CN101368166A - Method for preparing oligomycin A and special for bacterial strain thereof - Google Patents
Method for preparing oligomycin A and special for bacterial strain thereof Download PDFInfo
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- CN101368166A CN101368166A CNA2008102235006A CN200810223500A CN101368166A CN 101368166 A CN101368166 A CN 101368166A CN A2008102235006 A CNA2008102235006 A CN A2008102235006A CN 200810223500 A CN200810223500 A CN 200810223500A CN 101368166 A CN101368166 A CN 101368166A
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Abstract
The invention provides a method for producing oligomycin A and a special bacterial strain thereof. The method includes the following steps: 1), strain culture: the bacterial strain Streptomyces avermitilis L033 CGMCC No.2678 which can produce oligomycin A is cultured by fermentation to obtain mycelium; 2), the mycelium is extracted through organic solvent to obtain oligomycin A. The bacterial strain is S.avermitilis L033 CGMCC No.2678. The bacterial strain of the invention can produce rich sporus, which has fast growth rate and is convenient for industrial production. The bacterial strain of the invention has high yield of oligomycin A and the yield can be up to 1461 Mug/ml of fermentation liquid.
Description
Technical field
The present invention relates to the production method of oligomycin and produce bacterial strain, particularly relate to a kind of method and special strain therefore thereof of producing Oligomycin A.
Background technology
Oligomycin has the various biological activity, for example has intensive anti-mycotic activity, anti-tumor activity and respiration inhibition effect.
But Oligomycin A BC mixture strongly inhibited aspergillus niger (Aspergillus niger), porous wood mould (Tolypocladium inflatum), Fusarium ocsisporum, crescent Curvularia lunata (Curvularia lunata) and Trichoderma alba, especially the scorching blastomycete of human disease's mycoderma (Blastomyces dermatitidis) had significant inhibition active (Smith et al., 1954; Grammatikova et al., 2003).Usually, the anti-mycotic activity intensity of Oligomycin A, B and C is A〉B〉C (Marty and McCoy, 1959).Oligomycin A is to plant pathogen Botrytis cinerea bacterium (Botrytis cinerea), dosporium cucumerinumand its (Cladosporiumcucumerinum), cucumber anthracnose (Colletotrichum lagenarium), the minimum inhibitory concentration (MIC) of Pyricularia oryzae (Magnaporthe grisea) and pumpkin parasitica (Phytophthora capsici) is 3～5 μ g/ml (Kim et al., 1999).
In with 37,000 compounds the antitumour activity test to human 60 tumor cell lines of plastosome as target, oligomycin is one of 37 kinds of best compounds of effect.The tumor cell line R-HepG2 cell of anti-Zorubicin can produce P-glycoprotein, and the Zorubicin of accumulation lacks than parental cell.Oligomycin can stop the activity of P-glycoprotein, makes the more Zorubicins of R-HepG2 cell accumulation, thus the programmed death of trigger cell (Li et al., 2004).Korystov etc. found in 2003, and the mixture of Oligomycin A BC begins to suppress the survival of mouse P388 lymphoid leukemia cell during for 3pg/ml in concentration, makes cell survival rate drop to 54% when 30pg/ml (being 38pM).These concentration ratios suppress various cellular respiration desired concns fully, and (80～300nM) (Currie et al., 1965) will be hanged down more than 1000 times.Oligomycin A is similar to the mixture of the growth-inhibiting effect of P388 cell and Oligomycin A BC, but inhibition concentration is lower, be Oligomycin A just begins to suppress cell when 1pg/ml survival, when 10pg/ml, reach maximal percentage inhibition (Korystov et al., 2003).Therefore, Oligomycin A has application potential as antineoplastic agent.
