CN114524861A - Antifungal lipopeptide and application of streptomycete in preparation of antifungal lipopeptide - Google Patents
Antifungal lipopeptide and application of streptomycete in preparation of antifungal lipopeptide Download PDFInfo
- Publication number
- CN114524861A CN114524861A CN202210078489.9A CN202210078489A CN114524861A CN 114524861 A CN114524861 A CN 114524861A CN 202210078489 A CN202210078489 A CN 202210078489A CN 114524861 A CN114524861 A CN 114524861A
- Authority
- CN
- China
- Prior art keywords
- lipopeptide
- antifungal
- culture medium
- streptomyces
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108010028921 Lipopeptides Proteins 0.000 title claims abstract description 121
- 230000000843 anti-fungal effect Effects 0.000 title claims abstract description 38
- 229940121375 antifungal agent Drugs 0.000 title claims abstract description 38
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 241001655322 Streptomycetales Species 0.000 title claims description 15
- 241000187747 Streptomyces Species 0.000 claims abstract description 25
- 244000053095 fungal pathogen Species 0.000 claims abstract description 14
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 120
- 239000001963 growth medium Substances 0.000 claims description 36
- 238000010828 elution Methods 0.000 claims description 27
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 22
- 239000000126 substance Substances 0.000 claims description 21
- 238000000746 purification Methods 0.000 claims description 19
- 239000012071 phase Substances 0.000 claims description 16
- 238000000926 separation method Methods 0.000 claims description 16
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 15
- 238000010521 absorption reaction Methods 0.000 claims description 15
- 239000006228 supernatant Substances 0.000 claims description 15
- 230000001580 bacterial effect Effects 0.000 claims description 13
- 239000000287 crude extract Substances 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 11
- 238000004811 liquid chromatography Methods 0.000 claims description 11
- 239000000243 solution Substances 0.000 claims description 11
- 238000012258 culturing Methods 0.000 claims description 10
- 239000003480 eluent Substances 0.000 claims description 10
- 238000004108 freeze drying Methods 0.000 claims description 10
- 238000002347 injection Methods 0.000 claims description 10
- 239000007924 injection Substances 0.000 claims description 10
- 239000012074 organic phase Substances 0.000 claims description 10
- 239000000706 filtrate Substances 0.000 claims description 9
- 239000002244 precipitate Substances 0.000 claims description 9
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 8
- 238000003825 pressing Methods 0.000 claims description 8
- 238000007710 freezing Methods 0.000 claims description 7
- 230000008014 freezing Effects 0.000 claims description 7
- 238000002414 normal-phase solid-phase extraction Methods 0.000 claims description 7
- 239000000047 product Substances 0.000 claims description 7
- 230000004913 activation Effects 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 6
- 238000000622 liquid--liquid extraction Methods 0.000 claims description 6
- 238000000638 solvent extraction Methods 0.000 claims description 6
- 230000002401 inhibitory effect Effects 0.000 claims description 5
- 238000007747 plating Methods 0.000 claims description 5
- 238000006748 scratching Methods 0.000 claims description 5
- 230000002393 scratching effect Effects 0.000 claims description 5
- 238000002791 soaking Methods 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 238000010257 thawing Methods 0.000 claims description 5
- 240000008067 Cucumis sativus Species 0.000 claims description 3
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 claims description 3
- 240000008790 Musa x paradisiaca Species 0.000 claims description 3
- 235000018290 Musa x paradisiaca Nutrition 0.000 claims description 3
- 240000007594 Oryza sativa Species 0.000 claims description 3
- 235000007164 Oryza sativa Nutrition 0.000 claims description 3
- 235000009566 rice Nutrition 0.000 claims description 3
- 239000012141 concentrate Substances 0.000 claims description 2
- 239000003814 drug Substances 0.000 claims description 2
- 240000001462 Pleurotus ostreatus Species 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 230000008021 deposition Effects 0.000 claims 1
- 229940079593 drug Drugs 0.000 claims 1
- 230000002906 microbiologic effect Effects 0.000 claims 1
- 244000005700 microbiome Species 0.000 claims 1
- 238000011085 pressure filtration Methods 0.000 claims 1
- 238000011160 research Methods 0.000 abstract description 8
- 239000000284 extract Substances 0.000 abstract description 7
- 230000003042 antagnostic effect Effects 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 5
- 230000007246 mechanism Effects 0.000 abstract description 5
- 238000000034 method Methods 0.000 abstract description 5
- 239000000178 monomer Substances 0.000 abstract description 5
- 238000002474 experimental method Methods 0.000 abstract description 4
- 230000015572 biosynthetic process Effects 0.000 abstract description 3
- 230000000443 biocontrol Effects 0.000 abstract description 2
- 239000002207 metabolite Substances 0.000 abstract description 2
- 150000002500 ions Chemical class 0.000 description 19
- AXMACOGXBDPVJT-UHFFFAOYSA-N 3-[6-(3-amino-1-hydroxy-3-oxopropyl)-12-(2-amino-2-oxoethyl)-9-benzyl-21-(carboxymethyl)-15,18-bis(hydroxymethyl)-10,13,25-trimethyl-24-(12-methyltetradecanoylamino)-2,5,8,11,14,17,20,23-octaoxo-1-oxa-4,7,10,13,16,19,22-heptazacyclopentacos-3-yl]propanoic Chemical compound CN1C(=O)C(CC(N)=O)N(C)C(=O)C(CO)NC(=O)C(CO)NC(=O)C(CC(O)=O)NC(=O)C(NC(=O)CCCCCCCCCCC(C)CC)C(C)OC(=O)C(CCC(O)=O)NC(=O)C(C(O)CC(N)=O)NC(=O)C1CC1=CC=CC=C1 AXMACOGXBDPVJT-UHFFFAOYSA-N 0.000 description 6
- 108010085983 lipopeptin A Proteins 0.000 description 6
