CN110846292A - Extraction method of horseradish peroxidase - Google Patents
Extraction method of horseradish peroxidase Download PDFInfo
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- CN110846292A CN110846292A CN201911296591.0A CN201911296591A CN110846292A CN 110846292 A CN110846292 A CN 110846292A CN 201911296591 A CN201911296591 A CN 201911296591A CN 110846292 A CN110846292 A CN 110846292A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
- C12Y111/01—Peroxidases (1.11.1)
- C12Y111/01007—Peroxidase (1.11.1.7), i.e. horseradish-peroxidase
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Abstract
The invention relates to the technical field of horseradish peroxidase extraction, in particular to a method for extracting horseradish peroxidase, which comprises the following steps: step 1: selecting, cleaning, crushing, centrifuging and removing residues; selecting fresh and clean horseradish, crushing by using a crusher, firstly carrying out coarse filtration by using nylon gauze, and then further removing slag by using a centrifugal mode; step 2: primary impurity removal; centrifuging the turbid solution obtained in the step 1 at 3500r/min for 15min, removing precipitated impurity proteins, salting out by using 53-72 concentration ammonium sulfate, and centrifuging the precipitated part in a freezing centrifuge at 12000r/min for 20 min; and step 3: desalting; adding the precipitate obtained in the step (2) into an ice salt bath, dialyzing by using a 14000 molecular dialysis membrane to remove salt, and obtaining a supernatant for later use; and 4, step 4: secondary impurity removal; and (3) adding acetone into the supernatant obtained in the step (3), centrifuging for 20min at 4200r/min in a refrigerated centrifuge, standing, dissolving in distilled water, dialyzing to remove acetone, and solving the problems that the existing horseradish peroxidase extraction technology is low in purification purity and generates a large amount of waste materials.
Description
Technical Field
The invention relates to the technical field of horseradish peroxidase extraction, and particularly relates to a method for extracting horseradish peroxidase.
Background
Peroxidase (HRP, EC 1.11.1.7) is generally derived from Horseradish (Armoracia rusticana) (perennial herbaceous plants growing in Europe and parts of Asia), so the Peroxidase is called Horseradish Peroxidase, is a common enzyme in clinical test reagents, is not only used for a plurality of biochemical detection projects, but also widely applied to immune experimental detection and the like, and is used as a key component of an immune color development system and is of great importance for the influence of experimental results and related product quality.
The horseradish peroxidase HRP has high specific activity, stable property and small molecular weight, so the horseradish peroxidase HRP is widely applied to various fields of life science. HRP is widely distributed in the plant world, is high in content, is glycoprotein formed by combining colorless zymoprotein and brown ferriporphyrin, and has the sugar content of 18%. HRP is composed of at least 15 isozymes (A1-A3, B1-B3, C1, C2, D, E1-E6), wherein the content of horseradish peroxidase C (HRP C) is the highest; the molecular weight of HRP is 40,000, the isoelectric point is PH 3-9 (acidic (A type), neutral (B and C type), and alkaline (D and E type)), and the optimum PH for enzyme catalysis is slightly different due to different hydrogen donors, but is mostly about PH 5. The enzyme is dissolved in water and a solution of ammonium sulfate at a saturation of less than 58%. The prosthetic group and the enzyme protein of HRP have maximum absorption spectra of 403nm and 275nm, respectively, and the purity of the enzyme is generally expressed as the ratio RZ (Reinheit Zahl Germany) of OD403nm/OD275 nm. The RZ value of the high-purity enzyme is about 3.0 (up to 3.4). The smaller the RZ value, the more non-enzyme proteins.
As early as 80 years ago, HRP has been purified and widely used in various fields of biology; most of the traditional HRP production methods are ammonium sulfate and ethanol precipitation, and the method not only produces a large amount of waste materials to pollute the environment, has low recovery rate and low purity of the enzyme, but also greatly reduces the activity of the enzyme in the purification process; in many cases, HRP needs to be further purified by affinity adsorption or chromatographic separation techniques, and once a scholarer uses the chromatography techniques of phaseolin a (con a) and phenoxyhydroxamic acid (BHA) to purify HRP, respectively, but the problem of nonspecific binding of protein (HRP is a glycoprotein) cannot be effectively avoided, and the purity of the produced HRP is not high.
