CN108823181A - The extracting method of mixed coloured cowpea esterase and its application in organophosphorus pesticide detection - Google Patents
The extracting method of mixed coloured cowpea esterase and its application in organophosphorus pesticide detection Download PDFInfo
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- CN108823181A CN108823181A CN201810448436.5A CN201810448436A CN108823181A CN 108823181 A CN108823181 A CN 108823181A CN 201810448436 A CN201810448436 A CN 201810448436A CN 108823181 A CN108823181 A CN 108823181A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
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Abstract
The present invention extracts mixed coloured cowpea esterase using the method for crushing centrifugation from mixed coloured cowpea, and purified with the purifying process of ethanol precipitation to mixed coloured cowpea esterase crude liquid after being saltoutd by the different ammonium sulfate of concentration, according to enzyme inhibition principle, acetic acid and alpha-Naphthol are generated using mixed coloured cowpea esterase hydrolyzed substrate α-naphthaleneaceticacidester, alpha-Naphthol and solid indigo plant B salt action generate the azo-compound of aubergine.Influence of the pesticide to enzymatic activity is detected by production quick measuring card, after mixed coloured cowpea esterase is inhibited by organophosphorus pesticide, will affect chromogenic reaction, therefore, pesticide residue situation is indicated according to the chromogenic reaction power of generation.
Description
Technical field
The present invention relates to the extracting method of technical field of pesticide more particularly to a kind of mixed coloured cowpea enzyme and its in organophosphorus pesticide
Application in residue detection.
Background technique
Pesticide is essential in agricultural production, and China is large agricultural country and pesticide producing and uses big country, could not
Recognize, pesticide pest control, go in terms of all play important function, but pesticide is with a kind of toxic and can be residual
Soil, the chemical substance in vegetables are stayed in, exceedingly using the health care belt of the mankind can be given to carry out immeasurable harm, and it is remaining
Pesticide in the soil can destroy ecological environment, influence to cultivate environment.Relevant report explains that a large amount of pesticide in China does not obtain
It utilizes, is mainly diffused into atmospheric environment through pesticide residue, and pass through precipitation contamination system, contaminated soil, finally by plant roots
Portion is absorbed into enrichment, and enters human body by food chain, causes the generation of the events such as human body pesticide poisoning.
In addition, organophosphorus pesticide successfully becomes the maximum pesticide of China's usage amount, extensively since organo-chlorine pesticide is disabled
Organophosphorus pesticide kind in general use belongs to high toxicity pesticide, and a large amount of uses will cause many potentially hazardous, such as pollution ring
Border, phytotoxicity and person poultry poisoning etc..
Currently, mainly there are four major class pesticides in China in agricultural production, it is organochlorine class, organic phosphates, amino first respectively
Esters, quasi- chrysanthemum deinsectization ester pesticides, wherein organophosphorus pesticide category organophosphorus ester compound belongs to organophosphorus ester or vulcanization
Acid esters compound is in yellow or brown oil fat-soluble liquid more, and minority is crystalline solid, volatile, meets alkali and easily decomposes.Mesh
The preceding type used is more, including thimet (3911), demeton (1059), parathion (1605), Mortopl, metrifonate, Rogor,
Marathon (4049), parathion-methyl (parathion-methyl), thiometon, DDVP, demeton-methyl (demeton-methyl), oxidation
Rogor, Azodrin etc..After the percutaneous skin of organic phosphorus desinsection, mucous membrane, alimentary canal, respiratory tract absorb, it is distributed each internal organs of whole body quickly, with
It is minimum in concentration highest in liver, muscle and brain.Organophosphorus pesticide employment mechanism is the activity for inhibiting neurotic enzyme, makes neurotic enzyme
It cannot hydrolyze, so as to cause corresponding poisoning symptom.
Detection for pesticide residue has relevant research both at home and abroad, and correlative study is it is found that the inspection of residue of pesticide at present
Survey depend on gas chromatography, high performance liquid chromatography, gas/liquid phase chromatograph-mass spectrometer coupling method and capillary electrophoresis,
Immunization, biosensor etc., the above detection method detection sensitivity and accuracy are higher, but gas chromatograph need to be used etc.
