CN102174543A - Dioxygenase gene, clone expression thereof and conversion of pyrene and benzo [a] pyrene - Google Patents

Abstract

The invention relates to a dioxygenase gene, clone expression thereof and conversion of pyrene and benzo [a] pyrene by virtue of engineering bacteria. The dioxygenase can not only convert tetracyclic polycyclic aromatic hydrocarbon-pyrene but also convert pentacyclic benzo [a] pyrene, thus being of certain value in the genetic engineering bacteria restoration research of epipodium polycyclic aromatic hydrocarbon.

Description

A kind of dioxygenase gene and clonal expression thereof and to the conversion of pyrene and benzo [a] pyrene

Technical field

The invention belongs to environmental organic pollutant microorganism recovery technique field, be particularly related to a kind of dioxygenase gene and clonal expression thereof and the conversion of pyrene and benzo [a] pyrene.

Background technology

Polycyclic aromatic hydrocarbons is the organic pollutant that a class extensively exists in environment, and wherein numerous species all has carcinogenic, teratogenesis, mutagenic " three cause " effect.And high ring polycyclic aromatic hydrocarbon has lower water-soluble and stronger adsorptivity because of it, and it is not high to make it in environment biological effectiveness, thereby causes persistence residual.In physical environment, the main cancellation of polycyclic aromatic hydrocarbons is microbiological deterioration, comprises the effect of fungi and bacterium.Fungi is not thorough to the degraded of polycyclic aromatic hydrocarbons, often just converts it into a water-soluble higher product.Relative fungi, bacterium then has the ability that the thorough mineralising of polycyclic aromatic hydrocarbons can be become carbonic acid gas, therefore has more reparation potential.

Bacterium mineralising polycyclic aromatic hydrocarbons is a biochemistry metabolism process by multiple enzyme fellowship.Wherein relate to multiple biochemical reactions such as two oxygenations, dehydrogenation, the two oxygenations of open loop.In this mineralising approach, by dioxygenase the catalytic pair of oxygenation step be the first step in the polycyclic aromatic hydrocarbons mineralisation process, also be the rate-limiting step in the polycyclic aromatic hydrocarbons mineralising.Therefore the two oxygenation control from view of profit of polycyclic aromatic hydrocarbons the mineralisation process of whole polycyclic aromatic hydrocarbons.Therefore, obtain the dioxygenase that high ring polycyclic aromatic hydrocarbon is carried out two oxygenation effects and have the meaning of important theoretical research and practical application.

Summary of the invention

Technical purpose of the present invention is to clone the dioxygenase gene that can transform high ring polycyclic aromatic hydrocarbon, this enzyme is expressed and utilize engineering bacteria verify its to pyrene and benzo [ a] transformation of pyrene.

In order to realize technical purpose of the present invention, technical program of the present invention lies in:

One, a kind of dioxygenase gene is characterized in that this enzyme gene has the nucleotide sequence shown in the SEQ ID NO:1; Its corresponding amino acid sequence is made up of the Dare's basic sequence shown in small subunit sequence shown in the SEQ ID NO:2 and the SEQ ID NO:3.

Two, the cloning and expression of dioxygenase gene of the present invention specifically may further comprise the steps:

(1) with pyrene degradation bacteria mycobacterium MycobacteriumSp. the NJS-P genome is a template, and according to the dioxygenase conserved sequence of prior art, the design degenerated primer amplifies the gene fragment that comprises the dioxygenase gene entire reading frame by round pcr, carries out the TA clone, checks order; Design primer again, from the TA carrier that comprises the dioxygenase gene entire reading frame, amplify the dioxygenase gene entire reading frame, obtain the nucleotide sequence shown in SEQ ID NO:1, its corresponding amino acid sequence is made up of the Dare's basic sequence shown in small subunit sequence shown in the SEQ ID NO:2 and the SEQ ID NO:3;

(2) the TA carrier that will contain the dioxygenase gene entire reading frame carries out enzyme and cuts, and is connected with the pET15b expression vector that same enzyme is cut, make up the dioxygenase expression vector PdoAB-15b; And then with plasmid PdoAB-15b and pBRCD cotransformation that electron carrier protein is provided make up genetic engineering bacterium in e. coli bl21 (DE3), and abduction delivering goes out the PdoAB soluble proteins in substratum, is dioxygenase of the present invention.

Three, engineering bacteria of the present invention is to the conversion of pyrene and benzo [a] pyrene.

