CN105779556A - Method for preparing 16alpha, 17alpha-epoxy-11alpha-hydroxy-pregna-1,4-diene-3,20-dione through combined microbial fermentation - Google Patents
Method for preparing 16alpha, 17alpha-epoxy-11alpha-hydroxy-pregna-1,4-diene-3,20-dione through combined microbial fermentation Download PDFInfo
- Publication number
- CN105779556A CN105779556A CN201410779441.6A CN201410779441A CN105779556A CN 105779556 A CN105779556 A CN 105779556A CN 201410779441 A CN201410779441 A CN 201410779441A CN 105779556 A CN105779556 A CN 105779556A
- Authority
- CN
- China
- Prior art keywords
- epoxy
- hydroxy
- alpha
- diene
- diketone
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for preparing 16alpha, 17alpha-epoxy-11alpha-hydroxy-pregna-1,4-diene-3,20-dione by taking 16alpha,17alpha-epoxy progesterone as a starting material through one-step mixed fermentation of aspergillus ochraceus and arthrobacter simplex.
Description
Technical field
The present invention relates to the production method of steroidal drug, utilize microbial association transformation fermentation specifically
16 α, 17 α-epoxy progesterone prepares 16 α, 17 α-epoxy-11 Alpha-hydroxy-pregnant steroid-1,4-diene-3,20-
The method of diketone.
Background technology
Steroid drugs is a class important in medicine, has anti-inflammatory, anti-immunity, contraception, anticancer, interior point of regulation
The multiple effect such as secrete, be widely used in rheumatism, cardiovascular disease, Collagen illness, leukemic lymphoblastoid, human body device
The diseases such as official's transplanting, bacterial encephalitis, skin disease, endocrinopathy, geriatric disease, hormone-dependent neoplasm
Sick treatment.
During steroid hormone medicine produces, multiple groups of steroidal parent nucleus need to carry out through chemical method and microorganism
Modify, wherein C11'alpha '-hydroxylation and C1,2It is important Steroid Transformation reaction that dehydrogenation introduces double bond, for steroidal medicine
Synthesis and the structure of modification of thing are significant, and microorganism is mainly passed through in this two classes conversion reaction the most industrial
Substep converts.
The microorganism being presently used for the reaction of steroidal C11 'alpha '-hydroxylation mainly has bread mold, green muscardine fungus, reddish brown song
Mould, aspergillus flavus, mould, Nocard's bacillus etc..Bread mold, conspicuous aspergillus, green muscardine fungus are that studies in China is more in recent years
Steroidal C11 'alpha '-hydroxylation microorganism.But document report (Zhao Weiwei, Master's thesis " reddish brown song in 2012
The catalysis steroidal 11 'alpha '-hydroxylation repercussion study of mould fermentation method ", microbiology circulate a notice of 26 (6) P417 in 1999)
The Aspergillus ochraceus phase in post-conversion produces the brick-red pigment being difficult to separate with product, and it is unstable that green muscardine fungus there is also activity
Determine, convert nonspecific shortcoming, limit its industrial applications.In industrial production, domestic commonly use black root
Mould carry out C11 'alpha '-hydroxylation reaction, but existing technique conversion ratio is the highest.
It is presently used for steroidal C1,2Dehydrogenation introduces the microorganism of double bond reaction mainly to be had: Arthrobacter simplex, branch
Bacillus, bacillus, corynebacteria etc..
16 α, 17 α-epoxy progesterone (16 α, 17 α-Epoxyprogester-one, EP) full name is
The pregnant Gona-4-ene-3 of 16 α, 17 α-epoxy, 20-diketone, have another name called fertile formula oxide.Yellow with 16 α, 17 α-epoxy
Body ketone is that substrate utilization microorganism carries out C11'alpha '-hydroxylation is an important reaction in steroidal bioconversion,
The C arrived11'alpha '-hydroxylation product 16 α, 17 α-epoxy-11 Alpha-hydroxy-pregnant Gona-4-ene-3,20-diketone is steroid
Body synthesis important intermediate.16 α, 17 α-epoxy-11 Alpha-hydroxy-pregnant Gona-4-ene-3,20-diketone or its derive
Thing is through Microbial biomass C1,2The reaction such as dehydrogenation can synthesize dexamethasone, prednisolone, prednisone and fluocinolone acetonide etc.
Medicine.
In currently available technology, 11 hydroxylatings of steroidal Microbial biomass C and the dehydrogenation of C1,2 position mainly divide two
Step is carried out, and back needs to post-process after having reacted, and increases product post processing cost and fermentation energy
Consumption, and reduce product yield.Chinese patent CN201210316179.2 reports and utilizes Aspergillus ochraceus and letter
The method that single-unit bacillus microorganisms associating transformation fermentation 4-AD prepares 11 α-OH-ADD, but through test of many times
Finding that the method is difficult to repeat, the yield of target product and content are all not less than 50%.Associating transformation fermentation is in skill
There is great difficulty in art, mainly due to growth and the fermentation condition of different bacterium, such as: temperature, culture medium,
PH value is different, is difficult to make two kinds of bacterium each play maximum enzyme vigor under same fermentation condition.Therefore, find
The culture medium being suitable for different bacterium fermentation is extremely important.
There is no at present with 16 α, 17 α-epoxy progesterone is substrate utilization microbial association transformation fermentation one step system
Standby 16 α, the document report of 17 α-epoxy-11 Alpha-hydroxy-pregnant steroid-1,4-diene-3,20-diketone.
Summary of the invention
It is less that the technical problem to be solved in the present invention is to provide a kind of fermentation energy consumption, cost-effective and improve product and receive
Microbial association transformation fermentation 16 α, 17 α of rate-epoxy progesterone (I) prepare 16 α, 17 α-epoxy-11 α-
The method of hydroxyl-pregnant steroid-1,4-diene-3,20-diketone (II).
The reaction scheme of the present invention is as follows:
The present invention relates to a kind of is starting material with 16 α, 17 α-epoxy progesterone, utilizes microbial association fermentation system
Standby 16 α, 17 α-epoxy-11 Alpha-hydroxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3, the method for 20-diketone, it is characterized in that through reddish brown
Aspergillus Aspergillus ochraceus ATCC18500 and Arthrobacter simplex Arthrobacter simplex
AS1.94 mixing one-step fermentation prepares 16 α, 17 α-epoxy-11 Alpha-hydroxy-pregnant steroid-1,4-diene-3,20-two
Ketone.
Described one is starting material with 16 α, 17 α-epoxy progesterone, utilizes microbial association fermentation preparation
16 α, 17 α-epoxy-11 Alpha-hydroxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3, the method for 20-diketone, it is characterized in that 1L water
In, the component of fermentation medium and weight are glucose 20g-30g, sucrose 10g-20g, dried silkworm chrysalis meal 5g-10g,
Corn steep liquor 20g-30g, diammonium hydrogen phosphate 2g-5g, ammonium dihydrogen phosphate 2g-5g, bubble enemy 0.1g-0.4g.
Described one is starting material with 16 α, 17 α-epoxy progesterone, utilizes microbial association fermentation preparation
16 α, 17 α-epoxy-11 Alpha-hydroxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3, the method for 20-diketone, it is characterized in that 1L water
In, the component of fermentation medium and weight are glucose 20g, sucrose 20g, dried silkworm chrysalis meal 5g, corn steep liquor 20g,
Diammonium hydrogen phosphate 5g, ammonium dihydrogen phosphate 2g, bubble enemy 0.2g.
