CN107058137A - A kind of wet graceful mould and its method for producing wortmannin - Google Patents
A kind of wet graceful mould and its method for producing wortmannin Download PDFInfo
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Abstract
The invention provides a kind of 1507 N13 of wet graceful mould (Penicillium wortmannii), and by cultivating the method that it prepares wortmannin.Wet graceful mould (Penicillium wortmannii) 1507 N13 were preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms center " on 01 09th, 2017, and deposit number is CGMCC NO.13366.
Description
Technical field
The present invention relates to a kind of new wet graceful mould, pass through the method for fermented and cultured bacterium generation wortmannin.
Background technology
The molecular formula of wortmannin (Wortmannin) is C23H24O8, molecular weight is 428.43, its structure such as following formula institute
Show：
Wortmannin is a steroid class antibiotic, and it mainly has suppression to make phosphatidylinositol 3-kinase (PI3K)
With, therefore, therapeutic action particularly to tumour many on the research that it treats phosphatidylinositol 3-kinase dependence disease.
Wortmannin can be combined with permeabilized cells, and PI3K 110kD catalytic subunits, and specificity suppresses PI3K, suppression
PI3K/Akt signal pathways processed, including common suppression Akt phosphorylation etc..Wortmannin can also suppress myosin simultaneously
Light chain kinase (myosin light chain kinase, MLCK) etc..Wortmannin has without obvious antibacterial actions
There is very strong suppression fungi activity.But because it is also limited in vitro study at present to the toxic action of cell, not yet develops into and face
Bed application.
In the prior art, it is initially from Penicillium wormannin that P.W.Brian etc., which reports wortmannin,
Isolated natural products in Klocker fermentation culture medium, and the form of wortmannin production bacterium has been carried out in detail
Description (Trans.Brit.mycol.Soc.40 (3), 365-368 (1957))；US5378725 and CN1111127A are disclosed
Wortmannin production bacterium A24603.1 fermented and cultured and the separation and purifying of wortmannin, A24603.1 produce wet graceful mould
The potency of element is 151 μ g/mL, because its potency is low, wortmannin yield is small, and existing wortmannin producing strains are present not
The problem of being adapted to industrialized production；Such as WO2007120364 and WO2003024183 only report wet graceful mould in some documents
Element is isolated from fungi Penicillium wormannin fermentation medium, and is given birth to for the physiology of producing strains
Changing the concrete composition and content of feature, condition of culture, solid, seed and fermentation medium does not have new report.As can be seen here,
The production bacterium of wortmannin is more original in disclosed patent and document, and fermentation level is low, then exploitation new strains and fermentation
Technology is necessary, therefore finds a kind of work of new efficient wortmannin production bacterium still in proceeding.
The content of the invention
Being of the purpose of the present invention provides a kind of new efficient wortmannin production bacterium, and the bacterium is wet graceful mould
(Penicillium wortmannii) 1507-N13, its deposit number is CGMCC No.13366.
The wet graceful mould 1507-N13 of present invention microorganism fungus kind was preserved in China Microbiological bacterium on 01 09th, 2017
Plant preservation administration committee's common micro-organisms center (address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, the micro- life of the Chinese Academy of Sciences
Thing research institute), deposit number is CGMCC No.13366, and Classification And Nomenclature is wet graceful mould Penicillium wortmannii,
And register on the books, it was demonstrated that survival.
The present invention also aims to there is provided the wet graceful mould 1507-N13 (CGMCC No.13366) of one kind prepare it is wet
Application in graceful penicillin or pharmaceutical composition containing wortmannin.
Present invention also offers a kind of preparation method of wortmannin, this method includes using wet graceful mould 1507-N13
(CGMCC No.13366) in the nutrient medium containing assimilable carbon source and/or nitrogen source, carry out fermented and cultured the step of.
In preferred embodiment, above-mentioned assimilable carbon source be selected from glucose, lactose, mannitol, sorbierite, sucrose,
The combination of one of maltodextrin, cornstarch, glycerine, soya-bean oil, methyl oleate or above-mentioned substance.
In preferred embodiment, above-mentioned assimilable nitrogen source is selected from extraction from yeast powder, dusty yeast, beef extract, soybean protein
The combination of one of peptone, peptone, soybean cake powder, cottonseed meal, groundnut meal, seitan powder, Dried Corn Steep Liquor Powder, wheat bran or above-mentioned substance.