Concrete grammar is:

1) centrifugal collection is through protein induced engineering bacteria, cleans also suspended bacteria body again with the M9 substratum of about 1/10 volume;

2) by 10% amount resuspended thalline is inserted in silicone oil-water diphasic system, its transformation system component comprise 25 mL M9 substratum (containing penbritin and gentamicin) and 5 mL contain the pyrene of 100 mg/L or the benzo of 50 mg/L [ a] pyrene;

3) cultivated 2 days for 28 ℃.

Beneficial effect of the present invention is:

Method for transformation involved in the present invention is full cell transformation system, has exempted the extraction step of enzyme; And the dioxygenase that the present invention obtains not only can transform Fourth Ring polycyclic aromatic hydrocarbons-pyrene, and can also transform benzo [a] pyrene at five rings, has certain researching value aspect the genetic engineering bacterium reparation research of high ring polycyclic aromatic hydrocarbon.

Description of drawings

Fig. 1 is dioxygenase of the present invention conversion mass spectrometric detection figure to pyrene.

Fig. 2 be dioxygenase of the present invention to benzo [ a] the conversion mass spectrometric detection figure of pyrene.

Embodiment

Related genetic manipulation method is with reference to " molecular cloning experiment guide " (Huang Peitang etc. translate, Science Press for the third edition, J. Sa nurse Brooker D.W. Russell work) among the embodiment

Embodiment 1

Present embodiment illustrates the preparation method of dioxygenase gene sequence of the present invention.

With pyrene degradation bacteria mycobacterium MycobacteriumSp. (this bacterial strain was stored in Chinese typical culture collection center to NJS-P on January 10th, 2011, abbreviate CCTCC as, being numbered CCTCC NO:M 2011011) genome is template, according to the dioxygenase of having reported (ncbi database: conserved sequence http://www.ncbi.nlm.nih.gov/), the design degenerated primer, amplify the gene fragment that comprises the dioxygenase gene entire reading frame by round pcr, carry out the TA clone, check order; Design primer again, amplify the dioxygenase gene entire reading frame from the TA carrier that comprises the dioxygenase gene entire reading frame, concrete operations are as follows:

1) extracts mycobacterium NJS-P genomic dna.

2) design degenerated primer PdoAB-F and PdoAB-R amplifies by round pcr with high-fidelity enzyme PrimerSTAR HS (Takara) CueOGene complete is read frame.Primer sequence is:

pdoAB-F: 5’-TAGGATCCAGAGGAGTTCGRTKTGATGAAC-3’

pdoAB-R: 5’-TAGAATTCCAGAAKCKTCATCRAGCAC-3’。

3) pcr amplification product is added A and handle, use p EASY-T1 Simple Cloning Kit (Transgen) carries out TA clone, order-checking.

4) design degenerated primer again Ex-pdoAB-F and Ex-pdoAB-R, introduce respectively at upstream and downstream primer two ends NcoI and BamThe HI restriction enzyme site, amplify the dioxygenase gene entire reading frame among the TA clone who from step 3, is obtained with high-fidelity enzyme PrimerSTAR HS (Takara), obtain the nucleotide sequence shown in SEQ ID NO:1, this dioxygenase gene has the aminoacid sequence of the big small subunit shown in SEQ ID NO:2 and SEQ ID NO:3 respectively as can be known.Wherein, primer sequence is:

Ex-pdoAB-F: 5’- GTAT CCATGG GCAACGCGGTCGCGGTGGAC-3’

Ex-pdoAB-R: 5’- AC GGATCC TCATCGAGCACCGCCGCGGAACTG-3’。

Embodiment 2

The method that the present embodiment explanation is expressed dioxygenase on the basis of embodiment 1.

The TA carrier that will contain the dioxygenase gene entire reading frame carries out enzyme to be cut, and is connected with the pET15b expression vector (Novagen) that same enzyme is cut, make up the dioxygenase expression vector PdoAB-15b.And then with plasmid PdoAB-15b and provide pBRCD (being so kind as to give) cotransformation of electron carrier protein in e. coli bl21 (DE3) (available from the full formula in Beijing King Company) by Dr. Jouanneau, make up genetic engineering bacterium, abduction delivering goes out the PdoAB soluble proteins in substratum, and concrete operations are as follows:

1) extracts TA cloned plasmids and the expression vector plasmid pET15b that the 4th step obtained among the embodiment 1.

2) use NcoI and BamHI(Fermentas) two plasmids are carried out enzyme and cut, reclaim required dna fragmentation.

3) in DNA: the ratio of carrier=1:3, with dna ligase (Takara) with the endonuclease bamhi connection of spending the night, make up the bacterial laccase expression vector PdoAB-15b.

