CN102174607A - Method for increasing output of active metabolites of microorganisms - Google Patents
Method for increasing output of active metabolites of microorganisms Download PDFInfo
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- CN102174607A CN102174607A CN2011100453474A CN201110045347A CN102174607A CN 102174607 A CN102174607 A CN 102174607A CN 2011100453474 A CN2011100453474 A CN 2011100453474A CN 201110045347 A CN201110045347 A CN 201110045347A CN 102174607 A CN102174607 A CN 102174607A
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- 244000005700 microbiome Species 0.000 title claims abstract description 34
- 239000002207 metabolite Substances 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 24
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims abstract description 28
- 241000233866 Fungi Species 0.000 claims abstract description 16
- 241000894006 Bacteria Species 0.000 claims abstract description 8
- 241000186361 Actinobacteria <class> Species 0.000 claims abstract description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 9
- 150000001875 compounds Chemical class 0.000 claims description 7
- 239000000758 substrate Substances 0.000 claims description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical group CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- 239000013543 active substance Substances 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 5
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 3
- GSNUFIFRDBKVIE-UHFFFAOYSA-N DMF Natural products CC1=CC=C(C)O1 GSNUFIFRDBKVIE-UHFFFAOYSA-N 0.000 claims description 2
- 238000004090 dissolution Methods 0.000 claims description 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 2
- 229910052736 halogen Inorganic materials 0.000 claims description 2
- 150000002367 halogens Chemical class 0.000 claims description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 2
- 230000006698 induction Effects 0.000 claims description 2
- 238000011081 inoculation Methods 0.000 claims description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 2
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 2
- 229920000136 polysorbate Polymers 0.000 claims description 2
- 125000001424 substituent group Chemical group 0.000 claims description 2
- 238000000855 fermentation Methods 0.000 abstract description 8
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- 239000000411 inducer Substances 0.000 abstract 1
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- 238000011160 research Methods 0.000 description 7
- 238000000605 extraction Methods 0.000 description 6
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- 241000588882 Beijerinckia Species 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
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- 101100412856 Mus musculus Rhod gene Proteins 0.000 description 2
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- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 241000124001 Alcyonacea Species 0.000 description 1
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- 241000196324 Embryophyta Species 0.000 description 1
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 1
- PCNDJXKNXGMECE-UHFFFAOYSA-N Phenazine Natural products C1=CC=CC2=NC3=CC=CC=C3N=C21 PCNDJXKNXGMECE-UHFFFAOYSA-N 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- KYQCOXFCLRTKLS-UHFFFAOYSA-N Pyrazine Natural products C1=CN=CC=N1 KYQCOXFCLRTKLS-UHFFFAOYSA-N 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
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- 241000209140 Triticum Species 0.000 description 1
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- 238000009825 accumulation Methods 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for increasing the output of active metabolites of microorganisms. When the microorganisms including bacteria, fungi, actinomycetes and the like are subjected to fermentation culture, a proper amount of aromatic compounds of benzene ring can be added to serve as inducers, so that the components and the contents of the metabolites of the microorganisms can be changed, and the output of the active metabolites of the microorganisms can be increased. The method has the advantages of low cost, moderate conditions, high efficiency and the like, and has high popularization and application values.
Description
Technical field
The present invention relates to a kind of method that improves microorganism active meta-bolites output.
Background technology
Microorganism comprises bacterium, fungi, actinomycetes etc., and resource is extremely abundant.In recent decades, the research of microbial metabolites, development rapidly, existing large quantities of novel structures, the meta-bolites with potent physiologically active are found in succession, show good prospects for application.The microorganism active meta-bolites has been considered to the important source of following new drug.
For impelling the production by biological meta-bolites, often all use eutrophic substratum to come microorganism is carried out fermentation culture.Yet for educable microorganism, its nutritional needs great majority are still mystery, or solve comprehensive not enough.The kinds of culture medium of microorganism is various, selects which kind of substratum and cultural method actually, often has very big subjectivity, randomness, even makes Optimizing Conditions of Fermentation work also be in unavoidably blindly or half-blindness order state.In addition, on specific substratum, will produce the compound of which kind of structure type, the prediction of also having no way of for the bacterial strain of a certain kind; The research of active metabolite much is to be based upon on the large-scale bacterial strain screening basis repetitive research, wasting of resources showed increased at present; Under conventional culture condition, a lot of active metabolites yield poorly, and complex structure also is not easy to synthetic, and there is the resource bottleneck problem in the research and development of medicine.For solving the problem that these highlight day by day, press for people and further investigate microorganism, the culture technique and the metabolic rule that comprise bacterium, fungi, actinomycetes etc., fully excavate their the synthetic potentiality of source of students, but realize artificial adjustment, have a High-efficient Production of chemical structure diversity active metabolite.
