CN109456986B

CN109456986B - Dioxygenase Snpd with chirality selectivity for intermediates of aryloxyphenoxypropionic acid herbicides and its encoding gene and application

Info

Publication number: CN109456986B
Application number: CN201811276486.6A
Authority: CN
Inventors: 蒋建东; 黄俊伟; 陈典
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2018-10-30
Filing date: 2018-10-30
Publication date: 2021-06-01
Anticipated expiration: 2038-10-30
Also published as: CN109456986A

Abstract

The invention discloses a dioxygenase Snpd with chirality selectivity to an intermediate of aryloxyphenoxypropionic acid herbicides, its encoding gene and application. A dioxygenase gene snpd with stereoselectivity, the nucleotide sequence is: SEQ ID NO.1, and the encoded dioxygenase Snpd, the amino acid sequence is: SEQ ID NO.2. The present invention successfully clones an α-ketoglutarate-dependent chiral selective dioxygenase gene snpd through whole genome sequencing and gene comparison method, and this gene is the first published gene capable of degrading S-2-(1- Naphthyloxy)propionic acid and S-2-(2,4-dichlorophenoxy)propionic acid, S-2-(4-hydroxyphenoxy)propionic acid and the herbicide 2,4-dichlorophenoxy The dioxygenase of acetic acid intermediate has very important theoretical value and application prospect for the production of high optical purity R-type herbicide.

Description

Dioxygenase Snpd with chiral selectivity on aryloxy phenoxy propionic acid herbicide intermediate, and coding gene and application thereof

Technical Field

The invention belongs to the bioengineering technology, is applied to the field of enzymatic resolution of low-activity isomers of aryloxy phenoxy propionic acid herbicide intermediates and synthesis of optically pure effective isomers, and relates to a dioxygenase Snpd with alpha-ketoglutaric acid dependent type and chiral selectivity of low-activity S-isomers, and a coding gene and application thereof.

Background

Chirality is an essential property of a three-dimensional molecule, and a compound is called a chiral compound if it cannot coincide with its mirror image. Chiral pesticides are important components of chiral compounds, and currently, chiral pesticides account for about 28% of the global pesticide market, while chiral pesticides in China are used in higher amounts, accounting for about 40%. Each enantiomer of the same chiral pesticide has enantioselectivity not only for the insecticidal and herbicidal activity of a target organism, but also for non-target organisms. For example, the herbicidal activity of the herbicide metolachlor is mainly derived from the S-enantiomer, which has a mutagenic effect on mice. However, due to the difficulty and cost of the production process, most of the chiral pesticides are produced and used in the form of racemate, so that the enantiomer which has no herbicidal and insecticidal activity and high toxicity is greatly remained in the environment, and serious threat is caused to the ecological environment. Therefore, the preparation of optically pure chiral pesticides has been receiving increasing attention in recent years. At present, the descemization method of chiral pesticide mainly comprises a physical method, a chemical method and a biological method: 1) the physical method mainly comprises a selective adsorption resolution method and a crystallization method. The method has low production efficiency and narrow application range. 2) The chemical method mainly comprises chemical resolution, asymmetric synthesis and the like. The method is complex to operate, needs to consume a large amount of chiral sources and reagents, and is high in cost. 3) The biological method is to utilize biological enzyme to catalyze one enantiomer monomer in the racemate stereoselectively, promote the monomer to react and convert the monomer into other compounds, and further realize the separation of the enantiomer monomer in the racemate. Compared with physical methods and chemical methods, biological methods have more advantages. The biological enzyme has high stereoselectivity, can be selectively converted into an optical pure chiral compound, has the advantages of high optical purity of the product, less side reaction, mild reaction condition, small environmental pollution and the like, and has great development potential and application value.

The aryloxy phenoxy propionic acid herbicide is one of important herbicide types for preventing and killing gramineous weeds, and the weeding mechanism is that the synthesis of fatty acid in plants is inhibited by inhibiting the activity of acetyl coenzyme A carboxylase in gramineous plants, so that the growth and development of the plants are influenced, and the weeding purpose is achieved. The herbicide has two optical isomers, the activity is almost totally concentrated in R-isomer, and S-isomer has no activity or extremely low activity and large toxic and side effects. Numerous studies have shown that aryloxyphenoxypropionic acid herbicides can cause acute and chronic damage to aquatic animals and plants and land non-target animals and plants. Therefore, the optically pure R-aryloxy phenoxy propionic acid herbicide intermediate obtained by efficiently splitting or converting the racemate by using the chiral specific biological enzyme has very important scientific significance and application prospect, and the key is to obtain the optically pure R-intermediate. However, no chiral-specific biological enzymes have been reported which can be used for the resolution or conversion of racemic aryloxyphenoxypropionic acid herbicide intermediates.

