CN100362916C - Application of chymotrypsin as degradation agent for organic pesticide - Google Patents
Application of chymotrypsin as degradation agent for organic pesticide Download PDFInfo
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- CN100362916C CN100362916C CNB2006100852469A CN200610085246A CN100362916C CN 100362916 C CN100362916 C CN 100362916C CN B2006100852469 A CNB2006100852469 A CN B2006100852469A CN 200610085246 A CN200610085246 A CN 200610085246A CN 100362916 C CN100362916 C CN 100362916C
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Abstract
The present invention relates to a new purpose of chymotrypsin, particularly to the application of chymotrypsin as a degradation agent for organic pesticides. Because the chymase can be directly used for degrading organic pesticides and can be produced in bulk through gene engineering, the chymotrypsin provides a new purpose for being applied in bulk to cleaning residual pesticides of the surfaces of agricultural products, governing soil and waste water which are polluted by the pesticides and detoxifying people and livestock which are poisoned by the pesticides.
Description
Technical field
The present invention relates to a kind of new purposes of chymotrypsin, specifically is the application of chymotrypsin as degradation agent for organic pesticide.
Background technology
Using organic insecticide (agricultural chemicals) is to eliminate agricultural and sanitary insect pest, ensures the cost-effective means of increasing crop yield, stable yields and human health.The organic insecticide that uses is based on organic phosphor and chrysanthemum ester class at present.For a long time, because too much mistake use of insecticide excessively causes the insecticide in agricultural product, soil and the water source residual in a large number, pollution is on the rise.Seek effective method degraded insecticide, the harm that reduces environment and health is the problem that presses for solution at present.
The biological treatment strategy has bigger potentiality to eradicating and reducing pesticide residues.For example, can be with the agricultural product, water source and the soil that pollute by containing the biomass generator of enzyme, the degraded insecticide, thus reduce its residual level and time.
Chymotrypsin belongs to the serine stretch protein enzyme family, and physiologic function comprises digestion, blood coagulation, immune response, signal conduction, the hormone activation of the albumen of ingesting and grow, and it is in insect larvae midgut great expression, the digestion of the albumen that participates in ingesting.Studies show that, the parasporal crystal protein of pathogene bacillus thuringiensis,Bt in its body of lepidopterid midgut serine protease degradable, thus the opposing bacillus thuringiensis,Bt is to the lethality of insect.The 1st page in the specification of applicant's Chinese patent application (application number is 02112820.0) formerly, therefore mention that " chymotrypsin gene is high expressed in anti-decis Culex pipiens pallens, infers that Dilutus culex chymotrypsin gene may be relevant with the anti-decis pesticide resistance of Culex pipiens pallens." still, this application can not show fully that only according to the characteristics of chymotrypsin gene high expressed in anti-decis Culex pipiens pallens the anti-decis pesticide resistance of this gene (Dilutus culex chymotrypsin) and Culex pipiens pallens is relevant, remain further to be furtherd investigate.
Summary of the invention
The new purposes that the purpose of this invention is to provide a kind of chymotrypsin.
Technical scheme of the present invention is: chymotrypsin is as the application of degradation agent for organic pesticide.
Said organic insecticide can be but is not limited to organic phosphates, chrysanthemum ester insecticide in the technique scheme.
Said organophosphorus insecticides can be but is not limited to malathion, basudin insecticide in the technique scheme, and chrysanthemum ester insecticide can be but be not limited to decis, beta-cypermethrin pesticide.
Said chymotrypsin is preferred but be not limited to Dilutus culex chymotrypsin, ox source property chymotrypsin in the technique scheme.
Reorganization of inventor's applying gene and protein expression technology, will be in the resistance to insecticides mosquito chymotrypsin gene fragment cloning of high expressed in pET-32a (+) plasmid, make up pET-32a (+)/NYD-Ch recombinant plasmid, be converted into Escherichia coli E.coli BL21 (DE3) and abduction delivering; Expression product obtains recombinant protein behind the nickel metal chelate affinity chromatography, renaturation is an activated protein; Chymotrypsin activated protein and decis are carried out biologicall test to the mosquito four-age larva jointly, as a result the LC of mosquito sensitivity and resistant strain
50Improve 0.022 and 2.8ug/ml respectively, show that the chymotrypsin of expression can make the decis degraded.
