CN109182147B - Penicillium and method for producing fumagillin by using same - Google Patents
Penicillium and method for producing fumagillin by using same Download PDFInfo
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- CN109182147B CN109182147B CN201811192509.5A CN201811192509A CN109182147B CN 109182147 B CN109182147 B CN 109182147B CN 201811192509 A CN201811192509 A CN 201811192509A CN 109182147 B CN109182147 B CN 109182147B
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/80—Penicillium
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/336—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having three-membered rings, e.g. oxirane, fumagillin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/14—Fungi; Culture media therefor
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- 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 discloses a novel Penicillium (Penicillium sp.) HS-NF-684Z with a preservation number as follows: CGMCC No. 14144; also disclosed is a method for preparing fumagillin or a pharmaceutical composition containing fumagillin by culturing it.
Description
Technical Field
The invention relates to the technical field of microbial engineering, in particular to novel penicillium and application thereof in preparation of fumagillin.
Background
Fumagillin (formula I), originally used to kill the nosema parasite of bees, was later found to be useful in the treatment of nosema and/or cryptosporidiosis caused by intestinal infections in humans, which is quite fatal to immunocompromised patients. In recent years, with intensive research on fumagillin and analogues thereof, it has been reported that fumagillin and analogues thereof have anti-angiogenic activity and can block the formation of blood vessels by binding to human methionine aminopeptidase-2 (MetAP-2). Thus, fumagillin and its analogs are widely used as angiogenesis inhibitors for the treatment of cancer.
Hanson et al report that fumagillin was originally a natural product isolated from fermentation cultures of Aspergillus fumigatus, and in subsequent studies it was disclosed that fumagillin was the first to be produced (Ebleand Hanson, 1951; McCowen et al, 1951); J.C.Frisvad (Personia, vol.14, No.2, 177-182, 1990) et al disclosed that fumagillin was first found in fermentation cultures of Penicillium scabrosum and described in detail about the morphology of this strain, Z.Barbor kov et al (Journal of microbiology, technology and Food, 2012:1(4) 466) -disclosed that P.477 FCB 353 was culture optimized to yield fumagillin of 110 mg/L. it was necessary to find a new strain that was fermented to produce fumagillin at a high level.
Disclosure of Invention
An object of the present invention is to provide a novel fumagillin-producing strain which is penicillium sp (penicillium sp.)
HS-NF-684Z with preservation number of CGMCC No. 14144.
The microbial strain of the Penicillium HS-NF-684Z is preserved in the China general microbiological culture Collection center (the address: West Lu No.1, northwest of the republic of Kyoho, Beijing, China, institute of microbiology, national academy of sciences) within 24 months and 24 days in 2017, the preservation number is CGMCC No.14144, the classification name is Penicillium sp, and the registration proves the survival.
The invention also aims to provide application of the penicillium HS-NF-684Z (CGMCC NO.14144) in preparation of fumagillin or a pharmaceutical composition containing fumagillin.
The invention also provides a preparation method of fumagillin, which comprises the step of carrying out aerobic fermentation on penicillium HS-NF-684Z (CGMCC No.14144) in a nutrient medium containing assimilable carbon sources and/or nitrogen sources.
In a preferred embodiment, the assimilable carbon source is selected from one of starch, dextrin, glucose, sucrose, lactose, maltose, industrial molasses, glycerol, sorbitol, mannitol or a combination thereof, preferably starch, dextrin, glucose, sucrose.
In a preferred embodiment, the assimilable nitrogen source is selected from one of yeast extract powder, yeast extract, soybean cake powder, cottonseed cake powder, peanut cake powder, gluten powder, corn steep liquor dry powder, soybean meal, peptone, urea, ammonium salt or a combination thereof, preferably soybean cake powder, yeast extract powder, and yeast powder.
In a preferred embodiment, the nutrient medium further comprises an inorganic salt selected from one of trisodium citrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium sulfate, calcium carbonate, ferrous sulfate, zinc sulfate, copper sulfate, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride, manganese sulfate or a combination thereof, preferably calcium carbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, trisodium citrate.
