CN109182180B - A kind of application of the brown streptomycete of ash and its fermenting and producing bar bifilomycin A1 - Google Patents
A kind of application of the brown streptomycete of ash and its fermenting and producing bar bifilomycin A1 Download PDFInfo
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- CN109182180B CN109182180B CN201811072302.4A CN201811072302A CN109182180B CN 109182180 B CN109182180 B CN 109182180B CN 201811072302 A CN201811072302 A CN 201811072302A CN 109182180 B CN109182180 B CN 109182180B
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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
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
- C12P17/162—Heterorings having oxygen atoms as the only ring heteroatoms, e.g. Lasalocid
Abstract
The invention belongs to technical field of microbial fermentation, and in particular to a kind of brown streptomycete of ash for producing bar bifilomycin A1, and the application of its fermenting and producing bar bifilomycin A1 is further disclosed.The present invention screens to obtain the brown streptomycete of ash of plant height production bafliomycins compound by bacterial screening and mutation breeding, the bacterial strain can effectively improve the potency of bafliomycin A1 compound in fermentation liquid, in fermenting experiment, the potency that the brown streptomycete FIM-Ba150115 fermentation of ash produces bafliomycin A1 is up to 615.87mg/L or so, the extraction purification work for largely facilitating bafliomycin A1, can satisfy industrialization demand.
Description
Technical field
The invention belongs to technical field of microbial fermentation, and in particular to a kind of brown strepto- of ash for producing bar bifilomycin A1
Bacterium, and the application of its fermenting and producing bar bifilomycin A1 is further disclosed.
Background technique
Bafliomycins is that lipid resists in a kind of polyene macrocyclic containing 16 membered ring skeletons generated by actinomycete fermentation
It is raw plain, extremely special in chemical structure, the hemiacetal knot in most of such compounds in side chain structure all containing a hexatomic ring
Structure.Reported bafilomycins compound has bafilomycin A1, A2, B1, B2, C1, C2, D, E, F, K and L etc..
Bafilomycins has the multiple biological activities such as antibacterium, antimycotic, desinsection, antitumor and immunosupress, it may also be used for controls
Treat osteoporosis, deafness etc..And existing research shows Bafilomycins series compound, especially bafilomycinA1 tool
There are specific autophagy inhibiting effect and preferable Synergistic action, can be used as antitumor lead compound research, spread out for formulating
Raw new anti-tumor drug；And Bafilomycin A1 has the killing children acute bone-marrow-derived lymphocyte leukaemia of specificity thin
The new role of born of the same parents can provide new formula for preparation treatment leukaemia novel drugs.
Bafilomycin A1 is a kind of vacuole H+-ATP enzyme (vacuolar-type H+-ATPase, V-ATPase)
Specific inhibitor can have stronger inhibiting effect to V-ATPase in extremely low nmol/L level.V-ATPase is extensive
It is distributed in the Endomembrane systems such as Eukaryotic vacuole, trans- golgiosome, lysosome, endosome, secretory granules, and root
According to the small acid difference of organelle, independent function is respectively played, is a kind of new drug target.Violetta N is equal to
2010 the study found that the bafilomycin A1 of low dosage can maintain normal autophagy process to a certain extent, this makes
It is possibly realized using neurodegenerative diseases such as bafilomycin A1 treatment parkinsonisms.Hans Peter in 2013 etc.
Report bafilomycin A1 can be used as antibody drug (antibody-drug conjugate, ADC) for treating tumour.
YUAN N in 2015 etc. reports that bafilomycin A1 passes through inhibition cell autophagy and promotion Apoptosis at low concentrations (1nM)
Two kinds of approach inhibit and kill B-lineage Acute Lymphocyte Leukemia cell.It can be seen that bafilomycins is latent with very strong application
Power.
However, the production of the bafilomycins series compound of oneself report at present, majority are obtained with microbe fermentation method,
But whole fermentation yield is not very high.Gerherd Werner in 1984 etc. divides from 10L streptomyces griseus fermentation liquid
From obtaining bafilomycin A1 45mg, bafilomycinA2 36mg, bafilomycinB1 92mg, bafilomycinB2
41mg, bafilomycinC1 120mg, bafilomycinC2 24mg, whole fermentation titer are extremely low；Gavin in 2010
The report such as Carr, it is 8 isolated from marine actinomycete Streptomyces sp.RJA71 and RJA635
Bafilomycins compound, wherein bafilomycin F-J etc. 5 are novel compound, and other 3 are
bafilomycinA1,B1,D；The isolated bafilomycin of 9.6L tunning from bacterial strain YIM56209 such as Yu in 2011
A1 9.8mg；Isolated 3 macrolides activity from the fermentation liquid of marine actinomycete Y12-26 such as Wei Gang in 2011
Compound bafilomycin D (14.7mg), bafilomycin A1 (15.8mg), bafilomyein K (9.6mg)；2012
Patent CN103829351B report Ka Wuer streptomycete (Streptomyces cavourensis) in year Pan Huaqi etc. is fermented
Bafilomycin B1 and the bafilomycin C1 of generation, content reach 23.45mg/L and 6.603mg/L respectively；Poplar in 2013
The towering people etc. are isolated and purified to obtain Bafilomycin D sterling about 14.7mg from the 40L fermentation liquid of marine actinomycete Y-0117.With
The microbial fermentation production method of these upper published report bafilomycins series compounds, common problem are
It is that yield is all relatively low, is not enough to reach industrialized level.And causing one of this problem is mainly existing fermentation strain
Fermentative activity and stability it is poor, it is seen then that filter out the strong bacterial strain of fermentative activity for bafilomycins series compound
Fermenting and producing have great importance.
