CN1181198C - Preparing method for acetylcholinesterase - Google Patents
Preparing method for acetylcholinesterase Download PDFInfo
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- CN1181198C CN1181198C CNB021344647A CN02134464A CN1181198C CN 1181198 C CN1181198 C CN 1181198C CN B021344647 A CNB021344647 A CN B021344647A CN 02134464 A CN02134464 A CN 02134464A CN 1181198 C CN1181198 C CN 1181198C
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- acetylcholinesterase
- ammonium sulfate
- enzyme liquid
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Abstract
The present invention relates to a method for preparing acetylcholinesterase which is used for detecting the residual amount of pesticide in fruits and vegetables. In the present invention, animal blood is used as an enzyme source. The method comprises the following processing steps that a coarse extracting solution of acetylcholinesterase is extracted from the enzyme source, acetylcholinesterase is purified by an ammonium sulfate deposition method, and ammonium sulfate is removed by a dialysis method; then, enzyme liquid is concentrated and dried so as to obtain a purified acetylcholinesterase product. The method for preparing acetylcholinesterase has the advantages of wide raw material source for animal blood, simple and stable preparation process and low production cost. The acetylcholinesterase product prepared by the method has the advantages of high purity and high sensitivity for detecting residual pesticide after detection. The purity of the coarse extracting solution of acetylcholinesterase, which is purified by the method, is improved by more than 4 times.
Description
Technical field
The present invention relates to a kind of preparation method who is used for the acetylcholinesterase of the fruits and vegetables agricultural chemicals determination of residual amount.
Background technology
Pesticide residue can cause the death of people's acute poisoning in the fruits and vegetables, and trace accumulation can cause chronic poisoning such as carcinogenic, teratogenesis, mutagenesis etc. again, and the serious harm people's is healthy.The vegetables of present listing, except ordinary methods such as dependence gas-chromatography detected, the rapid determination technology also was applied.The present rapid determination product that uses at home-agricultural chemicals speed test paper, its main raw material acetylcholinesterase is to rely on external import, these acetylcholinesterases are owing to the reason of products material and complicated process of preparation, cost an arm and a leg, make rapid determination product be difficult to popularization and application pesticide residue in the fruits and vegetables.
Summary of the invention
The objective of the invention is to propose a kind of fruits and vegetables pesticide residue rapid determination that can be used for, the preparation method of the simple and low production cost acetylcholinesterase of technology.
The present invention is a kind of preparation method who extracts acetylcholinesterase from animal blood, comprises following processing step:
(1) with fresh animal blood after solidifying under the room temperature, in 0～4 ℃ freezing 2～48 hours down, get its supernatant liquor;
(2) with the supernatant liquor of previous step gained with 2000～15000r.p.m, 0～4 ℃ is centrifugal, gets its supernatant liquor, promptly gets the acetylcholinesterase crude extract;
(3) with ammonium sulfate precipitation method purifying acetylcholinesterase:
A, add ammonium sulfate under slowly stirring in ice bath in the acetylcholinesterase crude extract, the saturation ratio that makes ammonium sulfate in the enzyme liquid is in 20%～90% scope;
B, above-mentioned gained solution are in 3000-15000r.p.m, and 0-4 ℃ of frozen centrifugation keeps precipitation;
The phosphoric acid buffer dissolution precipitation of c, adding pH7.5-8.0, and with pH7.5-8 phosphoric acid buffer dilution 2-10 doubly;
(4) take off ammonium sulfate with dialysis method: will go up the step and pack in the dialysis tubing, with the distill water dialysis of 20～30 times of its volumes, till dialyzate does not produce precipitation through the enzyme liquid of ammonium sulfate precipitation;
(5) the enzyme liquid with the previous step gained concentrates, and drying promptly gets the acetylcholinesterase goods of purifying.
The animal blood raw material that the present invention is used for extracting acetylcholinesterase can adopt the blood that extracts from big white mouse, chicken, duck, pig, ox, dog, swamp eel fish, trowel fish or rabbit.
(3) process steps in the described method is promptly used the technology of ammonium sulfate precipitation method purifying acetylcholinesterase, the results showed, makes the effect of saturation ratio in 40%～60% scope of ammonium sulfate in the enzyme liquid better.
(5) process steps in the described method can adopt vacuum freeze drier, concentrates enzyme liquid to solid under 0～4 ℃ of condition.Also can adopt dialysis method to concentrate enzyme liquid, enzyme liquid is packed in the dialysis tubing, make siccative, in 0～4 ℃ of concentrated enzyme liquid down with polyethylene glycol 6000.
