CN112080485A - Method for extracting ribonuclease from bovine pancreas - Google Patents
Method for extracting ribonuclease from bovine pancreas Download PDFInfo
- Publication number
- CN112080485A CN112080485A CN202010996781.XA CN202010996781A CN112080485A CN 112080485 A CN112080485 A CN 112080485A CN 202010996781 A CN202010996781 A CN 202010996781A CN 112080485 A CN112080485 A CN 112080485A
- Authority
- CN
- China
- Prior art keywords
- ribonuclease
- solution
- eluent
- bovine pancreas
- extracting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Images
Classifications
-
- 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)
- C12N9/22—Ribonucleases RNAses, DNAses
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The application relates to the technical field of bioengineering, and particularly discloses a method for extracting ribonuclease from bovine pancreas. The method comprises the following steps: sequentially carrying out acid extraction treatment, ammonium sulfate fractional salting-out, boiling for impurity removal treatment, ion exchange column chromatography, concentration and drying on the bovine pancreas to obtain a finished product of ribonuclease; wherein the boiling condition is boiling at 98-102 deg.C for 2-5 min. Therefore, the present application can improve the purity of the ribonuclease obtained.
Description
Technical Field
The application relates to the technical field of enzymology and separation and purification methods thereof, in particular to a method for extracting ribonuclease from bovine pancreas.
Background
The pancreas is one of the most practical biological materials for animal biochemical pharmacy, and the comprehensive utilization of pancreas resources is more and more paid attention by people. The pancreas contains a plurality of active enzymes including dnase, rnase, trypsin, etc., and different enzymes have different functions, but since many enzymes have similar molecular structures and physicochemical properties, when one enzyme is extracted alone, other enzymes need to be removed, and thus, extraction for different enzymes requires different processes for extraction.
Ribonuclease (also known as ribonuclease A, ribonuclease I, RNA enzyme) is a type of endoribonuclease that catalyzes the breakdown of ribonucleic acid (RNA). The first step in RNA degradation is the hydrolysis of the phosphodiester bonds between RNAs, and ribonucleases can specifically attack the 3' end of pyrimidine residues on RNAs, cleaving the phosphodiester bonds formed by adjacent RNAs, and thereby degrading the RNAs into small fragments. Ribonuclease is a protein family specific to vertebrates, can be used for biochemical research and determination of nucleic acid structures, can also be used in anti-inflammatory enzyme preparations, and pharmacosome can be used for treating trauma and arthralgia, inhibiting influenza and herpes virus, and is suitable for recordable pancreatitis and epidemic encephalitis.
The bovine pancreatic ribonuclease is the first ribonuclease applied to clinical treatment of animal tumors and leukemia, and achieves certain effect, most of ribonucleases are extracted from bovine pancreatic glands at present, although the sources of raw materials are rich, the technical requirement on purification is high, and the ribonuclease obtained by the traditional extraction method is low in purity and contains DNA, so that the using effect is influenced.
Therefore, the method for extracting the ribonuclease from the bovine pancreas is developed, and the purity of the ribonuclease obtained by the extraction method is high.
Disclosure of Invention
In order to overcome the defects in the prior art, the application provides a method for extracting ribonuclease from bovine pancreas, which can improve the purity of the obtained ribonuclease.
The application provides a method for extracting ribonuclease from bovine pancreas, which comprises the following steps:
sequentially carrying out acid extraction treatment, ammonium sulfate fractional salting-out, boiling for impurity removal treatment, ion exchange column chromatography, concentration and drying on the bovine pancreas to obtain a finished product of ribonuclease;
wherein, the boiling impurity removal treatment comprises the following steps: boiling at 98-102 deg.C for 2-5 min.
The bovine pancreatic ribonuclease contains 124 amino acids, about 13.7KDa, and 4 disulfide bonds, respectively at Cys26-Cys84, Cys40-Cys95, Cys58-Cys110, and Cys65-Cys 72; the number of disulfide bonds of the protein containing 100-200 amino acids is only 0.5, because the bovine pancreatic ribonuclease has more disulfide bonds, so that the bovine pancreatic ribonuclease has higher heat stability.
According to the technical scheme, the salting-out materials obtained by fractional salting-out of ammonium sulfate are boiled for 2-5min at 98-102 ℃, so that proteins which are not high in temperature resistance in the salting-out materials can be denatured, ribonuclease can be kept active, most of proteins are denatured and inactivated, and then the denatured proteins are filtered out, so that the purity of the extracted ribonuclease can be increased.