Poisonous phenyl ring aromatics, for example: benzene, toluene, neighbour and paraxylene, phenylformic acid, phenol, nitrobenzene, the chlorinated benzene class, naphthalene, the microbiological deterioration of methylnaphthalene etc. always is the focus of international research for many years.Have now found that a lot of bacteriums, fungi and actinomycetes can both rely on the enzyme of its generation, the phenyl ring aromatic compound is transformed or degrades.Common microorganism has: Rhod (Rhodococcus), Rhodopseudomonas (Pseudomonas), mycobacterium (Mycobacterium), Bacillus (Bacillus), Flavobacterium (Flavobacterium), Aeromonas (Aeromonas), Beijerinckia (Beijernckia), corynebacterium (Corynebacterium), cyanobacteria (Cyanobacteria), micrococcus sp (Micrococcus), Nocardia (Nocardia) and Vibrio (Vibrio), and multiple moulds such as yeast and white-rot fungi.These microorganisms have strong tolerance to the simple relatively phenyl ring aromatics of these structures, can start the regulation system of self, resist " toxicity " of these compounds, and realize this detoxification processes of degraded.
In general, the microbiological deterioration research document of at present relevant phenyl ring aromatics is a lot, but mainly be that the biologist is carrying out relevant research with the environmentalist, and work concentrate on the research of analysis, enzyme and the genes involved of Screening of Bioflocculant-producing Bacteria, degraded product and degradation pathway.
The phenyl ring aromatics can cause the oxidative stress of microorganism, makes cytolipin and albumen generation peroxidation.Cytolemma is the structure of a kind of mesomorphic phase (liquid-crystalline phase), has the dynamic adjustments function.Can pass through activity regulation of enzymes, to stop the peroxidation of lipid, increase the flowability of film, keep membrane structure, under oxidative stress status, can activate some and be combined in enzyme on the film, make that the content of unsaturated fatty acids increases significantly on the film, unsaturated fatty acids has the function of removing superfluous active oxygen.Very clear and definite, microorganism grows in the substratum that has added deleterious phenyl ring aromatics, can cause allergic reaction.Anaphylaxis is made up of multiple biochemical reactions, as form microbiotic, the accumulation protease inhibitor, the enzyme of synthetic and secretion degraded substrate etc., the automatic conditioned reaction of these of microorganism can stop cell to go to pot, and the expression of microorganism specific gene can be induced, and can open new source of students route of synthesis, the result will cause the composition of meta-bolites and content to change, and improves the output of microorganism active metabolite.As seen, the phenyl ring aromatics produces the microbic activity meta-bolites and has important regulation.Yet, to so far, the international and domestic variation that does not all have relevant report to relate to microbial metabolites.
Summary of the invention
The object of the present invention is to provide a kind of method of utilizing the phenyl ring aromatics to improve microorganism active meta-bolites output.
To achieve these goals, the present invention adopts following technical scheme:
A kind of method that improves microorganism active meta-bolites output, the method for employing substrate for induction, described substrate is the phenyl ring aromatics.
Described microorganism can be a bacterium, fungi, actinomycetes etc., common kind is: Rhod (Rhodococcus), Rhodopseudomonas (Pseudomonas), mycobacterium (Mycobacterium), Bacillus (Bacillus), Flavobacterium (Flavobacterium), Aeromonas (Aeromonas), Beijerinckia (Beijernckia), corynebacterium (Corynebacterium), cyanobacteria (Cyanobacteria), micrococcus sp (Micrococcus), Nocardia (Nocardia) and Vibrio (Vibrio), and multiple moulds such as yeast and white-rot fungi, these microorganisms can be from land, or separation obtains in the ocean environment.
Used substrate is the compound that contains one or two phenyl ring, on the phenyl ring substituting group can be arranged, and substituting group can be: methyl (CH
3), ethyl (CH
2CH
3), hydroxyl (OH), carboxyl (COOH), nitro (NO
2), halogen (fluorine, chlorine, bromine) etc., substituent number is one, two or 3.Shown in formula I, be some common aromatic compounds that contains one and two phenyl ring.