Described one is starting material with 16 α, 17 α-epoxy progesterone, utilizes microbial association fermentation preparation
16 α, 17 α-epoxy-11 Alpha-hydroxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3, the method for 20-diketone, it is characterized in that fermentation training
The pH supporting base is 5.7-6.5.
Described one is starting material with 16 α, 17 α-epoxy progesterone, utilizes microbial association fermentation preparation
16 α, 17 α-epoxy-11 Alpha-hydroxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3, the method for 20-diketone, it is characterized in that simply saving
The inoculum concentration of bacillus is 15%-20%, and the inoculum concentration of Aspergillus ochraceus is 25%-30%.
Described one is starting material with 16 α, 17 α-epoxy progesterone, utilizes microbial association fermentation preparation
16 α, 17 α-epoxy-11 Alpha-hydroxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3, the method for 20-diketone, it is characterized in that simply saving
The inoculum concentration of bacillus is 15%, and the inoculum concentration of Aspergillus ochraceus is 30%.
The Aspergillus ochraceus Aspergillus ochraceus ATCC18500 and arthrobacterium Arthrobacter of the present invention
Simplex AS1.94 is cultivated and seed culture by conventional inclined-plane, obtains fermentation and uses bacterium.
Aspergillus ochraceus Aspergillus ochraceus ATCC18500
Inclined-plane is cultivated
Slant medium: potato culture medium
Condition of culture: 28 DEG C
Incubation time: 5-7 days
Liquid seed culture medium
| Title | Consumption (g/L) |
| Glucose | 20 |
| Dried silkworm chrysalis meal | 0.5 |
| Ammonium sulfate | 1.1 |
| Corn steep liquor | 35 |
PH=6.5, spore suspension: with aseptic washing spore inclined-plane, making spore suspension, microscopy counts, spore
Sub-concentration 4～5 × 107Individual/Ml；Shake-flask seed is cultivated: inoculum concentration 10%, 180rpm, 28 DEG C, cultivates 24 little
Time；400L seeding tank: inoculum concentration 10%, 180rpm, air mass flow: 20m3/ hr, tank pressure: 0.05MPa,
Cultivating 20 hours, seed weight in wet base is 7%～10%.The computational methods of seed weight in wet base are: 4000rpm is centrifuged 10min,
Abandoning supernatant, collect thalline, weigh, thalline weight accounts for the percentage of culture medium gross weight.
Arthrobacter simplex Arthrobacter simplex AS1.94
Inclined-plane is cultivated
Slant medium: potato culture medium
Condition of culture: 31 DEG C
Incubation time: 5-7 days
Liquid seed culture medium
| Title | Consumption (g/L) |
| Glucose | 20 |
| Potassium dihydrogen phosphate | 1 |
| Corn steep liquor | 20 |
PH=7.0, spore suspension: with aseptic washing spore inclined-plane, making spore suspension, microscopy counts, spore
Sub-concentration 4～5 × 107Individual/Ml；Shake-flask seed is cultivated: inoculum concentration 10%, 180rpm, 33 DEG C, cultivates 24 little
Time；400L seeding tank: inoculum concentration 10%, 180rpm, air mass flow: 20m3/ hr, tank pressure: 0.05MPa,
Cultivating 22 hours, seed weight in wet base is 2%～3%.The computational methods of seed weight in wet base are: 4000rpm is centrifuged 10min,
Abandoning supernatant, collect thalline, weigh, thalline weight accounts for the percentage of culture medium gross weight.
Owing to the fermentation condition of fungi and bacterium is entirely different, Arthrobacter simplex is bacterium, grow and convert suitable
Preferably temperature is 30-34 DEG C, and suitable pH is 7.0-7.5；Aspergillus ochraceus is fungi, and the preference temperature that growth converts is
25-28 DEG C, suitable pH is 5.5-6.5.Therefore, combine the technological difficulties converted and key point is to optimize
To the fermentative medium formula being suitable for, in this fermentation medium, Aspergillus ochraceus can produce C11α hydroxylase, with
Time Arthrobacter simplex can generate dehydrogenase, and enzyme activity is no less than enzyme activity when individually converting, thus completes
Dehydrogenation and the bio combined conversion of upper hydroxyl.
Found by great many of experiments, the fermentation condition of the present invention, for Arthrobacter simplex Arthrobacter
simplex AS1.94 C1,2Position dehydrogenation one-step reaction or Aspergillus ochraceus Aspergillus ochraceus ATCC18500
C11Not optimal conditions for hydroxyl one-step reaction on position, but but it is applicable to Arthrobacter simplex Arthrobacter
Simplex AS1.94 and Aspergillus ochraceus Aspergillus ochraceusATCC18500 mixed fermentation, see comparison
Embodiment 1～8.Upper hydroxyl reaction and dehydrogenation reaction is carried out relative to being used alone above-mentioned two kind bacterium substep, this
Bright by two bacterium combined ferment 16 α, 17 α-epoxy progesterone obtain 11 Alpha-hydroxy-16 α, 17 α-epoxy-
Pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone, total recovery improves, decreases operating procedure, energy consumption and production cycle,
Reduce production cost and labour intensity, beneficially scientific management, meet present low energy consumption height output requirement.
Detailed description of the invention
Below will by embodiment, the invention will be further described, these descriptions are not to present invention
It is further limited.Person skilled should be understood that the equivalent that the technical characteristic to the present invention is made,
Or be correspondingly improved, within still falling within protection scope of the present invention.
The selection of embodiment 1 fermentation medium
Examine the fermentation medium impact on target product conversion ratio in the following embodiments.Inclined-plane, seed
Cultural method is as discussed in the summary of the invention section.
Embodiment 1-1
In 1L water, fermentation substrate 16 α, 17 α-epoxy progesterone 30g, the component glucose of fermentation medium
20g, sucrose 20g, dried silkworm chrysalis meal 5g, corn steep liquor 20g, diammonium hydrogen phosphate 5g, ammonium dihydrogen phosphate 2g, bubble enemy 0.2g,
PH is 6.0.Arthrobacter simplex two grades cultivation, after first order seed is cultivated 24 hours, is linked into two by inoculum concentration 10%
In level seed culture medium, after two grades are cultivated 22 hours, it is linked in above-mentioned fermentation medium according to inoculum concentration 15%；
After Aspergillus ochraceus first order seed is cultivated 24 hours, access in above-mentioned fermentation medium according to 30% inoculum concentration.Use 5
Ton fermentation tank converts, liquid amount 80%, tank pressure 0.05MPa, air mass flow 40m3/ hr, temperature 28 DEG C,
Stirring 180rpm, converts 72 hours, and sampling TLC analyzes, and substrate converts completely, and sample is extracted with ethyl acetate,
HPLC is sent by upper strata, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content
95.6%, 16 α, 17 α-epoxy progesterone content 2.5%, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-4-
Alkene-3,20-diketone content 0.5%, 16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content 0.6%.
Convert complete, 85 DEG C of zymotic fluid inactivations, it is cooled to room temperature, suction filtration.Filter cake ethyl acetate extracts, dense
Contracting, obtains white crystal, crude yield 89.5%, and 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-
Alkene-3,20-diketone content 97.7%.