Preferably, the boiling impurity removal treatment comprises:
mixing the salting-out material obtained by fractional salting-out of ammonium sulfate with water, boiling, cooling and filtering to obtain impurity-removed solution.
By adopting the technical scheme, the salting-out material is mixed with water to dissolve the ribonuclease in the water, then insoluble impurities are filtered, the protein which does not resist high temperature in the salting-out material is denatured by boiling, and the denatured protein is filtered, so that the impurity-removing liquid containing the ribonuclease with higher purity is obtained.
Preferably, the boiling impurity removal treatment comprises: and concentrating the impurity-removed solution until the protein content is 10-40 mg/mL.
By adopting the technical scheme, the impurity-removed solution is concentrated, and the content of protein in the impurity-removed solution is improved, so that the subsequent extraction of ribonuclease is facilitated.
Preferably, the acid extraction treatment comprises the following steps:
mincing fresh bovine pancreas gland, adding sodium chloride solution and filter aid for impregnation, wherein the weight of the sodium chloride solution is 4-6 times of the weight of the bovine pancreas gland, adjusting the pH value to 2.3-3.3, impregnating for 16-20h at the temperature of 16-20 ℃, and filtering to obtain acid extract.
By adopting the technical scheme, the filter resistance of the compressed filter cake is reduced by adding the filter aid, the structure of the filter cake is changed, and the rigidity and porosity of the filter cake are improved, so that the ribonuclease can better enter the acid extraction liquid through the filter cake, and the content of the ribonuclease in the acid extraction liquid is further improved; through the matching of the specific water adding amount and the pH value in the scheme, the ribonuclease can be better dissolved in the solution, so that the content of the ribonuclease in the acid extraction solution is improved; when the added sodium chloride solution is 5 times of the weight of the bovine pancreas, the effect of extracting the finished products of the ribonuclease in the bovine pancreas is the best, which is mainly shown in that the total weight of the obtained finished products of the ribonuclease is the largest, the purity of the obtained ribonuclease is the highest, and when the added sodium chloride solution is too high or too low in the acid extraction treatment, the effect of extracting the finished products of the ribonuclease is influenced.
Preferably, the concentration of the sodium chloride solution is 0.85-0.9%.
Preferably, the protein content of the supernatant is 8mg/mL or more, and the acid extract is filtered.
By adopting the technical scheme, the extraction effect of the ribonuclease can be improved by further limiting the concentration of the sodium chloride solution and the protein content of the supernatant.
Preferably, the ammonium sulfate fractional salting-out comprises:
salting out 70% and 95% saturation ammonium sulfate of the acid extract in sequence, filtering, and collecting the salting-out substances.
The ox pancreas contains a plurality of enzymes, and because the solubility and isoelectric point of different enzymes are different, the required ionic strength is different during precipitation, by adopting the technical scheme, the acid extraction solution is subjected to fractional salting-out, different enzyme preparations are salted out in batches, and then the ribonuclease in the acid extraction solution can be separated from other enzymes, so that the purity of the ribonuclease extracted in the later period is improved.
Preferably, the ion exchange column chromatography comprises the following steps:
(1) preparing the impurity removal solution and a phosphate buffer solution into a chromatographic solution for later use;
(2) a CM chromatographic column is balanced by phosphate buffer solution, then the chromatographic solution is loaded on the CM chromatographic column, and then is eluted by eluent a1 and eluent a2 in sequence, and eluent a1 and eluent a2 containing ribonuclease are collected to obtain a collecting solution;
wherein the eluent a1 is prepared by mixing the phosphate buffer solution and sodium chloride, the conductivity of the eluent a1 is 6.0-7.0ms/cm, the pH value is 7.0-7.1, and the concentration of the sodium chloride in the eluent a1 is 0.05 mol/L; mixing the phosphate buffer solution with sodium chloride to prepare the eluent a2, wherein the conductivity of the eluent a2 is 20.0-22.0ms/cm, the pH value is 7.0-7.1, and the concentration of the sodium chloride in the eluent a2 is 0.2 mol/L.
Preferably, eluting with eluent a1 until the effluent protein content is lower than 1mg/mL, eluting with eluent a2 until the effluent protein content is lower than 0.5mg/mL, and collecting the collected liquid.