Summary of the invention
For this purpose, technical problem to be solved by the present invention lies in provide a kind of brown strepto- of ash for producing bafilomycin A1
Bacterium, to solve the problems, such as that bafilomycin A1 compound fermentation titer is low in the prior art；
The invention solves second technical problem be to provide the ash brown streptomycete fermentation production
The application of bafilomycin A1.
In order to solve the above technical problems, a kind of brown streptomycete of ash of the present invention, classification naming are
Streptomyces griseobrunneus FIM-Ba150115, has been preserved in China Committee for Culture Collection of Microorganisms
Common micro-organisms center (abbreviation CGMCC), depositary institution address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, deposit number
For CGMCC No.15241, the deposit date is on January 18th, 2018.The bacterial strain is bafliomycins A1 compound high yield
Bacterial strain.
The invention also discloses application of the brown streptomycete of ash in fermenting and producing bafliomycins compound.
The bafliomycins compound is bafliomycins A1.
Application of the described brown streptomycete of ash in fermenting and producing bafliomycins compound, i.e., will be described greyish brown
Brown streptomycete, which is inoculated in suitable fermentation medium, carries out fermented and cultured.
The fermentation medium includes the component of following mass content: soluble starch 3.0-4.0%, peptone 1.5-
3%, yeast powder 1-2%, Dried Corn Steep Liquor Powder 0.5-1.5%, calcium carbonate 0.2-0.3% adjust pH value 7.0-7.5.
The fermentation medium further includes the precursor of 0.025-0.3%, the precursor include isobutanol, isobutyric acid,
Ammonium acetate, isoleucine, leucine and/or valine.
The condition of the fermented and cultured includes: control revolving speed 200-400rpm, ventilatory capacity 0.08-1.2vvm, in 25-30 DEG C
Carry out fermented and cultured.
Application of the brown streptomycete of ash in fermenting and producing bafliomycins compound, further including will be described
The brown streptomycete of ash is inoculated in progress seed liquor culture in seed culture medium, and the seed culture medium includes following mass content
Component: soluble starch 2.0%, peptone 2.0%, yeast powder 1.0%, calcium carbonate 0.2% adjust pH value 7.2.
Application of the brown streptomycete of ash in fermenting and producing bafliomycins compound, further including will be described
The brown streptomycete of ash, which is inoculated in ISP2 slant medium, to be saved, and the ISP2 slant medium includes following mass content
Component: glucose 0.4%, malt extract 1.0%, yeast extract 0.4%, agar 1.8%, adjust pH value 7.2.
The present invention screens to obtain the greyish brown of plant height production bafliomycins compound by bacterial screening and mutation breeding
Brown streptomycete, which can effectively improve the potency of bafliomycin A1 compound in fermentation liquid, in fermenting experiment, ash
The potency that brown streptomycete FIM-Ba150115 fermentation produces bafliomycin A1 is up to 615.87mg/L or so, greatly favorably
It works in the extraction purification of bafliomycin A1, can satisfy industrialization demand.
Detailed description of the invention
In order to make the content of the present invention more clearly understood, it below according to specific embodiments of the present invention and combines
Attached drawing, the present invention is described in further detail, wherein
Fig. 1 is the chadogram of FIM-Ba150115 bacterial strain；
Fig. 2 is the canonical plotting that HPLC method detects bafilomycin A1；
Fig. 3 is reference substance (A) and fermentation broth sample bafilomycin A1 (B) ultraviolet absorption peak map；
Fig. 4 is the HPLC-Q-TOF-MS map of fermented cpds Ba0115；
Fig. 5 is the infared spectrum of fermented cpds Ba0115；
Fig. 6 is fermented cpds Ba0115's1H-NMR spectrum；
Fig. 7 is fermented cpds Ba0115's13C-NMR spectrogram.
