CN101935669B - Detoxication engineering bacteria, and preparation method and application thereof - Google Patents

Abstract

The invention discloses detoxication engineering bacteria, and a preparation method and application thereof. The invention provides a recombinant plasmid which is obtained by introducing coded genes of detoxication esterase and coded genes of hydrolase into an enzyme cutting site in plasmid pETDuet-1. The engineering bacteria of the invention can be obtained by introducing the recombinant plasmid into host bacteria. The engineering bacteria comprise the coded genes of the detoxication esterase and the hydrolase and can realize inducible expression of the detoxication esterase and the hydrolase. Crude enzyme solution obtained by fermentation of the engineering bacteria can also be protected. The engineering bacteria and the crude enzyme solution thereof can simultaneously degrade organic acid esters such as malathion, parathion, high-efficiency cypermethrin, acetofenate and the like and organophosphorus pesticides, and can be used for detoxication of organophosphorus poisoned warm-blooded animals. The invention provides a new way for biological pollution treatment of toxic and harmful compounds such as pesticides remained in water sources, soil and the like by using natural resources of eukaryote and prokaryote.

Description

A kind of detoxifcation engineering bacteria and its preparation method and application

Technical field

The present invention relates to a kind of detoxifcation engineering bacteria and its preparation method and application.

Background technology

Modern agricultural production is that the hope that satisfies people's grain high yield has been brought into play huge effect; once being regarded as third world countries lifts oneself out of poverty and hungry miraculous cure; yet in the face of the agricultural byproducts quality in the modern agriculture descends; species diversity reduces; in the face of being becoming increasingly rampant of diseases and pests of agronomic crop; the waste of agricultural resource; the degeneration of agricultural environment; in the face of being growing on and on of African crisis in food; people are after joyful; produced gradually a kind of puzzlement; a kind of doubt; the pros and cons of beginning re-examine modern agriculture; re-recognize the intension of Agricultural resources and environment, again think deeply the direction of agri-scientific research and decision-making.

The application of sterilant (being agricultural chemicals) is a double-edged sword.On the one hand; farm crop are eaten in invading that insect is a large amount of; for example in India; almost 30% farm crop are invaded food by insect; and the global crisis that is short of food requires farm crop to get bumper crops to satisfy human survivability requirement; thereby more and more extensive as the application of the protection employed sterilant of farm crop on agricultural, accounted for 3% of worldwide consumption.On the other hand, the excessive use of sterilant causes environment to be subject to pesticidal contamination, causes simultaneously insecticide resistance and animal and the interior residue of pesticide of farming object of the insect of global range.This contradiction will be the sharp-pointed problem that will need to be resolved hurrily future.

Sterilant commonly used mainly contains organic phosphates, amino formate and pyrethroid three major types, all can cause neurotoxicity to Mammals.Being prevalent in the detoxification that the esterase in the various tissues in the insect body (Esterase is called for short EST) participates in the ester insecticides, is can be to one of important lytic enzyme of sterilant detoxifcation.Different lytic enzymes has separately specific site of action, and interacts with it, and the key between macromolecular cpd is hydrolyzed, and makes it to become micromolecular compound or nontoxic ion.Now separating the phosphoric triesterase that obtains in the microorganism cells is the organophosphor hydrolytic enzyme (OPH) of a kind of bacterium, can effectively be hydrolyzed organophosphorus compound, significantly reduce its toxicity, but it can not hydrolysising carboxy acid ester's key, thereby does not have the toxicity reduction effect for the sterilant of amino formate and pyrethroid.