Precision instrument, the high request of instrument make Detecting Pesticide that can not come into laboratory, come into vegetables and fruits planting greenhouse, come into people
Daily life.In addition, sample needed for the above detection method is also required to extremely complex pre-treatment, pole also is brought to Pesticides Testing
Big inconvenience increases detection difficulty to testing staff.
Secondly, there are also a kind of detection methods in major Pesticides Testing, i.e., detection method is inhibited by the enzyme of primary study,
Currently, enzyme inhibition is mainly used for the detection of organophosphorus insecticide and carbamate chemicals for agriculture.Enzyme inhibition can be divided into two
Class, one kind are animal cholinester enzyme inhibitions, and another kind of is vegetable esters enzyme inhibition.Enzyme inhibition, which has, detects quick, operation
Simply, the advantages that at low cost, the concern by various circles of society.Before this, we are mostly with enzyme acetylcholine detection pesticide for detection side
Method, but its enzyme source is mostly the expensive materials such as sardine and fly head, but also testing cost is higher and materials are difficult.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of extracting method of mixed coloured cowpea enzyme and its in organophosphorus pesticide
Application in residue detection.
In order to solve the above technical problems, the technical solution adopted by the present invention is that：A kind of extracting method of mixed coloured cowpea enzyme, including
Following steps：
(1), it crushes：20 grams of mixed coloured cowpeas are weighed with electronic balance, is crushed, is sieved with 100 mesh sieve with pulverizer, be placed in beaker；
(2), it mixes：120mL 0.1mol/L is added to beaker, 6.5 phosphate buffer of pH, vibrate or blend 10~
30min；
(3), it stands, filter：In 4 DEG C of refrigerators soaked overnight, with 4 layers of filtered through gauze；
(4), it is centrifuged：Under the conditions of 4 DEG C, 10000r/min refrigerated centrifuge 10min collects supernatant liquor, as crude enzyme liquid；
(5) purifying：
A, 10mL is taken from thick esterase, and the saturation degree of ammonium sulfate to 25% is added, and is placed in 4 DEG C of refrigerator and is stood 12h
Afterwards；
B, it takes and stands liquid in centrifuge tube, under the conditions of 4 DEG C, 10000r/min refrigerated centrifuge 10min discards precipitating, takes
Supernatant；
C, it is added to supernatant to 60% saturation degree of ammonium sulfate, lower sediment, mixed coloured cowpea as after purification are collected in centrifugation
Esterase.
The present invention also provides a kind of methods of quickly detection pedo relict organophosphorus pesticide content, include the following steps：
(1) production of the standard curve of alpha-Naphthol：The alpha-Naphthol dehydrated alcohol standard solution for taking 1mmol/L, uses pipettor
The accurate alpha-Naphthol dehydrated alcohol standard solution for drawing 0,200,400,600,800,1000,1200 μ L, adds phosphate buffer
To 4mL, then plus 0.5mL consolidate indigo plant B salting liquid, be uniformly mixed, alpha-Naphthol dehydrated alcohol standard solution is not added as control group,
The absorbance of the above reaction solution is read at 595nm, using concentration as ordinate, absorbance is abscissa, standard curve of mapping to obtain,
Slope is：
(2) measurement of protein content：Using bovine serum albumin as standard protein, the standard protein for being configured to 0.1mg/mL is molten
Liquid takes 12 test tubes, is separately added into the standard protein solution of 0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, uses
0.15mg/mlNaCl supplies 1mL, and the absorbance value that 4mL Coomassie brilliant blue reagent measures enzyme solution under 595nm wavelength is added,
The average value for taking 2 groups of measurements, using protein content as abscissa, absorbance is the standard curve that ordinate draws protein content；With
The protein content of Coomassie brilliant blue colorimetric method method measurement enzyme solution；
(3) general esterase activity measures：1.95mL phosphate buffer, 0.05mL α-naphthaleneaceticacidester are sequentially added in test tube
Enzyme solution after acetone soln and 0.5mL dilution；It mixes, after reacting 10min in 30 DEG C of water bath with thermostatic control, 0.5mL is added and consolidates indigo plant B
Salting liquid reacts 10min in 30 DEG C of water bath with thermostatic control, and solution absorbance is then measured at 595nm；According under corresponding pH value
