CN101293131B - Complex enzyme preparation for eliminating residual pesticides and application thereof - Google Patents
Complex enzyme preparation for eliminating residual pesticides and application thereof Download PDFInfo
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- CN101293131B CN101293131B CN200710093807.4A CN200710093807A CN101293131B CN 101293131 B CN101293131 B CN 101293131B CN 200710093807 A CN200710093807 A CN 200710093807A CN 101293131 B CN101293131 B CN 101293131B
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- pesticide
- hydrolase
- enzyme
- complex enzyme
- agricultural chemicals
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Landscapes
- Agricultural Chemicals And Associated Chemicals (AREA)
- Enzymes And Modification Thereof (AREA)
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
Technical field
The present invention relates to a kind of complex enzyme formulation, particularly eliminate complex enzyme formulation and the application thereof of remains of pesticide in vegetables, tealeaves and melon and fruit.
Background technology
The population that accounts for the world 20% is supported in the soil in China Xu Yizhan world 7%, thereby relies on very much agricultural chemicals.Particularly, in order to reduce the loss causing because of disease and pest, used a large amount of pesticides.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 selectively, often non-target organism is caused to direct murder by poisoning.In addition, because agricultural chemicals is compared with difficult degradation, have part to remain in crop, herbage, soil and water, and with food chain transport and enrichment, the agricultural chemicals harm that the mankind of the extreme higher position in food chain are subject to is the most serious.
Agricultural chemicals mainly contains people's harm: acute poisoning, slow poisoning, Delayed onset neurotoxic, carcinogenic, teratogenesis and mutagenesis, genotoxicity, as damage sperm, make to become pregnant and fecundity reduction, and can cause embryo developmenting disorder, filial generation depauperation or death etc.
The Pesticide Residue of China vegetables, fruit is quite serious, even has because of the edible vegetables containing remains of pesticide, the report that fruit causes acute poisoning.The method that conventionally adopts now clear water washing, soaks or clean with cleaning agent is removed vegetables, the residual agricultural chemicals of fruit surface.Agricultural chemicals is generally insoluble in water, only with clear water, cleans 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 with sewage again entered environment circulate and accumulate.Also can adopt soda lye wash, agricultural chemicals easily decomposes under alkali condition, but alkalescence is crossed strong nutrition and the local flavor that easily destroys vegetables, fruit, 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 their easier natural degradations or by enzyme hydrolysis with the organochlorine of having forbidden.
Organophosphorus pesticide mostly is phosphoric acid ester or group thiophosphate, contains three phosphide keys.Its mechanism of toxication is phosphorylated acetylcholinesterase, makes body lose the ability of decomposing acetylcholine.The acetylcholine of accumulating makes cholinergic nerve be subject to continuing impulsion, causes first a series of nervous system poisoning symptoms of excited rear exhaustion, when serious, can cause death.As long as a phosphide key of fracture, organophosphorus pesticide just loses the ability of acetylcholine esterase inhibition, loses or greatly reduces the toxic action to animal.
The structure of carbamate chemicals for agriculture is based on eserine, and skeleton is carbamic acid, and conventional is N-formamido group formic acid esters.Its toxicity mechanism is also acetylcholine esterase inhibition activity, blocks normal nerve conduction.But the mechanism with organic phosphates is different: 1. it does not need through metabolism activation, can be directly and acetylcholinesterase form loose complex, suppress the latter's activity.2. with acetylcholinesterase Reversible binding, the compound of formation can decompose rapidly, therefore symptom is lighter, recovers very fast.After ester linkage breaking, carbamate chemicals for agriculture loses toxic action.
Pyrethroid pesticide is develop on the basis of pyrethrum ester structure efficient, wide spectrum, knock down fast, the agent of neural intoxicating insects.Tradition pyrethroid is carboxylate, by alcohol and chrysanthemumic acid condensation, is formed.Chrysanthemumic acid is partly generally the dimethylcyclopropane containing different side chain molecules, and alcohol moiety is generally the aromatic rings with cyano group.The intoxicating mechanism of pyrethroid is by postponing closing of neuron voltage-gated sodium channel, causes axoneure sodium, calcium ionic current to increase, and causes that in born of the same parents, sodium, calcium ion concentration increase, film Na
+-K
+-ATP enzyme and Ca
2+-atpase activity and Functional change, cause central lesion.The same with carbamate chemicals for agriculture, after ester linkage breaking, pyrethroid pesticide loses toxic action.