Oligomycin is the inhibitor of mammalian cell oxidative phosphorylation.It is joint line plastochondria F effectively
0F
1The function subunit F of atp synthase
0, the configuration of atp synthase is changed, thereby the proton stream that has suppressed mitochondrial membrane space is back to mitochondrial matrix, consequently the synthetic of ATP is blocked, cause the deficiency of biological metabolism institute energy requirement, so oligomycin has very strong toxicity (Pinna et al., 1967) to Mammals.It is 80～300nM (molecular-weight average by Oligomycin A BC mixture is 791 calculating, then is equivalent to 0.0632～0.237 μ g/ml) (Currie et al., 1965) that Oligomycin A BC mixture suppresses various cellular respiration desired concns fully.Because the toxicity of oligomycin makes them not be employed clinically.Yet as the inhibitor of atp synthase, oligomycin has important scientific meaning.Lardy etc. are the pioneer personages (Lardy et al., 1958) who oligomycin is applied to scientific experiment work, and this is that illustrating of oxidative phosphorylation process made huge contribution (Lardy et al., 1969 afterwards; 1975).The change of cell biological energy is relevant with the numerous disease process, and the enzyme-F1F0-ATP synthase that eukaryotic cell is produced most of ATP is regulated, and is expected to be used for these treatment of diseases (Johnson et al., 2006).Plastosome is the key adjusting factor of programmed death, this means that plastosome can be used as the target of cancer therapy.Therefore, oligomycin as plastosome oxidative phosphorylation inhibitor, be widely used in oxidative phosphorylation, with the correlative study of disorderly diseases associated (Angelin et al., 2007) of mitochondrial function and programmed death etc. in the middle of (Dairaku et al., 2004; Karawajew et al., 2005; Hanada et al., 2006).The oligomycin that has the important application meaning in scientific research is sold as chemical reagent already, costs an arm and a leg, and has certain economic and is worth.In each component of Oligomycin A, B and C, the anti-mycotic activity and the anti-tumor activity of Oligomycin A are the strongest.Therefore, except that Oligomycin A BC mixture, Oligomycin A is a component that is widely used in scientific research most.Because Oligomycin A is higher than the production cost of Oligomycin A BC mixture, therefore commercially available price is higher.
Oligomycin A is the microbiotic that separates first oligomycin family that obtains, and produces (Smith et al., 1954) by streptomyces diastatochromogenes (S.diastatochromogenes).Afterwards, people are from other streptomycetes, Avid kyowamycin (Streptomyces avermitilis) (Ikedaet al. for example, 1993a), S.libani (Kim et al., 1999) and light gray streptomycete (S.Griseolus) (Grammatikova et al., 2003), also separate and having obtained Oligomycin A.In the wild-type Avid kyowamycin bacterial strain of having reported, the output of Oligomycin A very low (Ikeda et al., 1993a; 1993b).
Summary of the invention
At the problems referred to above, the object of the invention provides a kind of bacterial strain that can produce Oligomycin A.
Another object of the present invention provides the method that bacterial strain of the present invention is produced Oligomycin A of using.
(Avid kyowamycin is also referred to as deinsectization streptomycete to the Avid kyowamycin of high yield Oligomycin A provided by the invention (Streptomycesavermitilis) L033 bacterial strain; The Latin title is called for short S.avermitilis), be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on September 26th, 2008, deposit number is CGMCCNo.2678.
The L033 bacterial strain is general well-grown on organic substratum, forms abundant aerial hyphae and substrate mycelium.The visible down base silk of opticmicroscope non-cracking, no tabula.By making the electron microscopic sample line scanning electron microscopic observation of going forward side by side, the fibrillae of spores of visible L033 bacterial strain twist, every spore chain can contain greater than 35 spores.Spherical in shape, the avette or column of spore, smooth surface, size are 0.65-1.0 * 0.75-1.6 μ m.Hyphal diameter is 0.75～1.5 μ m.Sole carbon source utilization test shows that the L033 bacterial strain can utilize L (+)-pectinose, D-wood sugar, glucose, D-fructose, L (+)-rhamnosyl, raffinose, N.F,USP MANNITOL, inositol, citric acid is received, the D-semi-lactosi, glycerine, maltose, lactose, the D-seminose can not utilize sucrose, melizitose, L (-)-sorbose, synanthrin.Milk is peptonized, but milk is solidified.Gelatine liquefication can be made, melanochrome and hydrogen sulfide can be produced, can not decomposition of cellulose.The result that antibiotics resistance is measured shows that the L033 bacterial strain is to apramycin, thiostrepton, Streptomycin sulphate, kantlex and paraxin sensitivity, and is insensitive to penbritin and nalidixic acid.
