CN108998480B - Method for preparing phenol compounds by using microorganisms - Google Patents
Method for preparing phenol compounds by using microorganisms Download PDFInfo
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- CN108998480B CN108998480B CN201810925939.7A CN201810925939A CN108998480B CN 108998480 B CN108998480 B CN 108998480B CN 201810925939 A CN201810925939 A CN 201810925939A CN 108998480 B CN108998480 B CN 108998480B
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- gliocladium roseum
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- 150000002989 phenols Chemical class 0.000 title claims abstract description 11
- 238000000034 method Methods 0.000 title claims abstract description 9
- 244000005700 microbiome Species 0.000 title claims abstract description 6
- YQUVCSBJEUQKSH-UHFFFAOYSA-N 3,4-dihydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C(O)=C1 YQUVCSBJEUQKSH-UHFFFAOYSA-N 0.000 claims abstract description 15
- UYEMGAFJOZZIFP-UHFFFAOYSA-N 3,5-dihydroxybenzoic acid Chemical compound OC(=O)C1=CC(O)=CC(O)=C1 UYEMGAFJOZZIFP-UHFFFAOYSA-N 0.000 claims abstract description 15
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 claims abstract description 15
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 15
- RGHHSNMVTDWUBI-UHFFFAOYSA-N 4-hydroxybenzaldehyde Chemical compound OC1=CC=C(C=O)C=C1 RGHHSNMVTDWUBI-UHFFFAOYSA-N 0.000 claims abstract description 14
- 150000001875 compounds Chemical group 0.000 claims abstract description 12
- 229940090248 4-hydroxybenzoic acid Drugs 0.000 claims abstract description 7
- 239000000287 crude extract Substances 0.000 claims abstract description 7
- 238000010898 silica gel chromatography Methods 0.000 claims abstract description 6
- 241001149472 Clonostachys rosea Species 0.000 claims description 13
- 239000007788 liquid Substances 0.000 claims description 5
- 238000004440 column chromatography Methods 0.000 claims description 4
- 238000010829 isocratic elution Methods 0.000 claims description 4
- 239000003208 petroleum Substances 0.000 claims description 4
- 238000010828 elution Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 2
- 238000009629 microbiological culture Methods 0.000 claims description 2
- -1 phenol compound Chemical class 0.000 claims description 2
- HWJHWSBFPPPIPD-UHFFFAOYSA-N ethoxyethane;propan-2-one Chemical compound CC(C)=O.CCOCC HWJHWSBFPPPIPD-UHFFFAOYSA-N 0.000 claims 1
- 238000000855 fermentation Methods 0.000 abstract description 10
- 230000004151 fermentation Effects 0.000 abstract description 10
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- 229940079593 drug Drugs 0.000 abstract description 2
- 238000004237 preparative chromatography Methods 0.000 abstract description 2
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 abstract 1
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 6
- 241000896533 Gliocladium Species 0.000 description 6
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 4
- OKKJLVBELUTLKV-MZCSYVLQSA-N Deuterated methanol Chemical compound [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 4
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- 235000002595 Solanum tuberosum Nutrition 0.000 description 3
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- 239000002609 medium Substances 0.000 description 3
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- WCGUUGGRBIKTOS-GPOJBZKASA-N (3beta)-3-hydroxyurs-12-en-28-oic acid Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CC[C@@H](C)[C@H](C)[C@H]5C4=CC[C@@H]3[C@]21C WCGUUGGRBIKTOS-GPOJBZKASA-N 0.000 description 2