In preferred embodiment, the nutrient medium also includes inorganic salts, and the inorganic salts are selected from ammonium chloride, nitric acid
Ammonium, Triammonium citrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium sulfate, calcium carbonate, ferrous sulfate, zinc sulfate, copper sulphate, chlorination
The combination of one of sodium, potassium chloride, calcium chloride, magnesium sulfate, iron chloride, manganese sulfate or above-mentioned substance.
In preferred embodiment, the nutrient medium contains 10.0~50.0g/L of glycerine, 5.0~50.0g/ of lactose
L, 5.0~50.0g/L of sucrose, 5.0~20.0g/L of soybean cake powder, 5.0~20.0g/L of dusty yeast, cottonseed meal 5.0~
30.0g/L, 0.5~5.0g/L of potassium dihydrogen phosphate, 1.0~5.0g/L of ammonium sulfate, 1.0~5.0g/L of calcium carbonate, manganese sulfate 0.01
~1.0g/L.
In preferred embodiment, the temperature of the fermented and cultured is 20 DEG C -30 DEG C, preferably 24 DEG C -26 DEG C；Medium pH
For 5.0-8.0, preferably 6.0-7.0；Incubation time is 24-240 hours, preferably 72-192 hours.
In preferred embodiment, the wet graceful mould 1507-N13 (CGMCC No.13366) is inoculated with by seed liquor
The fermented and cultured is carried out into the culture medium；
Wherein, the seed liquor is that wet graceful mould 1507-N13 (CGMCC No.13366) is carried out into seed culture to obtain
's；The condition of the seed culture is：The temperature of seed culture is 20 DEG C -30 DEG C, preferably 24 DEG C -26 DEG C；Medium pH is
5.0-8.0, preferably 5.0-7.0；Incubation time is 24-80 hours, preferably 24-60 hours.
In preferred embodiment, described seed culture medium contain 0~20.0g/L of sucrose, glucose 10.0~
50.0g/L, 10.0~30.0g/L of cottonseed meal, 5.0~15.0g/L of dusty yeast, 1.0~10.0g/L of potassium dihydrogen phosphate, sulfuric acid
0.5~1.5g/L of ammonium, 0.5~2.0g/L of calcium carbonate.
Wortmannin of the present invention is detected by the following method：
Chromatographic column：C18 analytical columns (5 μm, 4.6 × 150mm)
Sample size：10μL
Mobile phase：Methanol：Water：Formic acid=50：50：0.1(v/v/v)
Flow velocity：1mL/min
Detection wavelength：254nm
Wet graceful mould (Penicillium wortmannii) 1507-N13 (CGMCC No.13366) the production energy of the present invention
Power is high；And, its ability for producing wortmannin has increased significantly than other strains in the prior art, is advantageously implemented
Industrialized production.
Wet graceful mould (Penicillium wortmannii) 1507-N13 (CGMCC No.13366) of the present invention it is main
Biological property is：Spore is oval, and colony colour is yellow, and edge is white, and reverse side white, tiling, corrugationless has water
Pearl produces, and 1.0~2.0cm of diameter is difficult picking.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
Below in conjunction with specific embodiment, the invention will be further described.It should be understood that following examples are merely to illustrate this
Invention is not for restriction the scope of the present invention.
Embodiment 1：Bacterium source
Wet graceful mould 1507-N13 (CGMCC No.13366) systems of the present invention are from the City of Taizhou of coastal area of southeastern China
Isolated original strain in nine peak mountain soil earth, then the mutant strain obtained by mutagenesis.
Original strain is in PDA slant mediums after 30 DEG C of cultures 12 days, aseptically with inoculation shovel by mycelium
Scrape, mycelium is ground in rub oral examination tube and is suspended in sterilized water, bacteria suspension is made, for NTG (nitrosoguanidine) mutagenic treatment.
NTG crystal 2mg are weighed, are dissolved in 2mL sterile Tris buffers (pH6.0).It is molten with pipette, extract 1ml NTG
Liquid is added to 1ml bacteria suspensions, in 30 DEG C of oscillation treatment 40min.Remaining is comprised the following steps that：
(1) mycelial preparation and culture
Solid culture based formulas：PDA, after being sterilized 30 minutes through 121 DEG C, is cooled to 50-60 DEG C and pours into flat board, will be treated
Bacteria suspension through appropriate dilution, draw 0.1mL bacteria suspensions and be coated on PDA plate, do not make the bacteria suspension of mutagenic treatment also through suitable
When dilution spread in PDA plate as control, 30 DEG C of constant incubator cultures are placed in, bacterium colony culture 7 days, mycelia is ripe.
