CN110283804A

CN110283804A - Esterase gene and coding albumen and application

Info

Publication number: CN110283804A
Application number: CN201910648324.9A
Authority: CN
Inventors: 万群; 徐文君; 李易芯; 余向阳
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2019-07-18
Filing date: 2019-07-18
Publication date: 2019-09-27

Abstract

The present invention provides a kind of esterase genes with wide spectrum degradation and its coding albumen and application, the esterase to derive from the esterase gene of bacillus subtilis, can be applied to a variety of phthalic acids of degrading；The present invention also constructs the engineering strain and plasmid that can express above-mentioned esterase, realizes heterogenous expression of the enzyme in Escherichia coli, lays a good foundation for its industrialized production.

Description

Esterase gene and coding albumen and application

Technical field

The invention belongs to technical field of bioengineering, be related to esterase gene, esterase, recombinant plasmid, engineering strain and It is applied.

Background technique

Esterase and lipase is all carboxylic acid hydrolase, is divided into 8 races.Esterase (Esterases) can be hydrolyzed less than 10 The ester bond of the short chain fatty acids of a carbon atom, and lipase (Lipases) can then hydrolyze the long-chain rouge more than 10 carbon atoms The ester bond of fat acid.Lipase and esterase are important industrialization enzyme preparation kind, can be reacted with catalyzing hydrolysis, transesterification, ester conjunction etc., It is widely used in the industry such as fats and oils processing, food, medicine, daily use chemicals.The esterase and lipase of separate sources has different catalysis Feature and catalysis activity.Wherein, the scale for the esterase and lipase with esterification or transesterification function of organic synthesis It produces product chemicals fine for Enzyme catalyzed synthesis and chipal compounds is significant.

The key effect of esterase is for synthetic drug intermediate, and the drug synthesized at present can be used for curing colon cancer, high Cholesterolemia etc..Esterase can also be used to production insecticide, and premise compound (+)-frans-ChA of Dalmatian chrysanthemum vinegar is in esterase Participation under synthesize.Industrially, various products are produced using the good regiospecificity of esterase and enantioselectivity, such as refined Work product, medicine, food etc..Esterase may also participate in drug, the metabolism of insecticide pollution.During degrading pesticide, esterase passes through The ester group for hydrolyzing pesticide, destroys the structure of pesticide, to reduce its toxicity.

Phthalic acid ester (Phthalateester, PAEs) is a substance the most used in plasticiser, is adjacent The compound (as shown in table 1 below) formed after the alcohol of phthalic acid and 4-15 carbon is esterified, 14 kinds of phthalic acid esters in table 1 The number of branched carbon atoms is respectively less than 8:

1 phthalate compound of table

The compound presents colourless or faint yellow at normal temperatures and pressures, and quality is oily thick liquid, and fluid temperature Range is wider, there is biggish mobility, and smell is special, is insoluble in water phase, is soluble in the organic phases such as methanol, acetonitrile, octanol-water Distribution coefficient (K_OW) high, fusing point is low, and boiling point is high, and stable structure, chemical structural formula is by a rigid plane basic ring and two plastic lines Property aliphatic side chains composition, belong to alkyl aromatic ester.Because of the property such as its compatibility, oil resistivity, electrical insulating property, cold resistance, processability Can be beneficial, therefore be widely used in plastic processes with high molecular material auxiliary agent, with reinforced plastics flexibility, in rubber The plastic products such as glue, adhesive, medical instrument, cosmetics, glue, carwash interior trim, furniture, pesticide, fragrance, preservative film, toy Middle application.

However, PAEs is toxic effect, it can promote peroxidation and generate cytotoxicity, and body is caused to be exempted from The harm such as epidemic disease power is low, leads to obesity, allergy, coronary heart disease, liver damage.Furthermore PAEs is also referred to as Environmental Hormone by people, tool There is estrogenic effect, severe jamming endocrine influences reproductive system, and the discovery DEHP exposure such as Lin Lin may increase pregnant early stage woman The probability that the weaker sex produces.