Show that with the laggard promoting the circulation of qi analysis of hplc of NYD-Ch expressing protein effect decis along with the increase of NYD-Ch concentration, decis content descends gradually, shows that chymotrypsin has direct degradation to decis; Mass spectral analysis shows that further NYD-Ch can be decomposed into decis dibromoethane dimethyl cyclopropane carboxylic acid nontoxic to mammal (Br2CA) and phenoxy group benzoic acid (3-PBA).In 24 hours, 10mg NYD-Ch expressing protein degradable 4.03ug decis, degradation rate reaches 80.6%.
Above-mentioned chymotrypsin also shows degradation to beta-cypermethrin, malathion, basudin insecticide.Ox source property chymotrypsin and NYD-Ch belong to the serine protease superfamily member, and the amino acid comparison demonstrates than higher autoploidy.Further carry out insecticide degraded mensuration is also obtained analog result with ox source property chymotrypsin.
The organic insecticide because chymotrypsin can directly be degraded, and can produce in a large number by gene engineering, it is as the application of degradation agent, be appreciated that, said application can specifically be but be not limited to following mode, as chymotrypsin being made an addition in existing vegetables and fruits cleaning agent or the cleaning agent, administering with chymotrypsin and detoxified by organic insecticide contaminated soil and waste water and the people, animal of organic insecticide being poisoned with chymotrypsin etc.
Beneficial effect of the present invention is embodied in, a kind of new purposes of chymotrypsin is provided, for large-scale application in the residual pesticide of removing the agricultural product surface, administer by the soil of pesticide pollution and waste water, to insecticide poisoning after the detoxifcation of people, animal new approach is provided.
Description of drawings
Fig. 1 is the structure schematic diagram of reorganization prokaryotic expression plasmid pET-32a (+)/NYD-Ch.
Fig. 2 is that NYD-Ch expresses in E.coli BL21 (DE3), the SDS-PAGE of purifying analyzes;
Wherein M is the molecular weight of albumen standard; 1 is unloaded bacterium contrast; 2 is the NYD-Ch reorganization bacterium contrast of not inducing; 3 is the NYD-Ch reorganization bacterium of abduction delivering; 4 is the NYD-Ch recombinant expression protein of purifying.
Fig. 3 is the Western Blot identification and analysis of NYD-Ch recombinant protein;
Wherein M is the molecular weight of albumen standard; 1 is the NYD-Ch recombinant protein of abduction delivering; 2 is unloaded contrast.
Fig. 4 is the influence of pH to the NYD-Ch enzyme activity.
Fig. 5 is that the GC of NYD-Ch and catabolite thereof analyzes;
Wherein A is NYD-Ch; B is a decis; C is the catabolite after NYD-Ch and the decis effect.
Embodiment
The present invention is further described below in conjunction with drawings and Examples.
Employed in the present invention term unless other explanation is arranged, generally has the implication of those of ordinary skills' common sense.
Below in conjunction with specific embodiment, and comparable data is described the present invention in further detail.Should be understood that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.
In following embodiment, various processes of Xiang Ximiaoshuing and method are not conventional methods as known in the art.The source of agents useful for same, trade name and be necessary to list its constituent person indicate when occurring first that all used thereafter identical reagent if no special instructions, and is all identical with the content of indicating first.
1) adopts method of gene recombination, make up NYD-Ch prokaryotic expression carrier and abduction delivering
According to the Dilutus culex chymotrypsin gene full length sequence (referring to the GENBANK registration number: AF468495) design primer:
Upstream primer L:5 '-CGGAATTCAACATGGCATCGAAACTGACGGT-3 '
Downstream primer R:5 '-ACTAGCGGCCGCTTA AGCATCCTGACGCAGCTG-3 '
In upstream primer, introduce EcoR I restriction enzyme site (GAATTC), and introduce protectiveness base CG in the upstream of restriction enzyme site; Introduce Not I restriction enzyme site (GCGGCCGC) in the downstream primer, protectiveness base ACTA is introduced in its outside, thereby the enzyme that guarantees restriction endonuclease is cut efficient.