By adopting the technical scheme, the ribonuclease with negative charges is combined by the anion exchange matrix, so that the ribonuclease stays on the CM chromatographic column, and then the ribonuclease is eluted by the eluent a1 and the eluent a2, so that the ribonuclease can be obtained; and eluting the chromatographic column twice through eluent a1 and eluent a2, thereby eluting the ribonuclease and improving the content of the ribonuclease.
Preferably, the conductivity of the phosphate buffer solution is 1.6-2.1ms/cm, and the pH value is 6-6.1; the protein content in the chromatographic solution is 8-14mg/mL, the conductivity is 3.0-4.0ms/cm, and the pH value is 6.00-6.10.
By adopting the technical scheme, the binding rate of the ribonuclease and the anion exchange matrix can be improved through the configuration of the chromatographic solution, so that the extraction purity of the ribonuclease at the later stage is improved.
Preferably, the collected liquid is concentrated by an ultrafiltration membrane until the protein content is more than 40mg/mL, and then freeze-dried to obtain the finished product of the ribonuclease.
In summary, the present application has the following beneficial effects:
1. according to the method, through boiling for impurity removal treatment, proteins which are not high in temperature resistance can be denatured, so that most of the proteins are denatured and inactivated, and the denatured proteins are filtered, so that the purity of the ribonuclease can be increased;
2. according to the method, the extraction purity of the ribonuclease in the bovine pancreatic gland is improved by specific selection of ammonium sulfate saturation in fractional salting-out; meanwhile, through the selection of acid extraction treatment conditions and ion exchange column chromatography treatment conditions, the method improves the extraction purity of the ribonuclease in the bovine pancreatic gland, and the DNA residue in the finished ribonuclease product is not detected.
Drawings
Fig. 1 is a flow chart of a method provided herein.
Detailed Description
The present application will be described in further detail with reference to the following drawings and examples. It should be understood that the composition and method described in the examples are only for illustrating the present invention and not for limiting the present invention, and that simple modifications of the preparation method of the present invention based on the concept of the present invention are within the scope of the claimed invention.
Examples
Referring to FIG. 1, the following is an example of a process for extracting ribonuclease from bovine pancreas, in which fresh bovine pancreas is commercially available, perlite is used as a filter aid, municipal water is used as water, and sodium chloride, perlite, ammonium sulfate, sodium monohydrogen phosphate and sodium dihydrogen phosphate are all commercially available.
Example 1: comprises the following steps:
s1, acid extraction treatment:
1kg of fresh bovine pancreas is crushed, sodium chloride solution and perlite are added, the weight of the sodium chloride solution is 4 times of the weight of the bovine pancreas, the concentration of the sodium chloride solution is 0.864 percent, the pH value is adjusted to be 2.3 through sulfuric acid solution, the mixture is soaked for 16 to 20 hours at the temperature of between 16 and 20 ℃, the protein content in the supernatant is more than 8mg/mL, and the acid extract is obtained through filtration.
S2, fractional salting-out by ammonium sulfate:
(1) adding ammonium sulfate into the acid extraction solution to 70% saturation, standing for 2-3 hr until crystals are completely separated out, and filtering to obtain filtrate; (2) adding ammonium sulfate into the filtrate to 95% saturation, standing for 2-3 hr until crystal is completely separated out, filtering, and collecting the filter residue as salting-out material.
S3, boiling for impurity removal:
mixing the salted-out material with water, boiling for 2min, cooling, filtering again to obtain impurity-removed solution, and concentrating the impurity-removed solution until the protein content is 10-40mg/mL for later use.
S4, ion exchange column chromatography:
(1) 0.169g of sodium monohydrogen phosphate and 2.257g of sodium dihydrogen phosphate are added with water to be constant volume to 1L, and are prepared into phosphate buffer solution with the phosphate concentration of 0.02mol/mL, the pH value is adjusted to 6-6.1, and the conductivity is 1.6-2.1 ms/cm.
Mixing the impurity-removed solution and phosphate solution to obtain chromatography solution with protein content of 10-14mg/mL, conductivity of the chromatography solution of 3.0-4.0ms/cm, and adjusting pH to 6.00-6.10 with sodium hydroxide.
