CN110117311B - Method for separating and purifying bacillomycin from bacillus amyloliquefaciens - Google Patents
Method for separating and purifying bacillomycin from bacillus amyloliquefaciens Download PDFInfo
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- CN110117311B CN110117311B CN201910360461.2A CN201910360461A CN110117311B CN 110117311 B CN110117311 B CN 110117311B CN 201910360461 A CN201910360461 A CN 201910360461A CN 110117311 B CN110117311 B CN 110117311B
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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
A method for separating and purifying bacillomycin from bacillus amyloliquefaciens comprises the following steps: adsorbing the BaX030 fermentation supernatant through macroporous adsorption resin, eluting with ethanol, and concentrating under reduced pressure to obtain a crude extract; subjecting the crude extract to RPC reversed phase chromatographic column, performing primary chromatographic separation on Ä KTA avant 25, collecting and combining fractions, and concentrating under reduced pressure to obtain bacitracin Lb crude product; and performing secondary purification by using an RPC reverse phase chromatographic column, and concentrating under reduced pressure to obtain the target compound bacitracin Lb. The invention has the advantages that the bacitracin Lb is separated and purified by adopting a macroporous resin adsorption method, the process is simple and easy to implement, the bacitracin Lb is easy to recycle and low in production cost and is suitable for industrial production, and the obtained lipopeptide compound bacitracin Lb has broad-spectrum anti-tumor activity.
Description
Technical Field
The invention belongs to the technical field of industrial microorganisms, and particularly relates to a method for quickly separating and purifying bacillus Lb (bacillus Lb) antitumor active substances from bacillus amyloliquefaciens X030.
Background
Bacillus amyloliquefaciens can produce various lipopeptide antibiotics, such as surfactin (surfactin), iturin (iturin A and bacillomycin) and fengycin (fengycins), and has a structure comprising 1 cyclic polypeptide consisting of 7-10 amino acids and 1C 13 -C 18 Is an important source of clinically used agents or bioactive molecules. Bactericin Lb belongs to iturin family lipopeptide, and is a cyclic heptapeptide composed of asparagine (Asn), tyrosine (Tyr), serine (Ser), glutamic acid (Glu) and threonine (Thr)And a beta-sheet fatty acid chain is attached at the threonine (Thr) tail. Bacillamycin Lb has been recognized as one of the lipopeptides that exert important antifungal effects in controlling plant diseases such as rice sheath blight, tomato gray mold, and barley powdery mildew. In addition, the iturin family lipopeptide has potential application value in the fields of biomedicine, aquatic science, endogenous molecular probes of peptides and proteins and the like.
Research reports that most of lipopeptides are separated and purified mainly by separation methods such as acid precipitation, acetone precipitation, methanol extraction and the like. However, these methods use large amounts of acetone, are costly and not environmentally friendly; the acid precipitation method has the disadvantages of limited suitable chromatographic column and slow preparation speed. Therefore, a method which is simple and feasible in process, suitable for industrial production and capable of rapidly separating and purifying the bacitracin Lb antitumor active substance is urgently needed.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects of the prior art and provides a method for separating and purifying the bacillomycin from the bacillus amyloliquefaciens, which has high separation speed, does not use harmful organic solvents and is simple and easy to implement.
The technical scheme adopted by the invention for solving the technical problems is that the method for separating and purifying the bacillomycin from the bacillus amyloliquefaciens comprises the following steps:
(1) Activating the macroporous adsorption resin with absolute ethyl alcohol, and then washing with double distilled water until no alcohol smell exists to obtain activated macroporous adsorption resin;
further, the macroporous adsorption resin is common resin DM301. The dosage of the fermentation supernatant is 20-50g per liter, and the adsorption time is 24-48h.
