CN115109023B - Macrolide compound FWYZ52-A, fermentation strain, fermentation method and application thereof - Google Patents
Macrolide compound FWYZ52-A, fermentation strain, fermentation method and application thereof Download PDFInfo
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 78
- 239000003120 macrolide antibiotic agent Substances 0.000 title claims abstract description 61
- 238000000855 fermentation Methods 0.000 title claims abstract description 38
- 230000004151 fermentation Effects 0.000 title claims abstract description 38
- 238000000034 method Methods 0.000 title claims abstract description 12
- 241000187708 Micromonospora Species 0.000 claims abstract description 24
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 13
- 241000187723 Micromonospora sp. Species 0.000 claims abstract description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 22
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- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 1
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- ZUHRLTIPDRLJHR-UHFFFAOYSA-N Rosamicin Natural products CCC1OC(=O)CC(O)C(C)C(OC2OC(C)CC(C2O)N(C)C)C(CC=O)CC(C)C(=O)C=CC3OC3C1C ZUHRLTIPDRLJHR-UHFFFAOYSA-N 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
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- 229930192649 bafilomycin Natural products 0.000 description 1
- XDHNQDDQEHDUTM-UHFFFAOYSA-N bafliomycin A1 Natural products COC1C=CC=C(C)CC(C)C(O)C(C)C=C(C)C=C(OC)C(=O)OC1C(C)C(O)C(C)C1(O)OC(C(C)C)C(C)C(O)C1 XDHNQDDQEHDUTM-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 230000003115 biocidal effect Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
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- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- ZGXMNNDLHXNOQU-UHFFFAOYSA-N levantilide B Natural products CCC(=O)CCCC1OC(=O)C=CC=CCC(O)CC(O)C(C)C(O)C(C)CC(=CC(C)CC1C)C ZGXMNNDLHXNOQU-UHFFFAOYSA-N 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 229940041033 macrolides Drugs 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
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- 239000013642 negative control Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- IUPCWCLVECYZRV-JZMZINANSA-N rosaramicin Chemical compound O([C@@H]1[C@@H](C)[C@H](O)CC(=O)O[C@@H]([C@H]([C@@H]2O[C@@]2(C)/C=C/C(=O)[C@H](C)C[C@@H]1CC=O)C)CC)[C@@H]1O[C@H](C)C[C@H](N(C)C)[C@H]1O IUPCWCLVECYZRV-JZMZINANSA-N 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
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- 238000012163 sequencing technique Methods 0.000 description 1
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- 230000001225 therapeutic effect Effects 0.000 description 1
- 241001446247 uncultured actinomycete Species 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D313/00—Heterocyclic compounds containing rings of more than six members having one oxygen atom as the only ring hetero atom
-
- 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
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/22—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom rings with more than six members
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- 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
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/08—Oxygen as only ring hetero atoms containing a hetero ring of at least seven ring members, e.g. zearalenone, macrolide aglycons
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- Engineering & Computer Science (AREA)
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- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Communicable Diseases (AREA)
- Biomedical Technology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Oncology (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Saccharide Compounds (AREA)
Abstract
The invention belongs to the technical field of microorganisms and novel medicines and pesticides, in particular relates to a novel macrolide compound FWYZ52-A and application thereof, and further discloses a marine micromonospora capable of fermenting the compound and a method for preparing the macrolide compound FWYZ52-A based on the strain for fermentation. The macrolide compound FWYZ52-A is a novel structural substance and has inhibitory activity on staphylococcus aureus in bacteria. The isolated and screened marine Micromonospora sp. FIMYZ52 with inhibitory activity against bacteria and oxidation can be used for preparing a macrolide compound FWYZ52-A with inhibitory activity against staphylococcus aureus by fermentation.
Description
Technical Field
The invention belongs to the technical field of microorganisms and novel medicines and pesticides, in particular relates to a novel macrolide compound FWYZ52-A and application thereof, and further discloses a marine micromonospora capable of fermenting the compound and a method for preparing the macrolide compound FWYZ52-A based on the strain for fermentation.
Background
Micromonospora belongs to the family Micromonosporaceae of the order Actinomycetales, and its strains are widely available, both from freshwater lakes and isolated from marine environments. By the statistics of Talukdar et al, a total of nearly 700 antibiotics were derived from the metabolite of Micromonospora. Thus, pseudomonas is an important treasury of bioactive secondary metabolites (Qi et al 2020), and has important significance for the development of a variety of novel antibiotic drugs.
