CN101293131A - Complex enzyme preparation for eliminating residual pesticides and application thereof - Google Patents

Complex enzyme preparation for eliminating residual pesticides and application thereof Download PDF

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CN101293131A
CN101293131A CNA2007100938074A CN200710093807A CN101293131A CN 101293131 A CN101293131 A CN 101293131A CN A2007100938074 A CNA2007100938074 A CN A2007100938074A CN 200710093807 A CN200710093807 A CN 200710093807A CN 101293131 A CN101293131 A CN 101293131A
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enzyme
hydrolase
gene
pesticide
carbamatic
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CN101293131B (en
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苏勇
杨柏成
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Aolian Health Technology (Jiangsu) Co.,Ltd.
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SHANGHAI PUTIANXIN BIOLOGICAL TECHNOLOGY Co Ltd
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Abstract

The purpose of the invention is to provide a compound enzymatic preparation which has a wide degrading spectrum and is capable of clearing residual pesticide on common vegetables and fruits, and decomposes the pesticide remained on the vegetables and the fruits into a nonpoisonous or low-toxic outcome and reduces the poisoning effect of the pesticide to a man and a non-target organism as well as the pollution to the environment. The invention also provides the application of the compound enzymatic preparation. The compound enzymatic preparation clearing the residual pesticide of the invention comprises organophosphorus hydrolase, carbamate hydrolase and pyrethroid hydrolase. Preferably, the three enzymes are derived from a gene expression of a microorganism. Compared with the traditional method, the method has the advantages of simplicity, safety and moderate required reaction condition, and can not influence the taste and the nutrition of the vegetables and the fruits; the compound enzymatic preparation is extremely easy to be cleaned and really clears the residual pesticide at the same time.

Description

A kind of complex enzyme formulation and application of eliminating remains of pesticide
Technical field
The present invention relates to a kind of complex enzyme formulation, particularly eliminate the complex enzyme formulation and the application thereof of remains of pesticide in vegetables, tealeaves and the melon and fruit.
Background technology
China needs to support the population that accounts for the world 20% with the soil that accounts for the world 7%, thereby relies on very much agricultural chemicals.Particularly, used a large amount of pesticides in order to reduce the loss that due to illness insect pest causes.But agricultural chemicals suppresses the generation of disease and pest on the one hand; On the other hand, it has also brought great ecology and social concern.
The equal tool toxicity of most agricultural chemicals, and lack selectivity, often non-target organism is caused direct murder by poisoning.In addition, because agricultural chemicals is than difficult degradation, have part to remain in crop, herbage, soil and the water body, and with food chain transport and enrichment, the agricultural chemicals harm that the mankind of the extreme higher position that is in food chain are subjected to is the most serious.
Agricultural chemicals mainly contains people's harm: acute poisoning, slow poisoning, tardy property neurotoxic, carcinogenic, teratogenesis and mutagenesis, genotoxicity, as damage sperm, make and become pregnant and the fecundity reduction, and can cause embryo developmenting disorder, filial generation depauperation or death etc.
The residues of pesticides problem of China vegetables, fruit is quite serious, even has because of the edible vegetables of remains of pesticide, the report that fruit causes acute poisoning of containing.Usually adopt the clear water washing now, soak or remove vegetables, the residual agricultural chemicals of fruit surface with the method that cleaning agent cleans.Agricultural chemicals generally is insoluble in water, only cleans with clear water and can not obtain satisfied effect.
Though cleaning agent can be removed remains of pesticide more effectively, really do not remove remains of pesticide, agricultural chemicals enters environment circulation and accumulation once more with sewage.Also can adopt soda lye wash, agricultural chemicals decomposes under alkali condition easily, but alkalescence is crossed strong nutrition and the local flavor that destroys vegetables, fruit easily, and alkali is also relatively limited to the decomposition of agricultural chemicals in addition.
At present, most widely used general three class agricultural chemicals are organophosphor, carbamate and pyrethroid, and they all have neurotoxicity to mammal.Compare with the organochlorine of having forbidden, their are than easy natural degradation or by enzyme hydrolysis.
Organophosphorus pesticide mostly is phosphoric acid ester or group thiophosphate, contains three phosphide keys.Its mechanism of toxication is the phosphorylated acetylcholinesterase, the ability that makes the body forfeiture decompose acetylcholine.The acetylcholine of accumulating makes cholinergic nerve be subjected to continuing impulsion, causes the depleted a series of nervous system poisoning symptoms in excited earlier back, can cause death when serious.As long as phosphide key of fracture, organophosphorus pesticide just loses the ability of acetylcholine esterase inhibition, forfeiture or reduce toxic action to animal greatly.
