Method for extracting SOD (superoxide dismutase) by high-temperature protein cellulose removal column chromatography

Abstract

The invention relates to a method for extracting SOD (superoxide dismutase) by utilizing high-temperature protein cellulose removal column chromatography, and belongs to the technical field of SOD extraction.The method comprises the following steps that S1, a part of plasma is extracted and put into a beaker, and a copper sulfate solution is added into the plasma; s2, adding water which is 2-3 times of the plasma into a beaker; s3, the beaker is heated, and stirring is conducted during heating; s4, the heated solution is filtered, and blood residues are filtered out; s5, taking the filtrate with the volume of 500 to 1000 [mu] / ml; s6, concentrating the filtrate into a solution with the concentration of 10000 to 50000 u / ml by using an ultrafilter; s7, adding ammonia water into the solution; s8, adjusting the pH (Potential of Hydrogen) value to 4.8 to 5; s9, the solution is heated again, during heating, 1% of a protective agent is added into the solution, the protective agent is a 20% solution prepared by adding water into sulfate, the temperature is increased to 70-80 DEG C, filtering is conducted with filter paper, and filtrate is taken; and S10, adsorbing the SOD in the solution to the cellulose column. The sulfate solution is used as a protective agent, so that when the temperature is raised to 80 DEG C, a large amount of protein can be removed, and SOD does not lose activity.

Description

Technical Field

[0001] The invention relates to the technical field of SOD extraction, in particular to a method for extracting SOD by utilizing high-temperature protein removal cellulose column chromatography. Background Art

[0002] SOD is the abbreviation of Super Oxide Dismutase, and its Chinese name is superoxide dismutase. It is an important antioxidant enzyme in organisms and is widely distributed in various organisms, such as animals, plants, and microorganisms. SOD has special physiological activity and is the primary substance for removing free radicals in organisms. The level of SOD in organisms means a direct indicator of aging and death. It has been confirmed that there are more than 60 diseases caused by oxygen free radicals. It can fight and block the damage caused by oxygen free radicals to cells, and repair damaged cells in time, and restore the damage to cells caused by free radicals. Due to the pressure of modern life, environmental pollution, various radiations and excessive exercise, a large number of oxygen free radicals will be formed; therefore, the position of SOD in the biological antioxidant mechanism is becoming more and more important.

[0003] At present, there are many methods for extracting SOD from animal blood. Among them, patent CN201010241815.0 discloses a large-scale industrial method for extracting superoxide dismutase from cattle and sheep blood. Fresh cattle blood or sheep blood is subjected to anticoagulation, hypotonic hemolysis, ultrafiltration, rapid temperature rise and fall, precipitation, and three-stage temperature treatment by a mixer with a heat source / cold source: the first stage: 45-55°C; the second stage: 55-60°C; the third stage: 60-65°C, salting out, ultrafiltration and concentration, freeze drying and other production steps to obtain SOD with higher purity.

[0004] However, when heated to 50-65°C, a large amount of protein will still remain. If the heating continues, SOD will be inactivated. Summary of the invention

[0005] The purpose of the present invention is to solve the problems existing in the background technology and propose a method for extracting SOD by high temperature protein removal cellulose column chromatography by using sulfate solution as a protective agent so that a large amount of protein can be removed when the temperature rises to 80°C without losing the activity of SOD.

[0006] The technical solution of the present invention is a method for extracting SOD by high temperature protein removal cellulose column chromatography, the method comprising the following steps: S1. Extract a portion of plasma and put it into a beaker. Add copper sulfate solution to the plasma. S2. Add 2-3 times the amount of water as plasma into the beaker; S3, heating the beaker and stirring during heating; S4, filtering the heated solution to remove blood residue; S5, take 500u / ml-1000u / ml of filtrate; S6, using an ultrafilter to concentrate the filtrate to a solution of 10000u / ml-50000u / ml; S7, adding ammonia water to the solution to adjust the pH value to 8-9.5 without inactivating SOD; S8, add copper sulfate to adjust the pH value to 4.8-5, filter and take the filtrate, and then use an ultrafilter to concentrate the filtrate to 100000u / ml-200000u / ml; S9, heat the solution again, add 1% of protective agent to the solution during heating, the protective agent is sulfate and water is added to make a 20% solution, the temperature is raised to 70℃-80℃, filter with filter paper, and take the filtrate; S10, using an upper chromatography column to allow the SOD in the solution to be adsorbed onto the cellulose column; S11, washing away the impurity proteins on the cellulose column; S12, using 200 microliters of eluent, wash SOD from the cellulose column to 800,000u / ml-1,000,000u / ml; S13, placing the solution into a dialysis bag, and dialyzing it into a dialysis solution to a pH of 4.7; S14. Filter and freeze-dry the dialyzed SOD, and the enzyme activity reaches 6000u / mg-10000u / mg.