Specific embodiment
1 bacterium source of embodiment
This laboratory in Sediments of The East China Sea, obtains more plants through separation, culture, fermentation, determination of activity and puts in August, 2012
Line bacterium, through Preliminary Determination, wherein a streptomycete has stronger bacteriostatic activity to aspergillus niger.
By to the bacterial strain natural separation purifying, fermentation and after extraction process obtain compound Ba0115, through mass spectrum core
The identification such as magnetic and bar bifilomycin A1 (bafilomycin A1) homogeneity.But original strain fermenting and producing bafilomycin A1
Potency is lower, and the present embodiment carries out the mutagenesis of bacterial strain using the bacterial strain as starting strain.
Spore suspension preparation: being inoculated in 28 DEG C of culture 10d in the inclined-plane ISP2 or so for starting strain, and fresh inclined-plane is taken to be added
10mL sterile saline is gently scraped with inoculation shovel, pours into the sterile flasks oscillation 20min with bead, rear sterilizing
The double-deck lens wiping paper filters mycelia, and it is spare to leave spore suspension.
ARTP (atmospheric pressure at room plasma) mutagenesis: the operating power 100W of setting ARTP mutation breeding instrument, gas source is argon
Gas, throughput 10L/min, irradiation distance between plasma emission source and sample are 2mm, irradiation time is set as 0,30,60,
90,120,150S；It takes the spore suspension 10ul prepared to be uniformly applied on sterile metal slide glass, then puts it into ARTP instrument
Sample stage；Plasma irradiating is carried out by program, is put slide glass to raw equipped with 1ml with aseptic nipper after sample treatment
In the EP pipe for managing salt water, EP pipe is shaken into 1min on the oscillator, the microorganism being attached on fungus slide glass is eluted to liquid
In, form new bacteria suspension；New bacteria suspension is suitably diluted, 0.1ml dilution spread plate is taken, is placed in 32 DEG C of constant temperature
12d is cultivated in incubator, cultured plate is for calculating lethality and bacterial screening.
Single colonie under picking difference ARTP Induced dosage totally 591 plants of switching ISP2 inclined-plane cultures.By shake flask fermentation,
HPLC detects bafilomycin A1 content in fermentation liquid, and wherein strain number is in Ba076, Ba115, Ba367 fermentation liquid
Bafilomycin A1 potency improves 5 times or so of mutant strain, and the fermentation titer of bafilomycin A1 respectively reaches
310mg/L、308mg/L、298mg/L。
Continuous 5 generation secondary culture is carried out to above three plants of superior strains respectively, investigates mutant strain genetic stability, experiment
As a result it see the table below 1, mutant strain Ba115 genetic stability is preferable as the result is shown, it is further saved, number is named as FIM-
Ba150115。
The experiment of 1 high productive mutant genetic stability of table
Feature is learned in the morphology of 2 streptomycete FIM-Ba150115 of embodiment and culture
Identified, streptomycete FIM-Ba150115 described in the present embodiment is the positive through Gram's staining, synthesizes one in Gao Shi
After growing 7d on the culture mediums such as number agar and ISP2, substrate mycelium physically well develops, no tabula, not broken, and aerial hyphae growth is good
Well, multi-branched, fibrillae of spores are straight or flexible.
Seven kinds of culture mediums such as ISPl, ISP2, ISP3, ISP4 are respectively adopted, after 30 DEG C of culture 14d, observation mattress filament
Color and pigment situation, as a result see the table below shown in 2.
2 FIM-Ba150115 cultural characteristic of table
| Culture medium | Growing state | Aerial hyphae | Substrate mycelium | Soluble pigment |
| ISP1 | ++ | It is greyish white | Grey | Nothing |
| ISP2 | +++ | Ash is brown | Taupe | Nothing |
| ISP3 | + | Ash | It is poor | Nothing |
| ISP4 | + | Simple ash | It is poor | Nothing |
| ISP5 | +~++ | It is brown | Grey | Nothing |
| ISP6 | +++ | Ash is brown | Brown | Nothing |
| ISP7 | + | Brown | It is colourless | Nothing |
Remarks: +++: it is good；++: it is medium；+: it is poor.
The Physiology and biochemistry Property Identification of the bacterial strain is carried out according to the standard method in streptomyces category.It is identified, it protects
Deposit the biochemical reactions of the bacterial strain of bacterial strain FIM-Ba150115 are as follows: gelatin liquefaction, dark brown element, milk are solidified and peptonized, forms sediment
Powder hydrolyzes, and grown on cellulose is bad, nitrate reduction, generates Melanoidins and H2S can utilize D-Glucose, D- wood
Sugar, D-Fructose, PEARLITOL 25C, inositol, and L-arabinose, sucrose, rhamnose, gossypose cannot be utilized.