Research of the degrading enzyme to the PAEs that degrades is translated into gradually currently with degrading genes are filtered out in microbial DNA Increase, but since enzyme has specificity, so it is selective to the substrate of effect.Esterase is also the same in this way, at present The esterase of report can only have degradation capability to a few PAEs, and the esterase gene EstZ1 filtered out such as Peng Zhengcong can drop 50mg/mlDEP and DiBP is solved, EstZ22 has degradation property to DEP, DiBP, DBP and DCHP of 50mg/ml, but unknown Really point out degradation rate result (the esterase zymologic property in Peng Zheng bacillus source and to phthalate degradation it is preliminary Research [D] Yunnan Normal University, 2016.)；Wu etc. filters out one from acinetobacter Acinetobactersp.M673 Hydrolase gene, expression product can degrade 0.1mMDBP (WuJ, LiaoX, YuF, etal.Cloningofadibutylphthalate hydrolasegenefromAcinetobactersp.strainM673a ndfunctionalanalysis ofitsexpressionproductinEscherichiacoli[J] .AppliedMicrobiologyand Biotechnology,2013,97(6):2483-2491.)；The esterase base of square moon identification Because degradation rate of the Est-3563 translation product to 1mMDMP, DEP and DBP is 10-30% (Fang Yue plasticiser degradation bacteria The performance and its esterase zymology Quality Research [D] .2017. of Acinetobactersp.LMB-5).

Can degrade the PAEs that 14 kinds of branched carbon numbers are 1-8 in table 1 simultaneously at present, and the ester that degradation efficiency is relatively high Enzyme has not been reported.

Summary of the invention

In view of the above-mentioned problems, the present invention provides the esterase of wide spectrum degradation PAEs a kind of, the esterase can degrade 14 kinds it is common PAEs provides raw material sources for industrialized production esterase, the present invention is implemented as follows:

Firstly, the present invention provides a kind of nucleotide sequence esterase gene EST-T2 as shown in SEQ ID NO.1, the ester The esterase protein amino acid sequence of enzyme gene coding is as shown in SEQ ID NO.2.

Secondly, the present invention provides amino acid sequence esterases as shown in SEQ ID NO.2 in degradation phthalic acid ester In application；It is preferred that phthalic acid of the number of branched carbon atoms less than 8, such as: repefral (DMP), neighbour Diethyl phthalate (DEP), dimethoxyethyl phthalate (DMEP) diallyl phthalate (DAP), adjacent benzene Dioctyl phthalate dipropyl (DPRP), diisopropyl phthalate (DIPRP), dibutyl phthalate (DBP), O-phthalic Sour diisobutyl ester (DIBP), diamyl phthalate (DPP), phthalic acid tolyl butyl ester (BBP), phthalic acid two Cyclohexyl (DCHP), the own ester (DEHP) of dihexyl phthalate (DHP) phthalic acid two (2- ethyl), phthalic acid Di-n-octyl (DNOP) etc..

Third, the present invention also provides a kind of recombinant plasmid, the recombinant plasmid include expression vector and nucleotide sequence such as Esterase gene shown in SEQ ID NO.1；The expression vector is this field conventional plasmid carrier, such as carrier pET32b；The present invention The genetic engineering bacterium containing the recombinant plasmid is additionally provided simultaneously, the preferred Escherichia coli of the engineering bacteria.

Specifically, above-mentioned recombinant plasmid, engineered strain, esterase crude enzyme liquid obtain by the following method: (1) sharp Recombinant expression is synthesized with pET32b prokaryotic expression carrier with nucleotide sequence esterase EST-T2 gene as shown in SEQ ID NO.1 Plasmid pET32b-EST-T2；Recombinant expression plasmid is imported into conversion Escherichia coli TOP10 competent cell, is coated on containing 50 The LB plate of μ g/ml ampicillin (final concentration), 37 DEG C of overnight incubations, next day, picking positive colony are inoculated in containing 50 μ g/ In the LB liquid medium of ml ampicillin (final concentration), after 37 DEG C of overnight incubations, is extracted and recombinated with plasmid extraction kit Plasmid simultaneously carries out the bis- enzyme identifications of BamHI and HindIII, and 37 DEG C of water-baths are incubated for 3h, 1% agarose electrophoresis, while using pET32b (+) empty plasmid is as control；Clip size after digestion is correctly recombinated with the consistent i.e. acquisition digestion of EST-T2 gene size Plasmid；