China produces and uses the big country of agricultural chemicals especially as an agriculture production big country.When a large amount of use agricultural chemicals are subjected to the disease and pest loss to alleviate agricultural-food, also brought day by day serious Pesticide Residue.The residual food of long-term edible high farming can form chronic nervosa and poison, and serious meeting causes the illnesss such as senile dementia.Because using, China's agricultural chemicals lacks standardized administration, Practice for Pesticide Residue in Agricultural Products generally exceeds standard, and causes the agricultural products in China foreign export to suffer heavy losses, traditional exported product, such as fruit, tealeaves and herbal medicine etc. in enormous quantities the return of goods occurs repeatedly, export volume is seriously restricted.In addition, the vegetables that high farming is residual and fruit also can be given the people healthyly brings great harm, add up according to hygiene department, 1～October in 1999, receive altogether 78 of food poisoning reports, Poisoning Number 4394 people, 79 people are dead, wherein cause 1108 people that poison by agricultural chemicals, dead 59 people surpass 70% of death toll.Statistics also shows, in recent years in the food poisoning number, the shared ratio of the poisoning that is caused by pesticide residue is more and more higher.

Summary of the invention

The purpose of this invention is to provide a kind of detoxifcation engineering bacteria and its preparation method and application.

The invention provides a kind of recombinant plasmid, is to import the recombinant plasmid that the encoding gene of the encoding gene of detoxifcation esterase and lytic enzyme obtains in the multiple clone site of plasmid pETDuet-1.

Described recombinant plasmid specifically can be pETDuet-E4-mpd.PETDuet-E4-mpd is with among the plasmid pETDuet-1, and the small segment between BamHI and HindIII restriction enzyme site replaces with the encoding gene of detoxifcation esterase, and the small segment between BglII and XhoI restriction enzyme site replaces with the encoding gene of lytic enzyme, the recombinant plasmid that obtains.

Described detoxifcation esterase can be following (a) or protein (b):

(a) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 1;

(b) with the aminoacid sequence of sequence 1 through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and have detoxifcation esterase function by the derivative protein of sequence 1;

Described lytic enzyme can be following (c) or protein (d):

(c) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 3;

(d) with the aminoacid sequence of sequence 3 through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and have hydrolytic enzyme functional by the derivative protein of sequence 3.

The encoding gene of described detoxifcation esterase specifically can be following 1) or 2) or 3) or 4) dna molecular:

1) its encoding sequence be in the sequence table sequence 2 from the dna molecular shown in 5 ' terminal the 8th to 1666 Nucleotide;

2) dna molecular shown in the sequence 2 in the sequence table;

3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of encoding said proteins;

4) with 1) or 2) or 3) dna sequence dna that limits has 90% above homology, and the identical function protein DNA molecule of encoding;

The encoding gene of described lytic enzyme specifically can be following 5) or 6) or 7) or 8) dna molecular:

5) its encoding sequence be in the sequence table sequence 4 from the dna molecular shown in 5 ' terminal the 11st to 901 Nucleotide;

6) dna molecular shown in the sequence 4 in the sequence table;

7) under stringent condition with 5) or 6) the dna sequence dna hybridization that limits and the dna molecular of encoding said proteins;

8) with 5) or 6) or 7) dna sequence dna that limits has 90% above homology, and the identical function protein DNA molecule of encoding.

Described detoxifcation esterase (E4) but encoding gene both synthetic obtain, also can from the black peach aphid of organophosphorus resistance, clone and obtain.Described lytic enzyme (mpd) but gene both synthetic obtain, also can from bacterium, clone and obtain.

The present invention also protects described recombinant plasmid is imported the detoxifcation engineering bacteria that Host Strains obtains.

Described Host Strains specifically can be e. coli bl21 (DE3).

Described detoxifcation engineering bacteria specifically can be colon bacillus (Escherichia coli) pET30-mpd bacterial strain.This project bacterium has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center and (has been called for short CGMCC on 08 23rd, 2006, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.1785.

The present invention also protects a kind of method for preparing detoxication enzyme liquid, is to induce described detoxifcation engineering bacteria to express with the isopropylthio semi-lactosi, obtains detoxication enzyme liquid.In the described method, the temperature of abduction delivering specifically can be 14-25 ℃, and the time specifically can be 16-22 hour.

The detoxication enzyme liquid that described method prepares also belongs to protection scope of the present invention.