Total DNA with the L033 bacterial strain is a template, with the sequence synthetic primer 1492R of the gene order conservative region of E.coli 16S rRNA (5 '-GGTTACCTTGTTACGACTT-3) and Eubac27F (5 '-AGAGTTTGATCCTGGCTCAG-3 ') be primer, carried out the amplification of the almost completely sequence of 16S rDNA.The PCR product is about 1.5kb, with the expection consistent.Through order-checking as can be known, this PCR product total length 1487bp.Data among this sequence and the GenBank are carried out BLAST analyze, the 16S rDNA sequence of the bacterial strain that will be closely related compares, and the result shows the 16S rDNA sequence and the S.avermitilis ATCC31267 of L033 bacterial strain
T16S rDNA sequence the most similar, homology is 99.93%, and other neighbours' homology is between 98.23～98.97%.These sequences are carried out alignment analyze, remove gap and uncertain position, in L033,1461 definite nucleotide sequences are used to calculate evolutionary distance.Adopt CLUSTAL software to carry out the multisequencing coupling and arrange, the phylogenetic tree (as Fig. 1) that makes up by the adjacent method in the MEGA3.1 software.L033 bacterial strain and S.avermitilis ATCC 31267 as seen from the figure
TSibship nearest, their clusters are on cluster.The number of registration of L033 bacterial strain 16S rDNA sequencing result in the GenBank database is EU621830.
The strain classification status can be finally determined in the dna homology analysis, also is the essential index that gen et sp nov is determined.The type strain ATCC 31267 that concerns known kind of nearest S.avermitilis in L033 bacterial strain to be measured and the phylogeny branch has carried out the DNA cross experiment.The result shows that the dna homology of these two bacterial strains is 100%, shows that the L033 bacterial strain belongs to kind of a S.avermitilis, with its called after S.avermitilis L033.
The invention provides a kind of method of producing Oligomycin A, comprise the steps:
1) strain culturing: Avid kyowamycin (S.avermitilis) the L033 bacterial strain (CGMCC No.2678) that will produce Oligomycin A obtained thalline in 24～216 hours 28 ℃ of condition bottom fermentations cultivations;
2) organic solvent lixiviate mycelium obtains Oligomycin A.
The substratum of described fermentation culture contains following substances: Zulkovsky starch 70g, dry yeast 16g, MgSO
47H
2O 0.5g, K
2HPO
43H
2O 0.5g, KCl 4g, CaCO
32g, distilled water 1000ml, pH are 7.0～7.2.
In order to make the better effects if of fermentation, before carrying out liquid culture, frozen bacterial classification also activates through dull and stereotyped the cultivation, plate culture medium commonly used contains following substances: yeast extract paste 4g, Zulkovsky starch 4g, malt extract 10g, agar 20g, distilled water 1000ml, pH are 7.0～7.2.
In process of production, the described mycelium that obtains generally can adopt centrifugation, as obtaining mycelium in that 3000rpm is centrifugal.
The process that described organic solvent lixiviate mycelium obtains Oligomycin A is to add organic solvent such as acetone, ethyl acetate etc. in mycelium, lixiviate 30 minutes to 24 hours, suction filtration obtains being dissolved in the solution of the Oligomycin A of organic solvent, remove organic solvent after, obtain the thick product of Oligomycin A.Preferred 24 hours of extraction time.The preferred acetone of organic solvent, its add-on can be controlled in acetone and mycelial volume ratio is 2:1.
The mode of removing organic solvent in the Oligomycin A solution can adopt underpressure distillation.Through after the underpressure distillation, reclaim aqueous residue and also make it directly separate out solid matter, reclaim this solid matter and it is further purified, can obtain the crude product of Oligomycin A.Or aqueous residue extracted with ethyl acetate, carry out underpressure distillation again and remove ethyl acetate, also can obtain the crude product of Oligomycin A.
Bacterial strain of the present invention can produce abundant spore, and fast growth is convenient to suitability for industrialized production; The productive rate of bacterial strain production Oligomycin A of the present invention is higher, and output can reach 1461 μ g/ml fermented liquids.
Avid kyowamycin of the present invention (Streptomyces avermitilis) L033 bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on September 26th, 2008, and deposit number is CGMCC No.2678.