Among them, macrolides produced by Micromonospora have been reported to have a variety of active substances, and among them, there are Levantilide A and Levantilide C which exhibit moderate activity against malignant cell proliferation against various cell lines such as tumor cell human leukemia tumor (HL-60), human breast tumor (MDA-MB-231), human colon cancer (SW 620) and human liver cancer (SMMC 7721), bafilomycin which can inhibit malignant cell growth as an insecticide, rosamicin which has a broad spectrum of antibacterial activity, juvenicidin C which can enhance the activity of QR1 enzyme and the folding level of glutathione, and the like. Johannes F.Imhoff et al in 2011 found novel macrolide compounds Levantilide A and Levantilide B from metabolites of Micromonospora sp.Strain M71-A77 from a deep sea environment, which have moderate antiproliferative activity against various tumor cells; 2013 Peng Fei et al found that the novel macrolide compound Levantilide C from the metabolite of marine Micromonospora fiM07-0019 also has anti-proliferation activity against various tumor cells. However, none of the above Levantilides compounds have been reported to have antibacterial ability.
In view of the above, the novel macrolide active substances are extracted from the metabolites of the Micromonospora, which is beneficial to the continued expansion of the current drug reservoir and provides guarantee for the healthy life of human beings.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a novel macrolide compound FWYZ52-A, wherein the compound FWYZ52-A has antibacterial performance against staphylococcus aureus;
the second technical problem to be solved by the invention is to provide a marine micromonospora strain which can be used for producing the macrolide compound FWYZ52-A by fermentation;
the third technical problem to be solved by the invention is to provide a method for preparing the macrolide compound FWYZ52-A based on the fermentation of the marine micromonospora strain;
the fourth technical problem to be solved by the present invention is to provide the use of the macrolide compound FWYZ52-A.
In order to solve the technical problems, the macrolide compound FWYZ52-A disclosed by the invention has a structure shown in the following formula (I):
the invention also discloses a marine Micromonospora strain FIMYZ52 which is classified and named as actinomycetes Micromonospora sp, and is preserved in China general microbiological culture Collection center (CGMCC) No.24479 in the 3 rd month of 2022.
The invention also discloses application of the marine micromonospora strain in preparing the macrolide compound FWYZ52-A by fermentation.
The invention also discloses a method for preparing the macrolide compound FWYZ52-A, which comprises the step of inoculating the marine micromonospora strain into a proper fermentation medium for fermentation culture.
Specifically, the preparation method of the macrolide compound FWYZ52-A comprises the following steps:
(1) Taking marine Micromonospora sp. FIMYZ52 preserved by an inclined plane, inoculating into a liquid seed culture medium for constant temperature culture, and collecting seed liquid for later use;
the components of the liquid seed medium include: 1-2wt% of soluble starch, 0.1-1wt% of glucose, 0.1-1wt% of yeast extract and MgSO 4 ·7H 2 O 0.01-0.1wt％，NaCl 0.01-0.1wt％，(NH 4 ) 2 SO 4 0.01-0.1wt％，CaCO 3 0.05-0.2wt％，pH 6.0-8.5；
Preferably, the components of the liquid seed medium include: 1.5wt% of soluble starch, 0.5wt% of glucose, 0.5wt% of yeast extract and MgSO (MgSO) 4 ·7H 2 O 0.05wt％，NaCl 0.05wt％，(NH 4 ) 2 SO 4 0.05wt％，CaCO 3 0.1wt％，pH 6.0-8.5；
(2) Transferring the seed liquid into a fermentation medium for constant temperature culture to obtain fermentation liquid containing a required macrolide compound FWYZ52-A;
the components of the fermentation medium include: 3-5wt% of soluble starch, 0.1-1wt% of glucose, 2-3wt% of soybean cake powder, 0.1-1wt% of yeast powder and MgSO 4 ·7H 2 O 0.01-0.1wt％、K 2 HPO 4 0.01-0.1wt％、CaCO 3 0.05 to 0.2 weight percent of distilled water, and the pH value is 6.0 to 8.5;
preferably, the components of the fermentation medium include: 4wt% of soluble starch, 0.5wt% of glucose, 2.5wt% of soybean cake powder, 0.5wt% of yeast powder and MgSO 4 ·7H 2 O 0.05wt％、K 2 HPO 4 0.05wt％、CaCO 3 0.1wt% of distilled water and pH of 6.0-8.5.
