CN117003827A - Separation and purification method of sialic acid glycopeptide - Google Patents
Separation and purification method of sialic acid glycopeptide Download PDFInfo
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- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 title claims abstract description 38
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
- C07K9/001—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence having less than 12 amino acids and not being part of a ring structure
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The application discloses a method for separating and purifying sialic acid glycopeptides, and relates to the technical field of peptides. The application sequentially adopts Sha Fuya bacillus and Quartz ® The method does not use toxic substances phenol in the whole process, but adopts the mixed degreasing solution and ethyl acetate, has high degreasing efficiency, only needs 1 hour, the product yield can reach 75%, the SGP purity can reach more than 98%, the whole product preparation can be completed only by 1 day, and the operation is convenient and fastThe method is efficient, is not only suitable for laboratory preparation, but also is easy to be amplified to industrial production.
Description
Technical Field
The application relates to the technical field of peptides, in particular to a separation and purification method of sialic acid glycopeptides.
Background
Sialoglycopeptide (SGP) is a hexapeptide (kva kt) with an intact sialylated N-sugar chain, whose sugar chain is a complex sugar chain composed of eleven monosaccharides, and has a relative molecular mass of 2866. The structure of the polypeptide is very similar to that of a human N-sugar chain, and the polypeptide is a glycopeptide with high humanization degree. Such sialoglycopeptides play an important role in many biological processes. Researches show that the glycopeptide can effectively inhibit rotavirus @Rotavirus) Adhesion to human epithelial cells and salmonella enteritidisSalmonella enteritidis) Is infected with (a) a polypeptide. More importantly, the sugar chain carried by sialic acid glycopeptides has been used many times for synthesizing various glycopeptides and glycoproteins having important functions as glycosyl donors for endoglycosidases. Therefore, sialoglycopeptides have important roles in the research of glycoprotein sugar chain structure and function and the development of biological medicine. Along with the wide application of sialic acid glycopeptides in the fields of glycoprotein synthesis, modification and the like, the preparation and the production of the glycopeptides are attracting attention in the field of glycobiology. Although a variety of oligosaccharides can now be synthesized by chemical methods, the current technology is still unable to synthesize such intact sialylated N-glycosides. Thus, only such sialoglycopeptides can be isolated and purified from natural products.
The separation and purification methods of Sialoglycopeptide (SGP) mainly include the following methods. AkiraSeko reported for the first time a method for isolation and purification of SGP from unfertilized egg yolk. The method comprises the steps of removing lipid in egg yolk by phenol, separating by a molecular sieve Sephadex G-50 twice, desalting by Sephadex G-25, purifying by an ion exchange column, desalting by Sephadex G-25 again, and freeze-drying to obtain a sample. Chinese patent CN102382178B discloses a separation method of sialic acid glycopeptide, which comprises the steps of treating egg yolk with phenol, performing rotary evaporation to obtain a crude product of sialic acid glycopeptide, separating a sample obtained in the previous step by using molecular sieve sephadex G-50, adsorbing a concentrated sample by using activated carbon, eluting by using acetonitrile solution to obtain an eluent containing the sialic acid glycopeptide, and finally performing freeze drying to obtain a pure product of the sialic acid glycopeptide. Chinese patent CN109824762A discloses a method for preparing Sialoglycopeptide (SGP) by large-scale separation and purification, which comprises the steps of treating egg yolk with phenol, freeze-drying to obtain crude product of sialoglycopeptide, loading medical absorbent cotton into cotton hydrophilic chromatographic column for separation and purification, and freeze-drying to obtain pure product of sialoglycopeptide.
The separation and purification methods of the sialic acid glycopeptides (SGP) adopt toxic compound phenol to carry out degreasing treatment on egg yolk, so that the environmental damage is caused, and the safety of a final product is influenced; when other solvents are used for treatment, the problems of more operation steps, high cost, long period and low sample yield are faced, and especially the degreasing time in hours is very short, so that the product acquisition efficiency is seriously affected, and the industrial production is not facilitated.
Disclosure of Invention
The application aims to provide a method for separating and purifying sialic acid glycopeptides. The application sequentially adopts Sha Fuya bacillus and Quartz ® The mixed degreasing solution composed of Boost lipase and ethyl acetate are used for degreasing raw materials, ethanol is further used for extraction, then a polymer pre-packed column PolyRP30-300 is used for separation and purification, only water is used for elution in the process, and the pure product of sialic acid glycopeptide is finally obtained through freeze-drying, so that the method has the characteristics of safe and efficient degreasing process and mild elution condition.