The structure of carbamate chemicals for agriculture is based on eserine, and skeleton is a carbamic acid, and commonly used is N-formamido group formic acid esters.Its toxicity mechanism also is acetylcholine esterase inhibition activity, blocks normal nerve conduction.But machine-processed different with organic phosphates: 1. it does not need through metabolism activation, can be directly and acetylcholinesterase form loose complex, suppress the latter's activity.2. combine with acetylcholinesterase is reversible, the compound of formation can decompose rapidly, so symptom is lighter, recovers very fast.Carbamate chemicals for agriculture loses toxic action behind the ester linkage breaking.
Pyrethroid pesticide is develop on the basis of pyrethrum ester structure efficient, wide spectrum, knock down fast, the agent of neural intoxicating insects.The tradition pyrethroid is carboxylate, is formed by pure and mild chrysanthemumic acid condensation.Chrysanthemumic acid partly is generally the dimethylcyclopropane that contains different side chain molecules, and alcohol moiety is generally the aromatic rings of band cyano group.The intoxicating mechanism of pyrethroid is by postponing closing of the valtage-gated property of neuron sodium channel, cause axoneure sodium, calcium ionic current to increase, causing that sodium, calcium ion concentration increase film Na in the born of the same parents +-K +-ATP enzyme and Ca 2+-atpase activity and function change, and cause central lesion.The same with carbamate chemicals for agriculture, pyrethroid pesticide promptly loses toxic action behind the ester linkage breaking.
From china academia journal data storehouse, incomparably database and Wei Pu full-text database are collected the article of 1998-2006 year all about vegetables, fruit residues of pesticides.Reject to repeat document, extract the article of wherein quantitative analysis residues of pesticides out, data are wherein added up, conclude.The roughly present situation of residues of pesticides is (seeing Table 1) in present stage of china vegetables, the fruit: three kinds of organophosphorus pesticides that recall rate is the highest are acephatemet, omethoate and parathion; Two kinds of carbamates chemicals for agriculture that recall rate is the highest are furans pellet and sevin; Three kinds of pyrethroid pesticides that recall rate is the highest are cypermethrin, fenvalerate and decis.Recall rate is relative recall rate, is 100 in the highest the sort of of recall rate in every class agricultural chemicals.
Table 1.1998-2006 year China's Vegetable fruit agricultural chemicals recall rate statistics
Figure A20071009380700041
Degrading pesticide makes the method for its inefficacy mainly contain chemical degradation, light degradation and microbial degradation.The common chemical edman degradation Edman has oxidizing process, alkali hydrolysis method and acid-hydrolysis method, though the effective degrading pesticide of chemical method is only applicable to the processing of insecticide factory's factory effluent, and acid, the easy corrosion pipeline of alkali, the cost height has certain risk.Light degradation is to utilize daylight degrading plant surface, soil surface and airborne agricultural chemicals.Different compounds stability under illumination is different, and intensity of illumination, soil types also can influence degradation effect, thereby photodegradative effect is comparatively limited.Microorganism in the environment can be degraded and be transformed agricultural chemicals.In fact, degraded and the conversion of agricultural chemicals in environment mainly caused by microorganism.Microorganism is finished by a series of enzymatic reactions the metabolism of agricultural chemicals is many, comprises hydrolysis.
But, the removing of remains of pesticide on none suitable vegetables of said method and the melon and fruit.Required acid, alkali and the oxidizing condition of chemical method has been enough to destroy the nutritional labeling of vegetable melon and fruit; The light degradation method needs for a long time; The microbiological treatment degrading pesticide also has infringement to vegetable melon and fruit.Even without influence, the vegetable melon and fruit of handling is estimated to be difficult to be accepted by people like this.
Therefore, press for can be easy, safe removing vegetables, melon and fruit on the method and the product of remains of pesticide, to satisfy the needs of people's daily life.
Summary of the invention
One of the object of the invention is for the complex enzyme formulation of remains of pesticide on wide removing vegetables of a kind of spectrum of degrading and the melon and fruit is provided, to remain in the product that agricultural chemicals on vegetables and the fruit is decomposed into nontoxic or low toxicity, reduce it to murder by poisoning of people and non-target organism and to the pollution of environment.
Two of the object of the invention is to provide the application of above-mentioned complex enzyme formulation.
A first aspect of the present invention, a kind of complex enzyme formulation of eliminating remains of pesticide is provided, comprise organophosphor hydrolytic enzyme, carbamatic hydrolase, pyrethroid ester hydrolase, the mass ratio of described organophosphor hydrolytic enzyme, carbamatic hydrolase, pyrethroid ester hydrolase is 1: 27～40: 120～240.Described complex enzyme formulation is the aqueous solution or cushioning liquid, and the pH value of reaction system is 7.5～8.5.
