CN102660516A - Purification method of acetylcholinesterase from earthworm serum - Google Patents
Purification method of acetylcholinesterase from earthworm serum Download PDFInfo
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- 241000361919 Metaphire sieboldi Species 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 25
- 210000002966 serum Anatomy 0.000 title claims abstract description 21
- 238000000746 purification Methods 0.000 title abstract description 6
- 102000012440 Acetylcholinesterase Human genes 0.000 title abstract 4
- 108010022752 Acetylcholinesterase Proteins 0.000 title abstract 4
- 229940022698 acetylcholinesterase Drugs 0.000 title abstract 4
- 239000000243 solution Substances 0.000 claims abstract description 19
- 239000000284 extract Substances 0.000 claims abstract description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 13
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims abstract description 12
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 10
- IDOQDZANRZQBTP-UHFFFAOYSA-N 2-[2-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol Chemical compound CC(C)(C)CC(C)(C)C1=CC=CC=C1OCCO IDOQDZANRZQBTP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 229920004929 Triton X-114 Polymers 0.000 claims abstract description 8
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 5
- 239000000287 crude extract Substances 0.000 claims abstract description 5
- 238000000605 extraction Methods 0.000 claims abstract description 4
- 101001091385 Homo sapiens Kallikrein-6 Proteins 0.000 claims description 6
- 102100034866 Kallikrein-6 Human genes 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 4
- 238000000136 cloud-point extraction Methods 0.000 claims description 4
- 239000013543 active substance Substances 0.000 claims description 3
- 238000002203 pretreatment Methods 0.000 claims description 3
- 239000002689 soil Substances 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 abstract description 20
- 102000004190 Enzymes Human genes 0.000 abstract description 20
- 238000002360 preparation method Methods 0.000 abstract description 4
- 239000007853 buffer solution Substances 0.000 abstract description 3
- 239000005457 ice water Substances 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 229940088598 enzyme Drugs 0.000 abstract 2
- 238000007865 diluting Methods 0.000 abstract 1
- 230000035945 sensitivity Effects 0.000 abstract 1
- 239000004094 surface-active agent Substances 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 5
- 239000008280 blood Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 238000001502 gel electrophoresis Methods 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 108090000371 Esterases Proteins 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 230000007830 nerve conduction Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- GGRLKHMFMUXIOG-UHFFFAOYSA-M 2-acetyloxyethyl(trimethyl)azanium;hydroxide Chemical compound [OH-].CC(=O)OCC[N+](C)(C)C GGRLKHMFMUXIOG-UHFFFAOYSA-M 0.000 description 2
- 241000287828 Gallus gallus Species 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 2
- 229960001231 choline Drugs 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000002523 gelfiltration Methods 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 239000008057 potassium phosphate buffer Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 235000021419 vinegar Nutrition 0.000 description 2
- 239000000052 vinegar Substances 0.000 description 2
- 241000272525 Anas platyrhynchos Species 0.000 description 1
- 241000252229 Carassius auratus Species 0.000 description 1
- 102000010911 Enzyme Precursors Human genes 0.000 description 1
- 108010062466 Enzyme Precursors Proteins 0.000 description 1
- 241000646357 Monopterus cuchia Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- 239000003905 agrochemical Substances 0.000 description 1
- RQGNEYFWHWFECS-UHFFFAOYSA-N amino formate Chemical class NOC=O RQGNEYFWHWFECS-UHFFFAOYSA-N 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 238000003453 ammonium sulfate precipitation method Methods 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
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- 238000001035 drying Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 235000012055 fruits and vegetables Nutrition 0.000 description 1
- 231100000652 hormesis Toxicity 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 235000014666 liquid concentrate Nutrition 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- -1 octyl phenyl Chemical group 0.000 description 1
- 230000000590 parasiticidal effect Effects 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 235000015277 pork Nutrition 0.000 description 1
- 230000001242 postsynaptic effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 238000009987 spinning Methods 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 235000015099 wheat brans Nutrition 0.000 description 1
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Abstract
The invention provides a purification method of acetylcholinesterase from earthworm serum. The method comprises the following steps: placing earthworm in clean water for 24 hours to eliminate excrement in the earthworm body, and storing the earthworm in a refrigerator for later use; homogenizing with phosphoric acid buffer solution in an ice-water bath condition to prepare homogenate, centrifuging to obtain crude enzyme extract, and storing in the refrigerator; adding surfactant Triton X-114 solution and citric acid buffer solution into 100mu L of crude extract, and diluting with secondary water until the volume is 10mL, sufficiently oscillating and shaking uniformly; placing the solution in a constant-temperature water bath with the water bath temperature of 35 DEG C to balance for 9 minutes; and centrifuging for layering to obtain a lower product which is the purified acetylcholinesterase product. The purification extraction method of the acetylcholinesterase from earthworm serum has wide enzyme sources and low price, high sensitivity, simple and stable preparation process and low production cost.