From china academia Periodical Database Based, all places database and Wei Pu full-text database, collect 1998-2006 year all about the article of 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 present stage of china vegetables, pesticide residues in fruits is (in Table 1): 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 Furadan and sevin; Three kinds of pyrethroid pesticides that recall rate is the highest are cypermethrin, fenvalerate and decis.Recall rate is relative recall rate, and the highest that of the recall rate in every class agricultural chemicals of take is 100.
Table 1.1998-2006 year China's Vegetable fruit agricultural chemicals recall rate statistics
Degrading pesticide makes the method for its inefficacy mainly contain chemical degradation, light degradation and microbial degradation.Conventional chemical degradation method has oxidizing process, alkali hydrolysis method and acid-hydrolysis method, although 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, cost is high, has certain risk.Light degradation is to utilize sunlight degradation plant surface, soil surface and airborne agricultural chemicals.Different compounds is different at illumination stability inferior, and intensity of illumination, soil types also can affect degradation effect, thereby photodegradative effect is comparatively limited.Microorganism in 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 completes by a series of enzymatic reactions the metabolism of agricultural chemicals is many, comprises hydrolysis.
But, the removing of remains of pesticide on none applicable vegetables of said method and 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; Light degradation method needs for a long time; Microbiological treatment degrading pesticide, also has infringement to vegetable melon and fruit.Even without impact, the vegetable melon and fruit of processing is like this estimated to be difficult to be accepted by people.
Therefore, in the urgent need to method and the product of remains of pesticide on removing vegetables that can be easy, safe, melon and fruit, to meet 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 a kind of the degrade wide removing vegetables of spectrum and melon and fruit is provided, by remaining in agricultural chemicals on vegetables and fruit and be decomposed into the product of nontoxic or low toxicity, reduce it to the murder by poisoning of people and non-target organism and the pollution to 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, caha gene outcome; Pyrethroid ester hydrolase is 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 pyrethroid ester hydrolase is by estP gene expression.
Optimally, organophosphor hydrolytic enzyme is by opd gene (genbank accession number: M20392) express, carbamatic hydrolase is by cehA gene (genbank accession number: Ab069723) express, pyrethroid ester hydrolase is by estP gene (genbank accession number: AY995176) express.
Described complex enzyme formulation, its reaction system also contains final concentration and is 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 pesticide degradable enzyme and acceptable carrier or the excipient of effective dose conventionally.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, are diluted with water working concentration during use.Solid formulation can will be prepared into tablet, granule by conventional method after the mixing such as hydrolase, starch, and freeze-drying after also hydrolase can being dissolved, is 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, also can independent packaging, in use mixing.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 for remains of pesticide on degrade vegetables and melon and fruit, described agricultural chemicals includes 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 targetedly degraded spectrum wide, three kinds of pesticide degradable 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 of the most popular three types of current state's energy of simultaneously degrading, provides a kind of scheme of removing easily residues of pesticides can to the users such as family, restaurant;
(2) three kinds of enzymes can produce synergy, and the degraded by organophosphor hydrolytic enzyme to organophosphorus pesticide has overcome the inhibition of organophosphorus pesticide to carbamate and pyrethroid ester hydrolase;
(3) by the ratio optimization of three kinds of enzymes, additive preferably, reaction condition preferably, complex enzyme formulation enzyme of the present invention is lived high, remove residues of pesticides effect excellent;
(4) complex enzyme formulation of the present invention, the microbial degradation enzyme adopting adopts genetic engineering means preparation, and output is high, and cost is low, is conducive to 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 with vegetable and fruit, clean and remove, and is not infected with taste and nutrition that vegetable melon and fruit does not affect it yet.
Accompanying drawing explanation
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 activity and the pH relation of carbamatic hydrolase.