（Ⅰ）
The compound that adds can be above-mentioned pure compound, also can be the mixture that a plurality of compounds are formed.
Above-mentioned employing phenyl ring aromatics improves the method for microorganism active meta-bolites output, comprises following concrete steps:
(1) cultivation of microorganism:
Cultured microorganism strains in the inclined-plane is chosen in the fermention medium, and the carbon source of substratum, nitrogenous source, inorganic salt, pH value, culture temperature can be set arbitrarily.Can select for use the liquid or solid substratum to cultivate.Fermentation unit can be selected common glass culturing bottle for use, also can select the laboratory bio-reactor for use, perhaps the industrial fermentation jar.
(2) the adding method of phenyl ring aromatics:
Add the phenyl ring aromatics in fermention medium, the interpolation time is to add when the preparation substratum, or adds after the microorganism strains inoculation again;
Benzene ring type compounds adds after adopting dissolution with solvents, or directly adds, and also can add tensio-active agent simultaneously; Described solvent is preferably DMSO, DMF, ethanol, methyl alcohol, acetone.Described tensio-active agent is preferably tween.
Benzene ring type compounds can disposablely add, also can be on a small quantity, add in batches.
The addition of benzene ring type compounds is every liter of fermention medium 0.01 ~ 100 gram.
(3) after microbial fermentation stopped, meta-bolites can adopt organic solvent repeatedly to extract, and extraction liquid obtains the meta-bolites extract through normal pressure or decompression cryoconcentration; Described organic solvent is preferably ethyl acetate, propyl carbinol, methylene dichloride or chloroform.Also can be with nutrient solution by being filled with the Filter column of polymeric adsorbent, again with solvent with the meta-bolites wash-out that is adsorbed, concentrate.
(4) the meta-bolites extract can carry out chromatographic separation in silicagel column, with petroleum ether-ethyl acetate-methyl alcohol gradient elution, purifying.Compacting is equipped with chromatogram or high pressure liquid chromatography purifying in also can adopting.
Compare with other the technology of having reported of raising microbiological active material output, the present invention has following beneficial effect: the present invention only needs to add an amount of phenyl ring aromatics in existing substratum, just can induce the microbe high-yield active metabolite.Used phenyl ring aromatics can be degraded by microorganisms and be avirulent water soluble component, even eventual degradation is CO
2And H
2O.And by can recycling with solvent extraction that conversion is degraded.It is low that entire operation has a cost, mild condition, advantage such as efficient and environmentally friendly, application value height.
Embodiment
The invention will be further described below in conjunction with embodiment.
Embodiment 1 dimethylbenzene is induced fungi
AspergillusSp. the active pyrazine compound of high yield
One, thalassiomycetes
AspergillusSp. cultivation
Thalassiomycetes
AspergillusSp. be isolating from the soft coral of the South Sea, this bacterium culture medium is: glucose 10 grams, peptone 5 grams, yeast extract paste 2 grams, thick sea salt 23 grams, 1 liter in water, pH=7.5.Fungi
AspergillusSp. in this substratum, 28 ℃, 120 change the per minute shake-flask culture.Cultivate 60 liters altogether, be divided into 2 groups.
Two, inductor dimethylbenzene adds fungi
AspergillusSp. the method for nutrient solution
Above-mentioned fungi
AspergillusSp., choose 30 liters of 1 cultivation groups wherein,, add dimethylbenzene, 2 grams per liters at the 5th day that cultivates.Continue to be cultured to the 10th day, add the dimethylbenzene of 2 grams per liters again.
Three, fungi
AspergillusSp. the extraction of meta-bolites under above-mentioned two kinds of culture condition, separation and evaluation:
1. the extraction of meta-bolites
After the cultivation group of the dimethylbenzene that does not add dimethylbenzene and added is cultivated 20 days respectively, stop fermentation.Respectively nutrient solution is extracted 3 times with ethyl acetate, extracting solution concentrates, and obtains extract.The 30 liters of nutrient solutions of cultivation group that do not add dimethylbenzene obtain 6.6 gram extracts through extraction, and the 30 liters of nutrient solutions of cultivation group that added dimethylbenzene obtain 9.8 gram extracts through extraction.