- 239000012634 fragment Substances 0.000 description 4
- 238000001514 detection method Methods 0.000 description 3
- 150000004665 fatty acids Chemical group 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 2
- 108010019477 S-adenosyl-L-methionine-dependent N-methyltransferase Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000008485 antagonism Effects 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 description 2
- SCIFESDRCALIIM-UHFFFAOYSA-N n-methylphenylalanine Chemical compound CNC(C(O)=O)CC1=CC=CC=C1 SCIFESDRCALIIM-UHFFFAOYSA-N 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 108010000785 non-ribosomal peptide synthase Proteins 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 229930001119 polyketide Natural products 0.000 description 2
- 125000000830 polyketide group Chemical group 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 238000004885 tandem mass spectrometry Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- LNSMPSPTFDIWRQ-VKHMYHEASA-N (2s)-4-amino-2-(methylamino)-4-oxobutanoic acid Chemical compound CN[C@H](C(O)=O)CC(N)=O LNSMPSPTFDIWRQ-VKHMYHEASA-N 0.000 description 1
- VQINCDSXSZLRHY-VKHMYHEASA-N (2s)-5-amino-2-(hydroxyamino)-5-oxopentanoic acid Chemical compound NC(=O)CC[C@H](NO)C(O)=O VQINCDSXSZLRHY-VKHMYHEASA-N 0.000 description 1
- RXZBMPWDPOLZGW-XMRMVWPWSA-N (E)-roxithromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=N/OCOCCOC)/[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 RXZBMPWDPOLZGW-XMRMVWPWSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 235000009024 Ceanothus sanguineus Nutrition 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 240000003553 Leptospermum scoparium Species 0.000 description 1
- 235000015459 Lycium barbarum Nutrition 0.000 description 1
- 241000168254 Siro Species 0.000 description 1
- 241001546732 Streptomyces sp. XY006 Species 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 108010002015 fengycin Proteins 0.000 description 1
- CUOJDWBMJMRDHN-VIHUIGFUSA-N fengycin Chemical compound C([C@@H]1C(=O)N[C@H](C(=O)OC2=CC=C(C=C2)C[C@@H](C(N[C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C)C(=O)N2CCC[C@H]2C(=O)N[C@@H](CCC(N)=O)C(=O)N1)[C@@H](C)O)=O)NC(=O)[C@@H](CCCN)NC(=O)[C@H](CCC(O)=O)NC(=O)C[C@H](O)CCCCCCCCCCCCC)[C@@H](C)CC)C1=CC=C(O)C=C1 CUOJDWBMJMRDHN-VIHUIGFUSA-N 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000007269 microbial metabolism Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 229960000988 nystatin Drugs 0.000 description 1
- VQOXZBDYSJBXMA-NQTDYLQESA-N nystatin A1 Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/CC/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 VQOXZBDYSJBXMA-NQTDYLQESA-N 0.000 description 1
- 238000004262 preparative liquid chromatography Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 229960005224 roxithromycin Drugs 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
- A01N37/46—N-acyl derivatives
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses antifungal lipopeptide and application of streptomyces in preparation of antifungal lipopeptide, the scheme ingeniously extracts lipopeptide A and lipopeptide B through metabolites of streptomyces, and the monomer of the lipopeptide A and the lipopeptide B with the purity higher than 95% can be prepared by combining the preparation method of the scheme, so that the method is of important help for subsequent experiments of analyzing the mechanism of the antagonistic pathogenic fungi and the plant biocontrol effect; meanwhile, the difference of the antagonistic mechanisms of lipopeptide A and lipopeptide B can also point out the direction for the subsequent biosynthesis research of lipopeptide A and lipopeptide B.
Description
Technical Field
The invention relates to the field of biotechnology, and in particular relates to application of antifungal lipopeptide and streptomyces in preparation of antifungal lipopeptide.
Background
Lipopeptide substances are strong surfactants generated by microbial metabolism, and are amphiphilic molecules consisting of a polypeptide ring formed by a plurality of amino acids and a hydrophobic fatty acid side chain. Lipopeptide substances comprise surfactant, fengycin, iturin, roxithromycin, lipopeptide A and the like. Lipopeptides have been reported to possess a variety of biological activities, including antibacterial, antifungal, antitumor and antiviral activities. The synthesis of lipopeptide substances is encoded and synthesized by non-ribosomal peptide synthetases (non-ribosomal peptide synthetases) and polyketides (polyketides synthases), and the genes generally exist in clusters, so the substances mostly exist in the form of homologues at the same time, and the separation and purification of the structural analogues are very difficult, so the current research is mostly about the inhibitory activity of lipopeptide complexes, but the slight change of the structures of the substances can cause great difference in the activity, so the development of a method for preparing lipopeptide monomers is particularly important.
The inventor separates a Streptomyces sp.XY006 strain in a laboratory, and researches that main secondary metabolites of the Streptomyces are lipopeptide A (lipopeptin A) and lipopeptin B (lipopeptin B) which is different from the lipopeptin A by one methylene group (provisionally named), and earlier experiments show that the lipopeptin A and the lipopeptin B are structurally different by only one methylene group but have obvious activity difference. And experiments prove that the mixed initial extract of the lipopeptide A and the lipopeptide B has obvious inhibition effect on various important agricultural pathogenic fungi, and corresponding research reports are not disclosed at present. On the basis, in order to further research the antagonistic mechanism, the applicant prepares the active monomers of the lipopeptides A and B by a series of separation and purification methods.