Described engineering bacteria, described detoxication enzyme liquid can be applicable to degrading pesticide.Described agricultural chemicals can be organic acid acetic agricultural chemicals or organophosphorus pesticide.Described organic acid acetic agricultural chemicals specifically can be SevinCarbaryl, effective cypermethrin or acetofenate.Described organophosphorus pesticide specifically can be Malathion or thiophos, such as parathion-methyl.

Described detoxifcation engineering bacteria and described detoxication enzyme liquid can be applicable to the medicine for the preparation of the warm-blooded animal detoxifcation.

Engineering bacteria of the present invention has the function of detoxifcation esterase and lytic enzyme simultaneously, and the degraded spectrum of pesticide residue is widened, and can fall simultaneously organic acid acetic agricultural chemicals and organophosphorus pesticide, can be used for the detoxifcation of organophosphate poisoning warm-blooded animal.Engineering bacteria of the present invention, the amalgamation and expression amount of detoxifcation esterase gene accounts for 56% of total protein concentration.Engineering bacteria of the present invention, in the time of 30 minutes, i.e. the Malathion of degradable 67.0%, 66.7% thiophos.The present invention is for utilizing eucaryon and procaryotic natural resources to provide a new way to remaining in the compound polluted biological treatings of poisonous and harmful such as water source, Pesticide Residue in Soil.The present invention helps to solve the pollution problem of the pesticide residue of China, helps to eliminate loss that pesticide residual contamination causes national economy and to the harm of people ' s health, is significant and is worth.

Description of drawings

Fig. 1 is that crude enzyme liquid is to the degraded of SevinCarbaryl.

Fig. 2 is that crude enzyme liquid is to the degraded of Malathion.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.

Ex.Taq archaeal dna polymerase, various restriction enzyme BamHI, BglII, XhoI and HindIII, T4DNA ligase enzyme are the TaKaRa product, and all the other reagent are analytical pure.Plasmid pETDuet-1 is available from Novagen company, and as the carrier of expressing mpd gene and E4 gene, this plasmid is E.coli DH5 α propagation in intestinal bacteria.

Escherichia coli strain BL21 (DE3) is available from Novagen company.

The structure of embodiment 1, engineering bacteria

One, the structure of recombinant plasmid (pETDuet-E4-mpd)

1, the preparation of hydrolase gene

DNA shown in the artificial synthesized sequence 4, sequence 4 is lytic enzyme (mpd) gene from 5 ' terminal the 11st to 901 Nucleotide, and its upstream is the BamHI site, and the downstream is the HindIII site.

2, the preparation of detoxifcation esterase gene

DNA shown in the artificial synthesized sequence 2, sequence 2 is detoxifcation esterase (E4) gene from 5 ' terminal the 8th to 1666 Nucleotide, and its upstream is the BglII site, and the downstream is the XhoI site.

3, the structure of recombinant plasmid (pETDuet-E4-mpd)

1. cut the hydrolase gene of step 1 with restriction enzyme BamHI and HindIII enzyme.

2. use restriction enzyme BamHI and HindIII digested plasmid pETDuet-1.

3. step enzyme is 1. cut enzyme that product is connected with step and cut product and connect, obtain recombinant plasmid pETDuet-mpd.

4. cut the detoxifcation esterase gene of step 2 with restriction enzyme BglII and XhoI enzyme.

5. cut the recombinant plasmid pETDuet-mpd that 3. step obtains with restriction enzyme BglII and XhoI enzyme.

6. step enzyme is 4. cut enzyme that product is connected with step and cut product and connect, obtain recombinant plasmid pETDuet-E4-mpd.

4, the evaluation of recombinant plasmid (pETDuet-E4-mpd)

To the recombinant plasmid pETDuet-E4-mpd evaluation of checking order.Qualification result shows: mpd gene and E4 gene have inserted respectively the corresponding restriction enzyme site of pETDuet plasmid, and reading frame is correct, have obtained the purpose recombinant plasmid.

Two, the extraction of the preparation of engineering bacteria and crude enzyme liquid

1, the preparation of engineering bacteria

With the Transformation Program conversion e. coli bl21 (DE3) of recombinant plasmid pETDuet-E4-mpd with reference to pET System Manual, obtain engineering bacteria.

