CN109971813A - A kind of technique preparing bisnoralchol - Google Patents
A kind of technique preparing bisnoralchol Download PDFInfo
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- CN109971813A CN109971813A CN201811546481.0A CN201811546481A CN109971813A CN 109971813 A CN109971813 A CN 109971813A CN 201811546481 A CN201811546481 A CN 201811546481A CN 109971813 A CN109971813 A CN 109971813A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
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- 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
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Abstract
The present invention relates to bisnoralchol preparation technical fields, and disclose a kind of technique for preparing bisnoralchol, mycobacteria living body strain including choosing in suitable actinomyces carries out multiple mutagenesis and prepares C1, 2 dehydrogenases and C22 dehydrogenase double defect bacterial strain, by C1, 2 dehydrogenases and C22 dehydrogenase double defect bacterial strain are put into fermentation medium, it will be in suitable fermentation liquid implantation glass bottle, and fermentation liquid is stirred and heating water bath, extract suitable dilute sulfuric acid, dilute sulfuric acid is neutralized to pH3~3.5, the pearlite filtering aid of 3-6ml is added, 1~2h of heating stirring, aluminium polychloride and polyacrylamide is added, persistently stir 2~3h, stop heating, hot water is discharged, 45~50 DEG C are down to cold bath.This prepares the technique of bisnoralchol, and by C1,2 dehydrogenases and C22 dehydrogenase double defect bacterial strain can quickly and effectively prepare bisnoralchol, and the bisnoralchol purifying prepared and purification degree are high, use convenient for people.
Description
Technical field
The present invention relates to bisnoralchol preparation technical field, specially a kind of technique for preparing bisnoralchol.
Background technique
Steroid drugs is wide in variety and widely used major class drug, and clinically demand is only second to antibiotic, mainly
For treating rheumatism, angiocarpy, Collagen illness, leukemic lymphoblastoid, tumour, bacterial encephalitis, skin disease, endocrine mistake
The diseases such as tune, geriatric disease.
Bisnoralchol (21-hydroxy-20-methylpregn-4-en-3-one) molecular formula is C22H34O2, and molecular weight is
330.5, be a kind of important intermediate of steroid hormone class drug, have important commercial value, currently, China prepare it is double down
When alcohol, generally prepared using steroid drugs, and steroid drugs synthesis step is more, reaction is complicated, and the distant effect of group
It is fairly obvious, difficulty is isolated and purified, is not easy to people's use, therefore propose a kind of technique for preparing bisnoralchol.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of technique for preparing bisnoralchol, have quickly prepare it is double down
Alcohol, and the advantages that bisnoralchol purifying and high purification degree solves bisnoralchol on the market now and prepares cumbersome, and preparation purity is low
The problem of.
Bisnoralchol, and the purpose that bisnoralchol purifies and purification degree is high can be quickly prepared for realization is above-mentioned, the present invention provides
Following technical solution: a kind of technique preparing bisnoralchol includes the following steps:
Step 1: the mycobacteria living body strain chosen in suitable actinomyces carries out multiple mutagenesis preparation C1,2 dehydrogenases
And C22 dehydrogenase double defect bacterial strain, by C1,2 dehydrogenases and C22 dehydrogenase double defect bacterial strain are put into fermentation medium
In, fermentation liquid is made；
Step 2: by suitable fermentation liquid implantation glass bottle, and fermentation liquid is stirred and heating water bath；
Step 3: extracting suitable dilute sulfuric acid, dilute sulfuric acid is neutralized to pH3~3.5,3-6ml pearlite filtering aid is added,
Aluminium polychloride and polyacrylamide is added in 1~2h of heating stirring, persistently stirs 2~3h, stops heating, and hot water is discharged, and use is cold
Water-bath is down to 45~50 DEG C, refilters to obtain yellow powder solid；
Step 4: yellow powder solid is injected into the vial in step 2, it is stirred, then is infused into vial
Enter methanol solution, is continuously stirred at room temperature；
It is drained Step 5: mixed solution is filtered, filtrate is distilled by normal pressure, remove methanol, then cool down,
It filters and dries to obtain crude product；
Step 6: injecting suitable methanol solution into triangular flask and will enter crude product into triangular flask, then carry out water-bath
Heating and stirring add suitable active carbon and place 2~3h, then carry out filters pressing, filtrate is passed through until crude product is completely dissolved
Air-distillation is reduced to half, places separate crystallization at normal temperature, is finally filtered, is drained and drying obtains bisnoralchol and refines
Finished product.
Preferably, the temperature of the fermentation medium is 30~35 DEG C, and the fermentation medium is sodium nitrate, biphosphate
Potassium, magnesium sulfate, corn pulp, soya-bean cake oil are made.