Specifically, the preparation method of the macrolide compound FWYZ52-A comprises the following steps:
in the step (1), the temperature of the culture step is 25-35 ℃ and the culture time is 2-3 days;
in the step (2), the temperature of the culture step is 25-35 ℃ and the culture time is 3-5 days.
Specifically, the preparation method of the macrolide compound FWYZ52-A further comprises the steps of extracting and purifying the macrolide compound FWYZ52-A, and specifically comprises the following steps:
extracting: carrying out solid-liquid separation on the collected fermentation liquor and mycelium, wherein the fermentation liquor is adsorbed by macroporous resin HP20, and the crude extract A is obtained after the adsorption, the desorption, the recovery and the concentration of ethanol; extracting mycelium with alcohol solvent, collecting extractive solution, and concentrating under reduced pressure to obtain crude extract B;
purifying: combining the obtained crude extract A and the crude extract B, and purifying by normal phase silica gel column chromatography according to the volume ratio of 10:0-0:10, carrying out gradient elution by using a petroleum ether-ethyl acetate solvent, detecting by using thin layer chromatography, and collecting eluent containing a macrolide compound FWYZ52-A; and then go through C 18 Performing reversed phase column chromatography, gradient eluting with 50% -95% methanol-water solution, detecting by high performance liquid chromatography, and collecting eluate containing compound FWYZ52-A; then through the preparation of C 18 And (3) performing reversed-phase high-pressure liquid chromatography, and performing gradient elution by using 50-60% of methanol-water solution to obtain the required macrolide compound FWYZ52-A pure product.
Specifically, the mass ratio of the macroporous resin HP20 to the fermentation broth is 1:10-1:30, mixing, adsorbing with a resin column, washing with distilled water, removing impurities with 10-20% ethanol, desorbing with 100% ethanol, and recovering crude extract A of FW YZ52 after ethanol solvent; extracting mycelium with ethanol or methanol for 3 times, concentrating the soaking solution under reduced pressure to obtain extract, and obtaining crude extract B.
The invention also discloses application of the macrolide compound FWYZ52-A in preparing antibacterial preparations for medical, veterinary or agricultural non-therapeutic purposes.
Specifically, the antibacterial agent comprises staphylococcus aureus bacteriostat.
The macrolide compound FWYZ52-A provided by the invention has a 20-membered ring and a hydroxyl-containing short fatty chain, is a novel structural substance, has inhibitory activity on staphylococcus aureus in bacteria through verification, provides a lead compound for researching and developing a novel Levantilides series active drug, and has important value for developing and utilizing Chinese ocean drug resources.
The invention separates and screens a strain of antibacterial marine Micromonospora sp FIMYZ52 from marine actinomycetes, and can be used for preparing a macrolide compound FWYZ52-A with inhibitory activity of staphylococcus aureus by fermentation.
The preparation method of the macrolide compound FWYZ52-A is based on the marine Micromonospora sp. FiMYZ52 for fermentation, and the macrolide compound FWYZ52-A pure product is obtained through fermentation liquor extraction and purification, and the fermentation efficiency and the extraction efficiency are ideal.
Drawings
In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings, in which,
FIG. 1 is a schematic diagram of a phylogenetic tree of a marine Micromonospora strain of the invention based on the 16S rRNA gene sequence;
FIG. 2 is a high resolution mass spectrum of the macrolide compound FWYZ52-A of the invention;
FIG. 3 is a hydrogen nuclear magnetic resonance image of the macrolide compound FWYZ52-A of the invention 1 H-spectrum;
FIG. 4 is a carbon nuclear magnetic resonance image of the macrolide compound FWYZ52-A of the present invention 13 C spectrum;
FIG. 5 shows the macrolide compound FWYZ52-A of the present invention 1 H- 1 HCOSY profile;
FIG. 6 is a HSQC-related map of the macrolide compound FWYZ52-A of the present invention;
FIG. 7 is a HMBC pattern of the macrolide compound FWYZ52-A of the invention.