Disclosure of Invention
In view of the above, the present invention aims to provide an antifungal lipopeptide and an application of streptomyces in preparation of the antifungal lipopeptide, wherein the antifungal lipopeptide is reliable in implementation, efficient in preparation process, convenient and controllable.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:
an antifungal lipopeptide represented by formula C53H82N10O19The structural formula is as follows:
based on the above, the invention also provides an application of streptomyces in preparation of antifungal lipopeptide, which comprises the steps of freezing streptomyces and a culture medium thereof, then unfreezing and filtering the streptomyces under pressure to obtain a bacterial solution, concentrating and extracting the obtained bacterial solution with alcohol to obtain a crude extract, performing volume extraction on the crude extract by redissolving, n-butyl alcohol and the like for multiple times, collecting an organic phase for concentration, adjusting the concentrate to a preset concentration, passing through a solid phase extraction column, performing multi-stage gradient elution, collecting the eluate, separating and purifying the eluate to obtain the antifungal lipopeptide, wherein the antifungal lipopeptide comprises lipopeptide A and lipopeptide B, and the chemical formula of the lipopeptide A is C54H84N10O19The structural formula is as follows:
the chemical formula of the lipopeptide B is C53H82N10O19The structural formula is as follows:
as a possible implementation mode, the Streptomyces is Streptomyces XY006 which is named Streptomyces sp, is stored in the common microorganism center of the china culture collection and management committee on 12-13 th 2021, and has the storage address of No. 3 of western road 1 on north chen of yang-ward area in beijing and the storage number of CGMCC No. 24090.
As a preferred implementation choice, preferably, the application specifically includes the following steps:
(1) after the streptomycete XY006 is subjected to plate scratching and activation on an MS culture medium, the streptomycete XY006 is cultured for 3 days at the temperature of 30 ℃; then picking a single colony in a MYG culture medium, culturing for 2 days at 28 ℃ and 200rpm, then transferring the single colony to the MYG culture medium according to the amount of 1.0% for culturing, performing shake culture on the MYG culture medium for 6 days at 28 ℃ and 200rpm, then performing 6000g centrifugal treatment, discarding part of supernatant, and applying a bacterial liquid to MS solid culture medium plating; 100 mu L of the culture medium is evenly coated on each dish and cultured for seven days at the temperature of 30 ℃;
(2) transferring the cultured streptomycete XY006 to a low temperature of-80 ℃, freezing for 24h, then completely thawing at room temperature, wrapping by using a plurality of layers of gauze, applying pressure to extrude bacterial liquid, soaking the residual culture medium residues in methanol for 4h, wrapping by using the plurality of layers of gauze, applying pressure to filter, finally combining the two filtrates, and concentrating the filtrate to a preset volume by using a rotary evaporator; centrifuging at 12000g for 10min, collecting supernatant, adding 70% methanol at 4 deg.C, standing for extraction, centrifuging at 12000g for 10min, collecting supernatant, concentrating to preset volume by rotary evaporator, freeze drying in freeze dryer to obtain crude extract, and storing at-20 deg.C;
(3) completely re-dissolving the crude extract with pure water, performing isovolumetric liquid-liquid extraction with n-butanol, extracting for three times, combining three organic phases, standing overnight, collecting the organic phase, concentrating to dryness, re-dissolving with pure water, freeze-drying to obtain product A, and storing at-20 deg.C;
(4) adjusting the product A to a sample concentration of 500mg/mL, extracting the product A through a solid phase extraction column by a sample volume of 3-5 mL, performing multi-stage gradient elution, and collecting an eluate;
(5) and re-dissolving the eluate, centrifuging to remove precipitate, and separating and purifying by liquid chromatography to obtain antifungal lipopeptide.
As a preferred embodiment, it is preferred that the eluents of the multi-step gradient elution in step (4) are sequentially 100% H2O, 20% MeOH, 40% MeOH, 60% MeOH, 80% MeOH, 100% MeOH, wherein the MeOH is not in a 100% solution by mass, and the balance is water.
As a preferred embodiment, in step (5), the eluate is redissolved, centrifuged to remove the precipitate, and then the solution is subjected to liquid chromatography using methanol and water as mobile phases and acetonitrile and water as mobile phases to obtain the antifungal lipopeptide.
As a preferable implementation option, in the step (5), the conditions for performing the separation and purification treatment by liquid chromatography using methanol and water as mobile phases are preferably as follows: the chromatographic column is Xbridge Prep OBDTMC18 with the specification of 5 μm and the specification of 19X 150mm, the injection concentration of 200mg/mL and the injection volume of 200 μ L; the flow rate of the mobile phase is 5mL/min, the absorption wavelength is 200-300 nm, one tube is collected every minute under the condition of a preset HPLC elution gradient, eluents of the same absorption peak are combined according to the position of the absorption peak, and then concentrated and dried through a rotary evaporator, and then stored at the temperature of-20 ℃.
As a preferable implementation option, in the step (5), the conditions for performing the separation and purification treatment by liquid chromatography using acetonitrile and water as mobile phases are preferably as follows: the chromatographic column is Xbridge Prep OBDTMC18 with the specification of 5 μm, 19 × 150mm, the injection concentration of 200mg/mL and the injection volume of 200 μ L; the flow rate of the mobile phase is 5mL/min, the absorption wavelength is 200-300 nm, under the condition of a preset HPLC elution gradient, eluent is collected according to the position of an absorption peak, and then concentrated and dried by a rotary evaporator, weighed and stored at the temperature of-20 ℃.