Preferably, organophosphor hydrolytic enzyme, the ratio of carbamatic hydrolase and pyrethroid ester hydrolase is 1: 40: 240.
Described organophosphor hydrolytic enzyme, carbamatic hydrolase and pyrethroid ester hydrolase are the gene expression that origin comes from microorganism, and organophosphor hydrolytic enzyme is selected from a kind of or combination of opd gene, opdA gene, mpd gene, ophC2 gene expression product; Carbamatic hydrolase is selected from a kind of or combination in cehA gene, the caha gene outcome; The pyrethroid ester hydrolase is the estP gene expression product.
More preferably, organophosphor hydrolytic enzyme is by opd gene and/or ophC2 gene expression, and carbamatic hydrolase is by cehA gene and/or caha gene expression, and the pyrethroid ester hydrolase is by estP gene expression.
Optimally, organophosphor hydrolytic enzyme is by opd gene (genbank accession number: M20392) express, (the genbank accession number: Ab069723) express, the pyrethroid ester hydrolase is by estP gene (genbank accession number: AY995176) express by the cehA gene for carbamatic hydrolase.
Described complex enzyme formulation, its reaction system also contain final concentration and be ten thousand/and zero to two APES (op-10) and/or final concentration are 1/10000th to 5/10000ths coconut oil alkyl diethanolamide (6502).
Complex enzyme formulation of the present invention contains degradation of pesticide enzyme and the acceptable carrier or the excipient of effective dose usually.This class carrier includes but not limited to: water, buffer solution, glycerine, sucrose, starch ... and combination.Compound formulation of the present invention can be made into liquid or solid-state.Liquid formulation available water or phosphate buffer solution are prepared by conventional method, for example, can make concentrated solution, and thin up is to working concentration during use.Solid formulation can be prepared into tablet, granule by conventional method with mixing such as hydrolase, starch back, also hydrolase dissolving back freeze-drying can be prepared into freeze dried powder etc.The additive of complex enzyme formulation of the present invention includes but not limited to detergent, bivalent metal ion etc., can mix with digestive enzyme in advance, but also independent packaging mixes in use.The present invention is not limited to a certain concrete preparation type.
A second aspect of the present invention provides the application of above-mentioned complex enzyme formulation, and remains of pesticide on be used to degrade vegetables and the melon and fruit, described agricultural chemicals include but not limited to, organophosphorus insecticide and/or carbamate chemicals for agriculture and/or pyrethroid pesticide.
The present invention is according to domestic agricultural chemicals behaviour in service, selects the degraded spectrum wide targetedly, three kinds of degradation of pesticide enzymes that derive from microorganism that degrading activity is strong, and the proportioning of proportioning, hydrolase and the additive of preferred three kinds of enzymes is prepared into complex enzyme formulation.Its beneficial effect is embodied in:
(1) this complex enzyme formulation degraded spectrum is wide, and spreadability is strong, and the agricultural chemicals that the present state that can degrade simultaneously can be most popular three types provides a kind of scheme of removing residues of pesticides easily can for users such as family, restaurant;
(2) three kinds of enzymes can produce synergy, by the degraded of organophosphor hydrolytic enzyme to organophosphorus pesticide, have overcome the inhibition of organophosphorus pesticide to carbamate and pyrethroid ester hydrolase;
(3) preferred by to preferred, the reaction condition of the ratio optimization of three kinds of enzymes, additive, it is high to make complex enzyme formulation enzyme of the present invention live, and removes residues of pesticides effect excellence;
(4) complex enzyme formulation of the present invention, the microbial degradation enzyme that is adopted adopt the genetic engineering means preparation, the output height, and cost is low, helps large-scale promotion.
(5) complex enzyme formulation of the present invention adopts enzyme to send out degrading pesticide residues, not only removes effectively, and simple, safety, required reaction condition is gentle and very easily clean with vegetable and fruit and remove, and not being infected with vegetable melon and fruit does not influence its taste and nutrition yet.
Description of drawings
Fig. 1 is the activity of organophosphor hydrolytic enzyme and the relation of pH.
Fig. 2 is the activity of pyrethroid ester hydrolase and the relation of pH.
Fig. 3 is the activity and the pH relation of carbamatic hydrolase.
Fig. 4 adds the influence of detergent to the organophosphorus pesticide hydrolase.
Fig. 5 adds the influence of detergent to the carbamatic hydrolase activity.
Fig. 6 adds the influence of detergent to the pyrethroid hydrolytic enzyme activities.