Embodiment 1-2
In 1L water, fermentation substrate 16 α, 17 α-epoxy progesterone 30g, the component glucose of fermentation medium
30g, sucrose 20g, dried silkworm chrysalis meal 5g, corn steep liquor 20g, diammonium hydrogen phosphate 5g, ammonium dihydrogen phosphate 2g, bubble enemy 0.2g,
PH is 6.0.Arthrobacter simplex two grades cultivation, after first order seed is cultivated 24 hours, is linked into two by inoculum concentration 10%
In level seed culture medium, after two grades are cultivated 22 hours, it is linked in above-mentioned fermentation medium according to inoculum concentration 15%；
After Aspergillus ochraceus first order seed is cultivated 24 hours, access in above-mentioned fermentation medium according to 30% inoculum concentration.Use 5
Ton fermentation tank converts, liquid amount 80%, tank pressure 0.05MPa, air mass flow 40m3/ hr, temperature 28 DEG C,
Stirring 180rpm, converts 72 hours, and sampling TLC analyzes, and substrate converts completely, and sample is extracted with ethyl acetate,
HPLC is sent by upper strata, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content
93.1%, 16 α, 17 α-epoxy progesterone content 2.6%, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-4-
Alkene-3,20-diketone content 0.9%, 16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content 0.7%.
Convert complete, 85 DEG C of zymotic fluid inactivations, it is cooled to room temperature, suction filtration.Filter cake ethyl acetate extracts, dense
Contracting, obtains white crystal, crude yield 89.2%, and 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-
Alkene-3,20-diketone content 95.7%.
Embodiment 1-3
In 1L water, fermentation substrate 16 α, 17 α-epoxy progesterone 30g, the component glucose of fermentation medium
20g, sucrose 10g, dried silkworm chrysalis meal 5g, corn steep liquor 20g, diammonium hydrogen phosphate 5g, ammonium dihydrogen phosphate 2g, bubble enemy 0.2g,
PH is 6.0.Arthrobacter simplex two grades cultivation, after first order seed is cultivated 24 hours, is linked into two by inoculum concentration 10%
In level seed culture medium, after two grades are cultivated 22 hours, it is linked in above-mentioned fermentation medium according to inoculum concentration 15%；
After Aspergillus ochraceus first order seed is cultivated 24 hours, access in above-mentioned fermentation medium according to 30% inoculum concentration.Use 5
Ton fermentation tank converts, liquid amount 80%, tank pressure 0.05MPa, air mass flow 40m3/ hr, temperature 28 DEG C,
Stirring 180rpm, converts 72 hours, and sampling TLC analyzes, and substrate converts completely, and sample is extracted with ethyl acetate,
HPLC is sent by upper strata, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content
92.6%, 16 α, 17 α-epoxy progesterone content 2.7%, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-4-
Alkene-3,20-diketone content 0.8%, 16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content 0.9%.
Convert complete, 85 DEG C of zymotic fluid inactivations, it is cooled to room temperature, suction filtration.Filter cake ethyl acetate extracts, dense
Contracting, obtains white crystal, crude yield 89.9%, and 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-
Alkene-3,20-diketone content 94.3%.
Embodiment 1-4
In 1L water, fermentation substrate 16 α, 17 α-epoxy progesterone 30g, the component glucose of fermentation medium
20g, sucrose 20g, dried silkworm chrysalis meal 10g, corn steep liquor 20g, diammonium hydrogen phosphate 5g, ammonium dihydrogen phosphate 2g, bubble enemy 0.2g,
PH is 6.0.Arthrobacter simplex two grades cultivation, after first order seed is cultivated 24 hours, is linked into two by inoculum concentration 10%
In level seed culture medium, after two grades are cultivated 22 hours, it is linked in above-mentioned fermentation medium according to inoculum concentration 15%；
After Aspergillus ochraceus first order seed is cultivated 24 hours, access in above-mentioned fermentation medium according to 30% inoculum concentration.Use 5
Ton fermentation tank converts, liquid amount 80%, tank pressure 0.05MPa, air mass flow 40m3/ hr, temperature 28 DEG C,
Stirring 180rpm, converts 72 hours, and sampling TLC analyzes, and substrate converts completely, and sample is extracted with ethyl acetate,
HPLC is sent by upper strata, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content
92.1%, 16 α, 17 α-epoxy progesterone content 3.0%, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-4-
Alkene-3,20-diketone content 0.5%, 16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content 0.7%.
Convert complete, 85 DEG C of zymotic fluid inactivations, it is cooled to room temperature, suction filtration.Filter cake ethyl acetate extracts, dense
Contracting, obtains white crystal, crude yield 89.1%, and 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-
Alkene-3,20-diketone content 94.8%.
Embodiment 1-5
In 1L water, fermentation substrate 16 α, 17 α-epoxy progesterone 30g, the component glucose of fermentation medium
20g, sucrose 20g, dried silkworm chrysalis meal 5g, corn steep liquor 30g, diammonium hydrogen phosphate 5g, ammonium dihydrogen phosphate 2g, bubble enemy 0.2g,
PH is 6.0.Arthrobacter simplex two grades cultivation, after first order seed is cultivated 24 hours, is linked into two by inoculum concentration 10%
In level seed culture medium, after two grades are cultivated 22 hours, it is linked in above-mentioned fermentation medium according to inoculum concentration 15%；
After Aspergillus ochraceus first order seed is cultivated 24 hours, access in above-mentioned fermentation medium according to 30% inoculum concentration.Use 5
Ton fermentation tank converts, liquid amount 80%, tank pressure 0.05MPa, air mass flow 40m3/ hr, temperature 28 DEG C,
Stirring 180rpm, converts 72 hours, and sampling TLC analyzes, and substrate converts completely, and sample is extracted with ethyl acetate,
HPLC is sent by upper strata, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content
92.8%, 16 α, 17 α-epoxy progesterone content 2.9%, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-4-
Alkene-3,20-diketone content 0.6%, 16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content 0.8%.
Convert complete, 85 DEG C of zymotic fluid inactivations, it is cooled to room temperature, suction filtration.Filter cake ethyl acetate extracts, dense
Contracting, obtains white crystal, crude yield 90.5%, and 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-
Alkene-3,20-diketone content 93.9%.
Embodiment 1-6
In 1L water, fermentation substrate 16 α, 17 α-epoxy progesterone 30g, the component glucose of fermentation medium
20g, sucrose 20g, dried silkworm chrysalis meal 5g, corn steep liquor 20g, diammonium hydrogen phosphate 2g, ammonium dihydrogen phosphate 2g, bubble enemy 0.2g,
PH is 6.0.Arthrobacter simplex two grades cultivation, after first order seed is cultivated 24 hours, is linked into two by inoculum concentration 10%
In level seed culture medium, after two grades are cultivated 22 hours, it is linked in above-mentioned fermentation medium according to inoculum concentration 15%；
After Aspergillus ochraceus first order seed is cultivated 24 hours, access in above-mentioned fermentation medium according to 30% inoculum concentration.Use 5
Ton fermentation tank converts, liquid amount 80%, tank pressure 0.05MPa, air mass flow 40m3/ hr, temperature 28 DEG C,
Stirring 180rpm, converts 72 hours, and sampling TLC analyzes, and substrate converts completely, and sample is extracted with ethyl acetate,
HPLC is sent by upper strata, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content
93.2%, 16 α, 17 α-epoxy progesterone content 3.0%, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-4-
Alkene-3,20-diketone content 0.4%, 16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content 0.7%.
Convert complete, 85 DEG C of zymotic fluid inactivations, it is cooled to room temperature, suction filtration.Filter cake ethyl acetate extracts, dense
Contracting, obtains white crystal, crude yield 89.4%, and 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-
Alkene-3,20-diketone content 95.4%.
