CN117487877A - Reaction system and method for producing 11 alpha-OH-canrenone - Google Patents
Reaction system and method for producing 11 alpha-OH-canrenone Download PDFInfo
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- CN117487877A CN117487877A CN202311322488.5A CN202311322488A CN117487877A CN 117487877 A CN117487877 A CN 117487877A CN 202311322488 A CN202311322488 A CN 202311322488A CN 117487877 A CN117487877 A CN 117487877A
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- 229960005057 canrenone Drugs 0.000 title claims abstract description 46
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 33
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 11
- 238000000855 fermentation Methods 0.000 claims abstract description 53
- 230000004151 fermentation Effects 0.000 claims abstract description 53
- 241000223250 Metarhizium anisopliae Species 0.000 claims abstract description 28
- UJVLDDZCTMKXJK-WNHSNXHDSA-N canrenone Chemical compound C([C@H]1[C@H]2[C@@H]([C@]3(CCC(=O)C=C3C=C2)C)CC[C@@]11C)C[C@@]11CCC(=O)O1 UJVLDDZCTMKXJK-WNHSNXHDSA-N 0.000 claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 17
- 239000000725 suspension Substances 0.000 claims description 28
- 239000007788 liquid Substances 0.000 claims description 26
- 239000002609 medium Substances 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 238000001914 filtration Methods 0.000 claims description 12
- 239000008223 sterile water Substances 0.000 claims description 12
- 239000000843 powder Substances 0.000 claims description 10
- 239000001963 growth medium Substances 0.000 claims description 9
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 claims description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 8
- 239000008103 glucose Substances 0.000 claims description 8
- 239000000787 lecithin Substances 0.000 claims description 8
- 229940067606 lecithin Drugs 0.000 claims description 8
- 235000010445 lecithin Nutrition 0.000 claims description 8
- 241000931705 Cicada Species 0.000 claims description 4
- 241000382353 Pupa Species 0.000 claims description 4
- 235000019764 Soybean Meal Nutrition 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 4
- 239000004455 soybean meal Substances 0.000 claims description 4
- 229920001817 Agar Polymers 0.000 claims description 3
- 244000061456 Solanum tuberosum Species 0.000 claims description 3
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 238000011218 seed culture Methods 0.000 claims description 3
- 238000002425 crystallisation Methods 0.000 claims description 2
- 230000008025 crystallization Effects 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 239000000758 substrate Substances 0.000 abstract description 4
- 238000012258 culturing Methods 0.000 description 17
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 15
- 238000002360 preparation method Methods 0.000 description 15
- 239000000243 solution Substances 0.000 description 15
- 239000002054 inoculum Substances 0.000 description 10
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- 238000004128 high performance liquid chromatography Methods 0.000 description 6
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- 238000011068 loading method Methods 0.000 description 5
- 230000001105 regulatory effect Effects 0.000 description 5
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- 238000005406 washing Methods 0.000 description 5
- 241000122824 Aspergillus ochraceus Species 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 241000235527 Rhizopus Species 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 150000003431 steroids Chemical class 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- LUJVUUWNAPIQQI-UHFFFAOYSA-N (+)-androsta-1,4-diene-3,17-dione Natural products O=C1C=CC2(C)C3CCC(C)(C(CC4)=O)C4C3CCC2=C1 LUJVUUWNAPIQQI-UHFFFAOYSA-N 0.000 description 1
- LHNVKVKZPHUYQO-SRWWVFQWSA-N 16-alpha,17-Epoxypregn-4-ene-3,20-dione Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1C[C@H]3O[C@@]3(C(=O)C)[C@@]1(C)CC2 LHNVKVKZPHUYQO-SRWWVFQWSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 102000003979 Mineralocorticoid Receptors Human genes 0.000 description 1
- 108090000375 Mineralocorticoid Receptors Proteins 0.000 description 1
- 102000008109 Mixed Function Oxygenases Human genes 0.000 description 1
- 108010074633 Mixed Function Oxygenases Proteins 0.000 description 1