16SrDNA sequence analysis is carried out to the preservation bacterial strain: extracting genome from new fresh thalli with lysozyme Method
DNA carries out 16SrDNA amplification using universal primer, and PCR product is through detection, after purification directly with Taq Deoxy
Terminator Cyele Sequencing Kit sequencing, electrophoresis and number Applied Biosystems DNA Sequencer
(model377) automatic to carry out.The 16SrDNA sequence surveyed proofreaded, spliced after with related species in GenBank database
Sequence carries out BLAST comparison, and the 16SrDNA chadogram of bacterial strain FIM-Ba150115 sees attached drawing 1.
According to the morphological feature of degree bacterial strain, cultural characteristic, physiological and biochemical property and the analysis of 16SrDNA sequence synthesis, it was demonstrated that
The bacterial strain FIM-Ba150115 that the present invention screens is accredited as one plant of new taupe streptomycete (Streptomyces
griseobrunneus).It is common that above-mentioned streptomycete FIM-Ba150115 is preserved in China Committee for Culture Collection of Microorganisms
Microorganism center (road Chaoyang District Beijing great Tun first 3, Institute of Microorganism, Academia Sinica, referred to as CGMCC), register into
The number of volume is CGMCC No.15241, and the deposit date is on January 18th, 2018.The bacterial strain is bafliomycins A1 chemical combination
Object superior strain.
The detection of bafilomycin A1 in 3 fermentation liquid of embodiment
To the detection of bafilomycin A1 content in fermentation liquid according in the prior art in the following each embodiments of the present invention
HPLC method carries out.
Fermented sample preparation: taking bacterial strain fermentation liquor 10mL and the mixing of 20mL methanol is added, and oscillation mixes 20min, and centrifugation is standby
With, and using the detection of HPLC method progress bafilomycin A1.
Efficient liquid phase chromatographic analysis conditions Column testing conditions include:
Ultimate LP-C18 analytical column (5 μm, 4.6 × 250mm)；
Mobile phase: volume ratio is the methanol-water solution of 85:15；
Flow velocity: 1.0mL/min；
Sample volume: 10 μ L；
Detection wavelength: 247nm；
Using the time: 30min；
Using calculated by peak area content.
Bafilomycin A1 reference substance and fermentation broth sample to be measured is purple through photodiode array detector 210-400
Outer scanning, the maximum absorption band both observed are measured by chromatographic condition, if in fermentation liquid sample chromatogram respective peaks and
Bafilomycin A1 chromatographic peak retention time of reference substance is identical, then judges the peak for bafilomycin A1 compound.
Standard curve making
Bafilomycin A1 standard items 12.00mg is accurately weighed in 10mL volumetric flask, is dissolved with methanol, is made into concentration
For the standard solution of 1200 μ g/mL, accurately drawn using doubling dilution the standard solution methanol dilution at 18.75,
37.5, the serial solution of 75,150,300,600 μ g/mL takes 10 μ L to inject HPLC instrument respectively, with concentration (C, μ g/mL) for horizontal seat
Mark, integrating peak areas value (A) are ordinate, equation of linear regression are as follows: y=30013x+53907, R2=1 (as shown in Figure 2).
The result shows that bafilomycin A1 concentration is in the range of 18.75-600 μ g/mL, concentration and peak area line
Sexual intercourse is good, also turns out that it is feasible for detecting the content of bafilomycin A1 in fermentation liquid using above-mentioned HPLC method in the present invention
's.
4 high performance liquid chromatography of embodiment-level four bars flight time tandem mass spectrum (HPLC-Q-TOF-MS) analysis
To the structure detection of tunning bafilomycin A1 according in the prior art in the following each embodiments of the present invention
HPLC-Q-TOF-MS method carries out, and specific chromatographic condition includes:
Chromatographic column: Agilent Eclipse Plus C18 RRHD chromatographic column (2.1 × 50mm, 1.8 μm)；
Mobile phase: 0.1% formic acid-aqueous solution: methanol (volume ratio 20:80)；
Flow velocity: 0.4mL/min；
Column temperature: 40 DEG C；
Sample volume: 1 μ L.
The Mass Spectrometry Conditions are as follows:
Flight time mass spectrum uses electron spray positive ion mode；
Sheath temperature degree: 350 DEG C；
Sheath gas: 11.0L/min；
Spray nozzle voltage: 1000V；
Capillary voltage: 3500V；
Atomization gas pressure: 35psi；
Dry temperature degree: 320 DEG C；
Dry gas stream speed: 8.0L/min；
Spray chamber's electric current: 4.39 μ A；
Capillary tube current: 0.068 μ A；
Fragmentor voltage: 175V；
Skimmer voltage: 65V；
Eight grades of bar voltages: 750V；
Mass spectroscopy data scanning mode: full scan type collection；
Data acquisition range m/z:100-800.
In order to further confirm sample, ESI cation is carried out to target mass-to-charge ratio using HPLC-Q-TOF-MS analytical technology
Selection monitoring, identifies its structure.