(2) preparation and culture of seed liquor
Seed culture based formulas (g/L)：Sucrose 10.0g/L, glucose 25.0g/L, cottonseed meal 10.0g/L, dusty yeast
15.0g/L, potassium dihydrogen phosphate 5.0g/L, ammonium sulfate 1.0g/L, calcium carbonate 1.0g/L, pH6.0.Shaking flask liquid amount is 20mL/
250mL, 121 DEG C sterilize 30 minutes.0.5 bacterium colony of each seed flask access or so, 20~30 DEG C of cultivation temperature, bottle swingging machine shakes
Width 5cm, rotating speed 250rpm, are cultivated 36 hours.
(3) preparation and culture of wortmannin fermentation medium
Glycerine 20.0g/L, lactose 40.0g/L, sucrose 30.0g/L, soybean cake powder 10.0g/L, dusty yeast 15.0g/L, cotton
Seed cake powder 10.0g/L, potassium dihydrogen phosphate 0.5g/L, ammonium sulfate 2.0g/L, calcium carbonate 2.0g/L, manganese sulfate 0.05g/L, pH6.5,
Shaking flask liquid amount is 25mL/250mL, and 121 DEG C sterilize 30 minutes.Seed liquor is accessed with the inoculum concentration of 10% (percent by volume)
Fermentation medium, 25 DEG C of cultivation temperature, bottle swingging machine amplitude 5cm, rotating speed 250rpm are cultivated 98 hours.
1000 plants of the single bacterium colony after NTG mutagenesis is selected, shake flask fermentation is carried out, HPLC detects the production of wortmannin
Amount.The i.e. wet graceful mould 1507-N13 (CGMCC No.13366) of mutant strain of wortmannin most high yield is produced in screening.It is led
The biological property is wanted to be：Spore is oval, and colony colour is yellow, and edge is white, and reverse side white, tiling, corrugationless has
The globule is produced, and 1.0~2.0cm of diameter is difficult picking.
Embodiment 2：Strain idenfication
Reference《Common mycology》、《Common bacteria system identification handbook》、《Molecular Cloning:A Laboratory guide》And《Middle traditional Chinese medicines
Allusion quotation》Relevant content in books such as (2015 editions) is tested；Color judges to be compareed with reference to the color of RAL K7 colour atlas series.
1. bacterium numbering：1507-N13(CGMCC No.13366).
2. cultural characteristic：Using 5 kinds of culture mediums of Cha Shi, PDA, SDA, MEA and MEPG, after 28 DEG C are cultivated 5~7 days, observation
Mycelial color and pigment situation.The cultural characteristic of bacterial strain is shown in Table 1.
3. physiological and biochemical test：In addition to temperature is tested, it is 28 DEG C and cultivates 5~7 days.
A) utilization of carbon source：Using culture medium based on ISP9, the final concentration of various carbon sources is 1.0%, is shown in Table 2.
B) inorganic nitrogen-sourced utilization：Using culture medium based on ISP9, the concentration of potassium nitrate and ammonium sulfate is
0.1%, it is shown in Table 2.
C) Degrading experiment and NaCl tolerance tests use basal medium for GYEA (pH6.8), the concentration of various degradation products
And Degrading experiment the results are shown in Table 3；NaCl tolerance experimental results are shown in Table 7.
D) oxidizing ferment and catalase test, pH experiments and humid test use PDA culture medium.Oxidizing ferment and peroxide
Change hydrogen enzyme test the results are shown in Table 4, pH result of the tests and be shown in Table 5, and humid test the results are shown in Table 6.
E) M.R, V-P and urase experiment are used《Common bacteria system identification handbook》Method, it the results are shown in Table 4.