Alpha-Naphthol standard curve and the absorbance measured, calculating general esterase activity is
In formula：E is esterase activity contained by every milliliter of enzyme solution, U/mL
OD595For the absorbance measured at wavelength 595nm after reaction
D is the extension rate of enzyme solution
K is the slope of the standard curve of alpha-Naphthol under corresponding pH
U is enzyme activity unit, and 1U schedules under prescribed conditions, to be catalyzed per minute needed for 1 μm of obtained ol alpha-Naphthol
Enzyme amount；
(4) measurement of Rate activity：
Rate activity=total activity (U)/total protein (mg)
(5) pesticide sensitivity testing：
According to the measuring method of general esterase activity, the buffer without pesticide is changed into buffer containing different pesticides,
Measuring concentration is the metrifonate organophosphorus pesticide under 0mg/mL, 0.5mg/mL, 1.0mg/mL, 1.5mg/mL, 2.0mg/mL to flower
The inhibiting rate of cowpea esterase, using organophosphorus pesticide concentration as abscissa, inhibiting rate is that ordinate draws curve, with inhibiting rate I table
Show esterase to the susceptibility of organophosphorus pesticide；
Inhibiting rate calculates according to the following formula：
Inhibiting rate I (%)=(EDo not inhibit-EInhibit)/EDo not inhibit× 100%；
(6) drafting of pesticide sensitivity curves
By the linear relationship of the concentration of measurement inhibiting rate I and organic agricultural chemicals, regression equation is established, organic agriculture can be calculated
The concentration of medicine.
Preferably, the concentration of phosphate buffer described in step (2) is 0.0 4mol/L, pH 6.5.
Preferably, the alpha-Naphthol dehydrated alcohol standard solution preparation method of 1mmol/L described in step (1) is：It will
0.093105 g alpha-Naphthol, which is dissolved in 500mL dehydrated alcohol, to be made.
Further, linear equation described in step (6) is：Y=0.544x+56.14, y are inhibiting rate (y), and x is agriculture
Medicine (metrifonate) concentration (mg/mL).
The present invention also provides a kind of methods of Organophosphorus Pesticide Residues in quickly detection soil, include the following steps：
(1), sample-adding piece production：With the chromatographic paper disk of scissors clip diameter 1cm, the mixed coloured cowpea ester of 40 μ L after purification is pipetted
Enzyme solutions are carefully added drop-wise to disk center, are dried at room temperature for, are fabricated to sample-adding piece；
(2), substrate piece makes：With the chromatographic paper disk of scissors clip diameter 1cm；Pipetting 40 μ L mass concentrations is 0.016
The α-naphthaleneaceticacidester acetone soln of mol/L is carefully added drop-wise to disk center, up to substrate piece after drying under room temperature；
(3), colour developing item production：A length of 3cm is obtained with scissors clip, the chromatographic paper disk of wide 1cm, wherein one end is diameter 1
The disk of mm；The 100 μ L of solid blue B solution for pipetting 1.56mg/mL, is carefully added drop-wise in the scraps of paper, develops the color after the scraps of paper are dry
Item；
Will colour developing item, substrate piece, sample-adding piece by being sequentially sequentially placed between plastic packaging film upper and lower level from top to bottom, enzyme center
It is aligned with well and substrate piece center, substrate piece center is aligned with the center of circle of half nose circle of detector bar；The alignment of plastic packaging film upper and lower level,
Plastic packaging machine is preheated to 60~70 DEG C, and after plastic packaging, quick measuring card completes；
It (4), is respectively pure pesticide, 5 times of pesticide dilution, 10 times of pesticide dilution, 20 times of pesticide dilution, pesticide dilution by concentration
100 times, pesticide dilute 1000 times, pesticide dilute 10000 times of distilled water metrifonate be added drop-wise to quick measuring card well, visual observations
Gradation of color is fabricated to colorimetric card；
(5), sample to be tested is dripped on quick measuring card, forms corresponding color, it, can by being compared with the color on colorimetric card
The quickly residual content of the organophosphorus pesticide of measurement sample.
Not only general esterase activity and Rate activity are high for the mixed coloured cowpea enzyme that the present invention selects, but also are easily obtained, and it is convenient to extract, at
This is low；And test method provided by the present invention is at low cost, surveys inspection quickly, and it is practical, it can be in large-scale application.