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- 229940125904 compound 1 Drugs 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000002024 ethyl acetate extract Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 229910001385 heavy metal Inorganic materials 0.000 description 2
- 238000005805 hydroxylation reaction Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
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- 239000000843 powder Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- GHMLBKRAJCXXBS-UHFFFAOYSA-N resorcinol Chemical compound OC1=CC=CC(O)=C1 GHMLBKRAJCXXBS-UHFFFAOYSA-N 0.000 description 2
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- 229910002027 silica gel Inorganic materials 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- PLSAJKYPRJGMHO-UHFFFAOYSA-N ursolic acid Natural products CC1CCC2(CCC3(C)C(C=CC4C5(C)CCC(O)C(C)(C)C5CCC34C)C2C1C)C(=O)O PLSAJKYPRJGMHO-UHFFFAOYSA-N 0.000 description 2
- 229940096998 ursolic acid Drugs 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- BNGCIRYZSMUYSN-NRQMUMKPSA-N (1S,2S,3S,11R,14S)-3-[(1S,2S,3S,11R,14S)-14-ethyl-2-hydroxy-18-methyl-13,17-dioxo-15,16-dithia-10,12,18-triazapentacyclo[12.2.2.01,12.03,11.04,9]octadeca-4,6,8-trien-3-yl]-2-hydroxy-14,18-dimethyl-15,16-dithia-10,12,18-triazapentacyclo[12.2.2.01,12.03,11.04,9]octadeca-4,6,8-triene-13,17-dione Chemical compound N([C@@H]1N2C(=O)[C@]3(C)SS[C@]2(C(N3C)=O)[C@H]2O)C3=CC=CC=C3[C@]12[C@@]12[C@H](O)[C@@]3(C(=O)N4C)SS[C@@]4(CC)C(=O)N3[C@H]2NC2=CC=CC=C12 BNGCIRYZSMUYSN-NRQMUMKPSA-N 0.000 description 1
- ZGZLUDDEYVIROA-FGZHOGPDSA-N (5ar,10bs)-10b-(1h-indol-3-yl)-2-methyl-5a,6-dihydro-1h-pyrazino[1',2':1,5]pyrrolo[2,3-b]indole-1,3,4(2h,10bh)-trione Chemical compound C1=CC=C2C([C@@]34C=C5C(=O)N(C(C(=O)N5[C@H]4NC=4C3=CC=CC=4)=O)C)=CNC2=C1 ZGZLUDDEYVIROA-FGZHOGPDSA-N 0.000 description 1
- MFEVGQHCNVXMER-UHFFFAOYSA-L 1,3,2$l^{2}-dioxaplumbetan-4-one Chemical compound [Pb+2].[O-]C([O-])=O MFEVGQHCNVXMER-UHFFFAOYSA-L 0.000 description 1
- 241000123650 Botrytis cinerea Species 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- 102000012286 Chitinases Human genes 0.000 description 1
- 108010022172 Chitinases Proteins 0.000 description 1
- 244000241257 Cucumis melo Species 0.000 description 1
- 235000015510 Cucumis melo subsp melo Nutrition 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- YKOPWPOFWMYZJZ-FMMUPTMQSA-N Echinocystic acid Natural products C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)C[C@H](O)[C@@]5(C(O)=O)CCC(C)(C)C[C@H]5C4=CC[C@@H]3[C@]21C YKOPWPOFWMYZJZ-FMMUPTMQSA-N 0.000 description 1
- YKOPWPOFWMYZJZ-UHFFFAOYSA-N Echinocystsaeure Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CC(O)C5(C(O)=O)CCC(C)(C)CC5C4=CCC3C21C YKOPWPOFWMYZJZ-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- JKLISIRFYWXLQG-UHFFFAOYSA-N Epioleonolsaeure Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C)(C)CC5C4CCC3C21C JKLISIRFYWXLQG-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000223195 Fusarium graminearum Species 0.000 description 1
- 229910000003 Lead carbonate Inorganic materials 0.000 description 1
- YBRJHZPWOMJYKQ-UHFFFAOYSA-N Oleanolic acid Natural products CC1(C)CC2C3=CCC4C5(C)CCC(O)C(C)(C)C5CCC4(C)C3(C)CCC2(C1)C(=O)O YBRJHZPWOMJYKQ-UHFFFAOYSA-N 0.000 description 1
- MIJYXULNPSFWEK-UHFFFAOYSA-N Oleanolinsaeure Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C)(C)CC5C4=CCC3C21C MIJYXULNPSFWEK-UHFFFAOYSA-N 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 241000813090 Rhizoctonia solani Species 0.000 description 1
- PRYIVLXRWBBBJH-UHFFFAOYSA-N Sch 52900 Natural products C=1C=CC=C2C=1NC1N3C(=O)C(N(C4=O)C)(C)SSC34C(O)C21C12C(O)C3(C(=O)N4C)SSC4(C(O)C)C(=O)N3C2NC2=CC=CC=C12 PRYIVLXRWBBBJH-UHFFFAOYSA-N 0.000 description 1