- 241000952054 Rhizopus sp. Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
- AEMFNILZOJDQLW-QAGGRKNESA-N androst-4-ene-3,17-dione Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CCC2=C1 AEMFNILZOJDQLW-QAGGRKNESA-N 0.000 description 1
- LUJVUUWNAPIQQI-QAGGRKNESA-N androsta-1,4-diene-3,17-dione Chemical compound O=C1C=C[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CCC2=C1 LUJVUUWNAPIQQI-QAGGRKNESA-N 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
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- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229960001208 eplerenone Drugs 0.000 description 1
- JUKPWJGBANNWMW-VWBFHTRKSA-N eplerenone Chemical compound C([C@@H]1[C@]2(C)C[C@H]3O[C@]33[C@@]4(C)CCC(=O)C=C4C[C@H]([C@@H]13)C(=O)OC)C[C@@]21CCC(=O)O1 JUKPWJGBANNWMW-VWBFHTRKSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- OSVMTWJCGUFAOD-KZQROQTASA-N formestane Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CCC2=C1O OSVMTWJCGUFAOD-KZQROQTASA-N 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 229940044551 receptor antagonist Drugs 0.000 description 1
- 239000002464 receptor antagonist Substances 0.000 description 1
- 230000036454 renin-angiotensin system Effects 0.000 description 1
- 239000003270 steroid hormone Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
- C12P33/20—Preparation of steroids containing heterocyclic rings
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
- C12P33/06—Hydroxylating
- C12P33/08—Hydroxylating at 11 position
- C12P33/10—Hydroxylating at 11 position at 11 alpha-position
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
Abstract
The invention discloses a reaction system and a method for producing 11 alpha-OH-canrenone, wherein the reaction system comprises the following components: canrenone and metarhizium anisopliae fermentation broth. The reaction system and the method provided by the invention are simple and convenient to operate, low in equipment requirement and high in substrate conversion rate.
Description
Technical Field
The invention belongs to the technical field of application microorganisms, and relates to a reaction system and a method for producing 11 alpha-OH-canrenone.
Background
Canrenone (canrenone) is a steroid hormone which is used clinically as a non-selective aldosterone receptor antagonist to treat cardiovascular diseases and effectively blocks the action of the renin-angiotensin-aldosterone system (RAAS), but is likely to cause overdose and mortality in the treatment of heart diseases. Another drug, eplerenone, can greatly reduce this risk and can be synthesized via the intermediate 11 a-OH-canrenone.
At present, the 11 alpha-OH-canrenone is produced by converting canrenone through microorganisms, and researches and reports are carried out mainly by utilizing aspergillus ochraceus, rhizopus and the like. Patent application CN104046675A utilizes the enzyme solution of Rhizopus (Rhizopus sp. UJ-0602) to produce 11 alpha-OH-canrenone, and patent application CN103255192A utilizes Aspergillus ochraceus (Aspergillus ochraceus TCCC 41060) to produce 11 alpha-OH-canrenone efficiently by high-density fermentation. Meanwhile, patent application CN102876582a discloses that metarhizium anisopliae mutant (Metarhizium anisopliae) 11490 can effectively introduce 11 alpha hydroxyl on 19-desmethyl-13-ethyl-androsta-4-ene-3, 17-dione, androsta-4-ene-3, 17-dione and androsta-1, 4-diene-3, 17-dione. Researches have also reported that metarhizium anisopliae can also introduce 11 alpha hydroxyl on steroid substrates 16 alpha, 17 alpha epoxyprogesterone, and has wide steroid substrate selectivity. However, the synthesis of 11 alpha-OH-canrenone by using Metarhizium anisopliae to convert canrenone has been reported in a few cases.
The above prior art has the following problems: the patent application CN104046675A needs to crush thalli by ultrasonic waves to obtain enzyme solution containing hydroxylase, then convert, the feeding amount is 5g/L, and the purity of converted 84h,11 alpha-OH-canrenone reaches 96.0 percent. In the patent application CN103255192A, glucose is fed in the fermentation process to realize high-density fermentation, and the conversion is 58 hours and the conversion rate is 97% when the feeding amount is 15 g/L. Complicated operation, high requirements on equipment and increased cost.
