CN113248571B - Method for separating and purifying bacitracin by using thermal response type polymer NPE-108 - Google Patents
Method for separating and purifying bacitracin by using thermal response type polymer NPE-108 Download PDFInfo
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- CN113248571B CN113248571B CN202110743115.XA CN202110743115A CN113248571B CN 113248571 B CN113248571 B CN 113248571B CN 202110743115 A CN202110743115 A CN 202110743115A CN 113248571 B CN113248571 B CN 113248571B
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- 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
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; and (3) reverse extraction: adding water with a volume half of the volume of the NPE-108 into the lower 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 the bacitracin into 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, belonging 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 aqueous solution of NPE-108 is heated, the temperature is above the cloud point of 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-aqueous phase system with short phase forming time, low preparation cost of phase forming components and mature synthesis process is a problem to be urgently solved for the industrial separation of biomolecules at present.
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:
bacitracin was extracted forward by mixing NPE-108 with bacitracin fermentation broth at a volume ratio of 1 2 The concentration in the system is 4 mmol/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 to carry 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). After two back 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; during the back extraction, NPE-108 and water can form phase within 10 min, and 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 transfer of bacitracin in the fermentation liquor between the NPE-108 phase and the water phase can be realized by adjusting the pH of the system, the recovery of NPE-108 can be realized by adjusting the temperature, the downstream separation cost is reduced, the operation is simple and convenient, and the 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 the 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 positive extraction bacitracin is 1.01, and the 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 added 2 The distribution effect on bacitracin is good, but the precipitation is generated at the bottom of the graduated tube and part of bacitracin is contained in the precipitation, and then the precipitation on MnCl 2 The concentration is further optimized in MnCl 2 At 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 system 2 When the concentration is 4mM, the positive extraction effect of bacitracin is best, 1/K and the extraction recovery rate (ER) f ) The highest can reach 7.65 and 81.15 percent.
TABLE 1 MnCl 2 Effect of concentration on bacitracin Positive extraction
After screening various salt ions, low concentration of divalent cation (Mn) was found 2+ ) Can improve the distribution effect of bacitracin. And areAnd 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 MnCl 2 (concentration in the system is 4 mmol/L), shaking 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 the two phases by 10 times by ultrapure water, filtering the two phases 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 HPLC. The ratio of the bacitracin concentrations of the upper phase and the lower phase is the partition coefficient K, and under the extraction condition, the partition coefficient of the bacitracin is 0.12.
EXAMPLE 2 NPE-108/Water recoverable two-aqueous phase System for Bacto peptide reverse extraction experiments
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 upper phase and the lower phase respectively 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 mass of bacitracin in the upper phase after back extraction to the mass 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;
a reverse extraction step: adding water with a volume half of that of NPE-108 into the lower 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 MnCl 2 The concentration thereof in the system was 4mmol/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 (3) taking out an upper phase obtained in the reverse extraction step, adding water according to the volume ratio of NPE-108 to water being 2.
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