Detailed description of the invention
Fig. 1 is the canonical plotting of alpha-Naphthol of the present invention；
Fig. 2 is present protein content standard curve graph；
Fig. 3 is quick measuring card structural schematic diagram of the present invention；
Fig. 4 is pesticide sensitivity curve figure of the present invention.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, but do not constitute limiting the scope of the invention.
The present invention generates acetic acid and alpha-Naphthol, alpha-Naphthol and solid indigo plant B using mixed coloured cowpea esterase hydrolyzed substrate α-naphthaleneaceticacidester
The azo-compound of salt action generation aubergine.After enzyme is inhibited by organophosphorus pesticide, chromogenic reaction will affect, therefore can root
Pesticide residue situation is indicated according to the chromogenic reaction power of generation.
Naphthols and color developing agent consolidate indigo plant B salt action in the reaction, and system color becomes rose-red from yellow, eventually forms purplish red
Color azo-compound, to complete chromogenic reaction.The repressed degree of enzyme is different, and the yield of aubergine azo-compound is different,
The depth of solution colour is also different.The Rate activity that plant esterase can be measured out according to the principle, compares when being inhibited by pesticide
Enzymatic activity and enzymatic activity when not inhibited by pesticide, can find out the inhibiting rate of pesticide.It is similar to acetylcholinesterase, Ke Yitong
The relationship between persticide residue and inhibition of enzyme activity rate is crossed, measures the size of inhibiting rate to show the remaining poison of organophosphorus pesticide
Property degree.
Mixed coloured cowpea ester enzyme viability
The plant esterase of separate sources its general esterase activity, Rate activity and to the sensitivity of different pesticides not
Together.
Minimum detection value and susceptibility of the different plant source esterase of table 1 to various pesticides
As seen from the above table, mixed coloured cowpea esterase is higher to the susceptibility of organophosphorus pesticide, has reached 40.433 detected level, this
Outside, the general esterase activity of mixed coloured cowpea and Rate activity are extremely significant higher than other plant esterases and housefly.Its general esterase activity is
14.695U, Rate activity 1.219U/mg, and be easily obtained, it is convenient to extract, and it is at low cost, therefore in this, as Detecting Pesticide
It is suitable with plant esterase.
Embodiment 1
1.1 experimental materials and reagent
Mixed coloured cowpea (Taobao), 0.1mol/L, 6.5 phosphate buffer of pH, 0.04mol/L, 6.5 phosphate-buffered of pH
Liquid, α-naphthyl acetate (chemistry is pure), dehydrated alcohol (chemistry is pure), solid indigo plant B salt (chemistry is pure), ammonium sulfate (chemistry is pure), distillation
Water, 1mmol/L alpha-Naphthol dehydrated alcohol standard solution (chemistry is pure), α-naphthaleneaceticacidester acetone soln：0.016mol/L：Use acetone
(99%) it prepares, 4 DEG C are kept in dark place, Coomassie brilliant blue reagent, bovine serum albumin, NaCl solution
1.2 reagents are equipped with
(1) substrate piece dropping liquid is equipped with
It is substrate piece dropping liquid that 14.8mg (0.014g) acetic acid-α-naphthylacetate, which is dissolved in 50mL acetone,.
(2) developing solution is equipped with
15.6mg (0.015g) solid indigo plant B salt is dissolved in 10mL distilled water, the rate oscillation 30min with 180 min is
Developing solution.
(3) outfit of 1mmoL/L alpha-Naphthol dehydrated alcohol standard solution
0.093105g alpha-Naphthol is dissolved in 500mL dehydrated alcohol
(4) outfit of buffer
The preparation of mother liquor
0.2M Na2HPO4:Weigh 71.6g Na2HPO4-12H2O is dissolved in 1000mL
0.2M NaH2PO4:Weigh 31.2g NaH2PO4-2H2O is dissolved in 1000mL
The preparation of PB (pH=6.5)
First match 0.2M PB (pH=6.5,100mL):Take the Na of 68.5mL 0.2mol/L2HPO4With 31.5mL 0.2mol/L
NaH2PO4Mixing；
0.1M PB (pH=6.5):500mL 0.2M PB is taken, 1000mL is diluted with water to；
0.04M PB (pH=6.5):200mL 0.2M PB is taken, 1000mL is diluted with water to.
