CN109182147A - A kind of mould and its method for producing fumidil - Google Patents
A kind of mould and its method for producing fumidil Download PDFInfo
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Abstract
The invention discloses a kind of new mould (Penicillium sp.) HS-NF-684Z, deposit numbers are as follows: CGMCC No.14144；Also disclose the method for preparing fumidil or the pharmaceutical composition containing fumidil by cultivating it.
Description
Technical field
The present invention relates to microbial engineering fields, and in particular to a kind of new mould and its in preparing fumidil
Application.
Background technique
Fumidil (Formulas I) is initially used to kill the microsporidian helminth of honeybee, and discovery can be used for treating later
Microsporidiosis caused by people's enteric infection and (or) Cryptosporidiosis, and this disease is quite to cause for immune deficient patients
Life.In recent years, it with going deep into fumidil and the like research, has been reported that and shows that fumidil and the like has
There is the activity of anti-angiogenesis, it can be by being combined with human methionine aminopeptidase-2 (MetAP-2), to block the shape of blood vessel
At.Therefore, fumidil and the like is widely used in the angiogenesis inhibitors for the treatment of cancer.
In the prior art, it is from aspergillus fumigatus Aspergillus that F.R.Hanson etc., which reports fumidil initially,
Isolated natural products in the fermentation culture medium of fumigatus, and production technology (Eble is disclosed in subsequent research
and Hanson,1951；McCowen et al.,1951)；J.C.Frisvad (Persoonia, vol.14, no.2,177-
182,1990) it etc. discloses and finds that cigarette is bent from the fermentation culture medium of Penicillium notatum Penicillium scabrosum a kind of for the first time
Mycin, and the form of the bacterial strain is described in detail；(the Journal of such as Z.Barbor á kov á
Microbiology, Biotechnology and Food Sciences, 2012:1 (4) 466-477) it discloses
P.scabrosum FCB 353 passes through culture optimization, and the potency for producing fumidil can achieve 110mg/L.It can be seen that existing
The production bacterium of fumidil used in technology is more original, and fermentation level is low, therefore finds a kind of new efficient aspergillus fumigatus
Element production bacterium is necessary.
Summary of the invention
One of the objects of the present invention is to provide a kind of new fumidils to produce bacterium, which is mould (Penicillium
sp.)
HS-NF-684Z, deposit number are CGMCC No.14144.
The microorganism fungus kind of mould HS-NF-684Z of the present invention was preserved in Chinese microorganism strain on 07 24th, 2017
Preservation administration committee common micro-organisms center (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the micro- life of the Chinese Academy of Sciences
Object research institute), deposit number is CGMCC No.14144, entitled mould of classifying (Penicillium sp.), and registering on the books,
Prove survival.
The object of the invention is also to provide a kind of mould HS-NF-684Z (CGMCC NO.14144) to prepare cigarette song
The application of mycin or the pharmaceutical composition containing fumidil.
The present invention also provides a kind of preparation method of fumidil, this method includes using mould HS-NF-684Z
(CGMCC No.14144) is the step of containing in the nutrient medium of assimilable carbon source and/or nitrogen source, carrying out aerobic fermentation.
In preferred embodiments, above-mentioned assimilable carbon source is selected from starch, dextrin, glucose, sucrose, lactose, wheat
The combination of one of bud sugar, industrial molasses, glycerol, sorbierite, mannitol or above-mentioned substance, preferred starch, dextrin, glucose,
Sucrose.
In preferred embodiments, above-mentioned assimilable nitrogen source is selected from extraction from yeast powder, yeast powder, yeast extract, soya bean
One of cake powder, cottonseed meal, groundnut meal, seitan powder, Dried Corn Steep Liquor Powder, dregs of beans, peptone, urea, ammonium salt or above-mentioned object
The combination of matter, preferably soybean cake powder, yeast extract, extraction from yeast powder, yeast powder.
In preferred embodiments, above-mentioned nutrient medium further includes inorganic salts, and the inorganic salts are selected from citric acid three
Sodium, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium sulfate, calcium carbonate, ferrous sulfate, zinc sulfate, copper sulphate, sodium chloride, potassium chloride,
The combination of one of calcium chloride, magnesium sulfate, iron chloride, manganese sulfate or above-mentioned substance, preferably calcium carbonate, potassium dihydrogen phosphate, phosphoric acid hydrogen
Dipotassium, magnesium sulfate, trisodium citrate.
