 μm of nylon leaching film,
High performance liquid chromatography (HPLC) is detected.HPLC chromatogram condition are as follows: chromatographic column is Syncronis C18 (Thermo Fisher
Scientific) reversed-phase column, specification are 250mm × 4.6mm × 5 μm；Mobile phase is methanol: water: acetic acid (60:39:1 [v/v/
v]；Column temperature is 30 DEG C；Flow velocity is 1.0mLmin-1；Detection wavelength is 289nm；Applied sample amount is 20 μ L.It can from liquid chromatogram
To find out bacterial strain B2 degradation S-2- (4- hydroxyphenoxy) propionic acid, there is a product peak at 3.043min, but bacterial strain B2 cannot drop
Solve R-2- (4- hydroxyphenoxy) propionic acid (Fig. 1).
Basal salt media (1L) formula are as follows: 1.0g NaCl, 1.0g NH4NO3, 1.5g K2HPO4, 0.5g KH2PO4,
0.2g MgSO4, add deionized water to 1L, pH 7.0.
LB culture medium (1L) formula are as follows: 5.0g NaCl, 5.0g yeast powder, 10g peptone, addition plus deionized water to 1L,
pH 7.0。
There is the sample at product peak to liquid phase detection, metabolite is analyzed and identified with HPLC-MS/MS.HPLC
Condition are as follows: UltiMate 3000RSLC (Thermo Fisher Scientific, the U.S.), chromatographic column are Kinetex C18
(100mm × 2.1mm, partial size are 2.6 μm), mobile phase condition are as follows: 0-3min, methanol: water: acetic acid is 30:69:1 (v/v/v),
3-15min, eluent gradient increase to methanol: water: acetic acid is 75:24:1 (v/v/v), and maintains 15min constant, Zhi Houliu
Dynamic phase methanol, water, proportion of acetic acid are down to 30:69:1 (v/v/v) and maintain 5min constant.Detection wavelength is 289nm, and sample volume is
10μL.Mass spectrometer is TripleTOF 5600 (AB SCIEX, the U.S.), and analysis ion source is positive ion detection mode.
It is 109.0305 that the mass spectrogram of HPLC-MS/MS, which shows that the first mass spectrometric figure (see Fig. 2) of its product shows that it has m/z,
Anion peak.Thus it is speculated that the biochemical reaction of degradation S-2- (4- hydroxyphenoxy) propionic acid is that disconnected ehter bond generates hydroquinone
(Fig. 3).
The clone of embodiment 2S-2- (4- hydroxyphenoxy) propionic acid degradation gene and functional verification
The comparison and lookup of 2.1 dioxygenase genes
By consulting pertinent literature, the dioxygenase gene of the report tfdA of the similar reaction of catalysis is found out.Utilize TfdA egg
Bai Xulie and bacterial strain B2 genome alignment, as a result there is the protein sequence of similitude in bacterial strain B2 genome than to 3.
The heterogenous expression of 2.2 dioxygenase genes speculated and functional verification
Using bacterial strain B2 genomic DNA as template, design primer is used to expand the dioxygenase gene segment of supposition.It is used
Primer is as follows:
Used primer is tested in 1 functional verification of table
Amplification system:
Amplification program
A.95 DEG C initial denaturation 3min；
B.95 DEG C denaturation 15sec, 60 DEG C of annealing 15sec, 72 DEG C of extension 1.0min, carry out 30 circulations.
C.72 DEG C whole extension 5min, is cooled to room temperature.
PET28a (+) plasmid of extraction Nde Ι and Xho I double digestion.
Digestion system:
Above-mentioned reaction system is purified in 37 DEG C of waters digestion 4h with gel purification kit.
The above-mentioned expression vector pET28a (+) linearized and amplified fragments are passed through into ClonExpress II One
Step cloning kit kit is attached.
Recombining reaction system:
10min is reacted in 50 DEG C of waters, is immediately placed on cooled on ice.
Above-mentioned recombinant products are converted into BL21 (DE3), are constructed recombinant strains (Fig. 4).Each recombinant strains
It cultivates in LB liquid medium to OD600About 0.6 or so, 1mM IPTG is added in 16 DEG C, 150rpm/min induction 12 is small
When, 12000rpm/min is centrifuged 10min, collects thallus, and thallus, ultrasonication 15min, 12000rpm/ is resuspended with PBS buffer solution
Min is centrifuged 30min, collects supernatant (i.e. the thick enzyme of recombinant strains), verifies the thick enzyme of each recombinant strains to S-2- (4- hydroxyl
Phenoxy group) propionic acid degradation capability.