Disclosure of Invention
In order to solve the problems of complicated operation, high requirement on equipment and high cost in the existing 11 alpha-OH-canrenone synthesis technology, the invention provides a reaction system and a method for producing 11 alpha-OH-canrenone.
In order to solve the above technical problems, a first aspect of the present invention provides a reaction system for producing 11 α -OH-canrenone, the reaction system comprising: canrenone and metarhizium anisopliae fermentation broth.
In some embodiments of the invention, the reaction system further comprises lecithin; and/or the preservation number of the metarhizium anisopliae is CGMCC No. 3.17278.
In some preferred embodiments of the invention, the canrenone is dosed at a mass fraction of 1.5% -2%, such as 1.5%, 1.8% or 2%; and/or the mass fraction of lecithin is 1.5% -2%, such as 1.5% or 2%.
In order to solve the above technical problems, a second aspect of the present invention provides a method for producing 11 α -OH-canrenone by using the reaction system according to the first aspect of the present invention.
In some preferred embodiments of the invention, the conditions of production are selected from one or more of the following:
the canrenone is added in the form of dry powder; and/or the number of the groups of groups,
the temperature is 25-30deg.C, such as 28deg.C, 29 deg.C or 30deg.C;
the rotation speed is 200-300rpm, such as 200rpm or 250rpm;
introducing air at 2-5L/min, such as 3L/min;
the conversion is carried out for 40-50h, for example 42h or 48h.
In some embodiments of the invention, the metarhizium anisopliae fermentation broth is obtained by inoculating seed liquor into a fermentation medium for fermentation.
In some preferred embodiments of the invention, the seed solution is obtained by inoculating a spore suspension into a seed culture medium and culturing; the spore suspension is obtained, for example, by mixing sterile water with spores.
In some more preferred embodiments of the invention, the spores are obtained from metarhizium anisopliae inoculated into a slant culture.
In some embodiments of the invention, the fermentation is carried out at 25-30deg.C, 200-300rpm,2-3L/min with air, and culturing for 15-20h; and/or the number of the groups of groups,
the culture condition of the seed liquid is 25-30 ℃,150-250rpm, and the seed liquid is cultured for 15-20h; and/or the number of the groups of groups,
the culture condition of the slant strain is 25-30 ℃, and the slant strain is cultured for 5-10 days at constant temperature.
In some embodiments of the invention, the components of the seed medium and/or fermentation medium include: 10g/L of glucose, 10g/L of soybean meal, 5g/L of cicada pupa powder and the balance of water.
In some preferred embodiments of the invention, the composition of the slant medium comprises: 200g/L of potato, 20g/L of glucose, 15-20g/L of agar and the balance of water.
In some embodiments of the invention, the seed solution has an inoculation volume ratio of 5% to 10%; and/or the number of the groups of groups,
the inoculation volume ratio of the spore suspension is 4% -6%; and/or the number of the groups of groups,
the spore suspension had a concentration of 8X 10 6 -1.5×10 8 And each mL.
In some embodiments of the invention, the method further comprises a step of purifying;
in some preferred embodiments of the invention, the purification is selected from one or more of extraction, concentration, crystallization and filtration.
In order to solve the technical problems, a third aspect of the invention provides an application of metarhizium anisopliae or the reaction system according to the first aspect of the invention in the production of 11 alpha-OH-canrenone.
In some preferred embodiments of the present invention, the metarhizium anisopliae has a preservation number of CGMCC No. 3.17278.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that:
the method directly utilizes the metarhizium anisopliae GD-6 to produce the 11 alpha-OH-canrenone, and the product purity is up to 98.7% after 48 hours of conversion when the feeding amount is 20g/L, so that the method is more efficient, the operation is simpler, and the requirement on equipment is low.