5 fermented and cultured of embodiment
Prepare ISP2 solid slope culture medium: glucose 0.4%, malt extract 1.0%, yeast extract 0.4%, fine jade
Rouge 1.8%, distilled water are prepared, and pH7.2 is adjusted.By taupe streptomycete FIM-Ba150115 (deposit number CGMCC of the present invention
No.15241) be inoculated in the above-mentioned inclined-plane ISP2, after in 28 DEG C of constant temperature incubation 10-12d, and in 4 DEG C of preservations.
Shake-flask seed liquid culture medium is prepared: soluble starch 2.0%, peptone 2.0%, yeast powder 1.0%, CaCO3
0.2%, tap water is prepared, and adjusts pH7.2.
Shake-flask seed liquid culture: above-mentioned seed culture medium 80ml, 121 DEG C of autoclave sterilizations are packed into 500mL triangular flask
30min is inoculated with slant pore suspension after cooling, and in 250rpm, 28-32 DEG C of culture 48h of shaking table, seed liquor is made.
Shake-flask seed liquid 600mL is prepared by above-mentioned formula, it is (practical to be inoculated in 20L seeding tank according to 3% inoculum concentration later
Liquid amount 12L) in, seed tank culture is carried out, controls 28 DEG C of temperature, revolving speed 200-400rpm, tank presses 0.03-0.05Mpa, leads to
Tolerance is 1:1vvm, is cultivated 48 hours, and fermentation tank seed liquor is made.
Fermentation tank culture medium: soluble starch 3.0%, peptone 1.5%, yeast powder 1.0%, Dried Corn Steep Liquor Powder 0.5%,
Calcium carbonate 0.3%, tap water are prepared, and pH7.2 is adjusted.
The good seed liquor of above-mentioned seed tank culture is inoculated into 100L fermentor (practical liquid amount with 5.0% inoculum concentration
In 75L), fermentation revolving speed control 200-400rpm, ventilatory capacity control 0.08-1.2vvm, incubation time 96-120h.
Fermentation process, which took fermentation liquid 5ml every 12 hours and 10ml methanol is added, mixes oscillation 30min, 8000rpm/min
Be centrifuged 10min, after take supernatant 0.22um membrane filtration, according in embodiment 3 method carry out HPLC detection,
Bafilomycin A1 reference substance and fermentation broth sample to be measured is purple through photodiode array detector 210-400
The maximum absorption band of the two is observed in outer scanning, and scanning result is as shown in Figure 3.The result shows that the two ultraviolet absorpting spectrum basic one
It causes, there is maximum absorption band at 247.4nm.It is measured by chromatographic condition, respective peaks and bafilomycin in the liquid chromatography that ferments
A1 chromatographic peak retention time of reference substance is identical, judges to contain bafilomycin A1 compound in the present embodiment shake flask fermentation liquid.
It is 320mg/L according to the potency that 3 standard curve of embodiment measures final product bafilomycin A1.
Extraction, purifying and the structural confirmation of 6 tunning of embodiment
Fermentation liquid about 70L in Example 5 after fermentation is directly added into twice 95% of industrial alcohol stirring and impregnates
2h is centrifuged 20min with frame tripod pendulum type batch centrifugal 3000r/min afterwards, abandons bacteria residue afterwards, and ethyl alcohol impregnates clear liquid with the speed of 0.5BV/h
HP20 large pore resin absorption column is splined on to be adsorbed, then respectively with the water of 6 times of column volumes, 40% ethanol-water solution come into
Row elution, flow velocity 1BV/h remove partial impurities pigment；Afterwards with 95% ethanol elution of 4 times of column volumes, eluent is collected, then
Solvent evaporated is concentrated with Rotary Evaporators, obtains paste crude extract about 70g.
The a small amount of methanol ultrasonic dissolution of take cream shape crude extract, is sufficiently stirred mixing, rear vacuum with G200-300 mesh silica gel afterwards
Dry removal organic solvent.It will be filled into silica gel (G200-300 mesh) column upper layer with the silica gel of crude product, then uses petroleum respectively
Ether: ethyl acetate=8:1, petroleum ether: ethyl acetate=4:1, petroleum ether: ethyl acetate=2:1 carries out gradient as mobile phase
Elution, Fractional Collections eluent.Collection liquid detects active principle using HPLC, merges same composition, and rear vacuum-concentrcted is dense
Contracting liquid is dissolved in after a small amount of DMSO the DAC dynamic axial compression system that is splined on, and (filler C18, mobile phase are 70% acetonitrile-water, stream
Fast 10ml/min, Detection wavelength 247nm).Merge target product after HPLC is detected, after vacuum drying, obtains compound Ba0115
Sterling, and the property of the compound is identified.