In order to achieve the above purpose, the application adopts the following technical scheme:
in a first aspect of the present application, there is provided a method for separation and purification of sialoglycopeptide comprising the steps of;
(1) Degreasing:
concentration is set to 10 7~ 10 8 CFU/ml Sha Fuya cell bacillus liquid and Quartz ® Mixing Boost lipase according to a volume ratio of 1:1 to form a mixed degreasing solution, dispersing yolk powder in water, adding the mixed degreasing solution into the mixed degreasing solution, stirring for 0.5-1 h at 35-45 ℃, dissolving the filtered solid in ethyl acetate, stirring for 0.5-1 h at room temperature, filtering, and discardingRemoving supernatant, and reserving sediment for standby; the Sha Fuya bacillus strain is numbered as CICC 21077;
(2) Obtaining crude sialic acid glycopeptide:
re-suspending the precipitate obtained in the step (1) by using a cold ethanol solution with the volume of 1-5 times, stirring for 1-5 hours, centrifuging to remove protein precipitate, and collecting supernatant; performing reduced pressure treatment at 40-50 ℃; concentrating until the content of sialic acid glycopeptide is 100-200 mg/ml;
(3) Sialic acid glycopeptide purification:
weighing the polymer pre-packed column PolyRP30-300 by using purified water with the volume of 1-5 times, loading the sample, eluting the sample by using purified water after loading the sample, and collecting eluent;
(4) Freeze-drying the sample;
and (3) putting the eluent into a vacuum freeze dryer for freeze drying for 24 hours to obtain a freeze-dried pure product containing sialic acid glycopeptide.
In the step (1), the yolk powder and the mixed degreasing solution are mixed in a mass-volume ratio of 5-15 g to 100 ml.
In the step (1), the yolk powder mixture and water are mixed and dissolved in a mass-volume ratio of 1g to 5-20 ml.
Further, in step (1), the filtered solid was mixed with ethyl acetate in an amount of 1g: and mixing the materials in a mass-volume ratio of 1-5 mL.
In the step (2), the temperature of the cold ethanol solution is-10 ℃ and the concentration is 25-60 v/v%.
Further, in the step (2), the centrifugal speed is 5000-10000 rpm, and the centrifugal time is 10-30 min.
Further, in the step (3), the sample loading flow rate is 50-100 ml/min.
Further, in the step (3), the loading capacity is 10-50 mg/ml.
The application has the beneficial effects that:
1. the application utilizes the characteristic of strong activity and hydrophilicity of polymer chromatographic packing, increases the affinity of separated substances, has excellent separation performance, higher loading capacity and yield, the loading capacity is 30mg/ml, the yield can reach 75%, and the SGP purity can reach more than 98%. In addition, in the elution process, the method directly uses purified water, and the elution condition is mild.
2. At present, 2 methods for preparing SGP are mainly available, one is that fresh egg yolk is treated by phenol, and then is subjected to gel filtration and a series of desalination treatment by an anion-cation exchange column; the other method is that fresh egg yolk is treated by phenol, and then is desalted by gel filtration and an active carbon column is used for replacing an anion-cation exchange column; the method of the application does not use toxic substances phenol in the whole process, but adopts Sha Fuya bacillus and Quartz ® The mixed degreasing solution composed of the Boost lipase and ethyl acetate are used for degreasing raw materials, and ethanol is further used for extraction, so that the efficiency is high, only 1 hour is needed, and the pollution to products and the environmental pollution can be reduced.
3. The application has low operation condition requirement and high product acquisition efficiency, can finish the preparation of the product only by 1 day, is not only suitable for laboratory preparation, but also is easy to be amplified to industrial production.