4.28S rDNA sequence analyses：The new fresh thalli of PDA cultures is collected, STb gene mould is extracted using liquid nitrogen wall-breaking method
Plate, carries out 28S rDNA gene ITS regions using Fungi Identification PCR Kit (TaKaRa) and expands, PCR productions
After thing is detected through agarose electrophoresis, sequencing is directly carried out, the purifying of PCR primer is with being sequenced by Nanjing Jin Sirui biotechnologies
Co., Ltd is carried out.The 28S rDNA gene ITS regional sequences that bacterial strain 1507-N13 is surveyed are more true through BLAST after check and correction
Its fixed homology.
Experimental result
1. cultural characteristic：Bacterial strain 1507-N13 (CGMCC No.13366) cultural characteristic is shown in Table 1.
Cultural characteristics of the bacterial strain 1507-N13 of table 1 (CGMCC No.13366) on 5 kinds of culture mediums
2. physiological and biochemical property：It is shown in Table 2~table 7.
The bacterial strain 1507-N13 of table 2 (CGMCC No.13366) carbon source and the utilization power of nitrogen source
The bacterial strain 1507-N13 of table 3 (CGMCC No.13366) Degrading experiment result
| Degradation product | Degradate concentrations | As a result * | Degradation product | Degradate concentrations | As a result |
| Adenine | 0.5% | 4 ,- | Casein | 1.0% | 4 ,- |
| Guanine | 0.5% | 4 ,- | Tyrosine | 1.0% | 4 ,- |
| Xanthine | 0.4% | 4 ,- | Tween-40 | 1.0% | 3 ,- |
| Xylan | 0.4% | 4 ,- | Tween-60 | 1.0% | 3 ,- |
| Hypoxanthine | 0.4% | 4 ,- | Tween-80 | 1.0% | 3 ,- |
Physiological and biochemical property main the bacterial strain 1507-N13 of table 4 (CGMCC No.13366)
| Pilot project | As a result | Pilot project | As a result | Pilot project | As a result |
| Gelatin liquefaction | + | Milk is peptonized | - | Cellulose utilization | - |
| Starch Hydrolysis | - | Nitrate reduction | - | Catalase | - |
| Milk solidifies | - | Hydrogen sulfide is produced | - | ||
| V.P is tested | - | M.R is tested | + |
The pH experiments of the bacterial strain 1507-N13 of table 5 (CGMCC No.13366) growths
| pH | 3.5 | 4.0 | 4.5 | 5.0 | 5.5 | 6.0 | 6.5 | 7.0 | 7.5 |
| Growing state | 3 | 3 | 4 | 4 | 4 | 4 | 4 | 4 | 4 |
The humid test of the bacterial strain 1507-N13 of table 6 (CGMCC No.13366) growths
| Temperature (DEG C) | 7 | 14 | 28 | 37 | 45 |
| Growing state | 0 | 4 | 4 | 0 | 0 |
The bacterial strain 1507-N13 of table 7 (CGMCC No.13366) is to NaCl tolerance
| NaCl concentration | 1% | 4% | 7% | 10% |
| Strain growth situation | 4 | 0 | 0 | 0 |
* note：In table 2-7：0, no growth；1, grow very weak；2, it can grow, there is a small amount of spore；3, well-grown has a large amount of
Spore；4, growth is best, there is abundant spore；+, it is positive；-, it is negative.
3.28S rDNA sequence analyses：Bacterial strain 1507-N13 (CGMCC No.13366) 28S ITS sequences and GenBank
Middle correlated series carries out BLAST comparisons, the results are shown in Table 8 (the higher bacterial strain of homology is only listed in table).
The bacterial strain 1507-N13 of table 8 (CGMCC No.13366) and related strain homology
Strain idenfication result
It is sequenced by the 28S rDNA ITS regions to bacterial strain 1507-N13 (CGMCC No.13366), with GenBank numbers
Homologous sequence BLAST is carried out according to related kind, the sequence of category in storehouse to compare, and is as a result shown：Bacterial strain 1507-N13 (CGMCC
No.13366) and Penicillium (Penicillium sp.SGE10) has very high homology, highest is up to 99.6%, while to bacterial strain
The experiment of 1507-N13 (CGMCC No.13366) appearance features, finds the bacterial strain and Penicillium (Penicillium sp.)
Classify relevant parameter closely, therefore bacterial strain 1507-N13 is accredited as Penicillium (Penicillium sp.) bacterial strain.