With the strain in the engineering bacteria: colon bacillus (Escherichia coli) pET30-mpd bacterial strain, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 08 23rd, 2006 and (be called for short CGMCC, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.1785.

2, the extraction of crude enzyme liquid

1. from the mono-clonal bacterium colony of the dull and stereotyped picking Pet30-mpd bacterial strain of fresh conversion, add the LB substratum (containing 50 μ g/ml penbritins) of 2ml, 37 ℃ of concussions were cultivated 8-10 hour;

2. the LB substratum (containing 50 μ g/ml penbritins) that the 2ml nutrient solution is injected 50ml continues to cultivate; Concentration OD when cell ₆₀₀Adding 500 μ l IPTG (concentration is 100mM) at=0.6 o'clock, to make the IPTG final concentration be 1mM, induced 16～22 hours at 14～25 ℃.

3. with nutrient solution at 5000 * g centrifugal 5 minutes; Collect the Pet30-mpd bacterial strain, again regulating concentration with PBS is OD ₆₀₀=1, centrifugal 5 minutes of 5000 * g;

4. again collect the Pet30-mpd bacterial strain, again add PBS (volume be step 3. PBS 1/10), making bacterium liquid cycles of concentration is 10 times, adding N,O-Diacetylmuramidase in the bacterium liquid, to make its final concentration be 100 μ g/ml, add 1%TritonX-100 (volume be step 3. PBS 1/10), 30 ℃ of temperature were bathed 15 minutes; Ultrasonic treatment is processed under condition of ice bath, and centrifugal removal precipitates again, and supernatant liquor is the crude enzyme liquid of Pet30-mpd bacterial strain.

Three, the preparation of contrast bacterium and the extraction of crude enzyme liquid

1, the preparation of contrast bacterium I

1. cut 2 pcr amplification product of step 2 with restriction enzyme BglII and XhoI enzyme.

2. use restriction enzyme BglII and XhoI digested plasmid pETDuet-1.

3. step enzyme is 1. cut enzyme that product is connected with step and cut product and connect, obtain recombinant plasmid pETDuet-E4.

4. with the Transformation Program conversion e. coli bl21 (DE3) of recombinant plasmid pETDuet-E4 with reference to pET System Manual, obtain contrasting bacterium I.

Extract the crude enzyme liquid (method is with 2 of step 2) of contrast bacterium I.

2, the preparation of contrast bacterium II

1. cut the pcr amplification product of step 1 with restriction enzyme BamHI and HindIII enzyme.

2. use restriction enzyme BamHI and HindIII digested plasmid pETDuet-1.

3. step enzyme is 1. cut enzyme that product is connected with step and cut product and connect, obtain recombinant plasmid pETDuet-mpd.

4. with the Transformation Program conversion e. coli bl21 (DE3) of recombinant plasmid pETDuet-mpd with reference to pET System Manual, obtain contrasting bacterium II.

Extract the crude enzyme liquid (method is with 2 of step 2) of contrast bacterium II.

3, the preparation of contrast bacterium III

With the Transformation Program conversion e. coli bl21 (DE3) of plasmid pETDuet-1 with reference to pET System Manual, obtain contrasting bacterium III.

Extract the crude enzyme liquid (method is with 2 of step 2) of contrast bacterium III.

Four, the target protein expression amount compares in the crude enzyme liquid

Pet30-mpd bacterial strain (engineering bacteria), contrast bacterium I, contrast bacterium II and contrast bacterium III are carried out SDS-PAGE to be analyzed.