Drawings
FIG. 1 is a diagram of the structure of sialoglycopeptides;
FIG. 2 is an elution profile of a protein purifier for detecting polymeric filler PolyRP 30-300;
FIG. 3 is a SGP standard profile;
FIG. 4 is a crude SGP spectrum without purification treatment of polymeric filler PolyRP 30-300;
FIG. 5 is a graph of a sample of sialoglycopeptide after purification.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
The test materials used in the examples of the present application, which are not specifically described, are all conventional in the art and are commercially available. The yolk powder used in the application is purchased from Anhui Rongda poultry industry stock company; SGP standards were purchased from belvedere technologies, inc, product number: a01622106. Sha Fuya Bacillus cellBacillus safensis) Purchased from China center for type culture Collection of microorganisms, and the strain number is CICC 21077; lipase Quartz ® Boost purchase from novelian; polymer pre-loaded columns PolyRP30-300 (columns used were of the size: diameter =50×250 mm) were purchased from Sedan technologies Inc. of Suzhou. The column adopted by the application is polymer pre-packed column PolyRP30-300, wherein the PolyRP polymer filler adopts polystyrene/divinylbenzene (PS/DVB) with high crosslinking degree as a matrix, has strong rigidity, contains phenyl functional groups in a fixed phase, directly serves as reverse phase separation filler by utilizing the strong hydrophobic property of the matrix skeleton, and has the advantages of uniform particle size distribution and controllable particle size.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present application, the technical scheme of the present application will be described in detail with reference to specific embodiments.
Example 1
To examine the effect of the mixing ratio of egg yolk powder and ethyl acetate on SGP extraction purity and yield, the egg yolk powder solid after enzyme treatment and filtration was mixed with ethyl acetate at 1g: mixing (1-5 mL) in a mass-volume ratio, wherein the mass-volume ratio comprises the following specific steps:
(1) Degreasing
The concentration of the bacterial liquid is 10 7 CFU/ml Sha Fuya Bacillus and Quartz ® Mixing the Boost lipase according to a volume ratio of 1:1 to form a mixed degreasing solution, mixing the yolk powder with the mixed degreasing solution according to a mass volume ratio of 15g to 100ml, mixing and dissolving the yolk powder mixture with water according to a mass volume ratio of 1g to 20ml, stirring for 0.5h at 40 ℃, and mixing the filtered solid with ethyl acetate according to a mass volume ratio of 1g: mixing (1-5 mL) in a mass-volume ratio, stirring for 30min at room temperature, filtering, discarding the supernatant, and reserving a precipitate for standby;
(2) Obtaining crude sialic acid glycopeptide
The precipitate obtained in step (1) was resuspended in 3 volumes of cold ethanol solution (temperature-10 ℃ C., concentration 40 v/v%), stirred for 1.5 hours, centrifuged at 8000rpm for 20min to remove protein precipitate, and the supernatant was collected. Decompressing at 40 ℃; concentrating to sialic acid glycopeptide content of 150mg/ml.
(3) Sialic acid glycopeptide purification
The polymer pre-packed column PolyRP30-300 is balanced by 5 times volume of purified water, the pre-packed column is connected with a protein purifier, the flow rate is set to be 75ml/min, the sample is loaded with 30mg/ml loading, the purified water is used for eluting after the sample is loaded, and the eluent is collected.
(4) Lyophilization of samples
And (3) putting the eluent into a vacuum freeze dryer for freeze drying for 24 hours to obtain a freeze-dried pure product containing sialic acid glycopeptide.
The results of example 1 are shown in Table 1, wherein the enzyme-treated yolk powder was mixed with ethyl acetate in an amount of 1g:3mL, the purification purity of SGP reaches 97.8%, and after the volume ratio is continuously increased, the extraction purity and the yield of SGP are not obviously changed, which indicates that the yolk powder and ethyl acetate are mixed in a ratio of 1g:3mL, and can completely remove lipid in the yolk powder, and has highest yield. Therefore 3 times ethyl acetate was chosen for resuspension.
Table 1: influence of different proportions of filtered yolk powder and ethyl acetate on SGP separation and purification
Example 2
In order to examine the influence of different ethanol concentrations on the extraction purity and yield of the purified SGP, the concentration of a cold ethanol solution during resuspension is set to be 25-60 v/v%, and the specific steps are as follows:
(1) Degreasing
The concentration of the bacterial liquid is 10 7 CFU/ml Sha Fuya Bacillus and Quartz ® Mixing the Boost lipase according to a volume ratio of 1:1 to form a mixed degreasing solution, mixing the yolk powder with the mixed degreasing solution according to a mass volume ratio of 15g to 100ml, mixing and dissolving the yolk powder mixture with water according to a mass volume ratio of 1g to 20ml, stirring for 0.5h at 40 ℃, and mixing the filtered solid with ethyl acetate according to a mass volume ratio of 1g: mixing 3mL of the materials in a mass-volume ratio, stirring for 30min at room temperature, filtering, discarding the supernatant, and reserving the precipitate for standby;
(2) Obtaining crude sialic acid glycopeptide
And (3) re-suspending the precipitate obtained in the step (1) by using a 3-time volume cold ethanol solution (the temperature is-10 ℃ and the concentration is 25-60 v/v%), stirring for 1.5 hours, centrifuging at 8000rpm for 20min to remove protein precipitate, and collecting supernatant. Decompressing at 40 ℃; concentrating to sialic acid glycopeptide content of 150mg/ml.