The obtained degradation gene and enzyme with chiral specificity to the intermediate of the aryloxy-phenoxy-propionic acid herbicide can be used for producing high-efficiency, low-toxicity and high-optical-purity single R-type intermediate by modern enzyme catalysis and conversion technology, and can be used for producing R-type aryloxy-phenoxy-propionic acid herbicides (such as galingale, clodinafop-propargyl, fenoxaprop-p-ethyl, fluazifop-p-butyl, quizalofop-p-ethyl and the like).

Disclosure of Invention

The invention aims to provide a dioxygenase gene snpd with stereoselectivity, a protein coded by the same and application thereof. The enzyme Spnd coded by the gene can specifically degrade S-aryloxy phenoxy propionic acid herbicides (such as S-Geranium, S-clodinafop-propargyl, S-fenoxaprop-P-ethyl, S-fluazifop-p-butyl and S-quizalofop-p-ethyl) and S-type intermediate S-2- (1-naphthyloxy) propionic acid of S-napropamide and the like. Therefore, the dioxygenase Snpd can be applied to the production process of napropamide and partial aryloxy phenoxy propionic acid herbicides, specifically removes S-isomer with higher toxicity, and obtains R-isomer with low toxicity, high herbicidal activity and high optical purity.

Another purpose of the invention is to provide the application of the gene.

A dioxygenase gene snpd with stereoselectivity has a nucleotide sequence: SEQ ID NO. 1.

The amino acid sequence of the dioxygenase Snpd coded by the alpha-ketoglutarate dependent dioxygenase gene is as follows: SEQ ID NO. 2.

Contains the alpha-ketoglutarate dependent dioxygenase gene snpd recombinant expression vector.

The recombinant expression vector is preferably characterized in that the dioxygenase gene snpd is inserted between NdeI and XhoI sites of pET-28a (+), and an N-terminal histidine tag purified protein is reserved.

A genetically engineered bacterium containing the alpha-ketoglutarate dependent dioxygenase gene snpd.

The expression strain of the genetic engineering bacteria is preferably escherichia coli BL21(DE 3).

The invention relates to application of alpha-ketoglutarate dependent dioxygenase, a recombinant expression vector and a genetic engineering bacterium in degrading or converting S-2- (1-naphthoxy) propionic acid, 2, 4-dichlorophenoxyacetic acid, S-2- (2, 4-dichlorophenoxy) propionic acid and S-2- (4-hydroxyphenoxy) propionic acid.

The invention has the following beneficial effects:

1. the invention successfully clones an alpha-ketoglutarate dependent chiral selective dioxygenase gene snpd by a whole genome sequencing and gene comparison method, the gene is the first disclosed gene capable of degrading S-napropamide degradation intermediate products S-2- (1-naphthoxy) propionic acid and aryloxy phenoxy propionic acid herbicide intermediates S-2- (2, 4-dichlorophenoxy) propionic acid, S-2- (4-hydroxyphenoxy) propionic acid and herbicide 2, 4-dichlorophenoxy acetic acid intermediates, the full length (from an initiation codon to a termination codon) of the gene is 891bp, and 296 amino acids are coded.

2. The Snpd pure enzyme provided by the invention can completely degrade 0.2mM S-2- (1-naphthoxy) propionic acid and aryloxy phenoxy propionic acid herbicide intermediates S-2- (2, 4-dichlorophenoxy) propionic acid, S-2- (4-hydroxyphenoxy) propionic acid and herbicide 2, 4-dichlorophenoxyacetic acid intermediates within 1h, can be used for optimizing the synthesis process of alachlor, 2- (2, 4-dichlorophenoxy) propionic acid and 2- (4-hydroxyphenoxy) propionic acid, and has very important theoretical value and application prospect in producing high-optical-purity R-type herbicides.