Description of drawings
Fig. 1 is the phylogenetic tree that reaches relevant bacterial strain according to the L033 bacterial strain that 16S rRNA gene order makes up.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
The separation screening of embodiment 1 Avid kyowamycin L033 bacterial strain
1, sample collecting
Take pedotheque from GuangZhou, Guangdong Province city Huadu District.
2, the separation of bacterial strain and screening
Preparation Bennett ' s substratum (yeast extract paste 1.0g, extractum carnis 1.0g, hydrolyzed casein 2.0g, glucose 10.0g, agar 20g, distilled water 1000ml, pH7.3), 115 ℃, high pressure steam sterilization 20min falls dull and stereotyped.
Carry out plate isolation after adopting ordinary method to the soil sample serial dilution, in 28 ℃ cultivate 4,7,14,21 days after picking actinomycetes bacterium colony, a strain bacterium called after L033 wherein, preservation behind the purifying.
The cultivation of embodiment 2 bacterial strains
The preparation plate culture medium: yeast extract paste 4g, Zulkovsky starch 4g, malt extract 10g, agar 20g, distilled water 1000ml, pH are 7.2.Plate culture medium is at 121 ℃ of 20min that sterilize down, dull and stereotyped, and cooling back access L033 bacterial strain was cultivated 7 days under 28 ℃.
The obtaining liq substratum: Zulkovsky starch 70g, dry yeast 16g, MgSO47H2O0.5g, K2HPO43H2O 0.5, KCl 4g, CaCO3 2g, distilled water 1000ml, pH are 7.2, the 100ml substratum is packed into sterilize in the 500ml culturing bottle.Cut a ferfas from plate culture, be inoculated in the aseptic liquid nutrient medium, carry out shaking table and cultivate under 28 ℃, rotating speed is 170rpm.Cultivate and begin to produce Oligomycin A after 24 hours, after this grow with time, the output of Oligomycin A constantly increases, and the generation of Oligomycin A enters lag phase after cultivating 216 hours, can stop fermentation.
The extraction of embodiment 3 Oligomycin A crude products
With fermented liquid centrifugal 10min under 3000rpm, abandon supernatant liquor, collect mycelium.Add acetone (acetone and mycelial volume ratio are 2:1) in mycelium, stir with agitator, left standstill 24 hours, suction filtration reclaims filtrate, obtains the acetone soln of Oligomycin A.If still have Oligomycin A to remain in the cell residue, can repeat the aforesaid operations step, till no longer Oligomycin A can being extracted.
Resulting acetone soln is carried out underpressure distillation, reclaim aqueous residue.With ethyl acetate aqueous residue is extracted, again through underpressure distillation, reclaim resistates, can obtain the crude extract of Oligomycin A, productive rate is the thick product of 3.6g Oligomycin A/L fermented liquid.
The extraction of embodiment 4 Oligomycin A crude products
With fermented liquid centrifugal 10min under 3000rpm, abandon supernatant liquor, collect mycelium.Add acetone (acetone and mycelial volume ratio are 2:1) in mycelium, stir with agitator, left standstill 24 hours, suction filtration reclaims filtrate, obtains the acetone soln of Oligomycin A.If still have Oligomycin A to remain in the cell residue, can repeat the aforesaid operations step, till no longer Oligomycin A can being extracted.
Resulting acetone soln is carried out underpressure distillation, reclaim aqueous residue.Aqueous residue is spent the night in 18 ℃ of placements, rice white solid and brown oil occur.This aqueous residue is divided into 3 layers behind the centrifugal 20min under 3000rpm, the upper strata is a brown oil, and the middle layer is the rice white solid, and lower floor is clarifying brown water.Analyze through HPLC, find that Oligomycin A mainly is present in the rice white solid.Reclaim these solid matters, to wherein adding normal hexane-ethanol (96:4) solution, stir, centrifugal removal insolubles obtains clarifying supernatant liquor.After normal hexane-ethanolic soln is removed in underpressure distillation, obtain the crude extract of Oligomycin A, productive rate is the thick product of 1.01g Oligomycin A/L fermented liquid.