(3) Sialic acid glycopeptide purification
The polymer pre-packed column PolyRP30-300 is balanced by 5 times volume of purified water, the pre-packed column is connected with a protein purifier, the flow rate is set to be 75ml/min, the sample is loaded with 30mg/ml loading, the purified water is used for eluting after the sample is loaded, and the eluent is collected.
(4) Lyophilization of samples
And (3) putting the eluent into a vacuum freeze dryer for freeze drying for 24 hours to obtain a freeze-dried pure product containing sialic acid glycopeptide.
The results of example 2 are shown in Table 2, where different ethanol concentrations had no effect on the purity of SGP after purification, and the yield was mainly affected, and the SGP yield was highest when 40 v/v% ethanol was used to extract SGP from egg yolk powder. Therefore, 40 v/v% ethanol was selected to extract SGP from the lipid-depleted yolk powder.
Table 2: effect of different concentrations of ethanol used in SGP extraction on separation and purification
Example 3
In order to examine the influence of different loading amounts on the purity and yield of the purified SGP, the loading amount is set to be 10-50 mg/ml, and the specific steps are as follows:
(1) Degreasing
The concentration of the bacterial liquid is 10 7 CFU/ml Sha Fuya Bacillus and Quartz ® Mixing the Boost lipase according to a volume ratio of 1:1 to form a mixed degreasing solution, mixing the yolk powder with the mixed degreasing solution according to a mass volume ratio of 15g to 100ml, mixing and dissolving the yolk powder mixture with water according to a mass volume ratio of 1g to 20ml, stirring for 0.5h at 40 ℃, and mixing the filtered solid with ethyl acetate according to a mass volume ratio of 1g:3mL of the mixture is mixed in a mass-volume ratio, stirred for 30min at room temperature and passedFiltering, discarding supernatant, and reserving precipitate for standby;
(2) Obtaining crude sialic acid glycopeptide
The precipitate obtained in step (1) was resuspended in 3 volumes of cold ethanol solution (temperature-10 ℃ C., concentration 40 v/v%), stirred for 1.5 hours, centrifuged at 8000rpm for 20min to remove protein precipitate, and the supernatant was collected. Decompressing at 40 ℃; concentrating to sialic acid glycopeptide content of 150mg/ml.
(3) Sialic acid glycopeptide purification
The polymer pre-packed column PolyRP30-300 is balanced by using a water with the volume of 5 times, the pre-packed column is connected with a protein purifier, the flow rate is set to be 75ml/min, the sample is loaded at the loading capacity of 10-50 mg/ml, the purified water is used for eluting after the sample is loaded, and the eluent is collected.
(4) Lyophilization of samples
And (3) putting the eluent into a vacuum freeze dryer for freeze drying for 24 hours to obtain a freeze-dried pure product containing sialic acid glycopeptide.
The results of comparative example 3 are shown in Table 3, the purity and yield of SGP after purification are greatly affected by different loading levels, and the purity and yield are higher when the loading level is lower, and 30mg/ml of loading level is selected to improve the production efficiency.
Table 3: effect of Polymer PolyRP30-300 Supports on SGP separation and purification
The experiment of example 4 was performed by using the best conditions obtained from the experimental results of examples 1-3.