Embodiment 1-7
In 1L water, fermentation substrate 16 α, 17 α-epoxy progesterone 30g, the component glucose of fermentation medium
20g, sucrose 20g, dried silkworm chrysalis meal 5g, corn steep liquor 20g, diammonium hydrogen phosphate 5g, ammonium dihydrogen phosphate 5g, bubble enemy 0.2g,
PH is 6.0.Arthrobacter simplex two grades cultivation, after first order seed is cultivated 24 hours, is linked into two by inoculum concentration 10%
In level seed culture medium, after two grades are cultivated 22 hours, it is linked in above-mentioned fermentation medium according to inoculum concentration 15%；
After Aspergillus ochraceus first order seed is cultivated 24 hours, access in above-mentioned fermentation medium according to 30% inoculum concentration.Use 5
Ton fermentation tank converts, liquid amount 80%, tank pressure 0.05MPa, air mass flow 40m3/ hr, temperature 28 DEG C,
Stirring 180rpm, converts 72 hours, and sampling TLC analyzes, and substrate converts completely, and sample is extracted with ethyl acetate,
HPLC is sent by upper strata, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content
92.5%, 16 α, 17 α-epoxy progesterone content 2.8%, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-4-
Alkene-3,20-diketone content 0.7%, 16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content 0.8%.
Convert complete, 85 DEG C of zymotic fluid inactivations, it is cooled to room temperature, suction filtration.Filter cake ethyl acetate extracts, dense
Contracting, obtains white crystal, crude yield 90.1%, and 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-
Alkene-3,20-diketone content 94.4%.
Embodiment 1-8
In 1L water, fermentation substrate 16 α, 17 α-epoxy progesterone 30g, the component glucose of fermentation medium
20g, sucrose 20g, dried silkworm chrysalis meal 5g, corn steep liquor 20g, diammonium hydrogen phosphate 5g, ammonium dihydrogen phosphate 2g, bubble enemy 0.4g,
PH is 6.0.Arthrobacter simplex two grades cultivation, after first order seed is cultivated 24 hours, is linked into two by inoculum concentration 10%
In level seed culture medium, after two grades are cultivated 22 hours, it is linked in above-mentioned fermentation medium according to inoculum concentration 15%；
After Aspergillus ochraceus first order seed is cultivated 24 hours, access in above-mentioned fermentation medium according to 30% inoculum concentration.Use 5
Ton fermentation tank converts, liquid amount 80%, tank pressure 0.05MPa, air mass flow 40m3/ hr, temperature 28 DEG C,
Stirring 180rpm, converts 72 hours, and sampling TLC analyzes, and substrate converts completely, and sample is extracted with ethyl acetate,
HPLC is sent by upper strata, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content
92.9%, 16 α, 17 α-epoxy progesterone content 2.8%, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-4-
Alkene-3,20-diketone content 0.4%, 16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content 0.9%.
Convert complete, 85 DEG C of zymotic fluid inactivations, it is cooled to room temperature, suction filtration.Filter cake ethyl acetate extracts, dense
Contracting, obtains white crystal, crude yield 90.5%, and 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-
Alkene-3,20-diketone content 94.5%.
Embodiment 1-9
In 1L water, fermentation substrate 16 α, 17 α-epoxy progesterone 30g, the component glucose of fermentation medium
25g, sucrose 15g, dried silkworm chrysalis meal 8g, corn steep liquor 25g, diammonium hydrogen phosphate 3.5g, ammonium dihydrogen phosphate 3.5g, bubble
Enemy 0.1g, pH are 6.0.Arthrobacter simplex two grades cultivation, after first order seed is cultivated 24 hours, by inoculum concentration 10%
It is linked in secondary seed medium, after two grades are cultivated 22 hours, is linked into above-mentioned fermentation according to inoculum concentration 15%
In culture medium；After Aspergillus ochraceus first order seed is cultivated 24 hours, access above-mentioned fermentation medium according to 30% inoculum concentration
In.5 tons of fermentation tanks are used to convert, liquid amount 80%, tank pressure 0.05MPa, air mass flow 40m3/ hr, temperature
Spending 28 DEG C, stir 180rpm, convert 72 hours, sampling TLC analyzes, and substrate converts completely, sample acetic acid
Ethyl ester extracts, and send HPLC by upper strata, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-
Diketone content 91.9%, 16 α, 17 α-epoxy progesterone content 2.6%, 11 Alpha-hydroxy-16 α, 17 α-ring
Oxygen-DELTA4-pregn-3,20-dione content 0.7%, 16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-bis-
Ketone content 0.8%.
Convert complete, 85 DEG C of zymotic fluid inactivations, it is cooled to room temperature, suction filtration.Filter cake ethyl acetate extracts, dense
Contracting, obtains white crystal, crude yield 89.2%, and 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-
Alkene-3,20-diketone content 92.7%.
The selection of embodiment 2 inoculum concentration
Inclined-plane, seed culture method are as discussed in the summary of the invention section.
Embodiment 2-1
In 1L water, fermentation substrate 16 α, 17 α-epoxy progesterone 30g, the component glucose of fermentation medium
20g, sucrose 20g, dried silkworm chrysalis meal 5g, corn steep liquor 20g, diammonium hydrogen phosphate 5g, ammonium dihydrogen phosphate 2g, bubble enemy 0.2g,
PH is 6.0.Arthrobacter simplex two grades cultivation, after first order seed is cultivated 24 hours, is linked into two by inoculum concentration 10%
In level seed culture medium, after two grades are cultivated 22 hours, it is linked in above-mentioned fermentation medium according to inoculum concentration 20%；
After Aspergillus ochraceus first order seed is cultivated 24 hours, access in above-mentioned fermentation medium according to 30% inoculum concentration.Use 5
Ton fermentation tank converts, liquid amount 80%, tank pressure 0.05MPa, air mass flow 40m3/ hr, temperature 28 DEG C,
Stirring 180rpm, converts 72 hours, and sampling TLC analyzes, and substrate converts completely, and sample is extracted with ethyl acetate,
HPLC is sent by upper strata, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content
93.9%, 16 α, 17 α-epoxy progesterone content 2.8%, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-4-
Alkene-3,20-diketone content 0.9%, 16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content 0.8%.
Convert complete, 85 DEG C of zymotic fluid inactivations, it is cooled to room temperature, suction filtration.Filter cake ethyl acetate extracts, dense
Contracting, obtains white crystal, crude yield 88.5%, and 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-
Alkene-3,20-diketone content 94.6%.
Embodiment 2-2
In 1L water, fermentation substrate 16 α, 17 α-epoxy progesterone 30g, the component glucose of fermentation medium
20g, sucrose 20g, dried silkworm chrysalis meal 5g, corn steep liquor 20g, diammonium hydrogen phosphate 5g, ammonium dihydrogen phosphate 2g, bubble enemy 0.2g,
PH is 6.0.Arthrobacter simplex two grades cultivation, after first order seed is cultivated 24 hours, is linked into two by inoculum concentration 10%
In level seed culture medium, after two grades are cultivated 22 hours, it is linked in above-mentioned fermentation medium according to inoculum concentration 15%；
After Aspergillus ochraceus first order seed is cultivated 24 hours, access in above-mentioned fermentation medium according to 25% inoculum concentration.Use 5
Ton fermentation tank converts, liquid amount 80%, tank pressure 0.05MPa, air mass flow 40m3/ hr, temperature 28 DEG C,
Stirring 180rpm, converts 72 hours, and sampling TLC analyzes, and substrate converts completely, and sample is extracted with ethyl acetate,
HPLC is sent by upper strata, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content
94.1%, 16 α, 17 α-epoxy progesterone content 2.9%, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-4-
Alkene-3,20-diketone content 0.6%, 16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content 0.9%.