Description
Technical field
The present invention relates to the extracting process of acetyl cholesterase, particularly a kind of from earthworm serum the method for purifying E.C. 3.1.1.7.
Background technology
E.C. 3.1.1.7 is a kind of key enzyme in the biological nerve conduction, in nerve conduction, plays vital function.This enzyme liberating vagusstoff stops neurotransmitter for the excited hormesis of postsynaptic membrane, guarantees the normal conduction in vivo of nerve signal molecule.The action site that organophosphorus and amino formate compounds are arranged on the E.C. 3.1.1.7; When this two compounds with after E.C. 3.1.1.7 combines; Will cause the phosphorylated and the carbamylization at enzyme catalysis position; Its acetylizing to vagusstoff can't be carried out, thereby destroyed normal nerve conduction effect.Thereby the inactivation of enzyme causes biological poisoning, utilizes this principle can play parasiticidal effect.The activity of E.C. 3.1.1.7 receives the amount of inhibition degree and agricultural chemicals to present linear relationship, thereby the activity of available AChE receives the inhibition degree to detect the remains of pesticide amount in the fruits and vegetables.Therefore, highly sensitive, highly purified E.C. 3.1.1.7 batch preparations become the emphasis problem of enzyme inhibition method research.
The source of E.C. 3.1.1.7 is a lot; The liver shape of animal; All there is more rich this enzyme source in brain in the blood, have the scholar to extract E.C. 3.1.1.7 with pork liver, wheat bran, Medulla Carassius auratus, earthworm and chicken blood respectively; Obtain the activity of enzyme and the comfort level of large-scale operation from extraction, use earthworm bigger advantage to be arranged as the enzyme source.Ding Shihua etc. (use and the environmental organism journal, 1997,3,246-251) adopt Triton X-100 earthworm serum extract after the processing of PEG2000-potassium phosphate buffer, ion exchange chromatography and gel-filtration, obtain electrophoretically pure choline vinegar enzyme.But this method purifying leaching process is complicated, and the purifying multiple is low, the separation of the esterase of being unrealized.Existing " a kind of preparation method who from animal blood, extracts E.C. 3.1.1.7 " (number of patent application is 02134464) comprises the steps: the blood of big white mouse, chicken, duck, ox, dog, swamp eel fish, eel is solidified under the greenhouse; Freezing, get its supernatant, the supernatant spinning obtains the crude extract of E.C. 3.1.1.7; Use the ammonium sulfate precipitation method purifying; Take off ammonium sulfate with dialysis method, enzyme liquid concentrate drying obtains the E.C. 3.1.1.7 goods of purifying.
The existing method of from earthworm serum, extracting E.C. 3.1.1.7 is: Ding Shihua etc. (use and the environmental organism journal; 1997; 3; 246-251) in earthworm serum, add the abundant homogenate of buffer solution of sodium phosphate, adopt Triton X-100 extract after the processing of PEG2000-potassium phosphate buffer, ion exchange chromatography and gel-filtration, obtain electrophoretically pure choline vinegar enzyme.This procedure is complicated, and the purifying multiple is low, and the separation of the esterase of being unrealized can weaken the vigor of enzyme.
Summary of the invention
The technical problem that the present invention will solve is to above-mentioned present situation, the method for purifying E.C. 3.1.1.7 from earthworm serum that aims to provide that a kind of cost is low, easy to operate, practicality is stronger.
For solving this technical problem, the technical scheme that the present invention adopts is following:
A kind of from earthworm serum the method for purifying E.C. 3.1.1.7, earthworm pre-treatment: earthworm is placed 24h in the clear water, get rid of intravital ight soil, be stored in 4 ℃ of refrigerators it for use; Use pH8.0, the phosphate buffer solution of 0.1mol/L, homogenate is processed in homogenate under the ice-water bath condition, and centrifugal (10000r/min) 20min gets thick zyme extract under 4 ℃, is kept in the refrigerator.(0.09%, w/v) solution and 0.6mL pH4.1 citric acid solution fully vibrate and shake up to 10mL with secondary water constant volume to add tensio-active agent Triton X-114 in the thick zyme extract of 100 μ L.It is balance 9min in 35 ℃ of Water Tanks with Temp.-controlled that its above-mentioned solution is placed on bath temperature, then with its taking-up, with the centrifugal 5min of 4000r/min, after the layering its upper solution is toppled over, and lower floor is E.C. 3.1.1.7 goods behind the purifying.
The present invention is to provide the purifying extracting process of earthworm serum E.C. 3.1.1.7, its enzyme source is cheaply extensive, and highly sensitive, preparation technology's simple and stable, and production cost is low.
This method uses gentle reagent Triton X-114 to extract the E.C. 3.1.1.7 in the serum, compares with the chromatography gel method, the vigor of enzyme is not had influence, and can keep the full structure of enzyme, helps enzymatic structure research; Moreover this method utilizes the iso-electric point of E.C. 3.1.1.7 in the earthworm serum to realize the separation and the purifying of enzyme, can other proteolytic enzyme and esterase be separated, and makes the purifying multiple of enzyme higher.
Triton X-114 is a kind of non-ionic surfactant, name of product: Triton X-114/ Triton X-114, and another name: the octyl phenyl Soxylat A 25-7,4-(1,1,3,3-Tetramethylbutyl) cyclohexyl-polyethylene glycol.
Description of drawings:
Fig. 1 extracts the schema of purifying E.C. 3.1.1.7 from earthworm serum.
The E.C. 3.1.1.7 that Fig. 2 cloud point extraction is purified into is through the SDS-gel electrophoresis figure.
1 is Marher; 2 is the resultant electrophorogram in the purified back of E.C. 3.1.1.7; 3 is the thick E.C. 3.1.1.7 electrophorogram that extracts.
Embodiment
Referring to Fig. 1, a kind of from earthworm serum the method for purifying E.C. 3.1.1.7:
1) earthworm pre-treatment: put into clear water 24h after the earthworm of buying cleaned, make the intravital ight soil of its emptying, be stored in 4 ℃ of refrigerators for use;
2) the thick extraction of earthworm serum: with phosphoric acid buffer (pH8.0; 0.1mol/L) for preparing the solution of crude extract; According to homogenate than the homogenate of processing for 1:1 (earthworm quality (g)/phosphoric acid buffer volume (mL)); Centrifugal (10000r/min) 20min abandoning supernatant under 4 ℃, what obtain is exactly thick zyme extract, is kept in the refrigerator.；
3) cloud point extraction process: the centrifuge tube of taking 10mL; The crude extract that then adds 100 μ L earthworm serum is thick zyme extract; Add tensio-active agent Triton X-114 (0.09% again; W/v) solution and the 0.6mL pH4.1 citric acid solution pH value of coming regulator solution is fully vibrated and is shaken up to 10mL with secondary water constant volume.
4) with the 3rd) to be placed on bath temperature be balance 9min in 35 ℃ of Water Tanks with Temp.-controlled to the solution that obtains of step; Then with its taking-up; Under the speed with 4000r/min centrifugal 5 minutes, after the layering its upper solution to be toppled over away, lower floor's solution is E.C. 3.1.1.7 purifying extracting solution.
The mensuration of degree of purification
The degree of purification of E.C. 3.1.1.7 can be measured with the SDS-gel electrophoresis figure, and is as shown in Figure 2.1 is Marher among Fig. 2; 2 is the resultant electrophorogram in the purified back of E.C. 3.1.1.7; 3 is the thick E.C. 3.1.1.7 electrophorogram that extracts.As can beappreciated from fig. 2; There is not purified proenzyme liquid many bands of a spectrum to be arranged through the SDS-detected through gel electrophoresis; Mainly contain two through bands of a spectrum bar number in the electrophorogram behind the cloud point extraction purifying; Corresponding molecular weight meets the molecular weight of E.C. 3.1.1.7 respectively at 50-60KDa and 110-120KDa, and it is pure to explain that E.C. 3.1.1.7 liquid can reach electrophoresis.