Detailed Description
The invention screens and obtains a strain from marine Micromonospora sp. FIMYZ52, which belongs to Micromonospora in actinomycetes through 16S rRNA gene analysis, and in addition, a macrolide compound FWYZ52-A with a new structure having inhibitory activity on staphylococcus aureus in bacteria is obtained by extracting and separating from a fermentation broth of the Micromonospora sp. FIMYZ 52.
EXAMPLE 1 identification of Strain FIMYZ52
And extracting the genome DNA of the screened strain Micromonospora sp.FIMYZ52 by adopting an enzymolysis method, storing the obtained DNA sample at-20 ℃, and taking the DNA sample as a DNA template to carry out 16S rRNA gene PCR amplification, and sequencing the PCR product.
A schematic diagram of a phylogenetic tree of the marine Micromonospora strains based on the 16S rRNA gene sequence is shown in FIG. 1. Blast alignment and homology analysis were performed on the 16S rDNA sequences of the tested strains with the existing sequences in the GenBank database. The result is 96.93% similar to Micromonospora sp; the accession number is: KP900787.1 and 16S partial sequences have the total length of 1454bp, and the specific sequence is shown as SEQ ID No. 1.
The strain FIMYZ52 is judged to be Micromonospora, the classification of the strain is named as actinomycete Micromonospora sp, and the strain is preserved in China general microbiological culture Collection center (China Committee) for culture Collection of microorganisms, 3 months and 7 days in 2022, the preservation address is North Chen Xie No.1, 3 in the Chaoyang area of Beijing city, and the preservation number is CGMCC No.24479.
EXAMPLE 2 fermentative preparation of novel Structure macrolide Compound FWYZ52-A
Inoculating the screened marine Micromonospora sp.FIMYZ52 to an ISP2 agar slant culture, inoculating a liquid seed culture medium, culturing for 2 days at the temperature of 30 ℃, and then mixing the seeds with an artificial seed culture medium according to the volume ratio of 1:10, mixing at 30 ℃, shaking and culturing for 4 days, and collecting fermentation products.
The seed culture medium comprises the following components: 1.5wt% of soluble starch, 0.5wt% of glucose, 0.5wt% of yeast extract and MgSO 4 ·7H 2 O 0.05wt％、NaCl 0.05wt％、(NH 4 ) 2 SO 4 0.05wt％、CaCO 3 0.1wt% of distilled water, and pH is 6.0-8.5;
the components of the selected fermentation medium are as follows: 4wt% of soluble starch, 0.5wt% of glucose, 2.5wt% of soybean cake powder, 0.5wt% of yeast powder and MgSO 4 ·7H 2 O 0.05wt％、K 2 HPO 4 0.05wt％、CaCO 3 0.1wt% of distilled water and pH of 6.0-8.5.
Solid-liquid separation is carried out on fermentation liquor and mycelium, the fermentation liquor is adsorbed by macroporous resin HP20, and the mass ratio of the resin to the fermentation liquor is 1:20, mixing, adsorbing with a resin column, washing with distilled water, removing impurities with 10-20% ethanol, desorbing with 100% ethanol, and recovering crude extract A of ethanol solvent-derived compound FWYZ52-A; extracting mycelium with ethanol or methanol for 3 times, concentrating the soaking solution under reduced pressure to obtain extract, and obtaining crude extract B.
Combining the crude extract A and the crude extract B, separating by normal phase silica gel column chromatography and reverse phase C18 column chromatography, and separating by petroleum ether: gradient elution with ethyl acetate solvent (volume ratio of 10:0-0:10), thin layer chromatography detection, and collecting eluate containing macrolide compound FWYZ52-A; and then go through C 18 Reversed phase column chromatography with methanol: gradient elution is carried out by water (volume ratio is 50-95%), high performance liquid chromatography detection is carried out, and FWYZ52-A substances are collected; through the preparation of C 18 Preparing reversed phase high pressure liquid phase chromatography (50-60% methanol water) by gradient elution, concentrating and evaporating to dryness 60% methanol water section to obtain pure product of macrolide compound FWYZ52-A with antibacterial activity.
EXAMPLE 3 structural resolution of Compound FWYZ52-A
From the fermentation broth of the new strain, a new macrolide compound is isolated, and the structure of the macrolide compound FWYZ52-A is identified by MS and NMR techniques.