Convert complete, 85 DEG C of zymotic fluid inactivations, it is cooled to room temperature, suction filtration.Filter cake ethyl acetate extracts, dense
Contracting, obtains white crystal, crude yield 88.6%, and 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-
Alkene-3,20-diketone content 95.8%.
Embodiment 2-3
In 1L water, fermentation substrate 16 α, 17 α-epoxy progesterone 30g, the component glucose of fermentation medium
20g, sucrose 20g, dried silkworm chrysalis meal 5g, corn steep liquor 20g, diammonium hydrogen phosphate 5g, ammonium dihydrogen phosphate 2g, bubble enemy 0.2g,
PH is 6.0.Arthrobacter simplex two grades cultivation, after first order seed is cultivated 24 hours, is linked into two by inoculum concentration 10%
In level seed culture medium, after two grades are cultivated 22 hours, it is linked in above-mentioned fermentation medium according to inoculum concentration 15%；
After Aspergillus ochraceus first order seed is cultivated 24 hours, access in above-mentioned fermentation medium according to 30% inoculum concentration.Use 5
Ton fermentation tank converts, liquid amount 80%, tank pressure 0.05MPa, air mass flow 40m3/ hr, temperature 28 DEG C,
Stirring 180rpm, converts 72 hours, and sampling TLC analyzes, and substrate converts completely, and sample is extracted with ethyl acetate,
HPLC is sent by upper strata, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content
95.2%, 16 α, 17 α-epoxy progesterone content 2.5%, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-4-
Alkene-3,20-diketone content 0.5%, 16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content 0.6%.
Convert complete, 85 DEG C of zymotic fluid inactivations, it is cooled to room temperature, suction filtration.Filter cake ethyl acetate extracts, dense
Contracting, obtains white crystal, crude yield 89.7%, and 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-
Alkene-3,20-diketone content 96.1%.
Embodiment 2-4
In 1L water, fermentation substrate 16 α, 17 α-epoxy progesterone 30g, the component glucose of fermentation medium
20g, sucrose 20g, dried silkworm chrysalis meal 5g, corn steep liquor 20g, diammonium hydrogen phosphate 5g, ammonium dihydrogen phosphate 2g, bubble enemy 0.2g,
PH is 6.0.Arthrobacter simplex two grades cultivation, after first order seed is cultivated 24 hours, is linked into two by inoculum concentration 10%
In level seed culture medium, after two grades are cultivated 22 hours, it is linked in above-mentioned fermentation medium according to inoculum concentration 20%；
After Aspergillus ochraceus first order seed is cultivated 24 hours, access in above-mentioned fermentation medium according to 25% inoculum concentration.Use 5
Ton fermentation tank converts, liquid amount 80%, tank pressure 0.05MPa, air mass flow 40m3/ hr, temperature 28 DEG C,
Stirring 180rpm, converts 72 hours, and sampling TLC analyzes, and substrate converts completely, and sample is extracted with ethyl acetate,
HPLC is sent by upper strata, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content
93.6%, 16 α, 17 α-epoxy progesterone content 2.7%, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-4-
Alkene-3,20-diketone content 0.7%, 16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content 0.8%.
Convert complete, 85 DEG C of zymotic fluid inactivations, it is cooled to room temperature, suction filtration.Filter cake ethyl acetate extracts, dense
Contracting, obtains white crystal, crude yield 88.8%, and 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-
Alkene-3,20-diketone content 94.8%.
The selection of embodiment 3 fermentation medium pH
Inclined-plane, seed culture method are as discussed in the summary of the invention section.
Embodiment 3-1
In 1L water, fermentation substrate 16 α, 17 α-epoxy progesterone 30g, the component glucose of fermentation medium
20g, sucrose 20g, dried silkworm chrysalis meal 5g, corn steep liquor 20g, diammonium hydrogen phosphate 5g, ammonium dihydrogen phosphate 2g, bubble enemy 0.2g,
PH is 5.7.Arthrobacter simplex two grades cultivation, after first order seed is cultivated 24 hours, is linked into two by inoculum concentration 10%
In level seed culture medium, after two grades are cultivated 22 hours, it is linked in above-mentioned fermentation medium according to inoculum concentration 15%；
After Aspergillus ochraceus first order seed is cultivated 24 hours, access in above-mentioned fermentation medium according to 30% inoculum concentration.Use 5
Ton fermentation tank converts, liquid amount 80%, tank pressure 0.05MPa, air mass flow 40m3/ hr, temperature 28 DEG C,
Stirring 180rpm, converts 72 hours, and sampling TLC analyzes, and substrate converts completely, and sample is extracted with ethyl acetate,
HPLC is sent by upper strata, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content
95.3%, 16 α, 17 α-epoxy progesterone content 2.6%, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-4-
Alkene-3,20-diketone content 0.5%, 16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content 0.7%.
Convert complete, 85 DEG C of zymotic fluid inactivations, it is cooled to room temperature, suction filtration.Filter cake ethyl acetate extracts, dense
Contracting, obtains white crystal, crude yield 89.5%, and 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-
Alkene-3,20-diketone content 96.3%.
Embodiment 3-2
In 1L water, fermentation substrate 16 α, 17 α-epoxy progesterone 30g, the component glucose of fermentation medium
20g, sucrose 20g, dried silkworm chrysalis meal 5g, corn steep liquor 20g, diammonium hydrogen phosphate 5g, ammonium dihydrogen phosphate 2g, bubble enemy 0.2g,
PH is 6.5.Arthrobacter simplex two grades cultivation, after first order seed is cultivated 24 hours, is linked into two by inoculum concentration 15%
In level seed culture medium, after two grades are cultivated 22 hours, it is linked in above-mentioned fermentation medium according to inoculum concentration 20%；
After Aspergillus ochraceus first order seed is cultivated 24 hours, access in above-mentioned fermentation medium according to 30% inoculum concentration.Use 5
Ton fermentation tank converts, liquid amount 80%, tank pressure 0.05MPa, air mass flow 40m3/ hr, temperature 28 DEG C,
Stirring 180rpm, converts 72 hours, and sampling TLC analyzes, and substrate converts completely, and sample is extracted with ethyl acetate,
HPLC is sent by upper strata, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content
95.2%, 16 α, 17 α-epoxy progesterone content 2.7%, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-4-
Alkene-3,20-diketone content 0.4%, 16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content 0.6%.
Convert complete, 85 DEG C of zymotic fluid inactivations, it is cooled to room temperature, suction filtration.Filter cake ethyl acetate extracts, dense
Contracting, obtains white crystal, crude yield 90.2%, and 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-
Alkene-3,20-diketone content 96.4%.
Embodiment 3-3
In 1L water, fermentation substrate 16 α, 17 α-epoxy progesterone 30g, the component glucose of fermentation medium
20g, sucrose 20g, dried silkworm chrysalis meal 5g, corn steep liquor 20g, diammonium hydrogen phosphate 5g, ammonium dihydrogen phosphate 2g, bubble enemy 0.2g,
PH is 5.0.Arthrobacter simplex two grades cultivation, after first order seed is cultivated 24 hours, is linked into two by inoculum concentration 15%
In level seed culture medium, after two grades are cultivated 22 hours, it is linked in above-mentioned fermentation medium according to inoculum concentration 20%；
After Aspergillus ochraceus first order seed is cultivated 24 hours, access in above-mentioned fermentation medium according to 30% inoculum concentration.Use 5
Ton fermentation tank converts, liquid amount 80%, tank pressure 0.05MPa, air mass flow 40m3/ hr, temperature 28 DEG C,
Stirring 180rpm, converts 72 hours, and sampling TLC analyzes, and substrate converts completely, and sample is extracted with ethyl acetate,
HPLC is sent by upper strata, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content
78.4%, 16 α, 17 α-epoxy progesterone content 9.5%, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-4-
Alkene-3,20-diketone content 4.5%, 16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content 5.6%.
