CN110551704A - Method for separating and purifying angiotensin converting enzyme from pig lungs - Google Patents
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Abstract
The invention discloses a method for separating and purifying angiotensin converting enzyme from pig lung, which comprises the steps of pretreating pig lung to obtain primary supernatant S 1, adjusting pH to acidity, standing in water bath, centrifuging, filtering with silk cloth, taking supernatant to obtain secondary supernatant S 2, salting out, adding solid salt, stirring, standing, centrifuging, filtering with silk cloth to obtain tertiary supernatant S 3, slowly adding salt, stirring, standing, centrifuging, collecting precipitate, dissolving in buffer solution to obtain crude angiotensin converting enzyme solution, separating with an ultrafiltration membrane, and collecting non-permeate liquid, namely high-purity angiotensin converting enzyme.
Description
Technical Field
The invention relates to the field of substance separation and purification, in particular to a method for separating and purifying angiotensin converting enzyme from pig lungs.
Background
With the rapid development of economy and the change of living habits of people, chronic non-infectious diseases have become public health problems affecting the health of people. Hypertension is a non-infectious chronic disease and is a very important risk factor for patients with stroke, heart failure and coronary artery disease. Estimated from data from 195 countries and regions, in 2015, there were 8.74 billion adults with a Systolic Blood Pressure (SBP) of 140 mmhg or higher. It is expected that by 2025 about 15.6 million people will suffer from hypertension worldwide. Hypertension has now become a major global health problem. In the pathological basis of hypertension, angiotensin and bradykinin play an important role in the renin-angiotensin system (RAS) to regulate blood pressure homeostasis, and kallikrein-kinin system (KKS) is closely related to the RAS to regulate blood pressure synergistically.
in the RAS system, Angiotensin Converting Enzyme (ACE) is the most important regulatory enzyme. ACE is widely present in different tissues of mammals, with lung capillary endothelial cells having the highest ACE activity. ACE can convert inactive angiotensin I (AngI) into angiotensin II (AngII) capable of vasoconstriction, and degrade bradykinin into inactive fragments, and is a key enzyme in screening drugs for treating hypertension.
the commercial ACE is expensive and not beneficial to the smooth research of ACE inhibitory peptide, and the separation and purification of the ACE are mainly carried out by a method combining ammonium sulfate fractional precipitation, affinity chromatography, ion exchange column chromatography, hydroxyapatite column chromatography and gel filtration chromatography at present, so that the purity of the obtained ACE is not high enough, the separation and purification process is complicated, the operation is complex, the consumed time is long, and the cost is high.
CN 108977423A discloses a preparation process for separating and purifying angiotensin converting enzyme from pig lung by sodium chloride salting-out and DEAE anion exchange column chromatography. The application of the patent utilizes sodium chloride to salt out and remove foreign proteins, multiple times of centrifugation are needed in the salting-out process, the operation is more complicated, and the yield of target proteins is reduced. In addition, the DEAE anion exchange column chromatography method is long in time consumption, high in cost and high in loss.
Disclosure of Invention
In view of the above problems, the inventors of the present invention have found, after intensive research, that the purification speed can be increased by using an acid precipitation method, the time consumption is short, the cost is low, the yield is high, and the obtained crude enzyme solution contains less impure proteins and target proteins with higher purity. Compared with sodium chloride salting-out technology, the ammonium sulfate salting-out technology adopted by the invention is simple to operate and high in yield of the target protein. Compared with dialysis, the ultrafiltration separation technology selected by the invention is short in time consumption, and can realize removal of the foreign protein and concentration of the target protein in one step. The combined technique of acid precipitation, ammonium sulfate salting-out, and ultrafiltration separation of the present invention enables high-purity (electrophoretically pure at the level of counterstaining) angiotensin-converting enzyme to be obtained, and the present invention has been completed based on this.