The pure crystalline of embodiment 5 Oligomycin As obtains
Carry out being further purified of Oligomycin A with preparation HPLC, column temperature is an envrionment temperature, and moving phase is normal hexane-ethanol (95:5) solution, and flow velocity is 25ml/min.Collect the elution peak of Oligomycin A, merge, carry out underpressure distillation, obtain white solid to remove organic solvent.In this solid residue, drip an amount of normal hexane-ethanol (95:5) solution, stir, occur the pure white crystallization immediately.Normal hexane-ethanol (95:5) solution washing 1 time is used in centrifugal recovery crystallization, centrifugal again recovery crystallization, and make residual organic solvents volatilization fully in air, and obtaining the pure crystallization of Oligomycin A at last, productive rate is the pure crystallization of 20.45mg Oligomycin A/L fermented liquid.
Claims (9)
1. an Avid kyowamycin bacterial strain (Streptomyces avermitilis) L033, preserving number is CGMCC No.2678.
2. a method that adopts the described bacterial classification of claim 1 to produce Oligomycin A is characterized in that, comprises the steps:
1) strain culturing: bacterial strain Avid kyowamycin (S.avermitilis) the L033 CGMCC No.2678 fermentation culture that will produce Oligomycin A obtains mycelium;
2) obtain Oligomycin A with organic solvent lixiviate mycelium.
3. the method for production Oligomycin A as claimed in claim 2 is characterized in that, the bacterial strain of described step 1) was cultivated 24～216 hours at 28 ℃ of condition bottom fermentations.
4. the method for production Oligomycin A as claimed in claim 2 is characterized in that, the substratum of described fermentation culture contains following substances: Zulkovsky starch, dry yeast, MgSO
47H
2O, K
2HPO
43H
2O, KCl, CaCO
3, distilled water; PH is 7.0～7.2.
5. the method for production Oligomycin A as claimed in claim 2 is characterized in that, before cultivating, described bacterial strain activates through dull and stereotyped the cultivation.
6. the method for production Oligomycin A as claimed in claim 5 is characterized in that, the used plate culture medium of described activation contains following substances: yeast extract paste, Zulkovsky starch, malt extract, agar, distilled water; PH is 7.0～7.2.
7. the method for production Oligomycin A as claimed in claim 2, it is characterized in that, described step 2) process that organic solvent lixiviate mycelium obtains Oligomycin A in is to add organic solvent in mycelium, lixiviate 30 minutes to 24 hours, suction filtration obtains being dissolved in the solution of the Oligomycin A of organic solvent, after removing organic solvent, obtain the thick product of Oligomycin A.
8. as the method for claim 2 or 7 described production Oligomycin As, it is characterized in that the volume ratio of described organic solvent and mycelial add-on is 2:1.
9. as the method for claim 2 or 7 described production Oligomycin As, it is characterized in that described organic solvent is acetone or ethyl acetate.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104877925A (en) * | 2014-02-28 | 2015-09-02 | 中国科学院沈阳应用生态研究所 | Actinoalloteichus sp., three antifungal maclafungin compounds, and preparation method and application of compounds |
| CN111647408A (en) * | 2020-06-19 | 2020-09-11 | 河北圆成环保科技股份有限公司 | Multifunctional microbial agent for environmental protection and preparation method thereof |
| CN115643955A (en) * | 2022-11-06 | 2023-01-31 | 华中农业大学 | Application of adriamycin in inhibiting phytopathogen |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| RU2160780C1 (en) * | 1999-05-17 | 2000-12-20 | Мосин Владимир Александрович | Strain streptomyces avermitilis pbm 0004 producing oligomycins |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104877925A (en) * | 2014-02-28 | 2015-09-02 | 中国科学院沈阳应用生态研究所 | Actinoalloteichus sp., three antifungal maclafungin compounds, and preparation method and application of compounds |
| CN111647408A (en) * | 2020-06-19 | 2020-09-11 | 河北圆成环保科技股份有限公司 | Multifunctional microbial agent for environmental protection and preparation method thereof |
| CN115643955A (en) * | 2022-11-06 | 2023-01-31 | 华中农业大学 | Application of adriamycin in inhibiting phytopathogen |
| CN115643955B (en) * | 2022-11-06 | 2024-04-26 | 华中农业大学 | Application of doxorubicin in inhibiting plant pathogenic bacteria |
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