Identified, compound Ba0115 is white to faint yellow unsetting powder, is soluble in methanol, ethyl alcohol, acetone, acetic acid
The organic solvents such as ethyl ester, chloroform.The maximal ultraviolet absorption peak of compound Ba0115 is in wavelength 247nm, and minimal absorption peak is in wavelength
261nm, ultraviolet absorption peak and infrared signature absorption peak feature and bafilomycin A1 are almost the same.
Compound Ba0115 is analyzed by mass spectrometry according to method in embodiment 4, ion stream is extracted and shows bafilomycin
A1 reference substance is consistent with bafilomycin A1 appearance time in fermentation liquid, and mass spectrogram is as shown in figure 4, the sodium of sample as the result is shown
Adduct ion peak [M+Na]+It is 645.3978, potassium adduct ion peak [M+K]+It is 661.3708, prompting its molecular weight is 622.83,
It is consistent with known bafilomycin A1 relative molecular mass.
The infared spectrum of compound Ba0115 as shown in figure 5,1H-NMR spectrum as shown in fig. 6,13C-NMR spectrogram such as Fig. 7 institute
Show, compound Ba0115 and bafilomycin A1's1H-NMR (400MHz) and13C-NMR (100MHz) data such as the following table 3 institute
Show.
3 compound Ba0115 and bafilomycin A1's of table1H-NMR (400MHz) and13C-NMR (100MHz) data
Pass through1H-NMR and13The data of C-NMR compare, compound Ba0115 and bafilomycin A1's1H-NMR and13The data of C-NMR are coincide substantially.
In summary HPLC-Q-TOF-MS, UV, IS and1H-NMR and13C-NMR analysis, determines and obtains in fermentation liquid of the present invention
Compound Ba0115 and bafilomycin A1 homogeneity are obtained, target product can be made.
7 shake flask fermentation of embodiment
ISP2 solid slope, which is prepared, according to method in embodiment 5 carries out the preservation of bacterial strain inclined-plane.
Shake-flask seed liquid culture medium is prepared according to method in embodiment 5, and carries out seed liquor culture, seed liquor is made.
The present embodiment Medium of shaking flask fermentation: soluble starch 3.0%, peptone 1.5%, yeast powder 1.0%, corn pulp
Dry powder 0.5%, calcium carbonate 0.3%, tap water are prepared, and pH7.2 is adjusted.
Shake flask fermentation culture: above-mentioned fermentation medium 80mL, 121 DEG C of autoclave sterilizations are packed into 500mL triangular flask
30min, is inoculated with cultured seed liquor after cooling, inoculum concentration 5% is collected in shaking table 250rpm, 28-32 DEG C of culture 120h
Fermentation liquid.
It taking fermentation liquid 5ml that 10mL methanol is added after fermentation and mixes oscillation 30min, 8000rpm/min is centrifuged 10min,
After take supernatant 0.22um membrane filtration, according to record method in embodiment 3 to fermentation liquid carry out HPLC detection, measure fermentation liquid
The potency of middle target product bafilomycin A1 is 310mg/L.
8 shake flask fermentation of embodiment
In the present embodiment bacterial strain save and seed liquor preparation step in culture medium selection and condition of culture with 5 phase of embodiment
Together.
The present embodiment Medium of shaking flask fermentation: soluble starch 3.0%, peptone 2.0%, yeast powder 1.5%, corn pulp
Dry powder 1.0%, calcium carbonate 0.2%, tap water are prepared, and pH7.2 is adjusted.Shake flask fermentation culture, system are carried out according to method in embodiment 5
Obtain fermentation liquid.
After fermentation, collecting takes fermentation liquid to carry out HPLC detection, measures target product in fermentation liquor bafilomycin
The potency of A1 is 384.73mg/L.
9 shake flask fermentation of embodiment
In the present embodiment bacterial strain save and seed liquor preparation step in culture medium selection and condition of culture with 5 phase of embodiment
Together.
The present embodiment Medium of shaking flask fermentation: soluble starch 3.0%, peptone 3.0%, yeast powder 1.5%, corn pulp
Dry powder 1.0%, calcium carbonate 0.2%, tap water are prepared, and pH7.2 is adjusted.And shake flask fermentation culture is carried out according to method in embodiment 5,
Fermentation liquid is made.
After fermentation, collecting takes fermentation liquid to carry out HPLC detection, measures target product in fermentation liquor bafilomycin
The potency of A1 is 392.79mg/L.
10 shake flask fermentation of embodiment
In the present embodiment bacterial strain save and seed liquor preparation step in culture medium selection and condition of culture with 5 phase of embodiment
Together.
The present embodiment Medium of shaking flask fermentation: soluble starch 4.0%, peptone 2.0%, yeast powder 2.0%, corn pulp
Dry powder 1.0%, calcium carbonate 0.2%, tap water are prepared, and pH7.2 is adjusted.Shake flask fermentation culture, system are carried out according to method in embodiment 5
Obtain fermentation liquid.