The present invention successfully separation of pure has dissolved the active E.C. 3.1.1.7 of maintenance constitutive enzyme.The E.C. 3.1.1.7 that from earthworm serum, obtains can know that through the gel electrophoresis method analysis it has the protein of two kinds of different molecular weights, and respectively at 50-60KDa and 110-120KDa, this also meets the report of some documents.In a word, present method has can extract E.C. 3.1.1.7 and purity is higher and keep advantage such as enzymic activity quickly and easily from earthworm serum, be a kind of important method of separating E.C. 3.1.1.7 in the purification animal body.
Claims (1)
1. the method for a purifying E.C. 3.1.1.7 from earthworm serum is characterized in that comprising the steps:
1) earthworm pre-treatment: earthworm is placed 24h in the clear water, get rid of intravital ight soil, be stored in 4 ℃ of refrigerators it for use;
2) the thick extraction of earthworm serum: with phosphoric acid buffer (pH8.0; 0.1mol/L) for preparing the solution of crude extract; Than the homogenate of processing for 1:1 [earthworm quality (g)/phosphoric acid buffer volume (mL)], centrifugal (10000r/min) 20min gets thick zyme extract under 4 ℃ according to homogenate;
3) cloud point extraction process: the centrifuge tube of taking 10mL; Then add the thick zyme extract of 100 μ L; (0.09%, w/v) solution and 0.6mL pH4.1 citric acid solution fully vibrate and shake up to 10mL with secondary water constant volume to add tensio-active agent Triton X-114 again;
4) with the 3rd) to be placed on bath temperature be balance 9 minutes in 35 ℃ of Water Tanks with Temp.-controlled to the solution that obtains of step, then it is taken out, in the whizzer of 4000r/min centrifugal 5 minutes, after the layering its upper solution to be toppled over, lower floor is E.C. 3.1.1.7 behind the purifying.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 201210144988 CN102660516B (en) | 2012-05-10 | 2012-05-10 | Purification method of acetylcholinesterase from earthworm serum |
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| CN 201210144988 CN102660516B (en) | 2012-05-10 | 2012-05-10 | Purification method of acetylcholinesterase from earthworm serum |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106906193A (en) * | 2017-04-12 | 2017-06-30 | 武汉中科志康生物科技有限公司 | A kind of method that acetylcholinesterase is extracted in the blood from horse |
| CN109781718A (en) * | 2019-01-17 | 2019-05-21 | 广东省食品工业研究所有限公司 | A kind of preparation method of the compound enzyme solution for fast detecting pesticide residue |
-
2012
- 2012-05-10 CN CN 201210144988 patent/CN102660516B/en not_active Expired - Fee Related
Non-Patent Citations (5)
| Title |
|---|
| CLEMENT BORDIER: "Phase Separation of Integral Membrane Proteins in Triton X-114 Solution", 《THE JOURNALO F BIOLOGICACLH EMISTRY》 * |
| NIGEL M. HOOPER ET AL: "Glycosyl-phosphatidylinositol-anchored membrane proteins can be distinguished from transmembrane polypeptide-anchored proteins by differential solubilization and temperature-induced phase separation in Triton X-114", 《BIOCHEM. J.》 * |
| 刘畅 等: "鸭血浆胆碱酯酶的分离纯化和性质的研究", 《食品科学》 * |
| 裴瑞瑞 等: "牛血清乙酰胆碱脂酶的分离纯化", 《农业环境科学学报》 * |
| 高守红 等: "浊点萃取法在生物医药中的应用", 《中国医药工业杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106906193A (en) * | 2017-04-12 | 2017-06-30 | 武汉中科志康生物科技有限公司 | A kind of method that acetylcholinesterase is extracted in the blood from horse |
| 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 |
| CN109781718A (en) * | 2019-01-17 | 2019-05-21 | 广东省食品工业研究所有限公司 | A kind of preparation method of the compound enzyme solution for fast detecting pesticide residue |
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