Drawings

FIG. 1 HPLC chromatogram of the degradation of R-2- (4-hydroxyphenoxy) propionic acid, S-2- (4-hydroxyphenoxy) propionic acid by strain B2.

FIG. 2 LC-MS/MS primary spectrum of the product obtained by degrading S-2- (4-hydroxyphenoxy) propionic acid with the chiral-selective dioxygenase Snpd and the substrate S-2- (4-hydroxyphenoxy) propionic acid.

FIG. 3 is a schematic diagram of the conversion of aryloxyphenoxypropionic acid herbicides by the chiral-selective dioxygenase Snpd.

FIG. 4 is a schematic diagram showing the expression strategy of the chiral selective dioxygenase gene snpd in BL21(pET-29a (+)).

FIG. 5 SDS-PAGE of the chiral selective dioxygenase Snpd. 1: protein marker, 2: IPTG-induced crude BL21(SnaH), 3: crude enzyme transpermeate, 4: 50mM imidazole eluent, 5: 100mM imidazole eluent, 6: 150mM imidazole eluent, 7: 200mM imidazole eluent, 8: 250mM imidazole eluent, 9: 300mM imidazole eluent.

Biological material preservation information

B2, classified and named as Sphingobium sp.strain B2, which is preserved in China center for type culture Collection, the preservation address is Wuhan university, China, the preservation date is 2018, 10 and 16 days, and the preservation number is CCTCC NO: M2018684.

The specific implementation mode is as follows:

EXAMPLE 1 determination of the degradation of S-2- (4-hydroxyphenoxy) propionic acid metabolite by Strain B2

Determination of degradation products: inoculating the strain B2 into 100mL LB liquid culture medium, culturing at 30 deg.C and 180rpm/min to exponential phase, centrifuging at 6000rpm/mim for 10min to collect thallus, washing thallus with sterile basic salt culture medium for 2 times, resuspending in 10mL basic salt culture medium, inoculating into 100mL inorganic salt liquid culture medium, adjusting OD₆₀₀About 1.0, 0.2mM S-2- (4-hydroxyphenoxy) propionic acid was added thereto, and shaking-cultured at 30 ℃ and 180 rpm/min. Sampling every 3h at regular intervals, adding methanol with the same volume into the timing sampling culture solution, shaking and mixing uniformly, centrifuging at 12000rpm/min for 5min, filtering the sample by using a 0.22 mu m nylon filter membrane, and detecting by using High Performance Liquid Chromatography (HPLC). The HPLC chromatographic conditions are as follows: the chromatographic column is a Syncronis C18(Thermo Fisher Scientific) reversed phase column with specification of 250mm × 4.6mm × 5 μm; the mobile phase is methanol: water: acetic acid (60:39:1[ v/v ]](ii) a The column temperature is 30 ℃; the flow rate was 1.0 mL/min^-1(ii) a The detection wavelength is 289 nm; the loading was 20. mu.L. From the liquid chromatogram, it can be seen that strain B2 degraded S-2- (4-hydroxyphenoxy) propionic acid, with a product peak at 3.043min, but strain B2 was unable to degrade R-2- (4-hydroxyphenoxy) propionic acid (FIG. 1).

The basic salt culture medium (1L) comprises the following components: 1.0g NaCl, 1.0g NH₄NO₃，1.5g K₂HPO₄，0.5g KH₂PO₄，0.2g MgSO₄Deionized water was added to 1L, pH 7.0.

The LB medium (1L) formula is as follows: 5.0g NaCl, 5.0g yeast powder, 10g peptone, add deionized water to 1L, pH 7.0.

And (4) analyzing and identifying the metabolic products by using HPLC-MS/MS on the sample with the product peak detected by the liquid phase. The HPLC conditions were as follows: UltiMate 3000RSLC (Thermo Fisher Scientific, usa), Kinetex C18(100mm × 2.1mm, 2.6 μm particle size) column, mobile phase conditions: 0-3min, methanol: water: the acetic acid is 30: 69: 1(v/v/v), 3-15min, mobile phase gradient increased to methanol: water: the acetic acid is 75: 24: 1(v/v/v) and maintained for 15min, after which the mobile phase methanol, water, acetic acid ratio was reduced to 30: 69: 1(v/v/v) and maintained for 5 min. The detection wavelength is 289nm, and the sample injection amount is 10 muL. The mass spectrometer was TripleTOF 5600(AB SCIEX, usa), and the analytical ion source was in positive ion detection mode.

