CN106906193B - Method for extracting acetylcholinesterase from horse blood - Google Patents
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- 210000004369 blood Anatomy 0.000 title claims abstract description 35
- 239000008280 blood Substances 0.000 title claims abstract description 35
- 102000012440 Acetylcholinesterase Human genes 0.000 title claims abstract description 30
- 108010022752 Acetylcholinesterase Proteins 0.000 title claims abstract description 30
- 229940022698 acetylcholinesterase Drugs 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 25
- 239000011259 mixed solution Substances 0.000 claims abstract description 64
- 239000000872 buffer Substances 0.000 claims abstract description 29
- 239000012264 purified product Substances 0.000 claims abstract description 25
- 239000000047 product Substances 0.000 claims abstract description 21
- 238000001042 affinity chromatography Methods 0.000 claims abstract description 13
- REQCZEXYDRLIBE-UHFFFAOYSA-N procainamide Chemical compound CCN(CC)CCNC(=O)C1=CC=C(N)C=C1 REQCZEXYDRLIBE-UHFFFAOYSA-N 0.000 claims abstract description 13
- 229960000244 procainamide Drugs 0.000 claims abstract description 13
- 239000000126 substance Substances 0.000 claims abstract description 9
- 238000001035 drying Methods 0.000 claims abstract description 8
- 238000002156 mixing Methods 0.000 claims abstract description 8
- 238000003756 stirring Methods 0.000 claims abstract description 8
- 239000006228 supernatant Substances 0.000 claims abstract description 8
- 238000000605 extraction Methods 0.000 claims abstract description 7
- 239000002202 Polyethylene glycol Substances 0.000 claims description 7
- 238000000502 dialysis Methods 0.000 claims description 7
- 238000007710 freezing Methods 0.000 claims description 7
- 230000008014 freezing Effects 0.000 claims description 7
- ZPIRTVJRHUMMOI-UHFFFAOYSA-N octoxybenzene Chemical compound CCCCCCCCOC1=CC=CC=C1 ZPIRTVJRHUMMOI-UHFFFAOYSA-N 0.000 claims description 7
- 229920001223 polyethylene glycol Polymers 0.000 claims description 7
- 239000008363 phosphate buffer Substances 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 abstract description 7
- 108090000790 Enzymes Proteins 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 7
- 229940088598 enzyme Drugs 0.000 abstract description 7
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 239000000243 solution Substances 0.000 description 20
- 239000008055 phosphate buffer solution Substances 0.000 description 6
- 238000009777 vacuum freeze-drying Methods 0.000 description 5
- 238000001514 detection method Methods 0.000 description 3
- JOYRKODLDBILNP-UHFFFAOYSA-N Ethyl urethane Chemical compound CCOC(N)=O JOYRKODLDBILNP-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 2
- 229960004373 acetylcholine Drugs 0.000 description 2
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- XAFOVULQFHVXSZ-UHFFFAOYSA-N [4-[(4-oxocyclohexa-2,5-dien-1-ylidene)amino]phenyl] acetate Chemical compound C1=CC(OC(=O)C)=CC=C1N=C1C=CC(=O)C=C1 XAFOVULQFHVXSZ-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 230000001713 cholinergic effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002964 excitative effect Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- RJNPPEUAJCEUPV-UHFFFAOYSA-N naphthalen-2-yl acetate Chemical compound C1=CC=CC2=CC(OC(=O)C)=CC=C21 RJNPPEUAJCEUPV-UHFFFAOYSA-N 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000007830 nerve conduction Effects 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- 230000001242 postsynaptic effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 210000000225 synapse Anatomy 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01007—Acetylcholinesterase (3.1.1.7)
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
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Abstract
The invention discloses a method for extracting acetylcholinesterase from horse blood, which comprises the following steps: adding fresh horse blood into the mixed buffer extract, stirring and mixing for 15-20min to obtain a first mixed solution; standing the first mixed solution for 12-24 hours to obtain a second mixed solution; homogenizing the second mixed solution for 8-12min to obtain a third mixed solution; centrifuging the third mixed solution for 23-28min, and collecting supernatant to obtain a fourth mixed solution; purifying the fourth mixed solution by Procainamide affinity chromatography to obtain a primary purified substance; purifying the primary purified substance by using a SephadexG-200 gel column to obtain a final purified substance; and concentrating and drying the final purified product to obtain the product. The acetylcholinesterase obtained by the extraction method has the advantages of high enzyme activity, high purity and high sensitivity, and the prepared product is powdery, is easy to store and has wide market prospect.