The result shows: the efficient that the Pet30-mpd bacterial strain inducing is expressed the detoxifcation esterase is consistent with the efficient that contrasts bacterium I (pETDuet-E4); The efficient that the Pet30-mpd bacterial strain inducing is expressed lytic enzyme is consistent with the efficient that contrasts bacterium II (pETDuet-mpd); The efficient that contrast bacterium III expresses detoxifcation esterase and lytic enzyme is all very low.Be that the Pet30-mpd bacterial strain can effectively be expressed detoxifcation esterase and lytic enzyme simultaneously, the time that so just can realize an abduction delivering fermentation culture obtains 2 kinds of enzymes simultaneously, thereby the needed various starting material of abduction delivering fermentation culture production engineering bacterium have been greatly reduced, shortened the time, save the energy, reduced cost.

The crude enzyme liquid of embodiment 2, Pet30-mpd bacterial strain is to the Degradation of SevinCarbaryl and Malathion

One, the crude enzyme liquid of Pet30-mpd is to the degraded of SevinCarbaryl (carbaryl)

Measure the degraded of SevinCarbaryl according to the method for Hayatsu and Nagata (1993), specific as follows:

Damping fluid: 10mM KH ₂PO ₄/ KOH damping fluid; PH7.5.

Developer (DBLS): the solid blue B (Fast Blue B salt) of 4.5mg is dissolved in the 4.5ml water, then adds 10.5ml 5%SDS solution.

Reaction system (20ml): the crude enzyme liquid of the Pet30-mpd bacterial strain that adding Carbaryl and embodiment 1 prepare in damping fluid contains 130 μ M Carbaryl and 5ml crude enzyme liquid (107U ml in the reaction system ^-1).

Control systems (20ml): replace crude enzyme liquid with deionized water, other same reaction system.

Temperature of reaction is 37 ℃, every interval 30min sampling 2ml.The DBLS solution that adds immediately the 0.5ml new system in the sample, content with the spectrophotometric determination naphthyl alcohol (generates naphthyl alcohol after the SevinCarbaryl hydrolysis, the color producing reaction of naphthyl alcohol and DBLS solution has maximum absorption band at the wavelength of 590nm, the concentration of naphthyl alcohol is directly proportional with absorption value), according to the palliating degradation degree of the cubage SevinCarbaryl of naphthyl alcohol.Test arranges three repetitions, results averaged.The results are shown in Figure 1.

The result shows, the crude enzyme liquid of Pet30-mpd (engineering bacteria) 64% SevinCarbaryl of degrading in 2.5hr.

Two, the crude enzyme liquid of Pet30-mpd is to the degraded of Malathion (Malathion)

Measure the degraded of Malathion according to the method for Leng and Qiao (1986), specific as follows:

Damping fluid: 10mM KH ₂PO ₄/ KOH damping fluid; PH7.5.

Reaction system (20ml): the Malathion is dissolved in the 15ml damping fluid, and making its concentration is 10.9 μ M, crude enzyme liquid (the 107U ml of the Pet30-mpd bacterial strain that adding 5ml embodiment 1 prepares ^-1).

Control systems (20ml): replace crude enzyme liquid with deionized water, other same reaction system.

Temperature of reaction is 37 ℃, gets the 1ml sample every 15min.Add the 1ml sherwood oil in the sample and extract, extracting solution with anhydrous sodium sulfate drying after, use again the normal hexane extracting, the extract gas chromatographic analysis.The stratographic analysis instrument is the 5890II of Hewlett-Packard type gas-chromatography (Hewlett-Packard 5890 series II), the NPD detector, capillary column SGE company product, column length 30m, internal diameter 0.53mm, thickness 0.5 μ m, 300 ℃ of detector temperatures, 250 ℃ of injector temperatures, 200 ℃ of column temperatures, post flow 2.79ml/min, linear velocity 29.7cm/sec, splitting ratio 2.2: 1, air pressure 58psi, hydrogen pressure 21psi, nitrogen pressure 52psi, air flow quantity 81ml/min, hydrogen flowing quantity 3ml/min, nitrogen flow 30ml/min, retention time 11min.Test arranges three repetitions, results averaged.The results are shown in Figure 2.

The result shows, the crude enzyme liquid of Pet30-mpd (engineering bacteria) 80% Malathion of degrading in 1.25hr.