(2) the correct recombinant plasmid of double digestion is selected, it is thin that recombinant plasmid is imported conversion Escherichia coli TOP10 competence Born of the same parents obtain transformed bacteria solution；The transformed bacteria solution is coated on the LB plate containing 50 μ g/ml ampicillins (final concentration), 37 DEG C Overnight incubation；Next day picking positive colony is inoculated in the LB liquid medium containing 50 μ g/ml ampicillins (final concentration) In, 37 DEG C of overnight incubations obtain recombination engineered strain；

(3) engineering strain is inoculated into the LB liquid medium containing 50 μ g/mL ampicillins (final concentration), 37 DEG C shaken overnight culture；Next day, 1:100 ratio is forwarded in 100mLLB culture medium by volume, 37 DEG C of shaken cultivations to OD₆₀₀ When value is 0.3, IPTG to final concentration of 0.2mmol/L is added, 30 DEG C of inductions are for 24 hours；Then thallus is collected, PBS buffer solution is added, Ultrasonic disruption cell obtains esterase crude enzyme liquid.

Application of the above-mentioned esterase in degradation phthalic acid ester, the specific method is as follows: by the esterase crude enzyme liquid of acquisition Middle addition PBS buffer solution (50molL^-1, pH7.5), it is then added and is dissolved in acetonitrile 14 kinds of PAEs (each PAEs concentration is 10mg/L), 1h is reacted in 37 DEG C of waters bath with thermostatic control, that is, completes the catalysis to PAEs.Crude enzyme liquid and PBS buffer solution volume ratio preferably 1: 4。

In addition, on the basis of the application discloses esterase gene sequence, nucleotide sequence, those skilled in the art can be with The esterase is synthesized by conventional method in that art, and 14 kinds of PAEs in table 1 are added in esterase, can be used to adjacent benzene two of degrading Formic acid esters.

Compared with existing esterase, esterase gene provided by the invention derives from bacillus subtilis, can be applied to drop simultaneously Solve a variety of phthalic acids；The present invention also constructs the engineering strain and plasmid that can express above-mentioned esterase, realizes this Heterogenous expression of the enzyme in Escherichia coli, lays a good foundation for the industrialized production of the esterase.

Detailed description of the invention

The SDS-PAGE figure that Fig. 1 is the esterase EST-T2 of purifying of the invention.

A figure: swimming lane M is Marker, and swimming lane 1 is bacteria suspension total protein before inducing, and swimming lane 2 is the total egg of bacteria suspension after induction It is white；B figure: swimming lane M is Marker, and swimming lane 1 is bacteria suspension centrifugation total protein before inducing, swimming lane 2 be after induction bacteria suspension from The heart precipitates total protein, and swimming lane 3 is bacteria suspension centrifuged supernatant total protein before inducing, after swimming lane 4 is bacteria suspension centrifugation after induction Supernatant total protein.

Fig. 2 is the effect diagram of 14 kinds of PAEs of esterase EST-T2 degradation of the invention.

Specific embodiment

Embodiment is related to the preparation method of culture medium, and following concentration mentioned that are formulated are final concentration:

LB plate formula: tryptone (Tryptone) 10g/L, yeast extract (Yeastextract) 5g/L, chlorination Sodium (NaCl) 10g/L, 15g/L agar powder finally adjusts pH=7.4, the steam sterilizing 20min under 15psi high pressure with NaOH； 10ml falls 1 culture dish, after culture medium pours into culture dish, opens lid, it is ultraviolet it is lower according to 10-15 minutes after it is spare.

LB liquid medium formula: tryptone (Tryptone) 10g/L, yeast extract (Yeastextract) 5g/ L, sodium chloride (NaCl) 10g/L finally adjust pH=7.4 with NaOH, spare after steam sterilizing 20min under 15psi high pressure.