Preferably, the water bath heating temperature of the fermentation liquid is 70~80 DEG C.
Preferably, the additional amount of the aluminium polychloride is 25-35mL, and the additional amount of the polyacrylamide is 1-2g.
Preferably, the volume of the methanol solution is 3L, and the concentration volume ratio of methanol and water in the methanol solution is
80%.
Preferably, the water bath heating temperature of the crude product and methanol solution is 55-65 DEG C.
Preferably, the C1,2 dehydrogenases and C22 dehydrogenase double defect bacterial strain pass through ultraviolet light-by mycobacteria
LiCl and the preparation of nitrosoguanidine complex mutation.
Compared with prior art, the present invention provides a kind of technique for preparing bisnoralchol, have with next the utility model has the advantages that
This prepares the technique of bisnoralchol, carries out multiple mutagenesis preparation by using the mycobacteria living body strain in actinomyces
Simultaneously fermentation liquid is made by fermentation culture in C1,2 dehydrogenases and C22 dehydrogenase double defect bacterial strain, by by dilute sulfuric acid, treasure
Pearl rock filter aid, aluminium polychloride and polyacrylamide carry out Hybrid Heating stirring and mixed solution are made, by mixed solution and hair
Zymotic fluid, which is mixed and added into methanol solution, can be made bisnoralchol crude product into crossing stirring, filtering, distillation and drying again, then by thick
It is added that methanol solution is heated, stirred, filter press drying can obtain bisnoralchol purification finished product in product, so as to can be fast
It is fast effectively to prepare bisnoralchol, and the bisnoralchol purifying prepared and purification degree are high, use convenient for people.
Specific embodiment
Here is that technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Embodiment 1
A kind of technique preparing bisnoralchol, includes the following steps:
Step 1: the mycobacteria living body strain chosen in suitable actinomyces carries out multiple mutagenesis preparation C1,2 dehydrogenases
And C22 dehydrogenase double defect bacterial strain, by C1,2 dehydrogenases and C22 dehydrogenase double defect bacterial strain are put into fermentation medium
In, fermentation liquid is made；
Step 2: by suitable fermentation liquid implantation glass bottle, and fermentation liquid is stirred and heating water bath；
Step 3: extracting the dilute sulfuric acid of 5ml, dilute sulfuric acid is neutralized to pH3, the pearlite filtering aid of 3ml is added, heating is stirred
1h is mixed, aluminium polychloride and polyacrylamide is added, persistently stirs 2h, stops heating, hot water is discharged, is down to 45 with cold bath
DEG C, it refilters to obtain yellow powder solid；
Step 4: yellow powder solid is injected into the vial in step 2, it is stirred, then is infused into vial
Enter methanol solution, is continuously stirred at room temperature；
It is drained Step 5: mixed solution is filtered, filtrate is distilled by normal pressure, remove methanol, then cool down,
It filters and dries to obtain crude product；
Step 6: injecting suitable methanol solution into triangular flask and crude product being added into triangular flask, then carry out water-bath
Heating and stirring add suitable active carbon and place 2h, then carry out filters pressing until crude product is completely dissolved, and filtrate are passed through normal
Pressure distillation is reduced to half, places separate crystallization at normal temperature, is finally filtered, is drained and drying obtains bisnoralchol and is refined into
Product.
Wherein, C1 in step 1,2 dehydrogenases and C22 dehydrogenase double defect bacterial strain pass through ultraviolet light-by mycobacteria
LiCl and the preparation of nitrosoguanidine complex mutation, the temperature of fermentation medium are 32 DEG C, and the fermentation medium is sodium nitrate, phosphorus
Acid dihydride potassium, magnesium sulfate, corn pulp, soya-bean cake oil are made, and the water bath heating temperature of the fermentation liquid in step 2 is 75 DEG C, step 3
The additional amount of kind aluminium polychloride is 30mL, and the additional amount of polyacrylamide is 1.3g, and the volume of methanol solution is in step 4
3L, the concentration volume ratio of methanol and water in methanol solution are 80%, the heating water bath temperature of crude product and methanol solution in step 6
Degree is 60 DEG C.