The detoxifcation experiment that embodiment 3, chicken Chlorpyrifos 94 poison

Grow up aircraft carrier chicken (Gallus gallus) (at 12 monthly ages, chicken is heavily approximately about 1.5kg) available from Beijing Agricultural University's experiment chicken house.Chicken is bought rear independent nursing back, just begins test after the week that conforms at least.Freely take food and drink water, experimental session, the hen house temperature is controlled at about 20 ℃, and illumination every day is 10h approximately.

Experimental chicken is divided into seven groups, 10 every group:

Test one group: give simultaneously Chlorpyrifos 94 (in the capsulae vacuus of packing into, dosage is 170mg/kg, and is oral) and Pet30-mpd (engineering bacteria) (the 0.5g wet thallus is packed in the capsulae vacuus, and is oral);

Test two groups: give first Chlorpyrifos 94 (in the capsulae vacuus of packing into, dosage is 170mg/kg, and is oral), give Pet30-mpd (engineering bacteria) (the 0.5g wet thallus is packed in the capsulae vacuus, and is oral) behind the 40min;

Contrast one group: give simultaneously Chlorpyrifos 94 (in the capsulae vacuus of packing into, dosage is 170mg/kg, and is oral) and contrast bacterium I (the 0.5g wet thallus is packed in the capsulae vacuus, and is oral);

Contrast two groups, give first Chlorpyrifos 94 (in the capsulae vacuus of packing into, dosage is 170mg/kg, and is oral), contrast bacterium I (the 0.5g wet thallus is packed in the capsulae vacuus, and is oral) behind the 40min;

Contrast three groups: give simultaneously Chlorpyrifos 94 (in the capsulae vacuus of packing into, dosage is 170mg/kg, and is oral) and contrast bacterium III (the 0.5g wet thallus is packed in the capsulae vacuus, and is oral);

Contrast four groups: give first Chlorpyrifos 94 (in the capsulae vacuus of packing into, dosage is 170mg/kg, and is oral), contrast bacterium III (the 0.5g wet thallus is packed in the capsulae vacuus, and is oral) behind the 40min;

Contrast five groups: give Chlorpyrifos 94 (in the capsulae vacuus of packing into, dosage is 170mg/kg, and is oral);

The acute poisoning situation of the tested chicken of close observation after the administration was weighed after administration in 10 days, put to death tested chicken.

Testing one group of one group and contrast does not have obvious acute poisoning and delayed neurotoxicity symptom, recovers gradually normal behind administration 3h.When experiment finished, the body weight of chicken did not obviously descend.Test two groups, two groups of contrasts, three groups of contrasts, four groups of contrasts and contrast five groups and all after administration, occurred the acute poisoning symptom in the 40min.

Testing two groups of acute poisoning symptoms that occur is languishment, mouthful tells mucus, give engineering bacteria after symptom begin to alleviate, until recover normal, do not have obvious delayed neurotoxicity symptom.Contrasting two groups of acute poisoning symptoms that occur is languishment, mouthful tells mucus, contrast bacterium I after symptom begin to alleviate, until recover normal, do not have obvious delayed neurotoxicity symptom.When experiment finished, the body weight of chicken did not obviously descend.

Contrast three groups, four groups of contrasts and contrast five groups and serious acute poisoning symptom and delayed neurotoxicity symptom have all occurred, the part experimental chicken is dead, and delayed neurotoxicity symptom has in various degree appearred in the experimental chicken of survival, and body weight significantly descends.

Embodiment 4, crude enzyme liquid are to the Degradation of parathion-methyl

Get 9 disposal plastic cups, be divided into three groups, 3 every group.Add 50ml water in each cup, add again 20 milligrams parathion-methyl, then be handled as follows respectively:

Process one: the crude enzyme liquid of the Pet30-mpd (engineering bacteria) that adding 20 μ l embodiment 1 make in cup;

Process two: in cup, add 20 μ l deionized waters;

Process three: the crude enzyme liquid of the contrast bacterium III that adding 20 μ l embodiment 1 make in cup.

