Method for separating and purifying bacitracin by using thermo-responsive copolymer NPE-108

Abstract

The invention relates to a method for separating and purifying bacitracin by using a thermo-responsive copolymer NPE-108, which comprises the following steps: a forward extraction step: uniformly mixing NPE-108 and bacitracin fermentation liquor according to a volume ratio of 1: 4, adding divalent manganese salt, adjusting the pH value to 8.5, uniformly oscillating, standing in a constant-temperature water bath kettle at 30 DEG C until two phases with a clear interface is formed, and taking out the upper phase; and a reverse extraction step: adding water of which the volume is half of that of NPE-108 into the upper phase obtained in the forward extraction step, uniformly mixing, adjusting the pH value to 5.5, standing in a water bath kettle at 60 DEG C until two phases with a clear interface are formed, and enriching bacitracin in the upper phase. The thermo-responsive copolymer NPE-108 is mixed with water to form a recoverable thermo-responsive two-aqueous-phase system, and the method has the advantages of mild operation conditions, environmental friendliness and recoverable phase forming components, and is beneficial to large-scale bacitracin separation and application.

Description

technical field

[0001] The invention relates to the technical field of two-water phase extraction, in particular to the application of a recyclable two-water-phase system in the separation of bacitracin in fermentation broth, and belongs to the downstream separation technical field. Background technique

[0002] Block polyether NPE-108 is a heat-responsive polymer, and EOPO with different cloud points can be synthesized by adjusting the addition ratio of EO and PO during the synthesis process. When the NPE-108 aqueous solution is heated, the temperature is above the cloud point of NPE-108, and the polymer NPE-108 will separate from water to form two phases with a clear interface, and this method can also be used to recover NPE-108 from the phase system and carry out Reuse, this is NPE-108 / water recyclable two-phase aqueous system.

[0003] Due to its high water content, mild extraction conditions, and the ability to protect the active groups of biomolecules, the two-water p...

Examples

Embodiment 1

[0021] Example 1 NPE-108 / water recyclable two-phase system for forward extraction of bacitracin

[0022] Add 7mL NPE-108 to a 50mL graduated centrifuge tube, then add 28mL bacitracin fermentation broth, mix well, add MnCl 2 (the concentration in the system is 4mmol / L), shake evenly, adjust the pH of the system to 8.5, place the centrifuge tube in a constant temperature water bath at 30°C and let it stand until two phases with a clear interface are observed, take it out and centrifuge , take out 100 μL from the upper and lower phases with a pipette gun, dilute 10 times with ultrapure water, filter through a 0.45 μm water filter membrane, and measure the concentration of bacitracin in the upper and lower phases in the system by HPLC. The ratio of the concentrations of bacitracin in the upper and lower phases is the distribution coefficient K, and under this extraction condition, the distribution coefficient of bacitracin is 0.12.

Embodiment 2

[0023] Example 2 Back extraction experiment of bacitracin in NPE-108 / water recyclable two-phase system

[0024] Take out the upper phase (fermentation broth enriched phase) in Example 1, add water to the graduated centrifuge tube for the first reverse extraction, the volume of water is 1 / 2 of the volume of NPE-108 initially added, mix and oscillate evenly Finally, adjust the pH of the system to 5.5, place it in a constant temperature water bath at 60 °C and let it stand until two phases with a clear interface are observed, take them out and centrifuge, take out 100 μL from the upper and lower phases with a pipette gun, and wash with ultrapure water Dilute 10 times, filter through a 0.45 μm water filter, and measure the concentration of bacitracin in the upper and lower phases of the system by HPLC.

[0025] The upper phase after the back extraction is taken out, and water is added to the centrifuge tube for the second back extraction. The conditions of the back extraction are ...

