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CN-121991177-A - Purification method and application of bacitracin A

CN121991177ACN 121991177 ACN121991177 ACN 121991177ACN-121991177-A

Abstract

The invention discloses a purification method and application of bacitracin A, and relates to the field of biology. The invention develops a purification method of bacitracin A, which realizes the preparation of bacitracin A with higher purity through liquid chromatography separation, the purity of bacitracin A can reach more than 95%, the separation and purification process of bacitracin A with high purity is simplified to a great extent, the reagent loss in the separation and purification stage is reduced, and the method has the advantages of high separation purity, less consumption of organic solvent, simple device, simple and convenient process operation, environmental protection and the like, and is suitable for technological production.

Inventors

  • YANG MENGRUI
  • WU YUXIN
  • LI LIANG
  • ZHOU JIAN
  • LI FUKAI
  • LI KAI
  • WANG TONGTONG

Assignees

  • 中国农业科学院农业质量标准与检测技术研究所

Dates

Publication Date
20260508
Application Date
20260310

Claims (10)

  1. 1. A method for purifying bacitracin a, comprising the steps of: carrying out first liquid chromatographic separation on a solution containing a bacitracin A crude product to be purified, and collecting an eluent; The conditions of the first liquid chromatography comprise that the chromatographic column is a reversed phase C18 semi-prepared column, the mobile phase A is an ammonium formate aqueous solution, the mobile phase B is acetonitrile, and isocratic elution is adopted.
  2. 2. The purification method according to claim 1, wherein the concentration of the aqueous ammonium formate solution is 40-60 mmol/L and the pH is 3.8-4.2; Optionally, the size of the reversed phase C18 semi-prepared column is (1-15) mm× (200-300) mm, the particle size is 1-10 μm, the pore diameter is 250-350A, and the column temperature is 30-50 ℃; Alternatively, the reverse phase C18 semi-prepared column is Watertight XBridge Peptide BEH C, 10mm 250 mm,5 μm, 300A, or Athena C18-BIO,10mm 250 mm,5 μm, 300A.
  3. 3. The purification method according to claim 1, wherein the sample loading concentration of the first liquid chromatography is 20-100 mg/mL.
  4. 4. The method according to claim 1, wherein the isocratic elution procedure comprises maintaining the volume percentage of the mobile phase B at 20% -30% and the flow rate at 3-4 mL/min for 0-50 min.
  5. 5. The method according to claim 1, further comprising subjecting the eluate of the first liquid chromatography or a lyophilized product thereof to a second liquid chromatography, and collecting the eluate; optionally, the conditions of the second liquid-phase chromatographic separation comprise that the chromatographic column is an inverse C18 semi-prepared column, the mobile phase A is 40-60 mmol/L ammonium formate aqueous solution, the mobile phase B is acetonitrile, and gradient elution is adopted; Optionally, the size of the reversed phase C18 semi-prepared column is (1-15) mm× (200-300) mm, the particle size is 1-10 μm, the pore diameter is 250-350A, and the column temperature is 30-50 ℃; Alternatively, the inverted C18 semi-prepared column is waters XBridge Peptide BEH C, 10mm x 250mm, 5 μm,300 a.
  6. 6. The method of claim 5, wherein the gradient elution is performed as follows: 0-30 min, wherein the volume percentage of the mobile phase B is increased from 20-25% to 28-32%; 30-31 min, wherein the volume percentage of the mobile phase B is increased from 28-32% to 85-95%; 31-36 min, wherein the volume percentage of the mobile phase B is maintained at 85% -95%; 36-37 min, wherein the volume percentage of the mobile phase B is reduced from 85-95% to 20-25%; And 37-40 min, wherein the volume percentage of the mobile phase B is maintained at 20% -25%.
  7. 7. The purification method according to claim 1, wherein the loading concentration of the second liquid chromatography is 35-60 mg/mL; optionally, the loading amount of the second liquid chromatography separation is 10-50 mu L.
  8. 8. The method according to any one of claims 5 to 7, further comprising freeze-drying the eluate of the first liquid chromatography and/or the second liquid chromatography.
  9. 9. Use of the purification method according to any one of claims 1 to 8 for the preparation of a product for inhibiting a pathogen, a medicament for preventing and/or treating an infection caused by a pathogen; Optionally, the pathogen comprises at least one of staphylococcus aureus and escherichia coli.
  10. 10. Use of the purification method according to any one of claims 1 to 8 for the preparation of cosmetics, foods or medical devices.