The physical and chemical properties of the macrolide compound FWYZ52-A are brown amorphous solids, and the molecular formula is as follows: c (C) 30 H 50 O 7 High resolution mass spectrometry: measurement value: m/z 545.3492[ M+Na ]] + Theoretical value M/z545.3449[ M+Na ]] + The unsaturation degree is 6. Solubility: is soluble in organic solvents such as methanol, acetonitrile, ethyl acetate, dimethyl sulfoxide, and the like.
FWYZ52-A 1 H nuclear magnetic resonance spectrum (DMSO-d 6, 600 MHz) delta 7.06 (dd, j=15.2, 10.9hz, 1H), 6.31 (dd, j=16.3, 10.9hz, 1H), 6.12 (ddd, j=14.9, 9.0,5.5hz, 1H), 5.77 (d, j=15.3 hz, 1H), 4.74 (d, j=7.6 hz, 2H), 4.71 (d, j=6.5 hz, 2H), 3.95 (d, j=7.4 hz, 2H), 3.92 (s, 1H), 3.82 (dt, j=9.1, 4.0hz, 1H), 2.95 (d, j=7.5 hz, 1H), 2.56-2.51(m,1H),2.46–2.41(m,3H),2.39(t,J＝7.4Hz,1H),1.72(q,J＝7.5,6.4Hz,1H),1.51(s,3H),1.48-1.38(m,5H),1.30(m,2H),1.24(d,J＝4.6Hz,2H),1.21(d,J＝15.7Hz,1H),1.04(s,3H),0.90(t,J＝7.3Hz,3H),0.87(d,J＝7.0Hz,3H),0.83(d,J＝6.7Hz,3H),0.54(d,J＝6.4Hz,3H)。
FWYZ52-A 13 C nuclear magnetic resonance spectrum (DMSO-D6, 150 MHz): 13 C NMR(151MHz,DMSO)δ210.8,166.3,144.3,140.7,133.8,132.5,130.3,119.7,78.0,77.6,77.1,70.9,67.1,41.4,41.3,41.1,41.0,40.0,35.0,34.1,33.5,31.3,30.1,29.0,21.2,20.2,20.1,18.7,16.3,7.7。
meanwhile, the invention also determines a plurality of nuclear magnetic resonance patterns of the compound FWYZ52-A, and the nuclear magnetic resonance patterns are shown in the accompanying figures 2-7, so that the attribution of all carbon atoms and hydrogen atoms of the compound and the chemical structure of the compound are determined, and the compound is determined to be a macrolide compound with a novel structure, and the structural formula is characterized in the following table 1.
TABLE 1 FWYZ52-A Compounds 1 H and 13 c (DMSO-6) ascription
In summary, the structural formula of the compound FWYZ52-A obtained by extraction and purification is as follows:
EXAMPLE 4 determination of antibacterial Activity of macrolide Compound FWYZ52-A
In this example, in vitro bacteria and fungus inhibition tests were performed on the macrolide compound FWYZ52-A, and the inhibition activity of the compound FWYZ52-A on bacteria and bacteria was measured by using a paper-agar diffusion test (paper-agar disk diffusion assay), and the result shows that the compound FWYZ52-A has the effect of inhibiting bacteria, namely staphylococcus aureus.
First, escherichia coli and Staphylococcus aureus were treated with 10 -8 Colony density per ml was inverted on MH plates; candida albicans and aspergillus niger 10 percent -8 Spores density per ml were poured onto a sand plate. FWYZ52-A is dissolved in methanol solution, 8 mu l of a sample to be tested is taken and placed on a round filter paper sheet with the diameter of 6mm, after methanol on the filter paper sheet can be completely generated, the filter paper sheet carrying the sample is stuck on a flat plate containing test bacteria (escherichia coli, staphylococcus aureus, candida albicans and aspergillus niger) with the concentration, and meanwhile, the methanol solution is used as a negative control, and the culture is carried out for 24-48 hours at the constant temperature of 28 ℃. The diameter of the inhibition zone is observed and recorded, and the larger the diameter of the inhibition zone is, the stronger the antibacterial activity of the strain is.
Experimental results show that the macrolide compound FWYZ52-A shows antibacterial activity against staphylococcus aureus, and the diameter of a bacteriostasis ring is 7-8mm. Thus, the compound FWYZ52-A is expected to be a lead compound for antibacterial activity.