Based on the above, the invention also provides an agricultural pathogenic fungi inhibiting medicament, which comprises antifungal lipopeptide, wherein the antifungal lipopeptide comprises lipopeptide A and lipopeptide B, wherein the chemical formula of the lipopeptide A is C54H84N10O19The structural formula is as follows:
the chemical formula of the lipopeptide B is C53H82N10O19The structural formula is as follows:
as a preferred embodiment, it is preferred that the agricultural pathogenic fungi include rice blast, banana wilt, cucumber wilt, anthracnose pathogenic fungi;
as a preferred embodiment, preferably, the preparation method of the antifungal lipopeptide comprises:
(1) after the streptomycete XY006 is subjected to plate scratching and activation on an MS culture medium, the streptomycete XY006 is cultured for 3 days at the temperature of 30 ℃; then picking a single colony in a MYG culture medium, culturing for 2 days at 28 ℃ and 200rpm, then transferring the single colony to the MYG culture medium according to the amount of 1.0% for culturing, performing shake culture on the MYG culture medium for 6 days at 28 ℃ and 200rpm, then performing 6000g centrifugal treatment, discarding part of supernatant, and applying a bacterial liquid to MS solid culture medium plating; 100 mu L of the culture medium is evenly coated on each dish and cultured for seven days at the temperature of 30 ℃;
(2) transferring the cultured streptomycete XY006 to a low temperature of-80 ℃, freezing for 24h, then completely thawing at room temperature, wrapping by using a plurality of layers of gauze, applying pressure to extrude bacterial liquid, soaking the residual culture medium residues in methanol for 4h, wrapping by using the plurality of layers of gauze, applying pressure to filter, finally combining the two filtrates, and concentrating the filtrate to a preset volume by using a rotary evaporator; then centrifuging at 12000g for 10min, taking the supernatant, adding 70% methanol at the final concentration, standing and extracting at 4 ℃, centrifuging at 12000g for 10min, keeping the supernatant, concentrating to a preset volume by a rotary evaporator, and freeze-drying in a freeze-drying instrument to obtain the antifungal lipopeptide.
The study of lipopeptide A in the scheme is originally appeared in 80 years of the 20 th century, and is the only study report so far, and no related study of lipopeptide B is found in the published literature, and almost no study about the antagonism of lipopeptide A and B on important pathogenic fungi in agriculture exists.
By adopting the technical scheme, compared with the prior art, the invention has the beneficial effects that: the scheme ingeniously extracts the lipopeptide A and the lipopeptide B through metabolites of streptomyces, and the monomer of the lipopeptide A and the lipopeptide B with the purity higher than 95 percent can be prepared by combining the preparation method of the scheme, so that the method is of great help for subsequent experiments of analyzing the mechanism of the antagonistic pathogenic fungi of the lipopeptide A and the lipopeptide B and the biocontrol effect of plants; meanwhile, the difference of antagonistic mechanisms of lipopeptide A and lipopeptide B can also indicate the direction for the subsequent biosynthesis research of lipopeptide A and lipopeptide B.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without any creative effort.
FIG. 1 is a total ion flow chromatogram of the active layer after n-butanol extraction of the crude extract;
FIG. 2 is a total ion current chromatogram of six active layers after separation on a Poly-Sery HLB solid phase extraction cartridge;
FIG. 3 is a total ion flux chromatogram of lipopeptides A and B;
FIG. 4 is a secondary mass spectrum of lipopeptide A (m/z 1177.6021) and lipopeptide B (m/z 1163.5846);
FIG. 5 is a comparative characterization plot of the inhibitory effect of the primary extracts of lipopeptides A and B on major agro-pathogenic fungi.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples. It is to be noted that the following examples are only illustrative of the present invention, and do not limit the scope of the present invention. Similarly, the following examples are only some but not all examples of the present invention, and all other examples obtained by those skilled in the art without any inventive work are within the scope of the present invention.
The scheme discloses an application of streptomyces in preparation of antifungal lipopeptide, which comprises the following steps:
1 amplification culture of Streptomyces XY006
Streptomyces XY006 (hereinafter referred to as XY006) is separated from leaves of an Tieguanyin tea tree in a tea garden of agriculture and forestry university of Fujian, is named as Streptomyces sp after strain identification, is preserved in China general microbiological culture Collection center on 12.13.2021, and has the preservation address of No. 3 Siro No. 1 of the North Cheng of the sunny region in Beijing, and the preservation number is CGMCC No. 24090.
And (3) amplification culture: carrying out plate scratching activation on XY006 on an MS culture medium, culturing for three days at 30 ℃, selecting a single strain to be placed in a MYG (3mL) culture medium, carrying out shake culture at 28 ℃ and 200rpm for two days, transferring the single strain to the MYG (250mL) culture medium according to 1% of strain inoculation amount, carrying out shake culture at 28 ℃ and 200rpm for six days, carrying out 6000g centrifugation, discarding part of supernatant, and using a bacterium solution for MS solid culture medium plating; each dish was 100. mu.L, coated uniformly, and incubated at 30 ℃ for seven days.