The specific embodiment
The degradation of pesticide enzyme
The enzymic degradation residues of pesticides have the reaction condition gentleness, the characteristics that degradation efficiency is high.Selectable degradation of pesticide enzyme comprises: microbe-derived, insect is originated, the degradation of pesticide enzyme of animal origin.
Can be by the enzyme product of simulation insect detoxifcation mechanism exploitation degrading pesticide, i.e. the detoxifcation system of reconstruct insect.The enzyme that insect participates in detoxifcation mainly contains mixed-function oxidase (MFO), esterase and glutathione-S-transferase (GST) etc., and wherein the effect of MFO needs NADPH, and GST plays a role then needs glutathione.In the insect such as anopheles, diamondback moth and aphid of carbamate chemicals for agriculture resistance, carboxy-lesterase is to cause drug-fast reason.But the ability of insect carboxy-lesterase hydrolysis agricultural chemicals very limited (malathion exception); resistant insects is not by expressing the carboxy-lesterase that hydrolysing activity improves greatly; but combine with pesticide by expressing a large amount of carboxy-lesterases; make pesticide can not arrive target, thereby target-acetylcholinesterase is avoided suppressing in the protective.In addition, the carboxy-lesterase pyrethroid of also degrading to a certain extent, but insect mainly is because Na on the neu to the resistance mechanism of pyrethroid +The sensitiveness of passage reduces.Thereby the detoxifcation system of reconstruct insect is removed residual agricultural chemicals and uneconomical.
The phosphoric triesterase that obtains in the animal body also can decompose organophosphorus pesticide, and the esterase of acquisition also has the ability of hydrolysis carbamate and pyrethroid, but by contrast, they are lower to the capacity of decomposition of agricultural chemicals.
By contrast, advantages such as active high, the easy screening of microbe-derived hydrolase tool become the focus of seeking the degradation of pesticide enzyme.Promptly screen the bacterial strain of specific agricultural chemicals of to degrade, and therefrom separate corresponding hydrolase gene from occurring in nature.Studying more and fruitful is that organophosphorus pesticide hydrolase: opd finds and study the most thorough organophosphor hydrolytic enzyme gene the earliest, finds many genes that can the hydrolysis organophosphors afterwards again successively, as opaA, opdA etc.The domestic report that discovery organophosphor hydrolytic enzyme gene is also arranged, as discovery ophC2 such as Fan Yunliu, Li Shunpeng etc. find the mpd gene.Also has the carbamate chemicals for agriculture hydrolase gene, as pcd, cehA, mcd, cahA etc. and some pyrethroid ester hydrolases, as estP.
Complex enzyme formulation
Current many relevant patents that solves the residues of pesticides problem mainly concentrate on screening agricultural chemicals hydrolase gene, especially organophosphorus pesticide hydrolase gene, and remove residual agricultural chemicals in vegetables, the fruit with the gene that obtains.Only residual carbamate and pyrethroid are not almost removed effect with organophosphor hydrolytic enzyme.The composite use of esterase of also useful organophosphor hydrolytic enzyme and insect, but as previously mentioned, the mechanism of the esterase of insect detoxifcation mainly is combination, and hydrolysis is less important.
Because vegetable and fruit is numerous in variety, the pesticide variety that uses in the production process has nothing in common with each other, so the residues of pesticides that run in the daily life often are not a kind of, but different types of Multiple Pesticides.Thereby adopt the method for an a kind of or class agricultural chemicals of single degraded can not fine solution residues of pesticides problem, must adopt compound formulation with degraded broad spectrum activity.
In the period of the 1998-2006,3 kinds of organophosphorus pesticides that recall rate is the highest in China vegetables, fruit are acephatemet, omethoate and parathion according to investigations; Two kinds of carbamates chemicals for agriculture are furans pellet and sevin; Three kinds of pyrethroid pesticides are cypermethrin, fenvalerate and decis, therefore, and should be with these agricultural chemicals as the preferential object that decomposes.
Complex enzyme formulation of the present invention selects to comprise machine phosphorus hydrolase, carbamatic hydrolase, pyrethroid ester hydrolase.More preferably, the organophosphor hydrolytic enzyme of three kinds of enzyme source microorganisms, carbamatic hydrolase and pyrethroid ester hydrolase.
Selectable enzyme class is a lot, such as organophosphor hydrolytic enzyme selections such as Opd, Opda, OphC2 is just arranged.Different enzymes has different substrate spectrum and active, does not have all agricultural chemicals of a kind of enzyme fine hydrolysis of energy.Therefore, should preferentially select modal remains of pesticide kind is had the enzyme or the enzyme combination of degraded advantage.Simultaneously, because the different different reaction conditions of enzyme require, it is also by no means easy to coordinate these conditions.Need between the broad spectrum activity of enzyme and activity, look for an equalization point, with a kind of or a few enzyme realization decomposition to a certain class or a few class remainss of pesticide.