(2) And (2) balancing the CM chromatographic column by using a phosphate buffer solution, wherein the volume of the phosphate buffer solution is 5 times of that of the CM chromatographic column, loading the chromatographic solution onto the CM chromatographic column, eluting by using an eluent a1 until the protein content is lower than 1mg/mL, then eluting by using an eluent a2 until the protein content is lower than 0.5mg/mL, and mixing the eluent a1 containing ribonuclease and the eluent a2 to obtain a collecting solution.
Wherein the diameter of the CM chromatographic column is 30CM, the medium height of the CM chromatographic column is 16CM, and the flow rate is 1L/mim; eluent a1 is a mixture of phosphate buffer solution and sodium chloride solution, the final concentration of phosphate in eluent a1 is 20mmol/L, the concentration of sodium chloride in eluent a1 is 0.05mol/L, the conductivity of eluent a1 is 6.0-7.0ms/cm, and the pH value is 7.0-7.1; eluent a2 is a mixture of phosphate buffer solution and sodium chloride, the final concentration of phosphate in eluent a2 is 20mmol/L, the concentration of sodium chloride in eluent a2 is 0.2mol/L, the conductivity of eluent a1 is 20.0-22.0ms/cm, and the pH value is 7.0-7.1.
S5, concentrating and drying:
concentrating the eluent a through a hollow fiber ultrafiltration membrane until the protein content is more than 40mg/mL, and then carrying out freeze drying to obtain a finished product of the ribonuclease.
Example 2: the difference from example 1 is that:
and S1, acid extraction, wherein the weight of the sodium chloride solution is 5 times that of the bovine pancreas gland, and the pH value is adjusted to 2.8 by a sulfuric acid solution.
S3, boiling for 3min to remove impurities.
Example 3: the difference from example 1 is that:
and S1, acid extraction, wherein the weight of the sodium chloride solution is 6 times that of the bovine pancreas gland, and the pH value is adjusted to 3.3 by a sulfuric acid solution.
S3, boiling for 5min to remove impurities.
Example 4: the difference from example 2 is that:
and S1, acid extraction treatment, wherein the weight of the sodium chloride solution is 4 times that of the bovine pancreas gland.
Example 5: the difference from example 2 is that:
and S1, acid extraction treatment, wherein the weight of the sodium chloride solution is 6 times that of the bovine pancreas gland.
Example 6: the difference from example 2 is that:
s1, acid extraction treatment, wherein the weight of the sodium chloride solution is 4.7 times of that of the bovine pancreas gland.
Example 7: the difference from example 2 is that:
and S1, acid extraction, wherein the weight of the sodium chloride solution is 5.3 times of that of the bovine pancreas gland.
Example 8: the difference from example 2 is that:
and S1, acid extraction treatment, namely adding an acid solution until the pH value is 2.3.
Example 9: the difference from example 2 is that:
and S1, acid extraction treatment, namely adding an acid solution until the pH value is 3.3.
Example 10: the difference from example 2 is that:
s2, fractional salting-out by ammonium sulfate:
(1) adding ammonium sulfate into the filtrate concentrate to 70% saturation, standing until crystallization is completely separated out, and filtering to obtain filtrate;
(3) adding ammonium sulfate into the filtrate to 90% saturation, standing until crystallization is completely separated out, filtering, and collecting the filter residue as salting-out substance.
Example 11: the difference from example 2 is that:
s2, fractional salting-out by ammonium sulfate:
(1) adding ammonium sulfate into the acid extraction solution to 80% saturation, standing until crystals are completely separated out, and filtering to obtain filtrate;
(2) adding ammonium sulfate to the filtrate to 90% saturation, standing until crystallization is completely separated out, filtering, and collecting the filter residue as salting-out substance.
Example 12: the difference from example 2 is that:
s2, fractional salting-out by ammonium sulfate:
(1) adding ammonium sulfate into the acid extraction solution to 80% saturation, standing until crystals are completely separated out, and filtering to obtain filtrate;
(2) adding ammonium sulfate into the filtrate to 95% saturation, standing until crystallization is completely separated out, filtering, and collecting the filter residue as salting-out substance.
Example 13: the difference from example 2 is that:
and S4, performing ion exchange column chromatography, namely eluting only through the eluent a1 in the step (2) until the protein content is lower than 0.5mg/mL, and collecting the eluent a1 of the ribonuclease to obtain a collected liquid.