2 preparation of crude extract
Transferring the cultured XY006 to a refrigerator with the temperature of-80 ℃, freezing for 24h at low temperature, completely thawing at room temperature, extruding bacterial liquid by four layers of gauze, soaking culture medium residues in methanol (MeOH) for 4h, filtering by the four layers of gauze, combining filtrates obtained in two times, and concentrating to a small volume (about 10 times of concentration multiple) by a rotary evaporator. Centrifuging at 12000g for 10min, collecting supernatant, adding 70% methanol, standing at 4 deg.C for extraction, centrifuging at 12000g for 10min, collecting supernatant, concentrating to small volume with rotary evaporator, freeze drying with freeze dryer, weighing, and storing in-20 deg.C refrigerator.
3 separation and purification of antibacterial active substance
3.1 liquid-liquid extraction
After the crude extract is completely redissolved by pure water, the same volume of liquid-liquid extraction is carried out by normal butanol. Extracting for three times, combining organic phases of the three times, standing overnight, collecting the organic phase, concentrating to dryness, redissolving with pure water, freeze drying, and storing in a refrigerator at-20 deg.C.
Waters UPLC-SYNAPT G2-Si HDMS QTOF-MS (UPLC-QTOF/MS) measures the active layer purity as shown in FIG. 1. As can be seen from the figure, lipopeptide A peaked at 3.09min, lipopeptide B peaked at 2.67 min, and the peak times of the two substances were close. A plurality of hetero-peaks exist before and after the peak positions of the lipopeptides A and B, which indicates that the purity of an active layer subjected to liquid-liquid extraction is lower and further purification is still needed.
3.2 Poly-Sery HLB solid-phase extraction column separation and purification
3.2.1 column activation: 2mL of methanol and 2mL of pure water were added in this order.
3.2.2 Loading: and (3) when the sample loading concentration is 500mg/mL and the sample loading volume is 3-5 mL, completely passing through the column, and starting elution.
3.2.3 elution: the gradient elution is performed according to the following table.
TABLE 1 elution gradient of CNW Poly-Sery HLB solid phase extraction cartridge
3.2.4 collecting the elution layer: the six elution layers were concentrated to small volume, freeze dried and weighed.
3.2.575% chromatographic pure methanol dissolves the active layer, 10000g of the solution is centrifuged to remove the precipitate, the solution is diluted to 0.1mg/mL, and the active layer is screened after UPLC-QTOF/MS detection, and the result is shown in figure 2.
As can be seen, lipopeptides A and B were not detected in both the 100% water and 100% methanol elution layers, and lipopeptides A and B were detected in the remaining four elution layers, with a slight difference in the proportions of the two species in each of the elution layers, predominantly lipopeptide A in the 60% methanol and 80% methanol elution layers, and relatively uniformly in the 20% methanol and 40% methanol elution layers. The purity of lipopeptides A and B was calculated by peak area integration, and the purity of lipopeptides A and B in these four active layers was significantly improved over that obtained by liquid-liquid extraction.
3.3 preparative liquid chromatography methanol separation purification
Respectively re-dissolving the active layers obtained by 3.2 separation and purification, centrifuging by 10000g to remove precipitates, and performing liquid chromatography separation and purification by taking methanol and water as mobile phases.
Separation and purification conditions: chromatographic column XBridge Prep OBDTMC18(5 μm, 19X 150mm), injection concentration 200mg/mL, injection volume 200 μ L. The mobile phase is methanol and water, the flow rate is 5mL/min, the absorption wavelength is 200-300 nm, the HPLC elution gradient is shown in the following table, one tube is collected every minute, eluent with the same absorption peak is combined according to the position of the absorption peak, a rotary evaporator is used for concentrating and drying, weighing is carried out, and the mixture is stored in a refrigerator at the temperature of-20 ℃.
TABLE 2 HPLC SEPARATION-PURIFICATION ASOLUTION GRADIENT
Dissolving each elution layer by 75% chromatographic pure methanol, centrifuging at 10000g to remove precipitate, diluting to 0.01mg/mL, and screening the active layer by UPLC-QTOF/MS detection.
3.4 separation and purification of acetonitrile by liquid chromatography
Respectively re-dissolving the active layers obtained by screening in the step 3.3, centrifuging by 10000g to remove precipitates, and respectively carrying out liquid chromatography separation and purification by taking Acetonitrile (ACN) and water as mobile phases.
Separation and purification conditions: chromatographic column XBridge Prep OBDTMC18(5 μm, 19X 150mm), sample concentration 200mg/mL, sample volume 200 μ L. The mobile phase is acetonitrile and water, the flow rate is 5mL/min, the absorption wavelength is 200-300 nm, the HPLC elution gradient is shown in the table, the eluent is collected according to the position of an absorption peak, the eluent is concentrated and dried by a rotary evaporator, the weight is weighed, and the eluent is stored in a refrigerator at the temperature of-20 ℃.
TABLE 3 HPLC SEPARATION-PURIFICATION ELUTION GRADIENT
Redissolving each active layer by using 75% chromatographic pure methanol, centrifuging 10000g to remove precipitates, diluting to 0.01mg/mL, and screening the active layers by UPLC-QTOF/MS detection, wherein the result is shown in Table 4, nine active layers with the purity of more than 90% are obtained, two of the active layers are active monomers of lipopeptide A and lipopeptide B, the total ion chromatogram is shown in figure 3, 70% methanol is a solvent blank, MS blank is a culture medium blank, and the purity of both lipopeptide A and B is more than 95% through peak area integral calculation.
