CN113248571A - Method for separating and purifying bacitracin by utilizing thermal response polymer NPE-108 - Google Patents
Method for separating and purifying bacitracin by utilizing thermal response polymer NPE-108 Download PDFInfo
- Publication number
- CN113248571A CN113248571A CN202110743115.XA CN202110743115A CN113248571A CN 113248571 A CN113248571 A CN 113248571A CN 202110743115 A CN202110743115 A CN 202110743115A CN 113248571 A CN113248571 A CN 113248571A
- Authority
- CN
- China
- Prior art keywords
- bacitracin
- npe
- water
- phase
- phases
- 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.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/50—Cyclic peptides containing at least one abnormal peptide link
- C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
- C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
- C07K7/58—Bacitracins; Related peptides
Landscapes
- 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)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a method for separating and purifying bacitracin by utilizing a thermal response polymer NPE-108, which comprises the following steps: a forward extraction step: uniformly mixing NPE-108 and bacitracin fermentation liquor according to the volume ratio of 1:4, adding divalent manganese salt, adjusting the pH to 8.5, uniformly oscillating, standing in a constant-temperature water bath at 30 ℃ until two phases with clear interfaces are formed, and taking out an upper phase; and (3) reverse extraction: adding water with a volume half of that of NPE-108 into the upper phase obtained in the forward extraction step, uniformly mixing, adjusting the pH to 5.5, standing in a water bath kettle at 60 ℃ until two phases with clear interfaces are formed, and enriching bacitracin in the upper phase. The thermal response type polymer NPE-108 used in the invention is mixed with water to form a recoverable thermal response type two-water phase system, has the advantages of mild operation conditions, environmental friendliness and recoverable component phase components, and is beneficial to large-scale bacitracin separation and application.
Description
Technical Field
The invention relates to the technical field of two-water phase extraction, in particular to application of a recyclable two-water phase system in separation of bacitracin in fermentation liquor, and belongs to the technical field of downstream separation.
Background
The block polyether NPE-108 is a thermal response polymer, and EOPO with different cloud points can be synthesized by adjusting the adding ratio of EO and PO in the synthesis process. When the NPE-108 aqueous solution is heated, the temperature is above the cloud point of the NPE-108, the polymer NPE-108 is separated from water to form two phases with clear interfaces, and the NPE-108 can be recovered from the phase system by the method for recycling, namely the NPE-108/water recoverable two-water phase system.
The two-water phase system has the advantages of high water content, mild extraction conditions and capability of protecting the active groups of the biomolecules, so that the two-water phase system is easier to separate and apply the biomolecules industrially.
The two-aqueous phase extraction technology has been widely studied in the separation and purification of bioactive substances such as enzymes, proteins, antibodies, antibiotics, etc. due to its unique advantages (mild operating conditions, protection of active groups of biomolecules, etc.). However, this separation and purification means has been used only in the laboratory stage at present, and has not been industrially used. The industrial application of the two-water phase system is limited mainly because the polymer synthesis cost in the two-water phase system constructed by two response type polymers is high and the phase forming time is long.
Therefore, the development of a recyclable two-water phase system with short phase forming time, low preparation cost of the phase forming components and mature synthesis process is a problem to be solved urgently for the industrialization of biomolecule separation.
Disclosure of Invention
The invention mainly aims at the problems, provides a process for separating bacitracin by a recyclable two-water phase system, only uses a thermal response polymer NPE-108, and the polymer aqueous solution can be separated from water by temperature induction, thereby realizing the recycling of the polymer, greatly reducing the separation cost of bacitracin in fermentation liquor, having simple operation and being easy for industrialized application.
In order to achieve the above object, the present invention provides a process for extracting bacitracin in a NPE-108/water recoverable two-aqueous phase system, comprising:
forward extracting bacitracin by mixing NPE-108 with bacitracin fermentation broth at a volume ratio of 1:4, and adding divalent manganese salt (preferably MnCl)2The concentration in the system is 4mmol/L), the oscillation is uniform, the pH value of the system is adjusted to 8.5, and the centrifuge tube is placed in a constant temperature water bath kettle at 30 ℃ for standing until two phases with clear interfaces are observed. Under this condition, bacitracin is mainly distributed in the NPE-108 rich phase;
and (2) carrying out reverse extraction on bacitracin, taking out the upper phase after the forward extraction, adding water into a graduated centrifuge tube for carrying out first reverse extraction, wherein the volume of the water is 1/2 of the volume of the NPE-108 initially added, mixing and oscillating uniformly, adjusting the pH of the system to 5.5, placing the system in a constant-temperature water bath kettle at 60 ℃ for standing until two phases with clear interfaces are observed, taking out the two phases, centrifuging, taking out the upper phase after the reverse extraction, adding water (the volume ratio of the NPE-108 to the water is 2:1) into the centrifuge tube for carrying out second reverse extraction, adjusting the pH of the system to 5.5, and placing the centrifuge tube in the constant-temperature water bath kettle at 60 ℃ for standing until two phases with clear interfaces are observed. After two reverse extractions, bacitracin in the lower phase was back extracted into the aqueous upper phase.