In a preferred embodiment, the nutrient medium contains 40-1000 g/L of sucrose, 20-1000 g/L of dextrin, 10-80 g/L of glycerol, 1-15 g/L of yeast extract powder, 5-20 g/L of yeast powder, 5-20 g/L of yeast extract, 1-10 g/L of magnesium sulfate, 1-5 g/L of potassium dihydrogen sulfate and 0-50 g/L of calcium carbonate.
In a preferred embodiment, the temperature of the aerobic fermentation is 20-30 ℃, preferably 23-28 ℃; the pH of the culture medium is 5.0-8.0, preferably 5.0-7.0; the culture time is 24-226 hours, preferably 72-168 hours; the oxygen throughput is from 0.1 to 2.0vvm, preferably from 0.5 to 2.0 vvm.
In a preferred embodiment, said penicillium HS-NF-684Z is said fermentatively cultured by seed liquid inoculation into said nutrient medium; wherein the seed solution is obtained by seed culture of Penicillium HS-NF-684Z (CGMCC No.14144) in a seed culture medium; the seed culture conditions are as follows: the temperature of seed culture is 20-30 ℃, preferably 23-28 ℃; the pH of the culture medium is 5.0-8.0, preferably 5.0-7.0; the culture time is 24 to 80 hours, preferably 24 to 60 hours.
In a preferred embodiment, the seed culture medium contains 5-20 g/L of glucose, 5-20 g/L of starch, 5-20 g/L of soybean cake powder, 1-10 g/L of yeast extract powder, 1-20 g/L of calcium carbonate, 1-10 g/L of magnesium sulfate and 1-10 g/L of potassium dihydrogen sulfate.
The fumagillin of the invention is subjected to HP L C detection under the following conditions:
chromatographic column Accchrom C18(4.6 × 250mm, 5 μm)
Column temperature: 25 ℃;
the injection volume is 5 mu L;
the flow rate is 1m L/min;
detection wavelength: 350 nm;
analysis time: 40 min;
mobile phase:
mobile phase A: 0.1% (volume percent) aqueous acetic acid;
mobile phase B: 0.1% (volume percent) acetonitrile acetate 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 characteristics of the Penicillium (Penicillium sp.) HS-NF-684Z (CGMCC NO.14144) are as follows: the spore is round, the spore is blue-green, the colony color is yellow, the reverse side is yellow brown, the surface is radial furrow and velvet, and the diameter is 20-22 mm.
The Penicillium sp HS-NF-684Z (CGMCC NO.14144) is a brand-new fumagillin producing strain, the capability of producing fumagillin is greatly improved compared with other strains in the prior art, the titer can reach 1000 mg/L, and the industrial production is favorably realized.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Wherein the used fungus DNA extraction kit is purchased from Beijing Sanbo Polygala tenuifolia Biotechnology Limited liability company;
purification and recovery of PCR products the SanPrep column type PCR product purification kit used was purchased from Biotechnology engineering (Shanghai) Ltd.
The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative of the present invention only and are not intended to limit the scope of the present invention.
Example 1: the source of the strain
The penicillium HS-NF-684Z (CGMCC No.14144) is a penicillium separated from soil of a certain hillside of a Qingcheng mountain of city, Sichuan province in China.
Cross sampling is carried out on soil in the Qingcheng mountain area, 5 sampling points are randomly selected, 10g of soil sample is taken at each point, the soil samples are put into a conical flask, 10g of sample is taken after uniform mixing, the sample is added into the conical flask filled with 90m L sterile water (a magnetic stirrer is arranged in the flask), vortex stirring is carried out for 30 minutes, the mixture is fully and uniformly mixed to prepare suspension, namely 10-1And (4) bacterial suspension. Mixing the suspension with sterile water according to a volume ratio of 1: 9 to 10-2，10-3，10-4，10-5Taking bacterial suspension with different dilution multiples of 0.1m L, coating the bacterial suspension on a Czapek-Dox Medium (Czapek-Dox Medium) plate, lightly coating the bacterial suspension on the surface of a culture Medium by using a sterile coating rod, standing the bacterial suspension for 30 minutes at room temperature, and then placing the bacterial suspension in a constant temperature incubator at 25 ℃, observing and recording the color, transparency, surface and edge morphology of a bacterial colony after the bacterial colony grows out, finally selecting 1000 bacterial strains, inoculating the bacterial strains into the Czapek Medium to prepare a slant, and carrying out fermentation verification, selecting a ring of the bacterial strains cultured by using an inoculating ring, inoculating the ring of the bacterial strains in the slant culture into 250m L conical flasks containing 30m L seed culture Medium, carrying out shake culture for 1 day at 25 ℃, absorbing 1m L, inoculating the bacterial strains into 250m L conical flasks containing 20m L fermentation Medium, carrying out shake culture for 2 days at 25 ℃, and detecting the content of the fumagillin in the obtained fermentation broth by using HP L C, and selecting the highest-yield strain, namely high-yield Penicillium-NF-CGMCC-14144 (.
The formula (g/L) of the seed culture medium comprises 10 g/L of glucose, 10 g/L of starch, 10 g/L of soybean cake powder, 5 g/L of yeast extract powder, 15 g/L of calcium carbonate, 1.5 g/L of magnesium sulfate and 1 g/L of potassium dihydrogen sulfate, and water is added to the mixture until the volume is 1000m L and the pH value is 6.0 +/-0.1.
The formula (g/L) of the fermentation medium comprises 40 g/L of sucrose, 40 g/L of dextrin, 30 g/L of glycerol, 5 g/L of yeast extract powder, 10 g/L of yeast powder, 10 g/L of yeast extract, 30 g/L of calcium carbonate, water and constant volume of 1000m L, and the pH value is 6.0 +/-0.1.
Example 2: strain identification
Experiments are carried out according to relevant contents in books such as Chinese funguses, common fungology, common bacteria system identification handbook, molecular cloning experimental guideline, Chinese pharmacopoeia (2015 edition) and the like, and color judgment is carried out according to colors of RA L K7 color card series for comparison.
1. And (3) numbering strains: HS-NF-684Z (CGMCC No. 14144).
2. The cultural characteristics of the strain: culturing in CA medium (Czapek Agar) at 25 deg.C for 12 days, culturing in CYA medium (Czapekyeast Autolysate Agar) at 5 deg.C/25 deg.C/37 deg.C for 7 days, culturing in G25N medium (25% Glycerol Agar) at 25 deg.C for 7 days, and observing the form, color and pigment of mycelium, wherein the culture characteristics of the strain are shown in Table 1. After culturing in 4 culture media of PDA, SDA, MEA and MEPG at 25 ℃ for 5-7 days, observing the color and pigment condition of the mycelium, wherein the culture characteristics of the strains are shown in Table 2.
3. Physiological and biochemical tests: except for the temperature experiment, the culture is carried out for 5-7 days at 25 ℃.
a) Utilization of carbon source: using ISP9 as the basal medium, the final concentration of each carbon source was 1.0%, as shown in Table 3.
b) Utilization of inorganic nitrogen source: using ISP9 as the basal medium, the potassium nitrate and ammonium sulfate concentrations were 0.1% each, as shown in Table 3.
c) The basic culture medium adopted in the degradation test and the NaCl tolerance test is GYEA (pH6.8), and the concentration of various degradation products and the degradation test results are shown in Table 4; the results of the NaCl tolerance experiments are shown in Table 8.
d) The oxidase and catalase tests, the pH tests and the temperature tests all adopt PDA culture medium. The oxidase and catalase test results are shown in Table 5, the pH test results are shown in Table 6, and the temperature test results are shown in Table 7.
e) M.R, V-P and urease experiments are carried out by the method of handbook of identifying common bacteria systems, and the results are shown in Table 5.
Results of the experiment
1. The cultural characteristics of the strain: the culture characteristics of HS-NF-684Z (CGMCC No.14144) are shown in tables 1 and 2.
TABLE 1 culture characteristics of the strain HS-NF-684Z (CGMCC No.14144) on CA/CYA/G25N medium
TABLE 2 culture characteristics of the strain HS-NF-684Z (CGMCC No.14144) on PDA, SDA, MEA and MEPG 4 media
2. Physiological and biochemical characteristics: see tables 3-8.
TABLE 3 utilization of carbon and nitrogen sources of strain HS-NF-684Z (CGMCC No.14144)
| Carbon source | Growth conditions | Carbon source | Growth conditions | Inorganic nitrogen source | Growth conditions |
| D-glucose | 4 | Salicin | 3 | Ammonium sulfate | + |
| D-raffinose | 3 | D-lactose | 3 | Potassium nitrate | + |
| D-xylose | 2 | Galactose | 4 | ||
| D-sorbitol | 4 | Inositol | 4 | ||
| L-arabinose | 2 | Mannitol | 4 | ||
| Glycerol | 4 | Glycine | 2 | ||
| Maltose | 4 | Xylan | 4 | ||
| D-fructose | 4 | Inulin powder | 2 | ||
| D-sucrose | 4 |
TABLE 4 degradation test results of the strain HS-NF-684Z (CGMCC No.14144)
| Degradation product | Concentration of degradants | Results | Degradation product | Concentration of degradants | Results |
| Adenine | 0.5％ | 4，+ | Casein protein | 1.0％ | 4，- |
| Guanine and its preparing process | 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 Main physiological and biochemical characteristics of the strain HS-NF-684Z (CGMCC No.14144)
| Test items | Results | Test items | Results | Test items | Results |
| Liquefaction of gelatin | + | Milk peptone | - | Cellulose utilization | - |
| Starch hydrolysis | - | Nitrate reduction | - | Catalase enzyme | - |
| Milk coagulation | + | Hydrogen sulfide generation | - | ||
| V.P experiment | - | M.R experiment | - |
TABLE 6 pH test for the growth of the strain HS-NF-684Z (CGMCC No.14144)
| pH | 3.5 | 4.0 | 4.5 | 5.0 | 5.5 | 6.0 | 6.5 | 7.0 | 7.5 |
| Growth conditions | 3 | 3 | 3 | 4 | 4 | 4 | 4 | 4 | 4 |
TABLE 7 temperature test for the growth of the strain HS-NF-684Z (CGMCC No.14144)
| Temperature (. degree.C.) | 7 | 14 | 28 | 37 | 45 |
| Growth conditions | 2 | 3 | 4 | 0 | 0 |
TABLE 8 tolerance of the strain HS-NF-684Z (CGMCC No.14144) to NaCl
| Concentration of NaCl | 1％ | 4％ | 7％ | 10％ |
| Growth of the Strain | 4 | 4 | 3 | 2 |
Injecting: in tables 1 to 8: 0, no growth; 1, very weak growth; 2, the fertilizer can grow and has a small amount of spores; 3, the growth is good, and a large number of spores exist; 4, the growth is best, and spores are abundant; positive; negative.
Example 3: 18s rDNA sequence analysis and strain identification
Experiments were performed with reference to the book "molecular cloning, laboratory Manual". Collecting fresh thallus of the strain, extracting total DNA of the thallus by adopting a fungus DNA extraction kit, amplifying an 18S rDNA sequence by adopting a universal primer NS1(SEQ ID NO:1)/NS8(SEQ ID NO:2), wherein an amplification system and a PCR reaction program are shown in a table 9, a PCR product is detected by adopting 0.8% agarose gel electrophoresis, a SanPrep column type PCR purification product kit is adopted for purifying and recycling the PCR product, and the purified PCR product is directly sent to Nanjing Jinsry biotechnology limited company for sequence determination.
TABLE 9PCR amplification System and reaction procedure
The tested 18s rDNA sequence (SEQ ID NO:3) is subjected to proofreading and then is subjected to homologous sequence B L AST comparison with related species and genus sequences in a GenBank database to determine the classification status of the strain.
The strain HS-NF-684Z (CGMCC No.14144)18s rDNA sequence is compared with related sequences in GenBank database by B L AST, and the result is shown in Table 10 (only strains with higher homology are listed in the table).
TABLE 10 homology results for strain HS-NF-684Z and related strains
| Bacterial species name | GenBank No. | Homology of |
| 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 the strain HS-NF-684Z (CGMCC No.14144) shows that: the strain HS-NF-684Z (CGMCC No.14144) and the Penicillium (Penicillium sp.) have very high homology, the highest homology reaches 99.4%, and the morphological and cultural characteristic experiment results of the strain HS-NF-684Z (CGMCC No.14144) show that the characteristics of the strain and the Penicillium (Penicillium sp) are very similar, so that the strain HS-NF-684Z (CGMCC No.14144) is identified as the Penicillium (Penicillium sp.).
The comparison between the penicillium HS-NF-684Z (CGMCC No.14144) and other fumagillin producing bacteria is as follows:
frisvad et al (Personia, vol.14, No.2, 177-182, 1990) disclose Penicilium scabrosum cultured on CYA medium at 25 ℃ for 7 days with colony diameter of 26-32mm, few radial grooves and frequent effusion in the central area; the penicillium HS-NF-684Z (CGMCC No.14144) is cultured for 7 days at 25 ℃ on a CYA culture medium, the diameter of a bacterial colony is 22mm, radial grooves are obvious, and less exudates exist in the central area.
The fumagillin-producing bacteria disclosed by Hanson et al (Journal of Bacteriology, 1949, 58 (4): 527- & 529) and Wen Yang et al (Current Microbiology, 2003, 46 (1): 0024- & 0027) are Aspergillus fumigatus; the strain HS-NF-684Z (CGMCC No.14144) is identified as Penicillium sp through 18s rDNA sequence comparison.
Z.Barbor-kov-et al (Journal of Microbiology, Biotechnology and foodsiences, 2012:1 (4): 466-477) disclose the titer of fumagillin produced by fumagillin producing strain P.scabrosum FCB 353 as 110 mg/L, while the titer of fumagillin produced by penicillium HS-NF-684Z (CGMCC No.14144) of the invention is as high as 1000 mg/L.
In combination with the morphology, culture characteristics and DNA sequence identification result of the strain HS-NF-684Z (CGMCC No.14144), the strain HS-NF-684Z (CGMCC No.14144) belongs to Penicillium (Penicillium sp.), and is different from known fumagillin producing bacteria, so that the strain HS-NF-684Z (CGMCC No.14144) is a brand new strain.
Example 4: shake flask fermentation for preparing fumagillin
(1) Preparation and culture of slant colony
The slant culture medium is potato glucose agar culture medium (g/L), potato 200 g/L, glucose 20 g/L, agar 20 g/L, distilled water 1000m L, and natural pH.
Sterilizing with high pressure steam at 121 deg.C for 20min, placing the slant when the culture medium is cooled to 50-60 deg.C, inoculating a ring spore on a superclean bench with sterile inoculating loop, uniformly coating, and culturing at 25 deg.C in dark place for 3-5 days.
(2) Preparation and culture of seed culture medium
The formula (g/L) of the seed culture medium comprises 10 g/L of glucose, 10 g/L of starch, 10 g/L of soybean cake powder, 5 g/L of yeast extract powder, 15 g/L of calcium carbonate, 1.5 g/L of magnesium sulfate and 1 g/L of potassium dihydrogen sulfate, and water is added to the mixture until the volume is 1000m L and the pH value is 6.0 +/-0.1.
The liquid filling amount of the shake flask is 30m L/bottle, the high-pressure steam is used for sterilization at 121 ℃, the inoculation amount of the spores is 10 min5-106U/m L at 25 + -1 deg.C and 250rpm, and shaking the shaker for 24 hours.
(3) Preparation and culture of fermentation medium
The formula (g/L) of the fermentation medium comprises 80 g/L of sucrose, 20 g/L0 of dextrin, 40 g/L of glycerol, 5 g/L of yeast extract powder, 10 g/L of yeast powder, 10 g/L of yeast extract, 1.5 g/L of magnesium sulfate, 1 g/L of potassium dihydrogen sulfate and 30 g/L of calcium carbonate, and water is added to the mixture to reach the volume of 1000m L and the pH value of 6.0 +/-0.1.
The liquid loading in a shake flask is 20m L/bottle, high-pressure steam is used for sterilization at 121 ℃, the inoculation amount of the seed liquid is 5 percent (volume percentage), the culture temperature is 25 +/-1 ℃, the rpm is 250, the shake culture is carried out for 168 hours in a shaking table to obtain fumagillin fermentation liquid, and the fermentation unit is 1000 mg/L through HP L C detection.
Example 5: preparation of fumagillin in fermentation tank
(1) Preparation and culture of seed liquid in seed tank
9L of seed culture medium (the proportion of the seed culture medium is shown in the step (2) in the example 4) is put into a 15L seed tank, 0.1 percent (volume percentage) of molinate is added as an antifoaming agent), steam is used for sterilization at 121 ℃ for 20min, after the temperature is cooled to 25 ℃, 30m L of shake flask seed liquid is inoculated, the culture temperature is 25 +/-1 ℃, the stirring speed is 100 plus materials and 600rpm, the oxygen introducing amount is 0.5-2.0vvm, and the seed is cultured for 24 h.
(2) Preparation and culture of fermentation broth of fermentation tank
30L of fermentation medium (the proportion of the fermentation medium is shown in the step (3) in the example 4, and 0.05 percent (volume percentage) of foam killer is added) is put into a 50L fermentation tank, steam is used for sterilization at 121 ℃, the fermentation medium is used for 20min, after being cooled to 25 ℃, the fermentation medium is transferred into a 3L seed tank seed solution, the culture temperature is 25 +/-1 ℃, the stirring speed is 100 plus and 600rpm, the oxygen input is 0.5-2.0vvm, the fermentation medium is cultured for 120 hours to obtain fumagillin fermentation liquor, and the fermentation unit is 1010 mg/L detected by HP L C.
Example 6: extraction and preparation of fumagillin
30L of fumagillin fermentation liquor (the fumagillin titer is 1010 mg/L) in example 5 is taken, 45L methyl tert-butyl ether (MTBE) is added, the mixture is stirred for 2 hours, the mixture is kept stand for 8 hours, the MTBE extract phase 42L is collected, the MTBE or ETBE extract phase is concentrated to 1.1L, the concentration temperature is 30 ℃, then the temperature is reduced to 0 ℃, the mixture is stirred for 8 hours, and filtered to obtain 50.0g of wet solid, then the wet solid is dissolved by 200ml of mixed solvent of dichloromethane and methanol (the volume ratio of the dichloromethane to the methanol is 1: 1), the filtrate is filtered to obtain 220ml of filtrate, the filtrate is stirred under the conditions of the temperature of-10 ℃, the pressure of-0.08 MPa and the stirring speed of 100rmp, 66ml of methanol is added into the filtrate after 1 hour, 66ml of methanol is added into the filtrate every 1 hour, and the solid powder is filtered to obtain 41.1g after.
Example 7: identification of fumagillin
(1) The solid powder obtained in example 6 was subjected toMS analysis determined its molecular weight to be 458.55. By passing1H NMR and13CNMR analysis, which determines it to be fumagillin, the structure is as follows:
(2) the physical and chemical properties of fumagillin are as follows:
the characteristics are as follows: white to light yellow amorphous powder
Solubility: is easily soluble in methyl tert-butyl ether and dichloromethane, and is insoluble in water
The molecular formula is as follows: c26H34O7
Fumagillin in CDCl3The specific results of the nuclear magnetic data (hydrogen spectrum, 400 MHz; carbon spectrum, 100MHz) in (1) are shown in Table 11.
TABLE 11 fumagillin in CDCl3Nuclear magnetic data of
Sequence listing
<110> Zhejiang Haizheng pharmaceutical industry Co., Ltd
<120> penicillium and method for producing fumagillin by using same
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gtagtcatat gcttgtctc 19
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tccgcagctt cacctacgga 20
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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 (12)
1. Penicillium sp HS-NF-684Z with preservation number of CGMCC No. 14144.
2. Use of penicillium HS-NF-684Z according to claim 1 for the preparation of fumagillin or a pharmaceutical composition containing fumagillin.
3. A preparation method of fumagillin is characterized in that: comprising the step of aerobic fermentation with Penicillium HS-NF-684Z of claim 1 in a nutrient medium containing assimilable carbon and/or nitrogen sources;
the assimilable carbon source is selected from one of starch, dextrin, glucose, sucrose, lactose, maltose, industrial molasses, glycerol, sorbitol, mannitol or the combination of the above substances;
the assimilable nitrogen source is selected from one of yeast extract powder, yeast extract, soybean cake powder, cottonseed cake powder, peanut cake powder, gluten powder, corn steep liquor dry powder, soybean meal, peptone, urea and ammonium salt or the combination of the above substances;
the temperature of the aerobic fermentation is 20-30 ℃.
4. The method of claim 3, wherein; the assimilable carbon source is selected from starch, dextrin, glucose, sucrose; the assimilable nitrogen source is selected from soybean cake powder, yeast extract powder and yeast powder.
5. The method of claim 3, wherein: the nutrient medium also comprises inorganic salt, wherein the inorganic salt is selected from one of trisodium citrate, monopotassium phosphate, dipotassium phosphate, ammonium sulfate, calcium carbonate, ferrous sulfate, zinc sulfate, copper sulfate, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride and manganese sulfate or the combination of the substances.
6. The method of claim 5, wherein: the inorganic salt is selected from calcium carbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, and trisodium citrate.
7. The method of claim 3, wherein the nutrient medium comprises sucrose 40-1000 g/L, dextrin 20-1000 g/L, glycerin 10-80 g/L, yeast extract powder 1-15 g/L, yeast powder 5-20 g/L, yeast extract 5-20 g/L, magnesium sulfate 1-10 g/L, potassium dihydrogen sulfate 1-5 g/L, and calcium carbonate 0-50 g/L.
8. The method of claim 3, wherein: the temperature of the aerobic fermentation is 23-28 ℃; the pH value of the culture medium is 5.0-8.0; the culture time is 24-226 hours; the oxygen introduction amount is 0.1-2.0 vvm.
9. The method of claim 8, wherein: the pH value of the culture medium is 5.0-7.0; the culture time is 72-168 hours; the oxygen introduction amount is 0.5-2.0 vvm.
10. The method according to any one of claims 3-9, wherein: the penicillium HS-NF-684Z is inoculated into the nutrient medium through seed liquid to be subjected to fermentation culture;
wherein the seed solution is obtained by seed culture of Penicillium HS-NF-684Z of claim 1 in a seed culture medium; the seed culture conditions are as follows: the temperature of seed culture is 20-30 ℃; the pH value of the culture medium is 5.0-8.0; the culture time is 24-80 hours.
11. The method of claim 10, wherein: the temperature of the seed culture is 23-28 ℃; the pH value of the culture medium is 5.0-7.0; the culture time is 24-60 hours.
12. The method of claim 10, wherein the seed medium comprises glucose 5-20 g/L, starch 5-20 g/L, soybean meal 5-20 g/L, yeast extract powder 1-10 g/L, calcium carbonate 1-20 g/L, magnesium sulfate 1-10 g/L, and potassium dihydrogen sulfate 1-10 g/L.
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| CN201811192509.5A CN109182147B (en) | 2018-10-13 | 2018-10-13 | Penicillium and method for producing fumagillin by using same |
| PCT/CN2019/110893 WO2020074009A1 (en) | 2018-10-13 | 2019-10-12 | Penicillium sp. and method for producing fumagillin |
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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 |
| CN110563569A (en) * | 2019-09-26 | 2019-12-13 | 青岛农业大学 | polyketide with trans-decalin ring and preparation method and application thereof |
| 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 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| 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;Barborakova,et al;《Journal of Microbiology,Biotechnology and Food Science》;20121231;第1卷(第4期);第466-477页 * |
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