The building of the clone, expression vector of 1 esterase EST-T2 gene of embodiment

(a) according to the genome sequence of bacillus subtilis endophyte HB-T2, clone's degradation PAE esterase gene EST- T2, nucleotide sequence is as shown in Seq ID NO.1.

Respectively as shown in SEQ ID NO.3 and SEQ ID NO.4, two primers are respectively provided with upstream and downstream primer sequence BamHI and HindIII restriction enzyme site expands the complete sequence of EST-T2 gene.

Amplification system: (Beijing Qing Kexin industry Bioisystech Co., Ltd kit (TSE003):: 2 × TSINGKE^TM MasterMix25ul；10uMprimerF/R1ul；DNA<50ng；ddH₂Oupto50ul)；Amplification program: 94 DEG C of initial denaturations 5min；Loop parameter is 94 DEG C of denaturation 30s, 57 DEG C of annealing 30s, 72 DEG C of extension 90s；Run 30 circulations；72 DEG C of extensions 10min, 4 DEG C of stable 1min reactions terminate.

By after purification PCR product (Beijing Qing Kexin industry Bioisystech Co., Ltd: PCR purification kit TSP301, Purification step is carried out referring to kit specification) and pET32b carrier (Invitrogen) carries out BamHI simultaneously and HindIII is bis- Then digestion is attached, the recombinant plasmid of building is named as pET32b-EST-T2.

Double digestion system (20 μ l): 10Xbuffer:2ul；BamHI(Takara:1010S)1ul； HindIII (Takara:1060S)1ul；PCR product (500ng) after purification, pET32b carrier (1ug), with ddH₂O complements to 20 μ l；

Double enzyme digestion reaction: 37 DEG C of reaction 2h obtain recombinant plasmid 32b-EST-T2.

(b) recombinant expression plasmid 32b-EST-T2 is imported into conversion Escherichia coli TOP10 competent cell (purchased from Tiangeng life Change Science and Technology Ltd., model C B104, concrete operation step is carried out according to product description), then by the large intestine bar after conversion Bacterium is coated on the LB plate containing ampicillin (50 μ g/ml), 37 DEG C of overnight incubations, and picking positive colony is inoculated in containing ammonia In the LB liquid medium of parasiticin (50 μ g/ml), after 37 DEG C of overnight incubations, with plasmid extraction kit, (Guangzhou Dongsheng is raw Object Science and Technology Ltd.) it extracts recombinant plasmid (concrete operations are illustrated to carry out by kit) and carries out the bis- enzymes of BamHI and HindIII Identification (is purchased from NEB company, specific authentication step prepares 50 μ l endonuclease reaction systems according to operating instruction), and 37 DEG C of water-baths are incubated for 3h, 1% agarose electrophoresis, while using pET32b (+) empty plasmid as control, molecular size is consistent with expection (it is expected that size is EST-T2 gene is 54kD, in addition carrier protein, the two adds up to size to be 72kD), show that building is correct.

(c) double digestion is identified that correct plasmid delivers sequencing company sequencing, sequencing is identified that correct plasmid is named as PET32b-EST-T2, plasmid is transformed into e. coli bl21 competent cell, and (Tiangeng biochemistry Co., Ltd product, is specifically pressed Operating instruction carries out), the bacterium solution after conversion is coated on the LB plate containing ampicillin (50 μ g/ml), 37 DEG C were cultivated Night；Next day picking positive colony is inoculated in the LB liquid medium containing ampicillin (50 μ g/ml), and 37 DEG C were cultivated Night, recombinant bacterium are named as BL21 (pET32b-EST-T2)