Wet graceful mould 1507-N13 (CGMCC No.13366) of the invention and other wortmannin producing strains relatively such as
Under：
P.W.Brian etc. reports (Trans.Brit.mycol.Soc.40 (3), 365-368 (1957) Penicillium
Bacterium colony is fairly small on CheckShi culture mediums, mycelium by wormannin Klocker (nineteen fifty-two is with Penicillium sp. preservations)
Mainly lurid, bacterium colony reverse side is in orange red or light brown, there is fold, slightly towering；And the wet graceful mould of the present invention
1507-N13 (CGMCC No.13366) colony diameter 10mm on CheckShi culture mediums, bacterium colony is in glassy yellow, and bacterium colony reverse side is in
White, corrugationless, tiling.
The potency of US5378725 and CN1111127A wortmannin production bacterium A24603.1 production wortmannin is
151μg/mL；And the potency of wet graceful mould 1507-N13 (CGMCC No.13366) the production wortmannin of the present invention is up to 3500 μ
g/mL。
With reference to the morphology, cultural characteristic, life of foregoing wet graceful mould 1507-N13 (CGMCC No.13366) of the invention
Manage biochemical character and DNA sequence dna qualification result understands that wet graceful mould 1507-N13 (CGMCC No.13366) of the invention belongs to
Wet graceful mould Penicillium wortmannii, and it is different from known wortmannin producing strains, therefore the present invention is wet graceful
Mould 1507-N13 (CGMCC No.13366) is one plant of brand-new strain.
Embodiment 3：Prepare wortmannin
(1) solid culture based formulas and culture
Solid culture based formulas：PDA, after being sterilized 30 minutes through 121 DEG C, cools down stand-by, access 0.1mL bacteria suspensions culture 7
My god.
(2) seed culture based formulas and culture
Seed culture based formulas (g/L)：Sucrose 10.0g/L, glucose 25.0g/L, cottonseed meal 10.0g/L, dusty yeast
15.0g/L, potassium dihydrogen phosphate 5.0g/L, ammonium sulfate 1.0g/L, calcium carbonate 1.0g/L, pH6.0.Shaking flask liquid amount is 20mL/
250mL, 121 DEG C sterilize 30 minutes.0.5 bacterium colony of each seed flask access or so, 20~30 DEG C of cultivation temperature, bottle swingging machine shakes
Width 5cm, rotating speed 250rpm, are cultivated 36 hours.
(3) preparation and culture of wortmannin fermentation medium
Glycerine 40.0g/L, lactose 50.0g/L, dusty yeast 25g/L, cottonseed meal 10.0g/L, potassium dihydrogen phosphate 0.5g/L,
Ammonium sulfate 2.0g/L, calcium carbonate 2.0g/L, manganese sulfate 0.05g/L, pH6.5, shaking flask liquid amount is 25mL/250mL, and 121 DEG C go out
Bacterium 30 minutes.Seed liquor is accessed into fermentation medium, 25 DEG C of cultivation temperature, bottle swingging machine with the inoculum concentration of 10% (percent by volume)
Amplitude 5cm, rotating speed 250rpm, are cultivated 96 hours.Detected through HPLC, wortmannin potency is 3156 μ g/mL.
Embodiment 4：Prepare wortmannin
(1) step (1) in solid culture based formulas and condition of culture equivalent integers 3
(2) step (2) in seed culture based formulas and condition of culture equivalent integers 3
(3) preparation and culture of wortmannin fermentation medium
Glycerine 20.0g/L, lactose 50.0g/L, sucrose 30.0g/L, soybean cake powder 15.0g/L, dusty yeast 15.0g/L, cotton
Seed cake powder 10.0g/L, potassium dihydrogen phosphate 0.8g/L, ammonium sulfate 1.5g/L, calcium carbonate 1.5g/L, manganese sulfate 0.05g/L, pH6.5,
Shaking flask liquid amount is 25mL/250mL, and 121 DEG C sterilize 30 minutes.Seed liquor is accessed with the inoculum concentration of 10% (percent by volume)
Fermentation medium, 25 DEG C of cultivation temperature, bottle swingging machine amplitude 5cm, rotating speed 250rpm are cultivated 100 hours.Detected through HPLC, Wo Man
Penicillin potency is 3344 μ g/mL.