Example 4
(1) Degreasing
The concentration of the bacterial liquid is 10 7 CFU/ml Sha Fuya Bacillus and Quartz ® Mixing the Boost lipase according to a volume ratio of 1:1 to form a mixed degreasing solution, mixing the yolk powder with the mixed degreasing solution according to a mass volume ratio of 15g to 100ml, mixing and dissolving the yolk powder mixture with water according to a mass volume ratio of 1g to 20ml, stirring for 0.5h at 40 ℃, and mixing the filtered solid with ethyl acetate according to a mass volume ratio of 1g:3mL of the materials are mixed according to the mass-volume ratio and stirred for 30min at room temperature,filtering, discarding supernatant, and reserving precipitate for standby;
(2) Obtaining crude sialic acid glycopeptide
The precipitate obtained in step (1) was resuspended in 3 volumes of cold ethanol solution (temperature-10 ℃ C., concentration 40 v/v%), stirred for 1.5 hours, centrifuged at 8000rpm for 20min to remove protein precipitate, and the supernatant was collected. Decompressing at 40 ℃; concentrating to sialic acid glycopeptide content of 150mg/ml.
(3) Sialic acid glycopeptide purification
The polymer pre-packed column PolyRP30-300 is balanced by 5 times volume of purified water, the pre-packed column is connected with a protein purifier, the flow rate is set to be 75ml/min, the sample is loaded with 30mg/ml loading, the purified water is used for eluting after the sample is loaded, and the eluent is collected.
(4) Lyophilization of samples
And (3) putting the eluent into a vacuum freeze dryer for freeze drying for 24 hours to obtain a freeze-dried pure product containing sialic acid glycopeptide.
Comparative example 1: degreasing treatment with Sha Fuya Bacillus only
The difference from example 4 is that in step (1), only a bacterial liquid concentration of 10 was used 7 CFU/ml Sha Fuya Bacillus as degreasing solution, yolk powder was mixed with degreasing solution at 15g:100ml mass-volume ratio without using ethyl acetate. Degreasing treatment time is 1h.
Comparative example 2: using Quartz only ® Degreasing treatment by Boost lipase
The difference from example 4 is that in step (1), only Quartz was used ® Boost lipase as degreasing solution, yolk powder was mixed with the degreasing solution in a mass-to-volume ratio of 15g:100ml, without using ethyl acetate. Degreasing treatment time is 1h.
Comparative example 3: degreasing with ethyl acetate alone
The difference from example 4 is that in step (1), the yolk powder was not treated with the mixed degreasing solution, and the yolk powder was mixed with water at a mass-to-volume ratio of 15g:100 ml. Degreasing treatment time is 1h.
Test examples
The sialoglycopeptide was isolated and purified by the methods of examples 1 to 4 and comparative examples 1 to 3 to obtain a lyophilized pure product of the sialoglycopeptide. Degreasing the yolk powder by using a method of comparative examples 1-3, detecting by a first Soxhlet extraction method of GB 5009.6-2016, and calculating a degreasing effect according to the content of fat in a sample; the obtained product was identified by using an instrument, and a High Performance Liquid Chromatograph (HPLC) was used, which was manufactured by Shimadzu corporation, and a reverse C-18 column was Shimadzu, shim-pack PREP-ODS (H) (4.6X1250 mm); chromatographic conditions: column oven temperature: linear gradient (using a two-phase procedure, mobile phase a was 0.1% aqueous trifluoroacetic acid, mobile phase B was 0.09% acetonitrile containing trifluoroacetic acid, 4.5% mobile phase B at 0min, 67.5% mobile phase B at 20 min) was eluted for 20min, uv detector 214nm detection, LC-Solution analysis data.
Experimental results
The structure of the sialoglycopeptide obtained using example 4 is shown in FIG. 1. In example 4, the elution pattern of the polymer filler PolyRP30-300 is shown in figure 2 (at 1000ml, the curves represented by three peaks from top to bottom are sequentially 280nm ultraviolet detection, 200nm ultraviolet detection and conductivity), the polymer filler PolyRP30-300 is used for detecting that SGP crude products contain more impurities (figure 4) before being treated by the polymer filler PolyRP30-300, and the purified sialoglycopeptide sample is purer (figure 5) and meets the experimental design requirement.