Fig. 4 adds the impact of detergent on organophosphorus pesticide hydrolase.
Fig. 5 adds the impact of detergent on carbamatic hydrolase activity.
Fig. 6 adds the impact of detergent on pyrethroid hydrolytic enzyme activities.
The specific embodiment
Pesticide degradable enzyme
Enzymic degradation residues of pesticides have reaction condition gentleness, the feature that degradation efficiency is high.Selectable pesticide degradable enzyme comprises: microbe-derived, insect is originated, the pesticide degradable enzyme of animal origin.
Can be by simulated hexapod the detoxify enzyme product of mechanism exploitation degrading pesticide, i.e. the removing toxic substances system of reconstruct insect.The enzyme that insect participates in removing toxic substances 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 needs glutathione.The insect of carbamate chemicals for agriculture resistance as anopheles, diamondback moth and aphid in, carboxy-lesterase is to cause drug-fast reason.But the ability very limited (malathion exception) of insect carboxy-lesterase hydrolysis agricultural chemicals; resistant insects is not the carboxy-lesterase greatly improving by expressing hydrolysing activity; but be combined with pesticide by expressing a large amount of carboxy-lesterases; make pesticide can not arrive target, thereby in protective, target-acetylcholinesterase is avoided suppressing.In addition, the carboxy-lesterase pyrethroid of also degrading to a certain extent, but insect is mainly because Na on neu to the resistance mechanism of pyrethroid
+the Reduced susceptibility of passage.Thereby the removing toxic substances system of reconstruct insect is removed residual agricultural chemicals uneconomical.
From the phosphoric triesterase obtaining in animal body, also can decompose organophosphorus pesticide, 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, the advantages such as microbe-derived hydrolase tool activity is high, easy screening, become the focus of finding pesticide degradable enzyme.From can the degrade bacterial strain of specific pesticide of occurring in nature screening, and separated corresponding hydrolase gene therefrom.Studying more and fruitful is that organophosphorus pesticide hydrolase: opd finds and study the most thorough organophosphor hydrolytic enzyme gene the earliest, finds successively again afterwards many genes that can be hydrolyzed organophosphor, as opaA, opdA etc.The domestic report that is also found organophosphor hydrolytic enzyme gene, as discovery ophC2 such as Fan Yunliu, Li Shunpeng etc. find mpd gene.Also has 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 Pesticide Residue mainly concentrate on screening agricultural chemicals hydrolase gene, especially organophosphorus pesticide hydrolase gene, and remove residual agricultural chemicals in vegetables, fruit with the gene obtaining.Only with organophosphor hydrolytic enzyme, residual carbamate and pyrethroid are not almost removed to effect.Also the composite use of esterase of useful organophosphor hydrolytic enzyme and insect, but as previously mentioned, the mechanism of the esterase of insect removing toxic substances is mainly combination, and hydrolysis is less important.
Because vegetable and fruit is numerous in variety, the pesticide variety using in production process is different, so the residues of pesticides that run in daily life are not often 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 Pesticide Residue, must adopt the compound formulation with degraded broad spectrum activity.
Between 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 Furadan and sevin; Three kinds of pyrethroid pesticides are cypermethrin, fenvalerate and decis, therefore, and object that should be using these agricultural chemicals as preferential decomposition.
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 just has the selections such as Opd, Opda, OphC2.Different enzymes has different substrate spectrum and active, there is no all agricultural chemicals of a kind of enzyme fine hydrolysis of energy.Therefore, should preferentially select modal remains of pesticide kind to have enzyme or the enzyme combination of degraded advantage.Meanwhile, due to the different reaction condition of different enzyme requires, coordinate these conditions also by no means easy.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 a large amount of research work, finds (the genbank accession number: M20392), ceha (genbank accession number: Ab069723) and estp (genbank accession number: the agricultural chemicals that AY995176) three kinds of enzymes combinations can the listed need of fine degraded be preferentially degraded with existing opd.The reason of combination is not that the enzyme that can decompose simply three class agricultural chemicals 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, ceha and the removing of the incompatible realization of estp genome to the preferential degrading pesticide of listed need of microorganism.
