阅读说明：本技术 一种辣根过氧化物酶的提取方法 (Extraction method of horseradish peroxidase ) 是由 卓之明 于 2019-12-16 设计创作，主要内容包括：本发明涉及辣根过氧化物酶提取技术领域,具体为一种辣根过氧化物酶的提取方法,包括以下步骤：步骤1：挑选清洗、破碎、离心去渣；挑选新鲜干净的辣根,利用破碎机破碎,首先经过尼龙纱布进行粗过滤,然后利用离心方式进一步去渣；步骤2：一次去杂；步骤1的浊液以3500r/min离心15min,去除沉淀杂蛋白,利用53～72浓度的硫酸铵盐析,沉淀部分在冰冻离心机以12000r/min离心20min；步骤3：去盐；把步骤2沉淀加入冰盐浴中,利用14000分子透析膜透析去盐,取得上清液备用；步骤4：二次去杂；把步骤3中的上清液加入丙酮,在冰冻离心机中以4200r/min离心20min,静置,然后溶于蒸馏水中透析除丙酮,解决现有的辣根过氧化物酶提取技术提纯纯度较低,同时会产生大量的废料的问题。(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.)

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.

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.