In conclusion, an in vitro bacteria inhibition activity test of the macrolide compound shows that the compound FWYZ52-A has the effect of inhibiting staphylococcus aureus activity, so that a lead compound is provided for researching and developing a new bacteria inhibition medicament, and the compound has important value for developing and utilizing Chinese ocean medicament resources.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (10)
1. A macrolide compound FWYZ52-a, characterized by having a structure represented by the following formula (i):
2. a marine Micromonospora strain FIMYZ52 is classified and named as Micromonospora sp of actinomycetes, and is preserved in China general microbiological culture Collection center (CGMCC) No.24479 in the 3 rd month of 2022.
3. Use of a marine micromonospora strain according to claim 2 for the fermentative preparation of the macrolide compound FWYZ52-a according to claim 1.
4. A process for preparing the macrolide compound FWYZ52-a according to claim 1, which comprises the step of inoculating the marine micromonospora strain according to claim 2 in a suitable fermentation medium for fermentation culture.
5. The process for producing macrolide compound FWYZ52-A according to claim 4, which comprises the following steps:
(1) Taking the sea Micromonospora sp.FIMYZ52 preserved by an inclined plane, inoculating into a liquid seed culture medium for constant temperature culture, and collecting seed liquid for later use;
the components of the liquid seed medium include: 1-2wt% of soluble starch, 0.1-1wt% of glucose, 0.1-1wt% of yeast extract and MgSO 4 ·7H 2 O 0.01-0.1wt％，NaCl 0.01-0.1wt％，(NH 4 ) 2 SO 4 0.01-0.1wt％，CaCO 3 0.05-0.2wt％，pH 6.0-8.5；
(2) Transferring the seed liquid into a fermentation medium for constant temperature culture to obtain fermentation liquid containing a required macrolide compound FWYZ52-A;
the components of the fermentation medium include: 3-5wt% of soluble starch, 0.1-1wt% of glucose, 2-3wt% of soybean cake powder, 0.1-1wt% of yeast powder and MgSO 4 ·7H 2 O 0.01-0.1wt％、K 2 HPO 4 0.01-0.1wt％、CaCO 3 0.05-0.2wt% and distillingWater is prepared, and the pH value is 6.0-8.5.
6. The process for producing macrolide compound FWYZ52-A according to claim 5, wherein:
in the step (1), the temperature of the culture step is 25-35 ℃ and the culture time is 2-3 days;
in the step (2), the temperature of the culture step is 25-35 ℃ and the culture time is 3-5 days.
7. The method for preparing the macrolide compound FWYZ52-A according to any one of claims 4 to 6, wherein the method further comprises the steps of extracting and purifying the macrolide compound FWYZ52-A, and specifically comprises the following steps:
extracting: carrying out solid-liquid separation on the collected fermentation liquor and mycelium, wherein the fermentation liquor is adsorbed by macroporous resin HP20, and the crude extract A is obtained after the adsorption, the desorption, the recovery and the concentration of ethanol; extracting mycelium with alcohol solvent, collecting extractive solution, and concentrating under reduced pressure to obtain crude extract B;
purifying: combining the obtained crude extract A and the crude extract B, and purifying by normal phase silica gel column chromatography according to the volume ratio of 10:0-0:10, carrying out gradient elution by using a petroleum ether-ethyl acetate solvent, detecting by using thin layer chromatography, and collecting eluent containing a macrolide compound FWYZ52-A; and then go through C 18 Performing reversed phase column chromatography, gradient eluting with 50% -95% methanol-water solution, detecting by high performance liquid chromatography, and collecting eluate containing compound FWYZ52-A; then through the preparation of C 18 And (3) performing reversed-phase high-pressure liquid chromatography, and performing gradient elution by using 50-60% of methanol-water solution to obtain the required macrolide compound FWYZ52-A pure product.
8. The method for producing a macrolide compound FWYZ52-a according to claim 7, wherein the mass ratio of the macroporous resin HP20 to the fermentation broth is 1:10-1:30.
9. use of the macrolide compound FWYZ52-a according to claim 1 for the preparation of a medical, veterinary or agricultural antibacterial agent.
10. The use according to claim 9, wherein the antibacterial agent comprises a staphylococcus aureus antibacterial agent.
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