Description
Technical Field
The invention relates to an enzyme extraction method, in particular to a method for extracting acetylcholinesterase from horse blood.
Background
Acetylcholinesterase is a key enzyme in biological nerve conduction, and in cholinergic synapses, the enzyme degrades acetylcholine, terminates the excitatory action of neurotransmitters on postsynaptic membranes, and ensures the normal transmission of nerve signals in organisms. Acetylcholinesterase has received increasing attention in recent years from researchers due to its high rate of hydrolysis of acetylcholine. Acetylcholinesterase has wide application in biological prevention and treatment, pesticide residue detection, molecular biology and treatment of various neurological diseases. Recent studies have shown that ethyl carbamate can inhibit the activity of acetylcholinesterase to some extent, so that acetylcholinesterase may be applied to the detection of ethyl carbamate.
Acetylcholinesterase is widely present in various animal and plant tissues, and for animals, it is mainly concentrated in liver, blood and brain tissues. The existing extraction method of acetylcholinesterase generally has the defect of poor enzyme activity.
Disclosure of Invention
The present invention aims at providing a method for extracting acetylcholinesterase from horse blood, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for extracting acetylcholinesterase from horse blood comprises the following steps:
1) taking fresh horse blood, adding the mixed buffer extract, stirring and mixing for 15-20min at the ice bath temperature of 4 ℃ and the rotation speed of 100-200rpm to obtain a first mixed solution;
2) standing the first mixed solution at 0-4 ℃ for 12-24 hours to obtain a second mixed solution;
3) homogenizing the second mixed solution at ice bath temperature of 4 ℃ and rotation speed of 300-400rpm for 8-12min to obtain a third mixed solution;
4) freezing and centrifuging the third mixed solution at 4 deg.C and 7500-8000rpm for 23-28min, and collecting supernatant to obtain a fourth mixed solution;
5) purifying the fourth mixed solution by Procainamide affinity chromatography to obtain a primary purified substance;
6) purifying the primary purified substance by using a SephadexG-200 gel column to obtain a final purified substance;
7) and concentrating and drying the final purified product to obtain the product.
As a further scheme of the invention: in the step 1), the adding amount of the mixed buffer extracting solution is 3-5 times of the volume of the used fresh horse blood.
As a still further scheme of the invention: in the step 1), the mixed buffer extracting solution contains 0.5-0.8% of polyethylene glycol octyl phenyl ether by mass percent, and the balance is phosphate buffer solution.
As a still further scheme of the invention: in the step 1), the pH value of the mixed buffer extracting solution is 7.2-7.6.
As a still further scheme of the invention: in step 5), the pH of the system was 7.3.
As a still further scheme of the invention: in step 6), the pH of the system was 7.5.
As a still further scheme of the invention: in step 7), the final purified product is concentrated by dialysis at 4 ℃ and then vacuum freeze-dried.
Compared with the prior art, the invention has the beneficial effects that:
the acetylcholinesterase obtained by the extraction method has the advantages of high enzyme activity, high purity and high sensitivity, and the prepared product is powdery, is easy to store and has wide market prospect.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to specific embodiments.