1.3 instrument
Electronic balance, pulverizer (100 mesh), high speed freezing centrifuge, thermostat water bath, spectrophotometer, chromatographic paper
2 experimental methods
2.1 flower cowpea esterase crude enzyme liquid preparations
2.1.1 crushing
20 grams of mixed coloured cowpeas are weighed with electronic balance, is crushed, is sieved with 100 mesh sieve with pulverizer, be placed in beaker.
2.1.2 mixing
120mL 0.1mol/L is added, 6.5 phosphate buffer of pH vibrates or blend 10~30min.
2.1.3 standing, filtering
In 4 DEG C of refrigerators soaked overnight, with 4 layers of filtered through gauze.
2.1.4 centrifugation
Under the conditions of 4 DEG C, 10000r/min refrigerated centrifuge 10min collects supernatant, as crude enzyme liquid.
The purifying of 2.2 mixed coloured cowpea esterases
(1) 10mL is taken from thick esterase, and the saturation degree of ammonium sulfate to 25% is added (25 DEG C, every 1000mL solution is reinforced
Body ammonium sulfate 144g), it is placed on after standing 12h in 4 DEG C of refrigerator；
(2) it takes and stands liquid in centrifuge tube, under the conditions of 4 DEG C, 10000r/min refrigerated centrifuge 10min discards precipitating, takes
Supernatant；
(3) it is added that (25 DEG C, be adjusted to 60% on 25%, only needs every 1000mL to 60% saturation degree of ammonium sulfate to supernatant
Solution reinforcing body ammonium sulfate 230g), it is centrifuged (4 DEG C, 10000r/min, 15min), collects lower sediment, flower as after purification
Cowpea esterase.
The enzyme activity determination of 2.3 mixed coloured cowpea esterases
2.3.1 the production of the standard curve of alpha-Naphthol
The alpha-Naphthol dehydrated alcohol standard solution for taking 1mmol/L, accurately draw 0 with pipettor, 200,400,600,800,
1000, the alpha-Naphthol dehydrated alcohol standard solution of 1200 μ L adds phosphate buffer (0.0 4mol/L, pH 6.5) to 4mL,
Again plus 0.5mL consolidates indigo plant B salting liquid, is uniformly mixed.Alpha-Naphthol dehydrated alcohol standard solution is not added as control group, at 595nm
The absorbance for reading the above reaction solution, using concentration as ordinate, absorbance is abscissa, standard curve of mapping to obtain, and slope is：
2.5.2 the measurement of protein content
Using bovine serum albumin as standard protein, be configured to the standard protein solution of 0.1mg/mL, take 12 test tubes, respectively plus
The standard protein solution for entering 0 mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL is supplied with 0.15mg/mlNaCl to 1 mL,
The absorbance value that 4mL Coomassie brilliant blue reagent measures enzyme solution under 595nm wavelength is added, the average value of 2 groups of measurements is taken, with albumen
Content is abscissa, and absorbance is the standard curve that ordinate draws protein content.Enzyme is measured with Coomassie brilliant blue colorimetric method method
The protein content of liquid.
2.5.3 general esterase activity measures
It is dilute that 1.95mL phosphate buffer, 0.05mL α-naphthaleneaceticacidester acetone soln and 0.5mL are sequentially added in test tube
Enzyme solution after releasing.It mixes, after reacting 10min in 30 DEG C of water bath with thermostatic control, 0.5mL is added and consolidates indigo plant B salting liquid, 30 DEG C of thermostatted water
10min is reacted in bath, and solution absorbance is then measured at 595nm.According under corresponding pH value alpha-Naphthol standard curve and survey
The absorbance obtained, calculating esterase activity is
In formula：E is esterase activity contained by every milliliter of enzyme solution, U/mL
OD595For the absorbance measured at wavelength 595nm after reaction
D is the extension rate of enzyme solution
K is the slope of the standard curve of alpha-Naphthol under corresponding pH
U is enzyme activity unit, and 1U schedules under prescribed conditions, to be catalyzed per minute needed for 1 μm of obtained ol alpha-Naphthol
Enzyme amount.