In order to achieve the above object, the present invention provides a method for separating and purifying angiotensin converting enzyme from pig lungs, comprising:
Pre-treating, namely cleaning the pig lung, removing lung organs and outer membrane tissues, cooling at low temperature, placing the pig lung in a buffer solution for tissue homogenate, adding a penetration enhancer, stirring at low temperature, filtering to obtain a filtrate, centrifuging the filtrate to obtain a supernatant, and obtaining a first-level supernatant S 1;
Acid precipitation, namely adjusting the pH of the primary supernatant S 1 to acidity, standing in a water bath, centrifuging, filtering with silk cloth, and taking the supernatant to obtain a secondary supernatant S 2;
Salting out, namely slowly adding solid salt into the secondary supernatant S 2, stirring, standing, centrifuging, filtering by using silk cloth to obtain a tertiary supernatant S 3, slowly adding salt into the tertiary supernatant S 3, stirring, standing, centrifuging, collecting precipitates, dissolving the precipitates in a buffer solution to obtain a crude angiotensin converting enzyme solution;
and (3) ultrafiltration separation: and separating the crude angiotensin converting enzyme solution by using an ultrafiltration membrane, and collecting the non-permeate liquid, namely the high-purity angiotensin converting enzyme.
Further, in the pretreatment, the low-temperature cooling temperature is-25 ℃ to 0 ℃;
Optionally, the volume dosage of the buffer solution is 2-6 times of the mass (ml/g) of the pig lung.
Further, in the pretreatment, the penetration enhancer is any one of polyethylene glycol octyl phenyl ether, ethyl phenyl polyethylene glycol and n-octyl-beta-D-glucoside, and the volume dosage of the penetration enhancer is 0.5-1.0% (ml/g) of the mass of the pig lung.
Further, in the pretreatment, the stirring temperature at the low temperature is 0-10 ℃, and the stirring time at the low temperature is 0.5-2 hours.
further, in the acid precipitation, the pH value is adjusted to be acidic, and the pH value is adjusted to be 3.0-4.0; the pH of the supernatant is generally 8.0. It is usually prepared by using an acidic buffer.
Optionally, the temperature of the water bath is 30-50 ℃, and the standing time is 3-5 h;
Optionally, the silk cloth is 2-6 layers; preferably, the silk cloth is 4 layers.
further, in the salting out step, the solid salt is solid (NH 4) 2 SO 4;
Optionally, the addition amount of the solid salt 1 is 1.0-2.0 mol/L;
Optionally, the stirring time is 0.5-2 h;
optionally, standing for 2-4 h;
optionally, the silk cloth is 2-6 layers; preferably, the silk cloth is 4 layers;
Optionally, the salt 2 is added in an amount to enable the final concentration to reach 2.0-3.0 mol/L;
Optionally, the using amount of the buffer solution is 2-6 times of the mass of the pig lung.
Further, in the pretreatment and salting-out step, the buffer is Tris-HCL buffer or phosphate buffer.
Further, in the ultrafiltration separation, the molecular weight of the ultrafiltration membrane is 100 kDa.
the buffer solution can be various existing weak alkaline buffer solutions, and specifically can be any one of Tris-HCL buffer solution and phosphate buffer solution independently. In addition, in order to obtain higher purity and yield, the buffers are preferably precooled buffers, and the temperatures of the buffers are preferably 0-10 ℃ respectively and independently. It should be noted that, in this context, unless otherwise specified, when referring to the ratio of the amount of pig lung to the amount of other substances, the amount of pig lung refers to the amount of pig lung dissolved in buffer.
The penetration enhancer is a nonionic surfactant, can dissolve cell membrane by dissolving lipid components on the cell membrane, can be various existing solvents capable of promoting protein in pig lung tissue to permeate into buffer solution, and is preferably at least one selected from polyethylene glycol octyl phenyl ether (Triton x-100), ethyl phenyl polyethylene glycol (Nonidet-P40), and n-octyl-beta-D-glucoside (1-O-n-octyl-beta-D-glucopyranoside). Wherein, the number average molecular weight of the polyethylene glycol octyl phenyl ether can be 646.86, the number average molecular weight of the ethyl phenyl polyethylene glycol can be 680, and the number average molecular weight of the n-octyl-beta-D-glucoside can be 292.37.
In the invention, the pretreatment is used for removing fat, residual lung organs and outer membrane tissues in pig lungs.
In the invention, the acid precipitation is used for removing proteins with molecular weights of 250kDa, 220kDa, 150kDa, 140kDa, 120kDa, 90kDa, 72kDa and 68kDa in pig lungs and further removing residual fat, lung organs and outer membrane tissues.