After fermentation, collecting takes fermentation liquid to carry out HPLC detection, measures target product in fermentation liquor bafilomycin
The potency of A1 is 371.79mg/L.
11 shake flask fermentation of embodiment
In the present embodiment bacterial strain save and seed liquor preparation step in culture medium selection and condition of culture with 5 phase of embodiment
Together.
The present embodiment Medium of shaking flask fermentation: soluble starch 4.0%, peptone 3.0%, yeast powder 2.0%, corn pulp
Dry powder 1.5%, calcium carbonate 0.2%, tap water are prepared, and pH7.2 is adjusted.Shake flask fermentation culture, system are carried out according to method in embodiment 5
Obtain fermentation liquid.
After fermentation, collecting takes fermentation liquid to carry out HPLC detection, measures target product in fermentation liquor bafilomycin
The potency of A1 is 371.13mg/L.
12 shake flask fermentation of embodiment
In the present embodiment bacterial strain save and seed liquor preparation step in culture medium selection and condition of culture with 5 phase of embodiment
Together.
The present embodiment Medium of shaking flask fermentation: soluble starch 4.0%, peptone 3.0%, yeast powder 2.0%, corn pulp
Dry powder 1.5%, calcium carbonate 0.2%, ammonium acetate 0.05%, tap water are prepared, and pH7.2 is adjusted.It is shaken according to method in embodiment 5
Bottle fermented and cultured, is made fermentation liquid.
After fermentation, collecting takes fermentation liquid to carry out HPLC detection, measures target product in fermentation liquor bafilomycin
The potency of A1 is 432.08mg/L.
13 shake flask fermentation of embodiment
In the present embodiment bacterial strain save and seed liquor preparation step in culture medium selection and condition of culture with 5 phase of embodiment
Together.
The present embodiment Medium of shaking flask fermentation: soluble starch 4.0%, peptone 3.0%, yeast powder 2.0%, corn pulp
Dry powder 1.5%, calcium carbonate 0.2%, leucine 0.1%, tap water are prepared, and pH7.2 is adjusted.It is shaken according to method in embodiment 5
Bottle fermented and cultured, is made fermentation liquid.
After fermentation, collecting takes fermentation liquid to carry out HPLC detection, measures target product in fermentation liquor bafilomycin
The potency of A1 is 446.82mg/L.
14 shake flask fermentation of embodiment
In the present embodiment bacterial strain save and seed liquor preparation step in culture medium selection and condition of culture with 5 phase of embodiment
Together.
The present embodiment Medium of shaking flask fermentation: soluble starch 4.0%, peptone 3.0%, yeast powder 2.0%, corn pulp
Dry powder 1.5%, calcium carbonate 0.2%, isoleucine 0.2%, tap water are prepared, and pH7.2 is adjusted.It is carried out according to method in embodiment 5
Fermentation liquid is made in shake flask fermentation culture.
After fermentation, collecting takes fermentation liquid to carry out HPLC detection, measures target product in fermentation liquor bafilomycin
The potency of A1 is 462.58mg/L.
15 shake flask fermentation of embodiment
In the present embodiment bacterial strain save and seed liquor preparation step in culture medium selection and condition of culture with 5 phase of embodiment
Together.
The present embodiment Medium of shaking flask fermentation: soluble starch 4.0%, peptone 3.0%, yeast powder 2.0%, corn pulp
Dry powder 1.5%, calcium carbonate 0.2%, valine 0.025%, tap water are prepared, and pH7.2 is adjusted.It is carried out according to method in embodiment 5
Fermentation liquid is made in shake flask fermentation culture.
After fermentation, collecting takes fermentation liquid to carry out HPLC detection, measures target product in fermentation liquor bafilomycin
The potency of A1 is 468.14g/L.
16 shake flask fermentation of embodiment
In the present embodiment bacterial strain save and seed liquor preparation step in culture medium selection and condition of culture with 5 phase of embodiment
Together.
The present embodiment Medium of shaking flask fermentation: soluble starch 4.0%, peptone 3.0%, yeast powder 2.0%, corn pulp
Dry powder 1.5%, calcium carbonate 0.2%, valine 0.05%, tap water are prepared, and pH7.2 is adjusted.It is shaken according to method in embodiment 5
Bottle fermented and cultured, is made fermentation liquid.
After fermentation, collecting takes fermentation liquid to carry out HPLC detection, measures target product in fermentation liquor bafilomycin
The potency of A1 is 448.68mg/L.
17 shake flask fermentation of embodiment
In the present embodiment bacterial strain save and seed liquor preparation step in culture medium selection and condition of culture with 5 phase of embodiment
Together.