After investigating the influence of ethyl acetate addition in different proportions on SGP separation and purification during yolk powder resuspension, the influence of different concentrations of ethanol used during SGP extraction on SGP separation and purification and the influence of polymer PolyRP30-300 loading on SGP separation and purification, the inventor further investigated the influence of a degreasing treatment method on degreasing effect. The method of comparative examples 1-3 did not allow complete degreasing of the yolk powder after 1h of degreasing treatment; the degreasing amounts of comparative examples 1 to 3 were 7.4%, 49.1% and 32.6%, respectively, as measured (whereas the method of example 4 was used, the degreasing treatment was performed by mixing the degreasing solution for 30 minutes, and the degreasing treatment was performed by using ethyl acetate for 30 minutes, and after a total of 1 hour, complete degreasing was achieved). After further lengthening the degreasing time, the method of comparative example 1 was used, i.e., onlySha Fuya bacillus is treated, and 14.2 hours are needed for complete degreasing; using the method of comparative example 2, i.e., using Quartz ® Boost lipase is used for treatment, and the total degreasing time is 2.3 hours; using the procedure of comparative example 3, i.e., treatment with ethyl acetate, 3.2 hours was required for complete degreasing; in the method of the embodiment 3, degreasing can be completed only by 1h, and the actual effect of the methods of the comparative examples 1-3 is not as high as that of the method of sequentially using the mixed degreasing solution and the ethyl acetate.
The application adopts the steps of degreasing raw materials by using mixed lipase and ethyl acetate, and compared with the method which singly uses Sha Fuya bacillus and Quartz ® The Boost lipase and ethyl acetate have the advantages of high elution efficiency, short treatment time and less toxic substance contact, then the polymer pre-packed column PolyRP30-300 is adopted for separation and purification, only water is used for elution in the process, and the pure product of the sialoglycopeptide is finally obtained through freeze-drying, the yield can reach 75%, and the SGP purity after purification is close to 99%.
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (8)
1. The separation and purification method of the sialic acid glycopeptide is characterized by comprising the following steps of;
(1) Degreasing:
concentration is set to 10 7~ 10 8 CFU/ml Sha Fuya cell bacillus liquid and Quartz ® Mixing Boost lipase according to a volume ratio of 1:1 to form a mixed degreasing solution, dispersing yolk powder in water, adding the mixed degreasing solution into the mixed degreasing solution, stirring for 0.5-1 h at 35-45 ℃, dissolving the filtered solid in ethyl acetate, stirring for 0.5-1 h at room temperature, filtering, discarding the supernatant, and reserving the precipitate for later use; the Sha Fuya bacillus strain is numbered as CICC 21077;
(2) Obtaining crude sialic acid glycopeptide:
re-suspending the precipitate obtained in the step (1) by using a cold ethanol solution with the volume of 1-5 times, stirring for 1-5 hours, centrifuging to remove protein precipitate, and collecting supernatant; performing reduced pressure treatment at 40-50 ℃; concentrating until the content of sialic acid glycopeptide is 100-200 mg/ml;
(3) Sialic acid glycopeptide purification:
weighing the polymer pre-packed column PolyRP30-300 by using purified water with the volume of 1-5 times, loading the sample, eluting the sample by using purified water after loading the sample, and collecting eluent;
(4) Freeze-drying the sample;
and (3) putting the eluent into a vacuum freeze dryer for freeze drying for 24 hours to obtain a freeze-dried pure product containing sialic acid glycopeptide.
2. The method for separating and purifying sialoglycopeptide according to claim 1, wherein in step (1), yolk powder and a mixed degreasing solution are mixed at a mass-volume ratio of 5-15 g/100 ml.
3. The method for separating and purifying a sialoglycopeptide according to claim 1, wherein in the step (1), the yolk powder mixture and water are mixed and dissolved in a mass-volume ratio of 1g:5 to 20 ml.
4. The method for separating and purifying a sialoglycopeptide according to claim 1, wherein in step (1), the filtered solid and ethyl acetate are mixed in an amount of 1g: and mixing the materials in a mass-volume ratio of 1-5 mL.
5. The method for separating and purifying sialoglycopeptide according to claim 1, wherein in the step (2), the temperature of the cold ethanol solution is-10 ℃ and the concentration is 25-60 v/v%.
6. The method for separating and purifying a sialoglycopeptide according to claim 1, wherein in the step (2), the centrifugal speed is 5000 to 10000rpm, and the centrifugal time is 10 to 30 minutes.
7. The method for separating and purifying sialoglycopeptide according to claim 1, wherein in the step (3), the loading flow rate is 50-100 ml/min.
8. The method for separating and purifying sialoglycopeptide according to claim 1, wherein in the step (3), the loading amount is 10-50 mg/ml.
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| KR102454000B1 (en) * | 2021-11-17 | 2022-10-14 | 대한민국 | Bacillus species having antimicrobial activity and uses thereof |
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