The present inventor is by number of research projects, finds (the genbank accession number: M20392), ceha (genbank accession number: Ab069723) and estp (genbank accession number: AY995176) three kinds of enzymes combinations agricultural chemicals of can the listed need of fine degraded preferentially degrading with existing opd.Zu He reason is not that the enzyme that can decompose three class agricultural chemicals simply mixes like this, and one of advantage that it is maximum is to eliminate the inhibition of organophosphorus pesticide to carbamate and pyrethroid ester hydrolase.As preferred embodiment, the present invention selects to derive from opd, the ceha of microorganism and the incompatible realization of the estp genome removing to the preferential degrading pesticide of listed need.
Because various enzymes are more different than living, the using dosage of various agricultural chemicals is also different, thereby can not simply several enzymes be mixed.Determine that in the practical residue limit of agricultural chemicals the ratio of enzyme is best in theory, but because the stability of various agricultural chemicals is different, the Time To Market difference of vegetable and fruit, the actual residual ratio of agricultural chemicals varies, and is difficult to draw one and has general data.The application dosage relative fixed of agricultural chemicals, thereby, can it calculate the ratio of various enzymes in the complex enzyme formulation for reference.According to investigations, the application dosage of agricultural chemicals is all at the same order of magnitude, and several agricultural chemicals of this that we considered are like this at least, thereby determines that the principal element of various enzyme ratios is the ratio work of enzyme.When the organophosphor hydrolytic enzyme that is adopted among the present invention (opd expression), carbamatic hydrolase (ceh expression) and pyrethroid ester hydrolase (estP expression) are substrate with the listed three class agricultural chemicals of the present invention, its average specific is lived and is respectively 1000-1200IU/mg, 30-45IU/mg and 5-10IU/mg approximately.Thereby the ratio of three kinds of hydrolases is 1 in the present invention: 27-40: 120-240.
In addition, agricultural chemicals mostly is water-insoluble materials, often is adsorbed on vegetables, fruit surface, is difficult for interacting with hydrolase.Add detergent and help agricultural chemicals to break away from, also help agricultural chemicals to be dispersed in the aqueous solution, promote agricultural chemicals and hydrolase to interact from vegetables, fruit surface.Thereby, also contain the detergent that can promote that agricultural chemicals is eliminated in the complex enzyme reaction system.More preferably, the detergent of selecting permission in abluent for foodstuff, to use.More preferably, the detergent of interpolation is APES (op-10) and coconut oil alkyl diethanolamide (6502).
The preparation of degradation of pesticide enzyme
Microbe-derived degradation of pesticide enzyme can obtain by cultivating the microorganism that produces this kind enzyme, also can prepare by gene engineering method.For example, can be cloned in the production of carrying out recombinant protein in prokaryotic expression carrier such as coli expression carrier or carrier for expression of eukaryon such as the Yeast expression carrier by the gene of the degradation of pesticide enzyme of will encoding, this is that this area staff knows.
The enzyme of external elimination remains of pesticide, the requirement of its purity is low far beyond the protein medicine.For simplifying purifying process, reduce cost, can add the label of being convenient to purifying at albumen n end or C end, as FLAG, 6 * His etc. are wherein more ripe with 6 * His, effective.As preferably, the present invention is chosen in the N end of enzyme and introduces 6 * His label.
Escherichia coli have advantages such as fast growth, simple to operate, technology maturation, thereby more preferably, the present invention produces hydrolase by gene is cloned in the prokaryotic expression carrier in Escherichia coli.
Opd gene of the present invention, cehA gene, cehA gene are synthesized by Shanghai bioengineering Co., Ltd, expression plasmid pET30a (+) purchases the company in Novagen, e. coli bl21 (DE3) is purchased the company in Stratagene, and restriction enzyme is purchased in Shanghai bioengineering Co., Ltd.Adopt the technique for gene engineering method, obtain the proteinase expression product of said gene.Parathion, sevin and cypermethrin are purchased in the Shanghai Pesticide Research Institute, and detergent APES (op-10), coconut oil alkyl diethanolamide (6502) are homemade, and other reagent are homemade AR.
In order to further specify effect of the present invention and method, now be illustrated with concrete embodiment.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, " molecular cloning: laboratory manual " (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or " national agricultural industry criteria NY/T761-2004 " method and condition of limiting, or the condition of advising according to manufacturer.It should be understood that these embodiment only are used for the purpose of illustration, never limit protection scope of the present invention.