The inducing expression and purifying of 2 esterase EST-T2 albumen of embodiment

Recombinant bacterium BL21 (pET32b-EST-T2) is inoculated into the training of the 4mlLB liquid containing ampicillin (50 μ g/mL) It supports in base, 37 DEG C of shaken overnight cultures；Next day is forwarded in 100mLLB fluid nutrient medium in 1:100 ratio, 37 DEG C of oscillation trainings It supports to OD₆₀₀When value is 0.5, IPTG to final concentration 0.2mmol/L is added, 30 DEG C of inductions are for 24 hours；By the Escherichia coli of expression 4 At DEG C after 8000rpm centrifugation, liquid is discarded supernatant, the PBS buffer solution of 20mL is added by thallus resuspension and is centrifuged 3 times repeatedly again, finally Thallus is resuspended the PBS buffer solution that 20mL is added, and the broken (ultrasound condition are as follows: super of Ultrasonic Cell Disruptor is utilized under ice-water bath 2 s of sound time, intermittent time 8s, work are set as 10min at total time), complete crude enzyme liquid preparation.It will be before induction and after induction Crude enzyme liquid and crude enzyme liquid 8000rpm are centrifuged the supernatant precipitating progress SDS-PAGE electrophoresis after 10m.Electrophoretogram is as shown in Figure 1.Figure In 1A: swimming lane M is Marker, and swimming lane 1 is bacteria suspension total protein before inducing, and swimming lane 2 is bacteria suspension total protein after induction；Figure 1B In: swimming lane M is Marker, and swimming lane 1 is bacteria suspension centrifugation total protein before inducing, and swimming lane 2 is heavy for bacteria suspension centrifugation after induction Shallow lake total protein, swimming lane 3 are bacteria suspension centrifuged supernatant total protein before inducing, and swimming lane 4 is supernatant after bacteria suspension centrifugation after induction Liquid total protein.

Electrophoresis result, which is shown, has successfully induced esterase EST-T2 albumen, and esterase EST-T2 is present in supernatant, lures The albumen size expressed after leading is 73kDa (the as 54kDa+ carrier protein 19kDa of esterase EST-T2).

The analysis of 3 esterase EST-T2 of embodiment degradation PAEs spectrum

Crude enzyme liquid 1mL made of Example 2, which is added in 4mLPBS buffer, forms 5mL reaction system, and addition is dissolved in The mixed solution of 14 kinds of PAEs makes the various PAEs concentration in reaction system be 10mg/L in table 1 in acetonitrile, then 37 1h is reacted in DEG C water bath with thermostatic control, GC-MS is utilized to verify PAEs content.Using the crude enzyme liquid of pET32b zero load as blank control, each Processing is repeated 3 times.

The extraction and analytical method of 14 kinds of PAEs: the acetonitrile of equivalent is added into reaction system to be measured, in 150 r/min shaking tables Concussion extraction 40min, takes out and 15gNaCl is added, and vortex 2min makes to be layered, and draws 1mL supernatant in 10mL glass centrifuge tube In, 10 times are diluted after being settled to 1mL with trifluoroacetic acid aqueous solution after being dried with nitrogen, crosses 0.22 μm of organic phase filter membrane, are measured to GC-MS. 14 kinds of PAEs contents are detected using GC-MS, testing conditions are shown in Table 2 and table 3.

2 14 kinds of PAEs gas chromatographic detection conditions of table

The mass spectrometry parameters of 3 14 kinds of PAEs of table

Note: " * " indicates quota ion

Degradation rate calculation formula are as follows:

In the present embodiment, the extraction and analytical method of PAEs is the conventional method of this field, and specific steps also may refer to Document " is offered: Wang H, Liang H, Gao D W.Occurrence and risk assessment of phthalate esters(PAEs)in agricultural soils of the Sanjiang Plain, northeast China[J] .Environmental Science and Pollution Research, 2017,24 (24): 1-10.) " in report.

Testing result as shown in Fig. 2, discovery esterase EST-T2 is 87-99% to the PAEs degradation rate of 10ppmmg/L, Middle DMP:97%；DEP:97%；DAP:97%；DIPRP:98%；DMEP:99%；DPRP:95%；DBP:97%；DIBP: 96%；DPP:98%；BBP:98%；DCHP:91%；DHP:98%；DEHP:88%；DNOP:92%.This example demonstrates that ester Enzyme EST-T2 to table 1 list 14 in PAEs all have degradation effect, expanded the application field of esterase.