The mass spectrum of HPLC-MS/MS shows that the primary mass spectrum (see FIG. 2) of the product shows that the product has a negative ion peak with m/z of 109.0305. Therefore, it is assumed that the biochemical reaction for degrading S-2- (4-hydroxyphenoxy) propionic acid is to break the ether bond to produce hydroquinone (FIG. 3).

Example 2 cloning and functional verification of S-2- (4-hydroxyphenoxy) propionic acid-degrading Gene

2.1 comparison and search of dioxygenase genes

By referring to the relevant literature, the reported dioxygenase gene tfdA catalyzing similar reactions was found. The TfdA protein sequence was aligned with the genome of strain B2, resulting in 3 protein sequences with similarity in the genome of strain B2.

2.2 heterologous expression and functional verification of the putative dioxygenase Gene

The genomic DNA of the strain B2 was used as a template to design primers for amplifying the putative dioxygenase gene fragment. The primers used are as follows:

TABLE 1 primers used in functional verification experiments

An amplification system:

amplification procedure

a.95 ℃ pre-denaturation for 3 min;

b.95 ℃ denaturation 15sec, 60 ℃ annealing 15sec, 72 ℃ extension 1.0min, 30 cycles.

c.72 ℃ Final extension for 5min, cool to room temperature.

The extracted pET28a (+) plasmid was digested simultaneously with Nde I and Xho I.

Enzyme digestion system:

the above reaction system was digested in water at 37 ℃ for 4h and purified with gel purification kit.

The linearized expression vector pET28a (+) was ligated to the amplified fragment using the Clonexpress II One Step cloning kit.

A recombination reaction system:

reacting in water at 50 deg.C for 10min, and immediately cooling on ice.

The recombinant product was transformed into BL21(DE3) to construct a recombinant expression strain (FIG. 4). Each recombinant expression strain was cultured in LB liquid medium to OD₆₀₀About 0.6, adding 1mM IPTG, inducing at 16 ℃ and 150rpm/min for 12 hours, centrifuging at 12000rpm/min for 10min, collecting thalli, resuspending the thalli by PBS buffer solution, carrying out ultrasonic crushing for 15min, centrifuging at 12000rpm/min for 30min, collecting supernatant (namely recombinant expression strain crude enzyme), and verifying the degradation capability of each recombinant expression strain crude enzyme on S-2- (4-hydroxyphenoxy) propionic acid.

Through enzyme activity verification, a recombinant strain crude enzyme can degrade S-2- (4-hydroxyphenoxy) propionic acid, and the gene is named as snpd.

2.2 expression and purification of snpd in E.coli BL21(DE3-pET-28a (+) -snpd)

The recombinant expression strain is transferred into 20mL liquid LB (containing 50mg/L Kan), when the strain grows to an exponential phase, the culture solution is transferred (the inoculum size is 4 percent, v/v) into 100mL LB (containing 50mg/L Kan) liquid culture medium to be cultured to OD₆₀₀About 0.6, adding 1mM IPTG, inducing at 16 ℃ for 12h, centrifuging at 12000rpm/min for 10min, collecting thalli, resuspending the thalli by PBS buffer solution, carrying out ultrasonication for 15min, centrifuging for 30min, collecting supernatant, purifying Snpd by using a nickel ion affinity chromatography column, detecting the purification effect by SDS-PAGE protein electrophoresis, and ensuring that the size of a band is consistent with the size predicted by theory (figure 5).

2.3 substrate spectra of Snpd

Adding 0.2mM alpha-ketoglutaric acid and 0.1mM Fe into 1mL enzyme activity reaction system²⁺0.5mM Vc, 10. mu.g Snpd pure enzyme and 0.2mM substrate, reacting for 72h, placing in boiling water for 1min, and terminating the reaction. After cooling, the reaction solution was added with methanol of the same volume, mixed well, centrifuged at 12000rpm/min for 5min, filtered through a 0.22 μm nylon membrane filter, and each substrate degradation was detected by HPLC (Table 2).