The preparation of embodiment 1, opd gene outcome
(1) expression and purifying
A, according to the gene order that genbank accession number M20392 announces, introduce Nde I site at 5 ' end, after terminator codon, add Eco RI site design opd expressing gene;
B, gene is cloned between the Nde I and Eco RI site of pET30a (+) plasmid the construction expression plasmid;
C, use CaCl 2Method changes expression plasmid over to e. coli bl21 (DE3) bacterial strain, selects the clone of that resistance of card, obtains engineering bacteria;
The engineering bacteria of D, inoculation step (2) in the LB culture medium that contains 50 μ g/ml kanamycins, 37 ℃ of shaking table overnight incubation.Transferred in the fresh LB culture medium that contains kanamycins by 1: 100, continue to be cultured to OD600 ≈ 0.8, adding IPTG is 0.5mmol/L abduction delivering 4 hours to working concentration, and centrifugal collection thalline is standby, abandons supernatant;
E, usefulness PB (50mmol/L, pH7.5) resuspended thalline, the ultrasonic disruption cell, 18000 * g collected supernatant in centrifugal 15 minutes, PB (50mmol/L will be used on the supernatant, pH7.5) the Ni-Chelating Sepharose FastFlow post of pre-equilibration, then with same PB balance Ni-Chelating Sepharose FastFlow post again, with elution buffer (50mmol/L PB, the 100mmol/L imidazoles, pH7.5) wash-out is collected eluting peak, use Sephadex G-25 to 50mmol/L PB the eluting peak of collecting, the pH8.0 desalination.
Target protein solution 75ml is gathered in the crops in this test altogether.
(2) purity is identified
The gel scanning method is determined the purity of protein of expression product, and the Bradford method is determined its concentration, and multiply by the concentration that corresponding purity is converted pure product with gained concentration.
After measured, the opd gene outcome purity of this preparation is 82.0%, and concentration is 3.2mg/ml.
(3) determination of activity
Measure active as follows: the fundamental reaction system is that (50mmol/L pH9.0), adds about 1 μ g enzyme to the 1ml Tris-HCl buffer solution that contains 2.5mmol/L substrate parathion, and reaction is 10 minutes under the room temperature.The trichloroacetic acid cessation reaction that adds 1ml10%.Set up control group, except that not enzyme-added, other and test group are in full accord.
After reaction finishes, according to the organophosphor peak of national agricultural industry criteria NY/T761-2004 with gc analysis control group and test group.Being calculated as follows enzyme lives: (1-A1/A0)/and enzyme amount * 100%.Wherein A1 is a test group organophosphor peak area, and A0 is a control group organophosphor peak area.
After measured, the ratio of the opd gene outcome of this preparation is lived and is 1000-1200IU/mg.
The preparation of embodiment 2, cehA gene outcome
According to the gene order that genbank accession number: Ab069723 announces, introduce Nde I site at 5 ' end, after terminator codon, add Eco RI site design cehA expressing gene;
Press embodiment 1 identical method and prepare, characterize the cehA gene expression product, target protein solution 67ml. is gathered in the crops in acquisition altogether altogether; Purity is 85.4%, and concentration is 2.7mg/ml.
Measure active as follows: the fundamental reaction system is that (50mmol/L pH7.5), adds about 5 μ g enzymes to the 1ml PB buffer solution that contains the corresponding substrate sevin of 2mmol/L, and room temperature reaction was used the dilute sulfuric acid cessation reaction after 10 minutes.Set up control group too.After reaction finishes, press the carbamate peak area of NY/T 761-2004, with embodiment 1 method calculated activity with gc analysis control group and test group.
The ratio of the cehA gene outcome of this preparation is lived and is 30-45IU/mg.
The preparation of embodiment 3, estP gene outcome
According to the gene order that genbank accession number: AY995176 announces, introduce Nde I site at 5 ' end, after terminator codon, add Eco RI site design cehA expressing gene;
Press embodiment 1 identical method and prepare, characterize the cehA gene expression product, gather in the crops target protein solution 90ml. altogether; Purity is 74.2%, and concentration is 1.9mg/ml.
Measure active as follows: the fundamental reaction system is that (20mmol/L pH8.0), adds about 10 μ g enzymes to the 1ml Tris-HCl buffer solution that contains the corresponding substrate cypermethrin of 2mmol/L, and room temperature reaction is used the trichloroacetic acid cessation reaction after 10 minutes, also set up control group.The same, press the peak area of NY/T 761-2004, with embodiment 1 method calculated activity with the pyrethroid pesticide of gc analysis control group and test group.
The ratio of the estP gene outcome of this preparation is lived and is 5-10IU/mg.