Embodiment 2
A kind of technique preparing bisnoralchol, includes the following steps:
Step 1: the mycobacteria living body strain chosen in suitable actinomyces carries out multiple mutagenesis preparation C1,2 dehydrogenases
And C22 dehydrogenase double defect bacterial strain, C1, the mutagenesis method of 2 dehydrogenases and C22 dehydrogenase double defect bacterial strain can be with
Mutagenesis is carried out using nitrous acid, wherein the concentration of nitrous acid is 0.05mol/L, 15 minutes is kept the temperature at 25 DEG C, by C1,2 dehydrogenases
And C22 dehydrogenase double defect bacterial strain is put into fermentation medium, and fermentation liquid is made；
Step 2: by suitable fermentation liquid implantation glass bottle, and fermentation liquid is stirred and heating water bath, heating temperature
Degree is 78 DEG C, is heated 5 minutes, using stirring clockwise when stirring；
Step 3: extracting the dilute sulfuric acid of 8ml, dilute sulfuric acid is neutralized to pH3.3, the super-cell of 6g is added, heated
1h is stirred, aluminium polychloride and polyacrylamide is added, wherein aluminium polychloride is 40mL, polyacrylamide 1.7g, is continued
2h is stirred, heating is stopped, hot water is discharged, is down to 48 DEG C with cold bath, refilters to obtain yellow powder solid；
Step 4: yellow powder solid is injected into the vial in step 2, it is stirred, then is infused into vial
Enter methanol solution, the volume of methanol solution is 3L, and the concentration volume ratio of methanol and water in methanol solution is 80%, at room temperature
Lasting stirring；
It is drained Step 5: mixed solution is filtered, filtrate is distilled by normal pressure, remove methanol, then cool down,
It filters and dries to obtain crude product；
Step 6: injecting the methanol solution of 2.5L into triangular flask and crude product being added into triangular flask, then carry out water-bath
Heating and stirring, heating temperature is 65 DEG C, until crude product is completely dissolved, adds suitable active carbon and places 2h, then pressed
Filtrate is reduced by air-distillation to half, places separate crystallization at normal temperature, be finally filtered, drain and dry by filter
Obtain bisnoralchol purification finished product.
Embodiment 3
A kind of technique preparing bisnoralchol, includes the following steps:
Step 1: the mycobacteria living body strain chosen in suitable actinomyces carries out multiple mutagenesis preparation C1,2 dehydrogenases
And C22 dehydrogenase double defect bacterial strain, C1, the mutagenesis method of 2 dehydrogenases and C22 dehydrogenase double defect bacterial strain can be with
Mutagenesis is carried out using EMS mother liquor, after twenty minutes, 50 times of normal saline dilutions are added in the mutagenesis of EMS mother liquor, and be repeatedly centrifuged,
Then C1,2 dehydrogenases and C22 dehydrogenase double defect bacterial strain are put into fermentation medium by washing with terminating reaction, send out
The temperature of ferment culture medium be 32 DEG C, the fermentation medium be glucose, sodium nitrate, potassium dihydrogen phosphate, magnesium sulfate, brewer's wort,
Soya-bean cake oil is made, and fermentation liquid is made in the fermentation medium；
Step 2: by the fermentation liquid implantation glass bottle of 20ml, and fermentation liquid is stirred and heating water bath, heating temperature
Degree is 74 DEG C；
Step 3: extracting the dilute sulfuric acid of 10ml, dilute sulfuric acid is neutralized to pH3.5, the pearlite filtering aid of 6ml is added, adds
Aluminium polychloride and polyacrylamide is added in thermal agitation 2h, and wherein aluminium polychloride is 35mL, and polyacrylamide 2.0g is held
Continuous stirring 3h, stops heating, hot water is discharged, is down to 50 DEG C with cold bath, refilters to obtain yellow powder solid；
Step 4: yellow powder solid is injected into the vial in step 2, it is stirred, then is infused into vial
Enter methanol solution, the volume of methanol solution is 2L, and the concentration volume ratio of methanol and water in methanol solution is 65%, at room temperature
Lasting stirring；
It is drained Step 5: mixed solution is filtered, filtrate is distilled by normal pressure, remove methanol, then cool down,
It filters and dries to obtain crude product；
Step 6: injecting suitable methanol solution into triangular flask and crude product being added into triangular flask, then carry out water-bath
Heating and stirring, heating temperature is 62 DEG C, until crude product is completely dissolved, the active carbon for adding 50g places 3h, then is pressed
Filtrate is reduced by air-distillation to half, places separate crystallization at normal temperature, be finally filtered, drain and dry by filter
Obtain bisnoralchol purification finished product.