Embodiment 3

[0026] The preparation experiment of embodiment 3 methylene disalicylic acid bacitracin

[0027] Methylenedisalicylic acid is dissolved with NaOH and its pH is adjusted to 8.5, and the upper phase and methylenesalicylic acid solution after two back extractions in Example 2 are mixed to adjust the pH of the mixed solution to 4.5, namely Precipitation of bacitracin methylenedisalicylate can be observed. After centrifugation, the supernatant is removed, and the precipitate is placed in a freeze-drying oven (5 hours) to freeze-dry to obtain bacitracin methylenedisalicylate powder. Weigh 6 mg of bacitracin powder, dissolve it in 1 mL of NaOH solution, and detect the content of bacitracin by HPLC. The final concentration of bacitracin is 3.42 mg / mL, and the purity of bacitracin is 57%.

technical field

[0001] The invention relates to the technical field of two-water phase extraction, in particular to the application of a recyclable two-water-phase system in the separation of bacitracin in fermentation broth, and belongs to the downstream separation technical field. Background technique

[0002] Block polyether NPE-108 is a heat-responsive polymer, and EOPO with different cloud points can be synthesized by adjusting the addition ratio of EO and PO during the synthesis process. When the NPE-108 aqueous solution is heated, the temperature is above the cloud point of NPE-108, and the polymer NPE-108 will separate from water to form two phases with a clear interface, and this method can also be used to recover NPE-108 from the phase system and carry out Reuse, this is NPE-108 / water recyclable two-phase aqueous system.

[0003] Due to its high water content, mild extraction conditions, and the ability to protect the active groups of biomolecules, the two-water p...

Examples

Embodiment 1

[0021] Example 1 NPE-108 / water recyclable two-phase system for forward extraction of bacitracin

[0022] Add 7mL NPE-108 to a 50mL graduated centrifuge tube, then add 28mL bacitracin fermentation broth, mix well, add MnCl 2 (the concentration in the system is 4mmol / L), shake evenly, adjust the pH of the system to 8.5, place the centrifuge tube in a constant temperature water bath at 30°C and let it stand until two phases with a clear interface are observed, take it out and centrifuge , take out 100 μL from the upper and lower phases with a pipette gun, dilute 10 times with ultrapure water, filter through a 0.45 μm water filter membrane, and measure the concentration of bacitracin in the upper and lower phases in the system by HPLC. The ratio of the concentrations of bacitracin in the upper and lower phases is the distribution coefficient K, and under this extraction condition, the distribution coefficient of bacitracin is 0.12.

Embodiment 2

[0023] Example 2 Back extraction experiment of bacitracin in NPE-108 / water recyclable two-phase system

[0024] Take out the upper phase (fermentation broth enriched phase) in Example 1, add water to the graduated centrifuge tube for the first reverse extraction, the volume of water is 1 / 2 of the volume of NPE-108 initially added, mix and oscillate evenly Finally, adjust the pH of the system to 5.5, place it in a constant temperature water bath at 60 °C and let it stand until two phases with a clear interface are observed, take them out and centrifuge, take out 100 μL from the upper and lower phases with a pipette gun, and wash with ultrapure water Dilute 10 times, filter through a 0.45 μm water filter, and measure the concentration of bacitracin in the upper and lower phases of the system by HPLC.

[0025] The upper phase after the back extraction is taken out, and water is added to the centrifuge tube for the second back extraction. The conditions of the back extraction are ...

Embodiment 3

[0026] The preparation experiment of embodiment 3 methylene disalicylic acid bacitracin

[0027] Methylenedisalicylic acid is dissolved with NaOH and its pH is adjusted to 8.5, and the upper phase and methylenesalicylic acid solution after two back extractions in Example 2 are mixed to adjust the pH of the mixed solution to 4.5, namely Precipitation of bacitracin methylenedisalicylate can be observed. After centrifugation, the supernatant is removed, and the precipitate is placed in a freeze-drying oven (5 hours) to freeze-dry to obtain bacitracin methylenedisalicylate powder. Weigh 6 mg of bacitracin powder, dissolve it in 1 mL of NaOH solution, and detect the content of bacitracin by HPLC. The final concentration of bacitracin is 3.42 mg / mL, and the purity of bacitracin is 57%.

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