Because various enzyme specific activity are different, the using dosage of various agricultural chemicals is also different, thereby can not simply several enzymes be mixed.In the practical residue limit of agricultural chemicals, determine that the ratio of enzyme is best in theory, but because the stability of various agricultural chemicals is different, the Time To Market of vegetable and fruit is different, the residual ratio of reality of agricultural chemicals varies, and is difficult to draw that has general data.The application dosage of agricultural chemicals is relatively fixing, thereby, can it be the ratio with reference to calculating various enzymes in complex enzyme formulation.According to investigations, the application dosage of agricultural chemicals is all at the same order of magnitude, and these several agricultural chemicals that at least we consider are like this, thereby determine that the principal element of various enzyme ratios is specific activity of enzyme.The organophosphor hydrolytic enzyme adopting in the present invention (opd expression), carbamatic hydrolase (ceh expression) and pyrethroid ester hydrolase (estP expression) be take the listed three class agricultural chemicals of the present invention during as substrate, its average specific activity reduction of a fraction is not 1000-1200IU/mg, 30-45IU/mg and 5-10IU/mg.Thereby the ratio of three kinds of hydrolases is 1:27-40:120-240 in the present invention.
In addition, agricultural chemicals mostly is water-insoluble materials, is often adsorbed on vegetables, fruit surface, is difficult for interacting with hydrolase.Add detergent and contribute to agricultural chemicals to depart from from vegetables, fruit surface, also contribute to pesticide dispersion in the aqueous solution, promote agricultural chemicals and hydrolase to interact.Thereby, in complex enzyme reaction system, also contain the detergent that can promote that agricultural chemicals is eliminated.More preferably, the detergent of selecting permission to use in abluent for foodstuff.More preferably, the detergent of interpolation is APES (op-10) and coconut oil alkyl diethanolamide (6502).
The preparation of pesticide degradable enzyme
Microbe-derived pesticide degradable enzyme, the microorganism that can produce this kind of enzyme by cultivation obtains, and also can prepare by gene engineering method.For example, can be by the Gene cloning of coding pesticide degradable enzyme be carried out to the production of recombinant protein at prokaryotic expression carrier in as Yeast expression carrier as coli expression carrier or carrier for expression of eukaryon, this is that this area staff knows.
The enzyme of external elimination remains of pesticide, the requirement of its purity is low far beyond protein pharmaceuticals.For simplifying purifying process, reduce costs, can add the label of being convenient to purifying at albumen n end or C end, as FLAG, 6 * His etc., 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 the advantages such as fast growth, simple to operate, technology maturation, thereby, more preferably, the present invention by by Gene cloning in prokaryotic expression carrier, in Escherichia coli, produce hydrolase.
Opd gene of the present invention, cehA gene, cehA gene are synthesized by Shanghai bioengineering Co., Ltd, expression plasmid pET30a (+) is purchased from Novagen company, e. coli bl21 (DE3) is purchased from Stratagene company, and restriction enzyme is purchased from Shanghai bioengineering Co., Ltd.Adopt technique for gene engineering method, obtain the proteinase expression product of said gene.Parathion, sevin and cypermethrin are purchased from Shanghai Pesticide Research Institute, and detergent APES (op-10), coconut oil alkyl diethanolamide (6502) are domestic, and other reagent are domestic AR.
In order to further illustrate effect of the present invention and method, now with concrete embodiment, be illustrated.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, such as people such as Sambrook, < < molecular cloning: laboratory manual > > (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or method and the condition of the agricultural industry criteria NY/T761-2004 > > of < < country restriction, or the condition of advising according to manufacturer.It should be understood that these embodiment, only for the object of illustration, never limit the scope of the invention.