TABLE 4 high-purity active layer obtained by multiple isolation and purification
3.5 structural characterization of lipopeptides A and B
For parent ion [ M + H ] of lipopeptide A]+(m/z 1177.6021) MS/MS analysis was performed, and the ion fragment information is shown in FIG. 4. The secondary mass spectrum information is uploaded to a GNPS (Global Natural Products society) database, and the ion fragments are matched with Lipopeptin A. The research of the literature finds that the polypeptide is related to lipopeptin A (C) in the research of Nishii et al54H84N10O19) Consistent, M/z 1199([ M + Na ]]+) It is assumed that the fragmentation pattern is such that fragment ion 162.0910 corresponds to methylphenylalanine (MePhe) in lipopeptide A, ions 1177.6021 and 1049.5417 differ by 128, methylasparagine (MeAsn) in lipopeptide A, ions 306.1458 and 162.0910 differ by 144, hydroxyglutamine (HyGln) in lipopeptide A, ions 435.1892 and 306.1376 differ by 129, glutamic acid (Glu) in lipopeptide A, ions 760.4507 and 435.1892 differ by 325, fatty acid and threonine (C15 FA + Thr) in lipopeptide A, the Chemical structure of lipopeptide A is shown in Formula I, and Chemical Formula for Chemical mula of lipopeptide A is C54H84N10O19。
For parent ion [ M + H ] of lipopeptide B]+(m/z 1163.5846) MS/MS analysis was performed, and the ion fragment information is shown in FIG. 4. The parent ion differs from the parent ion of lipopeptide A by 14, which may differ in structure by one-CH2The major fragment ions are 162.0916, 306.1454, 435.1884, 746.4335, 861.4587, 948.4932, and 1035.5247, and fragment ions 746.4335, 861.4587, 948.4932, and 1035.5247 differ from fragment ions 760.4507, 875.4780, 962.5101, and 1049.5417 of lipopeptide a by 14. Uploading the secondary mass spectrum information of the lipopeptide A to a GNPS database, wherein the matched substance is Lipopeptin B, and the structure of the Lipopeptin B is different from that of Lipopeptin A by a methylene-CH on a fatty acid chain2-. The Chemical structural Formula of the lipopeptide B is shown as a Formula II, and the Chemical Formula of the lipopeptide B is C53H82N10O19(ii) a The current document retrieval is not sentLipopeptide B is now described.
4 inhibiting action of lipopeptide A and B on important pathogenic fungus in agriculture
The initial extracts of lipopeptide a and lipopeptide B prepared in step 2 (purity control fig. 1) were applied to antagonize major agricultural pathogenic fungi, and the test results are shown in fig. 5, where the Positive Control (PC) is nystatin, the Negative Control (NC) is medium blank, and the treatment (T) is the initial extract of lipopeptide a and B. The results show that the primary extract with lipopeptide A and B as main components has obvious antagonism on pathogenic fungi including rice blast, banana wilt, cucumber wilt, anthracnose and the like.
The above description is only a partial embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent devices or equivalent processes performed by the present invention or directly or indirectly applied to other related technical fields using the contents of the present specification and the accompanying drawings are included in the scope of the present invention.
Claims (10)
1. An antifungal lipopeptide, wherein: having a chemical formula of C53H82N10O19The structural formula is as follows:
2. the application of streptomycete in preparation of antifungal lipopeptide is characterized in that the streptomycete and a culture medium thereof are subjected to freezing treatment, then are subjected to unfreezing and pressure filtration treatment to obtain bacterial liquid, the obtained bacterial liquid is concentrated and alcohol-extracted to obtain a crude extract, the crude extract is subjected to redissolving and n-butyl alcohol isovolumetric extraction for a plurality of times, an organic phase is collected and concentrated, the concentrate is adjusted to a preset concentration, passes through a solid phase extraction column, is subjected to multi-stage gradient elution treatment, and is subjected to multi-stage gradient elution treatmentCollecting eluate, separating and purifying to obtain antifungal lipopeptide, wherein the antifungal lipopeptide comprises lipopeptide A and lipopeptide B, and the chemical formula of the lipopeptide A is C54H84N10O19The structural formula is as follows:
the chemical formula of the lipopeptide B is C53H82N10O19The structural formula is as follows:
3. the use of the Streptomyces as claimed in claim 2 in the preparation of antifungal lipopeptides, wherein the Streptomyces is Streptomyces XY006, which is named Streptomyces sp, and which has been deposited at the general microbiological center of the china committee for culture collection of microorganisms at 12.13.2021, with the deposition address of No. 3, CGMCC No.24090, north chen, yang ward, beijing.