The present invention utilizes a thermo-responsive polymer NPE-108, a block polymer copolymerized from the monomers Ethylene Oxide (EO) and Propylene Oxide (PO). NPE-108 can be separated from water through temperature induction to form a two-water phase system, and the transfer of bacitracin between fermentation liquor and NPE-108 is realized by adjusting the pH value in the extraction process.
The invention provides a preparation method of bacitracin methylene disalicylate, which comprises the steps of dissolving methylene disalicylate by NaOH, adjusting the pH value to 8.5, mixing an upper phase obtained after two times of reverse extraction with the methylene disalicylate solution, adjusting the pH value of the mixed solution to 4.5, observing precipitation of bacitracin methylene disalicylate, centrifuging, removing a supernatant, and freeze-drying the precipitate in a freeze-drying box (5 hours) to obtain bacitracin methylene disalicylate powder.
The thermal response polymer NPE-108 used in the invention is a commercial commodity, the upper phase after the forward extraction is finished and the lower phase after the reverse extraction is finished are heated at the temperature of 60 ℃ to separate the NPE-108 from water, the aqueous solution is removed and then is placed in an oven to be dried to constant weight, and the recovery of the NPE-108 is realized.
The beneficial effects of the invention are as follows: the invention designs a technological process for separating bacitracin by an NPE-108/water recoverable two-water phase system. The two-water phase system can quickly form two phases at low temperature (30 ℃), and has a certain protection effect on the activity of bacitracin which is not high in temperature resistance. During forward extraction, bacitracin fermentation liquor and NPE-108 can form a phase within 10 minutes, and bacitracin in the fermentation liquor can be transferred into the NPE-108 phase by adjusting the pH of a system; when the NPE-108 and water are in phase within 10 minutes during reverse extraction, the bacitracin in the NPE-108 phase is transferred to the water phase by adjusting the pH of the system, and the NPE-108 is recovered by heating. The method has the main advantages that the bacitracin in the fermentation liquor can be transferred between the NPE-108 phase and the water phase by adjusting the pH of the system, the NPE-108 can be recovered by adjusting the temperature, the downstream separation cost is reduced, the operation is simple and convenient, and the large-scale production is easy.
Drawings
FIG. 1 is a graph showing the results of the effect of salt species and concentration on bacitracin extraction.
Detailed Description
For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the invention, and not to limit the scope of the claims.
Bacitracin, a polypeptide material, is relatively hydrophilic and relatively difficult to partition in NPE-108/water two-phase aqueous systems. Repeated experiments show that when NPE-108/water two-phase system is directly used for distribution, namely no salt is added in the system, the distribution coefficient (1/K) of the forward extraction bacitracin is 1.01, which indicates that bacitracin is almost evenly distributed in the upper phase and the lower phase under the condition. When inorganic salt is added into a two-water phase system, the phase equilibrium state of the whole system is changed, salt ions are unevenly distributed in two phases, the phase-to-phase potential difference of the system is changed, and the phase-to-phase potential difference directly influences the uneven distribution of biomolecules in the two phases.
Thus, the present invention selects the exploration of salt ions to improve bacitracin partitioning, and the results are shown in FIG. 1. As can be seen from FIG. 1, 10mM MnCl was added2The distribution effect on bacitracin is good, but the precipitation is generated at the bottom of the graduated tube and contains partial bacitracin, and then MnCl is treated2The concentration is further optimized in MnCl2At concentrations below 10mM, no precipitate was formed at the bottom of the graduated tube. As can be seen from Table 1 below, when MnCl is present in the system2When the concentration is 4mM, the positive extraction effect of bacitracin is best, 1/K and the extraction recovery rate (ER)f) Up to 7.65 and 81.15%.