In the invention, the salting-out function is to enrich the target protein ACE and further remove residual fat, lung organs and outer membrane tissues.
in the present invention, the ultrafiltration separation is performed to remove the hetero-proteins having molecular weights of 100kDa, 58kDa and 50kDa and the low molecular weight peptides and salts.
The prior art for separating and purifying angiotensin converting enzyme adopts a column chromatography method, and has the disadvantages of complex operation, low yield, high cost and low enzyme activity. In order to solve the technical problem, the invention creatively provides a combined technical scheme of acid precipitation, salting out and ultrafiltration separation to realize simple and low-cost purification of the angiotensin converting enzyme with a single electrophoresis band from pig lungs, and the method has high protein recovery rate and high enzyme activity recovery rate.
drawings
FIG. 1 is an SDS-PAGE electrophoresis of ACE purification, wherein lane M is a standard protein, lane 1 is a pre-treated product, lane 2 is a product after acid precipitation, lane 3 is a product after salting out, lane 4 is a product after ultrafiltration separation, and the gel is stained.
FIG. 2 is an SDS-PAGE of purified ACE where M is standard protein, lane 1 is the pre-treated product, lane 2 is purified ACE and the gel is stained.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
1. And (2) pretreatment, namely cleaning 200g of pig lung, removing lung organs and outer membrane tissues, prefreezing the pig lung at the temperature of-20 ℃ until the tissues are fully cooled, placing the pig lung in 600mL of precooled Tris-HCL (20mmoL/L, pH 8.0 and 4 ℃) buffer solution for tissue homogenate, adding 1.5mL of Triton x-100 (the number average molecular weight is 646.86), slowly stirring the mixture for 0.5h at the temperature of 4 ℃, filtering the obtained filtrate, centrifuging the filtrate (6000g, 4 ℃, 20min) to obtain 500mL of supernatant (S 1), wherein the protein content is 5650.00mg, and the enzyme activity is 44.36U.
2. Acid precipitation, the supernatant of S 1 is adjusted to pH 3.45 with an acidic buffer such as hydrochloric acid, left to stand in a water bath at 37 ℃ for 5 hours, centrifuged (12000g, 4 ℃, 30min), and the four layers of silk cloth are filtered to obtain 480mL of supernatant (S 2).
3. Salting out, namely slowly adding solid (NH 4) 2 SO 4 into S 2 until the final concentration reaches 1.6mol/L, stirring for 0.5h, standing for 2h, centrifuging (12000g, 4 ℃, 30min), filtering through four layers of silk cloth to obtain 460mL of supernatant (S 3), slowly adding solid (NH 4) 2 SO 4 into S 3 until the final concentration reaches 2.6mol/L, stirring for 0.5h, standing for 2h, centrifuging (12000g, 4 ℃, 30min) to obtain 4g of precipitate, and dissolving the precipitate into 20mL of precooled Tris-HCL (20mmoL/L, pH 8.0, 4 ℃) buffer solution to obtain a crude angiotensin converting enzyme solution.
4. and (3) ultrafiltration separation: separating the crude angiotensin converting enzyme solution by using a 100kDa ultrafiltration membrane, and collecting the non-permeate liquid, namely the high-purity angiotensin converting enzyme, wherein the protein content is 510.20mg, and the enzyme activity is 10.52U.
The results of SDS-PAGE electrophoretic staining analysis of the product obtained from the separation and purification process of this example are shown in FIG. 1. it can be seen from FIG. 1 that the pretreatment product (lane 1) contains 11 hetero-bands and the 180kDa band (ACE) is less distinct, the acid precipitation product (lane 2) contains only 3 hetero-proteins, which effectively removes a large amount of hetero-proteins, salting-out (lane 3) can effectively enrich ACE, and the ultrafiltration separation product (lane 4) contains only the 180kDa (ACE) band and the band is distinct.