The preparation method of acetylcholinesterase provided by the present invention, its animal blood raw material sources are extensive, preparation technology's simple and stable, production cost is lower.With the prepared acetylcholinesterase product of the inventive method, product purity height, after measured, be used for that Determination of Residues Pesticide is had higher sensitivity, improve more than 4 times through the acetylcholinesterase remolding sensitivity enzyme extract behind the purifying of the present invention.
Embodiment
Embodiment one
The processing step that extracts acetylcholinesterase from chicken blood is as follows:
(1) under the new freshly-slaughtered poultry blood room temperature after the condensation, places 4 ℃ in refrigerator freezing 2 hours down, discard precipitation, get its supernatant liquor;
(2) with supernatant liquor with 8000r.p.m, 4 ℃ of following frozen centrifugations 15 minutes discard precipitation again, its supernatant liquor is the acetylcholinesterase crude extract;
(3) ammonium sulfate precipitation method purifying acetylcholinesterase:
A, get acetylcholinesterase crude extract 10ml, under ice bath slowly stirs, add saturation ratio and be 60% ammonium sulfate 3.61 grams (according to ammonium sulfate saturation ratio scale);
B, to above-mentioned gained solution in 11000r.p.m, 4 ℃ of following frozen centrifugations 10 minutes, the supernatant liquor that inclines obtains the ammonium sulfate saturation ratio and is 60% precipitation;
C, add 1ml, pH8 phosphoric acid buffer dissolution precipitation, and with 4 times of pH8 phosphoric acid buffer dilutions;
(4) dialysis method is sloughed ammonium sulfate: above-mentioned enzyme liquid through ammonium sulfate precipitation is packed in the dialysis tubing, with the distill water dialysis of long-pending 25 times of enzyme liquid 24 hours, change water 3-5 time therebetween, until using 1%BaCl
2Can't measure till the sulfate radical;
(5) concentrate acetylcholinesterase: use vacuum freeze drier, under 4 ℃ of conditions, concentrate enzyme liquid, be drying to obtain the acetylcholinesterase product of purifying then to solid.Preserve the acetylcholinesterase product sealing back (0 ℃) under low temperature of gained.
The purifying acetylcholinesterase product of pressing this routine prepared is according to indophenols acetic ester explicit representation, and its activity is after measured: 0.397.Sensitivity determination result to this product is as follows:
Working sample: the purifying acetylcholinesterase that present method is extracted from chicken serum.
Measuring method: indophenols acetic ester development process.On the round filter paper that contains a certain amount of enzyme liquid behind the ammonium sulfate precipitation purifying, add 0.2 milliliter 500,250,100, the methylamine phosphine solution of 50 mcg/ml respectively, contrast adds the 0.2mlpH8 phosphoric acid buffer, after leaving standstill 1 minute, contain filter paper (containing 1.5 milligrams of indophenols acetic ester in the every milliliter of benzene) doubling of staining agent with another, allow it react 1.5 minutes, observe colour-change, be blue for not suppressed fully by the methylamine phosphine, fade for part suppresses, be white in color to being suppressed fully.
Reaction system: 30 μ l enzyme liquid+0.2ml methylamine phosphine solution+30 μ l staining agents.
Measurement result: the detection limit by the acetylcholinesterase liquid of 60% sulfuric acid amine saturation ratio purifying is 100 mcg/ml.Purified acetylcholinesterase sensitivity is filled than enzyme extract and is improved 4 times.
Embodiment two
The processing step that extracts acetylcholinesterase from pig blood is as follows:
(1) under the fresh pig blood room temperature after the condensation, places 4 ℃ in refrigerator freezing 2 hours down, discard precipitation, get its supernatant liquor;
(2) with supernatant liquor with 8000r.p.m, 4 ℃ of following frozen centrifugations 15 minutes discard precipitation again, its supernatant liquor is the acetylcholinesterase crude extract;
(4) ammonium sulfate precipitation method purifying acetylcholinesterase:
A, get acetylcholinesterase crude extract 10ml, under ice bath slowly stirs, add saturation ratio and be 50% ammonium sulfate 2.91 grams (according to ammonium sulfate saturation ratio scale);
B, to above-mentioned gained solution in 11000r.p.m, 4 ℃ of following frozen centrifugations 10 minutes, the supernatant liquor that inclines obtains the ammonium sulfate saturation ratio and is 50% precipitation;
C, add 1ml, pH8 phosphoric acid buffer dissolution precipitation, and with 4 times of pH8 phosphoric acid buffer dilutions;
(4) dialysis method is sloughed ammonium sulfate: above-mentioned enzyme liquid through ammonium sulfate precipitation is packed in the dialysis tubing, with the distill water dialysis of long-pending 30 times of enzyme liquid 24 hours, change water 3-5 time therebetween, until using 1%BaCl
2Can't measure till the sulfate radical;
(5) concentrate acetylcholinesterase: adopt dialysis method to concentrate enzyme liquid, enzyme liquid is packed in the dialysis tubing, make siccative with polyethylene glycol 6000, consumption is 5 a milliliters/gram (W/V), in 4 ℃ of following concentrated enzyme liquid, then concentrated solution is drying to obtain the acetylcholinesterase product of purifying.Preserve the acetylcholinesterase product sealing back (0 ℃) under low temperature of gained.