Embodiment 5 prepares wortmannin
(1) step (1) in solid culture based formulas and condition of culture equivalent integers 3
(2) preparation and culture of seed liquor
Seed culture based formulas：Glucose 35.0g/L, cottonseed meal 10.0g/L, dusty yeast 15.0g/L, potassium dihydrogen phosphate
3.0g/L, ammonium sulfate 1.0g/L, calcium carbonate 1.0g/L, pH6.0.Shaking flask liquid amount is 20mL/250mL, and 121 DEG C sterilize 30 points
Clock.20~30 DEG C of cultivation temperature, bottle swingging machine amplitude 5cm, rotating speed 250rpm are cultivated 36 hours.
(3) step (3) in fermentative medium formula and condition of culture equivalent integers 3
Detected through HPLC, wortmannin potency is 3271 μ g/mL.
Embodiment 6：Prepare and isolate and purify wortmannin
(1) preparation of seeding tank seed liquor
10L seed culture medium (step (2) in the formula be the same as Example 3 of seed culture medium) is put into 15L seeding tanks,
The PPG of addition 0.05% is used as defoamer simultaneously), sterilizing is used under the conditions of steam sterilizing, 121 DEG C 30 minutes, after cooling, is connect
Enter 200ml shake-flask seed liquids, 25 ± 1 DEG C of cultivation temperature, 100~500rpm of speed of agitator, 0.5~1.0vvm of throughput, dissolved oxygen
20~70%, cultivate 36 hours.
(2) preparation and culture of fermentation tank culture medium
The formula of fermentation medium is identical with step (3) fermentating formula in previous embodiment 3, but to add 0.05%PPG
As defoamer, fermentation tank loading amount is 30L/50L, pH7.0, in steam sterilizing 30 minutes at 121 DEG C, after cooling, and access is about
3.5L seed tank culture liquid, 25 ± 1 DEG C of fermentation temperature, 200~500rpm of speed of agitator, throughput is 0.5~1.0vvm, fermentation
Culture 103 hours.Detected through HPLC, wortmannin potency is 3510 μ g/mL.
(3) zymotic fluid is extracted
Fermentation obtains 30L zymotic fluids, is extracted 2 times with isometric ethyl acetate, by gained acetic acid ethyl acetate extract at 45 DEG C
Under the conditions of be concentrated under reduced pressure removal ethyl acetate until dry, obtain 76g oily maters.
(4) silicagel column column chromatography is purified
The grease of gained is dissolved into upper silicagel column (particle diameter 100-200 mesh) with 5L chloroforms and carries out column chromatography, chloroform is used:Second
Acetoacetic ester=85:15 (V/V) are eluted, and collect the eluent containing wortmannin.
(5) Structural Identification of the purifying of crude product and sterling
Eluent containing wortmannin is concentrated to dryness, then with ethyl acetate 500mL dissolvings, added after filter paper filtering
1000mL n-hexanes, are transferred in 4 DEG C of refrigerators after being well mixed and refrigerate.After 2 hours, by refrigeration liquid filtering, obtained acicular crystal
Vacuum drying chamber is transferred to, 50 DEG C are dried in vacuo 5 hours.
Above-mentioned crystal determines that it is wortmannin by Spectrum Analysis such as NMR, MS, and its structure is as follows：
The physicochemical property of wortmannin is as follows：
Character:White needle-like crystals
Dissolubility:Chloroform, acetone, methanol are easily soluble in, it is water insoluble
Molecular formula:C23H24O8
ESIMS m/z:427.12[M-H]-
The wortmannin of table 9 is in CDCl3In nuclear magnetic data (hydrogen compose, 400MHz；Carbon is composed, 100MHz)
The above results show that the wet graceful mould 1507-N13 (CGMCC No.13366) of the present invention produces the potency of wortmannin
Up to 3500 μ g/mL (CGMCC No.13366), the wet graceful mould 1507-N13 (CGMCC No.13366) of the present invention produces wet graceful green grass or young crops
The ability of mycin has increased significantly compared with other strains, is advantageously implemented wortmannin industrialized production.
Claims (10)
1. a kind of wet graceful mould (Penicillium wortmannii) 1507-N13, its deposit number is CGMCC
No.13366。
2. wet graceful mould 1507-N13 according to claim 1 is preparing wortmannin or containing wortmannin
Application in pharmaceutical composition.
3. a kind of preparation method of wortmannin, it is characterised in that：Including using wet graceful mould as claimed in claim 1
1507-N13 is in the nutrient medium containing assimilable carbon source and/or nitrogen source, the step of carrying out fermented and cultured.