- BNGCIRYZSMUYSN-UHFFFAOYSA-N Sch 52901 Natural products OC1C2(C(N3C)=O)SSC3(C)C(=O)N2C2NC3=CC=CC=C3C21C12C(O)C3(C(=O)N4C)SSC4(CC)C(=O)N3C2NC2=CC=CC=C12 BNGCIRYZSMUYSN-UHFFFAOYSA-N 0.000 description 1
- 241000221696 Sclerotinia sclerotiorum Species 0.000 description 1
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 235000010724 Wisteria floribunda Nutrition 0.000 description 1
- LUTSRLYCMSCGCS-BWOMAWGNSA-N [(3s,8r,9s,10r,13s)-10,13-dimethyl-17-oxo-1,2,3,4,7,8,9,11,12,16-decahydrocyclopenta[a]phenanthren-3-yl] acetate Chemical compound C([C@@H]12)C[C@]3(C)C(=O)CC=C3[C@@H]1CC=C1[C@]2(C)CC[C@H](OC(=O)C)C1 LUTSRLYCMSCGCS-BWOMAWGNSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 150000001721 carbon Chemical class 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 244000053095 fungal pathogen Species 0.000 description 1
- ZGZLUDDEYVIROA-UHFFFAOYSA-N gliocladin C Natural products C1=CC=C2C(C34C=C5C(=O)N(C(C(=O)N5C4NC=4C3=CC=CC=4)=O)C)=CNC2=C1 ZGZLUDDEYVIROA-UHFFFAOYSA-N 0.000 description 1
- 229930188952 gliocladine Natural products 0.000 description 1
- 230000033444 hydroxylation Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000005065 mining Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- MCHWKJRTMPIHRA-UHFFFAOYSA-N n-(pyrrolidin-2-ylmethyl)aniline Chemical compound C1CCNC1CNC1=CC=CC=C1 MCHWKJRTMPIHRA-UHFFFAOYSA-N 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 229940100243 oleanolic acid Drugs 0.000 description 1
- AICOOMRHRUFYCM-ZRRPKQBOSA-N oxazine, 1 Chemical compound C([C@@H]1[C@H](C(C[C@]2(C)[C@@H]([C@H](C)N(C)C)[C@H](O)C[C@]21C)=O)CC1=CC2)C[C@H]1[C@@]1(C)[C@H]2N=C(C(C)C)OC1 AICOOMRHRUFYCM-ZRRPKQBOSA-N 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- HZLWUYJLOIAQFC-UHFFFAOYSA-N prosapogenin PS-A Natural products C12CC(C)(C)CCC2(C(O)=O)CCC(C2(CCC3C4(C)C)C)(C)C1=CCC2C3(C)CCC4OC1OCC(O)C(O)C1O HZLWUYJLOIAQFC-UHFFFAOYSA-N 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- PRYIVLXRWBBBJH-HRANWFEFSA-N sch-52900 Chemical compound S([C@](N(C1=O)C)(C)C(=O)N2[C@H]3NC=4C5=CC=CC=4)S[C@]21[C@@H](O)[C@]53[C@@]12[C@H](O)[C@@]3(C(=O)N4C)SS[C@@]4(C(O)C)C(=O)N3[C@H]2NC2=CC=CC=C12 PRYIVLXRWBBBJH-HRANWFEFSA-N 0.000 description 1
- 229910052711 selenium Inorganic materials 0.000 description 1
- 239000011669 selenium Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 235000014347 soups Nutrition 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
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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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/42—Hydroxy-carboxylic acids
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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Abstract
The invention discloses a method for preparing phenol compounds by using microorganisms, which belongs to the technical field of microbial chemistryGliocladium roseum4 phenol compounds are obtained by separating the crude extract of the CGMCC 3.3657 fermentation liquor ethyl acetate and are respectively identified as p-hydroxybenzaldehyde (1), p-hydroxybenzoic acid (2), 3, 4-dihydroxybenzoic acid (3) and 3, 5-dihydroxybenzoic acid (4) by nuclear magnetic resonance spectrum. The secondary metabolite is separated by adopting a medium-pressure preparative chromatography and a silica gel column chromatography, the compound structure identification is carried out by a nuclear magnetic resonance technology, the 4 compounds are separated from the strain for the first time, the yield is high, the strain is easy to obtain, the application value of the strain is further excavated, and a new technology is provided for producing related medicines.