Claims (5)
1. preparation method who extracts acetylcholinesterase from animal blood comprises following processing step:
(1) with fresh animal blood after solidifying under the room temperature, in 0～4 ℃ freezing 2～48 hours down, get its supernatant liquor;
(2) with the supernatant liquor of previous step gained with 2000～15000r.p.m, 0～4 ℃ is centrifugal, gets its supernatant liquor, promptly gets the acetylcholinesterase crude extract;
(3) with ammonium sulfate precipitation method purifying acetylcholinesterase:
A, add ammonium sulfate under slowly stirring in ice bath in the acetylcholinesterase crude extract, the saturation ratio that makes ammonium sulfate in the enzyme liquid is in 20%～90% scope;
B, above-mentioned gained solution are in 3000-15000r.p.m, and 0-4 ℃ of frozen centrifugation keeps precipitation;
The phosphoric acid buffer dissolution precipitation of c, adding pH7.5-8.0, and with pH7.5-8 phosphoric acid buffer dilution 2-10 doubly;
(4) take off ammonium sulfate with dialysis method: will go up the step and pack in the dialysis tubing, with the distill water dialysis of 20～30 times of its volumes, till dialyzate does not produce precipitation through the enzyme liquid of ammonium sulfate precipitation;
(5) the enzyme liquid with the previous step gained concentrates, and drying promptly gets the acetylcholinesterase goods of purifying.
2. the preparation method of acetylcholinesterase according to claim 1, the animal blood raw material that it is characterized in that being used for extracting acetylcholinesterase adopts the blood that extracts from big white mouse, chicken, duck, pig, ox, dog, swamp eel fish, trowel fish or rabbit.
3. the preparation method of acetylcholinesterase according to claim 1 is characterized in that the saturation ratio that makes ammonium sulfate in the enzyme liquid is in 40%～60% scope with ammonium sulfate precipitation method purifying acetylcholinesterase.
4. the preparation method of acetylcholinesterase according to claim 1 is characterized in that (5) step of technology is adopted vacuum freeze drier, concentrates enzyme liquid to solid under 0～4 ℃ of condition.
5. the preparation method of acetylcholinesterase according to claim 1 is characterized in that (5) step of technology adopts dialysis method to concentrate enzyme liquid, and enzyme liquid is packed in the dialysis tubing, makes siccative with polyethylene glycol 6000, in 4 ℃ of concentrated enzyme liquid down.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021344647A CN1181198C (en) | 2002-07-29 | 2002-07-29 | Preparing method for acetylcholinesterase |
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| Application Number | Priority Date | Filing Date | Title |
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| CNB021344647A CN1181198C (en) | 2002-07-29 | 2002-07-29 | Preparing method for acetylcholinesterase |
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| CN1472316A CN1472316A (en) | 2004-02-04 |
| CN1181198C true CN1181198C (en) | 2004-12-22 |
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| CNB021344647A Expired - Fee Related CN1181198C (en) | 2002-07-29 | 2002-07-29 | Preparing method for acetylcholinesterase |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100390276C (en) * | 2006-04-11 | 2008-05-28 | 修志明 | Process for extracting esterase from animals' livers |
| CN101190330A (en) | 2006-11-30 | 2008-06-04 | 深圳市鼎兴生物医药技术开发有限公司 | Application of cholinesterase in antagonistic tachykinin medicine |
| CN101581670A (en) * | 2009-06-11 | 2009-11-18 | 王振宇 | Method for preparing acetylcholinesterase fluorescence biosensor |
| CN101974498A (en) * | 2010-10-18 | 2011-02-16 | 上海应用技术学院 | Method for extracting acetylcholinesterase and application thereof |
| CN106906193B (en) * | 2017-04-12 | 2020-04-10 | 武汉中科志康生物科技有限公司 | Method for extracting acetylcholinesterase from horse blood |
| CN108169220B (en) * | 2017-11-24 | 2021-02-12 | 武汉市农业科学院 | Horse serum cholinesterase-based pesticide residue rapid detection card and production process and use method thereof |
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2002
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