2.5.4 the measurement of Rate activity
Rate activity=total activity (U)/total protein (mg)
2.5.5 pesticide sensitivity testing
According to the measuring method of general esterase activity, the buffer without pesticide is changed into buffer containing different pesticides,
Measuring concentration is the metrifonate organophosphorus pesticide under 0mg/mL, 0.5mg/mL, 1.0mg/mL, 1.5mg/mL, 2.0mg/mL to flower
The inhibiting rate of cowpea esterase, using organophosphorus pesticide concentration as abscissa, inhibiting rate is that ordinate draws curve, with inhibiting rate I table
Show esterase to the susceptibility of organophosphorus pesticide.
Inhibiting rate calculates according to the following formula：
Inhibiting rate I (%)=(EDo not inhibit-EInhibit)/EDo not inhibit× 100%
3 experimental results and analysis
The standard curve of 3.1 alpha-Naphthols
7 15mm*150mm test tubes are taken, reagent is added according to following table respectively after number；
The standard curve of 2 alpha-Naphthol of table
3.1.1 the drafting of the standard curve of alpha-Naphthol：As shown in Figure 1；
3.2 protein content standard curves：As shown in Figure 2；
The measurement of 3.3 general esterase activities
1 test tube is taken, sequentially adds reagent according to following table；
The measurement of 3 general esterase activity of table
3.3.1 the calculating of general esterase activity
3.6.1 is shown in the measurement of pesticide concentration.
3.3.2 the measurement of specific enzyme activity power
It can be acquired by protein standard curve, as A=3.812, protein content 1.066mg
Specific enzyme activity power is：503.79/1.066=472.598
The detection blocking that the application-of 3.4 mixed coloured cowpea esterases quickly detects organophosphorus pesticide is standby
3.4.1 being loaded piece production
With the chromatographic paper disk of scissors clip diameter 1cm, the mixed coloured cowpea esterase solution of 40 μ L after purification is pipetted, it is careful to be added dropwise
To disk center, it is dried at room temperature for, is fabricated to sample-adding piece.
3.4.2 substrate piece makes
With the chromatographic paper disk of scissors clip diameter 1cm.Pipette α-acetic naphthalene that 40 μ L mass concentrations are 0.016mol/L
Ester acetone soln is carefully added drop-wise to disk center, up to substrate piece after drying under room temperature.
3.4.3 develop the color item production
A length of 3cm is obtained with scissors clip, the chromatographic paper disk of wide 1cm, wherein one end is the disk of diameter 1mm.It pipettes
The 100 μ L of solid blue B solution of 1.56mg/mL, is carefully added drop-wise in the scraps of paper, up to colour developing item after the scraps of paper are dry.
Will colour developing item, substrate piece, sample-adding piece by being sequentially sequentially placed between plastic packaging film upper and lower level from top to bottom, enzyme center
It is aligned with well and substrate piece center, substrate piece center is aligned with the center of circle of half nose circle of detector bar.The alignment of plastic packaging film upper and lower level,
Plastic packaging machine is preheated to 60~70 DEG C, and after plastic packaging, quick measuring card completes, as shown in figure 3,1 expression colour developing item in attached drawing, 2
Indicate substrate piece, 3 indicate sample-adding piece, and 4 indicate plastic packaging film.
3.5 detection
3.5.1 testing principle
α-naphthaleneaceticacidester can be reacted with the product alpha-Naphthol of mixed coloured cowpea esterase with solid indigo plant B generates aubergine product, and organic
Phosphorus pesticide can inhibit the activity of mixed coloured cowpea esterase, so that aubergine product be made to reduce, colour developing item colour developing is light violet magenta or colourless.
Based on this principle, it can tentatively judge whether contain organophosphorus pesticide in test sample.
3.5.2 detection method
It is respectively pure pesticide, 5 times of pesticide dilution, 10 times of pesticide dilution, 20 times of pesticide dilution, pesticide dilution 100 by concentration
Times, pesticide dilute 1000 times, pesticide dilute 10000 times, the metrifonate of distilled water be added drop-wise to quick measuring card well, visual observations face
The depth degree of color.
The toxic degree of subsequent quick survey detection pesticide, is that will be added drop-wise to colorimetric card to test sample mouth, then sticks into colorimetric
Row comparison.