TABLE 1 MnCl2Effect of concentration on bacitracin Positive extraction
After screening various salt ions, low concentration of divalent cation (Mn) was found2+) Can improve the distribution effect of bacitracin. And the purity of the finally obtained bacitracin methylene disalicylate is obviously improved. Compared with the traditional bacitracin methylene disalicylate premix (the bacitracin content is about 15%), the bacitracin methylene disalicylate premix is prepared by separating bacitracin in fermentation liquor to obtain bacitracin methylene disalicylate powder, weighing 6mg of the powder to dissolve the bacitracin powder, and detecting the content of the bacitracin by HPLC (high performance liquid chromatography), wherein the final bacitracin accounts for 3.42mg and the purity is 57%.
EXAMPLE 1 NPE-108/Water recoverable two-aqueous phase System for Forward extraction experiments of bacitracin
Adding 7mL of NPE-108 into a 50mL graduated centrifuge tube, adding 28mL of bacitracin fermentation liquor, uniformly mixing, and adding MnCl2(concentration in the system is 4mmol/L), oscillating uniformly, adjusting pH of the system to 8.5, placing the centrifuge tube in a constant temperature water bath kettle at 30 ℃ for standing until two phases with clear interfaces are observed, taking out the two phases, centrifuging, taking out 100 mu L of each of the upper phase and the lower phase by a pipette gun, diluting with ultrapure water by 10 times, filtering by a 0.45 mu m water system filter membrane, and measuring bacitracin concentration of the upper phase and the lower phase in the system by HPLCAnd (4) degree. The ratio of the bacitracin concentrations in the upper and lower phases is the partition coefficient K, and under the extraction conditions, the partition coefficient of bacitracin is 0.12.
EXAMPLE 2 NPE-108/Water recoverable two-aqueous phase System reverse extraction experiments of bacitracin
Taking out the upper phase (fermentation liquor enrichment phase) in example 1, adding water into a graduated centrifuge tube for first reverse extraction, wherein the volume of the water is 1/2 of the volume of the initially added NPE-108, mixing and oscillating uniformly, adjusting the pH of the system to 5.5, placing the system in a constant temperature water bath kettle at 60 ℃ for standing until two phases with clear interfaces are observed, taking out the two phases, centrifuging, taking out 100 mu L of the two phases from the upper phase and the lower phase by using a pipette gun, diluting the two phases by 10 times by using ultrapure water, filtering the two phases by using a 0.45 mu m water system filter membrane, and measuring the bacitracin concentration of the upper phase and the lower phase in the system by using HPLC.
And taking out the upper phase after the back extraction, and adding water into the centrifuge tube to perform secondary back extraction, wherein the back extraction conditions are completely the same as the conditions of the first back extraction. After completion of the extraction, the resultant was centrifuged, 100. mu.L of each of the upper and lower phases was taken out by a pipette gun, diluted 10-fold with ultrapure water, filtered through a 0.45 μm aqueous membrane, and the bacitracin concentrations in the upper and lower phases in the system were measured by HPLC. The extraction recovery rate of bacitracin is the ratio of the total amount of substance of bacitracin in the upper phase after back extraction to the amount of substance of bacitracin in the lower phase after forward extraction. Under the condition of the reverse extraction, the total extraction recovery rate can reach 95.78 percent after two times of reverse extraction.
Example 3 preparation experiment of Methylenedisalicylate peptide
Dissolving methylene disalicylic acid by NaOH, adjusting the pH value to 8.5, mixing the upper phase obtained after twice reverse extraction in example 2 with the methylene disalicylic acid solution to adjust the pH value of the mixed solution to 4.5, observing the precipitation of the methylene disalicylic acid bacitracin, centrifuging, removing the supernatant, and freeze-drying the precipitate in a freeze-drying oven (5 hours) to obtain the methylene disalicylic acid bacitracin powder. 6mg of bacitracin methylene disalicylate powder is weighed, dissolved by 1mL of NaOH solution, and the bacitracin content is detected by HPLC, wherein the final bacitracin concentration is 3.42mg/mL, and the purity of the bacitracin is 57%.
EXAMPLE 4 recovery experiment of NPE-108 thermo-responsive Polymer
And (3) placing the upper phase after the forward extraction in example 1 and the lower phase after the reverse extraction in example 2 in a constant-temperature water bath kettle at 60 ℃, standing for 1 hour, observing that NPE-108 and water are separated into two phases, taking out the water phase, and placing in an oven (150 ℃, 3 hours) to dry to constant weight, wherein the recovery rate of the NPE-108 reaches 98.42 percent finally.