The result of the SDS-PAGE electrophoretic staining analysis of ACE separated and purified in this example is shown in fig. 2, and it can be seen from fig. 2 that lane 1 contains many hetero-bands and the 180kDa band (ACE) is less obvious, and lane 2 contains only the 180kDa (ACE) band and the band is obvious, i.e. after treatment, the hetero-protein is effectively removed, and the ACE is highly purified. The protein band at a molecular weight of about 180kDa is the ACE band, consistent with the reported ACE molecular weight.
the combined technical scheme of acid precipitation, salting out and ultrafiltration separation realizes simple and low-cost purification of the angiotensin converting enzyme with a single electrophoresis band from pig lungs, the protein recovery rate of the embodiment is up to 9.03% (510.20mg/5650.00mg), and the enzyme activity recovery rate is up to 23.72% (10.52U/44.36U).
example 2
1. And (2) pretreatment, namely cleaning 200g of pig lung, removing lung organs and outer membrane tissues, prefreezing the pig lung at-25 ℃ until the tissues are fully cooled, placing the pig lung in 400mL of precooled Tris-HCL (20mmoL/L, pH 8.0 and 4 ℃) buffer solution, homogenating the tissues, adding 2mL of Nonidet-P40 (number average molecular weight is 680), slowly stirring the mixture for 1 hour at 10 ℃, filtering the obtained filtrate, centrifuging the filtrate (6000g, 4 ℃ and 20min) to obtain 450mL of supernatant (S 1), wherein the protein content is 5085.00mg, and the enzyme activity is 39.92U.
2. and (4) acid precipitation, namely adjusting the pH of the supernatant of S 1 to 3, standing in a water bath at 30 ℃ for 4 hours, centrifuging (12000g, 4 ℃, 30min), and filtering four layers of silk cloth to obtain 420mL of supernatant (S 2).
3. Salting out, namely slowly adding solid (NH 4) 2 SO 4 into S 2 until the final concentration reaches 1.0mol/L, stirring for 2h, standing for 3h, centrifuging (12000g, 4 ℃, 30min), filtering by four layers of silk cloth to obtain 400mL of supernatant (S 3), slowly adding solid (NH 4) 2 SO 4 into S 3 until the final concentration reaches 2.0mol/L, stirring for 1h, standing for 2h, centrifuging (12000g, 4 ℃, 30min) to obtain 3g of precipitate, and dissolving the precipitate into 15mL of precooled Tris-HCL (20 ═ mmL/L, pH 8.0, 4 ℃) buffer solution to obtain a crude angiotensin converting enzyme solution.
4. And (3) ultrafiltration separation: separating the crude angiotensin converting enzyme solution by using a 100kDa ultrafiltration membrane, and collecting the non-permeate liquid, namely the high-purity angiotensin converting enzyme, wherein the protein content is 410.00mg, and the enzyme activity is 8.33U.
The combined technical scheme of acid precipitation, salting out and ultrafiltration separation realizes simple and low-cost purification of the angiotensin converting enzyme with a single electrophoresis band from pig lungs, the protein recovery rate of the embodiment is up to 8.06%, and the enzyme activity recovery rate is up to 20.88%.
Example 3
1. Pretreating raw materials, namely cleaning 200g of pig lung, removing lung organs and outer membrane tissues, pre-freezing the pig lung at 0 ℃ until the tissues are fully cooled, placing the pig lung in 1200mL of precooled Tris-HCL (20mmoL/L, pH 8.0 and 4 ℃) buffer solution, homogenating the tissues, adding 7.5mL of n-octyl-beta-D-glucoside (the number average molecular weight is 292.37), slowly stirring the mixture at 0 ℃ for 2 hours, filtering the mixture to obtain filtrate, centrifuging the filtrate (6000g, 4 ℃ and 20min) to obtain 1180mL of supernatant (S 1), wherein the protein content is 5700.00mg, and the enzyme activity is 46.62U.
2. Acid precipitation, adjusting the pH of the supernatant of S 1 to 4, standing in a water bath at 50 ℃ for 3h, centrifuging (12000g, 4 ℃, 30min), and filtering with four layers of silk cloth to obtain 1000mL of supernatant (S 2).
3. salting out, namely slowly adding solid (NH 4) 2 SO 4 into S 2 until the final concentration reaches 2mol/L, stirring for 1h, standing for 4h, centrifuging (12000g, 4 ℃, 30min), filtering four layers of silk cloth to obtain 960mL of supernatant (S 3), slowly adding solid (NH 4) 2 SO 4 into S 3 until the final concentration of the solution reaches 3.0mol/L, stirring for 2h, standing for 3h, centrifuging (12000g, 4 ℃, 30min) to obtain 4.5g of precipitate, and dissolving the precipitate into 22.5mL of precooled Tris-HCL (20mmoL/L, pH 8.0, 4 ℃) buffer solution to obtain a crude angiotensin converting enzyme solution.