3.6 pesticide sensitivity testings
5 test tubes are taken, sequentially add reagent according to following table；
4 pesticide sensitivity testing of table
(reference group is phosphate buffer)
3.6.1 the drafting of pesticide sensitivity curve
Pesticide sensitivity curve as shown in figure 4,
Linear equation can be obtained from standard curve：
Y=0.544x+56.14 R2=0.9892
Y is inhibiting rate (y), and x is pesticide (metrifonate) concentration (mg/mL)
It obtains x=(y-56.14)/0.544
3.6.2 analysis of experimental results
It is available from figure 4, the size of the concentration of pesticide and the inhibiting rate of enzyme at related ratio, i.e., in sample pesticide it is dense
The increase of degree, enzyme inhibition level are bigger.
With the reduction of pesticide concentration in sample, the aubergine in quick measuring card colour developing card is deeper；Therefore quick measuring card detection colour developing
There is correlativity with enzyme solution concentration.
The above is a preferred embodiment of the present invention, it is noted that for those skilled in the art
For, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also considered as
Protection scope of the present invention.
Claims (6)
1. a kind of extracting method of mixed coloured cowpea enzyme, which is characterized in that include the following steps：
(1), it crushes：20 grams of mixed coloured cowpeas are weighed with electronic balance, is crushed, is sieved with 100 mesh sieve with pulverizer, be placed in beaker；
(2), it mixes：120mL 0.1mol/L is added to beaker, 6.5 phosphate buffer of pH vibrates or blend 10~30min；
(3), it stands, filter：In 4 DEG C of refrigerators soaked overnight, with 4 layers of filtered through gauze；
(4), it is centrifuged：Under the conditions of 4 DEG C, 10000r/min refrigerated centrifuge 10min collects supernatant liquor, as crude enzyme liquid；
(5) purifying：
A, 10mL is taken from thick esterase, and the saturation degree of ammonium sulfate to 25% is added, and is placed on after standing 12h in 4 DEG C of refrigerator；
B, it takes and stands liquid in centrifuge tube, under the conditions of 4 DEG C, 10000r/min refrigerated centrifuge 10min discards precipitating, takes supernatant
Liquid；
C, it is added to supernatant to 60% saturation degree of ammonium sulfate, lower sediment, mixed coloured cowpea ester as after purification are collected in centrifugation
Enzyme.
2. a kind of method of quickly detection pedo relict organophosphorus pesticide content, which is characterized in that include the following steps：
(1) production of the standard curve of alpha-Naphthol：The alpha-Naphthol dehydrated alcohol standard solution of 1mmol/L is taken, it is accurate with pipettor
The alpha-Naphthol dehydrated alcohol standard solution for drawing 0,200,400,600,800,1000,1200 μ L, adds phosphate buffer extremely
4mL, then plus 0.5mL consolidate indigo plant B salting liquid, be uniformly mixed, alpha-Naphthol dehydrated alcohol standard solution is not added as control group,
The absorbance of the above reaction solution is read at 595nm, using concentration as ordinate, absorbance is abscissa, standard curve of mapping to obtain,
Slope is：
(2) measurement of protein content：Using bovine serum albumin as standard protein, it is configured to the standard protein solution of 0.1mg/mL,
12 test tubes are taken, the standard protein solution of 0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL is separately added into, uses 0.15mg/
MlNaCl supplies 1mL, and the absorbance value that 4mL Coomassie brilliant blue reagent measures enzyme solution under 595nm wavelength is added, takes 2 groups of surveys
Fixed average value, using protein content as abscissa, absorbance is the standard curve that ordinate draws protein content；It is bright with coomassie
The protein content of blue colorimetric method method measurement enzyme solution；
(3) general esterase activity measures：1.95mL phosphate buffer, 0.05mL α-naphthaleneaceticacidester acetone are sequentially added in test tube
Enzyme solution after solution and 0.5mL dilution；It mixes, after reacting 10min in 30 DEG C of water bath with thermostatic control, it is molten that addition 0.5mL consolidates indigo plant B salt
Liquid reacts 10min in 30 DEG C of water bath with thermostatic control, and solution absorbance is then measured at 595nm；According to α-naphthalene under corresponding pH value
Phenol standard curve and the absorbance measured, calculating general esterase activity is
In formula：E is esterase activity contained by every milliliter of enzyme solution, U/mL
OD595For the absorbance measured at wavelength 595nm after reaction