Embodiment 4, reaction system pH are to the influence of three kinds of enzymatic activitys
Press embodiment 1-3 measuring method for activity ready reaction system, but buffer solution is changed to phosphate buffer (pH=5.5,6.0,6.5 of 50mmol/L, 7.0,7.5,8.0,8.5), substrate is selected parathion, sevin and cypermethrin respectively, and enzyme is respectively opd, cehA and estP.The mensuration enzyme is lived, and the high activity that obtains with each enzyme is 100 to draw activity-pH curves respectively, sees Fig. 1-3.
The result shows that three kinds of enzymes are all to have higher relatively enzymatic activity at pH between 8.0-8.5.
Embodiment 5, detergent are to the influence of hydrolytic enzyme activities
With embodiment 1-3 ready reaction system, buffer solution is 50mM PB (pH 8.0), removes to contain corresponding substrate (parathion, sevin and cypermethrin), in contrast group; Detergent---working concentration is 5/10000ths APES (op-10), as experimental group in every pipe adding on the control group basis.
Took out a pipe, cessation reaction every five minutes.
Detecting residual agricultural chemicals with embodiment 1-3, with the peak area of remains of pesticide the time is mapped, is 100 with not enzyme-added pipe, the results are shown in Figure 4-6.
Embodiment 6, detergent are to the influence of hydrolytic enzyme activities
With embodiment 5, the test group detergent changes working concentration into and is ten thousand/ coconut oil alkyl diethanolamide (6502), obtain the result similar to embodiment 5.
Embodiment 7, eliminate remains of pesticide on vegetables, the melon and fruit with complex enzyme formulation
The enzyme solutions 1 μ l, 50 μ l, the 5ml that in volume is the 50mM PB (pH 8.0) of 1000ml, add embodiment 1-3 preparation respectively, the mass ratio 1: 40: 240 of three kinds of hydrolases that makes embodiment 1-3 preparation; Adding working concentration in above-mentioned solution is that working concentration is 5/10000ths APES (op-10), makes composite enzyme solution.
Organophosphorus pesticide, carbamate pesticide and pyrethroid pesticide are mixed respectively by its using dosage, and every group of agricultural chemicals sprays 6 in romaine lettuce respectively, 6 of naked oats dishes, 6 of cauliflowers, 6 of spray apples.Spray afternoon on the previous day, sampling in morning next day.
It is two groups that above-mentioned all kinds of vegetables are divided equally, uses the determined complex enzyme formulation solution of previous embodiment room temperature immersion treatment 15 minutes for one group, and another group is not then handled.Carry out pesticide residue analysis according to NY/T 761-2004.
The result shows that undressed vegetables, melon and fruit detect strong residues of pesticides, and treated vegetables, melon and fruit do not detect residues of pesticides or residual very low.
The present invention is not restricted to the described embodiments; that describes in the foregoing description and the specification just illustrates principle of the present invention; without departing from the spirit and scope of the present invention, the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.The claimed scope of the present invention is defined by appending claims and equivalent thereof.

Claims (8)

1, a kind of complex enzyme formulation of eliminating remains of pesticide is characterized in that, described complex enzyme formulation comprises organophosphor hydrolytic enzyme, carbamatic hydrolase, pyrethroid ester hydrolase, and its mass ratio is followed successively by 1: 27～40: 120～240.
2, enzyme preparation as claimed in claim 1 is characterized in that, described complex enzyme formulation is the aqueous solution or cushioning liquid, and the pH value of reaction system is 7.5～8.5.
3, enzyme preparation as claimed in claim 1 is characterized in that, described organophosphor hydrolytic enzyme, and the ratio of carbamatic hydrolase and pyrethroid ester hydrolase is 1: 40: 240.
4, enzyme preparation as claimed in claim 1, it is characterized in that, described organophosphor hydrolytic enzyme, carbamatic hydrolase and pyrethroid ester hydrolase origin come from the gene code of microorganism and express, and organophosphor hydrolytic enzyme is selected from a kind of or combination of opd gene, opdA gene, mpd gene, ophC2 gene expression product; Carbamatic hydrolase is selected from a kind of or combination in cehA gene, the caha gene outcome; The pyrethroid ester hydrolase is the estP gene expression product.
5, enzyme preparation as claimed in claim 4 is characterized in that, organophosphor hydrolytic enzyme is by opd gene and/or ophC2 gene expression, and carbamatic hydrolase is by cehA gene and/or caha gene expression, and the pyrethroid ester hydrolase is by estP gene expression.
6, enzyme preparation as claimed in claim 4 is characterized in that, organophosphor hydrolytic enzyme is by opd gene expression, genbank accession number: M20392; Carbamatic hydrolase is by cehA gene expression, genbank accession number: Ab069723; The pyrethroid ester hydrolase is by the estP gene, genbank accession number: AY995176.