Example 14: the difference from example 2 is that:
and S4, performing ion exchange column chromatography, namely eluting only through the eluent a2 in the step (2) until the protein content is lower than 0.5mg/mL, and collecting the eluent a2 of the ribonuclease to obtain a collected liquid.
Comparative example 1: a method for extracting ribonuclease from bovine pancreas is different from the method in example 2 in that: no boiling for removing impurities.
Comparative example 2: a method for extracting ribonuclease from bovine pancreas is different from the method in example 2 in that: s3, boiling for 1min to remove impurities.
Comparative example 3: a method for extracting ribonuclease from bovine pancreas is different from the method in example 2 in that: s3, boiling for 6min to remove impurities.
All of the finished ribonucleases obtained in examples 1 to 14 and comparative examples 1 to 3 were white crystalline powders.
Performance test
The detection method comprises the following steps:
the method for measuring the protein content in the finished product of the ribonuclease is an ultraviolet spectrophotometry:
(1) 0.1mL, 0.2mL, 0.3mL, 0.4mL, and 0.5mL of 5.00mg/mL standard protein solutions were pipetted into 5 10mL cuvettes, diluted to the mark with 0.9% sodium chloride solution, and shaken well. And respectively measuring the absorbance A of each standard solution at 280nm by using a 1cm quartz cuvette and a 0.9% sodium chloride solution as a reference, recording data and drawing a standard curve.
(2) Diluting 1mg of finished ribonuclease product to 10 times by using 0.9% sodium chloride to obtain a solution to be detected, uniformly mixing, using a 1cm quartz cuvette and using 0.9% sodium chloride solution as a reference, respectively measuring the absorbance A1 of each standard solution at 280nm, measuring the absorbance at 280nm, and performing parallel measurement on three parts to obtain a mean value. And (4) finding out the concentration of the solution to be detected on the standard curve, and calculating the protein content in the finished product of the ribonuclease.
The method for measuring the specific activity of the ribonuclease in the finished product comprises the following steps:
(1) reagent preparation
Reagent a (sodium acetate buffer at a concentration of 0.1mol/mL, pH 5.0): the sodium acetate solution of 8.2g/L is prepared by dissolving anhydrous sodium acetate in purified water, and the pH is adjusted to 5.0 by 2mol/mL acetic acid under the condition of 25 ℃.
Reagent B (0.1% RNA solution): the RNA was dissolved in a reagent A to a concentration of 1mg/mL, and the mixture was shaken sufficiently to dissolve the RNA, but stirring was not carried out, and the solution took 30min, and the substrate concentration was confirmed before detection after the completion of the dissolution.
Reagent C (RNase a stock solution): preparing RNase A stock solution with the enzyme activity of 50-75Kunitz unit/mL by using cold deionized water.
And (3) reagent D: before use, the reagent C is prepared into RNase A solution with the enzyme activity of 0.50-0.75Kunitz unit/mL by using cold deionized water.
And (3) reagent E: before use, the reagent C is prepared into RNase A solution with the enzyme activity of 0.20-0.30Kunitz unit/mL by using cold deionized water.
(2) Substrate concentration confirmation method
Blank control: taking 1.5mL of reagent A and 1.5mL of purified water to mix evenly
Substrate solution: 1.5mL of RNA solution and 1.5mL of purified water are mixed uniformly
The substrate solution is first zeroed at A300nm with a blank and then measured for absorbance between 0.705 and 0.755, if necessary, adjusted using reagent A or solid RNA.
(3) Measurement of
Total hydrolytic activity assay (Ef):
sucking RNA (reagent B) and RNase A (reagent D) by using a pipette 1.50mL to 1cm respectively, covering the cuvette, shaking uniformly, and testing at the wavelength of 300nm, wherein the internal temperature of the spectrophotometer is kept at 25 +/-0.1 ℃ during the test, the absorbance of the cuvette is read every 1min, and the hydrolysis reaction is finished about 120min or until delta A300nm is less than or equal to 0.002 and is kept stable within 5min (the blank control absorbance is subtracted from the final absorbance). Three controls were run in parallel, and the final results of the three runs should be 90% consistent.
Hydrolysis rate determination (Eo):
sample 1: and (3) sucking 1.50mL of reagent B and 1.30mL of purified water by using a pipette, uniformly mixing, preheating to 25 ℃, then adding 0.2mL of reagent E, and uniformly mixing to obtain a sample 1.