The preparation of embodiment 1, opd gene outcome
(1) expression and purifying
A, the gene order of announcing according to genbank accession number M20392, introduce Nde I site at 5 ' end, adds EcoRI site design opd expressing gene after terminator codon;
B, by Gene cloning between the Nde I and EcoRI site of pET30a (+) plasmid, construction expression plasmid;
C, use CaCl
2method proceeds to e. coli bl21 (DE3) bacterial strain by expression plasmid, selects the clone of that resistance of card, obtains engineering bacteria;
The engineering bacteria of D, inoculation step (2) is in containing in the LB culture medium of 50 μ g/ml kanamycins, 37 ℃ of shaking table overnight incubation.By 1:100, transfer in containing in the fresh LB culture medium of kanamycins, 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, use PB (50mmol/L, pH7.5) resuspended thalline, ultrasonic disruption cell, 18000 * g collects supernatant for centrifugal 15 minutes, PB (50mmol/L will be used on 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, 100mmol/L imidazoles, pH7.5) wash-out, collects eluting peak, use Sephadex G-25 to 50mmol/L PB the eluting peak of collecting, pH8.0 desalination.
Target protein solution 75ml is gathered in the crops in this test altogether.
(2) Purity
Gel image scanning method is determined the purity of protein of expression product, and Bradford method is determined its concentration, and by gained concentration, is multiplied by the concentration of corresponding purity conversion sterling.
After measured, this opd gene outcome purity of preparing is 82.0%, and concentration is 3.2mg/ml.
(3) determination of activity
Measure as follows active: fundamental reaction system is the Tris-HCl buffer solution (50mmol/L, pH9.0) that 1ml contains 2.5mmol/L substrate parathion, adds approximately 1 μ g enzyme, reacts 10 minutes under room temperature.The trichloroacetic acid cessation reaction that adds 1ml10%.Set up control group, except not enzyme-added, other and test group are in full accord.
After reaction finishes, the organophosphor peak according to national agricultural industry criteria NY/T761-2004 by gc analysis control group and test group.Being calculated as follows enzyme lives: (1-A1/A0)/enzyme amount * 100%.Wherein A1 is test group organophosphor peak area, and A0 is control group organophosphor peak area.
After measured, the specific activity of this opd gene outcome of preparing is 1000-1200IU/mg.
The preparation of embodiment 2, cehA gene outcome
The gene order of announcing according to genbank accession number: Ab069723, introduces Nde I site at 5 ' end, adds Eco RI site design cehA expressing gene after terminator codon;
Press method preparation, sign cehA gene expression product that embodiment 1 is identical, target protein solution 67ml. is gathered in the crops in acquisition altogether altogether; Purity is 85.4%, and concentration is 2.7mg/ml.
Measure as follows active: fundamental reaction system is the PB buffer solution (50mmol/L, pH7.5) that 1ml contains the corresponding substrate sevin of 2mmol/L, adds approximately 5 μ g enzymes, and room temperature reaction, after 10 minutes, is used dilute sulfuric acid cessation reaction.Set up too control group.After reaction finishes, the carbamate peak area by NY/T761-2004 by gc analysis control group and test group, with embodiment 1 method calculated activity.
The specific activity of the cehA gene outcome that this is prepared is 30-45IU/mg.
The preparation of embodiment 3, estP gene outcome
The gene order of announcing according to genbank accession number: AY995176, introduces Nde I site at 5 ' end, adds Eco RI site design cehA expressing gene after terminator codon;
Press method preparation, sign cehA gene expression product that embodiment 1 is identical, gather in the crops altogether target protein solution 90ml.; Purity is 74.2%, and concentration is 1.9mg/ml.
Measure as follows active: fundamental reaction system is the Tris-HCl buffer solution (20mmol/L that 1ml contains the corresponding substrate cypermethrin of 2mmol/L, pH8.0), add approximately 10 μ g enzymes, room temperature reaction is used trichloroacetic acid cessation reaction after 10 minutes, also set up control group.The same, the peak area by NY/T761-2004 with the pyrethroid pesticide of gc analysis control group and test group, with embodiment 1 method calculated activity.
The specific activity of the estP gene outcome that this is prepared is 5-10IU/mg.
Embodiment 4, the reaction system pH impact on 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 respectively parathion, sevin and cypermethrin, and enzyme is respectively opd, cehA and estP.Mensuration enzyme is lived, and the high activity of take respectively that each enzyme obtains is 100 drafting activity-pH curves, sees Fig. 1-3.
Result demonstration, three kinds of enzymes are all to have relatively high enzymatic activity at pH between 8.0-8.5.