The existing horseradish peroxidase extraction technology has low purification purity and can generate a large amount of waste materials.
Disclosure of Invention
The invention aims to solve the technical problems that the existing horseradish peroxidase extraction technology has low purification purity and generates a large amount of waste materials.
In order to solve the technical problems, the invention provides a method for extracting horseradish peroxidase, which comprises the following steps:
step 1: selecting, cleaning, crushing, centrifuging and removing residues; selecting fresh and clean horseradish, crushing by using a crusher, firstly carrying out coarse filtration by using nylon gauze, and then further removing slag by using a centrifugal mode;
step 2: primary impurity removal; centrifuging the turbid solution obtained in the step 1 at 3500r/min for 15min, removing precipitated impurity proteins, salting out by using 53-72 concentration ammonium sulfate, and centrifuging the precipitated part in a freezing centrifuge at 12000r/min for 20 min;
and step 3: desalting; adding the precipitate obtained in the step (2) into an ice salt bath, dialyzing by using a 14000 molecular dialysis membrane to remove salt, and obtaining a supernatant for later use;
and 4, step 4: secondary impurity removal; adding acetone into the supernatant obtained in the step 3, centrifuging for 20min at 4200r/min in a refrigerated centrifuge, standing, dissolving in distilled water, and dialyzing to remove acetone;
and 5: purifying; and 4, adding zinc sulfate into the enzyme solution, centrifuging, dialyzing with water, and desalting.
Preferably, the sulfate concentration in the step 2 is 65%.
Preferably, the acetone in the step 4 adopts an acetone reagent at the temperature of-20 ℃.
Preferably, the concentration of zinc ions in the step 5 is 8 mol/L.
Preferably, the centrifugal rotating speed in the step 5 is 4800 r/min.
The invention has the advantages that:
1. the method has the advantages of high purification speed, high precision, cost saving, no generation of a lot of wastes, and greatly improved enzyme activity compared with the traditional method.
Detailed Description
The present invention is further described in order to make the technical means, the creation features, the achievement purposes and the effects of the present invention easy to understand.
A method for extracting horseradish peroxidase comprises the following steps:
step 1: selecting, cleaning, crushing, centrifuging and removing residues; selecting fresh and clean horseradish, crushing by using a crusher, firstly carrying out coarse filtration by using nylon gauze, and then further removing slag by using a centrifugal mode;
step 2: primary impurity removal; centrifuging the turbid solution obtained in the step 1 at 3500r/min for 15min, removing precipitated impurity proteins, salting out by using 53-72 concentration ammonium sulfate, and centrifuging the precipitated part in a freezing centrifuge at 12000r/min for 20 min;
and step 3: desalting; adding the precipitate obtained in the step (2) into an ice salt bath, dialyzing by using a 14000 molecular dialysis membrane to remove salt, and obtaining a supernatant for later use;
and 4, step 4: secondary impurity removal; adding acetone into the supernatant obtained in the step 3, centrifuging for 20min at 4200r/min in a refrigerated centrifuge, standing, dissolving in distilled water, and dialyzing to remove acetone;
and 5: purifying; and 4, adding zinc sulfate into the enzyme solution, centrifuging, dialyzing with water, and desalting.
In one embodiment of the present invention, the sulfate concentration in step 2 is 65%.
As a specific embodiment of the invention, the acetone in the step 4 adopts an acetone reagent at the temperature of-20 ℃.
As a specific embodiment of the invention, the concentration of zinc ions in the step 5 is 8 mol/L.
As a specific embodiment of the invention, the centrifugal speed in the step 5 is 4800 r/min.