(2) Transferring Bacillus amyloliquefaciens BaX030 (the preservation number is CCTCC No. M2014159, see CN 104630111A) into an LB culture medium, performing shaking culture for 44-48h under the conditions of 30-32 ℃ and 120-130rpm/min, centrifuging, collecting fermentation supernatant, adding the fermentation supernatant into the activated macroporous adsorption resin obtained in the step (1), and placing the activated macroporous adsorption resin at the temperature of 4-6 ℃ for adsorption for 24-48h to obtain adsorption resin I;
the LB culture medium comprises the following substances in percentage by weight: 1.0-1.2% NaCl,0.5-0.6% yeast extract, 1.0-1.2% tryptone;
(3) Eluting the adsorption resin I obtained in the step (2) with 30-50% ethanol by volume concentration [ the dosage of 30-50% ethanol is calculated by adding 100-250mL of 30-50% ethanol into each liter of fermentation supernatant obtained in the step (2) ], continuously eluting with 55-75% ethanol by volume concentration [ the dosage of 55-75% ethanol is calculated by adding 200-500mL of 55-75% ethanol solution into each liter of fermentation supernatant obtained in the step (2) ], concentrating the obtained 55-75% ethanol eluent under reduced pressure (concentrating to rotary evaporation at the temperature of 40-60 ℃) to obtain a crude extract;
dissolving the collected eluates with PBS, and allowing to act on human liver cancer SMMC-7721 cells for 24h, wherein the result shows that 75% ethanol eluate has anti-tumor activity;
(4) Re-dissolving the crude extract prepared from the 55-75% ethanol eluent obtained in the step (3) by using an acetonitrile solution with the mass concentration of 5-10%, and loading the re-dissolved crude extract on RESOURCE TM RPC 3mL column, with 2-80wt% acetonitrile: linearly eluting with double distilled water solution at ultraviolet absorption wavelength OD 280 At nm, the absorption peak A11 has anti-tumor activity;
(5) Collecting peak A11, vacuum drying, re-dissolving with acetonitrile solution with mass concentration of 5-10%, and loading onto RESOURCE TM RPC 3mL column, with 15-55wt% acetonitrile: linearly eluting with double distilled water solution at ultraviolet absorption wavelength OD 280 At nm, the absorption peak A11 has anti-tumor activity, namely anti-tumor lipopeptide bacillomycin Lb (bacillus Lb).
The molecular weight of the anti-tumor lipopeptide bacillomycin Lb obtained by the invention is 1035.5427 ([ M + H ]] + ) Completely different from BaX030 antineoplastic active substance molecular weight 122.14 in the granted patent of separation method and application of a strain of bacillus amyloliquefaciens and active metabolites thereof.
The antitumor lipopeptide bacillomycin Lb obtained by the invention acts on human breast cancer cells (MDA-MB-231 and MCF-7), mouse breast cancer cells (4T-1), human liver cancer cells (SMMC-7721), mouse melanoma cells (B16) and mouse colon cancer cells (CT 26), so that the tumor cells shrink and become round, vacuole degeneration is presented, and the antitumor lipopeptide bacillomycin Lb has antitumor activity.
The antitumor lipopeptid bacillomycin Lb obtained by the method has small molecular weight and medium polarity, and can achieve the effect of separating other metabolites by a medium-polarity macroporous resin adsorption method. The method does not use harmful organic solvent, is simple and feasible, and can achieve the purpose of quickly separating and purifying the anti-tumor active substance baccatin Lb.
The bacillus amyloliquefaciens X030 used by the invention is a biocontrol bacterium separated from Arachis hypogaea Hayata by the inventor, and the preservation number of the strain is (CCTCC No. M2014159). The experiment of the subject group shows that the strain can secrete active small molecules with strong antibacterial and antitumor effects. According to the invention, a resin adsorption and rapid separation and purification system is adopted to obtain a BaX030 metabolic antitumor active product with high purity, and the product is identified as bacitracin Lb through nuclear magnetic resonance and has broad-spectrum antitumor activity.
Drawings
FIG. 1 is a first isolation chromatogram of antitumor lipopeptide bacillomycin Lb, with cell activity tracing inserted;
FIG. 2 is a second separation chromatogram of antitumor lipopeptide bacillomycin Lb, with cell activity tracking and HPLC analysis shown in the inset;
FIG. 3 is a chart of anti-tumor lipopeptide bacillomycin Lb HPLC analysis;
FIG. 4 is a diagram of anti-tumor lipopeptide bacillomycin Lb MALDI matrix assisted laser desorption ionization mass spectrometry;
FIG. 5 is a first-order mass spectrum (molecular weight 122.14) of BaX030 as an antitumor agent in granted patent "A Bacillus amyloliquefaciens strain and method for separating active metabolites thereof and use thereof";
FIG. 6 is a graph showing the effect of anti-tumor lipopeptide bacillomycin Lb on cell morphology of breast cancer, liver cancer, melanoma, and colon cancer.
The specific implementation mode is as follows:
the following examples are intended to further illustrate the invention, but are not intended to limit the invention.
In the present specification, the percentages refer to mass percentages unless otherwise indicated.