2. the separation of meta-bolites
Silica gel column chromatography method is adopted in the separation of meta-bolites, with petroleum ether-ethyl acetate-methyl alcohol gradient elution; Wherein with the volume ratio sherwood oil: ethyl acetate=3:1 wash-out obtains 3,6-diisobutyl-2 (1
H)-pyrazine ketone, with the volume ratio sherwood oil: ethyl acetate=1:1 wash-out obtains 3-isobutyl--6-(1-hydroxy-2-methyl propyl group)-2 (1
H)-pyrazine ketone.
3,6-diisobutyl-2 (1
H)-pyrazine ketone 3-isobutyl--6-(1-hydroxy-2-methyl propyl group)-2 (1
H)-pyrazine ketone
Never add in the cultivation group of dimethylbenzene, separate obtaining 3,6-diisobutyl-2 (1
H(3,6-diisobutyl-2 (1 for)-pyrazine ketone
H15 milligrams of)-pyrazinone), 3-isobutyl--6-(1-hydroxy-2-methyl propyl group)-2 (1
H)-pyrazine ketone (3-isobutyl-6-(1-hydroxyl-2-methylpropyl)-2 (1
H43 milligrams of)-pyrazinone).In having added the cultivation group of dimethylbenzene, separate obtaining 3,6-diisobutyl-2 (1
H615 milligrams of)-pyrazine ketone, 3-isobutyl--6-(1-hydroxy-2-methyl propyl group)-2 (1
H1126 milligrams of)-pyrazine ketone.
3-isobutyl--6-(1-hydroxy-2-methyl propyl group)-2 (1
H)-pyrazine ketone is important active substance, has study of anti-atherogenic effect, and promotes plant, as the effect of the growth of soybean, wheat.
3. the evaluation of meta-bolites
The physical constant and the spectral data of main metabolites are as follows:
3,6-diisobutyl-2 (1
H)-pyrazine ketone: white solid, m.p. 127.5-129 ℃; IR (KBr),
ν/ cm
-1: 3429,3068,2959,2909,2870,1645,1530,1470,1384,1263,1238,1172,1100,957,889,806.
1H NMR (300MHz, CDCl
3, TMS): δ 13.2 (s, 1H), 7.27 (s, 1H), 2.72 (d, J=7.2 Hz, 2H), 2.62 (d, J=7.2 Hz, 2H), 2.22 (nonets, J=7.2 Hz, 1H), 2.21 (nonets, J=7.2 Hz, 1H), 0.99 (d, J=7.2 Hz, 6H), 0.96 (d, J=7.2 Hz, 6H).
13C?NMR(75MHz,?CDCl
3,?TMS):?δ?153.4,?152.1,?136.2,?124.1,?41.8,?36.8,?27.0,?26.8,?22.5（2C）,?22.4（2C）。
3-isobutyl--6-(1-hydroxy-2-methyl propyl group)-2 (1
H)-pyrazine ketone: white solid, m.p. 153-154 ℃.IR?(KBr),?
ν/cm
-1:?3411,?3054,?2964,?2929,?2872,?1650,?1624,?1531,?1469,?1427,?1384,?1364,?1348,?1307,?1237,?1164,?1133,?1071,?1035,?1015,?977,?866,?830,?801,?752.?
1H?NMR(300MHz,?CDCl
3,?TMS):?δ?11.38(s,?1H),?7.26(s,?1H),?4.27(d,?J=7.0?Hz,?1H),?3.32?(s,?1H),?2.71?(dd,?J=14.0,?7.0?Hz,?1H),?2.64?(dd,?J=14.0,?7.0?Hz,?1H),?2.20?(nonets,?J=7.0?Hz,?1H),?2.04?(octet,?J=7.0?Hz,?1H),?1.05?(d,?J=7.0?Hz,?3H),?0.96?(d,?J=7.0?Hz,?3H),?0.96?(d,?J=7.0?Hz,?3H),?0.91?(d,?J=7.0?Hz,?3H)。
13C?NMR(75MHz,?CDCl
3,?TMS)：158.6,?157.3,?138.3,?121.7,?74.4,?41.7,?34.2,?26.9,?22.6,?22.6,?18.9,?17.7。
By fungi
AspergillusSp. add dimethylbenzene in the nutrient solution, make 3,6-diisobutyl-2 (1
HThe productive rate of)-pyrazine ketone is brought up to 20.5 mg/litre by 0.5 mg/litre, has improved 41 times; 3-isobutyl--6-(1-hydroxy-2-methyl propyl group)-2 (1
HThe productive rate of)-pyrazine ketone is brought up to 37.54 mg/litre by 1.43 mg/litre, has improved 26.2 times.