4. method according to claim 3, it is characterised in that：The assimilable carbon source is selected from glucose, lactose, sweet dew
The combination of one of alcohol, sorbierite, sucrose, maltodextrin, cornstarch, glycerine, soya-bean oil, methyl oleate or above-mentioned substance.
5. method according to claim 3, it is characterised in that：The assimilable nitrogen source is selected from extraction from yeast powder, yeast
Powder, beef extract, soy peptone, peptone, soybean cake powder, cottonseed meal, groundnut meal, seitan powder, Dried Corn Steep Liquor Powder, bran
The combination of one of skin or above-mentioned substance.
6. the method according to claim any one of 3-5, it is characterised in that：The nutrient medium also includes inorganic salts,
The inorganic salts are selected from ammonium chloride, ammonium nitrate, Triammonium citrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium sulfate, calcium carbonate, sulphur
One of sour ferrous iron, zinc sulfate, copper sulphate, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, iron chloride, manganese sulfate or above-mentioned substance
Combination.
7. the method according to claim any one of 3-5, it is characterised in that：The nutrient medium contain glycerine 10.0~
50.0g/L, 5.0~50.0g/L of lactose, 5.0~50.0g/L of sucrose, 5.0~20.0g/L of soybean cake powder, dusty yeast 5.0~
20.0g/L, 5.0~30.0g/L of cottonseed meal, 0.5~5.0g/L of potassium dihydrogen phosphate, 1.0~5.0g/L of ammonium sulfate, calcium carbonate
1.0~5.0g/L, 0.01~1.0g/L of manganese sulfate.
8. the method according to claim any one of 3-5, it is characterised in that：The temperature of the fermented and cultured is 20 DEG C -30
DEG C, preferably 24 DEG C -26 DEG C；Medium pH is 5.0-8.0, preferably 6.0-7.0；Incubation time is 24-240 hours, preferably 72-
192 hours.
9. the method according to claim any one of 3-8, it is characterised in that：The wet graceful mould 1507-N13 is by planting
Sub- liquid, which is seeded in the nutrient medium, carries out the fermented and cultured；
Wherein, the seed liquor is that the wet graceful mould 1507-N13 described in claim 1 is carried out into seed in seed culture medium
What culture was obtained；The condition of the seed culture is：The temperature of seed culture is 20 DEG C -30 DEG C, preferably 24 DEG C -26 DEG C；Culture
Base pH is 5.0-8.0, preferably 5.0-7.0；Incubation time is 24-80 hours, preferably 24-60 hours.
10. method according to claim 9, it is characterised in that：Described seed culture medium contain 0~20.0g/L of sucrose,
10.0~50.0g/L of glucose, 10.0~30.0g/L of cottonseed meal, 5.0~15.0g/L of dusty yeast, potassium dihydrogen phosphate 1.0~
10.0g/L, 0.5~1.5g/L of ammonium sulfate, 0.5~2.0g/L of calcium carbonate.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182147A (en) * | 2018-10-13 | 2019-01-11 | 浙江海正药业股份有限公司 | A kind of mould and its method for producing fumidil |
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| JPH03215423A (en) * | 1990-01-18 | 1991-09-20 | Kyowa Hakko Kogyo Co Ltd | Vasodilator |
| US5378725A (en) * | 1993-07-19 | 1995-01-03 | The Arizona Board Of Regents | Inhibition of phosphatidylinositol 3-kinase with wortmannin and analogs thereof |
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2017
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH03215423A (en) * | 1990-01-18 | 1991-09-20 | Kyowa Hakko Kogyo Co Ltd | Vasodilator |
| US5378725A (en) * | 1993-07-19 | 1995-01-03 | The Arizona Board Of Regents | Inhibition of phosphatidylinositol 3-kinase with wortmannin and analogs thereof |
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| Title |
|---|
| BY P. W.等: "WORTMANNIN, AN ANTIBIOTIC PRODUCED BY PENICILLIUM WORTMANNI", 《TRANS. BRIT. MYCOL. SOC.》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182147A (en) * | 2018-10-13 | 2019-01-11 | 浙江海正药业股份有限公司 | A kind of mould and its method for producing fumidil |
| CN109182147B (en) * | 2018-10-13 | 2020-07-14 | 浙江海正药业股份有限公司 | Penicillium and method for producing fumagillin by using same |
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