Convert complete, 85 DEG C of zymotic fluid inactivations, it is cooled to room temperature, suction filtration.Filter cake ethyl acetate extracts, dense
Contracting, obtains white crystal, crude yield 89.7%, and 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-
Alkene-3,20-diketone content 80.7%.
Embodiment 3-4
In 1L water, fermentation substrate 16 α, 17 α-epoxy progesterone 30g, the component glucose of fermentation medium
20g, sucrose 20g, dried silkworm chrysalis meal 5g, corn steep liquor 20g, diammonium hydrogen phosphate 5g, ammonium dihydrogen phosphate 2g, bubble enemy 0.2g,
PH is 7.0.Arthrobacter simplex two grades cultivation, after first order seed is cultivated 24 hours, is linked into two by inoculum concentration 15%
In level seed culture medium, after two grades are cultivated 22 hours, it is linked in above-mentioned fermentation medium according to inoculum concentration 20%；
After Aspergillus ochraceus first order seed is cultivated 24 hours, access in above-mentioned fermentation medium according to 30% inoculum concentration.Use 5
Ton fermentation tank converts, liquid amount 80%, tank pressure 0.05MPa, air mass flow 40m3/ hr, temperature 28 DEG C,
Stirring 180rpm, converts 72 hours, and sampling TLC analyzes, and substrate converts completely, and sample is extracted with ethyl acetate,
HPLC is sent by upper strata, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content
75.6%, 16 α, 17 α-epoxy progesterone content 10.6%, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid
-4-alkene-3,20-diketone content 6.5%, 16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content
6.6%.
Convert complete, 85 DEG C of zymotic fluid inactivations, it is cooled to room temperature, suction filtration.Filter cake ethyl acetate extracts, dense
Contracting, obtains white crystal, crude yield 90.5%, and 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-
Alkene-3,20-diketone content 79.5%.
Comparative example 1
Under microbial association fermentation condition of the present invention, utilize Aspergillus ochraceus Aspergillus ochraceus
ATCC18500 is that substrate carries out C with 16 α, 17 α-epoxy progesterone11α hydroxylating prepares 11 Alpha-hydroxies
-16 α, 17 α-epoxy-DELTA4-pregn-3,20-dione
Experiment: inclined-plane, seed culture method as discussed in the summary of the invention section, fermentation medium such as embodiment 1-1
Described, fermented and cultured substrate is 16 α, 17 α-epoxy progesterone, content 30g/L, inoculates Aspergillus ochraceus
Aspergillus ochraceus ATCC18500 carries out C11α hydroxylating, 28 DEG C, 180rpm, convert 72 little
Time, sample and send HPLC to detect, 11 Alpha-hydroxy-16 α, 17 α-epoxy-DELTA4-pregn-3,20-dione content
75%.
Convert complete, 85 DEG C of zymotic fluid inactivations, it is cooled to room temperature, suction filtration.Filter cake ethyl acetate extracts, dense
Contracting, obtains 11 Alpha-hydroxy-16 α, 17 α-epoxy-DELTA4-pregn-3,20-dione crude product, yield 86.5%.
Comparative example 2
Under Aspergillus ochraceus Aspergillus ochraceus ATCC18500 mono-bacterium optimization of fermentation conditions, utilize reddish brown
Aspergillus Aspergillus ochraceus ATCC18500 is that substrate is carried out with 16 α, 17 α-epoxy progesterone
C11α hydroxylating prepares 11 Alpha-hydroxy-16 α, 17 α-epoxy-DELTA4-pregn-3,20-dione
Experiment: as discussed in the summary of the invention section, fermentation medium is as follows for inclined-plane, seed culture method: in 1L water,
Glucose 30g, dried silkworm chrysalis meal 0.5g, ammonium sulfate 1.1g, corn steep liquor 35g, pH6.5, fermented and cultured substrate is 16 α,
17 α-epoxy progesterone, content 30g/L, inoculates Aspergillus ochraceus Aspergillus ochraceus ATCC18500
Carry out C11α hydroxylating, 28 DEG C, 180rpm, convert 72 hours, sampling send HPLC to detect, 11 Alpha-hydroxy-16 α,
17 α-epoxy-pregnant Gona-4-ene-3,20-diketone content 83.2%.
Convert complete, 85 DEG C of zymotic fluid inactivations, it is cooled to room temperature, suction filtration.Filter cake ethyl acetate extracts, dense
Contracting, obtains 11 Alpha-hydroxy-16 α, 17 α-epoxy-DELTA4-pregn-3,20-dione crude product, yield 88%.
Comparative example 3
Under microbial association fermentation condition of the present invention, utilize Aspergillus ochraceus Aspergillus ochraceus
ATCC18500 is with 16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3, and 20-diketone is that substrate carries out C11α hydroxylating
Prepare 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone.
Inclined-plane, seed culture method as discussed in the summary of the invention section, fermentation medium as described in embodiment 1-1,
Fermented and cultured substrate is 16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone, and content 30g/L connects
Plant Aspergillus ochraceus Aspergillus ochraceus ATCC18500 and carry out C11α hydroxylating, 28 DEG C, 180rpm,
Converting 72 hours, sampling send HPLC to detect, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene
-3,20-diketone content 48.6%.
Convert complete, 85 DEG C of zymotic fluid inactivations, it is cooled to room temperature, suction filtration.Filter cake ethyl acetate extracts, dense
Contracting, obtains 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone crude product, yield
85.9%.
Comparative example 4
Under Aspergillus ochraceus Aspergillus ochraceus ATCC18500 mono-bacterium optimization of fermentation conditions, utilize reddish brown
Aspergillus Aspergillus ochraceus ATCC18500 is with 16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene
-3,20-diketone is that substrate carries out C11α hydroxylating prepare 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-
Diene-3,20-diketone
As discussed in the summary of the invention section, fermentation medium is as follows for inclined-plane, seed culture method: in 1L water, grape
Sugar 35g, dried silkworm chrysalis meal 0.2g, ammonium sulfate 1.0g, corn steep liquor 40g, pH6.5, fermented and cultured substrate is 16 α,
17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone, content 30g/L, inoculates Aspergillus ochraceus Aspergillus
Ochraceus ATCC18500 carries out C11α hydroxylating, 28 DEG C, 180rpm, convert 72 hours, other conditions
Constant, sampling send HPLC to detect, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-
Diketone content 57.4%.
Convert complete, 85 DEG C of zymotic fluid inactivations, it is cooled to room temperature, suction filtration.Filter cake ethyl acetate extracts, dense
Contracting, obtains 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone crude product, yield
87.2%.
Comparative example 5
Under microbial association fermentation condition of the present invention, utilize Arthrobacter simplex Arthrobacter simplex
AS1.94 is that substrate carries out C with 16 α, 17 α-epoxy progesterone1,216 α, 17 α-epoxy-pregnant are prepared in position dehydrogenation
Steroid-Isosorbide-5-Nitrae-diene-3,20-diketone
Inclined-plane, seed culture method as discussed in the summary of the invention section, fermentation medium as described in embodiment 1-1,
Fermented and cultured substrate is 16 α, 17 α-epoxy progesterone, content 30g/L, inoculates Arthrobacter simplex
Arthrobacter simplex AS1.94 carries out dehydrogenation conversion, 33 DEG C, and 180rpm converts 72 hours, takes
Sample send HPLC to detect, 16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content 72.1%.