The present embodiment Medium of shaking flask fermentation: soluble starch 4.0%, peptone 3.0%, yeast powder 2.0%, corn pulp
Dry powder 1.5%, calcium carbonate 0.2%, valine 0.1%, tap water are prepared, and pH7.2 is adjusted.It is shaken according to method in embodiment 5
Bottle fermented and cultured, is made fermentation liquid.
After fermentation, collecting takes fermentation liquid to carry out HPLC detection, measures target product in fermentation liquor bafilomycin
The potency of A1 is 529.95mg/L.
18 shake flask fermentation of embodiment
In the present embodiment bacterial strain save and seed liquor preparation step in culture medium selection and condition of culture with 5 phase of embodiment
Together.
The present embodiment Medium of shaking flask fermentation: soluble starch 4.0%, peptone 3.0%, yeast powder 2.0%, corn pulp
Dry powder 1.5%, calcium carbonate 0.2%, valine 0.2%, tap water are prepared, and pH7.2 is adjusted.It is shaken according to method in embodiment 5
Bottle fermented and cultured, is made fermentation liquid.
After fermentation, collecting takes fermentation liquid to carry out HPLC detection, measures target product in fermentation liquor bafilomycin
The potency of A1 is 615.87mg/L.
19 shake flask fermentation of embodiment
In the present embodiment bacterial strain save and seed liquor preparation step in culture medium selection and condition of culture with 5 phase of embodiment
Together.
The present embodiment Medium of shaking flask fermentation: soluble starch 4.0%, peptone 3.0%, yeast powder 2.0%, corn pulp
Dry powder 1.5%, calcium carbonate 0.2%, valine 0.3%, tap water are prepared, and pH7.2 is adjusted.It is shaken according to method in embodiment 5
Bottle fermented and cultured, is made fermentation liquid.
After fermentation, collecting takes fermentation liquid to carry out HPLC detection, measures target product in fermentation liquor bafilomycin
The potency of A1 is 536.21mg/L.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Claims (8)
1. a kind of brown streptomycete of ash of high yield bafliomycins A1 compound, classification naming Streptomyces
Griseobrunneus FIM-Ba150115 has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms
The heart, deposit number are CGMCC No.15241, and the deposit date is on January 18th, 2018.
2. application of the brown streptomycete of ash described in claim 1 in fermenting and producing bafliomycins A1 compound.
3. application according to claim 2, which is characterized in that the brown streptomycete of ash described in claim 1 to be inoculated in
It is suitable for carrying out fermented and cultured in fermentation medium.
4. application according to claim 3, which is characterized in that the fermentation medium includes the group of following mass content
Point: soluble starch 3.0-4.0%, peptone 1.5-3%, yeast powder 1-2%, Dried Corn Steep Liquor Powder 0.5-1.5%, calcium carbonate
0.2-0.3% adjusts pH value 7.0-7.5.
5. application according to claim 4, which is characterized in that before the fermentation medium further includes 0.025-0.3%
Body object, the precursor include isobutanol, isobutyric acid, ammonium acetate, isoleucine, leucine and/or valine.
6. application according to claim 5, which is characterized in that the condition of the fermented and cultured includes: control revolving speed 200-
400rpm, ventilatory capacity 0.08-1.2vvm, in 25-30 DEG C of progress fermented and cultured.
7. according to the described in any item applications of claim 3-6, which is characterized in that further including will be described in claim 1 greyish brown
Brown streptomycete is inoculated in progress seed liquor culture in seed culture medium, and the seed culture medium includes the group of following mass content
Point: soluble starch 2.0%, peptone 2.0%, yeast powder 1.0%, calcium carbonate 0.2% adjust pH value 7.2.
8. according to the described in any item applications of claim 3-6, which is characterized in that further including will be described in claim 1 greyish brown
Brown streptomycete is inoculated in ISP2 slant medium and is saved, and the ISP2 slant medium includes the group of following mass content
Point: glucose 0.4%, malt extract 1.0%, yeast extract 0.4%, agar 1.8% adjust pH value 7.2.
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| CN104817547A (en) * | 2015-03-17 | 2015-08-05 | 农业部环境保护科研监测所 | Novel bafilomycin, extraction microorganism and extraction method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104817547A (en) * | 2015-03-17 | 2015-08-05 | 农业部环境保护科研监测所 | Novel bafilomycin, extraction microorganism and extraction method thereof |
Non-Patent Citations (2)
| Title |
|---|
| Analysis of a draft genome sequence of Kitasatospora cheerisanensis KCTC 2395 producing bafilomycin antibiotics;Jae Yoon Hwang等;《Journal of Microbiology》;20141204;第53卷(第1期);第84-89页，参见全文 * |
| 链霉菌BAF-0711产生的次级代谢产物Bafilomycins的研究;张欣;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20170715;第E079-1页，参见摘要，第4-5页，第15页图5和图6 * |
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