In preferred embodiments, the nutrient medium contains sucrose 40-1000g/L, dextrin 20-1000g/L, sweet
Oily 10-80g/L, extraction from yeast powder 1-15g/L, yeast powder 5-20g/L, yeast extract 5-20g/L, magnesium sulfate 1-10g/L, sulfuric acid two
Hydrogen potassium 1-5g/L, calcium carbonate 0-50g/L.
In preferred embodiments, the temperature of the aerobic fermentation is 20-30 DEG C, preferably 23-28 DEG C；Medium pH is
5.0-8.0 preferably 5.0-7.0；Incubation time is 24-226 hours, preferably 72-168 hours；Oxygen-supply quantity is 0.1-2.0vvm, excellent
Select 0.5-2.0vvm.
In preferred embodiments, the mould HS-NF-684Z is that the nutrient medium is seeded to by seed liquor
The middle progress fermented and cultured；Wherein, the seed liquor is by mould HS-NF-684Z (CGMCC No.14144) in seed
Carry out what seed culture obtained in culture medium；The condition of the seed culture are as follows: the temperature of seed culture is 20 DEG C -30 DEG C, excellent
Select 23 DEG C -28 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 embodiments, the seed culture medium contains glucose 5-20g/L, starch 5-20g/L, soya bean
Cake powder 5-20g/L, extraction from yeast powder 1-10g/L, calcium carbonate 1-20g/L, magnesium sulfate 1-10g/L, potassium dihydrogen sulfate 1-10g/L.
Fumidil of the present invention carries out HPLC detection by the following conditions:
Chromatographic column: Accchrom C18 (4.6 × 250mm, 5 μm)
Column temperature: 25 DEG C；
Sampling volume: 5 μ L；
Flow velocity: 1mL/min；
Detection wavelength: 350nm；
Analysis time: 40min；
Mobile phase:
Mobile phase A: 0.1% (percent by volume) acetic acid aqueous solution；
Mobile phase B: 0.1% (percent by volume) acetic acid acetonitrile solution；
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 00:00 | 65 | 35 |
| 20:00 | 10 | 90 |
| 30:00 | 10 | 90 |
| 34:01 | 65 | 35 |
| 36:00 | 65 | 35 |
The main biological property of mould (Penicillium sp.) HS-NF-684Z (CGMCC NO.14144) of the present invention
Be: spore is rounded, and spore face is in blue-green, and colony colour is yellow, and reverse side yellowish-brown, radial rill, suede is presented in surface
Shape, diameter 20-22mm.
Mould (Penicillium sp.) HS-NF-684Z (CGMCC NO.14144) of the present invention is that one plant of completely new cigarette is bent
Mycin producing strains, and other strains have increased significantly its ability for producing fumidil than in the prior art, potency can
Up to 1000mg/L is advantageously implemented industrialized production.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material as used in the following examples, reagent etc., is commercially available unless otherwise specified.
Wherein fungi DNA extraction kit wins Radix Polygalae biotechnology Co., Ltd purchased from Beijing three；
SanPrep pillar PCR product purification kit used in PCR product purification and recovery be purchased from raw work bioengineering (on
Sea) limited liability company.
Below in conjunction with specific embodiment, the invention will be further described.It should be understood that following embodiment is merely to illustrate this
The range of invention rather than limiting the invention.
Embodiment 1: bacterium source
Mould HS-NF-684Z (CGMCC No.14144) is one plant from Chinese Sichuan Province Chengdu Green City Mountain hillside
Isolated mould in soil.
In Green City Mountain, regional soil carries out ALTERNATE SAMPLING, takes 5 sampled points at random, each point takes pedotheque 10g, is put into
In conical flask, sample 10g is taken after mixing, and being added in the conical flask that one is fitted into 90mL sterile water (has a magnetic in bottle
Power blender), whirlpool stirs 30 minutes, and it mixes well it and suspension is made, as 10-1Bacteria suspension.With dilution spread plate
Above-mentioned suspension and sterile water are diluted to 10 according to volume ratio 1:9 by method-2, 10-3, 10-4, 10-5Concentration takes different extension rates
Bacteria suspension 0.1mL is coated in Czapek's medium (Czapek-Dox Medium) plate, with sterile spreading rod in culture base table
Face is gently coated with, and is stood 30 minutes at room temperature and is placed on 25 DEG C of constant incubators.After bacterium colony is grown, bacterium colony face is observed and recorded
Color, transparency, bacterium colony surface, edge configuration.Final 1000 plants of strain inoculateds of picking are carried out in Czapek's medium bevel
Fermentation verifying.With one ring of thallus of oese picking inclined-plane culture, it is inoculated in the 250mL cone containing 30mL seed culture medium respectively
In shape bottle, under the conditions of 25 DEG C after shake culture 1 day, then 1mL culture transferring is drawn in the 250mL taper containing 20mL fermentation medium
In bottle, under the conditions of 25 DEG C after shake culture 2 days, the content of fumidil in gained fermentation liquid is detected through HPLC, is picked out most
Superior strain, that is, mould HS-NF-684Z (CGMCC No.14144).