Sequence table

<110>Jiangsu Province Agriculture Science Institute

<120>esterase gene and coding albumen and application

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1467

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

atgactcatc aaatagtaac gactcaatac ggcaaagtaa aaggcacaac ggaaaacggc 60

gtacataagt ggaaaggcat cccctatgcc aagccgcctg tcggacaatg gcgttttaaa 120

gcacctgagc cgcctgaagt gtgggaagat gtgcttgatg ccacagcgta cggctctatt 180

tgcccgcagc cgtctgattt gctgtcactt tcgtatactg agctgccccg ccagtccgag 240

gattgcttgt atgtcaatgt atttgcgcct gacaccccaa gtaaaaatct tcctgtcatg 300

gtgtggattc acggaggcgc tttttatcta ggagcgggca gtgagccatt gtatgacgga 360

tcaaaacttg cggcacaggg agaagtcatt gtcgttacat tgaactatcg gctggggccg 420

tttggctttt tgcacttgtc ttcatttaat gaggcgtatt ctgataacct tgggctttta 480

gaccaagccg ccgcgctgaa atgggtgcga gagaatattt cagcgtttgg cggtgatccc 540

gataacgtaa cagtatttgg agaatccgcc ggcgggatga gcattgccgc gctgcttgct 600

atgcctgcgg caaaaggcct gttccagaaa gcaatcatgg aaagcggcgc ttctcgaacg 660

atgacgaaag aacaagcggc gagcacctcg gcagcctttt tacaggtcct tgggattaac 720

gagggccaac tggataaatt gcatacggtt tctgcggaag atttgctaaa agcggctgat 780

cagcttcgga ttgcagaaaa agaaaatatc tttcagctgt tcttccagcc cgcccttgat 840

ccgaaaacgc tgcctgaaga accagaaaaa gcgatcgcag aaggggctgc ttccggtatt 900

ccgctattaa ttggaacaac ccgtgatgaa ggatatttat ttttcacccc ggattcagac 960

gttcattctc aggaaacgct tgatgcagcg ctcgagtatt tactagggaa gccgctggca 1020

gagaaagttg ccgatttgta tccgcgttct ctggaaagcc aaattcatat gatgactgat 1080

ttattatttt ggcgccctgc cgtcgcctat gcatccgcac agtctcatta cgcccctgtc 1140

tggatgtaca ggttcgattg gcacccgaag aagccgccgt acaataaagc gtttcacgca 1200

ttagagcttc cttttgtctt tggaaatctg gacggattgg aacgaatggc aaaagcggag 1260

attacggatg aggtgaaaca gctttctcac acgatacaat cagcgtggat cacgttcgcc 1320

aaaacaggaa acccaagcac cgaagctgtg aattggcctg cgtatcatga agaaacgaga 1380

gagacgctga ttctagactc agagattacg atcgaaaacg atcccgaatc tgaaaaaagg 1440

cagaagctat tcccttcaaa aggagaa 1467

<210> 2

<211> 489

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 2

Met Thr His Gln Ile Val Thr Thr Gln Tyr Gly Lys Val Lys Gly Thr

1 5 10 15

Thr Glu Asn Gly Val His Lys Trp Lys Gly Ile Pro Tyr Ala Lys Pro

20 25 30

Pro Val Gly Gln Trp Arg Phe Lys Ala Pro Glu Pro Pro Glu Val Trp

35 40 45

Glu Asp Val Leu Asp Ala Thr Ala Tyr Gly Ser Ile Cys Pro Gln Pro

50 55 60

Ser Asp Leu Leu Ser Leu Ser Tyr Thr Glu Leu Pro Arg Gln Ser Glu

65 70 75 80

Asp Cys Leu Tyr Val Asn Val Phe Ala Pro Asp Thr Pro Ser Lys Asn

85 90 95

Leu Pro Val Met Val Trp Ile His Gly Gly Ala Phe Tyr Leu Gly Ala

100 105 110

Gly Ser Glu Pro Leu Tyr Asp Gly Ser Lys Leu Ala Ala Gln Gly Glu

115 120 125

Val Ile Val Val Thr Leu Asn Tyr Arg Leu Gly Pro Phe Gly Phe Leu