Sample 2: and sucking 1.50mL of reagent B and 1.35mL of purified water by using a pipette, uniformly mixing, preheating to 25 ℃, then adding 0.15mL of reagent E, and uniformly mixing to obtain a sample 2.
Sample 3: and (3) sucking 1.50mL of reagent B and 1.40mL of purified water by using a pipette, uniformly mixing, preheating to 25 ℃, then adding 0.10mL of reagent E, and uniformly mixing to obtain a sample 3.
After mixing the decrease in A300nm was detected and recorded for sample 1, sample 2 and sample 3, and the change in absorbance per minute over ten minutes was recorded.
And (3) calculating:
the method for detecting the DNA residue in the finished product of the ribonuclease is an electrophoretic gradient method. The test results are shown in Table 1.
TABLE 1 results of measurement of total weight, protein content, specific activity of enzymes in protein and DNA residue of finished products of ribonuclease in examples 1 to 14 and comparative examples 1 to 3
As shown in Table 1, the RNase products obtained in example 2 were the most, the protein content was the highest, the specific enzyme activity of the RNase was the highest, and no DNA remained, as compared with examples 1 to 3.
By comparing example 2 with comparative example 1, although the ribonuclease product obtained in comparative example 1 is more than the ribonuclease product obtained in example 2, the ribonuclease product in comparative example 1 contains DNA residues, and when the ribonuclease product contains impurities such as DNA, the use effect of the ribonuclease product is poor. By comparing example 2 with comparative example 2, the boiling time in comparative example 2 is short, which increases the total weight of the finished ribonuclease, but it cannot be guaranteed that proteins other than ribonuclease in the salted-out material are denatured, which results in the remaining of DNA. By comparing example 2 with comparative example 3, the boiling time in comparative example 3 is too long, which causes denaturation of part of ribonuclease, resulting in a decrease in the total weight of the extracted ribonuclease finished product, and a decrease in the enzymatic specific activity of ribonuclease in the ribonuclease finished product.
Compared with the examples 4-5, the finished products of ribonuclease obtained in example 2 have the most amount, the highest protein content and the highest specific activity of ribonuclease, so that when the added sodium chloride solution in example 2 is 5 times of the weight of the bovine pancreas, the best effect of extracting the finished products of ribonuclease from the bovine pancreas is achieved, which is mainly shown in that the total weight of the obtained finished products of ribonuclease is the most amount, the purity of the obtained ribonuclease is the highest, and when the added sodium chloride solution in the acid extraction treatment is too high or too low, the effect of extracting the finished products of ribonuclease is affected, but the specific reason for causing the phenomenon is unknown. Therefore, by setting examples 6 to 7 and further conducting experiments on the amount of the sodium chloride solution added, experiments showed that the finished RNase product obtained in example 2 was still superior to that obtained in examples 6 to 7. The dissolution of solute is a reversible process, and the water consumption and the material quantity are large when the concentration is too low. Too high to be dissolved conveniently
Compared with examples 8-9, the finished products of ribonuclease obtained in example 2 have the most amount, the highest protein content and the highest specific activity of ribonuclease, so that the effect of extracting the finished products of ribonuclease from bovine pancreas is the best when the pH value in the acid extraction treatment is 2.8 in example 2, and the effect of extracting the finished products of ribonuclease is affected when the pH value is too high or too low.
The total weight of the finished products of ribonuclease obtained in examples 10-12 was reduced and the specific enzyme activity of ribonuclease was reduced compared to example 2, thereby reducing the total weight of the finished products of ribonuclease obtained in examples 10-12 and the purity of ribonuclease. Therefore, in the extraction method, 70% and 95% saturation ammonium sulfate salting-out is sequentially carried out on the acid extraction solution, and the obtained finished product of the ribonuclease has the best effect.
In example 13, elution was carried out with only eluent a1, resulting in a significant reduction in the total weight of the finished ribonuclease obtained in example 13, as compared with example 2; in example 14, elution was carried out using only the eluent a2, which resulted in a decrease in the specific enzyme activity of ribonuclease in the finished ribonuclease obtained in example 14, and thus a decrease in the purity of ribonuclease in the finished ribonuclease.
The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.