In conclusion this prepares the technique of bisnoralchol, by using the mycobacteria living body strain in actinomyces through excessive
Mutagenesis, which prepares C1,2 dehydrogenases and C22 dehydrogenase double defect bacterial strain and culture solution is made by fermentation medium, again is packed into glass
In glass bottle, then heating water bath and stirring are carried out, dilute sulfuric acid is neutralized to pH3~3.5, pearlite filtering aid, polyaluminium is added
Aluminium and polyacrylamide, heating stirring, then cooled down with cold bath, yellow powder solid is obtained by filtration, yellow powder solid is infused
Enter into vial, methanol solution injected into vial, be continuously stirred at room temperature, mixed solution is filtered drain and
Distillation removes methanol, then cools down, filters and dry to obtain crude product, suitable methanol solution is injected into triangular flask and by crude product
It will enter into triangular flask, then carry out heating water bath and stirring, and until crude product is completely dissolved, add suitable active carbon, then into
Row filters pressing places separate crystallization at normal temperature by filtrate by air-distillation, is finally filtered, drains and drying obtains pair
It drops alcohol and refines finished product.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (7)
1. a kind of technique for preparing bisnoralchol, characterized by the following steps:
Step 1: choosing mycobacteria living body strain in suitable actinomyces carries out multiple mutagenesis preparation C1,2 dehydrogenases and
C22 dehydrogenase double defect bacterial strain, by C1,2 dehydrogenases and C22 dehydrogenase double defect bacterial strain are put into fermentation medium,
Fermentation liquid is made；
Step 2: by suitable fermentation liquid implantation glass bottle, and fermentation liquid is stirred and heating water bath；
Step 3: extracting suitable dilute sulfuric acid, dilute sulfuric acid is neutralized to pH3~3.5, the pearlite filtering aid of 3-6ml is added, adds
Aluminium polychloride and polyacrylamide is added in 1~2h of thermal agitation, persistently stirs 2~3h, stops heating, and hot water is discharged, uses cold water
Bath is down to 45~50 DEG C, refilters to obtain yellow powder solid；
Step 4: yellow powder solid is injected into the vial in step 2, it is stirred, then injects first into vial
Alcoholic solution is continuously stirred at room temperature；
It is drained Step 5: mixed solution is filtered, filtrate is distilled by normal pressure, remove methanol, then cool down, filter
And it dries and obtains crude product；
Step 6: injecting suitable methanol solution into triangular flask and crude product being added into triangular flask, then carry out heating water bath
And stirring adds suitable active carbon and places 2~3h, then carry out filters pressing, filtrate is passed through normal pressure until crude product is completely dissolved
Distillation is reduced to half, places separate crystallization at normal temperature, is finally filtered, is drained and drying obtains bisnoralchol and is refined into
Product.
2. a kind of technique for preparing bisnoralchol according to claim 1, it is characterised in that: the temperature of the fermentation medium
It is 30~35 DEG C, the fermentation medium is sodium nitrate, and potassium dihydrogen phosphate, magnesium sulfate, corn pulp, soya-bean cake oil is made.
3. a kind of technique for preparing bisnoralchol according to claim 1, it is characterised in that: the heating water bath of the fermentation liquid
Temperature is 70~80 DEG C.
4. a kind of technique for preparing bisnoralchol according to claim 1, it is characterised in that: the addition of the aluminium polychloride
Amount is 25-35mL, and the additional amount of the polyacrylamide is 1-2g.
5. a kind of technique for preparing bisnoralchol according to claim 1, it is characterised in that: the volume of the methanol solution is
3L, the concentration volume ratio of methanol and water in the methanol solution are 80%.
6. a kind of technique for preparing bisnoralchol according to claim 1, it is characterised in that: the crude product and methanol solution
Water bath heating temperature is 55-65 DEG C.
7. a kind of technique for preparing bisnoralchol according to claim 1, it is characterised in that: the C1,2 dehydrogenases and
C22 dehydrogenase double defect bacterial strain is prepared by mycobacteria by ultraviolet light-LiCl and nitrosoguanidine complex mutation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201811546481.0A CN109971813A (en) | 2018-12-18 | 2018-12-18 | A kind of technique preparing bisnoralchol |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811546481.0A CN109971813A (en) | 2018-12-18 | 2018-12-18 | A kind of technique preparing bisnoralchol |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115029368A (en) * | 2022-06-24 | 2022-09-09 | 中国科学院上海高等研究院 | Gene engineering bacterium for producing dideoxy alcohol and application thereof |
-
2018
- 2018-12-18 CN CN201811546481.0A patent/CN109971813A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115029368A (en) * | 2022-06-24 | 2022-09-09 | 中国科学院上海高等研究院 | Gene engineering bacterium for producing dideoxy alcohol and application thereof |
| CN115029368B (en) * | 2022-06-24 | 2023-10-31 | 中国科学院上海高等研究院 | Genetically engineered bacterium for producing bisnoralcohol and application thereof |
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Application publication date: 20190705 |