TABLE 2 substrate spectra of the chiral-selective dioxygenase Snpd

2.4 Activity assay of Snpd for degrading Rac-2- (1-naphthyloxy) propionic acid, S-2- (1-naphthyloxy) propionic acid, Rac-2- (2, 4-dichlorophenoxy) propionic acid, S-2- (2, 4-dichlorophenoxy) propionic acid, Rac-2- (4-hydroxyphenoxy) propionic acid, S-2- (4-hydroxyphenoxy) propionic acid and 2, 4-dichlorophenoxyacetic acid.

Enzyme activity reaction system (1 mL): (pH7.4) PBS buffer, 0.2mM Rac-2- (1-naphthalene)Oxy) propionic acid (or 0.2mM S-2- (1-naphthyloxy) 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-dichlorophenoxyacetic acid), Snpd pure enzyme (purified in FIG. 5), 0.2mM alpha-ketoglutaric acid, 0.1mM Fe²⁺0.5mM Vc, 37 for 20min, each reaction being started with the addition of the enzyme, and after 20min being left in boiling water for 1min, the reaction is terminated. Cooling the reaction solution, adding methanol with the same volume, mixing uniformly, centrifuging at 12000rpm/min for 5min, filtering through a nylon membrane filter with the diameter of 0.22 mu m, and detecting the generation amount of a product by HPLC. One unit of enzyme activity is defined as: the amount of enzyme required to catalyze the formation of 1. mu. mol of product per minute of the substrate at a pH of 7.4 and a temperature of 37 ℃.

The enzymology experiment shows that the specific enzyme activities of Snpd on Rac-2- (1-naphthoxy) propionic acid and S-2- (1-naphthoxy) propionic acid are 2.78U/mg and 2.35U/mg in sequence, the specific activities on Rac-2- (2, 4-dichlorophenoxy) propionic acid and S-2- (2, 4-dichlorophenoxy) propionic acid are 0.747U/mg and 0.756U/mg in sequence, the specific activities on Rac-2- (4-hydroxyphenoxy) propionic acid and S-2- (4-hydroxyphenoxy) propionic acid are 0.306U/mg and 0.378U/mg in sequence, and the specific activity on 2, 4-dichlorophenoxyacetic acid is 0.159U/mg.

Sequence listing

<110> Nanjing university of agriculture

<120> dioxygenase Snpd with chiral selectivity on aryloxyphenoxypropionic acid herbicide intermediate, and coding gene and application thereof

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 891

<212> DNA

<213> Sphingobacterium 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 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

1. A dioxygenase gene snpd with stereoselectivity is characterized in that the nucleotide sequence is shown as SEQ ID N0.1.

2. The dioxygenase Snpd encoded by the dioxygenase gene Snpd according to claim 1, characterized in that the amino acid sequence is shown in SEQ ID N0.2.

3. A recombinant expression vector comprising the dioxygenase gene snpd according to claim 1.

4. The recombinant expression vector of claim 3, wherein the dioxygenase gene snpd is inserted between NdeI and XhoI sites of pET-28a (+) and purified by leaving a histidine tag at the N-terminal.

5. A genetically engineered bacterium comprising the dioxygenase gene snpd according to claim 1.

6. The genetically engineered bacterium of claim 5, wherein the host bacterium is Escherichia coli BL21(DE 3).

7. Use of the dioxygenase gene snpd according to claim 1 for degrading or converting S-2- (1-naphthoxy) propionic acid, 2, 4-dichlorophenoxyacetic acid, S-2- (2, 4-dichlorophenoxy) propionic acid and S-2- (4-hydroxyphenoxy) propionic acid.

8. Use of the dioxygenase Snpd according to claim 2 for the degradation or conversion of S-2- (1-naphthoxy) propionic acid, 2, 4-dichlorophenoxyacetic acid, S-2- (2, 4-dichlorophenoxy) propionic acid and S-2- (4-hydroxyphenoxy) propionic acid.

9. Use of the recombinant expression vector of claim 3 or 4 for degrading or transforming S-2- (1-naphthoxy) propionic acid, 2, 4-dichlorophenoxyacetic acid, S-2- (2, 4-dichlorophenoxy) propionic acid and S-2- (4-hydroxyphenoxy) propionic acid.

10. Use of the genetically engineered bacteria of claim 5 or 6 for degrading or transforming S-2- (1-naphthoxy) propionic acid, 2, 4-dichlorophenoxyacetic acid, S-2- (2, 4-dichlorophenoxy) propionic acid and S-2- (4-hydroxyphenoxy) propionic acid.