Embodiment 2 benzene are induced fungi
ChondrostereumSp. the antitumor sesquiterpenoids of high yield
One, thalassiomycetes
ChondrostereumSp. fermentation culture
Fermention medium consists of: glucose 10g, and peptone 5g, yeast extract 2g, seawater 1L, pH7.5, in room temperature 25-28 ℃, 120 change the per minute shake-flask culture.Cultivate 60 liters altogether, be divided into 2 groups.
Two, benzene adds fungi as inductor
ChondrostereumSp. method
Above-mentioned thalassiomycetes is chosen 30 liters of 1 cultivation groups wherein, at the 10th day that cultivates, gets 30 gram benzene, is dissolved in 100 milliliters of acetone, adds in the culturing bottle equably.Continue again to cultivate 10 days.
Three, above-mentioned cultured filtering fermentation liquor, the filtering thalline is collected nutrient solution.The nutrient solution volume ratio is the ethyl acetate extraction 3 times of 1:1, united extraction liquid, and 40 ℃ of water-bath rotary evaporations concentrate and obtain general extractives.The cultivation group extract that does not add benzene is 13.1 grams, and the cultivation group extract that has added benzene is 14.8 grams.
Four, extract separates with the preparation high performance liquid phase, and eluent is that (40/60, V/V), but purifying obtains white solid sesquiterpenoid Hirsutanol A to water/acetonitrile.Hirsutanol A is the powerful antitumor activity material.
Hirsutanol?A
Purified preparation obtains totally 193 milligrams of Hirsutanol A, productive rate 6.43 mg/litre in the blank cultivation group that does not add benzene; The cultivation group that has added benzene obtains totally 487 milligrams of Hirsutanol A, productive rate 16.23 mg/litre; Productive rate has improved 2.52 times.
Claims (7)
1. method that improves microorganism active meta-bolites output is characterized in that adopting the method for substrate for induction, and described substrate is the phenyl ring aromatics.
2. the method for claim 1 is characterized in that, described microorganism is bacterium, fungi or actinomycetes.
3. the method for claim 1 is characterized in that, described phenyl ring aromatics is the compound that contains one or two phenyl ring.
4. method as claimed in claim 3 is characterized in that, on the described phenyl ring substituting group is arranged, and substituting group is: methyl, ethyl, hydroxyl, carboxyl, nitro or halogen, substituent number are one, two or 3.
5. the method for claim 1 is characterized in that, described phenyl ring aromatics is to add when the preparing microorganism substratum, or adds after the microorganism strains inoculation again;
Benzene ring type compounds adds after adopting dissolution with solvents, or directly adds; Benzene ring type compounds is disposable adding or adds in batches on a small quantity; Described solvent is DMSO, DMF, ethanol, methyl alcohol or acetone.
6. the method for claim 1 is characterized in that, adds tensio-active agent when adding substrate; Described tensio-active agent is a tween.
7. the method for claim 1 is characterized in that, the addition of described phenyl ring aromatics is that the fermention medium of every liter of microorganism adds 0.01 ~ 100 gram.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020045225A1 (en) * | 2000-08-09 | 2002-04-18 | Cawley James J. | Microbial conversion of bicyclic heteroaromatic compounds |
| CN1880309A (en) * | 2004-09-29 | 2006-12-20 | 汕头市双骏生物科技有限公司 | Novel compound, bacteria strain and method for producing novel compound using the strain |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020045225A1 (en) * | 2000-08-09 | 2002-04-18 | Cawley James J. | Microbial conversion of bicyclic heteroaromatic compounds |
| CN1880309A (en) * | 2004-09-29 | 2006-12-20 | 汕头市双骏生物科技有限公司 | Novel compound, bacteria strain and method for producing novel compound using the strain |
Non-Patent Citations (2)
| Title |
|---|
| JENS BITZER ET AL: "new aminophenoxazinones from a marine halomonas sp.: fermentation, structure elucidation, and biological activity", 《THE JOURNAL OF ANTIBIOTICS》 * |
| RONALDP.DE VIRES ET AL: "the aspergillus niger faeB gene encodes a second feruloyl esterase involved in pectin and xylan degradation and is specifically induced in the presence of aromatic compounds", 《BIOCHEM J》 * |
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