It is verified by enzyme activity, has the thick enzyme of recombinant bacterial strain that can degrade S-2- (4- hydroxyphenoxy) propionic acid, by this
Unnamed gene is snpd.
Expression and purifying of the 2.2snpd in E.coli BL21 (DE3-pET-28a (+)-snpd)
Recombinant strains are transferred in 20mL liquid LB (Kan containing 50mg/L), to bacterial strain length to exponential phase, then will training
It cultivates in nutrient solution switching (inoculum concentration 4%, v/v) to 100mL LB (Kan containing 50mg/L) fluid nutrient medium to OD600For 0.6 left side
The right side adds 1mM IPTG, and 16 DEG C of inductions 12h, 12000rpm/min are centrifuged 10min, collect thallus, thallus is resuspended with PBS buffer solution,
Ultrasonication 15min is centrifuged 30min, collects supernatant, is purified with nickel ion affinity chromatograph column to Snpd, SDS-PAGE egg
The size of white electrophoresis detection purification effect, stripe size and theoretical prediction is consistent (Fig. 5).
The substrate of 2.3Snpd is composed
0.2mM α-ketoglutaric acid, 0.1mM Fe are added in 1mL enzyme activity reaction system2+, 0.5mM Vc, 10 μ g Snpd be pure
The substrate of enzyme and 0.2mM places 1min after reacting 72h in boiling water, terminates reaction.Isometric first is added after reaction solution is cooling
Alcohol mixes well, and 12000rpm/min is centrifuged 5min, crosses 0.22 μm of nylon membrane filter, detects each degradation of substrates feelings by HPLC
Condition (table 2).
The substrate of 2 chiral selectivity dioxygenase Snpd of table is composed
2.4Snpd degradation Rac-2- (1- naphthoxy) propionic acid, S-2- (1- naphthoxy) propionic acid, Rac-2- (2,4- dichloro-benzenes
Oxygroup) propionic acid, S-2- (2,4- dichlorophenoxy) propionic acid, Rac-2- (4- hydroxyphenoxy) propionic acid, S-2- (4- hydroxy benzenes oxygen
Base) propionic acid and 2,4 dichlorophenoxyacetic acid vitality test.
Enzyme activity reaction system (1mL): (pH7.4) PBS buffer solution, 0.2mM Rac-2- (1- naphthoxy) propionic acid (or 0.2mM
S-2- (1- naphthoxy) propionic acid or Rac-2- (2,4- dichlorophenoxy) propionic acid or S-2- (2,4- dichlorophenoxy) propionic acid, or
Rac-2- (4- hydroxyphenoxy) propionic acid or S-2- (4- hydroxyphenoxy) propionic acid or 2,4- dichlorphenoxyacetic acid), Snpd is pure
Enzyme (Fig. 5 purifying gained), 0.2mM α-ketoglutaric acid, 0.1mM Fe2+, 0.5mM Vc, 37 reaction 20min, each reaction is to be added
Enzyme starts timing, places 1min after 20min in boiling water, terminates reaction.Isometric methanol is added after reaction solution is cooling, it is sufficiently mixed
Even, 12000rpm/min is centrifuged 5min, crosses 0.22 μm of nylon membrane filter, and HPLC detects the production quantity of product.One enzyme activity list
Position is defined as: under the conditions of pH7.4,37 DEG C of temperature, enzyme amount needed for catalysis substrate per minute generates 1 μm of ol product.
Enzymatic assay shows Snpd to Rac-2- (1- naphthoxy) propionic acid, and the specific enzyme activity of S-2- (1- naphthoxy) propionic acid is successively
For 2.78U/mg, 2.35U/mg, to Rac-2- (2,4- dichlorophenoxy) propionic acid and S-2- (2,4- dichlorophenoxy) propionic acid
Rate activity is followed successively by 0.747U/mg, 0.756U/mg, to Rac-2- (4- hydroxyphenoxy) propionic acid and S-2- (4- hydroxy benzenes oxygen
Base) Rate activity of propionic acid is followed successively by 0.306U/mg and 0.378U/mg, and the Rate activity to 2,4- dichlorphenoxyacetic acid is 0.159U/
mg。
Sequence table
<110>Agricultural University Of Nanjing
<120>there is the dioxygenase Snpd and its coding base of chiral selectivity to fragrant phenoxy phenoxy propionic acid herbicide intermediate
Cause and application
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 891
<212> DNA
<213>Sphingobacterium (Sphingobium sp.)