[0007] Preferably, boiling water is added to the copper sulfate solution to dissolve it and prepare a 10% copper sulfate solution.

[0008] Preferably, in step S1, the content of the copper sulfate solution is 0.8%-1%.

[0009] Preferably, the ultrafilter has a molecular weight cut-off of 6,000.

[0010] Preferably, in step S9, acetic acid solution is used to adjust the pH value.

[0011] Preferably, the cellulose column is soaked in deionized water.

[0012] Preferably, after soaking, the cellulose column is fished out, cleaned, and loaded into a collection column.

[0013] Compared with the prior art, the present invention has the following beneficial technical effects: In the present invention, 1% copper sulfate solution is added to the extracted plasma, and tap water is added, and then heated to 65°C-75°C and maintained for 30 minutes, and then the blood residue is filtered out, and 500u / ml-1000u / ml of the filtrate is taken, and the filtrate is concentrated to 10000u / ml-50000u / ml of the solution by using a hollow fiber ultrafilter with a molecular weight cutoff of 6000, and the pH value is adjusted to 8-9.5 with one equivalent of amine water. When adjusting the pH value, the higher the pH value, the better, under the premise of ensuring that SOD is not inactivated.

[0014] Then adjust the pH value to below 5 with one equivalent of copper sulfate, filter with filter paper, take the filtrate, concentrate it to 100000u / ml-200000u / ml with ultrafilter, adjust the pH to 4.7 with one equivalent of acetic acid, adsorb SOD to the column with a balanced cellulose column, and after the column is full, wash away the impurities with acetic acid balance solution, and use 200 millimolar eluent to wash SOD off the column to 800000u / ml-1000000u / ml. Install the dialysis bag, put it in the dialysis solution and dialyze it to pH4.7, filter and freeze-dry the dialyzed SOD, and the enzyme activity reaches 6000u / mg-10000u / mg. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0016] Figure 1 A detection report of superoxide dismutase in an embodiment of the present invention; Figure 2 It is the detection report of SOD in the embodiment of the present invention; Figure 3 It is a SOD acute toxicity test result record table of the embodiment of the present invention; Figure 4 This is a superoxide dismutase test report according to an embodiment of the present invention. DETAILED DESCRIPTION

[0017] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings.

[0018] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0019] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor is it an embodiment that is mutually exclusive with other embodiments, either individually or selectively.

[0020] Secondly, the present invention is described in detail with reference to the schematic diagram. When describing the embodiments of the present invention in detail, for the sake of convenience, the cross-sectional diagrams showing the device structure will not be partially enlarged according to the general scale, and the schematic diagrams are only examples, which should not limit the scope of protection of the present invention. In addition, in actual production, the three-dimensional dimensions of length, width and depth should be included.

[0021] Embodiment 1 like Figure 1-4 As shown, the present invention proposes a method for extracting SOD by high temperature protein removal cellulose column chromatography, the method comprising the following steps: S1. Extract a portion of plasma and put it into a beaker, add copper sulfate solution to the plasma; add boiling water to the copper sulfate solution to make a 10% copper sulfate solution with a content of 0.8%-1%. By adding copper sulfate solution to the plasma and heating it, SOD is effectively released from red blood cells, and the purity of SOD is significantly improved through subsequent filtration and ultrafiltration steps.

[0022] S2. Add 2-3 times the amount of water as plasma into the beaker; S3, heating the beaker and stirring during heating; S4, filtering the heated solution to remove blood residue; S5, take 500u / ml-1000u / ml of filtrate; S6. Use an ultrafilter to concentrate the filtrate to a solution of 10000u / ml-50000u / ml; the molecular weight cutoff of the ultrafilter is 6000.

[0023] S7, adding ammonia water to the solution to adjust the pH value to 8-9.5 without inactivating SOD; S8, add copper sulfate to adjust the pH value to 4.8-5, filter and take the filtrate, and then use an ultrafilter to concentrate the filtrate to 100000u / ml-200000u / ml; S9, heat the solution again, add 1% of protective agent to the solution during heating, the protective agent is sulfate and water is added to make a 20% solution, the temperature is raised to 70℃-80℃, filter with filter paper, and take the filtrate; S10, using an upper chromatography column to allow the SOD in the solution to be adsorbed onto the cellulose column; S11, washing away the impurity proteins on the cellulose column; S12, using 200 microliters of eluent, wash SOD from the cellulose column to 800,000u / ml-1,000,000u / ml; S13, placing the solution into a dialysis bag, and dialyzing it into a dialysis solution to a pH of 4.7; S14. Filter and freeze-dry the dialyzed SOD, and the enzyme activity reaches 6000u / mg-10000u / mg.