Behind the 30min, each the cup in add respectively 20 4 age mosquito larvae, detected result after 24 hours.

Because the crude enzyme liquid of engineering bacteria can be degraded parathion-methyl, the larva in the cup of processing one all is not killed; And process two and the larva of processing in three all be killed.Experimental result shows, this crude enzyme liquid parathion-methyl of can effectively degrading, thus make parathion-methyl lose toxic action to mosquito larvae.

Sequence table

＜110〉Institute of Zoology, Academia Sinica

＜120〉a kind of detoxifcation engineering bacteria and its preparation method and application

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aagtaaagca gcccagcata tagctgcaaa gaatacggca cctgtatatt tctatgaatt 1320

tggctacagt ggtaattatt cttacgtagc ttttttcgat ccaaaatcat attcccgggg 1380

ttcaagcccg actcatggcg atgaaaccag ctatgtatta aaaatggatg gtttctacgt 1440

ttacgacaat gaagaagata gaaagatgat caaaactatg gttaatattt gggcaacttt 1500

tatcaaatct ggagtaccag atactgaaaa ttcagaaatt tggttacctg tttctaagaa 1560

tctagcagat cctttcaggt tcactaagat tactcaacaa caaacatttg aagccagaga 1620

acaatcaacc acgggaatta tgaattttgg agtagcttac cattaactcg ag 1672

<210>3

<211>296

<212>PRT

＜213〉artificial sequence

<220>

<223>

<400>3

Ala Ala Pro Gln Val Arg Thr Ser Ala Pro Gly Tyr Tyr Arg Met Leu

1 5 10 15

Leu Gly Asp Phe Glu Ile Thr Ala Leu Ser Asp Gly Thr Val Ala Leu

20 25 30

Pro Val Asp Lys Arg Leu Asn Gln Pro Ala Pro Lys Thr Gln Ser Ala

35 40 45

Leu Ala Lys Ser Phe Gln Lys Ala Pro Leu Glu Thr Ser Val Thr Gly

50 55 60

Tyr Leu Val Asn Thr Gly Ser Lys Leu Val Leu Val Asp Thr Gly Ala

65 70 75 80

Ala Gly Leu Phe Gly Pro Thr Leu Gly Arg Leu Ala Ala Asn Leu Lys

85 90 95

Ala Ala Gly Tyr Gln Pro Glu Gln Val Asp Glu Ile Tyr Ile Thr His

100 105 110

Met His Pro Asp His Val Gly Gly Leu Met Val Gly Glu Gln Leu Ala

115 120 125

Phe Pro Asn Ala Val Val Arg Ala Asp Gln Lys Glu Ala Asp Phe Trp

130 135 140

Leu Ser Gln Thr Asn Leu Asp Lys Ala Pro Asp Asp Glu Ser Lys Gly

145 150 155 160

Phe Phe Lys Gly Ala Met Ala Ser Leu Asn Pro Tyr Val Lys Ala Gly

165 170 175

Lys Phe Lys Pro Phe Ser Gly Asn Thr Asp Leu Val Pro Gly Ile Lys

180 185 190

Ala Leu Ala Ser His Gly His Thr Pro Gly His Thr Thr Tyr Val Val

195 200 205

Glu Ser Gln Gly Gln Lys Leu Ala Leu Leu Gly Asp Leu Ile Leu Val

210 215 220

Ala Ala Val Gln Phe Asp Asp Pro Ser Val Thr Thr Gln Leu Asp Ser

225 230 235 240

Asp Ser Lys Ser Val Ala Val Glu Arg Lys Lys Ala Phe Ala Asp Ala

245 250 255

Ala Lys Gly Gly Tyr Leu Ile Ala Ala Ser His Leu Ser Phe Pro Gly

260 265 270

Ile Gly His Ile Arg Ala Glu Gly Lys Gly Tyr Arg Phe Val Pro Val

275 280 285

Asn Tyr Ser Val Val Asn Pro Lys

290 295

<210>4

<211>907

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>4

ggatcccatg gccgcaccgc aggtgcgcac ctcggccccc ggctactacc ggatgctgct 60

gggcgacttc gaaatcaccg cgctgtcgga cggcacggtg gcgctgccgg tcgacaagcg 120

gctgaaccag ccggccccga agacgcagag cgcgctggcc aagtccttcc agaaagcgcc 180

gctcgaaacc tcggtcaccg gttacctcgt caacaccggc tccaagctgg tgctggtgga 240

caccggcgcg gccggcctgt tcggccccac cctgggccgg ctggcggcca acctcaaggc 300