Embodiment 5, the impact of detergent on hydrolytic enzyme activities
With embodiment 1-3 ready reaction system, buffer solution is 50mM PB (pH8.0), except containing corresponding substrate (parathion, sevin and cypermethrin), as a control group; On control group basis, every pipe adds detergent---and working concentration is 5/10000ths APES (op-10), as experimental group.
Every five minutes, take out a pipe, cessation reaction.
With embodiment 1-3, detect residual agricultural chemicals, with the peak area of remains of pesticide, the time is mapped, the not enzyme-added pipe of take is 100, the results are shown in Figure 4-6.
Embodiment 6, the impact of detergent on hydrolytic enzyme activities
With embodiment 5, 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, with complex enzyme formulation, eliminate the remains of pesticide on vegetables, melon and fruit
The enzyme solutions 1 μ l, 50 μ l, the 5ml that in the 50mM PB that is 1000ml at volume (pH8.0), add respectively embodiment 1-3 to prepare, make the mass ratio 1:40:240 of three kinds of hydrolases prepared by embodiment 1-3; In above-mentioned solution, adding working concentration 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 respectively 6, romaine lettuce, 6 of Lettuces, 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 processed.According to NY/T761-2004, carry out pesticide residue analysis.
Result shows, 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 in above-described embodiment and description, describes 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 (4)
1. a complex enzyme formulation of eliminating remains of pesticide, it is characterized in that, described complex enzyme formulation comprises organophosphor hydrolytic enzyme, carbamatic hydrolase, pyrethroid ester hydrolase, its mass ratio is followed successively by 1: 27～and 40: 120～240, described organophosphor hydrolytic enzyme is by opd gene expression, genbank accession number: M20392; Carbamatic hydrolase is by cehA gene expression, genbank accession number: Ab069723; Pyrethroid ester hydrolase is by estP gene expression, genbank accession number: AY995176; Also containing final concentration be 5/10000ths APES and/or final concentration be ten thousand/ coconut oil alkyl diethanolamide.
2. complex enzyme formulation as claimed in claim 1, is characterized in that, described complex enzyme formulation is the aqueous solution or cushioning liquid, and pH value is 7.5～8.5.
3. complex enzyme formulation as claimed in claim 1, is characterized in that, described organophosphor hydrolytic enzyme, and the mass ratio of carbamatic hydrolase and pyrethroid ester hydrolase is 1: 40: 240.
4. the application of the complex enzyme formulation as described in claim 1～3 any one, for remains of pesticide on the vegetables of degrading, tealeaves and melon and fruit, described agricultural chemicals is 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 |
| CN102485876A (en) * | 2009-11-20 | 2012-06-06 | 党永富 | Residual pesticide remover for vegetables and fruits |
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| 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 |
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| CN110240306B (en) * | 2019-05-30 | 2021-12-14 | 西安建筑科技大学 | Method for reducing toxicity of wastewater containing organophosphorus pesticides |
| 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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| CN1059931A (en) * | 1991-09-21 | 1992-04-01 | 广州白云科技开发中心 | Vegetables, fruit cleaning agent of remanent pesticide |
| WO2003066874A1 (en) * | 2002-02-06 | 2003-08-14 | Commonwealth Scientific And Industrial Research Organisation | Degradation of hydrophobic ester pesticides and toxins |
| CN1651571A (en) * | 2004-05-21 | 2005-08-10 | 中国科学院动物研究所 | Super engineering bactrerin, its expressed detoxicating enzyme, construction method and application |
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|---|---|---|---|---|
| CN1059931A (en) * | 1991-09-21 | 1992-04-01 | 广州白云科技开发中心 | Vegetables, fruit cleaning agent of remanent pesticide |
| WO2003066874A1 (en) * | 2002-02-06 | 2003-08-14 | Commonwealth Scientific And Industrial Research Organisation | Degradation of hydrophobic ester pesticides and toxins |
| CN1651571A (en) * | 2004-05-21 | 2005-08-10 | 中国科学院动物研究所 | Super engineering bactrerin, its expressed detoxicating enzyme, construction method and application |
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