Drawings
FIG. 1 shows a specific purification step after the conversion.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
EXAMPLE 1 preparation of 11 alpha-OH-canrenone by Metarhizium anisopliae fermentation
1. Preparing inclined plane strains: inoculating Metarhizium anisopliae (CGMCC 3.17278) into slant culture medium (PDA): 200g/L of potato, 20g/L of glucose, 15-20g/L of agar and natural pH. Culturing at 28deg.C for 7 days until mycelia are fully covered on the surface, and the mycelia start to turn green to obtain slant strain.
2. Spore suspension preparation: adding sterile water into slant strain, washing spores, filtering mycelium with 6 layers of mirror cleaning paper to obtain spore suspension, and regulating spore suspension concentration to 2.46×10 with sterile water 7 And each mL.
3. Seed liquid preparation: the spore suspension was inoculated into a seed culture medium (glucose 10g/L, soybean meal 10g/L, cicada pupa meal 5 g/L) at an inoculum size of 5% by volume, and the liquid loading was 50mL/250mL Erlenmeyer flask. Culturing at 29 deg.C and 170rpm for 16 hr to obtain seed solution.
4. Preparing fermentation liquid: the seed solution was inoculated into a fermentation medium (glucose 10g/L, soybean meal 10g/L, cicada pupa powder 5 g/L) at an inoculum size of 5% by volume. The volume ratio of the fermentation medium to the fermentation tank is 70%, the temperature is 28 ℃, the rpm is 200, and air is introduced for culturing for 16 hours at 2L/min to obtain fermentation liquor.
5. The transformation process comprises the following steps: and directly adding canrenone dry powder into the fermentation liquor, wherein the feeding concentration is 1.5%, and simultaneously adding 1.5% lecithin, and introducing air at the temperature of 28 ℃ and at the speed of 200rpm for conversion for 42 hours at the speed of 3L/min to obtain 11 alpha-hydroxylated canrenone. And extracting, concentrating, crystallizing and filtering by using ethyl acetate after the conversion is finished to obtain 11 alpha-OH-canrenone, wherein the specific operation is shown in figure 1. Compared with the prior reported enzyme liquid method and high-density fermentation method, the method does not need the auxiliary of unconventional equipment.
Hplc detection: instrument: HPLC LC-40DXR Shimadzu
The detection method comprises the following steps:
(1) Preparing a solution:
samples were taken and solutions at 0.5mg/ml were prepared with acetonitrile-water (60:40) solutions, respectively.
(2 chromatographic conditions:
chromatographic column: c18 250 x 4.6mm 5um
Flow rate: 1.0ml/min
Column temperature: room temperature
Wavelength: 280nm of
Sample injection amount: 20ul
Mobile phase: acetonitrile: water = 60:40 (v/v)
Dilution liquid: acetonitrile: water = 60:40
The purity was 95.3% by HPLC.
EXAMPLE 2 preparation of 11 alpha-OH-canrenone by Metarhizium anisopliae fermentation
1. Preparing inclined plane strains: inoculating Metarhizium anisopliae to slant culture medium, culturing at 28deg.C for 8 days until mycelium is fully distributed on the surface, and the mycelium starts to turn green to obtain slant strain.
2. Spore suspension preparation: adding sterile water into slant strain, washing spores, filtering mycelium with 6 layers of mirror cleaning paper to obtain spore suspension, and regulating spore suspension concentration to 8.2X10 with sterile water 6 And each mL.
3. Seed liquid preparation: the spore suspension was inoculated into the seed medium in an inoculum size of 6% by volume, and the liquid loading was 50mL/250mL Erlenmeyer flask. Culturing at 30 deg.c and 200rpm for 18 hr to obtain seed liquid.
4. Preparing fermentation liquid: the seed solution was inoculated into the fermentation medium in an inoculum size of 8% by volume. The volume ratio of the fermentation medium to the fermentation tank is 60%, the temperature is 30 ℃, the air is introduced for culturing for 18 hours at 200rpm and 2L/min, and the fermentation liquid is obtained.
5. The transformation process comprises the following steps: and directly adding dry canrenone powder into the fermentation broth, wherein the feeding concentration is 1.8%, 2% lecithin is added simultaneously, the temperature is 30 ℃, the speed is 200rpm, and air is introduced into the fermentation broth for 48 hours at 3L/min to obtain 11 alpha-hydroxylated canrenone. And extracting, concentrating, crystallizing and filtering by using ethyl acetate after the conversion is finished to obtain 11 alpha-OH-canrenone, wherein the specific operation is shown in figure 1. Purity by HPLC 98.3%.