130 135 140

His Leu Ser Ser Phe Asn Glu Ala Tyr Ser Asp Asn Leu Gly Leu Leu

145 150 155 160

Asp Gln Ala Ala Ala Leu Lys Trp Val Arg Glu Asn Ile Ser Ala Phe

165 170 175

Gly Gly Asp Pro Asp Asn Val Thr Val Phe Gly Glu Ser Ala Gly Gly

180 185 190

Met Ser Ile Ala Ala Leu Leu Ala Met Pro Ala Ala Lys Gly Leu Phe

195 200 205

Gln Lys Ala Ile Met Glu Ser Gly Ala Ser Arg Thr Met Thr Lys Glu

210 215 220

Gln Ala Ala Ser Thr Ser Ala Ala Phe Leu Gln Val Leu Gly Ile Asn

225 230 235 240

Glu Gly Gln Leu Asp Lys Leu His Thr Val Ser Ala Glu Asp Leu Leu

245 250 255

Lys Ala Ala Asp Gln Leu Arg Ile Ala Glu Lys Glu Asn Ile Phe Gln

260 265 270

Leu Phe Phe Gln Pro Ala Leu Asp Pro Lys Thr Leu Pro Glu Glu Pro

275 280 285

Glu Lys Ala Ile Ala Glu Gly Ala Ala Ser Gly Ile Pro Leu Leu Ile

290 295 300

Gly Thr Thr Arg Asp Glu Gly Tyr Leu Phe Phe Thr Pro Asp Ser Asp

305 310 315 320

Val His Ser Gln Glu Thr Leu Asp Ala Ala Leu Glu Tyr Leu Leu Gly

325 330 335

Lys Pro Leu Ala Glu Lys Val Ala Asp Leu Tyr Pro Arg Ser Leu Glu

340 345 350

Ser Gln Ile His Met Met Thr Asp Leu Leu Phe Trp Arg Pro Ala Val

355 360 365

Ala Tyr Ala Ser Ala Gln Ser His Tyr Ala Pro Val Trp Met Tyr Arg

370 375 380

Phe Asp Trp His Pro Lys Lys Pro Pro Tyr Asn Lys Ala Phe His Ala

385 390 395 400

Leu Glu Leu Pro Phe Val Phe Gly Asn Leu Asp Gly Leu Glu Arg Met

405 410 415

Ala Lys Ala Glu Ile Thr Asp Glu Val Lys Gln Leu Ser His Thr Ile

420 425 430

Gln Ser Ala Trp Ile Thr Phe Ala Lys Thr Gly Asn Pro Ser Thr Glu

435 440 445

Ala Val Asn Trp Pro Ala Tyr His Glu Glu Thr Arg Glu Thr Leu Ile

450 455 460

Leu Asp Ser Glu Ile Thr Ile Glu Asn Asp Pro Glu Ser Glu Lys Arg

465 470 475 480

Gln Lys Leu Phe Pro Ser Lys Gly Glu

485

<210> 3

<211> 33

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

cgcggatccc atgactcatc aaatagtaac gac 33

<210> 4

<211> 32

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

cccaagcttc ttctcctttt gaagggaata gc 32

Claims

1. a kind of nucleotide sequence esterase gene as shown in SEQ ID NO.1.

2. the esterase of esterase gene coding as described in claim 1, amino acid sequence is as shown in SEQ ID NO.2.

3. application of the esterase as claimed in claim 2 in degradation phthalic acid ester.

4. application as claimed in claim 3, which is characterized in that the number of the branched carbon atoms of the phthalic acid ester is less than 8。

5. a kind of recombinant plasmid, which is characterized in that the recombinant plasmid includes expression vector and esterase base described in claim 1 Cause.

6. recombinant plasmid as claimed in claim 5, which is characterized in that the expression vector is pET32b.

7. a kind of engineering bacteria containing recombinant plasmid described in claim 5.