Claims (5)
1. A method for extracting horseradish peroxidase is characterized in that: the method comprises the following steps:
step 1: selecting, cleaning, crushing, centrifuging and removing residues; selecting fresh and clean horseradish, crushing by using a crusher, firstly carrying out coarse filtration by using nylon gauze, and then further removing slag by using a centrifugal mode;
step 2: primary impurity removal; centrifuging the turbid solution obtained in the step 1 at 3500r/min for 15min, removing precipitated impurity proteins, salting out by using 53-72 concentration ammonium sulfate, and centrifuging the precipitated part in a freezing centrifuge at 12000r/min for 20 min;
and step 3: desalting; adding the precipitate obtained in the step (2) into an ice salt bath, dialyzing by using a 14000 molecular dialysis membrane to remove salt, and obtaining a supernatant for later use;
and 4, step 4: secondary impurity removal; adding acetone into the supernatant obtained in the step 3, centrifuging for 20min at 4200r/min in a refrigerated centrifuge, standing, dissolving in distilled water, and dialyzing to remove acetone;
and 5: purifying; and 4, adding zinc sulfate into the enzyme solution, centrifuging, dialyzing with water, and desalting.
2. The method for extracting horseradish peroxidase according to claim 1, which is characterized in that: the sulfate concentration in step 2 was 65%.
3. The method for extracting horseradish peroxidase according to claim 1, which is characterized in that: in the step 4, acetone reagent with the temperature of-20 ℃ is adopted.
4. The method for extracting horseradish peroxidase according to claim 1, which is characterized in that: the concentration of zinc ions in the step 5 is 8 mol/L.
5. The method for extracting horseradish peroxidase according to claim 1, which is characterized in that: and the centrifugal rotating speed in the step 5 is 4800 r/min.
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| CN201911296591.0A CN110846292A (en) | 2019-12-16 | 2019-12-16 | Extraction method of horseradish peroxidase |
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| CN201911296591.0A CN110846292A (en) | 2019-12-16 | 2019-12-16 | Extraction method of horseradish peroxidase |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1928078A (en) * | 2006-10-10 | 2007-03-14 | 山西职工医学院 | Extraction method of peroxidase |
| CN102250850A (en) * | 2011-07-11 | 2011-11-23 | 湖南人文科技学院 | Method for extracting peroxidase from sweet potato root tuber |
| CN102618514A (en) * | 2012-04-18 | 2012-08-01 | 孙海霞 | Method for ultrafiltration extraction of horse radish peroxidase |
| CN104017783A (en) * | 2014-05-13 | 2014-09-03 | 安徽味仙食品有限公司 | Method for extracting catalase from crop |
| KR101629439B1 (en) * | 2014-06-26 | 2016-06-10 | 을지대학교 산학협력단 | Skin Barrier Protein ELISA Kit of Atopic Dermatitis Patient Using Non-invasive Sample Extraction Method |
| CN108721943A (en) * | 2018-06-19 | 2018-11-02 | 长春万成生物电子工程有限公司 | The preparation method of Electrospun adsorption column and horseradish peroxidase |
-
2019
- 2019-12-16 CN CN201911296591.0A patent/CN110846292A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1928078A (en) * | 2006-10-10 | 2007-03-14 | 山西职工医学院 | Extraction method of peroxidase |
| CN102250850A (en) * | 2011-07-11 | 2011-11-23 | 湖南人文科技学院 | Method for extracting peroxidase from sweet potato root tuber |
| CN102618514A (en) * | 2012-04-18 | 2012-08-01 | 孙海霞 | Method for ultrafiltration extraction of horse radish peroxidase |
| CN104017783A (en) * | 2014-05-13 | 2014-09-03 | 安徽味仙食品有限公司 | Method for extracting catalase from crop |
| KR101629439B1 (en) * | 2014-06-26 | 2016-06-10 | 을지대학교 산학협력단 | Skin Barrier Protein ELISA Kit of Atopic Dermatitis Patient Using Non-invasive Sample Extraction Method |
| CN108721943A (en) * | 2018-06-19 | 2018-11-02 | 长春万成生物电子工程有限公司 | The preparation method of Electrospun adsorption column and horseradish peroxidase |
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