In conclusion, the NPE-108/water-recoverable two-water-phase system used in the invention has the advantages of short phase forming time, simple and convenient operation and high recovery rate of phase-forming components, and compared with the traditional bacitracin methylene disalicylate premix (bacitracin content is about 15%), the bacitracin in the fermentation liquor is separated by adopting the method, the purity of the finally obtained bacitracin methylene disalicylate powder reaches 57%, and the product purity is improved.
In this specification, the invention has been described with reference to specific embodiments thereof. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention. The description is thus to be regarded as illustrative instead of limiting.
Claims (4)
1. A method for separating and purifying bacitracin by using a thermo-responsive polymer NPE-108, comprising:
a forward extraction step: uniformly mixing NPE-108 and bacitracin fermentation liquor according to the volume ratio of 1:4, adding divalent manganese salt, adjusting the pH to 8.5, uniformly oscillating, standing in a constant-temperature water bath at 30 ℃ until two phases with clear interfaces are formed, and taking out an upper phase;
and (3) reverse extraction: adding water with a volume half of that of NPE-108 into the upper phase obtained in the forward extraction step, uniformly mixing, adjusting the pH to 5.5, standing in a water bath kettle at 60 ℃ until two phases with clear interfaces are formed, and enriching bacitracin in the upper phase.
2. The method for separating and purifying bacitracin using NPE-108, a thermo-responsive polymer according to claim 1, wherein two distinct phases are formed within 10 minutes in both the forward extraction step and the backward extraction step.
3. The method for separating and purifying bacitracin of claim 1 wherein the divalent manganese salt added in the forward extraction step is MnCl2The concentration thereof in the system was 4 mmol/L.
4. The method for separating and purifying bacitracin using NPE-108, a thermo-responsive polymer according to claim 1 further comprising a secondary back extraction step of: and taking out the upper phase obtained in the reverse extraction step, adding water according to the volume ratio of NPE-108 to water of 2:1, carrying out secondary reverse extraction, adjusting the pH value to 5.5, standing in a water bath kettle at 60 ℃ until two phases with clear interfaces are formed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110743115.XA CN113248571B (en) | 2021-06-30 | 2021-06-30 | Method for separating and purifying bacitracin by using thermal response type polymer NPE-108 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110743115.XA CN113248571B (en) | 2021-06-30 | 2021-06-30 | Method for separating and purifying bacitracin by using thermal response type polymer NPE-108 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113248571A true CN113248571A (en) | 2021-08-13 |
| CN113248571B CN113248571B (en) | 2022-10-18 |
Family
ID=77190324
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110743115.XA Active CN113248571B (en) | 2021-06-30 | 2021-06-30 | Method for separating and purifying bacitracin by using thermal response type polymer NPE-108 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113248571B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114230639A (en) * | 2021-12-22 | 2022-03-25 | 齐鲁制药（内蒙古）有限公司 | Preparation method of bacitracin methylene disalicylate |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101830979A (en) * | 2010-04-16 | 2010-09-15 | 中南民族大学 | Method for separating and purifying serum albumin by using liquid-solid extraction system |
| CN102224160A (en) * | 2008-11-25 | 2011-10-19 | 通用电气健康护理生物科学股份公司 | Aqueous two phase extraction augmented precipitation process for purification of therapeutic proteins |
| CN105566458A (en) * | 2016-02-23 | 2016-05-11 | 华北制药集团新药研究开发有限责任公司 | Method for preparing bacitracin and method for preparing zinc salt of bacitracin |
| CN112321704A (en) * | 2020-11-20 | 2021-02-05 | 华东理工大学 | Using liquid thermo-responsive polymer EO20PO80Separating and purifying monoclonal antibody |
| CN112375109A (en) * | 2020-11-20 | 2021-02-19 | 华东理工大学 | Separation and purification of spiramycin by using alkylphenol polyoxyethylene polyoxypropylene ether NPE-108 |
| CN112851754A (en) * | 2021-01-06 | 2021-05-28 | 铜仁职业技术学院 | Method for separating and purifying surfactin by extraction-back extraction |