Example 1
A method for extracting acetylcholinesterase from horse blood comprises the following steps:
1) adding fresh horse blood into a mixed buffer extracting solution, stirring and mixing for 15min at an ice bath temperature of 4 ℃ and a rotating speed of 100rpm to obtain a first mixed solution, wherein the adding amount of the mixed buffer extracting solution is 3 times of the volume of the used fresh horse blood; the mixed buffer extracting solution contains 0.5 percent of polyethylene glycol octyl phenyl ether by mass percent, and the balance is phosphate buffer solution; the pH of the mixed buffer extracting solution is 7.2;
2) standing the first mixed solution at 0 ℃ for 12 hours to obtain a second mixed solution;
3) homogenizing the second mixed solution at ice bath temperature of 4 deg.C and rotation speed of 300rpm for 8min to obtain a third mixed solution;
4) freezing and centrifuging the third mixed solution at 4 deg.C and 7500rpm for 23min, and collecting supernatant to obtain fourth mixed solution;
5) purifying the fourth mixed solution by a Procainamide affinity chromatography to obtain a primary purified product, wherein the pH value of a Procainamide affinity chromatography system is 7.3;
6) purifying the primary purified product by using a SephadexG-200 gel column to obtain a product, wherein the pH value of a SephadexG-200 gel column system is 7.5;
7) and (3) concentrating and drying the final purified product to obtain the product, specifically, concentrating the final purified product at 4 ℃ by a dialysis method, and then carrying out vacuum freeze drying to obtain the product.
Example 2
A method for extracting acetylcholinesterase from horse blood comprises the following steps:
1) adding fresh horse blood into a mixed buffer extracting solution, stirring and mixing for 16min at an ice bath temperature of 4 ℃ and a rotating speed of 150rpm to obtain a first mixed solution, wherein the adding amount of the mixed buffer extracting solution is 3 times of the volume of the used fresh horse blood; the mixed buffer extracting solution contains 0.6 mass percent of polyethylene glycol octyl phenyl ether and the balance of phosphate buffer solution; the pH of the mixed buffer extracting solution is 7.3;
2) standing the first mixed solution at 0 ℃ for 15 hours to obtain a second mixed solution;
3) homogenizing the second mixed solution at ice bath temperature of 4 deg.C and rotation speed of 320rpm for 9min to obtain a third mixed solution;
4) freezing and centrifuging the third mixed solution at 7600rpm at 4 deg.C for 24min, and collecting supernatant to obtain fourth mixed solution;
5) purifying the fourth mixed solution by a Procainamide affinity chromatography to obtain a primary purified product, wherein the pH value of a Procainamide affinity chromatography system is 7.3;
6) purifying the primary purified product by using a SephadexG-200 gel column to obtain a product, wherein the pH value of a SephadexG-200 gel column system is 7.5;
7) and (3) concentrating and drying the final purified product to obtain the product, specifically, concentrating the final purified product at 4 ℃ by a dialysis method, and then carrying out vacuum freeze drying to obtain the product.
Example 3
A method for extracting acetylcholinesterase from horse blood comprises the following steps:
1) adding fresh horse blood into a mixed buffer extracting solution, stirring and mixing for 18min at an ice bath temperature of 4 ℃ and a rotating speed of 150rpm to obtain a first mixed solution, wherein the adding amount of the mixed buffer extracting solution is 4 times of the volume of the used fresh horse blood; the mixed buffer extracting solution contains 0.7 mass percent of polyethylene glycol octyl phenyl ether and the balance of phosphate buffer solution; the pH of the mixed buffer extracting solution is 7.4;
2) standing the first mixed solution at the temperature of 2 ℃ for 18 hours to obtain a second mixed solution;
3) homogenizing the second mixed solution at ice bath temperature of 4 deg.C and rotation speed of 350rpm for 10min to obtain a third mixed solution;
4) freezing and centrifuging the third mixed solution at 4 deg.C and 7800rpm for 25min, and collecting supernatant to obtain fourth mixed solution;
5) purifying the fourth mixed solution by a Procainamide affinity chromatography to obtain a primary purified product, wherein the pH value of a Procainamide affinity chromatography system is 7.3;
6) purifying the primary purified product by using a SephadexG-200 gel column to obtain a product, wherein the pH value of a SephadexG-200 gel column system is 7.5;
7) and (3) concentrating and drying the final purified product to obtain the product, specifically, concentrating the final purified product at 4 ℃ by a dialysis method, and then carrying out vacuum freeze drying to obtain the product.
Example 4
A method for extracting acetylcholinesterase from horse blood comprises the following steps:
1) adding fresh horse blood into the mixed buffer extract, and stirring and mixing for 19min at the ice bath temperature of 4 ℃ and the rotation speed of 180rpm to obtain a first mixed solution, wherein the adding amount of the mixed buffer extract is 5 times of the volume of the used fresh horse blood; the mixed buffer extracting solution contains 0.7 mass percent of polyethylene glycol octyl phenyl ether and the balance of phosphate buffer solution; the pH of the mixed buffer extracting solution is 7.5;
2) standing the first mixed solution for 20 hours at 4 ℃ to obtain a second mixed solution;
3) homogenizing the second mixed solution at ice bath temperature of 4 deg.C and rotation speed of 400rpm for 10min to obtain a third mixed solution;
4) freezing and centrifuging the third mixed solution at 4 deg.C and 8000rpm for 26min, and collecting supernatant to obtain fourth mixed solution;
5) purifying the fourth mixed solution by a Procainamide affinity chromatography to obtain a primary purified product, wherein the pH value of a Procainamide affinity chromatography system is 7.3;
6) purifying the primary purified product by using a SephadexG-200 gel column to obtain a product, wherein the pH value of a SephadexG-200 gel column system is 7.5;
7) and (3) concentrating and drying the final purified product to obtain the product, specifically, concentrating the final purified product at 4 ℃ by a dialysis method, and then carrying out vacuum freeze drying to obtain the product.
Example 5
A method for extracting acetylcholinesterase from horse blood comprises the following steps:
1) adding fresh horse blood into the mixed buffer extract, and stirring and mixing for 20min at the ice bath temperature of 4 ℃ and the rotation speed of 200rpm to obtain a first mixed solution, wherein the adding amount of the mixed buffer extract is 5 times of the volume of the used fresh horse blood; the mixed buffer extracting solution contains 0.8 mass percent of polyethylene glycol octyl phenyl ether and the balance of phosphate buffer solution; the pH of the mixed buffer extracting solution is 7.6;
2) standing the first mixed solution at 4 ℃ for 24 hours to obtain a second mixed solution;
3) homogenizing the second mixed solution at ice bath temperature of 4 deg.C and rotation speed of 400rpm for 12min to obtain a third mixed solution;
4) freezing and centrifuging the third mixed solution at 4 deg.C and 8000rpm for 28min, and collecting supernatant to obtain fourth mixed solution;
5) purifying the fourth mixed solution by a Procainamide affinity chromatography to obtain a primary purified product, wherein the pH value of a Procainamide affinity chromatography system is 7.3;
6) purifying the primary purified product by using a SephadexG-200 gel column to obtain a product, wherein the pH value of a SephadexG-200 gel column system is 7.5;
7) and (3) concentrating and drying the final purified product to obtain the product, specifically, concentrating the final purified product at 4 ℃ by a dialysis method, and then carrying out vacuum freeze drying to obtain the product.
The horse blood is extracted by the extraction method of the embodiment 1-5 of the invention, the activity is determined by an acetic acid- β -naphthyl ester color development method, the activity of the obtained acetylcholinesterase is 85.25-90.57U/mg, the sensitivity of the obtained acetylcholinesterase is determined by an indophenol acetate color development method, and the detection limit is 10-15 mug/mL.
The acetylcholinesterase obtained by the extraction method has the advantages of high enzyme activity, high purity and high sensitivity, and the prepared product is powdery, is easy to store and has wide market prospect.