<400> 1
atgccattcc aggtcaatca gctgcatccg atctttgcgg cagaaatgtt cggttgcgac 60
atcctcaagc cggtgacgga tgaaacgaga aatgccgtcg aagatgcgat ggccaaatat 120
gccgtgctgg ttatccgcga tcaggcggcc gcgagtgacg aggatcaggt ccgctttgct 180
aaggcattcg ggcccttgga gttgccgccc gatctcggca tgtcggagag gacgaggccg 240
acgcgcgtcc atccgaaact ctacgacgtg tcgaacttgg acgagaacgg caatctggac 300
aaacccgata cgctgcgccg caagttcgcc aagggcaacg agctcttcca tacggacagc 360
tcattcaatg acctgccgac caaatggtcg atgttgttgg cccacgtcgt cacgcccacg 420
gggggcaata ccgaatttgt cgatacacgg gcagcctatg atgcgctgtc gccttcgatg 480
aaggagcagg tcgaaccact gagcgtcatt cacagtctca cctactcgcg cgaaaggggt 540
ggcctcaccg gcacgactgt tttcgatcgc gcctttccgc cggtgacaca gccacttgta 600
cggacaagcg catcgggccg taaggctctc tatatcggcg cacacgccgc aaccgttgtc 660
ggcatggatg aggcggccgg gcggaccctg ctcgacaagt tgatctcgga tgccagccga 720
ccggagtata tatattcaca caaatggctg cccggcgacc tcgttatctg ggacaatcgc 780
tgcactatgc atcgcgcgac cgaatatgcg tacatggagg accggcggga tctgcgtcgg 840
gcgaccatca acgaatttgg cgaggatcgc gtcggcgcgc gaccgatcta g 891
<210> 2
<211> 296
<212> PRT
<213>Sphingobacterium (Sphingobium sp.)
<400> 2
Met Pro Phe Gln Val Asn Gln Leu His Pro Ile Phe Ala Ala Glu Met
1 5 10 15
Phe Gly Cys Asp Ile Leu Lys Pro Val Thr Asp Glu Thr Arg Asn Ala
20 25 30
Val Glu Asp Ala Met Ala Lys Tyr Ala Val Leu Val Ile Arg Asp Gln
35 40 45
Ala Ala Ala Ser Asp Glu Asp Gln Val Arg Phe Ala Lys Ala Phe Gly
50 55 60
Pro Leu Glu Leu Pro Pro Asp Leu Gly Met Ser Glu Arg Thr Arg Pro
65 70 75 80
Thr Arg Val His Pro Lys Leu Tyr Asp Val Ser Asn Leu Asp Glu Asn
85 90 95
Gly Asn Leu Asp Lys Pro Asp Thr Leu Arg Arg Lys Phe Ala Lys Gly
100 105 110
Asn Glu Leu Phe His Thr Asp Ser Ser Phe Asn Asp Leu Pro Thr Lys
115 120 125
Trp Ser Met Leu Leu Ala His Val Val Thr Pro Thr Gly Gly Asn Thr
130 135 140
Glu Phe Val Asp Thr Arg Ala Ala Tyr Asp Ala Leu Ser Pro Ser Met
145 150 155 160
Lys Glu Gln Val Glu Pro Leu Ser Val Ile His Ser Leu Thr Tyr Ser
165 170 175
Arg Glu Arg Gly Gly Leu Thr Gly Thr Thr Val Phe Asp Arg Ala Phe
180 185 190
Pro Pro Val Thr Gln Pro Leu Val Arg Thr Ser Ala Ser Gly Arg Lys
195 200 205
Ala Leu Tyr Ile Gly Ala His Ala Ala Thr Val Val Gly Met Asp Glu
210 215 220
Ala Ala Gly Arg Thr Leu Leu Asp Lys Leu Ile Ser Asp Ala Ser Arg
225 230 235 240
Pro Glu Tyr Ile Tyr Ser His Lys Trp Leu Pro Gly Asp Leu Val Ile
245 250 255
Trp Asp Asn Arg Cys Thr Met His Arg Ala Thr Glu Tyr Ala Tyr Met
260 265 270
Glu Asp Arg Arg Asp Leu Arg Arg Ala Thr Ile Asn Glu Phe Gly Glu
275 280 285
Asp Arg Val Gly Ala Arg Pro Ile
290 295
<210> 3
<211> 43
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gtgccgcgcg gcagccatat gatgccattc caggtcaatc agc 43
<210> 4
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gtggtggtgg tggtgctcga gctagatcgg tcgcgcgcc 39
<210> 5
<211> 45
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
gtgccgcgcg gcagccatat gatgactgcc catatcaaca atttc 45
<210> 6
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gtggtggtgg tggtgctcga gtcactcggc cgggaccag 39
<210> 7
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gtgccgcgcg gcagccatat gttgaagccg atttcgccc 39
<210> 8
<211> 44
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
gtggtggtgg tggtgctcga gtcaatattt ccgcatatgt tcgg 44
Claims (10)
1. a kind of double oxygenation gene snpd with stereoselectivity, it is characterised in that nucleotide sequence such as SEQ ID N0.1 institute
Show.
2. the dioxygenase Snpd of double oxygenation gene snpd codings described in claim 1, it is characterised in that amino acid sequence is such as
Shown in SEQ ID N0.2.
3. containing the recombinant expression carrier of double oxygenation gene snpd described in claim 1.
4. recombinant expression carrier according to claim 3, it is characterised in that be to insert the dioxygenase gene snpd
Enter between the site NdeI and XhoI of pET-28a (+), and retains obtained by the histidine tag purifying protein of N-terminal.
5. containing the genetic engineering bacterium of double oxygenation gene snpd described in claim 1.
6. genetic engineering bacterium according to claim 5, it is characterised in that host strain is e. coli bl21 (DE3).
7. double oxygenation gene snpd described in claim 1 are degrading or are converting S-2- (1- naphthoxy) propionic acid, 2,4 dichloro benzene
Application in fluoroacetic acid, S-2- (2,4 dichloro benzene oxygroup) propionic acid and S-2- (4- hydroxyphenoxy) propionic acid.
8. dioxygenase Snpd as claimed in claim 2 is degrading or is converting S-2- (1- naphthoxy) propionic acid, 2,4 dichloro benzene oxygen
Application in acetic acid, S-2- (2,4 dichloro benzene oxygroup) propionic acid and S-2- (4- hydroxyphenoxy) propionic acid.
9. S-2- (1- naphthoxy) propionic acid, 2,4 dichloro benzene is being degraded or converted to recombinant expression carrier described in claim 3 or 4
Application in fluoroacetic acid, S-2- (2,4 dichloro benzene oxygroup) propionic acid and S-2- (4- hydroxyphenoxy) propionic acid.
10. S-2- (1- naphthoxy) propionic acid, 2,4 dichloro benzene is being degraded or converted to genetic engineering bacterium described in claim 5 or 6
Application in fluoroacetic acid, S-2- (2,4 dichloro benzene oxygroup) propionic acid and S-2- (4- hydroxyphenoxy) propionic acid.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111321092A (en) * | 2020-02-17 | 2020-06-23 | 南京农业大学 | (R, S) -2, 4-dichlorprop degrading strain and dioxygenase gene for specifically degrading (S) -type isomer thereof |
| CN111378601A (en) * | 2020-02-25 | 2020-07-07 | 南京农业大学 | Halogenated phenol degradation strain and microbial inoculum produced by same |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103981195A (en) * | 2013-12-17 | 2014-08-13 | 南京农业大学 | Dioxygenase gene pbaAaAbAcAd and coding protein and application thereof |
| CN107012132A (en) * | 2016-10-12 | 2017-08-04 | 南京工业大学 | A kind of fragrant phenoxy phenoxy propionic acid herbicide hydrolysis esterase and its application |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103981195A (en) * | 2013-12-17 | 2014-08-13 | 南京农业大学 | Dioxygenase gene pbaAaAbAcAd and coding protein and application thereof |
| CN107012132A (en) * | 2016-10-12 | 2017-08-04 | 南京工业大学 | A kind of fragrant phenoxy phenoxy propionic acid herbicide hydrolysis esterase and its application |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111321092A (en) * | 2020-02-17 | 2020-06-23 | 南京农业大学 | (R, S) -2, 4-dichlorprop degrading strain and dioxygenase gene for specifically degrading (S) -type isomer thereof |
| CN111321092B (en) * | 2020-02-17 | 2022-05-24 | 南京农业大学 | (R, S) -2, 4-dichlorprop degrading strain and dioxygenase gene for specifically degrading (S) -type isomer thereof |
| CN111378601A (en) * | 2020-02-25 | 2020-07-07 | 南京农业大学 | Halogenated phenol degradation strain and microbial inoculum produced by same |
| CN111378601B (en) * | 2020-02-25 | 2021-11-16 | 南京农业大学 | Halogenated phenol degradation strain and microbial inoculum produced by same |
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