4. The use of the streptomyces strain of claim 3 in the preparation of antifungal lipopeptides, comprising the steps of:
(1) after the streptomycete XY006 is subjected to plate scratching and activation on an MS culture medium, the streptomycete XY006 is cultured for 3 days at the temperature of 30 ℃; then picking a single colony in a MYG culture medium, culturing for 2 days at 28 ℃ and 200rpm, then transferring the single colony to the MYG culture medium according to the amount of 1.0% for culturing, performing shake culture on the MYG culture medium for 6 days at 28 ℃ and 200rpm, then performing 6000g centrifugal treatment, discarding part of supernatant, and applying a bacterial liquid to MS solid culture medium plating; 100 mu L of the culture medium is evenly coated on each dish and cultured for seven days at the temperature of 30 ℃;
(2) transferring the cultured streptomycete XY006 to a low temperature of-80 ℃, freezing for 24h, then completely thawing at room temperature, wrapping by using a plurality of layers of gauze, applying pressure to extrude bacterial liquid, soaking the residual culture medium residues in methanol for 4h, wrapping by using the plurality of layers of gauze, applying pressure to filter, finally combining the two filtrates, and concentrating the filtrate to a preset volume by using a rotary evaporator; centrifuging at 12000g for 10min, collecting supernatant, adding 70% methanol at 4 deg.C, standing for extraction, centrifuging at 12000g for 10min, collecting supernatant, concentrating to preset volume by rotary evaporator, freeze drying in freeze dryer to obtain crude extract, and storing at-20 deg.C;
(3) fully redissolving the crude extract by pure water, performing liquid-liquid extraction with n-butyl alcohol in the same volume for three times, combining organic phases of the three times, standing overnight, collecting the organic phase again, concentrating the organic phase to dryness, redissolving the organic phase by pure water, performing freeze drying to obtain a product A, and storing the product A at-20 ℃;
(4) adjusting the product A to a sample concentration of 500mg/mL, extracting the product A through a solid phase extraction column by a sample volume of 3-5 mL, performing multi-stage gradient elution, and collecting an eluate;
(5) and re-dissolving the eluate, centrifuging to remove precipitate, and separating and purifying by liquid chromatography to obtain antifungal lipopeptide.
5. The use of the Streptomyces strain according to claim 4 for the preparation of antifungal lipopeptides wherein in step (4) the elution solutions of the multi-step gradient elution are sequentially 100% H2O, 20% MeOH, 40% MeOH, 60% MeOH, 80% MeOH, 100% MeOH, wherein the MeOH is not in a 100% solution by mass, and the balance is water.
6. The use of the Streptomyces strain according to claim 4 in the preparation of antifungal lipopeptides wherein in step (5) the antifungal lipopeptides are prepared by redissolving the eluate, centrifuging to remove the precipitate, and purifying the solution by liquid chromatography using methanol and water as mobile phases and acetonitrile and water as mobile phases.
7. The Streptomyces shimeji of claim 6 is in true breedingThe application of the lipopeptide in preparation is characterized in that in the step (5), the conditions of the liquid chromatography with methanol and water as mobile phases for separation and purification treatment are as follows: the chromatographic column is Xbridge Prep OBDTMC18 with the specification of 5 μm, 19 × 150mm, the injection concentration of 200mg/mL and the injection volume of 200 μ L; the flow rate of the mobile phase is 5mL/min, the absorption wavelength is 200-300 nm, one tube is collected every minute under the condition of a preset HPLC elution gradient, the eluents of the same absorption peak are combined according to the position of the absorption peak, and then the eluent is concentrated and dried by a rotary evaporator and stored at the temperature of-20 ℃.
8. The use of the Streptomyces strain according to claim 6 in the preparation of antifungal lipopeptides, wherein in step (5), the conditions for the separation and purification by liquid chromatography with acetonitrile and water as mobile phases are as follows: the chromatographic column is Xbridge Prep OBDTMC18 with the specification of 5 μm, 19 × 150mm, the injection concentration of 200mg/mL and the injection volume of 200 μ L; the flow rate of the mobile phase is 5mL/min, the absorption wavelength is 200-300 nm, under the condition of a preset HPLC elution gradient, eluent is collected according to the position of an absorption peak, and then concentrated and dried by a rotary evaporator, weighed and stored at the temperature of-20 ℃.
9. An agricultural pathogenic fungus inhibiting drug, which comprises antifungal lipopeptide, wherein the antifungal lipopeptide comprises lipopeptide A and lipopeptide B, wherein the chemical formula of the lipopeptide A is C54H84N10O19The structural formula is as follows:
the chemical formula of the lipopeptide B is C53H82N10O19The structural formula is as follows:
10. the agricultural pathogenic fungus suppressing agent as claimed in claim 9, wherein said agricultural pathogenic fungi include rice blast, banana wilt, cucumber wilt, anthracnose pathogenic fungi; the preparation method of the antifungal lipopeptide comprises the following steps:
(1) performing plate scratching activation on a MS culture medium by streptomycete XY006, and then culturing for 3 days at the temperature of 30 ℃; then picking a single colony in a MYG culture medium, culturing for 2 days at 28 ℃ and 200rpm, then transferring the single colony to the MYG culture medium according to the amount of 1.0% for culturing, performing shake culture on the MYG culture medium for 6 days at 28 ℃ and 200rpm, then performing 6000g centrifugal treatment, discarding part of supernatant, and applying a bacterial liquid to MS solid culture medium plating; 100 mu L of the culture medium is evenly coated on each dish and cultured for seven days at the temperature of 30 ℃;