While the preferred embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art.
Claims (7)
1. A method for extracting acetylcholinesterase from horse blood is characterized by comprising the following steps:
1) taking fresh horse blood, adding the mixed buffer extract, stirring and mixing for 15-20min at the ice bath temperature of 4 ℃ and the rotation speed of 100-200rpm to obtain a first mixed solution;
2) standing the first mixed solution at 0-4 ℃ for 12-24 hours to obtain a second mixed solution;
3) homogenizing the second mixed solution at ice bath temperature of 4 ℃ and rotation speed of 300-400rpm for 8-12min to obtain a third mixed solution;
4) freezing and centrifuging the third mixed solution at 4 deg.C and 7500-8000rpm for 23-28min, and collecting supernatant to obtain a fourth mixed solution;
5) purifying the fourth mixed solution by Procainamide affinity chromatography to obtain a primary purified substance;
6) purifying the primary purified substance by using a SephadexG-200 gel column to obtain a final purified substance;
7) and concentrating and drying the final purified product to obtain the product.
2. The method for extracting acetylcholinesterase from horse blood as claimed in claim 1, wherein in step 1), the amount of mixed buffer extract added is 3-5 times of the volume of fresh horse blood.
3. The method for extracting acetylcholinesterase from horse blood as claimed in claim 2, wherein in step 1), the mixed buffer extract contains 0.5-0.8% by weight of polyethylene glycol octyl phenyl ether, and the balance is phosphate buffer.
4. The method for extracting acetylcholinesterase from horse blood as claimed in claim 3, wherein in step 1), the pH of the mixed buffer extract is 7.2-7.6.
5. The method for extracting acetylcholinesterase from horse blood according to any one of claims 1-4, wherein the pH of the system in step 5) is 7.3.
6. The method for extracting acetylcholinesterase from horse blood according to any one of claims 1-4, wherein in step 6), the pH of the system is 7.5.
7. A process for the extraction of acetylcholinesterase from horse blood according to any one of claims 1-4, wherein the final purified product is concentrated by dialysis at 4 ℃ in step 7), and then vacuum freeze-dried.
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| CN201710242170.4A CN106906193B (en) | 2017-04-12 | 2017-04-12 | Method for extracting acetylcholinesterase from horse blood |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1472316A (en) * | 2002-07-29 | 2004-02-04 | 广东省微生物研究所 | Preparing method for acetylcholinesterase |
| CN101126082A (en) * | 2007-07-18 | 2008-02-20 | 华中农业大学 | Extraction method for acetylcholinesterase and application thereof |
| CN102660516A (en) * | 2012-05-10 | 2012-09-12 | 浙江师范大学 | Purification method of acetylcholinesterase from earthworm serum |
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2017
- 2017-04-12 CN CN201710242170.4A patent/CN106906193B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1472316A (en) * | 2002-07-29 | 2004-02-04 | 广东省微生物研究所 | Preparing method for acetylcholinesterase |
| CN101126082A (en) * | 2007-07-18 | 2008-02-20 | 华中农业大学 | Extraction method for acetylcholinesterase and application thereof |
| CN102660516A (en) * | 2012-05-10 | 2012-09-12 | 浙江师范大学 | Purification method of acetylcholinesterase from earthworm serum |
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Effective date of registration: 20240618 Address after: Building B1, 1034, No. 76-4 Chuangyu Road, Ningxi Street, Zengcheng District, Guangzhou City, Guangdong Province, 511300 (self compiled) Patentee after: Zhongke Zhikang (Guangzhou) Science and Technology Co.,Ltd. Country or region after: China Address before: Room 203, 2nd Floor, Building 16, Zone A, Gaonong Biological Park Headquarters, No. 888 Gaoxin Avenue, Donghu High tech Zone, Wuhan City, Hubei Province, 430206 Patentee before: WUHAN ZHONGKE ZHIKANG BIOTECHNOLOGY CO.,LTD. Country or region before: China |