Description
Technical Field
The invention relates to the technical field of microbial chemistry, in particular to a method for preparing phenol compounds by using gliocladium roseum.
Background
Gliocladium roseum (Gliocladium roseum) (II)Gliocladium roseumBainier.) belongs to deuteromycota, hyphomycetes, moniliformes, moniliaceae and gliocladium, is a soil inhabitation bacterium and a heavy parasitic bacterium with wide distribution, and has the advantages of high growth speed, large spore production, wide host range, strong parasitic capacity, various antagonistic mechanisms and the like. Gliocladium roseum is one of the biological control factors with great potential in agricultural biological control. The microbial pesticide can generate cell wall degrading mildew such as chitinase, cellulase, protease and the like, so that the cell walls of various pathogenic fungi harmful to melons, fruits, vegetables, ornamental plants and rice, such as sclerotinia sclerotiorum, fusarium graminearum, rhizoctonia solani, botrytis cinerea and the like are infected, the biological control effect is achieved, and the problems of environmental pollution, pesticide residue, drug resistance of germs and the like caused by chemical control are avoided. It also has potential application value in heavy metal bioremediation. Has the research findingsGliocladium roseumCan convert insoluble lead carbonate into soluble lead oxalate and can repair the pollution of heavy metals around a mining site. The gliocladium roseum secondary metabolite has remarkable nematicidal ability.
The student Dong et al isolated a series of nematicidal compounds gliocladine A-E, vertecillin A, 11' -deoxyvertecillin A, sch52900 and sch52901 from a solid fermentation of Gliocladium roseum wheat, strain number YMF 1.00133. The student Song et al continued Dong's research fromGliocladium roseumTwo nematicidal active compounds, namely gliocladin C and 5-n-heterocyclic resorcinol, are obtained by separating YMF1.00133 wheat solid fermentation products. Gliocladium roseumThe fungus also has good application prospect in the aspect of microbial transformation.
Gliocladium roseumSpherical, crystalline, monodisperse selenium nanoparticles can be biosynthesized by an economical, environment-friendly, green and sustainable method. Andre's Garc ı' -a-Granados was usedGliocladium roseumThe CECT2733 microbial transformation method realizes the transformation of C-11 hydroxylation of sesquiterpene-4-hydroxysuccinic acid-1, 6-diketone which is difficult to realize by a chemical method to obtain three new compounds. Used by the learner FuGliocladium roseumCGMCC 3.3657 realizes hydroxylation reaction on saturated carbon of ursolic acid which is a natural product with a complex structure, and the biotransformation new product has better anti-HCV activity than the ursolic acid. Fu also findsGliocladium roseumThe CGMCC 3.3657 can be used as a new method for modifying the oleanolic acid pentacyclic triterpenoid A ring by the microbial transformation of echinocystic acid.
The gliocladium roseum has wide application, can further research the application value of the gliocladium roseum, and provides help for the development of new drugs and the search of novel compounds.
Disclosure of Invention
The invention is characterized by the fact that gliocladium roseum is treatedGliocladium roseumThe liquid fermentation product of CGMCC 3.3657 was studied, and 4 phenolic compounds were isolated from its ethyl acetate extract: p-hydroxybenzaldehyde, p-hydroxybenzoic acid, 3, 4-dihydroxybenzoic acid, 3, 5-dihydroxybenzoic acid, and the chemical formula is as follows:
the name of the gliocladium roseum strain isGliocladium roseum，Is preserved in China general microbiological culture Collection center with the preservation number of CGMCC 3.3657.