Convert complete, 85 DEG C of zymotic fluid inactivations, it is cooled to room temperature, suction filtration.Filter cake ethyl acetate extracts, dense
Contracting, obtains 16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone crude product, yield 85.2%.
Comparative example 6
Under Arthrobacter simplex Arthrobacter simplex AS1.94 mono-bacterium optimization of fermentation conditions, utilize letter
Single-unit bacillus Arthrobacter simplex AS1.94 is that substrate carries out C with 16 α, 17 α-epoxy progesterone1,2
16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone are prepared in position dehydrogenation.
As discussed in the summary of the invention section, fermentation medium is as follows for inclined-plane, seed culture method: in 1L water, grape
Sugar 20g, corn steep liquor 20g, dipotassium hydrogen phosphate 1g, pH7.0, substrate is 16 α, and 17 α-epoxy progesterone contains
Amount 30g/L, inoculation Arthrobacter simplex Arthrobacter simplex AS1.94 carry out dehydrogenation conversion,
33 DEG C, 180rpm, convert 72 hours, sampling send HPLC to detect, 16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-
Diene-3,20-diketone content 82.3%.
Convert complete, 85 DEG C of zymotic fluid inactivations, it is cooled to room temperature, suction filtration.Filter cake ethyl acetate extracts, dense
Contracting, obtains 16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone crude product, yield 87.8%.
Comparative example 7
Under microbial association fermentation condition of the present invention, utilize Arthrobacter simplex Arthrobacter simplex
AS1.94 is that substrate carries out C with 11 Alpha-hydroxy-16 α, 17 α-epoxy-DELTA4-pregn-3,20-dione1,2Position is de-
Hydrogen prepares 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone.
Inclined-plane, seed culture method as discussed in the summary of the invention section, fermentation medium as described in embodiment 1-1,
Substrate is 11 Alpha-hydroxy-16 α, 17 α-epoxy-DELTA4-pregn-3,20-dione, content 30g/L, inoculation letter
Single-unit bacillus Arthrobacter simplex AS1.94 carries out dehydrogenation conversion, 33 DEG C, 180rpm, converts 72
Hour, sampling send HPLC to detect, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-
Diketone content 80.5%.
Convert complete, 85 DEG C of zymotic fluid inactivations, it is cooled to room temperature, suction filtration.Filter cake ethyl acetate extracts, dense
Contracting, obtains 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone crude product, yield
86.1%.
Comparative example 8
Under Arthrobacter simplex Arthrobacter simplex AS1.94 mono-bacterium optimization of fermentation conditions, utilize letter
Single-unit bacillus Arthrobacter simplex AS1.94 is with 16 α, 17 α-epoxy-11 Alpha-hydroxy-pregnant steroid-4-
Alkene-3,20-diketone is that substrate carries out C1,211 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-1 is prepared in position dehydrogenation,
4-diene-3,20-diketone.
As discussed in the summary of the invention section, fermentation medium is as follows for inclined-plane, seed culture method: in 1L water, 1L
In water, glucose 25g, corn steep liquor 22g, dipotassium hydrogen phosphate 1.5g, pH7.5, fermented and cultured substrate is
16 α, 17 α-epoxy-11 Alpha-hydroxies-DELTA4-pregn-3,20-dione, content 30g/L, inoculates Arthrobacter simplex
Arthrobacter simplex AS1.94 carries out dehydrogenation conversion, 33 DEG C, and 180rpm converts 72 hours, takes
Sample send HPLC to detect, 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone content
84.4%.
Convert complete, 85 DEG C of zymotic fluid inactivations, it is cooled to room temperature, suction filtration.Filter cake ethyl acetate extracts, dense
Contracting, obtains 11 Alpha-hydroxy-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3,20-diketone crude product, yield
88.2%.
It is substrate with 16 α, 17 α-epoxy progesterone, converts preparation 11 Alpha-hydroxies by microorganism two step
-16 α, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3, the approach of 20-diketone have two kinds: (1) 16 α, 17 α-
Epoxy progesterone utilizes hydroxyl on Aspergillus ochraceus Aspergillus ochraceus ATCC18500 microorganism, then
16 α that will obtain, 17 α-epoxy-11 Alpha-hydroxy-pregnant Gona-4-ene-3,20-diketone utilizes Arthrobacter simplex
Arthrobacter simplex AS1.94 C1,2Position dehydrogenation obtains target product, such as comparative examples 2 and right
Combination according to embodiment 8.(2) 16 α, 17 α-epoxy progesterone utilizes Arthrobacter simplex Arthrobacter
simplex AS1.94 C1,2Position dehydrogenation, 16 α that then will obtain, 17 α-epoxy-pregnant steroid-Isosorbide-5-Nitrae-diene
-3,20-diketone utilizes hydroxyl on Aspergillus ochraceus Aspergillus ochraceus ATCC18500 microorganism to obtain target
Product, such as comparative examples 6 and the combination of comparative examples 4.
The fermentation condition of comparative examples 2,4,6,8 is for Arthrobacter simplex Arthrobacter under different substrates
Simplex AS1.94 C1,2 position dehydrogenation or Aspergillus ochraceus Aspergillus ochraceus ATCC18500 C11
The optimal conditions of hydroxyl on position.Each substep converts under optimization of fermentation conditions, and changing effect is better than of the present invention
Associating transformation fermentation condition (seeing comparative examples 1,3,5,7).Although the fermentation condition of the present invention,
For Arthrobacter simplex Arthrobacter simplex AS1.94 C1,2 position dehydrogenation one-step reaction or Aspergillus ochraceus
Not optimal conditions for hydroxyl one-step reaction on Aspergillus ochraceus ATCC18500 C11 position, but
It is but to be applicable to Arthrobacter simplex Arthrobacter simplex AS1.94 and Aspergillus ochraceus Aspergillus
Ochraceus ATCC18500 combines transformation fermentation.Relative to be used alone above-mentioned two kind bacterium substep carry out on
Hydroxyl reaction and dehydrogenation reaction (such as comparative examples 2 and the combination of comparative examples 8), the present invention is joined by two bacterium
Close fermentation 16 α, 17 α-epoxy progesterone and obtain 16 α, 17 α-epoxy-11 Alpha-hydroxy-pregnant steroid-1,4-diene
-3,20-diketone total recovery improves, and decreases a step culture medium sterilization, decreases the filtration of a step post processing, carries
Take and refined, decrease energy consumption and production cycle, reduce production cost and labour intensity.
Claims (6)
1. with 16 α, 17 α-epoxy progesterone is starting material, utilizes microbial association fermentation preparation 16 α, 17 α-ring
Oxygen-11 Alpha-hydroxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3, the method for 20-diketone, it is characterized in that through Aspergillus ochraceus Aspergillus
Ochraceus ATCC18500 and Arthrobacter simplex Arthrobacter simplex AS1.94 mixing one-step fermentation system
Standby 16 α, 17 α-epoxy-11 Alpha-hydroxy-pregnant steroid-1,4-diene-3,20-diketone.
One the most according to claim 1 is starting material with 16 α, 17 α-epoxy progesterone, utilizes microorganism to join
Close fermentation preparation 16 α, 17 α-epoxy-11 Alpha-hydroxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3, the method for 20-diketone, it is characterized in that 1L
In water, the component of fermentation medium and weight are glucose 20g-30g, sucrose 10g-20g, dried silkworm chrysalis meal 5g-10g,
Corn steep liquor 20g-30g, diammonium hydrogen phosphate 2g-5g, ammonium dihydrogen phosphate 2g-5g, bubble enemy 0.1g-0.4g.