4) with plasmid PdoAB-15b with and the pBRCD cotransformation to expressive host bacterium e. coli bl21 (DE3), obtain genetic engineering bacterium.

5) with containing the two anti-Luria-Bertani(LB of penbritin and gentamicin) the flat board activation engineering bacteria that spends the night, picking list bacterium colony is in 100 mL LB liquid nutrient mediums (containing microbiotic), and 37 ℃ are cultured to OD ₆₀₀=0.6～0.8, add 100 mg/L IPTG, spend the night in 25～28 ℃ and induce the PdoAB enzyme protein expression.

Embodiment 3

Present embodiment illustrates the method for transformation of described engineering bacteria to pyrene and benzo [a] pyrene.

4) centrifugal collection is through protein induced engineering bacteria, cleans also suspended bacteria body again with the M9 substratum of about 1/10 volume;

5) by 10% amount resuspended thalline is inserted in silicone oil-water diphasic system, its transformation system component comprise 25 mL M9 substratum (containing penbritin and gentamicin) and 5 mL contain the pyrene of 100 mg/L or the benzo of 50 mg/L [ a] pyrene;

6) cultivated 2 days for 28 ℃.

After product is through the silane derivatize, adopt GC/MS to carry out product analysis, concrete operations are as follows:

1) conversion finishes, and conversion fluid equal-volume ethyl acetate extracts at twice.

2) combining extraction liquid with the organic solvent evaporate to dryness, adds 1 mL n-hexane dissolution with rotary evaporation method (30 ℃).Lysate is crossed 0.22 μ m filter membrane, dries up with nitrogen again, finally uses 100 μ L n-hexane dissolutions.

3) add 100 μ L TMS silylating reagents (supelco), carried out the silane derivatize in 1 hour in 60 ℃.

4) the silanization product carries out product analysis with gas phase-mass spectrometry, detects (seeing attached Fig. 1 and 2) with the ion selection mode.It is 290(monohydroxy product that the converted product of pyrene is selected ion) and 380(dihydrodiol product); Benzo [ a] to select ion be 340(monohydroxy product for the converted product of pyrene).

Sequence table

＜110〉Nanjing Soil Inst., Chinese Academy of Sciences

＜120〉a kind of dioxygenase gene and clonal expression thereof and to the conversion of pyrene and benzo [a] pyrene

＜130〉Nanjing Soil Inst., Chinese Academy of Sciences

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 1950

<212> DNA

<213> Artificial Sequence

<220>

＜223〉dioxygenase gene pdoAB

<400> 1

atgaacgcgg tcgcggtgga ccgggatacc cgtgagtccg tcgaggactt catgtttcgg 60

gaagcggagt tgctcgatgg aggacagttc cgggactggt tgggcctgct tgaccccgac 120

atccggtatg tggttccggt gcgcaccacc cgggaggact ccgcgggttg ggtgagcgcg 180

atcgcgcact ggaacgacga ttacacgggc ttggagatgc gggtcctgcg cagcgagacg 240

gagttctcat gggcggagtc acctcgatcc cggacccgcc acttcgtgtc caatatccgc 300

accactgccg gacctggggc tgatgagttg accgtgcggt cgaacctgtt gttcttccgc 360

agccgcggag ctagcggacg gtgggagttg ctgtcggctg aacgcgtcga cgtgctgcgc 420

cggagcgacg ggtcgctgcg gctcgctcgg cgtgaggtct tgctcgacca ttcgacgctg 480

cctatcgaca acctgtcggt agtcctgtag ccacgctctc tcgccgtcgt cgtccttgaa 540

agcactgcct gtccagaaag gtccgatcac atgaccaccg aaacaaccga gaccaccgga 600

accgctggca cgagtgatcg ctacctgcgg cgcgcgttgc gcgacgtagc ggacgggctc 660

aaggtcgggc gactacccgc acgcgtcgtc agcgatccga cgctccatac gattgagatg 720

gagcggatct tcgggcgcgc ctgggtgttc ctcgcgcacg agtcggagtt ggccaagtcc 780

ggcgactttg tggtgcgtta tatcggggcc gattcggtga tcgtctgccg ggacgcctcc 840

ggccagatcc aagcgctgtc aaattcttgt cgacaccgtg gcgctctcgt gtgccgtgcg 900

gagacgggaa acaccgccca cttccagtgc ccttaccacg gctgggtgta cagcaatacc 960

ggggatctcg tcggtgtgcc agcgatgagg gaggcctatc ccggcggttt cgataagacg 1020

cagtggggat tgcgccacat cccccatgtc gactcgtatg ccggattcat cttcggtagc 1080

gtcgatccga aggcgccgaa cctgaccgac tatctcggcg acacgacgtt ttacctcgac 1140

ctcattgcga agaagaccgc gggcggtctg gaggtgatag gggcgccgca tcggtggact 1200

atgtctgcga actggaagac ggccgccgac aatttcgtcg gcgactccta ccacaccctt 1260

ttcgctcacc gctctatggt ggagctgggc atggctccgg gtgacccgaa cttcgcgagc 1320

gcacccgcgg agatctcgct gcagaacggt cacggcgtag gtgtgctcgg gtttccgccc 1380

acgctcgccg atttcccaga atacgaggga taccccgacg aagtcgtcga ccagatggcg 1440

gcgtcctacc cgtctttggc gcacaaggac atgatgcgac gctcagcctt cattcacggc 1500

acggtgttcc caaatttgtc cttcatcaat gtgaccatcg cgcctgacca catgtcgccc 1560

cccaccccgt ttatcacgtt ccggttgtgg cagccgctat ctcatgaccg gatggaggtc 1620

ctttcctggt tcctggtcga acgcgatgct ccggagtggc tacgcgaggc gtcgcaggcc 1680

tcctatgtca acaacttcgg tccggccggt gttttcgaac aggacgacgc ggaggcatgg 1740

aaggccatca cggaatctgt ccagggtccg ttcgccggtg aaggactcct gaactacgag 1800

atgggcatgg acctgactcc acttaatgac tggccaggac cgggcgaagc cctcccgagt 1860

gggtacgccg agcagaacca gcgacggttc tgggggcgat ggctggacta catgagccaa 1920

cctgcccagt tccgcggcgg tgctcgatga 1950

<210> 2

<211> 169

<212> PRT

<213> Artificial Sequence

<220>

＜223〉PdoB small subunit sequence

<400> 2

Met Asn Ala Val Ala Val Asp Arg Asp Thr Arg Glu Ser Val Glu Asp

1 5 10 15

Phe Met Phe Arg Glu Ala Glu Leu Leu Asp Gly Gly Gln Phe Arg Asp

20 25 30

Trp Leu Gly Leu Leu Asp Pro Asp Ile Arg Tyr Val Val Pro Val Arg

35 40 45

Thr Thr Arg Glu Asp Ser Ala Gly Trp Val Ser Ala Ile Ala His Trp

50 55 60

Asn Asp Asp Tyr Thr Gly Leu Glu Met Arg Val Leu Arg Ser Glu Thr

65 70 75 80

Glu Phe Ser Trp Ala Glu Ser Pro Arg Ser Arg Thr Arg His Phe Val

85 90 95

Ser Asn Ile Arg Thr Thr Ala Gly Pro Gly Ala Asp Glu Leu Thr Val

100 105 110

Arg Ser Asn Leu Leu Phe Phe Arg Ser Arg Gly Ala Ser Gly Arg Trp

115 120 125

Glu Leu Leu Ser Ala Glu Arg Val Asp Val Leu Arg Arg Ser Asp Gly

130 135 140

Ser Leu Arg Leu Ala Arg Arg Glu Val Leu Leu Asp His Ser Thr Leu

145 150 155 160

Pro Ile Asp Asn Leu Ser Val Val Leu

165

<210> 3

<211> 459

<212> PRT

<213> Artificial Sequence

<220>

＜223〉PdoA Dare basic sequence

<400> 3

Met Thr Thr Glu Thr Thr Glu Thr Thr Gly Thr Ala Gly Thr Ser Asp

1 5 10 15

Arg Tyr Leu Arg Arg Ala Leu Arg Asp Val Ala Asp Gly Leu Lys Val

20 25 30

Gly Arg Leu Pro Ala Arg Val Val Ser Asp Pro Thr Leu His Thr Ile

35 40 45

Glu Met Glu Arg Ile Phe Gly Arg Ala Trp Val Phe Leu Ala His Glu

50 55 60

Ser Glu Leu Ala Lys Ser Gly Asp Phe Val Val Arg Tyr Ile Gly Ala

65 70 75 80