The preparation method comprises the following steps: preparing PDA culture solution, inoculating the gliocladium roseum, culturing for 15-20 days, filtering the fermented liquid, extracting with ethyl acetate, vacuum concentrating to obtain crude extract, and treating with medium pressure C18Performing column chromatography with methanol-water gradient elution, and performing silica gel column chromatography with petroleum ether and acetone isocratic elution.
The invention is characterized in thatGliocladium roseum4 phenol compounds are obtained by separating the crude extract of the CGMCC 3.3657 fermentation liquor ethyl acetate and are respectively identified as p-hydroxybenzaldehyde (1), p-hydroxybenzoic acid (2) and,3, 4-dihydroxybenzoic acid (3), 3, 5-dihydroxybenzoic acid (4). The secondary metabolite is separated by adopting a medium-pressure preparative chromatography and a silica gel column chromatography, the compound structure identification is carried out by a nuclear magnetic resonance technology, the 4 compounds are separated from the strain for the first time, the yield is high, the strain is easy to obtain, the application value of the strain is further excavated, and a new technology is provided for producing related medicines.
Detailed Description
The present invention is further described with reference to the following examples, but the present invention is not limited to the following examples, and it is anticipated that one skilled in the art may make various modifications in combination with the prior art.
Example 1
The invention discloses Gliocladium roseumGliocladium roseum4 phenolic compounds are separated from the ethyl acetate extract of the liquid fermentation product of CGMCC 3.3657: p-hydroxybenzaldehyde, p-hydroxybenzoic acid, 3, 4-dihydroxybenzoic acid, 3, 5-dihydroxybenzoic acid, and the chemical formula is as follows:
1. materials and methods
1.1 Instrument and Material Agilent DD 2400-MR NMR Nuclear magnetic resonance apparatus, TMS as internal Standard (Agilent Co., USA), Sepacore Medium pressure preparation System (Switzerland B Ü CHI Co., Ltd.), reversed phase C18The device comprises a filler (ODS, Fuji silicon chemical Co., Ltd., Japan), thin-layer chromatography silica gel GF 254 and column chromatography silica gel (300-400 meshes, Qingdao ocean chemical Co., Ltd., China), a rotary evaporator (Shanghai Yangrong biochemical instrument factory), a BS-2F constant-temperature shaking table (Changzhou Huaguan instrument manufacturing Co., Ltd.), a biological purification table (Suzhou purification instrument Co., Ltd.), and a three-phase ultraviolet analyzer (Shanghai Jiapeng science and technology Co., Ltd.).
Gliocladium roseumCGMCC 3.3657 is purchased from China center for preservation of microbial strains, and PDA is stored in refrigerator at 4 deg.C.
1.2 liquid fermentation culture of bacterial strain and extraction of secondary metabolite peeling and cutting potato into small pieces, adding into water, boiling for about 30 min, filtering potato soup with 8 layers of gauze, adding glucose, mixing, packaging (potato: glucose: distilled water =1: 0.1: 5), and sterilizing at 121 deg.C for 20 min. Will be protected in a sterile operating tableStored inGliocladium roseumAfter twice activation of CGMCC 3.3657, three small pieces of mycelia together with the culture medium are selected and inoculated into a 100mL conical flask containing 40 mL of LPDA culture solution, and the culture solution is cultured in a shaking table at the temperature of 150 r/min and 28 ℃ for 4 days to prepare mother solution. The mother liquor was inoculated into a 1L cone containing 400 mL of culture medium in the same sterile operating station, and a total of 30L of culture medium was subjected to fermentation culture under the same conditions for 15 days. After fermentation, mycelium and fermentation liquor are filtered by a filter flask, extracted for three times by ethyl acetate, and concentrated under reduced pressure to obtain 6.0g of crude extract.