Seed culture based formulas (g/L): glucose 10g/L, starch 10g/L, soybean cake powder 10g/L, extraction from yeast powder 5g/
L, calcium carbonate 15g/L, magnesium sulfate 1.5g/L, potassium dihydrogen sulfate 1g/L add water to be settled to 1000mL, pH 6.0 ± 0.1.
Fermentative medium formula (g/L): sucrose 40g/L, dextrin 40 g/L, glycerol 30g/L, extraction from yeast powder 5g/L, yeast
Powder 10g/L, yeast extract 10g/L, calcium carbonate 30g/L add water to be settled to 1000mL, pH 6.0 ± 0.1.
Embodiment 2: strain idenfication
Referring to " Chinese fungi will ", " common mycology ", " common bacteria system identification handbook ", " Molecular Cloning: A Laboratory refers to
South " and the books such as " Chinese Pharmacopoeia " (2015 editions) in related content tested；Color judgement is referring to RAL K7 colour atla series
Color is compareed.
1, bacterium numbering: HS-NF-684Z (CGMCC No.14144).
2, it the culture feature of bacterial strain: using 25 DEG C of CA culture medium (Czapek Agar) culture 12 days, is cultivated using CYA
5 DEG C/25 DEG C/37 DEG C of base (CzapekYeast Autolysate Agar) are cultivated 7 days, using G25N culture medium (25%
Glycerol Gitrate Agar) 25 DEG C culture 7 days, observe mycelial form, color and pigment situation, the culture of bacterial strain
Feature is shown in Table 1.Using 4 kinds of culture mediums of PDA, SDA, MEA and MEPG, 25 DEG C after culture 5~7 days, observe mycelial color and
The cultural characteristic of pigment situation, bacterial strain is shown in Table 2.
3, physiological and biochemical test: in addition to temperature experiment, it is 25 DEG C and cultivates 5~7 days.
A) carbon source using: using ISP9 as basis culture medium, the final concentration of various carbon sources is 1.0%, is shown in Table 3.
B) it is inorganic nitrogen-sourced using: using ISP9 as basis culture medium, the concentration of potassium nitrate and ammonium sulfate is
0.1%, it is shown in Table 3.
C) Degrading experiment and NaCl tolerance test use basal medium for GYEA (pH6.8), the concentration of various degradation products
And Degrading experiment the results are shown in Table 4；NaCl tolerance experimental results are shown in Table 8.
D) oxidizing ferment and catalase test, pH test and humid test are all made of PDA culture medium.Oxidizing ferment and peroxide
Change hydrogen enzyme test the results are shown in Table 5, pH test result and be shown in Table 6, and humid test the results are shown in Table 7.
E) M.R, V-P and urase experiment use " common bacteria system identification handbook " method, and the result is shown in tables 5.
Experimental result
1, the culture feature of bacterial strain: the cultural characteristic of HS-NF-684Z (CGMCC No.14144) is shown in Tables 1 and 2.
Cultural characteristic of the 1 bacterial strain HS-NF-684Z of table (CGMCC No.14144) on CA/CYA/G25N culture medium
Training of the 2 bacterial strain HS-NF-684Z of table (CGMCC No.14144) on 4 kinds of culture mediums of PDA, SDA, MEA and MEPG
Support feature
2, physiological and biochemical property: it is shown in Table 3- table 8.