Claims (10)
1. A method for extracting ribonuclease from bovine pancreas, comprising:
sequentially carrying out acid extraction treatment, ammonium sulfate fractional salting-out, boiling for impurity removal treatment, ion exchange column chromatography, concentration and drying on the bovine pancreas to obtain a finished product of ribonuclease;
wherein, the boiling impurity removal treatment comprises the following steps: boiling at 98-102 deg.C for 2-5 min.
2. The method for extracting ribonuclease from bovine pancreas according to claim 1, wherein the boiling decontamination treatment comprises:
mixing the salting-out material obtained by fractional salting-out of ammonium sulfate with water, boiling, cooling and filtering to obtain impurity-removed solution.
3. The method for extracting ribonuclease from bovine pancreas according to claim 2, wherein the boiling decontamination treatment further comprises:
and concentrating the impurity-removed solution until the protein content is 10-40 mg/mL.
4. The method for extracting ribonuclease from bovine pancreas according to claim 1, wherein the acid extraction treatment comprises:
mincing fresh bovine pancreas gland, adding sodium chloride solution and filter aid for impregnation, wherein the weight of the sodium chloride solution is 4-6 times of the weight of the bovine pancreas gland, adjusting the pH value to 2.3-3.3, impregnating for 16-20h at the temperature of 16-20 ℃, and filtering to obtain acid extract.
5. The method for extracting ribonuclease from bovine pancreas as claimed in claim 4, wherein the concentration of sodium chloride solution is 0.85-0.9%.
6. The method for extracting ribonuclease from bovine pancreas as claimed in claim 4, wherein the protein content in the supernatant is 8mg/mL or more, and the acid extract is filtered.
7. The method for extracting ribonuclease from bovine pancreas as claimed in claim 4, wherein the ammonium sulfate fractional salting-out comprises:
salting out 70% and 95% saturation ammonium sulfate of the acid extract in sequence, filtering, and collecting the salting-out substances.
8. The method for extracting ribonuclease from bovine pancreas as claimed in claim 2, wherein the ion exchange column chromatography comprises the following steps:
(1) preparing the impurity removal solution and a phosphate buffer solution into a chromatographic solution for later use;
(2) a CM chromatographic column is balanced by phosphate buffer solution, then the chromatographic solution is loaded on the CM chromatographic column, and then is eluted by eluent a1 and eluent a2 in sequence, and eluent a1 and eluent a2 containing ribonuclease are collected to obtain a collecting solution;
wherein the eluent a1 is prepared by mixing the phosphate buffer solution and sodium chloride, the conductivity of the eluent a1 is 6.0-7.0ms/cm, the pH value is 7.0-7.1, and the concentration of the sodium chloride in the eluent a1 is 0.05 mol/L; mixing the phosphate buffer solution with sodium chloride to prepare the eluent a2, wherein the conductivity of the eluent a2 is 20.0-22.0ms/cm, the pH value is 7.0-7.1, and the concentration of the sodium chloride in the eluent a2 is 0.2 mol/L.
9. The method for extracting ribonuclease from bovine pancreas as claimed in claim 8, wherein the phosphate buffer has a conductivity of 1.6-2.1ms/cm, a pH of 6-6.1; the protein content in the chromatographic solution is 8-14mg/mL, the conductivity is 3.0-4.0ms/cm, and the pH value is 6.00-6.10.
10. The method for extracting ribonuclease from bovine pancreas as claimed in claim 8, wherein the collected liquid is concentrated by ultrafiltration membrane until the protein content is more than 40mg/mL, and then freeze-dried to obtain finished ribonuclease.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010996781.XA CN112080485A (en) | 2020-09-21 | 2020-09-21 | Method for extracting ribonuclease from bovine pancreas |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010996781.XA CN112080485A (en) | 2020-09-21 | 2020-09-21 | Method for extracting ribonuclease from bovine pancreas |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112080485A true CN112080485A (en) | 2020-12-15 |
Family
ID=73738597
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010996781.XA Withdrawn CN112080485A (en) | 2020-09-21 | 2020-09-21 | Method for extracting ribonuclease from bovine pancreas |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112080485A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1173558A (en) * | 1968-08-28 | 1969-12-10 | Inst Khim Prirodnykh Soedineny | A Process for Preparing Pure Ribonuclease. |
| CN101538561A (en) * | 2009-04-17 | 2009-09-23 | 广州大学 | Method for extracting CEL I nuclease in celery |
| CN104212779A (en) * | 2014-08-29 | 2014-12-17 | 宁波美成生物科技有限公司 | Novel ribonuclease A and purification production process thereof |
| CN106520814A (en) * | 2016-12-28 | 2017-03-22 | 苏州旷世骏弛生物科技有限公司 | Thermo-stable nuclease and preparation method thereof |
| CN109370996A (en) * | 2018-12-18 | 2019-02-22 | 枣庄全鼎生物科技有限公司 | A kind of catalase sublimate method of purification |
-
2020
- 2020-09-21 CN CN202010996781.XA patent/CN112080485A/en not_active Withdrawn
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1173558A (en) * | 1968-08-28 | 1969-12-10 | Inst Khim Prirodnykh Soedineny | A Process for Preparing Pure Ribonuclease. |
| CN101538561A (en) * | 2009-04-17 | 2009-09-23 | 广州大学 | Method for extracting CEL I nuclease in celery |
| CN104212779A (en) * | 2014-08-29 | 2014-12-17 | 宁波美成生物科技有限公司 | Novel ribonuclease A and purification production process thereof |
| CN106520814A (en) * | 2016-12-28 | 2017-03-22 | 苏州旷世骏弛生物科技有限公司 | Thermo-stable nuclease and preparation method thereof |
| CN109370996A (en) * | 2018-12-18 | 2019-02-22 | 枣庄全鼎生物科技有限公司 | A kind of catalase sublimate method of purification |
Non-Patent Citations (4)
| Title |
|---|
| 刘德富等: "猪胰五种酶制品的联产", 《食品科学》 * |
| 欧阳平凯: "《生物分离原理及技术》", 28 February 1999, 化学工业出版社 * |
| 王沛: "《制药工艺学》", 31 August 2017, 华南理工大学出版社 * |
| 赵景联: "《科技树致富新技术—实用生化产品制造50例》", 31 March 1994, 西安交通大学出版社 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP3585892B1 (en) | Methods for purification of messenger rna | |
| Held et al. | Reconstitution of Escherichia coli 30 S ribosomal subunits from purified molecular components | |
| US5596092A (en) | Extraction of genomic DNA from blood using cationic detergents | |
| Hedenskog et al. | Reduction of the nucleic acid content of single‐cell protein concentrates | |
| Monier et al. | [53] Isolation and characterization of 5 S RNA from Escherichia coli | |
| CN109593756B (en) | An extractive solution and its application in preserving tissue or cell and extracting RNA | |
| CN112080485A (en) | Method for extracting ribonuclease from bovine pancreas | |
| BLÜTHMANN et al. | Non‐histone Chromosomal Proteins: Their Isolation and Role in Determining Specificity of Transcription in vitro | |
| US5061627A (en) | Method for preparing enzymes from crustaceans | |
| CN116162618A (en) | Method for separating DNA and RNA from nucleic acid solution and reagent combination | |
| CN115433724B (en) | Method for extracting 5-nucleotidase from pig liver | |
| JP4662846B2 (en) | Alcohol lowering agent | |
| CN112457377B (en) | Periplaneta americana polypeptide and application thereof | |
| CN109260231B (en) | Preparation method of earthworm extract with cough stopping, phlegm eliminating, anti-inflammatory and antimicrobial functions | |
| JP2002512255A (en) | Method for recovering and purifying recombinant protein from cells | |
| CN1298943A (en) | Process for extracting Fe-contained SOD from spirulina | |
| CA2620881A1 (en) | Method for the selective enrichment of double-stranded dna from nucleic acid mixtures | |
| CN112812969A (en) | System purification method for recombinant expression polypeptide in genetic engineering | |
| CN114990183B (en) | Method for preparing cordycepin and antihypertensive peptide based on rice cordyceps militaris fruiting body | |
| CN115521926A (en) | Production process of ribonuclease | |
| Anandaraj et al. | A simple method for large-scale preparation of chick embryo tRNA | |
| US6982158B2 (en) | Extraction and purification of phosphodiesterase 1 | |
| CN1488639A (en) | Method for separating RNA from tissue sample | |
| CN115058415A (en) | Rapid, high-quality and universal genome DNA extraction kit and DNA extraction method | |
| CN115851704A (en) | Method and kit for efficiently extracting exosome microRNA |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20201215 |
|
| WW01 | Invention patent application withdrawn after publication |