1.3 separation and purification of crude extract 6.0g of crude extract subjected to Medium pressure C18Column chromatography (50 mm x 460 mm) methanol-water (10% → 100%,v/v) The gradient elution was roughly divided into 6 fractions (Fr. 1-6). Fr. 3 was purified by silica gel column chromatography (petroleum ether: acetone =2: 1,v/v) Isocratic elution gave compound 3(17 mg) and compound 4(80 mg), Fr. 4 was purified by silica gel column chromatography (petroleum ether: acetone =1.5: 1,v/visocratic elution) to give compound 1(25 mg) and compound 2(749.2 mg).
2. Results
We are directed to fungiGliocladium roseumCGMCC 3.3657 is fermented and cultured to accumulate secondary metabolites, 4 phenolic compounds are separated from the secondary metabolites and are respectively identified as: p-hydroxybenzaldehyde (1), p-hydroxybenzoic acid (2), 3, 4-dihydroxybenzoic acid (3), and 3, 5-dihydroxybenzoic acid (4).
2.1 yellow crystals, 25 mg of Compound 1.13C-NMR(400 MHz, C3D6O)δ:191.16(CHO),163.88(C-4), 132.79(C-2, 6), 115.76(C-3, 5). The above data and literature [11]Consistent reports have established that compound 1 is p-hydroxybenzaldehyde.
2.2 Compound 2 as a white powder, 749.2 mg.13C-NMR(400 MHz, C3D6O)δ:167.27(C-7), 161.82(C-4), 131.87(C-2, 6), 121.57(C-1), 115.10(C-3, 5). The above data and reference [12 ]]Essentially, compound 2 was determined to be p-hydroxybenzoic acid.
2.3 Compound 3 as a white powder, 17 mg.13C-NMR(400 MHz, CD3OD)δ:168.08(COOH), 151.08(C-4), 145.90(C-3), 123.97(C-6), 123.40(C-1), 117.80(C-2),116.03 (C-5). The above data and literature [13 ]]The results are reported to be consistent with each other,the compound 3 was determined to be 3, 4-dihydroxybenzoic acid.
2.4 Compound 4 yellow crystals, 80 mg.13C-NMR(400 MHz, CD3OD)δ:168.91(COOH), 158.05(C-3, 5), 132.15(C-1), 107.91(C-2, 6), 106.97 (C-4). The above data and literature [14]Consistent reports were made to identify compound 4 as 3, 5-dihydroxybenzoic acid.
Claims (2)
1. A method for preparing phenol compounds by using microorganisms is characterized in that the microorganisms are gliocladium roseum, and the strain name isGliocladium roseum，The compound is preserved in China general microbiological culture collection center with the preservation number of CGMCC 3.3657, and the phenolic compounds are p-hydroxybenzoic acid, p-hydroxybenzaldehyde, 3, 4-dihydroxybenzoic acid and 3, 5-dihydroxybenzoic acid.
2. The method for producing a phenol compound by using a microorganism according to claim 1, wherein the production method comprises: preparing PDA culture solution, inoculating the gliocladium roseum, culturing for 15-20 days, filtering the fermented liquid, extracting with ethyl acetate, vacuum concentrating to obtain crude extract, and treating with medium pressure C18Performing column chromatography with methanol-water gradient elution, and performing silica gel column chromatography with petroleum ether-acetone isocratic elution.
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| CN102199079A (en) * | 2011-01-31 | 2011-09-28 | 中国科学院海洋研究所 | Phenol compound and preparation method and application thereof |
| CN103764819A (en) * | 2011-06-23 | 2014-04-30 | Rho可再生股份有限公司 | Recombinant production systems for aromatic molecules |
| CN108315361A (en) * | 2018-04-20 | 2018-07-24 | 安徽大学 | Method for synthesizing p-hydroxybenzaldehyde by using microorganisms |
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| CN102199079A (en) * | 2011-01-31 | 2011-09-28 | 中国科学院海洋研究所 | Phenol compound and preparation method and application thereof |
| CN103764819A (en) * | 2011-06-23 | 2014-04-30 | Rho可再生股份有限公司 | Recombinant production systems for aromatic molecules |
| CN108315361A (en) * | 2018-04-20 | 2018-07-24 | 安徽大学 | Method for synthesizing p-hydroxybenzaldehyde by using microorganisms |
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