Adopt PCR method amplification NYD-Ch full length sequence (813bp), make up procaryotic clone carrier pGEM-T/NYD-Ch, make up schematic diagram as shown in Figure 1, pass through double digestion after PCR and order-checking evaluation are errorless, to expression vector pET-32a (+), structure prokaryotic expression carrier pET-32a (+)/NYD-Ch also is converted among the competence bacteria BL21 (DE3) with genes of interest segment subclone.The reorganization bacterium that transforms is induced 4 hours to express destination protein with 1mMIPTG at 37 ℃.Expression product is through the SDS-PAGE proteins gel electrophoresis, and coomassie brilliant blue staining is judged the molecular weight size.As shown in Figure 2, at the 50kD place obvious expression albumen is arranged as can be known, consistent with theoretic NYD-Ch molecular weight of albumen.
2) adopt metal chelate affinity chromatography method purifying NYD-Ch expressing protein and evaluation
Expression product is behind centrifugal collection total bacterial protein, ultrasonication cell, with the destination protein of Ni-NTA purifying expression.Identify with Western blot method specific expressed, as shown in Figure 3, occur on the NC film as can be known 50kD the colour developing band and default quite, unloaded control group does not see that the colour developing band occurs.
3) adopt enzymatic substrate method, measure the activity of expressing protein with specific substrate
Select for use BTEE and S (Ala) 2ProPhe-pNA as the chymotrypsin specific substrate, under different pH, the NYD-Ch expressing protein is identified.Is 4.0,5.0,5.5,6.0,6.5 with enzyme liquid and substrate at pH, 7.0,7.5,8.0,9.0,10.0 buffer solution in 30 ℃ of reaction 20min, each corresponding pH gradient is contrast with the substrate of enzyme-added liquid not respectively, by measuring the enzyme activity under the condition of different pH, determines the optimal reaction PH of enzyme.As shown in Figure 4, the maximum vigor of NYD-Ch obtains by S (Ala) 2ProPhe-pNA, and the vigor of enzyme increases along with increasing of pH, and is higher in pH is the scope of 8.00-11.0, reaches the highest at 10.0 o'clock.
Select for use serpin PMSF, chymase inhibitors TPCK by the inhibition experiment of avtive spot being determined the characteristic of albumen.Result such as table 1, the vigor of PMSF, TPCK energy obvious suppression NYD-Ch confirms that NYD-Ch has the chymotrypsin activity as can be known.
Table 1 inhibitor is to the influence of expressing protein NYD-Ch and ox chymotrypsin vigor
| Inhibitor | Concentration (M) | Residual vigor (%) | |
| The ox chymotrypsin | NYD-Ch | ||
| PMSF |
10
-3 10
-4 10
-3 10
-4The |
2 2.5 6 10.3 100 | 2.5 17 6 5 100 |
4) with gas-chromatography connexus spectrum analysis method, measure the degradation rate of NYD-Ch to decis
In six duplicate samples, respectively add 0,0.1,0.5,1,5 respectively, the decis (purity＞98%, the excellent Ke Fu of French Russell company product) of the NYD-Ch expressing protein of 10mg and 5ug adds water and be settled to 1ml, hatches 24h for 29 ℃.The acetone that adds 1ml in the sample liquid, vortex vibration 30s.Add the abundant mixing of 2ml cyclohexane, extraction, 2, the centrifugal 10min of 000rpm/min draws upper strata liquid; After repeating to extract 2 times with cyclohexane, merge all upper strata liquid, in 60 ℃ of water-baths, dry up solvent with nitrogen.With the infiltration of 0.2ml cyclohexane, dissolution residual substance, with the content of chromatographic determination decis on the 0.6ul sample size, find that the degradation rate and the enzyme concentration of decis presents good correlation in the 0.5-10mg scope, experimental result is seen Fig. 5 and table 2.GC/MS analyzes, and expressing protein NYD-Ch can make decis be degraded to Br2CA and 3-PBA.
Table 2 various dose expressing protein NYD-Ch is to the degradation rate of decis
| Chymotrypsin concentration (mg/ml) | 0 | 0.1 | 0.5 | 1 | 5 | 10 |
| Decis amount (ug) degradation rate (%) that peak area is measured | 1213799 0 | 1109147 4.57 8.6 | 1058606 4.36 12.8 | 956900 3.94 21.2 | 674677 2.78 44.4 | 242760 0.97 80.6 |
Metabolite behind the embodiment 2 NYD-Ch degraded decis is to the toxicity test of the different strain four-age larvas of Culex pipiens pallens
Experiment divides responsive and resistance group, and final volume is 300ml.
Responsive group is measured:
The A group:
Decis is to the toxicity test of sensitive strain Culex pipiens pallens four-age larva
Decis (ppm) 0 0.001 0.005 0.01 0.05 0.1 0.5
Four-age larva (bar) 50 50 50 50 50 50 50
Act on after 24 hours record larva death toll
The B group
Metabolite is to the toxicity test of responsive Culex pipiens pallens four-age larva behind the NYD-Ch effect decis
Decis (ppm) 0 0.001 0.005 0.01 0.05 0.1 0.5
NYD-Ch(mg/ml) 1 1 1 1 1 1 1
At 28 ℃, act on after 24 hours, add four-age larva
Four-age larva (bar) 50 50 50 50 50 50 50
Act on after 24 hours record larva death toll
The C group
Decis (ppm) 0 0.001 0.005 0.01 0.05 0.1 0.5
The NYD-Ch of deactivation (mg/ml) 1111111
At 28 ℃, act on after 24 hours, add four-age larva
Four-age larva (bar) 50 50 50 50 50 50 50
Act on after 24 hours record larva death toll
The D group
The toxicity test in four ages of NYD-Ch antagonism strain Culex pipiens pallens
NYD-Ch(mg/ml) 0 10 100 250 500 1000
Four-age larva (bar) 50 50 50 50 50 50
Act on after 24 hours record larva death toll
The mensuration of resistant strain
The A group:
The toxicity test of decis antagonism strain Culex pipiens pallens four-age larva
Decis (ppm) 0 0.1 0.5 1.0 5.0 10.0 20.0
Four-age larva (bar) 50 50 50 50 50 50 50
Act on after 24 hours record larva death toll
The B group
The toxicity test of metabolite antagonism Culex pipiens pallens four-age larva behind the NYD-Ch effect decis
Decis (ppm) 0 0.1 0.5 1.0 5.0 10.0 20.0
NYD-Ch(mg/ml) 10 10 10 10 10 10 10
At 28 ℃, act on after 24 hours, add four-age larva
Four-age larva (bar) 50 50 50 50 50 50 50
Act on after 24 hours record larva death toll
The C group
Decis (ppm) 0 0.1 0.5 1.0 5.0 10.0 20.0
The NYD-Ch of deactivation (mg/ml) 10 10 10 10 10 10 10
At 28 ℃, act on after 24 hours, add four-age larva
Four-age larva (bar) 50 50 50 50 50 50 50
Act on after 24 hours record larva death toll
The D group
The toxicity test of NYD-Ch antagonism strain Culex pipiens pallens four-age larva
NYD-Ch (mg/ml) 0 10 100 250 500 1000
Four-age larva (bar) 50 50 50 50 50 50
Act on after 24 hours record larva death toll
Experimental result sees Table 3.
Table 3 decis and metabolite thereof are to the toxicity test of Culex pipiens pallens four-age larva
| LC 50(ug/ml) | ||
| Resistant strain | Sensitive strain | |
| Decis decis+NYD-Ch NYD-Ch | 5.3 8.1 ＞1000 | 0.043 0.065 ＞1000 |
Embodiment 3 ox of the present invention source property chymotrypsins are to the degradation of decis
In four duplicate samples, respectively add 5,10,50 respectively, the ox source property chymotrypsin (Ameresco company product) of 100U and the decis of 5ug add water and are settled to 1ml, hatch 24h for 29 ℃.The acetone that adds 1ml in the sample liquid, vortex vibration 30s.Add the abundant mixing of 2ml cyclohexane, extraction, 2, the centrifugal 10min of 000rpm/min draws upper strata liquid; After repeating to extract 2 times with cyclohexane, merge all upper strata liquid, in 60 ℃ of water-baths, dry up solvent with nitrogen.With the infiltration of 0.2ml cyclohexane, dissolution residual substance,, find that the degradation rate and the enzyme concentration of decis presents good correlation in the 5-100U scope with the content of chromatographic determination decis on the 0.6ul sample size.GC/MS analyzes, and expressing protein NYD-Ch can make decis be degraded to Br2CA and 3-PBA.Experimental result sees Table 4.
Table 4 various dose ox source property chymotrypsin is to the degradation rate of decis
| Chymotrypsin concentration (U/ml) | 5 | 10 | 50 | 100 | The correlation r 0.9682 of the degradation rate of chymotrypsin concentration and decis |
| The decis amount of measuring (ug) | 4.67 | 4.42 | 2.77 | 2.06 | |
| The degradation rate of decis (%) | 6.6 | 11.6 | 44.6 | 58.8 |
Embodiment 4: by method substantially the same manner as Example 2, after acting on malathion, basudin, beta-cypermethrin respectively with Dilutus culex chymotrypsin, measure different insecticides and catabolite thereof toxic action, all show the good degradation effect similar to embodiment 2 to Culex pipiens pallens 4 instar larvaes.
The experiment mosquito is a sensitive strain, and final volume is 200ml.
A group: malathion 0.07ug/ml
NYD-Ch (mg/ml) 0 0.05 0.1 0.15 0.2
Four-age larva (bar) 50 50 50 50 50
Act on after 24 hours record larva death toll
B group: basudin 0.5ug/ml
NYD-Ch (mg/ml) 0 0.05 0.1 0.15 0.2
Four-age larva (bar) 50 50 50 50 50
Act on after 24 hours record larva death toll
C group: beta-cypermethrin 0.04ug/ml
NYD-Ch(mg/ml) 0 0.05 0.1 0.15 0.2
Four-age larva (bar) 50 50 50 50 50
Act on after 24 hours record larva death toll
Experimental result sees Table 5.
Different organic insecticides of table 5 and metabolite thereof are to the toxicity test of Culex pipiens pallens four-age larva
| NYD-Ch(mg/ml) | 0 | 0.05 | 0.1 | 0.15 | 0.2 |
| A group mosquito death toll (bar) B group mosquito death toll (bar) C group mosquito death toll (bar) | 47 12 5 | 8 5 4 | 4 3 0 | 1 2 1 | 2 0 0 |
。
Claims (8)
1. chymotrypsin is as the application of degradation agent for organic pesticide, and wherein said organic insecticide is chrysanthemum ester insecticide or organophosphorus insecticides.
2. according to the application of the said chymotrypsin of claim 1 as degradation agent for organic pesticide, it is characterized in that: wherein said organic insecticide is a chrysanthemum ester insecticide.
3. according to the application of the said chymotrypsin of claim 2 as degradation agent for organic pesticide, it is characterized in that: wherein said chrysanthemum ester insecticide is the decis insecticide.
4. according to the application of the said chymotrypsin of claim 2 as degradation agent for organic pesticide, it is characterized in that: wherein said chrysanthemum ester insecticide is a beta-cypermethrin pesticide.
5. according to the application of the said chymotrypsin of claim 1 as degradation agent for organic pesticide, it is characterized in that: wherein said organic insecticide is an organophosphorus insecticides.
6. according to the application of the said chymotrypsin of claim 5 as degradation agent for organic pesticide, it is characterized in that: wherein said organophosphorus insecticides is the malathion insecticide.
7. according to the application of the said chymotrypsin of claim 5 as degradation agent for organic pesticide, it is characterized in that: wherein said organophosphorus insecticides is the basudin insecticide.
8. according to the application of the said chymotrypsin of one of claim 1-7 as the insecticide degradation agent, it is characterized in that: wherein said chymotrypsin is Dilutus culex chymotrypsin or ox source property chymotrypsin.
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| CNB2006100852469A CN100362916C (en) | 2006-06-07 | 2006-06-07 | Application of chymotrypsin as degradation agent for organic pesticide |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02250823A (en) * | 1989-03-24 | 1990-10-08 | Tsumura & Co | Microcapsule agent and production thereof |
| WO2002098232A1 (en) * | 2001-06-04 | 2002-12-12 | Nobex Corporation | Mixtures of insulin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same |
| CN1385531A (en) * | 2002-03-29 | 2002-12-18 | 南京医科大学 | Dilutus culex chymotrypsin gene |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02250823A (en) * | 1989-03-24 | 1990-10-08 | Tsumura & Co | Microcapsule agent and production thereof |
| WO2002098232A1 (en) * | 2001-06-04 | 2002-12-12 | Nobex Corporation | Mixtures of insulin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same |
| CN1385531A (en) * | 2002-03-29 | 2002-12-18 | 南京医科大学 | Dilutus culex chymotrypsin gene |
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