The carbon source of 3 bacterial strain HS-NF-684Z of table (CGMCC No.14144) and the utilization power of nitrogen source
| Carbon source | Growing state | Carbon source | Growing state | It is inorganic nitrogen-sourced | Growing state |
| D-Glucose | 4 | Salicin | 3 | Ammonium sulfate | + |
| D- gossypose | 3 | D- lactose | 3 | Potassium nitrate | + |
| D- xylose | 2 | Galactolipin | 4 | ||
| D-glucitol | 4 | Inositol | 4 | ||
| L-arabinose | 2 | Mannitol | 4 | ||
| Glycerol | 4 | Glycine | 2 | ||
| Maltose | 4 | Xylan | 4 | ||
| D-Fructose | 4 | Inulin | 2 | ||
| D- sucrose | 4 |
The Degrading experiment result of 4 bacterial strain HS-NF-684Z of table (CGMCC No.14144)
| 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% | 4 ,- |
| Xylan | 0.4% | 4 ,- | Tween-60 | 1.0% | 4 ,- |
| Hypoxanthine | 0.4% | 4 ,+ | Tween-80 | 1.0% | 4 ,- |
Table 5 bacterial strain HS-NF-684Z (CGMCC No.14144) main physiological and biochemical property
| Pilot project | As a result | Pilot project | As a result | Pilot project | As a result |
| Gelatin liquefaction | + | Milk peptonizes | - | Cellulose utilization | - |
| Starch Hydrolysis | - | Nitrate reduction | - | Catalase | - |
| Milk solidification | + | Hydrogen sulfide generates | - | ||
| V.P experiment | - | M.R experiment | - |
The pH test of 6 bacterial strain HS-NF-684Z of table (CGMCC No.14144) growth
| pH | 3.5 | 4.0 | 4.5 | 5.0 | 5.5 | 6.0 | 6.5 | 7.0 | 7.5 |
| Growing state | 3 | 3 | 3 | 4 | 4 | 4 | 4 | 4 | 4 |
The humid test of 7 bacterial strain HS-NF-684Z of table (CGMCC No.14144) growth
| Temperature (DEG C) | 7 | 14 | 28 | 37 | 45 |
| Growing state | 2 | 3 | 4 | 0 | 0 |
8 bacterial strain HS-NF-684Z of table (CGMCC No.14144) is to the tolerance of NaCl
| NaCl concentration | 1% | 4% | 7% | 10% |
| Strain growth situation | 4 | 4 | 3 | 2 |
* note: in table 1-8: 0, no growth；1, it grows 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；, negative.
The analysis of embodiment 3:18s rDNA sequence and strain idenfication
It is tested referring to the related content in " Molecular Cloning:A Laboratory guide " book.The new fresh thalli of bacterial strain is collected, then
Thallus total DNA is extracted using fungal DNA extraction kits, using universal primer NS1 (SEQ ID NO:1)/NS8 (SEQ ID
NO:2 18S rDNA sequence amplification) is carried out, amplification system and PCR response procedures are as shown in table 9, and PCR product detection uses 0.8%
Agarose gel electrophoresis, PCR product purification and recovery use SanPrep pillar PCR purified product kit, and PCR after purification is produced
Object directly send Nanjing Genscript Biotechnology Co., Ltd. to carry out sequencing.
Table 9PCR amplification system and response procedures
The 18s rDNA sequence (SEQ ID NO:3) surveyed is after check and correction to GenBank database related kind, category sequence
Column carry out homologous sequence BLAST comparison, to determine the classification position of the bacterial strain.
In bacterial strain HS-NF-684Z (CGMCC No.14144) 18s rDNA sequence and GenBank database correlated series into
Row BLAST is compared, and the results are shown in Table 10 (the higher bacterial strain of homology is only listed in table).
The homology result of table 10 bacterial strain HS-NF-684Z and related strain
| Strain name | GenBank No. | Homology |
| Penicillium freii(IBT 3464) | AJ005446.1 | 99.4% |
| Penicillium sp.enrichment culture clone NJ-F4 | GU190185.1 | 99.3% |
| Penicillium solitumstrain 20-01 | JN642222.1 | 99.3% |
| Penicillium expansum | AB028137.1 | 99.3% |
The 18s rDNA sequence alignment result of bacterial strain HS-NF-684Z (CGMCC No.14144) is shown: bacterial strain HS-NF-
684Z (CGMCC No.14144) and Penicillium (Penicillium sp.) have very high homology, highest to reach
99.4%, characterization experiments are learned in conjunction with the morphology of bacterial strain HS-NF-684Z (CGMCC No.14144) and culture as a result, discovery should
Bacterial strain and the feature of mould (Penicilliurn sp.) classification are closely similar, therefore by bacterial strain HS-NF-684Z (CGMCC
No.14144) it is accredited as Penicillium (Penicillium sp.).
Mould HS-NF-684Z (CGMCC No.14144) of the present invention and other fumidil producing strains are compared as follows:
J.C.Frisvad etc. (Persoonia, vol.14, no.2,177-182,1990) discloses Penicillium
Scabrosum is cultivated 7 days, colony diameter 26-32mm for 25 DEG C on CYA culture medium, and seldom radial rill, central area often goes out
Existing diffusate；And mould HS-NF-684Z (CGMCC No.14144) of the present invention is cultivated 7 days for 25 DEG C on CYA culture medium, bacterium colony
Diameter 22mm, hence it is evident that radial rill, the rare diffusate in central area.
F.R.Hanson etc. (Journal of Bacteriology, 1949,58 (4): 527-529) and Wen Yang etc.
It is aspergillus fumigatus that fumidil disclosed in (Current Microbiology, 2003,46 (1): 0024-0027), which produces bacterium,
Aspergillus fumigatus；And the bacterial strain HS-NF-684Z (CGMCC No.14144) in the present invention passes through 18s rDNA
Sequence alignment is accredited as Penicillium Penicillium sp..
(Journal of Microbiology, the Biotechnology and Food such as Z.Barbor á kov á
Sciences, 2012:1 (4): 466-477) disclose the fumidil production production fumidil of bacterium P.scabrosum FCB 353
Potency be 110mg/L；And the potency that mould HS-NF-684Z (CGMCC No.14144) of the present invention produces fumidil is up to
1000mg/L。
In conjunction with morphology, cultural characteristic and the DNA of aforementioned bacterial strain HS-NF-684Z (CGMCC No.14144) of the invention
Sequence Identification result is it is found that bacterial strain HS-NF-684Z (CGMCC No.14144) of the invention belongs to Penicillium (Penicillium
Sp.), and it is different from known fumidil producing strains, therefore mould HS-NF-684Z of the present invention (CGMCC No.14144)
It is one plant of completely new strain.
Embodiment 4: shake flask fermentation prepares fumidil
(1) preparation and culture of inclined-plane bacterium colony
Slant medium uses potato dextrose agar (g/L): potato 200g/L, glucose 20g/L, fine jade
Rouge 20g/L, distilled water 1000mL, natural pH.
121 DEG C of high pressure steam sterilization, 20min is cooled to 50-60 DEG C to culture medium and puts inclined-plane, makes in superclean bench
It is inoculated with a ring spore with aseptic inoculation ring, coating is uniformly placed on 25 DEG C and is protected from light culture 3-5 days.
(2) preparation and culture of seed culture medium
Seed culture based formulas (g/L): glucose 10g/L, starch 10g/L, soybean cake powder 10g/L, extraction from yeast powder 5g/
L, calcium carbonate 15g/L, magnesium sulfate 1.5g/L, potassium dihydrogen sulfate 1g/L add water to be settled to 1000mL, pH 6.0 ± 0.1.
Shaking flask liquid amount is 30mL/ bottles, 121 DEG C of high steam sterilizings, 20min.Spore inoculating amount is 105-106c.f.u/
ML, cultivation temperature are 25 ± 1 DEG C, 250rpm, shaking table shaken cultivation 24 hours.
(3) preparation and culture of fermentation medium
Fermentative medium formula (g/L): sucrose 80g/L, dextrin 20g/L, glycerol 40g/L, extraction from yeast powder 5g/L, yeast
Powder 10g/L, yeast extract 10g/L, magnesium sulfate 1.5g/L, potassium dihydrogen sulfate 1g/L, calcium carbonate 30g/L add water to be settled to 1000mL,
pH 6.0±0.1。
Shaking flask liquid amount is 20mL/ bottles, 121 DEG C of high steam sterilizings, 20min.Seed liquor inoculum concentration is 5% (volume hundred
Divide ratio), cultivation temperature is 25 ± 1 DEG C, 250rpm, obtains fumidil fermentation liquid within shaking table shaken cultivation 168 hours.It is examined through HPLC
It surveys, fermentation unit 1000mg/L.
Embodiment 5: fermentor prepares fumidil
(1) preparation and culture of seeding tank seed liquor
The seed culture medium (seed culture medium proportion is shown in step (2) in embodiment 4) of 9L is put into 15L seeding tank, and
0.1% (percent by volume) GPE is added as defoaming agent), 121 DEG C of steam sterilizings, 20min, after being cooled to 25 DEG C, access
30mL shake-flask seed liquid, cultivation temperature are 25 ± 1 DEG C, speed of agitator 100-600rpm, oxygen-supply quantity 0.5-2.0vvm, and culture is for 24 hours.
(2) preparation and culture of ferment tank liquid
In 50L fermentor put into 30L fermentation medium (fermentation medium proportion sees step (3) in embodiment 4, and
0.05% (percent by volume) GPE is added as defoaming agent), 121 DEG C of steam sterilizings, 20min after being cooled to 25 DEG C, is moved into
3L seeding tank seed liquor, cultivation temperature are 25 ± 1 DEG C, speed of agitator 100-600rpm, oxygen-supply quantity 0.5-2.0vvm, culture 120
Hour obtains fumidil fermentation liquid.It is detected through HPLC, fermentation unit 1010mg/L.
Embodiment 6: extraction prepares fumidil
Fumidil fermentation liquid (fumidil potency is 1010mg/L) 30L of Example 5, is added 45L methyl- tert fourth
Base ether (MTBE) stirs 2h, stands 8h, and liquid separation collects MTBE extraction phase 42L, MTBE or ETBE extraction phase is concentrated into 1.1L,
Thickening temperature is 30 DEG C, is then cooled to 0 DEG C, stirs 8h, 50.0g wet solid is obtained by filtration.Then, by wet solid dichloromethane
Mixed solvent 200ml (volume ratio of methylene chloride and methanol is 1:1) dissolution of alkane and methanol, filters to obtain 220ml filtrate, filtrate
It is stirred under the conditions of -10 DEG C of temperature, pressure -0.08Mpa, stirring rate 100rmp, the methanol of 66ml is added after 1h into filtrate,
The methanol of 66ml is added in every 1h later, and solid powder 41.1g is obtained by filtration after 8h.
Embodiment 7: the identification of fumidil
(1) 6 obtained solid powder of embodiment is analyzed by MS and determines that its molecular weight is 458.55.Pass through1H NMR and13C
NMR analysis, determines that it is fumidil, structure is as follows:
(2) physicochemical property of fumidil is as follows:
Character: white to faint yellow unformed powdered
Dissolubility: being soluble in methyl tertiary butyl ether(MTBE), methylene chloride, not soluble in water
Molecular formula: C26H34O7
Fumidil is in CDCl3In nuclear magnetic data (hydrogen spectrum, 400MHz；Carbon spectrum, 100MHz) concrete outcome be shown in Table 11.
11 fumidil of table is in CDCl3In nuclear magnetic data
Sequence table
<110>Haizheng Medicine Stock Co., Ltd., Zhejiang Prov
<120>a kind of mould and its method for producing fumidil
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213>artificial sequence primer NS1 (artificial sequence)
<400> 1
gtagtcatat gcttgtctc 19
<210> 2
<211> 20
<212> DNA
<213>artificial sequence primer NS8 (artificial sequence)
<400> 2
tccgcagctt cacctacgga 20
<210> 3
<211> 1830
<212> DNA
<213>mould (Penicillium sp.)
<400> 3
tcgagctcgg tacccgggga tcctctagag attgtagtca tatgcttgtc tcaaagatta 60
agccatgcat gtctaagtat aagcaacttg tactgtgaaa ctgcgaatgg ctcattaaat 120
cagttatcgt ttatttgata gtaccttact acatggatac ctgtggtaat tctagagcta 180
atacatgcta aaaaccccga cttcaggaag gggtgtattt attagataaa aaaccaacgc 240
ccttcggggc tccttggtga atcataataa cttaacgaat cgcatggcct tgcgccggcg 300
atggttcatt caaatttctg ccctatcaac tttcgatggt aggacagtgg cctaccatgg 360
tggcaacggg taacggggaa ttagggttcg attccggaga gggagcctaa gaaacggcta 420
ccacatccaa ggaaggcagc aggcgcgcaa attacccaat cccgatacgg ggaggtagtg 480
acaataaata ctgatacggg gctcttttgg gtctcgtaat tggaatgaga acaatttaaa 540
tcccttaacg aggaacaatt ggagggcaag tctggtgcca gcagccgcgg taattccagc 600
tccaatagcg tatattaaag ttgttgcagt taaaaagctc gtagttgaac cttgggcctg 660
gctggccggt ccgcctcacc gcgagtactg gtccggctgg gcctttcctt ctggggaacc 720
tcatggcctt cactggctgt ggggggaacc aggactttta ctgtgaaaaa attagagtgt 780
tcaaagcagg cctttgctcg aatacattag cacggaataa tagaatagga cgtgcggttc 840
tattttgttg gtttctagga ccgccgtaat gattaatagg gatagtcggg ggcgtcagta 900
ttcagctgtc agaggtgaaa ttcttggatt tgctgaagac taactactgc gaaagcattc 960
gccaaggatg ttttcattaa tcagggaacg aaagttaggg gatcgaagac gatcagatac 1020
cgtcgtagtc ttaaccataa actatgccga ctagggatcg gacgggattc tataatgacc 1080
cgttcggcac cttacgagaa atcaaagttt ttgggttctg gggggagtat ggtcgcaagg 1140
ctgaaactta aagaaattga cggaagggca ccacaaggcg tggagcctgc ggcttaattt 1200
gactcaacac ggggaaactc accaggtcca gacaaaataa ggattgacag attgagagct 1260
ctttcttgat cttttggatg gtggtgcatg gccgttctta gttggtggag tgatttgtct 1320
gcttaattgc gataacgaac gagacctcgg cccttaaata gcccggtccg catttgcggg 1380
ccgctggctt cttaggggga ctatcggctc aagccgatgg aagtgcgcgg caataacagg 1440
tctgtgatgc ccttagatgt tctgggccgc acgcgcgcta cactgacagg gccagcgagt 1500
acatcacctt aaccgagagg tttgggtaat cttgttaaac cctgtcgtgc tggggataga 1560
gcattgcaat tattgctctt caacgaggaa tgcctagtag gcacgagtca tcagctcgtg 1620
ccgattacgt ccctgccctt tgtacacacc gcccgtcgct actaccgatt gaatggctca 1680
gtgaggcctt gggactggct caggagggtt ggcaacgacc ccccagagcc ggaaacttgg 1740
tcgaactcgg tcatttagag gaagtaaaag tcgtaacaag gtttccgtag gtgaagctgc 1800
ggaaatcgtc gacctgcagg catgcaagct 1830
Claims (10)
1. a kind of mould (Penicillium sp.) HS-NF-684Z, deposit number is CGMCC No.14144.
2. mould HS-NF-684Z according to claim 1 is preparing fumidil or containing the medicine group of fumidil
Close the application of object.
3. a kind of preparation method of fumidil, it is characterised in that: including using mould HS-NF- as described in claim 1
684Z is the step of containing in the nutrient medium of assimilable carbon source and/or nitrogen source, carrying out aerobic fermentation.
4. according to the method described in claim 3, it is characterized by: the assimilable carbon source is selected from starch, dextrin, grape
The combination of one of sugar, sucrose, lactose, maltose, industrial molasses, glycerol, sorbierite, mannitol or above-mentioned substance, preferably forms sediment
Powder, dextrin, glucose, sucrose.
5. according to the method described in claim 3, it is characterized by: the assimilable nitrogen source is selected from extraction from yeast powder, yeast
Powder, yeast extract, soybean cake powder, cottonseed meal, groundnut meal, seitan powder, Dried Corn Steep Liquor Powder, dregs of beans, peptone, urea, ammonium salt
One of or above-mentioned substance combination, preferably soybean cake powder, yeast extract, extraction from yeast powder, yeast powder.
6. according to the described in any item methods of claim 3-5, it is characterised in that: the nutrient medium further includes inorganic salts,
The inorganic salts be selected from trisodium citrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium sulfate, calcium carbonate, ferrous sulfate, zinc sulfate,
The combination of one of copper sulphate, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, iron chloride, manganese sulfate or above-mentioned substance, preferably carbonic acid
Calcium, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, trisodium citrate.
7. according to the described in any item methods of claim 3-6, it is characterised in that: the nutrient medium contains sucrose 40-
1000g/L, dextrin 20-1000g/L, glycerol 10-80g/L, extraction from yeast powder 1-15g/L, yeast powder 5-20g/L, yeast extract 5-
20g/L, magnesium sulfate 1-10g/L, potassium dihydrogen sulfate 1-5g/L, calcium carbonate 0-50g/L.
8. according to the described in any item methods of claim 3-7, it is characterised in that: the temperature of the aerobic fermentation is 20-30 DEG C,
It is preferred that 23-28 DEG C；Medium pH is 5.0-8.0, preferably 5.0-7.0；Incubation time is 24-226 hours, and preferably 72-168 is small
When；Oxygen-supply quantity is 0.1-2.0vvm, preferably 0.5-2.0vvm.
9. according to the described in any item methods of claim 3-8, it is characterised in that: the mould HS-NF-684Z is to pass through seed
Liquid, which is seeded in the nutrient medium, carries out the fermented and cultured；
Wherein, the seed liquor is that mould HS-NF-684Z described in claim 1 is carried out seed training in seed culture medium
It supports and obtains；The condition of the seed culture are as follows: the temperature of seed culture is 20 DEG C -30 DEG C, preferably 23 DEG C -28 DEG C；Culture medium
PH is 5.0-8.0, preferably 5.0-7.0；Incubation time is 24-80 hours, preferably 24-60 hours.
10. according to the method described in claim 9, it is characterized by: the seed culture medium contain glucose 5-20g/L,
Starch 5-20g/L, soybean cake powder 5-20g/L, extraction from yeast powder 1-10g/L, calcium carbonate 1-20g/L, magnesium sulfate 1-10g/L, sulphur
Acid dihydride potassium 1-10g/L.
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| PCT/CN2019/110893 WO2020074009A1 (en) | 2018-10-13 | 2019-10-12 | Penicillium sp. and method for producing fumagillin |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111004106A (en) * | 2019-09-26 | 2020-04-14 | 青岛农业大学 | Polyketide with trans-decalin ring and preparation method and application thereof |
| WO2020074009A1 (en) * | 2018-10-13 | 2020-04-16 | 浙江海正药业股份有限公司 | Penicillium sp. and method for producing fumagillin |
| WO2020074008A1 (en) * | 2018-10-13 | 2020-04-16 | 浙江海正药业股份有限公司 | Fumagillin extraction and purification method |
| CN115287371A (en) * | 2022-02-15 | 2022-11-04 | 中国食品药品检定研究院 | Method for detecting genomic DNA of penicillium chrysogenum |
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| GB8511095D0 (en) * | 1985-05-01 | 1985-06-12 | Fujisawa Pharmaceutical Co | Immunosuppressant |
| KR20010011248A (en) * | 1999-07-27 | 2001-02-15 | 복성해 | Cis-Fumagillin, a Novel Angiogenesis Inhibitor and Anti-angiogenic Composition Containing Same |
| CN107058137A (en) * | 2017-06-14 | 2017-08-18 | 浙江海正药业股份有限公司 | A kind of wet graceful mould and its method for producing wortmannin |
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| 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 |
| CN109053638B (en) * | 2018-10-13 | 2020-07-31 | 浙江海正药业股份有限公司 | Extraction and purification method of fumagillin |
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2018
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| GB8511095D0 (en) * | 1985-05-01 | 1985-06-12 | Fujisawa Pharmaceutical Co | Immunosuppressant |
| KR20010011248A (en) * | 1999-07-27 | 2001-02-15 | 복성해 | Cis-Fumagillin, a Novel Angiogenesis Inhibitor and Anti-angiogenic Composition Containing Same |
| CN107058137A (en) * | 2017-06-14 | 2017-08-18 | 浙江海正药业股份有限公司 | A kind of wet graceful mould and its method for producing wortmannin |
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| BARBORAKOVA,ET AL: "Effect of glucose concentration and growth conditions on the fungal biomass ph of media and production of fumagillin by a non-pathogenic strain Penicillium Scabrosum", 《JOURNAL OF MICROBIOLOGY,BIOTECHNOLOGY AND FOOD SCIENCE》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020074009A1 (en) * | 2018-10-13 | 2020-04-16 | 浙江海正药业股份有限公司 | Penicillium sp. and method for producing fumagillin |
| WO2020074008A1 (en) * | 2018-10-13 | 2020-04-16 | 浙江海正药业股份有限公司 | Fumagillin extraction and purification method |
| CN111004106A (en) * | 2019-09-26 | 2020-04-14 | 青岛农业大学 | Polyketide with trans-decalin ring and preparation method and application thereof |
| CN111004106B (en) * | 2019-09-26 | 2020-12-18 | 青岛农业大学 | Polyketide with trans-decalin ring and preparation method and application thereof |
| CN115287371A (en) * | 2022-02-15 | 2022-11-04 | 中国食品药品检定研究院 | Method for detecting genomic DNA of penicillium chrysogenum |
| CN115287371B (en) * | 2022-02-15 | 2023-12-01 | 中国食品药品检定研究院 | Method for detecting genomic DNA of penicillium chrysogenum |
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| CN109182147B (en) | 2020-07-14 |
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