Example (b):
the embodiment comprises the following steps:
1. preparation of crude lipopeptide extract
(1) Activating DM301 macroporous adsorbent resin (Shanghai Miteng Biotech Co., ltd.) with anhydrous ethanol, and washing with double-distilled water until no alcohol smell exists to obtain activated DM301 macroporous adsorbent resin;
(2) Transferring 1wt% of Bacillus amyloliquefaciens BaX030 (CCTCC No: M2014159, see CN 104630111A) into an LB culture medium (1 wt% NaCl,0.5wt% yeast extract and 1wt% tryptone), carrying out shaking culture for 40-48h under the conditions of 30 ℃ and 120rpm/min, centrifugally collecting fermentation supernatant, adding the fermentation supernatant into the activated DM301 macroporous adsorption resin obtained in the step (1), wherein the weight of the activated DM301 macroporous adsorption resin is 5 times of the volume of fermentation liquid, and placing the activated DM301 macroporous adsorption resin at 4 ℃ for adsorption for 48h to obtain adsorption resin I;
(3) Eluting the adsorption resin I obtained in the step (2) by using 50% ethanol by volume concentration [ the dosage of 50% ethanol is calculated by adding 100mL of 50% ethanol into each liter of fermentation supernatant obtained in the step (2) ], continuously eluting by using 75% ethanol by volume concentration [ the dosage of 75% ethanol is calculated by adding 500mL of 75% ethanol solution into each liter of fermentation supernatant obtained in the step (2) ], and concentrating the obtained 75% ethanol eluent under reduced pressure to obtain a crude extract;
dissolving the collected eluates with PBS, and allowing to act on human liver cancer SMMC-7721 cells for 24h, wherein the result shows that 75% ethanol eluate has anti-tumor activity;
2. isolation of crude bacitracin Lb
(4) Re-dissolving the crude extract prepared from the 75% ethanol eluent obtained in the step (3) by using an acetonitrile solution with the mass concentration of 5%, and loading the re-dissolved crude extract on RESOURCE TM RPC 3mL column, linear elution with 2% -80% acetonitrile, OD at UV absorption wavelength 280 Collecting the eluted sample at nm in an equal volume of 2mL, vacuum drying, dissolving with PBS, respectively acting on SMMC-7721 cells, and having an absorption peak A11 with anti-tumor activity;
3. purification of bacitracin Lb
(5) A large amount of active peak A11 is collected, and after vacuum drying,redissolving with 2wt% acetonitrile solution, loading onto RPC 3mL chromatographic column, linearly eluting with 15% -55% acetonitrile solution, and measuring OD at ultraviolet absorption wavelength 280 At nm, collecting the eluted sample in an equal volume of 2mL, vacuum drying, dissolving with PBS, and respectively acting on SMMC-7721 cells, wherein the absorption peak A11 has antitumor activity, namely antitumor lipopeptid bacitracin Lb (bacillus Lb).
4. Antitumor spectrum of bacitracin Lb
Weighing 1.034-1.1mg bacitracin Lb, dissolving in 1-1.2mL PBS, acting on human breast cancer cells (MDA-MB-231 and MCF-7), mouse breast cancer cells (4T-1), human liver cancer cells (SMMC-7721), mouse melanoma cells (B16) and mouse colon cancer cells (CT 26) at a concentration of 5-40 μ M, and observing cell vacuolation or shrinkage and rounding, IC (integrated circuit) under an inverted microscope 50 The value was 15-25. Mu.M.
Claims (5)
1. A method for separating and purifying bacillomycin from bacillus amyloliquefaciens is characterized by comprising the following steps:
(1) Activating the macroporous adsorbent resin with absolute ethyl alcohol, and washing with double distilled water until no alcohol smell exists to obtain activated DM301 macroporous adsorbent resin;
(2) Transferring Bacillus amyloliquefaciens BaX030 into an LB culture medium, carrying out shake culture on 44-48h under the conditions of 30-32 ℃ and 120-130rpm/min, centrifuging, collecting fermentation supernatant, adding the fermentation supernatant into the activated DM301 macroporous adsorption resin obtained in the step (1), and placing the activated DM301 macroporous adsorption resin at 4-6 ℃ for adsorbing 24-48h to obtain adsorption resin I;
the collection number of the bacillus amyloliquefaciens BaX030 is CCTCC No. M2014159;
the LB culture medium comprises the following substances in percentage by weight: 1.0-1.2% NaCl,0.5-0.6% yeast extract, 1.0-1.2% tryptone;
(3) Eluting the adsorption resin I obtained in the step (2) by using 30-50% ethanol, continuously eluting by using 55-75% ethanol, and concentrating the obtained 55-75% ethanol eluent under reduced pressure to obtain a crude extract; the dosage of 30-50% ethanol is calculated by adding 100-250ml30-50% ethanol into each liter of fermentation supernatant obtained in the step (2); the dosage of 55-75% ethanol is calculated by adding 200-500ml of 55-75% ethanol solution into each liter of fermentation supernatant obtained in the step (2);
(4) Re-dissolving the crude extract prepared from the 55-75% ethanol eluent obtained in the step (3) by using an acetonitrile solution with the mass concentration of 5-10%, and loading the re-dissolved crude extract on RESOURCE TM RPC 3mL chromatography column, purified using 2-80wt% acetonitrile: linearly eluting with double distilled water solution at ultraviolet absorption wavelength OD 280 At nm, the absorption peak A11 has anti-tumor activity;
(5) Collecting a large amount of peak A11, vacuum drying to obtain bacitracin Lb crude product, re-dissolving with acetonitrile solution with mass concentration of 5-10%, and loading onto RESOURCE TM RPC 3mL column purified using 15-55wt% acetonitrile: linearly eluting with double distilled water solution at ultraviolet absorption wavelength OD 280 At nm, the absorption peak A11 has anti-tumor activity, namely anti-tumor lipopeptide bacillomycin Lb.
2. The method for separating and purifying bacillomycin from bacillus amyloliquefaciens according to claim 1, wherein in the step (1), the macroporous absorption resin is a common resin DM301.
3. A method for separating and purifying bacillomycin from Bacillus amyloliquefaciens according to claim 1 or 2, wherein in the step (1), the dosage of the macroporous adsorption resin is 20-50g and the adsorption time is 24-48h per liter of fermentation supernatant.
4. A method for separating and purifying bacillomycin from Bacillus amyloliquefaciens according to claim 1 or 2, wherein in the step (3), concentration is rotary evaporation at 40-60 ℃.
5. A process for the isolation and purification of bacilycin from Bacillus amyloliquefaciens as claimed in claim 1 or claim 2 wherein in step (5) crude bacilycin Lb is passed through RPC reverse phase chromatography column RESOURCE TM RPC 3mL, 15 μm, 6.4X 100 mm in Ä KTA avant 25 for secondary purification.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630111A (en) * | 2015-02-09 | 2015-05-20 | 湖南师范大学 | Bacillus amyloliquefaciens and separation method and application of active metabolites thereof |
| CN105985919A (en) * | 2015-02-12 | 2016-10-05 | 上海医药工业研究院 | Bacillus and application thereof |
| CN108250270A (en) * | 2016-12-29 | 2018-07-06 | 天津领世生物科技开发有限公司 | A kind of method of the enrichment extraction Daptomycin from zymotic fluid |
| CN108611388A (en) * | 2018-04-11 | 2018-10-02 | 大连民族大学 | A method of improving bacillus amyloliquefaciens Q-426 Cyclic lipopeptide antibiotic yield |
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| WO2013086003A1 (en) * | 2011-12-05 | 2013-06-13 | The Ohio State University | Antimicrobial agent, bacterial strain, biosynthesis, and methods of use |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630111A (en) * | 2015-02-09 | 2015-05-20 | 湖南师范大学 | Bacillus amyloliquefaciens and separation method and application of active metabolites thereof |
| CN105985919A (en) * | 2015-02-12 | 2016-10-05 | 上海医药工业研究院 | Bacillus and application thereof |
| CN108250270A (en) * | 2016-12-29 | 2018-07-06 | 天津领世生物科技开发有限公司 | A kind of method of the enrichment extraction Daptomycin from zymotic fluid |
| CN108611388A (en) * | 2018-04-11 | 2018-10-02 | 大连民族大学 | A method of improving bacillus amyloliquefaciens Q-426 Cyclic lipopeptide antibiotic yield |
Non-Patent Citations (2)
| Title |
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| Induction of apoptosis in human cancer cells by a Bacillus lipopeptide bacillomycin D;Sachin N. Hajare等;《Biochimie》;20130614;第95卷;1722-1731 * |
| 解淀粉芽孢杆菌Q-426 Bacillomycin D 的分离纯化及其抗肿瘤活性;权春善等;《生物工程学报》;20180225;第34卷(第2期);235-245 * |
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Effective date of registration: 20231008 Address after: 518000, 23rd Floor, Sannuo Smart Building, No. 3388 Binhai Avenue, Binhai Community, Yuehai Street, Nanshan District, Shenzhen, Guangdong Province Patentee after: Qiansheng (Shenzhen) Kechuang Group Co.,Ltd. Address before: 410081 No. 36, Mount Lu, Changsha, Hunan, Yuelu District Patentee before: HUNAN NORMAL University |