[0024] In step S9, an acetic acid solution is used to adjust the pH value.

[0025] In this embodiment, 1% copper sulfate solution is added to the extracted plasma, and tap water is added, and then heated to 65°C-75°C and stirred evenly, and then the blood residue is filtered out, and 500u / ml-1000u / ml of the filtrate is taken, and the filtrate is concentrated to 10000u / ml-50000u / ml using a hollow fiber ultrafilter with a molecular weight cutoff of 6000, and the pH value is adjusted to 8-9.5 with one equivalent of amine water. When adjusting the pH value, the higher the pH value, the better, under the premise of ensuring that SOD is not inactivated; Then adjust the pH value to below 5 with one equivalent of copper sulfate, filter with filter paper, take the filtrate, concentrate it to 100000u / ml-200000u / ml with ultrafilter, adjust the pH to 4.7 with one equivalent of acetic acid, adsorb SOD to the column with a balanced cellulose column, and after the column is full, wash away the impurities with acetic acid balance solution, and use 200 millimolar eluent to wash SOD off the column to 800000u / ml-1000000u / ml. Install the dialysis bag, put it in the dialysis solution and dialyze it to pH4.7, filter and freeze-dry the dialyzed SOD, and the enzyme activity reaches 6000u / mg-10000u / mg.

[0026] SOD is a substance wrapped in red blood cells. After being separated from the blood residue and concentrated, the SOD solution contains a lot of protein that is difficult to remove. The protein is removed using cellulose column chromatography.

[0027] Practice has proved that the method of removing protein by heating is very effective. Under normal circumstances, when the temperature reaches 70°C, most of the protein can be separated, but some of the protein cannot be separated. When the temperature is raised, SOD will lose its activity.

[0028] After many experiments, the SOD solution was concentrated to 100,000 / ml-200,000 / ml using an ultrafilter, and sulfate solution was used as a protective agent. The temperature could be raised to 80°C to remove a large amount of protein without losing the activity of SOD, thereby ensuring that SOD still maintains its original activity. Compared with the extraction process in the prior art, efficient extraction of SOD can be achieved.

[0029] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited thereto, and various changes can be made within the knowledge scope of technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A method for extracting SOD using high temperature protein removal cellulose column chromatography, characterized in that: The method comprises the following steps: S1. Extract a portion of plasma and put it into a beaker. Add copper sulfate solution to the plasma. S2. Add 2-3 times the amount of water as plasma into the beaker; S3, heating the beaker and stirring during heating; S4, filtering the heated solution to remove blood residue; S5, take 500u / ml-1000u / ml of filtrate; S6, using an ultrafilter to concentrate the filtrate to a solution of 10000u / ml-50000u / ml; S7, adding ammonia water to the solution to adjust the pH value to 8-9.5 without inactivating SOD; S8, add copper sulfate to adjust the pH value to 4.8-5, filter and take the filtrate, and then use an ultrafilter to concentrate the filtrate to 100000u / ml-200000u / ml; S9, heat the solution again, add 1% of protective agent to the solution during heating, the protective agent is sulfate and water is added to make a 20% solution, the temperature is raised to 70℃-80℃, filter with filter paper, and take the filtrate; S10, using an upper chromatography column to allow the SOD in the solution to be adsorbed onto the cellulose column; S11, washing away the impurity proteins on the cellulose column; S12, using 200 microliters of eluent, wash SOD from the cellulose column to 800,000u / ml-1,000,000u / ml; S13, placing the solution into a dialysis bag, and dialyzing it into a dialysis solution to a pH of 4.7; S14. Filter and freeze-dry the dialyzed SOD, and the enzyme activity reaches 6000u / mg-10000u / mg.

2. The method for extracting SOD by high temperature protein removal cellulose column chromatography according to claim 1, characterized in that: Add boiling water to the copper sulfate solution to make a 10% copper sulfate solution.

3. The method for extracting SOD by high temperature protein removal cellulose column chromatography according to claim 2, characterized in that: In step S1, the content of the copper sulfate solution is 0.8%-1%.

4. The method for extracting SOD by high temperature protein removal cellulose column chromatography according to claim 3, characterized in that: The molecular weight cut-off of the ultrafilter was 6000.

5. The method for extracting SOD by high temperature protein removal cellulose column chromatography according to claim 4, characterized in that: In step S9, an acetic acid solution is used to adjust the pH value.

6. The method for extracting SOD by high temperature protein removal cellulose column chromatography according to claim 5, characterized in that: The cellulose column was soaked in deionized water.

7. The method for extracting SOD by high temperature protein removal cellulose column chromatography according to claim 6, characterized in that: After soaking, remove the cellulose column, clean it, and place it into the collection column.

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