EXAMPLE 3 preparation of 11 alpha-OH-canrenone by Metarhizium anisopliae fermentation
1. Preparing inclined plane strains: inoculating Metarhizium anisopliae to slant culture medium, culturing at 29 deg.C for 8 days until mycelium is fully distributed on the surface, and the mycelium starts to turn green to obtain slant strain.
2. Spore suspension preparation: adding sterile water into slant strain, washing spores, filtering mycelium with 6 layers of mirror cleaning paper to obtain spore suspension, and regulating spore suspension concentration to 1.23×10 with sterile water 8 And each mL.
3. Seed liquid preparation: the spore suspension was inoculated into the seed medium in an inoculum size of 4% by volume, and the liquid loading was in a 50mL/250mL Erlenmeyer flask. Culturing at 29 deg.C and 220rpm for 17 hr to obtain seed solution.
4. Preparing fermentation liquid: the seed solution was inoculated into the fermentation medium in an inoculum size of 8% by volume. The volume ratio of the fermentation medium to the fermentation tank is 70%, the temperature is 29 ℃, the air is introduced for culturing for 17 hours at 200rpm and 3L/min, and the fermentation liquid is obtained.
5. The transformation process comprises the following steps: and directly adding dry canrenone powder into the fermentation liquor, adding 2% of lecithin at the feeding concentration, and introducing air at 29 ℃ and 250rpm for conversion for 48 hours at 3L/min to obtain 11 alpha-hydroxylated canrenone. And extracting, concentrating, crystallizing and filtering by using ethyl acetate after the conversion is finished to obtain 11 alpha-OH-canrenone, wherein the specific operation is shown in figure 1. Purity by HPLC 98.7%.
EXAMPLE 4 preparation of 11 alpha-OH-canrenone by Metarhizium anisopliae fermentation
1. Preparing inclined plane strains: inoculating Metarhizium anisopliae to slant culture medium, culturing at 28deg.C for 7 days until mycelium is fully distributed on the surface, and the mycelium starts to turn green to obtain slant strain.
2. Spore suspension preparation: adding sterile water into slant strain, washing spores, filtering mycelium with 6 layers of mirror cleaning paper to obtain spore suspension, and regulating spore suspension concentration to 9.2X10 with sterile water 7 And each mL.
3. Seed liquid preparation: the spore suspension was inoculated into the seed medium in an inoculum size of 6% by volume, and the liquid loading was 50mL/250mL Erlenmeyer flask. Culturing at 30 deg.c and 200rpm for 18 hr to obtain seed liquid.
4. Preparing fermentation liquid: the seed solution was inoculated into the fermentation medium in an inoculum size of 5% by volume. The volume ratio of the fermentation medium to the fermentation tank is 70%, the temperature is 28 ℃, the rpm is 200, and air is introduced for culturing for 18 hours at 2L/min to obtain fermentation liquor.
5. The transformation process comprises the following steps: directly adding canrenone dry powder into fermentation liquor, wherein the feeding concentration is 1.5%, dissolving a substrate by using DMF, converting for 48 hours at 30 ℃ at 200rpm by introducing air at 3L/min, and obtaining 11 alpha-hydroxylated canrenone. And extracting, concentrating, crystallizing and filtering by using ethyl acetate after the conversion is finished to obtain 11 alpha-OH-canrenone, wherein the specific operation is shown in figure 1. The purity of the product was 80.85% by HPLC.
EXAMPLE 5 preparation of 11 alpha-OH-canrenone by Metarhizium anisopliae fermentation
1. Preparing inclined plane strains: inoculating Metarhizium anisopliae to slant culture medium, culturing at 29 deg.C for 8 days until mycelium is fully distributed on the surface, and the mycelium starts to turn green to obtain slant strain.