cgcaggctat cagcccgagc aggtcgacga gatctacatc acccacatgc accccgacca 360

cgtgggcggc ttgatggtgg gtgagcaact ggcgttcccg aacgcggtgg tgcgtgcgga 420

ccagaaagaa gccgatttct ggctcagcca gaccaacctc gacaaggccc cggacgacga 480

gagcaaaggc ttcttcaaag gcgccatggc ctcgctgaac ccctatgtga aggccggcaa 540

gttcaagcct ttctcgggga acaccgacct ggtgcccggc atcaaagcgc tggccagcca 600

cggccacacc ccgggccaca ccacctacgt ggtcgaaagc caggggcaaa agctcgccct 660

gctcggcgac ctgatactcg tcgccgcggt gcagttcgac gaccccagcg tcacgaccca 720

gctcgacagc gacagcaagt ccgtcgcggt ggagcgcaag aaggccttcg cggatgccgc 780

caagggcggc tacctgatcg cggcgtccca cctgtcgttc cccggcatcg gccacatccg 840

cgccgaaggc aagggctacc gtttcgtgcc ggtgaactac tcggtcgtca accccaagtg 900

aaagctt 907

Claims (10)

1. recombinant plasmid is that multiple clone site in plasmid pETDuet-1 imports the recombinant plasmid that the encoding gene of the encoding gene of detoxifcation esterase and lytic enzyme obtains;

The protein that described detoxifcation esterase is comprised of the aminoacid sequence shown in the sequence in the sequence table 1;

The protein that described lytic enzyme is comprised of the aminoacid sequence shown in the sequence in the sequence table 3.

2. recombinant plasmid as claimed in claim 1 is characterized in that:

The encoding gene of described detoxifcation esterase is that sequence 2 is from the dna molecular shown in 5 ' terminal the 8th to 1666 Nucleotide;

The encoding gene of described lytic enzyme be in the sequence table sequence 4 from the dna molecular shown in 5 ' terminal the 11st to 901 Nucleotide.

3. a detoxifcation engineering bacteria is that claim 1 or 2 described recombinant plasmids are imported the detoxifcation engineering bacteria that Host Strains obtains.

4. detoxifcation engineering bacteria as claimed in claim 3, it is characterized in that: described Host Strains is e. coli bl21 (DE3).

5. detoxifcation engineering bacteria as claimed in claim 4, it is characterized in that: described detoxifcation engineering bacteria is that deposit number is colon bacillus (Escherichia coli) the pET30-mpd bacterial strain of CGMCC No.1785.

6. a method for preparing detoxication enzyme liquid is to induce the arbitrary described detoxifcation engineering bacteria of claim 3-5 to express with isopropyl-β-D-thiogalactoside(IPTG), obtains detoxication enzyme liquid.

7. method as claimed in claim 6, it is characterized in that: the temperature of described expression is 14-25 ℃, the time is 16-22 hour.

8. the detoxication enzyme liquid for preparing of claim 6 or 7 described methods.

9. the arbitrary described engineering bacteria of claim 3-5 or the application of the described detoxication enzyme liquid of claim 8 in degrading pesticide.

10. application as claimed in claim 9 is characterized in that: described agricultural chemicals is organic acid acetic agricultural chemicals or organophosphorus pesticide; Described organic acid acetic agricultural chemicals is SevinCarbaryl, effective cypermethrin or acetofenate; Described organophosphorus pesticide is Malathion or thiophos; Described thiophos is parathion-methyl.

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