4. And (3) ultrafiltration separation: separating the crude angiotensin converting enzyme solution by using a 100kDa ultrafiltration membrane, and collecting the non-permeate liquid, namely the high-purity angiotensin converting enzyme, wherein the protein content is 541.50mg, and the enzyme activity is 11.28U.
The combined technical scheme of acid precipitation, salting out and ultrafiltration separation realizes simple and low-cost purification of the angiotensin converting enzyme with a single electrophoresis band from pig lungs, the protein recovery rate of the embodiment is up to 9.50%, and the enzyme activity recovery rate is up to 24.20%.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.
Claims (8)
1. A method for separating and purifying angiotensin converting enzyme from pig lungs, which comprises the following steps:
Pre-treating, namely cleaning the pig lung, removing lung organs and outer membrane tissues, cooling at low temperature, placing the pig lung in a buffer solution for tissue homogenate, adding a penetration enhancer, stirring at low temperature, filtering to obtain a filtrate, centrifuging the filtrate to obtain a supernatant, and obtaining a first-level supernatant S 1;
Acid precipitation, namely adjusting the pH of the primary supernatant S 1 to acidity, standing in a water bath, centrifuging, filtering with silk cloth, and taking the supernatant to obtain a secondary supernatant S 2;
Salting out, namely slowly adding solid salt into the secondary supernatant S 2, stirring, standing, centrifuging, filtering by using silk cloth to obtain a tertiary supernatant S 3, slowly adding salt into the tertiary supernatant S 3, stirring, standing, centrifuging, collecting precipitates, dissolving the precipitates in a buffer solution to obtain a crude angiotensin converting enzyme solution;
And (3) ultrafiltration separation: and separating the crude angiotensin converting enzyme solution by using an ultrafiltration membrane, and collecting the non-permeate liquid, namely the high-purity angiotensin converting enzyme.
2. The method for separating and purifying angiotensin converting enzyme from pig lung according to claim 1, wherein the temperature of the low-temperature cooling is-25 ℃ to 0 ℃ in the pretreatment;
Optionally, the volume dosage of the buffer solution is 2-6 times of the mass of the pig lung.
3. The method for separating and purifying angiotensin converting enzyme from pig lungs according to claim 1, wherein in the pretreatment, the penetration enhancer is any one of polyethylene glycol octyl phenyl ether, ethyl phenyl polyethylene glycol and n-octyl-beta-D-glucoside, and the volume dosage of the penetration enhancer is 0.5% -1.0% of the mass of the pig lungs.
4. The method for separating and purifying angiotensin converting enzyme from pig lung according to claim 1, wherein the stirring temperature at low temperature is 0-10 ℃ and the stirring time at low temperature is 0.5-2 h in the pretreatment.
5. The method for separating and purifying angiotensin converting enzyme from pig lung according to claim 1, wherein in the acid precipitation, the pH adjustment to acidity is performed to adjust the pH to 3.0-4.0;
optionally, the temperature of the water bath is 30-50 ℃, and the standing time is 3-5 h;
Optionally, the silk cloth is 2-6 layers; preferably, the silk cloth is 4 layers.
6. The method according to claim 1, wherein in the salting-out step, the solid salt is solid (NH 4) 2 SO 4;
Optionally, the addition amount of the solid salt 1 is 1.0-2.0 mol/L;
Optionally, the stirring time is 0.5-2 h;
Optionally, standing for 2-4 h;
Optionally, the silk cloth is 2-6 layers; preferably, the silk cloth is 4 layers;
Optionally, the salt 2 is added in an amount to enable the final concentration to reach 2.0-3.0 mol/L;
Optionally, the using amount of the buffer solution is 2-6 times of the mass of the pig lung.
7. the method according to claim 1, wherein in the pretreatment and salting-out step, the buffer is Tris-HCL buffer or phosphate buffer.
8. The method of claim 1, wherein the ultrafiltration membrane has a molecular weight of 100kDa during the ultrafiltration separation.
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