D is the extension rate of enzyme solution
K is the slope of the standard curve of alpha-Naphthol under corresponding pH
U is enzyme activity unit, and it is enzyme amount needed for being catalyzed 1 μm of obtained ol alpha-Naphthol per minute under prescribed conditions that 1U, which is scheduled,；
(4) measurement of Rate activity：
Rate activity=total activity (U)/total protein (mg)
(5) pesticide sensitivity testing：
According to the measuring method of general esterase activity, changes the buffer without pesticide into buffer containing different pesticides, measure
Concentration is the metrifonate organophosphorus pesticide under 0mg/mL, 0.5mg/mL, 1.0mg/mL, 1.5mg/mL, 2.0mg/mL to mixed coloured cowpea
The inhibiting rate of esterase, using organophosphorus pesticide concentration as abscissa, inhibiting rate is that ordinate draws curve, indicates ester with inhibiting rate I
Susceptibility of the enzyme to organophosphorus pesticide；
Inhibiting rate calculates according to the following formula：
Inhibiting rate I (%)=(EDo not inhibit-EInhibit)/EDo not inhibit× 100%；
(6) drafting of pesticide sensitivity curves
By the linear relationship of the concentration of measurement inhibiting rate I and organic agricultural chemicals, regression equation is established, organic agricultural chemicals can be calculated
Concentration.
3. the method for quick detection pedo relict organophosphorus pesticide content according to claim 2, it is characterised in that：Step
(2) concentration of the phosphate buffer described in is 0.04mol/L, pH 6.5.
4. the method for quick detection pedo relict organophosphorus pesticide content according to claim 2, it is characterised in that：Step
(1) the alpha-Naphthol dehydrated alcohol standard solution preparation method of the 1mmol/L described in is：0.093105g alpha-Naphthol is dissolved in
It is made in 500mL dehydrated alcohol.
5. the method for quick detection pedo relict organophosphorus pesticide content according to claim 2, it is characterised in that：Step
(6) linear equation described in is：Y=0.544x+56.14, y are inhibiting rate (y), and x is pesticide concentration (mg/mL).
6. a kind of method of Organophosphorus Pesticide Residues in quickly detection soil, which is characterized in that include the following steps：
(1), sample-adding piece production：With the chromatographic paper disk of scissors clip diameter 1cm, it is molten to pipette the mixed coloured cowpea esterase of 40 μ L after purification
Liquid is carefully added drop-wise to disk center, is dried at room temperature for, is fabricated to sample-adding piece；
(2), substrate piece makes：With the chromatographic paper disk of scissors clip diameter 1cm；Pipetting 40 μ L mass concentrations is 0.016mol/L
α-naphthaleneaceticacidester acetone soln, be added drop-wise to disk center, it is dry under room temperature after up to substrate piece；
(3), colour developing item production：A length of 3cm is obtained with scissors clip, the chromatographic paper disk of wide 1cm, wherein one end is diameter 1mm's
Disk；The 100 μ L of solid blue B solution for pipetting 1.56mg/mL, is added drop-wise in the scraps of paper, up to colour developing item after the scraps of paper are dry；
Will colour developing item, substrate piece, sample-adding piece by being sequentially sequentially placed between plastic packaging film upper and lower level from top to bottom, enzyme center with plus
Sample hole and the alignment of substrate piece center, substrate piece center is aligned with the center of circle of half nose circle of detector bar；The alignment of plastic packaging film upper and lower level, plastic packaging
Machine is preheated to 60~70 DEG C, and after plastic packaging, quick measuring card completes；
It (4), is respectively pure pesticide, 5 times of pesticide dilution, 10 times of pesticide dilution, 20 times of pesticide dilution, pesticide dilution 100 by concentration
Times, pesticide dilute 1000 times, pesticide dilute 10000 times of distilled water metrifonate be added drop-wise to quick measuring card well, visual observations face
The depth degree of color, is fabricated to colorimetric card；
(5), sample to be tested is dripped on quick measuring card, forms corresponding color, it, can be quick by being compared with the color on colorimetric card
Measure the residual content of the organophosphorus pesticide of sample.
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| CN114235792A (en) * | 2021-11-30 | 2022-03-25 | 广东省科学院测试分析研究所(中国广州分析测试中心) | Rapid detection method for pyrethroid pesticide residue |
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