-
2021
- 2021-06-30 CN CN202110743115.XA patent/CN113248571B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102224160A (en) * | 2008-11-25 | 2011-10-19 | 通用电气健康护理生物科学股份公司 | Aqueous two phase extraction augmented precipitation process for purification of therapeutic proteins |
| CN101830979A (en) * | 2010-04-16 | 2010-09-15 | 中南民族大学 | Method for separating and purifying serum albumin by using liquid-solid extraction system |
| CN105566458A (en) * | 2016-02-23 | 2016-05-11 | 华北制药集团新药研究开发有限责任公司 | Method for preparing bacitracin and method for preparing zinc salt of bacitracin |
| CN112321704A (en) * | 2020-11-20 | 2021-02-05 | 华东理工大学 | Using liquid thermo-responsive polymer EO20PO80Separating and purifying monoclonal antibody |
| CN112375109A (en) * | 2020-11-20 | 2021-02-19 | 华东理工大学 | Separation and purification of spiramycin by using alkylphenol polyoxyethylene polyoxypropylene ether NPE-108 |
| CN112851754A (en) * | 2021-01-06 | 2021-05-28 | 铜仁职业技术学院 | Method for separating and purifying surfactin by extraction-back extraction |
Non-Patent Citations (2)
| Title |
|---|
| DAISONG HOU ET AL.: ""Synthesis of two thermo-sensitive copolymers forming aqueous two-phase systems"", 《SEPARATION AND PURIFICATION TECHNOLOGY》 * |
| 房秀敏: ""聚醚型非离子表面活性剂的浊点及其影响因素"", 《精细石油化工》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114230639A (en) * | 2021-12-22 | 2022-03-25 | 齐鲁制药（内蒙古）有限公司 | Preparation method of bacitracin methylene disalicylate |
| CN114230639B (en) * | 2021-12-22 | 2024-03-12 | 齐鲁制药（内蒙古）有限公司 | Preparation method of bacitracin methylene disalicylate |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113248571B (en) | 2022-10-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kula et al. | Purification of enzymes by liquid-liquid extraction | |
| CN110003323B (en) | Method for separating and purifying protein by aqueous two-phase system | |
| Mattiasson | Applications of aqueous two-phase systems in biotechnology | |
| Tanuja et al. | Aqueous two-phase extraction for downstream processing of amyloglucosidase | |
| CN113248571B (en) | Method for separating and purifying bacitracin by using thermal response type polymer NPE-108 | |
| CN103772497A (en) | Ultrafiltration membrane separation method for obtaining royal jelly major protein and active filtrate in royal jelly | |
| CN109932463B (en) | Endogenous peptide enrichment method based on nanoporous organic covalent framework material | |
| CN110882357A (en) | High-purity bamboo leaf flavonoid powder and preparation method thereof | |
| Probst et al. | Newly synthesized mammalian cell DNA: studies on the reasons for the increased affinity to nitrocellulose | |
| CN101830979B (en) | Method for separating and purifying serum albumin by using liquid-solid extraction system | |
| CN112851754B (en) | Extraction-back extraction separation and purification method for surfactin | |
| Luong et al. | Recent developments in downstream processing based on affinity interactions | |
| CN112375109B (en) | Separation and purification of spiramycin by using alkylphenol polyoxyethylene polyoxypropylene ether NPE-108 | |
| CN102925416A (en) | Method for extracting superoxide dismutase from blood of mammal | |
| CN109133254B (en) | Method for removing phthalic acid in wastewater by modified calcite | |
| CN112321704A (en) | Using liquid thermo-responsive polymer EO20PO80Separating and purifying monoclonal antibody | |
| CN113121352A (en) | Method for separating and purifying chlorogenic acid by using novel composite imprinted polymer | |
| EP3816177A1 (en) | Method for isolating polypeptide, method for producing polypeptide, and apparatus for purifying polypeptide | |
| WO2015099550A1 (en) | Magnetic liquid-liquid extraction for purification and partitioning of substances | |
| CN113166732A (en) | Purification method for vaccine viruses using affinity chromatography | |
| CN113999886B (en) | Method for extracting chicken bile acid by one-pot enzymolysis | |
| CN114563489B (en) | Method for detecting recombinant protein charge variant and application thereof | |
| CN115058414B (en) | Method for purifying plasmid DNA | |
| CN103988972A (en) | Desalting and protein recovering method of salted egg white | |
| CN113444196B (en) | Extraction adsorption resin and synthesis method and application thereof |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |