CN116121324A - Method for separating cyclic lipopeptide Baelezcin A from bacillus bailii - Google Patents
Method for separating cyclic lipopeptide Baelezcin A from bacillus bailii Download PDFInfo
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Abstract
The invention discloses a method for separating cyclic lipopeptide Baelezcin A from bacillus bailii, which comprises the following steps: fermenting bacillus belicus to obtain fermentation liquor; centrifuging the fermentation liquor, collecting supernatant, acidifying the supernatant, centrifuging and collecting precipitate; adding methanol into the washed precipitate for extraction to obtain lipopeptide crude extract AME; dissolving AME with methanol, and performing reverse silica gel column chromatography to obtain AME-4; dissolving AME with methanol, and performing hydroxypropyl sephadex column chromatography to obtain AME-4-5; AME-4-5 is prepared into solution by methanol, and purified by a high performance liquid chromatograph and a C18 column to obtain the compound cyclic lipopeptide Baelezcin A. The method is simple and easy to operate, and the obtained cyclic lipopeptide Baelezcin A has no report yet and has very important significance for researching effective components of natural products of microorganisms.
Description
Technical Field
The invention relates to the technical field of compound lifting, in particular to a method for separating cyclic lipopeptide Baelezcin A from bacillus bailii.
Background
In recent years, fungi, bacteria and actinomycetes have been widely used in research on biocontrol, and bacillus stands out as an excellent biocontrol bacterium. Bacillus is ubiquitous in nature, can produce highly resistant endospores under adverse conditions, is an important microbial resource, has the advantages of inhibiting diseases, promoting plant growth, being strong in reproductive capacity, stimulating metabolite diversity and the like, and is widely studied by scholars at home and abroad. It is unclear which of the active secondary metabolites of bacillus act in a large number, and specifically which of the substance components act.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the related art to some extent. To this end, the object of the present invention is to provide a method for isolating the cyclic lipopeptide Baelezcin A from Bacillus bailii.
To achieve the above object, an embodiment of the present invention provides a method for isolating a cyclic lipopeptide Baelezcin a from bacillus beleiensis, comprising the steps of:
(1) Inoculating bacillus belicus into a fermentation medium for fermentation to obtain fermentation liquor;
(2) Centrifuging the fermentation liquor to remove thalli, collecting supernatant, acidifying the supernatant, centrifuging, collecting precipitate, and washing the precipitate;
(3) Adding methanol into the washed precipitate for extraction for 3 times, each time for 12 hours, combining organic phases, and removing the organic phase solvent by rotary evaporation to obtain lipopeptide crude extract AME;
(4) Dissolving lipopeptide crude extract AME with methanol, and performing reverse silica gel column chromatography to obtain extract AME-4;
(5) Dissolving extract AME with methanol, and subjecting to hydroxypropyl dextran gel column chromatography to obtain extract AME-4-5;
(6) Preparing extract AME-4-5 into solution with methanol, and purifying with high performance liquid chromatograph and C18 column to obtain cyclic lipopeptide Baelezcin A.
According to the method for separating the cyclic lipopeptide Baelezcin A from the bacillus beleiensis, disclosed by the embodiment of the invention, the method is simple and easy to operate, and the obtained cyclic lipopeptide Baelezcin A has no report yet, has potential activity and application in the aspects of being used as a biosurfactant, a biocontrol microbial agent and the like, and has very important significance for researching the effective components of natural products of microorganisms.
In addition, the method for separating the cyclic lipopeptide Baelezcin A from bacillus bailii according to the embodiment of the invention can also have the following additional technical characteristics:
alternatively, in step (4), 5 tubes were collected per system, 200mL per tube, according to a methanol-water elution of 25%,50%,75% and 100% methanol, with a flow rate set to 10 mL/min; the first two tubes of the 100% methanol component of the fourth system collected were subjected to rotary evaporation to remove the organic phase solvent, thus obtaining extract AME-4.
Optionally, in step (5), the setting procedure is: 15-20s/drop, connecting 200 test tubes every 30min, cutting 200 test tubes into 8 components, collecting the fifth component, and removing organic phase solvent by rotary evaporation to obtain extract AME-4-5.
Optionally, in the step (6), the sample injection amount is 100 mu L, 1mL/min of 66.0% acetonitrile water is adopted, 0.1% trifluoroacetic acid is added for eluting, and 210nm wavelength detection is adopted; collecting the large peak of 33-37min to obtain the compound cyclic lipopeptide Baelezcin A.
Optionally, in step (1), the fermentation is performed for 72 hours.
Optionally, in the step (2), acidifying to the supernatant, adding 6mol/L HCl to adjust the pH value to 2, and acidifying for 24 hours.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a high performance liquid chromatogram of a compound according to an embodiment of the invention;
FIG. 2 is an ESI-MS spectrum of Baelezcin A according to an embodiment of the invention;
FIG. 3 is a diagram of the chemical structure and key two-dimensional nuclear magnetic resonance correlation of Baelezcin A according to an embodiment of the present invention;
FIG. 4 is an infrared spectrum of Baelezcin A according to an embodiment of the invention;
FIG. 5 is a graph showing the effect of Baelezcin A on Botrytis cinerea growth according to an embodiment of the present invention.
Detailed Description
The technical scheme of the invention is described below through specific examples. It is to be understood that the mention of one or more method steps of the present invention does not exclude the presence of other method steps before and after the combination step or that other method steps may be interposed between these explicitly mentioned steps; it should also be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Moreover, unless otherwise indicated, the numbering of the method steps is merely a convenient tool for identifying the method steps and is not intended to limit the order of arrangement of the method steps or to limit the scope of the invention in which the invention may be practiced, as such changes or modifications in their relative relationships may be regarded as within the scope of the invention without substantial modification to the technical matter.
In order to better understand the above technical solution, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the invention are shown, it should be understood that the invention may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
Wherein, bacillus belicus, latin name is Bacillus velezensis, and is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of 20398 at the month 17 of 2020, and the preservation unit address is North Chen West Lu No. 1 and 3 in the Chaoyang area of Beijing city.
The invention will now be described with reference to specific examples, which are intended to be illustrative only and not limiting in any way.
EXAMPLES isolation of the Cyclic lipopeptides Baelezcin A from Bacillus bailii
1) Shake flask fermentation culture was performed using a shaking incubator (ZQZY-BF 9.9, shanghai know Chu instrument) at 37℃and 200rpm for 72 hours, 10L of the fermented Bacillus bailii broth was centrifuged at 12000rpm and 4℃for 10 minutes to remove the cells, and 6mol/L HCl was added to the supernatant to adjust the pH to 2. After 24h, the acidified supernatant was centrifuged under the same conditions and the bottom pellet was collected.
2) After washing the precipitate with sterile distilled water, 100% methanol was added for extraction 3 times each for 12 hours, the organic phases were combined and the organic solvent was removed by rotary evaporation to obtain 2.2097g of lipopeptide crude extract (AME).
3) The lipopeptide crude extract was prepared with 25% methanol (methanol: water=1:3, v/v) (dissolution, reverse-phase silicse:Sup>A GE column chromatography (Huiksep P0100; glass column: 25 cm. Times. Phi.5 cm; bed volume about 500 m; packing: YMC. Times. GE ODS-A-HG,50 μm), methanol-water system elution (25%, 50%,75% and 100% methanol), flow rates were set at 10 m/min, 5 tubes were collected per system, 200m per tube; the first two tubes of the 100% methanol component of the fourth system collected were freed from the organic phase solvent by rotary evaporation to give AME-4 (598.6 mg).
4) Component AME-4 (598.6 mg) from step 3) was dissolved with a small amount of 100% methanol. Eluting with hydroxypropyl dextran gel column chromatography (column height: 2m; diameter: 2.5cm; packing: sephadex LH-20, 25-100 μm), collecting sample with automatic collector, and setting the procedures as follows: 15-20s/drop, connecting one test tube every 30min, and connecting 200 test tubes in total; the 200 tube was cut into 8 fractions, wherein the fifth fraction (105-125 tube) was collected and the organic phase solvent was removed by rotary evaporation to give AME-4-5 (83.2 mg).
5) AME-4-5 (83.2 mg) collected in step 4) was prepared as a 5mg/mL solution with 100% methanol and finely separated using an Agilent 1260 index II high performance liquid chromatograph (The Agilent 1260infinity II Quaternary Pump VL G7111A solution transfer unit and 1260infinity II DAD WR G7115A high sensitivity diode array detector) equipped with a ulmate LP C18 column (Welch, shanghai, china,5 μm,4.6 x 250 mm). The sample injection amount is 100 mu L, 1mL/min of 66.0% acetonitrile water is adopted, 0.1% trifluoroacetic acid is added for eluting, and 210nm wavelength detection is adopted; collecting large peaks about 33-37min, and concentrating to obtain cyclic lipopeptide Baelezcin A (about 24.5mg, purity over 95% and liquid chromatogram shown in figure 1).
The obtained cyclic lipopeptide Baelezcin A is white powder, and has molecular formula C as determined by positive ion resolution mass spectrum 52 H 91 N 7 O 13 ([M+H] + M/z measured 1022.6742, calculated 1022.6753), m/z1,022.6742 was used as a precursor ion for further ESI-MS/MS analysis. M/z1022.674 [ M+H ] as determined by mass spectrometry] + A kind of electronic device x The product pattern of Max is shown in FIG. 2. Found in spectra y Max ion at m/z1,004.641 corresponds to H lost from m/z1022.674 2 O (-18 Da). FIG. 2A also shows that the molecular mass is based x ion and sum y The main fragment ion peak of the ion series. Product(s) x ion m/z 909.58 is interpreted as the loss of one Leu amino acid residue from the peptide and the fragment ion of m/z 796.51 is generated by the loss of 2 Leu amino acid residues from the peptide. Other fragment ions (m/z 667.440,554.356,441.269,342.203 and 213.160) in FIG. 2B can be used to determine the remaining peptide sequence using similar calculations. Product(s) y ions show x The peptide sequences of the ions were identical and the fatty acid (m/z 213.160) and seven amino acid sequences (Glu-Val-Leu-Leu-Glu-Leu-Leu) in FIG. 1 were explained. Thus, baelezcin A was initially speculated to be a Cyclic Lipopeptide (CLP). TABLE 1Baelezcin A 1 H NMR 13 All characteristic signals (δ, ppm) for CLP were determined in the C NMR spectrum: in CD 3 Recorded in OD 1 H NMR showedThere is a long aliphatic chain (CH 2 in δH2.27-1.37) and a peptide backbone consisting of 7 "-NH-" with an alpha-H signal between δH2.03-4.78. Resonance at δH2 5.29 indicates the presence of a further "-OH", and a signal of "-CH3" is observed at δH2 0.89-1.01. In addition, baelezcin A 13 The C NMR spectrum shows the presence of 10 carbonyl groups at δC174.0-176.8, which is attributable to amino acids and fatty acids. For a pair of 1 H- 1 H COSY、 1 H- 13 CHSQC 1 H- 13 Further analysis of the spectra of C HMBC enabled us to determine cyclic lipopeptides with a Baelezcin a structure linked by two Glu, one Val, four Leu and saturated linear β -OH-C13 fatty acid residues (fig. 3A), part of which key two-dimensional nuclear magnetic resonance correlations are shown in fig. 3B. Shown by IR chart of IR spectrum at 3319.38cm -1 (peptide "-NH-"), 2962.56 and 2925.91cm -1 (fatty acid residues) and 1649.08cm -1 (peptide bond "-c=o-") has strong absorption signals, respectively (fig. 4). This also supports the results of nuclear magnetic resonance and MS analysis.
Table 1Baelezcin A C, H Attribute
Example 2 control of Boelezcin A
1. A pellet (7.0 mm diameter) of Botrytis cinerea (ACCC 37273Botrytis cinerea Pers.ex Fr.) was inoculated in the center of PDA solid medium containing 0, 25 and 50mg/L Baelezcin A and cultured at 25 ℃. Colony diameters were measured every 24h. And the inhibition ratio was calculated. In addition, spores of Botrytis cinerea were incubated in PDB liquid medium containing 0, 25 and 50mg/mL Baelezcin A concentration gradient for 10h, and the germination rate and the length of the shoot tubes were examined by microscopic observation.
As shown in FIG. 5, baelezcin A significantly inhibited the growth of Botrytis cinerea mycelium, and as shown in FIGS. 5A and B, colony diameters decreased with increasing Baelezcin A dose, significantly inhibited the growth of Botrytis cinerea mycelium (P < 0.05). After 50mg/L Baelezcin A is incubated for 72 hours, the inhibition effect on hypha growth is still strong, the colony diameter is about 36% of that of a control, and the hypha area is reduced by 87.4%. After careful examination of the mycelium morphology, agar plates containing Baelezcin A were found to show shorter mycelium, non-diffuse globules at the colony edges, and the blank was radial (FIG. 5A).
As shown in Table 2, baelezcin A (mg/L) reduced the germination rate and shoot tube length of Botrytis cinerea spores.
TABLE 2 influence of Baelezcin A on Botrytis cinerea spore germination Rate and bud tube Length
Note that: the different letters in the row indicate that there was a statistical difference (P < 0.05) in the Duncan multiplex assay.
In summary, according to the embodiments of the present invention, a novel compound, the cyclic lipopeptide Baelezcin A, was isolated from Bacillus Bei Lai, and the isolation procedure was simple and easy to operate; the novel compound is not reported yet, and can prevent the occurrence of gray mold after fruit and vegetable picking.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms should not be understood as necessarily being directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Further, one skilled in the art can engage and combine the different embodiments or examples described in this specification.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (6)
1. A method for isolating the cyclic lipopeptide Baelezcin a from bacillus beleiensis, comprising the steps of:
(1) Inoculating bacillus belicus into a fermentation medium for fermentation to obtain fermentation liquor;
(2) Centrifuging the fermentation liquor to remove thalli, collecting supernatant, acidifying the supernatant, centrifuging, collecting precipitate, and washing the precipitate;
(3) Adding methanol into the washed precipitate for extraction for 3 times, each time for 12 hours, combining organic phases, and removing the organic phase solvent by rotary evaporation to obtain lipopeptide crude extract AME;
(4) Dissolving lipopeptide crude extract AME with methanol, and performing reverse silica gel column chromatography to obtain extract AME-4;
(5) Dissolving extract AME with methanol, and subjecting to hydroxypropyl dextran gel column chromatography to obtain extract AME-4-5;
(6) Preparing extract AME-4-5 into solution with methanol, and purifying with high performance liquid chromatograph and C18 column to obtain cyclic lipopeptide Baelezcin A.
2. The method according to claim 1, wherein in step (4), the flow rate is set to 10mL/min according to methanol-water elution of 25%,50%,75% and 100% methanol, and each system is collected in 5 tubes of 200mL per tube; the first two tubes of the 100% methanol component of the fourth system collected were subjected to rotary evaporation to remove the organic phase solvent, thus obtaining extract AME-4.
3. The method of claim 1, wherein in step (5), the program is set to: 15-20s/drop, connecting 200 test tubes every 30min, cutting 200 test tubes into 8 components, collecting the fifth component, and removing organic phase solvent by rotary evaporation to obtain extract AME-4-5.
4. The method of claim 1, wherein in step (6), the sample injection amount is 100 μl, 1mL/min of 66.0% acetonitrile water is used, 0.1% trifluoroacetic acid is added for eluting, and 210nm wavelength detection is performed; collecting the large peak of 33-37min to obtain the compound cyclic lipopeptide Baelezcin A.
5. The method of claim 1, wherein in step (1), the fermentation is performed for 72 hours.
6. The method according to claim 1, wherein in the step (2), the supernatant is acidified to a pH of 2 by adding 6mol/L HCl, and the mixture is acidified for 24 hours.
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| CN110117556A (en) * | 2019-03-27 | 2019-08-13 | 海南大学 | A kind of efficient biocontrol microorganisms HN-2 and zymotechnique for producing lipopeptid substance |
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