7, the composition of enzyme preparation formation as claimed in claim 1, it is characterized in that, also comprise detergent in the described enzyme preparation composition, include but not limited to for, contain final concentration and be 1/10000th to two APES, and/or final concentration is 1/10000th to 5/10000ths coconut oil alkyl diethanolamide (6502).
8, as the application of each the described complex enzyme formulation among the claim 1-7, remains of pesticide on be used to degrade vegetables, tealeaves and the melon and fruit, described agricultural chemicals include but not limited to, organophosphorus insecticide, carbamate chemicals for agriculture and pyrethroid pesticide.
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CN101429515B (en) * 2008-12-17 2010-09-01 南京农业大学 Pyrethroid insecticide hydrolytic enzyme genes
WO2011060610A1 (en) * 2009-11-20 2011-05-26 Dang Yongfu Pesticide remover for fruit and vegetable
CN102423019A (en) * 2011-11-17 2012-04-25 宁波海通食品科技有限公司 Physical and chemical treatment method for degradation of specific pesticide-containing vegetable raw material before processing, and quality guaranteeing
CN101732819B (en) * 2008-11-14 2012-07-25 中国科学院沈阳应用生态研究所 Organic phosphorus degrading enzyme preparation and preparation method thereof
CN102618399A (en) * 2012-03-19 2012-08-01 江苏爱特福84股份有限公司 Composite capable of removing pesticide residue and disinfecting and preparation method thereof
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WO2016015668A1 (en) * 2014-08-01 2016-02-04 南京农业大学 Carbamate pesticide degrading enzyme cfh, coding gene cfd thereof and applications of both
CN107988175A (en) * 2017-11-03 2018-05-04 常州市万昌化工有限公司 A kind of preparation method of pyrethroid degradation agent
CN108850785A (en) * 2018-06-27 2018-11-23 罗瑞格斯（广东）生物科技有限公司 A kind of Methods of Degrading Pesticide Residues for vegetable and fruit
CN110240306A (en) * 2019-05-30 2019-09-17 西安建筑科技大学 A method of reducing wastewater toxicity containing organophosphorus pesticide
CN111019930A (en) * 2019-12-30 2020-04-17 武汉新华扬生物股份有限公司 Enzyme protective agent, composite enzyme liquid for pesticide degradation, preparation method of composite enzyme liquid and washing combined liquid
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CN101429515B (en) * 2008-12-17 2010-09-01 南京农业大学 Pyrethroid insecticide hydrolytic enzyme genes
WO2011060610A1 (en) * 2009-11-20 2011-05-26 Dang Yongfu Pesticide remover for fruit and vegetable
CN101935669B (en) * 2009-12-16 2013-01-02 中国科学院动物研究所 Detoxication engineering bacteria, and preparation method and application thereof
CN102423019A (en) * 2011-11-17 2012-04-25 宁波海通食品科技有限公司 Physical and chemical treatment method for degradation of specific pesticide-containing vegetable raw material before processing, and quality guaranteeing
CN102618399A (en) * 2012-03-19 2012-08-01 江苏爱特福84股份有限公司 Composite capable of removing pesticide residue and disinfecting and preparation method thereof
WO2016015668A1 (en) * 2014-08-01 2016-02-04 南京农业大学 Carbamate pesticide degrading enzyme cfh, coding gene cfd thereof and applications of both
CN104174607A (en) * 2014-08-06 2014-12-03 镇江市丹徒区茗缘茶叶专业合作社 Cleaning method for tea leaves
CN104560469A (en) * 2014-12-15 2015-04-29 深圳市中健科技产业投资有限公司 Fruit and vegetable agriculture remanet removing agent and preparation method thereof
CN107988175A (en) * 2017-11-03 2018-05-04 常州市万昌化工有限公司 A kind of preparation method of pyrethroid degradation agent
CN108850785A (en) * 2018-06-27 2018-11-23 罗瑞格斯（广东）生物科技有限公司 A kind of Methods of Degrading Pesticide Residues for vegetable and fruit
CN110240306A (en) * 2019-05-30 2019-09-17 西安建筑科技大学 A method of reducing wastewater toxicity containing organophosphorus pesticide
CN111019930A (en) * 2019-12-30 2020-04-17 武汉新华扬生物股份有限公司 Enzyme protective agent, composite enzyme liquid for pesticide degradation, preparation method of composite enzyme liquid and washing combined liquid
CN113189069A (en) * 2021-04-27 2021-07-30 华南农业大学 Pesticide-target protein screening method based on fluorescence analysis

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