Asp Ser Val Ile Val Cys Arg Asp Ala Ser Gly Gln Ile Gln Ala Leu

85 90 95

Ser Asn Ser Cys Arg His Arg Gly Ala Leu Val Cys Arg Ala Glu Thr

100 105 110

Gly Asn Thr Ala His Phe Gln Cys Pro Tyr His Gly Trp Val Tyr Ser

115 120 125

Asn Thr Gly Asp Leu Val Gly Val Pro Ala Met Arg Glu Ala Tyr Pro

130 135 140

Gly Gly Phe Asp Lys Thr Gln Trp Gly Leu Arg His Ile Pro His Val

145 150 155 160

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

165 170 175

Asn Leu Thr Asp Tyr Leu Gly Asp Thr Thr Phe Tyr Leu Asp Leu Ile

180 185 190

Ala Lys Lys Thr Ala Gly Gly Leu Glu Val Ile Gly Ala Pro His Arg

195 200 205

Trp Thr Met Ser Ala Asn Trp Lys Thr Ala Ala Asp Asn Phe Val Gly

210 215 220

Asp Ser Tyr His Thr Leu Phe Ala His Arg Ser Met Val Glu Leu Gly

225 230 235 240

Met Ala Pro Gly Asp Pro Asn Phe Ala Ser Ala Pro Ala Glu Ile Ser

245 250 255

Leu Gln Asn Gly His Gly Val Gly Val Leu Gly Phe Pro Pro Thr Leu

260 265 270

Ala Asp Phe Pro Glu Tyr Glu Gly Tyr Pro Asp Glu Val Val Asp Gln

275 280 285

Met Ala Ala Ser Tyr Pro Ser Leu Ala His Lys Asp Met Met Arg Arg

290 295 300

Ser Ala Phe Ile His Gly Thr Val Phe Pro Asn Leu Ser Phe Ile Asn

305 310 315 320

Val Thr Ile Ala Pro Asp His Met Ser Pro Pro Thr Pro Phe Ile Thr

325 330 335

Phe Arg Leu Trp Gln Pro Leu Ser His Asp Arg Met Glu Val Leu Ser

340 345 350

Trp Phe Leu Val Glu Arg Asp Ala Pro Glu Trp Leu Arg Glu Ala Ser

355 360 365

Gln Ala Ser Tyr Val Asn Asn Phe Gly Pro Ala Gly Val Phe Glu Gln

370 375 380

Asp Asp Ala Glu Ala Trp Lys Ala Ile Thr Glu Ser Val Gln Gly Pro

385 390 395 400

Phe Ala Gly Glu Gly Leu Leu Asn Tyr Glu Met Gly Met Asp Leu Thr

405 410 415

Pro Leu Asn Asp Trp Pro Gly Pro Gly Glu Ala Leu Pro Ser Gly Tyr

420 425 430

Ala Glu Gln Asn Gln Arg Arg Phe Trp Gly Arg Trp Leu Asp Tyr Met

435 440 445

Ser Gln Pro Ala Gln Phe Arg Gly Gly Ala Arg

450 455

Claims (3)

1. a dioxygenase gene is characterized in that this gene has the nucleotide sequence shown in the SEQ ID NO:1; Its corresponding amino acid sequence is made up of the Dare's basic sequence shown in small subunit sequence shown in the SEQ ID NO:2 and the SEQ ID NO:3.

2. the cloning and expression of dioxygenase gene according to claim 1 is characterized in that specifically may further comprise the steps:

(1) with pyrene degradation bacteria mycobacterium MycobacteriumSp. the NJS-P genome is a template, and according to the dioxygenase conserved sequence of prior art, the design degenerated primer amplifies the gene fragment that comprises the dioxygenase gene entire reading frame by round pcr, carries out the TA clone, checks order; Design primer again, from the TA carrier that comprises the dioxygenase gene entire reading frame, amplify the dioxygenase gene entire reading frame;

(2) the TA carrier that will contain the dioxygenase gene entire reading frame carries out enzyme and cuts, and is connected with the pET15b expression vector that same enzyme is cut, make up the dioxygenase expression vector PdoAB-15b; And then with plasmid PdoAB-15b and pBRCD cotransformation that electron carrier protein is provided make up genetic engineering bacterium in e. coli bl21 (DE3), and abduction delivering goes out the PdoAB soluble proteins in substratum, is dioxygenase albumen.

3. the engineering bacteria of expression dioxygenase according to claim 2 to pyrene and benzo [ a] conversion of pyrene, it is characterized in that described concrete grammar is:

(1) centrifugal collection is through protein induced engineering bacteria, cleans also suspended bacteria body again with the M9 substratum of 1/10 volume;

(2) by 10% amount resuspended thalline is inserted in silicone oil-water diphasic system, its transformation system component comprise 25 mL contain the benzo of the M9 substratum of penbritin and gentamicin and pyrene that 5 mL contain 100 mg/L or 50 mg/L [ a] pyrene;

Cultivated 2 days for (3) 28 ℃.

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