(2) transferring the cultured streptomycete XY006 to a low temperature of-80 ℃, freezing for 24h, then completely thawing at room temperature, wrapping by using a plurality of layers of gauze, applying pressure to extrude bacterial liquid, soaking the residual culture medium residues in methanol for 4h, wrapping by using the plurality of layers of gauze, applying pressure to filter, finally combining the two filtrates, and concentrating the filtrate to a preset volume by using a rotary evaporator; then centrifuging at 12000g for 10min, taking the supernatant, adding 70% methanol at the final concentration, standing and extracting at 4 ℃, centrifuging at 12000g for 10min, keeping the supernatant, concentrating to a preset volume by a rotary evaporator, and freeze-drying in a freeze-drying instrument to obtain the antifungal lipopeptide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210078489.9A CN114524861B (en) | 2022-01-24 | 2022-01-24 | Application of antifungal lipopeptides and streptomycetes in preparation of antifungal lipopeptides |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210078489.9A CN114524861B (en) | 2022-01-24 | 2022-01-24 | Application of antifungal lipopeptides and streptomycetes in preparation of antifungal lipopeptides |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114524861A true CN114524861A (en) | 2022-05-24 |
| CN114524861B CN114524861B (en) | 2024-02-20 |
Family
ID=81619982
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210078489.9A Active CN114524861B (en) | 2022-01-24 | 2022-01-24 | Application of antifungal lipopeptides and streptomycetes in preparation of antifungal lipopeptides |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114524861B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024074628A1 (en) * | 2022-10-07 | 2024-04-11 | Syngenta Crop Protection Ag | Fungicidal compound |
| WO2024074637A1 (en) * | 2022-10-07 | 2024-04-11 | Syngenta Crop Protection Ag | Means and methods for controlling pathogens and pests in plants |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101851654A (en) * | 2009-04-03 | 2010-10-06 | 中国科学院沈阳应用生态研究所 | Method for separating and purifying antifungal lipopeptide |
| CN103288928A (en) * | 2012-03-02 | 2013-09-11 | 中国科学院沈阳应用生态研究所 | Bacillus mojavensis, antifungal lipopeptide compound, and preparation as well as application thereof |
| CN103773712A (en) * | 2013-12-06 | 2014-05-07 | 天津大学 | Antifungal peptide high-yield strain and method for preparing antibacterial peptide |
-
2022
- 2022-01-24 CN CN202210078489.9A patent/CN114524861B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101851654A (en) * | 2009-04-03 | 2010-10-06 | 中国科学院沈阳应用生态研究所 | Method for separating and purifying antifungal lipopeptide |
| CN103288928A (en) * | 2012-03-02 | 2013-09-11 | 中国科学院沈阳应用生态研究所 | Bacillus mojavensis, antifungal lipopeptide compound, and preparation as well as application thereof |
| CN103773712A (en) * | 2013-12-06 | 2014-05-07 | 天津大学 | Antifungal peptide high-yield strain and method for preparing antibacterial peptide |
Non-Patent Citations (2)
| Title |
|---|
| PAULINA BEKIESCH等: "Viennamycins: Lipopeptides Produced by a Streptomyces sp", J NAT PROD ., vol. 83, no. 8, pages 2381 - 2389 * |
| WENNA SHAN等: "Draft Genome Sequence of Streptomyces sp. XY006, an Endophyte Isolated from Tea (Camellia sinensis)", GENOME ANNOUNC., vol. 5, no. 37, pages 00971 - 17 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024074628A1 (en) * | 2022-10-07 | 2024-04-11 | Syngenta Crop Protection Ag | Fungicidal compound |
| WO2024074637A1 (en) * | 2022-10-07 | 2024-04-11 | Syngenta Crop Protection Ag | Means and methods for controlling pathogens and pests in plants |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114524861B (en) | 2024-02-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Sebastianes et al. | 3-Hydroxypropionic acid as an antibacterial agent from endophytic fungi Diaporthe phaseolorum | |
| CN114524861A (en) | Antifungal lipopeptide and application of streptomycete in preparation of antifungal lipopeptide | |
| CN107964557B (en) | Fermentation method for increasing yield of antibacterial lipopeptide of bacillus | |
| CN104513300A (en) | Bio-control bacillus subtilis surfactin separation and purification method | |
| CN103497980A (en) | Preparation method for Nigericin | |
| CN112795609B (en) | Method for efficiently preparing cyclodepsipeptide, cyclodepsipeptide and application | |
| CN113005048B (en) | Streptomyces nigricans CYS22, metabolite thereof and application thereof | |
| Kuncharoen et al. | Characterisation of two polyketides from Streptomyces sp. SKH1-2 isolated from roots of Musa (ABB) cv.‘Kluai Sao Kratuep Ho’ | |
| CN115466772A (en) | Ergoline type steroid compound and preparation method and application thereof | |
| CN115894401A (en) | Tetracyclic sesterterpene compounds and synthetic gene cluster thereof | |
| US9624266B2 (en) | Brachiatin D and process for production thereof | |
| KR102063801B1 (en) | Preparing method of dibutyl succinate | |
| CN109265461B (en) | Paenibacillus kribbensis metabolite and application thereof in biocontrol | |
| CN112608953A (en) | Preparation method of anisomycin, detection method of anisomycin and antibacterial application | |
| CN114574386B (en) | Bacillus licheniformis and application thereof | |
| CN115043719B (en) | Polyketide from fungus, preparation method and application thereof | |
| CN115504990B (en) | Sugar-spiro-macrolide compound FW-5-39 and preparation method and application thereof | |
| CN113151000B (en) | Marine fungus and application thereof in production of dibutyl phthalate | |
| CN104774773A (en) | Polygonum cuspidatum endogenous bacterial strain aspergillus fumigates J3 bacterial strain for converting polydatin | |
| CN111004197A (en) | Sorbicillinoid derivatives, and preparation method and application thereof | |
| CN103524600B (en) | New cyclic lipopeptide microbiotic Roc mycin (locillomycin) A, B, C and manufacture method thereof | |
| Shenin et al. | Isolation and characterization of antibiotics produced by Streptomyces chrysomallus R-21 and S. globisporus L-242 strains | |
| CN114805343B (en) | Compound, preparation method and application thereof, and microorganism producing compound | |
| CN115925635B (en) | Fusarium tricarum fermentation extract, preparation of metabolite and application of metabolite in resisting Psa | |
| CN112694406B (en) | Preparation method and application of two dimeric hexyl itaconic acid derivatives |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |