CN107058137B - Penicillium wortmannii and method for producing wortmannin by penicillium wortmannii - Google Patents
Penicillium wortmannii and method for producing wortmannin by penicillium wortmannii Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
Abstract
The invention provides Penicillium wortmannii (Penicillium wortmannii)1507-N13 and a method for preparing wortmannin by culturing the same. The Penicillium wortmannii (Penicillium wortmannii)1507-N13 is preserved in China general microbiological culture Collection center (CGMCC) in 2017 at 09.01.M.and the preservation number is CGMCC NO. 13366.
Description
Technical Field
The invention relates to a novel penicillium wortmannii, a method for producing wortmannin by fermenting and culturing the same.
Background
The molecular formula of Wortmannin (Wortmannin) is C23H24O8Molecular weight is 428.43, and the structure is shown as the following formula:
wortmannin is a steroid antibiotic, which mainly inhibits phosphatidylinositol-3 kinase (PI3K), so that many researches on its therapeutic effect on phosphatidylinositol-3 kinase dependent diseases, especially on tumor, are conducted.
Wortmannin can permeate cells, is combined with a 110kD catalytic subunit of PI3K, specifically inhibits PI3K, inhibits a PI3K/Akt signal pathway, and comprises common inhibition of Akt phosphorylation and the like. Wortmannin also inhibits myostatin chain kinase (MLCK), etc. Wortmannin has no obvious antibacterial effect, but has strong fungus inhibiting activity. However, the toxic effect on cells is limited to in vitro studies at present, and the clinical application of the compound is not developed.
In the prior art, P.W.Brian et al reported that wortmannin was originally a natural product isolated from a fermentation culture of Penicillium wortmannin Klocker, and detailed descriptions of the morphology of wortmannin producing bacteria were provided (trans.Brit. mycol. Soc.40(3), 365-; US5378725 and CN1111127A disclose the fermentation culture of wortmannin producing bacteria A24603.1 and the separation and purification of wortmannin, the titer of the wortmannin produced by A24603.1 is 151 mug/mL, and the existing wortmannin producing bacteria have the problem that the prior wortmannin producing bacteria are not suitable for industrial production due to low titer and small yield of wortmannin; some documents, such as WO2007120364 and WO2003024183, only report that wortmannin is isolated from the fermentation medium of the fungus Penicillium wortmannin, and no new report is made on the physiological and biochemical characteristics of the producing strain, the culture conditions, the specific composition and content of the solid, the seed and the fermentation medium. It can be seen that the wortmannin producing bacteria disclosed in the patent and literature are relatively original and have low fermentation level, so that it is necessary to develop new strains and fermentation technology, and the work for finding a new and efficient wortmannin producing bacteria is still going on.
Disclosure of Invention
The invention aims to provide a new efficient wortmannin producing strain, which is wortmannium (Penicillium wortmannii)1507-N13 with the preservation number of CGMCC No. 13366.
The microbial strain of the Penicillium wortmannii 1507-N13 is preserved in the common microbial center of China Committee for culture Collection of microorganisms (address: West Lu No.1 Hospital, China academy of sciences, North Kyoho, Beijing) on 09.2017, the preservation number is CGMCC No.13366, the microbial strain is classified and named as Penicillium wortmannii, and the microbial strain is registered in a book to prove survival.
The invention also aims to provide application of the penicillium wortmannii 1507-N13(CGMCC No.13366) in preparing wortmannin or a pharmaceutical composition containing the wortmannin.
The invention also provides a preparation method of wortmannin, which comprises the step of carrying out fermentation culture on wortmannin 1507-N13(CGMCC No.13366) 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 glucose, lactose, mannitol, sorbitol, sucrose, maltodextrin, corn starch, glycerol, soybean oil, methyl oleate or a combination thereof.
In a preferred embodiment, the assimilable nitrogen source is selected from one of yeast extract powder, yeast powder, beef extract, soy peptone, soybean cake powder, cottonseed cake powder, peanut cake powder, gluten powder, corn steep liquor dry powder, bran, or a combination thereof.
In a preferred embodiment, the nutrient medium further comprises an inorganic salt selected from one of ammonium chloride, ammonium nitrate, triammonium 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.
In a preferred embodiment, the nutrient medium contains 10.0-50.0 g/L of glycerol, 5.0-50.0 g/L of lactose, 5.0-50.0 g/L of sucrose, 5.0-20.0 g/L of soybean cake powder, 5.0-20.0 g/L of yeast powder, 5.0-30.0 g/L of cottonseed cake powder, 0.5-5.0 g/L of monopotassium phosphate, 1.0-5.0 g/L of ammonium sulfate, 1.0-5.0 g/L of calcium carbonate and 0.01-1.0 g/L of manganese sulfate.
In a preferred embodiment, the temperature of the fermentation culture is 20 ℃ to 30 ℃, preferably 24 ℃ to 26 ℃; the pH of the culture medium is 5.0-8.0, preferably 6.0-7.0; the incubation time is 24-240 hours, preferably 72-192 hours.
In a preferred embodiment, said Penicillium wortmannii 1507-N13(CGMCC No.13366) is subjected to said fermentation culture by seed liquid inoculation into said medium;
wherein the seed solution is obtained by seed culture of Penicillium wortmannii 1507-N13(CGMCC No. 13366); the seed culture conditions are as follows: the temperature of seed culture is 20-30 ℃, preferably 24-26 ℃; 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 0-20.0 g/L of sucrose, 10.0-50.0 g/L of glucose, 10.0-30.0 g/L of cottonseed cake powder, 5.0-15.0 g/L of yeast powder, 1.0-10.0 g/L of monopotassium phosphate, 0.5-1.5 g/L of ammonium sulfate and 0.5-2.0 g/L of calcium carbonate.
The wortmannin of the invention is detected by the following method:
a chromatographic column: c18 analytical column (5 μm, 4.6X 150mm).
Sample introduction amount: 10 μ L
Mobile phase: methanol: water: formic acid 50: 50: 0.1(v/v/v)
Flow rate: 1mL/min
Detection wavelength: 254nm
The Penicillium wortmannii (Penicillium wortmannii)1507-N13(CGMCC No.13366) has high production capacity; and the capability of producing wortmannin is greatly improved compared with other strains in the prior art, and the method is favorable for realizing industrial production.
The main biological characteristics of the Penicillium wortmannii (Penicillium wortmannii)1507-N13(CGMCC No.13366) are as follows: the spores are oval, the colony color is yellow, the edge is white, the reverse side is white, the spores are tiled and have no wrinkles, water drops are generated, the diameter is 1.0-2.0 cm, and the spores are not easy to pick.
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.
The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.
Example 1: the source of the strain
The wortmannium penicillium 1507-N13(CGMCC No.13366) is an original strain separated from soil of Jiufeng mountain in Taizhou city of Zhejiang province in coastal region of southeast China, and is a mutant strain obtained by mutagenesis.
After culturing the original strain in PDA slant culture medium at 30 deg.C for 12 days, scraping off mycelium under aseptic condition with inoculating shovel, grinding mycelium in ground test tube and suspending in sterile water to obtain bacterial suspension for NTG (nitrosoguanidine) mutagenesis treatment.
2mg of NTG crystals were weighed and dissolved in 2mL of sterile Tris buffer (pH 6.0). Pipette 1ml of NTG solution into 1ml of the bacterial suspension, and shake-treat at 30 ℃ for 40 min. The other specific steps are as follows:
(1) preparation and culture of mycelia
The solid culture medium formula comprises: PDA is sterilized at 121 ℃ for 30 minutes, cooled to 50-60 ℃ and poured into a flat plate, the treated bacterial suspension is diluted properly, 0.1mL of bacterial suspension is absorbed and coated on the PDA flat plate, the bacterial suspension which is not subjected to mutagenesis treatment is also diluted properly and coated on the PDA flat plate as a control, the PDA flat plate is placed in a constant-temperature incubator at 30 ℃ for culture, bacterial colonies are cultured for 7 days, and hyphae are mature.
(2) Preparation and culture of seed liquid
Seed culture medium formula (g/L): 10.0g/L of sucrose, 25.0g/L of glucose, 10.0g/L of cottonseed cake powder, 15.0g/L of yeast powder, 5.0g/L of monopotassium phosphate, 1.0g/L of ammonium sulfate, 1.0g/L of calcium carbonate and pH 6.0. The liquid loading of the shake flask is 20mL/250mL, and the shake flask is sterilized at 121 ℃ for 30 minutes. And inoculating about 0.5 bacterial colony to each seed shake flask, culturing at 20-30 ℃ at an amplitude of 5cm in a shake flask machine at a rotation speed of 250rpm for 36 hours.
(3) Preparation and culture of wortmannin fermentation medium
20.0g/L of glycerol, 40.0g/L of lactose, 30.0g/L of sucrose, 10.0g/L of soybean cake powder, 15.0g/L of yeast powder, 10.0g/L of cottonseed cake powder, 0.5g/L of monopotassium phosphate, 2.0g/L of ammonium sulfate, 2.0g/L of calcium carbonate, 0.05g/L of manganese sulfate, pH6.5, the liquid filling amount of a shake flask is 25mL/250mL, and the shake flask is sterilized at 121 ℃ for 30 minutes. Inoculating the seed liquid into a fermentation culture medium at an inoculation amount of 10% (volume percentage), culturing at 25 deg.C with a shaking machine with an amplitude of 5cm and a rotation speed of 250rpm for 98 hr.
1000 single colonies subjected to NTG mutagenesis are selected, shake flask fermentation is carried out, and the yield of wortmannin is detected by HPLC. The mutant strain with the highest yield of wortmannin, namely wortmannin 1507-N13(CGMCC No.13366), is screened. The main biological characteristics are as follows: the spores are oval, the colony color is yellow, the edge is white, the reverse side is white, the spores are tiled and have no wrinkles, water drops are generated, the diameter is 1.0-2.0 cm, and the spores are not easy to pick.
Example 2: strain identification
Experiments are carried out according to the relevant contents in books such as general mycology, common bacteria system identification manual, molecular cloning experimental guidelines and Chinese pharmacopoeia (2015 edition); color determination is compared with the colors of the RAL K7 color chip series.
1. And (3) numbering strains: 1507-N13(CGMCC No. 13366).
2. The culture characteristics are as follows: and 5 culture media of Chashi, PDA, SDA, MEA and MEPG are adopted, and after the culture media are cultured for 5-7 days at the temperature of 28 ℃, the color and the pigment condition of the mycelium are observed. The culture characteristics of the strains are shown in Table 1.
3. Physiological and biochemical tests: except for the temperature experiment, the culture is carried out for 5-7 days at 28 ℃.
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 2.
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 2.
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 3; the results of the NaCl tolerance experiments are shown in Table 7.
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 4, the pH test results are shown in Table 5, and the temperature test results are shown in Table 6.
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 4.
4.28S rDNA sequence analysis: collecting fresh thallus cultured by PDA, extracting total DNA template by liquid nitrogen wall breaking method, carrying out 28S rDNA gene ITS region amplification by using Fungi Identification PCR Kit (TaKaRa), directly carrying out sequence determination after agarose electrophoresis detection of PCR product, and purifying and sequencing the PCR product by Nanjing Jinsry biological technology limited company. The ITS region sequences of the 28S rDNA gene measured in the strain 1507-N13 were aligned and compared with BLAST to determine the homology.
Results of the experiment
1. The culture characteristics are as follows: the culture characteristics of the strain 1507-N13(CGMCC No.13366) are shown in Table 1.
TABLE 1 culture characteristics of the strains 1507-N13(CGMCC No.13366) on 5 media
2. Physiological and biochemical characteristics: see tables 2-7.
TABLE 2 utilization of carbon and nitrogen sources by Strain 1507-N13(CGMCC No.13366)
TABLE 3 degradation test results of Strain 1507-N13(CGMCC No.13366)
| 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％ | 3，- |
| Xylan | 0.4％ | 4，- | Tween-60 | 1.0％ | 3，- |
| Hypoxanthine | 0.4％ | 4，- | Tween-80 | 1.0％ | 3，- |
TABLE 4 Main physiological and biochemical characteristics of Strain 1507-N13(CGMCC No.13366)
| 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 5 pH test for growth of Strain 1507-N13(CGMCC No.13366)
| pH | 3.5 | 4.0 | 4.5 | 5.0 | 5.5 | 6.0 | 6.5 | 7.0 | 7.5 |
| Growth conditions | 3 | 3 | 4 | 4 | 4 | 4 | 4 | 4 | 4 |
TABLE 6 temperature test for growth of Strain 1507-N13(CGMCC No.13366)
| Temperature (. degree.C.) | 7 | 14 | 28 | 37 | 45 |
| Growth conditions | 0 | 4 | 4 | 0 | 0 |
TABLE 7 tolerance of strain 1507-N13(CGMCC No.13366) to NaCl
| Concentration of NaCl | 1％ | 4％ | 7％ | 10％ |
| Growth of the Strain | 4 | 0 | 0 | 0 |
Injecting: in tables 2 to 7: 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.
3.28S rDNA sequence analysis: BLAST comparison of the 28S ITS sequence of strain 1507-N13(CGMCC No.13366) with the related sequences in GenBank gave the results shown in Table 8 (only the more homologous strains are listed in the Table).
TABLE 8 homology of strains 1507-N13(CGMCC No.13366) and related strains
Identification result of strain
By sequencing the 28S rDNA ITS region of strain 1507-N13(CGMCC No.13366), and performing BLAST comparison with the homologous sequences of related species and genera in the GenBank database, the results show that: the strain 1507-N13(CGMCC No.13366) has high homology with Penicillium (Penicillium sp. SGE10) and the highest homology reaches 99.6%, and the apparent characteristic test of the strain 1507-N13(CGMCC No.13366) shows that the strain is very close to the classification related parameters of Penicillium (Penicillium sp.), so the strain 1507-N13 is identified as the Penicillium (Penicillium sp.) strain.
The comparison between the penicillium wortmannii 1507-N13(CGMCC No.13366) and other wortmannin producing bacteria is as follows:
P.W.Brian et al (trans.Brit.mycol.Soc.40(3),365- "368 (1957) Penicillium workmanin Klocker (Penicillium sp. preservation 1952) reported that colonies on the test medium were quite small, the mycelia were mostly pale yellow, and the reverse side of the colonies was orange red or light brown, wrinkled, and slightly tall, while the Penicillium wortmannii 1507-N13(CGMCC No.13366) of the present invention had a colony diameter of 10mm on the test medium, the colonies were bright yellow, and the reverse side of the colonies was white, non-wrinkled, and flat.
The titer of wortmannin produced by wortmannin producing bacteria A24603.1 of US5378725 and CN1111127A is 151 mug/mL; the titer of wortmannin produced by the penicillium wortmannii 1507-N13(CGMCC No.13366) of the invention is as high as 3500 mu g/mL.
In combination with the morphological, culture, physiological and biochemical characteristics and DNA sequence identification results of the wortmannium 1507-N13(CGMCC No.13366) of the invention, the wortmannium 1507-N13(CGMCC No.13366) of the invention belongs to the wortmannium Penicillium wortmannii and is different from known wortmannium bacteria, so the wortmannium 1507-N13(CGMCC No.13366) of the invention is a brand new strain.
Example 3: preparation of wortmannin
(1) Solid culture medium formula and culture
The solid culture medium formula comprises: PDA, sterilized at 121 deg.C for 30 min, cooled for use, inoculated with 0.1mL of bacterial suspension and cultured for 7 days.
(2) Seed culture medium formula and culture
Seed culture medium formula (g/L): 10.0g/L of sucrose, 25.0g/L of glucose, 10.0g/L of cottonseed cake powder, 15.0g/L of yeast powder, 5.0g/L of monopotassium phosphate, 1.0g/L of ammonium sulfate, 1.0g/L of calcium carbonate and pH 6.0. The liquid loading of the shake flask is 20mL/250mL, and the shake flask is sterilized at 121 ℃ for 30 minutes. And inoculating about 0.5 bacterial colony to each seed shake flask, culturing at 20-30 ℃ at an amplitude of 5cm in a shake flask machine at a rotation speed of 250rpm for 36 hours.
(3) Preparation and culture of wortmannin fermentation medium
40.0g/L of glycerol, 50.0g/L of lactose, 25g/L of yeast powder, 10.0g/L of cottonseed cake powder, 0.5g/L of monopotassium phosphate, 2.0g/L of ammonium sulfate, 2.0g/L of calcium carbonate, 0.05g/L of manganese sulfate, pH6.5, the liquid filling amount of a shake flask is 25mL/250mL, and the shake flask is sterilized at 121 ℃ for 30 minutes. Inoculating the seed liquid into a fermentation culture medium at an inoculation amount of 10% (volume percentage), culturing at 25 deg.C with a shaking machine with an amplitude of 5cm and a rotation speed of 250rpm for 96 hr. The wortmannin titer is 3156 mu g/mL through HPLC detection.
Example 4: preparation of wortmannin
(1) Solid Medium formulation and culture conditions were the same as those in the step (1) of example 3
(2) Seed Medium formulation and culture conditions were the same as those in the step (2) of example 3
(3) Preparation and culture of wortmannin fermentation medium
20.0g/L of glycerol, 50.0g/L of lactose, 30.0g/L of sucrose, 15.0g/L of soybean cake powder, 15.0g/L of yeast powder, 10.0g/L of cottonseed cake powder, 0.8g/L of monopotassium phosphate, 1.5g/L of ammonium sulfate, 1.5g/L of calcium carbonate and 0.05g/L of manganese sulfate, the pH value is 6.5, the liquid filling amount of a shake flask is 25mL/250mL, and the shake flask is sterilized at 121 ℃ for 30 minutes. Inoculating the seed liquid into a fermentation culture medium at an inoculation amount of 10% (volume percentage), culturing at 25 deg.C with a shaking machine with an amplitude of 5cm and a rotation speed of 250rpm for 100 hr. The wortmannin titer is 3344 mug/mL by HPLC detection.
Example 5 preparation of wortmannin
(1) Solid Medium formulation and culture conditions were the same as those in the step (1) of example 3
(2) Preparation and culture of seed liquid
The seed culture medium formula comprises: 35.0g/L of glucose, 10.0g/L of cottonseed cake powder, 15.0g/L of yeast powder, 3.0g/L of monopotassium phosphate, 1.0g/L of ammonium sulfate and 1.0g/L of calcium carbonate, and the pH value is 6.0. The liquid loading of the shake flask is 20mL/250mL, and the shake flask is sterilized at 121 ℃ for 30 minutes. The culture temperature is 20-30 ℃, the amplitude of the shaking machine is 5cm, the rotating speed is 250rpm, and the culture is carried out for 36 hours.
(3) The fermentation medium formulation and culture conditions were the same as those in the step (3) of example 3
The wortmannin titer is 3271 mug/mL by HPLC detection.
Example 6: preparation, isolation and purification of wortmannin
(1) Preparation of seed liquid in seed tank
Putting 10L of seed culture medium (the formula of the seed culture medium is the same as the step (2) in the example 3) into a 15L seeding tank, simultaneously adding 0.05% of PPG (PPG) as an antifoaming agent), performing steam sterilization for sterilization, performing 30 minutes at 121 ℃, cooling, then adding 200ml of shake flask seed solution, performing culture at the temperature of 25 +/-1 ℃, stirring at the rotating speed of 100-500 rpm, the ventilation amount of 0.5-1.0 vvm, and performing culture for 36 hours with dissolved oxygen of 20-70%.
(2) Preparation and culture of fermentation tank culture medium
The formulation of the fermentation medium was the same as that in the step (3) of example 3, except that 0.05% PPG was added as an antifoaming agent, the fermentation tank was filled at 30L/50L and pH7.0, steam sterilized at 121 ℃ for 30 minutes, cooled, and inoculated into about 3.5L of a seed tank culture medium at a fermentation temperature of 25. + -. 1 ℃, a stirring rotation speed of 200 to 500rpm, and an aeration rate of 0.5 to 1.0vvm, and subjected to fermentation culture for 103 hours. The wortmannin titer is 3510 mug/mL through HPLC detection.
(3) Extraction of fermentation broth
Fermenting to obtain 30L fermentation liquid, extracting with equal volume of ethyl acetate for 2 times, and concentrating the obtained ethyl acetate extractive solution at 45 deg.C under reduced pressure to remove ethyl acetate until dry to obtain 76g oily substance.
(4) Purifying by silica gel column chromatography
The resulting oil was dissolved in 5L chloroform and subjected to column chromatography on a silica gel column (particle size 100-200 mesh), eluted with chloroform-ethyl acetate 85:15(V/V), and the wortmannin-containing eluate was collected.
(5) Purification of crude product and structural identification of pure product
Concentrating the eluate containing wortmannin to dryness, dissolving with 500mL of ethyl acetate, filtering with filter paper, adding 1000mL of n-hexane, mixing uniformly, and refrigerating in a refrigerator at 4 ℃. After 2 hours, the cold storage solution was filtered, and the obtained needle-like crystals were transferred to a vacuum drying oven and vacuum-dried at 50 ℃ for 5 hours.
The crystal is determined to be wortmannin through spectrum analysis such as NMR, MS and the like, and the structure of the crystal is as follows:
the physicochemical properties of wortmannin are as follows:
white acicular crystal
Solubility, is easily soluble in chloroform, acetone and methanol, and insoluble in water
Molecular formula C23H24O8
ESIMS m/z:427.12[M-H]-
TABLE 9 wortmannin in CDCl3Nuclear magnetic data of (hydrogen spectrum, 400 MHz; carbon spectrum, 100MHz)
The results show that the titer of wortmannin produced by the penicillium wortmannii 1507-N13(CGMCC No.13366) is as high as 3500 mug/mL (CGMCC No.13366), the ability of the penicillium wortmannii 1507-N13(CGMCC No.13366) to produce wortmannin is greatly improved compared with other strains, and the industrial production of the wortmannin is favorably realized.
Claims (17)
1. Penicillium wortmannii (Penicillium wortmannii)1507-N13 with the preservation number of CGMCCNo.13366.
2. Use of wortmannin 1507-N13 according to claim 1 for the preparation of wortmannin or a pharmaceutical composition containing wortmannin.
3. A preparation method of wortmannin is characterized by comprising the following steps: comprising the step of carrying out fermentative culture using Penicillium wortmannii 1507-N13 according to claim 1 in a nutrient medium containing assimilable carbon and/or nitrogen sources.
4. The method of claim 3, wherein: the assimilable carbon source is selected from one of glucose, lactose, mannitol, sorbitol, sucrose, maltodextrin, corn starch, glycerol, soybean oil, methyl oleate or a combination thereof.
5. The method of claim 3, wherein: the assimilable nitrogen source is selected from one of yeast extract powder, yeast powder, beef extract, peptone, soybean cake powder, cottonseed cake powder, peanut cake powder, gluten powder, corn steep liquor dry powder and bran or the combination of the above substances.
6. The method of claim 5, wherein: the peptone is soybean peptone.
7. The method according to any one of claims 3-6, wherein: the nutrient medium also comprises inorganic salt, wherein the inorganic salt is selected from one of ammonium chloride, ammonium nitrate, triammonium 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 and manganese sulfate or the combination of the substances.
8. The method according to any one of claims 3-6, wherein: the nutrient medium contains 10.0-50.0 g/L of glycerol, 5.0-50.0 g/L of lactose, 5.0-50.0 g/L of sucrose, 5.0-20.0 g/L of soybean cake powder, 5.0-20.0 g/L of yeast powder, 5.0-30.0 g/L of cottonseed cake powder, 0.5-5.0 g/L of monopotassium phosphate, 1.0-5.0 g/L of ammonium sulfate, 1.0-5.0 g/L of calcium carbonate and 0.01-1.0 g/L of manganese sulfate.
9. The method according to any one of claims 3-6, wherein: the temperature of the fermentation culture is 20-30 ℃; the pH value of the culture medium is 5.0-8.0; the culture time is 24-240 hours.
10. The method of claim 9, wherein: the temperature of the fermentation culture is 24-26 ℃.
11. The method of claim 9, wherein: the pH value of the culture medium is 6.0-7.0.
12. The method of claim 9, wherein: the culture time is 72-192 hours.
13. The method according to any one of claims 3-6, wherein: the penicillium wortmannii 1507-N13 is subjected to fermentation culture by inoculating seed liquid into the nutrient medium;
wherein the seed liquid is obtained by seed culture of the penicillium wortmannii 1507-N13 in a seed culture medium according to claim 1; 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.
14. The method of claim 13, wherein: the temperature of the seed culture is 24-26 ℃.
15. The method of claim 13, wherein: the pH value of the culture medium is 5.0-7.0.
16. The method of claim 13, wherein: the culture time is 24-60 hours.
17. The method of claim 13, wherein: the seed culture medium contains 0-20.0 g/L of sucrose, 10.0-50.0 g/L of glucose, 10.0-30.0 g/L of cottonseed cake powder, 5.0-15.0 g/L of yeast powder, 1.0-10.0 g/L of monopotassium phosphate, 0.5-1.5 g/L of ammonium sulfate and 0.5-2.0 g/L of calcium carbonate.
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Citations (2)
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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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- 2017-06-14 CN CN201710447621.8A patent/CN107058137B/en active Active
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| 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 |
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| Secovironolide, a novel furanosteroid scaffold with a five-membered B ring from the endophytic fungus Talaromyces wortmannii LGT-4;Hai-E. Ding等;《Tetrahedron Letters》;20151019;第56卷;摘要，第6754页左栏第1段至第6757页右栏最后1段 * |
| Wortmannin, a Microbial Product Inhibitor of Myosin Light Chain Kinase;Satoshi Nakanishi等;《THE JOURNAL OF BIOLOGICAL CHEMISTRY》;19920205;第267卷(第4期);摘要，第2157页左栏最后1段至第2162页右栏最后1段 * |
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