Description

Purification method and application of bacitracin A Technical Field The invention relates to the field of biology, in particular to a purification method and application of bacitracin A. Background The antimicrobial peptide is also called antimicrobial peptide and antibiotic peptide, is a kind of small molecular polypeptide produced by encoding specific genes of biological cells, and the generation and release of the small molecular polypeptide are components of inflammatory reaction of organisms and are important molecular barriers for host defense against invasion of pathogenic microorganisms. The antibacterial peptide has wide inhibition effect on bacteria, fungi, parasites, viruses, tumor cells and the like, and is subjected to intensive research. Such as cecropin in insect antimicrobial peptides, thionine in plant antimicrobial peptides, bacitracin in microbial antimicrobial peptides, nisin, and the like. Wherein, bacitracin has stronger inhibition effect on gram-positive bacteria and gram-negative bacteria. In clinical aspects, bacitracin is mainly used for gram-positive bacteria infection with penicillin resistance, external application for skin infection and the like, can be used as an antibiotic feed additive in animal breeding industry, and has the characteristics of no residue, no incompatibility, no drug resistance, long-term use and the like when added in proper amount. Bacitracin is thus one of the best alternatives to existing antibiotics. Bacitracin is mainly a thiazoline ring-containing polypeptide substance produced by bacillus licheniformis and bacillus subtilis, contains A, B, C, D, E, F, G and other isomers, and the active ingredients gradually decrease from A to G, so that bacitracin A is the main active ingredient of bacitracin. Bacitracin a consists of 12 amino acids (L-isoleucine-D-glutamate-L-lysine-D-ornithine-L-aspartate-L-ornithine-D-ornithine-L-glycine-L-histidine-D-aspartate-L-threonine-L-leucine) and has a molecular formula of C66H103N17O16S and a relative molecular weight of 1422.72 Da. Bacitracin a can spontaneously oxidize under alkaline conditions to form bacitracin F without antibacterial activity, and thus bacitracin a is stable under acidic or neutral conditions. At present, the separation and purification of bacitracin A have the defects of low biological potency of products, environmental pollution caused by using a large amount of toxic reagents and the like. In view of this, the present invention has been made. Disclosure of Invention The invention aims to provide a purification method and application of bacitracin A. The invention is realized in the following way: In a first aspect, embodiments of the present invention provide a method for purifying bacitracin a, comprising the steps of: carrying out first liquid chromatographic separation on a solution containing a bacitracin A crude product to be purified, and collecting an eluent; The conditions of the liquid chromatography comprise that the chromatographic column is a reversed phase C18 semi-prepared column, the mobile phase A is an ammonium formate aqueous solution, the mobile phase B is acetonitrile, and isocratic elution is adopted. In a second aspect, embodiments of the present invention provide the use of a purification method as described in the previous embodiments for the preparation of a medicament for the prevention and/or treatment of an infection caused by a pathogen. In a third aspect, embodiments of the present invention provide the use of a purification method as described in the previous embodiments for the preparation of a cosmetic, food or medical device. The invention has the following beneficial effects: The embodiment of the invention develops a purification method of bacitracin A, which realizes the preparation of bacitracin A with higher purity through liquid chromatography separation under specific conditions, and the purity of bacitracin A can reach more than 95%, so that the separation and purification process of bacitracin A with high purity is greatly simplified, the loss of reagents in the separation and purification stage is reduced, and the method has the advantages of high separation purity, less consumption of organic solvents, simple device, simple and convenient process operation, environmental protection and the like, and is suitable for technological production. Drawings In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. FIG. 1 is a chromatogram of a crude bacitracin A product prepared in accordance with the present invention after separation by a se