One the most according to claim 2 is starting material with 16 α, 17 α-epoxy progesterone, utilizes microorganism to join
Close fermentation preparation 16 α, 17 α-epoxy-11 Alpha-hydroxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3, the method for 20-diketone, it is characterized in that 1L
In water, the component of fermentation medium and weight are glucose 20g, sucrose 20g, dried silkworm chrysalis meal 5g, corn steep liquor 20g,
Diammonium hydrogen phosphate 5g, ammonium dihydrogen phosphate 2g, bubble enemy 0.2g.
4. it is starting material according to the arbitrary described one of claim 2～3 with 16 α, 17 α-epoxy progesterone, utilizes micro-
Bio combined fermentation preparation 16 α, 17 α-epoxy-11 Alpha-hydroxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3, the method for 20-diketone, it is special
Levy be the pH of fermentation medium be 5.7-6.5.
5. it is starting material according to the arbitrary described one of claims 1 to 3 with 16 α, 17 α-epoxy progesterone, utilizes micro-
Bio combined fermentation preparation 16 α, 17 α-epoxy-11 Alpha-hydroxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3, the method for 20-diketone, it is special
Levy be the inoculum concentration of Arthrobacter simplex be 15%-20%, the inoculum concentration of Aspergillus ochraceus is 25%-30%.
One the most according to claim 5 is starting material with 16 α, 17 α-epoxy progesterone, utilizes microorganism to join
Close fermentation preparation 16 α, 17 α-epoxy-11 Alpha-hydroxy-pregnant steroid-Isosorbide-5-Nitrae-diene-3, the method for 20-diketone, it is characterized in that letter
The inoculum concentration of single-unit bacillus is 15%, and the inoculum concentration of Aspergillus ochraceus is 30%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410779441.6A CN105779556A (en) | 2014-12-15 | 2014-12-15 | Method for preparing 16alpha, 17alpha-epoxy-11alpha-hydroxy-pregna-1,4-diene-3,20-dione through combined microbial fermentation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410779441.6A CN105779556A (en) | 2014-12-15 | 2014-12-15 | Method for preparing 16alpha, 17alpha-epoxy-11alpha-hydroxy-pregna-1,4-diene-3,20-dione through combined microbial fermentation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105779556A true CN105779556A (en) | 2016-07-20 |
Family
ID=56374828
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410779441.6A Pending CN105779556A (en) | 2014-12-15 | 2014-12-15 | Method for preparing 16alpha, 17alpha-epoxy-11alpha-hydroxy-pregna-1,4-diene-3,20-dione through combined microbial fermentation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105779556A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636287A (en) * | 2016-12-30 | 2017-05-10 | 山东宝利甾体生物科技有限公司 | Method for preparing 16,17[alpha]-epoxyprogesterone by virtue of biological process |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101760496A (en) * | 2008-11-06 | 2010-06-30 | 天津金耀集团有限公司 | Biological dehydrogenation preparation method of steroid drug intermediate |
| CN102827913A (en) * | 2012-08-31 | 2012-12-19 | 湖南诺凯生物医药有限公司 | Method for preparing 11 alpha-OH-ADD by mixed fermentation of microbes |
| CN103255194A (en) * | 2013-03-25 | 2013-08-21 | 天津科技大学 | Method for raising conversion rate of 15 alpha, 17 alpha-epoxyprogesterone |
| CN103966299A (en) * | 2014-04-28 | 2014-08-06 | 河北众盛生物科技有限公司 | Preparation method for hydroxylation of 11 alpha of important intermediate of steroidal hormone substance |
-
2014
- 2014-12-15 CN CN201410779441.6A patent/CN105779556A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101760496A (en) * | 2008-11-06 | 2010-06-30 | 天津金耀集团有限公司 | Biological dehydrogenation preparation method of steroid drug intermediate |
| CN102827913A (en) * | 2012-08-31 | 2012-12-19 | 湖南诺凯生物医药有限公司 | Method for preparing 11 alpha-OH-ADD by mixed fermentation of microbes |
| CN103255194A (en) * | 2013-03-25 | 2013-08-21 | 天津科技大学 | Method for raising conversion rate of 15 alpha, 17 alpha-epoxyprogesterone |
| CN103966299A (en) * | 2014-04-28 | 2014-08-06 | 河北众盛生物科技有限公司 | Preparation method for hydroxylation of 11 alpha of important intermediate of steroidal hormone substance |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636287A (en) * | 2016-12-30 | 2017-05-10 | 山东宝利甾体生物科技有限公司 | Method for preparing 16,17[alpha]-epoxyprogesterone by virtue of biological process |
| CN106636287B (en) * | 2016-12-30 | 2021-01-05 | 山东宝利甾体生物科技有限公司 | Method for preparing 16,17 alpha-epoxy progesterone by biological method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102827913B (en) | Method for preparing 11 alpha-OH-ADD by mixed fermentation of microbes | |
| CN101760496A (en) | Biological dehydrogenation preparation method of steroid drug intermediate | |
| CN101659928A (en) | Novel bacteriological culture medium | |
| CN106434401B (en) | A kind of preparation method of yeast strain and ergot yeast powder rich in ergosterol | |
| CN102061320B (en) | Preparation method of 11 alpha,17 alpha-dyhydroxyl-androst-4-ene-3,20-dione | |
| CN102703339A (en) | High-yield arginine deiminase bacterial strain and method for producing L-citrulline by same | |
| CN109234346A (en) | A kind of fermentation process producing vitamin B2 | |
| CN102399702B (en) | Aspergillus niger and application thereof as well as citric acid preparation method through fermentation | |
| CN102533570B (en) | Aspergillus niger, application of Aspergillus niger and method for preparing citric acid by fermentation | |
| CN102851330A (en) | Method for preparing citric acid through fermentation | |
| CN102533877A (en) | Method for preparing citric acid by fermentation | |
| CN102168024B (en) | Meterhizium anisopliae and application thereof | |
| CN106929441A (en) | A kind of cultural method of enterococcus faecalis | |
| CN105779554A (en) | Method for preparing 11alpha, 17alpha-dyhydroxyl-pregn-1,4-diene-3,20-dione through microbial co-fermentation | |
| CN105779555A (en) | 11beta-hydroxy-1,4-diene-3,20-dione steroid prepared through combined fermentation of absidia and arthrobacter | |
| CN102443611B (en) | Production method of citric acid | |
| CN105779556A (en) | Method for preparing 16alpha, 17alpha-epoxy-11alpha-hydroxy-pregna-1,4-diene-3,20-dione through combined microbial fermentation | |
| CN106148203A (en) | A kind of Absidia coerulea and application technology thereof | |
| CN104694609A (en) | Method for improving conversion efficiency of converting phytosterol into androstenedione | |
| CN106754401A (en) | A kind of production method of hirsutella sinensis fungal | |
| CN108085359B (en) | Production method of 11 α -hydroxy-4-ene-3, 17-androstenedione | |
| CN113136358B (en) | Aerobic co-culture probiotics fermentation process for improving ginsenoside yield | |
| CN104862238B (en) | One Accharomyces cerevisiae and its application | |
| CN101760437B (en) | Bread yeast with high nucleic acid content and preparation method thereof | |
| CN106957882A (en) | A kind of method of preparation of citric acid by fermentation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160720 |