2. Spore suspension preparation: adding sterile water into slant strain, washing spores, filtering mycelium with 6 layers of mirror cleaning paper to obtain spore suspension, and regulating spore suspension concentration to 1.23×10 with sterile water 8 And each mL.
3. Seed liquid preparation: the spore suspension was inoculated into the seed medium in an inoculum size of 4% by volume, and the liquid loading was in a 50mL/250mL Erlenmeyer flask. Culturing at 29 deg.C and 220rpm for 17 hr to obtain seed solution.
4. Preparing fermentation liquid: the seed solution was inoculated into the fermentation medium in an inoculum size of 8% by volume. The volume ratio of the fermentation medium to the fermentation tank is 70%, the temperature is 29 ℃, the air is introduced for culturing for 17 hours at 200rpm and 3L/min, and the fermentation liquid is obtained.
5. The transformation process comprises the following steps: directly adding canrenone dry powder into fermentation liquor, adding material concentration is 2%, adding no lecithin, 29 ℃, introducing air at 250rpm for conversion for 48 hours at 3L/min, and obtaining 11 alpha-hydroxylated canrenone. And extracting, concentrating, crystallizing and filtering by using ethyl acetate after the conversion is finished to obtain 11 alpha-OH-canrenone, wherein the specific operation is shown in figure 1. The purity was 62.9% by HPLC.
Claims (10)
1. A reaction system for producing 11 a-OH-canrenone, the reaction system comprising: canrenone and metarhizium anisopliae fermentation broth.
2. The reaction system of claim 1, wherein the reaction system further comprises lecithin; and/or the preservation number of the metarhizium anisopliae is CGMCC No. 3.17278;
preferably, the mass fraction of the canrenone is 1.5% -2%; and/or, the mass fraction of the lecithin is 1.5% -2%.
3. A method for producing 11 a-OH-canrenone, characterized in that 11 a-OH-canrenone is produced using the reaction system of claim 1 or 2;
preferably, the conditions of production are selected from one or more of the following:
the canrenone is added in the form of dry powder;
the temperature is 25-30 ℃;
the rotating speed is 200-300rpm;
introducing air at 2-5L/min;
the conversion is carried out for 40-50h.
4. The method according to claim 3, wherein the metarhizium anisopliae fermentation broth is obtained by inoculating seed liquor into a fermentation medium for fermentation;
preferably, the seed solution is obtained by inoculating spore suspension into a seed culture medium for culture; the spore suspension is obtained, for example, by mixing sterile water with spores;
more preferably, the spores are obtained by inoculating metarhizium anisopliae into slant culture.
5. The method according to claim 4, wherein the fermentation is carried out at 25-30 ℃ at 200-300rpm with air at 2-3L/min for 15-20h; and/or the number of the groups of groups,
the culture condition of the seed liquid is 25-30 ℃,150-250rpm, and the seed liquid is cultured for 15-20h; and/or the number of the groups of groups,
the culture condition of the slant strain is 25-30 ℃, and the slant strain is cultured for 5-10 days at constant temperature.
6. The method of claim 4 or 5, wherein the components of the seed medium and/or fermentation medium comprise: 10g/L of glucose, 10g/L of soybean meal, 5g/L of cicada pupa powder and the balance of water;
preferably, the composition of the slant culture medium comprises: 200g/L of potato, 20g/L of glucose, 15-20g/L of agar and the balance of water.
7. The method of any one of claims 4-6, wherein the seed solution is inoculated at a volume ratio of 5% -10%; and/or the number of the groups of groups,
the inoculation volume ratio of the spore suspension is 4% -6%; and/or the number of the groups of groups,
the spore suspension had a concentration of 8X 10 6 -1.5×10 8 And each mL.
8. The method of any one of claims 3-7, further comprising the step of purifying;
preferably, the purification is selected from one or more of extraction, concentration, crystallization and filtration.
9. Use of metarhizium anisopliae or the reaction system according to claim 1 or 2 for the production of 11 alpha-OH-canrenone.
10. The use according to claim 9, wherein the metarhizium anisopliae has a preservation number of CGMCC No. 3.17278.
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